565 Particular Shape, Lack Laminations, and Have Rough Surfaces Absence of Obvious Tissue Degeneration, the Cause of Pulpal (Fig

CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 565 particular shape, lack laminations, and have rough surfaces absence of obvious tissue degeneration, the cause of pulpal (Fig. 12-53). Laminated stones appear to grow by the addition calcification is enigmatic. It is often difficult to assign the term of collagen fibrils to their surface, whereas unlaminated stones dystrophic calcification to pulp stones because they so often develop by way of the mineralization of preformed collagen occur in apparently healthy pulps, suggesting that functional fiber bundles. In the latter type, the mineralization front seems stress need not be present for calcification to occur. Calcifica- to extend out along the coarse fibers, making the surface of tion in the mature pulp is often assumed to be related to the the stones appear fuzzy (Fig. 12-54). Often these coarse aging process, but in a study involving 52 impacted canines fiber bundles appear to have undergone hyalinization, thus from patients between 11 and 76 years of age, there was a resembling old scar tissue. constant incidence of concentric denticles for all age groups, Pulp stones may also form around epithelial cells (i.e., rem- indicating no relation to aging.276 Diffuse calcifications, on the nants of Hertwig’s epithelial root sheath). Presumably the epi- other hand, increased in incidence to age 25 years; thereafter thelial remnants induce adjacent mesenchymal stem cells to they remained constant in successive age groups. differentiate into odontoblasts. Characteristically these pulp At times, numerous concentric pulp stones with no appar- stones are found near the root apex and contain dentinal ent cause are seen in all the teeth of young individuals. In such tubules. cases, the appearance of pulp stones may be ascribed to indi- The cause of pulpal calcification is largely unknown. Calci- vidual biologic characteristics (e.g., tori, cutaneous nevi).276 fication may occur around a nidus of degenerating cells, blood Although soft-tissue collagen does not usually calcify, it is thrombi, or collagen fibers. Many authors believe that this common to find calcification occurring in old hyalinized scar represents a form of dystrophic calcification. In this type of tissue in the skin. This may be due to the increase in the extent calcification, calcium is deposited in tissues that are degenerat- of cross-linking between collagen molecules (because increased ing. Calcium phosphate crystals may be deposited within the cross-linkage is thought to enhance the tendency for collagen cells themselves. Initially this takes place within the mitochondria because of the increased membrane permeability to calcium resulting from a failure to maintain active transport systems within the cell membranes. Thus, degenerating cells serving as a nidus may initiate calcification of a tissue. In the

FIG. 12-54 High-power view of a pulp stone from Fig. 12-53, showing the FIG. 12-52 Pulp stones occupying much of the pulp chamber. relationship of mineralization fronts to collagen fibers.

FIG. 12-53 Rough surface form of pulp stone. Note hyalinization of collagen fibers. 566 PART II The Advanced Science of Endodontics

FIG. 12-55 A, Calcific metamorphosis of pulp tissue after luxation of tooth as a result of trauma. Note presence of soft-tissue inclusion. B, High-power view showing cementoblasts (arrows) lining cementum (C), which has been deposited on the dentin walls.

A B

fibers to calcify). A relationship may exist between pathologic the pulp canal is still large enough to instrument. In a classic alterations in collagen molecules within the pulp and pulpal study of luxated teeth, Andreasen7 found that only 7% of the calcification. pulps that underwent calcific metamorphosis exhibited - sec Calcification replaces the cellular components of the ondary infection. Because the success rate for nonsurgical end- pulp and may possibly hinder the blood supply, although odontic therapy, not only in general399 but also for obliterated concrete evidence for this strangulation theory is lacking. Idio- teeth,67 is considered high, prophylactic intervention does not pathic pulpal pain was classically attributed to the presence of seem to be warranted. pulp stones. Modern knowledge of mechanisms of nociceptor activation coupled with the observation that pulp stones are so frequently observed in teeth lacking a history of pain have AGE CHANGES largely discounted this hypothesis. Therefore, from a clinical Continued formation of secondary dentin throughout life grad- perspective, it would be unlikely that a patient’s unexplained ually reduces the size of the pulp chamber and root canals, pain symptoms are due to pulpal calcifications, no matter how although the width of the cementodentinal junction appears dramatic they may appear on a radiograph. to stay relatively the same.109,342 In addition, certain regressive Luxation of teeth as a result of trauma may result in changes in the pulp appear to be related to the aging process calcific metamorphosis, a condition that can, in a matter of (see also Chapter 26). There is a gradual decrease in the cel- months or years, lead to partial or complete radiographic lularity and a concomitant increase in the number and thick- obliteration of the pulp chamber. The cause of radiographic ness of collagen fibers, particularly in the radicular pulp. The obliteration is excessive deposition of mineralized tissue thick collagen fibers may serve as foci for pulpal calcification resembling cementum or, occasionally, bone on the dentin (see Fig. 12-53). The odontoblasts decrease in size and number, walls, also referred to as internal ankylosis (Fig. 12-55). Histo- and they may disappear altogether in certain areas of the pulp, logic examination invariably reveals the presence of some soft particularly on the pulpal floor over the bifurcation or trifurca- tissue, and cells resembling cementoblasts can be observed tion areas of multirooted teeth. lining the mineralized tissue. This calcific metamorphosis With age there is a progressive reduction in the number of of the pulp has also been reported in replanted teeth of nerves102 and blood vessels.23,25 Evidence also suggests that the rat.275 aging results in an increase in the resistance of pulp tissue to Clinically, the crowns of teeth affected by calcific metamor- the action of proteolytic enzymes,420 hyaluronidase, and siali- phosis may show a yellowish hue compared with adjacent dase,25 suggesting an alteration of both collagen and proteogly- normal teeth. This condition usually occurs in teeth with cans in the pulps of older teeth. The main changes in dentin incomplete root formation. Trauma results in disruption of associated with aging are an increase in peritubular dentin, blood vessels entering the tooth, thus producing pulpal dentinal sclerosis, and the number of dead tracts.*342 Dentinal infarction. The wide periapical foramen allows connective sclerosis produces a gradual decrease in dentinal permeability tissue from the periodontal ligament to proliferate and replace as the dentinal tubules become progressively reduced in the infarcted tissue, bringing with it cementoprogenitor and diameter.350 osteoprogenitor cells capable of differentiating into either cementoblasts or osteoblasts or both. When calcific metamorphosis is noted on a patient’s radio- *The term dead tract refers to a group of dentinal tubules in which odontoblast pro- graph, it is sometimes suggested that the tooth be treated cesses are absent. Dead tracts are easily recognized in ground sections because the endodontically because the pulp is expected to be secondarily empty tubules refract transmitted light, and the tract appears black in contrast to the infected, and endodontic therapy should be performed while light color of normal dentin. CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 567

REFERENCES

1. Aars H, Brodin P, Anderson E: A study of cholinergic and 22. Berggreen E, Heyeraas KJ: Role of K+ATP channels, 46. Byers MR, Närhi MVO: Nerve supply of the pulpodentin β-adrenergic components in the regulation of blood flow endothelin A receptors, and effect of angiotensin II on complex and response to injury. In Hargreaves K, Goodis in the tooth pulp and gingiva of man, Acta Physiol Scand blood flow in oral tissues, J Dent Res 82:33,2003. H, editors: Seltzer and Bender’s dental pulp, Chicago, 148:441, 1993. 23. Bernick S, Nedelman C: Effect of aging on the human 2002, Quintessence Publishing. 2. Aars H, Gazelius B, Edwall L, Olgart L: Effects of pulp, J Endod 1:88, 1975. 47. Byers MR, Schatteman GC, Bothwell MA: Multiple autonomic reflexes on tooth pulp blood flow in man, Acta 24. Bevilacqua MP, et al: Interleukin-1 activation of vascular functions for NGF-receptor in developing, aging and Physiol Scand 146:423, 1992. endothelium. Effects on procoagulant activity and injured rat teeth are suggested by epithelial, 3. Accorinte ML, Loguercio AD, Reis A, et al: Response of leukocyte adhesion, Am J Pathol 121:394, 1985. mesenchymal and neural immunoreactivity, Development human dental pulp capped with MTA and calcium 25. Bhussary BR: Modification of the dental pulp organ during 109:461, 1990. hydroxide powder, Oper Dent 33:488, 2008. development and aging. In Finn SB, editor: Biology of the 48. Byers MR, Sugaya A: Odontoblast process in dentin 4. Amess TR, Matthews B: The effect of topical application dental pulp organ: a symposium, Birmingham, 1968, revealed by fluorescent Di-I, J Histochem Cytochem of lidocaine to dentin in the cat on the response of University of Alabama Press. 43:159, 1995. intra-dental nerves to mechanical stimuli. In Shimono M, 26. Biesterfeld RC, Taintor JF, Marsh CL: The significance of 49. Byers MR, Suzuki H, Maeda T: Dental neuroplasticity, Maeda T, Suda H, Takahashi K, editors: Proceedings of alterations of pulpal respiration: a review of the literature, neuro-pulpal interactions and nerve regeneration, Microsc the international conference on dentin/pulp complex, J Oral Pathol 8:129, 1979. Res Tech 60:503, 2003. Tokyo, 1996, Quintessence Publishing. 27. Bisgaard H, Kristensen J: Leukotriene B4 produces 50. Byers MR, Taylor PE: Effect of sensory denervation on the 5. Andelin WE, Shabahang S, Wright K, Torabinejad M: hyperalgesia in humans, Prostaglandins 30:791, 1985. response of rat molar pulp to exposure injury, J Dent Res Identification of hard tissue after experimental pulp 28. Bishop MA, Malhotra MP: An investigation of lymphatic 72:613, 1993. capping using dentin sialoprotein (DSP) as a marker, vessels in the feline dental pulp, Am J Anat 187:247, 51. Byers MR, Taylor PE, Khayat BG, et al: Effects of injury J Endod 29:646, 2003. 1990. and inflammation on pulpal and periapical nerves, 6. Anderson LC, Vakoula A, Veinote R: Inflammatory 29. Bishop MA, Yoshida S: A permeability barrier to J Endod 16:78, 1990. hypersensitivity in a rat model of trigeminal neuropathic lanthanum and the presence of collagen between 52. Byers MR, Westenbroek RE: Odontoblasts in developing, pain, Arch Oral Biol 48:161, 2003. odontoblasts in pig molars, J Anat 181:29, 1992. mature and ageing rat teeth have multiple phenotypes 7. Andreasen JO: Luxation of permanent teeth due to 30. Bletsa A, et al: Cytokine signalling in rat pulp interstitial that variably express all nine voltage-gated sodium trauma: a clinical and radiographic follow-up study fluid and transcapillary fluid exchange during channels, Arch Oral Biol 56:1199, 2011. of 189 injured teeth, Scand J Dent Res 78:273, lipopolysaccharide-induced acute inflammation, J Physiol 53. Byers MR, Wheeler EF, Bothwell M: Altered expression of 1970. 573 (Pt 1):225, 2006. NGF and P75 NGF-receptor by fibroblasts of injured teeth 8. Anneroth G, Norberg KA: Adrenergic vasoconstrictor 31. Bongenhielm U, Haegerstrand A, Theodorsson E, Fried K: precedes sensory nerve sprouting, Growth Factors 6:41, innervation in the human dental pulp, Acta Odontol Scand Effects of neuropeptides on growth of cultivated rat molar 1992. 26:89, 1968. pulp fibroblasts, Regul Pept 60:2391, 1995. 54. Camps J, Pashley DH: In vivo sensitivity to air blasts and 9. Arbuckle JB, Docherty RJ: Expression of tetrodotoxin- 32. Borda E, Furlan C, Orman B, et al: Nitric oxide synthase scratching of human root dentin, J Periodontol 74:1589, resistant sodium channels in capsaicin-sensitive dorsal and PGE2 reciprocal interactions in rat dental pulp: 2003. root ganglion neurons of adult rats, Neurosci Lett 185:70, cholinoceptor modulation, J Endod 33:142, 2007. 55. Camps J, Salomon JP, Van Meerbeek B, et al: Dentin 1995. 33. Botero TM, Shelburne CE, Holland GR, Hanks CT, et al: deformation after scratching with clinically-relevant forces, 10. Artese L, Rubini C, Ferrero G, et al: Vascular endothelial TLR4 mediates LPS-induced VEGF expression in Arch Oral Biol 48:527, 2003. growth factor (VEGF) expression in healthy and inflamed odontoblasts, J Endod 32:951, 2006. 56. Caraivan O, Manolea H, Corlan Puscu D, et al: human dental pulps, J Endod 28:20, 2002. 34. Bowles WR, Flores CM, Jackson DL, Hargreaves KM: beta Microscopic aspects of pulpal changes in patients with 11. Avery JK: Structural elements of the young normal human 2-Adrenoceptor regulation of CGRP release from chronic marginal periodontitis, Rom J Morphol Embryol pulp, Oral Surg Oral Med Oral Pathol 32:113, 1971. capsaicin-sensitive neurons, J Dent Res 82:308, 2003. 53:725, 2012. 12. Awawden L, Lundy FT, Shaw C, et al: Quantitative 35. Brännström M: The transmission and control of dentinal 57. Casasco A, Calligaro A, Casasco M, et al: analysis of substance P, neurokinin A, and calcitonin pain. In Grossman LJ, editor: Mechanisms and control of Immunohistochemical localization of endothelin-like gene-related peptide in pulp tissue from painful and pain, New York, 1979, Masson Publishing USA. immunoreactivity in human tooth germ and mature dental healthy human teeth, Int Endod J 36:30, 2002. 36. Brännström M: Communication between the oral cavity pulp, Anat Embryol (Berl) 183:515, 1991. 13. Banchs F, Trope M: Revascularization of immature and the dental pulp associated with restorative treatment, 58. Chaudhary P, Martenson ME, Baumann TK: Vanilloid permanent teeth with apical periodontitis: new treatment Oper Dent 9:57, 1984. receptor expression and capsaicin excitation of rat protocol? J Endod 30:196, 2004. 37. Brännström M, Aström A: A study of the mechanism of dental primary afferent neurons, J Dent Res 80:1518, 14. Basbaum AI, Fields HL: Endogenous pain control systems: pain elicited from the dentin, J Dent Res 43:619,1964. 2001. brainstem spinal pathways and endorphin circuitry, Ann 38. Breschi L, Lopes M, Gobbi P, et al: Dentin proteoglycans: 59. Chiang CY, Park SJ, Kwan CL, et al: NMDA receptor Rev Neurosci 7:309, 1984. an immunocytochemical FEISEM study, J Biomed Mater mechanisms contribute to neuroplasticity induced in 15. Baume LJ: The biology of pulp and dentine. In Myers HM, Res 61:40, 2002. caudalis nociceptive neurons by tooth pulp stimulation, editor: Monographs in oral science, vol 8, Basel, 1980, 39. Brown AC, Yankowitz D: Tooth pulp tissue pressure and J Neurophysiol 80:2621, 1998. S Karger AG. hydraulic permeability, Circ Res 15:42, 1964. 60. Chu SC, et al: Induction of vascular endothelial growth 16. Beck P, Handwerker H: Bradykinin and serotonin effects 40. Butler WT, D’Sousa RN, Bronckers AL, et al: Recent factor gene expression by proinflammatory cytokines in on various types of cutaneous nerve fibers, Pflugers Arch investigations on dentin specific proteins, Proc Finn Dent human pulp and gingival fibroblasts, J Endod 30:704, 347:209, 1974. Soc 88(suppl 1):369, 1992. 2004. 17. Bender IB, Landau MA, Fonsecca S, Trowbridge HO: The 41. Byers MR: Dynamic plasticity of dental sensory nerve 61. Costos CAS, Hebling J, Hanks CT: Current status of pulp optimum placement-site of the electrode in electric pulp structure and cytochemistry, Arch Oral Biol 39(suppl):13S, capping with dentin adhesive systems: a review, Dent testing of the 12 anterior teeth, J Am Dent Assoc 1994. Mater 16:188, 2000. 118:305, 1989. 42. Byers MR: Neuropeptide immunoreactivity in dental 62. Coure E: Ultrastructural changes during the life cycle of 18. Bergenholtz G: Evidence for bacterial causation of adverse sensory nerves: variations related to primary odontoblast human odontoblasts, Arch Oral Biol 31:643, 1986. pulpal responses in resin-based dental restorations, Crit function and survival. In Shimono M, Takahashi K, editors: 63. Cousins M, Power I: Acute and postoperative pain. In Wall Rev Oral Biol Med 11:467, 2000. Dentin/pulp complex, Tokyo, 1996, Quintessence P, Melzack R, editors: Textbook of pain, Edinburgh, 2002, 19. Berggreen E, Haug SR, Mkony LE, Bletsa A: Publishing. Churchill Livingstone, p 456. Characterization of the dental lymphatic system and 43. Byers MR, Chudler EH, Iadarola MJ: Chronic tooth pulp 64. Cox CF, Bogen G, Kopel HM, Ruby JP: Repair of pulpal identification of cells immunopositive to specific lymphatic inflammation causes transient and persistent expression injury by dental materials, Chap. 14. In Hargreaves K, markers, Eur J Oral Sci 117:34, 2009. of Fos in dynorphin-rich regions of rat brainstem, Brain Goodis H, editors: Seltzer and Bender’s dental pulp, 20. Berggreen E, Heyeraas KJ: The role of sensory Res 861:191, 2000. Chicago, 2002, Quintessence Publishing. neuropeptides and nitric oxide on pulpal blood flow 44. Byers MR, Narhi MV, Mecifi KB: Acute and chronic 65. Cox CF, White KC, Ramus DL, et al: Reparative dentin: and tissue pressure in the ferret, J Dent Res 78:1535, reactions of dental sensory nerve fibers to cavities and factors affecting its deposition, Quintessence Int 23:257, 1999. desiccation in rat molars, Anat Rec 221:872, 1988. 1992. 21. Berggreen E, Heyeraas KJ: Effect of the sensory 45. Byers MR, Narhi MVO: Dental injury models: experimental 66. Csillag M, Berggreen E, Fristad I, et al: Effect of electrical neuropeptide antagonists h-CGRP(8) and SR 140.33 on tools for understanding neuroinflammatory interactions tooth stimulation on blood flow and immunocompetent pulpal and gingival blood flow in ferrets, Arch Oral Biol and polymodal nociceptor functions, Crit Rev Oral Biol cells in rat dental pulp after sympathectomy, Acta Odontol 45:537, 2000. Med 10:4, 1999. Scand 62:305, 2004. 568 PART II The Advanced Science of Endodontics

67. Cvek M, Granath L, Lundberg M: Failures and healing in 93. Firestone AR, Wheatley AM, Thüer UW: Measurement of 117. Gilron I, Max MB, Lee G, et al: Effects of the endodontically treated non-vital anterior teeth with blood perfusion in the dental pulp with laser Doppler 2-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid/ posttraumatically reduced pulpal lumen, Acta Odontol flowmetry, Int J Microcirc Clin Exp 17:298, 1997. kainate antagonist LY293558 on spontaneous and evoked Scand 40:223, 1982. 94. Fish WE: An experimental investigation of enamel, dentine postoperative pain, Clin Pharmacol Ther 68:320, 2000. 68. Dahl E, Major IA: The fine structure of the vessels in the and the dental pulp, London, 1932, John Bale, Sons & 118. Gloe T, Pohl U: Laminin binding conveys mechanosensing human dental pulp, Acta Odontol Scand 31:223, 1973. Danielson. in endothelial cells, News Physiol Sci 17:166, 2002. 69. Dahl T, Sabsay B, Veis A: Type I collagen-phosphophoryn 95. Fisher AK: Respiratory variations within the normal dental 119. Gold M: Tetrodotoxin-resistant Na currents and interactions: specificity of the monomer-monomer binding, pulp, J Dent Res 46:424, 1967. inflammatory hyperalgesia, Proc Natl Acad Sci U S A J Struct Biol 123:162, 1998. 96. Fisher AK, Schumacher ER, Robinson NR, Sharbondy GP: 96:7645, 1999. 70. Dallel R, Clavelou P, Woda A: Effects of tractotomy on Effects of dental drugs and materials on the rate of 120. Gold MS, Reichling DB, Shuster MJ, Levine JD: nociceptive reactions induced by tooth pulp stimulation in oxygen consumption in bovine dental pulp, J Dent Res Hyperalgesic agents increase a tetrodotoxin-resistant Na+ the rat, Exp Neurol 106:78, 1989. 36:447, 1957. current in nociceptors, Proc Natl Acad Sci U S A 93:1108, 71. Davidson RM: Neural form of voltage-dependent sodium 97. Fisher AK, Walters VE: Anaerobic glycolysis in bovine 1996. current in human cultured dental pulp cells, Arch Oral Biol dental pulp, J Dent Res 47:717, 1968. 121. Goldberg M, Farges J-C, Lacerda-Pinheiro S, et al: 39:613, 1994. 98. Fitzgerald M, Chiego DJ, Heys DR: Autoradiographic Inflammatory and immunological aspects of dental pulp 72. Diamond J: The effect of injecting acetylcholine into analysis of odontoblast replacement following pulp repair, Pharmacol Res 58:137, 2008. normal and regenerating nerves, J Physiol (Lond) exposure in primate teeth, Arch Oral Biol 35:707, 122. Goldberg M, Six N, Decup F, et al: Bioactive molecules 145:611, 1959. 1990. and the future of pulp therapy, Am J Dent 16:66, 2003. 73. Diamond RD, Stanley HR, Swerdlow H: Reparative dentin 99. Follenfant R, Nakamura-Craig M, Henderson B, Higgs GA: 123. Goldberg M, Takagi M: Dentine proteoglycans: formation resulting from cavity preparation, J Prosthet Inhibition by neuropeptides of interleukin-1ß-induced, composition, ultrastructure and functions, Histochem J Dent 16:1127, 1966. prostaglandin-independent hyperalgesia, Br J Pharmacol 25:781, 1993. 74. Diogenes A, Akopian AN, Hargreaves KM: NGF 98:41, 1989. 124. Goodis H, Bowles W, Hargreaves K: Prostaglandin E2 upregulates TRPA1: implications for orofacial pain, J Dent 100. Fouad A: Molecular mediators of pulpal inflammation. In enhances bradykinin-evoked iCGRP release in bovine Res 86:550, 2007. Hargreaves KM, Goodis HE, editors: Seltzer and Bender’s dental pulp, J Dent Res 79:1604, 2000. 75. Dionne R: Oral sedation, Compend Contin Educ Dent dental pulp, Chicago, 2002, Quintessence Publishing, 125. Gordon SM, Dionne RA, Brahim J, et al: Blockade of 19:868, 1998. p 247. peripheral neuronal barrage reduces postoperative pain, 76. Dray A: Inflammatory mediators of pain, Br J Anaesth 101. Fraser JR, Kimpton WG, Laurent TC, et al: Uptake and Pain 70:209, 1997. 75:125, 1995. degradation of hyaluronan in lymphatic tissue, Biochem J 126. Gotjamanos T: Cellular organization in the 77. D’Souza RN, Bachman T, Baumgardner KR, et al: 256:153, 1988. subodontoblastic zone of the dental pulp. II. Period and Characterization of cellular responses involved in reparative 102. Fried K: Changes in pulp nerves with aging. Proc Finn mode of development of the cell-rich layer in rat molar dentinogenesis in rat molars, J Dent Res 74:702, 1995. Dent Soc 88(suppl 1):517, 1992. pulps, Arch Oral Biol 14:1011, 1969. 78. Dworkin SF: Anxiety and performance in the dental 103. Fried K, Sessle BJ, Devor M: The paradox of pain from 127. Gould HJ, England JD, Soignier RD, et al: Ibuprofen

environment: an experimental investigation, J Am Soc tooth pulp: low-threshold “algoneurons”? Pain 152:2685, blocks changes in Nav1.7 and 1.8 sodium channels Psychosom Dent Med 14:88, 1967. 2011. associated with complete Freund’s adjuvant-induced 79. Ebihara A, Tokita Y, Izawa T, Suda H: Pulpal blood flow 104. Fristad I, Heyeraas KJ, Kvinnsland I: Nerve fibres and cells inflammation in rat, J Pain 5:270, 2004. assessed by laser Doppler flowmetry in a tooth with a immunoreactive to neurochemical markers in developing 128. Gould HJ 3rd, Gould TN, Paul D, et al: Development of horizontal root fracture, Oral Surg Oral Med Oral Path rat molars and supporting tissues, Arch Oral Biol 39:633, inflammatory hypersensitivity and augmentation of sodium 81:229, 1996. 1994. channels in rat dorsal root ganglia, Brain Res 824:296, 80. Eda S, Saito T: Electron microscopy of cells displaced into 105. Fristad I, Jacobsen EB, Kvinnsland IH: Coexpression of 1999. the dentinal tubules due to dry cavity preparation, J Oral vasoactive intestinal polypeptide and substance P in 129. Gregg JM, Dixon AD: Somatotopic organization of the Pathol 7:326, 1978. reinnervating pulpal nerves and in trigeminal ganglion trigeminal ganglion, Arch Oral Biol 18:487, 1973. 81. Edwall B, Gazelius B, Fazekas A, et al: Neuropeptide Y neurones after axotomy of the inferior alveolar nerve in 130. Grossman ES, Austin JC: Scanning electron microscope (NPY) and sympathetic control of blood flow in oral the rat, Arch Oral Biol 43:183, 1998. observations on the tubule content of freeze-fractured mucosa and dental pulp in the cat, Acta Physiol Scand 106. Fristad I, Kvinnsland IH, Jonsson R, Heyeraas KJ: Effect of peripheral vervet monkey dentine (Cercopithecus 125:253, 1985. intermittent long-lasting electrical tooth stimulation on pygerythrus), Arch Oral Biol 28:279, 1983. 82. Edwall L, Kindlová M: The effect of sympathetic nerve pulpal blood flow and immunocompetent cells: a 131. Gunji T: Morphological research on the sensitivity of stimulation on the rate of disappearance of tracers from hemodynamic and immunohistochemical study in young dentin, Arch Histol Jpn 45:45, 1982. various oral tissues, Acta Odontol Scand 29:387, 1971. rat molars, Exp Neurol 146:230, 1997. 132. Hagermark O, Hokfelt T, Pernow B: Flare and itch 83. Embery G: Glycosaminoglycans of human dental pulp, 107. Fristad I, Vandevska-Radunovic V, Fjeld K, et al: NK1, produced by substance P in human skin, J Invest J Biol Buccale 4:229, 1976. NK2, NK3 and CGRP1 receptors identified in rat oral soft Dermatol 71:233, 1979. 84. Embery G, Hall R, Waddington R, et al: Proteoglycans in tissues, and in bone and dental hard tissue cells, Cell 133. Hahn C-L, Falkler WA Jr, Siegel MA: A study of T cells dentinogenesis, Crit Rev Oral Biol Med 12:331, 2001. Tissue Res 311:383, 2003. and B cells in pulpal pathosis, J Endod 15:20, 1989. 85. England MC, Pellis EG, Michanowicz AE: Histopathologic 108. Fuss Z, Trowbridge H, Bender IB, et al: Assessment of 134. Hals E, Tonder KJ: Elastic pseudoelastic tissue in study of the effect of pulpal disease upon nerve fibers of reliability of electrical and thermal pulp testing agents, arterioles of the human and dog dental pulp, Scand the human dental pulp, Oral Surg Oral Med Oral Pathol J Endod 12:301, 1986. J Dent Res 89:218, 1981. 38:783, 1974. 109. Gani O, Visvisian C: Apical canal diameter in the first 135. Hamersky PA, Weimer AD, Taintor JF: The effect of 86. England S: Molecular basis of peripheral hyperalgesia. In upper molar at various ages, J Endod 10:689, 1999. orthodontic force application on the pulpal tissue Wood J, editor: Molecular basis of pain induction, ed 1, 110. Garant PR: The organization of microtubules within rat respiration rate in the human premolar, Am J Orthod New York, 2000, Wiley-Liss, p 261. odontoblast processes revealed by perfusion fixation with 77:368, 1980. 87. Engström C, Linde A, Persliden B: Acid hydrolases in the glutaraldehyde, Arch Oral Biol 17:1047, 1972. 136. Han SS: The fine structure of cells and intercellular odontoblast-predentin region of dentinogenically active 111. Garberoglio R, Brännström M: Scanning electron substances of the dental pulp. In Finn SB, editor: Biology teeth, Scand J Dent Res 84:76, 1976. microscopic investigation of human dentinal tubules, Arch of the dental pulp organ, Birmingham, 1968, University of 88. Evans D, Reid T, Strang R, Stirrups D: A comparison of Oral Biol 21:355, 1976. Alabama Press, p 103. laser Doppler flowmetry with other methods of assessing 112. Gedney JJ, Logan H, Baron RS: Predictors of short-term 137. Hargreaves KM: Pain mechanisms of the pulpodentin vitality in traumatized anterior teeth, Endod Dent and long-term memory of sensory and affective complex. In Hargreaves KM, Goodis HE, editors: Seltzer Traumatol 15:284, 1999. dimensions of pain, J Pain 4:47, 2003. and Bender’s dental pulp, Chicago, 2002, Quintessence 89. Fava L: A comparison of one versus two appointment 113. George CH, Kendall JM, Evans WH: Intracellular trafficking Publishing Company, p 181. endodontic therapy in teeth with non-vital pulps, Int pathways on assembly of connexins into tight junctions, 138. Hargreaves KM, Bowles WR, Jackson DL: Intrinsic Endod J 22:179, 1989. J Biol Chem 274:8678, 1999. regulation of CGRP release by dental pulp sympathetic 90. Fearnhead RW: Innervation of dental tissues. In Miles 114. Gerli R, Secciani I, Sozio F, et al: Absence of lymphatic fibers, J Dent Res 82:398, 2003. AEW, editor: Structural and chemical organization of the vessels in human dental pulp: a morphological study, Eur 139. Hargreaves KM, Dionne RA, Mueller GP, et al: Naloxone, teeth, vol 1, New York, 1967, Academic Press. J Oral Sci 118:110, 2010. fentanyl, and diazepam modify plasma beta-endorphin 91. Ferrara N: Vascular endothelial growth factor, Eur J 115. Gibbs JL, Hargreaves KM: Neuropeptide Y Y1 receptor levels during surgery, Clin Pharmacol Ther 40:165, Cancer 32A:2413, 1996. effects on pulpal nociceptors, J Dent Res 87:948, 2008. 1986. 92. Finkelman RD, Mohan S, Jennings JC, et al: Quantitation 116. Gilbert TM, Pashley DH, Anderson RW: Response of 140. Hargreaves KM, Keiser K: Local anesthetic failure in of growth factors IGF-1, SGF/IGF-11 and TGF-b in human pulpal blood flow to intra-arterial infusion of endothelin, endodontics: mechanisms and management, Endod dentin, J Bone Miner Res 5:717, 1990. J Endod 18:228, 1992. Topics 1:26, 2002. CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 569

141. Hargreaves KM, Milam SB: Mechanisms of pain and 165. Jacobsen EB, Heyeraas KJ: Pulp interstitial fluid pressure and neuronal co-expression with neuropeptide Y following analgesia. In Dionne R, Phero J, editors: Management of and blood flow after denervation and electrical tooth inferior alveolar nerve axotomy in the rat, J Peripher Nerv pain and anxiety in dental practice, New York, 2001, stimulation in the ferret, Arch Oral Biol 42:407, 1997. Syst 3:79, 1998. Elsevier, p 18. 166. Janig W, Kollman W: The involvement of the sympathetic 187. Kim S: Regulation of pulpal blood flow, J Dent Res 142. Hargreaves KM, Swift JQ, Roszkowski MT, et al: nervous system in pain, Fortschr Arzneimittelforsch 64(Spec No):590, 1985. Pharmacology of peripheral neuropeptide and 34:1066, 1984. 188. Kim S, Dorscher-Kim JE, Liu M: Microcirculation of the inflammatory mediator release, Oral Surg Oral Med Oral 167. Jebeles JA, Reilly JS, Gutierrez JF, et al: Tonsillectomy dental pulp and its autonomic control, Proc Finn Dent Soc Pathol 78:503, 1994. and adenoidectomy pain reduction by local bupivacaine 85:279, 1989. 143. Harris R, Griffin CJ: Fine structure of nerve endings in the infiltration in children, Int J Ped Otorhinolaryngology 189. Kim S, Dorscher-Kim JE, Liu M, et al: Functional human dental pulp, Arch Oral Biol 13:773, 1968. 25:149, 1993. alterations in pulpal microcirculation in response to 144. Hartmann A, Azerad J, Boucher Y: Environmental effects 168. Jeske NA, Diogenes A, Ruparel NB, et al: A-kinase various dental procedures and materials, Proc Finn Dent on laser Doppler pulpal blood-flow measurements in man, anchoring protein mediates TRPV1 thermal hyperalgesia Soc 88(suppl 1):65, 1992. Arch Oral Biol 41:333, 1996. through PKA phosphorylation of TRPV1, Pain 138:604, 190. Kim S, Edwall L, Trowbridge H, Chien S: Effects of local 145. Hashioka K, Suzuki K, Yoshida T, et al: Relationship 2008. anesthetics on pulpal blood flow in dogs, J Dent Res between clinical symptoms and enzyme-producing bacteria 169. Johnsen D, Johns S: Quantitation of nerve fibers in the 63:650, 1984. isolated from infected root canals, J Endod, 20:75, 1994. primary and permanent canine and incisor teeth in man, 191. Kim S, Liu M, Simchon S, et al: Effects of selected 146. Hattler AB, Listgarten MA: Pulpal response to root planing Arch Oral Biol 23:825, 1978. inflammatory mediators on blood flow and vascular in a rat model, J Endod 10:471, 1984. 170. Johnsen DC, Harshbarger J, Rymer HD: Quantitative permeability in the dental pulp, Proc Finn Dent Soc 147. Haug SR, Heyeraas KJ: Effects of sympathectomy on assessment of neural development in human premolars, 88(suppl 1):387, 1992. experimentally induced pulpal inflammation and periapical Anat Rec 205:421, 1983. 192. Kim S, Schuessler G, Chien S: Measurement lesions in rats, Neuroscience 120:827, 2003. 171. Johnson G, Brännström M: The sensitivity of dentin: of blood flow in the dental pulp of dogs with the 148. Haug SR, Heyeraas KJ: Modulation of dental inflammation changes in relation to conditions at exposed tubule 133xenon washout method, Arch Oral Biol 28:501, by the sympathetic nervous system, J Dent Res 85:488, apertures, Acta Odontol Scand 32:29, 1974. 1983. 2006. 172. Jones PA, Taintor JF, Adams AB: Comparative dental 193. Kim S, Trowbridge HO, Dorscher-Kim JE: The influence of 149. Hermanstyne TO, Markowitz K, Fan L, Gold MS: material cytotoxicity measured by depression of rat incisor 5-hydroxytryptamine (serotonin) on blood flow in the dog Mechanotransducers in rat pulpal afferents, J Dent Res pulp respiration, J Endod 5:48, 1979. pulp, J Dent Res 65:682, 1986. 87:834, 2008. 173. Jontell M, Okiji T, Dahlgren U, Bergenholtz G: Immune 194. Kim SK, Ang L, Hsu YY, et al: Antagonistic effect of 150. Heyeraas KJ: Pulpal hemodynamics and interstitial fluid defense mechanisms of the dental pulp, Crit Rev Oral Biol D-myo-inositol-1,2,6-trisphosphate (PP56) on pressure: balance of transmicrovascular fluid transport, Med 9:179, 1998. neuropeptide Y-induced vasoconstriction in the feline J Endod 15:468, 1989. 174. Juan H, Lembeck F: Action of peptides and other dental pulp, Arch Oral Biol 41:791, 1996. 151. Heyeraas KJ, Berggreen E: Interstitial fluid pressure in analgesic agents on paravascular pain receptors of the 195. Kimberly CL, Byers BR: Inflammation of rat molar pulp normal and inflamed pulp, Crit Rev Oral Biol Med 10:328, isolated perfused rabbit ear, Naunyn Schmiedebergs Arch and periodontium causes increased calcitonin-gene- 1999. Pharmacol 283:151, 1974. related peptide and axonal sprouting, Anat Rec 222:289, 152. Heyeraas KJ, Jacobsen EB, Fristad I: Vascular and 175. Katoh Y, Yamaguchi R, Shinkai K, et al: Clinicopathological 1988. immunoreactive nerve fiber reactions in the pulp after study on pulp-irritation of adhesive resinous materials 196. Kramer IRH: The distribution of blood vessels in the stimulation and denervation: proceedings of the (report 3). Direct capping effects on exposed pulp of human dental pulp. In Finn SB, editor: Biology of the International Conference, in Dentin/Pulp Complex. Macaca fascicularis, Jpn J Conserv Dent 40:163, 1997. dental pulp organ, Birmingham, 1968, University of Shimono M, Maeda T, Suda H, Takahashi K, editors. 176. Kaufman E, Hargreaves KM, Dionne RA: Comparison of Alabama Press, p 361. Tokyo, 1996, Quintessence Publishing, p 162. oral triazolam and nitrous oxide with placebo and 197. Kress M, Sommer C: Neuroimmunology and pain: 153. Heyeraas KJ, Kim S, Raab WH, et al: Effect of electrical intravenous diazepam for outpatient premedication, Oral peripheral effects of proinflammatory cytokines. In Brune tooth stimulation on blood flow, interstitial fluid pressure Surg Oral Med Oral Pathol 75:156, 1993. K, Handwerker H, editors: Hyperalgesia: molecular and substance P and CGRP-immunoreactive nerve fibers 177. Kayaoglu G, Orstavik D: Virulence factors of Enterococcus mechanisms and clinical implications, Seattle, WA, 2003, in the low compliant cat dental pulp, Microvasc Res faecalis: relationship to endodontic disease, Crit Rev Oral IASP Press, p 57. 47:329, 1994. Biol Med 15:308, 2004. 198. Kroeger DC, Gonzales F, Krivoy W: Transmembrane 154. Heyeraas KJ, Kvinnsland I: Tissue pressure and blood flow 178. Kaye H, Herold RC: Structure of human dentine. I. Phase potentials of cultured mouse dental pulp cells, Proc Soc in pulpal inflammation, Proc Finn Dent Soc 88(suppl contrast, polarization, interference, and bright field Exp Biol Med 108:134, 1961. 1):393, 1992. microscopic observations on the lateral branch system, 199. Kumazawa T, Mizumura K: Thin-fiber receptors 155. Hikiji A, Yamamoto H, Sunakawa M, Suda H: Increased Arch Oral Biol 11:355, 1966. responding to mechanical, chemical and thermal blood flow and nerve firing in the cat canine tooth in 179. Kelley KW, Bergenholtz G, Cox CF: The extent of the stimulation in the skeletal muscle of the dog, Arch Physiol response to stimulation of the second premolar pulp, Arch odontoblast process in rhesus monkeys (Macaca mulatta) 273:179, 1977. Oral Biol 45:53, 2000. as observed by scanning electron microscopy, Arch Oral 200. Kvinnsland IH, Luukko K, Fristad I, et al: Glial cell 156. Hill R: NK1 (substance P) receptor antagonists—why are Biol 26:893, 1981. line-derived neurotrophic factor (GDNF) from adult rat they not analgesic in humans? [see comment], Trends 180. Kerezoudis NP, Funato A, Edwall L, et al: Activation of tooth serves a distinct population of large-sized trigeminal Pharmacol Sci 21:244, 2000. sympathetic nerves exerts an inhibitory influence on neurons, Eur J Neurosci 19:2089, 2004. 157. Hirvonen T, Närhi M, Hakumäki M: The excitability of dog afferent nerve-induced vasodilation unrelated to 201. Langeland K, Langeland LK: Histologic study of 155 pulp nerves in relation to the condition of dentine surface, vasoconstriction in rat dental pulp, Acta Physiol Scand impacted teeth, Odontol Tidskr 73:527, 1965. J Endod 10:294, 1984. 147:27, 1993. 202. Langeland K, Langeland LK: Pulp reactions to cavity and 158. Holland GR: The extent of the odontoblast process in the 181. Kerezoudis NP, Olgart L, Edwall L: CGRP(8) reduces the crown preparations, Aust Dent J 15:261, 1970. cat, Arch Anat 121:133, 1976. duration but not the maximal increase of antidromic 203. Lantelme RL, Handleman SL, Herbison RJ: Dentin 159. Holland GR: The odontoblast process: form and function, vasodilation in dental pulp and lip of the rat, Acta Physiol formation in periodontally diseased teeth, J Dent Res J Dent Res 64(special issue):499, 1985. Scand 151:73, 1994. 55:48, 1976. 160. Holland GR: Morphological features of dentine and pulp 182. Kerezoudis NP, Olgart L, Edwall L: Involvement of 204. Latremoliere A, Woolf CJ: Central sensitization: a related to dentine sensitivity, Arch Oral Biol 39(suppl):3S, substance P but not nitric oxide or calcitonin gene-related generator of pain hypersensitivity by central neural 1994. peptide in neurogenic plasma extravasation in rat incisor plasticity, J Pain 10:895, 2009. 161. Ianiro SR, Jeansonne BG, McNeal SF, Eleazer PD: The pulp and lip, Arch Oral Biol 39:769, 1994. 205. Laurent TC, et al: The catabolic fate of hyaluronic acid, effect of preoperative acetaminophen or a combination of 183. Khan A, Diogenes A, Jeske N, et al: Tumor necrosis factor Connect Tissue Res 15:33, 1986. acetaminophen and ibuprofen on the success of inferior alpha enhances the sensitivity of trigeminal ganglion 206. Lechner JH, Kalnitsky G: The presence of large alveolar nerve block for teeth with irreversible pulpitis, neurons to capsaicin, Neuroscience 155:503, 2008. amounts of type III collagen in bovine dental pulp J Endod 33:11, 2007. 184. Khan AA, McCreary B, Owatz CB, et al: The development and its significance with regard to the mechanism of 162. Ibricevic H, Heyeraas KJ, Pasic Juhas E, et al: of a diagnostic instrument for the measurement of dentinogenesis, Arch Oral Biol 26:265, 1981. Identification of alpha 2 adrenoceptors in the blood mechanical allodynia, J Endod 33:663, 2007. 207. Lepinski AM, Hargreaves KM, Goodis HE, Bowles WR: vessels of the dental pulp, Int Endod J 24:279, 1991. 185. Khan AA, Owatz CB, Schindler WG, et al: Measurement of Bradykinin levels in dental pulp by microdialysis, J Endod 163. Ikeda H, Tokita Y, Suda H: Capsaicin-sensitive A fibers in mechanical allodynia and local anesthetic efficacy in 26:744, 2000. cat tooth pulp, J Dent Res 76:1341, 1997. patients with irreversible pulpitis and acute periradicular 208. Lesot H, Osman M, Ruch JV: Immunofluorescent 164. Inoue H, Kurosaka Y, Abe K: Autonomic nerve endings in periodontitis, J Endod 33:796, 2007. localization of collagens, fibronectin and laminin during the odontoblast/predentin border and predentin of the 186. Khullar SM, Fristad I, Brodin P, Kvinnsland IH: terminal differentiation of odontoblasts, Dev Biol 82:371, canine teeth of dogs, J Endod 18:149, 1992. Upregulation of growth associated protein 43 expression 1981. 570 PART II The Advanced Science of Endodontics

209. Levi-Montalcini R: The nerve growth factor: its mode of 233. Maixner W, Dubner R, Kenshalo DR Jr, et al: Responses placebo premedication therapy on the depth of anesthesia action on sensory and sympathetic nerve cells, Harvey of monkey medullary dorsal horn neurons during the during treatment of inflamed teeth, Oral Surg Oral Med Lect 60:217, 1966. detection of noxious heat stimuli, J Neurophysiol 62:437, Oral Pathol 102:399, 2006. 210. Levine J, Moskowitz M, Basbaum A: The contribution of 1989. 255. Mullaney TP, Howell RM, Petrich JD: Resistance of nerve neurogenic inflammation in experimental arthritis, J 234. Malmberg AB, Yaksh TL: Spinal nitric oxide synthesis fibers to pulpal necrosis, Oral Surg 30:690, 1970. Immunol 135:843, 1985. inhibition blocks NMDA-induced thermal hyperalgesia and 256. Murray PE, About I, Lumley PJ, et al: Human odontoblast 211. Levine J, Taiwo Y: Inflammatory pain. In Wall P, Melzack produces antinociception in the formalin test in rats, Pain cell numbers after dental injury, J Dent 28:277, 2000. R, editors: Textbook of pain, Edinburgh, 1994, 54:291, 1993. 257. Murray PE, Hafez AA, Windsor LJ, et al: Comparison of Churchill-Livingston. 235. Malmberg AB, Yaksh TL: Cyclooxygenase inhibition and pulp responses following restoration of exposed and 212. Levine JD, Gordon NC, Fields HL: The mechanism of the spinal release of prostaglandin E2 and amino acids non-exposed cavities, J Dent 30:213, 2002. placebo analgesia, Lancet 2:654, 1978. evoked by paw formalin injection: a microdialysis study in 258. Murray PE, Lumley PJ, Ross HF, Smith AJ: Tooth slice 213. Lewin G, Rueff A, Mendell L: Peripheral and central unanesthetized rats, J Neurosci 15:2768, 1995. organ culture for cytotoxicity assessment of dental mechanisms of NGF-induced hyperalgesia, Eur J Neurosci 236. Maltos KL, Menezes GB, Caliari MV, et al: Vascular and materials, Biomaterials 21:1711, 2000. 6:1903, 1994. cellular responses to pro-inflammatory stimuli in rat dental 259. Naftel JP, et al: Course and composition of the nerves 214. Light A: The initial processing of pain and its descending pulp, Arch Oral Biol 49:443, 2004. that supply the mandibular teeth of the rat, Anat Rec control: spinal and trigeminal systems, Basel, 1992, 237. Mangkornkarn C, Steiner JC: In vivo and in vitro 256:433, 1999. Karger. glycosaminoglycans from human dental pulp, J Endod 260. Nagaoka S, Miyazaki Y, Liu HJ, et al: Bacterial invasion 215. Lilja J: Innervation of different parts of the predentin and 18:327, 1992. into dentinal tubules of human vital and nonvital teeth, dentin in a young human premolar, Acta Odontol Scand 238. Marbach JJ, Raphael KG: Phantom tooth pain: a new look J Endod 21:70, 1995. 37:339, 1979. at an old dilemma. Pain Med 1:68, 2000. 261. Nair PN, et al: Histological, ultrastructural and quantitative 216. Lilja J, Noredenvall K-J, Brännström M: Dentin sensitivity, 239. Marfurt CF, Zaleski EM, Adams CE, Welther CL: investigations on the response of healthy human pulps to odontoblasts and nerves under desiccated or infected Sympathetic nerve fibers in rat orofacial and cerebral experimental capping with mineral trioxide aggregate: a experimental cavities, Swed Dent J 6:93, 1982. tissues as revealed by the HRP-WGA tracing technique: a randomized controlled trial, Int Endod J 41:128, 2008. 217. Lilja JJ, Kivisto KT, Backman JT, Neuvonen PJ: Effect of light and electron microscopic study, Brain Res 366:373, 262. Nakanishi T, Shimizu H, Hosokawa Y, et al: An grapefruit juice dose on grapefruit juice-triazolam 1986. immunohistological study on cyclooxygenase-2 in human interaction: repeated consumption prolongs triazolam 240. Marion D, Jean A, Hamel H, et al: Scanning electron dental pulp, J Endod 27:385, 2001. half-life, Eur J Clin Pharmacol 56:411, 2000. microscopic study of odontoblasts and circumferential 263. Närhi M: Activation of dental pulp nerves of the cat and 218. Lindahl O: Pain-a chemical explanation, Acta Rheumatol dentin in a human tooth, Oral Surg Oral Med Oral Pathol the dog with hydrostatic pressure, Proc Finn Dent Soc Scand 8:161, 1962. 72:473, 1991. 74(suppl 5):1, 1978. 219. Linde A: A study of the dental pulp glycosamino-glycans 241. Marriott D, Wilkin GP, Coote PR, Wood JN: Eicosanoid 264. Narhi M: The characteristics of intradental sensory units from permanent human teeth and rat and rabbit incisors, synthesis by spinal cord astrocytes is evoked by and their responses to stimulation, J Dent Res 64:564, Arch Oral Biol 18:49, 1973. substance P; possible implications for nociception and 1985. 220. Linde A: The extracellular matrix of the dental pulp and pain, Adv Prostaglandin Thromboxane Leukot Res 265. Narhi M, Jyväsjärvi E, Virtanen A, et al: Role of intradental dentin, J Dent Res 64(special issue):523, 1985. 21B:739, 1991. A and C type nerve fibers in dental pain mechanisms, 221. Linden GJ, Curtis TM, About I, et al: Human odontoblasts 242. Martin A, Gasse H, Staszyk C: Absence of lymphatic Proc Finn Dent Soc 88:507, 1992. express functional thermo-sensitive TRP channels: vessels in the dog dental pulp: an immunohistochemical 266. Närhi M, Jyväsjärvi E, Hirronen T: Activation of implications for dentin sensitivity, Pain 152: 2211-2223, study, J Anat 217:609, 2010. heat-sensitive nerve fibers in the dental pulp of the cat, 2011. 243. Martin H, Basbaum AI, Kwiat GC, et al: Leukotriene and Pain 14:317, 1982. 222. Liu L, Simon SA: Capsaicin, acid and heat-evoked prostaglandin sensitization of cutaneous high-threshold 267. Närhi M, Virtanen A, Kuhta J, Huopaniemi T: Electrical currents in rat trigeminal ganglion neurons: relationship to C- and A-delta mechanoreceptors in the hairy skin of rat stimulation of teeth with a pulp tester in the cat, Scand functional VR1 receptors, Physiol Behav 69:363, 2000. hindlimbs, Neuroscience 22:651, 1987. J Dent Res 87:32, 1979. 223. Lohinai Z, Balla I, Marczis J, et al: Evidence for the role of 244. Matsuo S, Ichikawa H, Henderson TA, et al: trkA 268. Närhi M, Yamamoto H, Ngassapa D: Function of nitric oxide in the circulation of the dental pulp, J Dent modulation of developing somatosensory neurons in intradental nociceptors in normal and inflamed teeth. In Res 74:1501, 1995. oro-facial tissues: tooth pulp fibers are absent in trkA Shimono M, Maeda T, Suda H, Takahashi K, editors: 224. Lohinai Z, Szekely AD, Benedek P, Csillag A: Nitric oxide knockout mice, Neuroscience 105:747, 2001. Dentin/pulp complex, Tokyo, 1996, Quintessence synthetase containing nerves in the cat and dog dental 245. Matthews B, Andrew D: Microvascular architecture and Publishing, p 136. pulps and gingiva, Neurosci Lett 227:91, 1997. exchange in teeth, Microcirculation 2:305, 1995. 269. Närhi M, Yamamoto H, Ngassapa D, Hirvonen T: The 225. Lundberg JM, Änggård A, Fahrenkrug J, et al: Vasoactive 246. Matthews B, Vongsavan N: Interactions between neural neurophysiological basis and the role of inflammatory intestinal polypeptide in cholinergic neurons of exocrine and hydrodynamic mechanisms in dentine and pulp, Arch reactions in dentine hypersensitivity, Arch Oral Biol glands: functional significance of coexisting transmitters Oral Biol 39(suppl 1):87S, 1994. 39(suppl):23S, 1994. for vasodilation and secretion, Proc Natl Acad Sci U S A 247. McGrath PA, Gracely RH, Dubner R, Heft MW: Non-pain 270. Nassar M, Stirling L, Forlani G, et al: Nociceptor-specific 77:1651, 1980. and pain sensations evoked by tooth pulp stimulation, gene deletion reveals a major role for NAv 1.7 (PN1) in 226. Lundberg JM, Fried G, Fahrenkrug J, et al: Subcellular Pain 15:377, 1983. acute and inflammatory pain, Proc Natl Acad Sci U S A fractionation of cat submandibular gland: comparative 248. Meller ST, Dykstra C, Gebhart GF: Production of 101:12706, 2004. studies on the distribution of acetylcholine and vasoactive endogenous nitric oxide and activation of soluble 271. Nawroth PP, Stern DM: Modulation of endothelial cell intestinal polypeptide (VIP), Neuroscience 6:1001, 1981. guanylate cyclase are required for N-methyl-D-aspartate- hemostatic properties by tumor necrosis factor, J Exp 227. Lundgren T, Nannmark U, Linde A: Calcium ion activity produced facilitation of the nociceptive tail-flick reflex, Eur Med 163:740, 1986. and pH in the odontoblast-predentin region: ion-selective J Pharmacol 214:93, 1992. 272. Neumann S, Doubell TP, Leslie T, Woolf CJ: Inflammatory microelectrode measurements, Calcif Tissue Int 50:134, 249. Meyer MW, Path MG: Blood flow in the dental pulp of pain hypersensitivity mediated by phenotype switch in 1992. dogs determined by hydrogen polarography and myelinated primary sensory neurons, Nature 384:360, 228. Luthman J, Luthman D, Hökfelt T: Occurrence and radioactive microsphere methods, Arch Oral Biol 24:601, 1996. distribution of different neurochemical markers in the 1979. 273. Ngassapa D, Närhi M, Hirvonen T: The effect of serotonin human dental pulp, Arch Oral Biol 37:193, 1992. 250. Meyer R, Campbell J: Myelinated nociceptive afferents (5-HT) and calcitonin gene-related peptide (CGRP) on the 229. Madison S, Whitsel EA, Suarez-Roca H, Maixner W: account for the hyperalgesia that follows a burn to the function of intradental nerves in the dog, Proc Finn Dent Sensitizing effects of leukotriene B4 on intradental primary hand, Science 213:1527, 1981. Soc 88(suppl 1):143, 1992. afferents, Pain 49:99, 1992. 251. Mirza AF, Mo J, Holt JL, et al: Is there a relationship 274. Nicol GD, Vasko MR: Unraveling the story of NGF- 230. Maeda T, Honma S, Takano Y: Dense innervation of between throbbing pain and arterial pulsations? mediated sensitization of nociceptive sensory neurons: ON radicular human dental pulp as revealed by J Neurosci 32:7572, 2012. or OFF the Trks? Mol Interv 7:26, 2007. immunocytochemistry for protein gene-product 9.5, Arch 252. Mitsiadis TA, De Bari C, About I: Apoptosis in 275. Nishioka M, Shiiya T, Ueno K, Suda H: Tooth replantation Oral Biol 39:563, 1994. developmental and repair-related human tooth in germ-free and conventional rats, Endod Dent Traumatol 231. Magloire H, Couble ML, Thivichon-Prince B, et al: remodeling: a view from the inside, Exp Cell Res 14:163, 1998. Odontoblast: a mechano-sensory cell, J Exp Zool (Mol Dev 314:869, 2008. 276. Nitzan DW, Michaeli Y, Weinreb M, Azaz B: The effect of Evol) 312B:416, 2009. 253. Mjör IA, Nordahl I: The density and branching of dentinal aging on tooth morphology: a study on impacted teeth, 232. Magloire H, Maurin JC, Couble ML, et al: Topical review. tubules in human teeth, Arch Oral Biol 41:401, 1996. Oral Surg Oral Med Oral Pathol 61:54, 1986. Dental pain and odontoblasts: facts and hypotheses, 254. Modaresi J, Dianat O, Mozayeni MA: The efficacy 277. O’Neil RG, Brown RC: The vanilloid receptor family of J Orofacial Pain 24:335, 2010. comparison of ibuprofen, acetaminophen-codeine, and calcium-permeable channels: molecular integrators of CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 571

microenvironmental stimuli, News Physiol Sci 18:226, 301. Pohto P, Antila R: Innervation of blood vessels in the 325. Schüpbach P, Lutz F, Finger WT: Closing of dentin tubules 2003. dental pulp, Int Dent J 22:228, 1972. by Gluma desensitizer, Eur J Oral Sci 105:414, 1997. 278. Ochoa JL, Torebjork E, Marchettini P, Sivak M: 302. Pomonis JD, Rogers SD, Peters CM, et al: Expression and 326. Scott JN, Weber DF: Microscopy of the junctional region Mechanism of neuropathic pain: cumulative observations, localization of endothelin receptors: implications for the between human coronal primary and secondary dentin, new experiments, and further speculation. In Fields HL, involvement of peripheral glia in nociception, J Neurosci J Morphol 154:133, 1977. Dubner R, Cervero F, editors: Advances in pain research 21:999, 2001. 327. Seltzer S, Bender IB, Ziontz M: The interrelationship of and therapy, New York, 1985, Raven Press, p 431. 303. Prati C, Cervellati F, Sanasi V, Montebugnoli L: Treatment pulp and periodontal disease, Oral Surg 16:1474, 1963. 279. Okeson JP: Assessment of orofacial pain disorders. In of cervical dentin hypersensitivity with resin adhesives: 4 328. Senger DR, Galli SJ, Dvorak AM, et al: Tumor cells Okeson JP, editors: Orofacial pain: guidelines for week evaluation, Arch Dent 14:378, 2001. secrete a vascular permeability factor that promotes assessment, diagnosis, and management, Chicago, 1996, 304. Prescott RS, Alsanea R, Fayad MI, et al: In vivo generation accumulation of ascites fluid, Science 219:983, 1983. Quintessence Publishing, p 35. of dental pulp-like tissue by using dental pulp stem cells, 329. Sessle BJ: Recent developments in pain research: central 280. Okiji T, Kawashima N, Kosaka T, et al: An a collagen scaffold, and dentin matrix protein 1 after mechanisms of orofacial pain and its control, J Endodon immunohistochemical study of the distribution of subcutaneous transplantation in mice, J Endod 34:421, 12:435, 1986. immunocompetent cells, especially macrophages and Ia 2008. 330. Sessle BJ: The neurobiology of facial and dental pain: antigen-presenting cells of heterogeneous populations, in 305. Qian XB, Naftel JP: Effects of neonatal exposure to present knowledge, future directions, J Dent Res 66:962, normal rat molar pulp, J Dent Res 71:1196, 1992. anti-nerve growth factor on the number and size 1987. 281. Okiji T, Morita I, Sunada I, et al: Involvement of distribution of trigeminal neurones projecting to the molar 331. Sessle BJ: Acute and chronic craniofacial pain: brainstem arachidonic acid metabolites in increases in vascular dental pulp in rats, Arch Oral Biol 41:359, 1996. mechanisms of nociceptive transmission and permeability in experimental dental pulpal inflammation in 306. Rapp R, el-Labban NG, Kramer IR, Wood D: Ultrastructure neuroplasticity, and their clinical correlates, Crit Rev Oral the rat, Arch Oral Biol 34:523, 1989. of fenestrated capillaries in human dental pulps, Arch Oral Biol Med 11:57, 2000. 282. Olgart L, Kerezoudis NP: Nerve-pulp interactions, Arch Biol 22:317, 1977. 332. Sessle BJ, Greenwood LF: Inputs to trigeminal brain stem Oral Biol 39(suppl):47S, 1994. 307. Reader A, Foreman DW: An ultrastructural qualitative neurones from facial, oral, tooth pulp and 283. Olgart LM, Edwall L, Gazelius B: Neurogenic mediators in investigation of human intradental innervation, J Endod pharyngolaryngeal tissues: I. Responses to innocuous and control of pulpal blood flow, J Endod 15:409, 1989. 7:493, 1981. noxious stimuli, Brain Res 117:211, 1976. 284. Olgart LM, Edwall L, Gazelius B: Involvement of afferent 308. Renton T, Yiangou Y, Plumpton C, et al: Sodium channel 333. Sessle BJ, Hu JW, Amano N, Zhong G: Convergence of nerves in pulpal blood-flow reactions in response to Nav1.8 immunoreactivity in painful human dental pulp, cutaneous, tooth pulp, visceral, neck and muscle afferents clinical and experimental procedures in the cat, Arch Oral BMC Oral Health 5:5, 2005. onto nociceptive and non-nociceptive neurones in Biol 36:575, 1991. 309. Roberts-Clark D, Smith AJ: Angiogenic growth factors in trigeminal subnucleus caudalis (medullary dorsal horn) 285. Olgart LM, Gazelius B, Brodin E, Nilsson G: Release of human dentine matrix, Arch Oral Biol 42:1013, 2000. and its implications for referred pain, Pain 27:219, 1986. substance P-like immunoreactivity from the dental pulp, 310. Rodd HD, Boissonade FM: Innervation of human tooth 334. Shortland PJ, Jacquin MF, De Maro JA, et al: Central Acta Physiol Scand 101:510, 1977. pulp in relation to caries and dentition type, J Dent Res projections of identified trigeminal primary afferents after 286. Orchardson R, Cadden SW: An update on the physiology 80:389, Jan 2001. molar pulp differentiation in adult rats, Somatosens Mot of the dentine-pulp complex, Dent Update 28:200, 208, 311. Rodd HD, Boissonade FM: Comparative immunohistochemical Res 12:227, 1995. 2001. analysis of the peptidergic innervation of human primary and 335. Shulman K, Rosen S, Tognazzi K, et al: Expression of 287. Orchardson R, Gillam DG: Managing dentin permanent tooth pulp, Arch Oral Biol 47:375, 2002. vascular permeability factor (VPF/VEGF) is altered in many hypersensitivity, J Am Dent Assoc 137:990; quiz 1028, 312. Rodd HD, Boissonade FM, Day PF: Pulpal status of glomerular diseases, J Am Soc Nephrol 7:661, 1996. 2006. hypomineralized permanent molars, Pediatr Dent 29:514, 336. Shuttleworth CA, Ward JL, Hirschmann PN: The presence 288. Owatz CB, Khan AA, Schindler WG, et al: The incidence of 2007. of type III collagen in the developing tooth, Biochim mechanical allodynia in patients with irreversible pulpitis, 313. Roy ML, Narahashi T: Differential properties of Biophys Acta 535:348, 1978. J Endod 33:552, 2007. tetrodotoxin-sensitive and tetrodotoxin-resistant sodium 337. Sigal MJ, Pitaru S, Aubin JE, Ten Cate AR: A combined 289. Oxlund H, Manschot J, Viidik A: The role of elastin in the channels in rat dorsal root ganglion neurons, J Neurosci scanning electron microscopy and immunofluorescence mechanical properties of skin, J Biomech 21:213, 1988. 12:2104, 1992. study demonstrating that the odontoblast process extends 290. Paine MF, Widmer WW, Hart HL, et al: A furanocoumarin- 314. Ruparel NB, Patwardhan AM, Akopian A, Hargreaves KM: to the dentinoenamel junction in human teeth, Anat Rec free grapefruit juice establishes furanocoumarins as the Homologous and heterologous desensitization of capsaicin 210:453, 1984. mediators of the grapefruit juice-felodipine interaction and mustard oil responses utilize different cellular 338. Smith AJ, Garde C, Cassidy N, et al: Solubilization of [erratum appears in Am J Clin Nutr 84:264, 2006], pathways in nociceptors, Pain 135:271, 2008. dentin extracellular matrix by calcium hydroxide, J Dent Am J Clin Nutr 83:1097, 2006. 315. Rutherford B: BMP-7 gene transfer into inflamed Res 74:829, 1995 (abstract). 291. Pashley DH: Dynamics of the pulpodentin complex, Crit ferret-dental pulps, Eur J Oral Sci 109:422, 2001. 339. Smith AJ, Sloan AJ, Matthews JB, et al: Reparative Rev Oral Biol Med 7:104, 1996. 316. Rutherford B, Fitzgerald M: A new biological approach to processes in dentine and pulp. In Addy M, Embery G, 292. Pashley DH: Potential treatment modalities for dentin vital pulp therapy, Crit Rev Oral Biol Med 6:218, 1995. Edger WM, Orchardson R, editors: Tooth wear and hypersensitivity—in office products. In Addy M, 317. Rutherford RB, Spanberg L, Tucker M, et al: The sensitivity: clinical advances in restorative dentistry, 2000, Orchardson R, editors: Tooth wear and sensitivity, London, time-course of the induction of reparative dentine Martin Dunitz. 2000, Martin-Dunitz Publishers, p 351. formation in moneys by recombinant human osteogenic 340. Smith AJ, Tobias RS, Cassidy N, et al: Odontoblast 293. Patwardhan A, Cerka K, Vela J, Hargreaves KM: protein-1, Arch Oral Biol 39:833, 1994. stimulation in ferrets by dentine matrix components, Arch Trigeminal nociceptors express prostaglandin receptor 318. Sakamoto N, Nakajima T, Ikunaga K, et al: Identification of Oral Biol 39:13, 1994. subtypes EP2 and EP3, J Dent Res 87:262, 2008. hyaluronidase activity in rabbit dental pulp, J Dent Res 341. Souza PP, Fukada SY, Cunha FQ, et al: Regulation of 294. Penfield W, Rasmussen G: The cerebral cortex of man, 60:850, 1981. angiotensin II receptors levels during rat induced pulpitis. New York, 1950, Macmillan. 319. Sakurai K, Okiji T, Suda H: Co-increase of nerve fibers and Regul Pept 140:27, 2007. 295. Perl E: Causalgia, pathological pain and adrenergic HLA-DR- and/or factor XIIIa-expressing dendritic cells in 342. Stanley HR, White CL, McCray L: The rate of tertiary receptors, Proc Natl Acad Sci U S A 96:7664, 1999. dentinal caries-affected regions of the human dental pulp: an (reparative) dentin formation in the human tooth, Oral 296. Perl ER: Ideas about pain, a historical view [review] [140 immunohistochemical study, J Dent Res 78:1596, 1999. Surg 21:180, 1966. refs], Nature Revi Neuroscie 8:71, 2007. 320. Sasaki S: Studies on the respiration of the dog tooth 343. Steen KH, Reeh PW, Anton F, Handwerker HO: Protons 297. Petty B, Cornblath DR, Adornato BT, et al: The effect of germ, J Biochem (Tokyo) 46:269, 1959. selectively induce lasting excitation and sensitization to systemically administered recombinant human nerve 321. Sasano T, Kuriwada S, Sanjo D: Arterial blood pressure mechanical stimulation of nociceptors in rat skin in vitro, growth factor in healthy human subjects, Ann Neurol regulation of pulpal blood flow as determined by laser J Neurosci 21:86, 1992. 36:244, 1994. Doppler, J Dent Res 68:791, 1989. 344. Stenvik A, Iverson J, Mjör IA: Tissue pressure and 298. Poggi P, Casasco A, Marchetti C, et al: Ultrastructural 322. Sasano T, Kuriwada S, Shoji N, et al: Axon reflex histology of normal and inflamed tooth pulps in Macaque localization of elastin-like immunoreactivity in the vasodilatation in cat dental pulp elicited by noxious monkeys, Arch Oral Biol 17:1501, 1972. extracellular matrix around human small lymphatic stimulation of the gingiva, J Dent Res 73:1797, 1994. 345. Stern D, Nawroth P, Handley D, Kisiel W: An endothelial vessels, Lymphology 28:189, 1995. 323. Sasano T, Shoji N, Kuriwada S, et al: Absence of cell-dependent pathway of coagulation, Proc Natl Acad 299. Pohto P: Sympathetic adrenergic innervation of permanent parasympathetic vasodilatation in cat dental pulp, J Dent Sci U S A 82:2523, 1985. teeth in the monkey (Macaca irus), Acta Odontol Scand Res 74:1665, 1995. 346. Sunakawa M, Tokita Y, Suda H: Pulsed Nd:YAG laser 30:117, 1972. 324. Schaible H, Schmidt R: Discharge characteristics of irradiation of the tooth pulp in the cat. II. Effect of 300. Pohto P, Antila R: Demonstration of adrenergic nerve receptors with fine afferents from normal and inflamed scanning lasing, Lasers Surg Med 26:477, 2000. fibres in human dental pulp by histochemical fluorescence joints: influence of analgesics and prostaglandins, Agents 347. Sundqvist G, Lerner UH: Bradykinin and thrombin method, Acta Odontol Scand 26:137, 1968. Actions 19:99, 1986. synergistically potentiate interleukin 1 and tumour 572 PART II The Advanced Science of Endodontics

necrosis factor induced prostanoid biosynthesis in human 373. Trowbridge HO, Silver DR: Review of current approaches 399. Weiger R, Axmann-Kremar D, Lost C: Prognosis of dental pulp fibroblasts, Cytokine 8:168, 1996. to in-office management of tooth hypersensitivity, Dent conventional root canal treatment reconsidered, Endodon 348. Sweitzer SM, Schubert P, DeLeo JA: Propentofylline, a Clin North Am 16:561, 1990. Dent Traumatol 14:1, 1998. glial modulating agent, exhibits antiallodynic properties in 374. Trowbridge HO, Stewart JCB, Shapiro IM: Assessment of 400. Weinstock A, Weinstock M, Leblond CP: Autoradiographic a rat model of neuropathic pain, J Pharmacol Exp Ther indurated, diffusely calcified human dental pulps. In detection of 3H-fucose incorporation into glycoprotein by 297:1210, 2001. Proceedings of the International Conference on Dentin/ odontoblasts and its deposition at the site of the 349. Swift ML, Byers MR: Effects of aging responses of nerve Pulp Complex, Tokyo, 1996, Quintessence Publishing, calcification front in dentin, Calcif Tissue Res 8:181, fibers to pulpal inflammation in rat molars analyzed by p 297. 1972. quantitative immunohistochemistry, Arch Oral Biol 37:901, 375. Turner D, Marfurt C, Sattelburg C: Demonstration of 401. Weinstock M, Leblond CP: Synthesis, migration and 1992. physiological barrier between pulpal odontoblasts and its release of precursor collagen by odontoblasts as 350. Tagami J, Hosoda H, Burrow MF, Nakajima M: Effect of perturbation following routine restorative procedures: a visualized by radioautography after 3H-proline aging and caries on dentin permeability, Proc Finn Dent horseradish peroxidase tracing study in the rat, J Dent administration, J Cell Biol 60:92, 1974. Soc 88(suppl 1):149, 1992. Res 68:1262, 1989. 402. Wells J, Bingham V, Rowland K, Hatton J: Expression of

351. Takahashi K, Kishi Y, Kim S: A scanning electron 376. Turner DF: Immediate physiological response of Nav1.9 channels in human dental pulp and trigeminal microscope study of the blood vessels of dog pulp using odontoblasts, Proc Finn Dent Soc 88(suppl 1):55, 1992. ganglion, J Endod 33:1172, 2007. corrosion resin casts, J Endod 8:131, 1982. 377. Uddman R, et al: Occurrence of VIP nerves in mammalian 403. Wiig H, Aukland K, Tenstad O: Isolation of interstitial fluid 352. Tanaka T: The origin and localization of dentinal fluid in dental pulps, Acta Odontol Scand 38:325, 1980. from rat mammary tumors by a centrifugation method, developing rat molar teeth studied with lanthanum as a 378. van Amerongen JP, Lemmens IG, Tonino GJ: The Arch Physiol Heart Circ Physiol 284:H416-H424, 2003. tracer, Arch Oral Biol 25:153, 1980. concentration, extractability and characterization of 404. Wiig H, Gyenge C, Iversen PO, et al: The role of the 353. Telles PD, Hanks CT, Machado MA, Nör JE: Lipoteichoic collagen in human dental pulp, Arch Oral Biol, 28:339, extracellular matrix in tissue distribution of macromolecules acid up-regulates VEGF expression in macrophages and 1983. in normal and pathological tissues: potential therapeutic pulp cells, J Dent Res 82:466, 2003. 379. Van Hassel HJ: Physiology of the human dental pulp, Oral consequences, Microcirculation 15:283, 2008. 354. Thomas HF: The extent of the odontoblast process in Surg Oral Med Oral Path 32:126, 1971. 405. Willingale HL, Gardiner NJ, McLymont N, et al: human dentin, J Dent Res 58(D):2207, 1979. 380. Van Hassel HJ, Brown AC: Effect of temperature changes Prostanoids synthesized by cyclo-oxygenase isoforms in 355. Thomas HF, Payne RC: The ultrastructure of dentinal on intrapulpal pressure and hydraulic permeability in dogs, rat spinal cord and their contribution to the development tubules from erupted human premolar teeth, J Dent Res Arch Oral Biol 14:301, 1969. of neuronal hyperexcitability, Br J Pharmacol 122:1593, 62:532, 1983. 381. van Wijk AJ, Hoogstraten J: Reducing fear of pain 1997. 356. Thomas JJ, Stanley HR, Gilmore HW: Effects of gold foil associated with endodontic therapy, Int Endod J 39:384, 406. Winter HF, Bishop JG, Dorman HL: Transmembrane condensation on human dental pulp, J Am Dent Assoc 2006. potentials of odontoblasts, J Dent Res 42:594, 1963. 78:788, 1969. 382. Veerayutthwilai O, Byers MR, Pham TT, et al: Differential 407. Wong M, Lytle WR: A comparison of anxiety levels 357. Tokita Y, Sunakawa M, Suda H: Pulsed ND: YAG laser regulation of immune responses by odontoblasts, Oral associated with root canal therapy and oral surgery irradiation of the tooth pulp in the cat. I. Effect of spot Microbiol Immunol 22:5, 2007. treatment, J Endod 17:461, 1991. lasing, Lasers Surg Med 26:477, 2000. 383. Vickers ER, Cousins MJ: Neuropathic orofacial pain part 408. Woodnutt DA, Wager-Miller J, O’Neill PC, et al: Neurotrophin 358. Tominaga M, Numazaki M, Iida T, et al: Molecular 1: prevalence and pathophysiology, Aust Endod J 26:19, receptors and nerve growth factor are differentially expressed mechanisms of TRPV1-mediated thermal hypersensitivity, 2000. in adjacent nonneuronal cells of normal and injured tooth vol 30, p 37. Seattle, 2003, IASP Press. 384. Vongsavan N, Matthews B: The permeability of cat pulp, Cell Tissue Res 299:225, 2000. 359. Tönder KH, Naess G: Nervous control of blood flow in the dentine in vivo and in vitro, Arch Oral Biol 36:641, 1991. 409. Woolf C: Transcriptional and posttranslational plasticity dental pulp in dogs, Acta Physiol Scand 104:13, 1978. 385. Vongsavan N, Matthews B: Fluid flow through cat dentine and the generation of inflammatory pain, Proc Natl Acad 360. Tönder KJ: Blood flow and vascular pressure in the dental in vivo, Arch Oral Biol 37:175, 1992. Sci U S A 96:7723, 1999. pulp. Summary, Acta Odontol Scand 38:135, 1980. 386. Vongsavan N, Matthews B: The vascularity of dental pulp 410. Wright EF: Referred craniofacial pain patterns in patients 361. Tönder KJ: Effect of vasodilating drugs on external carotid in cats, J Dent Res, 71:1913, 1992. with temporomandibular disorder [see comment][erratum and pulpal blood flow in dogs: “stealing” of dental 387. Vongsavan N, Matthews B: The relation between fluid flow appears in J Am Dent Assoc 131:1553, 2000], J Am perfusion pressure, Acta Physiol Scand 97:75, 1976. through dentine and the discharge of intradental nerves, Dent Assoc 131:1307, 2000. 362. Tönder KJH, Kvininsland I: Micropuncture measurements Arch Oral Biol 39(suppl):140S, 1994. 411. Yaksh TL: Central pharmacology of nociceptive of interstitial fluid pressure in normal and inflamed dental 388. Vongsavan N, Matthews B: The relationship between the transmission. In Wall P, Melzack R, editors: Textbook of pulp in cats, J Endod 9:105, 1983. discharge of intradental nerves and the rate of fluid flow pain, Edinburgh, 2002, Churchill Livingstone, p 285. 363. Tonioli M, Patel T, Diogenes A, et al: Effect of through dentine in the cat, Arch Oral Biol 52:640, 2007. 412. Yamada T, Nakamura K, Iwaku M, Fusayama T: The neurotrophic factors on bradykinin expression in rat 389. Wakisaka S: Neuropeptides in the dental pulp: their extent of the odontoblast process in normal and carious trigeminal sensory neurons determined by real-time distribution, origins and correlation, J Endod 16:67, 1990. human dentin, J Dent Res 62:798, 1983. polymerase chain reaction, J Endod 30:263, 2004. 390. Wakisaka S, Ichikawa H, Akai M: Distribution and origins 413. Yamaguchi M, Kojima T, Kanekawa M, et al: 364. Torabinejad M, Cymerman JJ, Frankson M, et al: of peptide- and catecholamine-containing nerve fibres in Neuropeptides stimulate production of interleukin-1 beta, Effectiveness of various medications on postoperative pain the feline dental pulp and effects of cavity preparation on interleukin-6, and tumor necrosis factor-alpha in human following complete instrumentation, J Endod 20:345, 1994. these nerve fibres, J Osaka Univ Dent Sch 26:17, 1986. dental pulp cells, Inflamm Res 53:199, 2004. 365. Torebjörk HE, Hanin RG: Perceptual changes 391. Wakisaka S, Sasaki Y, Ichikawa H, Matsuo S: Increase in 414. Yamamura T: Differentiation of pulpal wound healing, accompanying controlled preferential blocking of A and C c-fos-like immunoreactivity in the trigeminal nucleus J Dent Res 64(special issue):530, 1985. fiber responses in intact human skin nerves, Exp Brain complex after dental treatment, Proc Finn Dent Soc 415. Yang BH, Piao ZG, Kim Y-B: Activation of vanilloid Res 16:321, 1973. 88(suppl 1):551, 1992. receptor 1 (VR1) by eugenol, J Dent Res 82:781, 2003. 366. Torneck CD: Dentin-pulp complex. In Ten Cate AR, editor: 392. Wall PD: Alterations in the central nervous system after 416. Yonehara N, Amano K, Kamisaki Y: Involvement of the Oral histology: development, structure, and function, ed 5, deafferentation: connectivity control. In Bonica JJ, NMDA-nitric oxide pathway in the development of St Louis, 1998, Mosby, p 150. Lindblom U, Iggo A, editors: Advances in pain research hypersensitivity to tactile stimulation in dental injured rats, 367. Torneck CD, Kwan CL, Hu JW: Inflammatory lesions of the and therapy, vol 5, New York, 1983, Raven Press, p 677. Jpn J Pharmacol 90:145, 2002. tooth pulp induce changes in brainstem neurons of the rat 393. Walton R, Fouad A: Endodontic interappointment 417. Yu CY, Boyd NM, Cringle SJ: An in vivo and in vitro trigeminal subnucleus oralis, J Dent Res 75:553, 1996. flare-ups: a prospective study of incidence and related comparison of the effects of vasoactive mediators on 368 Tran XV, Gorin C, Willig C, et al: Effect of a calcium- factors, J Endod 18:172, 1992. pulpal blood vessels in rat incisors, Arch Oral Biol 47:723, silicate-based restorative cement on pulp repair, J Dent 394. Wang C, Li GW, Huang LY: Prostaglandin E2 potentiation 2002. Res 91:1166, 2012. of P2X3 receptor mediated currents in dorsal root 418. Yu CY, Boyd NM, Cringle SJ, et al: Oxygen distribution 369. Trantor IR, Messer HH, Birner R: The effects of ganglion neurons, Mol Pain 3:22, 2007. and consumption in rat lower incisor pulp, Arch Oral Biol neuropeptides (calcitonin-gene-related peptide and 395. Warfvinge J, Dahlen G, Bergenholtz G: Dental pulp response 47:529, 2002. substance P) on cultured human pulpal cells, J Dent Res to bacterial cell wall material, J Dent Res 64:1046, 1985. 419. Zerari-Mailly F, Braud A, Davido N, et al: Glutamate 74:1066, 1995. 396. Warren C, Mok L, Gordon S, et al: Quantification of neural control of pulpal blood flow in the incisor dental pulp of 370. Trowbridge HO: Pathogenesis of pulpitis resulting from protein in extirpated tooth pulp, J Endod 34:7, 2008. the rat, Eur J Oral Sci 120:402, 2012. dental caries, J Endod 7:52, 1981. 397. Watkins LR, Milligan ED, Maier SF: Glial activation: a 420. Zerlotti E: Histochemical study of the connective tissue of 371. Trowbridge HO: Review of dental pain–histology and driving force for pathological pain, Trends Neurosci the dental pulp, Arch Oral Biol 9:149, 1964. physiology, J Endod 12:445, 1986. 24:450, 2001. 421. Zhang J, Kawashima N, Suda H, et al: The existence of 372. Trowbridge HO, Franks M, Korostoff E, Emling R: Sensory 398. Weber DK, Zaki AL: Scanning and transmission electron CD11c+ sentinel and F4/80+ interstitial dendritic cells in response to thermal stimulation in human teeth, J Endod microscopy of tubular structure presumed to be human dental pulp and their dynamics and functional properties, 6:405, 1980. odontoblast processes, J Dent Res 65:982, 1986. Int Immunol 18:1375, 2006. CHAPTER 13

Pulpal Reactions to Caries and Dental Procedures

ASHRAF FOUAD | LINDA G. LEVIN

CHAPTER OUTLINE Pulpal Reaction to Caries Direct Pulp Capping with Mineral Trioxide Aggregate Neurogenic Mediators The Use of Hemostatic Agents and Disinfectants on Direct Correlation Between Clinical Symptoms and Actual Pulp Exposures Pulpal Inflammation Pulpal Reactions to Laser Procedures Dentin Hypersensitivity and Its Management Lasers in the Prevention, Diagnosis, and Treatment of Caries Pulpal Reactions to Local Anesthetics Lasers in the Treatment of Dentin Hypersensitivity Pulpal Reactions to Restorative Procedures Use of Lasers as a Protective Measure for Dentin, Under The Degree of Inflammation of the Pulp Preoperatively Traditional Cavity Preparation The Amount of Physical Irritation Caused by the Procedure Pulpal Reactions to Vital Bleaching Techniques The Proximity of the Restorative Procedures to the Dental Pulpal Reactions to Periodontal Procedures Pulp and the Surface Area of Dentin Exposed Mechanical Irritants: Orthodontic Movement The Permeability of Dentin and the Odontoblastic Layer Pulpal Reactions to Orthodontic/Orthognathic Surgery Between the Area Being Restored and the Pulp Biomechanical Irritation: Parafunctional Habits The Age of the Patient Pulpal Reactions to Implant Placement and Function Pulpal Reactions to Restorative Materials

The dental pulp is a dynamic tissue that responds to external constituents of dentin mediate the effects of dental caries on stimuli in many ways. However, there are certain unique fea- the pulp. Three basic reactions tend to protect the pulp against tures about the dental pulp response that distinguish it from caries: (1) a decrease in dentin permeability, (2) tertiary dentin other connective tissues in the body. The pulp’s exposure to formation, and (3) inflammatory and immune reactions.133 dental caries, a prevalent chronic infectious disease, its encase- These responses occur concomitantly, and their robustness is ment in an unyielding environment after complete tooth matu- highly dependent on the aggressive nature of the advancing ration, and the scarcity of collateral circulation render it lesion as well as host responses such as the age of the patient susceptible to injury and complicate its regeneration. More- (see also Chapter 26). over, the pulp is endowed with a rich neurovascular supply In the advancing infection front of the carious lesion, mul- that regulates the effects of inflammation that may ultimately tiple intrinsic and extrinsic factors are released that stimulate lead to rapid degeneration and necrosis. The treatment of nearby pulpal tissue. Bacterial proteolytic enzymes, toxins, and dental caries and other tooth abnormalities involves removal metabolic by-products have been thought to initiate pulpal of the enamel and dentin, the hardest tissues in the body, thus reactions, yet the buffering capacity of dentin and dentinal adding to the irritation of the pulp. This chapter discusses the fluid likely attenuate these deleterious effects. This protective response of the pulp to all of these variables and presents function is significantly reduced when the remaining dentin advances in our understanding of dental procedures and their thickness is minimal.237 When relatively unhindered access to effects on the pulp. pulpal tissue is present, both bacterial metabolites and their cell wall components induce inflammation. In initial-to-moderate lesions, current evidence suggests that acidic by-products PULPAL REACTION TO CARIES of the carious process act indirectly by degrading the dentin Dental caries is a localized, destructive, and progressive infec- matrix and thereby liberating bioactive molecules previously tion of dentin, which, if left unchecked, can result in pulpal sequestered during dentinogenesis. Once liberated, these necrosis and potential tooth loss. Both bacterial by-products molecules again assume their role in dentin formation, this and products from the dissolution of the organic and inorganic time stimulatory for tertiary dentinogenesis.238 This theory is

573 574 PART II The Advanced Science of Endodontics

A B

FIG. 13-1 A, Whitlockite crystals occlude the dentinal tubules in sclerotic dentin. B, Dentinal sclerosis is radiographically apparent beneath a deep class II lesion. (A, From Yoshiyama M, Masada J, Uchida A, Ishida H: Scanning electron microscopic characterization of sensitive vs. insensitive human radicular dentin, J Dent Res 68:1498, 1989.)

supported by the findings that demineralized dentin matrix implanted at the site of pulpal exposure can induce dentinogenesis.266 Furthermore, placement of purified dentin matrix proteins on exposed dentin or exposed pulp stimulates tertiary dentin formation, indicating that these molecules can act directly or across intact dentin.239,265 More information on growth factors embedded in dentin is reviewed in the chapter on regenerative endodontics (see Chapter 10). Evidence offers several candidate molecules that stimulate reparative dentinogenesis. Heparin-binding growth factor, transforming growth factor (TGF)-β1, TGF-β3, insulin-like TD growth factors I and II, platelet-derived growth factor, bone morphogenetic protein-2 (BMP-2) and angiogenic growth factors have been shown to be embedded in dentin and stimulatory for dentinogenesis in vitro. The TGF-β superfamily in particular seems to be important in the signaling process for odontoblast differentiation from mesenchymal stem or progenitor cells as well as primary and tertiary dentinogenesis. As the predominant isoform, TGF-β1 is equally distributed in the 41 soluble and insoluble fractions of dentin matrix. During the FIG. 13-2 Reactionary dentinogenesis (TD). Note the tubular morphology carious dissolution of dentin, it is believed that the soluble and the discontinuity of the tubules at the interface of secondary and reaction- pool of TGF-β1 can diffuse across intact dentin while the ary dentin. Resident odontoblasts are still present. insoluble pool is immobilized on insoluble dentin matrix and serves to stimulate odontoblasts much like membrane-bound TGF-βs during odontogenesis.237,240 precipitation of mineral released during the demineralization Despite the research interest in tertiary dentinogenesis, it is process156,262 (Fig. 13-1). neither the first nor necessarily the most effective pulpally The formation of tertiary dentin occurs over a longer period mediated defense against invading pathogens. A combination than does that of sclerotic dentin, and its resultant character is of an increased deposition of intratubular dentin and the direct highly dependent on the stimulus. Mild stimuli activate resi- deposition of mineral crystals into the narrowed dentin tubules dent quiescent odontoblasts whereupon they elaborate the to decrease dentin permeability is the first defense to caries and organic matrix of dentin. This type of tertiary dentin is referred is called dentin sclerosis. It occurs by a combination of increased to as reactionary dentin and can be observed when initial dentin deposition of intratubular dentin and tubule occlusion by demineralization occurs beneath the noncavitated enamel precipitated crystals. This results in an effective decrease in lesion.151 Mediators present during the carious process induce dentin permeability underneath the advancing carious lesion.206 a focal upregulation of matrix production by resident odonto- In vitro studies with cultured tooth slices implicate TGF-β1 as blasts. The resultant dentin is similar in morphology to physi- a central player in the increased deposition of intratubular ologic dentin and may only be apparent due to a change in the dentin.236 The deposition of whitlockite crystals in the tubular direction of the new dentinal tubules (Fig. 13-2). In contrast, lumen most likely results from a similar stimulation of in aggressive lesions the carious process may prove cytocidal vital associated odontoblasts, possibly in combination with to subjacent odontoblasts and require repopulation of the CHAPTER 13 Pulpal Reactions to Caries and Dental Procedures 575

A FIG. 13-4 The early pulpal response to caries is represented by a focal accumulation of chronic inflammatory cells. Note that peripheral to the inflammation the pulpal parenchyma is relatively unaffected.

FIG. 13-3 Reparative dentin; the strong stimulus of the impinging infection is cytocidal for odontoblasts. The resultant dentin is irregular with soft tissue inclusions. Once the odontoblast TLR is stimulated by a pathogen, proinflammatory cytokines, chemokines, and antimicrobial peptides disrupted odontoblast layer with differentiating progenitors. are elaborated by the odontoblast, resulting in recruitment and The organization and composition of the resultant matrix are stimulation of immune effector cells as well as direct bacterial a direct reflection of the differentiation state of the secretory killing.74,76,129 cells. This accounts for the heterogeneity of reparative dentin, Many cells produce chemokines at low levels constitutively. where the morphology can range from organized tubular Unstimulated odontoblasts express genes coding for CCl2, dentin to more disorganized irregular fibrodentin. Fibrodentin, CXCL12, and CXCL14, three genes known to code for factors due to its irregular configuration and tissue inclusions, is more chemotactic for immature dendritic cells.42 They also produce permeable than physiologic dentin275 (Fig. 13-3). CCL26, a natural antagonist for CCR1, CCR2, and CCR5 that Although dentin can provide a physical barrier against are chemokines normally produced by monocytes and den- noxious stimuli, the pulpal immune response provides humoral dritic cells.287 Stimulation with bacterial cell wall constituents and cellular challenges to invading pathogens. In the progress- has been shown to upregulate the expression of multiple che- ing carious lesion, the host immune response increases in mokine genes including CXCL12, CCL2, CXCL9, CX3CL1, intensity as the infection advances. It has been shown that CCL8, CXCL10, CCL16, CCL5, CXCL2, CCL4, CXCL11, and titers of T-helper cells, B-lineage cells, neutrophils, and mac- CCL3, and nine chemokine receptor genes including CXCR4, rophages are directly proportional to lesion depth in human CCR1, CCR5, CX3CR1, CCR10, and CXCR3, suggesting that teeth.118 The disintegration of large amounts of dentin, however, odontoblasts sense pathogens and express factors that recruit is not necessary to elicit a pulpal immune response. This is immune effector cells42,75,106,152 (Fig. 13-5). These data suggest supported by the observation that a pulpal inflammatory a scenario whereby stimulated odontoblasts express high levels response can be seen beneath noncavitated lesions and nonco- of chemokines such as IL-8 (CXCL8) that act in concert with alesced pits and fissures.34 the release of formerly sequestered growth factors from carious The early inflammatory response to caries is characterized dentin that induce a focal increase in dendritic cell numbers by the focal accumulation of chronic inflammatory cells with the additional release of chemotactic mediators.77,240 The (Fig. 13-4). This is mediated initially by odontoblasts and later subsequent influx of immune effector cells is composed of by dendritic cells. As the most peripheral cell in the pulp, the lymphocytes, macrophages, and plasma cells. This cellular odontoblast is positioned to encounter foreign antigens first infiltrate is accompanied by localized capillary sprouting in and initiate the innate immune response. Pathogen detection response to angiogenic factors as well as co-aggregation of in general is accomplished via specific receptors called pattern nerve fibers and human leukocyte antigen-DR (HLA-DR)- recognition receptors (PRRs).120 These receptors recognize positive dendritic cells.280,281 pathogen associated molecular patterns (PAMPs) on invading As the carious lesion progresses, the density of the chronic organisms and initiate a host defense through the activation of inflammatory infiltrate as well as that of dendritic cells in the the NF-κB pathway.101 One class of the PAMP recognition mol- odontoblast region increases. Pulpal dendritic cells are respon- ecules is the Toll-like receptor family (TLRs). Odontoblasts sible for antigen presentation and stimulation of T lympho- have been shown to increase expression of certain TLRs cytes. In the uninflamed pulp they are scattered throughout in response to bacterial products. Under experimental condi- the pulp. With caries progression they aggregate initially in the tions, odontoblasts expression of TLR3, 5, and 9 increased in pulp and subodontoblastic regions, then extend into the odon- response to lipoteichoic acid, whereas lipopolysaccharide toblast layer, and eventually migrate into the entrance to increased TLR2 and 4 expression.66,107,183 It was also shown that tubules beside the odontoblast process282 (Fig. 13-6). Two dis- TGFβ-1 inhibits the expression of TLR2 and 4 by odontoblasts tinct populations of dendritic cells have been identified in the in response to gram-positive and gram-negative bacteria.27,107 dental pulp. CD11c+ is found in the pulp/dentin border and 576 PART II The Advanced Science of Endodontics

A B

FIG. 13-5 Odontoblasts exposed to LPS in an in vitro culture model express IL-8, as evidenced by immunos- taining with anti–IL-8 antibodies.

(GM-CSF) and osteopontin by dendritic cells and macrophages represents a mechanism whereby they contribute to odontoblast differentiation.227 Pulpal Schwann cells have also been shown to produce molecules in response to caries, which indicates the acquisition of the ability for antigen presentation. Evidence suggests that odontoblasts also play a role in the humoral immune response to caries. IgG, IgM, and IgA have been localized in the cytoplasm and cell processes of odontoblasts in human carious dentin, suggesting that these cells actively transport antibodies to the infection front.189 In the incipient lesion, antibodies accumulate in the odontoblast layer and with lesion progression can be seen in the dentinal tubules. Eventually this leads to a focal concentration of antibodies beneath the advancing lesion.188 In the most advanced phase of carious destruction, the humoral immune response is accompanied by immunopatho- logic destruction of pulpal tissue. In animal studies where monkeys were hyperimmunized to bovine serum albumin (BSA), there was an observed increase in pulpal tissue destruction subsequent to antigenic challenge across freshly cut dentin.20 The odontoblasts also appear to be involved in the production of innate antimicrobial molecules such as human FIG. 13-6 Dental caries stimulates the accumulation of pulpal dendritic beta defensing-2 (HBD2). Therefore, interleukin (IL)-1 and cells in and around the odontoblastic layer. (Reprinted with permission from tumor necrosis factor (TNF)-alpha as well as bacterial lipo- Mats Jontell.) polysaccharide (LPS) were responsible for significant increases in HBD2 in response to caries.106 In summary, it appears the odontoblasts play a central role in orchestrating local and che- subjacent to pits and fissures. F4/80+ dendritic cells are motactic inflammatory responses to dental caries (Fig. 13-7). concentrated in the perivascular spaces in the subodontoblas- Pulpal exposure in primary and immature permanent teeth tic zone and inner pulp.287 CD11c+ dendritic cells express can lead to a proliferative response or hyperplastic pulpitis. Toll-like receptors 2 and 4 and are CD205 positive. F4/80+ Exuberant inflammatory tissue proliferates through the expo- dendritic cells have migratory ability. As they migrate from the sure and forms a “pulp polyp” (Fig. 13-8). It is presumed that central pulp they increase in size and become CD86 positive. a rich blood supply allows this proliferative response. Conven- The close spatial relationship between odontoblasts and tional root canal therapy or progressive vital pulp therapy is dendritic cells under the carious lesion has led to speculation indicated. that dendritic cells may play a role in odontoblast differentiation or secretory activity in the immune defense and in dentinogenesis. Recent studies have demonstrated that pulp NEUROGENIC MEDIATORS dendritic cell can migrate to regional lymph nodes, for antigen Neurogenic mediators are involved in the pulpal response to presentation.24a In vitro studies have suggested that the irritants and, like immune components, they can mediate secretion of granulocyte-macrophage colony-stimulating factor pathology as well as the healing response (see also Chapter 12). CHAPTER 13 Pulpal Reactions to Caries and Dental Procedures 577

Caries Bacteria

Dendritic cells Macrophages Odontoblasts

Pathogen pattern recognition receptors e.g., TLR4, TLR2 Chemokines Migrating immune cells e.g., HBD2, IL-8, CCLs (1–5, 7–8, 11, 13, 15–17, 19–21, 24–26), CXCLs (1–3, 5–6, 9–14) Pro-inflammatory cytokines e.g., IL-1β, TNF-α, IL-1α Antimicrobial peptides e.g., HBD1, HBD2

FIG. 13-7 Innate immunity in the odontoblast layer (ODL). Bacterial components from caries activate cytokine/ chemokine release from odontoblasts, dendritic cells, or macrophages via Toll-like receptors (TLRs). Proinflam- matory cytokines released from these cells act as autocrine and paracrine signals to amplify cytokine responses. including antimicrobial peptide, cytokine, and chemokine production. The release of chemokines creates a migration gradient for immune cells to ODL while antimicrobial peptides reduce bacterial load. (From Horst OV, Horst JA, Samudrala R, Dale BA: Caries induced cytokine network in the odontoblast layer of human teeth, BMC Immunol 12:9, Fig. 4C, 2011. Reprinted with permission.)

extreme and persistent circumstances. Stimulation of sympathetic nerves in response to the local release of mediators such as norepinephrine, neuropeptide Y, and adenosine triphosphate (ATP) has been shown to alter pulpal blood flow. Receptor field studies as well as anatomic studies have shown sprouting of afferent fibers in response to inflammation.36 Neuropeptides can act to modulate the pulpal immune response. It has been demonstrated that SP acts as a chemotactic and stimulatory agent for macrophages and T lymphocytes. The result of this stimulation is increased production of arachidonic acid metabolites, stimulation of lymphocytic mitosis, and production of cytokines. CGRP demonstrates immunosuppres- sive activity, which is evidenced by a diminution of class II antigen presentation and lymphocyte proliferation. SP and CGRP are mitogenic for pulpal and odontoblast-like

FIG. 13-8 A proliferative response to caries in a young tooth, typically cells; thereby they initiate and propagate the pulpal healing referred to as proliferative pulpitis, hyperplastic pulpitis, or pulp polyp. (Cour- response.260 CGRP has been shown to stimulate the production tesy Dr. Howard Strassler, University of Maryland, with permission.) of bone morphogenic protein by human pulpal cells. The result of this stimulation has been postulated to induce tertiary dentinogenesis.38 Substance P appears to increase in the dental External stimulation of dentin causes the release of pro- pulp and periodontal ligament as a result of acutely induced inflammatory neuropeptides from pulpal afferent nerves.36,130 occlusal trauma,45 which may be related to the pain associated Substance P (SP), calcitonin gene–related peptide (CGRP), with concussion traumatic injury. neurokinin A (NKA), neurokinin Y, and vasoactive intestinal It has been shown that there may be gender differences in peptide are released and effect vascular events such as vasodila- CGRP release in the dental pulp.29,159 In one study, serotonin tion and increased vascular permeability. This results in a net (a peripheral pronociceptive mediator) induced a significant increase in tissue pressure that can progress to necrosis in increase in capsaicin-evoked CGRP release in dental pulps 578 PART II The Advanced Science of Endodontics

obtained from female but not male patients.159 This interplay including partial necrosis histologically, but with little or no of inflammatory mediators may explain some of the gender clinical symptoms—the so-called painless pulpitis.64,100,169,232 differences in clinical presentation with dental pain. Moreover, the density of nerve fibers224 and the vascularity225 in inflamed pulp do not correlate with clinical symptoms in primary and permanent teeth. It has been reported that the CORRELATION BETWEEN incidence of painless pulpitis that leads to pulp necrosis and CLINICAL SYMPTOMS AND asymptomatic apical periodontitis is about 40% to 60% of all pulpitis cases.168 ACTUAL PULPAL INFLAMMATION Objective clinical findings are essential for determining the From a clinical perspective, it would be most helpful to the vitality of the pulp and whether the inflammation has extended clinician to be able to diagnose pulpal conditions from a profile into the periapical tissues (see also Chapter 1). Lack of response of symptoms with which a patient presents. If symptoms are to electric pulp testing generally indicates that the pulp has not conclusive, a number of objective tests should aid the clini- become necrotic.221,232 Thermal pulp testing is valuable for cian in reaching a definitive diagnosis of the pulpal pathologic reproducing a symptom of thermal sensitivity and allowing the status. In actuality, combinations of subjective and objective practitioner to assess the reaction of the patient to a stimulus findings are frequently insufficient in reaching definitive diag- and the duration of the response. However, pulp testing cannot nosis of the status of the dental pulp. This is particularly true determine the degree of pulpal inflammation.64,232 These studies in cases of vital inflamed pulp, where it is difficult for the show that irreversible pulpal inflammation can be diagnosed practitioner to determine clinically whether the inflammation with some certainty only in cases where, in addition to being is reversible or irreversible. responsive to pulp testing, the pulp develops severe spontane- Many practitioners rely on painful symptoms to determine ous symptoms. Pulp necrosis could be predictably diagnosed the status of the pulp. Several studies have examined this ques- by a consistent negative response to pulp tests, preferably to tion in some detail. A number of classic studies were performed both cold and electrical tests to avoid false responses.210,211 Pulp in which the subjective and objective clinical findings related necrosis could be verified by a test cavity or lack of- hemor to carious teeth were recorded prior to extracting the teeth and rhagic pulp tissue upon access preparation. It should be noted, examining them histologically. The underlying hypothesis in however, that the latter sign should be assessed cautiously. these studies was that the more severe the clinical symptoms, Occasionally, the pulp space is very small, such as in older the more intense pulpal inflammation and destruction was individuals with calcified canals, and hemorrhage upon access evident histologically. These studies showed that in the vital to the pulp may not be clinically appreciable. Conversely, pulp, clinical symptoms generally did not correlate with gross cases with pulp necrosis and acute periapical infections histologic findings.100,169,232 Furthermore, carious pulp expo- may have hemorrhagic purulent drainage through the large sure was associated with severe inflammatory response or liq- pulp space upon access preparation, particularly after initial uefactive necrosis, regardless of symptoms (Fig. 13-9). These instrumentation. histologic changes ranged in extent from being present only at The lack of correlation between the histologic status of the the site of the exposure to deep into the root canals.232 In a few pulp and clinical symptoms may be explained by advances in studies prolonged or spontaneous severe symptoms were asso- the science of pulp biology. Studies have shown that numerous ciated with chronic partial, total pulpitis, or pulp necrosis.64,232 molecular mediators may act in synchrony to initiate, promote However, in these as well as other studies it was common to or modulate the inflammatory response in the dental pulp. The find cases with evidence of severe inflammatory responses nature and quantity of these inflammatory mediators cannot be determined from histologic analysis, without the use of specialized staining techniques. Many of these molecular mediators tend to reduce the pain threshold, either directly by acting on peripheral nerve cells or by promoting the inflammatory process. Thus, a number of these mediators were shown to be elevated in human pulp diagnosed with painful pulpitis. These mediators include prostaglandins,51,223 the vasoactive amine bradykinin,150 tumor necrosis factor alpha,139 neuropeptides such as substance P,30 CGRP and neurokinin A,13 and catecholamines.185 In fact, it was even shown that when patients have painful pulpitis, the crevicular fluid related to the affected teeth has significantly increased neuropeptides compared to the levels in contralateral teeth.13 In another study, trained volunteers stimulated an incisor with a constant current three fold the threshold value for 90 seconds.12 This resulted in a significant increase in crevicular matrix metalloproteinase 8 (MMP-8), one of the collagenases involved in tissue destruction. FIG. 13-9 Histologic photomicrograph of a molar with carious pulp expo- It has also been determined that peripheral opioid receptors sure. The exposure had been capped but had failed and the patient presented are present in the dental pulp,119 and these could play a role in with symptoms. The photomicrograph shows an area of necrosis and exten- why many cases with irreversible pulpitis are asymptomatic. As sive inflammation throughout the coronal pulp. (Courtesy Dr. Larz Spangberg, noted before, carious teeth are frequently not associated with University of Connecticut, with permission.) significant symptoms. However, they still have a significant CHAPTER 13 Pulpal Reactions to Caries and Dental Procedures 579 amount of inflammation. The pulp in teeth with mild to moder- the placement of passive molecules or crystals may provide ate caries has increased neuropeptide Y,68 and its Y1 receptor,69 only temporary relief, thus there has been the need to provide compared to that in normal teeth. Neuropeptide Y is a neu- biocompatible materials that bond to the root surface in order rotransmitter for the sympathetic nervous system and is thought to provide a more lasting solution. One such material was a to act as a modulator of neurogenic inflammation. Likewise, calcium sodium phosphosilicate bioactive glass,157 which was the levels of vasoactive intestinal peptide (VIP), although not developed into a commercial product (SootheRx, NovaMin its receptor VPAC1, seemed to increase in the pulp of moder- Technology Inc., Alachua, FL). Another product uses a com- ately carious teeth.67 bination of a calcium oxalate and an acid-etched bonding With the advances in molecular biology, efficient detection material to seal the dentinal tubules (BisBlock, Bisco Inc., of hundreds of molecular mediators simultaneously by their Schaumberg, IL). A concern has been raised that the acidic gene expression has become a reality. Current research seeks pH during etching may cause dissolution of the oxalate to examine which genes are specifically expressed or upregu- crystals, thus interfering with the effectiveness of the mate- lated in the pulp, in response to the carious lesion. In this rial.279 However, one study found that BisBlock and two regard, preliminary studies have shown that various cyto- other products—Seal&Protect (Dentsply Professional, York, kines and other inflammatory mediators are upregulated Pennsylvania) and Vivasens (Ivoclar Vivadent AG, Schaan, underneath a carious lesion in a manner that correlates with Liechtenstein)—were effective compared to placebo several the depth of caries.164 Several researchers have used gene weeks after treatment.198 In the long term, the development of microarrays to obtain an accurate mapping of candidate genes smear layer, such as from tooth brushing, dentin sclerosis, that show elevated expression in inflamed pulp and the odon- reactionary dentin, and the blockage of tubules with large toblastic cell layer.165,194,195 In addition, research has revealed endogenous macro molecules, is all thought to reduce the the differential expression of microRNAs (miRNAs) in the problem203 (see animation from the online edition of this healthy and diseased dental pulp.288 MiRNAs are noncoding chapter). A practice-based, randomized clinical trial compared RNA molecules that regulate gene expression in complex the effectiveness of non-desensitizing toothpaste (Colgate inflammatory responses and may eventually assist in clinical Cavity Protection Regular, Colgate-Palmolive, New York, New predictions of pulpal status. Therefore, more accurate chair- York), desensitizing toothpaste (Colgate Sensitive Fresh Stripe, side diagnostic methods are potentially feasible to develop, Colgate-Palmolive), and a professionally applied desensitizing especially a method that involves sampling from crevicular agent (Seal & Protect).89 The findings showed a significant fluid, dentinal fluid, or the pulp directly. For this reason, reduction of dentin hypersensitivity in the desensitizing thera- more research is needed to determine the key mediators that pies compared to the non-desensitizing group that was a much would predict survival or degeneration of the dental pulp in more significant reduction in the professionally applied desen- difficult diagnostic cases. sitizing agent over a 6-month period.

DENTIN HYPERSENSITIVITY AND PULPAL REACTIONS TO ITS MANAGEMENT LOCAL ANESTHETICS Dentin hypersensitivity is a special situation in which a signifi- An intact pulpal blood flow is critical for maintaining the cant, chronic, pulpal pain arises, which does not seem to be health of the dental pulp. Because the dental pulp is enclosed associated with irreversible pulpal pathosis in the majority of in a rigid chamber and is supplied by few arterioles through cases. Dentin hypersensitivity is characterized by brief sharp the apical foramina, it cannot benefit from collateral circulation pain arising from exposed dentin in response to stimuli, typi- or volumetric changes that compensate for changes in blood cally thermal, evaporative, tactile, osmotic, or chemical, that flow in other soft tissues. Furthermore, reduction in blood flow cannot be ascribed to any other form of dental defect or patho- has the compounding effect of reducing the clearance of large sis.104 Facial root surfaces in canines, premolars, and molars molecular weight toxins or waste products,201 thus causing are particularly affected, especially in areas of periodontal irreversible pulpal pathosis. Vasoconstrictors are added to local attachment loss. Dentin hypersensitivity may be related to anesthetics to enhance the duration of anesthesia. However, excessive abrasion during tooth brushing, periodontal disease, vasoconstrictors in local anesthetics could negatively impact or erosion from dietary or gastric acids,2,3,47 and it may increase the health of the pulp if they reduce blood flow, particularly if following scaling and root planing.47,274 The dentin is hyper- the pulp is inflamed preoperatively. Earlier studies have docu- sensitive, most likely due to the lack of protection by cemen- mented that vasoconstrictors in local anesthetics do reduce tum, loss of smear layer by acidic dietary fluids, and the pulpal blood flow in experimental animals when administered hydrodynamic movement of fluid in dentinal tubules.4,33 The by infiltration and nerve block132 (Fig. 13-12), and that this degree of inflammation in the pulp in cases of dentin hyper- effect was more severe with periodontal ligament injections131 sensitivity is not well characterized, because the condition is (Fig. 13-13). More recently, clinical trials were conducted in usually not severe enough to warrant tooth extraction or end- which subjects were given infiltration of different local anes- odontic therapy. However, patent dentinal tubules are present thetics with or without epinephrine at a concentration of in areas of hypersensitivity284 (Fig. 13-10) and may result in 1 : 100,000 and the pulpal blood flow was measured by laser increased irritation and localized reversible inflammation of Doppler flowmetry. In groups that received the epinephrine, the pulp at the sites involved. there were consistently significant reductions in pulpal blood The application of neural modulating agents such as potas- flow,5,49,177 even if the infiltration was palatal to maxillary pre- sium nitrate,163 or tubule blocking agents such as strontium molars.213 Interestingly, in one study the reduction in pulpal chloride, oxalates or dentin bonding agents (Fig. 13-11),4,202 blood flow with epinephrine infiltration was more than the usually alleviates the condition, at least temporarily. However, reduction in gingival blood flow and did not return to baseline 580 PART II The Advanced Science of Endodontics

FIG. 13-10 SEM image of an exposed dentin surface of a hypersensitive area. A, A large proportion of dentinal tubules (arrows) are shown to be open. B, SEM image of a fractured dentinal tubule of a hypersensitive area. The lumen of the dentinal tubule is partitioned by membranous structures (arrow). C, SEM image of exposed dentin surface of a naturally desensitized area. The lumens of dentinal tubules A 5 mm B 1 mm (arrows) are mostly occluded, and the surface is extremely smooth. D, SEM image of a fractured dentinal tubule of a naturally desensitized area. Rhombohedral platelike crystals of from 0.1 to 0.3 µm (arrow) are present. (From Yoshiyama M, Masada J, Uchida A, Ishida H: Scanning electron microscopic characterization of sensitive vs. insensitive human radicular dentin, J Dent Res 68:1498, 1989. Reprinted with permission.)

1 mm C 5 mm D

125 n 5 5 Maxillary canine teeth

225

250

275 Percent change in pulp blood flow 2100 01 5 10 15 20 75 Time (min)

FIG. 13-11 Smear layer treated with 30% dipotassium oxalate for 2 FIG. 13-12 Effects of infiltration anesthesia (i.e., 2% lidocaine with minutes plus 3% monopotassium and monohydrogen oxalate for 2 minutes. 1 : 100,000 epinephrine) on pulpal blood flow in the maxillary canine teeth of Dentin surface is completely covered with calcium oxalate crystals (original dogs. There is a drastic decrease in pulpal blood flow soon after the injection. magnification ×1900). (From Pashley DH, Galloway SE: The effects of oxalate Arrow indicates the time of injection. Bars depict standard deviation. (From treatment on the smear layer of ground surfaces of human dentine, Arch Oral Kim S, Edwall L, Trowbridge H, Chien S: Effects of local anesthetics on pulpal Biol 30:731, 1985. Reprinted with permission.) blood flow in dogs, J Dent Res 63:650, 1984. Reprinted with permission.) CHAPTER 13 Pulpal Reactions to Caries and Dental Procedures 581

(mean 6 SEM, n 5 20) 24 weeks between the groups that did or did not receive 120 intrapulpal anesthesia, and when given, in the groups where the anesthetic contained or did not contain epinephrine.254 0 PULPAL REACTIONS TO 220 Canine teeth RESTORATIVE PROCEDURES A large body of literature exists on the effects of restorative 240 procedures on the dental pulp. This topic, understandably, has been important for practicing dentists for many years. Restor- 260 ative procedures are performed primarily to treat an infectious disease, dental caries, which itself causes significant irritation Premolar teeth 280 of the pulp. They may also be performed to help restore missing teeth; correct developmental anomalies; address fractures, Percent change in pulpal blood flow cracks, or failures of previous restorations; or a myriad of other 2100 0 5 10 20 30 60 abnormalities. One key requirement of a successful restorative procedure is to cause minimal additional irritation of the pulp Time (min) so as not to interfere with normal pulpal healing. When pulp FIG. 13-13 Effects of ligamental injection (i.e., 2% lidocaine with vitality is to be maintained during a restorative procedure, then 1 : 100,000 epinephrine) on pulpal blood flow in the mandibular canine and a provisional diagnosis of reversible pulpitis rather than irre- premolar teeth of dogs. Injection was given in the mesial and distal sulcus of versible pulpitis must preexist. Therefore, it would be most premolar teeth. Injection caused total cessation of pulpal blood flow, which desirable to perform a minimally traumatic restorative proce- lasted about 30 minutes in the premolar teeth. Arrow indicates time of injec- dure, which would not potentially convert the diagnosis to tion. (From Kim S: Ligamental injection: a physiological explanation of its irreversible pulpitis. As discussed previously, irreversible pul- efficacy, J Endod 12:486, 1986. Reprinted with permission.) pitis may present clinically with severe spontaneous postoperative pain, but it may also be asymptomatic, leading to the asymptomatic demise of the pulp. The additive effects of restor- values after 1 hour of injection.5 Similar reductions in pulpal ative procedures are particularly critical in borderline cases, blood flow were reported when inferior alveolar nerve block such as those of moderately symptomatic teeth with deep caries injections of lidocaine and 1 : 100,000 or 1 : 80,000 epinephrine but no pulp exposure. There are still many factors whose influ- were administered.187 It is important to note a limitation of ence on the response of the dental pulp to the cumulative effects studies using laser Doppler flowmetry, which is that a large of caries, microleakage, restorative procedures, and materials is proportion of the signal measured may be from sources other not well understood. It is generally accepted that the effects of than the dental pulp.212,241 Thus, the monitoring of minor pulpal insults, be they from caries, restorative procedures, or changes in pulpal blood flow must be interpreted with caution, trauma, are cumulative—that is, with each succeeding irrita- particularly if the rubber dam or a similar barrier was not tion, the pulp has a diminished capacity to remain vital. As a used.99 Human studies on the effects of periodontal ligament part of informed consent, the clinician is often faced with the or intraosseous injections on pulpal blood flow are not avail- task of outlining possible risks of restorative treatment. One able, but from animal studies it is probable that these supple- study from a hospital in Hong Kong addressed the fate of pulps mental anesthetic techniques cause a more severe reduction or beneath single-unit metal-ceramic (MC) crowns or MC bridge even transient cessation of pulpal blood flow.133 It was also abutments.48 Patients who had received either treatment were shown that intraosseous injection of Depo-Medrol™ (a corti- invited to attend a recall appointment that involved both clini- costeroid) in patients with symptomatic irreversible pulpitis cal and radiographic examinations. Researchers examined 122 causes a significant reduction of prostaglandin E2 in the pulp teeth with preoperatively vital pulps treated with single-unit 1 day after administration, indicating that this route of injec- MC crowns and 77 treated as bridge abutments. The mean tion results in significant permeation into the pulpal tissues.116 observation period was 14 years for the former and 15.6 years Taken together, these findings suggest that local anesthesia for the latter. Pulpal necrosis had occurred in 15.6% of the teeth with vasoconstrictors may compromise the inflamed pulp’s treated with single-unit crowns, whereas 32.5% of the pulps in ability to recover from inflammation, particularly if it is severely the bridge retainer groups had become necrotic. There was a inflamed, or if the tooth is subjected to extensive restorative significantly higher percentage of pulpal necrosis in anterior procedures, and if the anesthetic is delivered via a periodontal teeth that served as bridge abutments (54.5% of anterior abut- ligament or an intraosseous route. However, it is important to ment teeth examined). In general, however, the available evi- realize that this hypothesis should be supported or refuted by dence indicates that the effects of dental procedures on the pulp prospective randomized clinical trials. depend on the following factors. Intrapulpal anesthesia is often used as a last resort, when pulpal anesthesia is insufficient during root canal therapy. The effect of intrapulpal anesthesia on the pulp in these cases is The Degree of Inflammation not considered, as the pulp will be removed. However, occa- of the Pulp Preoperatively sionally a pulpotomy is performed to maintain pulpal vitality, As stated previously, the dental pulp is compromised in its such as in children where the tooth has an immature apex. One ability to respond to external irritants because it is enclosed in study has shown that intrapulpal anesthesia can be used in a noncompliant environment and because it lacks collateral these cases, with no clinical differences on follow-up of over circulation. Thus, the more severe the pulp is inflamed, the less 582 PART II The Advanced Science of Endodontics

will be its ability to respond to further irritation, such as in the form of restorative procedures.147 X5Degree of R. D.5Remaining pulpal response dentin thickness (MM) Most research studies designed to evaluate the effects of restorative procedures (or materials) on the pulp are conducted on human or experimental animal teeth with normal pulp. Furthermore, many of the animal research projects have Decreasing been performed on anesthetized animals without the use of R. D. local anesthesia, which as stated previously, reduces pulpal blood flow. Therefore, the results of these studies may not 3.0 MM reveal the true effects of these procedures when the carious lesion already causes inflammation of the pulp, and pulpal blood flow is reduced by local anesthetic with vasoconstric- 6000 or 20,000 rpm X 2.0 MM tors. A study that evaluated the response of the pulp to capping procedures as a function of duration of exposure

in human molars with uninflamed pulp caused significant dis- X5Degree of R. D.5Remaining placement of odontoblastic nuclei, pulp inflammation, and pulpal response dentin thickness (MM) even areas of necrosis related to the areas that were dried.57 X5Burn lesion However, another study showed that the effects of desiccation are transient in that within 7 to 30 days there is autolysis of 32 Decreasing the aspirated cells and formation of reactionary dentin. The R. D. pulp in cases with aspirated odontoblasts, following desiccation for 1 minute, was not sensitive to clinical scraping with an explorer. The sensitivity was restored with rehydration of 3.0 MM the cavities and was increased in other cases where pulp inflammation was induced by microbial contamination.154 In this study, despite the lack of sensitivity in desiccated cavities, X 2.0 MM neural elements were seen histologically to be pushed into the tubules like the odontoblastic nuclei. The disruption of the XX 1.5 MM odontoblastic layer and peripheral neural elements in the pulp with desiccation was also observed in a rat model using axonal XXX 1.0 MM transport of radioactive protein.37 XXX Burn lesion XXXX Biologic and Chemical irritation Dental caries is clearly an infectious disease in which microorganisms and their virulence determinants constantly irritate the pulp, even at the early stages, long before pulp exposure.34 However, despite the elimination of visible caries during cavity preparation, the cavity floor is undoubtedly left with some contamination by caries bacteria. Although the rubber dam should be used with any cavity preparation to prevent cavity

FIG. 13-15 Without adequate water coolant, larger cutting tools (e.g., no. contamination with salivary microorganisms, the use of water 37 diamond point) create typical burn lesions within the pulp when the remain- coolants allows the cavity to be contaminated with bacteria ing dentin thickness becomes less than 1.5 mm. (From Stanley HR, Swerdlow from water lines. Concerns about residual cavity contamina- H: An approach to biologic variation in human pulpal studies, J Prosthet Dent tion prompted some to use cavity disinfection with caustic 14:365, 1964. Reprinted with permission.) chemicals. Chemicals such as hydrogen peroxide, sodium hypochlorite, or calcium hydroxide solutions have been proposed for this purpose, although they may exert a toxic effect.54 An earlier study showed that the amounts of residual bacteria following adequate restoration are not significant.170 Once dentin is exposed, there is a constant outward flow of dentinal fluid that minimizes the inward flow of any noxious agents.275 This may aid in the reduction of irritation from residual microbial factors in dentinal tubules. In contemporary practice, most chemical irritation during restorative procedures results from the application of etching agents, especially strong acids, in the form of total dentin etch, particularly if capping of exposed pulp is performed.93,197 Etching is performed to remove the smear layer, promote physical adhesion of bonding agents to dentin by forming resin tags in the dentinal tubules, and permeate the newer unfilled resin primers into the unmineralized surface layer of collagen to

FIG. 13-16 Burn lesion with necrosis and expanding abscess formation in form the so-called hybrid layer. a 10-day specimen. Cavity preparation dry at 20,000 rpm with remaining If the cavity is relatively superficial and is adequately sealed dentin thickness is 0.23 mm. (From Swerdlow H, Stanley HR Jr: Reaction of with a restorative resin, then etching of dentin is probably not the human dental pulp to cavity preparation. I. Effect of water spray at detrimental to the pulp because of the narrow diameter of 28 20,000 rpm, J Am Dent Assoc 56:317, 1958. Reprinted with permission.) dentinal tubules and their low density in peripheral dentin. In fact, one study documented that histologic evidence of bacteria in human cavities restored with composite was signifi- excessive heat with other irritation factors and its proximity to cantly less if the cavity had been etched with phosphoric acid the pulp may induce pathologic changes. than if it were etched with 17% EDTA or nonetched.176 Pulpal inflammation in this study was not correlated with the etching Desiccation treatments but with bacterial presence; thus, in cases of etching Desiccation during cavity and crown preparation has long been with phosphoric acid, if bacteria were also present, severe known to cause aspiration of odontoblastic nuclei into dentinal pulpal inflammation and necrosis could be seen. tubules and pulpal inflammation.31 One study showed that as Self-etching formulations have become popular because little as 30 seconds of continuous air drying of class V cavities they eliminate the separate etching step involved in total-etch 584 PART II The Advanced Science of Endodontics

procedures. Some have speculated that the bonding of self- estimated that the area occupied by tubule lumina at the DEJ etching systems may be poorer than total-etch systems because was 1% of the total surface area at the DEJ and 22% at the of the weaker acidity of the acidic primers of self-etching pulp.203 Thus, it is not surprising that several studies have systems when compared to that of total-etch systems.28 shown that pulpal inflammation in response to restorative pro- However, studies have shown no significant differences between cedures increases with the reduction in RDT.159,175 One study the two adhesive systems in postoperative sensitivity,208 long- examined the differential effects on the rat pulp of the prepara- term in vivo degradation,141 or long-term in vitro bond tion method, remaining dentin thickness, coolant, drill speed, strength.10 One clinical study showed no differences between conditioning with EDTA, and filling materials.174 Subsequent the two systems with respect to bacterial leakage and the to the cavity preparations a tooth slice was obtained and main- inflammatory response in the pulp.181 The most important vari- tained ex vivo as an organ culture for up to 2 weeks. The results able that affected the pulp in this study was the amount of showed that the remaining dentin thickness was the most bacterial leakage with either system. important factor in pulpal injury. Other factors that may contribute to pulpal irritation during With the passage of time following cavity preparation, there resin placement from chemical/biologic irritants include un is reduction in the permeability of RDT.204 This may be due to polymerized monomer and polymerization shrinkage. Higher rapid deposition of reactionary dentin, the migration of large concentrations of monomeric resin components were shown proteins into the tubules, or the diminution of tubule diameter to exert an inhibitory effect on T lymphocytes and spleen as dentin becomes more sclerotic. Using a primate model, it cells,124 and monocytes/macrophages149,216,217,231 in vitro. These was shown that the basic rate of secondary dentin deposition components may leach directly into the pulp in deep cavities was about 0.8 µm/day and that this rate increased to an average and cause chemical irritation.59,110 Shrinkage during polymer- of 2.9 µm/day following restorative procedures. Interestingly, ization of composites may induce internal stresses on dentin in this study dentin deposition was also more rapid next to and create voids that allow microleakage. Shrinkage of resins shallow cavities than deep cavities277; however, another study is estimated to range from 0.6% to 1.4%, and should be mini- showed that total reactionary dentin deposited was thicker in mized during placement by incremental curing and possibly deeper and wider cavities.173 starting the restoration with flowable resins.28 Clinically, it has been observed that postoperative sensitiv- In summary, the available evidence indicates that chemicals ity is common with many restorative procedures. Following involved in modern restorative procedures may irritate the resin composite restorations on patients, it was shown that pulp if placed directly on an exposure, or if there is microbial postoperative sensitivity was related to the depth of the cavity, leakage along the tooth/restoration interface. but not to the presence or absence of liners or bases.268 In addition to the depth or the width of a large cavity prepara- The Proximity of the Restorative tion, a crown preparation exposes more dentinal tubules to microbial or chemical irritation. During crown fabrication, there Procedures to the Dental Pulp and are added irritation factors such as length of time of the prepara- the Surface Area of Dentin Exposed tion, impression techniques, and the imperfect adaptation of It has been known for several decades that as the carious lesion temporary restorations, causing microleakage during the tempo- progresses toward the pulp, particularly when the remaining rization period. Because of the precise engineering requirements dentin thickness (RDT) is less than 0.5 mm, there is an increas- of some restorations, some providers may be inclined to reduce ingly severe pulpal reaction, with a greater likelihood of the the coolant during crown preparation steps such as finalizing the pulp undergoing irreversible pathosis.220 The diameter and finishing lines. However, crown preparations without coolants density of dentinal tubules increase closer to the pulp (Fig. have been shown to dramatically reduce pulpal blood flow in an 13-17). Based on the dentinal tubule density at the DEJ (about animal model (Fig. 13-18).133 There are few studies available on 65,000/mm2) and the pulp (about 15,000/mm2),84,88 it was the direct effects of modern crown and bridge techniques on the

FIG. 13-17 Schematic of convergence of tubules toward the pulp. A, Periphery of the dentin. Most surface area is occupied by intertubular dentin (), with a few tubules surrounded by hypermineralized A peritubular dentin (). B, Near the pulp, the increase Top in tubule diameter has occurred largely at the Bottom expense of the peritubular dentin. This substrate has B high protein content. As the remaining dentin is made thinner (from A to B), the permeability of the dentin increases, because both the diameter and the density of dentinal tubules are increased. (From Bouillaguet S: Biological risks of resin-based materi- B als to the dentin-pulp complex, Crit Rev Oral Biol Med 15:47, 2004. Reprinted with permission.)

A CHAPTER 13 Pulpal Reactions to Caries and Dental Procedures 585 pulp. However, some long-term outcome studies have docu- and that permeability depends on factors such as the location mented that the incidence of pulp necrosis following crown within the same tooth, the age of the patient, and the presence placement ranges from 10% to 50%.48,79,269 of pathologic conditions such as dental caries. Fundamentally, the permeability of dentin depends on the collective sum of The Permeability of Dentin and the the permeability of individual tubules at a particular site in the tooth. The tubular diameter increases from about 0.6 to 0.8 µm Odontoblastic Layer Between the Area 200 close to the DEJ to about 3 µm at the pulp. Given that bacte- Being Restored and the Pulp rial cells are about 0.5 to 1 µm in diameter, it is evident The permeability of dentin plays an important role in the that in deep cavity preparations, particularly when total-etch ingress of potential irritants to the pulp. It is clear from research procedures are employed, bacteria can migrate through the done since the 1980s that dentin is not uniformly permeable remaining dentin into the pulp. With age the width of peritubular dentin increases, causing a reduction in tubular lumen or sclerosis. Caries causes demin-

With H2O spray eralization in superficial dentin, which is associated with rem- ineralization and the formation of caries crystals within the 100 tubules of inner undemineralized dentin (Fig. 13-19). This causes a decrease in permeability in dentin subjacent to the 75 carious lesion202 and could be considered a protective mechanism, as it may delay the progress of the carious lesion. 50 It was shown that irritation from cavity preparation increased the permeability of the odontoblastic cell layer only at the site of the cavity preparation.117,263 In addition to the 25

(15-mm microspheres) physical barrier to permeability and the production of reactionary or reparative dentin, the odontoblastic layer may, in fact,

Percent of control pulpal blood 0 contribute to the host response of the dental pulp by express- Control One-half Completed 1 hour ing important inflammatory mediators152,196 or recognizing bac- preparation preparation later teria through Toll-like receptors.22,66,74,123 FIG. 13-18 Effects of crown preparation in dogs, with and without water and air spray (at 350,000 rpm), on pulpal blood flow. Tooth preparation without water and air spray caused a substantial decrease in pulpal blood The Age of the Patient (see also Chapter 26) flow, whereas that with water and air spray caused insignificant changes in Resting pulpal blood flow (PBF), as well as the changes in PBF the flow. in response to cold application, will decrease with age.112 Age

C A 1 mm 2 mm 500 nm A H T H SP

H Pd

U A U B C U

FIG. 13-19 TEM of undemineralized specimens of resin-bonded, caries-affected dentin. A, Stained TEM of undemineralized specimens following the application of the self-etch ABF system to caries-affected dentin. The hybrid layer (H ; between arrows) was about 3 µm thick, and the underlying undemineralized dentin (U) was highly porous (arrowhead). The dentinal tubule was covered with a smear plug (SP) and was partially obliterated with large caries crystals (pointer). A, filled adhesive. B, Stained section of the total-etch single bond adhesive bonded to caries-affected dentin. A hybrid layer (H) between 15 and 19 µm thick could be seen, with a partially demineralized zone (open arrows) above the undemineralized caries affected dentin (U). T, dentinal tubule. C, composite. C, Higher magnification of the basal part of the unusually thick hybrid layer (H), shown in (A). Banded collagen fibrils (open arrow) were separated by unusually wide and porous interfibrillar spaces (open arrowheads). A partially demineralized zone (Pd) was present along the demineralization front. This zone was not seen in phosphoric- acid-etched sound dentin (B). U, undemineralized caries-affected dentin. (From Yoshiyama M, Tay FR, Doi J, et al: Bonding of self-etch and total-etch adhesives to carious dentin, J Dent Res 81:556, 2002. Reprinted with permission.) 586 PART II The Advanced Science of Endodontics

may also be associated with reduction in pulpal neuropep- containing extruded globules of resin material.114,115 Histologic tides.85 However, studies show no differences between young examination of these pulps shows macrophages and giant cells and old pulp in the regenerative capacity of odontoblast-like surrounding the resin particles. Lastly, resin monomers have cells and in the presence of cells positive for class II major been shown to decrease the activity of immunocompetent cells histocompatibility complex, heat shock protein 25, or nestin, in a dose-dependent manner in in vitro functional assays.124 when subjected to cavity preparation.128 An examination of Although all of these effects are documented, their extent and young versus old normal human pulp showed that young pulp therefore morbidity on the dental pulp is speculative and had increased expression of biologic factors related to cell dif- doubtless does not act solely to effect pulpal demise. As previ- ferentiation, proliferation, and the immune response, whereas ously noted, most restorative materials are placed adjacent to older pulp had increased factors related to apoptosis.259 These pulps that are previously compromised by bacterial insult, and analyses cannot translate to the pulp’s ability to deal with irrita- disease, debridement, and restoration of the tooth have cumu- tion and its sequelae. Thus, the net result of the ability of the lative effects on the dental pulp. pulp to cope with external stimulation or irritation in humans Although pulpal irritation is largely considered to be a nega- with advancing age is not clear. tive sequela, the irritant potential of certain restorative materials is central to their usefulness in restorative dentistry. Calcium PULPAL REACTIONS TO hydroxide is one of the oldest and most widely used medicaments for stimulation of dentinal bridge formation subsequent RESTORATIVE MATERIALS to microscopic or gross pulpal exposure. The low-grade pulpal The effects of restorative materials on the dental pulp have irritation that it induces is important for dentinal bridge forma- been investigated and seem to relate directly to the perme tion in exposures.61,230 The degree of inflammation is depen- ability of the associated dentin. The degree of dentin permea- dent on the preparation of calcium hydroxide used. Aqueous bility, however, is often variable and is governed by several suspensions of calcium hydroxide applied to exposed pulps factors including age and caries status.251 The most important cause superficial necrosis of pulpal tissue followed by low- variable in dentin permeability to restorative materials is the grade inflammatory changes. Within 30 days the tissue subja- thickness of dentin between the floor of the cavity preparation cent to the necrotic zone has reorganized and resumed normal and the pulp.1 architecture. Hard-setting calcium hydroxide preparations as Given the importance of dentin permeability, there are direct well as mineral trioxide aggregates are effective in eliciting pulpal effects of any given restorative material that are governed dentinal bridge formation with a much smaller to nonexistent by the composition of the material and associated eluted or necrotic zone.102 This is preferable in vital pulp therapies degraded products. Unbound components of resin materials such as the Cvek pulpotomy, where maintenance of the and preparative agents such as acid etchants can affect the maximum amount of vital pulp tissue is desirable and the subjacent pulp by inducing an inflammatory response.87,93,215 extent of pulpal inflammation is minimal60 (Fig. 13-20). The indirect effects of desiccation or demineralization of The irritation potential of calcium hydroxide across intact dentin as well as the direct effects of the material itself when in dentin is dependent on factors such as the remaining dentin contact with pulpal tissue mediate this inflammatory response. thickness and permeability. Application of calcium hydroxide Studies have shown that the certain cytotoxic components of to intact dentin appears to induce sclerosis by promoting resin monomers (e.g., triethylene glycol dimethacrylate and crystal precipitation within the tubules accompanied by reduc- 2-hydroxyethyl methacrylate) readily penetrate dentin.81 Simi- tions in permeability.172 larly, eugenol and components of Ledermix (triamcinolone and Although the irritation potential of calcium hydroxide plays demeclocycline) have been shown to pass through dentin into a role in its effectiveness, the high pH of this material can liber- the subjacent pulp.109,111 In vivo data show that these chemicals ate bioactive molecules from dentin. Numerous authors have have an effect on the pulp; however, the effect seems to be short demonstrated that dentin matrix proteins like TGF-β1 and lived and, in the absence of bacteria, it is reversible.21 adrenomedullin are liberated by both calcium hydroxide and The mechanisms whereby restorative materials exert an mineral trioxide aggregate.92,257 Once liberated they are able to injurious effect on the dental pulp vary. Evidence exists that facilitate hard tissue formation yet again. This offers another supports direct and, in some instances, prolonged cytotoxicity, explanation for the ability of these materials to induce hard stimulation of hypersensitivity reactions, or impairment of the tissue formation in vivo. Acid etching or the use of chelating host immune response to bacteria.229 Some of the components agents such as EDTA represent a common step in the place- of resin restorations are released at cytotoxic levels after polym- ment of bonded restorations. The etching process removes erization is completed, leading to chronic stimulation and a mineral and exposes collagen fibrils. As with a high pH, resultant prolonged inflammatory response.80 Furthermore, sequestered bioactive molecules are released and can act on even subtoxic concentrations of certain agents are capable pulpal cells. These include growth factors and metalloprotein- of eliciting allergic reactions in humans.110 Primates hyper ases, which can be inductive for healing but can also degrade immunized with BSA showed significant pulpal damage with the bond of the restoration.205 EDTA has been shown to be even repeated antigenic challenge in class V cavity preparations, more effective at extracting noncollagenous proteins from suggesting a role for antigen-antibody complex–mediated dentin than either calcium hydroxide or MTA but less so than hypersensitivity in tissue destruction.24 In a separate study, citric acid.92,257 exposure to dentin primers elicited a delayed-type hypersensi- Investigations into the pulpal response to glass-ionomer tivity reaction in guinea pigs.127 These studies taken together materials have shown that both the lowered pH of the material present a compelling argument for immune-mediated pulpal and the release of high concentrations of fluoride can inflict tissue damage subsequent to exposure to restorative materials. damage to pulp tissues.126 In direct contact with pulpal cells, Foreign body reactions have also been described in pulps glass ionomers are toxic.143 Direct pulp capping studies have CHAPTER 13 Pulpal Reactions to Caries and Dental Procedures 587

A B

FIG. 13-20 Calcium hydroxide produces an inflammatory response that stimulates dentinal bridge formation. The dentin bridge forms lower in the tooth with calcium hydroxide paste (A) versus hard-setting calcium hydroxide (B). shown it to be inferior to calcium hydroxide.63,105 When applied DIRECT PULP CAPPING WITH to intact dentin and in the absence of bacteria, there is a tran- MINERAL TRIOXIDE AGGREGATE sient inflammatory response and reparative dentin is formed.55 In addition to the direct chemical effects of restorative mate- Direct capping of pulp exposures is indicated in pulps that rials, there are indirect factors that contribute to pulpal irrita- were previously healthy and exposed by trauma or dental tion. The technique sensitivity of certain materials predisposes restorative procedures.250 Although calcium hydroxide has his- them to faulty bonds to tooth structure that can translate to torically been the preferred dressing agent on mechanically dentin hypersensitivity, recurrent disease, and pulpal inflam- exposed pulps, the use of mineral trioxide aggregate (MTA) mation or necrosis. Much attention has been given to the has been proposed, even on carious pulp exposures.16,26,166 Pro- interface created between resin-bonded materials and dentin. spective animal studies and human case reports have evaluated During the etching process, the more highly mineralized peri- the ability of MTA to allow for the formation of a reparative tubular dentin is preferentially dissolved, leaving free collagen dentin bridge and maintain continued pulp vitality.73,83,137 fibrils and opening lateral tubular branches.93,94,171 Applied Although the results are generally favorable, one concern is of resin infiltrates the exposed collagen mesh, creating a layer 5 tooth discoloration if the gray MTA formulation is used on to 10 µm thick referred to as the hybrid layer.178 This layer, anterior teeth. along with the resin permeating exposed tubules, forms the In one clinical study, MTA was used as a pulp capping mate- bond between the resin and dentin. If the preparation is too rial for carious pulp exposures.26 Forty patients, ages 7 to 45, dry, the collagen fibrils collapse and the resin cannot effectively who were diagnosed with reversible pulpitis had caries removed permeate the mesh, which results in a defective bond. As the using a caries detection dye and sodium hypochlorite for optimal degree of hydration of the preparation surface can vary hemostasis. The treatment was performed in two visits to allow from material to material, resin restoration placement is tech- the MTA to set up and to confirm pulp sensibility to pulp tests nique sensitive. This same principle is applicable to the prac- in the second visit. Success was determined radiographically, tice of bonding fractured tooth fragments where the segment with subjective symptoms, pulp testing with cold, and contin- has become dehydrated while outside of the mouth. Current ued root formation on immature teeth. Outcomes were mea- protocols recommend rehydration of the segment prior to sured over a period of up to 9 years postoperatively and showed bonding, thus increasing the mechanical and presumably the an overall success of 97%, with all the teeth in the immature microbial seal.78 This is particularly important with a compli- root group showing success. In another clinical study in which cated crown fracture where the pulpal protection by intact dentists and students participated in the treatment, pulp dentin is absent. capping of carious pulp exposures was compared between Some restorative materials rely on their medicinal proper- MTA and calcium hydroxide.166 At follow-up, 122 cases of ties as well as their ability to seal a cavity preparation. Materials patients with a mean age of 40 years were available. The containing zinc oxide and eugenol (ZOE) fall into this cate- success with MTA was 78% and that with calcium hydroxide gory. ZOE is used for a variety of purposes in dentistry largely was 60%, a difference that was statistically significant. More because of its anesthetic and antiseptic properties. It has been recently, the same group reported an update of their findings shown to block transmission of action potentials in nerve with a sample size of 229 teeth, which were evaluated from fibers and to suppress nerve excitability in the pulp when 24 to 123 months following treatment. The overall success applied to deep excavations.261 In addition, ZOE has good rates were 80.5% of teeth in the MTA group (137/170) and 59% adaptation to dentin and inhibits bacterial growth on cavity of teeth in the CH group (35/59), which was a statistically walls. These properties have made this a favored material for significant difference. The outcome was also significantly temporary fillings but not for long-term restorations, as ZOE better in any group if the tooth was promptly restored. Within temporaries have been shown to leak after only a few weeks the parameters of these studies, it seems that white MTA is a in situ.286 suitable capping agent for pulp exposures of healthy or 588 PART II The Advanced Science of Endodontics

reversibly inflamed pulps, including cases with asymptomatic carious exposures. However, given that the prognosis of root canal therapy in this last category has been shown to be pre- Dental Laser Wavelengths on the Electromagnetic Spectrum dictably high in many studies, the patient needs to be educated carefully of the options, and consent must be documented. Other calcium silicate–based materials are gaining popular- ity as pulp capping agents and appear to have properties and 400nm 750nm effects similar to MTA.11,184 Biodentine deserves special refer- Invisible ionizing radiation Visible Invisible thermal radiation ence because in addition to the biocompatibility, it has physical properties that are similar to dentin.39,142 X-Rays Ultraviolet Near Infrared Mid Infrared

100nm 1,000nm 2,000nm 3,000nm CO Argon Nd:YAG 2 1,064nm 9300, 9600 514nm Ho:YAG 10,500nm THE USE OF HEMOSTATIC AGENTS Argon HeNe 2,120nm 488nm 632nm Er:YAG AND DISINFECTANTS ON DIRECT AIGa AIGaAS Er,Cr:YSGG 2,940nm PULP EXPOSURES 820nm 830,980nm 2790nm FIG. 13-21 Currently available dental wavelengths on the electromagnetic There is still debate as to whether the outcome of pulp capping spectrum. Note all of the wavelengths are nonionizing. (From Coluzzi DJ: An for exposure depends on the toxicity of medicaments and overview of laser wavelengths used in dentistry, Dent Clin North Am 44:753, materials placed on vital pulp tissues, the ability of these mate- 2000. Reprinted with permission.) rials to induce mineralization, or their ability to seal the cavity from further bacterial ingress. It is likely a combination of these factors, as was shown in the clinical trial with MTA mentioned It has the longest wavelength (10,600 nm). It cannot be deliv- before.26 Another factor in the prognosis of direct pulp caps is ered in an optic fiber, thus it must be used in a hollow-tube-like the ability to control hemorrhage at the exposure site.243 Given wave guide in continuous gated-pulse mode. This means that the difficulty in creating a bacteria-free operating environment the operator does not feel a solid resistance when using this during tooth preparation, the ideal hemostatic agent also laser. Er:YAG, Nd:YAG or Ho:YAG lasers all have an active would have the ability to kill bacteria. medium of a solid crystal of yttrium-aluminum-garnet, which One study compared the effects of two hemostatic/ is impregnated in erbium, neodymium, or holmium, respec- disinfectant agents on the healing of experimental pulp expo- tively. Er:YAG has a wavelength of 2940 nm and is delivered sures created in human third molar teeth and capped with using a solid optic fiber. It has a high affinity for water and calcium hydroxide.234 Pulp exposures were made in 45 maxil- hydroxyapatite, thus it can be used to remove caries and cut lary wisdom teeth scheduled for extraction for orthodontic dentin with coolant. It can also be used on soft tissue. Ho:YAG reasons. Teeth were randomly assigned to receive hard-setting laser has a wavelength of 2120 nm and has high affinity to calcium hydroxide pulp caps after a 30-second surface treat- water but not to tooth structure, thus it is used primarily for ment with one of three agents: 0.9% saline, 2% chlorhexidine, soft tissue surgery. Nd:YAG laser is also delivered fiberoptically, or 5.25% sodium hypochlorite. Although the 7-day saline has a wavelength of 1064 nm, and has been used extensively specimens showed slightly less inflammatory response, there in dentistry because it has a high affinity for water and pig- were no statistically significant differences between the groups mented tissues and offers good hemostasis; thus is used exten- with respect to all dependent measures over the course of the sively in surgery.52 In addition, there are some low-power study. Complete healing was seen in 88% of all specimens at output lasers such as HeNe (helium-neon) (632 nm), and 90 days. GaAlAs (gallium-aluminum-arsenide) (diode; semiconductor) The pulps in these teeth were previously uninjured, and the (720-904 nm) lasers, which have been used in laser Doppler exposures were made in a clean environment. Therefore, it flowmetry and in treating dentin hypersensitivity.134 seems that a mechanical exposure of a healthy pulp created To summarize the available data, a discussion of the applica- during cavity preparation could be disinfected with either 2% tion of lasers for two specific purposes follows. chlorhexidine or 5.25% sodium hypochlorite, capped with a hard-setting calcium hydroxide formulation, and expected to have a favorable prognosis for healing. Lasers in the Prevention, Diagnosis, and Treatment of Caries PULPAL REACTIONS Laser irradiation of deep susceptible pits and fissures may reduce the incidence of dental caries. Once caries develops, TO LASER PROCEDURES some lasers may be effective in removing the carious lesion and Numerous studies have been published on the effect of using sparing undemineralized dentin, because of their differential lasers on enamel, dentin, and pulp (see also Chapter 8). Laser absorption by water and hydroxyapatite. Furthermore, if caries use on hard tissue has been a popular area of research because exposes the pulp in young teeth, particularly those with imma- of the potential benefits of efficiency, reduced sensitivity, dis- ture apices, lasers may be able to effectively excise coronal infection, and precision. Several types of laser technologies are infected pulp in pulpotomy, because of their hemostatic and available that depend on the wavelength, active medium, emis- antibacterial properties. All these potential uses prompted a sion mode, delivery system, power output, and duration of large number of investigations on the effectiveness of lasers in application. The main types available in dentistry today are these applications.

shown in Fig. 13-21. The CO2 laser is historically the oldest Some clinicians have proposed using lasers to enhance adhe- type used on soft tissues and thus has been the most studied. sion of pit and fissure sealants; however, this was shown to not CHAPTER 13 Pulpal Reactions to Caries and Dental Procedures 589

A B C

FIG. 13-22 A, Dental caries. B, Pulsed Nd:YAG ablation by-products (160 mJ, 10 Hz). C, Debris removed with acid etch and polishing. Enamel surface was faceted for reflection spectroscopy. (From Harris DM, White JM, Goodis H, et al: Selective ablation of surface enamel caries with a pulsed Nd:YAG dental laser, Lasers Surg Med 30:342, 2002. Reprinted with permission.) add any advantage following acid etching—a necessity for adhesion.160 Laser fluorescence is used by the DIAGNOdent and DIAGNOdent pen, which are laser devices that have been introduced for the diagnosis of noncavitated caries. Although these devices initially showed some promising results,161 more recent work shows that they are best used as adjunctive devices to radiography and visual examination.7,53,144 Laser ablation of superficial carious lesions may be more conservative than bur preparation. A recent controlled clinical trial supports this tenet, when a free-running pulsed Nd:YAG laser was used to ablate superficial pit and fissure caries in third molars scheduled for extraction98 (Fig. 13-22). In this study there were no histologic differences in the pulp response between the two groups. From the perspective of the effects on the pulp, most laser A applications that are employed in cutting or modifying cavities in dentin or acting directly on the pulp tissue are important. Earlier studies showed reduced permeability of dentin in vitro with a XeCl excimer laser (a laser with a relatively short wavelength of 308 nm in the ultraviolet range).242 The apparent fusion of tubules in superficial layers of dentin was shown 278 to occur with CO2, Nd:YAG, and Er:YAG lasers in vitro. The pulpal responses to Nd:YAG and CO2 lasers were not favorable. It was shown that Nd:YAG and to a lesser degree

CO2 lasers may be associated with charring and significant inflammation in the pulp compared with the Er:YAG laser256,278 (Fig. 13-23). More recently it was reported that water cooling was necessary for laser ablation as it is for high-speed bur preparations.43 Studies have shown that the Er:YAG laser appears to induce similar responses in the pulp to those seen B with high-speed bur preparations, at the level of light micros- 72,252,253,278 FIG. 13-23 A, B, Histopathologic picture of Nd:YAG laser specimen with copy analysis. However, the findings with electron an increasing power. There is direct relationship between the degree of microscopy were different: it was reported that while shallow pathologic changes and the increasing power of the laser. In fact, 1.5W and cavities ablated in rat molars using Er:YAG lasers did not the greater power cause permanent damage to the pulp. show changes from base-line using light microscopy, transmission electron microscopy showed disruption and degeneration of pulpal peripheral nerve endings and of myelin in cavity preparation compared with traditional methods at sheath in the immediate postoperative period (Fig. 13-24).113 this time. This may explain the reduced sensitivity that accompanies For pulpotomy procedures, such as in primary teeth, per- laser cavity preparations. Thus, in summary, it does not manent teeth with immature apices, or pulps that are exposed appear that the use of lasers provides predictable advantages due to fracture and treated promptly, the lasers, particularly 590 PART II The Advanced Science of Endodontics

author reported a reduction of hypersensitivity in 73% of mild cases, 19% of moderate cases, and 14% of severe cases after 4 months using a GaAlAs laser.134 Low-output lasers do not have any effects on the morphology of enamel or dentin, but they are thought to cause a transient reduction in action potential mediated by pulpal C-fibers, but not δA -fibers,276 although this finding was not consistent.192 Nd:YAG lasers have also been used in dentin hypersensitivity. Because of the higher power output, these lasers cause superficial occlusion of dentinal tubules of up to 4 µm,158 in addition to action potential blockage within the pulp in vitro or in experimental animals.191,192 However, a placebo-controlled clinical trial has shown that both Nd:YAG and placebo caused a significant reduction in dentin hypersensitivity for up to 4 months postoperatively but were not different from each other.153 More recent clinical trials A show that although lasers are useful for managing hypersensitivity, they do not appear to be better than less costly and more readily available alternatives.82,267 * Use of Lasers as a Protective Measure for Dentin, Under Traditional Cavity Preparation One clinical study has proposed that if lasers are used to prepare cavities or used on prepared dentin after traditional cavity preparation with burs, this would protect the dentin, as its permeability and bacterial content may decrease. In this study, two patients were scheduled to have six teeth extracted during orthodontic treatment.90 In the teeth that had laser irradiation (GaA1As laser, lambda = 660 nm, power of 30 mW and energy dose of 2 J/cm2) and were examined with transmission electron microscopy (TEM) after 28 days, B the odontoblastic process had increased contact with the extracellular matrix, and the collagen fibrils appeared more FIG. 13-24 A, Control; normal odontoblastic process (arrow) and a few organized than those of the control group (traditional bur nerve terminals (arrowheads) are seen in a dental tubule of rat upper first preparation only). The study concluded that laser irradiation molar. B, Six hours after Er:YAG laser irradiation; disrupted cell membrane of accelerates the recovery of the dental tissues in the predentin a nerve terminal that contains some granular vesicles (arrow) and shrinkage region. of an odontoblastic process (arrowhead) are noted in dentinal tubules just under the ablated area. An asterisk indicates the irradiated side (TEM, ×13,700, bar = 1 µm). (From Inoue H, Izumi T, Ishikawa H, Watanabe K: Short-term histomorphological effects of Er:YAG laser irradiation to rat coronal PULPAL REACTIONS TO VITAL dentin-pulp complex, Oral Surg Oral Med Oral Pathol Oral Radiol Endod BLEACHING TECHNIQUES 97:246, 2004. Reprinted with permission.) Vital bleaching techniques employ strong oxidizing agents, namely 10% carbamide peroxide and hydrogen peroxide, to

CO2 lasers, may be useful in achieving precise surgical excision bleach enamel of teeth with vital pulp. There have been con- of coronal pulp and immediate hemostasis. A controlled clini- cerns about the potential for pulpal irritation during these

cal study showed that CO2 laser pulpotomy was comparable to procedures because of the long duration that the chemicals traditional methods in experimental pulpotomies of primary are in contact with the teeth, particularly if dentin with open teeth scheduled for extraction for orthodontic reasons.70 tubules or cracks is present. Histologic or histochemical anal-

However, an animal study in which CO2 and Nd:YAG lasers ysis of the pulp following bleaching for up to 2 weeks showed were compared revealed poor response with both lasers minor inflammatory changes in the pulp of the bleached compared with calcium hydroxide.125 An animal study using teeth that were reversible.9,86 One clinical report documented the Er:YAG laser showed that the results are dependent on the that if 16% carbamide peroxide was used, gingival irritation power settings, in that lower energy delivered with this laser was evident; however, no changes in pulp vitality or in symp- produced favorable results.135 toms were noted. Even in patients who develop symptoms postoperatively, these tend to be reversible and could be prevented by treating the teeth with fluorides and by correcting Lasers in the Treatment restorative deficiencies preoperatively.180 Clinical symptoms of Dentin Hypersensitivity are likely to be due to increases in neuropeptides, such as Earlier studies have shown effectiveness ranging from 5% to substance P, in the pulp.44 An earlier clinical trial showed that 100% of low-output lasers on dentin hypersensitivity.134 One vital bleaching using 10% carbamide peroxide in a custom CHAPTER 13 Pulpal Reactions to Caries and Dental Procedures 591

a A D

B C

FIG. 13-25 Mandibular first molar of 49-year-old female, no pain. Clinically caries free. Calculus, periodontal disease, and bone loss from two thirds to three fourths of root. A, Note narrowness of pulp chamber seen throughout all serial sections (original magnification ×8.5). B, Bacterial plaque and adjacent dentinal tubules with bacteria (oblique arrow in A) (original magnification ×400). C, Farthest penetration of bacteria in dentinal tubules (vertical arrow in A) (original magnification ×400). D, Where dentinal tubules invaded by bacteria terminate in the pulp (horizontal arrow in A), a small but dense accumulation of lymphocytes and macrophages appears (a, artifact; pulp tissue torn away from dentin during processing) (original magnification ×400). (From Langeland K: Tissue response to dental caries, Endod Dent Traumatol 3:149, 1987. Reprinted with permission.) tray for 6 weeks was safe for the pulp health for up to 10 the root surface to the oral cavity. Occasionally, pulpal inflam- years postoperatively, although the bleaching effectiveness mation secondary to severe periodontitis is observed.148 Some may decline with time.222 In one clinical study, premolars reports have described bacterial infiltration through dentinal scheduled for orthodontic extraction had 38% H2O2 bleach- tubules of exposed root surface causing mild inflammatory ing gel with and without a halogen light source applied. changes in the pulp (Fig. 13-25).146 However, in a case report There were no histologic effects on the dental pulp at 2 to 15 of 25 teeth that were extracted in a patient with extensive days after bleaching.136 When the study was performed on periodontal disease, none of the pulp tissue had significant incisors with the same treatment for 45 minutes, areas of inflammatory changes.258 It is much more common for pulp coagulation necrosis could be seen in the pulp.56 Therefore, necrosis, or for failure of healing of periapical lesions, to caution should be exercised when this caustic agent is used present clinically with signs of periodontal disease than for for extended bleaching. periodontal disease to cause pulpal pathosis. Primary endodontic, secondary periodontal pathosis is particularly evident PULPAL REACTIONS TO if a perforation occurs in the pulp chamber or coronal third of the root during endodontic treatment and is not promptly PERIODONTAL PROCEDURES treated in cases of cracked teeth/vertical root fractures or in In an intact nontraumatized tooth, the dental pulp maintains cases of congenital tooth defects such as palatal groove defects. its healthy existence throughout life because it is insulated Thus, it is more likely for microbial irritants to move outward from microbial irritation in the oral cavity (see also Chapter from a necrotic pulp to cause periodontal breakdown than for 17). Periodontal disease causes attachment loss, which exposes them to move inward from a periodontal pocket to cause 592 PART II The Advanced Science of Endodontics

irreversible pathosis in the pulp. The reasons for this observa- orthodontic force.95 Biochemical and molecular markers tion are not fully understood. However, if the assumption is confirm that apoptosis and necrosis of pulpal cells is also that bacteria may migrate through patent dentinal tubules in increased subsequent to movement.209 However, it was shown these situations, as shown in Fig. 13-25, then it may be that that the inflammatory mediators IL-1 alpha and TNF-alpha the outward dentinal fluid flow in teeth with vital pulp con- show a minor increase in the pulp during orthodontic move- tributes to the resistance to ingress of bacteria in sufficient ment, compared to the increase in periodontal tissues.25 Histo- amounts to cause a clinically significant disease process. Once logic examination of pulps in teeth subjected to intrusive the pulp degenerates, dentinal fluid flow no longer exists. forces showed vascular congestion and dilatation as well as Thus, microbial irritants from the pulp may promote pocket vacuolization of the odontoblastic layer.245-247 Most if not all of formation and periodontal bone loss,121 and the prognosis of these effects are due to circulatory changes, and the consensus endodontic and periodontal treatment may be related,122 as the is that they are transient provided that the movement forces microbial factors may pass across dentin more readily. are not excessive.140 It was shown, however, that pulp necrosis Periodontal scaling and root planing may result in removal of teeth that were undergoing orthodontic treatment, and had of cementum and exposure of dentin to the oral cavity. Fre- also been traumatized prior to that, is significantly increased quently, this treatment results in dentin hypersensitivity, as compared to teeth that had either of these conditions but not discussed previously. In theory, periodontal disease and its the other, particularly in lateral incisors, and if the traumatized treatment should be associated with an increased incidence of tooth had pulp canal obliteration.18,19 As noted previously, sub- pulpal pathosis. In an older study, investigators induced peri- stance P appears to increase in the dental pulp as a result of odontal disease in a primate model using ligatures, and they acutely induced occlusal trauma.45 Also, CGRP increases under compared the effects on the pulp of periodontal disease with the influence of orthodontic forces.46 or without scaling.22 In teeth with periodontal disease, mild chronic inflammatory changes were observed in 29% of the PULPAL REACTIONS TO ORTHODONTIC/ teeth and could be seen in areas of the pulp related to bone loss. One of 40 teeth developed pulp necrosis. In teeth that ORTHOGNATHIC SURGERY received scaling, a similar percentage of 32% developed the It has been known for decades that osteotomies in the maxilla same mild inflammation and none developed pulp necrosis. In or mandible may cause disruption in the blood supply to teeth a later clinical study,23 52 periodontitis patients who had 672 in the area of the surgery, with resultant inflammation or necro- teeth with vital pulp were followed up and maintained every sis.15,138,179,207 Occasionally, the teeth affected show postopera- 3 to 6 months for 4 to 13 years (mean 8.7 years). Of those tive manifestations common with traumatic injuries, such as teeth, 255 were bridge abutments. The results showed a sig- pulp canal obliteration.271 Animal studies have shown that if a nificantly higher chance of pulpal complications in teeth that safe distance of 5 to 10 mm is maintained between the site of were bridge abutments than in teeth that were not abutments the surgery and the teeth, minimal disruption occurs.65,283 A (15% versus 3%; P < 0.01). Considering that both types of teeth number of studies have documented, using laser Doppler flow- had similar degrees of periodontal disease, the authors con- metry, actual reduction in pulpal blood flow (PBF) immediately cluded that prosthodontic treatment is associated with pulpal following maxillary Le Fort I osteotomy,193,218,228 particularly if involvement more frequently than periodontal disease and its segmental osteotomy is performed.71 In most cases, the blood treatment. Another histologic analysis of 46 teeth with varying flow is regained within months of the surgery. A modification degrees of periodontal disease and coronal restorations reached of the Le Fort I osteotomy technique has been described in a similar conclusion.62 Furthermore, two comprehensive which the Le Fort I sectioning is combined with a horseshoe reviews of the topic concluded that although the potential palatal osteotomy to spare any disruption to the descending exists for periodontal disease and its treatment to cause pulpal palatine artery.96 An examination of PBF of maxillary teeth pathosis, particularly if large lateral or accessory canals are using laser Doppler flowmetry as well as the responsiveness to exposed, this occurrence is rare.97,226 electric pulp testing showed significant differences between the two surgical techniques in the postoperative recovery values MECHANICAL IRRITANTS: (Fig. 13-26). In cases where the surgery did not disrupt the palatine artery, the PBF in the anterior teeth consistently ORTHODONTIC MOVEMENT increased without disruption in the postoperative period. The most conspicuous pulpal change observed in response to It should also be noted that, occasionally, teeth are trauma- orthodontic forces is hemodynamic. Both human and animal tized during endotracheal intubation for surgery that requires studies have confirmed that both lateral and intrusive forces general anesthesia, when the surgery itself is not related to the increase pulpal blood flow.145,182,190 Furthermore, blood flow jaws or teeth.235 alterations are not confined to the tooth in active movement. Observed increases in blood flow are seen in teeth adjacent to BIOMECHANICAL IRRITATION: the focus of movement forces, implying that directed forces on one tooth could shunt blood to proximal vessels supplying PARAFUNCTIONAL HABITS other oral structures, including teeth. If orthodontic forces Occlusal loading of teeth affects deformation to varying are extreme, circulatory interruptions can occur, resulting in degrees.167 Whereas enamel is largely resistant to flexure, pulpal necrosis.35 the underlying dentin demonstrates considerable elastic and Biochemical, biologic, and histologic studies of the effects viscoelastic characteristics. As a result, defects in enamel of orthodontic movement have confirmed that metabolic as secondary to cracks, decay, or restorative preparation allow well as inflammatory changes can result. The dental pulp tissue cuspal flexure with subsequent pulpal responses, presumably respiration rate is depressed after short-term application of due to dentinal fluid flow secondary to compression and CHAPTER 13 Pulpal Reactions to Caries and Dental Procedures 593

(%) loading of restored teeth.103 A second source of pulpal pain is bacterial microleakage created by a gap at the restoration/ 100 dentin interface that is repeatedly opened during cycles of occlusal loading. If repeated cuspal flexure gives rise to a crack, 80 dentinal exposure to bacteria and their by-products is even * greater. It is likely that in vivo both dentinal fluid flow and 60 bacterial access to dentinal surfaces work together to produce * inflammation that is often manifested by thermal as wellas * * biting sensitivity for the patient. In the instance of parafunc- 40 * * tional habits, this is often combined with concussive periodontal forces to the periodontium that induce acute periradicular C-group 20 periodontitis, mobility, and radiographic changes. NC-group On the cellular level, studies have shown increased levels of substance P after experimentally induced occlusal trauma.45 0 Substance P is a modulator of both sensory and immune A Pre 1 2 3 4 5 6 7d 14d 3M 6M 12M function in the dental pulp. Alterations in physiologic levels (%) of this mediator could result in disruptions in the pain response as well as secondary stimulation of prostaglandin 100 E2. Furthermore, the immunostimulatory ability of neuropeptides could initiate and maintain a chronic pulpal inflam- 80 mation, possibly leading to necrosis. Although animal studies have suggested that pulp necrosis is a possible sequela to chronic occlusal trauma, there are no controlled clinical 60 * studies that confirm this. Dentinal cracks expose tubules unoccluded by a smear layer 40 and therefore offer a direct portal to the subjacent pulp. When dentinal tubules are freely exposed, there is an outward flow

20 C-group of dentinal fluid driven by relatively high pulpal tissue pres- NC-group sures. Dentinal fluid is composed of proteins such as fibrinogen and serum albumin, which can coagulate and effectively block 0 the tubule lumen thereby limiting fluid egress and resultant B Pre 1d 14d 3M 6M 12M dentin hypersensitivity. This phenomenon can occur within FIG. 13-26 A, The postoperative change of the mean pulpal blood flow in two days. As this serves as a short-term protective mechanism the upper incisors of the two groups (Pre, before the operation; d, day[s] after for the pulp, dentinal sclerosis and tertiary dentin formation the operation; M, months after the operation; bars, SD; *, P < .05; **, P < ultimately can provide greater protection for the pulp and .01). B, The postoperative change in the percentages of teeth (upper incisors) reduction of symptoms. Clinical interventions include the with positive pulpal sensitivity in the two groups (Pre, before the operation; application of materials that occlude the tubules and extra- d, day[s] after the operation; M, months after the operation; *, P < .05). (From coronal restorations to seal and prevent the propagation of Harada K, Sato M, Omura K: Blood-flow and neurosensory changes in the cracks. maxillary dental pulp after differing Le Fort I osteotomies, Oral Surg Oral Med Oral Pathol Oral Radiol Endod 97:12, 2004. Reprinted with permission.) PULPAL REACTIONS TO IMPLANT microleakage.103 The magnitude of the pulpal response is dic- PLACEMENT AND FUNCTION tated by the degree and chronicity of dentinal deformation. Osseointegrated implants are now a common option for the Multiple factors influence the degree of tooth deformation replacement of missing teeth. The placement of implants during occlusal loading. Investigators have noted that prepara- requires multifaceted preoperative radiographic techniques, tion geometry has a direct impact on cuspal flexure. The width including intraoral, tomographic, cephalometric, and pan- of the occlusal isthmus relative to the faciolingual dimension oramic imaging.264 This assures that implant placement fully of the tooth as well as the ablation of marginal ridges directly rests in bone and does not compromise neighboring structures, impact on the degree of cuspal flexure.199,219,270 MOD prepara- including teeth. The lack of attention to the three-dimensional tions have been shown to effect a 50% reduction in cuspal anatomy of the site of implant placement and the orientation stiffness and resistance to fracture. Physical properties of the of neighboring teeth may lead to the implant perforating the restorative material can also play a part in cuspal flexure. root and devitalizing the pulp.162,249 Studies have shown that polymerization shrinkage of certain It is usually recommended that implants not be placed resin composites can induce an inward deflection of cusps with directly at a site where a periradicular lesion exists, particularly resultant stresses on tooth structure.272,273 one with signs of purulence, as microbial irritants may inter- Symptomatology from cuspal flexure can result from two fere with osseointegration.214 However, some data suggest that primary sources. It has been theorized that cuspal flexure immediate implant placement in sites that have been ade- results in dentin deformation, thus promoting the dentinal quately debrided is successful.40 A systematic review generally fluid flow that activates nerve endings in the odontoblast layer corroborates this finding, provided that adequate preoperative, of the tooth. This is supported in part by an in vitro study that intraoperative, and postoperative antimicrobial measures are found that dentinal fluid flow could be induced by occlusal utilized.50 594 PART II The Advanced Science of Endodontics

Case reports have also claimed that teeth with periradicular and nonsurgical treatments.233 The results showed that the pres- lesions may reduce the success of neighboring implants even if ence of treated or untreated periradicular lesions did not affect adequate endodontic treatment is performed.248 To address this the long-term osseointegration of implants that were already issue, a study was reported in which implants were placed to osseointegrated. Clinical cases with complete resolution of peri- replace premolars in dogs; periradicular lesions were then apical lesions that also involve neighboring implants, following induced, and some were treated nonsurgically or with surgical adequate endodontic treatment, have been published.155,244

REFERENCES 1. About I, Murray PE, Franquin JC, et al: Pulpal 18. Bauss O, Rohling J, Rahman A, Kiliaridis S: The effect of 37. Byers MR, Narhi MV, Mecifi KB: Acute and chronic inflammatory responses following non-carious class V pulp obliteration on pulpal vitality of orthodontically reactions of dental sensory nerve fibers to cavities and restorations, Oper Dent 26:336, 2001. intruded traumatized teeth, J Endod 34:417, 2008. desiccation in rat molars, Anat Rec 221:872, 1988. 2. Absi EG, Addy M, Adams D: Dentine hypersensitivity— 19. Bauss O, Rohling J, Sadat-Khonsari R, Kiliaridis S: 38. Calland JW, Harris SE, Carnes DL Jr: Human pulp the effect of toothbrushing and dietary compounds on Influence of orthodontic intrusion on pulpal vitality of cells respond to calcitonin gene-related peptide in vitro, dentine in vitro: an SEM study, J Oral Rehabil 19:101, previously traumatized maxillary permanent incisors, J Endod 23:485, 1997. 1992. Am J Orthod Dentofacial Orthop 134:12, 2008. 39. Camilleri J: Investigation of Biodentine as dentine 3. Addy M, Pearce N: Aetiological, predisposing and 20. Bergenholtz G, Ahlstedt S, Lindhe J: Experimental replacement material, J Dent 41:600, 2013. environmental factors in dentine hypersensitivity, Arch pulpitis in immunized monkeys, Scand J Dent Res 40. Casap N, Zeltser C, Wexler A, et al: Immediate placement Oral Biol 39 (suppl):33S, 1994. 85:396, 1977. of dental implants into debrided infected dentoalveolar 4. Ahlquist M, Franzen O, Coffey J, Pashley D: Dental pain 21. Bergenholtz G, Cox CF, Loesche WJ, Syed SA: Bacterial sockets, J Oral Maxillofac Surg 65:384, 2007. evoked by hydrostatic pressures applied to exposed leakage around dental restorations: its effect on the 41. Cassidy N, Fahey M, Prime SS, Smith AJ: Comparative dentin in man: a test of the hydrodynamic theory of dentin dental pulp, J Oral Pathol 11:439, 1982. analysis of transforming growth factor-beta isoforms 1-3 sensitivity, J Endod 20:130, 1994. 22. Bergenholtz G, Lindhe J: Effect of experimentally induced in human and rabbit dentine matrices, Arch Oral Biol 5. Ahn J, Pogrel MA: The effects of 2% lidocaine with marginal periodontitis and periodontal scaling on the 42:219, 1997. 1:100,000 epinephrine on pulpal and gingival blood flow, dental pulp, J Clin Periodontol 5:59, 1978. 42. Caux C, Vanbervliet B, Massacrier C, et al: Regulation of Oral Surg Oral Med Oral Pathol Oral Radiol Endod 23. Bergenholtz G, Nyman S: Endodontic complications dendritic cell recruitment by chemokines, Transplantation 85:197, 1998. following periodontal and prosthetic treatment of patients 73:S7, 2002. 6. Al-Hiyasat AS, Barrieshi-Nusair KM, Al-Omari MA: The with advanced periodontal disease, J Periodontol 55:63, 43. Cavalcanti BN, Lage-Marques JL, Rode SM: Pulpal radiographic outcomes of direct pulp-capping procedures 1984. temperature increases with Er:YAG laser and high-speed performed by dental students: a retrospective study, J Am 24. Bergenholtz G, Warfvinge J: Migration of leukocytes in handpieces, J Prosthet Dent 90:447, 2003. Dent Assoc 137:1699, 2006. dental pulp in response to plaque bacteria, Scand J Dent 44. Caviedes-Bucheli J, Ariza-Garcia G, Restrepo-Mendez S, 7. Aljehani A, Yang L, Shi XQ: In vitro quantification of Res 90:354, 1982. et al: The effect of tooth bleaching on substance P smooth surface caries with DIAGNOdent and the 24a. Bhingare AC, Ohno T, Tomura M, et al: Dental pulp expression in human dental pulp, J Endod 34:1462, 2008. DIAGNOdent pen, Acta Odontol Scand 65:60, 2007. dendritic cells migrate to regional lymph nodes, J Dent 45. Caviedes-Bucheli J, Azuero-Holguin MM, Correa-Ortiz JA, 8. Amano T, Muramatsu T, Amemiya K, et al: Responses of Res 93(3):288, 2014. et al: Effect of experimentally induced occlusal trauma on rat pulp cells to heat stress in vitro, J Dent Res 85:432, 25. Bletsa A, Berggreen E, Brudvik P: Interleukin-1alpha and substance p expression in human dental pulp and 2006. tumor necrosis factor-alpha expression during the early periodontal ligament, J Endod 37:627, 2011. 9. Anderson DG, Chiego DJ Jr, Glickman GN, McCauley LK: phases of orthodontic tooth movement in rats, Eur J Oral 46. Caviedes-Bucheli J, Moreno JO, Ardila-Pinto J, et al: A clinical assessment of the effects of 10% carbamide Sci 114:423, 2006. The effect of orthodontic forces on calcitonin gene-related peroxide gel on human pulp tissue, J Endod 25:247, 26. Bogen G, Kim JS, Bakland LK: Direct pulp capping with peptide expression in human dental pulp, J Endod 1999. mineral trioxide aggregate: an observational study, J Am 37:934, 2011. 10. Armstrong SR, Vargas MA, Fang Q, Laffoon JE: Dent Assoc 139:305; quiz 305, 2008. 47. Chabanski MB, Gillam DG: Aetiology, prevalence and Microtensile bond strength of a total-etch 3-step, 27. Botero TM, Shelburne CE, Holland GR, et al: TLR4 clinical features of cervical dentine sensitivity, J Oral total-etch 2-step, self-etch 2-step, and a self-etch 1-step mediates LPS-induced VEGF expression in odontoblasts, Rehabil 24:15, 1997. dentin bonding system through 15-month water storage, J Endod 32:951, 2006. 48. Cheung GS, Lai SC, Ng RP: Fate of vital pulps beneath a J Adhes Dent 5:47, 2003. 28. Bouillaguet S: Biological risks of resin-based materials to metal-ceramic crown or a bridge retainer, Int Endod J 11. Asgary S, Eghbal MJ, Parirokh M, et al: A comparative the dentin-pulp complex, Crit Rev Oral Biol Med 15:47, 38:521, 2005. study of histologic response to different pulp capping 2004. 49. Chng HS, Pitt Ford TR, McDonald F: Effects of prilocaine materials and a novel endodontic cement, Oral Surg Oral 29. Bowles WR, Burke R, Sabino M, et al: Sex differences local anaesthetic solutions on pulpal blood flow in Med Oral Pathol Oral Radiol Endod 106:609, 2008. in neuropeptide content and release from rat dental pulp, maxillary canines, Endod Dent Traumatol 12:89, 1996. 12. Avellan NL, Sorsa T, Tervahartiala T, et al: Painful tooth J Endod 37:1098, 2011. 50. Chrcanovic BR, Martins MD, Wennerberg A: Immediate stimulation elevates matrix metalloproteinase-8 levels 30. Bowles WR, Withrow JC, Lepinski AM, Hargreaves KM: placement of implants into infected sites: a systematic locally in human gingival crevicular fluid, J Dent Res Tissue levels of immunoreactive substance P are review, Clin Implant Dent Relat Res 17(Suppl 1):e1, 2015. 84:335, 2005. increased in patients with irreversible pulpitis, J Endod 51. Cohen JS, Reader A, Fertel R, et al: A radioimmunoassay 13. Awawdeh L, Lundy FT, Shaw C, et al: Quantitative 29:265, 2003. determination of the concentrations of prostaglandins E2 analysis of substance P, neurokinin A and calcitonin 31. Brannstrom M: Dental and pulpal response. III. Application and F2alpha in painful and asymptomatic human dental gene-related peptide in pulp tissue from painful and of an air stream to exposed dentin. Long observation pulps, J Endod 11:330, 1985. healthy human teeth, Int Endod J 35:30, 2002. periods, Acta Odontol Scand 18:234, 1960. 52. Coluzzi DJ: An overview of laser wavelengths used in 14. Baldissara P, Catapano S, Scotti R: Clinical and 32. Brannstrom M: The effect of dentin desiccation and dentistry, Dent Clin North Am 44:753, 2000. histological evaluation of thermal injury thresholds in aspirated odontoblasts on the pulp, J Prosthet Dent 53. Costa AM, Bezzerra AC, Fuks AB: Assessment of the human teeth: a preliminary study, J Oral Rehabil 24:791, 20:165, 1968. accuracy of visual examination, bite-wing radiographs and 1997. 33. Brannstrom M: Smear layer: pathological and DIAGNOdent on the diagnosis of occlusal caries, Eur Arch 15. Banks P: Pulp changes after anterior mandibular treatment considerations, Oper Dent Supplement 3:35, Paediatr Dent 8:118, 2007. subapical osteotomy in a primate model, J Oral Maxillofac 1984. 54. Costa CA, Edwards CA, Hanks CT: Cytotoxic effects of Surg 5:39, 1977. 34. Brannstrom M, Lind PO: Pulpal response to early dental cleansing solutions recommended for chemical lavage of 16. Barrieshi-Nusair KM, Qudeimat MA: A prospective clinical caries, J Dent Res 44:1045, 1965. pulp exposures, Am J Dent 14:25, 2001. study of mineral trioxide aggregate for partial pulpotomy 35. Butcher EO, Taylor AC: The effects of denervation and 55. Costa CA, Giro EM, do Nascimento AB, et al: Short-term in cariously exposed permanent teeth, J Endod 32:731, ischemia upon the teeth of the monkey, J Dent Res evaluation of the pulpo-dentin complex response to a 2006. 30:265, 1951. resin-modified glass-ionomer cement and a bonding 17. Barthel CR, Rosenkranz B, Leuenberg A, Roulet JF: Pulp 36. Byers MR, Narhi MV: Dental injury models: experimental agent applied in deep cavities, Dent Mater 19:739, 2003. capping of carious exposures: treatment outcome after 5 tools for understanding neuroinflammatory interactions 56. Costa CA, Riehl H, Kina JF, et al: Human pulp responses and 10 years: a retrospective study, J Endod 26:525, and polymodal nociceptor functions, Crit Rev Oral Biol to in-office tooth bleaching, Oral Surg Oral Med Oral 2000. Med 10:4, 1999. Pathol Oral Radiol Endod 109:e59, 2010. CHAPTER 13 Pulpal Reactions to Caries and Dental Procedures 595

57. Cotton WR: Pulp response to an airstream directed into 80. Ferracane JL, Condon JR: Rate of elution of leachable 105. Holland R, de Souza V, Nery MJ, et al: Reaction of dogs’ human cavity preparations, Oral Surg Oral Med Oral components from composite, Dent Mater 6:282, 1990. teeth to root canal filling with mineral trioxide aggregate Pathol 24:78, 1967. 81. Fitzgerald M, Hanks CT: In vivo study of resin diffusion or a glass ionomer sealer, J Endod 25:728, 1999. 58. Cox CF, Bergenholtz G, Fitzgerald M, et al: Capping of through intact vital human dentin (abstract), J Dent Res 106. Horst OV, Horst JA, Samudrala R, Dale BA: Caries the dental pulp mechanically exposed to the oral 76:305, 1997. induced cytokine network in the odontoblast layer of microflora: a 5 week observation of wound healing in 82. Flecha OD, Azevedo CG, Matos FR, et al: Cyanoacrylate human teeth, BMC Immunol 12:9, 2011. the monkey, J Oral Pathol 11:327, 1982. versus laser in the treatment of dentin hypersensitivity: a 107. Horst OV, Tompkins KA, Coats SR, et al: TGF-beta1 59. Cox CF, Hafez AA, Akimoto N, et al: Biocompatibility of controlled, randomized, double-masked and non-inferiority inhibits TLR-mediated odontoblast responses to oral primer, adhesive and resin composite systems on clinical trial, J Periodontol 84:287, 2013. bacteria, J Dent Res 88:333, 2009. non-exposed and exposed pulps of non-human primate 83. Ford TR, Torabinejad M, Abedi HR, et al: Using mineral 108. Horsted P, Sandergaard B, Thylstrup A, et al: A teeth, Am J Dent 11:S55, 1998. trioxide aggregate as a pulp-capping material, J Am Dent retrospective study of direct pulp capping with calcium 60. Cvek M: A clinical report on partial pulpotomy and Assoc 127:1491, 1996. hydroxide compounds, Endod Dent Traumatol 1:29, capping with calcium hydroxide in permanent incisors 84. Fosse G, Saele PK, Eide R: Numerical density and 1985. with complicated crown fracture, J Endod 4:232, 1978. distributional pattern of dentin tubules, Acta Odontol 109. Hume WR: An analysis of the release and the diffusion 61. Cvek M, Granath L, Cleaton-Jones P, Austin J: Hard Scand 50:201, 1992. through dentin of eugenol from zinc oxide-eugenol tissue barrier formation in pulpotomized monkey teeth 85. Fried K: Changes in pulpal nerves with aging, Proc Finn mixtures, J Dent Res 63:881, 1984. capped with cyanoacrylate or calcium hydroxide for 10 Dent Soc 88:517, 1992. 110. Hume WR, Gerzia TM: Bioavailability of components of and 60 minutes, J Dent Res 66:1166, 1987. 86. Fugaro JO, Nordahl I, Fugaro OJ, et al: Pulp reaction to resin-based materials which are applied to teeth, Crit Rev 62. Czarnecki RT, Schilder H: A histological evaluation of the vital bleaching, Oper Dent 29:363, 2004. Oral Biol Med 7:172, 1996. human pulp in teeth with varying degrees of periodontal 87. Fujitani M, Inokoshi S, Hosoda H: Effect of acid etching 111. Hume WR, Kenney AE: Release of 3H-triamcinolone from disease, J Endod 5:242, 1979. on the dental pulp in adhesive composite restorations, Ledermix, J Endod 7:509, 1981. 63. do Nascimento AB, Fontana UF, Teixeira HM, Costa CA: Int Dent J 42:3, 1992. 112. Ikawa M, Komatsu H, Ikawa K, et al: Age-related changes Biocompatibility of a resin-modified glass-ionomer cement 88. Garberoglio R, Brannstrom M: Scanning electron in the human pulpal blood flow measured by laser applied as pulp capping in human teeth, Am J Dent microscopic investigation of human dentinal tubules, Arch Doppler flowmetry, Dent Traumatol 19:36, 2003. 13:28, 2000. Oral Biol 21:355, 1976. 113. Inoue H, Izumi T, Ishikawa H, Watanabe K: Short-term 64. Dummer PM, Hicks R, Huws D: Clinical signs and 89. Gibson M, Sharif MO, Smith A, et al: A practice-based histomorphological effects of Er:YAG laser irradiation to rat symptoms in pulp disease, Int Endod J 13:27, 1980. randomised controlled trial of the efficacy of three coronal dentin-pulp complex, Oral Surg Oral Med Oral 65. Duran S, Guven O, Gunhan O: Pulpal and apical changes interventions to reduce dentinal hypersensitivity, J Dent Pathol Oral Radiol Endod 97:246, 2004. secondary to segmental osteotomy in the mandible: an 41:668, 2013. 114. Inoue T, Miyakoshi S, Shimono M: Dentin/pulp adhesive experimental study, J Craniomaxillofac Surg 23:256, 90. Godoy BM, Arana-Chavez VE, Nunez SC, Ribeiro MS: resin interface: biological view from basic science to 1995. Effects of low-power red laser on dentine-pulp interface clinic. In Shimono M, Suda H, Takahashi K, editors: 66. Durand SH, Flacher V, Romeas A, et al: Lipoteichoic acid after cavity preparation: an ultrastructural study, Arch Oral Proceedings of the International Conference on Dentin/ increases TLR and functional chemokine expression while Biol 52:899, 2007. Pulp Complex 1995, Chiba, Japan, Chicago, 1996, reducing dentin formation in in vitro differentiated human 91. Goodis HE, Schein B, Stauffer P: Temperature changes Quintessence, pp 217-220. odontoblasts, J Immunol 176:2880, 2006. measured in vivo at the dentinoenamel junction and 115. Inoue T, Miyakoshi S, Shimono M: The in vitro and in vivo 67. El Karim IA, Lamey PJ, Ardill J, et al: Vasoactive intestinal pulpodentin junction during cavity preparation in the influence of 4-META/MMA-TBB resin components on polypeptide (VIP) and VPAC1 receptor in adult human Macaca fascicularis monkey, J Endod 14:336, 1988. dental pulp tissues, Adv Dent Res 15:101, 2001. dental pulp in relation to caries, Arch Oral Biol 51:849, 92. Graham L, Cooper PR, Cassidy N, et al: The effect of 116. Isett J, Reader A, Gallatin E, et al: Effect of an 2006. calcium hydroxide on solubilisation of bio-active dentine intraosseous injection of depo-medrol on pulpal 68. El Karim IA, Lamey PJ, Linden GJ, et al: Caries-induced matrix components, Biomaterials 27:2865, 2006. concentrations of PGE2 and IL-8 in untreated irreversible changes in the expression of pulpal neuropeptide Y, 93. Gwinnett AJ, Tay F: Early and intermediate time response pulpitis, J Endod 29:268, 2003. Eur J Oral Sci 114:133, 2006. of the dental pulp to an acid etch technique in vivo, Am J 117. Izumi T, Inoue H, Matsuura H, et al: Changes in the 69. El Karim IA, Lamey PJ, Linden GJ, Lundy FT: Dent 11:S35, 1998. pattern of horseradish peroxidase diffusion into predentin Neuropeptide Y Y1 receptor in human dental pulp cells 94. Gwinnett AJ, Tay FR, Pang KM, Wei SH: Quantitative and dentin after cavity preparation in rat molars, Oral of noncarious and carious teeth, Int Endod J 41:850, contribution of the collagen network in dentin Surg Oral Med Oral Pathol Oral Radiol Endod 92:675, 2008. hybridization, Am J Dent 9:140, 1996. 2001. 70. Elliott RD, Roberts MW, Burkes J, Phillips C: Evaluation of 95. Hamersky PA, Weimer AD, Taintor JF: The effect of 118. Izumi T, Kobayashi I, Okamura K, Sakai H: the carbon dioxide laser on vital human primary pulp orthodontic force application on the pulpal tissue Immunohistochemical study on the immunocompetent tissue, Pediatr Dent 21:327, 1999. respiration rate in the human premolar, Am J Orthod cells of the pulp in human non-carious and carious teeth, 71. Emshoff R, Kranewitter R, Brunold S, et al: Characteristics 77:368, 1980. Arch Oral Biol 40:609, 1995. of pulpal blood flow levels associated with non-segmented 96. Harada K, Sato M, Omura K: Blood-flow and neurosensory 119. Jaber L, Swaim WD, Dionne RA: Immunohistochemical and segmented Le Fort I osteotomy, Oral Surg Oral Med changes in the maxillary dental pulp after differing Le Fort localization of mu-opioid receptors in human dental pulp, Oral Pathol Oral Radiol Endod 105:379, 2008. I osteotomies, Oral Surg Oral Med Oral Pathol Oral Radiol J Endod 29:108, 2003. 72. Eversole LR, Rizoiu I, Kimmel AI: Pulpal response to cavity Endod 97:12, 2004. 120. Janeway CA Jr, Medzhitov R: Innate immune recognition, preparation by an erbium, chromium:YSGG laser-powered 97. Harrington GW, Steiner DR, Ammons WF: The Annu Rev Immunol 20:197, 2002. hydrokinetic system, J Am Dent Assoc 128:1099, 1997. periodontal-endodontic controversy, Periodontol 2000 121. Jansson L, Ehnevid H, Lindskog S, Blomlof L: Relationship 73. Faraco IM Jr, Holland R: Response of the pulp of dogs to 30:123, 2002. between periapical and periodontal status: a clinical capping with mineral trioxide aggregate or a calcium 98. Harris DM, White JM, Goodis H, et al: Selective ablation retrospective study, J Clin Periodontol 20:117, 1993. hydroxide cement, Dent Traumatol 17:163, 2001. of surface enamel caries with a pulsed Nd:YAG dental 122. Jansson L, Sandstedt P, Laftman AC, Skoglund A: 74. Farges JC, Keller JF, Carrouel F, et al: Odontoblasts in laser, Lasers Surg Med 30:342, 2002. Relationship between apical and marginal healing in the dental pulp immune response, J Exp Zoolog B Mol 99. Hartmann A, Azerad J, Boucher Y: Environmental effects periradicular surgery, Oral Surg Oral Med Oral Pathol Oral Dev Evol, 312B:425, 2009. on laser Doppler pulpal blood-flow measurements in man, Radiol Endod 83:596, 1997. 75. Farges JC, Keller JF, Carrouel F, et al: Odontoblasts in Arch Oral Biol 41:333 1996. 123. Jiang HW, Zhang W, Ren BP, et al: Expression of toll like the dental pulp immune response, J Exp Zool B Mol Dev 100. Hasler JE, Mitchell DF: Painless pulpitis, J Am Dent Assoc receptor 4 in normal human odontoblasts and dental pulp Evol 312B:425, 2009. 81:671, 1970. tissue, J Endod 32:747, 2006. 76. Farges JC, Keller JF, Carrouel F, et al: O34-pathogen 101. Hayden MS, West AP, Ghosh S: NF-kappaB and the 124. Jontell M, Hanks CT, Bratel J, Bergenholtz G: Effects of sensing by human odontoblasts, Bull Group Int Rech Sci immune response, Oncogene 25:6758, 2006. unpolymerized resin components on the function of Stomatol Odontol 49:90, 2010. 102. Heys DR, Cox CF, Heys RJ, Avery JK: Histological accessory cells derived from the rat incisor pulp, J Dent 77. Farges JC, Romeas A, Melin M, et al: TGF-beta1 induces considerations of direct pulp capping agents, J Dent Res Res 74:1162, 1995. accumulation of dendritic cells in the odontoblast layer, 60:1371, 1981. 125. Jukic S, Anic I, Koba K, et al: The effect of pulpotomy J Dent Res 82:652, 2003. 103. Hirata K, Nakashima M, Sekine I, et al: Dentinal fluid using CO2 and Nd:YAG lasers on dental pulp tissue, 78. Farik B, Munksgaard EC, Andreasen JO, Kreiborg S: movement associated with loading of restorations, J Dent Int Endod J 30:175, 1997. Drying and rewetting anterior crown fragments prior to Res 70:975, 1991. 126. Kanjevac T, Milovanovic M, Volarevic V, et al: Cytotoxic bonding, Endod Dent Traumatol 15:113, 1999. 104. Holland GR, Narhi MN, Addy M, et al: Guidelines for the effects of glass ionomer cements on human dental pulp 79. Felton D: Long-term effects of crown preparation on pulp design and conduct of clinical trials on dentine stem cells correlate with fluoride release, Med Chem vitality, J Dent Res 68:1009 (abstract 139), 1989. hypersensitivity, J Clin Periodontol 24:808, 1997. 8:40, 2012. 596 PART II The Advanced Science of Endodontics

127. Katsuno K, Manabe A, Itoh K, et al: A delayed monomers on THP-1 human monocytes, J Biomater Sci 172. Mjor IA, Finn SB, Quigley MB: The effect of calcium hypersensitivity reaction to dentine primer in the Polym Ed 10:1265, 1999. hydroxide and amalgam on non-carious, vital dentine, guinea-pig, J Dent 23:295, 1995. 150. Lepinski AM, Haegreaves KM, Goodis HE, Bowles WR: Arch Oral Biol 3:283, 1961. 128. Kawagishi E, Nakakura-Ohshima K, Nomura S, Ohshima Bradykinin levels in dental pulp by microdialysis, J Endod 173. Murray PE, About I, Lumley PJ, et al: Postoperative pulpal H: Pulpal responses to cavity preparation in aged rat 26:744, 2000. and repair responses, J Am Dent Assoc 131:321, 2000. molars, Cell Tissue Res 326:111, 2006. 151. Lesot H, Kubler MD: Experimental induction of odontoblast 174. Murray PE, Smith AJ, Garcia-Godoy F, Lumley PJ: 129. Keller JF, Carrouel F, Colomb E, et al: Toll-like receptor 2 differentiation and stimulation during reparative processes, Comparison of operative procedure variables on pulpal activation by lipoteichoic acid induces differential Cells Materials 3:201, 1993. viability in an ex vivo model, Int Endod J 41:389, 2008. production of pro-inflammatory cytokines in human 152. Levin LG, Rudd A, Bletsa A, Reisner H: Expression of IL-8 175. Murray PE, Smith AJ, Windsor LJ, Mjor IA: Remaining odontoblasts, dental pulp fibroblasts and immature by cells of the odontoblast layer in vitro, Eur J Oral Sci dentine thickness and human pulp responses, Int Endod J dendritic cells, Immunobiology 215:53, 2010. 107:131, 1999. 36:33, 2003. 130. Killough SA, Lundy FT, Irwin CR: Substance P expression 153. Lier BB, Rosing CK, Aass AM, Gjermo P: Treatment of 176. Murray PE, Smyth TW, About I, et al: The effect of etching by human dental pulp fibroblasts: a potential role in dentin hypersensitivity by Nd:YAG laser, J Clin Periodontol on bacterial microleakage of an adhesive composite neurogenic inflammation, J Endod 35:73, 2009. 29:501, 2002. restoration, J Dent 30:29, 2002. 131. Kim S: Ligamental injection: a physiological explanation of 154. Lilja J, Nordenvall KJ, Branstrom M: Dentin sensitivity, 177. Musselwhite JM, Klitzman B, Maixner W, Burkes EJ Jr: its efficacy, J Endod 12:486, 1986. odontoblasts and nerves under desiccated or infected Laser Doppler flowmetry: a clinical test of pulpal vitality, 132. Kim S, Edwall L, Trowbridge H, Chien S: Effects of local experimental cavities. A clinical, light microscopic and Oral Surg Oral Med Oral Pathol Oral Radiol Endod 84:411, anesthetics on pulpal blood flow in dogs, J Dent Res ultrastructural investigation, Swed Dent J 6:93, 1982. 1997. 63:650, 1984. 155. Lin S, Mayer Y: Treatment of a large periradicular lesion 178. Nakabayashi N, Kojima K, Masuhara E: The promotion of 133. Kim S, Trowbridge H, Suda H: Pulpal reaction to caries of endodontic origin around a dental implant with enamel adhesion by the infiltration of monomers into tooth and dental procedures. In Cohen S, Burns RC, editors: matrix protein derivative, J Periodontol 78:2385, 2007. substrates, J Biomed Mater Res 16:265, 1982. Pathways of the pulp, ed 8, St. Louis, 2002, Mosby, 156. Linde A: Dentin and entinogenesis. In Linde A, editor: 179. Nanda R, Legan HL, Langeland K: Pulpal and radicular pp 573-600. Noncollagenous proteins and proteoglycans in response to maxillary osteotomy in monkeys, Oral Surg 134. Kimura Y, Wilder-Smith P, Yonaga K, Matsumoto K: dentinogenesis, Boca Raton, FL,1984, CRC Press, Oral Med Oral Pathol 53:624, 1982. Treatment of dentine hypersensitivity by lasers: a review, pp 55-92. 180. Nathanson D: Vital tooth bleaching: sensitivity and pulpal J Clin Periodontol 27:715, 2000. 157. Litkowski LJ, Hack GD, Sheaffer HB, Greenspan DC: considerations, J Am Dent Assoc 128 (suppl):41S, 1997. 135. Kimura Y, Yonaga K, Yokoyama K, et al: Histopathological Occlusion of dentin tubules by 45s5 bioglass. Paper 181. Nayyar S, Tewari S, Arora B: Comparison of human pulp changes in dental pulp irradiated by Er:YAG laser: a presented at Bioceramics, Proceedings of the 10th response to total-etch and self-etch bonding agents, Oral preliminary report on laser pulpotomy, J Clin Laser Med international symposium on ceramics in medicine, October Surg Oral Med Oral Pathol Oral Radiol Endod 104:e45, Surg 21:345, 2003. 1997, Paris, France, 1997. 2007. 136. Kina JF, Huck C, Riehl H, et al: Response of human pulps 158. Liu HC, Lin CP, Lan WH: Sealing depth of Nd:YAG laser 182. Nixon CE, Saviano JA, King GJ, Keeling SD: after professionally applied vital tooth bleaching, Int Endod on human dentinal tubules, J Endod 23:691, 1997. Histomorphometric study of dental pulp during orthodontic J 43:572, 2010. 159. Loyd DR, Sun XX, Locke EE, et al: Sex differences in tooth movement, J Endod 19:13, 1993. 137. Koh ET, Ford TR, Kariyawasam SP, et al: Prophylactic serotonin enhancement of capsaicin-evoked calcitonin 183. Nomiyama K, Kitamura C, Tsujisawa T, et al: Effects of treatment of dens evaginatus using mineral trioxide gene-related peptide release from human dental pulp, lipopolysaccharide on newly established rat dental aggregate, J Endod 27:540, 2001. Pain 153:2061, 2012. pulp-derived cell line with odontoblastic properties, J 138. Kohn MW, White RP Jr: Evaluation of sensation after 160. Lupi-Pegurier L, Bertrand MF, Muller-Bolla M, et al: Endod 33:1187, 2007. segmental alveolar osteotomy in 22 patients, J Am Dent Comparative study of microleakage of a pit and fissure 184. Nowicka A, Lipski M, Parafiniuk M, et al: Response of Assoc 89:154, 1974. sealant placed after preparation by Er:YAG laser in human dental pulp capped with biodentine and mineral 139. Kokkas AB, Goulas A, Varsamidis K, et al: Irreversible but permanent molars, J Dent Child (Chic) 70:134, 2003. trioxide aggregate, J Endod 39:743, 2013. not reversible pulpitis is associated with up-regulation of 161. Lussi A, Hibst R, Paulus R: DIAGNOdent: an optical 185. Nup C, Rosenberg P, Linke H, Tordik P: Quantitation of tumour necrosis factor-alpha gene expression in human method for caries detection, J Dent Res 83 Spec No catecholamines in inflamed human dental pulp by pulp, Int Endod J 40:198, 2007. C:C80, 2004. high-performance liquid chromatography, J Endod 27:73, 140. Konno Y, Daimaruya T, Iikubo M, et al: Morphologic and 162. Margelos JT, Verdelis KG: Irreversible pulpal damage of 2001. hemodynamic analysis of dental pulp in dogs after molar teeth adjacent to recently placed osseointegrated 186. Nyborg H, Brannstrom M: Pulp reaction to heat, J intrusion with the skeletal anchorage system, Am J implants, J Endod 21:479, 1995. Prosthet Dent 19:605, 1968. Orthod Dentofacial Orthop 132:199, 2007. 163. McCormack K, Davies R: The enigma of potassium 187. Odor TM, Pitt Ford TR, McDonald F: Adrenaline in local 141. Koshiro K, Inoue S, Tanaka T, et al: In vivo degradation of ion in the management of dentine hypersensitivity: anaesthesia: the effect of concentration on dental pulpal resin-dentin bonds produced by a self-etch vs. a is nitric oxide the elusive second messenger? Pain 68:5, circulation and anaesthesia, Endod Dent Traumatol total-etch adhesive system, Eur J Oral Sci 112:368, 1996. 10:167, 1994. 2004. 164. McLachlan JL, Sloan AJ, Smith AJ, et al: S100 and 188. Okamura K: Histological study on the origin of dentinal 142. Koubi G, Colon P, Franquin JC, et al: Clinical evaluation of cytokine expression in caries, Infect Immun 72:4102, immunoglobulins and the change in their localization the performance and safety of a new dentine substitute, 2004. during caries, J Oral Pathol 14:680, 1985. Biodentine, in the restoration of posterior teeth: a 165. McLachlan JL, Smith AJ, Bujalska IJ, Cooper PR: 189. Okamura K, Maeda M, Nishikawa T, Tsutsui M: Dentinal prospective study, Clin Oral Investig 17:243, 2013. Gene expression profiling of pulpal tissue reveals the response against carious invasion: localization of 143. Koulaouzidou EA, Papazisis KT, Economides NA, et al: molecular complexity of dental caries, Biochim Biophys antibodies in odontoblastic body and process, J Dent Res Antiproliferative effect of mineral trioxide aggregate, zinc Acta 1741:271, 2005. 59:1368, 1980. oxide-eugenol cement, and glass-ionomer cement against 166. Mente J, Geletneky B, Ohle M, et al: Mineral trioxide 190. Olgart L, Gazelius B, Sundstrom F: Intradental nerve three fibroblastic cell lines, J Endod 31:44, 2005. aggregate or calcium hydroxide direct pulp capping: an activity and jaw-opening reflex in response to mechanical 144. Kuhnisch J, Bucher K, Henschel V, Hickel R: analysis of the clinical treatment outcome, J Endod deformation of cat teeth, Acta Physiol Scand 133:399, Reproducibility of DIAGNOdent 2095 and DIAGNOdent Pen 36:806, 2010. 1988. measurements: results from an in vitro study on occlusal 167. Messer HM: Permanent restorations and the dental pulp. 191. Orchardson R, Peacock JM, Whitters CJ: Effects of pulsed sites, Eur J Oral Sci 115:206, 2007. In Hargreaves KMG, Goodis HE, editors. Seltzer and Nd:YAG laser radiation on action potential conduction in 145. Kvinnsland S, Heyeraas K, Ofjord ES: Effect of Bender’s dental pulp, Carol Stream, IL, 2002, nerve fibres inside teeth in vitro, J Dent 26:421, 1998. experimental tooth movement on periodontal and pulpal Quintessence. 192. Orchardson R, Whitters CJ: Effect of HeNe and pulsed blood flow, Eur J Orthod 11:200, 1989. 168. Michaelson PL, Holland GR: Is pulpitis painful? Int Endod Nd:YAG laser irradiation on intradental nerve responses to 146. Langeland K: Tissue response to dental caries, Endod J 35:829, 2002. mechanical stimulation of dentine, Lasers Surg Med Dent Traumatol 3:149, 1987. 169. Mitchell DF, Tarplee RE: Painful pulpitis; a clinical and 26:241, 2000. 147. Langeland K, Dowden WE, Tronstad L, Langeland LK: microscopic study, Oral Surg Oral Med Oral Pathol 193. Ozturk M, Doruk C, Ozec I, et al: Pulpal blood flow: Human pulp changes of iatrogenic origin, Oral Surg Oral 13:1360, 1960. effects of corticotomy and midline osteotomy in surgically Med Oral Pathol 32:943, 1971. 170. Mjor IA: Bacteria in experimentally infected cavity assisted rapid palatal expansion, J Craniomaxillofac Surg 148. Langeland K, Rodrigues H, Dowden W: Periodontal preparations, Scand J Dent Res 85:599, 1977. 31:97, 2003. disease, bacteria, and pulpal histopathology, Oral Surg 171. Mjor IA, Ferrari M: Pulp-dentin biology in restorative 194. Paakkonen V, Ohlmeier S, Bergmann U, et al: Analysis of Oral Med Oral Pathol 37:257, 1974. dentistry. Part 6: reactions to restorative materials, gene and protein expression in healthy and carious tooth 149. Lefebvre CA, Wataha JC, Bouillaguet S, Lockwood PE: tooth-restoration interfaces, and adhesive techniques, pulp with cDNA microarray and two-dimensional gel Effects of long-term sub-lethal concentrations of dental Quintessence Int 33:35, 2002. electrophoresis, Eur J Oral Sci 113:369, 2005. CHAPTER 13 Pulpal Reactions to Caries and Dental Procedures 597

195. Paakkonen V, Vuoristo JT, Salo T, Tjaderhane L: 220. Reeves R, Stanley HR: The relationship of bacterial 244. Steiner DR: The resolution of a periradicular lesion Comparative gene expression profile analysis between penetration and pulpal pathosis in carious teeth, Oral Surg involving an implant, J Endod 34:330, 2008. native human odontoblasts and pulp tissue, Int Endod J Oral Med Oral Pathol 22:59, 1966. 245. Stenvik A: Pulp and dentine reactions to experimental 41:117, 2008. 221. Reynolds RL: The determination of pulp vitality by means tooth intrusion. (A histologic study—long-term effects), 196. Palosaari H, Wahlgren J, Larmas M, et al: The expression of thermal and electrical stimuli, Oral Surg Oral Med Oral Rep Congr Eur Orthod Soc 449, 1969. of MMP-8 in human odontoblasts and dental pulp cells is Pathol 22:231, 1966. 246. Stenvik A, Mjor IA: Pulp and dentine reactions to down-regulated by TGF-beta1, J Dent Res 79, 2000. 222. Ritter AV, Leonard RH Jr, St Georges AJ, et al: Safety and experimental tooth intrusion. A histologic study of the 197. Pameijer CH, Stanley HR: The disastrous effects of the stability of nightguard vital bleaching: 9 to 12 years initial changes, Am J Orthod 57:370, 1970. “total etch” technique in vital pulp capping in primates post-treatment, J Esthet Restor Dent 14:275, 2002. 247. Stenvik A, Mjor IA: The effect of experimental tooth [published erratum appears in Am J Dent 11:148, 1998], 223. Rodd HD, Boissonade FM: Substance P expression in intrusion on pulp and dentine, Oral Surg Oral Med Oral Am J Dent 11:S45, 1998. human tooth pulp in relation to caries and pain Pathol 32:639, 1971. 198. Pamir T, Dalgar H, Onal B: Clinical evaluation of three experience, Eur J Oral Sci 108:467, 2000. 248. Sussman HI: Endodontic pathology leading to implant desensitizing agents in relieving dentin hypersensitivity, 224. Rodd HD, Boissonade FM: Innervation of human tooth failure—a case report, J Oral Implantol 23:112; Oper Dent 32:544, 2007. pulp in relation to caries and dentition type, J Dent Res discussion 15, 1997. 199. Panitvisai P, Messer HH: Cuspal deflection in molars in 80:389, 2001. 249. Sussman HI: Tooth devitalization via implant placement: a relation to endodontic and restorative procedures, J Endod 225. Rodd HD, Boissonade FM: Vascular status in human case report, Periodontal Clin Investig 20:22, 1998. 21:57, 1995. primary and permanent teeth in health and disease, Eur J 250. Swift EJ Jr, Trope M: Treatment options for the exposed 200. Pashley D: Pulpdentin complex. In Hargreaves KM, Goodis Oral Sci 113:128, 2005. vital pulp, Pract Periodontics Aesthet Dent 11:735; quiz HE, editors: Seltzer and Bender’s The Dental Pulp, Carol 226. Rotstein I, Simon JH: Diagnosis, prognosis and 40, 1999. Stream, IL, 2002, Quintessence, p 66. decision-making in the treatment of combined periodontal- 251. Tagami J, Hosoda H, Burrow MF, Nakajima M: Effect of 201. Pashley DH: The influence of dentin permeability and endodontic lesions, Periodontol 2000 34:165, 2004. aging and caries on dentin permeability, Proc Finn Dent pulpal blood flow on pulpal solute concentrations, J Endod 227. Saito K, Nakatomi M, Ida-Yonemochi H, et al: The Soc 88 (suppl 1):149, 1992. 5:355, 1979. expression of GM-CSF and osteopontin in 252. Takamori K: A histopathological and immunohistochemical 202. Pashley DH: In vitro simulations of in vivo bonding immunocompetent cells precedes the odontoblast study of dental pulp and pulpal nerve fibers in rats after conditions, Am J Dent 4:237, 1991. differentiation following allogenic tooth transplantation in the cavity preparation using Er:YAG laser, J Endod 26:95, 203. Pashley DH: Dynamics of the pulpo-dentin complex, Crit mice, J Histochem Cytochem 59:518, 2011. 2000. Rev Oral Biol Med 7:104, 1996. 228. Sato M, Harada K, Okada Y, Omura K: Blood-flow change 253. Tanabe K, Yoshiba K, Yoshiba N, et al: 204. Pashley DH, Kepler EE, Williams EC, Okabe A: Progressive and recovery of sensibility in the maxillary dental pulp Immunohistochemical study on pulpal response in rat decrease in dentine permeability following cavity after a single-segment Le Fort I osteotomy, Oral Surg Oral molars after cavity preparation by Er:YAG laser, Eur J Oral preparation, Arch Oral Biol 28:853, 1983. Med Oral Pathol Oral Radiol Endod 95:660, 2003. Sci 110:237, 2002. 205. Pashley DH, Tay FR, Yiu C, et al: Collagen degradation by 229. Schmalz G, Krifka S, Schweikl H: Toll-like receptors, LPS, 254. Teixeira LS, Demarco FF, Coppola MC, Bonow ML: host-derived enzymes during aging, J Dent Res 83:216, and dental monomers, Adv Dent Res 23:302, 2011. Clinical and radiographic evaluation of pulpotomies 2004. 230. Schroder U: Effects of calcium hydroxide-containing performed under intrapulpal injection of anaesthetic 206. Pashley EL, Talman R, Horner JA, Pashley DH: pulp-capping agents on pulp cell migration, proliferation, solution, Int Endod J 34:440, 2001. Permeability of normal versus carious dentin, Endod Dent and differentiation, J Dent Res 64 Spec No:541, 1985. 255. Tjan AH, Grant BE, Godfrey MF 3rd: Temperature rise in Traumatol 7:207, 1991. 231. Schuster GS, Caughman GB, Rueggeberg FA, et al: the pulp chamber during fabrication of provisional crowns, 207. Pepersack WJ: Tooth vitality after alveolar segmental Alterations in cell lipid metabolism by glycol methacrylate J Prosthet Dent 62:622, 1989. osteotomy, J Oral Maxillofac Surg 1:85, 1973. (HEMA), J Biomater Sci Polym Ed 10:1121, 1999. 256. Tokita Y, Sunakawa M, Suda H: Pulsed Nd:YAG laser 208. Perdigao J, Geraldeli S, Hodges JS: Total-etch versus 232. Seltzer S, Bender IB, Ziontz M: The dynamics of pulp irradiation of the tooth pulp in the cat: I. Effect of spot self-etch adhesive: effect on postoperative sensitivity, inflammation: correlations between diagnostic data and lasing, Lasers Surg Med 26:398, 2000. J Am Dent Assoc 134:1621, 2003. actual histologic findings in the pulp, Oral Surg Oral Med 257. Tomson PL, Grover LM, Lumley PJ, et al: Dissolution of 209. Perinetti G, Varvara G, Festa F, Esposito P: Aspartate Oral Pathol 16:846, 1963. bio-active dentine matrix components by mineral trioxide aminotransferase activity in pulp of orthodontically treated 233. Shabahang S, Bohsali K, Boyne PJ, et al: Effect of teeth aggregate, J Dent 35:636, 2007. teeth, Am J Orthod Dentofacial Orthop 125:88, 2004. with periradicular lesions on adjacent dental implants, 258. Torabinejad M, Kiger RD: A histologic evaluation of 210. Peters DD, Baumgartner JC, Lorton L: Adult pulpal Oral Surg Oral Med Oral Pathol Oral Radiol Endod 96:321, dental pulp tissue of a patient with periodontal diagnosis. I. Evaluation of the positive and negative 2003. disease, Oral Surg Oral Med Oral Pathol 59:198, responses to cold and electrical pulp tests, J Endod 234. Silva AF, Tarquinio SB, Demarco FF, et al: The influence 1985. 20:506, 1994. of haemostatic agents on healing of healthy human dental 259. Tranasi M, Sberna MT, Zizzari V, et al: Microarray 211. Petersson K, Soderstrom C, Kiani-Anaraki M, Levy G: pulp tissue capped with calcium hydroxide, Int Endod J evaluation of age-related changes in human dental pulp, Evaluation of the ability of thermal and electrical tests to 39:309, 2006. J Endod 35:1211, 2009. register pulp vitality, Endod Dent Traumatol 15:127, 235. Simon JH, Lies J: Silent trauma, Endod Dent Traumatol 260. Trantor IR, Messer HH, Birner R: The effects of 1999. 15:145, 1999. neuropeptides (calcitonin gene-related peptide and 212. Polat S, Er K, Akpinar KE, Polat NT: The sources of laser 236. Sloan AJ, Smith AJ: Stimulation of the dentine-pulp substance P) on cultured human pulp cells, J Dent Res Doppler blood-flow signals recorded from vital and root complex of rat incisor teeth by transforming growth 74:1066, 1995. canal treated teeth, Arch Oral Biol 49:53, 2004. factor-beta isoforms 1-3 in vitro, Arch Oral Biol 44:149, 261. Trowbridge H, Edwall L, Panopoulos P: Effect of zinc 213. Premdas CE, Pitt Ford TR: Effect of palatal injections on 1999. oxide-eugenol and calcium hydroxide on intradental nerve pulpal blood flow in premolars, Endod Dent Traumatol 237. Smith AJ: Pulpal responses to caries and dental repair, activity, J Endod 8:403, 1982. 11:274, 1995. Caries Res 36:223, 2002. 262. Tsatsas BG, Frank RM: Ultrastructure of the dentinal 214. Quirynen M, Gijbels F, Jacobs R: An infected jawbone site 238. Smith AJ, Lesot H: Induction and regulation of crown tubular substances near the dentino-enamel junction, compromising successful osseointegration, Periodontol dentinogenesis: embryonic events as a template for dental Calcif Tissue Res 9:238, 1972. 2000 33:129, 2003. tissue repair? Crit Rev Oral Biol Med 12:425, 2001. 263. Turner DF, Marfurt CF, Sattelberg C: Demonstration of 215. Qvist V, Stoltze K, Qvist J: Human pulp reactions to resin 239. Smith AJ, Tobias RS, Plant CG, et al: Preliminary studies physiological barrier between pulpal odontoblasts and its restorations performed with different acid-etch restorative on the in vivo morphogenetic properties of dentine matrix perturbation following routine restorative procedures: a procedures, Acta Odontol Scand 47:253, 1989. proteins, Biomaterials 11:22, 1990. horseradish peroxidase tracing study in the rat, J Dent 216. Rakich DR, Wataha JC, Lefebvre CA, Weller RN: Effects of 240. Smith JG, Smith AJ, Shelton RM, Cooper PR: Recruitment Res 68:1262, 1989. dentin bonding agents on macrophage mitochondrial of dental pulp cells by dentine and pulp extracellular 264. Tyndall AA, Brooks SL: Selection criteria for dental implant activity, J Endod 24:528, 1998. matrix components, Exp Cell Res 318:2397, 2012. site imaging: a position paper of the American Academy 217. Rakich DR, Wataha JC, Lefebvre CA, Weller RN: Effect of 241. Soo-ampon S, Vongsavan N, Soo-ampon M, et al: The of Oral and Maxillofacial Radiology, Oral Surg Oral Med dentin bonding agents on the secretion of inflammatory sources of laser Doppler blood-flow signals recorded from Oral Pathol Oral Radiol Endod 89:630, 2000. mediators from macrophages, J Endod 25:114, 1999. human teeth, Arch Oral Biol 48:353, 2003. 265. Tziafas D: Basic mechanisms of cytodifferentiation and 218. Ramsay DS, Artun J, Bloomquist D: Orthognathic surgery 242. Stabholz A, Rotstein L, Neev J, Moshonov J: Efficacy of dentinogenesis during dental pulp repair, Int J Dev Biol and pulpal blood flow: a pilot study using laser Doppler XeCl 308-nm excimer laser in reducing dye penetration 39:281, 1995. flowmetry, J Oral Maxillofac Surg 49:564, 1991. through coronal dentinal tubules, J Endod 21:266, 1995. 266. Tziafas D, Kolokuris I: Inductive influences of 219. Reeh ES, Messer HH, Douglas WH: Reduction in tooth 243. Stanley HR: Pulp capping: conserving the dental demineralized dentin and bone matrix on pulp cells: an stiffness as a result of endodontic and restorative pulp—can it be done? Is it worth it? Oral Surg Oral Med approach of secondary dentinogenesis, J Dent Res 69:75, procedures, J Endod 15:512, 1989. Oral Pathol 68:628, 1989. 1990. 598 PART II The Advanced Science of Endodontics

267. Umberto R, Claudia R, Gaspare P, et al: Treatment of evoked in trigeminal caudal neurons by tooth pulp 284. Yoshiyama M, Masada J, Uchida A, Ishida H: Scanning dentine hypersensitivity by diode laser: a clinical study, stimulation, Lasers Surg Med 13:605, 1993. electron microscopic characterization of sensitive vs. Int J Dent 2012:858950, 2012. 277. Wennberg A, Mjor IA, Heide S: Rate of formation of insensitive human radicular dentin, J Dent Res 68:1498, 268. Unemori M, Matsuya Y, Akashi A, et al: Composite resin regular and irregular secondary dentin in monkey teeth, 1989. restoration and postoperative sensitivity: clinical follow-up Oral Surg Oral Med Oral Pathol 54:232, 1982. 285. Zach L, Cohen G: Pulp response to externally applied in an undergraduate program, J Dent 29:7, 2001. 278. Wigdor H, Abt E, Ashrafi S, Walsh JT Jr: The effect of heat, Oral Surg Oral Med Oral Pathol 19:515, 1965. 269. Valderhaug J, Jokstad A, Ambjornsen E, Norheim PW: lasers on dental hard tissues, J Am Dent Assoc 124:65, 286. Zaia AA, Nakagawa R, De Quadros I, et al: An in vitro Assessment of the periapical and clinical status of 1993. evaluation of four materials as barriers to coronal crowned teeth over 25 years, J Dent 25:97, 1997. 279. Yiu CK, King NM, Suh BI, et al: Incompatibility of oxalate microleakage in root-filled teeth, Int Endod J 35:729, 270. Vale WA:Cavity preparation, Ir Dent Rev 2:33, 1956. desensitizers with acidic, fluoride-containing total-etch 2002. 271. Vedtofte P: Pulp canal obliteration after Le Fort I adhesives, J Dent Res 84:730, 2005. 287. Zhang J, Kawashima N, Suda H, et al: The existence of osteotomy, Endod Dent Traumatol 5:274, 1989. 280. Yoshiba K, Yoshiba N, Iwaku M: Class II antigen- CD11c+ sentinel and F4/80+ interstitial dendritic cells in 272. Versluis A, Douglas WH, Cross M, Sakaguchi RL: Does an presenting dendritic cell and nerve fiber responses to dental pulp and their dynamics and functional properties, incremental filling technique reduce polymerization cavities, caries, or caries treatment in human teeth, Int Immunol 18:1375, 2006. shrinkage stresses? J Dent Res 75:871, 1996. J Dent Res 82:422, 2003. 287a. Zhang J, Zhu LX, Cheng X, et al: Peng: Promotion of 273. Versluis A, Tantbirojn D, Douglas WH: Distribution of 281. Yoshiba N, Yoshiba K, Iwaku M, Ozawa H: Dental Pulp Cell Migration and Pulp Repair by a transient properties during polymerization of a Immunohistochemical localizations of class II antigens and Bioceramic Putty Involving FGFR-mediated Signaling light-initiated restorative composite, Dent Mater 20:543, nerve fibers in human carious teeth: HLA-DR Pathways, B2 J Dent Res 2015 Feb 27. 2004. immunoreactivity in Schwann cells, Arch Histol Cytol pii: 0022034515572020. 274. von Troil B, Needleman I, Sanz M: A systematic review of 61:343, 1998. 288. Zhong S, Zhang S, Bair E, et al: Differential expression of the prevalence of root sensitivity following periodontal 282. Yoshiba N, Yoshiba K, Nakamura H, et al: microRNAs in normal and inflamed human pulps, J Endod therapy, J Clin Periodontol 29 (suppl 3):173; discussion Immunohistochemical localization of HLA-DR-positive cells 38:746, 2012. 95, 2002. in unerupted and erupted normal and carious human 275. Vongsavan N, Matthews RW, Matthews B: The teeth, J Dent Res 75:1585, 1996. permeability of human dentine in vitro and in vivo, Arch 283. Yoshida S, Oshima K, Tanne K: Biologic responses Oral Biol 45:931, 2000. of the pulp to single-tooth dento-osseous osteotomy, 276. Wakabayashi H, Hamba M, Matsumoto K, Tachibana H: Oral Surg Oral Med Oral Pathol Oral Radiol Endod 82:152, Effect of irradiation by semiconductor laser on responses 1996. CHAPTER 14

Microbiology of Endodontic Infections

JOSÉ F. SIQUEIRA, JR. | ISABELA N. RÔÇAS

CHAPTER OUTLINE Apical Periodontitis as an Infectious Disease Types of Endodontic Infections Routes of Root Canal Infection Diversity of the Endodontic Microbiota Mechanisms of Microbial Pathogenicity and Primary Intraradicular Infection Virulence Factors Microbial Composition and Diversity Spatial Distribution of the Microbiota: Anatomy Symptomatic Infections of Infection Geographic Influence Biofilm and Community-Based Microbial Pathogenesis Microbial Ecology and the Root Canal Ecosystem Biofilm and Bacterial Interactions Other Microorganisms in Endodontic Infections Biofilm Community Lifestyle Persistent/Secondary Endodontic Infections Resistance to Antimicrobial Agents Persistent/Secondary Infections and Treatment Failure Apical Periodontitis as a Biofilm-Related Disease Bacteria at the Root Obturation Stage Methods for Microbial Identification Microbiota in Endodontically Treated Teeth The Five Generations of Endodontic Microbiology Studies Extraradicular Infections Impact of Molecular Methods in Endodontic Microbiology

Apical periodontitis is essentially an inflammatory disease of The ultimate goal of endodontic treatment is either to microbial etiology primarily caused by infection of the root prevent the development of apical periodontitis or, in cases canal system.196 Although chemical and physical factors can where the disease is already present, to create adequate condi- induce periradicular inflammation, a large body of scientific tions for periradicular tissue healing. The intent is to repair evidence indicates that endodontic infection is essential to the and preserve the tooth and associated periradicular bone. progression and perpetuation of the different forms of apical Because apical periodontitis is an infectious disease, the ratio- periodontitis.16,86,120,250 Endodontic infection develops in root nale for endodontic treatment is to eradicate the occurring canals devoid of host defenses, as a consequence of either pulp infection or prevent microorganisms from infecting or reinfect- necrosis (as a sequel to caries, trauma, periodontal disease, or ing the root canal or the periradicular tissues. The cardinal invasive operative procedures) or pulp removal for treatment. principle of any health care profession is the thorough under- Although fungi and most recently archaea and viruses standing of disease etiology and pathogenesis, which provides have been found in association with endodontic infec- a framework for effective treatment. In this context, under- tions,57,177,232,238,268 bacteria are the major microorganisms impli- standing the microbiologic aspects of apical periodontitis is the cated in the pathogenesis of apical periodontitis. In advanced basis for endodontic practice and should be managed with an stages of the endodontic infectious process, bacterial organiza- evidence-based approach. This chapter focuses on diverse tions resembling biofilms can be observed adhered to the canal aspects of endodontic microbiology, including pathogenetic, walls.121,126,152,223 Consequently, apical periodontitis has been taxonomic, morphologic, and ecologic issues. included in the roll of biofilm-related oral diseases.92,228 Bacteria colonizing the root canal system enter in contact with the peri- APICAL PERIODONTITIS radicular tissues via apical/lateral foramina or root perforations. As a consequence of the encounter between bacteria and host AS AN INFECTIOUS DISEASE defenses, inflammatory changes take place in the periradicular The first recorded observation of bacteria in the root canal tissues and give rise to the development of apical periodontitis. dates back to the 17th century and the Dutch amateur micro- Depending on several bacterial and host-related factors, end- scope builder Antony van Leeuwenhoek (1632-1723). He odontic infections can lead to acute (symptomatic) or chronic reported that the root canals of a decayed tooth “were stuffed (asymptomatic) apical periodontitis. with a soft matter” and that “the whole stuff” seemed to be

599 600 PART II The Advanced Science of Endodontics

infectious cause of this disease. Anaerobic bacteria accounted Fig. 2 for more than 90% of the isolates. Findings from Sundqvist’s study also demonstrated that in the absence of infection, the Fig. 3 Fig. 4 necrotic pulp tissue itself and stagnant tissue fluid in the root canal cannot induce or perpetuate apical periodontitis lesions. Fig. 1 Möller and colleagues120 also provided strong evidence a about the microbial causation of apical periodontitis. Their b study using monkeys’ teeth demonstrated that only devitalized c pulps that were infected induced apical periodontitis lesions, whereas devitalized and noninfected pulps showed an absence of significant pathologic changes in the periradicular tissues. In addition to corroborating the importance of microorganisms for the development of apical periodontitis, this study also confirmed that necrotic pulp tissue per se is unable to induce FIG. 14-1 Drawings from Miller’s classic paper showing different bacterial forms in a root canal sample observed by microscopy. and maintain an apical periodontitis lesion. Microorganisms causing apical periodontitis are primarily organized in biofilms colonizing the root canal system. Nair126 alive.46 At that time, the role of Leeuwenhoek’s “animalcules” was possibly the first to observe intracanal bacterial organiza- in disease causation was unsuspected. It took almost 200 years tions adhered to the root canal walls, resembling biofilm until his observation was confirmed and a cause-and-effect structures. Several other morphologic studies found similar relationship between bacteria and apical periodontitis was sug- structures,121,190,223 but it was not until the study of Ricucci and gested. This occurred specifically in 1894, when Willoughby Siqueira152 that the high prevalence of bacterial biofilms were Dayton Miller, an American dentist working at the laboratory consistently revealed in association with primary and post- of Robert Koch in Berlin, Germany, published a milestone treatment apical periodontitis (see the section Spatial Distribu- study reporting the association between bacteria and apical tion of the Microbiota: Anatomy of Infection for a further periodontitis after an analysis of samples collected from root discussion of biofilms in endodontic infections). canals.115 By means of bacterioscopy of the canal samples, he found bacterial cells in the three basic morphologies known at the time: cocci, bacilli, and spirilla (or spirochetes) (Fig. 14-1). ROUTES OF ROOT CANAL INFECTION Morphologically, the endodontic microbiota was clearly differ- Under normal conditions, the pulpodentin complex is sterile ent in the coronal, middle, and apical parts of the root canal. and isolated from oral microbiota by overlying enamel, dentin, Spirochetes were found in high frequencies in abscessed cases, and cementum. In the event that the integrity of these natural and a pathogenic role was suspected for these bacteria. Most layers is breached (e.g., as a result of caries, trauma-induced of the bacteria Miller observed under light microscopy could fractures and cracks, restorative procedures, scaling and root not be cultivated using the technology available at that time. planning, attrition, abrasion) or naturally absent (e.g., because Those bacteria were conceivably anaerobic bacteria, which of gaps in the cemental coating at the cervical root surface), were only successfully cultivated about 50 to 100 years later the pulpodentin complex is exposed to the oral environment. with the advent of anaerobic culture techniques. However, it The pulpodentin complex is then challenged by microorgan- is now widely recognized that a large number of bacterial isms present in caries lesions, saliva bathing the exposed area, species living in diverse environments still remain to be culti- or dental plaque formed on the exposed area. Microorganisms vated by current technology,6,147 and the root canal is no excep- from subgingival biofilms associated with periodontal disease tion (discussed later in this chapter). Based on his findings, may also have access to the pulp via dentinal tubules at the Miller raised the hypothesis that bacteria were the causative cervical region of the tooth and lateral or apical foramina (see agents of apical periodontitis. also Chapter 25). Microorganisms may also have access to the Approximately 70 years after Miller’s classic study, his root canal any time during or after endodontic intervention, assumptions were confirmed by an elegant study from emphasizing the need for effective (fluid tight) use of the Kakehashi and colleagues.86 These authors investigated the rubber dam. response of exposed dental pulps to the oral cavity in conven- Whenever dentin is exposed, the pulp is put at risk of infec- tional and germ-free rats. Histologic evaluation was performed tion as a consequence of the permeability of normal dentin and revealed that pulp necrosis and apical periodontitis lesions dictated by its tubular structure135 (Fig. 14-2). Dentinal tubules developed in all conventional rats; however, the exposed pulps traverse the entire width of the dentin and have a conical con- of germ-free rats not only remained vital but also repaired formation, with the largest diameter located near the pulp themselves with hard-tissue formation. Dentin-like tissue (mean, 2.5 µm) and the smallest diameter in the periphery, sealed the exposure area and isolated the pulps again from the near the enamel or cementum (mean, 0.9 µm).62 The smallest oral cavity. tubule diameter is entirely compatible with the cell diameter The important role of bacteria in the etiology of apical of most oral bacterial species, which usually ranges from 0.2 periodontitis was further confirmed by Sundqvist’s classic to 0.7 µm. One might assume that once exposed, dentin offers study.250 This author applied advanced anaerobic culturing an unimpeded pathway for bacteria to reach the pulp via these techniques to the evaluation of bacteria occurring in the root tubules. However, it has been demonstrated that bacterial inva- canals of teeth whose pulps became necrotic after trauma. sion of dentinal tubules occurs more rapidly with a nonvital Bacteria were found only in the root canals of teeth exhibiting pulp than with a vital pulp.124 With a vital pulp, outward move- radiographic evidence of apical periodontitis, confirming the ment of dentinal fluid and the tubular contents (including CHAPTER 14 Microbiology of Endodontic Infections 601

A B

FIG. 14-2 A, Scanning electron micrographs of dentin showing tubules in cross-sectional view (magnification ×850). B, Longitudinal views (magnification ×130). odontoblast processes, collagen fibrils, and the sheathlike The egress of microorganisms and their products from lamina limitans that lines the tubules) influence dentinal per- infected root canals through apical, lateral, or furcation foram- meability and can conceivably delay intratubular invasion by ina, dentinal tubules, and iatrogenic root perforations can bacteria. Because of the presence of tubular contents, the func- directly affect the surrounding periodontal tissues and give rise tional or physiologic diameter of the tubules is only 5% to 10% to pathologic changes in these tissues. However, there is no of the anatomic diameter seen by microscopy.113 Other factors consensus as to whether the opposite is true—that is, whether such as dentinal sclerosis beneath a carious lesion, tertiary subgingival biofilms associated with periodontal disease can dentin, smear layer, and intratubular deposition of fibrinogen directly cause pulpal disease. Conceptually, microorganisms in also reduce dentin permeability and thereby limit or even subgingival plaque biofilms associated with periodontal disease impede bacterial progression to the pulp via dentinal tubules.137 could reach the pulp by the same pathways intracanal micro- Host defense molecules, such as antibodies and components organisms reach the periodontium and could thereby exert of the complement system, may also be present in the dentinal harmful effects on the pulp. However, it has been demonstrated fluid of vital teeth and can assist in the protection against deep that although degenerative and inflammatory changes of dif- bacterial invasion of dentin.3,130,131 As long as the pulp is vital, ferent degrees may occur in the pulp of teeth with associated dentinal exposure does not represent a significant route of marginal periodontitis, pulpal necrosis as a consequence of pulpal infection, except when dentin thickness is considerably periodontal disease only develops if the periodontal pocket reduced or when the dentin permeability is significantly reaches the apical foramen, leading to irreversible damage to increased. the main blood vessels that penetrate through this foramen98 Most of the bacteria in the carious process are nonmotile; (Fig. 14-3). After the pulp becomes necrotic, periodontal bac- they invade dentin by repeated cell division, which pushes cells teria can reach the root canal system via exposed dentinal into tubules. Bacterial cells may also be forced into tubules by tubules at the cervical area of the root or via lateral and apical hydrostatic pressures developed on dentin during mastica- foramina to establish an endodontic infectious process. tion.114 Bacteria inside tubules under a deep carious lesion can It has been claimed that microorganisms can reach the pulp reach the pulp even before frank pulpal exposure.81 As men- by anachoresis.73 Theoretically, microorganisms can be trans- tioned, it has been assumed that the pulp will not be infected ported in the blood or lymph to an area of tissue damage, if it is still vital. The few bacteria that reach the pulp may not where they leave the vessel, enter the damaged tissue, and be significant, because the vital pulp can eliminate sucha establish an infection.64,156 However, there is no clear evidence transient infection and rapidly clear or remove bacterial prod- showing that this process can represent a route for root canal ucts. This efficient clearance mechanism tends to prevent inju- infection. It has been revealed that bacteria could not be recov- rious agents from reaching a high enough concentration to ered from unfilled root canals when the bloodstream was induce significant inflammatory reactions.136 On the other experimentally infected, unless the root canals were overin- hand, if the vitality of the pulp is compromised and the defense strumented during the period of bacteremia, with resulting mechanisms are impaired, even a small amount of bacteria may injury to periodontal blood vessels and blood seepage into the initiate infection. canal.42 Another argument against anachoresis as a route for Direct exposure of the dental pulp to the oral cavity is the pulpal infection comes from the study by Möller and col- most obvious route of endodontic infection. Caries is the most leagues,120 who induced pulpal necrosis in monkeys’ teeth and common cause of pulp exposure, but bacteria may also reach reported that all cases of aseptic necrosis remained bacteria- the pulp via direct pulp exposure as a result of iatrogenic free after 6 to 7 months of observation. restorative procedures or trauma. The exposed pulp tissue Bacteria have been isolated from traumatized teeth with comes in direct contact with oral bacteria from carious lesions, necrotic pulps with apparently intact crowns.250,278 Although saliva, or plaque accumulated onto the exposed surface. Almost anachoresis has been suggested to be the mechanism through invariably, exposed pulps will undergo inflammation and which these traumatized teeth become infected,73 current evi- necrosis and become infected. The time elapsed between pulp dence indicates that the main pathway of pulpal infection in exposure and infection of the entire canal is unpredictable, but these cases is dentinal exposure due to enamel cracks.106,107 it is usually a slow process.38 Macro- and microcracks in enamel can be present in most teeth 602 PART II The Advanced Science of Endodontics

biofilm, calculus, or caries on the tooth crown; leaking rubber dam; contamination of endodontic instruments (e.g., after touching with the fingers); and contamination of irrigant solutions or other solutions of intracanal use (e.g., saline solution, distilled water, citric acid). Microorganisms can also enter the root canal system between appointments by leakage through the temporary restorative material; breakdown, fracture, or loss of the temporary restoration; fracture of the tooth structure; and in teeth left open for drainage. Microorganisms can penetrate the root canal system even after completion of the root canal obturation by leakage through the temporary or permanent restorative material; breakdown, fracture, or loss of the temporary/permanent restoration; fracture of the tooth struc- A ture; recurrent decay contaminating the root canal obturation; or delay in the placement of permanent restorations.205

MECHANISMS OF MICROBIAL PATHOGENICITY AND VIRULENCE FACTORS The ability of a microorganism to cause disease is regarded as its pathogenicity. Virulence denotes the degree of pathogenicity of a microorganism, and virulence factors are the microbial products, structural cellular components, or strategies that contribute to pathogenicity. One example of bacterial strategy that contributes to pathogenicity includes the ability to coaggregate and form biofilms, which confers protection against B microbial competitors, host defenses, and antimicrobial agents. Some microorganisms routinely cause disease in a given host FIG. 14-3 Periodontal disease (A) mostly affects the pulp vitality when the and are called primary pathogens. Other microorganisms cause subgingival biofilm reaches the apical foramen (B). disease only when host defenses are impaired and are called opportunistic pathogens. Bacteria that make up the normal microbiota are usually present as harmless commensals and (not only traumatized teeth) and do not necessarily end at the live in balance with the host. One of the greatest beneficial enamel-dentin junction; they can extend deep into the dentin.107 effects of human microbiota is probably the tendency to protect A large number of dentinal tubules can be exposed to the oral the host from exogenous infections by excluding other microenvironment by a single crack. These cracks can be clogged organisms. Nevertheless, in certain situations, the balance may with dental plaque and provide portals of entry for bacteria. If be disturbed by a decrease in the normal level of resistance, the pulp remains vital after trauma, bacterial penetration into and then the commensal bacteria are usually the first to take tubules is counteracted by the dentinal fluid and tubular con- advantage. Most bacteria involved with endodontic infections tents, as discussed earlier, and pulpal health is not usually are normal inhabitants of the oral microbiota that exploit jeopardized. But if the pulp becomes necrotic as a consequence changes in the balance of the host–bacteria relationship, of trauma, it loses the ability to protect itself against bacterial becoming opportunistic pathogens. invasion, and regardless of dentin thickness, dentinal tubules Bacteria involved with the pathogenesis of primary apical then will become true avenues through which bacteria can periodontitis may have participated in the early stages of pulp reach and colonize the necrotic pulp. inflammation and necrosis, or they may have gained entry into Whatever the route of bacterial access to the root canal, the root canal space any time after pulpal necrosis. In the necrosis of pulp tissue is a prerequisite for the establishment former situation, involved bacteria are usually those present in of primary endodontic infections. To reiterate: if the pulp is the advanced front of caries lesions and from saliva bathing the vital, it can protect itself against bacterial invasion and coloni- affected area. Bacteria in caries lesions form authentic biofilms zation. If the pulp becomes necrotic due to caries, trauma, adhered to dentin (Fig. 14-4). Diffusion of bacterial products operative procedures, or periodontal disease, then it can be through dentinal tubules induces pulpal inflammation long easily infected. This is because host defenses do not function before the tissue is exposed. After pulp exposure, the surface in the necrotic pulp tissue, and those in the periradicular of the tissue can also be colonized and covered by bacteria tissues do not reach deep into the root canal space. present in the caries biofilm. The exposed pulp tissue is in Another situation in which the root canal system is devoid direct contact with bacteria and their products and responds of host defenses relates to cases in which the pulp was removed with severe inflammation. Some tissue invasion by bacteria for treatment. Microbial penetration in the canal can occur may also occur. Bacteria in the battlefront have to survive the during treatment, between appointments, or even after root attack from the host defenses and at the same time acquire canal obturation. The main causes of microbial introduction nutrients to keep themselves alive. In this bacteria–pulp clash, into the canal during treatment include remnants of dental the latter invariably is “defeated” and becomes necrotic, so CHAPTER 14 Microbiology of Endodontic Infections 603 bacteria move forward and “occupy the territory”—that is, in the microbiota. Rearrangement in the proportions of the they colonize the necrotic tissue. These events advance through pioneer species and latecomers occurs, and as the environment tissue compartments, coalesce, and move toward the apical changes, some early colonizers are expected to no longer part of the canal until virtually the entire root canal is necrotic participate in the consortium of advanced disease. With the and infected (Fig. 14-5). At this stage, involved bacteria passage of time, the endodontic microbiota becomes more and can be regarded as the early root canal colonizers or pioneer more structurally and spatially organized. species. Some virulence attributes required for pathogens to thrive Early colonizers play an important role in the initiation of in other sites may be of no value for bacteria that reach the the apical periodontitis disease process. Moreover, they may root canal after necrosis—for instance, the ability to evade host significantly modify the environment, making it conducive to defenses. This is because latecomers face no significant opposi- the establishment of other bacterial groups. These new species tion from host defenses, which are no longer active in the canal may have access to the canal via coronal exposure or exposed after necrosis. Although colonization may appear an easy task dentinal tubules, establish themselves, and contribute to a shift for late colonizers, other environmental factors (e.g., interaction with pioneer species, oxygen tension, nutrient availability) will determine whether new species entering the canal will succeed in establishing themselves and join the early colonizers to make up a dynamic mixed community in the root canal. Ultimately, the root canals of teeth with radiographically detectable apical periodontitis lesions harbor both early colonizers that managed to stay in the canals and late colonizers that managed to adapt to the new but propitious environmental conditions.214 Bacteria colonizing the necrotic root canal induce damage to the periradicular tissues and give rise to inflammatory changes. In fact, periradicular inflammation can be observed even before the frontline of infection reaches the apical foramen.8,121,242,280 Bacteria exert their pathogenicity by wreak- ing havoc on the host tissues through direct or indirect mechanisms. Bacterial virulence factors that cause direct tissue harm include those that damage host cells or the intercellular matrix of the connective tissue. These factors usually involve secreted products, including enzymes, exotoxins, heat-shock proteins, and metabolic end products.214 Furthermore, bacterial struc- FIG. 14-4 Scanning electron micrograph showing a bacterial biofilm cover- tural components, including lipopolysaccharide (LPS), pepti- ing dentin in a deep carious lesion. Note the presence of different bacterial doglycan, lipoteichoic acid, fimbriae, flagella, outer membrane morphotypes (magnification ×3500). (From Torabinejad M, Walton RE: End- proteins and vesicles, lipoproteins, DNA, and exopolysaccha- odontics: principles and practice, ed 4, St. Louis, 2009, Saunders/Elsevier.) rides, can act as modulins by stimulating the development of

A B C D

FIG. 14-5 Dynamics of pulp response from caries exposure (A) to pulp inflammation (B) to pulp necrosis (C) to apical periodontitis formation (D). 604 PART II The Advanced Science of Endodontics

LTA Cytoplasmic membrane Cell wall PG Flagellum CM

Gram-positive

OMP LPS OM Chromosome LPtn Ribosomes PG CM Plasmids Exopolysaccharide (capsule) Fimbriae Gram-negative

FIG. 14-6 Bacterial cell and its structural components that can act as virulence factors. Right: A detailed scheme of the bacterial cell walls from gram-positive and gram-negative bacteria. CM, cytoplasmic membrane; LPS, lipopolysaccharide (endotoxin); LPtn, lipoproteins; LTA, lipoteichoic acid; OM, outer membrane; OMP, outer membrane protein; PG, peptidoglycan.

host immune reactions capable not only of defending the host form in different individuals is unlikely to follow a stereotyped against infection but also of causing severe tissue destruc- course with regard to the bacterial mediators involved.197 tion80,214,265 (Fig. 14-6). For instance, inflammatory and nonin- flammatory host cells can be stimulated by bacterial components SPATIAL DISTRIBUTION OF THE to release chemical mediators such as cytokines and prostaglandins, which are involved in the induction of bone resorp- MICROBIOTA: ANATOMY OF INFECTION tion characteristically observed in asymptomatic (chronic) Mounting evidence indicates that apical periodontitis, like apical periodontitis lesions.184 Another example of indirect caries and periodontal diseases, is also a biofilm-related damage caused by bacteria is the formation of purulent exudate disease. Morphologic studies have shown that the root canal in acute apical abscesses. Host defense mechanisms against microbiota in primary infections is dominated by bacterial bacteria emanating from the root canal appear to be the most morphotypes that include cocci, rods, filaments, and spirilla important factor involved in the formation of purulent exudate (spirochetes) (Fig. 14-7). Fungal cells are sporadically associated with abscesses. Formation of oxygen-derived free found190,223 (Fig. 14-8). Although planktonic bacterial cells sus- radicals, such as superoxide and hydrogen peroxide, alongside pended in a fluid phase and enmeshed in necrotic pulp tissue the release of lysosomal enzymes by polymorphonuclear leu- can be observed in the main root canal, most bacteria coloniz- kocytes, gives rise to destruction of the connective extracel- ing the root canal system usually grow in sessile multispecies lular matrix, leading to pus formation.264 Although direct biofilms adhered to the dentinal walls121,126,223 (Fig. 14-9). damage caused by bacterial products may certainly be involved Lateral canals, apical ramifications, and isthmuses connecting in the pathogenesis of apical periodontitis, bacterial indirect main canals may also be clogged with bacterial biofilms125,151 destructive effects seem to be more significant in this regard.197 (Figs. 14-10 and 14-11). Apical periodontitis is a multifactorial disease that is resul- Bacterial cells from endodontic biofilms are often seen tant of the interplay of many host and bacterial factors. Few if penetrating the dentinal tubules (Fig. 14-12). Dentinal tubule any of the putative endodontic pathogens are individually infection can occur in about 70% to 80% of the teeth evincing capable of inducing all of the events involved in the pathogen- apical periodontitis lesions.112,143 A shallow penetration is more esis of the different forms of apical periodontitis. Probably, the common, but bacterial cells can be observed reaching approxi- process requires an integrated and orchestrated interaction of mately 300 µm in some teeth223 (Fig. 14-13). Dividing cells are the selected members of the mixed endodontic microbiota and frequently observed within tubules during in situ investiga- their respective virulence attributes. Although LPS is undoubt- tions223 (see Fig. 14-13), indicating that bacteria can derive edly the most studied and quoted virulence factor, it sounds nutrients within tubules, probably from degrading odontoblas- simplistic to ascribe to this molecule all responsibility for tic processes, denatured collagen, bacterial cells that die during apical periodontitis causation. This statement is further rein- the course of infection, and intracanal fluids that enter the forced by the fact that some cases of primary infections and tubules by capillarity. many cases of secondary/persistent infections harbor exclu- Several putative endodontic pathogens have been shown to sively gram-positive bacteria. Therefore, the involvement of be capable of penetrating dentinal tubules in vitro, including other factors must not be overlooked. In fact, the pathogenesis Porphyromonas endodontalis, Porphyromonas gingivalis, Fuso- of different forms of apical periodontitis and even the same bacterium nucleatum, Actinomyces israelii, Propionibacterium CHAPTER 14 Microbiology of Endodontic Infections 605

FIG. 14-7 Mixed bacterial population colonizing the root canal wall. Cocci FIG. 14-8 Heavy colonization of yeast cells in the root canal of an extracted are the predominant forms, but rods, filaments, and spirochetes are also tooth with primary infection associated with apical periodontitis (magnification observed. In some areas, coccoid cells are relatively apart from each other ×300). Note that some cells are in the stage of budding. A daughter cell is (magnification ×2200). (From Siqueira JF Jr, Rôças IN, Lopes HP: Patterns of growing on the surface of the mother cell (insets: magnification ×2700 microbial colonization in primary root canal infections, Oral Surg Oral Med [bottom], magnification ×3500[top]). (From Siqueira JF Jr, Sen BH: Fungi in Oral Pathol Oral Radiol Endod 93:174, 2002.) endodontic infections, Oral Surg Oral Med Oral Pathol Oral Radiol Endod 97:632, 2004.)

A B

FIG. 14-9 A, Biofilm on the walls of a mesial root canal from a mandibular first molar (magnification ×100). The tooth was symptomatic, and an apical periodontitis lesion was present. Sections in B and C correspond to higher magnifications of the larger and smaller insets, respectively. Note the accumulation of polymorphonuclear neutrophils in the canal near the biofilm. (B, magnification ×400; C, magnification ×1000). Sections stained with a Taylor- modified Brown and Brenn technique. (Courtesy Dr. Domenico Ricucci.)

C acnes, Enterococcus faecalis, Candida albicans, and strepto- and streptococci. Using immunohistologic analysis, Matsuo cocci.107,141,201,224,273 In their clinical study, Peters and col- and associates112 observed the occurrence of F. nucleatum, leagues143 isolated and identified bacteria present in root dentin Pseudoramibacter alactolyticus, Eubacterium nodatum, Lactoba- at different depths, and the most common isolates belonged to cillus casei, and Parvimonas micra inside dentinal tubules the genera Prevotella, Porphyromonas, Fusobacterium, Veillon- from the canal walls of extracted infected teeth with apical ella, Peptostreptococcus, Eubacterium, Actinomyces, lactobacilli, periodontitis. 606 PART II The Advanced Science of Endodontics

Whereas bacteria present as planktonic cells in the main root canal may be easily accessed and eliminated by instruments and substances used during endodontic treatment, those organized in biofilms attached to the canal walls or located into isthmuses, lateral canals, and dentinal tubules are definitely more difficult to reach125,267 and may require special therapeutic strategies to be eradicated.

BIOFILM AND COMMUNITY-BASED MICROBIAL PATHOGENESIS Individual microorganisms proliferating in a habitat give rise to populations. Such populations often occur as microcolonies in the environment. Populations interact with one another to

B FIG. 14-12 Heavy infection of the root canal walls, mainly by cocci, but FIG. 14-10 A, Bacterial biofilm in the necrotic canal and in an apical some small rods are also seen. Cocci are penetrating into dentinal tubules ramification contiguous to inflamed periradicular tissues (magnification ×25). (magnification ×3500). (From Siqueira JF Jr, Rôças IN, Lopes HP: Patterns of B, Higher magnification of A (magnification ×100). Sections stained with a microbial colonization in primary root canal infections, Oral Surg Oral Med Taylor-modified Brown and Brenn technique. (Courtesy Dr. Domenico Ricucci.) Oral Pathol Oral Radiol Endod 93:174, 2002.)

A B

FIG. 14-11 Transverse sections of a maxillary second molar with fused mesial and palatal roots. A, Heavy bacterial infection of the canal, spreading to an isthmus (magnification ×25). B, Higher magnification of the isthmus clogged with bacteria (magnification ×400). Sections stained with a Taylor-modified Brown and Brenn technique. (Courtesy Dr. Domenico Ricucci.) CHAPTER 14 Microbiology of Endodontic Infections 607

FIG. 14-13 Cocci in dentinal tubules approximately 300 µm from the main root canal (magnification ×5000). (From Siqueira JF Jr, Rôças IN, Lopes HP: Patterns of microbial colonization in primary root canal infections, Oral Surg FIG. 14-14 Mixed bacterial biofilm adhered to the tooth surface (Brown Oral Med Oral Pathol Oral Radiol Endod 93:174, 2002.) and Brenn staining, magnification ×1000). form a community. Thus, community refers to a unified assem- ecology: the interplay of the different parts composing the blage of populations that coexist and interact at a given habitat. ecosystem will ultimately determine its properties. The community and habitat are part of a larger system called It has been recognized that the biofilm (dental plaque) an ecosystem, which can be defined as a functional self- associated with caries and periodontal diseases represents a supporting system that includes the microbial community and sophisticated community that exert functions essential for the its environment. In summary, the following hierarchy becomes biofilm architecture and physiology, with consequent pathoge- apparent: ecosystem, community, population, and the single netic implications. Recent evidence indicates that apical peri- individual (cell). odontitis can also develop as a result of collaborative activities Populations perform functions that contribute to the overall of a biofilm community established in the root canal system. community and maintain the ecologic balance of the ecosys- Community profiling studies revealed that bacterial compo- tem. Each population occupies a functional role (niche) within sition of the endodontic microbiota differs consistently between the community. There are a limited number of niches within individuals suffering from the same disease.32,108,168,229 This the community for which populations must compete. More indicates that apical periodontitis has a heterogeneous etiology, competent populations occupy the niches and displace those where multiple bacterial combinations can play a role in less competent. As discussed later, highly structured and spa- disease causation. Interindividual variability is even more pro- tially organized microbial communities may exhibit properties nounced when different geographic locations are studied.108,222 that are greater than the sum of the component populations. Moreover, community structure differs significantly between In reality, complex microbial communities have been shown to different disease forms (e.g., asymptomatic apical periodontitis be endowed with the ability to confront and withstand the versus acute apical abscess),179,183,229 suggesting existence of a challenges imposed by the environment by creating a mosaic pattern associated with each form. of microenvironments that enable the survival and growth of the community members. Biofilm and Bacterial Interactions Historically, microbiologists dealing with infectious diseases The community-forming ability can be regarded as essential have faced periods of “reductionism” and “holism.”95 Reduc- for microbial survival in virtually all environments. Indeed, the tionism is based on the idea that the whole can be understood majority of microorganisms in nature invariably grow and by examining smaller and smaller pieces of it, that is, all function as members of metabolically integrated communities, complex systems can be completely understood in terms of or biofilms.35,110 Biofilm can be defined as a sessile multicellular their individual components. Through reductionist approaches, microbial community characterized by cells that are firmly individual species are isolated from complex mixed communi- attached to a surface and enmeshed in a self-produced matrix ties and metabolically and genetically studied so as to allow of extracellular polymeric substance (EPS), usually a polysac- understanding of the community by examining every single charide35,47 (Fig. 14-14). The ability to form biofilms has been constituent. However, it has become quite apparent for the regarded as a virulence factor,76 and biofilm infections account microbiota associated with many human infectious diseases for an estimated 65% to 80% of bacterial infections that affect that the whole is very often greater than the simple sum of its humans in the developed world.34 Given its importance in parts. This concept has prompted microbiologists to adopt a varied aspects, there has been a high level of interest in the holistic approach to understand the community behavior asso- study of biofilm properties, not only in medical microbiology ciated with pathogenesis of many infectious diseases known to but also in different sectors of industrial and environmental have a polymicrobial etiology. Holism holds that any compo- microbiology. nent cannot be thoroughly understood except in their relation Bacterial cells in biofilms form microcolonies± ( 15% by to the whole. The holistic theory has been largely employed in volume) that are embedded and nonrandomly distributed in 608 PART II The Advanced Science of Endodontics the EPS matrix (±85% by volume) and separated by water of a broad habitat range for growth, increased metabolic diver- channels.37,47,240,244 Microcolonies are usually shaped as “towers” sity and efficiency, enhanced possibilities for genetic exchanges or “mushrooms.” Dental biofilms can reach up to 300 or more and bacterial intercommunications (quorum-sensing systems), cell layers in thickness.240 Individual microcolonies may consist and protection from external threats (competing microorgan- of a single bacterial species but more frequently are composed isms, host defenses, antimicrobial agents, and environmental of several different species in a mixed community. stress).111 As the biofilm matures on the surface, extracellular polysac- Biofilm organizations can also result in enhanced pathoge- charides are continually synthesized to form an extracellular nicity. To cause disease, bacteria must adhere to host surfaces, matrix that eventually may constitute as much as 85% of the obtain nutrients from the host and multiply, invade tissues, volume of the biofilm.34 Although the matrix is primarily com- overcome or evade the host defenses, and induce tissue damage. posed of polysaccharides, it can also contain proteins and A diverse range of virulence traits are required for these particu- nucleic acids.76 The matrix is not only important physically as lar stages of the disease process, and it is highly probable that part of the scaffold that determines the biofilm structure, but each will require the concerted action of bacteria in a commu- it is also biologically active and can retain nutrients, water, and nity. Similarly, it is possible that certain species can have more essential enzymes within the biofilm.5 The matrix can also than one role in disease, and different species can perform protect the biofilm community from exogenous threats and similar functions. This helps explain why communities with may participate in adherence to the surface. different bacterial composition can be found in different indi- Community members form distinct populations or micro- viduals with similar disease. In multispecies communities, a colonies separated by open water channels that traverse the broad spectrum of relationships may arise between the compo- biofilm matrix and create primitive circulatory systems.36 nent species, ranging from no effect or reduced pathogenicity Fluid in these channels carries substrate, end products of bac- to additive or synergistic pathogenic effects. Endodontic terial metabolism, and signal molecules involved in bacterial abscesses are examples of polymicrobial infections whereby interactions.20 Thus, vital nutrients and communication mol- bacterial species that individually have low virulence and ecules can diffuse, and wastes can be washed out through these are unable to cause disease can do so when in association channels. with others as part of a mixed consortium (pathogenic Microcolonies that form in the biofilm arise from surface synergism).21,39 colonization by planktonic (unattached) bacterial cells. During the early stages of biofilm formation, bacteria bind to many Resistance to Antimicrobial Agents host proteins and coaggregate with other bacteria. These inter- From a clinical standpoint, biofilm increased resistance to anti- actions lead to changes in growth rate, gene expression, and microbial agents is of special concern. Bacteria arranged in protein production. It has been demonstrated by proteomic biofilms are considered more resistant to antibiotics than the techniques or DNA arrays that genes expressed by cells in same cells grown in planktonic state. The antibiotic concentra- biofilms differed by 20% to 70% from those expressed by the tion required to kill bacteria in the biofilm is about 100 to 1000 same cells growing in planktonic culture.15,132,185 Thus, bacteria times higher than that needed to kill the same species in plank- in biofilms adopt a radically different phenotype compared tonic state.109 Several possible mechanisms are involved with with their planktonic counterparts. Within biofilms, some bac- biofilm resistance to antimicrobials. teria also use sophisticated systems of cell-cell communication (quorum sensing) to coordinate gene expression. Phenotypic Biofilm Structure May Restrict Penetration heterogeneity in biofilms is also observed as a result of expo- of Antimicrobial Agents sure of microcolonies to a variety of gradients (e.g., oxygen The agent may adsorb to and even inhibit the bacteria at the tension, pH, osmolarity, type and amounts of nutrients, cell biofilm surface, but cells deeply located in the biofilm may density), which contribute to form diverse microenvironments remain relatively unaffected. The matrix in biofilms can also throughout the biofilm structure. bind and retain neutralizing enzymes at concentrations that could inactivate the antimicrobial agent.243 Biofilm Community Lifestyle Many naturally occurring biofilms have a highly diverse micro- Altered Growth Rate of Biofilm Bacteria biota. These multispecies biofilms are not merely passive bacte- Many antibiotics can freely penetrate the biofilm matrix, but rial assemblages that are stuck to surfaces; they are complex cells are often still protected. The occurrence of starved bacte- biologic systems formed by populations (microcolonies) that ria entering the stationary phase in biofilms seems to be a are not randomly distributed but are spatially and functionally significant factor in the resistance of biofilm populations to organized throughout the community. Indeed, populations antimicrobials. Bacteria grow slowly under conditions of low are strategically positioned for optimal metabolic interaction, availability of nutrients in an established biofilm and as a con- and the resultant architecture favors the ecologic role of sequence are much less susceptible than faster-dividing cells. the community in the ecosystem. The properties displayed Most antibiotics require at least some degree of cellular activity by a multispecies biofilm community are mostly dictated by to be effective. Therefore, bacterial cells in stationary phase the interactions between populations, which create novel phys- might represent a general mechanism of antibiotic resistance iologic functions that cannot be usually observed with indi- in the biofilm.76 vidual components. As a result, biofilm communities have a collective physiology, responding in concert to environmental Presence of “Persister” Bacteria challenges. Increased tolerance of some biofilms to antibiotics may be The biofilm community lifestyle affords a number of largely due to the presence of a subpopulation of specialized advantages to colonizing bacteria, including establishment survivor cells known as persisters.89 It remains unclear whether CHAPTER 14 Microbiology of Endodontic Infections 609 these bacteria actually represent a distinct phenotype or are 7. Extraradicular biofilms were infrequent; they occurred simply the most resistant cells within a population.76 in only 6% of the cases. Except for one case, they were always associated with intraradicular biofilms. All cases APICAL PERIODONTITIS AS showing an extraradicular biofilm exhibited clinical symptoms. Thus, it seems that extraradicular infections A BIOFILM-RELATED DISEASE in the form of biofilms or planktonic bacteria are not a The evidence that apical periodontitis is a disease associated common occurrence, are usually dependent on the intra- with polymicrobial biofilms comes mostly from in situ- mor radicular infection, and are more frequent in symptom- phologic investigations.25,121,125,126,152,153,154,186,223 These studies atic teeth. have observed that bacteria colonizing the root canal system 8. Bacteria were also seen in the lumen of the main canal, of teeth with primary or posttreatment apical periodontitis ramifications, and isthmuses as flocs and planktonic usually formed sessile biofilm communities covering the walls cells, either intermixed with necrotic pulp tissue or of the main canal, apical ramifications, lateral canals, and possibly suspended in a fluid phase. Bacterial flocs are isthmuses. sometimes regarded as “planktonic biofilms” and may Although the concept of apical periodontitis as a biofilm- originate from growth of cell aggregates/coaggregates in related disease has been built upon these observations, the a fluid or they may have detached from biofilms.77 prevalence of biofilms and their association with diverse pre- Some criteria have been proposed to establish a causal link sentations of apical periodontitis were only recently disclosed between biofilms and a given infectious disease77,134,152: by Ricucci and Siqueira.152 These authors evaluated the preva- 1. Infecting bacteria are adhered to or associated with a lence of biofilms in untreated teeth with primary apical peri- surface. odontitis and treated teeth with posttreatment disease and 2. Direct examination of the infected tissue shows bacteria looked for associations between biofilms and clinical/ forming clusters or microcolonies encased in an extracel- histopathologic conditions. Some of the most important find- lular matrix. ings of their study are as follows: 3. The infection is generally confined to a particular site and 1. Intraradicular biofilms were generally observed in the although dissemination may occur, it is a secondary event. apical segment of approximately 80% of the root 4. The infection is difficult or impossible to eradicate with canals of teeth with primary or posttreatment apical antibiotics in spite of the responsible microorganisms being periodontitis. susceptible to killing in the planktonic cell state. 2. Morphology of endodontic biofilms differed consistently 5. Ineffective host clearance is evident, as suggested by the from individual to individual (e.g., thickness, morpho location of bacterial colonies in areas of the host tissue types, bacterial cells/extracellular matrix ratio). associated with host inflammatory cells. Accumulation of 3. Dentinal tubules underneath biofilms were often invaded polymorphonuclear neutrophils and macrophages sur- by bacterial cells from the bottom of the biofilm rounding bacterial aggregates/coaggregates in situ consider- community. ably increases the suspicion of biofilm involvement with 4. Biofilms were also commonly seen covering the walls of disease causation. apical ramifications, lateral canals, and isthmuses. 6. Elimination or significant disruption of the biofilm 5. Bacterial biofilms were more frequent in root canals structure and ecology leads to remission of the disease of teeth with large apical periodontitis lesions. Because process. it takes time for apical periodontitis to develop Based on findings from the Ricucci and Siqueira’s study,152 and become radiographically visible, one can surmise apical periodontitis can be considered to fulfill five of the six that large lesions represent a longstanding pathologic criteria. Bacterial aggregates/coaggregates are observed adhered process caused by an even “older” intraradicular infec- to or at least associated with the dentinal root canal walls tion. In longstanding infectious processes, involved (criterion 1). Bacterial colonies are often seen encased in an bacteria may have had enough time and conditions to amorphous extracellular matrix (criterion 2). Endodontic bio- adapt themselves to the environment and set a mature films are frequently confined to the root canal system, in only and organized biofilm community. The fact that the a few cases extending to the external root surface, but dissemi- apical root canal of teeth with large lesions harbors a nation through the lesion never occurred (criterion 3). In the large number of bacterial cells and species almost great majority of cases, biofilms are directly faced by an accu- always organized in biofilms may help explain the long- mulation of inflammatory cells, especially polymorphonuclear held concept that treatment outcome is influenced by neutrophils (criterion 5). lesion size.33,128 As for criterion 4, it is widely known that intraradicular 6. The prevalences of intraradicular biofilms in teeth asso- endodontic infections cannot be effectively treated by systemic ciated with apical cysts, abscesses, and granulomas were antibiotic therapy, even though most endodontic bacteria in 95%, 83%, and 69.5%, respectively. Biofilms were signifi- the planktonic cell state are susceptible to currently used anti- cantly associated with epithelialized lesions. Because biotics.13,66,90 The lack of efficacy of systemic antibiotics against apical cysts develop as a result of epithelial proliferation intraradicular infections is mainly because the drug does not in some granulomas,102 it may be anticipated that the reach endodontic bacteria that are located in an avascular older the apical periodontitis lesion, the greater the prob- necrotic space. The recognition of biofilms as the main mode ability of it becoming a cyst. Similar to teeth with large of bacterial establishment in the root canal system further lesions, the age of the pathologic process may also help strengthens the explanations for the lack of antibiotic effective- explain the high prevalence of biofilms in association ness against endodontic infections. Finally, there is a clear with cysts. potential for fulfillment of criterion 6, because biofilms are 610 PART II The Advanced Science of Endodontics

frequently observed in canals of treated teeth with posttreat- BOX 14-1 ment apical periodontitis,154 whereas teeth with a successful Advantages and Limitations of Culture Methods outcome show no biofilm infection of the root canal.149 Advantages Limitations METHODS FOR MICROBIAL Broad-range nature, Impossibility of culturing a large number IDENTIFICATION identification of of extant bacterial species unexpected species Not all viable bacteria can be recovered The endodontic microbiota has been traditionally investigated Allow quantification of Once isolated, bacteria require by microbiologic culture methods. Culture is the process of all major viable identification using a number of propagating microorganisms in the laboratory by providing cultivable techniques them with the required nutrients and proper physicochemical microorganisms in Misidentification of strains with conditions, including temperature, moisture, atmosphere, salt 237 samples ambiguous or aberrant phenotypic concentration, and pH. Essentially, culture analyses involve Allow determination behavior the following steps: sample collection and transport, disper- 251 of antimicrobial Low sensitivity sion, dilution, cultivation, isolation, and identification. End- susceptibilities Strict dependence on the mode of sample odontic samples are collected and transported to the laboratory of isolates transport in a viability-preserving, nonsupportive, anaerobic medium. Physiologic studies are Samples require immediate processing They are then dispersed by sonication or vortex mixing, possible Costly, time consuming, and laborious, as diluted, distributed onto various types of agar media, and cul- Pathogenicity studies for cultivation of anaerobes tivated under aerobic or anaerobic conditions. After a suitable are possible Specificity is dependent on experience of period of incubation, individual colonies are subcultivated and Widely available microbiologist identified on the basis of multiple phenotype-based aspects, Extensive expertise and specialized including colony and cellular morphology, gram-staining equipment needed to isolate pattern, oxygen tolerance, comprehensive biochemical charac- anaerobes terization, and metabolic end-product analysis by gas-liquid Takes several days to weeks to identify chromatography. The outer cellular membrane protein profile most anaerobes as examined by gel electrophoresis, fluorescence under ultraviolet light, and susceptibility tests to selected antibiotics can be needed for identification of some species.52 Marketed pack- aged kits that test for preformed enzymes have also been used for rapid identification of several species. grow, identification methods not based on cultivability are Culture analyses of endodontic infections have provided a required. substantial body of information about the etiology of apical In some situations, even the successful cultivation of a given periodontitis, composition of the endodontic microbiota in microorganism does not necessarily mean that this microor- different clinical conditions, effects of treatment procedures ganism can be successfully identified. Culture-dependent iden- in microbial elimination, susceptibilities of endodontic micro- tification is based on phenotypic traits observed in reference organisms to antibiotics, and so on. Advantages and limita- strains, with predictable biochemical and physical properties tions of culture methods are listed in Box 14-1; however, under optimal growth conditions. However, many phenotype- some important limitations of culture methods make a com- related factors can lead to difficulties in identification and even prehensive analysis of the endodontic microbiota difficult to to misidentification.14,18,210,260 As a consequence of all these achieve. factors, phenotype-based identification does not always allow The difficulties in culturing or identifying many microbial an unequivocal identification. species are of special concern. Unfortunately, not all microor- To sidestep the limitations of culturing, tools and proce- ganisms can be cultivated under artificial conditions, and this dures based on molecular biology have become available and is simply because the nutritional and physiologic needs of most have substantially improved the ability to achieve a more real- microorganisms are still unknown. Investigations of many istic description of the microbial world without the need for aquatic and terrestrial environments using culture-independent cultivation (Fig. 14-15). Molecular technology has also been methods have revealed that the cultivable members of these applied to reliably identify cultivated bacteria, including strains systems represent less than 1% of the total extant popula- with ambiguous or aberrant phenotypic behavior, rare isolates, tion.6,276 Furthermore, 50% to 80% of bacterial species compos- poorly described or uncharacterized bacteria, and newly named ing the microbiota associated with diverse human sites, species.19,48,144,211,241,258 including the oral cavity, represent unknown and still unculti- Molecular approaches for microbial identification rely on vated bacteria.1,2,44,49,94,138,146,245 certain genes that contain revealing information about the That a given species has not been cultivated does not microbial identity. Of the several genes that have been chosen imply that this species will remain indefinitely impossible to as targets for bacterial identification, the 16S rRNA gene (or cultivate. Myriad obligate anaerobic bacteria were uncultivable 16S rDNA) has been the most widely used because it is uni- in the early 1900s, but further developments in anaerobic versally distributed among bacteria, is long enough to be culturing techniques have to a large extent helped to solve this highly informative and short enough to be easily sequenced, problem. It must be assumed that no single method or culture possesses conserved and variable regions, and affords reliabil- medium is suitable for isolating the vast diversity of microor- ity for inferring phylogenetic relationships.279 Similarly, the 18S ganisms present in most environments.70 Because we remain rRNA gene of fungi and other eukaryotes has also been used relatively unaware of the requirements for many bacteria to extensively to identify these organisms. CHAPTER 14 Microbiology of Endodontic Infections 611

BOX 14-2 Broad-range analysis Advantages and Limitations of Molecular Sampling Biology Methods

Advantages Limitations Cloning Detect both cultivable and Most assays are qualitative as-yet-uncultivated species or or semiquantitative DGGE strains (exceptions: real-time PCR) Sequencing High specificity and accurate Most assays only detect one identification of strains with species or a few different ambiguous or aberrant species at a time phenotypic behavior (exceptions: broad-range T-RFLP analysis Detect species directly in clinical PCR, checkerboard, samples microarray) High sensitivity Most assays detect only the DNA extraction Rapid; most assays take no more target species and fail to than minutes to a few hours detect unexpected species Pyrosequencing PCR to identify a microbial species (exception: broad-range broad-range primers Do not require carefully PCR) controlled anaerobic Some assays can be laborious conditions during sampling and costly (e.g., broad- Species-specific analysis and transportation range PCR) Can be used during antimicrobial Biases in broad-range PCR Sampling Checkerboard DNA-DNA treatment introduced by Hybridization Anaerobic handling and expertise homogenization procedures, not required preferential DNA Samples can be stored frozen for amplification, and PCR (single, nested, multiplex) later analysis differential DNA extraction Species-specific primers Qualitative data DNA can be transported easily Hybridization assays using between laboratories whole genome probes Detect dead microorganisms detect only cultivable species Sequencing Can be very expensive

Real-time PCR Species-specific primers designed to detect specific target species directly in clinical Quantitative data DNA extraction samples. Many molecular methods for the study of microorganisms Reverse-capture exist; the choice of a particular approach depends on the ques- checkerboard tions being addressed.218 Broad-range PCR followed by cloning DNA-DNA hybridization PCR and sequencing can be used to unravel the breadth of microbial broad-range diversity in a given environment. Bacterial community struc- primers Microarray tures can be analyzed via pyrosequencing technology and by fingerprinting techniques such as denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length FIG. 14-15 Molecular biology methods used (or with potential to be used) polymorphism (T-RFLP). Fluorescence in situ hybridization in the study of endodontic infections. The choice for a particular technique (FISH) can measure the abundance of target species and will depend on the type of analysis to be performed. Some methods can be provide information on their spatial distribution in tissues. used for detection of target microbial species or groups and others for a Among other applications, DNA-DNA hybridization arrays broader analysis of the microbiota. (checkerboard techniques, DNA microarrays), species-specific single PCR, nested PCR, multiplex PCR, and quantitative real- Data from 16S rRNA gene sequences can be used for accu- time PCR can be used to survey large numbers of clinical rate and rapid identification of known and unknown bacterial samples for the presence of target species. Variations in PCR species, using techniques that do not require cultivation. The technology can also be used to type microbial strains. As with 16S RNA gene of virtually all bacterial species in a given envi- any other method, molecular methods have their own advan- ronment, including still uncultivated and uncharacterized bac- tages and limitations (Box 14-2). teria, can be amplified by polymerase chain reaction (PCR) using broad-range (or universal) primers that are complementary to conserved regions of this gene. Sequencing of the vari- The Five Generations of Endodontic able regions flanked by the broad-range primers will provide Microbiology Studies information for accurate bacterial identification. Primers or Microbiologic studies for identification of the species partici- probes that are complementary to variable regions can also be pating in endodontic infections can be chronologically 612 PART II The Advanced Science of Endodontics

TABLE 14-1 Generations of Studies for Microbiologic Identification in Endodontic Infections Study Generation Identification Method Nature Description and Findings First Culture Open ended Revealed many cultivable species in association with apical (broad range) periodontitis Second Molecular methods (e.g., PCR Closed ended Target cultivable bacteria and its derivatives, original (species Confirmed and strengthened data from first generation checkerboard assay) specific) Allowed inclusion of some culture-difficult species in the set of candidate endodontic pathogens Third Molecular methods (e.g., Open ended Allowed a more comprehensive investigation of the bacterial PCR-cloning-sequencing, (broad range) diversity in endodontic infections T-RFLP) Not only cultivable species but also as-yet-uncultivated and uncharacterized bacteria were identified Fourth Molecular methods (e.g., PCR, Closed ended Target cultivable and as-yet-uncultivated bacteria microarrays, reverse-capture (species Large-scale clinical studies to investigate prevalence and checkerboard) specific) association of species/phylotypes with endodontic infections Fifth Molecular methods (e.g., Open ended Permit a deep-coverage and more comprehensive analysis of the pyrosequencing) (broad range) diversity of endodontic infections

divided into five generations on the basis of the different stra- categories according to the time microorganisms entered the tegic approaches used.73 These generations are detailed in root canal system: primary infection, caused by microorganisms Table 14-1. that initially invade and colonize the necrotic pulp tissue (initial or “virgin” infection); secondary infection, caused by microorganisms not present in the primary infection but intro- Impact of Molecular Methods in duced in the root canal at some time after professional inter- Endodontic Microbiology vention (i.e., secondary to intervention); and persistent infection, Culture studies (first generation) identified a set of species caused by microorganisms that were members of a primary or thought to play an important role in the pathogenesis of apical secondary infection and in some way resisted intracanal anti- periodontitis.189,253 Further, not only have findings from culture- microbial procedures and were able to endure periods of nutri- based methods been confirmed, but they have also been signifi- ent deprivation in treated canals. Extraradicular infection in cantly supplemented with those from culture-independent turn is characterized by microbial invasion of the inflamed molecular biology techniques, which constitute the other four periradicular tissues and is a sequel to the intraradicular infec- generations of endodontic microbiology studies.216 Molecular tion. Extraradicular infections can be dependent on or inde- methods have confirmed and strengthened the association of pendent of the intraradicular infection. many cultivable bacterial species with apical periodontitis and have also revealed new suspected endodontic pathogens.212 The DIVERSITY OF THE list of candidate pathogens has expanded to include culture- difficult species or as-yet-uncultivated bacteria that had never ENDODONTIC MICROBIOTA been found in endodontic infections by culturing approaches. Microbiota is a collective term for microorganisms and should The results from molecular studies impact remarkably on the replace terms such as flora and microflora, which perpetuate an knowledge of bacterial diversity in endodontic infections. More outdated classification of microorganisms as plants.43 Diversity than 400 different bacterial species have already been detected refers to the number of different species present (richness) and in different types of endodontic infections.216 Of these, about their relative abundance (evenness) in a given ecosystem.83 45% were exclusively reported by molecular biology studies, The oral cavity harbors one of the highest accumulations of compared with 32% detected by culture studies alone.216 microorganisms in the body. Even though viruses, archaea, Twenty-three percent of the total bacterial species richness has fungi, and protozoa can be found as constituents of the oral been detected by application of both culture and molecular microbiota, bacteria are by far the most dominant inhabitants studies (Fig. 14-16). As a consequence, it becomes quite evident of the oral cavity. There are an estimated 10 billion bacterial that the endodontic microbiota has been refined and redefined cells in the oral cavity,116 and culture-independent studies by molecular methods.212 (microscopy and molecular biology technologies) have shown that over 50% to 60% of the oral microbiota still remains to be cultivated and fully characterized.2,44,239 A high diversity of TYPES OF ENDODONTIC INFECTIONS bacterial species has been revealed in the oral cavity by cultur- Endodontic infections can be classified according to the ana- ing approaches,122 but application of molecular biology tomic location as intraradicular or extraradicular infection. methods to the analysis of the bacterial diversity has revealed Intraradicular infection is caused by microorganisms colonizing a still broader and more diverse spectrum of extant oral the root canal system and can be subdivided into three bacteria.139 More than 1000 bacterial species/phylotypes have CHAPTER 14 Microbiology of Endodontic Infections 613

OVERALL

Actinobacteria Bacteroidetes Firmicutes Fusobacteria Proteobacteria Spirochaetes Synergistetes TM7 SR1

Taxa detected in molecular studies Taxa detected in both molecular and culture studies Taxa detected in culture studies

FIG. 14-16 Percentage distribution of bacterial species/phylotypes found in endodontic infections according to the detection method. Data refer to the percentage of species or phylotypes overall or in each of the nine phyla that have endodontic representatives.

FIG. 14-17 Bacterial phyla with their respective endodontic representatives. Right: Example species or phylotypes for each phylum. been found in the human oral cavity,44 but advanced DNA archaea are types of microorganisms that have been only occa- pyrosequencing studies have indicated that this number may sionally found in endodontic infections. still have been largely underestimated.4,72,88 Modern anaerobic Endodontic infections develop in a previously sterile place culture and sophisticated molecular biology techniques have that does not contain a normal microbiota. Any species found demonstrated that, at a broader taxonomic level, endodontic has the potential to be an endodontic pathogen or at least play bacteria fall into nine phyla—namely, Firmicutes, Bacteroide- a role in the ecology of the endodontic microbial community. tes, Spirochaetes, Fusobacteria, Actinobacteria, Proteobacteria, Culture and molecular studies reveal only prevalence of Synergistetes, TM7, and SR1123,148,162,178-180,213,216 (Fig. 14-17). species; consequently, only association can be inferred. Causa- However, data from studies using pyrosequencing technology tion is usually surmised on the basis of both frequency of reveal that several other phyla may have been overlooked by detection and potential pathogenicity (in animal models or previous identification techniques.82,100,183,198,202 Fungi and association with other human diseases), and several species 614 PART II The Advanced Science of Endodontics

Olsenella, Actinomyces, Peptostreptococcus, and Eubacte- rium).11,60,61,75,90,123,148,162,164,178,179,212,227,230,231,252,250,270 Bacterial prevalence in primary infections may vary from study to study as a function of several factors, such as sensitivity and specificity of the detection and identification methods, sampling technique, geographic location, and accuracy or divergence in clinical diagnosis and disease classification. Even so, the species most frequently detected are expected to be the same in most well- conducted studies. Figs. 14-19 to 14-21 display the most frequently detected species associated with asymptomatic apical periodontitis, symptomatic apical periodontitis, and acute apical abscesses, as revealed by studies from the authors’ group using a highly sensitive molecular biology technique. About 40% to 66% of the endodontic microbiota in primary infections is composed of species still uncultivated.123,148,179 As for their abundance in these infections, as-yet-uncultivated phylotypes corresponded to about 40% of the clones sequenced.179 Molecular studies investigating the breadth of

FIG. 14-18 Apical periodontitis due to primary intraradicular infection. Pulp bacterial diversity in infected root canals have disclosed is necrotic and lesion size is usually directly proportional to complexity of the the occurrence of uncultivated phylotypes belonging to microbiota involved. several genera, including Dialister, Prevotella, Solobacterium, Olsenella, Fusobacterium, Treponema, Eubacterium, Megasphaera, Veillonella, and Selenomonas, as well as phylotypes related to the family Lachnospiraceae or the TM7 and Synergistetes have emerged as candidate endodontic pathogens. The follow- phyla.123,148,160,161,172,178,179,182,213,221 Some uncultivated phylotypes ing sections discuss specific aspects of each type of endodontic can even be among the most prevalent bacteria in primary infection. intraradicular infections, and others may be associated with pain.179 Bacteroidetes clone X083 is one of the most prevalent PRIMARY INTRARADICULAR INFECTION phylotypes found in endodontic infections.162-164,219 Detection of as-yet-uncultivated phylotypes in samples from endodontic Microbial Composition and Diversity infections suggests that they can be previously unrecognized Primary intraradicular infection is infection of the necrotic bacteria that play a role in the pathogenesis of different forms pulp tissue (Fig. 14-18). It occurs in untreated teeth and is the of apical periodontitis. The fact that they have not yet been cause of primary apical periodontitis. Participating microor- cultivated and phenotypically characterized does not mean that ganisms may be involved in earlier stages of pulp invasion they are not important. (usually via caries) that culminated in inflammation and further necrosis, or they can be latecomers that took advantage Symptomatic Infections of the environmental conditions in the root canal after pulp Symptomatic apical periodontitis and acute apical abscesses necrosis. are typical examples of endodontic infections causing severe Primary infections are characterized by a mixed (multispe- symptoms. In these cases, the infection is located in the canal, cies) community conspicuously dominated by anaerobic bac- but it has also reached the periradicular tissues and, in teria. The number of bacterial cells may vary from 103 to 108 abscessed cases, can spread to other anatomic spaces. Acute per root canal.17,180,231,251,180,226,250,269 Molecular studies have dis- apical abscesses are caused by bacteria that egress from the closed a mean of 10 to 20 species/phylotypes per infected infected root canal and invade the periradicular tissues to canal.123,148,162,212,229 Canals of teeth with associated sinus tracts establish an extraradicular infection and evoke purulent may exhibit a number of species close to the top of this range.162 inflammation. Clinically, the disease leads to pain or swelling The size of apical periodontitis lesion has been shown to be and has the potential to diffuse to sinuses and other fascial proportional to the number of bacterial species and cells in spaces of the head and neck to form a cellulitis or other com- the root canal.162,227,250 A molecular study162 demonstrated that plications (Fig. 14-22). The microbiota involved in endodontic the number of taxa per canal was clearly in direct proportion abscesses is mixed and dominated by anaerobic bacte- to the lesion size: small lesions (< 5 mm) harbored about 12 ria41,90,96,155,179,217,229 (see Fig. 14-19). Direct comparisons using taxa, lesions from 5 to less than 10 mm harbored 16 taxa, and molecular technology reveal an average of 12 to 18 taxa per lesions over 10 mm harbored about 20 species. Some canals abscess case, compared with 7 to 12 taxa present in root canals associated with large lesions may harbor even more than 40 of teeth with asymptomatic lesions.179,229 Uncultivated phylo- taxa.162 Therefore, the larger the lesion, the higher the bacterial types constitute approximately 40% of the taxa found in diversity and density in the canal. abscesses and collectively represent more than 30% of the 16S The most prevalent named bacterial species detected in rRNA gene sequences retrieved in clone libraries.179 primary infections, including abscessed cases, belong to diverse Whereas microbial causation of apical periodontitis is genera of gram-negative bacteria (i.e., Fusobacterium, Dialister, well established, there is no strong evidence disclosing Porphyromonas, Prevotella, Tannerella, Treponema, Pyramidobacter, specific involvement of a single species with any particular sign Campylobacter, and Veillonella) and gram-positive (Parvimonas, or symptom of apical periodontitis. Some gram-negative Filifactor, Pseudoramibacter, Streptococcus, Propionibacterium, anaerobic bacteria have been suggested to be involved with CHAPTER 14 Microbiology of Endodontic Infections 615

FIG. 14-19 Prevalence of bacteria detected in primary infections of teeth with asymptomatic (chronic) apical periodontitis. Data from the authors’ studies using a taxon-specific nested-polymerase chain reaction protocol. (From references 159, 168, 211, and 210.) symptomatic lesions,67,71,169,179,250,266,281 but the same species affect virulence of the whole bacterial community. Indeed, it may also be present in somewhat similar frequencies in asymp- has been shown that different species combinations may result tomatic cases,11,61,75,85,230,231 so factors other than the mere pres- in different outcomes because of the network of interactions.170 ence of a given putative pathogenic species may play a role in The possibility exists that bacterial interactions result in com- the etiology of symptomatic endodontic infections.193,199 These munities that are more or less aggressive and consequently can factors include differences in virulence ability among strains cause host responses of corresponding intensity. This is con- of the same species, bacterial interactions resulting in additive sistent with the concept that the entire bacterial community is or synergistic effects among species in mixed infections, the unit of pathogenicity. number of bacterial cells (infectious load), environmental cues regulating expression of virulence factors, host resistance; and Geographic Influence concomitant herpesvirus infection. Association of some or all Findings from laboratories in different countries are often quite of these factors (instead of an isolated event) is likely to deter- different regarding the prevalence of the species involved in mine the occurrence and intensity of symptoms.193,199 endodontic infections. Although these differences may be Molecular studies using DGGE, T-RFLP, or pyrosequencing attributed to variations in identification methodologies, a geo- analysis have revealed that the structure of the endodontic graphic influence in the composition of the root canal micro- bacterial communities in symptomatic teeth, including abscess biota has been suspected. Studies using molecular biology cases, is significantly different from that of asymptomatic techniques directly compared the endodontic microbiota of teeth.179,183,229 Differences are represented by different domi- patients residing in distinct geographic locations and suggested nant species in the communities and larger numbers of species that significant differences in the prevalence of some important in symptomatic cases. Differences in the type and load of species can actually exist.10,157,204 In a more holistic approach, dominant species and the resulting bacterial interactions may analysis of the bacterial community profiles of the microbiota 616 PART II The Advanced Science of Endodontics

FIG. 14-20 Prevalence of bacteria detected in primary infections of teeth with symptomatic (acute) apical periodontitis. Data from the authors’ studies using a taxon-specific nested-polymerase chain reaction protocol. (From references 159, 168, 211, and 210.)

associated with endodontic infections from different countries with colonization not being a difficult task for virtually all oral also revealed a geography-related pattern, with several species bacterial species. Although a large number of bacterial species being exclusive for each location and others shared by the (about 100 to 200) can be found in the oral cavity of a particu- locations showing great differences in prevalence.108,222 The lar individual,139 only a limited assortment of these species factors that can lead to differences in the composition of (about 10 to 20) is consistently selected out for growth and the endodontic microbiota and the impact of these differences survival within a root canal containing necrotic pulp tissue on therapy, particularly in abscessed cases requiring systemic from the same individual. This indicates that ecologic deter- antibiotic therapy, remain elusive. minants operate in the necrotic canal and dictate which species will succeed in colonizing this previously sterile environment. The major ecologic factors that determine the composition of Microbial Ecology and the Root the root canal microbiota include oxygen tension, type and Canal Ecosystem amount of available nutrients, and bacterial interactions. Other A root canal with necrotic pulp provides a space for bacterial factors such as temperature, pH, and receptors for adhesins colonization and affords bacteria a moist, warm, nutritious, may also be involved. and anaerobic environment, which is by and large protected The root canal infection is a dynamic process, and different from the host defenses because of lack of active blood circula- bacterial species apparently dominate at different stages.259 tion in the necrotic pulp tissue. Also, the root canal walls are Shifts in the composition of the microbiota are largely due to nonshedding surfaces conducive to persistent colonization and changes in environmental conditions, particularly in regard formation of complex communities. The necrotic root canal to oxygen tension and nutrient availability. In the initial might be considered a fertile environment for bacterial growth, phases of the pulpal infectious process, facultative bacteria CHAPTER 14 Microbiology of Endodontic Infections 617

FIG. 14-21 Prevalence of bacteria detected in primary infections of teeth with acute apical abscesses. Data from the authors’ studies using a taxon-specific nested-polymerase chain reaction protocol. (From references 159, 168, 211, and 210.)

predominate.54 After a few days or weeks, oxygen is depleted within the root canal as a result of pulp necrosis and consumption by facultative bacteria. Further oxygen supply is inter- rupted with loss of blood circulation in the necrotic pulp. An anaerobic milieu develops and is highly conducive to the survival and growth of obligate anaerobic bacteria. With the passage of time, anaerobic conditions become even more pro- nounced, particularly in the apical third of the root canal; as a consequence, anaerobes will dominate the microbiota, out- numbering facultative bacteria (Fig. 14-23). The main sources of nutrients for bacteria colonizing the root canal system include (1) the necrotic pulp tissue, (2) proteins and glycoproteins from tissue fluids and exudate that seep into the root canal system via apical and lateral foramina, (3) components of saliva that may coronally penetrate into the root canal, and (4) products of the metabolism of other bacteria. Because the largest amount of nutrients is available in the main canal, the most voluminous part of the root canal system, most of the infecting microbiota (particu- FIG. 14-22 Acute apical abscess. The infection has spread to other ana- larly fastidious anaerobic species) is expected to be located tomic spaces to form cellulitis. (Courtesy Dr. Henrique Martins.) in this region. Bacterial species that can best utilize and 618 PART II The Advanced Science of Endodontics compete for nutrients in the root canal system will succeed in stage of the infectious process, bacteria that have a proteolytic colonization. capacity or establish a cooperative interaction with those that In addition to being influenced by variations in oxygen can utilize this substrate in the metabolism, start to dominate. levels, shifts in the composition of the microbiota colonizing Therefore, as the infectious process reaches the stage of the root canal system can also be dependent upon the dynam- induction of periradicular inflammation, proteins become the ics of nutrient utilization. Saccharolytic species dominate the principal nutrient source, particularly in the apical part of very early stages of the infectious process but are soon out- the canal, favoring the establishment of anaerobic species numbered by asaccharolytic species, which will dominate later that utilize peptides or amino acids in their metabolism (see stages.254 Even though the necrotic pulp tissue can be regarded Fig. 14-23). as a finite source of nutrients to bacteria (given the small Because primary endodontic infections are usually charac- volume of tissue that is progressively degraded), induction of terized by mixed communities, different bacterial species are periradicular inflammation guarantees a sustainable source in close proximity with one another, and interactions become of nutrients, particularly in the form of proteins and glyco inevitable. Thus, establishment of certain species in the root proteins present in the exudate that seep into the canal. At this canal is also influenced by interactions with other species. In this regard, early colonizers play an important role in dictating which species will live along with them in the community. Bacterial interactions can be positive or negative. Positive Apical region interactions enhance the survival capacity of the interacting Lower oxygen tension bacteria and enable different species to coexist in habitats G Nutrients from the periradicular tissues: proteins and glycoproteins where neither could exist alone. For instance, interbacterial R Lower bacterial counts nutritional interactions are important ecologic determinants Bacteria less accessible to that result in higher metabolic efficiency of the whole com- A treatment measures munity. Nutritional interactions are mainly represented by D food webs, including utilization of metabolic end products from one species by another and bacterial cooperation for the I Coronal region breakdown of complex host-derived substrates. Fig. 14-24 dis- E Higher oxygen tension plays a complex array of interbacterial nutritional interactions Nutrients from the oral cavity: that can take place in infected root canals, where the growth N carbohydrates Higher bacterial counts of some species can be dependent on products of the metabo- T Microorganisms more accessible lism of other species. Moreover, one species can provide to treatment growth conditions favorable to another—for example, by reducing oxygen tension in the environment and favoring the establishment of anaerobes, or by releasing some proteinases FIG. 14-23 Ecologic conditions in different areas of the root canal. A gradi- that can provide protection from host defenses. Negative inter- ent of oxygen tension and nutrients (type and availability) is formed. Conse- actions in turn act as feedback mechanisms that limit popula- quently, the microbiota residing in different parts can also differ in diversity, tion densities. Examples include competition (for nutrients density, and accessibility to treatment procedures. and space) and amensalism (when one species produces a

Eikenella corrodens

CO2

Fusobacterium NH4 Streptococcus Eubacterium Actinomyces Peptostreptococcus Vitamin K Veillonella Propionibacterium Formate, H Succinate 2 Formate

Porphyromonas Hemin Campylobacter Prevotella Tannerella forsythia

Isobutyrate Acetate

Succinate Treponema Pseudoramibacter alactolyticus

FIG. 14-24 Interbacterial nutritional interactions that can take place in infected root canals where growth of some species can be dependent on products of metabolism of other species. CHAPTER 14 Microbiology of Endodontic Infections 619 substance that inhibits another species, including antibiotics may cause an influx of virus-infected cells into the periradicu- such as penicillin). lar tissues. Reactivation of HCMV or EBV by tissue injury Many species adhere directly to host surfaces, whereas other induced by bacteria may evoke impairment of host immune species adhere to bacteria already attached to the surface. The response in the periradicular microenvironment, changing latter is called coaggregation and is a highly specific phenom- the potential of local defense cells to mount an adequate enon with regard to the partners involved.93 A given pair of response against infectious agents. In addition, herpesvirus species can attach to each other by means of specific receptor- may directly stimulate inflammatory cells to release proinflam- adhesin interactions, which are usually lectin-like interactions matory cytokines.117,275 Evidence of herpesvirus infection has (attachment of a specific protein on the surface of one species been observed in symptomatic apical periodontitis lesions,174,176 to a specific carbohydrate on the surface of the other). Coag- abscesses,29,57 large lesions,175-176 and lesions from HIV-positive gregation can favor colonization of host surfaces and also facili- patients.177 However, the role of herpesviruses in the pathogen- tate metabolic interactions between the partners. Coaggregation esis of apical periodontitis, if any, has still to be elucidated. has been demonstrated for several pairs of bacterial taxa found in endodontic infections.91 PERSISTENT/SECONDARY ENDODONTIC INFECTIONS Other Microorganisms As defined earlier in this chapter, persistent intraradicular in Endodontic Infections infections are caused by microorganisms that resisted intraca- Fungi nal antimicrobial procedures and survived in the treated canal. Fungi are eukaryotic microorganisms that may colonize the Involved microorganisms are remnants of a primary or second- oral cavity, especially Candida species, but they have been ary infection. The latter, in turn, is caused by microorganisms only occasionally detected in primary intraradicular infec- that at some time entered the root canal system secondary to tions.51,97,119,225 One molecular study12 has, however, reported clinical intervention. The moment can be during treatment, the occurrence of C. albicans in 21% of samples from primarily between appointments, or even after root canal filling. In any infected canals. Fungi are more frequently detected in canals circumstance, if penetrating microorganisms manage to adapt of teeth with posttreatment disease (discussed later). themselves to the new environment, surviving and flourishing, a secondary infection is established. Species involved can be Archaea oral microorganisms or not, depending on the source of sec- Archaea consist of a highly diverse group of prokaryotes dis- ondary infection. tinct from bacteria. Members of this domain have been tradi- Persistent and secondary infections are for the most part tionally recognized as extremophiles, but some of these clinically indistinguishable. Exceptions include infectious microorganisms have also been found to flourish in nonex- complications (such as an apical abscess) arising after the treat- treme environments, including the human body.50 To date, no ment of noninfected vital pulps or cases in which apical peri- member of the Archaea domain has been described as a human odontitis was absent at the time of treatment but present on pathogen. Although studies have failed to detect archaea in the follow-up radiograph. Both situations are typical examples samples from primary endodontic infections,165,220 others of secondary infections. Both persistent and secondary infec- detected a Methanobrevibacter oralis–like phylotype in some tions can be responsible for several clinical problems, includ- primarily infected canals.268,270 Because these microorganisms ing persistent exudation, persistent symptoms, interappointment have not been consistently detected in infected root canals, flare-ups, and failure of the endodontic treatment, character- their role, if any, in the pathogenesis of apical periodontitis is ized by a posttreatment apical periodontitis lesion. questionable.

Viruses Persistent/Secondary Infections Viruses are not cells but inanimate particles composed of a and Treatment Failure nucleic acid molecule (DNA or RNA) and a protein coat. On Although there is some suggestion in the literature that their own, they have no metabolism. In order to replicate the extraradicular infection or nonmicrobial factors may be viral genome, they need to infect living cells and use the cell’s involved,127,262 intraradicular infections, either persistent or machinery. Because viruses require viable host cells to infect secondary, can be regarded as the major causes of endodontic and replicate themselves, they cannot thrive in the root canal treatment failure (Fig. 14-25). This statement is supported with necrotic pulp. Viruses have been reported to occur in root by two strong evidence-based arguments. First, it has been canals only in teeth with vital pulps. For instance, the human demonstrated that there is an increased risk of adverse treat- immunodeficiency virus (HIV) has been detected in vital pulps ment outcome when bacteria are present in the canal at the of HIV-seropositive patients,65 and some herpesviruses have time of obturation.55,234,271 Second, most (if not all) root canal– been identified in both noninflamed and inflamed vital pulps.99 treated teeth evincing persistent apical periodontitis lesions Human cytomegalovirus (HCMV) and Epstein-Barr virus have been demonstrated to harbor an intraradicular infec- (EBV) have been detected in apical periodontitis lesions.175 It tion.101,103,145,154,159,181,210,255 Based on these arguments, studies has been hypothesized that HCMV and EBV may be implicated investigating bacteria remaining in the root canals at the obtu- in the pathogenesis of apical periodontitis as a direct result of ration stage disclose species that have the potential to influence virus infection and replication or as a result of virally induced the treatment outcome (outcome into perspective). On the other impairment of local host defenses, which might give rise to hand, studies dealing with the microbiota of root canal–treated overgrowth of pathogenic bacteria in the very apical part of the teeth with apical periodontitis show the association of species root canal.238 Bacterial challenge emanating from the canals with treatment failure, because the microorganisms detected 620 PART II The Advanced Science of Endodontics

Bacteria persisting in the root canal after chemomechanical procedures or intracanal medication will not always maintain an infectious process. This statement is supported by evidence that some apical periodontitis lesions healed even after bacteria were isolated from the canal at the obturation stage.55,234 There are some possible explanations215: ♦ Residual bacteria may die after obturation because of toxic effects of the filling material or sealer, access denied to nutrients, or disruption of bacterial ecology. ♦ Residual bacteria may be present in quantities and virulence subcritical to sustaining periradicular inflammation. ♦ Residual bacteria remain in locations where access to periradicular tissues is denied. Bacteria that resist intracanal procedures and are present in the canal at the time of obturation can influence the outcome of the endodontic treatment provided they do the following215: ♦ Have the ability to withstand periods of nutrient scarcity, scavenging for low traces of nutrients or assuming a dormant state or a state of low metabolic activity, to prosper again when the nutrient source is reestablished. FIG. 14-25 Posttreatment apical periodontitis lesions in root canal–treated ♦ Resist treatment-induced disturbances in the ecology of the teeth. In poorly treated canals, the microbiota is similar to primary infections. bacterial community, including disruption of quorum- In cases apparently well treated, fewer species are found. Regardless of sensing systems, food webs, genetic exchanges, and disor- treatment quality, persistent or secondary intraradicular infections are the ganization of protective biofilm structures. main causative agents of endodontic treatment failure. ♦ Reach a critical population density (load) necessary to inflict damage to the host. ♦ Have unrestrained access to the periradicular tissues through apical/lateral foramina or iatrogenic root are likely to be participating in the etiology of posttreatment perforations. disease (outcome already established). ♦ Possess virulence attributes that are expressed in the modified environment and reach concentrations adequate to Bacteria at the Root Obturation Stage directly or indirectly induce damage to the periradicular Diligent antimicrobial treatment may still fail to completely tissues. eliminate bacteria from the infected root canal system. This is In this context, it should not be forgotten that host resistance because persisting bacteria are either inaccessible or even resis- to infection is also an important and probably decisive coun- tant to treatment procedures. Whatever the cause of persis- teracting factor. tence, bacterial diversity and density in infected canals are substantially reduced after treatment. Root canal samples positive for bacterial growth after chemomechanical procedures, Microbiota in Endodontically followed or not by intracanal medication, have been shown to Treated Teeth harbor one to five bacterial species per case, and the number The microbiota in root canal–treated teeth with apical peri- of persistent bacterial cells usually varies from 102 to 105 per odontitis also exhibits decreased diversity in comparison to sample.24,180,207,208,234,269 primary infections. Canals apparently well treated harbor one No single species has been significantly found to persist to five species, but the number of species in canals with inad- after treatment procedures. Gram-negative bacteria, which are equate treatment can reach up to 10 to 20 species, which is common members of primary infections, are usually elimi- similar to untreated canals (see Fig. 14-25).145,168,181,210,255 The nated. Exceptions include some anaerobic rods, such as number of bacterial cells in treated teeth with posttreatment F. nucleatum, Prevotella species, and Campylobacter rectus, disease varies from 103 to 107 per canal.17,140,167,187 which are among the species found in postinstrumentation or Several culture and molecular biology studies have revealed postmedication samples.24,68,142,180,226,234 Most studies on this that E. faecalis is the most frequent species in root canal– subject have clearly revealed that when bacteria resist treat- treated teeth, with prevalence values reaching up to 90% of ment procedures, gram-positive bacteria are more frequently cases118,145,159,171,167,187,210,255,283 (Fig. 14-26). Root canal–treated present. Gram-positive facultatives or anaerobes often detected teeth are about nine times more likely to harbor E. faecalis than in these samples include streptococci, P. micra, Propionibacte- cases of primary infections.171 This suggests that other members rium species, P. alactolyticus, Actinomyces species, lactobacilli, of a mixed bacterial consortium commonly present in primary E. faecalis, and Olsenella uli.24,26-28,31,68,140,142,164-166,180,203,207,208,234,257 infections can inhibit this species, and that the bleak environ- This supports the notion that gram-positive bacteria can be mental conditions within obturated root canals do not prevent more resistant to antimicrobial treatment measures and have its survival. The fact that E. faecalis has been commonly recov- the ability to adapt to the harsh environmental conditions in ered from teeth treated at multiple visits or left open for drain- instrumented and medicated root canals. As-yet-uncultivated age233 suggests that this species is a secondary invader capable bacteria have also been detected in posttreatment samples,133,180 of colonizing the canal and resisting treatment. In other words, indicating that they may also resist antimicrobial treatment. E. faecalis may cause secondary infections that later become FIG. 14-26 Prevalence of Enterococcus faecalis in samples from root canal–treated teeth with apical periodontitis. Data from culture (yellow bars) and molecular (blue bars) studies follow. Molecular Studies 1. Foschi F, Cavrini F, Montebugnoli L, et al: Detection of bacteria in endodontic samples by polymerase chain reaction assays and association with defined clinical signs in Italian patients, Oral Microbiol Immunol 20:289, 2005. 2. Gomes BP, Pinheiro ET, Jacinto RC, et al: Microbial analysis of canals of root-filled teeth with periapical lesions using polymerase chain reaction, J Endod 34:537, 2008. 3. Gomes BP, Pinheiro ET, Sousa EL, et al: Enterococcus faecalis in dental root canals detected by culture and by polymerase chain reaction analysis, Oral Surg Oral Med Oral Pathol Oral Radiol Endod 102:247, 2006. 4. Kaufman B, Spangberg L, Barry J, Fouad AF: Enterococcus spp. in endodontically treated teeth with and without periradicular lesions, J Endod 31:851, 2005. 5. Rôças IN, Hulsmann M, Siqueira JF Jr: Microorganisms in root canal-treated teeth from a German population, J Endod 34:926, 2008. 6. Rôças IN, Siqueira JF Jr: Characterization of microbiota of root canal-treated teeth with posttreatment disease, J Clin Microbiol 50:1721, 2012. 7. Rôças IN, Siqueira JF Jr, Santos KR: Association of Enterococcus faecalis with different forms of periradicular diseases, J Endod 30:315, 2004. 8. Sedgley C, Nagel A, Dahlen G, et al: Real-time quantitative polymerase chain reaction and culture analyses of Enterococcus faecalis in root canals, J Endod 32:173, 2006. 9. Siqueira JF Jr, Rôças IN: Polymerase chain reaction-based analysis of microorganisms associated with failed endodontic treatment, Oral Surg Oral Med Oral Pathol Oral Radiol Endod 97:85, 2004. 10. Williams JM, Trope M, Caplan DJ, Shugars DC: Detection and quantitation of Enterococcus faecalis by real-time PCR (qPCR), reverse transcription-PCR (RT-PCR), and cultivation during endodontic treatment, J Endod 32:715, 2006. 11. Zoletti GO, Siqueira JF Jr, Santos KR: Identification of Enterococcus faecalis in root-filled teeth with or without periradicular lesions by culture-dependent and -independent approaches, J Endod 32:722, 2006. Culture Studies 1. Engström B: The significance of enterococci in root canal treatment, Odontol Rev 15:87, 1964. 2. Gomes BP, Pinheiro ET, Sousa EL, et al: Enterococcus faecalis in dental root canals detected by culture and by polymerase chain reaction analysis, Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontology 102:247, 2006. 3. Hancock HH 3rd, Sigurdsson A, Trope M, Moiseiwitsch J: Bacteria isolated after unsuccessful endodontic treatment in a North American population, Oral Surg Oral Med Oral Pathol Oral Radiol Endod 91:579, 2001. 4. Molander A, Reit C, Dahlen G, Kvist T: Microbiological status of root-filled teeth with apical periodontitis, Int Endod J 31:1, 1998. 5. Möller AJR: Microbial examination of root canals and periapical tissues of human teeth, Odontol Tidskr 74(suppl):1, 1966. 6. Peciuliene V, Balciuniene I, Eriksen HM, Haapasalo M: Isolation of Enterococcus faecalis in previously root-filled canals in a Lithuanian population, J Endod 26:593, 2000. 7. Pinheiro ET, Gomes BP, Ferraz CC, et al: Microorganisms from canals of root-filled teeth with periapical lesions, Int Endod J 36:1, 2003. 8. Sedgley C, Nagel A, Dahlen G, et al: Real-time quantitative polymerase chain reaction and culture analyses of Enterococcus faecalis in root canals, J Endod 32:173, 2006. 9. Sundqvist G, Figdor D, Persson S, Sjogren U: Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment, Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85:86, 1998. 10. Williams JM, Trope M, Caplan DJ, Shugars DC: Detection and quantitation of Enterococcus faecalis by real-time PCR (qPCR), reverse transcription-PCR (RT-PCR), and cultivation during endodontic treatment, J Endod 32:715, 2006. 11. Zoletti GO, Siqueira JF Jr, Santos KR: Identification of Enterococcus faecalis in root-filled teeth with or without periradicular lesions by culture-dependent and -independent approaches, J Endod 32:722, 2006. 622 PART II The Advanced Science of Endodontics

following findings from studies carried out in independent laboratories have questioned the status of E. faecalis as the main causative agent of endodontic failures: ♦ Despite being easily cultivated, E. faecalis is not detected in all studies evaluating the microbiota of root canal–treated teeth with posttreatment disease.30,172 ♦ Even when present, E. faecalis is rarely one of the most dominant species in retreatment cases.158,167,168,181 ♦ E. faecalis has been found not to be more prevalent in root canal–treated teeth with lesions when compared with treated teeth with no lesions.87,283 Other bacteria found in endodontically treated teeth with apical periodontitis include streptococci and some fastidious anaerobic species, such as P. alactolyticus, Propionibacterium species, Filifactor alocis, Dialister pneumosintes, Dialister invisus,

FIG. 14-27 Dentinal tubule infection by Enterococcus faecalis in dog’s Tannerella forsythia, P. micra, Prevotella intermedia, and Trepo- teeth after experimental infection. Notice that cells invaded the entire extent nema denticola7,69,118,145,158,167,168,181,210,213,255 (Fig. 14-28). Uncul- of some tubules up to the cementum (Brown and Brenn staining, ×1000). tivated phylotypes correspond to 55% of the taxa detected in treated canals, and collectively they can also be in high proportions, corresponding to about half of the 16S rRNA gene persistent. E. faecalis is considered a transient species in the sequences retrieved in clone libraries.181 Detection of unculti- oral cavity; its source may be food.282 vated phylotypes helps explain why culture studies fail to For a given microorganism to survive in endodontically detect bacteria in some treated root canals. treated teeth, it has to resist intracanal disinfection procedures The bacterial community profiles in treated cases vary from and adapt to the harsh environmental conditions caused by individual to individual, indicating that distinct bacterial com- treatment. The ability of E. faecalis to penetrate dentinal binations can play a role in treatment failure.168,181 All these tubules, sometimes to a deep extent74,201 (Fig. 14-27), can findings strongly suggest that the microbiota of root canal– enable it to escape the action of endodontic instruments and treated teeth with apical periodontitis is more complex than irrigants used during chemomechanical preparation.74,200 previously anticipated by culture studies. Moreover, its ability to form biofilms in root canals can be Fungi are only occasionally found in primary infections, but important for its resistance to and persistence after intracanal Candida species have been detected in root canal–treated teeth antimicrobial procedures.45 Enterococcus faecalis is also resis- in up to 18% of the cases.30,51,118,119,140,145,210,255 Fungi gain access tant to calcium hydroxide22; such an ability to resist high pH to root canals via contamination during endodontic therapy values seems to be related to a functioning proton pump that (secondary infection) or they overgrow after inefficient intra- drives protons into the cell to acidify the cytoplasm.53 Unlike canal antimicrobial procedures that cause an imbalance in the most putative endodontic pathogens frequently found in primary endodontic microbiota.232 Candida albicans is by far primary infections, E. faecalis may colonize root canals in the most commonly detected fungal species in root canal– single infections,255 and such relative independence of living treated teeth. This species has several properties that can be without deriving nutrients from other bacteria can be extremely involved in persistence following treatment, including its important for its establishment in treated root canals. Finally, ability to colonize and invade dentin191,192,224 and resistance to environmental cues can regulate gene expression in E. faecalis, calcium hydroxide.272,274 affording this bacterium the ability to adapt to varying (and adverse) conditions.84 Indeed, E. faecalis can enter a so-called viable but noncultivable (VBNC) state,104 which is a survival EXTRARADICULAR INFECTIONS mechanism adopted by several bacteria when exposed to Apical periodontitis lesions are formed in response to intrara- unfavorable environmental conditions.105 In the VBNC state, dicular infection and by and large constitute an effective barrier bacteria lose the ability to grow in culture media but maintain against spread of the infection to the alveolar bone and other viability and pathogenicity and sometimes can resume division body sites. In most situations, apical periodontitis inflamma- when optimal environmental conditions are restored. Studies tory lesions succeed in preventing microorganisms from invad- have demonstrated that E. faecalis has the ability to survive in ing the periradicular tissues. Nevertheless, in some specific environments with scarcity of nutrients and then flourish when circumstances, microorganisms can overcome this defense the nutrient source is reestablished.59 It also has the capacity barrier and establish an extraradicular infection. The most to recover from a prolonged starvation state in obturated common form of extraradicular infection is the acute apical canals,188 suggesting that viable cells of this species, entombed abscess, characterized by purulent inflammation in the perira- at the time of canal obturation, may provide a long-term nidus dicular tissues in response to a massive egress of virulent for subsequent infection. bacteria from the root canal. There are, however, other forms Taken together, all these properties help explain the signifi- of extraradicular infection, which have been discussed as one cantly high prevalence of E. faecalis in root canal–treated teeth. of the possible etiologies of persistence of apical periodontitis Although association of this species with posttreatment disease lesions in spite of diligent root canal treatment.194,263 These is suggested by epidemiologic studies and supported by the conditions entail the establishment of microorganisms in the species attributes that allow it to survive under unfavorable periradicular tissues either by adherence to the apical environmental conditions, causation is unproved. In fact, the external root surface in the form of extraradicular biofilm CHAPTER 14 Microbiology of Endodontic Infections 623

FIG. 14-28 Prevalence of microorganisms detected in root canal–treated teeth with posttreatment disease. Data from the authors’ studies using a taxon-specific polymerase chain reaction assay. (From references 218 and 210.) structures129,261 or by formation of cohesive actinomycotic col- dependent on the intraradicular infection; once the intrara- onies within the body of the inflammatory lesion.79 dicular infection is properly controlled by root canal treat- Therefore, extraradicular infection can develop as follows195: ment or tooth extraction and drainage of pus is achieved, ♦ It can be a result of direct advance of some bacterial species the extraradicular infection is handled by the host defenses that overcome host defenses concentrated near or beyond and usually subsides. Nonetheless, it should be appreciated the apical foramen, an extension of the intraradicular infec- that in some rare cases, bacteria that have participated in tious process, or bacterial penetration into the lumen of acute apical abscesses may persist in the periradicular pocket (bay) cysts, which are in direct communication tissues following resolution of the acute response and estab- with the apical foramen. The borderline between the infect- lish a persistent extraradicular infection associated with ing endodontic microbiota and the host defenses is often chronic periradicular inflammation, sometimes resulting in located intraradicularly, short of or at the apical foramen. an actively draining sinus tract. In some cases, however, microorganisms may reach the ♦ It can be a sequel to apical extrusion of debris during root periradicular tissues, and the borderline is then situated canal instrumentation (particularly after overinstrumenta- extraradicularly, beyond the boundaries of the apical tion). Bacteria embedded in dentinal chips can be physically foramen (Fig. 14-29). In the latter situation, the whole protected from the host defense cells and therefore can infectious process would be a continuum composed of an persist in the periradicular tissues and sustain periradicular intraradicular and an extraradicular segment, in which the inflammation. The virulence and quantity of the involved former fosters the latter (see Fig. 14-29). It has been sug- bacteria, as well as the host’s ability to deal with infection, gested that in some cases, the extraradicular segment may will be decisive factors dictating whether an extraradicular become independent of the intraradicular component of infection will develop or not. the infectious process, but this has not been confirmed by Conceivably, the extraradicular infection can be dependent on scientific findings. or independent of the intraradicular infection.194 Independent ♦ It can result from bacterial persistence in the apical extraradicular infections would be those that are no longer periodontitis lesion after remission of an acute apical fostered by the intraradicular infection and can persist even abscess. The acute apical abscess is for the most part clearly after successful eradication of the latter. So far, it has been 624 PART II The Advanced Science of Endodontics

A B

C D

FIG. 14-29 Scanning electron micrograph showing extensive bacterial colonization in the very apical part of the canal, near and at the apical foramen (A-B, magnification ×550 and magnification ×850, respectively). C, Higher magnification of left inset in B showing a bacterial biofilm adhered to the very apical canal walls (magnification ×3700). D, Higher magnification of right inset in B showing a fully developed “corncob” in tissue meshwork adjacent to the apical foramen (magnification ×4000). (Modified from Siqueira JF Jr, Lopes HP: Bacteria on the apical root surfaces of untreated teeth with periradicular lesions: a scanning electron microscopy study, Int Endod J 34:216, 2001.)

suggested that the main bacterial species implicated in inde- evaluate the bacteriologic conditions of the apical part of the pendent extraradicular infections are Actinomyces species and root canal. This makes it difficult to ascertain whether those Propionibacterium propionicum, in a pathologic entity named extraradicular infections were dependent on or independent of apical (or periapical, periradicular) actinomycosis23,79,236,256 (Fig. an intraradicular infection. Actually, findings from a study 14-30). These bacteria form cohesive colonies that may be evaluating both the resected root ends and the apical periodon- collectively resistant to phagocytosis.58 However, existence of titis lesions from treated teeth suggested that the large majority apical actinomycosis as a self-sustained pathologic entity, no of cases of extraradicular infections are maintained by a con- longer nurtured by the intraradicular infection, and its involve- comitant apical root canal infection.246 ment as an exclusive cause of treatment failure is speculative The incidence of extraradicular infections in untreated and remains to be proved.151,209 teeth is conceivably low.126,206 Extraradicular biofilms are It is still controversial whether chronic apical periodontitis infrequent, and when present they are virtually always associ- lesions can harbor bacteria for very long beyond initial tissue ated with intraradicular biofilms.152 This is in consonance invasion.9 Studies using culture-dependent247,262,277 or culture- with the high success rate of nonsurgical root canal treat- independent molecular biology methods, such as checker- ment.40,150,235 Even in root canal–treated teeth with recalci- board hybridization,63,249 fluorescence in situ hybridization trant lesions in which a higher incidence of extraradicular (FISH),248 clone library analysis,78 and pyrosequencing,173 have bacteria has been reported, a high rate of healing following reported the extraradicular occurrence of a complex microbi- retreatment56,235 indicates that the major cause of posttreat- ota associated with apical periodontitis lesions that did not ment disease is located within the root canal system, charac- respond favorably to the root canal treatment. Anaerobic bac- terizing a persistent or secondary intraradicular infection. teria have been reported as the dominant microorganisms in This has been confirmed by culture and by molecular and several of those lesions.247,249 Apart from a discussion about histobacteriologic studies investigating the microbiologic whether contamination can be effectively prevented during conditions of root canals associated with posttreatment apical surgical sampling of periradicular lesions, these studies did not periodontitis.103,118,145,154,168,210,255 CHAPTER 14 Microbiology of Endodontic Infections 625

A B C

FIG. 14-30 Bacterial aggregate in an epithelialized apical periodontitis lesion, suggestive of actinomycosis. This case presented a severe flare-up in the past, but the tooth was asymptomatic at the time of extraction. A, Section not passing through the canal; note cyst cavity and bacterial aggregation on the left (magnification ×25). B, Detail of area with colony (magnification ×100). C, Higher magnification of colony, which is surrounded by neutrophils (magnification ×400). Sections stained with a Taylor-modified Brown and Brenn technique. (Courtesy Dr. Domenico Ricucci.)

REFERENCES 1. Aas JA, Griffen AL, Dardis SR, et al: Bacteria of dental 14. Beighton D, Hardie JM, Whiley RA: A scheme for the apical periodontitis: an examination of endodontic biofilms caries in primary and permanent teeth in children and identification of viridans streptococci, J Med Microbiol in an endodontic retreatment failure, J Endod 35:1303, young adults, J Clin Microbiol 46:1407, 2008. 35:367, 1991. 2009. 2. Aas JA, Paster BJ, Stokes LN, et al: Defining the normal 15. Beloin C, Valle J, Latour-Lambert P, et al: Global impact 26. Chavez de Paz L, Svensater G, Dahlen G, Bergenholtz G: bacterial flora of the oral cavity, J Clin Microbiol 43:5721, of mature biofilm lifestyle on Escherichia coli K-12 gene Streptococci from root canals in teeth with apical 2005. expression, Mol Microbiol 51:659, 2004. periodontitis receiving endodontic treatment, Oral Surg 3. Ackermans F, Klein JP, Frank RM: Ultrastructural 16. Bergenholtz G: Micro-organisms from necrotic pulp of Oral Med Oral Pathol Oral Radiol Endod 100:232, 2005. localization of immunoglobulins in carious human dentine, traumatized teeth, Odontol Revy 25:347, 1974. 27. Chavez de Paz LE, Dahlen G, Molander A, et al: Bacteria Arch Oral Biol 26:879, 1981. 17. Blome B, Braun A, Sobarzo V, Jepsen S: Molecular recovered from teeth with apical periodontitis after 4. Ahn J, Yang L, Paster BJ, et al: Oral microbiome profiles: identification and quantification of bacteria from antimicrobial endodontic treatment, Int Endod J 36:500, 16S rRNA pyrosequencing and microarray assay endodontic infections using real-time polymerase chain 2003. comparison, PLoS ONE 6:e22788, 2011. reaction, Oral Microbiol Immunol 23:384, 2008. 28. Chavez de Paz LE, Molander A, Dahlen G: Gram-positive 5. Allison DG: The biofilm matrix, Biofouling 19:139, 2003. 18. Bosshard PP, Abels S, Altwegg M, et al: Comparison rods prevailing in teeth with apical periodontitis 6. Amann RI, Ludwig W, Schleifer KH: Phylogenetic of conventional and molecular methods for identification undergoing root canal treatment, Int Endod J 37:579, identification and in situ detection of individual microbial of aerobic catalase-negative gram-positive cocci in 2004. cells without cultivation, Microbiol Rev 59:143, 1995. the clinical laboratory, J Clin Microbiol 42:2065, 29. Chen V, Chen Y, Li H, et al: Herpesviruses in abscesses 7. Anderson AC, Hellwig E, Vespermann R, et al: 2004. and cellulitis of endodontic origin, J Endod 35:182, Comprehensive analysis of secondary dental root canal 19. Bosshard PP, Abels S, Zbinden R, et al: Ribosomal DNA 2009. infections: a combination of culture and culture- sequencing for identification of aerobic gram-positive rods 30. Cheung GS, Ho MW: Microbial flora of root canal-treated independent approaches reveals new insights, PLoS One in the clinical laboratory (an 18-month evaluation), J Clin teeth associated with asymptomatic periapical radiolucent 7:e49576, 2012. Microbiol 41:4134, 2003. lesions, Oral Microbiol Immunol 16:332, 2001. 8. Armada-Dias L, Breda J, Provenzano JC, et al: 20. Bowden GH: The microbial ecology of dental caries, 31. Chu FC, Leung WK, Tsang PC, et al: Identification of Development of periradicular lesions in normal and Microb Ecol Health Dis 12:138, 2000. cultivable microorganisms from root canals with apical diabetic rats, J Appl Oral Sci 14:371, 2006. 21. Brook I: Encapsulated anaerobic bacteria in synergistic periodontitis following two-visit endodontic treatment 9. Baumgartner JC: Microbiologic aspects of endodontic infections, Microbiol Rev Rev 50:452, 1986. with antibiotics/steroid or calcium hydroxide dressings, infections, J Calif Dent Assoc 32:459, 2004. 22. Byström A, Claesson R, Sundqvist G: The antibacterial J Endod 32:17, 2006. 10. Baumgartner JC, Siqueira JF Jr, Xia T, Rôças IN: effect of camphorated paramonochlorophenol, 32. Chugal N, Wang JK, Wang R, et al: Molecular Geographical differences in bacteria detected in camphorated phenol and calcium hydroxide in the characterization of the microbial flora residing at the endodontic infections using polymerase chain reaction, treatment of infected root canals, Endod Dent Traumatol apical portion of infected root canals of human teeth, J Endod 30:141, 2004. 1:170, 1985. J Endod 37:1359, 2011. 11. Baumgartner JC, Watkins BJ, Bae KS, Xia T: Association 23. Byström A, Happonen RP, Sjogren U, Sundqvist G: 33. Chugal NM, Clive JM, Spangberg LS: A prognostic model of black-pigmented bacteria with endodontic infections, Healing of periapical lesions of pulpless teeth after for assessment of the outcome of endodontic treatment: J Endod 25:413, 1999. endodontic treatment with controlled asepsis, Endod Dent effect of biologic and diagnostic variables, Oral Surg Oral 12. Baumgartner JC, Watts CM, Xia T: Occurrence of Candida Traumatol 3:58, 1987. Med Oral Pathol Oral Radiol Endod 91:342, 2001. albicans in infections of endodontic origin, J Endod 24. Byström A, Sundqvist G: The antibacterial action of 34. Costerton B: Microbial ecology comes of age and joins 26:695, 2000. sodium hypochlorite and EDTA in 60 cases of endodontic the general ecology community, Proc Natl Acad Sci U S A 13. Baumgartner JC, Xia T: Antibiotic susceptibility of bacteria therapy, Int Endod J 18:35, 1985. 101:16983, 2004. associated with endodontic abscesses, J Endod 29:44, 25. Carr GB, Schwartz RS, Schaudinn C, et al: Ultrastructural 35. Costerton JW: The biofilm primer, Berlin, Heidelberg, 2003. examination of failed molar retreatment with secondary 2007, Springer-Verlag. 626 PART II The Advanced Science of Endodontics

36. Costerton JW, Lewandowski Z, Caldwell DE, et al: 60. Foschi F, Cavrini F, Montebugnoli L, et al: Detection of 84. Jett BD, Huycke MM, Gilmore MS: Virulence of Microbial biofilms, Annu Rev Microbiol 49:711, 1995. bacteria in endodontic samples by polymerase chain enterococci, Clin Microbiol Rev 7:462, 1994. 37. Costerton JW, Stewart PS, Greenberg EP: Bacterial reaction assays and association with defined clinical 85. Jung IY, Choi BK, Kum KY, et al: Molecular epidemiology biofilms: a common cause of persistent infections, signs in Italian patients, Oral Microbiol Immunol 20:289, and association of putative pathogens in root canal Science 284:1318, 1999. 2005. infection, J Endod 26:599, 2000. 38. Cvek M, Cleaton-Jones PE, Austin JC, Andreasen JO: 61. Fouad AF, Barry J, Caimano M, et al: PCR-based 86. Kakehashi S, Stanley HR, Fitzgerald RJ: The effects of Pulp reactions to exposure after experimental crown identification of bacteria associated with endodontic surgical exposures of dental pulps in germ-free and fractures or grinding in adult monkeys, J Endod 8:391, infections, J Clin Microbiol 40:3223, 2002. conventional laboratory rats, Oral Surg Oral Med Oral 1982. 62. Garberoglio R, Brännström M: Scanning electron Pathol 20:340, 1965. 39. Dahlen G: Microbiology and treatment of dental abscesses microscopic investigation of human dentinal tubules, 87. Kaufman B, Spangberg L, Barry J, Fouad AF: and periodontal-endodontic lesions, Periodontol 2000 Arch Oral Biol 21:355, 1976. Enterococcus spp. in endodontically treated teeth with 28:206, 2002. 63. Gatti JJ, Dobeck JM, Smith C, et al: Bacteria of and without periradicular lesions, J Endod 31:851, 40. de Chevigny C, Dao TT, Basrani BR, Marquis V, et al: asymptomatic periradicular endodontic lesions identified 2005. Treatment outcome in endodontics: the Toronto by DNA-DNA hybridization, Endod Dent Traumatol 88. Keijser BJ, Zaura E, Huse SM, et al: Pyrosequencing study–phase 4: initial treatment, J Endod 34:258, 16:197, 2000. analysis of the oral microflora of healthy adults, J Dent 2008. 64. Gier RE, Mitchell DF: Anachoretic effect of pulpitis, J Dent Res 87:1016, 2008. 41. de Sousa EL, Ferraz CC, Gomes BP, et al: Bacteriological Res 47:564, 1968. 89. Keren I, Kaldalu N, Spoering A, et al: Persister cells and study of root canals associated with periapical abscesses, 65. Glick M, Trope M, Bagasra O, Pliskin ME: Human tolerance to antimicrobials, FEMS Microbiol Lett 230:13, Oral Surg Oral Med Oral Pathol Oral Radiol Endod immunodeficiency virus infection of fibroblasts of dental 2004. 96:332, 2003. pulp in seropositive patients, Oral Surg Oral Med Oral 90. Khemaleelakul S, Baumgartner JC, Pruksakorn S: 42. Delivanis PD, Fan VS: The localization of blood-borne Pathol 71:733, 1991. Identification of bacteria in acute endodontic infections bacteria in instrumented unfilled and overinstrumented 66. Gomes BP, Jacinto RC, Montagner F, et al: Analysis of and their antimicrobial susceptibility, Oral Surg Oral Med canals, J Endod 10:521, 1984. the antimicrobial susceptibility of anaerobic bacteria Oral Pathol Oral Radiol Endod 94:746, 2002. 43. Dethlefsen L, Eckburg PB, Bik EM, Relman DA: Assembly isolated from endodontic infections in Brazil during a 91. Khemaleelakul S, Baumgartner JC, Pruksakom S: of the human intestinal microbiota, Trends Ecol Evol period of nine years, J Endod 37:1058, 2011. Autoaggregation and coaggregation of bacteria associated 21:517, 2006. 67. Gomes BP, Lilley JD, Drucker DB: Clinical significance of with acute endodontic infections, J Endod 32:312, 2006. 44. Dewhirst FE, Chen T, Izard J, et al: The human oral dental root canal microflora, J Dent 24:47, 1996. 92. Kishen A, Haapasalo M: Biofilm models and methods of microbiome, J Bacteriol 192:5002, 2010. 68. Gomes BP, Lilley JD, Drucker DB: Variations in the biofilm assessment, Endod Topics 22:58, 2010. 45. Distel JW, Hatton JF, Gillespie MJ: Biofilm formation in susceptibilities of components of the endodontic 93. Kolenbrander PE, Andersen RN, Blehert DS, et al: medicated root canals, J Endod 28:689, 2002. microflora to biomechanical procedures, Int Endod J Communication among oral bacteria, Microbiol Rev Mol 46. Dobell C: Antony van Leeuwenhoek and his “little 29:235, 1996. Biol Rev 66:486, 2002. animals,” London, 1932, Staples Press. 69. Gomes BP, Pinheiro ET, Jacinto RC, et al: Microbial 94. Kumar PS, Griffen AL, Moeschberger ML, Leys EJ: 47. Donlan RM, Costerton JW: Biofilms: survival mechanisms analysis of canals of root-filled teeth with periapical Identification of candidate periodontal pathogens and of clinically relevant microorganisms, Clin Microbiol Rev lesions using polymerase chain reaction, J Endod 34:537, beneficial species by quantitative 16S clonal analysis, 15:167, 2002. 2008. J Clin Microbiol 43:3944, 2005. 48. Drancourt M, Bollet C, Carlioz A, et al: 16S ribosomal 70. Green BD, Keller M: Capturing the uncultivated majority, 95. Kuramitsu HK, He X, Lux R, et al: Interspecies interactions DNA sequence analysis of a large collection of Curr Opin Biotechnol 17:236, 2006. within oral microbial communities, Microbiol Rev Mol Biol environmental and clinical unidentifiable bacterial isolates, 71. Griffee MB, Patterson SS, Miller CH, et al: The Rev 71:653, 2007. J Clin Microbiol 38:3623, 2000. relationship of Bacteroides melaninogenicus to symptoms 96. Kuriyama T, Karasawa T, Nakagawa K, et al: Bacteriologic 49. Eckburg PB, Bik EM, Bernstein CN, et al: Diversity of the associated with pulpal necrosis, Oral Surg Oral Med Oral features and antimicrobial susceptibility in isolates from human intestinal microbial flora, Science 308:1635, Pathol 50:457, 1980. orofacial odontogenic infections, Oral Surg Oral Med Oral 2005. 72. Griffen AL, Beall CJ, Campbell JH, et al: Distinct and Pathol Oral Radiol Endod 90:600, 2000. 50. Eckburg PB, Lepp PW, Relman DA: Archaea and their complex bacterial profiles in human periodontitis and 97. Lana MA, Ribeiro-Sobrinho AP, Stehling R, et al: potential role in human disease, Infect Immun 71:591, health revealed by 16S pyrosequencing, ISME J 6:1176, Microorganisms isolated from root canals presenting 2003. 2012. necrotic pulp and their drug susceptibility in vitro, 51. Egan MW, Spratt DA, Ng YL, et al: Prevalence of yeasts 73. Grossman LI: Origin of microorganisms in traumatized, Oral Microbiol Immunol 16:100, 2001. in saliva and root canals of teeth associated with apical pulpless, sound teeth, J Dent Res 46:551, 1967. 98. Langeland K, Rodrigues H, Dowden W: Periodontal periodontitis, Int Endod J 35:321, 2002. 74. Haapasalo M, Ørstavik D: In vitro infection and disease, bacteria, and pulpal histopathology, Oral Surg 52. Engelkirk PG, Duben-Engelkirk J, Dowell VR Jr: Principles disinfection of dentinal tubules, J Dent Res 66:1375, Oral Med Oral Pathol 37:257, 1974. and practice of clinical anaerobic bacteriology, Belmont, 1987. 99. Li H, Chen V, Chen Y, et al: Herpesviruses in endodontic CA, 1992, Star Publishing. 75. Haapasalo M, Ranta H, Ranta K, Shah H: Black- pathoses: association of Epstein-Barr virus with 53. Evans M, Davies JK, Sundqvist G, Figdor D: Mechanisms pigmented Bacteroides spp. in human apical periodontitis, irreversible pulpitis and apical periodontitis, J Endod involved in the resistance of Enterococcus faecalis to Infect Immun 53:149, 1986. 35:23, 2009. calcium hydroxide, Int Endod J 35:221, 2002. 76. Hall-Stoodley L, Costerton JW, Stoodley P: Bacterial 100. Li L, Hsiao WW, Nandakumar R, et al: Analyzing 54. Fabricius L, Dahlén G, Ohman AE, Möller AJR: biofilms: from the natural environment to infectious endodontic infections by deep coverage pyrosequencing, Predominant indigenous oral bacteria isolated from diseases, Nat Rev Microbiol 2:95, 2004. J Dent Res 89:980, 2010. infected root canals after varied times of closure, Scand J 77. Hall-Stoodley L, Stoodley P: Evolving concepts in biofilm 101. Lin LM, Pascon EA, Skribner J, et al: Clinical, Dent Res 90:134, 1982. infections, Cell Microbiol 11:1034, 2009. radiographic, and histologic study of endodontic treatment 55. Fabricius L, Dahlén G, Sundqvist G, et al: Influence of 78. Handal T, Caugant DA, Olsen I, Sunde PT: Bacterial failures, Oral Surg Oral Med Oral Pathol 71:603, 1991. residual bacteria on periapical tissue healing after diversity in persistent periapical lesions on root-filled 102. Lin LM, Ricucci D, Lin J, Rosenberg PA: Nonsurgical root chemomechanical treatment and root filling of teeth, J Oral Microbiol 1:DOI:10.3402/jom.v1i0.1946, canal therapy of large cyst-like inflammatory periapical experimentally infected monkey teeth, Eur J Oral Sci 2009. lesions and inflammatory apical cysts, J Endod 35:607, 114:278, 2006. 79. Happonen RP: Periapical actinomycosis: a follow-up study 2009. 56. Farzaneh M, Abitbol S, Friedman S: Treatment outcome in of 16 surgically treated cases, Endod Dent Traumatol 103. Lin LM, Skribner JE, Gaengler P: Factors associated endodontics: the Toronto study. Phases I and II: 2:205, 1986. with endodontic treatment failures, J Endod 18:625, orthograde retreatment, J Endod 30:627, 2004. 80. Henderson B, Poole S, Wilson M: Bacterial modulins: a 1992. 57. Ferreira DC, Paiva SS, Carmo FL, et al: Identification of novel class of virulence factors which cause host tissue 104. Lleo MM, Bonato B, Tafi MC, et al: Resuscitation rate in herpesviruses types 1 to 8 and human papillomavirus in pathology by inducing cytokine synthesis, Microbiol Rev different enterococcal species in the viable but acute apical abscesses, J Endod 37:10, 2011. Rev 60:316, 1996. nonculturable state, J Appl Microbiol 91:1095, 2001. 58. Figdor D: Microbial aetiology of endodontic treatment 81. Hoshino E, Ando N, Sato M, Kota K: Bacterial invasion of 105. Lleo MM, Bonato B, Tafi MC, et al: Molecular vs culture failure and pathogenic properties of selected species non-exposed dental pulp, Int Endod J 25:2, 1992. methods for the detection of bacterial faecal indicators in [odontological dissertation no.79], Umea, Sweden, 2002, 82. Hsiao WW, Li KL, Liu Z, et al: Microbial transformation groundwater for human use, Lett Appl Microbiol 40:289, University of Umea. from normal oral microbiota to acute endodontic 2005. 59. Figdor D, Davies JK, Sundqvist G: Starvation survival, infections, BMC Genomics 13:345, 2012. 106. Love RM: Bacterial penetration of the root canal of intact growth and recovery of Enterococcus faecalis in human 83. Huston MA: Biological diversity, Cambridge, UK, 1994, incisor teeth after a simulated traumatic injury, Endod serum, Oral Microbiol Immunol 18:234, 2003. Cambridge University Press. Dent Traumatol 12:289, 1996. CHAPTER 14 Microbiology of Endodontic Infections 627

107. Love RM, Jenkinson HF: Invasion of dentinal tubules 131. Okamura K, Tsubakimoto K, Uobe K, et al: Serum proteins 154. Ricucci D, Siqueira JF Jr, Bate AL, Pitt Ford TR: Histologic by oral bacteria, Crit Rev Oral Biol Med 13:171, and secretory component in human carious dentin, J Dent investigation of root canal-treated teeth with apical 2002. Res 58:1127, 1979. periodontitis: a retrospective study from twenty-four 108. Machado de Oliveira JC, Siqueira JF Jr, Rôças IN, et al: 132. Oosthuizen MC, Steyn B, Theron J, et al: Proteomic patients, J Endod 35:493, 2009. Bacterial community profiles of endodontic abscesses analysis reveals differential protein expression by Bacillus 155. Robertson D, Smith AJ: The microbiology of the acute from Brazilian and USA subjects as compared by cereus during biofilm formation, Appl Environ Microbiol dental abscess, J Med Microbiol 58:155, 2009. denaturing gradient gel electrophoresis analysis, Oral 68:2770, 2002. 156. Robinson HBG, Boling LR: The anachoretic effect in Microbiol Immunol 22:14, 2007. 133. Paiva SS, Siqueira JF Jr, Rôças IN, et al: Molecular pulpitis: bacteriologic studies, J Am Dent Assoc 28:268, 109. Mah TF, O’Toole GA: Mechanisms of biofilm resistance to microbiological evaluation of passive ultrasonic activation 1941. antimicrobial agents, Trends Microbiol 9:34, 2001. as a supplementary disinfecting step: a clinical study, 157. Rôças IN, Baumgartner JC, Xia T, Siqueira JF Jr: 110. Marsh PD: Dental plaque as a microbial biofilm, Caries J Endod 39:190, 2013. Prevalence of selected bacterial named species and Res 38:204, 2004. 134. Parsek MR, Singh PK: Bacterial biofilms: an emerging link uncultivated phylotypes in endodontic abscesses from two 111. Marsh PD: Dental plaque: biological significance of a to disease pathogenesis, Annu Rev Microbiol 57:677, geographic locations, J Endod 32:1135, 2006. biofilm and community life-style, J Clin Periodontol 2003. 158. Rôças IN, Hulsmann M, Siqueira JF Jr: Microorganisms 32(suppl 6):7, 2005. 135. Pashley DH: Dentin-predentin complex and its in root canal-treated teeth from a German population, 112. Matsuo T, Shirakami T, Ozaki K, et al: An permeability: physiologic overview, J Dent Res 64 Spec J Endod 34:926, 2008. immunohistological study of the localization of bacteria No:613, 1985. 159. Rôças IN, Jung IY, Lee CY, Siqueira JF Jr: Polymerase invading root pulpal walls of teeth with periapical lesions, 136. Pashley DH: Clinical considerations of microleakage, chain reaction identification of microorganisms in J Endod 29:194, 2003. J Endod 16:70, 1990. previously root-filled teeth in a South Korean population, 113. Michelich V, Pashley DH, Whitford GM: Dentin 137. Pashley DH: Dynamics of the pulpo-dentin complex, J Endod 30:504, 2004. permeability: a comparison of functional versus Crit Rev Oral Biol Med 7:104, 1996. 160. Rôças IN, Siqueira JF Jr: Detection of novel oral species anatomical tubular radii, J Dent Res 57:1019, 1978. 138. Paster BJ, Boches SK, Galvin JL, et al: Bacterial diversity and phylotypes in symptomatic endodontic infections 114. Michelich VJ, Schuster GS, Pashley DH: Bacterial in human subgingival plaque, J Bacteriol 183:3770, including abscesses, FEMS Microbiol Lett 250:279, penetration of human dentin in vitro, J Dent Res 59:1398, 2001. 2005. 1980. 139. Paster BJ, Olsen I, Aas JA, Dewhirst FE: The breadth of 161. Rôças IN, Siqueira JF Jr: Characterization of Dialister 115. Miller WD: An introduction to the study of the bacterial diversity in the human periodontal pocket and species in infected root canals, J Endod 32:1057, 2006. bacterio-pathology of the dental pulp, Dent Cosmos other oral sites, Periodontol 2000 42:80, 2006. 162. Rôças IN, Siqueira JF Jr: Root canal microbiota of teeth 36:505, 1894. 140. Peciuliene V, Reynaud AH, Balciuniene I, Haapasalo M: with chronic apical periodontitis, J Clin Microbiol 46:3599, 116. Mims C, Nash A, Stephen J: Mims’ pathogenesis of Isolation of yeasts and enteric bacteria in root-filled teeth 2008. infectious diseases, ed 5, San Diego, 2001, Academic with chronic apical periodontitis, Int Endod J 34:429, 163. Rôças IN, Siqueira JF Jr: Prevalence of new Press. 2001. candidate pathogen: Prevotella baroniae, Prevotella 117. Mogensen TH, Paludan SR: Molecular pathways in 141. Perez F, Calas P, de Falguerolles A, Maurette A: Migration multisaccharivorax and as-yet-uncultivated Bacteroidetes virus-induced cytokine production, Microbiol Rev Mol Biol of a Streptococcus sanguis strain through the root clone X083 in primary endodontic infections, J Endod Rev 65:131, 2001. dentinal tubules, J Endod 19:297, 1993. 35:1359, 2009. 118. Molander A, Reit C, Dahlen G, Kvist T: Microbiological 142. Peters LB, van Winkelhoff AJ, Buijs JF, Wesselink PR: 164. Rôças IN, Siqueira JF Jr: Identification of bacteria status of root-filled teeth with apical periodontitis, Effects of instrumentation, irrigation and dressing with enduring endodontic treatment procedures by a combined Int Endod J 31:1, 1998. calcium hydroxide on infection in pulpless teeth with reverse transcriptase-polymerase chain reaction and 119. Möller AJR: Microbial examination of root canals and periapical bone lesions, Int Endod J 35:13, 2002. reverse-capture checkerboard approach, J Endod 36:45, periapical tissues of human teeth. Odontologisk Tidskrift 143. Peters LB, Wesselink PR, Buijs JF, van Winkelhoff AJ: 2010. 74(suppl):1, 1966. Viable bacteria in root dentinal tubules of teeth with apical 165. Rôças IN, Siqueira JF Jr: Comparison of the in vivo 120. Möller AJR, Fabricius L, Dahlén G, et al: Influence on periodontitis, J Endod 27:76, 2001. antimicrobial effectiveness of sodium hypochlorite and periapical tissues of indigenous oral bacteria and necrotic 144. Petti CA, Polage CR, Schreckenberger P: The role of 16S chlorhexidine used as root canal irrigants: a molecular pulp tissue in monkeys, Scand J Dent Res 89:475, rRNA gene sequencing in identification of microorganisms microbiology study, J Endod 37:143, 2011. 1981. misidentified by conventional methods, J Clin Microbiol 166. Rôças IN, Siqueira JF Jr: In vivo antimicrobial effects of 121. Molven O, Olsen I, Kerekes K: Scanning electron 43:6123, 2005. endodontic treatment procedures as assessed by microscopy of bacteria in the apical part of root canals in 145. Pinheiro ET, Gomes BP, Ferraz CC, et al: Microorganisms molecular microbiologic techniques, J Endod 37:304, permanent teeth with periapical lesions, Endod Dent from canals of root-filled teeth with periapical lesions, Int 2011. Traumatol 7:226, 1991. Endod J 36:1, 2003. 167. Rôças IN, Siqueira JF Jr: Characterization of microbiota 122. Moore WEC, Moore LVH: The bacteria of periodontal 146. Preza D, Olsen I, Aas JA, et al: Bacterial profiles of root of root canal-treated teeth with posttreatment disease, diseases, Periodontol 2000 5:66, 1994. caries in elderly patients, J Clin Microbiol 46:2015, J Clin Microbiol 50:1721, 2012. 123. Munson MA, Pitt-Ford T, Chong B, et al: Molecular and 2008. 168. Rôças IN, Siqueira JF Jr, Aboim MC, Rosado AS: cultural analysis of the microflora associated with 147. Rappe MS, Giovannoni SJ: The uncultured microbial Denaturing gradient gel electrophoresis analysis of endodontic infections, J Dent Res 81:761, 2002. majority, Annu Rev Microbiol 57:369, 2003. bacterial communities associated with failed endodontic 124. Nagaoka S, Miyazaki Y, Liu HJ, et al: Bacterial invasion 148. Ribeiro AC, Matarazzo F, Faveri M, et al: Exploring treatment, Oral Surg Oral Med Oral Pathol Oral Radiol into dentinal tubules of human vital and nonvital teeth, bacterial diversity of endodontic microbiota by cloning and Endod 98:741, 2004. J Endod 21:70, 1995. sequencing 16S rRNA, J Endod 37:922, 2011. 169. Rôças IN, Siqueira JF Jr, Andrade AFB, Uzeda M: 125. Nair PN, Henry S, Cano V, Vera J: Microbial status of 149. Ricucci D, Lin LM, Spangberg LS: Wound healing of Identification of selected putative oral pathogens in apical root canal system of human mandibular first molars apical tissues after root canal therapy: a long-term primary root canal infections associated with symptoms, with primary apical periodontitis after “one-visit” clinical, radiographic, and histopathologic observation Anaerobe 8:200, 2002. endodontic treatment, Oral Surg Oral Med Oral Pathol study, Oral Surg Oral Med Oral Pathol Oral Radiol Endod 170. Rôças IN, Siqueira JF Jr, Debelian GJ: Analysis of Oral Radiol Endod 99:231, 2005. 108:609, 2009. symptomatic and asymptomatic primary root canal 126. Nair PNR: Light and electron microscopic studies of root 150. Ricucci D, Russo J, Rutberg M, et al: A prospective cohort infections in adult Norwegian patients, J Endod 37:1206, canal flora and periapical lesions, J Endod 13:29, 1987. study of endodontic treatments of 1,369 root canals: 2011. 127. Nair PNR: On the causes of persistent apical periodontitis: results after 5 years, Oral Surg Oral Med Oral Pathol Oral 171. Rôças IN, Siqueira JF Jr, Santos KR: Association of a review, Int Endod J 39:249, 2006. Radiol Endod 112:825, 2011. Enterococcus faecalis with different forms of periradicular 128. Ng YL, Mann V, Gulabivala K: A prospective study of the 151. Ricucci D, Siqueira JF Jr: Apical actinomycosis as a diseases, J Endod 30:315, 2004. factors affecting outcomes of nonsurgical root canal continuum of intraradicular and extraradicular infection: 172. Rolph HJ, Lennon A, Riggio MP, et al: Molecular treatment: part 1: periapical health, Int Endod J 44:583, case report and critical review on its involvement with identification of microorganisms from endodontic 2011. treatment failure, J Endod 34:1124, 2008. infections, J Clin Microbiol 39:3282, 2001. 129. Noiri Y, Ehara A, Kawahara T, et al: Participation of 152. Ricucci D, Siqueira JF Jr: Biofilms and apical periodontitis: 173. Saber MH, Schwarzberg K, Alonaizan FA, et al: Bacterial bacterial biofilms in refractory and chronic periapical study of prevalence and association with clinical and flora of Dental periradicular lesions analyzed by the periodontitis, J Endod 28:679, 2002. histopathologic findings, J Endod 36:1277, 2010. 454-pyrosequencing technology, J Endod 38:1484, 2012. 130. Okamura K, Maeda M, Nishikawa T, Tsutsui M: Dentinal 153. Ricucci D, Siqueira J Jr: Fate of the tissue in lateral 174. Sabeti M, Simon JH, Slots J: Cytomegalovirus and response against carious invasion: localization of canals and apical ramifications in response to pathologic Epstein-Barr virus are associated with symptomatic antibodies in odontoblastic body and process, J Dent Res conditions and treatment procedures, J Endod 36:1, periapical pathosis, Oral Microbiol Immunol 18:327, 59:1368, 1980. 2010. 2003. 628 PART II The Advanced Science of Endodontics

175. Sabeti M, Slots J: Herpesviral-bacterial coinfection in 199. Siqueira JF Jr, Barnett F: Interappointment pain: 220. Siqueira JF Jr, Rôças IN, Baumgartner JC, Xia T: periapical pathosis, J Endod 30:69, 2004. mechanisms, diagnosis, and treatment, Endod Topics Searching for Archaea in infections of endodontic origin, 176. Sabeti M, Valles Y, Nowzari H, et al: Cytomegalovirus and 7:93, 2004. J Endod 31:719, 2005. Epstein-Barr virus DNA transcription in endodontic 200. Siqueira JF Jr, de Uzeda M: Disinfection by calcium 221. Siqueira JF Jr, Rôças IN, Cunha CD, Rosado AS: Novel symptomatic lesions, Oral Microbiol Immunol 18:104, hydroxide pastes of dentinal tubules infected with two bacterial phylotypes in endodontic infections, J Dent Res 2003. obligate and one facultative anaerobic bacteria, J Endod 84:565, 2005. 177. Saboia-Dantas CJ, Coutrin de Toledo LF, Sampaio-Filho 22:674, 1996. 222. Siqueira JF Jr, Rôças IN, Debelian GJ, et al: Profiling of HR, Siqueira JF Jr: Herpesviruses in asymptomatic apical 201. Siqueira JF Jr, de Uzeda M, Fonseca ME: A scanning root canal bacterial communities associated with chronic periodontitis lesions: an immunohistochemical approach, electron microscopic evaluation of in vitro dentinal tubules apical periodontitis from Brazilian and Norwegian subjects, Oral Microbiol Immunol 22:320, 2007. penetration by selected anaerobic bacteria, J Endod J Endod 34:1457, 2008. 178. Saito D, de Toledo Leonardo R, Rodrigues JLM, et al: 22:308, 1996. 223. Siqueira JF Jr, Rôças IN, Lopes HP: Patterns of microbial Identification of bacteria in endodontic infections by 202. Siqueira JF Jr, Fouad AF, Rôças IN: Pyrosequencing colonization in primary root canal infections, Oral Surg sequence analysis of 16S rDNA clone libraries, J Med as a tool for better understanding of human microbiomes, Oral Med Oral Pathol Oral Radiol Endod 93:174, 2002. Microbiol 55:101, 2006. J Oral Microbiol 4:DOI:10.3402/jom.v4i0.10743, 2012. 224. Siqueira JF Jr, Rôças IN, Lopes HP, et al: Fungal infection 179. Sakamoto M, Rôças IN, Siqueira JF Jr, Benno Y: 203. Siqueira JF Jr, Guimarães-Pinto T, Rôças IN: Effects of of the radicular dentin, J Endod 28:770, 2002. Molecular analysis of bacteria in asymptomatic and chemomechanical preparation with 2.5% sodium 225. Siqueira JF Jr, Rôças IN, Moraes SR, Santos KR: Direct symptomatic endodontic infections, Oral Microbiol hypochlorite and intracanal medication with calcium amplification of rRNA gene sequences for identification of Immunol 21:112, 2006. hydroxide on cultivable bacteria in infected root canals, selected oral pathogens in root canal infections, Int Endod 180. Sakamoto M, Siqueira JF Jr, Rôças IN, Benno Y: Bacterial J Endod 33:800, 2007. J 35:345, 2002. reduction and persistence after endodontic treatment 204. Siqueira JF Jr, Jung IY, Rôças IN, Lee CY: Differences in 226. Siqueira JF Jr, Rôças IN, Paiva SS, et al: Bacteriologic procedures, Oral Microbiol Immunol 22:19, 2007. prevalence of selected bacterial species in primary investigation of the effects of sodium hypochlorite and 181. Sakamoto M, Siqueira JF Jr, Rôças IN, Benno Y: endodontic infections from two distinct geographic chlorhexidine during the endodontic treatment of teeth Molecular analysis of the root canal microbiota associated locations, Oral Surg Oral Med Oral Pathol Oral Radiol with apical periodontitis, Oral Surg Oral Med Oral Pathol with endodontic treatment failures, Oral Microbiol Immunol Endod 99:641, 2005. Oral Radiol Endod 104:122, 2007. 23:275, 2008. 205. Siqueira JF Jr, Lima KC: Staphylococcus epidermidis and 227. Siqueira JF Jr, Rôças IN, Paiva SSM, et al: Cultivable 182. Sakamoto M, Siqueira JF Jr, Rôças IN, Benno Y: Diversity Staphylococcus xylosus in a secondary root canal infection bacteria in infected root canals as identified by 16S rRNA of spirochetes in endodontic infections, J Clin Microbiol with persistent symptoms: a case report, Aust Endod J gene sequencing, Oral Microbiol Immunol 22:266, 2007. 47:1352, 2009. 28:61, 2002. 228. Siqueira JF, Rôças IN, Ricucci D: Biofilms in endodontic 183. Santos AL, Siqueira JF Jr, Rôças IN, et al: Comparing the 206. Siqueira JF Jr, Lopes HP: Bacteria on the apical root infection, Endod Topics 22:33, 2010. bacterial diversity of acute and chronic dental root canal surfaces of untreated teeth with periradicular lesions: a 229. Siqueira JF Jr, Rôças IN, Rosado AS: Investigation of infections, PLoS One 6:e28088, 2011. scanning electron microscopy study, Int Endod J 34:216, bacterial communities associated with asymptomatic and 184. Sasaki H, Stashenko P: Interrelationship of the pulp and 2001. symptomatic endodontic infections by denaturing gradient apical periodontitis. In Hargreaves KM, Goodis HE, Tay FR, 207. Siqueira JF Jr, Magalhães KM, Rôças IN: Bacterial gel electrophoresis fingerprinting approach, Oral Microbiol editors: Seltzer and Bender’s dental pulp, ed 2, Chicago, reduction in infected root canals treated with 2.5% NaOCl Immunol 19:363, 2004. 2012, Quintessence Publishing, p 277. as an irrigant and calcium hydroxide/camphorated 230. Siqueira JF Jr, Rôças IN, Souto R, et al: Checkerboard 185. Sauer K, Camper AK, Ehrlich GD, et al: Pseudomonas paramonochlorophenol paste as an intracanal dressing, DNA-DNA hybridization analysis of endodontic infections, aeruginosa displays multiple phenotypes during J Endod 33:667, 2007. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 89:744, development as a biofilm, J Bacteriol 184:1140, 2002. 208. Siqueira JF Jr, Paiva SS, Rôças IN: Reduction in the 2000. 186. Schaudinn C, Carr G, Gorur A, et al: Imaging of cultivable bacterial populations in infected root canals by 231. Siqueira JF Jr, Rôças IN, Souto R, et al: Microbiological endodontic biofilms by combined microscopy (FISH/ a chlorhexidine-based antimicrobial protocol, J Endod evaluation of acute periradicular abscesses by DNA-DNA cLSM—SEM), J Microsc 235:124, 2009. 33:541, 2007. hybridization, Oral Surg Oral Med Oral Pathol Oral Radiol 187. Sedgley C, Nagel A, Dahlen G, et al: Real-time 209. Siqueira JF Jr, Ricucci D: Periapikale aktinomykose. Endod 92:451, 2001. quantitative polymerase chain reaction and culture mikrobiologie, pathogenese und therapie, Endodontie 232. Siqueira JF Jr, Sen BH: Fungi in endodontic infections, analyses of Enterococcus faecalis in root canals, J Endod 17:45, 2008. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 97:632, 32:173, 2006. 210. Siqueira JF Jr, Rôças IN: Polymerase chain reaction- 2004. 188. Sedgley CM, Lennan SL, Appelbe OK: Survival of based analysis of microorganisms associated with failed 233. Siren EK, Haapasalo MP, Ranta K, et al: Microbiological Enterococcus faecalis in root canals ex vivo, Int Endod J endodontic treatment, Oral Surg Oral Med Oral Pathol findings and clinical treatment procedures in endodontic 38:735, 2005. Oral Radiol Endod 97:85, 2004. cases selected for microbiological investigation, Int Endod 189. Seltzer S, Farber PA: Microbiologic factors in 211. Siqueira JF Jr, Rôças IN: Exploiting molecular methods to J 30:91, 1997. endodontology, Oral Surg Oral Med Oral Pathol 78:634, explore endodontic infections: Part 1-current molecular 234. Sjögren U, Figdor D, Persson S, Sundqvist G: Influence 1994. technologies for microbiological diagnosis, J Endod of infection at the time of root filling on the outcome of 190. Sen BH, Piskin B, Demirci T: Observation of bacteria and 31:411, 2005. endodontic treatment of teeth with apical periodontitis, fungi in infected root canals and dentinal tubules by SEM, 212. Siqueira JF Jr, Rôças IN: Exploiting molecular methods to Int Endod J 30:297, 1997. Endod Dent Traumatol 11:6, 1995. explore endodontic infections: Part 2-redefining the 235. Sjögren U, Hagglund B, Sundqvist G, Wing K: Factors 191. Sen BH, Safavi KE, Spangberg LS: Colonization of Candida endodontic microbiota, J Endod 31:488, 2005. affecting the long-term results of endodontic treatment, albicans on cleaned human dental hard tissues, Arch Oral 213. Siqueira JF Jr, Rôças IN: Uncultivated phylotypes and J Endod 16:498, 1990. Biol 42:513, 1997. newly named species associated with primary and 236. Sjögren U, Happonen RP, Kahnberg KE, Sundqvist G: 192. Sen BH, Safavi KE, Spangberg LS: Growth patterns of persistent endodontic infections, J Clin Microbiol 43:3314, Survival of Arachnia propionica in periapical tissue, Int Candida albicans in relation to radicular dentin, Oral Surg 2005. Endod J 21:277, 1988. Oral Med Oral Pathol Oral Radiol Endod 84:68, 1997. 214. Siqueira JF Jr, Rôças IN: Bacterial pathogenesis and 237. Slots J: Rapid identification of important periodontal 193. Siqueira JF Jr: Microbial causes of endodontic flare-ups, mediators in apical periodontitis, Braz Dent J 18:267, microorganisms by cultivation, Oral Microbiol Immunol Int Endod J 36:453, 2003. 2007. 1:48, 1986. 194. Siqueira JF Jr: Periapical actinomycosis and infection with 215. Siqueira JF Jr, Rôças IN: Clinical implications and 238. Slots J, Sabeti M, Simon JH: Herpesviruses in periapical Propionibacterium propionicum, Endod Topics 6:78, microbiology of bacterial persistence after treatment pathosis: an etiopathogenic relationship? Oral Surg Oral 2003. procedures, J Endod 34:1291, 2008. Med Oral Pathol Oral Radiol Endod 96:327, 2003. 195. Siqueira JF Jr: Reaction of periradicular tissues to root 216. Siqueira JF Jr, Rôças IN: Diversity of endodontic 239. Socransky SS, Gibbons RJ, Dale AC, et al: The microbiota canal treatment: benefits and drawbacks, Endod Topics microbiota revisited, J Dent Res 88:969, 2009. of the gingival crevice in man. 1. Total microscopic and 10:123, 2005. 217. Siqueira JF Jr, Rôças IN: The microbiota of acute apical viable counts and counts of specific organisms, Arch Oral 196. Siqueira JF Jr: Microbiology of apical periodontitis. In abscesses, J Dent Res 88:61, 2009. Biol 8:275, 1963. Ørstavik D, Pitt Ford T, editors: Essential endodontology, 218. Siqueira JF Jr, Rôças IN: Molecular analysis of endodontic 240. Socransky SS, Haffajee AD: Dental biofilms: ed 2, Oxford, UK, 2008, Blackwell Munksgaard, p 135. infections. In Fouad AF, editors: Endodontic microbiology, difficult therapeutic targets, Periodontol 2000 28:12, 197. Siqueira JF Jr: Treatment of endodontic infections, Ames, Iowa, 2009, Wiley-Blackwell, p 68. 2002. London, 2011, Quintessence Publishing. 219. Siqueira JF Jr, Rôças IN, Alves FR, Silva MG: Bacteria in 241. Song Y, Liu C, McTeague M, Finegold SM: 16S ribosomal 198. Siqueira JF Jr, Alves FR, Rôças IN: Pyrosequencing the apical root canal of teeth with primary apical DNA sequence-based analysis of clinically significant analysis of the apical root canal microbiota, J Endod periodontitis, Oral Surg Oral Med Oral Pathol Oral Radiol gram-positive anaerobic cocci, J Clin Microbiol 41:1363, 37:1499, 2011. Endod 107:721, 2009. 2003. CHAPTER 14 Microbiology of Endodontic Infections 629

242. Stashenko P, Wang CY, Riley E, et al: Reduction of 256. Sundqvist G, Reuterving CO: Isolation of Actinomyces 270. Vickerman MM, Brossard KA, Funk DB, et al: Phylogenetic infection-stimulated periapical bone resorption by the israelii from periapical lesion, J Endod 6:602, 1980. analysis of bacterial and archaeal species in symptomatic biological response modifier PGG glucan, J Dent Res 257. Tang G, Samaranayake LP, Yip HK: Molecular evaluation and asymptomatic endodontic infections, J Med Microbiol 74:323, 1995. of residual endodontic microorganisms after 56:110, 2007. 243. Stewart PS, Costerton JW: Antibiotic resistance of bacteria instrumentation, irrigation and medication with either 271. Waltimo T, Trope M, Haapasalo M, Ørstavik D: Clinical in biofilms, Lancet 358:135, 2001. calcium hydroxide or Septomixine, Oral Dis 10:389, 2004. efficacy of treatment procedures in endodontic infection 244. Stoodley P, Sauer K, Davies DG, Costerton JW: Biofilms 258. Tang YW, Ellis NM, Hopkins MK, et al: Comparison of control and one year follow-up of periapical healing, as complex differentiated communities, Annu Rev phenotypic and genotypic techniques for identification of J Endod 31:863, 2005. Microbiol 56:187, 2002. unusual aerobic pathogenic gram-negative bacilli, J Clin 272. Waltimo TM, Orstavik D, Siren EK, Haapasalo MP: In vitro 245. Suau A, Bonnet R, Sutren M, et al: Direct analysis of Microbiol 36:3674, 1998. susceptibility of Candida albicans to four disinfectants and genes encoding 16S rRNA from complex communities 259. Tani-Ishii N, Wang CY, Tanner A, Stashenko P: Changes their combinations, Int Endod J 32:421, 1999. reveals many novel molecular species within the human in root canal microbiota during the development of rat 273. Waltimo TM, Orstavik D, Siren EK, Haapasalo MP: gut, Appl Environ Microbiol 65:4799, 1999. periapical lesions, Oral Microbiol Immunol 9:129, 1994. In vitro yeast infection of human dentin, J Endod 26:207, 246. Subramanian K, Mickel AK: Molecular analysis of 260. Tanner A, Lai C-H, Maiden M: Characteristics of oral 2000. persistent periradicular lesions and root ends reveals a gram-negative species. In Slots J, Taubman MA, editors: 274. Waltimo TM, Siren EK, Orstavik D, Haapasalo MP: diverse microbial profile, J Endod 35:950, 2009. Contemporary oral microbiology and immunology, Susceptibility of oral Candida species to calcium hydroxide 247. Sunde PT, Olsen I, Debelian GJ, Tronstad L: Microbiota St Louis, 1992, Mosby, p 299. in vitro, Int Endod J 32:94, 1999. of periapical lesions refractory to endodontic therapy, 261. Tronstad L, Barnett F, Cervone F: Periapical bacterial 275. Wara-Aswapati N, Boch JA, Auron PE: Activation of J Endod 28:304, 2002. plaque in teeth refractory to endodontic treatment, Endod interleukin 1beta gene transcription by human 248. Sunde PT, Olsen I, Gobel UB, et al: Fluorescence in situ Dent Traumatol 6:73, 1990. cytomegalovirus: molecular mechanisms and relevance hybridization (FISH) for direct visualization of bacteria in 262. Tronstad L, Barnett F, Riso K, Slots J: Extraradicular to periodontitis, Oral Microbiol Immunol 18:67, 2003. periapical lesions of asymptomatic root-filled teeth, endodontic infections, Endod Dent Traumatol 3:86, 276. Ward DM, Weller R, Bateson MM: 16S rRNA sequences Microbiology 149:1095, 2003. 1987. reveal numerous uncultured microorganisms in a natural 249. Sunde PT, Tronstad L, Eribe ER, et al: Assessment 263. Tronstad L, Sunde PT: The evolving new understanding of community, Nature 345:63, 1990. of periradicular microbiota by DNA-DNA hybridization, endodontic infections, Endod Topics 6:57, 2003. 277. Wayman BE, Murata SM, Almeida RJ, Fowler CB: Endod Dent Traumatol 16:191, 2000. 264. Trowbridge HO, Emling RC: Inflammation. A review of the A bacteriological and histological evaluation of 58 250. Sundqvist G: Bacteriological studies of necrotic dental process, ed 5, Chicago, 1997, Quintessence. periapical lesions, J Endod 18:152, 1992. pulps [odontologic dissertation no.7], Umea, Sweden, 265. van Amersfoort ES, van Berkel TJC, Kuiper J: Receptors, 278. Wittgow WC Jr, Sabiston CB Jr: Microorganisms from 1976, University of Umea. mediators, and mechanisms involved in bacterial sepsis pulpal chambers of intact teeth with necrotic pulps, 251. Sundqvist G: Endodontic microbiology. In Spangberg LSW, and septic shock, Clin Microbiol Rev 16:379, 2003. J Endod 1:168, 1975. editors: Experimental endodontics, Boca Raton, 1990, 266. van Winkelhoff AJ, Carlee AW, de Graaff J: Bacteroides 279. Woese CR: Bacterial evolution, Microbiol Rev Rev 51:221, CRC Press, p 131. endodontalis and others black-pigmented Bacteroides 1987. 252. Sundqvist G: Associations between microbial species in species in odontogenic abscesses, Infect Immun 49:494, 280. Yamasaki M, Kumazawa M, Kohsaka T, et al: Pulpal and dental root canal infections, Oral Microbiol Immunol 1985. periapical tissue reactions after experimental pulpal 7:257, 1992. 267. Vera J, Siqueira JF Jr, Ricucci D, et al: One- versus exposure in rats, J Endod 20:13, 1994. 253. Sundqvist G: Taxonomy, ecology, and pathogenicity of the two-visit endodontic treatment of teeth with apical 281. Yoshida M, Fukushima H, Yamamoto K, et al: Correlation root canal flora, Oral Surg Oral Med Oral Pathol 78:522, periodontitis: a histobacteriologic study, J Endod 38:1040, between clinical symptoms and microorganisms isolated 1994. 2012. from root canals of teeth with periapical pathosis, J Endod 254. Sundqvist G, Figdor D: Life as an endodontic pathogen. 268. Vianna ME, Conrads G, Gomes BPFA, Horz HP: 13:24, 1987. Ecological differences between the untreated and Identification and quantification of archaea involved in 282. Zehnder M, Guggenheim B: The mysterious appearance root-filled root canals, Endod Topics 6:3, 2003. primary endodontic infections, J Clin Microbiol 44:1274, of enterococci in filled root canals, Int Endod J 42:277, 255. Sundqvist G, Figdor D, Persson S, Sjogren U: 2006. 2009. Microbiologic analysis of teeth with failed endodontic 269. Vianna ME, Horz HP, Gomes BP, Conrads G: In vivo 283. Zoletti GO, Siqueira JF Jr, Santos KR: Identification of treatment and the outcome of conservative re-treatment, evaluation of microbial reduction after chemo-mechanical Enterococcus faecalis in root-filled teeth with or without Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85:86, preparation of human root canals containing necrotic pulp periradicular lesions by culture-dependent and 1998. tissue, Int Endod J 39:484, 2006. -independent approaches, J Endod 32:722, 2006. CHAPTER 15

Pathobiology of Apical Periodontitis

LOUIS M. LIN | GEORGE T.-J. HUANG

CHAPTER OUTLINE Apical Periodontitis Cell Biology Prevalence Inflammatory Mediators Etiology Histopathology Infection: A Conflict Between Host and Parasites Clinical Features Pathogenesis Outcomes Innate Immune Response Asymptomatic Apical Periodontitis with Reactive Bone Adaptive/Specific Immune Response Formation: Condensing Osteitis or Chronic Focal Neurogenic Inflammation Sclerosing Osteomyelitis Diagnosis Cell Biology Correlation Between Clinical and Histologic Findings Histopathology Correlation Between Radiographic and Histologic Findings Clinical Features Histopathology Outcomes Symptomatic Apical Periodontitis Periapical Lesions of Nonendodontic Origin Cell Biology Extraradicular Endodontic Infection Inflammatory Mediators Apical Periodontitis and Systemic Diseases Histopathology Genetic and Systemic Disease Risk Factors of Persistent Clinical Features Apical Periodontitis Outcomes Genetic Risk Factors Asymptomatic Apical Periodontitis: Apical Granuloma, Systemic Disease Risk Factors Chronic Apical Periodontitis Wound Healing of Apical Periodontitis Cell Biology Periapical Wound Healing After Nonsurgical Root Inflammatory Mediators Canal Therapy Histopathology Periapical Wound Healing After Surgical Endodontic Therapy Clinical Features Can Radicular Cysts in Apical Periodontitis Lesions Regress Outcomes After Nonsurgical Endodontic Therapy? Asymptomatic Apical Periodontitis with Cyst Formation: Factors Influencing Periapical Wound Healing After Radicular Cyst, Chronic Apical Periodontitis with Endodontic Therapy Cyst Formation

Periradicular tissues consist of cementum, periodontal liga- as they have been shown to be associated with cementoblast ment, and alveolar bone. Cementum is a mineralized, avascular proliferation, migration, and differentiation during cementum connective tissue and consists of three different types. Acel- wound healing.88 lular afibrillar cementum covers the teeth at and along the The periodontal ligament is a soft, specialized connective cementoenamel junction. Acellular extrinsic fiber cementum tissue that connects the cementum to the alveolar bone. Peri- is confined to the coronal half of the root. Cellular intrinsic odontal ligament contains heterogeneous cell populations and fiber cementum is present on the apical half of the root where extracellular matrix (ECM).144,192 The cells of the periodontal no acellular extrinsic fiber cementum has been laid down.192 ligament include osteoblasts, osteoclasts, fibroblasts, epithelial Many growth factors, such as insulin-like growth factor-1 cell rests of Malassez, macrophages, cementoblasts, and undif- (IGF-1), fibroblast growth factors (FGFs), epidermal growth ferentiated mesenchymal cells (stem cells).192 Fibroblasts, factor (EGF), bone morphogenetic proteins (BMPs), trans- osteoblasts, and epithelial cells are differentiated cells that have forming growth factor-β (TGF-β), and platelet-derived growth retained the ability to undergo limited cell divisions and pro- factor (PDGF), are contained in the cementum matrix.47,88,164 liferation upon stimulation by appropriate signals. Multipotent These growth factors may be released under certain conditions, mesenchymal stem cells of the periodontal ligament are capable

630 CHAPTER 15 Pathobiology of Apical Periodontitis 631 of differentiating into cementoblast-like cells and periodontal bacterial biofilms in the complex root canal system canbe ligament cells as well as osteoblasts.112,182,233 The ECM of the greatly reduced, but not eliminated, by conventional endodon- periodontal ligament consists of collagen fibers, fibronectin, tic procedures such as mechanical instrumentation, antiseptic elastin, other noncollagenous proteins, and proteoglycans. The irrigation, and intracanal medication. If microbes in the root ECM serves as stratum for cell adhesion and promotes cell canal system are effectively eliminated or entombed within the spreading and cytoskeletal organization. The collagen fibers root canal by filling material, and the root canal system is (Sharpey fiber) of the periodontal ligament connect the tooth adequately sealed and protected from coronal microleakage, with the alveolar bone. The periodontal ligament is highly then periradicular tissues have the ability to restore their origi- vascularized and innervated. The tissue apical to the dentino- nal structures by means of a repair/regeneration process. Nev- cemental junction should be considered as part of the peri- ertheless, the presence of posttreatment apical periodontitis odontal ligament because cementum is not a normal component may be due to persistent microbial biofilms,76 and this recogni- of the pulp tissue. tion has spurred considerable research into treating biofilms Epithelial cell rests of Malassez (ERM), the remnants of the (see Chapters 13 and 14). Hertwig epithelial root sheath that disintegrates after tooth development, are present in the periodontal ligament near the APICAL PERIODONTITIS root surface in all teeth after root formation.209 They are nests of epithelial cells connected as a network and surrounded by Prevalence a basal lamina.192,230 ERM are quiescent in normal periodontal Epidemiologic study of apical periodontitis documents that the ligament192 but can be stimulated to proliferate in apical peri- prevalence of apical periodontitis varies among patients aged odontitis.158 They are believed to be the cellular source that 20 to 30 (33% prevalence of apical periodontitis), 30 to 40 when properly stimulated can form radicular cysts in certain (40%), 40 to 50 (48%), 50 to 60 (57%), and older than 60 years apical periodontitis lesions.158,184,200 of age (62%).203 Most studies on the prevalence of apical Alveolar bone or alveolar process is that part of bone of the periodontitis are from European and Scandinavian coun- jaws housing the sockets for the teeth. It consists of outer tries.70,117,237 According to a survey by the American Dental cortical plate, a central spongy or cancellous bone, and bone Association in 1990, an estimated 14 million root canal treat- lining the sockets.192 Bone matrix contains IGFs, TGF-β, BMPs, ments were performed in the United States alone.9 Apical peri- FGF, and PDGF.40,250 These growth factors are essential for odontitis is a prevalent health problem.71 osteoblast progenitor cell proliferation, migration, and differentiation during bone wound healing.161 The response of the periradicular tissues to various injuries ETIOLOGY is similar to that of other connective tissues elsewhere in the The etiology, pathogenesis, and histopathology of apical peri- body. The response is manifested as an inflammatory reaction odontitis are similar to that of marginal periodontitis (see also regulated by both innate and adaptive immune mechanisms. Chapter 25). Both diseases are caused by bacterial infection Although microbial infection of the pulp in the root canals is and involve pathologic changes of alveolar bone, periodontal the primary cause of apical periodontitis,119,178,184 the patho- ligament, and cementum. Marginal periodontitis affects coronal logic changes of the periapical tissues in apical periodontitis periodontal tissues, whereas apical periodontitis affects apical are usually not directly caused by microbes themselves, but periodontal tissues. Bone loss is one of characteristic features rather by their toxins, noxious metabolic by-products, and in both diseases: crestal bone is lost in marginal periodontitis, disintegrated pulp tissue in the root canal system. These irri- and apical bone undergoes resorption in apical periodontitis. tants are capable of inducing both innate and adaptive immune Apical periodontitis can be caused by both exogenous and responses; they can either activate nonantigenic pathways or endogenous factors. Exogenous factors include microbes and serve as antigens to activate adaptive responses. The subse- their toxins and noxious metabolic by-products, chemical quent inflammatory responses are diverse and can involve agents, mechanical irritation, foreign bodies, and trauma. changes in microvasculature, transmigration of blood-borne Endogenous factors include the host’s metabolic products, cells and plasma proteins out of the blood circulation into the such as urate and cholesterol crystals,188 as well as cytokines tissue space, and activation of sensory nerves. In addition, or other inflammatory mediators that activate osteoclasts.252 endothelial cells, mast cells, platelets, fibroblasts, neutrophils, These irritants can activate nonantigenic pathways or antigenic macrophages, dendritic cells, innate and adaptive immune pathways to induce innate and adaptive immunoinflammatory cells, immunoglobulins, inflammatory mediators, proinflam- responses, respectively. matory cytokines, chemokines, and neuropeptides are also In the root canal system, infection of the pulp tissue caused involved in the inflammatory response. Apical periodontitis by caries or other pathways is the primary cause of apical can be protective or destructive, depending on the dynamic periodontitis.119,178,184,261 The classic study by Kakehashi and interaction between microbial insult and the host’s defenses in colleagues119 demonstrated that pulp necrosis and periradicu- the periapical tissues. Unfortunately, the bacterial biofilm lar inflammation developed in conventional rats when the formed in the root canal system with necrotic pulp is protected pulps of teeth were exposed to oral microorganisms. However, from host’s defenses and antibiotic therapy because of a lack in germ-free laboratory rats, no pulp necrosis and periradicular of blood circulation in the root canal system. Consequently, inflammation occurred even when the pulps of teeth were any attempt of wounded periradicular tissues to repair/ exposed to the oral environment and packed with sterile food regenerate is futile, because bacterial toxins and noxious meta- debris. A similar response occurs in humans. Using bacterial bolic by-products in the root canal system continuously egress culturing, it has been demonstrated that traumatized human into the periapical area and irritate the periapical tissues. teeth with intact crowns and necrotic pulps without bacterial Emerging lines of evidence suggest that under most conditions, contamination did not show radiographic evidence of periapical 632 PART II The Advanced Science of Endodontics

bone destruction. In contrast, if bacteria were isolated from a periradicular infection. Periapical infection is related to both traumatized teeth with intact crowns and necrotic pulps, then virulence and the number and specific combinations of micro- radiographic evidence of periradicular bone destruction was organisms in the periapical tissues.264 Bacteria may be tempo- observed.261 These important findings have been replicated in rarily present in the inflamed periradicular tissues only to be nonhuman primate experiments. When the pulps of intact vital killed by the host’s defense mechanisms when the focus of teeth were intentionally devitalized under aseptic conditions infection in the root canal system is effectively eliminated by and left in the root canals with bacteria-tight, sealed coronal mechanical instrumentation, antiseptic irrigation, and intraca- restoration for 6 months to 1 year, no periradicular inflamma- nal medication. For instance, the majority of apical periodon- tory reaction was observed.156,178 Taken together, there is con- titis lesions with abscess formation or draining sinus tracts heal siderable evidence that bacteria constitute a major etiologic satisfactorily after nonsurgical root canal treatment without the factor in the development of apical periodontitis. need for systemic antimicrobial therapy.203 Bacterial toxins (e.g., lipopolysaccharide [LPS], lipoteichoic Primary root canal infection in untreated root canals is acid [LTA]) and noxious metabolic by-products that egress a polymicrobial mix with approximately equal proportions from the root canal system into the periapical tissues are of gram-positive and gram-negative species, dominated by capable of inducing a periapical inflammatory reaction.53,64,226,311 obligate anaerobes (see Chapter 14).262,264 In root-filled teeth These substances can activate the innate immune system via with apical periodontitis, gram-positive microorganisms, with receptors that recognize the stereotypic pathogen-associated a relatively equal distribution of facultative and anaerobic molecular patterns (PAMPs) that are found in the structure of species, appear to dominate other microorganisms.176,265 A these toxins. Different classes of microbes express different high prevalence of E. faecalis is frequently observed in filled molecular patterns that are recognized by different pattern root canals associated with persistent apical periodonti- recognition receptors (PRRs) or Toll-like receptors (TLRs) on tis.94,124,176,206,244,264 These issues are described in greater detail host cells, such as phagocytes, dendritic cells, and B lympho- in Chapter 14. cytes.1,171,172 PRRs or TLRs are encoded in the germline. In Physical (overinstrumentation, overfilling) and chemical mammalian species, there are at least 10 TLRs, and each (irrigants, intracanal medication, root canal filling materials) appears to have a distinct function in innate immune recogni- insults,230 as well as traumatic injury10,11 to the periapical tion.171 For example, LPS can stimulate sensory nerve fibers to tissues, can also cause apical periodontitis, depending on the release calcitonin gene–related peptide (CGRP) and substance severity of injury and cytotoxicity of the chemicals. Foreign P (SP)61,107 to cause vasodilation and increased vascular perme- bodies, such as root canal filling materials, have been ability. LPS and lipoproteins can also activate TLRs on den- shown to cause persistent periapical inflammation.133,187,205,313 dritic cells to stimulate T lymphocyte differentiation.4 Certain However, the possibility of bacterial contamination in foreign subtypes of TLRs recognize the common shared structural body–induced apical periodontitis lesions was not carefully features of various toxins (i.e., PAMPS). Because the TLRs are ruled out in many studies, so it is possible that the foreign synthesized before an infection, they are classified as part of bodies served as carriers for the microorganisms. In addition, the innate immune system. foreign bodies have the odd property of favoring infection,315 Apical periodontitis can be caused either by entry into the as they can lower the infectious dose of bacteria (LL) and cause periapical tissues of bacterial toxins, enzymes, and noxious granulocytes to develop phagocytic defect or loss of ammuni- metabolic by-products or by direct invasion of the periapical tion (LL, OK).314 Although most root canal filling materials are tissues by microbes originating from the root canal system. It not inert and are capable of inducing certain degrees of inflam- is important to differentiate between apical inflammation and mation, in general they are biocompatible and well tolerated apical infection. Apical inflammation is the periapical tissue by periapical tissues.99 reaction to irritants emerging from the root canal system that It has been demonstrated histologically that periodontal manifests as vasodilation, increased vascular permeability, and disease could cause inflammatory pulpal and periapical exudation. In contrast, apical infection is due to the physical disease.140 presence of pathogenic microorganisms in the periapical tissues that subsequently produce tissue damage. There can be INFECTION: A CONFLICT BETWEEN HOST infection without inflammation, for instance, in a severely immunocompromised patient. There can also be inflammation AND PARASITES without infection, such as in a myocardial infarct, cerebral Every infection is a race between the capabilities of the micro- infarct, and physical or chemical injury.165 In diseases caused organism to multiply, spread, and cause disease and the ability by infection, bacteria are usually present in the involved tissues of the host to control and finally eliminate the microorgan- or organs,294 such as acute necrotizing gingivitis, marginal peri- isms.294 The host has physical barriers—surface epithelium, odontitis, actinomycosis, tuberculosis, and bacterial bronchi- enamel, and dentin—as well as innate and adaptive immune tis. Although apical periodontitis is primarily an infectious defenses to prevent pulpal and periapical infection. Neverthe- disease, bacteria are usually not present in the periapical tissues less, parasites also possess weapons, leading to inhibition of but in the root canal system,139,184,296 except in certain cases of phagocytosis, inhibited lysosomal function, reduced killing by apical periodontitis associated with abscess formation,201,291,303 phagocytes, inactivation of complement system and immuno- or with a draining sinus tract,90,203,301 or extraradicular end- globulins, and specific mechanisms that permit invasion of the odontic infection.260,284 One major current hypothesis is that host’s physical barriers.294 Infection of a tissue can manifest apical periodontitis is triggered by entry into the periapical different histopathologic features as a result of specific host- tissues of bacterial toxins, enzymes, and noxious metabolic parasite interactions that occur. Many infections are asymp- by-products.269 The mere presence of bacteria (colonization) in tomatic in more than 90% of individuals.294 For instance, pulp some apical periodontitis lesions does not necessarily denote necrosis and chronic apical periodontitis caused by root canal CHAPTER 15 Pathobiology of Apical Periodontitis 633 infection are usually asymptomatic, and patients are often sur- TABLE 15-1 prised to find out that this infection has been present fora sufficient time to lead to destruction of periapical bone. There- Features of Innate and Adaptive Immunity fore, there is no simple correlation between infection and Property Innate Adaptive clinical symptoms of apical periodontitis, except in cases of symptomatic apical periodontitis and acute apical abscess. Recognition Structures shared by Antigens of microbes groups of related and of nonmicrobial microbes antigens (details of PATHOGENESIS (conserved molecular structure) When pulps are infected/inflamed, many innate and adaptive molecular patterns) immune cells release elevated amounts of various inflamma- Diversity Limited Very large tory mediators, including cytokines, chemokines, and neuro- Memory None Yes peptides. As the pulpal inflammation spreads, the inflammatory mediators begin to alter the physiology of the periapical tissues. Receptors Encoded in the Encoded in gene Clinically, the observable changes on radiographic examina- genome segments (somatic tion are widening of the periodontal ligament space or develop- recombination) ment of apical osteolytic lesions due to bone resorption. The Blood proteins Complement Antibodies loss of bone is mainly caused by activated osteoclasts. Many Cells Macrophages Lymphocytes cytokines, such as interleukin (IL)-1, IL-11, IL-17, and tumor Neutrophis Antigen-presenting necrosis factor α (TNF-α), are found to have the ability to cells induce osteoclast progenitor cell differentiation and activa- NK cells tion.24,252 The inflammation-induced bone resorption in the Action time Immediate activation Delayed activation of periapical tissues is accompanied by recruitment of immune of effectors effectors cells that essentially build a defensive line against the spread of microbial invasion from the root canal.170 The pathogenesis Response Costimulatory of apical periodontitis involves innate and adaptive immune molecules, responses as well as sensory nerve response in the periapical cytokines tissues. Immune cells present in human periradicular lesions (IL-1, IL-6) consist of lymphocytes, macrophages, plasma cells, neutro- IL-2 Clonal expansion phils, dendritic cells, and natural killer (NK) cells with the Chemokines Effector cytokines former two types as the majority.151,169 The characteristic (IL-8) (IL-4, IFNr) features of innate and adaptive immunity are summarized in Table 15-1. IFN, interferon; IL, interleukin; NK, natural killer. Data from Janeway CA, Medzhitov R: Innate immune recognition, Annu Rev Immunol Innate Immune Response 20:197, 2002; Abbas AK, Lichtman AH, Pober JS: Cellular and molecular immunology, ed 5, Philadelphia, 2003, Saunders. Specificity of Innate Immune Response In recent years, the concept of the nonspecific nature of innate immunity has changed since identification of a network of of four anaerobic pathogens: Prevotella intermedia, Fusobacte- germline-encoded receptors, the pattern-recognition receptors rium nucleatum, Streptococcus intermedius (G+), and Peptostrep- (PRRs) mentioned earlier, that recognize specific molecular tococcus micros (G+).106 In addition, LPS is shown to be capable motifs of microorganisms.5,171 PRRs can be expressed on the of inducing pain via direct activation of TLR4/CD14 expressed cell surface (macrophages, dendritic cells, neutrophils, NK on nociceptive sensory neurons.293 Thus, the TLR4 PRR recep- cells, B cells), in intracellular compartments, or secreted into tor is importantly involved in odontogenic infections. the blood and tissue fluids.115 There are numerous microbial Components such as lipoteichoic acid (LTA) of gram- constitutive and conserved products such as the pathogen- positive bacterial cell walls can also stimulate innate immunity associated molecular patterns (PAMPs), also noted earlier. in a way similar to LPS. TLR2 plays a major role in detecting Importantly, the PRRs of the innate immune system recognize gram-positive bacteria and is involved in the recognition of a PAMPs.5,171 variety of microbial components, including LTA, lipoproteins, The specificity of innate immunity is due to the recognition and peptidoglycan. The importance of TLR2 in the host defense of PAMPs of microorganisms by PRRs, such as Toll-like recep- against gram-positive bacteria has been demonstrated using tors (TLRs), of the host’s cells. Activation of PRRs triggers TLR2-deficient (TLR2−/−) mice, which were found to be highly numerous host responses, including opsonization, activation susceptible to challenge with Staphylococcus aureus or Strepto- of complement and coagulation cascades, phagocytosis, activa- coccus pneumoniae.66,270 LTA also stimulates leukocytes to tion of proinflammatory signaling pathways, and induction of release inflammatory mediators, including TNF-α, IL-1β, IL-6, apoptosis.115 For example, TLR4/CD14 is the receptor for the IL-8, and prostaglandin (PG) E2, which are known to play a gram-negative bacterial LPS. TLR4-mutated C3H/HeJ mice role in various phases of the inflammatory response. All of (LPS hyporesponsive) have reduced response to gram-negative these inflammatory mediators have been detected in periapical bacteria and are highly susceptible to infection by Salmonella samples, and each has a well-known tissue-damaging effect by typhimurium or Neisseria meningitidis.48 Importantly, there is a activating various host responses. reduced expression of IL-1 and IL-12 and decreased perira- The innate immune response to bacterial infection induces dicular bone destruction in TLR-4 deficient mice when teeth expression of proinflammatory cytokines, chemokines, and are subjected to pulpal exposures and infection with a mixture costimulators, which are essential for activation and influence 634 PART II The Advanced Science of Endodontics

of the nature of adaptive immune response.4,172 The innate encounter foreign antigens presented by antigen-presenting immune system is capable of recognizing nonself and self- cells. About 97% of T cells undergo apoptosis, and only a small antigens, whereas the adaptive immune system does not; percentage of these cells are exported to the periphery as thus, many autoimmune diseases are disorders of adaptive mature T cells.1 immunity.172 The interaction between TCR and the antigen peptide/MHC complex and costimulators activates T cells, leading to the Nonspecific Innate Immune Response synthesis of T-cell growth factor, IL-2, and its receptor that The primary nonspecific innate immune defense mechanism causes T-cell clonal expansion/proliferation. Some of these T in apical periodontitis is phagocytosis of microbes by special- cells differentiate into armed, effector T cells, and others ized phagocytes such as polymorphonuclear leukocytes become memory cells. There are a number of T-cell subpopula-

(PMNs) and macrophages. Tissue inflammation leads to the tions, categorized by their functions: (1) T helper cells (TH), recruitment of PMNs from the blood circulation into the peri- (2) T regulatory cells (Treg), (3) T suppressor cells (TS), and 1,56,290 radicular tissue. Activated PMNs exhibit an abrupt increase in (4) T cytotoxic (cytolytic) (TC) cells. Some of them oxygen consumption, the well-known respiratory burst, result- can be distinguished by their cell surface markers, cytokine ing in the release of oxygen radicals, a family of extremely profiles, or transcriptional factors. See also Chapter 13 for destructive, short-lived substances that destroy nearby micro- additional details. organisms and host cells.15 Phagocytosed microbes or foreign Upon antigen stimulation, naive CD4 T cells proliferate and

particles are exposed to a toxic environment containing spe- differentiate into TH0, which are subsequently committed to cific and azurophil granules and oxygen-derived free radicals develop into TH1 or TH2 cells. Monocytoid DC (dendritic cell) 194 and are eventually degraded. PMNs also possess an extracel- (DC1) induces TH1-type responses; plasmacytoid DC (DC2) lular killing mechanism via neutrophil extracellular traps selectively induces TH2 responses. Each subset of TH cells has (NETs), which are extracellular structures composed of chro- distinct functions and cytokine profiles. TH1 cells mainly matin with specific proteins from the neutrophilic granules. produce IL-2 and interferon (IFN)-γ, which activate macro- Upon activation (e.g., by IL-8, LPS, bacteria, fungi, activated phages and induce B cells to produce opsonizing antibody. TH2 platelets), neutrophils start a cellular program called apoptosis cells produce IL-4, -5, -10, and -13, which activate B cells to

that leads to their death and the formation of NETs, which have make neutralizing antibody. TH0 can also develop into TH17 antimicrobial activities.30,77 Besides their role in the innate under the activation of IL-6 and TGF-β and produce IL-17, a immunity as professional phagocytes, macrophages also serve powerful pro-inflammatory cytokine. Overall, TH1 and TH2 1 as antigen-presenting cells by expressing MHC (major histo- have mutually inhibitory effects. The development of CD4 TH compatibility complex) class II molecules that interact with cells involves the encounter of antigen presented by antigen- antigen-specific clones of T-helper lymphocytes. Circulating presenting cells (APCs) in association with class II MHC. All monocytes are the precursors of both tissue macrophages and cells express MHC class I, but only certain cells express class many dendritic cell subsets.1,126,234 The details of the immuno- II MHC. These class II MHC–expressing cells constitute the logic activities of neutrophils and macrophages in periapical body’s population of APCs and consist of (1) dendritic cells, pathosis are described in Chapters 12 and 13. (2) macrophages, (3) B cells, (4) vascular endothelial cells, and (5) epithelial cells. The former three are considered “profes- Adaptive/Specific Immune Response sional” APCs, because they are dedicated to this function. The The specificity of adaptive immunity is regulated at genetic latter two APCs are quiescent under normal conditions but can levels in B and T lymphocytes through a complex process be induced to express class II MHC when exposed to elevated leading to the generation of molecules that recognize and bind concentrations of IFN-γ.1,116 to foreign or self-antigens. These molecules are specific recep- Dendritic cells and macrophages phagocytose foreign anti- tors on T cells (T-cell antigen receptors or TCRs) and on B cells gens, whereas B cells utilize the membrane-bound immuno- (B-cell antigen receptors or BCRs; also termed immunoglobu- globulin to bind and internalize antigens. Other APCs lins). TCRs on T cells interact with antigens that are presented endocytose foreign antigens into the cytoplasm for antigen by antigen presenting cells expressing MHC molecules along processing. The processed antigens are partially degraded into with other accessory molecules, whereas BCRs on B cells inter- small peptides. Many of them are 10 to 30 amino acids long act with antigens directly. BCRs may be secreted in the blood and capable of binding onto the newly synthesized class II circulation or in the tissues as antibodies. The variable region MHC molecules before the antigen/class II MHC complex is of both TCR and BCR proteins are rearranged at the genomic transported to the surface of cells and presented to TCRs of level via genetic recombination of the V(D)J segments. The CD4+ T cells. estimated total diversity after this recombination for TCR is Although controversial, evidence has suggested that CD8+ 21 16 + + approximately 10 and for BCR is approximately 10 that TS and CD8 CTLs represent distinct subpopulations of CD8 + − generates the repertoire of different individual T and B cell T cells. TS are MHC class I–restricted CD8 /CD28 TS cells, clones.1,116 Each T or B cell clone generated in the bone marrow which act on antigen-presenting cells (APC) by a contact- carries a specific TCR and BCR. They undergo a positive and dependent manner, rendering them tolerogenic to TH cells. 50,316 negative selection process through which most clones are They inhibit the proliferation of TH cells. T cytotoxic cells + deleted via apoptosis because they bind to self-antigens. This (CD8 TC), also known as cytolytic T lymphocytes (CTLs), are a initial “negative screening” process reduces the potential for subset of T cells that kill target cells expressing MHC-associated autoimmune disorders. Only those that do not interact with peptide antigens. The majority of TC express CD8 and recog- self-antigens are released into the lymphatic system and nize antigens degraded in the cytosol and expressed on the blood circulation. The naive T cells circulate back and forth cell surface in association with class I MHC molecules of the between the lymphatic system and blood circulation until they target cells. Functional TC acquire specific membrane-bound CHAPTER 15 Pathobiology of Apical Periodontitis 635 cytoplasmic granules, including a membrane pore-forming neuropeptides in the process of inflammation is now known protein called perforin or cytolysin and enzymes called to be far more complex. In the development of chronic apical granzymes.1 periodontitis lesions, neuropeptides are also involved in The role of B cells in adaptive immunity is mainly the pro- immune regulation, bone resorption, and wound healing. duction of antibodies that constitute the host humoral immune At sufficient concentrations, SP increases the secretion of response. The V(D)J gene recombination occurs in both heavy IL-1, TNF-α, and IL-6 from macrophages and stimulates and light (κ and λ) chains. A recombinase system, which is an T-lymphocyte proliferation and enhances antigen-induced enzymatic complex consisting of several enzymes—including IFN-γ production by T cells.253 Certain neuropeptides, such as recombination activating gene 1 and 2 (RAG-1, RAG-2), ter- SP, upregulate immune and inflammatory responses, whereas minal deoxynucleotidyl transferase (TdT), DNA ligase IV, Ku other neuropeptides, such as vasoactive intestinal peptide proteins, and XRCC4—is essential for successful recombina- (VIP) and neuropeptide Y (NPY), inhibit inflammatory tion. This recombinase system is also used for TCR recombina- responses. Synergistic interactions among neuropeptides, such tion. Mature IgM/IgD coexpressing B cells undergo isotype as CGRP and other inflammatory mediators, eicosanoids, and switching via a process called switch recombination after bradykinin, suggest a complex interplay between these mol- encountering antigen. The rearranged V(D)J gene segment ecules in the immune response.83,211,253 recombines with a downstream C region gene (γ, ε, or α), and In chronic apical periodontitis lesions, specific receptors for the intervening DNA sequence is deleted. This gives rise to SP and CGRP are expressed in certain immune cells, including other classes of immunoglobulins (IgG, IgE, IgA) besides IgM. macrophages and lymphocytes. Both CGRP and VIP may play In addition to isotype switching, activated B cells also undergo a role in inhibiting bone resorption by suppressing osteoclastic somatic mutation in the V region gene, leading to affinity functions. The level of VIP in apical periodontitis lesions is maturation of antibodies and alternative splicing of VDJ RNA inversely related to lesion size. Osteoclast cell culture studies to membrane or secreted immunoglobulin mRNA. A large have demonstrated that the presence of greater concentrations quantity of antibody is secreted when B cells terminally dif- of VIP leads to a decrease in their ability to form lacunae of ferentiate into plasma cells.1,116 The ability of antigens to selec- osseous resorption, causing rapid cytoplasmatic contraction tively stimulate the differentiation of plasma cells supports the and reduced cell mobility. VIP exerts an effect on macrophages clinical finding that plasma cells isolated from periapical to block the production of TNF-α, IL-6, and IL-12, suggesting lesions secrete antibodies specific for the particular bacteria that VIP could have a role in controlling growth of apical peri- found in the adjacent root canal system. odontitis lesions.43 The immune response and the role of lymphocyte subpopu- The major innate and adaptive immune responses and lations in apical periodontitis lesions were investigated by neurogenic inflammation in the pathogenesis of apical employing lymphocyte-deficient rodents as study models. periodontitis caused by root canal infection are illustrated in T-cell deficiency appears to accelerate bone loss at the early Fig. 15-1. phase of apical periodontitis lesions (2 weeks) but does not affect the overall course of lesion development.271,295 Using RAG (recombination activating)-2 SCID mice (both T- and DIAGNOSIS B-cell-deficient), it was found that approximately a third of the RAG-2 mice developed endodontic abscesses, whereas no Correlation Between Clinical and immunocompetent controls had abscesses.273 In another study, Histologic Findings specific RAG 2 knockout (k/o) mice were used to determine Clinical diagnosis of inflammatory periapical disease is mainly which immune element was important for the defense mecha- based on clinical signs or symptoms, duration of disease, pulp nism in endodontic infection. The results demonstrated that B tests, percussion, palpation, and radiographic findings (see also cells, but not T cells, played a pivotal role in preventing dis- Chapter 1). In contrast, a histologic diagnosis is a morphologic semination of endodontic infection.105 Therefore, both T and and biologic description of cells and extracellular matrix of B cells mediate the observed immune responses in apical peri- diseased tissues. The clinical diagnosis represents a provisional odontitis lesions.170 diagnosis based on signs, symptoms, and testing results, whereas the histologic diagnosis is a definitive diagnosis of Neurogenic Inflammation tissue disease. Certain primary afferent nerve fibers, upon stimulation by Similar to pulpitis,231,238 apical periodontitis is not always various irritants, release neuropeptides, which cause vasodila- symptomatic or painful. Although many inflammatory media- tion, protein extravasation, and recruitment/regulation of tors (histamine, bradykinin, prostaglandins) and proinflamma- immune cells such as macrophages, neutrophils, mast cells, tory cytokines (IL-1, IL-6, TNF, nerve growth factor [NGF]), and lymphocytes. This phenomenon is termed neurogenic as well as neuropeptides (SP, CGRP), are capable of sensitizing inflammation.26,29 Pivotal neuropeptides in the induction of and activating nociceptive sensory nerve fibers,96,97 other medi- neurogenic inflammation are CGRP, for vasodilation, and SP, ators such as endogenous opioids and somatostatin released by for the induction of protein extravasation. Neuropeptides and inflammatory cells during inflammation are able to inhibit their receptors are widely distributed throughout the body. firing of sensory nerve fibers.102,236 Activation of nociceptive During inflammation, there is a sprouting of afferent fibers38,39,96 sensory nerve fibers may also be related to concentrations of and local increases in inflammatory mediators that trigger inflammatory mediators. The complexity of these findings sup- neuropeptide release, leading to neurogenic inflammation.26,43 ports the clinical observation that there is no good correlation Besides the cardinal functions of the key neuropeptides between clinical symptoms and histopathologic findings of that cause the first sign of inflammation—vasodilation apical periodontitis.27,159 For example, many teeth with apical and increased vascular permeability—the role of these periodontitis are free of symptoms. 636 PART II The Advanced Science of Endodontics

Root canal

Innate Immune Response Adaptive Immune Response

Apex PAMPs M␾ Ags IL-1 APC IL-6 Bact. Abs B cell IL-8 TNF TLR Inflam. mediators PMN Proinflam. cytokines IL-1 IL-6 BV TCD4 TNF TLR Vasodilation MC Vascular TH1 TH2 Histamine permeability

SP CGRP GM-CSF IL-4 Neurogenic Sensory N. IL-2 IL-5 inflammation TNF-2 IL-10 INF-Y GM-CSF OC IL-1 IL-6 OB RANKL

FIG. 15-1 Major innate and adaptive immune responses and neurogenic inflammation in the pathogenesis of apical periodontitis. APC, antigen presenting cell; GM-CSF, granulocyte/monocyte colony-stimulating factor; MC, mast cell; Mφ, macrophage; OB, osteoblast; OC, osteoclast; PAMPs, pathogen-associated molecular patterns; PMN, polymorphonuclear leukocyte; RANKL, receptor activator of nuclear factor κB ligand; TLR, Toll-like receptor. (Courtesy Dr. Lin.)

Correlation Between Radiographic and of periapical lesions. Thus, currently available diagnostic methods used in endodontics, such as percussion, palpation, Histologic Findings and pulp tests (cold, heat, electric), are not sensitive enough Radiography is designed to detect pathologic changes at tissue to provide histologic diagnosis of an inflammatory periapical not cellular levels. Even using sensitive imaging systems such disease. In fact, all endodontic tests are basically used to as ultrasound, cone-beam computed tomography, and other examine the functions of nociceptive sensory nerves and not technologies, it is impossible to detect the presence of inflam- the pathologic changes of the pulp or periapical tissues. Until matory cells or other subtle changes in the periapical tissues. we have more advanced and sophisticated clinical diagnostic Using conventional radiographic and histologic methods in the technologies, we will continue to face the problem of clinical same cadavers, evidence of inflammation was often observed diagnosis of inflammatory periapical disease. Nevertheless, the in the periapical tissues of endodontically treated teeth with treatment of various types of apical periodontitis lesions is normal radiographic features.17,36,85 This finding is supported basically the same: nonsurgical root canal therapy. Interest- by the fact that lesions localized in the cancellous bone may ingly, this treatment does provide a consistently good clinical not be visible radiographically unless they involve cortical outcome, as measured by either clinical signs of success or bone (see also Chapter 2).20,22,109 In addition, radiographic find- survival of the treated tooth. Future research should focus on ings are unable to predict asymptomatic apical periodontitis developing testing methods that provide greater insight into (granuloma) from asymptomatic apical periodontitis with the status of the periapical tissue. cyst formation (radicular cyst).141,267 Accordingly, radiographic findings and histopathologic features of apical periodontitis have a poor correlation based on available case series studies. HISTOPATHOLOGY The absence of clinical symptoms and negative periapical The study of the histopathology of diseased tissues and organs radiographic findings of endodontically involved teeth does has gone through an interesting evolution. It began at macro- not necessarily indicate absence of apical periodontitis.17 By scopic, light microscopic, and then electron microscopic obser- the same token, clinical success of endodontically involved vations of diseased tissues and organs. Nowadays, histopathology teeth (i.e., absence of signs and symptoms and negative peri- focuses on cellular and molecular biology of diseased tissues apical radiographic findings) after nonsurgical root canal and organs. Biochemical disorders occur inside the cells before therapy does not necessarily imply complete histologic healing light and electron microscopic observable morphologic changes CHAPTER 15 Pathobiology of Apical Periodontitis 637 of cell injury. Cell death (protein denaturation) occurs before many factors, such as severity of tissue injury, specific etiology, observable morphologic changes of cell necrosis. Gene trans- host’s resistance, and particular tissue involved, can modify the formation or mutation occurs before observable morphologic course and morphologic variations as well as cell biology changes of neoplastic cells.165 of both acute and chronic inflammatory responses.137,165 Based on etiology, clinical signs and symptoms, and dura- Acute and chronic inflammatory responses are in fact not rigid tion of the disease process, the World Health Organization phases of a single programmed event but two overlapping (WHO) classifies disease of periapical tissues into many cate- responses with partially different triggering mechanisms and gories.307 There are also many classifications of inflammatory programs.165 periradicular disease in several endodontic textbooks110,203,282 and by the American Board of Endodontists, depending on clinical manifestations and histologic appearances. In addition, SYMPTOMATIC APICAL PERIODONTITIS the American Association of Endodontists held a consensus It is a general belief that the development of apical periodon- conference on diagnostic terms in the fall of 2008. Tradition- titis follows total pulp necrosis. This belief is based on (1) the ally, there has been a lack of consensus on clinical diagnostic pulpal strangulation theory due to a generalized increase in terminology of pulpal and periapical disease in the endodontic pulpal interstitial pressure inside the uncompromised pulp community because of the paucity of studies with high levels space during pulpal inflammation that causes collapse of of evidence. Because this chapter focuses on pathobiology of venules and cessation of blood flow96 and (2) animal and apical periodontitis, inflammatory periapical disease will be human studies that concluded that uncontaminated necrotic classified based on histologic appearances and cell biology of pulps that are intentionally devitalized or accidentally trauma- the injured periapical tissues. To avoid confusion between his- tized are generally incapable of inducing periapical inflamma- tologic and clinical diagnosis, readers are encouraged to read tion unless they are infected.156,178,261 However, if the vital pulps the chapters related to clinical examination, radiographic inter- become infected due to caries or other pathways, periapical pretation, and clinical diagnosis of inflammatory periapical inflammation can develop even when inflamed, but vital tissue disease (see Chapters 1, 2, and 3). is still present in the apical portion of the root canal. Most of The histopathologic analysis of a diseased tissue or organ our information related to the histopathology of apical peri- only shows structural changes of cells and extracellular matrix odontitis comes from analysis of longstanding, chronic human at the time the tissue or organ is removed. Therefore, it does lesions caused by caries or from time-course studies of devel- not represent the complete kinetics or spectrum of disease opment of apical periodontitis induced by artificial root canal development. Histologic classification of apical periodontitis is infection in animals. In these instances, the moment of transi- based on types of cells participating in inflammatory responses tion from pulpitis to apical periodontitis was not captured. In in the periapical tissues. In general, inflammation can be fact, apical periodontitis has been demonstrated to be a divided into acute and chronic responses, depending on types direct extension of apical pulpitis into the periapical tissues of cells present at the site of injured tissue.137,165,248,299 Acute before total pulp necrosis caused by root canal infection inflammatory response is characterized by participation of (Fig. 15-2).135,154,155,214,215,268 For example, Kovacevic and col- neutrophilic leukocytes and chronic inflammatory response leagues135 studied the transition from pulpitis to apical peri- by participation of macrophages and lymphocytes. However, odontitis in dogs’ teeth by artificial exposure of pulps to the

PAP

Osteoclast

Internal resorption

A B FIG. 15-2 A, Inflammation of the pulp tissue in the apical root canal extends into the periapical tissues in a mature tooth (H&E, magnification: ×100). B, Arrow in (A). High magnification of the pulp tissue in the apical root canal in A. The pulp tissue is vital and infiltrated with chronic inflammatory cells. Note resorption of canal wall and multinucleated clast cells (H&E magnification: ×200). (Courtesy Dr. Domenico Ricucci, Rome, Italy.) 638 PART II The Advanced Science of Endodontics

oral cavity and observed that pulpitis was coupled with an In addition, activated mast cells secret cytokines (TNF-α, IL-1, acute apical periodontitis. Similarly, Kimberly and Byers128 IL-3, IL-4, IL-5, IL-6, IL-13), prostaglandins, and leukotrienes demonstrated that periapical changes, including sprouting of to enhance inflammatory defense mechanisms.1,37,126 nerve fibers, appeared 3 to 5 weeks following establishment of irreversible pulpitis subsequent to pulp exposure lesions in Endothelial Cells animals. Yamasaki and coworkers310 and Stashenko and col- Endothelial cells are important players in the inflammatory leagues255 also showed that periapical inflammatory infiltrates, response. Without the participation of endothelial cells, the increased osteoclast numbers, and bone destruction were host is unable to deliver its cellular and humoral defense com- apparent well in advance of total pulpal necrosis, with vital ponents from the circulating blood to the site of tissue injury. pulp tissue still present in the apical portion of the root canal. Inflammatory mediators, complement components, proinflam- The biologic basis for these observations appears to hinge on matory cytokines, nitric oxide, neuropeptides, and bacterial the apical development of pulpal infection/inflammation toxins can all affect endothelial cells, resulting in vasodilation leading to the diffusion of many inflammatory mediators, pro- and increased vascular permeability.1,137,165 IL-1 and TNF inflammatory cytokines, chemokines, and bacterial toxins into released by activated macrophages and NK cells can stimulate the periapical area154 prior to total pulpal necrosis. endothelial cells to express intercellular adhesion molecules The development of acute apical periodontitis largely (ICAMs), such as ICAM-1, ICAM-2, ICAM-3, vascular cell reflects the innate immune system and is the first line of active adhesion molecule (VCAM), and platelet endothelial cell adhe- defense against irritants from the root canal. Acute apical peri- sion molecule (PECAM), which enhance leukocyte adhesion odontitis is an immediate defense reaction to irritants and does to endothelial cells and transmigration through the blood not require exquisite specificity and memory. Characteristic vessels.1,137,148,165 IL-1, TNF, and LPS also can activate endothe- features of acute apical periodontitis are similar to the typical lial cells to synthesize chemokine (IL-8), a potent chemotactic acute inflammatory reaction and consist of vasodilation, mediator for neutrophils.1 increased vascular permeability, and transmigration of leukocytes from the blood vessels into perivascular tissue space. The Polymorphonuclear Neutrophilic Leukocytes beneficial actions of acute inflammation are (1) infiltration of Polymorphonuclear neutrophilic leukocytes (PMNs) are the leukocytes to the injured tissue to phagocytose and kill micro- principal effector cells in acute apical periodontitis. They are bial agents; (2) accumulation and activation of humoral factors derived from bone marrow stem cells. Neutrophils have a lobu- such as immunoglobulins, complement factors, and plasma lated nucleus and contain primary or azurophil (elastase and proteins in the injured tissue to recruit more neutrophils and myeloperoxidase) and secondary or specific (lysozyme and macrophages; and (3) neutralization or degradation of bacte- other protease) granules in their cytoplasm.126,137,165 Neutro- rial toxins and their harmful metabolic by-products.137,165 phils are only present in the blood circulation. They are the first leukocytes to transmigrate through the blood vessels into Cell Biology perivascular tissue space and then are directed toward the The inflammatory response is a dynamic interaction between wound or irritants, peaking at 24 to 48 hours. The transmigra- host defense mechanisms and microbial insults. The interlac- tion of neutrophilic leukocytes from the blood vessels into ing activation and control pathways of cellular and humoral perivascular space involves complex cellular and molecular components involved in the inflammatory response are biology. Following vasodilation and increased vascular perme- complex. The cells involved—neutrophils, monocytes/ ability, leukocyte margination, rolling, capture, and activation macrophages, platelets, mast cells, T lymphocytes, B lympho- in the blood vessel and then transmigration through the blood cytes, NK cells, dendritic cells, endothelial cells, fibroblasts, vessel are mediated by an intricate interaction of cell adhesion eosinophils, and basophils—each have numerous functions molecules expressed on leukocytes (L-selectin, integrins) and that are activated and modulated by a multiplicity of bio on endothelial cells (P- and E-selectins, ICAM, VCAM, chemical messengers.1,137,165 Cell activation means that the cell PECAM-1). Neutrophil rolling is mediated by interaction acquires the ability to perform one or more new functions or between leukocyte selectin ligands and P-selectins on endothe- to perform normal functions at a higher rate1; it often results lial cells. Leukocyte sticking is mediated by interaction between in transcription of new genes and synthesis of new proteins.1 leukocyte integrins and ICAMs and VCAMs on endothelial cells. Leukocyte transmigration is mediated by interaction Mast Cells between PECAM-1 on both leukocytes and endothelial cells.165 Histamine stored in the cytoplasmic granules of mast cells is Chemokines (IL-8) increase the affinity of leukocyte integrins the mediator that first appears in acute inflammation to induce for their ligands on endothelial cells.1 Once transmigrating vasodilation and increased vascular permeability; mast cells are through the junction between endothelial cells and basement the designated triggers of acute inflammation. They are widely membrane into the perivascular tissue space, neutrophilic leu- distributed in perivascular tissue spaces and originate in the kocytes are directed toward stimuli by chemotactic factors or bone marrow from precursor cells. Mature mast cells contain chemotaxins, such as bacterial products (fLMP), C3a, C5a, numerous cytoplasmic granules, which are the source of vaso- leukotriene B4 (LTB4), platelet-activating factor (PAF), fibri- active mediators. The preformed histamine is released by mast nopeptides, dead cells, and chemokines (IL-8) by receptor- cell degranulation and can be triggered by (1) physical stimuli mediated mechanisms.1,137,165,248 such as cold, heat, and mechanical trauma; (2) binding of IgE- Neutrophils can be activated by bacteria and pathogen- specific antigen to mast cells and membrane-bound IgE anti- associated molecular patterns (also known as the TLRs, bodies; (3) binding of complement components (C3a and C5a) described earlier). They can also be stimulated by IL-1, TNF, to their complementary receptors on mast cells; and (4) stimu- and chemokines produced by activated macrophages and NK lation by neuropeptide (SP) and cytokines (IL-1, IL-8).1,29,137,165 cells to enhance the phagocytic activity of infectious agents and CHAPTER 15 Pathobiology of Apical Periodontitis 639 synthesis of defensins, which are broad-spectrum antibiotics.1 intracellular microbes.1 Killing of phagocytosed microbes by Neutrophilic leukocytes are terminally differentiated cells and neutrophilic leukocytes and macrophages is mediated through short lived—within hours to a few days. Most neutrophilic oxygen-dependent and oxygen-independent mechanisms. leukocytes in acute inflammatory response die as a result of Oxygen-dependent mechanisms are more effective in killing all apoptosis or programmed cell death. The apoptotic neutrophils kinds of bacteria than oxygen-independent mechanisms.1,137,248 are phagocytosed by macrophages.89,137,165 However, some The effector molecules of an oxygen-dependent system are neutrophilic leukocytes die after a furious battle against micro- hydrogen peroxide, superoxide anion, hydroxyl radical, singlet bial infection and release intracellular proteolytic lysosomal oxygen, and hypochlorite. An oxygen-independent system is enzymes, oxygen-derived active metabolites, nitric oxide, also important in killing microbes and is dependent on lyso- proinflammatory cytokines, eicosanoids, and matrix metallo zymes, defensins, and lactoferrin contained in phagocyte proteinases into tissue to intensify inflammation and tissue granules.1,137,165,248 Some antimicrobial peptides and other damage.137,165 The release of lysosomal enzymes by neutrophils oxygen-independent mechanisms possessed by phagocytes are and macrophages can also occur by lysosomal suicide due to specialized for killing of certain groups of microbes.1,248 rupture of phagolysosome in the cytosol, regurgitation during Microbes phagocytosed by phagocytes are enclosed in phagocytosis of irritants, or frustrated phagocytosis of indigest- membrane-bound phagosomes in the cytosol. The phagosomes ible foreign bodies.137,165 The main effector functions of neu- fuse with lysosomes to form phagolysosomes. Irritants such as trophilic leukocytes are phagocytosis, killing of microbes, and microbes, foreign protein antigens, and dead cells inside the release of inflammatory mediators (including proinflammatory phagolysosome are destroyed or degraded by proteolytic cytokines) to recruit more leukocytes to prevent spread of enzymes stored in lysosomes.1 There are also granules that are infection. fused with the nascent phagosome and release their contents into the phagosome. Some of these granules have direct anti- Macrophages microbial action, such as defensins and the bactericidal Macrophages make their appearance as a second wave in acute permeability-increasing protein azurocidin. Others are prote- apical periodontitis within 48 to 96 hours. Macrophages are ases, such as elastase and cathepsins, lactoferrin, and peroxi- blood-borne but have a counterpart in the connective tissue.165 dase myeloperoxidase. Lysosomal enzymes, reactive oxygen Blood monocytes transmigrate through the blood vessel into intermediates, and nitric oxide released by neutrophils and the tissues and become macrophages. Mature macrophages macrophages indiscriminately kill not only bacteria but also have an irregular-shaped nucleus and contain abundant lyso- tissue cells. Much of periapical tissue damage that occurs somes and many phagocytic vesicles (phagosomes). Mono- during acute inflammation can be attributed to release of pro- cytes use mechanisms similar to neutrophils to adhere to teolytic lysosomal enzymes and matrix metalloproteinases endothelial cells in high endothelial venule–expressing adhe- from disintegrated neutrophilic leukocytes and macrophages sion molecules for mononuclear leukocytes (ICAM for macro- rather than to bacteria and their toxins.1,165 phages and VCAM for lymphocytes); they then transmigrate through the blood vessel and are directed toward the site of Platelets tissue injury by chemotactic factors.165 Platelets normally circulate in the blood but also play an Macrophages can be activated by bacteria, PAMPs, and important role in inflammation. They are small cytoplasmic interferon-r (INF-r) and produce numerous products: lyso- fragments derived from the megakaryocyte.126 Platelets somal enzymes, coagulation factors, bioactive lipids, reactive are essential for blood clotting, hemostasis, and fibrinolysis. oxygen species, chemokines, cytokines/growth factors, and Platelets produce vasoactive amines (PAF, serotonin), angiogenesis factors.137,165,248 Macrophages are the most chemokines, and growth factors (PGDF, FGF, TGF) during dynamic and versatile leukocytes. The main functions of mac- inflammation.165 rophages are numerous. They include phagocytosis of microbes and foreign bodies, production of inflammatory mediators, Natural Killer Cells initiation of the immune response, cleanup operation of NK cells may also be players in acute apical periodontitis. They necrotic cells and tissue, induction of systemic effects (fever, are a subset of lymphocytes found in blood and lymphoid acute phase reaction, cachexia), synthesis of molecules or cyto- tissues. NK cells are derived from the bone marrow stem cells kines affecting cell and vascular growth in wound healing, as but lack the specific T-cell receptor for antigen recognition.1 well as antibacterial and antiviral defenses.165 Tissue macro- NK cells also possess TLRs for microbial constitutive and con- phages are not terminally differentiated cells and are able to served products. The effector functions of NK cells are to lyse undergo mitosis. Their life span is from several weeks to virus-infected cells without expressing class 1 MHC molecules months. and to secret IFN-r to activate macrophages. Antibody-coated Activated neutrophilic leukocytes and macrophages are cells, cells infected by viruses, some intracellular bacteria, and capable of phagocytosing and killing pathogens. They recog- IL-2 released by activated macrophages can activate NK cells. nize microbes through TLRs and receptors for the Fc fragment Viruses have been isolated in apical periodontitis lesions.218,249 of immunoglobulin IgG as well as complement component NK cells kill target cells by antibody-dependent cell-mediated C3b on their cell surface. C3b opsonization and antibody cytotoxicity because NK cells express receptor for the Fc frag- coating of microbes enhance recognition and phagocytosis of ment of IgG.1 NK cells provide a link between the innate and pathogens by activated neutrophilic leukocytes and macro- adaptive immune systems.115 phages.1,137,165 Activated neutrophilic leukocytes are effective in phagocytosing and killing extracellular microbes, whereas Inflammatory Mediators macrophages activated by IFN-r produced by NK cells and Numerous biochemical mediators are involved in the acute

TH1 cells are more effective in phagocytosing and killing innate inflammatory response. They are mainly derived from 640 PART II The Advanced Science of Endodontics

TABLE 15-2 Major Mediators in Inflammation Effector Cells Effector Cells Mediator Source and Tissues Mediator Source and Tissues Vasodilator Chemokines Macrophages Endothelial cells Histamine Mast cells Endothelial cells Neutrophils Platelets Endothelial cells Fibroblasts Prostaglandins Leukocytes Endothelial cells Mast cells Leukocyte Activation and Chemotaxis Nitric oxide Endothelial cells Vascular smooth C3a, C5a Plasma Leukocytes Macrophages muscle Leukotriene B4 Mast cells Leukocytes CGRP Sensory nerve Endothelial cells Leukocytes Fibrin degradation Plasma Endothelial cells Chemokines (IL-8) Macrophages Leukocytes Neutrophils products Endothelial cells Increased Vascular Permeability Fibroblasts Bradykinin Plasma Endothelial cells Fibrinopeptides Plasma Leukocytes Leukotrienes C4, D4, E4 Leukocytes Endothelial cells Bacterial products (fMLP) Bacteria Leukocytes Mast cells TNF Activated Leukocytes PAF Leukocytes Endothelial cells macrophages Mast cells Dead cells Endothelial cells Opsonins C3a, C5a Plasma Endothelial cells C3b, C5b, Plasma Microbes Fibrinopeptides Plasma Endothelial cells immunoglobulins Substance P Sensory nerve Endothelial cells Tissue Damage Increased Expression of Endothelial Adhesion Molecules Lysosomal enzymes Neutrophils Cells and tissues (Selectin, ICAM, VCAM, PEAM) Macrophages TNF Activated Endothelial cells Free oxygen radicals Activated Cells and tissues macrophages leukocytes NK cells Nitric oxide Macrophages Cells and tissues IL-1 Activated Endothelial cells Bacterial products Bacteria Cells and tissues macrophages (LPS) NK cells CGRP, calcitonin gene–related peptide; fMLP, formyl-methionyl-leucyl-phenylalanine; IL-1, interleukin-1; PAF, platelet activation factor; NK cell, natural killer cell; TNF, tumor necrosis factor. Data from Kumar V, Abbas AK, Fausto N, et al: Robbins and Cotran pathologic basis of disease, ed 8, Philadelphia, 2010, Saunders; Slauson DO, Cooper BJ: Mechanisms of disease, ed 3, St Louis, 2002, Mosby; Majno G, Joris I: Cell, tissues, and disease, ed 2, Oxford, 2004, Oxford University Press; Abbas AK, Lichtman, Pober JS: Cellular and molecular immunology, ed 5, Philadelphia, 2003, Saunders.

plasma and cells. The main biologic functions of inflammatory acid and platelet activating factor. Arachidonic acid molecules mediators are to cause vasodilation and increased vascular can be processed along two pathways: the cyclooxygenase permeability and recruit inflammatory cells, mainly neutro- pathway, which leads to production of prostaglandins and philic leukocytes and macrophages from blood circulation to thromboxanes, and the lipoxygenase pathway, which produces the site of tissue injury. Some mediators can also cause tissue leukotrienes.137,165 damage. The major mediators involved in vascular changes, Complement components, such as C3a, C3b, C5a, C5b, and cell recruitment, and tissue damage in the acute inflammatory C5-C9, are products of the complement cascade, which can be response are listed in Table 15-2. activated by two pathways. The classic pathway is initiated All listed inflammatory mediators have been shown to be by activation of C1 by multimolecular aggregates of IgG or present in apical periodontitis.278,279 Bradykinin is the product IgM antibody complexed with specific antigen. The alternate of kinin-system activation; fibrinopeptides, the products of pathway is activated by microbial cell components (lipopoly- blood-clotting system activation; and fibrin degradation prod- saccharide, teichoic acid) and plasmin. C3a and C5a are ana- ucts, the products of fibrinolytic-system activation. Kinin, fibri- phylatoxins that stimulate mast cells and basophils to release nolytic, and clotting systems are initiated by activated Hageman histamine. They also cause phagocytes to release lysosomal factor. Prostaglandins and leukotrienes are the products of enzymes. C3b is an opsonin and can coat bacteria to enhance arachidonic acid metabolism. Activated phospholipase A2 phagocytosis by phagocytes. In addition, C3b can also bind splits cell membrane phospholipid molecules into arachidonic to antibody bound to antigen or microbes. C5a is a strong CHAPTER 15 Pathobiology of Apical Periodontitis 641 chemotaxin for neutrophils. C5b-C9 is a membrane attack Clinical Features complex and able to cause cell lysis if activated on the host cell As discussed earlier, there is no correlation between clinical and bacterial cell membrane.137,165 and radiographic findings and histologic appearance of inflam- Besides inflammatory mediators, the inflammatory response matory periapical disease. Teeth with acute apical periodontitis is also dependent upon the timing and extent of various cyto- are usually symptomatic and painful to bite and percussion, kine and chemokine secretions. IL-1, TNF, IL-6, IL-12, and which results from mechanical allodynia and hyperalgesia.125 IFN-γ are present in the acute inflammatory response.1,91 IL-6 Pain is induced by sensitization and activation of nociceptive is produced by activated mononuclear phagocytes, endothelial sensory nerve fibers by inflammatory mediators, proinflamma- cells, and fibroblasts in response to microbial infection and tory cytokines, nerve growth factor, and pressure.125 Sprouting other cytokines, such as IL-1 and TNF. IL-6 stimulates the of sensory nerve fibers in inflamed periapical tissues could also synthesis of acute-phase proteins by hepatocytes.1 The major increase receptive field size in teeth with apical periodonti- source of IL-12 is activated mononuclear phagocytes and den- tis.38,39 Radiographic examination usually does not show peri- dritic cells. IL-12 provides a link between innate and adaptive apical bone destruction of the involved tooth in acute apical immune responses.1 IFN-r is produced by activated NK cells, periodontitis, although occasional slight widening of the apical

TH1 cells, and cytotoxic T cells and can activate macrophages periodontal ligament space and loss of the apical lamina dura and enhance their microbial killing ability. IL-1β and TNF are of the involved tooth may be present. associated with apical bone resorption during chronic apical periodontitis.252 Outcomes Chemokines are a large family of structurally homologous The fundamental purpose of the acute inflammatory response cytokines that stimulate leukocyte movement and regulate the is to restore the structural and functional integrity of damaged transmigration of leukocytes from the blood vessel to tissue tissue by eliminating irritants as soon as possible.137,165,299 space.1 They are produced by leukocytes, endothelial cells, and Tissue damage can also occur in acute inflammation by release fibroblasts. The secretion of chemokines is induced by micro- of lysosomal enzymes, toxic oxygen radicals, and nitric bial infection, TNF, and IL-1.1,91,240 Different types of leukocytes acid from disintegrated neutrophilic leukocytes and macro- express different chemokine receptors.1 phages into tissue.165 Depending on the dynamic interaction Neuropeptides are released via axon reflexes of afferent between host defenses and microbial insults, acute apical peri- sensory neurons in response to various stimuli. Neuropeptides, odontitis can result in (1) restitution of normal periapical as mediators of the inflammatory process, are described in detail tissues if irritants are immediately eliminated by root canal in the Neurogenic Inflammation section of this chapter. Inflam- therapy, (2) abscess formation if massive invasion of periapical matory mediators such as histamine, kinins, prostaglandins, and tissues by highly pyogenic bacteria occurs, (3) organization by proinflammatory cytokines are capable of sensitizing and acti- scarring if extensive destruction of periapical tissues results, or vating sensory nerves to release neuropeptides.29,96,97,213,225 (4) progression to chronic apical inflammation if irritants continue to persist. Histopathology Abscess is a focal localized collection of purulent exudate The acute inflammatory response is practically immutable in in a tissue or an organ.137 It is a morphologic variation of acute all vascularized living tissues, largely due to the programmed and chronic inflammation.137,165,299 The development of an actions of the innate immune system. Initially, blood vessels abscess in apical periodontitis lesions is probably caused by are engorged by a local infiltration of inflammatory cells, invasion of a combination of specific pyogenic bacteria in the mainly activated neutrophilic leukocytes and some macro- inflamed periapical tissues.32,201,263,291 Neutrophilic leukocytes phages in the apical periodontal ligament of the infected/ are the predominant cells in acute apical periodontitis with inflamed root canal. In addition, sprouting of sensory nerve abscess formation. Abscess begins as a furious battle between fibers has been shown early in the inflamed periapical highly virulent pathogens and an army of neutrophilic leuko- tissues.39,128 Several studies have also demonstrated the pres- cytes. The pathogens produce massive toxins to kill neutro- ence of inflamed vital pulp tissue with intact nerve fibers in phils. As neutrophils attack the pathogens, they secrete the apical portion of the root canal in association with apical lysosomal enzymes that digest not only the dead cells but periodontitis.154,214,215,255,310 This explains the clinical observa- also some live ones. Many neutrophils die fighting against tion that a patient may experience some pain if an instrument pathogens. The resulting purulent fluid is poorly oxygenated is introduced into the canal short of the apex in some teeth and has a low pH. The bactericidal ability of leukocytes appears with apical periodontitis lesions. to be impaired because of the deprivation of oxygen and In primary acute apical periodontitis, apical bone destruc- interference of respiratory burst. The purpose of respiratory tion is usually not observed radiographically because the dura- burst is to generate bactericidal agents.15,165 However, the tion of acute response is short, and activated neutrophilic phagocytic activity of leukocytes is not impaired in anaerobic leukocytes and macrophages are not able to resorb bone. Only conditions.165 osteoclasts are capable of resorbing bone, and they have to Histologically, apical abscess formation is characterized by differentiate from the monocyte/macrophage cell lineage in the local collection of suppurative or purulent exudate composed blood circulation. However, in a rat model experiment, Stash- of dead and live neutrophilic leukocytes, disintegrated tissue enko and colleagues255 showed histologically that periapical cells, degraded extracellular matrix, and lysosomal enzymes inflammatory cell infiltration increases osteoclast numbers, released by dead neutrophilic leukocytes. It also contains dead and that bone destruction was apparent well in advance of total and live bacteria and bacterial toxins released by dead bacteria pulpal necrosis. in the inflamed periapical tissues. Abscess formation also Bacteria are usually not present in acute apical periodontitis involves destruction of periodontal ligament and sometimes lesions. periapical bone—especially in chronic apical periodontitis 642 PART II The Advanced Science of Endodontics

(or through incision and drainage when indicated) during root RC D RC canal therapy, the patient usually experiences an immediate relief of acute pain. Radiographically, the involved teeth may BA D show slight widening of the apical periodontal ligament space to loss of apical lamina dura. In chronic apical abscess, the 25 ␮m involved teeth may be symptomatic or asymptomatic. If an intraoral or extraoral draining sinus tract is present, swelling D is usually absent. Radiographic bone destruction is obvious in teeth with chronic apical abscess formation. BV In most cases, if the source of infection in the root canal is 3 ␮m eliminated by root canal therapy, the abscess will heal by reab- sorption of the pus in teeth with an acute apical abscess. AB Phagocytes will kill all bacteria in the abscess. The continued influx of leukocytes stops because the chemotactic stimuli

5 ␮m have been removed, and the existing neutrophilic leukocytes die of apoptosis. Finally, macrophages move in to clean up necrotic neutrophilic leukocytes and disintegrated tissue cells. Fl In chronic apical abscess formations, wound healing will take place mainly by means of regeneration and to some degree by tissue repair. However, if bacterial virulence and numbers of NG pathogens overwhelm the host’s defenses, the abscess may break through the cortical bone, periosteum, and oral mucosa or facial skin to develop an intraoral or extraoral draining sinus 75 ␮m tract. Sometimes, the uncontrolled abscess may spread along the fascial planes of the head and neck to develop serious cellulitis (see also Chapter 18).142,230

A 200 ␮m B 200 ␮m ASYMPTOMATIC APICAL PERIODONTITIS: FIG. 15-3 Structure of a secondary periapical abscess. A, Axial section of APICAL GRANULOMA, CHRONIC an abscessed apical periodontitis. The microabscess (AB) contains a focus of APICAL PERIODONTITIS neutrophils (NG inset in A). Note the phagocytosed bacteria in one of the If pathogens in the root canal are not eliminated, the symp- neutrophils (further magnified in large inset in B). A secondary abscess forms tomatic apical periodontitis may progress to become an asymp- when bacteria (BA in oval inset) from the apical root canal (RC) advance into tomatic apical periodontitis. Asymptomatic apical periodontitis the chronic apical periodontitis lesion (B). Note the tissue necrosis immediately is characterized by the persistence of inflammatory stimuli, in front of the apical foramen and the bacterial front in the body of the lesion adaptation of the host’s response to stimuli, presence of (arrowheads in lower inset). BV, blood vessels; D, dentin. (A, magnification: adaptive immune responses, and initiation of the repair 130; B, magnification: 100; oval inset, magnification: 400; inset in A, × × × process.137,165,248,299 Chronic inflammation is good news and bad magnification: 2680; upper inset in B, magnification: 4900; lower inset in × × news. The good news is that the host’s defenses are able to B, magnification: 250.) (From Nair PNR: Apical periodontitis: a dynamic × maintain an active defense against the invading microorgan- encounter between root canal infection and host response, Periodontol 2000 isms and toxins; the bad news is that the host response is 13:121, 1997.) inadequate to eliminate these factors.165 Asymptomatic apical periodontitis is a form of adaptive with abscess formation—and a surrounding layer of viable immune response that requires exquisite specificity and neutrophilic leukocytes and a band of fibrovascular granula- memory. The adaptive immune response enhances bacterial tion tissue. Both layers are thought to serve as protective killing compared with the innate immune response. Tradition- barriers to prevent the spread of infection. Epithelial cell pro- ally, the terms asymptomatic chronic apical periodontitis and peri- liferation is scanty in acute apical periodontitis with abscess apical granuloma are used interchangeably. A granuloma is a focal formation (Fig. 15-3).44,215,230 area of granulomatous inflammation, which is a histologic term Clinically, teeth with acute apical abscess formation usually for a chronic inflammatory reaction.137,165,294 Granulomatous have symptoms such as pain to biting and percussion. The inflammation is characterized by the presence of activated mac- periapical area of the involved tooth may be tender to palpa- rophages with modified epithelioid cells in diseases such as tion. Intraoral or extraoral swelling is often present. Because tuberculosis, leprosy, syphilis, cryptococcosis, sarcoidosis, rheu- of a sudden outpouring of suppurative exudate in the periapi- matic fever, and foreign body granuloma.101,137,165,304 The presence cal area, tissue pressure increases such that mechanical stimuli of poorly digestible irritants (nonantigenic or antigenic), T cell– are capable of activating terminals of nociceptive neurons in mediated immunity to irritants, or both appears to be necessary the inflamed periapical tissues. The severe pain of an acute for granuloma formation.1,137,165 A granuloma is relatively avas- apical abscess can be due to activation of nociceptors by cular,137,165 whereas a chronic apical periodontitis is very vascular. inflammatory mediators and sensitization to mechanical Histologically, some but not all chronic apical periodontitis stimuli due to increased interstitial pressures.232 If the periapi- lesions may show some features of granulomatous inflamma- cal purulent exudate can be evacuated through the root canal tion,137,165 so the terms apical granuloma and asymptomatic chronic CHAPTER 15 Pathobiology of Apical Periodontitis 643 apical periodontitis should not be used interchangeably. A granu- periodontitis, both osteoclast and osteoblast activity decrease,297 loma is best considered a histologic term used to describe a so the periapical osteolytic lesion remains stationary. Based on specific form of chronic inflammation such as foreign body magnified radiographic and automated image analysis, periapi- granuloma or immune granuloma.137,165 cal bone destruction was observed at 7 days after the pulps of The foreign body reaction is a specific subtype of chronic experimental teeth were exposed to oral microorganisms in inflammation.137,165,299 Foreign materials such as root canal animal studies. A period of rapid bone destruction took place filling materials, paper points, cotton fibers, and surgical between 10 and 20 days, with slower bone resorption there sutures can trigger a foreign body giant cell granuloma.133,187,313 after.297 The stationary phase of bone resorbing activity was If activated macrophages are unable to engulf large indigestible correlated to asymptomatic apical periodontitis.297 Increased foreign particles, they can fuse to form giant cells on the expression of bone resorptive cytokines such as IL-1, IL-6, and 298 surface of the particles and continuously release lysosomal TNF was related to the period of active bone resorption. TH enzymes, inflammatory mediators, and proinflammatory cyto- cells appear to outnumber TS cells during the active stage of kines as a result of frustrated phagocytosis. Giant cells appear periapical bone destruction in induced rat periapical lesions, 165 to be at least as active metabolically as a regular macrophage. but TS cells dominate TH cells during the stationary stage of In addition, foreign bodies can favor infection in several ways, bone destruction.256 as they can be a source of bacterial biofilm51,197 and they lower Bone resorption is caused by osteoclasts. The formation of the infectious dose of bacteria to induce infection. For example, osteoclasts involves differentiation of the osteoclast precursor if a small, sterile plastic cage is implanted under the skin of a from the monocyte-macrophage cell lineage in bone marrow. guinea pig, as few as 100 Staphylococcus aureus are sufficient Several cytokines and growth factors, such as granulocyte/ to infect the tissue, whereas even 109 bacteria (i.e., a million- macrophage colony-stimulating factor (GM-CSF), RANKL fold increase in dose) fails to produce an abscess in normal (receptor activator of nuclear factor κB ligand), osteoproteg guinea pig skin.315 Finally, foreign bodies can make the infec- erin (OPG), IL-1, IL-6, TNF, as well as prostaglandins, brady- tion hard to treat because bacteria in biofilms can switch on kinin, kallidin, and thrombin, have been shown to mediate appropriate genes and cover themselves with a thick layer of osteoclast progenitor cell differentiation.24,40,65,147,180,208,272 Para- biopolymer that is resistant to both host defense mechanisms thyroid hormone is capable of stimulating osteoblasts to syn- and antimicrobial agents.118,165 thesize GM-CSF and RANKL. Bone stromal cells and T cells also produce RANKL. Osteoclast progenitor cells express Cell Biology receptor activator of nuclear factor κB (RANK). OPG, a decoy Macrophages and Lymphocytes receptor for RANKL secreted by osteoblasts, negatively regu- Macrophages and lymphocytes are the primary players in lates the differentiation of osteoclasts by absorbing RANKL and asymptomatic apical periodontitis.3,52,79,122,132,258,259,280,312 Lym- reducing its ability to activate the RANK pathway.126 phocytes are blood-borne and have counterparts in the con- RANKL activates the RANK pathway on osteoclast pro nective tissue.165 Macrophages play a dual role in host defenses. genitor cells, resulting in differentiation of these cells along In the innate immune response, activated macrophages phago- the osteoclast lineage. Proinflammatory cytokines, IL-1, TNF, cytose microbes, dead cells, and foreign bodies and produce and IL-6 also mediate the differentiation of osteoclast pro inflammatory mediators and proinflammatory cytokines to genitor cells to osteoclasts. The differentiation of the mono- enhance the host defense against stimuli. In the adaptive nuclear osteoclast progenitor cells terminates with fusion to immune response, activated macrophages function as APCs. latent multinucleated osteoclasts, which are finally activated to They phagocytose and present the processed foreign antigens become bone-resorbing osteoclasts. The mature osteoclasts in association with MHC to T cells. Thus, activated macro- then attach to mineralized bone surface after osteoblasts have phages are effector cells of adaptive immune response. primed the unmineralized bone surface and released chemotac- Lymphocytes are the only cells in the body capable of tic factor to attract osteoclasts.190 Osteoclasts attach to bone by specifically recognizing and distinguishing different antigenic the vitronectin receptor (integrin superfamily), expressed pref- determinants; they are responsible for the two defining char- erentially in the sealing zone. Vitronectin has binding sites for acteristics of the adaptive immune response: specificity and the arginine-glycine-aspartic amino acid (RDG) sequences memory. The functions of lymphocytes were described earlier present in many extracellular matrix proteins, including osteo- in the section on the adaptive immune response. pontin, bone sialoprotein, and fibronectin, on the surface of the exposed mineralized bone.81 When bound to bone extracellular Dendritic Cells matrix, osteoclasts develop a ruffled border. Inside the ruffled Dendritic cells play a vital role in asymptomatic apical peri- border, osteoclasts use ATP to drive H+ pumps, leading to the odontitis. They have been shown to be present in apical peri- acidification of the extracellular compartment. They - subse odontitis lesions in rats.120,202 Dendritic cells are accessory quently secrete proteolytic lysosomal enzymes and carbonic immune cells derived from bone marrow stem cells and may anhydrase to degrade both the mineralized and unmineralized be related to the mononuclear phagocyte lineage.1 They func- components of bone.16,18,28,272 (See Fig. 15-5 for the mechanism tion as antigen-presenting cells for naive T lymphocytes and of bone resorption by osteoclasts in apical periodontitis.) are important for the initiation of adaptive immune responses The resorption of root cementum or dentin in apical peri- to protein antigen.1 Activated dendritic cells produce IL-12, odontitis lesions is less well understood than resorption of which is a key inducer of cell-mediated immunity. bone. Bone is constantly remolding (resorption and deposition) through physiologic and functional processes and thus is Osteoclasts much easier to study. In contrast, cementum and dentin Periapical bone destruction is a hallmark of asymptomatic are more stable. The cells responsible for dental hard-tissue apical periodontitis. During the chronic stage of apical resorption are called odontoclasts.219 It has been shown in 644 PART II The Advanced Science of Endodontics

External Necrosis root resorption

Microbes B N␾ M␾ Mono T

HSC

TNF-␣ IL-1 Th2 Th17 Th1 IL-6 TNF-R RANK DC

IL-6 TNF-␣ PGE2 GM-CSF TNF-␣ OCP IL-17 IL-1 IL-11 RANKL TNF-R OB OPG RANK MSC PreOB RANK

TGF-␤

OB OC Apoptosis FIG. 15-4 Bone resorption by osteoclasts in apical periodontitis. DC, dendritic cell; HSC, hematopoietic stem cell; Mφ, macrophage; MSC, mesenchymal stromal cell; Nφ, neutrophil; OB, osteoblast; OC, osteoclast; OCP, osteoclast precursor; OPG, osteoprotegerin; RANK, receptor activator of nuclear factor κB; RANKL, receptor activator of nuclear factor κB ligand. (Courtesy Dr. Huang.)

ultrastructure and gene expression studies that odontoclasts extent of cellular proliferation appears to be related to the and osteoclasts are similar.220,221 Therefore, it is believed that degree of inflammatory cell infiltration.214,215 the cellular mechanisms of bone, cementum, and dentin resorption are similar.221 Nevertheless, little is known about Fibroblasts how the precursors of the odontoclasts appear and what causes Fibroblasts are important cells in chronic inflammation and odontoclast differentiation and activation to resorb dentin and wound healing. They are derived from undifferentiated mesen- cementum. Bone resorption by osteoclasts in apical periodon- chymal cells and exist in all connective tissues. They synthe- titis is illustrated in Fig. 15-4. size and secret proteoglycans, glycoproteins, and precursor molecules of various types of collagens and elastin.126 In Epithelial Cell Rests of Malassez chronic inflammation, fibroblast migration and proliferation Proliferation of epithelial cell rests of Malassez (ERM) is evident are triggered by multiple growth factors (TGF-β, PDGF, EGF, in approximately 52% of inflammatory periapical lesions col- FGF) and the fibrogenic cytokines, IL-1 and TGF-α, produced lected from extracted teeth.186 It is a form of pathologic (inflam- by activated platelets, macrophages, endothelial cells, and matory) hyperplasia. This type of hyperplasia is caused by inflammatory cells.137,165 In turn, activated fibroblasts produce stimulation of growth factors and cytokines produced during an array of cytokines such as IL-1, IL-6, and granulocyte/ the inflammatory response. Hyperplasia is a self-limiting macrophage colony-stimulating factor, which influence leuko- process and is reversible when the causative stimulus is elimi- cyte development. Fibroblasts stimulated by inflammation also nated.137,165,248 During periapical inflammation, innate and produce matrix metalloproteinase to degrade the proteins that adaptive immune cells and stromal cells (e.g., activated macro- comprise the extracellular matrix.165,287 phages, neutrophilic leukocytes, NK cells, T cells, fibroblasts) Fibrovascular granulation tissue is also a prominent feature in the periapical tissues produce many inflammatory mediators of chronic apical periodontitis as a repair process. Migration (e.g., prostaglandin, histamine), proinflammatory cytokines and proliferation of endothelial cells from preexisting capillar- (e.g., IL-1, IL-6, TNF), and growth factors (e.g., PDGF, epider- ies and venules into the site of chronic inflammation is called mal growth factor [EGF], keratinocyte growth factor [KGF], neovascularization. It is mediated by angiogenesis factors, such FGF). These inflammatory mediators, proinflammatory cyto- as vascular endothelial growth factor (VEGF) and TGF-β, pro- kines,35,46,87,173,223 and growth factors78,111,160,177,198,276 are capable duced by activated macrophages, platelets, and endothe- of stimulating epithelial cell rests to proliferate (Fig. 15-5). The lium.165,248 Neovascularization supplies oxygen and nutrients CHAPTER 15 Pathobiology of Apical Periodontitis 645

OC Fibroblast

KGF IL-6 EGF TH2 IL-1 IGF TNF M⌽ PGs TGF-␣

TNF TGF-␣

TH1 ERM EO

PMN

FIG. 15-5 Schematic illustration of the major mechanisms that activate proliferation of epithelial cell rests in apical periodontitis. (From Lin LM, Huang GT-J, Rosenberg P: Proliferation of epithelial cell rests, formation of apical cysts, and regression of apical cysts after periapical wound healing, J Endod 33:908, 2007.) for the support of metabolically active macrophages and fibro- Simon and associates242 observed that projections, depressions, blasts during wound healing. and fibers of cementum were haphazardly arranged in root canal infection under scanning electron microscopy. There was Inflammatory Mediators an increase of mineralized projections, cementum lacunae, and Many inflammatory mediators present in symptomatic apical surface resorptions and a decrease in fibers. All these changes periodontitis are also expressed in asymptomatic apical peri- may provide a more favorable condition for attachment of odontitis. In addition, several different cytokines and growth bacterial biofilm on the apical external root surface in extra factors are secreted by cells such as activated macrophages, radicular infection. lymphocytes, dendritic cells, and fibroblasts, in the chronic/ It is a general belief that asymptomatic apical periodontitis adaptive inflammatory response described earlier. lesions are usually devoid of innervation, so local anesthesia may not be necessary if teeth with asymptomatic apical peri- Histopathology odontitis require root canal therapy. However, light and trans- Macrophages and lymphocytes are predominant cells in asymp- mission electron microscopic studies have demonstrated that tomatic apical periodontitis lesions (Fig. 15-6). Occasionally, teeth with chronic apical periodontitis were well innervated foamy macrophages and giant cells are seen, especially associ- (Fig. 15-7).153,168 This explains why instruments accidentally ated with cholesterol crystal deposits, which are products of introduced into inflamed periapical tissues without local anes- disintegrated cell membranes. Cholesterol crystals are present thesia can cause the patient pain. in approximately 18% to 44% of all apical periodontitis External root resorption that involves cementum or both lesions.33,286 Bone resorption is a hallmark of asymptomatic cementum and dentin usually occurs in asymptomatic apical apical periodontitis. Multinucleated osteoclasts may some- periodontitis lesions.143,292 Fortunately, cementum and dentin times be seen in resorptive Howship lacunae. Proliferation of appear to be less readily resorbed than bone by inflammatory epithelial cell rests is often present in asymptomatic apical processes.92 However, the radiographic presence of root “blunt- periodontitis lesions. ERM proliferate in three dimensions and ing” should be interpreted by the astute clinician as evidence form irregular strands or islands of epithelium, which are often for resorption of dentin and cementum, and the working infiltrated by varying degrees of inflammatory cells (see Fig. length of the instrumentation and obturation phases of non- 15-6, B). A key feature of asymptomatic apical periodontitis is surgical endodontic treatment should be adjusted by a corre- a proliferation of fibrovascular granulation tissue that is an sponding amount. attempt to prevent further spread of infection/inflammation Using light and transmission electron microscopy and and to repair wounded periapical tissues. microbiologic culturing, bacteria have been shown to Little information is available concerning cemental changes be present in many asymptomatic apical periodontitis in apical periodontitis. Using a scanning electron microscope, lesions.2,114,284,300 Inadvertent contamination is a major concern 646 PART II The Advanced Science of Endodontics

D D BA

D BA M

D M N

GR NM ly p

80 ␮m

nl BV EP

FIG. 15-7 Presence of intact myelinated and nonmyelinated nerve fibers in chronic apical periodontitis lesion. ly, Lymphocyte; M, myelinated nerve fiber; N, nucleus of Schwann cell; nl, neutrophilic leukocyte; NM, nonmyelinated nerve fiber;p, plasma cell (EM, magnification:× 1600). (From Lin L, Langeland 50 ␮m K: Innervation of inflammatory periapical lesions, Oral Surg Oral Med Oral A 250 ␮m B 300 ␮m Pathol Oral Radiol Endod 51:535, 1981.) FIG. 15-6 Chronic asymptomatic apical periodontitis without (A) and with (B) epithelium (EP). The root canal contains bacteria (BA) in A and B. The lesion gingival sulcus. This leads to the development of a deep, in A has no acute inflammatory cells, even at the mouth of the root canal with narrow pseudopocket that often mimics a periodontal pocket visible bacteria at the apical foramen (BA). Note the collagen-rich maturing or a vertical root fracture (see Chapter 25 for details). granulation tissue (GR) infiltrated with plasma cells and lymphocytes (inset in A and B). BV, blood vessels; D, dentin. (A, magnification:× 80; B, magnification: Outcomes ×60; inset in A, magnification: ×250; inset in B, magnification: ×400.) (From Asymptomatic apical periodontitis may result in (1) regenera- Nair PNR: Apical periodontitis: a dynamic encounter between root canal tion or repair of the periapical tissues after root canal therapy, infection and host response, Periodontol 2000 13:121, 1997.) (2) severe periapical tissue destruction, (3) acute exacerba- tion, (4) development of an abscess with an intraoral or extraoral draining sinus tract, or (5) development of a serious when culturing bacteria from these lesions. Under meticulous cellulitis. transmission electron microscopic examination, Nair183 was unable to observe bacteria in most asymptomatic apical periodontitis lesions. If bacteria are present in the inflamed periapi- ASYMPTOMATIC APICAL PERIODONTITIS cal tissues, they are usually found inside the phagocytes (Fig. WITH CYST FORMATION: RADICULAR 15-8).154 Importantly, the mere presence of bacteria in the CYST, CHRONIC APICAL PERIODONTITIS inflamed periapical tissues (colonization) does not necessarily WITH CYST FORMATION imply periapical infection. Bacteria have to be able to establish an infectious process, such as survival and tissue destruction, The radicular cyst is unique because no cysts in the body have in the inflamed periapical tissues to be considered etiologic similar pathogenesis. It is believed that the radicular cyst is factors for the resulting infection. Most apical periodontitis likely formed by inflammatory proliferation of epithelial cell lesions are not infected; nonsurgical root canal therapy of teeth rests in the inflamed periodontal ligament.158,189,200,274 The with apical periodontitis can achieve a high success rate, pro- radicular cyst is a pathologic cavity completely lined by non- vided root canal infection is controlled. keratinized stratified squamous epithelium of variable thickness in a three-dimensional structure in an apical periodontitis Clinical Features lesion. A radicular cyst can be a “pocket cyst” (i.e., attached Involved teeth are usually asymptomatic and show an ill- to the apical foramen) or a “true cyst” (no attachment to the defined or well-defined periapical radiolucent lesion. The root structure), but it cannot form by itself. Therefore, a radic- chronic apical abscess and associated sinus tract are usually ular cyst should not be considered a separate disease entity asymptomatic. Occasionally, the sinus tract from an apical from asymptomatic apical periodontitis. A radicular cyst abscess may drain along the root surface and opens into the (pocket or true) is classified as an inflammatory and nota CHAPTER 15 Pathobiology of Apical Periodontitis 647

Pv ls

ls A B FIG. 15-8 Bacteria phagocytosed by phagocytes in chronic apical periodontitis lesion. A, Bacteria (arrows) in phagosome of a neutrophil. ls, Lysosome; N, part of nucleus; Pv, phagocytic vacuole (EM, magnification:× 6300). B, Part of the cell structure of a macrophage. Note longitudinally and cross-cut bacteria cells with unit membrane (arrows) in phagocytic vacuoles (EM, magnification: ×10,000). (From Lin L, Langeland K: Light and electron microscopic study of teeth with carious pulp exposures, Oral Surg Oral Med Oral Pathol 51:292, 1981.) neoplastic lesion in the WHO Histological Typing of Odonto- expansion is likely caused by degradation of fibrous connective genic Tumors, Jaw Cysts and Allied Lesions.136 The prevalence tissue capsule by matrix metalloproteinases, which are pro- of radicular cysts in apical periodontitis lesions from extracted duced by activated neutrophils, fibroblasts, and macrophages275 teeth ranges from 15% to 20%.186 and bone resorption. The ERM have also been shown to be capable of secreting a bone resorbing factor.25 Most inflamma- Cell Biology tory mediators and proinflammatory cytokines that stimulate In addition to the presence of all chronic inflammatory cells, proliferation of epithelial cell rests also mediate bone resorp- the epithelial cells are the most prominent cell type found in tion in apical periodontitis lesions. asymptomatic apical periodontitis with cyst formation. ERM can be considered unipotent or restricted-potential stem cells. Inflammatory Mediators They can be stimulated to divide symmetrically and asym- Mediators are basically similar to those present in chronic metrically into basal cells (stem cells) and suprabasal squa- apical periodontitis. mous cells of lining epithelium of radicular cysts.158 Many theories about apical cyst formation have been proposed. The Histopathology nutritional deficiency theory assumes that when islands of Two types of cysts have been described in chronic apical peri- epithelium keep growing, the central cells of the epithelial odontitis lesions. The pocket cyst’s lumen opens into the root island will move farther away from their source of nutritional canal of the involved tooth (Fig. 15-9).185,186,241 The true cyst supply and undergo necrosis and liquefaction degeneration. is completely enclosed by a lining epithelium, and its lumen The products accumulated attract neutrophils and granulo- has no communication with the root canal of the involved cytes into the necrotic area. Microcavities then coalesce to form tooth (Fig. 15-10).185,186,241 Apical cysts are lined by hyperplas- a cyst cavity lined by stratified squamous epithelium.274 The tic, nonkeratinized stratified squamous epithelium of variable abscess theory postulates that when an abscess is formed in thickness, which is separated from the fibrovascular connective connective tissue, epithelial cells proliferate and line the tissue capsule by a basement membrane. Both the lining epi- abscess cavity because of their inherent tendency to cover thelium and the connective tissue capsule are usually infil- exposed connective-tissue surface.189,200 The theory of merging trated with inflammatory cells,235 indicating that inflammatory of epithelial strands proposes that proliferating epithelial cells are attracted to these tissues by chemotactic irritants, strands merge in all directions to form a three-dimensional either in the root canal system or in the periapical tissues. In spherelike structure composed of fibrovascular connective nonproliferating epithelium, there is less inflammatory cell tissue, with varying degrees of inflammatory cells trapped, that infiltration.44,230 Occasionally, mucous cell metaplasia or cili- gradually degenerates due to lack of blood supply. This leads ated respiratory-type cells are present in cystic epithelial to formation of a cyst cavity.158 Regardless of how a radicular lining.195 The epithelial cells of lining epithelium do not show cyst (pocket or true) is formed, it is likely caused by inflam- any characteristic features of neoplastic changes, such as pleo- matory proliferation (hyperplasia) of epithelial cell rests in morphism, lack of polarity, nuclear enlargement, large nucle- these lesions. oli, abnormal nuclear/cytoplasmic ratio, hyperchromatism, or It has been speculated that radicular cyst expansion is abnormal mitosis. Unlike odontogenic keratocysts and calcify- caused by increased osmotic pressure in the cyst cavity,277 but ing odontogenic cysts, the basal cells of radicular cystic lining this hypothesis overlooks the cellular aspects of cyst growth epithelium are incapable of proliferation by themselves without and the biochemistry of bone destruction.72,173 Radicular cyst stimulation of growth factors or cytokines released by innate D D

LU LU

FIG. 15-9 A, A well-developed pocket cyst in apical periodontitis lesion. B, Note the saclike epithelial lumen. C-D, Sequen- tial axial serial section passing through the EP apical foramen in the root canal plane (RC) A 1 mm B 500 ␮m shows the continuity of the lumen with the root canal. D, dentin (A, C, magnification: 16; B, D, magnification: 40). (From Nair RC × × RC PNR: Non-microbial etiology: foreign body reaction maintaining post-treatment apical periodontitis, Endod Topics 6:96, 2003.) D EP LU LU

C 1 mm D 500 ␮m

FIG. 15-10 A, Two distinct cysts lined by epithelium in apical periodontitis lesion. There is no evidence of communication with the foramen in serial sections (H&E, A B magnification: ×25). B, Cyst cavity on the left side in A. Accumulation of foamy macrophages (H&E, magnification: ×50; inset magnification: ×1000). C, Lower part of cavity in B. Dense infiltration of neutrophilic leukocytes (H&E, magnification: ×1000). D, Cyst cavity on the right side in A. Necrotic tissue debris in the lumen; infiltration of inflammatory cells in the epithelium (H&E, magnification: ×50). E, Cystic wall in D. Cyst’s lining epithelium is infiltrated with acute and chronic inflammatory cells. LU, Lumen (H&E, magnification: ×1000). F, Foamy macro- LU phages, neutrophilic leukocytes, and necrotic tissue inside the cyst (inset), but no bacteria (Brown & Brenn, magnifica- C D E tion: ×25, inset magnification: ×1000). G, Area indicated by an open arrow in F. Bacterial colonies in the apical foramen (Brown & Brenn, magnification: ×1000). (From Ricucci D, Pascon EA, Pitt Ford TR, Langeland K: Epithe- lium and bacteria in periapical lesions, Oral Surg Oral Med Oral Pathol Oral Radiol Endod 101:241, 2006.)

F G CHAPTER 15 Pathobiology of Apical Periodontitis 649

Lumen CC

A B C CC FIG. 15-11 A, A radicular cyst in which the lumen is completely filled with cholesterol crystals (H&E, magnification: ×25). B, High magnification of cholesterol crystals in A (H&E, magnification: ×100). C, High magnification of rectangular area in B. Multinucleated giant cells associated with cholesterol crystals (H&E, magnification:× 400). (Courtesy Dr. Ricucci, Rome, Italy.)

A B FIG. 15-12 A, An apical true cyst in apical periodontitis lesion. Bacterial colonies are seen in the foraminal areas (Brown & Brenn, magnification: ×25). B, Bacterial colonies in the cyst lumen, surrounded by inflammatory cells (Brown & Brenn, magnification: ×400). (From Ricucci D, Bergenholtz G: Histologic features of apical periodontitis in human biopsies, Endod Topics 8:68, 2004, Fig. 6.) and adaptive immune cells during periapical inflammation. Unfortunately, apical cysts cannot be diagnosed clinically and The lumen of cysts may contain inflammatory exudates, cho- can only be diagnosed after surgical biopsy or extraction of lesterol crystals, clear fluid, or bacterial colonies (Figs. 15-11 teeth with apical periodontitis. Clinical outcome studies have and 15-12).186,215 shown that after proper nonsurgical root canal therapy, teeth with apical periodontitis heal in 78% of cases.247 Therefore, it Clinical Features is speculated that some cysts, especially pocket cysts, may heal The involved tooth is usually asymptomatic. Periapical osteo- after nonsurgical root canal therapy.185 Apical true cysts are less lytic lesions of endodontically involved teeth may sometimes likely to heal after nonsurgical root canal therapy because of show a well-demarcated radiolucency surrounded by a rim of their self-sustaining nature; surgical intervention is neces- radiopaque border. sary.185 Nonetheless, similar to the periapical pocket cyst, a periapical true cyst is also formed within an apical periodon- Outcomes titis lesion and is not a neoplastic lesion. Any disease caused There is no direct evidence demonstrating whether asymptom- by inflammation/infection should be able to heal if the caus- atic apical periodontitis with cyst formation (pocket and true) ative irritant/irritants are removed, unless the irritant/irritants can or cannot regress after nonsurgical root canal therapy. are neoplasm-inducing agents or a carcinogen. 650 PART II The Advanced Science of Endodontics

beyond the scope of this chapter; the interested reader is ASYMPTOMATIC APICAL PERIODONTITIS encouraged to seek the cited references. WITH REACTIVE BONE FORMATION: CONDENSING OSTEITIS OR CHRONIC EXTRARADICULAR ENDODONTIC FOCAL SCLEROSING OSTEOMYELITIS INFECTION Asymptomatic apical periodontitis with reactive bone forma- Extraradicular endodontic infection implies that bacteria have tion is analogous to chronic osteomyelitis with proliferative established an infectious process outside the root canal system periostitis. The etiology and pathogenesis of these two diseases in the periapical area.243,260,284 Extraradicular endodontic infec- are not well understood. It is generally believed that both tion can only be diagnosed after surgical biopsy or microbio- lesions are caused by a long-term, low-grade inflammation/ logic sampling or molecular detection of therapy-resistant infection or a high resistance of local tissue to inflammation/ periapical lesions during apical surgery. The method of sample infection.195,210 Instead of bone resorption, the inflammation collection should be carefully reviewed, because contamina- induces reactive bone formation of alveolar trabecular or tion is a problem in bacteriologic study of apical periodontitis spongy bone around the periapex of endodontically involved lesions during apical surgery. teeth. Extraradicular endodontic infection can be a part of intraradicular infection or an independent entity without an Cell Biology intraradicular component. To be considered an independent As described previously, during the chronic stage of extraradicular infection, the presence of pathogens in the root apical periodontitis, both osteoclast and osteoblast activity canal system must be completely ruled out, because intra decreases.297 However, in asymptomatic apical periodontitis radicular bacteria pose more potential problems than extra with reactive bone formation, osteoblasts appear to be stimu- radicular bacteria in periapical inflammation. Bacteria in the lated to produce more bone. It is not clear what factor/factors complex root canal system are well protected from root canal stimulate osteoblasts to produce more bone mass. It could be procedures (i.e., mechanical instrumentation, antiseptic irriga- due to increased expression of growth factors/cytokines, such tion, intracanal medication), host defenses, and antimicrobial as TGF-β, BMP, PDGF, and transcription factor Cbfa1 (core- therapy because of a lack of blood circulation and areas resis- binding factor family).126 tant to access (e.g., isthmi; see Chapter 6). In our opinion, the independent extraradicular infection probably does not occur Histopathology often and is usually associated with apical actinomycosis.95,266 There is an excessive apposition of bone mass without bone Bacteria have been observed as biofilm on the apical external resorption in the apical area. As the bone marrow spaces root surface.243,283 In addition, viruses have also been isolated become smaller and obliterated, the bone resembles compact from apical periodontitis lesions.149,218,249 If microbiologic root bone that is infiltrated by a small number of lymphocytes. The canal cultures are not completely reliable (because bacteria compact bone has few lacunae, and many of them are empty may remain in the complex root canal system),222 then inter- of osteocytes. It has many prominent resting and reversal pretation of independent extraradicular infections as endodon- lines, similar to an idiopathic osteosclerosis or a Pagetoid tic treatment failures should be carefully evaluated. appearance.44,195,210 Although virulence and number of pathogens, as well as the host’s defenses, determine whether extraradicular infection Clinical Features will develop or not, the development of extraradicular infec- The lesion is usually observed in young patients, and the man- tion is still not fully understood. What are the predisposing dibular first molar is most commonly involved. The teeth often factors to extraradicular infection? How are bacteria able to have gross carious lesions and can be vital or nonvital. They evade the host’s powerful defenses in order to colonize and are usually asymptomatic. Radiographically, the lesion may establish an infectious process in the periapical tissues? Are all have a well-defined or ill-defined radiopaque mass associated extraradicular infections resistant to nonsurgical root canal with the apex of an endodontically involved tooth. The lamina therapy? For instance, are bacteria present in apical periodon- dura around the root apex is usually intact. titis with abscess formation?32,90,263,291 Importantly, most periapical abscesses heal after proper nonsurgical root canal Outcomes therapy. Similarly, most apical periodontitis lesions with drain- Most apical periodontitis lesions with reactive bone formation ing sinus tracts also heal after proper nonsurgical root canal demonstrate healing after nonsurgical root canal therapy. The therapy, because the primary source of infection in the root excessive compact bone in most cases will be remodeled to canal system is removed. The preponderance of evidence does normal appearance.69 not support the consistent presence of an independent periapical infection in endodontic infections. PERIAPICAL LESIONS OF NONENDODONTIC ORIGIN APICAL PERIODONTITIS AND Many periapical lesions are not of endodontic origin and should SYSTEMIC DISEASES be considered in the differential diagnosis of apical periodonti- Information concerning apical periodontitis and systemic dis- tis. These lesions include (but are not limited to) trauma,10,11 eases is limited. Clinical and radiographic studies have shown foreign bodies,133,187,313 host metabolic by-products,188 advanced that there is a greater prevalence of periapical lesions in diabet- periodontal disease,140 fibro-osseous lesions, and benign and ics than in nondiabetics.21 Type 2 diabetes mellitus is signifi- malignant tumors.195 A complete description of these lesions is cantly associated with an increased prevalence of apical CHAPTER 15 Pathobiology of Apical Periodontitis 651 periodontitis.229 Other findings demonstrated that type 2 dia- causes of genetic polymorphisms. Single nucleotide polymor- betes is associated with increased risk of a poor response by phisms (SNPs) that have a single base variation resulting from the periradicular tissues to odontogenic pathogens.31 In rodent insertion or deletion of a base are considered the most common models, diabetic conditions enhance the development of peri- type of genetic polymorphism.42 radicular lesions and increase mortality.73,113,131 Mice deficient Occurrence of SNP within the coding region of a gene may in P/E-selectin are shown to be more prone to infection- produce an altered protein, which may lead to altered function mediated early-onset periodontal disease.196 (termed functional SNPs). Occurrence of SNP within the pro- Genetic polymorphisms may also play a role in the indi- moter region of a gene may alter gene regulation, which may vidual’s response to various diseases. Haplotype analyses have cause reduction/inhibition or overexpression of gene expres- indicated that a variant of the IL-1β gene was likely to be most sion. Functional SNPs alter amino acid sequence or interfere important for early-onset periodontitis risk.62 In a Chinese with the transcriptional factor binding, whereas nonfunctional male population, it was reported that the polymorphisms of SNPs do not affect the regulation of protein expression. Many IL1A+4845 and IL1B-511 might play an important role in SNPs have no effect on cell function, but some could predis- determining generalized aggressive periodontitis susceptibility. pose people to disease or influence their response to drugs. A possible combined effect of the polymorphism of IL1B-511 Besides SNPs, there are single amino-acid polymorphisms and smoking on this type of periodontitis susceptibility was (SAAPs or SAPs) of proteins, which may result from RNA suggested.150 In a study on patients from Chile, the heterozy- editing independent of SNPs.308 gous inheritance of the IL1B+3954 gene was found signifi- A genetic polymorphism is defined as alleles or variants that cantly higher in periodontitis cases than in control patients. appear in at least 1% of the population. The 1% cutoff excludes The prevalence of a positive genotype (at least one allele of the mutations that may have occurred in a single family. In most two variants present at each locus) is significantly higher in polymorphisms, one allele dominates and appears with < 99% cases (26%) than in controls (10%) and significantly associated frequency (for example, 65%) in the population. In a biallelic with periodontitis, irrespective of smoking status and peri- situation, some authors term the dominant allele as normal odontitis severity.162 Those displaying high-producer IL6 geno- (N-allele or allele 1) and it occurs in < 99%, whereas the rarer type, intermediate and high-producer IL1B genotypes, and allele (R-allele or allele 2) is present in >1% in the popula- low-producer TNFA genotype tend to exhibit symptomatic tion.227,245 It has been long known that genetic polymorphisms dental abscesses.57 are associated with increased severity of inflammatory dis- Apical periodontitis was once considered a focus of infec- eases.98 Both endodontic and periodontic diseases are mainly tion, and the microorganisms from the focus could disseminate an infectious disease caused by microbial infection. They share through blood circulation to remote parts of the body, where common features in terms of inflammation and bone resorp- secondary disease occurred.108 Even though bacteremias do tion. Studies have implicated association of periodontitis or occur during root canal treatment of teeth with apical periodon- apical periodontitis with several gene polymorphisms such as titis when instrumentation is carried through the apical foramen IL-1, IL-6, IL-8, FcγR, tumor necrosis factor receptor superfam- into the periapical tissues, the incidence and magnitude of ily member 1B (TNFRSF1B), (selenoprotein S) SEPS1, and bacteremias is not clinically significant for a healthy person and matrix metalloproteinases (MMPs).7,45,129,134,174,175,309 in fact appears to be lower than that observed by flossing.19,58,59 It has long been observed that the proinflammatory cyto- Microorganisms in the blood circulation are rapidly eliminated kine interleukin-1 (IL-1) genotype is a severity factor in adult by the host’s humoral and cellular defense components within periodontal disease.134 The specific periodontitis-associated minutes.19,59 However, bacteremia may pose a potential danger IL-1 genotype has a variant in the IL-1B gene that is associated to immunocompromised patients or patients with congenital with high levels of IL-1 production. Gene polymorphism heart valve disease.58 No correlation between apical periodon- studies suggest that the frequency of IL-1β genotypes (IL- titis and coronal heart disease and rheumatoid arthritis has 1β+3953 restriction fragment length biallelic polymorphism) been observed.41,181 There is insufficient evidence to support is increased in patients with advanced adult periodontitis com- that apical periodontitis could serve as a focus of infection and pared to early and moderate combined, and to healthy con- cause significant systemic diseases.68,204 trols.84 A more recent study showed that IL-1β levels in gingival crevicular fluid is increased with severity of disease and correlates well with clinical signs of incipient disease.254 This GENETIC AND SYSTEMIC DISEASE suggests that some individuals, when challenged by bacterial RISK FACTORS OF PERSISTENT accumulations, may respond with a higher level of inflamma- APICAL PERIODONTITIS tory activities, therefore leading to more severe periodontitis. Both FcγRIIa (CD32) and FcγRIIIb (CD16) influence polymor- Genetic Risk Factors phonuclear leukocyte phagocytic function and therefore their Genetic polymorphism in the population plays an important polymorphisms may also influence inflammatory disease role in the variation of the susceptibility and development of development. It was reported that the FcγR IIIb-NA2 allotype diseases among individuals. Knowledge on the polymorphism represents a risk factor for recurrence of adult periodontitis,129 of key genes that are involved in disease development will help and the FcγRIIIa-158V allele and possibly FcγRIIIb-NA2 may be us to understand and establish prevention or treatment strate- associated with severity of chronic periodontitis in a Japanese gies for the diseases. Because of the completion of the Human population130; whereas the FcγRIIa-H/H131 genotype may be Genome Project, many advanced molecular technologies have associated with chronic periodontitis risk (and disease sever- been developed including high-throughput screening and ity) in Caucasian smokers.309 FcγRIIIa and FcγRIIIb genotypes sequencing which further help gather the information on poly- may impose an additional risk of periodontal bone loss in a morphism in human populations. There are different types and German population.174 652 PART II The Advanced Science of Endodontics

The polymorphisms of two other genes involved in inflam- increased hexosamine pathway flux.34 The tissue most mation, SEPS1 and TNFRSF1B, have been found to be involved frequently affected by hyperglycemia is microvasculature, in determining host individual susceptibility to aggressive peri- whereby endothelial cells are not able to transport intracellular odontitis, and there is the potential association between IL-6 glucose effectively.34 Because of damage to vasculature (athero- and FcγR polymorphisms and the aggressive periodontitis in a sclerosis), blood circulation, innate and adaptive immuno- Caucasian-Italian population.224 inflammatory mechanism, and phagocyte function are The genetic polymorphisms have been involved in the impaired,34,137 thus leading to pulp necrosis and predisposition development of apical periodontitis reports. Two genetic to pulpal infection and subsequent apical periodontitis.21 conditions—carriage of allele H131 of the FcγRIIa gene and a Sickle cell anemia is the prototype of hereditary globinopa- combination of this allele with allele NA2 of the FcγRIIIb thies, characterized by the production of structurally abnormal gene—have been reported to be associated with posttreatment hemoglobulin.137 The red blood cells are sickle shaped. Accu- apical periodontitis,245 whereas polymorphism in the FcγRIIIa mulation of distorted red cells could cause vasoocclusion, does not influence the patient’s response to endodontic treat- which could lead to anoxia, infarcts, and necrosis of tissue.100 ment of teeth with apical periodontitis in a Brazilian popula- The occurrence of asymptomatic pulp necrosis in clinically tion.246 So far there has been only one report on IL-1 intact permanent teeth has been reported.13,123,239 Pulp necrosis polymorphism association with apical periodontitis in an could predispose to pulpal infection and apical periodontitis American population.179 In this study, patients with persistent because of a lack of defense mechanism. apical periodontitis showed an increased prevalence in the Sjögren syndrome is a chronic, systemic autoimmune disor- genotype composed of allele 2 of IL-1β polymorphism when der.195 It involves salivary glands resulting in xerostomia. The compared with those who experienced complete healing after lack of salivary cleansing action predisposes the patient to dental acceptable root canal therapy (70.6% versus 24.6%). This caries. If the caries is not prevented or treated early, it would outcome suggests that specific genetic markers associated with impose risk of pulpal infection and apical periodontitis. IL-1β production may predict increased susceptibility to per- Radiation therapy of head and neck malignant lesions is sistent apical periodontitis. An earlier paper that studied a associated with a high occurrence of caries.104 If untreated, the Brazilian population did not find an association between IL1 radiation caries could lead to pulpal infection and subsequent polymorphism and persistent apical periodontitis; however, apical periodontitis. It has been shown that a radiation dose allele 1 (for IL-1A and IL-1B) was always detected higher in 66 to 72.2 Gy (gray) in the head and neck region was associ- diseased than in healthy/healing individuals, although not sta- ated with a high prevalence of caries and apical periodontitis tistically significant.245 This lack of statistical difference could because of a change in microflora favorable for carious be due to small sample size or different population pools. development.103 The polymorphism of the IL-8 gene, a potent chemokine However, there is no clear-cut direct causation or mecha- that attracts neutrophils to the site of infection, has also been nism between systemic disease risk factors and the incidence shown to influence the development of different forms of or persistence of apical periodontitis without prior caries and apical periodontitis.7 IL8/CXCL8-251 T allele, which is associ- pulp infection. ated with higher production of IL8/CXCL8, is also associated with a higher risk of developing acute suppurative form of apical periodontitis. IL8/CXCL8 -251 A allele, which is associ- WOUND HEALING OF APICAL ated with lower production of IL8/CXCL8, is associated with PERIODONTITIS chronic nonsuppurative form of apical periodontitis. SNP of other inflammation-associated genes, matrix metalloprotein- Periapical Wound Healing After ases (MMPs) 2 and MMP3, also show influence on periapical Nonsurgical Root Canal Therapy lesion formation.175 Thus, markers in MMP3 and MMP2 genes Understanding wound healing is as important as knowing the could also help predict host susceptibility to developing peri- pathogenesis of disease, because satisfactory wound healing is apical lesions and the healing response. the ultimate goal of treatment. If we are able to understand the mechanisms of periapical wound healing, we can design treat- Systemic Disease Risk Factors ment approaches that maximize favorable conditions for Many systemic diseases have been linked to periodontal wound healing to occur, such as effective disinfection of the disease127,199 because they contribute to either a decreased root canal system in nonsurgical root canal therapy, control of host’s resistance to infection or dysfunction in the connective periapical inflammation by medication, or incorporation of tissue of periodontium, increasing patient susceptibility to growth factors in bone grafts in surgical endodontic therapy immuno-inflammation-induced destruction.14 However, the (see also Chapters 12 and 21). similar systemic diseases do not appear to link to apical peri- Interestingly, healing begins as soon as inflammation starts. odontitis, possibly because periodontitis is an open lesion and When irritants (microbial and nonmicrobial) in the canal apical periodontitis is a closed lesion. systems or in the periapical tissues are eliminated by nonsurgi- Diabetes mellitus (DM), a systemic disease, has been well cal or surgical endodontic therapy, inflammatory mediators are documented to be associated with a high prevalence of apical no longer produced in the periapical tissues because of the periodontitis.21,74,113,163,167,228 DM is a chronic disorder of carbo- reduction of inflammatory cells. Inflammatory mediators hydrate, fat and protein metabolism, and a defective or deficient already present are inactivated by the body’s control mecha- insulin secretory response.137 Hyperglycemia is characteristic of nisms to prevent an inflammatory reaction from going DM and has profound effects on cell metabolisms, especially unchecked. This process precedes wound healing. Although a endothelial cells such as increased polyol pathway flux, great deal of information is known about what turns on the increased formation of advanced glycation end products, inflammation, relatively little is known about what turns off the hyperglycemia-induced activation of protein kinase C, and inflammatory system after elimination of irritants. Examples of CHAPTER 15 Pathobiology of Apical Periodontitis 653 host antiinflammatory control mechanisms are (1) enzyme study,12,145,146 no studies of wound healing of apical periodon- destruction of inflammatory activators; (2) natural inhibitors titis lesions with cyst formation after nonsurgical root canal of inflammatory mediators (opioids, somatostatin, glucocorti- therapy exist in the literature. coids); (3) relative balance between intracellular levels of cyclic Wound healing of apical periodontitis lesions after proper AMP (adenosine monophosphate) and cyclic GMP (guanosine nonsurgical root canal therapy follows the general principle of monophosphate); (4) the antiphlogistic role of histamine; (5) wound healing of connective tissues elsewhere in the body, inhibitors of the complement system287; and (6) antiinflamma- with the formation of fibrovascular granulation tissue, removal tory cytokines, such as IL-4, IL-10, IL-13, and TGF-β.93,193 In of necrotic tissue and dead bacteria by activated macrophages, addition, the major cellular inducer of inflammation, neutro- and finally repair or regeneration of the wounded tissue. philic leukocytes, undergo apoptosis,89 and the major cellular Healing of apical periodontitis lesions is largely accomplished player of wound healing, macrophages, secrete antiinflammatory by regeneration and to some degree by fibrosis. Local tissue molecules such as lipoxins, resolvins, and protectins.137,165,193 resident cells involved in periapical wound healing are osteo- The process of wound healing is tightly regulated by cell- blasts and bone marrow mesenchymal stem cells in alveolar to-cell cross-talk, cell-to–extracellular matrix interactions, and bone and multipotent stem cells in the periodontal ligament.233 cell surface receptors, as well as the temporal and spatial During periapical wound healing, many unwanted hyperplas- expression of a variety of cytokines, growth factors, and tic cells (e.g., endothelial cells, fibroblasts, epithelial cells) are neuropeptides and apoptosis (Table 15-3).29,86,89,257,302 All these deleted by apoptosis,60,86,89 and the extracellular matrix is cellular and humoral factors operate together in either an remodeled by metalloproteinases. Pathologic processes such as antagonistic or synergistic manner and are precisely orches- extensive fibrosis do not occur often, and the damaged periapi- trated during wound healing. This results in a highly organized cal tissues can be restored mostly to their original structure by response permitting regeneration of the original tissue archi- the process of regeneration. tecture. Wound healing appears to be a programmed event. The temporal and spatial relationship between alveolar Much information concerning the pathogenesis of apical peri- bone, cementum, and periodontal ligament during periapical odontitis has been gained from animal studies.75,119,281 Although wound healing after nonsurgical root canal therapy cannot be studies of wound healing of teeth with apical periodontitis clearly delineated. Nevertheless, wound healing appears to after nonsurgical root canal therapy and endodontic surgery recapitulate the embryonic morphogenesis of damaged tissues are available in a few animal experiments and a human or organs in many instances. The process of periapical wound healing after nonsurgical root canal therapy may be similar to wound healing following guided tissue regeneration in peri- TABLE 15-3 odontal therapy: regeneration of new cementum, new alveolar bone, and new periodontal ligament.67,285 Both nonsurgical root Important Growth Factors/Cytokines in canal therapy and guided tissue regeneration therapy in peri- Wound Healing odontal disease are intended to remove irritants and provide a Target Cells and Major favorable microenvironment conducive to regeneration of peri- Cytokines Major Source Effects odontal tissues damaged by apical periodontitis and marginal periodontitis, respectively. EGF Macrophages, platelets, Epithelial cells, fibroblasts, During periapical wound healing, the cells of viable peri- fibroblasts endothelial cells odontal ligament from adjacent root surfaces proliferate to FGF Macrophages, Endothelial cells cover the root surfaces in which the periodontal ligament was endothelial cells (angiogenesis), damaged by apical periodontitis and removed by macrophages. mesenchymal cells Proteins derived from the Hertwig epithelial root sheath (i.e., TGF-α Macrophages, platelets, Angiogenesis, fibroblasts enamel matrix proteins) are required for cementoblast differ- keratinocytes entiation from ectomesenchymal stem cells in the dental fol- licle during tooth development.251 The cells of the Hertwig TGF-β Macrophages, platelets Similar to EGF epithelial root sheath are absent in mature teeth.192,230 PDGF Macrophages, platelets, Chemoattractant for Nevertheless, the extracellular matrix and growth factors endothelial cells macrophages, fibroblasts of cementum (i.e., IGF-1, FGFs, EGF, BMP, TGF-β, PDGF) VEGF Macrophages, epithelial Angiogenesis sequestered after cemental resorption in mature teeth are cells capable of inducing proliferation, migration, attachment, and IGF Fibroblasts, epithelial Granulation tissue formation, differentiation of multipotent stem cells in the periodontal cells reepithelialization ligament into cementoblast-like cells and produce cementoid tissue on the root surface denuded of periodontal ligament.88,233 CSF Multiple cells Macrophages, granulation This is similar to reparative dentin formation by pulp stem tissue formation cells in direct pulp capping procedures where growth factors SP, CGRP Sensory nerve Endothelial cells, fibroblasts, such as TGF-β are released from dentin matrix binding keratinocytes sites.191,217,289 Root resorption that involves cementum or both CGRP, calcitonin gene–related peptide; CSF, colony-stimulating factor; EGF, epidermal cementum and dentin can only be repaired by cementoid growth factor; FGF, fibroblast growth factor; IGF, insulin-like growth factor; PDGF, tissue, because multipotent stem cells of the periodontal liga- platelet-derived growth factor; TGF, transforming growth factor; VEGF, vascular endo- ment are incapable of differentiating into odontoblasts that thelial growth factor. produce dentin.233 Modified from Majno G, Joris I: Cell, tissues, and disease, ed 2, Oxford, 2004, Oxford University Press; Slauson DO, Cooper BJ: Mechanisms of disease, ed 3, St Louis, 2002, Bone has a remarkable capacity for regeneration in Mosby; Werner S, Grose R: Regulation of wound healing by growth factors and cyto- response to injury. During periapical wound healing, the kines, Physiol Rev 83:835, 2003. osteoprogenitor cells or mesenchymal cells lining the surfaces 654 PART II The Advanced Science of Endodontics of endosteum—stimulated by TGF-β, BMPs, IGFs, PDGF, lesions might regress after nonsurgical root canal therapy by VEGF, and cytokines released by stromal cells, osteoblasts, the mechanism of apoptosis or programmed cell death, based platelets, and bone matrix after bone resorption—can undergo on molecular cell biology.158 In contrast, apical true cysts may proliferation and differentiation into osteoblasts and produce be less likely to heal after nonsurgical root canal therapy bone matrix.6,161 When one of the cortical bone plates (buccal because of their self-sustaining nature.185 Histologically, or lingual/palatal) is destroyed, osteoprogenitor cells in the inflammatory cell infiltration is always present in the lining inner layer of periosteum beneath the oral mucosa—stimulated epithelium or fibrous connective tissue capsule of apical true by TGF-β, BMPs, IGFs, PDGF, and VEGF—are also capable of cysts.185,214,215 This indicates there is continued presentation of proliferation and differentiation into osteoblasts and can irritants (such as bacteria)—present in the root canal system, produce bone matrix.126,161 If both buccal and lingual/palatal the periapical tissues, or the lumen of cysts—to attract inflam- cortical bone plates are destroyed by large apical periodontitis matory cells to the cystic lining epithelium or fibrous connec- lesions, it is possible that the lesion may be repaired with tive tissue capsule.186,214,215 It is not known if epithelial cells of fibrous scar tissue because of extensive destruction of the periapical true cysts alone are capable of acting as autocrine cells osteum beneath the oral mucosa.12 Accordingly, a guided tissue and secreting growth factors to sustain their own survival. It regeneration procedure using membrane barriers and bone is important to realize that the apical true cyst is completely grafts is recommended to prevent ingrowth of fibroblasts from different from the odontogenic keratocyst, which is self- periosteum or submucosa into the bony defect and to enhance sustaining because it is a neoplastic lesion. Biologically, it is periapical wound healing if periapical surgery is necessary.54,55 unlikely that the hyperplastic epithelial cells of inflammatory The cellular and molecular mechanisms of excess scar tissue apical true cysts would suddenly transform into cells that formation in periapical wound healing are not completely behave like self-sustaining neoplasms. Any disease caused by understood. Growth factors/cytokines may play an important infection should be able to regress after removal of its caus- role in regulating fibroblast gene expression and excess scar ative irritant(s), unless the irritants themselves are neoplasm- tissue formation.288 inducing agents or carcinogens, such as some viruses and The newly regenerated periodontal ligament will finally human malignant tumors.165,294 From pathogenesis, histology, undergo remodeling into a mature periodontal ligament, with and molecular cell biology, apical true cysts are similar to one group of collagen fibers (Sharpey fibers) inserted into the pocket cysts. Accordingly, apical true cysts, similar to pocket newly formed cementum and another group of collagen fibers cysts, may be able to regress after nonsurgical root canal inserted into the newly formed alveolar bone. Thereby, regen- therapy by the mechanism of apoptosis if root canal infection eration of damaged periapical tissues, cementum, periodontal is effectively under control.158 This prediction is consistent ligament, and alveolar bone is completed. with the high level of healing observed after nonsurgical root canal treatment. In apical periodontitis lesions with cyst formation, the Periapical Wound Healing After Surgical cysts have to regress before the periapical tissues can be Endodontic Therapy restored to their original architecture. It is not known what The mechanism of periapical wound healing after nonsurgical matrix serves as a scaffold for endothelial cells, fibroblasts, and surgical endodontic therapy is similar, but the kinetics of and osteoblasts to migrate into the lumen of regressing cysts healing of the periapical wound after endodontic surgery is after nonsurgical root canal therapy. Complete regression of faster than nonsurgical endodontic therapy.138 In surgical end- radicular cysts after nonsurgical root canal therapy could be odontic therapy, a clinician removes irritants, such as necrotic due to any of several possible scenarios. Regression of radicu- cells, tissue debris, and bacteria in the periapical lesions, which lar cysts and regeneration of bone may occur concurrently; or is called surgical debridement.157,165 In contrast, in nonsurgical during regression of radicular cysts, part of the cystic lining endodontic therapy, activated macrophages perform bacterial epithelium could disintegrate due to apoptosis of local epi- killing and cleanup of periapical lesions, which is called bio- thelial cells. Together with degradation of the basal lamina by logic debridement.165 Surgical debridement is very effective and matrix metalloproteinases, this could allow a fibrous connec- of course quite rapid, whereas biologic debridement takes tive tissue capsule to grow into the lumen of radicular cysts. time. However, endodontic surgery is more invasive. In addi- Eventually, the cystic lining epithelium will completely tion, proper case selection is more important in endodontic regress or become remnants of epithelial cell rests remaining surgery than in nonsurgical endodontic therapy. The distinc- in the periodontal ligament. tive difference between nonsurgical and surgical endodontic Taken together, our knowledge of cyst mechanisms and therapy is that the goal of nonsurgical endodontic therapy is healing offers tantalizing clinical implications and clearly sup- to remove primary microbial etiology from the root canal ports the conclusion that more studies are required to under- system, and the goal of surgical endodontic therapy is often to stand the complex mechanisms of regression of inflammatory seal microbial etiology within the root canal system by root- apical cysts, both pocket and true. end filling in most cases (see also Chapter 21).

Can Radicular Cysts in Apical Periodontitis FACTORS INFLUENCING Lesions Regress After Nonsurgical PERIAPICAL WOUND HEALING Endodontic Therapy? AFTER ENDODONTIC THERAPY Based on histology and cell biology, no studies have ever Local and systemic factors may affect periapical wound healing. shown that apical true cysts are different from apical pocket Infection will complicate and prevent wound healing, foreign cysts. It was suggested that pocket cysts in apical periodontitis bodies can impair wound healing,187,313 and nutrition can also CHAPTER 15 Pathobiology of Apical Periodontitis 655

affect wound healing.305 Diabetes was reported to reduce the jawbones,212,216,306 so nonsurgical root canal therapy is recom- likelihood of healing of apical periodontitis lesions after non- mended for these patients.121,152 However, for endodontic surgical root canal therapy.74 Impaired nonspecific immune surgery, the guidelines of the American Association of Oral and response and disorders of the vascular system appeared to have Maxillofacial Surgeons position paper on bisphosphonate- a significant influence on the success rate of nonsurgical related osteonecrosis of the jaws are highly recommended.8 For root canal therapy on teeth with apical periodontitis.166 asymptomatic patients who have taken an oral bisphosphonate However, immunocompromised patients, such as HIV patients, for less than 3 years and have no clinical risk factors, endodon- responded as well as counterpart patients after nonsurgical tic surgery is not contraindicated.8 Nevertheless, the patient’s endodontic therapy.49,82,207 Although smoking has not been physician should be consulted. Any kind of surgical procedure shown to be associated with increased incidence of apical peri- should be avoided for patients receiving intravenous bisphos- odontitis and prognosis of nonsurgical root canal therapy,23,63 phonate.8 To prevent the complication of osteonecrosis, if smoking may increase complications of periapical surgery, patients are going to receive radiation of jaws or bisphospho- such as pain and swelling.80 nate therapy, nonsurgical or surgical endodontic therapy Patients receiving radiotherapy of jaws and bisphospho- should be completed before the initiation of radiation or nate therapy have a risk of developing osteonecrosis of bisphosphonate therapy.8

REFERENCES 1. Abbas AK, Lichtman AH, Pober JS: Cellular and molecular 19. Baumgartner JC, Heggers JP, Harrison JW: The incidence 37. Burd PR, Rogers HW, Gordon JR, et al: Interleukin immunology, Philadelphia, 2007, Saunders. of bacteremias related to endodontic procedures. I. 3-dependent and -independent mast cells stimulated with 2. Abous-Rass M, Bogen G: Microorganisms in closed Non-surgical endodontics, J Endod 2:135, 1976. IgE and antigen express multiple cytokines, J Exp Med periapical lesions, Int Endod J 31:39, 1998. 20. Bender IB: Factors influencing the radiographic 170:245, 1989. 3. Akamine A, Hashiguchi I, Toriya Y, Maeda K: appearance of bone lesions, J Endod 8:161, 1982. 38. Byers MR, Narhi MVO: Dental injury models: experimental Immunohistochemical examination of the localization of 21. Bender IB, Bender AB: Diabetes mellitus and the dental tools for understanding neuroinflammatory nociceptor macrophages and plasma cells in induced rat’s periapical pulp, J Endod 29:383, 2003. functions, Crit Rev Oral Biol 10:4, 1999. lesions, Endod Dent Traumatol 10:121, 1994. 22. Bender IB, Seltzer S: Roentgenographic and direct 39. Byers MR, Taylor PE, Khayat BG, Kimberly CL: Effects of 4. Akira S, Takeda K, Kaisho T: Toll-like receptors: critical observation of experimental lesions in bone: 1, J Am Dent injury and inflammation on the pulp and periapical nerves, proteins linking innate and acquired immunity, Nature Assoc 62:152, 1961. J Endod 16:78, 1990. Immunol 2:675, 2001. 23. Bergstrom J, Babcan J, Eliasson S: Tobacco smoking 40. Canalis E, McCarthy TL, Centrella M: Growth factors and 5. Akira S, Uematsu S, Takeuchi O: Pathogen recognition and dental periapical condition, Eur J Oral Sci 112:115, cytokines in bone cell metabolism, Annu Rev Med 42:17, and innate immunity, Cell 124:783, 2006. 2004. 1991. 6. Al-Aql ZS, Alagl AS, Graves DT, et al: Molecular 24. Bezerra MC, Carvalho JF, Prokopowitsch AS, Pereira RM: 41. Caplan DJ, Chasen JB, Krall EA, et al: Lesions of mechanisms controlling bone formation during fracture RANK, RANKL and osteoprotegerin in arthritic bone loss, endodontic origin and risk of coronary heart disease, healing and distraction osteogenesis, J Dent Res 87:107, Braz J Med Biol Res 38:161, 2005. J Dent Res 85:996, 2006. 2008. 25. Birek C, Heersche JN, Jez D, Brunette DM: Secretion of a 42. Cargill M, Altshuler D, Ireland J, et al: Characterization of 7. Amaya MP, Criado L, Blanco B, et al: Polymorphisms of bone resorbing factor by epithelial cells cultured from single-nucleotide polymorphisms in coding regions of pro-inflammatory cytokine genes and the risk for acute porcine rests of Malassez, J Periodontal Res 18:75, human genes, Nat Genet 22:231, 1999. suppurative or chronic nonsuppurative apical periodontitis 1983. 43. Caviedes-Bucheli J, Muñoz HR, Azuero-Holguín MM, Ulate in a Colombian population, Int Endod J 46:71, 2013. 26. Birklein F, Schmelz M: Neuropeptides, neurogenic E: Neuropeptides in dental pulp: the silent protagonists, 8. American Association of Oral and Maxillofacial Surgeons: inflammation and complex regional pain syndrome J Endodon 34:773, 2008. Position paper on bisphosphonate-related osteonecrosis (CRPS), Neurosci Lett 437:199, 2008. 44. Cawson RA, Everson JW: Oral pathology and diagnosis, of the jaws. Rosemont, IL, 2006, AAOMS Board of 27. Block RM, Bushell A, Rodrigues H, Langeland K: A Philadelphia, 1987, Saunders. Trustees, September. histopathologic, histobacteriologic, and radiographic study 45. Chai L, Song YQ, Zee KY, Leung WK: SNPs of Fc-gamma 9. American Dental Association: 1990 survey of dental of periapical endodontic surgical specimens, Oral Surg receptor genes and chronic periodontitis, J Dent Res services rendered. Chicago, 1994, American Dental Oral Med Oral Pathol 42:656, 1976. 89:705, 2010. Association. 28. Boyle WJ, Simonet WS, Lacey DL: Osteoclast 46. Chedid M, Rubin JS, Csaky KG, Aaronson SA: Regulation 10. Andreasen FM: Transient apical breakdown and differentiation and activation, Nature 423:337, 2003. of keratinocyte growth factor gene expression by its relation to color and sensitivity changes after 29. Brian SD: Sensory peptides: their role in inflammation interleukin 1, J Biol Chem 269:10753, 1994. luxation injuries to teeth, Endod Dent Traumatol 2:9, and wound healing, Immunopharmacology 37:133, 47. Cochran DL, Wozney JM: Biological mediators for 1986. 1997. periodontal regeneration, Periodontol 2000 19:40, 1999. 11. Andreasen JO, Andreasen FM: Textbook and color atlas 30. Brinkmann V, Zychlinsky A: Beneficial suicide: why 48. Cook DN, Pisetsky DS, Schwartz DA: Toll-like receptors in of traumatic injuries to the teeth, ed 3, Copenhagen, neutrophils die to make NETs, Nat Rev Microbiol 5:577, the pathogenesis of human disease, Nat Immunol 5:975, 1994, Munksgaard. 2007. 2004. 12. Andreasen JO, Rud J: Modes of healing histologically 31. Britto LR, Katz J, Guelmann M, Heft M: Periradicular 49. Cooper H: Root canal treatment in patients with HIV after endodontic surgery in 70 cases, Int Oral Surg 1:148, radiographic assessment in diabetic and control infection, Int Endod J 26:369, 1993. 1972. individuals, Oral Surg Oral Med Oral Pathol Oral Radiol 50. Cortesini R, LeMaoult J, Ciubotariu R, Cortesini NS: 13. Andrews CH, England MC, Kemp WB: Sickle cell anemia: Endod 96:449, 2003. CD8+CD28- T suppressor cells and the induction of an etiological factor in pulp necrosis, J Endod 9:249, 32. Brook I, Fraizier EH, Gher ME: Aerobic and anaerobic antigen-specific, antigen-presenting cell-mediated

1986. microbiology of periapical abscess, Oral Microbiol suppression of TH reactivity, Immunol Rev 182:201, 2001. 14. Armitage GC: Periodontal diagnoses and classification of Immunol 6:123, 1991. 51. Costerton JW, Geesey GG, Chen KJ: How bacteria stick, periodontal disease, Periodontology 2000 34:9, 2004. 33. Browne RM: The origin of cholesterol in odontogenic cysts Sci Am 238:86, 1978. 15. Babior BM: The respiratory burst of phagocytes, J Clin in man, Arch Oral Biol 16:107, 1971. 52. Cymerman JJ, Cymerman DH, Walters I, Nevins AJ: Invest 73:599, 1984. 34. Brownless M: The pathobiology of diabetes complications: Human T lymphocyte subpopulations in chronic periapical 16. Baron R: Molecular mechanisms of bone resorption by the a unifying mechanism, Diabetes 54:1615, 2005. lesions, J Endod 10:9, 1984. osteoclasts, Anat Rec 224:317, 1989. 35. Brunette DM: Cholera toxin and dibutyl cyclic-AMP 53. Dahlen G, Magnusson BC, Moller A: Histological and 17. Barthel CR, Zimmer S, Trope M: Relationship of radiologic stimulate the growth of epithelial cells derived from histochemical study of the influence of lipopolysaccharide and histologic signs of inflammation in human root-filled epithelial cell rests from porcine periodontal ligament, extracted from Fusobacterium nucleatum on the periapical teeth, J Endod 30:75, 2004. Arch Oral Biol 29:303, 1984. tissues in the monkey Macaca fascicularis, Arch Oral Biol 18. Bartkiewicz M, Hernando N, Reddy SV, et al: 36. Brynolf I: A histological and roentgenological study of the 26:591, 1981. Characterization of the osteoclast vacuolar H+-ATPase periapical region of human upper incisors, Odontol Rev 54. Dahlin C, Gottlow J, Linde A, Nyman S: Healing of B-subunit. Gene 160:157, 1995. 18(suppl 11):1, 1967. maxillary and mandibular bone defects using a membrane 656 PART II The Advanced Science of Endodontics

technique: an experimental study in monkeys, Scand J 79. Gao Z, Mackenzie IC, Rittman BR, et al: 103. Hommez GG, De Meerkeer GO, De Neve WJ, De Meer Plast Reconstr Hand Surg 24:13, 1990. Immunocytochemical examination of immune cells in RJG: Effect of radiation dose on the prevalence of apical 55. Dahlin C, Linde A, Gottlow J, Nyman S: Healing of bone periapical granulomas and odontogenic cysts, J Oral periodontitis: a dosimetric analysis, Clin Oral Invest defects by guided tissue regeneration, Plast Reconstr Pathol 17:84, 1988. 16:1543, 2012. Surg 81:672, 1988. 80. Garcia B, Penerrocha M, Marti E, et al: Pain and swelling 104. Hong CHL, Napenas JJ, Hodgson BD, et al, editors: A 56. Damoiseaux J: Regulatory T cells: back to the future, after periapical surgery related to oral hygiene and systematic review of dental disease in patients undergoing Neth J Med 64:4, 2006. smoking, Oral Surg Oral Med Oral Pathol Oral Radiol cancer therapy, Support Care Cancer 18:1007, 2010. 57. de Sa AR, Moreira PR, Xavier GM, et al: Association of Endod 104:271, 2007. 105. Hou L, Sasakj H, Stashenko P: B-Cell deficiency CD14, IL1B, IL6, IL10 and TNFA functional gene 81. Giachelli CM, Steitz S: Osteopontin: a versatile regulator predisposes mice to disseminating anaerobic infections: polymorphisms with symptomatic dental abscesses, Int of inflammation and biomineralization, Matrix Biol 19:615, protection by passive antibody transfer, Infect Immun Endod J 40:563, 2007. 2000. 68:5645, 2000. 58. Debelian GJ, Olsen I, Transtad L: Systemic diseases 82. Glick M, Abel SN, Muzyka BC, DeLorenzo M: Dental 106. Hou L, Sasaki H, Stashenko P: Toll-like receptor caused by oral microorganisms, Endod Dent Traumatol complications after treating patients with AIDS, J Am Dent 4-deficient mice have reduced bone destruction following 10:5, 1994. Assoc 125:296, 1994. mixed anaerobic infection, Infect Immun 68:4681, 59. Debelian GJ, Olsen I, Transtad L: Bacteremia in 83. Gonzalez-Rey E, Chorny A, Delgado M: Regulation of 2000. conjunction with endodontic therapy, Endod Dent immune tolerance by anti-inflammatory neuropeptides, 107. Hou L, Wang X: PKC and PKA, but not PKG mediate 2+ Traumatol 11:142, 1995. Nat Rev Immunol 7:52, 2007. LPS-induced CGRP release and (Ca )I elevation in DRG 60. Desmouliere A, Redard M, Darby I, Gabbiani G: Apoptosis 84. Gore EA, Sanders JJ, Pandey JP, et al: Interleukin- neurons of neonatal rats, J Neurosci Res 66:592, 2001. mediates the decrease in cellularity during the transition 1beta+3953 allele 2: association with disease status in 108. Hunter W: Oral sepsis as a cause of disease, Br Med J between granulation tissue and scar, Am J Pathol adult periodontitis, J Clin Periodontol 25:781, 1998. 2:215, 1900. 146:56, 1995. 85. Green TL, Walton RE, Taylor JK, Merrell P: Radiographic 109. Huumonen S, Orstavik D: Radiological aspects of apical 61. Dickerson C, Undem B, Bullock B, Winchurch RA: and histologic findings of root canal treated teeth in periodontitis, Endod Topics 1:3, 2002. Neuropeptides regulation of proinflammatory cytokine cadaver, Oral Surg Oral Med Oral Pathol Oral Radiol 110. Ingle J, Bakland L, Baugartner C: Ingle’s endodontics, responses, J Leukocyte Biol 63:602, 1998. Endod 83:707, 1997. ed 6, Hamilton, 2008, BC Decker. 62. Diehl SR, Wang Y, Brooks CN, et al: Linkage 86. Greenhalgh DG: The role of apoptosis in wound healing, 111. Irwin CR, Schor SL, Ferguson NW: Expression of disequilibrium of interleukin-1 genetic polymorphisms with Int J Biochem Cell Biol 30:1019, 1998. EGF-receptor on epithelial and stromal cells of normal and early-onset periodontitis, J Periodontol 70:418, 1999. 87. Grossman RM, Kreuger J, Yourish D, et al: Interleukin-6 inflamed gingiva, J Periodont Res 26:388, 1991. 63. Duncan HF, Pitt Ford TR: The potential association is expressed in high levels in psoriatic skin and stimulates 112. Isaka J, Ohazama A, Kobayashi M, et al: Participation of between smoking and endodontic disease, Int Endo J proliferation of cultured human keratinocyte, Proc Nat periodontal ligament cells with regeneration of alveolar 39:843, 2006. Acad Sci U S A 86:6367, 1989. bone, J Periodontol 72:314, 2001. 64. Dwyer TG, Torabinejad M: Radiographic and histologic 88. Grzesik WJ, Narayanan AS: Cementum and periodontal 113. Iwama A, Nishigaki N, Nakamura K, et al: The effect of high evaluation of the effect of endotoxin on the periapical wound healing and regeneration, Crit Rev Oral Biol Med sugar intake on the development of periradicular lesions in tissues of the cat, J Endod 7:31, 1980. 13:474, 2002. rats with type 2 diabetes, J Dent Res 82:322, 2003. 65. Dziak R: Biochemical and molecular mediators of bone 89. Haanen C, Vermes IV: Apoptosis and inflammation, 114. Iwu C, MacFarlane TW, MacKenzie D, Stenhouse D: The metabolism, J Periodontol 64:407, 1993. Mediators Inflamm 4:5, 1995. microbiology of periapical granulomas, Oral Surg Oral 66. Echchannaoui H, Frei K, Schnell C, et al: Toll-like receptor 90. Haapasalo M, Ranta K, Ranta H: Mixed anaerobic Med Oral Pathol 69:502, 1990. 2-deficient mice are highly susceptible to Streptococcus periapical infection with sinus tract, Endod Dent Traumatol 115. Janeway CA, Medzhitov R: Innate immune recognition, pneumoniae meningitis because of reduced bacterial 3:83, 1987. Annu Rev Immunol 20:197, 2002. clearing and enhanced inflammation, J Infect Dis 91. Hahn C-L, Liewehr FR: Innate immune responses of the 116. Janeway CA, Travers P, Walport M, Shlomchik MJ: 186:798, 2002. dental pulp to caries, J Endod 33:643, 2007. Immunobiology: the immune system in health and 67. Egelberg J: Regeneration and repair of periodontal 92. Hammarstrom L, Lindskog S: General morphological disease, ed 6, New York, 2005, Garland Science. tissues, J Periodontal Res 22:233, 1987. aspects of resorption of teeth and alveolar bone, 117. Jimenez-Pinzon A, Segura-Egea JJ, Poyato-Ferrera M, 68. Ehrmann EH: Focal infection: the endodontic point of Int Endod J 18:93, 1985. et al: Prevalence of apical periodontitis and frequency of view, Oral Surg Oral Med Oral Pathol 44:628, 1977. 93. Hanada T, Yoshimura A: Regulation of cytokine signaling root-filled teeth in an adult Spanish population, Int Endod 69. Eliasson S, Halvarson C, Ljunheimer C: Periapical and inflammation, Cytokine Growth Factor Rev 13:413, J 37:167, 2004. condensing osteitis and endodontic treatment, Oral Surg 2002. 118. Johnson GM, Lee DA, Regelmann WE, et al: Interference Oral Med Oral Pathol 57:195, 1984. 94. Hancock HH III, Sigurdsson A, Trope M, Moiseiwitsch J: with granulocyte function by Staphylococcus epidermidis 70. Eriksen HM, Bjertness E: Prevalence of apical periodontitis Bacteria isolated after unsuccessful endodontic treatment slime, Infect Immun 54:13, 1986. and results of endodontic treatment in middle-aged adults in a North American population, Oral Surg Oral Med Oral 119. Kakehashi S, Stanley H, Fitzgerald R: The effect of in Norway, Dent Traumatol 7:1, 1991. Pathol Oral Radiol Endod 91:579, 2001. surgical exposures of dental pulps in germ-free and 71. Figdor D: Apical periodontitis: a very prevalent problem, 95. Happonen RP: Periapical actinomycosis: a follow-up study conventional laboratory rats, Oral Surg Oral Med Oral Oral Surg Oral Med Oral Pathol Oral Radiol Endod 94:65, of 16 surgically treated cases, Endod Dent Traumatol Pathol 20:340, 1965. 2002. 2:205, 1986. 120. Kaneko T, Okiji T, Kan L, et al: Ultrastructural analysis of 72. Formigli L, Orlandini SZ, Tonelli P, et al: Osteolytic 96. Hargreaves KM, Goodis HE: Dental pulp, Chicago, 2002, MHC class II molecule-expressing cells in experimentally processes in human radicular cysts: morphological Quintessence. induced periapical lesions in the rat, J Endod 27:337, and biochemical results, J Oral Pathol Med 24:216, 97. Hargreaves KM, Swift JQ, Roszkowski MT, et al: 2001. 1995. Pharmacology of peripheral neuropeptide and 121. Katz H: Endodontic implications of bisphosphonate- 73. Fouad AF, Barry J, Russo J, et al: Periapical lesion inflammatory mediator release, Oral Surg Oral Med Oral associated osteonecrosis of the jaws: a report of three progression with controlled microbial inoculation in a type Pathol 78:503, 1994. cases, J Endod 31:831, 2005. I diabetic mouse model, J Endod 28:8, 2002. 98. Hart TC, Kornman KS: Genetic factors in the pathogenesis 122. Kawashima N, Okiji T, Kosaka T, Suda H: Kinetics of 74. Fouad AF, Burleson J: The effect of diabetes mellitus on of periodontitis, Periodontol 2000 14:202, 1997. macrophages and lymphoid cells during the development endodontic treatment outcome: data from an electronic 99. Hauman CHJ, Love RM: Biocompatibility of dental of experimentally induced periapical lesions in rat molars, patient record, J Am Dent Assoc 134:43, 2003. materials used in contemporary endodontic therapy: a J Endod 22:311, 1996. 75. Fouad AF, Walton RE, Rittman BR: Induced periapical review. Part 2. Root-canal filling materials, Int Endod J 123. Kaya AD, Aktener BO, Unsal P: Pulp necrosis with sickle lesions in ferret canines: histological and radiographic 36:147, 2003. cell anemia, Int Endod J 37:602, 2004. evaluation, Dent Traumatol 8:56, 1992. 100. Hebble PR, Vercellotti GM, Nath KA: A systems 124. Kayaoglu G, Orstavik D: Virulence factors of Enterococcus 76. Friedman S: Considerations and concepts of case biology consideration of the vasculopathy of the sickle faecalis: relationship to endodontic disease, Crit Rev Oral selection in the management of post-treatment cell anemia: the need for multi-modality chemo- Biol Med 15:308, 2004. endodontic disease (treatment failure), Endod Topics prophylaxis, Cardiovasc Hematol Disord Drug Targets 125. Khan AA, Hargreaves KM: Dental pain. In Trup JC, 1:54, 2002. 9:271, 2009. Sommer C, Hugger A, editors: The puzzle of orofacial 77. Fuchs TA, Abed U, Goosmann C, et al: Novel cell death 101. Hirsh BC, Johnson WC: Concepts of granulomatous pain, Basel, 2007, Karger. program leads to neutrophil extracellular traps, J Cell Biol inflammation, Int J Dermatol 23:90, 1984. 126. Kierszenbaum AL: Histology and cell biology, St. Louis, 176:231, 2007. 102. Hirvonen T, Hippi P, Narhi M: The effect of an opioid 2002, Mosby. 78. Gao Z, Falitz CM, Mackenzie IC: Expression of antagonist and somatostatin antagonist on the nerve 127. Kim J, Amar S: Periodontal disease and systematic keratinocyte growth factor in periapical lesions, J Dent function in normal and inflamed dental pulps, J Dent Res conditions: a bidirectional relationship, Odontology 94:10, Res 75:1658, 1996. 77:1329, 1998. 2006. CHAPTER 15 Pathobiology of Apical Periodontitis 657

128. Kimberly CL, Byers MR: Inflammation of rat molar pulp generalized aggressive periodontitis in a Chinese of MMP2 and MMP3 genes to the development of and periodontium causes increased calcitonin population, J Periodontol 75:1627, 2004. periapical lesions and healing response, J Endod 38:604, gene-related peptide and axonal sprouting, Ant Rec 151. Liapatas S, Nakou M, Rontogianni D: Inflammatory 2012. 222:289, 1988. infiltrate of chronic periradicular lesions: an 176. Molander A, Reit C, Dahlen G, Kvist T: Microbiological 129. Kobayashi T, Westerdaal NA, Miyazaki A, et al: Relevance immunohistochemical study, Int Endod J 36:464, 2003. status of root-filled teeth with apical periodontitis, of immunoglobulin G Fc receptor polymorphism to 152. Lilly JP, Cox D, Arcuri M, Krell KV: An evaluation of root Int Endod J 31:1, 1998. recurrence of adult periodontitis in Japanese patients, canal treatment in patients who have received irradiation 177. Moldauer I, Velez I, Kuttler S: Upregulation of basic Infect Immun 65:3556, 1997. to the mandible and maxilla, Oral Surg Oral Med Oral fibroblast growth factor in human periapical lesions, 130. Kobayashi T, Yamamoto K, Sugita N, et al: The Fc gamma Pathol Oral Radiol Endod 86:224, 1998. J Endod 32:408, 2006. receptor genotype as a severity factor for chronic 153. Lin L, Langeland K: Innervation of inflammatory periapical 178. Moller AJR, Fabricius L, Dahlen G, et al: Influence on periodontitis in Japanese patients, J Periodontol 72:1324, lesions, Oral Surg Oral Med Oral Pathol 51:535, 1981. periapical tissues of indigenous oral bacteria and necrotic 2001. 154. Lin L, Langeland K: Light and electron microscopic study pulp tissue in monkeys, Scand J Dent Res 89:29, 1981. 131. Kohsaka T, Kumazawa M, Yamasaki M, Nakamura H: of teeth with carious pulp exposures, Oral Surg Oral Med 179. Morsani JM, Aminoshariae A, Han YW, et al: Genetic Periapical lesions in rats with streptozotocin-induced Oral Pathol 51:292, 1981. predisposition to persistent apical periodontitis, J Endod diabetes, J Endod 22:418, 1996. 155. Lin L, Shovlin F, Skribner J, Langeland K: Pulp biopsies 37:455, 2011. 132. Kopp W, Schwarting R: Differentiation of T lymphocyte from the teeth associated with periapical radiolucency, 180. Mundy GR: Inflammatory mediators and the destruction of subpopulations, macrophages, and HLA-DR-restricted J Endod 10:436, 1984. bone, J Periodont Res 26:213, 1991. cells of apical granulation tissue, J Endod 15:72, 1989. 156. Lin LM, Di Fiore PM, Lin JL, Rosenberg PA: Histological 181. Murray CA, Saunders WP: Root canal treatment and 133. Koppang HS, Koppang R, Solheim T, et al: Cellulose fibers study of periradicular tissue responses to uninfected and general health: a review of the literature, Int Endod J from endodontic paper points as an etiological factor in infected devitalized pulps in dogs, J Endod 32:34, 2006. 33:1, 2000. postendodontic periapical granulomas and cysts, J Endod 157. Lin LM, Gaengler P, Langeland K: Periapical curettage, 182. Nagatomo K, Komaki M, Sekiya Y, et al: Stem cell 15:369, 1989. Int Endod J 29:220, 1996. properties of human periodontal ligament cells, J 134. Kornman KS, Crane A, Wang HY, et al: The interleukin-1 158. Lin LM, Huang G T-J, Rosenberg P: Proliferation of Periodontal Res 41:303, 2006. genotype as a severity factor in adult periodontal disease, epithelial cell rests, formation of apical cysts, and 183. Nair PNR: Light and electron microscopic studies on root J Clin Periodontol 24:72, 1997. regression of apical cysts after periapical wound healing, canal flora and periapical lesions, J Endod 13:29, 1987. 135. Kovacevic M, Tamarut T, Jonjic N, et al: The transition J Endod 33:908, 2007. 184. Nair PNR: Apical periodontitis: a dynamic encounter from pulpitis to periapical periodontitis in dog’s teeth, Aust 159. Lin LM, Pascon EA, Skribner J, et al: Clinical, between root canal infection and host response, Endod J 34:12, 2008. radiographic, and histological study of endodontic Periodontol 2000 13:121, 1997. 136. Kramer IR, Pindberg JJ, Shear M: WHO histological typing treatment failures, Oral Surg Oral Med Oral Pathol 185. Nair PNR: New perspectives on radicular cysts: do they of odontogenic tumors, ed 2, Geneva, 1992, Springer 11:603, 1991. heal?, Int Endod J 31:155, 1998. Verlag. 160. Lin LM, Wang SL, Wu-Wang C, et al: Detection of 186. Nair PNR, Pajarola G, Schroeder HE: Types and incidence 137. Kumar V, Abbas AK, Fausto N, et al, editors: Robbins and epidermal growth factor receptor in inflammatory of human periapical lesions obtained with extracted teeth, Cotran pathologic basis of disease, ed 8, Philadelphia, periapical lesions, Int Endod J 29:179, 1996. Oral Surg Oral Med Oral Pathol 81:93, 1996. 2010, Saunders. 161. Linkhart TA, Mohan S, Baylink DJ: Growth factors for 187. Nair PNR, Sjogren U, Krey G, Sundqvist G: Therapy- 138. Kvist T, Reit C: Results of endodontic retreatment: a bone growth and repair, Bone 19:1S, 1996. resistant foreign body giant cell granuloma at the periapex randomized clinical study comparing surgical and 162. Lopez NJ, Jara L, Valenzuela CY: Association of of a root-filled human tooth, J Endod 26:225, 1990. nonsurgical procedures, J Endod 25:814, 1999. interleukin-1 polymorphisms with periodontal disease, 188. Nair PNR, Sjogren U, Sundqvist G: Cholesterol crystals as 139. Langeland K, Block RM, Grossman LI: A histopathological J Periodontol 76:234, 2005. an etiological factor in non-resolving chronic inflammation: and histobacteriologic study of 35 periapical endodontic 163. Lopez-Lopez J, Jane-Salas E, Estrugo-Devesa A, et al an experimental study in guinea pigs, Eur J Oral Sci surgical specimens, J Endod 3:8, 1977. editors: Periapical and endodontic status of type 2 106:644, 1998. 140. Langeland K, Rodrigues H, Dowden W: Periodontal diabetic patients in Catalonia, Spain: a cross-sectional 189. Nair PNR, Sundqvist G, Sjogren U: Experimental evidence disease, bacteria and pulpal histopathology, Oral Surg study, J Endod 37:598, 2011. supports the abscess theory of development of radicular Oral Med Oral Pathol 37:257, 1974. 164. MacNeil RL, Somerman MJ: Development and cysts, Oral Surg Oral Med Oral Pathol Oral Radiol Endod 141. Lalonde ER: A new rationale for the management of regeneration of the periodontium: parallels and contrasts, 106:294, 2008. periapical granulomas and cysts: an evaluation of Periodontol 19:8, 1999. 190. Nakamura I, Takahashi N, Sasaki T, et al: Chemical and histopathological and radiographic findings, J Am Dent 165. Majno G, Joris I: Cells, tissues, and disease, ed 2, Oxford, physical properties of extracellular matrix are required for Assoc 80:1056, 1970. 2004, Oxford University Press. the actin ring formation in osteoclasts, J Bone Miner Res 142. Laskin DM: Anatomic considerations in diagnosis and 166. Marending M, Peters OA, Zehnder M: Factors affecting 11:1873, 1996. treatment of odontogenic infections, J Am Dent Assoc the outcome of orthograde root canal therapy in a general 191. Nakashima M: Induction of dentin formation on 69:308, 1964. dentistry hospital practice, Oral Surg Oral Med Oral Pathol canine amputated pulp by recombinant human bone 143. Laux M, Abbott PV, Pajarola G, Nair PNR: Apical Oral Radiol Endod 99:119, 2005. morphogenic proteins (BMP)-2 and -4, J Dent Res inflammatory root resorption: a correlative radiographic 167. Marotta PS, Fontes TV, Armada L, et al, editors: Type 2 73:1515, 1994. and histological assessment, Int Endod J 33:483, 2000. diabetes mellitus and the prevalence of apical periodontitis 192. Nanci A: Ten Cate’s oral histology: development, 144. Lekic P, Rojas J, Birek C, et al: Phenotypic comparison of and endodontic treatment in an adult Brazilian population, structure, and function, ed 7, St. Louis, 2008, Mosby. periodontal ligament cells in vivo and in vitro, J J Endod 38:297, 2012. 193. Nathan C: Points of control in inflammation, Nature Periodontal Res 36:71, 2001. 168. Martinelli C, Rulli MA: The innervation of chronic 420:846, 2002. 145. Leonardo MR, Hemandez MEFT, Silva LAB, Tanomaru- inflammatory human periapical lesions, Arch Oral Biol 194. Nauseef WM: How human neutrophils kill and degrade Filho M: Effect of a calcium hydroxide-based root canal 12:593, 1967. microbes: an integrated view. Immunol Rev 219:88, dressing on periapical repair in dogs: a histological study, 169. Marton IJ, Kiss C: Characterization of inflammatory cell 2007. Oral Surg Oral Med Oral Pathol Oral Radiol Endod infiltrate in dental periapical lesions, Int Endod J 26:131, 195. Neville BW, Damm DD, Allen CM, Bouquot JE: Oral and 102:680, 2006. 1993. maxillofacial pathology, ed 3, St. Louis, 2009, Saunders. 146. Leonardo MR, Silva LAB, Utrilla LS, et al: Calcium 170. Marton IJ, Kiss C: Protective and destructive immune 196. Niederman R, Westernoff T, Lee C, et al: Infection- hydroxide root canal sealers—histologic evaluation of reactions in apical periodontitis, Oral Microbiol Immunol mediated early-onset periodontal disease in P/E-selectin- apical and periapical repair after endodontic treatment, 15:139, 2000. deficient mice, J Clin Periodontol 28:569, 2001. J Endod 23:232, 1997. 171. Medzhitove R: Toll-like receptors and innate immunity, 197. Noirti Y, Ehara A, Kawahara T, et al: Participation of 147. Lerner UH: Inflammation-induced bone remodeling in Nat Rev Immunol 1:135, 2001. bacterial biofilms in refractory and chronic periapical periodontal disease and the influence of post-menopausal 172. Medzhitov R, Janeway C Jr: Innate immunity, New Engl J periodontitis, J Endod 28:679, 2002. osteoporosis, J Dent Res 85:596, 2006. Med 343:338, 2000. 198. Nordlund L, Hormia M, Saxen L, Thesleff I: 148. Ley K: Molecular mechanisms of leukocyte recruitment 173. Meghji S, Qureshi W, Henderson B, Harris M: The role of Immunohistochemical localization of epidermal growth in the inflammatory process, Cardiovasc Res 32:733, endotoxin and cytokines in the pathogenesis of factor receptors in human gingival epithelia, J Periodont 1996. odontogenic cysts, Arch Oral Biol 41:523, 1996. Res 26:333, 1991. 149. Li H, Chen V, Chen Y, et al: Herpesviruses in endodontic 174. Meisel P, Carlsson LE, Sawaf H, et al: Polymorphisms of 199. Nualart Grosllmus ZC, Morales Chavez MC, Slvestre Donat pathosis: association of Epstein-Barr virus with irreversible Fc gamma-receptors RIIa, RIIIa, and RIIIb in patients with FJ: Periodontal disease associated to systemic genetic pulpitis and apical periodontitis, J Endod 35:23, 2009. adult periodontal diseases, Genes Immun 2:258, 2001. disorders, Med Oral Pathol Oral Cir Bucal 12:E211, 2007. 150. Li QY, Zhao HS, Meng HX, et al: Association analysis 175. Menezes-Silva R, Khaliq S, Deeley K, et al: Genetic 200. Oehlers FAC: Periapical lesions and residual dental cysts, between interleukin-1 family polymorphisms and susceptibility to periapical disease: conditional contribution Br J Oral Surg 8:103, 1970. 658 PART II The Advanced Science of Endodontics

201. Oguntebi B, Slee AM, Tanzer JM, Langeland K: 225. Schaffer M, Beiter T, Becker HD, Hunt TK: Neuropeptides: 250. Solheim E: Growth factors in bone, Int Orthop 22:410, Predominant microflora associated with human dental mediators of inflammation and tissue repair?, Arch Surg 1998. periradicular abscesses, J Clin Microbiol 15:964, 1982. 133:1107, 1998. 251. Sonoyama W, Seo B-M, Yamaza T, Shi S: Human 202. Okiji T, Kawashima N, Kosaka T, et al: Distribution of la 226. Schoenfeld SE, Greening AB, Glick DH, et al: Endotoxic Hertwig’s epithelial root sheath cells play crucial roles in antigen-expressing nonlymphoid cells in various stages of activity in periapical lesions, Oral Surg Oral Med Oral cementum formation, J Dent Res 86:594, 2007. induced periapical lesions in rat molars, J Endod 20:27, Pathol Oral Radiol Endod 53:82, 1982. 252. Stashenko F: The role of immune cytokines in the 1994. 227. Schork NJ, Fallin D, Lanchbury JS: Single nucleotide pathogenesis of periapical lesions, Endod Dent Traumatol 203. Orstavik D, Pitt Ford TR: Essential endodontology: polymorphisms and the future of genetic epidemiology, 6:89, 1990. prevention and treatment of apical periodontitis, ed 2, Clin Genet 58:250, 2000. 253. Stashenko P, Teles R, D’Souza R: Periapical inflammatory Philadelphia, 2008, Wiley-Blackwell. 228. Segura-Egea JJ, Castellanos-Cosano L, Machuca G, et al, responses and their modulation, Crit Rev Oral Biol Med 204. Pallasch TJ, Wahl MJ: Focal infection: new age or ancient editors: Diabetes mellitus, periapical inflammation and 9:498, 1998. history? Endod Topics 4:32, 2003. endodontic treatment outcome, Med Oral Pathol Cir Bucal 254. Stashenko P, Van Dyke T, Tully P, et al: Inflammation 205. Pascon EA, Leonardo MR, Safavi K, Langeland K: Tissue 17:e356, 2012. and genetic risk indicators for early periodontitis in adults, reaction to endodontic materials: methods, criteria, 229. Segura-Egea JJ, Jimenez A, Rios-Santos JV, et al: High J Periodontol 82:588, 2011. assessment, and observations, Oral Surg Oral Med Oral prevalence of apical periodontitis amongst type 2 diabetic 255. Stashenko P, Wang CY, Riley E, et al: Reduction of Pathol 2:222, 1991. patients, Int Endod J 38:564, 2005. infection-stimulated bone periapical bone resorption by 206. Peciuliene V, Reynaud AH, Balciuniene I, Haapasalo M: 230. Seltzer S: Endodontology, ed 2, Philadelphia, 1988, Lea & the biological response modifier PGG glucan, J Dent Res Isolation of yeasts and enteric bacteria in root-filled teeth Febiger. 74:323, 1995. with chronic apical periodontitis, Int Endod 34:429, 2001. 231. Seltzer S, Bender IB, Zionitz M: The dynamics of pulp 256. Stashenko P, Yu SM: T helper and T suppressor cell 207. Quesnell BT, Alves M, Hawkinson RW, et al: The effect of inflammation: correlation between diagnostic data and reversal during the development of induced rat periapical human immunodeficiency virus on endodontic treatment actual histological finding in the pulp. Part 1 and Part 2, lesions, J Dent Res 68:830, 1989. outcome, J Endod 31:633, 2005. Oral Surg Oral Med Oral Pathol 16:846, 1963. 257. Steed DL: The role of growth factors in wound healing, 208. Reddy SV, Roodman GD: Control of osteoclast differentiation, 232. Seltzer S, Naidorf IJ: Flare-ups in endodontics: 1. Surg Clin North Am 77:575, 1997. Crit Rev Eukaryotic Gene Expression 8:1, 1998. Etiological factors, J Endod 11:472, 1985. 258. Stern MH, Dreizen S, Mackler BF, et al: Quantitative 209. Reeve CM, Wentz FM: The prevalence, morphology, and 233. Seo B-M, Miura M, Gronthos S, et al: Investigation of analysis of cellular composition of human periapical distribution of epithelial cell rests in the human multipotent postnatal stem cells from human periodontal granulomas, J Endod 7:117, 1981. periodontal ligament, Oral Surg 15:785, 1962. ligament, Lancet 364:149, 2004. 259. Stern MH, Dreizen S, Mackler BF, et al: Isolation and 210. Regezi JA, Scubba JJ, Jordan RCK: Oral pathology: 234. Serbina NV, Jia T, Hohl TM, Pamer EG: Monocyte- characterization of inflammatory cells from the human clinical pathologic correlations, ed 5, St. Louis, 2008, mediated defense against microbial pathogens, Ann Rev periapical granuloma, J Dent Res 61:1408, 1982. Saunders. Immunol 26:421, 2008. 260. Sunde P, Olsen I, Debelian G, Transtad L: Microbiota 211. Reinke E, Fabry Z: Breaking or making immunological 235. Shear M: The histogenesis of dental cysts, Dent Pract of periapical lesions refractory to endodontic therapy, privilege in the central nervous system: the regulation 13:238, 1963. J Endod 28:304, 2002. of immunity by neuropeptides, Immunol Lett 104:102, 236. Shimono M, Suda H, Maeda T, editors: Dentin/pulp 261. Sundqvist G: Bacteriological studies of necrotic dental 2006. complex, New York, 1996, Quintessence. pulps, Sweden, 1976, Dissertation, Umea. 212. Reuther T, Schuster T, Mende U, Kubler A: 237. Sidaravicius B, Aleksejuniene J, Eriksen HM: Endodontic 262. Sundqvist G: Taxonomy, ecology, and pathogenicity of the Osteoradionecrosis of the jaws as a side effect of treatment and prevalence of apical periodontitis in an root canal flora, Oral Surg Oral Med Oral Pathol 78:522, radiotherapy of head and neck tumor patients—a report adult population of Vilnius, Lithuania, Dent Traumatol 1994. of a thirty year retrospective review, Int J Oral Maxillofacial 15:210, 2000. 263. Sundqvist G, Eckerbom MI, Larsson AP, Sjogren UT: Surg 32:289, 2003. 238. Sigurdsson A: Pulpal diagnosis, Endod Topics 5:12, Capacity of anaerobic bacteria from necrotic pulp to 213. Richardson JD, Vasko MR: Cellular mechanisms of 2003. induce purulent infection, Infect Immun 25:685, 1979. neurogenic inflammation, J Pharmacol Exp Ther 302:839, 239. Silva Costa CP, Abreu Fonseca EB, Carvalho Souza S de 264. Sundqvist G, Figdor D: Life as an endodontic pathogen, 2002. F: Association between sickle cell anemia and pulp Endod Topics 6:3, 2003. 214. Ricucci D, Bergenholtz G: Histologic features of apical necrosis, J Endod 39:177, 2013. 265. Sundqvist G, Figdor D, Persson S, Sjogren U: periodontitis in human biopsies, Endod Topics 8:68, 2004. 240. Silva TA, Garlet GP, Fukada SY, et al: Chemokines in oral Microbiological analysis of teeth with failed endodontic 215. Ricucci D, Pascon EA, Pitt Ford TR, Langeland K: inflammatory diseases: apical periodontitis and periodontal treatment and the outcome of conservative re-treatment, Epithelium and bacteria in periapical lesions, Oral Surg disease, J Dent Res 86:306, 2007. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85:86, Oral Med Oral Pathol Oral Radiol Endod 101:241, 2006. 241. Simon JHS: Incidence of periapical cysts in relation to root 1998. 216. Ruggiero SL, Drew SJ: Osteonecrosis of the jaws and canal, J Endod 6:116, 1980. 266. Sundqvist G, Reuterving CO: Isolation of Actinomyces bisphosphonate therapy, J Dent Res 86:1013, 2007. 242. Simon JHS, Yonemoto GS, Bakland LK: Comparison of israelii from periapical lesion, J Endod 6:60, 1980. 217. Rutherford RB, Spangberg L, Tucker M, et al: The cellular cementum in normal and diseased teeth—a 267. Syrjanen S, Tammisalo E, Lilja R, Syrjanen K: time-course of the induction of reparative dentin formation scanning electron microscope study, J Endod 7:370, Radiographical interpretation of the periapical cysts and in monkeys by recombinant human osteogenic protein-1, 1981. granulomas, Dentomaxillofac Radiol 11:89, 1982. Arch Oral Biol 39:833, 1994. 243. Siqueira JF, Lopes HP: Bacteria on the apical root 268. Tagger M, Massler M: Periapical tissue reactions after 218. Sabeti M, Simon JH, Howzari H, Slot J: Cytomegalovirus surfaces of untreated teeth with periradicular lesions: a pulp exposure in rat molars, Oral Surg Oral Med Oral and Epstein-Barr virus active infection in periapical lesions scanning electron microscopy study, Int Endod J 34:216, Pathol 39:304, 1975. of teeth with intact crowns, J Endod 29:321, 2003. 2001. 269. Takahashi K: Microbiological, pathological, inflammatory, 219. Sahara N, Toyoki A, Ashizawa Y, et al: Cytodifferentiation 244. Siqueira JF Jr, Rocas IN: Polymerase chain reaction- immunological and molecular biological aspects of of the odontoclast prior to the shedding of human based analysis of microorganisms associated with failed periradicular disease, Int Endod J 31:311, 1998. deciduous teeth: an ultrastructural and cytochemical endodontic treatment, Oral Surg Oral Med Oral Pathol 270. Takeuchi O, Hoshino K, Akira S: Cutting edge: study, Anat Rec 244:33, 1996. Oral Radiol Endod 97:85, 2004. TLR2-deficient and MyD88-deficient mice are highly 220. Sasaki T: Differentiation and functions of osteoclasts and 245. Siqueira JF Jr, Rocas IN, Provenzano JC, et al: susceptible to Staphylococcus aureus infection, J Immunol odontoclasts in mineralized tissue resorption, Microscopy Relationship between Fcgamma receptor and interleukin-1 165:5392, 2000. Res Tech 61:483, 2003. gene polymorphisms and post-treatment apical 271. Tani N, Kuchiba K, Osada T, et al: Effect of T-cell 221. Sasaki T, Sasaki T, Motegi N, et al: Dentin resorption periodontitis, J Endod 35:1186, 2009. deficiency on the formation of periapical lesions in mice: mediated by odontoclasts in physiological root resorption 246. Siqueira JF Jr, Rocas IN, Provenzano JC, Guilherme BP: histological comparison between periapical lesion of human deciduous teeth, Am J Anat 183:303, 1988. Polymorphism of the FcgammaRIIIa gene and formation in BALB/c and BALB/c nu/nu mice, J Endod 222. Sathorn C, Parashos P, Messer HH: How useful is root post-treatment apical periodontitis, J Endod 37:1345, 21:195, 1995. canal culturing in predicting treatment outcome? J Endod 2011. 272. Teitelbaum SL, Tondravi MM, Ross FP: Osteoclasts, 33:220, 2007. 247. Sjogren U, Hagglund B, Sundqvist G, Wing K: Factors macrophages, and the molecular mechanisms of bone 223. Saunder DN: Interleukin-1 in dermatological disease. In affecting the long-term results of endodontic treatment, resorption, J Leukocyte Biol 61:381, 1997. Bomford R, Henderson B, editors: Interleukin-1, J Endod 16:498, 1990. 273. Teles R, Wang CY, Stashenko P: Increased susceptibility inflammation and disease, North Holland, 1989, Elsevier. 248. Slauson DO, Cooper BJ: Mechanisms of disease, ed 3, of RAG-2 SCID mice to dissemination of endodontic 224. Scapoli C, Mamolini E, Carrieri A, et al: Gene-gene St. Louis, 2002, Mosby. infections, Infect Immun 65:3781, 1997. interaction among cytokine polymorphisms influence 249. Slots J, Nowzari H, Sabeti M: Cytomegalovirus infection in 274. Ten Cate AR: The epithelial cell rests of Malassez and the susceptibility to aggressive periodontitis, Genes Immun symptomatic periapical pathosis, Int Endod J 37:519, genesis of the dental cyst, Oral Surg Oral Med Oral Pathol 12:473, 2011. 2004. 34:56, 1972. CHAPTER 15 Pathobiology of Apical Periodontitis 659

275. Teronen O, Salo T, Laitinen J, et al: Characterization of dog dental pulp cells in vivo, Arch Oral Biol 43:431, 303. Williams BL, McCann GF, Schoenknecht FD: Bacteriology interstitial collagenases in jaw cyst wall, Eur J Oral Sci 1998. of dental abscesses of endodontic origin, J Clin Microbiol 103:141, 1995. 290. van Oosterhout AJ, Bloksma N: Regulatory T-lymphocytes 18:770, 1983. 276. Thesleff I: Epithelial cell rests of Malassez bind epidermal in asthma, Eur Respir J 26:918, 2005. 304. Williams GT, Williams WJ: Granulomatous growth factor intensely, J Periodont Res 22:419, 1987. 291. van Winkelhoff AJ, Carless AW, de Graaff J: Bacteroides inflammation—a review, J Clin Pathol 36:723, 1983. 277. Toller PA: The osmolality of fluids from cysts of the jaws, endodontalis and other black-pigmented Bacteroides species 305. Williams JZ, Barbul A: Nutrition and wound healing, Surg Br Dent J 129:275, 1970. in odontogenic abscesses, Infect Immun 49:494, 1985. Clin North Am 83:571, 2003. 278. Torabinejad M: Mediators of acute and chronic 292. Vier FV, Figueiredo JA: Prevalence of different periapical 306. Woo S-B, Hellstein JW, Kalmar JR: Systematic review: periradicular lesions, Oral Surg Oral Med Oral Pathol lesions associated with human teeth and their correlation bisphosphonates and osteonecrosis of the jaws, Ann Int 78:511, 1994. with the presence and extension of apical external root Med 144:753, 2006. 279. Torabinejad M, Eby WC, Naidorf IJ: Inflammatory and resorption, Int Endod J 35:710, 2002. 307. World Health Organization: Application of the international immunological aspects of the pathogenesis of human 293. Wadachi R, Hargreaves KW: Trigeminal nociceptors classification of disease to dentistry and stomatology, periapical lesions, J Endod 11:479, 1985. express TLR-4 and CD14: a mechanism for pain due to ed 3, Geneva, 1995, WHO. 280. Torabinejad M, Kettering J: Identification and relative infection, J Dent Res 85:49, 2006. 308. Wu JR, Zeng R: Molecular basis for population variation: concentration of B and T lymphocytes in human chronic 294. Wakelin D, Roitt I, Mims C, et al, editors: Mims’ medical from SNPs to SAPs, FEBS Lett 586:2841, 2012. periapical lesions, J Endod 11:122, 1985. microbiology, ed 4, Philadelphia, 2008, Mosby. 309. Yamamoto K, Kobayashi T, Grossi S, et al: Association of 281. Torabinejad M, Kiger RD: Experimentally induced 295. Wallstrom JB, Torabinejad M, Kettering J, McMillan P: Fcgamma receptor IIa genotype with chronic periodontitis alterations in periapical tissues of the cat, J Dent Res Role of T cells in the pathogenesis of periapical lesions: a in Caucasians, J Periodontol 75:517, 2004. 59:87, 1980. preliminary report, Oral Surg Oral Med Oral Pathol 310. Yamasaki M, Kumazawa M, Kohsaka T, et al: Pulp and 282. Torabinejad M, Walton RE: Endodontics: principles and 76:213, 1993. periapical tissue reactions after experimental pulpal practice, ed 4, St. Louis, 2009, Saunders. 296. Walton RE, Ardjmand K: Histological evaluation of the exposure in rats, J Endod 20:13, 1994. 283. Tronstad L, Barnett F, Cervone F: Periapical bacterial presence of bacteria in induced periapical lesions in 311. Yamasaki M, Nakane A, Kumazawa M, et al: Endotoxin plaque in teeth refractory to endodontic treatment, monkeys, J Endod 18:216, 1992. and gram-negative bacteria in the rat periapical lesion, Dent Traumatol 6:73, 1990. 297. Wang CY, Stashenko P: Kinetics of bone-resorbing activity J Endod 18:501, 1992. 284. Tronstad L, Barnett F, Riso K, Slots J: Extraradicular in developing periapical lesions, J Dent Res 70:1362, 312. Yu SM, Stashenko P: Phenotypic characterization of endodontic infection, Dent Traumatol 3:86, 1987. 1991. inflammatory cells in induced rat periapical lesions, 285. Trombelli L, Lee MB, Promsudthi A, et al: Periodontal 298. Wang CY, Tani-Ishii N, Stashenko P: Bone resorptive J Endod 13:535, 1987. repair in dogs: histologic observations of guided tissue cytokine gene expression in developing rat periapical 313. Yusulf H: The significance of the presence of foreign regeneration with a prostaglandin E1 analog/methacrylate lesions, Oral Microbiol Immunol 12:65, 1997. material periapically as a cause of failure of root composite, Clin Periodontol 26:381, 1999. 299. Warren JR, Scarpelli DG, Reddy JK, Kanwar YS: Essentials treatment, Oral Surg Oral Med Oral Pathol 54:566, 1982. 286. Trott JR, Chebeb F, Galindo Y: Factors related to of general pathology, New York, 1987, Macmillan 314. Zimmerli W, Lew PD, Walvogel FA: Pathogenesis of cholesterol formation in cysts and granulomas, J Cand Publishing. foreign body infection: evidence for a local granulocyte Dent Assoc 38:76, 1973. 300. Wayman BE, Murata SM, Almeida RJ, Fowler CB: A defect, J Clin Invest 73:1191, 1984. 287. Trowbridge H, Emling RC: Inflammation: a review of the bacteriological and histological evaluation of 58 315. Zimmerli W, Waldvogel FA, Vaudaux P, Nydegger UE: process, ed 5, Chicago, 1997, Quintessence Publishing. periradicular lesions, J Endod 18:152, 1992. Pathogenesis of foreign body infection: description and 288. Tuan T-L, Nichter LS: The molecular basis of keloid and 301. Weiger R, Manncke B, Werner H, Lost C: Microbial flora characteristics of an animal model, J Infect Dis 146:487, hypertrophic scar formation, Mol Med Today 4:19, 1998. of sinus tracts and root canals of non-vital teeth, Endod 1982. 289. Tzaifas D, Alvanou A, Papadimitrious S, et al: Effects of Dent Traumatol 11:15, 1995. 316. Zimring JC, Kapp JA: Identification and characterization of recombinant basic fibroblast growth factor, insulin-like 302. Werner S, Grose R: Regulation of wound healing by CD8+ suppressor T cells, Immunol Res 29:303, 2004. growth factor-II and transforming growth factor-β 1 on growth factors and cytokines, Physiol Rev 83:835, 2003. CHAPTER 16

Root Resorption

SHANON PATEL | CONOR DURACK | DOMENICO RICUCCI

CHAPTER OUTLINE General Histologic Features Clinical Features External Inflammatory Resorption Radiographic Features Introduction Management Etiology and Pathogenesis of External Inflammatory Internal Root Resorption Resorption Introduction Histologic Appearance Etiology and Pathogenesis of Internal Root Resorption Clinical Features Histologic Appearance Radiographic Features Clinical Features Management Radiographic Features and Diagnosis Follow-up and Prognosis of External Inflammatory Resorption Management External Cervical Resorption Chemomechanical Debridement of the Root Canal Introduction Obturation Etiology and Pathogenesis Summary Histologic Appearance

Dental resorption is the loss of dental hard tissues as a result of mononuclear precursor cells of the monocyte-macrophage of clastic activities.90 It may occur as a physiologic or patho- lineage derived from the spleen or bone marrow; osteoblasts logic phenomenon. Root resorption in the primary dentition and osteocytes, on the other hand, are derived from skeletal is a normal physiologic process except when the resorption precursor cells.77,101 Osteoclasts are recruited to the site of occurs prematurely.22,23 The initiating factors involved in injury or irritation by the release of many proinflammatory physiologic root resorption in the primary dentition are not cytokines. To perform their function, osteoclasts must attach completely understood, although the process appears to be themselves to the bone surface. On contact with mineralized regulated by cytokines and transcription factors that are similar extracellular matrices, the actin cytoskeleton of an actively to those involved in bone remodeling.60,123 Unlike bone, which resorbing osteoclast is reorganized to produce an organelle-free undergoes continuous physiologic remodeling throughout life, zone of sealing cytoplasm (clear zone) associated with the root resorption of permanent teeth does not occur naturally osteoclast’s cell membrane; this enables the osteoclast to and is invariably inflammatory in nature. Thus, root resorption achieve intimate contact with the hard tissue surface.92 The in the permanent dentition is a pathologic event; if left clear zone surrounds a series of fingerlike projections (podo- untreated, it may result in premature loss of the affected teeth. somes) of cell membrane, known as the ruffled border, beneath Root resorption may be broadly classified into two types, which bone resorption occurs. The resorptive area within the external and internal, based on the location of the resorption clear zone, therefore, is isolated from the extracellular environ- in relation to the root surface.8,105 Internal root resorption was ment, creating an acidic microenvironment for the resorption reported as early as 1830.19 Compared with external root of hard tissues. resorption, internal root resorption is a relatively rare occur- Odontoclasts, which are the cells that resorb dental hard rence, and its etiology and pathogenesis have not been com- tissues (Fig. 16-1), are morphologically similar to osteoclasts.49 pletely elucidated.72,91 Nevertheless, internal root resorption Odontoclasts differ from osteoclasts in that they are smaller, (IRR) poses diagnostic concerns to the clinician because it is have fewer nuclei, and have smaller sealing zones, possibly as often confused with external cervical resorption (ECR). An a result of differences in their respective resorption substrata.74 incorrect diagnosis may result in inappropriate treatment in Osteoclasts and odontoclasts resorb their target tissues in a certain cases.57,58,88,89,91 similar manner.92 The two cells have similar enzymatic properties,84 show similar cytologic features, and create resorption depressions, termed Howship lacunae, on the surface of the GENERAL HISTOLOGIC FEATURES mineralized tissues (see Fig. 16-1).92 Odontoclasts are polar- Osteoclasts are motile, multinucleated giant cells that are ized in relation to dental tissue and have a ruffled border, responsible for bone resorption. They are formed by the fusion located inside a clear zone, that is in intimate contact with their

660 CHAPTER 16 Root Resorption 661

FIG. 16-1 Light microscopy images of a deciduous tooth showing both physiologic and pathologic (inflammatory) resorption. A, Tooth 7.5 in a 9-year-old boy. Extensive exposure of a mesial pulp horn occurred during caries excavation, and pulp capping was performed. The patient was A B C brought to the dental office 4 months after the treatment because of severe spontaneous pain. The radiograph shows radiolucencies on both apices and interradicularly. The parents did not accept any treatment and requested extraction. B, Longitudinal section taken on a mesiodistal plane, passing approximately at the center of the mesial root. (Hematoxylin-eosin (H&E) stain; original magnification ×25.) C, Detail of the resorption area apically in B (original magnification ×100). D, Magnification of the area demarcated by the rectangle in C. Two odontoclasts are present in an area of dentin D E being resorbed, surrounded by fibroblasts and inflammatory cells (original magnification ×400). E, High-power view of the area indicated by the arrow in C. An odontoclast is in close contact with the dentin in a Howship lacuna. Its cytoplasm appears vacuolated and shows a more intense staining reaction than the cytoplasm of the adjacent cells (original magnification ×1000). F, High-power view of the upper odontoclast in D. The characteristic ruffled border can be distinguished (original F G magnification ×1000). G, Howship lacuna with an odontoclast in another area (original magnification ×400). H to J, Progressive magnification of the mesial root apex. The resorption process may be physiologic at this level (original magnification ×16, ×100, and ×400).

H I J 662 PART II The Advanced Science of Endodontics

dental substratum.67,92 Wesselink et al.119 demonstrated that arginine-glycine–aspartic acid (RGD) sequence of amino acids odontoclasts can simultaneously have two areas of ruffled by means of integrins.98 The latter are specific surface adhesion border, which can resorb bone and hard dental tissue concur- glycoprotein membrane receptors containing different α and β 67 92 rently. Based on this evidence, Jones and Boyd and Pierce subunits. In particular, αvβ3 integrin plays a key role in the have stated that odontoclasts and osteoclasts are effectively the adhesion of clastic cells.81 Extracellular matrix proteins con- same cell type, differing only in their respective substrata. taining the RGD peptide sequence, particularly osteopontin, Wedenberg and Yumita117 demonstrated that rat osteoclasts present on the surface of mineralized tissues and serve as resorbed fully mineralized human dentin ex vivo, but did not binding sites of clastic cells.65 The osteopontin molecule con- attach to demineralized dentin or predentin. These researchers tains different domains, with one domain binding to apatites concluded that osteoclasts and odontoclasts are probably the in the denuded dentin and another domain binding to integrin same cell. receptors in the plasma membranes of clastic cells. Thus, At high-power magnification, the cytoplasm of the odonto- osteopontin serves as a linker molecule that optimizes the clasts appears vacuolated and shows a more intense staining attachment of a clastic cell to mineralized tissues, mediating reaction than the cytoplasm of the adjacent cells. Where odon- the rearrangement of its actin cytoskeleton.33 It has been specu- toclasts are in contact with the tooth surface, the characteristic lated that the lack of RGD peptides in predentin reduces the brush border can be seen, but the hematoxyphilic zone lining binding of odontoclasts, thereby conferring resistance of the the lacunae is absent.49 When odontoclasts are observed with canal walls to IRR. an electron microscope, the most striking features are the large Once the clastic cells have established contact with the number of mitochondria and vacuoles in the cytoplasm and cementum, any subsequent resorption is self-limiting unless the scarcity of endoplasmic reticulum. An abundance of cyto- the bound cells are subject to continued stimulation.8 There- plasmic ribosomes can be seen. The nucleoli are fairly large fore, in addition to the precipitating events discussed and centrally located in the nucleus. Close to the nucleus, the previously, progressive root resorption requires a source of Golgi apparatus appears as a narrow zone of fine canals. Where stimulation for the resorbing cells. Stimulating factors vary and the odontoclasts are in contact with the tooth surface, the are related to the site and type of resorption, in addition to the ruffled border (corresponding to the brush border) may be cause of the predisposing damage to the predentin or pre observed with the light microscope. The ruffled border consists cementum. Examples include persistent pressure and forces of cytoplasmic folds that create a system of canals extending 2 associated with continued orthodontic treatment; persistent to 3 µm into the cytoplasm. In undecalcified sections, mineral impacted teeth; untreated cysts, granulomas, and tumors; end- crystals could be observed in these canals.49 odontic inflammation and/or infection; and periodontal inflam- Although mononuclear dendritic cells share a common mation and/or infection.50 hematopoietic lineage with the multinucleated osteoclasts, they have previously been regarded solely as immunologic EXTERNAL INFLAMMATORY RESORPTION defense cells. Recent studies have indicated that immature dendritic cells function also as osteoclast precursors and have Introduction the potential to transdifferentiate into osteoclasts.102 Because External inflammatory resorption (EIR) affects the surface of dendritic cells are present in the dental pulp, it is possible they the root and is a relatively frequent sequel to dental luxation4 also may function as precursors of odontoclasts. and avulsion14 injuries. It is a progressive condition with a From a molecular signaling perspective, the OPG/RANKL/ potentially precipitous onset, and it is capable of advancing RANK transcription factor system25 that controls clastic func- rapidly, such that an entire root surface may be resorbed tions during bone remodeling has also been identified in root within a few months if the tooth is left untreated.10,14 It also resorption.109 The system is responsible for the differentiation affects teeth diagnosed with chronic periapical periodontitis of clastic cells from their precursors by means of complex (Figs. 16-2 through 16-5).70 cell-cell interactions with osteoblastic stromal cells. Similar to The prevalence of EIR after luxation injuries ranges from periodontal ligament cells that are responsible for external almost 5%4 to 18%.35 It affects 30% of replanted avulsed root resorption,111 the human dental pulp has recently been teeth.11,12 EIR is the most common form of external resorption shown to express osteoprotegerin (OPG) and receptor activa- root resorption after luxation and avulsion injuries.35 tor of NF-κb ligand (RANKL) messenger ribonucleic acids Clinical treatment of EIR is based on effective removal of (mRNAs).110 Osteoprotegerin, a member of the tumor necrosis the causal agent; namely, infected necrotic pulpal tissue in the factor superfamily, has the ability to inhibit clastic functions root canal space.41 Treatment should be carried out as soon by acting as a decoy receptor that binds to RANKL and reduces as the resorptive process has been diagnosed. The earlier EIR its affinity for RANK receptors on the surface of clastic precur- is diagnosed and treated, the better the prognosis is for the sors. This results in inhibition of the regulation of clastic cell affected tooth.38 differentiation. Thus, it is possible that the OPG/RANKL/ The diagnosis of EIR in clinical situations is based solely on RANK system may be actively involved in the differentiation the radiographic demonstration of the process.3 In some cases, of odontoclasts during root resorption. the initial radiographic signs of EIR can be visualized as early We know that osteoclasts do not adhere to nonmineralized as 2 weeks after replantation of avulsed teeth.14 However, the collagen matrices.106 It has been suggested that the presence of limitations of conventional radiographic imaging in dentistry a noncollagenous, organic component in dentin (odontoblast have been well reported. The diagnostic yield of radiographs is layer and predentin) prevents (internal) resorption of the root reduced by adjacent anatomic noise,20,56,69,95 geometric distor- canal wall, and precementum prevents (external) resorption tion,56 and compression of three-dimensional (3D) structures of the external root surface.114,116 Similar to osteoclasts, odon- onto a two-dimensional (2D) shadowgraph.34,87,113 These limi- toclasts may bind to extracellular proteins containing the tations may result in late diagnosis of EIR after dental trauma. CHAPTER 16 Root Resorption 663

A B

C D

FIG. 16-2 External apical inflammatory resorption. A, Maxillary first molar in a 46-year-old female patient that was causing severe pain. The radiographs revealed material in the pulp chamber, a large distal caries, and apical radiolucencies. The tooth was extracted. B, Palatal root apex extracted with the pathologic tissue attached. Note the ingrowth of granulation tissue into the foramen and the massive resorption of the apical profile. Also note the resorption of lacunae on the left radicular profile. (H&E stain; original magnification ×25.) C, High magnification of the area of the external radicular profile indicated by the arrow in B. A resorption lacuna can be seen in the cementum. (H&E stain; original magnification ×400.) D, View from the center of the apical periodontitis lesion showing severe concentration of chronic inflammatory cells (mostly plasma cells) (original magnification ×400).

A B

FIG. 16-3 External apical inflammatory resorption. A, Mandibular second premolar extracted with the periapical lesion attached. This histologic section, which did not pass through the canal, shows extensive apical resorption. (H&E stain; original magnification ×25.) B, Section taken approximately 120 sections away encompasses the apical foramen. In addition to resorption, the opposite phenomenon can be observed; that is, a large calcification partly embedded in the right apical dentin wall (original magnification ×25). 664 PART II The Advanced Science of Endodontics

A B

FIG. 16-4 External apical inflammatory resorption. A, Mesial root of a mandibular first molar extracted with a periapical lesion attached. This section passes through the canal and the foramen. Extensive resorption of the foramen has occurred, and a calcification can be seen more coronally. Note that the tissue in the apical canal and in the resorptive defect appears structured and continuous with the apical periodontitis lesion. A thick biofilm can be discerned layering the root canal walls more coronally. (Taylor’s modified Brown & Brenn stain; original magnification ×25). B, Palatal root of a maxillary first molar extracted with the periapical lesion attached. A cyst cavity is present in the body of the lesion. Resorption of the foramen is present, and a portion of the right dentin wall has been replaced by bone (arrowheads). (H&E stain; original magnification ×25.)

A B 3yr 3mo

FIG. 16-5 Apical resorption. A, A 22-year-old male patient complained of pain on chewing. This was caused by a molar that had been treated endodontically 1 year earlier. A radiograph showed obturation material only at the root canal orifices and a periapical radiolucency on the mesial root. Endodontic retreatment was recommended, but the patient declined this treatment. B, The patient returned 3 years and 3 months later because of an abscess that was causing severe pain. A radiograph disclosed destructive mesial caries and large periapical radiolucencies on both roots, along with resorption of the whole apical third of the distal root.

When the occurrence of EIR is correlated with the specific years. In that cohort, there were no reported cases of EIR after types of luxation injury, it is clear that this type of resorption concussion injuries and only one case after subluxation inju- is often associated with the severity of the luxation injury. ries, which represented only 0.5% of the total number of sub- Andreasen and Vestergaard Pedersen4 prospectively followed luxated teeth. Six percent of extrusively luxated teeth and 3% up 637 permanent luxated teeth in 400 patients for up to 10 of laterally luxated teeth developed EIR. However, EIR was a CHAPTER 16 Root Resorption 665 healing complication in 38% of intrusive luxation injuries.4 and moderate to severe luxation injuries, which have the Crona-Larsson et al.35 also reported a higher prevalence of EIR potential to compromise pulp vitality.4 If the strength of the after more severe luxation injuries, with 60% of extrusively initial osteoclast attack is sufficient to expose patent dentinal luxated teeth and 22% of intruded teeth affected by the com- tubules beneath the cementum, a communication is created plication. They reported EIR to be a much more frequent between the pulp space and the external root surface and occurrence after lateral luxation (16.7%) and subluxation inju- adjacent periodontal tissues.7,15 Microbes and/or their toxins ries (3.8%) relative to the findings of Andreasen and Vester- (e.g., lipopolysaccharide, muramyl dipeptide, and lipoteichoic gaard Pedersen. acid),16 located in the root canal and the dentinal tubules, diffuse through the tubules and directly stimulate the resorbing osteoclasts. The resorption process intensifies and accelerates.7 Etiology and Pathogenesis of External The resorbed mineralized tissue is replaced by granulation Inflammatory Resorption tissue, which ultimately invades the pulp space if the process A specific set of conditions is required for the initiation and continues.16 progression of EIR. Under normal conditions, permanent teeth Pressure on the root surface during orthodontic treatment71 are resistant to resorption.115-117 Although the mechanisms that and from impacted teeth,122 cysts,104 and tumors73 may also protect teeth against root resorption have not been fully eluci- denude the protective precementum from the root surface and dated, it is generally accepted that odontoclasts do not adhere therefore initiate ERR. to the nonmineralized layers covering the external root surface and the root canal wall (i.e., the precementum and predentin, Histologic Appearance respectively).8,59,115-117 Similarly, it has been shown that, between The histologic appearance of EIR is characterized by saucer- episodes of physiologic resorption, the surface of bone is or bowl-shaped areas of resorption in both the cementum and impervious to osteoclastic activity because it is covered by a dentin, with concomitant inflammation in the adjacent peri- layer of nonmineralized collagen fibrils, to which the osteoclast odontal membrane. Howship lacunae are a common feature is incapable of binding.32 of the resorption cavities, and histologic sections show that Traumatic dental injuries (e.g., intrusion, lateral luxation, the lacunae sometimes are occupied by odontoclasts. The and avulsion) and subsequent replantation often result in con- inflammatory reaction in the periodontal membrane appears tusion injuries to the periodontal ligament (PDL).8 Damage to intense and consists of a mixed-cell infiltrate that includes the precementum, with a resultant breach in its integrity, is plasma cells, lymphocytes, and polymorphonuclear leuko- the precipitating factor in all types of external resorption.8,9 cytes in a granulation tissue matrix. Proliferation of capillar- In the subsequent wound healing process, necrotic PDL tissue ies in the areas of inflammation is also a feature.15 EIR can be remnants are excavated and removed by macrophages and identified histologically 1 week after experimental replanta- osteoclasts.44 A critical factor is that the precementum may be tion of teeth.6 stripped from the root surface during the injury and the damaged cementum and bone may also be phagocytosed, Clinical Features resulting in exposure of the underlying dentin to osteoclastic The tooth in question may look normal, but it will not respond and odontoclastic activity.44 External root resorption may positively to vitality testing. In advanced cases, signs of pulpal ensue, but the precise type depends on the severity of the initial and/or periapical periodontitis may be present (e.g., discolored injury, the stage of root development, and the pulp status of tooth, sinus present, and/or tenderness to percussion and/or the affected tooth.8,9 Once the clastic cells have established palpation). contact with the cementum or dentin, any subsequent resorption is self-limiting unless the cells are subject to continued Radiographic Features stimulation.7 As already mentioned, odontoclasts do not adhere As mentioned, diagnosis of EIR is based solely on radiographic to the nonmineralized layers covering the external root demonstration of the process.3,14 EIR is characterized radio- surface.8,59,115-117 Therefore, damage to the precementum, with graphically by radiolucent, concave, and sometimes ragged a resultant breach in the integrity of these layers, is the pre- bowl-shaped excavations along the root surface, with corre- cipitating factor in all types of ERR.8,9 Damage to the prece- sponding and associated radiolucencies in the adjacent alveolar mentum and underlying tooth structure allow circulating bone. Complete loss of the lamina dura is seen in the area of odontoclasts to bind to the underlying mineralized dentin and the resorption.15 The initial radiographic signs of EIR can often cementum.115 be seen as early as 3 to 4 weeks after a TDI involving the peri- The pathogenesis of EIR can be explained as follows. Con- odontal tissues15 and, if it will develop, EIR is always seen tusion injuries to the PDL, after a traumatic dental injury (TDI) within 1 year after the injury.5 involving the periodontal structures, initiate wound healing, EIR can have a rapid onset and aggressive progression, such during which osteoclasts and macrophages are attracted to the that complete resorption of an entire root can occur within 3 site of the injury to remove the damaged tissue. The initial months. The diagnostic potential of a number of radiographic injury causes a breach in the integrity of the protective prece- imaging systems has been investigated, with varying degrees mentum. This permits odontoclasts to bind to and resorb the of success. underlying mineralized cementum and dentin in a manner Conventional intraoral radiographic imaging (digital or film similar to the development of surface resorption.9 However, based) is currently the clinical reference standard for the detec- EIR differs from surface resorption in that it is a progressive tion of ERR after luxation and avulsion injuries.46,47 However, event that relies on microbial stimulation from the infected it has been well documented that this form of imaging is an necrotic pulp of the affected tooth for its progression.7 There- inadequate method of detecting simulated ERR, especially fore, it is more commonly associated with tooth avulsions11,12 when the cavity sizes are small.3 Clinical studies have also 666 PART II The Advanced Science of Endodontics

demonstrated that conventional radiography grossly underes- human ex vivo model. Although both imaging modalities were timates the extent of inflammatory root resorption.45 100% sensitive in the detection of the lesions, only CBCT Andreasen et al.3 performed ex vivo experiments investigat- could accurately assess the position of the defects on the root ing the diagnostic accuracy of conventional radiographic surface and their relationship to the root canal, even though imaging in the detection of simulated EIR and surface resorp- multiple angled periapical radiographs of the test teeth were tion. The simulated resorption cavities were located on the available to the examiners. mesial, distal, or lingual surfaces and in the cervical, middle, Alqerban et al.1 compared the ability of two CBCT systems or apical thirds. Nine preoperative radiographs and nine post- and conventional panoramic radiography to detect simulated operative radiographs of each specimen were taken, for a total external surface resorption lesions of varying sizes associated of 90 radiographs for the five specimens. The radiographs dif- with canine impaction. The authors reported that with small fered in the angulation between the specimen and the x-ray and medium field of view (FOV), CBCT systems were superior beam and also in the exposure time. to conventional panoramic radiography in the detection of More than 50% of the medium and large cavities were iden- simulated external resorption cavities regardless of the cavity tified, but none of the small cavities could be visualized on the size. There was no statistical difference between the diagnostic radiographs, regardless of the horizontal x-ray beam angle or ability of the CBCT systems. However, conventional panoramic film density. The trabecular arrangement (noise) of the alveolar radiography is rarely used in endodontic-specific investiga- bone concealed the small cavities. Simulated resorption cavi- tions. Intraoral radiography is currently the imaging technique ties on the proximal surfaces of the teeth were significantly of choice for assessing traumatically injured permanent teeth, easier to identify than lesions on the lingual aspect of the root. which may develop EIR.46,47 However, the position of the cavities along the root length A more recent study by the King’s College London (KCL) (coronal, middle, or apical third) had no bearing on the exam- group concluded that CBCT was a reliable and valid method iners’ ability to identify them. Cavities were more readily iden- of detecting simulated EIR, and performed significantly better tified on high-contrast films; the availability of preoperative than intraoral periapical radiography. Durack et al.43 found that radiographs and multiple angled views of the specimens changing the exposure parameters so as to halve the radiation increased the chances of identifying the cavities.3 dose did not have a negative effect on the diagnostic yield of Chapnick31 used an experimental design similar to that of the reconstructed images. Andreasen et al.3 to further examine the efficacy of conventional radiography in the detection of simulated ERR. Multiple Management angled radiographs and alterations in exposure parameters Clinical treatment of EIR is based on effective removal of the were also used in this study in an attempt to maximize the causal agent, the infected necrotic pulpal tissue in the root diagnostic yield of the radiographs. Although the examiners canal space. This arrests the resorption process and creates were able to identify some small simulated resorption cavities, an environment conducive to hard tissue repair of the these defects were significantly more difficult to detect than damaged root surface.36,37,41 Therefore, it is essential to initiate medium or large ones. In agreement with Andreasen et al.,3 root canal treatment as soon as radiographic signs of EIR are Chapnick concluded that conventional radiography is an inad- identified.38 An exception to this is replanted teeth with equate method of detecting early ERR. closed apices; in these cases, root canal treatment should be Goldberg et al.54 concluded that “radiology is not a very carried out 7 to 10 days after replantation, even if there are accurate procedure for achieving an early and precise diagnosis of no radiographic signs of EIR.47 The earlier the resorption is resorption defects.” diagnosed and treated, the better the prognosis is for the The studies discussed to this point all used film-based con- affected tooth. Failure to diagnose and treat the condition ventional, intraoral radiography, Borg et al24 concluded that may result in tooth loss. charged coupled device (CCD) and photostimulable phosphor Effective chemomechanical debridement of the root canal plate (PSP) digital systems are as sensitive as film-based radi- space is fundamental to the success of the root canal treatment ography in the detection of simulated ERR. In a similar inves- and the inhibition and cessation of EIR.37,41 In principle, the tigation, Kamburoğlu et al.68 found that, although the CCD specific root canal protocol used is irrelevant, as long as the system used in their study performed as well as their conven- biologic objectives are met. Long-term dressing of the root tional film radiographs, the PSP system was significantly less canal with calcium hydroxide may be beneficial in the treat- accurate at identifying the artificial cavities. In agreement with ment of established EIR; however, this protocol should be used Andreasen et al.3 and Goldberg et al.,54 this study further judiciously because of the associated risk of root fracture.13 reported that lesions created on the proximal root surfaces In many cases the EIR is extensive, rendering the tooth were easier to detect than those on the buccal/lingual surfaces unsalvageable and requiring extraction. and that the best results were achieved when multiple angled views and preoperative radiographs were available to the examiners at the same time.68 Follow-up and Prognosis of External Clinical studies directly comparing the ability of intraoral Inflammatory Resorption radiographs and cone beam computed tomography (CBCT) to Healing of EIR is characterized radiographically by cessation detect and diagnose EIR are limited. One clinical study reported of the resorption process, resolution of the radiolucency in that CBCT is superior to conventional radiography in diagnos- the adjacent bone, and reestablishment of the PDL space.14 ing and determining the extent of nonspecific inflammatory As mentioned previously, in untreated cases, EIR can progress resorption on root surfaces.45 D’Addazio et al.40 compared the so rapidly that an entire root can be resorbed within 3 ability of CBCT and periapical radiography to detect simulated months.14,37 The prognosis is especially poor for untreated external resorption cavities of about 2 mm in diameter in a immature teeth.14 CHAPTER 16 Root Resorption 667

EXTERNAL CERVICAL RESORPTION and 0.9% had a history of bleaching and orthodontic treatment only. Introduction Although the data provided in this study are valuable and External cervical resorption (ECR) is a form of root resorption arguably the most comprehensive information available on the that originates on the external root surface but may invade root potential causes of ECR, no definitive cause-and-effect rela- dentin in any direction and to varying degrees. ECR generally tionship has been established. In the cases affected by a com- develops immediately apical to the epithelial attachment of the bination of predisposing factors, it was impossible to determine tooth. In healthy teeth with a normal periodontal attachment, definitively whether the development of ECR was the result of this is in the tooth’s cervical region, a feature that gave rise to one specific event or a combination of factors, or if any of the the name. However, in teeth that have developed gingival potential causes identified were in fact contributory. In 15% of recession and lost periodontal support and/or have developed the patients examined in Heithersay’s study, no potential pre- a long junctional epithelium, the resorptive defect may arise at disposing factor was identified. Furthermore, intracoronal res- a more apical location. torations were attributed as possible predisposing factors only ECR has also been referred to as invasive cervical resorp- when no other potential cause could be identified.61 tion,62 supraosseous extracanal invasive resorption,48 peripheral There are conflicting views on the manner in which the inflammatory root resorption,53 and subepithelial external root resorptive process is sustained once the clastic cells have resorption.107 The authors of this chapter prefer the term exter- bound to the root dentin in ECR. One view is that microorgan- nal cervical resorption because it describes the nature and loca- isms originating from the gingival sulcus provide the stimulus tion of the lesions. for continued resorption.50,105 The opposing hypothesis, by Heithersay,62 suggests that ECR is a type of “benign Etiology and Pathogenesis proliferative fibrovascular or fibro-osseous disorder” in which The exact etiology and pathogenesis of ECR have not been microorganisms play no active role and are either absent from fully elucidated. It is accepted that the resorptive process is the the site of resorption or invade it only secondarily. same for ECR as it is for any other type of resorption: a breach in the protective non-mineralized layers must exist to allow Histologic Appearance the clastic cells to bind to the underlying dentin, and the same The histologic profile of ECR is similar to that of other forms cells must be stimulated to perpetuate the process. However, of resorption, with certain unique features reflecting the inva- in ECR only some of the factors that predispose the root surface sive nature of the process. In the early stages, granulation to clastic activity have been identified. (fibrovascular) tissue occupies the resorptive cavity, and odon- The anatomic profile of the cementoenamel junction (CEJ) toclasts may be evident in lacunae on the resorbing front of is variable, and the junction between the enamel and the the defect.61 Acute inflammatory cells are often absent from the cementum in this region is not contiguous in all teeth. This site of resorption in the early stages of ECR, but secondary may lead to exposed areas of unprotected dentin, which are bacterial colonization of the site of resorption may occur at a vulnerable to osteoclastic activity, in the cervical region of later stage.39,62 some teeth.83 The resorptive cavity advances toward the subjacent root Heithersay61 investigated the potential predisposing factors canal system and typically extends either circumferentially in 257 cases of ECR in 222 patients. Orthodontic treatment, around, or in an apicocoronal direction through, the radicular dental trauma, oral surgery, periodontal therapy, bruxism, dentin without communicating with the root canal. Narrow intracoronal restorations, delayed eruption, enamel stripping, “channels” of resorption extend through the dentin and may and dental developmental defects were all identified as poten- communicate with the periodontal ligament.61 However, per- tial predisposing factors, either alone or in combination. foration of the root canal wall usually occurs only at a late stage Orthodontic treatment was the most common, sole predis- because the predentin affords protection against the resorbing posing factor identified, with a history of treatment in 21% cells.61,64 Consequently, the pulpal tissue adjacent to the site of of the patients and 24% of the teeth assessed. Dental trauma resorption has a normal histologic appearance until the root was the only identifiable predisposing factor for ECR in 14% canal has been invaded.61 of the examined teeth. Oral surgical procedures (particularly As the lesion progresses, bonelike tissue is deposited in the those in which the cervical region of the affected tooth resorptive cavity in direct contact with the adjacent dentin; this was involved) were identified as the sole etiologic factor is an attempt to repair the previous tissue destruction.61 in 6% of the cases. Specific surgical procedures included extraction of partially and fully erupted third molar teeth Clinical Features adjacent to the affected tooth, exposure of unerupted canines The clinical features of ECR are variable (Figs. 16-6 and 16-7). or supernumerary teeth, transplantation of canine teeth, and The process is very often quiescent and asymptomatic, espe- surgical amputation of periodontally compromised teeth. cially in the earlier stages, and absence of clinical signs and Intracoronal bleaching was highlighted as the only evident symptoms is very common; the diagnosis is commonly made predisposing factor in 5% of patients affected. A combination as a result of a chance radiographic finding. A pink or red dis- of predisposing factors was identified in a number of cases. coloration may develop at the cervical region of the tooth; when For example, 4.3% of the affected teeth had a combined present, this often is the feature that alerts the patient or clini- history of orthodontic treatment and another potential pre- cian to the possible existence of a problem. The discoloration disposing factor, primarily dental trauma and/or intracoronal is due to the fibrovascular granulation tissue occupying the bleaching. In addition, of the patients assessed, 7.7% had resorptive defect, which has a reduced thickness of enamel and a history of bleaching and dental trauma; 1.8% had a history dentin at its peripheries because of the loss of hard tissue. The of dental trauma, bleaching, and orthodontic treatment; granulation tissue imparts a pink hue to the tooth, through the 668 PART II The Advanced Science of Endodontics

A B C

D E F G

FIG. 16-6 External cervical resorption (ECR). A, A 55-year-old female patient presented with an asymptomatic “pink spot.” She had no history of any predisposing factors. B, A periapical radiograph revealed radiolucent defects on the proximal aspects of the upper left central incisor; note the ragged borders. C, A reconstructed coronal cone beam computed tomography (CBCT) slice reveals the true extent of the ECR lesion. Note that the root canal wall appears to be intact. Inhibitory factors in the root canal wall/odontoblastic layer prevented the ECR lesion from penetrating the root canal. D, A 41-year-old male patient presented as a new patient. Routine radiographic examination revealed a poorly defined periapical radiolucency in the root of the upper right central incisor. The appearance is suggestive of ECR; the patient had orthodontic treatment in his early teens and remembered “knocking the tooth” at least twice when he was very young. Reconstructed sagittal (E) and coronal (F) CBCT slices revealed the true nature of the ECR lesion and showed that the lesion was not amenable to treatment. The treatment options were discussed with the patient, and it was decided to review the tooth periodically. G, The 4-year radiograph shows no change in size of this asymptomatic lesion.

thinned enamel and dentin, in the region of the resorption.90 The granulation tissue may perforate the enamel or dentin at Radiographic Features the gingival margin, giving the appearance of mild gingival The radiographic appearance of ECR depends on the location, hyperplasia. The discoloration, sometimes referred to as a “pink the extent of invasion, and the relative proportions of fibro- spot,” can be quite subtle and is often a chance finding by the osseous and fibrovascular tissue occupying the resorptive patient, the dentist or, increasingly, the dental hygienist. cavity. All ECR defects present as a radiolucency of varying However, it is a relatively rare feature of ECR. Furthermore, it radiodensity, often in but not confined to the cervical region must occur at a site where it is readily identifiable (e.g., labial of the affected tooth or teeth (see Figs. 16-6 and 16-7; also surface of an anterior tooth) to be noticed. Loss of periodontal Figs. 16-8 and 16-9). attachment may occur in the region of the resorption, and The lesion tends to be radiolucent when the defect is pre- probing of the resorptive defect or the associated periodontal dominantly fibrovascular, granulomatous tissue. However, in pocket causes the granulation tissue to bleed profusely.89 cases with some fibro-osseous inclusions (i.e., more longstand- As the process progresses, perforation of the root canal wall ing lesions), the radiolucency may adapt a more cloudy appear- and bacterial contamination of the pulp may occur. The ance. In advanced cases with extensive repair of the tissue affected tooth may develop pulpitis and the associated clinical destruction, significant deposition of fibro-osseous tissue gives symptoms. Pulp necrosis and chronic periapical periodontitis the defect a mottled radiographic appearance (Fig. 16-10). may eventually develop. Clinical signs and symptoms may be The margins of the lesion may vary from poorly to well the first indication of a problem with the affected tooth; they defined, depending on the depth of the defect and the propor- may include tooth discoloration, spontaneous localized pain, tion and distribution of osseous inclusions in the lesion. tenderness on mastication, tenderness to percussion, tender- Although lesions with irregular margins are more common, ness to palpation over the apical region of the tooth, a draining some ECR defects may have smooth and/or well-defined sinus, and/or buccal sulcus swelling. margins. CHAPTER 16 Root Resorption 669

A B

C D E

F G

FIG. 16-7 A, This patient’s upper left central incisor had a pink spot and was cavitated. B, A radiograph revealed an unusual presentation of ECR; the lesion is circular and has well-defined margins. Note that the outline of the root canal is visible and intact through the radiolucent lesion. C, The tooth was unrestorable and was extracted; note the large amount of granulation tissue. D, Three-dimensional reconstruction of the extracted tooth from microtomography data revealed bonelike tissue below the overlying granulation tissue. Note the intact root canal wall (red arrow). E, Coronal reconstruction from a microtomography scan reveals how the predentin (red arrow) prevented the ECR defect from invading the root canal. In addition, bonelike tissue can be seen (yellow arrow). Posttreatment view (F) and radiograph (G) after replacement of the upper left central incisor with an implant- retained crown. (From Patel S, Kanagasingam S, Pitt Ford T: External cervical resorption: a review, J Endod 35:616, 2009.) 670 PART II The Advanced Science of Endodontics

FIG. 16-8 External cervical resorption (ECR) with replacement. A, Radiograph of a maxillary central incisor of a A B 34-year-old female patient who recalled being struck in the face with a cricket ball when she was 11 years old. The dentist mistakenly diagnosed the resorptive defect as caries and attempted to manage it accordingly. The tooth was asymptomatic; however, a 4-mm periodontal probing depth was identified on the palatal aspect of the tooth. A cone beam computed tomography (CBCT) scan confirmed an ECR defect. The tooth was deemed unrestorable and was extracted with the patient’s consent. B, Palatal view of the tooth at the end of the demineralization process, while immersed in the clearing agent. The tooth was separated into four portions, which were embedded separately in paraffin blocks. C, CBCT axial section passing through the coronal third at the level of line 1 in B. The corresponding histologic section shows that most C of the dentin has been replaced by a bonelike tissue. The root canal is no longer present. (H&E stain; original magnification ×8.) D, CBCT axial section passing through the middle third at the level of line 2 in B. The root canal can be appreciated at this level, although reduced in size, but it appears to be encircled by bonelike tissue (original magnification ×8). E, CBCT axial section taken from the apical third at the level of line 3 in B. At this level the canal is the same size as that of the contralateral tooth. The histologic section confirmed the absence of bonelike tissue (original magnification ×8).

E CHAPTER 16 Root Resorption 671

F G

H I

FIG. 16-8, cont’d F, Detail from C showing the transition from dentin to bonelike tissue (original magnification ×100). G, Higher magnification of F. The metaplastic tissue does not show the lamellar structure typical of bone (original magnification ×400). H, Detail of the canal in D. The metaplastic bonelike tissue has concentrically replaced a consistent portion of dentin, leaving in place a reduced layer of the original dentin (original magnification ×25). I, High-power view confirms that the tissue surrounding the canal is dentin (original magnification ×400). Considerations: The resorption process, followed by replacement with a bonelike tissue, started in the cervical area and extended in an apical direction, tunneling dentin circumferentially up to the transition between the middle and apical thirds of the tooth.

Despite its often cervical location, ECR may commence the root canal; it is merely superimposed on the defect radio- apical to this region, reflecting the position of the epithelial graphically. In cases of IRR, it should be possible to trace the attachment of the affected tooth. In teeth with a normal peri- outline of the root canal through the resorptive defect because odontal attachment, the invasive nature of the process may the defect is an extension of the root canal wall and is con- result in the lesion extending some distance apical and/or tinuous with it.51 Although this is a useful diagnostic feature, coronal to the cervical location where it started. Furthermore, it does have some shortcomings. First, the outline of the root the tissue destruction at the site of onset may sometimes be canal wall may be obscured by calcified tissue in the resorp- minimal and/or not evident on conventional radiographs due tive defect (ECR or IRR). Second, when ECR has resulted in to its location on the root surface. In these instances the lesion extensive tissue destruction, perforation of the root canal wall may appear to have originated at a location where significant may have allowed communication between the canal wall and tissue destruction, evident radiographically, has occurred. This the external defect. may be some distance from the actual point of origin. This Parallax radiographs should always be used to obtain further feature has come to light only since the advent of assessment information about the nature of the resorptive process. In addi- of ECR with CBCT. tion to a paralleled periapical radiograph, another radiograph The radiographic features of ECR are very similar to those should be taken with a shift (parallax) in the horizontal angu- of Internal Root Resorption (IRR) (discussed later), and dif- lation of the x-ray tube in relation to the image receptor. In ferentiating between them, especially in the absence of clini- cases of ECR, the position of the resorptive defect moves rela- cal signs, may be challenging. It is useful to trace the outline tive to the root canal. If the lesion is located palatally/lingually, of the root canal walls as they approach and pass through the the defect moves in the same direction as the x-ray tube shift. resorption defect on the radiograph. In cases of ECR, the If the lesion is located buccally, it moves in the opposite direc- outline of the canal wall should be visible and intact and tion. This is sometimes referred to as the “same lingual, oppo- should maintain its course as it passes through the defect. site buccal” (SLOB) rule. In contrast, internal resorptive defects This is due to the fact that the resorptive lesion lies on the maintain their position relative to the root canal because the external surface of the root and is not in communication with defects are an extension of the root canal system. 672 PART II The Advanced Science of Endodontics

A B

C D

E F

FIG. 16-9 External cervical resorption with replacement. A, Mandibular first and second molars in a 27-year-old female patient. The patient had symptoms of irreversible pulpitis associated with the lower right second molar; the lower right first molar was asymptomatic. B, CBCT scan taken through the second molar at the level of the area indicated by the arrow in A. Note massive resorption involving the crown and root. C, Mesial portion of the crown of the second molar after clearing. D, Section taken on a buccolingual plane. Overview shows resorption and replacement on the lingual side, corresponding to the area indicated by the arrow in C. (H&E stain; original magnification ×6.) E, Detail of the area indicated by the arrow in C. Several areas of resorption, with replacement by metaplastic tissue, can be seen (original magnification ×100). F, Magnification of the area indicated by the right lower arrow in E. The metaplastic tissue closely resembles bone (original magnification ×400).

CBCT has allowed 3D assessment of the nature, position, diagnosing the presence and nature of the root resorption. and extent of the resorptive defect, eliminating diagnostic These researchers also concluded that the correct treatment confusion and providing essential information about the plan should incorporate the additional information provided restorability and subsequent management of the tooth. CBCT by CBCT.88 is particularly useful if the clinician is not sure whether the ECR cavity has perforated the root canal wall (and thus for Management determining the need for root canal treatment). A CBCT scan The fundamental treatment objectives in ECR are to excavate eliminates the need for exploratory treatment. A clinical the resorptive defect, halt the resorptive process, restore the study by Patel et al.88 comparing the ability of conventional hard tissue defect with an aesthetic filling material, and radiographs and CBCT to diagnose and to differentiate accu- prevent and monitor the tooth for recurrence. Endodontic rately between IRR and ECR showed that CBCT was signifi- treatment of the affected tooth is necessary when the resorptive cantly more accurate (100%) than periapical radiographs at process has perforated the root canal wall. Surgical access CHAPTER 16 Root Resorption 673

G H

I J FIG. 16-9, cont’d G, High-power view of the area indicated by the right upper arrow in E. Note the lacuna in dentin occupied by a multinucleated clastic cell (original magnification ×1000). H, High-power view of the metaplastic bone tissue in the area indicated by the arrow in F. A bone trabecula is being resorbed by a typical osteoclast (original magnification ×1000). I, High-power view of the area indicated by the left lower arrow in E. A Howship lacuna with odontoclasts can be seen (original magnification ×1000). J, High-power view of the area indicated by the left upper arrow in E. Note the islands of bone tissue, with typical osteocytes, surrounded by dentin (original magnification ×1000). Considerations: In this case the metaplastic tissue was similar to normal lamellar bone. It is interesting that the bone tissue was undergoing remodeling, as evidenced by the presence of osteoclasts. Osteoclasts and odontoclasts can be observed in the same area and show morphologic similarity. to the site of resorption is gained by raising a mucoperiosteal removal of the tissue with ultrasonic instruments. It may be flap, the dimensions of which should allow visualization of extremely difficult to differentiate between sound dentin and the full extent of the defect (see Fig. 16-10). Once access fibro-osseous deposits; therefore, use of the surgical operating has been achieved, the resorptive cavity is excavated. microscope is essential. Frequent intraoperative radiographs Fibrovascular granulomatous tissue is readily removed with may be necessary to ensure accurate removal of unwanted, a hand excavator. However, defects containing significant hard resorptive tissue and to prevent unnecessary removal of amounts of fibro-osseous tissue (especially when the latter is sound dental tissue. The value of a preoperative CBCT scan in contiguous with the adjacent dentin) require discriminate these cases cannot be overemphasized. The scan allows 674 PART II The Advanced Science of Endodontics

A B C

D E F

FIG. 16-10 Early internal root resorption. A, A 54-year-old male patient presented with a long story of pain on chewing and with cold stimuli. Recently the pain had become continuous and severe. The maxillary first molar, which had a mesial amalgam restoration, did not respond to sensitivity tests. A radiograph showed a periapical radiolucency on the palatal root. After removal of the restorative materials, a crack line involving the pulp chamber floor was diagnosed, and the tooth was extracted. B, Some pathologic tissue remained attached to the palatal root apex at extraction. A longitudinal section passing at the center of the canal demonstrated vital connective tissue in the apical third. (H&E stain; original magnification ×16.) C, Detail of the canal in B. Note the connective tissue with vessels and relatively few inflammatory cells, in addition to the resorption lacunae on the root canal walls (original magnification ×1000). D, High-power view of the area of the canal wall indicated by the upper arrow in C. Note the resorption lacuna with odontoclasts (original magnification ×1000). E and F, Progressive magnification of the area of the canal wall indicated by the lower arrow in C. Note the Howship lacuna housing clastic cells. Predentin is present in some areas of the wall, but it is absent in the areas with active resorption (original magnification ×400 and ×1000). accurate interpretation of the full extent of the defect in all treats small channels of resorption that are not accessible to planes. True measurements can be made from the scan and mechanical instrumentation.89 Once the defect has been exca- related to clinical reference points, reducing greatly the need vated and treated with trichloroacetic acid, any undermined for subjective tissue removal. dentin or enamel at the peripheries of the cavity are removed Once the resorptive tissue has been removed, the cavity may with a bur in a high-speed handpiece, and the cavity is restored be treated with a 90% aqueous solution of trichloracetic acid; with an aesthetically acceptable restorative material, such as this causes coagulation necrosis of the resorptive tissue without composite resin or glass ionomer cement. Biodentine may damaging the periodontal tissue.61 The acid also penetrates and prove to be a particularly suitable material for restoring these CHAPTER 16 Root Resorption 675 defects30,85 because it may combine acceptable aesthetics with in exposure of the underlying mineralized dentin to odonto- the ability to support PDL attachment. clasts (Fig. 16-11).106,115 Once the cavity has been restored, the mucoperiosteal flap The precise injurious events necessary to bring about such is replaced and secured in position. If perforation of the root damage have not been completely elucidated. Various etiologic canal wall has occurred, root canal treatment should be carried factors have been proposed for the loss of predentin, including out. Access to the root canal system should be gained under a trauma, caries and periodontal infections, excessive heat gen- rubber dam before the resorptive defect is assessed. The root erated during restorative procedures on vital teeth, calcium canal should be prepared in the area of the defect as normal, hydroxide procedures, vital root resections, anachoresis, ortho using saline as an irrigant. A tapered gutta-percha (GP) point dontic treatment, cracked teeth, or simply idiopathic dystro- then should be placed in the canal to maintain its patency phic changes in normal pulps.5,18,28,93,112 In a study of 25 teeth during the excavation and restoration of the resorptive defect with internal resorption, trauma was found to be the most and to provide a barrier against which the final restoration can common predisposing factor, responsible for 45% of the cases be condensed. The rubber dam is removed, and surgical treat- examined.29 The suggested etiologies in the other cases were ment of the resorptive defect can be carried out as described, inflammation as a result of carious lesions (25%) and carious/ without any risk of the resorptive debris entering the root canal periodontal lesions (14%). The cause of the internal resorption system. After repositioning of the mucoperiosteal flap, root in the remaining teeth was unknown. Other reports in the canal treatment can be completed in the normal manner, literature support the view that trauma5,116,118 and pulpal without fear of extrusion of infected tissue, irrigants, or medi- inflammation/infection58,118 are the major contributory factors caments into the periodontal tissues. in the initiation of internal resorption. Heithersay62 advocated a nonsurgical approach to the treat- Wedenberg and Lindskog115 reported that IRR could be a ment of ECR. In this technique, a 90% aqueous solution of transient or progressive event. In an in vivo primate study, the trichloroacetic acid is applied to the resorptive tissue in the root canals were accessed in 32 incisors with the predentin defect until coagulation necrosis occurs. The necrosed intentionally damaged. The access cavities in half of the teeth tissue is then excavated, and the defect is restored with glass were sealed; those in the other half were left open to the oral ionomer cement. cavity. The teeth were extracted at intervals of 1, 2, 6, and 10 The treatment of ECR depends on the severity, extent, and weeks. The authors noted only a transient colonization of the location of the resorptive defect and the restorability of the damaged dentin by multinucleated clastic cells in the teeth that tooth. Heithersay61 developed a four-stage classification system had been sealed (i.e., transient internal root resorption). Those for ECR based on the depth of penetration of the resorption in teeth were free of bacterial contamination, and no signs of a buccolingual and apicocoronal direction. He examined the active hard tissue resorption occurred. The teeth that were left prognosis of treatment in 101 cases of ECR in 94 patients using unsealed during the experimental period showed signs of the nonsurgical protocol referred to previously, and related the extensive bacterial contamination of pulpal tissue and dentinal success rates for treatment to the classification of the lesion.61 tubules. Those teeth demonstrated extensive and prolonged He reported a 100% success rate for class I and class II lesions, colonization of the damaged dentin surface by clastic cells and a 77.8% success rate for class III lesions, and a 12.5% success signs of mineralized tissue resorption (progressive internal root rate for class IV lesions. This emphasizes the poorer outcome resorption). As previously mentioned, damage to the odonto- that can be expected for more advanced cases. A major limita- blast layer and predentin of the canal wall is a prerequisite for tion of the Heithersay classification is that it is valid only if the initiation of internal root resorption.116 However, progression ECR lesion is confined to the proximal aspect of the tooth, of internal root resorption depends on bacterial stimulation of because lesions are assessed on 2D radiographs. If the ECR the clastic cells involved in hard tissue resorption. Without this lesion is located on and/or extends to the labial and/or buccal stimulation, the resorption is self-limiting.116 (proximal) aspects of the tooth, the true nature of the lesion For IRR to continue, the pulp tissue apical to the resorptive cannot be accurately assessed with radiographs.86,89 lesion must have a viable blood supply; this provides clastic cells and their nutrients, and the infected necrotic coronal pulp tissue provides stimulation for those clastic cells (see Fig. INTERNAL ROOT RESORPTION 16-11).105 Bacteria may enter the pulp canal through dentinal tubules, carious cavities, cracks, fractures, and lateral canals. Introduction In the absence of a bacterial stimulus, the resorption is tran- Internal root resorption (IRR) is a form of root resorption that sient and may not advance to the stage that can be diagnosed originates in and affects the root canal wall.9 It is further clas- clinically and radiographically. Therefore, the pulp apical to sified as either inflammatory or replacement. The replacement the site of resorption must be vital for the resorptive lesion to type is associated with the deposition of mineralized tissue in progress (see Fig. 16-11). If left untreated, internal resorption the root canal space after the initial loss of dentin (initial may continue until the inflamed connective tissue filling dentin loss is a feature of both types).9 Because their charac- the resorptive defect degenerates, advancing the lesion in an teristics are largely similar, the two types of IRR are discussed apical direction. Ultimately, if left untreated, the pulp tissue together. apical to the resorptive lesion undergoes necrosis and the bacteria infect the entire root canal system, resulting in apical 96 Etiology and Pathogenesis of Internal periodontitis. Root Resorption Histologic Appearance For IRR to occur, the outermost protective odontoblast layer Wedenberg and Zetterqvist118 reported on the histologic nature and the predentin of the canal wall must be damaged, resulting of IRR. The authors specifically examined the histologic, 676 PART II The Advanced Science of Endodontics

A B

C D E

FIG. 16-11 A and B, Parallax views of the maxillary left lateral incisor showing internal root resorption with necrosis. A gutta-percha point has been used to track the sinus. The reconstructed sagittal (C) and axial (D) slices from a CBCT scan reveal that the lesion has resorbed the palatal aspect of the root (arrows) and has nearly perforated the root wall. E, The tooth has been obturated with gutta-percha using a thermoplasticized technique. (From Patel S, Ricucci D, Durack C, Tay F: Internal root resorption: a review, J Endod 36:1107, 2010.)

enzyme histochemical, and scanning electron micrographic resorption lacunae. The odontoclasts showed evidence of (SEM) features of the resorptive process in a small sample of active resorption (see Fig. 16-11). They were accompanied by primary and secondary teeth extracted due to progressive IRR. mononuclear cells, believed to be odontoclast precursors, The histologic and enzyme histochemical profiles were identi- which populated the connective tissue adjacent to the site of cal for the two groups, but the resorptive process appeared to resorption. Both types of cell displayed tartarate-resistant acid occur more rapidly in the primary teeth. The pulpal tissue was phosphatase (TRAP) activity. populated to varying degrees in all teeth with an inflammatory Interestingly, the root canal wall was incompletely lined infiltrate composed predominantly of lymphocytes and macro- with a mineralized tissue resembling bone or cementum in all phages, with some neutrophils. The connective tissue in the of the examined teeth.118 Furthermore, islands of calcified pulp spaces was less vascular than healthy pulpal tissue and tissue of a similar nature occupied the pulpal space in three of resembled periodontal membrane connective tissue with rela- the cases. Islands of mineralized tissue occupying the root tively more cells and fibers. The odontoblast layer and preden- canal space are the defining feature of internal replacement tin were absent from the affected dentinal walls, which were resorption.9 The authors suggested that deposition of this min- populated by large, multinucleated odontoclasts occupying eralized tissue is likely to be part of a coupling process, at the CHAPTER 16 Root Resorption 677 end of a period of resorption, in which osteoblasts are attracted the resorption defect. An IRR cavity is continuous with the to the affected site and participate in bone formation.118 normal root canal walls because it is essentially an extension of them. As such, in teeth with single canals affected by IRR, Clinical Features the canal walls should not be traceable through the defect. The clinical features of IRR largely depend on the histologic This is in contrast to ECR, in which the lesion lies buccal or status of the affected pulp, the extent of the hard tissue destruc- palatal/lingual to the defect and is consequently superim- tion caused by the resorptive process, and the position of the posed on the canal system when viewed on conventional resorptive cavity in the root canal space. In the active stages radiographs. In this situation the canal walls should maintain of resorption, bacterial contamination of vital pulpal tissue their normal course as they pass through the resorption may cause an acute inflammatory response, leading to clinical defect, allowing them to be traced through it. However, it symptoms of pulpitis. With the onset of pulpal necrosis and should be noted that in teeth with multiple canals, a canal an established bacterial colonization of the root canal space, that has been unaffected by IRR may be superimposed onto clinical signs and symptoms associated with acute or chronic the IRR resorption defect on conventional radiographs. When apical periodontitis may develop. Sinus tract(s) may occur and the guidelines outlined by Gartner et al.51 are used, this may may be associated with suppuration in the periapical tissues or lead to misdiagnosis. possibly at the site of a perforation of the root canal wall caused Parallax radiographs must always be used to obtain further by the hard tissue destruction. Extensive resorption of the information about the resorptive process. In addition to a par- coronal pulp may result in a pink or red discoloration visible alleled periapical radiograph, a radiograph should be taken through the crown of the affected tooth; this is caused by with a shift in the horizontal angulation of the x-ray tube in granulomatous tissue extending into and occupying the resorp- relation to the image receptor. IRR lesions maintain their positive defect.75 Although often reported as a common clinical tion relative to the root canal system on the angled view. ECR indicator of the process, these pink spots actually are rare in lesions move in the same direction as the x-ray tube shift if cases of IRR. They may occur relatively more frequently in they are lingually/palatally positioned and move in the oppo- cases of ECR, but they are not very common with that resorp- site direction if they are buccally located.51 This diagnostic tion type, either. Often the affected tooth is asymptomatic, and technique, coupled with tracing of the root canal/pulp chamber clinical signs are absent. outline through the lesion, has been the most reliable aid in the differential diagnosis of IRR when conventional radiogra- Radiographic Features and Diagnosis phy is used. However, as discussed previously, the amount of The diagnosis of any type of root resorption depends on radio- information available from conventional radiographic imaging graphic demonstration of its presence. The two-dimensional is limited. This can lead to misdiagnoses and incorrect nature of conventional radiographic imaging makes the detec- treatment in the management of IRR and invasive cervical tion and differentiation of the various types of resorption chal- resorption. lenging. This is especially true when attempts are made to The use of CBCT as a diagnostic and treatment planning differentiate between IRR and ECR, which may have similar tool in the management of IRR has been reported in the litera- radiographic features.57,90,91,105 Much has been reported in the ture.21 Information such as the position, extent, and dimen- literature about the “typical” radiographic features of IRR. sions of an IRR lesion, in addition to the presence of any Gartner et al.51 reported that lesions of IRR present radio- associated perforation, can be obtained from a CBCT scan. The graphically as radiolucencies of uniform density that have a same scan can differentiate between ECR and IRR, removing smooth outline and are symmetrically distributed over the root any doubt about the diagnosis that may have arisen with the of the affected tooth (Fig. 16-12). The authors further reported conventional radiographic examination. that the outline of the root canal wall should not be traceable through the resorption defect because the root canal wall bal- Management loons out. Other authors have described IRR lesions as oval, Once a diagnosis of IRR has been made, the extent of the circumscribed radiolucencies in continuity with the root canal hard tissue destruction must be assessed and a clinical wall.82 Although certain cases of IRR may have some or all of decision must be made about the prognosis of the affected these radiographic features, many do not; each case should be tooth. If the affected tooth is salvageable and has a reasonable assessed individually before a diagnosis is made. prognosis, root canal treatment is necessary. As with any IRR can occur at any location in the root canal system and infected tooth, the main purpose of the root canal treatment may manifest radiographically as a radiolucency with variable is to remove the intraradicular bacteria and disinfect the root shape, radiodensity, outline, and symmetry in relation to the canal space. If the resorptive process is still active, the treat- root canal. Internal inflammatory root resorption lesions are ment serves an adjunctive purpose, which is to eliminate the more likely be uniformly radiolucent, whereas in internal vital apical tissue that is sustaining and stimulating the replacement (metaplastic) root resorption, the defect has a resorbing cells. somewhat mottled or clouded appearance as a result of the The nature of the resorptive process in cases of IRR presents radiopaque nature of the calcified material occupying the the endodontist with unique operative challenges. In teeth lesion (Fig. 16-13).91 ECR lesions may contain predominantly with active resorption, profuse bleeding from the granuloma- granulomatous tissue, predominantly calcified tissue, or a tous and inflamed pulpal tissues may impair visibility in the mixture of the two; therefore, they may have a radiodensity initial stages of treatment and may provide a stubborn source similar to either type of internal resorption, which compli- of mild hemorrhage when attempts are made to dry the canal cates the clinical differentiation of the disease process. As after chemomechanical preparation. Furthermore, the irregu- noted by Gartner et al.,51 the best practice is to trace larly concave nature of resorption defects makes them inacces- the outline of the root canal wall as it approaches and leaves sible to direct mechanical debridement. 678 PART II The Advanced Science of Endodontics

3 2 1

A B C

D E

FIG. 16-12 Light microscopy images of a tooth with internal (root canal) replacement resorption. The tooth belonged to a 44-year-old male patient who was referred to the first author for management of a perforated root. The tooth was asymptomatic on examination, but there was a history of previous trauma. A, Radiograph of a maxillary central incisor with a radiolucent lesion in the middle third of the root canal. The radiolucent lesion appears to be mottled, which suggests internal root resorption with metaplasia. B, Clinical radiograph of the tooth after extraction, taken at a 90-degree angle, showing the continuity of the resorptive lesion with the canal space. C, Cross section taken approximately at the level of line 1 in Fig. 16-12, B. The low-magnification overview shows that the dentin around the root canal had been replaced by an ingrowth of bone tissue, and the root appears to have been perforated on the distopalatal aspect. (H&E stain; ×8.) D, Higher magnification of Fig. 16-12, C. (H&E stain; ×16.) E, High magnification of the area demarcated by the rectangle in Fig. 16-12, D. The intraradicular dentin has been resorbed. (H&E stain; ×100.) F, High-magnification view taken from the right part of Fig. 16-12, C, showing that the resorbed dentin has been replaced by lamellar bone. Osteocytes are present in lacunae between the lamellae. A characteristic cross section of an osteon can be seen on the right (open arrows), with concentric lamellae surrounding a vascular structure. (H&E stain; ×100.) CHAPTER 16 Root Resorption 679

G H

I J FIG. 16-12, cont’d G, High-magnification view of the area indicated by the left open arrow in Fig. 16-12, E. A multinucleated resorbing cell (odontoclast) can be seen in a dentinal lacuna, indicating active resorption of the dentinal wall. (H&E stain; ×1000.) H, High-magnification view of the bone surface indicated by the right arrow in Fig. 16-12, E. The large cells are osteoblast-like cells. Once they produced mineralized tissue, they were embedded in the bone lacunae, assuming the characteristics of osteocytes. (H&E stain; ×1000.) I, Cross section taken approximately at the level of line 2 in Fig. 16-12, B. The root canal is still large at this level and is surrounded by a relatively thin layer of newly formed bone. (H&E stain; ×16.) J, Cross section taken approximately at the level of line 3 in Fig. 16-12, B. At this level the root canal appears consistently narrowed by a dense layer of newly formed bone. (H&E stain; ×16.) (From Patel S, Ricucci D, Durack C, Tay F: Internal root resorption: a review, J Endod 36:1107, 2010.)

Chemomechanical Debridement of adjunctive measure is used, microbes may persist in confined areas after chemomechanical debridement.26 As such, an intra- the Root Canal canal antibacterial medicament should be used to further The complex anatomic and morphologic features of root canal reduce the microbial load and improve the disinfection of the systems provide unique recesses that may harbor microorgan- root canal space.99 Calcium hydroxide is an antibacterial, inter- isms in infected teeth. Endodontic instruments and passively appointment, endodontic medicament that has been shown to delivered irrigants fail to penetrate into these secluded spaces eradicate bacteria persisting in the root canal space after root and niches.80,97,99 The use of ultrasonic instruments to aid the canal treatment.27,100 Also, when used in conjunction with penetration of endodontic irrigants has been shown to improve sodium hypochlorite, it potentiates the effect of that irrigant the removal of organic debris and biofilms from the root canal in the removal of organic debris from the root canal system.2,108 space.26 Given the inaccessibility of IRR defects to normal Based on this evidence the authors advocate the use of instrumentation and passive irrigation, ultrasonic activation of calcium hydroxide as an intracanal, antibacterial medicament irrigants should be considered an essential step in the treat- to supplement the conventional chemomechanical debridement of these cases (see Fig. 16-11). However, even when this ment of the root canal system.91 1 1

2 2

A B C D

E F G

H I J

FIG. 16-13 Light microscopy images of a variant of internal (root canal) replacement resorption with tunneling resorption. The lower right lateral incisor belonged to a 39-year-old former boxer who had suffered a jaw fracture in a boxing match in his early twenties and was placed in intermaxillary fixation. The patient developed symptoms 20 years later and complained of pain associated with his lower incisors. A, Radiograph of the mandibular right incisors. The lower right central incisor had asymptomatic apical periodontitis associated with a necrotic and infected pulp. The lower right lateral incisor showed a large area of internal root resorption. The tooth did not respond to sensitivity tests. B, Sagittal CBCT slice shows some calcified tissue in the resorptive defect. C, Cross section taken at the level of line 1 in Fig. 16-13, A and B. The overview shows that the canal was apparently empty at this level. (H&E stain; ×6.) D, High magnification of the area indicated by the arrow in Fig. 16-13, C. Lamellar bone filling an area of previous resorption. Note the osteon structure (arrow). (H&E stain; ×100.) E, Cross section taken at the level of line 2 in Fig. 16-13, A and B. Overview shows that the canal lumen was partly occupied by necrotic remnants, partly by bonelike tissue. (H&E stain; ×8.) F, High magnification of the lower part in Fig. 16-13, E. (H&E stain; ×50.) G, Higher magnification of Fig. 16-13, F. Bone trabeculae are surrounded by necrotic debris. (H&E stain; ×100.) H, Cross section taken from the same area as that in Fig. 16-13, E. (Taylor’s modified Brown & Brenn [TBB] stain; ×16.) I, High magnification of the area indicated by the arrow in Fig. 16-13, H. A fragment of bonelike tissue can be seen surrounded by bacteria-colonized necrotic tissues. (TBB stain; ×100; inset ×1000.) J, Longitudinal section passing approximately through the center of the root apex. Dentin walls have been resorbed and replaced by a bonelike tissue. (H&E stain; ×16.) (From Patel S, Ricucci D, Durack C, Tay F: Internal root resorption: a review, J Endod 36:1107, 2010.) CHAPTER 16 Root Resorption 681

Obturation GP can then be used as a barrier against which the MTA can One of the fundamental objectives of endodontic treatment is be packed. Hsiang-Chi Hsien et al.63 successfully used this to fill the disinfected root canal space completely with an technique to treat a perforating IRR defect in a maxillary appropriate material. In IRR, the hard tissue defects caused by incisor. Jacobowitz and de Lima66 described a case in which a the resorptive process are challenging to fill adequately. To maxillary central incisor with a large, perforating internal obturate the resorptive defect, the root-filling material must be resorption defect had a poor prognosis and initially was des- able to flow. Gutta-percha is widely regarded as the gold ignated for extraction. The tooth subsequently was success- standard filling material in endodontics. It can plasticize fully treated in an orthograde manner with white MTA and when pressure is applied, and it becomes flowable with the gutta-percha.66 application of heat. Gencoglu et al.52 examined the ability of Clinical situations arise in which a perforating resorptive different obturation systems and techniques to fill the defects defect causes extensive dental hard tissue destruction that fails in artificially created internal resorptive cavities ex vivo. They to respond to or is not amenable to repair with an orthograde found that Obtura II (ObII) and Microseal (MS) thermoplastic approach. Surgical treatment may be needed in these cases. For gutta-percha systems produced significantly better fills in sim- the reasons discussed already, MTA would be the material of ulated resorptive cavities than did Thermafill, soft core systems choice to repair these perforations. In cases that have not already (SCS), and cold lateral condensation (CLC). In a similar study been treated in a nonsurgical manner, the operator first must Goldberg et al.55 demonstrated that the Obtura II system filled access the root canal as for orthograde treatment. A well-fitting, simulated resorptive defects statistically better than CLC, tapered GP point or an appropriately sized finger spreader is Thermafill, and a hybrid technique. Gencoglu et al.52 reported then positioned in the canal to occlude it and to provide a barrier that the Obtura II and Microseal systems produced fills with against which the MTA can be packed once surgical access to greater gutta-percha–to-sealer (GP/sealer) ratios than did the the defect has been gained. The barrier also prevents inadvertent other techniques examined. These findings were corroborated deposition of the MTA into the apical third of the canal. The in the study by Goldberg et al.55 perforation is then exposed surgically and repaired with the Because root canal sealers shrink on setting120 and dissolve MTA. The canal can be shaped, disinfected, and obturated with and degrade to varying degrees in the presence of moisture,78 thermoplasticized GP once the MTA has set. fillings with higher GP/sealer ratios reduce the risk ofvoid If the resorptive process has caused sufficient tissue destruc- formation and leakage of contaminants into the root canal tion to render the tooth unrestorable, extraction is the most system, with potentially positive benefits for the treatment appropriate treatment option. If the tooth has been weakened outcome. by the disease process to the extent that fracture of the tooth Stamos and Stamos103 and Wilson and Barnes121 reported on root is likely, the patient may elect to have the tooth extracted. cases of internal resorption in which the Obtura system was The presence of a perforating resorptive defect is certainly not successfully used to obturate the canal. In light of the evidence a contraindication to treatment, but a perforation of significant presented, the Obtura and Microseal systems apparently can size will have a bearing on the decision to surgically treat or be expected to produce the best technical results for obturating extract the tooth. CBCT is an invaluable component of the canals with IRR. clinician’s armamentarium in the treatment of IRR.42 A CBCT When choosing the appropriate materials and methods for scan gives the clinician a 3D view of the tooth, the resorptive filling resorptive defects of IRR, the clinician first must estab- defect, and the adjacent anatomy. The clinician thus has the lish the presence and extent of any perforations in the wall of information necessary to determine a prognosis for the tooth the affected root. This information can be readily obtained and/or its amenability to surgical repair. If extraction of the from an appropriate CBCT scan. If perforation has occurred, affected tooth is indicated, the scan may be used as a diagnostic mineral trioxide aggregate (MTA) should be considered the and treatment planning tool for provision of a dental implant– material of choice to repair the root wall. MTA is biocompat- retained prosthesis. Bhuva et al.21 described a case in which ible,79 has superior sealing properties when used as a retro- CBCT was used as a treatment planning tool in the successful grade filling material,17 and has proved effective in the repair treatment of a case of IRR. As mentioned previously, CBCT has of lateral and furcal root perforations in animal studies.76 Fur- been found to be highly accurate in revealing the nature of the thermore, the material is well tolerated in the periapical tissues, resorptive lesion, and this leads to the selection of the most and when used as a root-end filling material in the absence suitable treatment plan. of infection, it supports almost complete regeneration of the adjacent periodontium.94 These are desirable properties in the context of perforation repair because of the very real possibility SUMMARY of unintentional extrusion of the material when a perforating ♦ The prevalence, etiology, and pathogenesis of various types internal resorptive defect is repaired in an orthograde manner. of root resorption are not fully understood, and more However,, the flow properties of MTA are significantly poorer research is required in these areas. than those of heated GP. Its use as an effective filling material ♦ More clinical data are required on the presenting features in IRR depends on adequate ultrasonic activation of the mate- of ECR because this particular type of resorption may vary rial to disperse it into the recesses of the defect.21 Use of a significantly in its presentation. dental operating microscope and the correct equipment to ♦ Early detection of root resorption is essential for successful deliver the material are essential. management and favorable outcomes. A hybrid technique to obturate canals affected by perforat- ♦ CBCT is an excellent diagnostic tool for confirming the ing internal resorption also may be used. In these cases presence of ECR and IRR and also for appreciating the true the canal apical to the resorption defect is filled with GP. The nature of these conditions and managing them. 682 PART II The Advanced Science of Endodontics

REFERENCES 1. Alqerban A, Jacobs R, Souza PC, Willems G: In-vitro 22. Bille ML, Kvetny MJ, Kjaer I: A possible association 43. Durack C, Patel S, Davies J, et al: Diagnostic accuracy of comparison of 2 cone beam computed tomography between early apical resorption of primary teeth and small volume cone beam computed tomography and systems and panoramic imaging for detecting simulated ectodermal characteristics of the permanent dentition, intraoral periapical radiography for the detection of canine impaction-induced external root resorption in Eur J Orthod 30:346, 2008. simulated external inflammatory root resorption, Int Endod maxillary lateral incisors, Am J Orthod Dentofacial Orthop 23. Bille ML, Nolting D, Kvetny MJ, Kjaer I: Unexpected early J 44:136, 2011. 136:764e1, 2009. apical resorption of primary molars and canines, Eur Arch 44. Ehnevid H, Lindskog S, Jansson L, Blomlöf L: Tissue 2. Andersen M, Lund A, Andreasen JO, Andreasen FM: In Paediatr Dent 8:144, 2007. formation on cementum surfaces in vivo, Swed Dent J vitro solubility of human pulp tissue in calcium hydroxide 24. Borg E, Källqvist A, Gröndahl K, Gröndahl H-G: Film and 17:1, 1993. and sodium hypochlorite, Endod Dent Traumatol 1:170, digital radiography for detection of simulated root 45. Estrela C, Reis Bueno M, Alencar AHG, et al: Method to 1992. resorption cavities, Oral Surg Oral Med Oral Pathol Oral evaluate inflammatory root resorption by using cone beam 3. Andreasen FM, Sewerin I, Mandel U, Andreasen JO: Radiol Endod 86:110, 1998. computed tomography, J Endod 35:1491, 2009. Radiographic assessment of simulated root resorption 25. Boyce BF, Xing L: Functions of RANKL/RANK/OPG in bone 46. Flores MT, Andersson L, Andreasen JO, et al: Guidelines cavities, Endod Dent Traumatol 3:21, 1987. modeling and remodeling, Arch Biochem Biophys for the management of traumatic dental injuries. I. 4. Andreasen FM, Vestergaard Pedersen B: Prognosis of 473:139, 2008. Fractures and luxations of permanent teeth, Dent luxated permanent teeth: the development of pulp 26. Burleson A, Nusstein J, Reader A, Beck M: The in vivo Traumatol 23:66, 2007. necrosis, Endod Dent Traumatol 1:207, 1985. evaluation of hand/rotary/ultrasound instrumentation in 47. Flores MT, Andersson L, Andreasen JO, et al: Guidelines 5. Andreason JO: Luxation of permanent teeth due to necrotic human mandibular molars, J Endod 33:782, for the management of traumatic dental injuries. II. trauma: a clinical and radiographic follow up study of 189 2007. Avulsion of permanent teeth, Dent Traumatol 23:130, injured teeth, Scand J Dent Res 19:273, 1970. 27. Byström A, Claesson R, Sundqvist G: The antibacterial 2007. 6. Andreasen JO: A time-related study of periodontal healing effect of camphorated paramonochlorophenol, 48. Frank AL, Blakland LK: Nonendodontic therapy for and root resorption activity after replantation of mature camphorated phenol and calcium hydroxide in the supraosseous extracanal invasive resorption, J Endod permanent incisors in monkeys, Swed Dent J 4:101, treatment of infected root canals, Endod Dent Traumatol 13:348, 1987. 1980. 1:170, 1985. 49. Furseth R: The resorption process of human teeth studied 7. Andreasen JO: Relationship between surface and 28. Cabrini R, Maisto O, Manfredi E: Internal resorption of by light microscopy, microradiography and electron inflammatory root resorption and changes in the pulp dentine: histopathologic control of eight cases after pulp microscopy, Arch Oral Biol 12:417, 1968. after replantation of permanent incisors in monkeys, amputation and capping with calcium hydroxide, Oral 50. Fuss Z, Tsesis I, Lin S: Root resorption: diagnosis, J Endod 7:294, 1981. Surg Oral Med Oral Pathol 10:90, 1957. classification and treatment choices based on stimulation 8. Andreasen JO: Review of the root resorption systems and 29. Çalişkan M, Türkün M: Prognosis of permanent teeth with factors, Ental Traumatol 19:175, 2003. models: etiology of root resorption and the homeostatic internal resorption: a clinical review, Endod Dent 51. Gartner AH, Mark T, Somerlott RG, Walsh LC: Differential mechanisms of the periodontal ligament. In Davidotch D, Traumatol 13:75, 1997. diagnosis of internal and external cervical resorption, editor: The biological mechanisms of tooth eruption and 30. Camilleri J: Investigation of Biodentine as dentine J Endod 2:329, 1976. root resorption, Birmingham, Ala, 1988, EBSCO Media. replacement material, J Dent 41:600, 2013. 52. Gencoglu N, Yildrim T, Garip Y, et al: Effectiveness of 9. Andreasen JO, Andreasen FM: Root resorption following 31. Chapnick L: External root resorption: an experimental different gutta percha techniques when filling traumatic dental injuries, Proceedings of the Finnish radiographic evaluation, Oral Surg Oral Med Oral Pathol experimental internal resorptive cavities, Int Endod J Dental Society 88:95, 1991. 67:578, 1989. 41:836, 2008. 10. Andreasen JO, Andreasen FM: Avulsions. In Andreasen 32. Chow J, Chambers TJ: An assessment of the prevalence 53. Gold SI, Hasselgren G: Peripheral inflammatory root JO, Andreasen FM, Andersson L, editors: Textbook and of organic matter on bone surfaces, Calcif Tissue Int resorption: a review of the literature with case reports, colour atlas of traumatic injuries to the teeth, ed 4, 50:118, 1992. J Clin Periodontol 19:523, 1992. Oxford, 2007, Blackwell Munksgaard. 33. Chung CJ, Soma K, Rittling SR, et al: OPN deficiency 54. Goldberg F, De Silvio A, Dreyer C: Radiographic 11. Andreasen JO, Borum MK, Andreasen FM: Replantation of suppresses appearance of odontoclastic cells and assessment of simulated external root resorption cavities 400 avulsed permanent incisors. Part 3. Factors related resorption of the tooth root induced by experimental force in maxillary incisors, Endod Dent Traumatol 14:133, to root growth, Endod Dent Traumatol 11:69, 1995. application, J Cell Physiol 214:614, 2008. 1998. 12. Andreasen JO, Borum MK, Jacobsen HL, Andreasen FM: 34. Cohenca N, Simon JH, Mathur A, Malfaz JM: Clinical 55. Goldberg F, Massone EJ, Esmoris M, Alfie D: Comparison Replantation of 400 avulsed permanent incisors. Part 2. indications for digital imaging in dentoalveolar trauma. of different techniques for obturating experimental internal Factors related to pulpal healing, Endod Dent Traumatol Part 2. Root resorption, Dent Traumatol 23:105, resorptive cavities, Endod Dent Traumatol 16:116, 2000. 11:59, 1995. 2007. 56. Gröndahl H-G, Huumonen S: Radiographic manifestations 13. Andreasen JO, Farik B, Munksgaard EC: Long-term 35. Crona-Larsson G, Bjarnasan S, Norén JG: Effect of of periapical inflammatory lesions, Endod Topics 8:55, calcium hydroxide as a root canal dressing may increase luxation injuries on permanent teeth, Endod Dental 2004. risk of root fracture, Dent Traumatol 18:134, 2002. Traumatol 7:199, 1991. 57. Gulabivala K, Searson LJ: Clinical diagnosis of internal 14. Andreasen JO, Hjørting-Hansen E: Replantation of teeth. 36. Cvek M: Treatment of non-vital permanent incisors with resorption: an exception to the rule, Int Endod J 28:255, I. Radiographic and clinical study of 110 human teeth calcium hydroxide. II. Effect on external root resorption in 1995. replanted after accidental loss, Acta Odontol Scand luxated teeth compared with effect of root filling with 58. Haapasalo M, Endal U: Internal inflammatory root 24:263, 1966. gutta percha, Odontol Revy 24:343, 1973. resorption: the unknown resorption of the tooth, Endod 15. Andreasen JO, Hjørting-Hansen E: Replantation of teeth. 37. Cvek M: Prognosis of luxated non-vital maxillary incisors Topics 14:60, 2006. II. Histological study of 22 replanted anterior teeth in treated with calcium hydroxide and filled with 59. Hammarström L, Lindskog S: General morphological humans, Acta Odontol Scand 24:287, 1966. gutta-percha: a retrospective clinical study, Endod Dent aspects of resorption of teeth and alveolar bone, Int 16. Andreasen JO, Løvschall H: Response of oral tissues to Traumatol 8:45, 1992. Endod J 18:93, 1985. trauma. In Andreasen JO, Andreasen FM, Andersson L, 38. Cvek M: Endodontic management and the use of calcium 60. Harokopakis-Hajishengallis E: Physiologic root resorption editors: Textbook and colour atlas of traumatic injuries to hydroxide in traumatized permanent teeth. In Andreasen in primary teeth: molecular and histological events, J Oral the teeth, ed 4, Oxford, 2007, Blackwell Munksgaard. JO, Andreasen FM, Andersson L, editors: Textbook and Sci 49:1, 2007. 17. Aqrabawi J: Sealing ability of amalgam, super EBA colour atlas of traumatic injuries to the teeth, ed 4, 61. Heithersay GS: Invasive cervical resorption: an analysis of cement and MTA when used as retrograde filling Oxford, 2007, Blackwell Munksgaard. potential predisposing factors, Quintessence Int 30:83, materials, Br Dent J 188:266, 2000. 39. Cvek M, Lindvall AM: External root resorption following 1999. 18. Ashrafi MH, Sadeghi EM: Idiopathic multiple internal bleaching of pulpless teeth with oxygen peroxide, Endod 62. Heithersay GS: Invasive cervical resorption, Endod Topics resorption: report of case, J Dent Child 47:196, 1980. Dent Traumatol 1:56, 1985. 7:73, 2004. 19. Bell T: The anatomy, physiology, and disease of the teeth, 40. D’Addazio PS, Campos CN, Özcan M, et al: A comparative 63. Hsien HC, Cheng YA, Lee Y, et al: Repair of perforating Philadelphia, 1830, Carey & Lee. study between cone-beam computed tomography and internal resorption with mineral trioxide aggregate: a case 20. Bender IB, Seltzer S: Roentgenographic and direct periapical radiographs in the diagnosis of simulated report, J Endod 29:538, 2003. observation of experimental lesions in bone. I, J Am Dent endodontic complications, Int Endod J 44:218, 2011. 64. Iqbal MK: Clinical and scanning electron microscopic Assoc 62:152, 1961. 41. Dumsha T, Hovland EJ: Evaluation of long-term calcium features of invasive cervical resorption in a maxillary 21. Bhuva B, Barnes JJ, Patel S: The use of limited cone hydroxide treatment in avulsed teeth: an in vivo study, Int molar, Oral Med Oral Pathol Oral Radiol Endod 103:e49, beam computed tomography in the diagnosis and Endod J 28:7, 1995. 2007. management of a case of perforating internal root 42. Durack C, Patel S: Cone beam computed tomography in 65. Ishijima M, Rittling SR, Yamashita T, et al: Enhancement resorption, Int Endod J 44:777, 2011. endodontics, Braz Dent J 23:179, 2012. of osteoclastic bone resorption and suppression of CHAPTER 16 Root Resorption 683

osteoblastic bone formation in response to reduced 84. Nilsen R, Magnusson BC: Enzyme histochemistry of 105. Tronstad L: Root resorption: etiology, terminology and mechanical stress do not occur in the absence of induced heterotopic bone formation in guinea pigs, Arch clinical manifestations, Endod Dent Traumatol 4:241, osteopontin, J Exp Med 193:399, 2001. Oral Biol 24:833, 1979. 1988. 66. Jacobowitz M, de Lima RKP: Treatment of inflammatory 85. Nowicka A, Lipski M, Parafiniuk M, et al: Response of 106. Trope M: Root resorption of dental and traumatic origin: internal root resorption with mineral trioxide aggregate: a human dental pulp capped with Biodentine and mineral classification based on etiology, Pract Periodontics Aesthet case report, Int Endod J 41:1, 2008. trioxide aggregate, J Endod 39:743, 2013. Dent 10:515, 1998. 67. Jones SJ, Boyd A: A resorption of dentine and cementum 86. Patel S, Dawood A: The use of cone beam computed 107. Trope M: Root resorption due to dental trauma, Endod in vivo and in vitro. In Davidotch Z, editor: The biological tomography in the management of external cervical Topics 1:79, 2002. mechanisms of tooth eruption and root resorption, resorption lesions, Int Endod J 40:730, 2007. 108. Türkün M, Cengiz T: The effects of sodium hypochlorite Birmingham, Ala, 1988, EBSCO Media. 87. Patel S, Dawood A, Whaites E, Pitt Ford T: New and calcium hydroxide on tissue dissolution and root canal 68. Kamburoğlu K, Tsesis I, Kfir A, Kaffe I: Diagnosis of dimensions in endodontic imaging. Part 1. Conventional cleanliness, Int Endod J 30:335, 1997. artificially induced external root resorption using and alternative radiographic systems, Int Endod J 42:447, 109. Tyrovola JB, Spyropoulos MN, Makou M, Perrea D: Root conventional intraoral film radiography, CCD, and PSP: an 2009. resorption and the OPG/RANKL/RANK system: a mini ex vivo study, Oral Surg Oral Med Oral Pathol Oral Radiol 88. Patel S, Dawood A, Wilson R, et al: The detection and review, J Oral Sci 50:367, 2008. Endod 106:885, 2008. management of root resorption lesions using intraoral 110. Uchiyama M, Nakamichi Y, Nakamura M, et al: Dental 69. Kundel HL, Revesz G: Lesion conspicuity, structured radiography and cone beam computed tomography: an in pulp and periodontal ligament cells support osteoclastic noise, and film reader error, Am J Roentgenol 126:1233, vivo investigation, Int Endod J 42:831, 2009. differentiation, J Dent Res 88:609, 2009. 1976. 89. Patel S, Kanagasingham S, Pitt Ford T: External cervical 111. Wada N, Maeda H, Tanabe K, et al: Periodontal ligament 70. Laux M, Abbott PV, Pajarola G, Nair PN: Apical resorption: a review, J Endod 35:616, 2009. cells secrete the factor that inhibits osteoclastic inflammatory root resorption: a correlative radiographic 90. Patel S, Pitt Ford T: Is the resorption external or internal?, differentiation and function: the factor is osteoprotegerin/ and histological assessment, Int Endod J 33:483, 2000. Dent Update 34:218, 2007. osteoclastogenesis inhibitory factor, J Periodont Res 71. Levander E, Malmgren O: Evaluation of the risk of root 91. Patel S, Ricucci D, Durak C, Tay F: Internal root 36:56, 2001. resorption during orthodontic treatment: a study of upper resorption: a review, J Endod 36:1107, 2010. 112. Walton RE, Leonard LA: Cracked tooth: an etiology incisors, Eur J Orthod 10:30, 1988. 92. Pierce AM: Experimental basis for the management of for “idiopathic” internal resorption?, J Endod 12:167, 72. Levin L, Trope M: Root resorption. In Hargreaves KM, dental resorption, Endod Dent Traumatol 5:255, 1989. 1986. Goodis HE, editors: Seltzer and Bender’s dental pulp, 93. Rabinowitch BZ: Internal resorption, Oral Surg Oral Med 113. Webber RL, Messura JK: An in vivo comparison of Chicago, 2002, Quintessence. Oral Pathol 33:263, 1972. diagnostic information obtained from tuned-aperture 73. Li BL, Long X, Wang S, et al: Clinical and radiologic 94. Regan JD, Gutmann JL, Witherspoon DE: Comparison of computed tomography and conventional dental features of desmoplastic ameloblastoma, J Oral Maxillofac Diaket and MTA when used as root-end filling materials radiographic imaging modalities, Oral Surg Oral Med Oral Surg 69:2173, 2011. to support regeneration of the periradicular tissues, Int Pathol Oral Radiol Endod 88:239, 1999. 74. Lindskog S, Blomlöf L, Hammarström L: Repair of Endod J 35:840, 2002. 114. Wedenberg C: Evidence for a dentin-derived inhibitor of periodontal tissues in vivo and in vitro, J Clin Periodontol 95. Revesz G, Kundel HL, Graber MA: The influence of macrophage spreading, Scand J Dent Res 95:381, 1987. 10:188, 1983. structured noise on the detection of radiologic 115. Wedenberg C, Linskog S: Experimental internal resorption 75. Lyroudia KM, Dourou VL, Pantelidou OC, et al: Internal abnormalities, Invest Radiol 6:479, 1974. in monkey teeth, Endod Dent Traumatol 6:221, 1985. root resorption studied by radiography, stereomicroscope 96. Ricucci D: Apical limit of root canal instrumentation and 116. Wedenberg C, Lindskog S: Evidence for a resorption and computerized 3D reconstructive method, Endod Dent obturation. Part 1. Literature review, Int Endod J 31:384, inhibitor in dentine, Eur J Oral Sci 95:205, 1987. Traumatol 18:148, 2002. 1998. 117. Wedenberg C, Yumita S: Evidence for an inhibitor of 76. Main C, Mirzayan N, Shabahang S, Torabinejad M: Repair 97. Ricucci D, Langeland K: Apical limit of root canal osteoclast attachment in dentinal matrix, Endod Dent of root perforations using mineral trioxide aggregate: a instrumentation and obturation. Part 2. A histological Traumatol 6:255, 1990. long term study, J Endod 30:80, 2004. study, Int Endod J 31:394, 1998. 118. Wedenberg C, Zetterqvist L: Internal resorption in human 77. McHugh KP, Shen Z, Crotti TN, et al: Role of cell matrix 98. Schaffner P, Dard MM: Structure and function of RGD teeth: a histological, scanning electron microscopic and interactions in osteoclast differentiation, Adv Exp Med Biol peptides involved in bone biology, Cell Mol Life Sci enzyme histochemical study, J Endod 6:255, 1987. 602:107, 2007. 60:119, 2003. 119. Wesselink PR, Beertsen W, Everts V: Resorption of the 78. McMichen FR, Pearson G, Rahbaran S, Gulabivala K: A 99. Siqueira JF, Rôças IN, Santos SRLD, et al: Efficacy of mouse incisor after the application of cold to the comparative study of selected physical properties of five instrumentation techniques and irrigation regimens in periodontal attachment apparatus, Calcif Tissue Int 39:11, root-canal sealers, Int Endod J 36:629, 2003. reducing the bacterial population within root canals, 1986. 79. Mitchell PJ, Pitt Ford TR, Torabinejad M, McDonald F: J Endod 3:181, 2002. 120. Wiener BH, Schilder H: A comparative study of important Osteoblast biocompatibility of mineral trioxide aggregate, 100. Sjogren U, Figdor D, Spångberg L, Sundqvist G: The physical properties of various root canal sealers. II. Biomaterials 20:167, 1999. antimicrobial effect of calcium hydroxide as a short-term Evaluation of dimensional changes, Oral Surg Oral Med 80. Nair PNR, Henry S, Cano V, Vera J: Microbial status of intracanal dressing, Int Endod J 24:119, 1991. Oral Pathol 32:928, 1971. apical root canal system of human mandibular first molars 101. Soltanoff CS, Yang S, Chen W, Li YP: Signaling networks 121. Wilson PR, Barnes IE: Treatment of internal root resorption with primary apical periodontitis after “one-visit” that control the lineage commitment and differentiation of with thermoplasticized gutta-percha: a case report, Int endodontic treatment, Oral Surg Oral Med Oral Pathol bone cells, Crit Rev Eukaryot Gene Expr 19:1, 2009. Endod J 20:94, 1987. Oral Radiol Endod 99:231, 2005. 102. Speziani C, Rivollier A, Gallois A, et al: Murine dendritic 122. Yamaoka M, Furusawa K, Ikeda M, Hasegawa T: Root 81. Nakamura I, Duong LE, Rodan SB, Rodan GA: Involvement cell transdifferentiation into osteoclasts is differentially resorption of mandibular second molar teeth associated of alpha(v)beta3 integrinsin osteoclast function, J Bone regulated by innate and adaptive cytokines, Eur J Immunol with the presence of the third molars, Aust Dent J Miner Metab 25:337, 2007. 37:747, 2007. 44:112, 1999. 82. Ne RF, Witherspoon DE, Gutmann JL: Tooth resorption, 103. Stamos DE, Stamos DG: A new treatment modality for 123. Yildirim S, Yapar M, Sermet U, et al: The role of dental Quintessence Int 30:9, 1999. internal resorption, J Endod 12:315, 1986. pulp cells in resorption of deciduous teeth, Oral Surg Oral 83. Neuvald L, Consolaro A: Cementoenamel junction: 104. Suei Y, Taguchi A, Nagasaki T, Tanimoto K: Radiographic Med Oral Pathol Oral Radiol Endod 105:113, 2008. microscopic analysis and external cervical resorption, findings and prognosis of simple bone cysts of the jaws, J Endod 26:503, 2000. Dentomaxillofac Radiol 39:65, 2010. CHAPTER 17

Diagnosis of Nonodontogenic Toothache

DONNA MATTSCHECK | ALAN S. LAW | DONALD R. NIXDORF

CHAPTER OUTLINE Review of Neuroanatomy Clinical Entities That Can Present as Toothache Somatic Structures Sources of Odontogenic Toothache Neural Structures Sources of Nonodontogenic Toothache Autonomic Nervous System Taking a Patient’s History Review of Neurophysiology Patient Examination Peripheral Sensitization Additional Tests Central Sensitization Case Studies Terminology Summary

An unthinking dentist is a bad dentist. Perfect technique five types of facial pain. Of these pains, the most common type misapplied is at least as unconscionable as sloppy (12.2%) was toothache.81 work. Although toothache is the most common pain entity occur- —Marjorie Jeffcoat ring in the facial region,81 many other types of pain can occur in the same general area. A primary responsibility of a dental A nonodontogenic toothache is, of course, an oxymoron. How practitioner is to diagnose pathologic entities associated with can one have a toothache that is not odontogenic in etiology? the oral cavity and masticatory apparatus. Many of these patho- The answer lies in the differentiation of people’s perceptions of logic entities have pain as a primary component of their pre- where they sense their pain, termed the site of the pain, from sentation. Because dental practitioners are sought out daily for the location of a pathophysiologic process giving rise to the the alleviation of odontogenic pain, it is imperative for them pain that may or may not be in the same region, termed the to have a basic working knowledge of other types of facial pain source of the pain. This concept of the attribution of pain to an in order to make an accurate diagnosis and properly select care anatomic region that is different from the location of the etio- for patients. It is paramount to realize that not all pain entities logic process is generically known as the referred pain phenom- presenting as toothache are of odontogenic origin. The present- enon and can occur in multiple areas of the body. Thus, a ing toothache may be a heterotopic symptom of another dis- nonodontogenic toothache has a source of pain that is not the order. A heterotopic symptom is perceived to originate from a tooth the patient has indicated, clearly demonstrating the diag- site that is different from the tissue that is actually the source nostic challenge (Fig. 17-1). of the pain. This is in contrast to primary pain, in which the Pain is common. It causes human suffering and has signifi- perceived site of pain is the actual tissue from which the pain cant socioeconomic effects. Pain is a motivator that provokes originates. Before discussing pain entities that mimic tooth- individuals to seek care. But protracted chronic pain debilitates ache, it is helpful to understand the neurobiologic mechanisms and can significantly impair the quality and productivity of a of orofacial pain. person’s life. One survey revealed that 66% of respondents reported experiencing pain or discomfort over a 6-month REVIEW OF NEUROANATOMY period. Significantly, 40% of respondents reported that this pain affected them to a “high degree.”19 A study published in Somatic Structures 2003 estimated the lost productive work time attributed to To understand the pathways by which orofacial pain occurs, common pain conditions among active workers to cost $61.2 one must first gain a basic understanding of the structures billion per year.127 One investigator reported that over a involved in its transmission to higher brain centers (see also 6-month period, 22% of Americans experienced at least one of Chapter 4). Structures of the orofacial region can be divided

684