CHAPTER 9 445

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563. Voigt JP, Goran ML, Flesher RM: The width of lingual 582. Weinstein RS, Jilka RL, Parfitt AM, Manolagas SC: 597. Yeomans JD, Urist MR: Bone induction by decalcified mandibular attached gingiva, J Periodontol 49:77, 1978. Inhibition of osteoblastogenesis and promotion of dentine implanted into oral, osseous and muscle tissues, 564. von Arx T: Failed root canals: the case for apicoectomy apoptosis of osteoblasts and osteocytes by Arch Oral Biol 12:999, 1967. (periradicular surgery), J Oral Maxillofac Surg 63:832, glucocorticoids. Potential mechanisms of their deleterious 598. Young MP, Korachi M, Carter DH, et al: The effects of an 2005. effects on bone, J Clin Investig 102:274, 1998. immediately pre-surgical oral rinse on the 565. von Arx T, Gerber C, Hardt N: Periradicular surgery of 583. Wessel JH, Dodson TB, Zavras AI: Zoledronate, smoking, bacterial contaminants of bone debris collected during molars: a prospective clinical study with a one-year and obesity are strong risk factors for osteonecrosis of the dental implant surgery, Clin Oral Implants Res 13:20, follow-up, Int Endod J 34:520, 2001. jaw: a case-control study, J Oral Maxillofac Surg 66:625, 2002. 566. von Arx T, Jensen SS, Hanni S, Friedman S: Five-year 2008. 599. Yucel EA, Oral O, Olgac V, Oral CK: Effects of fibrin glue longitudinal assessment of the prognosis of apical 584. Weston GD, Moule AJ, Bartold PM: A comparison in vitro on wound healing in oral cavity, J Dent 31:569, 2003. microsurgery, J Endod 38:570, 2012. of fibroblast attachment to resected root-ends, Int Endod 600. Yusuf H: The significance of the presence of foreign 567. 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Regenerative Endodontics

ANIBAL DIOGENES | STÉPHANE SIMON | ALAN S. LAW

CHAPTER OUTLINE Overview of Regenerative Dentistry Clinical Studies on Overview of Regenerative Endodontics Clinical Procedures Related to Regenerative Preclinical Studies on Regenerative Endodontics Endodontics Stem Cells Overview of Clinical Regenerative Endodontic Growth Factors/Morphogens Procedures (REPS) Scaffolds Example of a Revascularization Protocol Delivery System Clinical Measures of Treatment Outcome Translational Studies Summary Summary of Basic Research on Regenerative Endodontics

a -dentin complex in permanent teeth with pulpal necrosis OVERVIEW OF REGENERATIVE DENTISTRY (discussed later).172,173 The scope and clinical application of Advancements in tissue engineering are dramatically changing regenerative dental procedures have advanced to now include medicine and dentistry. Tissue engineering is an interdisciplin- guided tissue or bone regeneration (GTR, GBR) procedures ary field that applies the principles of engineering and the life and distraction osteogenesis,32,133,175 the application of platelet- sciences to restoring, maintaining, or replacing biologic func- rich plasma for bone augmentation,85 Emdogain for regenera- tion.127 It involves the interplay among stem cells, growth tion of periodontal tissues and pulp,7,45 recombinant human factors, and scaffolds (biologic matrices). It has become bone morphogenic protein (rhBMP) for augmentation of increasingly clear that the intentional manipulation of these bone,4,151 and clinical trials on the use of fibroblast growth three factors can lead to the regeneration of tissue function that factor 2 (FGF-2) for periodontal tissue regeneration.253 The would not otherwise occur if repair had taken place without potential of regenerative procedures in endodontics has been intervention.54 This relatively young field was first applied to emphasized by elegant studies demonstrating the regeneration medicine with many examples of of pulp, dentin, and enamel using scaffold materials and stem approaches used in the clinical practice.125,188,240 Although the cells.81,107,210,265 Thus, regenerative dental procedures are emerg- inclusion of tissue engineering in dentistry is more recent, it ing as a vital, evolving field of dental care, creating a paradigm is also fundamentally changing the way clinicians are treating shift in many dental specialties, including endodontics.152 This patients while providing a fertile research field that fosters chapter reviews the current status of regenerative endodontic future advancements and therapies. procedures with an emphasis on biologic principles and the Most of the history of dentistry is marked by the evolution advantages and limitations of currently available clinical of dental materials and techniques tailored to the replacement procedures. of lost or diseased tissues with inert materials. This prosthetic replacement of missing dental tissues has prevailed in dentistry Overview of Regenerative Endodontics since the primordial examples of dental treatments in ancient The developing dentition is at risk for pulpal necrosis due to civilizations.136,145,238 In contrast, the goal of regenerative den- trauma, caries, and developmental dental anomalies such as tistry is to induce biologic replacement of dental tissues and .13,16,122,144,220,225,260 Loss of an immature perma- their supporting structures. The potential for regenerative den- nent tooth in young patients with mixed dentition can be tistry is in large part due to advancements in biologic therapies devastating, leading to loss of function, malocclusion, and that apply principles of tissue engineering with the spatial and inadequate maxillofacial development. These teeth were tradi- temporal assembly of stem cells, growth factors, and scaffolds tionally treated with procedures using either to achieve the functional regeneration of a missing tissue. long-term calcium hydroxide treatment53,52 or immediate Pioneering work supporting the concept of regenerating placement of a mineral trioxide aggregate (MTA) apical plug.247 dental tissues was reported in the 1960s when Dr. B.W. Although these treatments often resolve the signs and symp- Hermann described the application of calcium hydroxide toms of pathosis, they provide little to no benefit for continued 219 33 (Ca[OH]2) for vital pulp therapy and Professor Nygaard- root development. Thus, immature teeth treated with these Østby evaluated a revascularization method for reestablishing procedures are considered in a state of arrested development,

447 448 PART II The Advanced Science of Endodontics

and no further root growth, normal pulpal nociception, and formed in 28 out of 35 teeth, whereas cellular cementum was immune defense should be expected. formed in 18 out of 35 teeth. Again, these protocols generated Regenerative endodontic procedures (REPS) have been acceptable clinical outcomes (e.g., healing of apical periodon- defined as biologically based procedures designed to replace titis, lack of symptoms, etc.) with only partial evidence of damaged structures such as dentin, root structures, and cells dental pulp phenotype. Collectively, these findings laid the of the pulp-dentin complex.152 This new treatment modality foundation for contemporary regenerative endodontics, dem- has emerged as an alternative that, in addition to healing apical onstrating that repair could take place following root canal periodontitis, aims to promote normal pulpal physiologic func- disinfection in immature teeth. tions. These include continued root development, immune The first case report of a “contemporary” regenerative end- competency, and normal nociception, as seen in some pub- odontic procedure occurred in 2001.62 Since then, there has lished cases.62 Thus, the ultimate goal of these procedures is been an exponential increase in published cases reporting to regenerate the components and normal function of the pulp- unprecedented clinical outcomes such as resolution of signs dentin complex. and symptoms of apical periodontitis, continued root develop- Regenerative endodontics is founded on the seminal work ment, and, in certain cases, normal nociceptive responses to of Dr. Nygaard-Østby, completed in the 1960s. He hypothe- vitality testing.62 Despite the lack of randomized clinical trials, sized that a blood clot could be the first step in the healing of these published clinical observations support the hypothesis a damaged dental pulp, similar to the role of the blood clot in that patients with otherwise limited treatment options benefit the healing process observed in other areas (e.g., alveolar bone from these procedures. Importantly, the field of regenerative following extraction).171 To test the hypothesis that the pres- endodontics has seen a dramatic increase in knowledge ence of a blood clot within a root canal system promotes gained from translational basic science studies evaluating the healing, mature teeth diagnosed with either vital or necrotic interplay of the tissue engineering components (stem cells, pulp received pulp space debridement followed by foraminal growth factors, and scaffolds) applied to the clinical need and enlargement, medicament dressing for the necrotic cases, challenges. evoked intracanal , and a kloroperka obturation placed coronal to the formed blood clot. Patients (n = 17) were fol- PRECLINICAL STUDIES ON lowed for various time periods (17 days to 3.5 years), and then the treated tooth was extracted and the newly formed tissues REGENERATIVE ENDODONTICS were histologically examined. The outcomes were similar for Applying the principles of tissue engineering to the develop- all teeth: (1) resolution of symptoms of inflammation related ment of regenerative endodontic procedures requires research to foraminal enlargement and over-instrumentation in as early on the correct spatial assembly of distinct stem cells, growth as 17 days; (2) resolution of signs and symptoms of pathosis factors/morphogens, and scaffolds to form a functional pulp- for the necrotic cases; and, in certain cases, (3) radiographic dentin complex.95,107,127,161 In this section, we review each of evidence of apical closure. For the histologic analysis, it was these critical components in turn. observed that there was ingrowth of connective tissue into the canal space and varied levels of mineralized tissue found along Stem Cells the canal walls as well as “islands” of mineralized tissue embed- Stem cells are defined as a distinct subpopulation of undiffer- ded within the newly formed tissue (Fig. 10-1). Because dental entiated cells with self-renewal and differentiation potential. pulp is a type of connective tissue with a rich supply of fibro- They can be classified as pluripotent or multipotent cells. Plu- blasts, this general finding was quite promising. However, the ripotent stem cells have the capacity of becoming specialized inclusion of undesired cell types (e.g., cementoblasts) and the cells and belong to all three germ layers. Embryonic stem cells lack of desired cell types (e.g., odontoblasts) indicate that this are the best example of pluripotent cells. There is a significant protocol did not lead to complete histologic regeneration of body of research on embryonic stem cells, but ethical, legal, dental pulp. Despite its shortcomings, this pioneer study laid and medical (tissue-rejection) issues can render these cell types the foundation for the subsequent studies in the field of regen- unsuitable for clinical applications.152 True pluripotent stem erative endodontics. cells can only be found in the developing embryo, and the In 1966, a study was published reporting that disinfection harvesting of these cells requires destruction of the embryo, could be established primarily with inter-appointment medica- hence the legal and ethical concerns with such practice. Dr. tion with polyantibiotic mixes (three different formulations Yamanka and colleagues reported the groundbreaking finding used in five cases).196 The investigators did not purposely that somatic cells can be transformed into pluripotent stem evoke intracanal bleeding in this study, but instrumented cells—namely, induced pluripotent stem cells (iPSC).177 The canals short of what they thought to be vital tissue, determined use of iPSCs does not have the same legal and ethical concerns by visualization of tissue and pain sensation upon instrumenta- as the use of embryonic stem cells, but iPSCs share the lack of tion. Signs and symptoms of disease and continued root control over their uninhibited proliferation and differentiation development were resolved for all reported cases. The study of embryonic stem cells. These cells tend to form teratomas represented the first reported case in which polyantibiotic after implanted into a host, a true testament of their high pro- pastes were used in immature necrotic teeth for disinfection liferative and differentiation capacities, but this makes them and to promote root development. Five years later, another unsuitable for immediate common clinical practice.177,221 On study was published that included the use of antibiotics in the the other hand, all adult mesenchymal stem cells are more disinfection protocol and the intentional promotion of intra- restricted in their capacity to differentiate, only forming tissues canal bleeding.173 Resolution of symptoms and continued root of mesenchymal origin, and therefore are classified as multi- development was a common finding. However, the histology potent.40 These cells can be found compartmentalized within of the extracted teeth demonstrated that connective tissue was tissues in “stem cell niches.” The mesenchymal tissues (e.g., CHAPTER 10 Regenerative Endodontics 449

2 mm

A B C

D Cementum E

FIG. 10-1 Radiographic and histologic findings from a central incisor with a necrotic pulp, from Nygaard-Østby. A, Evidence of a file going beyond the apex. There is also evidence of a radiolucency in the apical area. B, Second radiograph at 14 months, taken shortly before tooth was extracted, showing the short fill. C, Histologic section of same tooth, showing fibrous connective tissue has grown into the apical 2 mm of the tooth. D, Higher magnification (upper right) shows evidence of what appears to be cementum deposition on the canal wall and fibrous connective tissue in the pulp space. E, Evidence of collagen bundles in the canal space. (From Nygaard-Østby B: The role of the blood clot in endodontic therapy: an experimental histologic study, Acta Odontol Scand 19:323, 1961.) bone, dental pulp, periodontal ligament, etc.) appear to have (PSCs) (Fig. 10-2).66,132 Although stem cells have been identi- an enriched population of adult stem cells.40 These cells were fied in most oral tissues, the stem cells more likely tobe first found in bone marrow decades ago and were characterized involved in REPS are localized around the periapical region. as self-renewing and plastic-adherent, and they formed cell These include SCAP, PDLSCs, BMSCs, iPAPCs, and DPSCs (if colonies with a fibroblastic appearance.79,80 They were initially vital pulp is still present apically). called stromal stem cells but later received the now widely The apical papilla and its residing stem cells (SCAP) were accepted name mesenchymal stem cells (MSCs).40 Most stem first characterized in 2006.223 The apical papilla (Fig. 10-3) is cells found in the orofacial region are MSCs.66 a dense reservoir of undifferentiated MSCs with great prolifera- Different populations of adult stem cells have been identi- tive and odontogenic differentiation capacity.104,197 Importantly, fied in tissue compartments in the oral region. These include SCAP are regulated by Hertwig’s epithelial root sheath through stem cells of the apical papilla (SCAP), inflammatory periapical a series of complex epithelial-mesenchymal interactions that progenitor cells (iPAPCs), dental follicle stem cells (DFSCs), dictate root development and shape.249 Further, the close prox- dental pulp stem cells (DPSCs), periodontal ligament stem imity of the apical papilla to the apices of teeth in continuum cells (PDLSCs), bone marrow stem cells (BMSCs), tooth germ with the root canal space makes this rich source of stem cells progenitor cells (TGPCs), salivary gland stem cells (SGSCs), readily available for regenerative endodontic therapeutics. The stem cells from human exfoliated (SHED), IPAPCs represent another important potential source of stem oral epithelial stem cells (OESCs), gingival-derived mesenchy- cells for regenerative endodontics in teeth with well-established mal stem cells (GMSCs), and periosteal derived stem cells apical periodontitis.132,141 Lastly, stem cells of the periodontal 450 PART II The Advanced Science of Endodontics

ligament (PDL) and bone marrow should be also considered OESCs as stem cells sources for regenerative procedures because the GMSCs action of mechanical disruption of the apical tissue (evoked PSCs bleeding) could also trigger the release of these cells, albeit their relative abundance is thought to be significantly less than SCAP and IPAPCs. In 2011, a study was conducted to evaluate the presence of mesenchymal stem cells following the evoked- DPSCs 135 SHED bleeding step in regenerative procedures. It was found that TGPCs there is a substantial influx of mesenchymal stem cells into root canals during regenerative procedures resulting in an DFSCs increase greater than 700-fold in the expression of MSC markers (Fig. 10-4). In addition, the cells could be harvested from clinical samples and examined under confocal micros- SCAP copy (Fig. 10-5). This was the first demonstration that REPs 62 iPAPCs are stem cell-based therapies. Although this study did not evaluate whether the MSCs detected in REPs are derived from SGSCs the apical papilla, it was assumed that these cells were SCAP PDLSCs because the evoked bleeding step lacerated the apical papilla. However, these MSCs are a heterogeneous population of cells BMSCs that could come from any of the periradicular tissues after the

FIG. 10-2 Schematic drawing illustrating potential sources of postnatal mechanical step of evoking bleeding into the root canal system. stem cells in the oral environment. Cell types include tooth germ progenitor The delivery of substantial concentrations of MSCs into the cells (TGPCS); dental follicle stem cells (DFSCS); salivary gland stem cells root canal space, despite advanced apical periodontitis or (SGSCS); stem cells of the apical papilla (SCAP); dental pulp stem cells abscess, points to an impressive survival capacity of these cells. (DPSCS); inflamed periapical progenitor cells (IPAPCS); stem cells from human In these clinical presentations, low oxygen tension, low pH, exfoliated deciduous teeth (SHED); periodontal ligament stem cells (PDLSCS), and a high concentration of endotoxins and inflammatory bone marrow stem cells (BMSCS) and, as illustrated in the inset, oral epithelial mediators are expected.70,111,129,211 Indeed, the finding of high stem cells (OESCS); gingival-derived mesenchymal stem cells (GMSCS); and concentrations of the immune cell marker CD14 in those clini- periosteal stem cells (PSCS). (From Hargreaves KM, Diogenes A, Teixeira FB: cal samples indicates that there was still a substantial chronic Treatment options: biologic basis of regenerative endodontic procedures, inflammatory exudate present at the apical region of those J Endod 39:s30, 2013.) teeth. These findings raise the question of how MSCs such as SCAP can survive during apical periodontitis where a complex microflora, an array of inflammatory mediators, immune cells, and presumably low oxygen tension are commonly encoun- tered. The biologic reason for this apparent resilient survival,

A B C

FIG. 10-3 A-C, Dissection of an immature permanent tooth indicating the extent of the apical papilla. Note that this structure is likely lacerated during the evoked bleeding step of revascularization cases and thus cells from this structure, including mesenchymal stem cells of the apical papilla (SCAP), are likely to be delivered into the root canal space. Arrow in C denotes junction of apical papilla and dental pulp. (Courtesy Dr. Michael Henry.) CHAPTER 10 Regenerative Endodontics 451

Systemic blood is the fibroblast. Together with blood vessels, lymphatics, and 1000 neurons, this core tissue is embedded in an extracellular Intracanal saline ** matrix consisting of collagen and other fiber types (see also 800 Intracanal blood Chapter 12). Dental pulp stem cells (DPSCs) can be found throughout the dental pulp but are known to accumulate in the perivascular region and the cell-rich zone of Hohl adjacent to the odontoblastic layer.75,205 Thus, DPSCs from both sources are thought to be active participants in the process of reparative * dentinogenesis. Dental pulp stem cells are recruited to the site of injury 50 following a gradient of chemotactic agents released by resident immune cells and from the damaged dentin.2,227 The 40 reparative dentin formed by these cells is distinct from

systemic blood levels systemic blood * the primary, secondary, and reactionary dentin that has been 30 lost.8,159,259 It is often called “osteodentin” when found to be 20 disorganized, atubular, and having cellular inclusions. This Fold change in mRNA expression / process of cellular repair is enhanced by bioactive materials 10 (e.g., MTA and Biodentine). These materials increase the inher- ent mineralizing potential of the dental pulp when used in both 0 indirect and direct pulp applications.187 However, the process CD73 CD105 of tertiary dentinogenesis requires a vital pulp and the resolu- tion of the etiology (e.g., caries or trauma). This process FIG. 10-4 Evoked-bleeding step in endodontic regenerative procedures in immature teeth with open apices leads to significant increase in expression becomes disrupted when the pulp succumbs to injury, result- of undifferentiated mesenchymal stem cell markers in the root canal space. ing in liquefaction necrosis of the dental pulp. Regeneration in Systemic blood, saline irrigation, and intracanal blood samples were collected this case is only possible with the recruitment or delivery of autologous stem cells to the canal space following adequate during second visit of regenerative procedures. Real-time RT-PCR was per- 62 formed by using RNA isolated from each sample as template, with validated disinfection. specific primers for target genes and 18S ribosomal RNA endogenous control. Odontoblasts are one of the most specialized cells of the pulp dentin complex with dentinogenic, immunogenic, and Expression of mesenchymal stem cell markers CD73 and CD105 was upregu- 34,67,226 lated after the evoked-bleeding step in regenerative procedures. Data were possibly sensorial functions. Odontoblasts in the intact pulp-dentin complex are easily identified based on their loca- normalized to the housekeeping gene 18S levels and presented as mean ± standard deviation fold increase in relation to systemic blood levels for each tion and distinct morphologic characteristics (i.e., columnar gene and analyzed with one-way analysis of variance with Bonferroni post hoc polarized cell body with cellular projections into the dentinal tubules). However, it is far more challenging to identify and test (n = 8; *P < .05; **P < .01; n.s., not statistically significant). (From Lovelace TW, Henry MA, Hargreaves KM, Diogenes A: Evaluation of the characterize an odontoblast-like cell, mainly because these cells lack a primary odontoblast morphology and unique delivery of mesenchymal stem cells into the root canal space of necrotic 102 immature teeth after clinical regenerative endodontic procedure, J Endod markers that could be used for identification. Indeed, many 37:133, 2011.) markers used for the identification of odontoblast-like cells are also expressed in other mineralizing cell types such as osteo- blasts. For example, both odontoblasts-like cells and osteo- despite challenging conditions, may be explained by the rela- blasts are similar in the formation of mineralized nodules and tively low density of blood vessels in the apical papilla in in the expression of several proteins such as dentin sialoprotein comparison to the adjacent dental pulp, whereas the dental (DSP), although DSP levels are nearly 400 times greater in follicle surrounding the apical papilla is highly vascularized odontoblasts than in osteoblasts.244 Measuring only one or two and may act as a capillary bed to supply nutrients to SCAP.62 characteristics of a cell might not conclusively identify whether Indeed, the apical papilla was found to remain vital despite the cell is a true odontoblast. Even among odontoblasts, the complete pulpal necrosis and advanced apical periodontitis in phenotype varies in cells located in the apical (squamous an animal model of endodontic infection.169 Further, it has shape) versus coronal (tall columnar) pulpal tissue. Impor- been demonstrated that hypoxic environments enhance the tantly, molecular studies have identified many of the genes proliferation, survival, and angiogenic potential of dental stem selectively expressed in odontoblasts.134,178-180 Lastly, an inter- cells.5,19,57,106,201 Interestingly, similar enhancing effects were mediate filament protein called nestin has been shown to be observed when dental stem cells were exposed to bacterial preferentially expressed in odontoblasts or odontoblast-like by-products such as endotoxin.1 Thus, it appears that SCAP cells when in the active secretory function. Nestin expression and surrounding stem cells are equipped to survive and main- could be used in conjunction with other markers to better tain their potential for differentiation in adverse conditions identify odontoblast-like cells.3,4 This knowledge is expected such as apical periodontitis and apical abscesses. Nonetheless, to aid future studies characterizing the conditions necessary stem cells delivered into root canals after bleeding is evoked for mesenchymal cells of multiple origins to differentiate into from the periradicular tissues are likely from various apical odontoblast-like cells. It is likely that definitive cellular iden- sources or niches. tification depends on both the morphology of the cell and an The dental pulp can be viewed as a core of innervated assessment of the expression of multiple genes. and vascularized loose connective tissue surrounded by a At least five different types of postnatal mesenchymal stem layer of odontoblasts. The major cell type of this core region cells, in addition to DPSCs, have been reported to differentiate 452 PART II The Advanced Science of Endodontics

CD105 TO-PRO-3 Overlay

A 15um 15um 15um

CD73 TO-PRO-3 Overlay

B 15um 15um 15um

STRO-1 TO-PRO-3 Overlay

15um 15um 15um C

FIG. 10-5 Mesenchymal stem cells were delivered into root canal spaces during regenerative procedures in immature teeth with open apices. Cells collected from intracanal blood samples after the evoked-bleeding step or from systemic blood were stained with antibodies against CD105, CD73, or Stro-1 and evaluated with a laser- scanning confocal microscope. Cells in intracanal blood samples collected after the evoked-bleeding step showed expression of mesenchymal stem cell marker (A) and CD105 (green in A), CD73 (green in B), and STRO-1 (green in C), whereas nuclei appear blue as stained with TO-PRO-3. (From Lovelace TW, Henry MA, Hargreaves KM, Diogenes A: Evaluation of the delivery of mesenchymal stem cells into the root canal space of necrotic immature teeth after clinical regenerative endodontic procedure, J Endod 37:133, 2011.)

into odontoblast-like cells, including SHED,147 SCAP,124,125 is required to make regenerative procedures more predictable IPAPCs,132 DFPC,149 and BMMSC.22 One study demonstrated for immature teeth, and these procedures may transition to be that over-instrumentation into the periradicular tissues fol- applicable to fully formed teeth. lowed by bleeding into the canal space results in a robust influx of cells with mesenchymal stem cell markers in fully mature Growth Factors/Morphogens teeth, similar to that seen in immature teeth.135 Thus, it appears Dentine is composed of collagen fibers (90%, collagen type I) that MSCs from the apical region can be delivered into the root and noncollagenous matrix molecules (proteoglycans, phos- canal spaces in both immature and mature teeth. However, phoproteins, and phospholipids). The collagen fibers act as a there is growing evidence that MSCs have decreased prolifera- grid or matrix, and this structure behaves as a scaffold upon tive and differentiation potential with aging.117,132,162 Further which mineralization can occur. Dentine phosphoprotein research is required to elucidate the age limit for the use of (DPP) and dentine sialoprotein (DSP) are the most abundant autologous dental-derived stem cells, but these findings suggest dentine-specific proteins among the noncollagenous proteins that regenerative procedures may be applicable to mature fully of organic matrix.38 DSP resembles other sialoproteins such as formed teeth in adults. In fact, in a proof-of-concept case bone sialoprotein, but its precise function is still unclear; it report, resolution of apical periodontitis followed by narrowing may have a role in matrix mineralization.92 Both DSP and of the canal space and apical closure was seen in two fully DPP make up part of the small integrin-binding, ligand mature teeth in adult patients treated with regenerative end- N-linked glycoproteins (SIBLINGS), which include dentine odontic procedures.182 Thus, more research and development matrix acidic phosphoprotein 1 (DMP-1), bone sialoprotein, CHAPTER 10 Regenerative Endodontics 453

this process, dentin-derived growth factors are thought to play TABLE 10-1 a key role to be deciphered into the regulation of progenitor List of Growth Factors Found into the Mineralized cell recruitment, cell proliferation, and differentiation of new Dentin Matrix dentine-secreting cells.153,212 Indeed, the differentiation of new odontoblast-like cells has also been reported following pulp Growth Factors in Dentin Matrix capping with basic fibroblast growth factor (FGF), TGF-β1,131 Transforming growth factor beta-1 Cassidy et al., 199742 and BMP-7.86 (TGFβ-1) The sequestration of these growth factors in the dentine Transforming growth factor beta-2 Cassidy et al., 199742 matrix and their subsequent “fossilization” during the miner- (TGFβ-2) alization process appears key to the pulp healing process where Transforming growth factor beta-2 Cassidy et al., 199742 their release from the matrix may be responsible for various signaling events. These growth factors are extremely potent (TGFβ-3) and have a variety of cell signaling properties. However, their 230 Bone morphogenic protein –2 Thomadakis et al., 1999 precise localization in the dentine214 and their various biologic (BMP-2) roles remain to be elucidated. Bone morphogenic protein –4 About et al., 20004 It is possible to imagine opportunities for therapeutic stim- (BMP-4) ulation, inducing a targeted release of these proteins. For Bone morphogenic protein –7 Thomadakis et al., 1999230 example, treatment of dentin with EDTA solution has been (BMP-7) shown to dissolve the mineral phase, liberating growth factors that orchestrate the stimulation of progenitors or stem cell Insulin growth factor-1 (IGF-I) Finkleman et al., 199074 differentiation.216-218 Etching with orthophosphoric acid, used 74 Insulin growth factor-2 (IGF-II) Finkleman et al., 1990 for conditioning the dentine in bonding procedures, also pro- Hepatocyte growth factor (HGF) Tomson et al., 2013232 motes demineralization of the dentine and liberation of bio- 64,72 Vascular endothelial growth factor Roberts-Clark and Smith, logic factors. For a long time, calcium hydroxide has been (VEGF) 2000194 used as a protective lining, especially beneath amalgams fill- 155 ings, or as a canal disinfection medication. It has been shown Adrenomedullin (ADM) Musson et al., 2010 to have the ability to release bioactive components from the dentine, including growth factors.91 Unlike dentin-etching acids, which only have brief contact with the dentine, calcium hydroxide remains in place beneath restorations or in canals osteopontin, osteocalcin, and osteonectin. These proteins are allowing for a gentle and continued dissolution, thus releasing only a small part of the whole cocktail of noncollagenous pro- growth factors; its action is prolonged and potentially control- teins that form components of the dentine.87 lable depending on the form of the product. Lastly, calcium Research on dentine structure and composition has high- hydroxide, a by-product of the use of MTA and Biodentine, lighted that the matrix contains some components that may be appears to underlie release of bioactive dentin-derived growth important in regulating tissue due to their bioactive properties. factors by these two bioactive materials.231 Thus, clinicians may For this reason, dentine is today considered a reservoir take advantage of potent growth factors stored within dentin of growth factors and cytokines.212 These growth factors/ with the use of chemical treatments and materials that promote cytokines are secreted by the odontoblasts during primary den- the release of these factors. tinogenesis, becoming sequestered and “fossilized” into the dentine after biomineralization (Table 10-1). However, they Morphogens may become solubilized by demineralization of the matrix, It is also important to clearly keep in mind that a second level bacterial acid (caries decay), chemical treatment (EDTA rinsing of regulation exists during dental development (and thus solution, calcium hydroxide or acid etching for bonded resto- during pulp regeneration process)—transcription factors. rations), or restorative materials such as mineral trioxide Notably, Msx1 is expressed in polarized preodontoblasts, aggregate and Biodentine.213,231 whereas Msx2 is present in mature odontoblasts.25 Protein and These growth factors and their receptors have been shown transcripts for Msx1 have been identified in the pulp mesen- to be present at the enamel organ-dental papilla interface chyme at early stages of tooth development and their concen- by immunohistochemistry and in situ hybridization during trations decreased at the bell stage.49 The expression of these tooth development and have been implicated in odontoblast transcription factors is under the control of growth factors, and differentiation: they can ultimately have broad-ranging effects. Significantly, ♦ Growth hormone (GH) plays a paracrine or autocrine role BMP4 upregulates Msx1 and Msx2 expression. In turn, tran- in dental development.263 scription factors regulate further growth factor expression; ♦ IGF-1 and -2 (of the family of IGF: insulin-like growth for example, Msx1 upregulates BMP4 synthesis in the mesen- factor).25,42,112 chyme, and Msx2 regulates Runx2 and osteocalcin gene 56,228 239 30,31 ♦ TGFβ-1, -2, and -3 and BMP-2, -4, and -6 play a role expression during odontogenesis. in the polarization and the differentiation of odontoblasts.25 Growth factors and transcription factors are central to the Notably in adult pulp, TGFβ-1 plays an important role in cascade of molecular and cellular events during tooth develop- the regulation of the inflammatory response and tissue ment and are responsible for many of the temporospatial mor- regenerative processes.131 phologic changes observed in the developing tooth germ. For The dental pulp has well recognized regenerative potential these reasons, they are also likely involved in the regeneration observed in the process of reparative dentinogenesis.153,212 In process. 454 PART II The Advanced Science of Endodontics

It is also important to consider the nature of the signaling reported that patients taking long-term corticosteroids often process between the injurious agent and the pulp cells. Bacteria present with dramatic reduction of the radiographic size of the and their toxins are key candidates in the direct stimulation of pulp chamber and up to a fivefold increase in the thickness of pulp cells.65 Lipopolysaccharides (LPS) and other bacterial the predentin layer.164,165,248 Although these were medically toxins initiate intrapulpal inflammatory processes by activa- complex patients (e.g., those experiencing renal failure) taking tion of Toll-like receptors (e.g., TLR4 activation by LPS).65,226,242 multiple drugs, the use of corticosteroids appeared to be Importantly, both progenitor and dental stem cells have been associated with the observed increased activity of human odon- shown to express these receptors.34,35 Thus, stem cells within toblasts. Further, these unexpected “side-effects” were also the dental pulp or periradicular tissues are equipped to detect observed in a retrospective study evaluating the association of microorganisms. Exposure of these cells to microbial antigens pulp calcifications and the long-term use of statins.186 These has been shown to directly modulate the proliferation and dif- incidental effects of commonly prescribed medications were ferentiation potential of these cells.34,46,139,213,237 Lastly, cyto- further evaluated in translational studies that have extended kines commonly found in the inflammatory milieu (including this general observation by demonstrating that the application that of the dental pulp) have a profound effect on stem cells. of dexamethasone or statins greatly increased the differentia- For example, it has been shown that TNF-alpha stimulates tion of human dental pulp cells into odontoblast-like cells.102,176 differentiation of dental pulp cells toward an odontoblastic This was particularly evident when dexamethasone was com- 102 phenotype via MAP kinase pathway activation and p38 phos- bined with 1,25-dihydroxyvitamin D3. Merely changing the phorylation.183,209 Therefore, stem cell fate within the dental composition of growth factors completely altered the differen- pulp is ultimately dictated by a complex cascade of intracel- tiation of these cells, with the same population of cells able to lular signaling pathways activated by agents released from express markers of odontoblasts, chondrocytes, or adipocytes, microorganisms, dentin, and immune cells. depending on their exposure to different combinations of Interestingly, morphogens are not only naturally occurring growth factors.244 Such findings emphasize the importance of factors found within teeth. Several growth factors have also growth factors in guiding the differentiation of these cells. been evaluated for their ability to trigger the differentiation of Other studies have evaluated growth factors administered selected mesenchymal stem cell populations into odontoblast- alone or in various combinations for promoting differentiation like cells (Table 10-2). Interestingly, several case studies have of odontoblast-like cells.

TABLE 10-2 Effects of Selected Growth Factors on the Differentiation of Odontoblast-Like Cells Growth Factors Cell Source Phenotype Condition Authors Dexamethasone Human dental pulp Odontoblast-like In vitro × 8 weeks Huang et al., 2006102 102 Dexamethasone and Vitamin D3 Human dental pulp Odontoblast-like In vitro × 8 weeks Huang et al., 2006 Dexamethasone and Ascorbate- Human or rat dental pulp Odontoblast-like In vitro × 3 weeks Wei et al., 2007244 2-phosphate and Zhang et al., 2005266 β-Glycerophosphate Insulin and Indomethacin and 3- Human dental pulp Adipocyte In vitro × 19 days Wei et al., 2007244 Isobatyl-1-methylxanthine (IMBX) Dexamethasone and Insulin and Human dental pulp Chondrocyte In vitro × 8 weeks Wei et al., 2007130 Ascorbate-2-phosphate and Sodium pyruvate and TGF-β1 Growth/differentiation factor 11 Dental pulp Odontoblast-like In vitro/in vivo 10 days Nakashima et al., 200475 (Gdf11) Simvastatin (statins) Human dental pulp Odontoblast-like In vitro/in vivo Okamoto et al., 200988 LIM mineralization protein 1 (LMP-1) Human dental pulp Odontoblast-like In vitro/in vivo Wang et al., 2007129 Bone morphogenetic proteins Dental pulp Odontoblast-like In vitro Saito et al., 200499 Sloan et al., 2000107 Chen et al., 200819 TGF-β1-3 Rat/monkey dental pulp Odontoblast-like In vitro Sloan et al., 1999109 Demineralized dentin Human or rodent pulp Odontoblast-like In vitro/in vivo Smith et al., 1990111 Smith et al., 2001110 Tziafas, 2004123 Nerve growth factor (NGF) Immortalized apical Odontoblast-like In vitro Arany et al., 200911 papilla Fibroblast growth factor 2 Human dental pulp Odontoblast-like In vitro He et al., 200843 Dentin matrix protein 1 Rat dental pulp Odontoblast-like In vivo Almushayt et al., 20061 CHAPTER 10 Regenerative Endodontics 455

Several of the approaches using compounds later found to into the microenvironment, which may be detrimental to stem have growth factor–like effects have immediate clinical impli- cell survival. cations. First, it is unlikely that a single growth factor will Another approach for creating a scaffold involves the use of result in maximal differentiation, so combinations of growth autologous platelet-rich plasma (PRP). It requires minimal ex factors may be required for evaluation in clinical trials. Related vivo manipulation, being fairly easy to prepare in a dental to this point, many of the studied growth factors (e.g., dexa- setting. PRP is rich in growth factors, degrades over time, and methasone, insulin) are drugs already approved for human use forms a three-dimensional fibrin matrix.17,18,109,174 Platelet rich in other medical/dental applications. Second, the demonstra- fibrin (PRF) is an alternative to PRP, as it has athree- tion that statins promote the differentiation of an odontoblast- dimensional architecture conducive with stem cell prolifera- like phenotype suggests that patients clinically taking statins tion and differentiation and contains bioactive molecules.58,61 may also have narrowing of the pulp chamber space, similar These autologous scaffolds have been used successfully in to the findings previously described for corticosteroids. This regenerative cases.21,222,264,267 However, it should be emphasized would be an important future area of research. Third, clinicians that, despite their reported use, there are some drawbacks to have long used demineralized human bone to augment healing their clinical use: the process requires collection of intravenous after surgical procedures.190 Demineralized human bone is blood that can be challenging in children, the diversity and thought to contain a natural combination of appropriate concentration of growth factors within PRP and PRF prepara- growth factors and scaffolds, thereby providing an appropriate tions are not controllable,20,116,241 and they lack temporal deg- environment for osteoblast differentiation or function. Extend- radation control and the mechanical strength to support a ing this concept, several research groups have demonstrated coronal restoration. Thus, despite some desirable characteris- that demineralized human dentin has significant benefit for tics, other scaffold alternatives to PRP and PRF should be promoting the differentiation of odontoblast-like cells. Impor- carefully considered. tantly, translational studies in regenerative endodontics have Hydrogels are a class of scaffolds composed of three- demonstrated that irrigation of dentin with 17% EDTA increases dimensional hydrophilic polymers that absorb water or tissue the survival of stem cells105,142 and odontoblastic differentia- fluids up to several times their weight.73,250 These water-swollen tion,81,142 possibly due to the release of bioactive molecules materials are easily injectable in their colloidal form, undergo- from dentin.215 Collectively these findings suggest that EDTA ing gelation by chemical (e.g., changes in pH and osmolarity) irrigation of the dentinal walls as part of an REP could improve or physical (e.g., temperature change) cues. These materials clinical outcomes. are highly tunable, biocompatible, and can be designed to resemble naturally occurring extracellular matrices.250 They are Scaffolds of particular interest for regenerative endodontics because they An important component of tissue engineering is a physical can be easily injected into narrow root canal spaces and can be scaffold.160,245 Tissues are organized as three-dimensional struc- modified to deliver chemotactic and angiogenic agents to drive tures, and appropriate scaffolding is necessary to (1) provide stem cell homing and supportive angiogenesis.62,89 Hydrogels a spatially correct position of cell location and (2) regulate made of self-assembly peptides (e.g., Puramatrix)43 show great differentiation, proliferation, or metabolism while promoting potential to be used in endodontic tissue engineering because nutrient and gaseous exchanges. Extracellular matrix mole- their sequence includes short peptide sequences, similar to cules are known to control the differentiation of stem cells,189,254 those naturally occurring in tissues, enhancing cell attachment and an appropriate scaffold might selectively bind and localize and proliferation.84 cells, contain growth factors,251,252 and undergo biodegradation over time.261 Thus, a scaffold is far more than a simple lattice Delivery System to contain cells, but instead can be viewed as the blueprint of Even with selection of the appropriate cell source, growth the engineered tissue. factors, and scaffold, the resultant mixture must be delivered Scaffolds can be classified as either natural or synthetic. in a spatially appropriate fashion into the space of the root Examples of natural scaffolds include collagen,103,159 glycos- canal system. For example, nearly all cells of the body are aminoglycans, hyaluronic acid (HA), demineralized or native within 0.1 to 1 mm of a blood vessel in order to maintain dentin matrix,24,93,158,236,255 and fibrin.82 On the other hand, adequate diffusion of oxygen and nutrients.90,98 This represents examples of synthetic scaffolds include poly-L-lactic acid a challenge still to be overcome in the currently performed (PLLA),60 polyglycolic acid (PGA), polylactic-coglycolic acid regenerative endodontic procedures that recruit stem cells135 to (PLGA),68 polyepsilon caprolactone,256 hydroxyapatite/ a canal space devoid of lateral vascularity and several millime- tricalcium phosphate,11 bioceramics, and hydrogels such as ters away from apical blood vessels. If one were to inject cells, self-assembly peptide hydrogels.71,140 The great majority of cur- in a cell-based approach, along the entire coronal-apical extent rently published regenerative endodontic procedures involve of a root canal system, the majority of cells would be expected evoked bleeding and the formation of a blood clot to serve as to succumb to tissue hypoxia. Interestingly, it has been dem- a scaffold.62 Although it is relatively straightforward as it does onstrated that under hypoxic conditions, stem cells proliferate not require ex vivo manipulation, this simplistic approach is faster and release greater levels of angiogenic factors such as not without challenges. The blood clot is often difficult to vascular endothelial growth factor 1 (VEGF) that promote achieve, and it does not have many of the properties of the targeted angiogenesis into the engineered space.19 Thus, an ideal scaffold. These properties include easy delivery, adequate alternative approach would be to inject a scaffold with chemo- mechanical properties, controllable biodegradation, and incor- tactic factors into the root canal. This approach is called cell poration of growth factors.82 In addition, the blood clot con- homing, as cells are attracted to the scaffold along with sup- tains a great number of hematopoietic cells that eventually portive blood vessels in a progressive manner121; instead of undergo cell death, releasing their toxic intracellular enzymes being abruptly delivered to an avascular space (i.e., similar to 456 PART II The Advanced Science of Endodontics

A B

C D

FIG. 10-6 Schematic drawing illustrating bioengineering using a cell homing approach. An immature premolar with a necrotic pulp and apical lesion (panel A) is disinfected (panel B), followed by placement of a biodegradable scaffold (s) containing growth factors and chemotactic factors (g) to allow progressive proliferation and migration of apical stem cells into the canal space (panel C) leading to the population of the canal space with stem cells concomitantly with vascular supply and tissue organization (panel D). (From Diogenes A, Henry MA, Teixeira FB, Hargreaves KM: An update on clinical regenerative endodontics, Endod Topics 28:2, 2013.)

the current revascularization procedures), the cell-homing One study demonstrated that new dentin and pulplike approach can be applied in a cell-free83 (no cells implanted tissue could be generated in human root segments implanted along the chemotactic factors, see Fig. 10-6) or cell-based subcutaneously in immune-compromised mice.130 In this approach (cells are delivered in the chemotactic-containing study, root segments had one of the openings sealed with MTA scaffold).152 Because dental pulp can be approximated as a to mimic the coronal restoration of regenerative endodontic loose connective tissue core surrounded by a layer of odonto- cases. The canal space was filled with either SCAP or DPSCs blasts, the spatial arrangement of cells and growth factors in a PLGA-based scaffold. The implants were harvested 3 within the scaffold may be particularly important to promote months later and processed for immunohistochemical analysis. odontogenesis without having complete calcification of the The results indicated that there was a dramatic circumferential root canal system. Complete recapitulation of the pulp-dentin apposition of dentin-like material along the dentinal walls. The complex architecture requires additional research effort. new mineralized tissue was lined with polarized cells express- ing odontoblastic markers. In addition, the dentin-like tissue Translational Studies was largely atubular and displayed cellular inclusions similar Several elegant studies in regenerative endodontics have used to the histologic presentation of osteodentin. Importantly, various translational methodologies including evaluation of the cells from the engineered pulp were positive for human clinical samples,135 organotypic root canal models,142,235 tooth mitochondria, demonstrating that they originated from the slice models,60,88,102 whole tooth culture, and animal implanted human stem cells and not from the host (mouse). models.81,119,121,162,229,243 These studies have been crucial to Lastly, the root segments that were implanted without stem provide a strong scientific foundation for the field of regenera- cells had only connective tissue that did not resemble a tive endodontics while allowing for clinical treatment protocol pulplike tissue, nor did it have mineralized tissues and optimization and the development of new treatment strategies odontoblast-like cells. Thus, a pulp-dentin complex could be such as inclusion of scaffold and growth factors in regenerative engineered in human roots implanted subcutaneously in procedures.71,157 immune-deficient mice. CHAPTER 10 Regenerative Endodontics 457

C 50 µm

500 µm A

TD

10 µm 50 µm B D

FIG. 10-7 Regeneration of pulp tissue after autologous transplantation of mobilized dental pulp stem cells (MDPSCs) with G-CSF in pulpectomized teeth of young dogs on day 14. A-B, Regenerated pulp tissue. B, Odon- toblastic cells (black arrows) lining to newly formed osteodentin/tubular dentin (TD) along with the dentin. C, Immunohistochemical staining of BS-1 lectin. D, Immunohistochemical staining of PGP9.5. (From Nakashima M, Iohara K: Mobilized dental pulp stem cells for pulp regeneration, J Endod 40:S29, 2014.)

Nakashima and colleagues accomplished complete pulpal (Fig. 10-7). The impressive results of this cell-based approach regeneration in dogs.107 In this elegant study, dental pulp was have laid the foundation for the use of this technology in removed via a sterile pulpectomy procedure, followed by place- emerging clinical trials.163 ment of sorted CD105+ DPSCs in a collagen gel to the mid- It is noteworthy that all of the demonstrations of pulp root. The remaining coronal part of the canal was back-filled regeneration in animal models were in root canals without with the collagen gel containing the chemotactic factor stromal any history of infection and pulpal necrosis.107,108,130,159,200 Pre- derived factor 1 (SDF-1). Subsequent histology demonstrated viously infected root canals must be adequately disinfected in the formation of new pulp tissue with innervation, vasculariza- order to suppress chronic inflammation that is detrimental to tion, and odontoblast-like cells lining the dentinal walls. In regeneration.48 However, many irrigants and medicaments addition, the engineered pulp had protein and RNA expression have detrimental effects on stem cell survival and differentia- similar to the native dental pulp.107 tion.63 Several studies using organotypic root canal models In another important study, mobilized DPSCs were and animal models evaluated the combination and concentra- generated by selecting DPSCs that migrated toward a con­ tions of irrigants and medicaments that allowed for stem centration gradient of granulocyte-colony stimulating factor cell proliferation and differentiation.81,142,235 Thus, adequate (G-CSF).108,150,163 These selected cells were implanted in a col- disinfection and resolution of inflammation appears to be a lagen gel into pulpectomized root canals.108 Complete pulpal limiting factor in complete pulpal regeneration, despite regeneration was observed, with evidence of new dentin forma- exciting results from current advances in dental pulp tissue tion, blood vessels, and innervation in the engineered tissue engineering. 458 PART II The Advanced Science of Endodontics

maintenance and maturation of a regenerating tissue, it is Summary of Basic Research on noteworthy that some of the published cases report positive Regenerative Endodontics responses to pulp sensitivity tests such as cold or electric pulp Regeneration of a functional pulp-dentin complex relies on the tests.62 This is evidence that a space that was previously vacant foundation of tissue engineering and can be viewed as a func- (debrided root canal) may become populated with an inner- tion of the spatially correct delivery of appropriate stem cells vated tissue supported by vascularity. Taken together, the core and growth factors embedded within a scaffold. Although con- concepts of tissue engineering distinguish a regenerative treat- siderable research has used in vitro cell-culture methods to ment philosophy from a revascularization philosophy derived identify key factors regulating the differentiation of odontoblast- from certain trauma cases (which only occur in a low percent- like cells, emerging studies conducted in animal models are age of replanted teeth). Lastly, a number of procedures promising for regeneration of this pulp tissue. Preclinical have been performed with intentional manipulation of the studies involving surgical placement of a filled principles of tissue engineering such as use of autologous with a human stem cell/growth factor/scaffold combination platelet-rich plasma (PRP),234 platelet-rich fibrin (PRF),208 and into immunocompromised mice119,130 have permitted histo- exogenous growth factors and scaffolds.157 Thus, instead of logic analysis of neovascularization as well as the differentia- using different terms for each variant of these procedures, we tion and mineralization activity of newly formed odontoblasts. will refer to them simply as regenerative endodontic proce- The use of human cells in a mouse model permits histologic dures (REPS), which include past, present, and future proce- confirmation that the resulting odontoblast-like cells were of dures that aim for the functional regeneration of the pulp human origin. These novel findings provide strong evidence dentin–complex. that either human SCAP or DPSC cell sources, on a PLGA scaffold, were able to regenerate a vascularized tissue that had histologic evidence of odontoblast-like cell differentiation and Clinical Procedures Related to the spatially appropriate formation of dentin-like material onto Regenerative Endodontics the root canal walls. Although no specific growth factors were Clinicians face several challenges when presented with an added to this mixture, it is important to note that the root canal incompletely formed root in need of endodontic treatment.62 walls were treated with 17% EDTA, an irrigant known to Because the apex is not fully developed and often has a blun- expose endogenous growth-factor proteins embedded in the derbuss shape, cleaning and shaping of the apical portion of the dentinal walls.268 This and other related studies provide strong root canal system can be difficult. The process is further com- impetus for clinical translational research evaluating various plicated by the presence of thin, fragile dentinal walls that may potential regenerative endodontic therapies. be prone to fracture during instrumentation or obturation. In addition, the open apex increases the risk of extruding material CLINICAL STUDIES ON into the periradicular tissues. Traditionally, an immature tooth with an open apex is treated by apexification, which involves REGENERATIVE ENDODONTICS creating an apical barrier to prevent extrusion. In many To date, most case reports, case series, and retrospective studies cases, this entails an involved, long-term treatment with

published in regenerative endodontic have not fully incorpo- Ca(OH)2, resulting in the formation of a hard-tissue apical rated the tissue-engineering concepts described. Instead, most barrier.50-52,78,77,96,97,247 However, a disadvantage of the traditional of these reports present cases with variations of revasculariza- apexification procedures is that the short-term195 or long- 62 14,15,257,258 tion techniques. These procedures were initially performed term use of Ca(OH)2 has the potential to reduce root empirically with a strong focus on disinfection and the inten- strength.14,257,258 This finding is consistent with a large case series tional bleeding into the root canal. However, it became obvious using the traditional apexification protocol; it showed that a that these were in fact stem-cell based procedures with all three major reason for tooth loss following apexification was root components of the tissue engineering triad present: stem fracture.53 In a retrospective study, the use of calcium hydroxide cells,135 growth factors,26 and scaffolds.23,234 Important preclini- in apexification procedures resulted in the fracture of 23% cal studies previously described in this chapter have provided of the teeth treated during the follow-up period of up to the foundational framework for a paradigm shift. This shift 18 months.110 The advent of one-step apexification, by the cre- represents a clear departure from the traditional “disinfect the ation of artificial barriers using materials such as MTA,146,181,233 canals at all cost” to “disinfect while creating a microenviron- has greatly decreased the number of appointments and time to ment conducive for tissue engineering.” Varied terminologies completion. Importantly, one-step apexification has been shown have been given to these procedures, including revasculariza- to have as high a success rate as apexification with calcium tion,23 revitalization,234 and maturogenesis.6,101,115 Of these terms, hydroxide in resolving apical periodontitis (both symptoms and the most popularly used has been revascularization. This term radiographic presentation).247 However, apexification proce- is largely based on the trauma literature observation that dures do not generally result in further root development. A immature teeth could become revascularized after trauma. primary advantage of regenerative endodontic procedures in However, revascularization is a term better used for the rees- these cases is the greater likelihood there will be an increase in tablishment of the vascularity of an ischemic tissue, such as root length and root wall thickness, in addition to the possibility the dental pulp of an avulsed tooth. From this perspective, a that the patient will regain vitality responses. focus on revascularization would ignore the potential impor- There have been numerous published cases of regenerative tance of growth factors and scaffolds that are required endodontic procedures. Investigators and clinicians have used for histologic recapitulation of the pulp-dentin complex. a variety of medicaments to disinfect the canal space.62 Although we appreciate that angiogenesis and the establish- Approximately 51% of the cases included the use of a ment of a functional blood supply are key requirements in the triple antibiotic paste (a 1 : 1 : 1 mixture of ciprofloxacin/ CHAPTER 10 Regenerative Endodontics 459

metronidazole/minocycline), whereas 37% used Ca(OH)2 as an 1000-fold drop in bacteria. These findings were confirmed in intracanal medicament.62 another related dog study.229 This study provides strong support The development of the triple antibiotic paste was led in for the effectiveness of triple antibiotic paste in disinfection of large part by Hoshino and colleagues.100,202 They demonstrated immature teeth with apical periodontitis. the effectiveness of combinations of antibiotics (and in particu- As stated previously, calcium hydroxide has been the second lar the high efficacy of the combination of ciprofloxacin, met- most used intracanal medicament in published cases. This ronidazole, and minocycline) in eradicating bacteria from the application represents a new use of a long established intraca- 202 infected dentin of root canals. Astute practitioners realized nal medicament in endodontics. Although Ca(OH)2 appears to that the triple antibiotic paste could be a valuable adjunct for be less effective against some intracanal bacterial species than revascularization procedures, because it could be used to create antibiotic paste formulations,199 its use is associated with lower an environment favorable for the ingrowth of vasculature and cytotoxicity to stem cells,126,198 release of important bioactive regenerative cells by reducing or eradicating bacteria in the growth factors from the treated dentin,94 and greater survival canal space of teeth with necrotic pulps and incompletely and proliferation of stem cells in the presence of the condi- formed apices. The efficacy of the triple antibiotic paste in tioned dentin.167 Also, the relatively short-term use of this disinfecting necrotic root canal systems has been demonstrated medicament in regenerative procedures does not appear suf- in a preclinical model.246 In this dog study, 60 teeth were ficient to reduce fracture resistance.258 Another factor to con- accessed and infected by sealing dental plaque and sterile sider when choosing an intracanal medicament is the ability to saline on a cotton pellet into the pulp chamber for 6 weeks. remove the medicament from the canal space. One study that By the end of this period, each premolar was radiographically addressed this question incorporated radioactive tracers in confirmed to have apical periodontitis. The canals were then both calcium hydroxide paste (Ultracal, Ultradent, Inc.) and sampled at three time points: before and after irrigation with triple antibiotic paste (Champs Pharmacy, San Antonio, TX).28 1.25% NaOCl, and 2 weeks after the delivery of the triple The radiolabeled medicaments were placed in extracted teeth antibiotic paste into the root canal system using a Lentulo with standardized root canals. After 28 days of incubation, spiral. Before irrigation, all of the teeth had positive cultures canal spaces were irrigated with a standardized protocol using for anaerobic bacteria, with a mean colony-forming unit (CFU) different techniques. Surprisingly, greater than 80% of the count of 1.7 × 10.8 After irrigation with 1.25% NaOCl, 10% of triple antibiotic paste could not be removed from the tooth the teeth sampled cultured bacteria free. The mean CFU count (Fig. 10-8), and it was found not in the canal lumen, but was 1.4 × 104, or an approximate 10,000-fold reduction in greater than 350 µm into the dentinal tubules. In contrast, viable bacteria. After dressing with the triple antibiotic paste greater than 80% of calcium hydroxide was removed (see for 2 weeks, 70% of the teeth sampled cultured bacteria free. Fig. 10-8) with the remaining medicament present superficially The mean CFU count was only 26, which is about another within dentin.28 This is an important finding, given that drugs

100 100 *

80 removed 2 60

50 n.s 40 % labeled-TAP removed % labeled-TAP % labeled-Ca(OH) 20

0 0 A PP EA EV PUI B PP PUI

FIG. 10-8 TAP remains in dentin, whereas most Ca(OH)2 is eliminated after endodontic irrigation. Radiolabeled

TAP or Ca(OH)2 was placed within canals of standardized root segments and incubated for 28 days at 37° C. The canals were flushed with standardized volumes of EDTA and saline using either positive pressure with a side-vented needle (PP) or positive pressure with ultrasonic activation of irrigants (PUI). There was no difference in labeled TAP removal among groups with only approximately 20% of the medicament being removed by the

irrigation protocols (A). In contrast, > 80% of Ca(OH)2 was removed, with more efficient removal observed in canals irrigated with PUI (B). Data are presented as the mean percentage of total radiolabeled medicament removal ± standard error of the mean. *P < .05 tested by the Student t test (n = 12/group). (Modified with permission from Berkhoff JA, Chen PB, Teixeira FB, Diogenes A: Evaluation of triple antibiotic paste removal by different irrigation procedures, J Endod 40:1172, 2014.) 460 PART II The Advanced Science of Endodontics

20000 plug of MTA was placed over the blood clot or a collagen-based * internal matrix, followed by a bonded coronal restoration. Each of these steps will be discussed later in light of the current *** American Association of Endodontists (AAE) considerations 15000 n.s. for REPs.

Overview of Clinical Regenerative 10000 Endodontic Procedures (REPS) To date, most published clinical studies on REPS are composed of case reports and case series, with only one retrospective 110 157

Luminescence (RLU) cohort study and one prospective randomized clinical trial 5000 on regenerative endodontics treatment available; no random- ized controlled clinical trials have been published. Although case series do not provide definitive evidence to support a given treatment modality, they do have the advantage of being Control TAP DAP Ca(OH)2 TAP DAP conducted on actual patients and thus provide a higher level 1,000 mg/ml 1 mg/ml of evidence than do preclinical studies. Although techniques for regenerative endodontics have varied in published case

FIG. 10-9 Dentin conditioning for 7 days with medicaments used in REPs 62 has a profound effect on SCAP survival. Standardized dentin disks were reports and case series, there have been some consistent fea- treated for 7 days with TAP or double antibiotic paste (DAP) (concentrations tures worth noting. Nearly all reported cases involve patients 8 to 18 years old and teeth with immature apices with the of 1000 mg/mL or 1 mg/mL), Ca(OH)2 (Ultracal), or sterile saline (control). exception of two published cases performed in mature, fully SCAP in a Matrigel scaffold (BD Biosciences, Bedford, MA) was seeded into 182 the lumen of the disks after the medicaments were removed and cultured for formed teeth. Thus, the patient age appears to be an impor- 7 days. Cell viability (survival) was determined using a luminescent assay. tant factor in case selection because some studies suggest that younger patients have a greater healing capacity or stem cell SCAP culture on dentin treated with TAP or DAP at the concentration of 10,12,55,128,154 1000 mg/mL resulted in no viable cells. Conversely, dentin conditioning with regenerative potential. Another important factor TAP or DAP at the concentration of 1 mg/mL supported cell viability with no related to age is the stage of root development, because the difference from untreated dentin disks (control). Greater survival and prolifera- large diameter of the immature (open) apex may foster the ingrowth of tissue into the root canal space and may indicate tion were detected in the group treated with Ca(OH)2. Data are presented as a rich source of mesenchymal stem cells of the apical papilla mean ± standard deviation of relative luminescence units (n = 12/group). 104,197,224 *P < .05. ***P < .001. n.s., no statistical difference as tested by 1-way (SCAP; see Fig. 10-3). These tissues are likely lacerated analysis of variance. (From Althumairy RI, Teixeira FB, Diogenes A: Effect of during the evoked bleeding step and constitute a likely source of mesenchymal stem cells delivered into the root canal dentin conditioning with intracanal medicaments on survival of stem cells of 135 apical papilla, J Endod 40:521, 2014.) space. Another consistent finding reported in nearly every case is the lack of instrumentation of the dentinal walls related to concerns about the potential fracture of these thin, incom- pletely developed roots. The lack of instrumentation would be remaining within dentin are likely to have an effect on the fate expected to have the benefit of avoiding generation of a smear of stem cells in contact with the treated dentin. One study layer that could occlude the dentinal walls or tubules. On the revealed that triple antibiotic paste (TAP), when used at con- other hand, the lack of instrumentation could result in remain- centrations typically used in case reports (1 g/mL) and removed ing bacterial biofilms within dentinal tubes. This is an issue with standardized irrigation protocol, resulted in no SCAP that has not been evaluated in many cases, but the lack of canal survival.9 In contrast, dentin treated with calcium hydroxide wall instrumentation (and subsequent identification of bacteria promoted SCAP survival and proliferation, and dentin treated in the apical dentin) has been suggested as a reason for failure with TAP at a 1 mg/mL had no effect on SCAP survival (Fig. of a regeneration case.183 Nonetheless, these procedures are 10-9). Thus, clinicians must carefully evaluate the advantages marked by robust disinfection protocols. Sodium hypochlorite, and disadvantages of each intracanal medicament while observ- either alone or in combination with other irrigants, has been ing the ideal therapeutic concentration.63 used to disinfect the canal space in most cases. In a majority Regardless of the intracanal medicament used, regenerative of cases, a combination of triple antibiotic (minocycline, met- endodontic procedures have common features. Most of these ronidazole, and ciprofloxacin) was left in the canal space for published procedures reported minimal to no instrumenta- a period of days to weeks, so the disinfection protocol tion.62 This might be due, at least in part, to the concern of was primarily a chemical method rather than the chemome- further weakening fragile dentinal walls and the difficulty of chanical approach used in conventional nonsurgical endodon- mechanically debriding canals of such large diameters. Because tic therapy.62 In most cases, a blood clot formed in the canal.62 of the lack of mechanical debridement, clinicians relied on The formation of a blood clot might serve as a protein scaffold, copious irrigation for maximum antimicrobial and tissue dis- permitting the three-dimensional ingrowth of tissue. solution effects.62 Canals were then medicated for a period that Nearly all of these reports noted continued thickening of varied from days to several weeks. At the second visit, if signs the root walls and subsequent apical closure. Examples of and symptoms of disease had subsided, the medicament was increased root length and thickening of root walls following removed, the canal was dried, and intracanal bleeding was regenerative endodontic procedures are seen in Figs. 10-10 evoked in most, but not all, cases.62 In most cases, a coronal through 10-13. Because of the lack of histology in most clinical CHAPTER 10 Regenerative Endodontics 461

A Pre-op B 10 months

FIG. 10-10 Revascularization case illustrating treatment delivered to a 9-year-old male patient with a diagnosis of pulpal necrosis secondary to trauma, with a class 3 fracture in tooth #8 and a class 2 fracture in tooth #9 (A). The patient reported moderate to severe pain in both teeth. The teeth were isolated, accessed, and irrigated with 5% sodium hypochlorite, followed by placement of a mixture of ciprofloxacin, metronidazole, and minocycline for 55 days. Upon recall, the teeth were isolated, and the triple antibiotic paste was removed by irrigation. Bleeding was established in tooth #9 but not in tooth #8, where CollaCote was placed prior to mineral trioxide aggregate (MTA). The root canal systems were sealed with white MTA and a composite restoration (B). (Courtesy Dr. Alan Law.)

A Pre-op B 6 months

FIG. 10-11 Regenerative endodontic case illustrating treatment delivered to a 13-year-old female patient with a diagnosis of pulpal necrosis secondary to caries, with an unspecified prior history of trauma (A). The tooth was isolated, accessed, and irrigated with 5% sodium hypochlorite, followed by placement of a mixture of ciprofloxacin, metronidazole, and minocycline for 21 days. Upon recall, the tooth was isolated, and the triple antibiotic paste was removed by irrigation. Bleeding was established in both teeth, and the root canal system was sealed with white mineral trioxide aggregate and a composite restoration (B). (Courtesy Dr. Alan Law.) cases, it should be recognized that radiographic findings of regenerated pulp tissue may be in the canal space,123,206 and continued root wall thickness do not necessarily indicate that that the mineralized tissue along the dentinal walls appears to dentin was formed. Based on histologic results from preclinical be cementum-like or osteodentin.143,207 It should also be noted studies, it is possible that the radiographic appearance of that in some of the case reports,11,118 although the teeth were increased root wall thickness might be due to the ingrowth of nonresponsive to pulp testing, the authors suggested that vital cementum, bone, or a dentin-like material. Histologic evidence tissue was identified in the apical portion of the canal space. from human extracted teeth following REPS suggests that In these cases, necrotic tissue was removed until bleeding was 462 PART II The Advanced Science of Endodontics

A A1 B

C D E

FIG. 10-12 Regenerative endodontic case illustrating treatment delivered to a 9-year-old male patient with a diagnosis of pulpal necrosis secondary to trauma and periradicular chronic apical abscess on tooth #9 seen on preoperative perirapical radiograph (A) and CBCT (B). The patient was asymptomatic and has draining facial sinus tract. The tooth was treated with a regenerative procedure using double antibiotic paste (DAP), a mixture of ciprofloxacin and metronidazole for 1 month. At the second appointment, the sinus tract had resolved. Then, the tooth was isolated, and the double antibiotic paste was removed by irrigation with 20 mL 17% EDTA. Intracanal bleeding was evoked from the apical tissues using a precurved #25 hand-file extending approximately 2 mm beyond the root apex. A Collaplug barrier was placed at the midroot level, then covered with 3 mm white MTA. Fuji II LC glass ionomer was used as a coronal seal of the MTA. The access was then restored with a composite resin and polished (C). At the 1-year follow-up, the patient was asymptomatic, responsive to electrical pulp test (EPT), periodontal probings were no greater than 3 mm, and the tooth exhibited grade I mobility without coronal discoloration. Importantly, there was appreciable root maturation in addition to resolution of apical radiolucency seen on CBCT (D) and periapical radiograph (E). (Courtesy Dr. Obadah Austah.)

observed, then the canals were disinfected with antibiotic paste Although published revascularization cases have been treated 62 or Ca(OH)2. One could argue that by leaving vital pulp tissue with varying clinical protocols, they can be grouped by method in the apical segment of the canal, the resulting progression of of canal disinfection—namely, triple antibiotic paste, Ca(OH)2 root formation is more similar to apexogenesis than revascu- treatment, or formocresol treatment. This study applied a larization. Although the biologic process may differ between mathematical image-correction procedure that permitted the the revascularization and apexogenesis cases presented in these comparison of nonstandardized radiographs with subsequent case reports, the goals of the procedures are similarly advanta- statistical analysis of radiographic outcomes. The percentage geous and significant. In both procedures, there is healing of change in root dimensions was first compared in two negative the periradicular tissues and a progression of root development control groups (nonsurgical [NSRCT] and in a tooth that would otherwise have had a progression of MTA apexification) predicted to have little to no change in root pulpal and periradicular pathosis. dimensions. This provides an internal test that the mathemati- A retrospective study has compared the radiographic cal analysis was appropriate. The results indicate that these two changes in 48 revascularization cases to 40 control cases.33 negative control groups had minimal measured changes in root CHAPTER 10 Regenerative Endodontics 463

A B

C D E

F G H

FIG. 10-13 Regenerative endodontic therapy case illustrating treatment delivered to a 9-year-old female patient with a diagnosis of previously initiated endodontic therapy (pulpectomy) due to pain from pulpal inflam- mationn o #29. Bilateral examination revealed presence of a talon cusp (dens evaginatus), which may have been the f etiology o pulpal inflammation on #29. Radiographic examination revealed large periapical radiolucency on a periapical radiograph (A) and CBCT (B). The tooth was treated with a regenerative procedure using double antibiotic paste as an intracanal medicament for 48 days. On the second visit, the tooth was isolated, and the double antibiotic paste removed by saline irrigation followed by a final rinse with 17% EDTA. The canal was dried and bleeding was evoked followed by placement of an internal matrix of Collaplug 3 to 4 mm below the CEJ. Mineral trioxide aggregate (MTA) was then placed over it and the tooth was restored with Fuji II LC and Build It. There was complete resolution of apical radiolucency on 1-month recall (C), appreciable root development at 5-month recall (D), and complete root maturation seen at 1-year recall on both perirapical radiograph and CBCT (E and F, respectively). Greater root maturation is observed at 2.5-year recall periapical radiograph (G) and CBCT I (H n and I). I addition, the tooth responded to EPT on the 1- and 2.5-year recall visits. (Courtesy Dr. Nikita Ruparel.) 464 PART II The Advanced Science of Endodontics

*** * 80 *** *** 125 * 70 100 60

75 50

40 50 30 25 20 0 Percent change in root width 10 Percent change in root length

−25 2 0

Ca(OH) −10 2 MTA control Formocresol NSRCT control Triple antibiotic Ca(OH) MTA control Formocresol Retrospective analysis of the percentage of change in root FIG. 10-14 NSRCT control Triple antibiotic length from preoperative image to postoperative image, measured from the FIG. 10-15 Retrospective analysis of the percentage of change in dentinal cementoenamel junction (CEJ) to the root apex in 40 control patients and in wall thickness from preoperative image to postoperative image, measured at 48 patients following a revascularization procedure. ***P < 0.001 versus the apical third of the root (position of apical third defined in the preoperative mineral trioxide aggregate (MTA) apexification control group (n = 20) and image) in 40 control patients and in 48 patients following a revascularization NSRCT control group (n = 20). P < 0.05 versus MTA control group only. procedure. ***P 0.001 versus mineral trioxide aggregate (MTA) apexification Median values for each group are depicted by horizontal line, and individual < control group and NSRCT control group. P 0.05 versus NSRCT control group cases are indicated by the corresponding symbol. (From Bose R, Nummikoski < only. P 0.05 versus calcium hydroxide (Ca[OH] ) and formocresol groups. P, Hargreaves K: A retrospective evaluation of radiographic outcomes in < 2 P 0.05 versus NSRCT control group only. (From Bose R, Nummikoski P, immature teeth with necrotic root canal systems treated with regenerative < Hargreaves K: A retrospective evaluation of radiographic outcomes in imma- endodontic procedures, J Endod 35:1343, 2009.) ture teeth with necrotic root canal systems treated with regenerative endodon- tic procedures, J Endod 35:1343, 2009.)

width (Fig. 10-14) or root length (Fig. 10-15), with the antici- pated finding that instrumentation with files of greater taper by either MTA apexification (0.0%) or calcium hydroxide resulted in a slight but detectable loss of apical root wall width. apexification. In addition to measuring changes in root dimen- The results indicated that revascularization treatment with sions, the authors reported survival (defined as retention of the

either the triple antibiotic paste or Ca(OH)2 medicament pro- tooth in the arch at the time of the postoperative recall) rate duced significantly greater increases in root length compared of 100% at an average of 14 months after revascularization with either the MTA or NSRCT control groups. Also, treatment treatment. This compared favorably with survival rates of 95% with the triple antibiotic paste produced significantly greater for MTA apexification and 77% for calcium hydroxide apexifi- increases in root wall thickness compared with the MTA and cation. Although prospective randomized clinical trials with

NSRCT control groups. Treatment with Ca(OH)2 resulted in standardized radiographic assessment are clearly required, the significantly greater changes in root wall thickness compared results of this retrospective study are consistent with a robust with the NSRCT group, but no differences were observed outcome of revascularization procedures, particularly when between these medicaments and the MTA apexification group. medicated with either a triple antibiotic paste or a Ca(OH)2 Finally, the triple antibiotic paste produced significantly greater medicament. The demonstration of continued root develop- differences in root wall thickness compared with either the ment does not reveal whether this radiopaque material is

Ca(OH)2 or formocresol groups. In general, the formocresol dentin, cementum, or bone, so given the known reliance of group showed the smallest improvement in root length and stem cells on an appropriate scaffold and growth factor com- wall thickness. Secondary analyses indicated that a 12- to bination, this will be an important focus for future research 18-month recall is probably the minimal time to judge radio- efforts. graphic evidence of root development, although later time points (36 months) often demonstrate continued root develop- Example of a Revascularization Protocol ment. Jeeruphan and colleagues (2012) used a similar method Based on current research, several factors can be reviewed to measure changes in root length and width following apexi- when considering a protocol for revascularization treatment. fication versus revascularization.110 They reported that teeth The first issue is case selection; the best available evidence treated with revascularization showed a significantly greater indicates that this treatment should be considered for the percentage increase in root length (14.9%) compared with incompletely developed permanent tooth that has an open teeth treated by either MTA apexification (6.1%) or calcium apex and is negative to pulpal responsiveness testing. Although hydroxide apexification. They also reported that the revascu- the ultimate goal of this approach may include a tissue larization protocol produced significantly greater percentage engineering–based method of pulpal regeneration in the fully increases in root width (28.2%) compared with teeth treated developed permanent tooth, it should be recognized that CHAPTER 10 Regenerative Endodontics 465 current revascularization protocols have not been developed (2012)197 have shown that higher TAP at concentrations used or evaluated for these more challenging cases. Informed in many published case reports has a profound detrimental consent should include the number of appointments (at least effect on stem cell survival. Importantly, it had minimal to no two), the possible adverse effects (primarily potential staining deleterious effect when used at the concentrations of 0.1 or 1 of the crown), the potential lack of response to treatment and mg/mL, whereas these concentrations are several orders of alternative treatments, and possible posttreatment symptoms. magnitude greater than those required to eliminate bacteria Because the canal space will not be accessible following revas- from infected dentin.203 In addition, the same report showed cularization, teeth requiring retention in the canal space for that all concentrations of calcium hydroxide promoted stem the restoration are not good candidates for REPs. Clinical cell survival. Moreover, TAP has an indirect detrimental effect staining of the crown and any root structure above the gingival on stem cell survival, as cells do not survive when in contact margin appears to be due to the presence of minocycline.120,185,191 with dentin previously treated with TAP at the concentration This outcome can be minimized by using a delivery system of 1 g/mL.167 This effect is largely avoided if TAP is used at the that restricts the drug below the cementoenamel junction lower concentration of 1 mg/mL.167 Thus, The use of TAP at the (CEJ).191 When it does occur, it can often be reduced or elimi- concentration of 1 mg/mL or calcium hydroxide pastes as intra- nated by a walking bleach method with sodium perborate. The canal medicaments seems warranted; however, further investi- use of mineral trioxide aggregate (MTA; ProRoot, Dentsply gation is needed to determine appropriate formulations and Tulsa Dental, Tulsa, OK), in both gray and white form vari- lower concentrations of antibiotic pastes, if necessary, to be ants,27,36,39 may also cause tooth discoloration, which can also used. When performing REPs on teeth in aesthetic areas, prac- be reduced or eliminated by a walking bleach technique.36 titioners should consider eliminating minocycline from the Alternative treatments that should be discussed with the antibiotic paste, sealing the coronal dentin with a dentin patient and guardian would include MTA apexification, no bonding agent or composite,120 or using calcium hydroxide treatment, or extraction. paste. After antimicrobial medicament is placed, the tooth is At the first appointment (Fig. 10-16, A-E), the treatment then sealed with a sterile sponge and a temporary filling (e.g., alternatives, risks, and potential benefits should be described Cavit), and the patient is discharged for 3 to 4 weeks. to the patient and guardian after collecting clinical informa- At the second appointment (see Fig. 10-16, F-M), the tion and establishing pulpal and apical diagnoses. Following patient is evaluated for resolution of any signs or symptoms of informed consent, the tooth is anesthetized, isolated, and an acute infection (\swelling, sinus tract pain, etc.) that may accessed. Minimal instrumentation should be accomplished, have been present at the first appointment. The antimicrobial but the use of a small file to “scout” the root canal system and treatment is repeated if resolution has not occurred.47,113 In determine working length is important. If sensation is experi- most reported cases, the acute signs and symptoms resolved enced within the canal system, this may suggest that some after treatment with the intracanal medicament.62 Because residual vital pulp tissue remains.59 The root canal system revascularization-induced bleeding will be evoked at this should be copiously and slowly irrigated. Because of its proven appointment, the tooth should not be treated with a local efficacy as a canal disinfectant and a tissue dissolution agent, anesthetic containing a vasoconstrictor. Instead, 3% mepiva- sodium hypochlorite (NaOCl) has been the irrigant of choice caine can be used, which will facilitate the ability to trigger in most of the revascularization case reports.62 However, studies bleeding into the root canal system.185 Following isolation and have demonstrated that NaOCl is cytotoxic to stem cells.62,142,235 reestablishment of coronal access, the tooth should be copi- Importantly, a minimally deleterious effect was seen with the ously and slowly irrigated, possibly together with gentle agita- use of 1.5% NaOCl followed by 17% EDTA. Thus, a lower tion with a small hand file to remove the antimicrobial concentration (1.5%) of NaOCl should be considered as the medicament. When choosing an irrigant at the second appoint- standard irrigant for REPs. Moreover, the use of chlorhexidine ment, it is worth considering that irrigants such as NaOCl and should be limited or avoided because it does not have tissue chlorhexidine may be cytotoxic to stem cells either directly69 dissolution capability and has also been shown to be cytotoxic or indirectly after dentin has been exposed to the irrig- to stem cells.235 Because canal disinfection relies considerably ants.69,142,235 It has been demonstrated that exposure of dentin on chemical irrigants, it is important to place the needle into to 5% to 6% sodium hypochlorite leads to decreased stem cell the apical third and irrigate using needles with a closed end survival and odontoblastic differentiation.81,142,235 This indirect and side-port vents (e.g., Max-I-Probe needles), together with effect is likely related to various deleterious effects of sodium a slow rate of infusion, to help to reduce any irrigants passing hypochlorite on the dentin matrix leading to reduced cell through the open apex. attachment192 and a decrease in dentin matrix–derived growth The root canal system is then dried with sterile paper points, factors such as TGF-β1.268 Thus, it is prudent to avoid sodium and the antimicrobial medicament is delivered into the root hypochlorite on the second visit. Instead, irrigation with 17% canal space. The best available evidence would support the use EDTA may be advantageous because it has been shown to 26,268 of either a triple antibiotic paste or Ca(OH)2. Triple antibiotic release growth factors from dentin and promote stem cell 142 paste (TAP) and Ca(OH)2 have been shown to be effective (see survival and differentiation. In addition, Galler and col- Figs. 10-9 and 10-10). The TAP has the advantage of being leagues (2011), using dentin cylinders transplanted into a very effective antibiotic combination against endodontic immunocompromised mice, showed that dental pulp stem microorganisms203; its efficacy is supported by its use in most cells seeded onto EDTA-conditioned dentin differentiated into published cases.62 This combination is not approved by the U.S. odontoblast-like cells and had cellular processes extending Food and Drug Administration (FDA), however, and carries a into the dentin.81 On the other hand, stem cells differentiated potential for minocycline staining of the crown. In addition, into osteoclasts/odontoclasts caused resorption on the dentinal triple antibiotic paste has been shown to be cytotoxic to stem walls if dentin was previously treated with sodium hypochlo- cells. It is important to note that Ruparel and colleagues rite. Thus, the use of 17% EDTA as the last irrigant promotes 466 PART II The Advanced Science of Endodontics

A B C

FIG. 10-16 Example of a revasculariza- tion case. Treatment was delivered to a 12-year-old boy who suffered trauma on tooth# 9 2 years prior to the appointment. First appointment: Clinical examination revealed a pain upon percussion and pal- pation (A). A working length file was placed into the root canal system (B). The tooth was slowly irrigated with 20 mL of 1.5% sodium hypochlorite (NaOCl) followed by D E 20 mL of saline using a Max-I-Probe needle inserted to the apical third (C). The canals were dried and medicated with calcium hydroxide paste (Ultracal) (D). The patient returned asymptomatic 1 month later. The tooth was isolated, accessed, and the medicament removed by slow irri- gation with 1.5% NaOCl followed by 17% EDTA. After the canal was adequately dried, intracanal bleeding was achieved by lacerating the apical tissues (E). CollaPlug F G as seen in access (F). CollaPlug was placed below the CEJ (G) to serve as a matrix to position the white mineral trioxide aggre- gate coronal to the blood clot (H). The tooth was then sealed with a layer of Fuji IX (I), etched and restored with a composite (J). A final radiograph was taken (K). The tooth responded to electrical pulp tested at the 1-year recall, and it demonstrated apical closure and moderate dentinal wall thick- ening (L). (Courtesy Dr. Anibal Diogenes.) H I

J K L CHAPTER 10 Regenerative Endodontics 467 the attachment, proliferation, and odontoblastic differentiation BOX 10-1 of stem cells. Treatment Procedures for Regenerative After drying the canal system with sterile paper points, a Endodontics file is placed a few millimeters beyond the , and the apical tissue is lacerated with bleeding up to 3 mm from First Treatment Visit for Regenerative Endodontics the CEJ. A small piece of Colla-Plug (Zimmer Dental, 1. Informed consent, including explanation of risks and alternative Carlsbad, CA) may be inserted into the root canal system to treatments or no treatment. serve as a resorbable matrix to restrict excessive apical posi- 2. After ascertaining adequate local anesthesia, rubber dam tioning of the MTA. About 3 mm of MTA is then placed. MTA isolation is obtained. has been used in many of the case reports and may have advan- 3. The root canal systems are accessed and working length is tages over other materials because it creates a bacteria-tight determined (radiograph of a file loosely positioned at 1 mm from seal, is biocompatible, and has conductive and inductive prop- 118,148,166,170 root end). erties. However, the use of MTA, including white 4. The root canal systems are slowly irrigated first with 1.5% NaOCl MTA, has been associated with tooth discoloration; thus, its (20 mL/canal, 5 min) and then irrigated with saline (20 mL/canal, use should be avoided in aesthetic areas. Biodentin is a bioac- 5 min), with irrigating needle positioned about 1 mm from tive material with desirable handling characteristics and less 124 root end. susceptibility to staining. Although this material is relatively 5. Canals are dried with paper points. new, there have been encouraging studies demonstrating that 6. Calcium hydroxide or an antibiotic paste or solution (combined it induces stem cell proliferation and odontoblastic differentia- 137,138,262 total of 0.1 to 1 mg/mL) is delivered to canal system. tion. Indeed, the use of Biodentine in and 7. Access is temporarily restored. direct cases is associated with an adequate dentin bridge and continued root development.29,59,204 Thus, MTA and Final (Second) Treatment Visit for Regenerative Endodontics Biodentine appear suitable to be used in regenerative endodon- (Typically 2 to 4 Weeks after the First Visit) tic procedures. 1. A clinical exam is first performed to ensure that that there is no In addition to follow-up visits in the first several months moderate to severe sensitivity to palpation and percussion. If after REPs to assess for signs of healing, a 12- to 18-month such sensitivity is observed, or a sinus tract or swelling is noted, recall should be considered as a reasonable time point to radio- then the treatment provided at the first visit is repeated. graphically assess apical healing and root development.33 2. After ascertaining adequate local anesthesia with 3% Box 10-1 summarizes the steps in a regenerative endodontic mepivacaine (no epinephrine), rubber dam isolation is obtained. procedure. Importantly, these considerations are based on the 3. The root canal systems are accessed; the intracanal medicament best available evidence and are likely to change as the field is removed by irrigating with 17% ethylenediaminetetraacetic progresses. acid (EDTA) (30 mL/canal, 5 min) and then a final flush with saline (5 mL/canal, 1 min). Clinical Measures of Treatment Outcome 4. The canals are dried with paper points. The goal of conventional endodontic therapy is to maintain or 5. Bleeding is induced by rotating a precurved K-file size #25 at restore the health of periradicular tissues by preventing or 2 mm past the apical foramen with the goal of having the whole healing apical periodontitis. The goals of regenerative proce- canal filled with blood to the level of the cementoenamel dures extend beyond the goals of conventional endodontic junction. therapy and include continued root development and reestab- 6. Once a blood clot is formed, a premeasured piece of Collaplug lishment of pulpal vitality. As mentioned earlier, contemporary (Zimmer Dental Inc., Warsaw, IN) is carefully placed on top of the regenerative endodontic procedures are being modified and blood clot to serve as an internal matrix for the placement of evolving toward accomplishing these additional objectives, approximately 3 mm of white MTA (Dentsply, Tulsa, OK) or which are not achievable with conventional nonsurgical root Biodentin (Septodont). canal therapy. Accordingly, the definition of success for regen- 7. A (3- to 4-mm) layer of glass ionomer layer (e.g., Fuji IX, GC erative endodontic procedures has not been fully determined. America, Alsip, IL, or other) is flowed gently over the bioactive Also, assessments of clinical outcomes of REPs are not always coronal barrier and light cured for 40 secs. easily achievable. Although the resolution of signs and symp- 8. A bonded reinforced composite resin restoration (e.g., Z-100, 3M, toms of disease can be easily assessed by a clinical and radio- St Paul, MN, or other) is placed over the glass ionomer. graphic examination, evidence of root development and vitality 9. The case needs to be followed-up at 3 months, 6 months, and responses are more challenging. yearly after that for a total of 4 years. It is well established that the primary goal of endodontics is the prevention and healing of pulpal and periradicular inflammation (i.e., resolution of signs and symptoms of to 100%.44,110,156 Most of these studies did not directly compare disease). Immature teeth with pulpal necrosis have been tradi- apexification to regenerative endodontic procedures with the tionally treated with apexification procedures.78 A retrospective exception of one prospective clinical trial157 and one retrospec- study reported a success rate of 93.5% for apexification proce- tive study.110 Thus, the success rate for both apexification dures performed in one visit and 90.5% for those performed and regenerative endodontic procedures are similarly high in two-visit appointments.247 Another retrospective study (i.e., above 90%) based on most current evidence. Nonetheless, reported that apexification procedures resulted in complete larger randomized clinical trials with adequate follow-up healing of apical periodontitis in 85% of the treated teeth.99 For periods are warranted to provide greater insight in the long- teeth treated with regenerative procedures, the healing of term outcome for both apexification and regenerative apical periodontitis has been reported to vary from 90%114,157 procedures. 468 PART II The Advanced Science of Endodontics

Continued root development is another desirable outcome clinical outcome is an asymptomatic tooth that does not of regenerative endodontic procedures. Most published cases require retreatment and demonstrates continued root develop- of regenerative endodontics report continued root develop- ment and perhaps vitality responses. ment or apical closure.62 It is important to note that there were It is worth noting that the published revascularization cases highly varied treatment protocols in the case reports and case have shown increased root wall thickness that is limited to the series.62 In addition, the reports of continued root development midroot and apical root.62 There has been no demonstration of are often subjective, without an attempt to quantify root devel- increased root thickness in the cervical area, an area shown to opment. This shortcoming is due, at least in part, to the dif- be prone to fracture in immature teeth with a history of trauma ficulty in acquiring standardized radiographs in young patients and subsequent endodontic treatment. Future clinical studies undergoing rapid cranioskeletal development. A study reported should focus on not only on making these procedures more the methodology to perform computational digital correction predictable, but extending regeneration into the cervical area, and the quantification of root length and width on nonstan- potentially strengthening this area and decreasing the risk of dardized regenerative endodontics radiographs.33 Studies that root fracture. have used this methodology to quantify root development have collectively reported that apexification procedures did not promote any root development, whereas regenerative proce- SUMMARY dures allowed mean increases in root width of 25% to The field of regenerative endodontics is rapidly advancing. 35.5%33,110,114 and in root length of 11.3% to 14.9%.33,110,114 This This progress is based on the principles of tissue engineering methodology has been modified to measure changes in radio- —namely, the spatial delivery of appropriate cells, scaffolds, graphic root area (RRA).76 Regenerative endodontic procedures and growth factors. Similar to most rapidly developing fields, resulted in a 31.6% increase in RRA, whereas apexification the preclinical area of research has outpaced translational procedures, as with the other studies, had no effect.76 Thus, clinical studies. Preclinical studies have identified several regenerative endodontic procedures allowed continued root mesenchymal stem cell sources capable of differentiating development not seen with other treatment alternatives. into odontoblast-like cells, as well as candidate scaffolds and Although achieving regeneration of pulp tissue continues growth factors capable of guiding this development. The to be a preferred objective, an alternative acceptable outcome— initial preclinical animal studies indicate that using all three retention of a tooth with healed apical tissue—could be components of the tissue engineering triad (stem cells, growth considered satisfactory. A retrospective study by Jeeruphan factors, and scaffolds) can result in complete regeneration of and colleagues110 compared survival rates (defined as retention the pulp-dentin complex. There is a delicate balance between of the tooth in the arch at the time of the postoperative recall) disinfection of the root canal system and the interplay of of teeth that underwent revascularization procedures versus these three components that warrants more research. It is teeth that underwent calcium hydroxide or MTA apexification. likely that this combined approach of in vitro and in vivo They reported a 100% survival rate for revascularization- preclinical research will greatly advance our understanding treated teeth versus 95% for MTA apexification cases and of the conditions necessary to regenerate a functional pulp- 77.2% for calcium hydroxide apexification cases.110 Survival of dentin complex. teeth treated with these procedures is noteworthy, because The translational nature of regenerative endodontic research premature loss of these permanent teeth would likely have is allowing for changes to take place in the clinical practice in led to a loss of alveolar bone and compromised future replace- a relatively short time. This cross-talk between basic and clini- ment of the tooth. Retention of a tooth following a revascular- cal sciences is largely fueled by the realization that all three ization procedure, even in the absence of continued root components of the tissue engineering triad are already present development, would potentially allow for implant placement in revascularization procedures: stem cells, scaffold (blood (if necessary) following alveolar bone growth37,184 and should clot), and growth factors (from dentin and blood). Preclinical be considered an acceptable outcome for patients, parents, and studies that evaluated the effect of irrigants81,142,235 and medica- practitioners. ments167,197 on the survival of stem cells, release of growth Vitality responses have been reported in approximately 50% factors from dentin,231,268 and odontoblastic differentiation41,81 of the published cases.62 Responses to electric pulp tester are shaping the future generations of regenerative procedures. (EPT) are more commonly reported than cold responses. These Further, the incorporation of other scaffolds such as PRP, PRF, responses to vitality testing (with either cold or EPT), as well and gelatin sponges has been used in the clinical practice with as the lack of signs and symptoms of pathosis, suggest the encouraging results.123,143,157,208,234 Discussion about the require- presence of functioning tissue in the canal space. Vitality ments of an appropriate scaffold and growth factor was begun responses, in addition to continued root development, are a in the 1960s by Nygaard-Østby,173 who had no access to our desirable outcome because “normal” nociception is protective contemporary instruments, materials, and knowledge base of and suggest immune-competence of the vascularized tissue. tissue engineering. Although clinical revascularization proce- However, the lack of responses should not be interpreted as dures do not constitute the ideal regenerative treatment, it is failure because there have been several reports of cases dem- important to note that they do generate a scaffold (fibrin) and onstrating healing of apical periodontitis and appreciable root growth factors (from platelets and access to proteins embedded development in the absence of a positive response to vitality in the dentinal walls), and the clinical outcome results in con- testing. Although vitality response is a desirable outcome tinued radiographic root development of the immature perma- because it suggests a “functional regeneration,” its presence nent tooth with a diagnosis of pulpal necrosis. Thus, REPs likely depends on several factors, including the depth level of provide a treatment of high value in cases with an otherwise coronal restoration placement and the degree of intracanal poor prognosis. Histologic analyses of REPs conducted in mineralization. Thus, from a clinical perspective, the ideal patients123,143,171,206 or animals168,193,243 suggest that the increase CHAPTER 10 Regenerative Endodontics 469

in root dimensions is often due to deposition of cementum-like delivery systems that permit structural reinforcement of the material, osteodentin, dentin, or bone. cervical area (or, ideally, the pulp chamber) might provide Future clinical research will likely focus on further trans- clinical opportunities to regenerate lost tooth structure, lating basic research findings into improved regenerative pro- thereby permitting natural teeth to be retained instead of pos- cedures. For example, the formation of a cementum-like sible fracture or extraction. Finally, the ultimate and long- material on the dentinal walls may lead to studies evaluating term goal for regenerative endodontic procedures should be benefits of REPs for overall tooth resistance to fracture. In to treat the fully formed permanent tooth. Although this situ- addition, it is clear that the multipotent nature of many mes- ation is more complex than the immature tooth with an open enchymal stem cell types could contribute to the finding apex and a ready source of stem cells, it provides the unique of cementum deposition. Controlled differentiation of stem potential of saving the natural dentition while restoring the cells into odontoblasts is an important area of research and sensory, immunologic, and defensive properties of the pulp- amenable to tissue-engineering concepts. The development of dentin complex.

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243. Wang X, Thibodeau B, Trope M, et al: Histologic 252. Yamada Y, Ueda M, Naiki T, et al: Autogenous injectable 261. Young CS, Terada S, Vacanti JP, et al: Tissue engineering characterization of regenerated tissues in canal space bone for regeneration with mesenchymal stem cells and of complex tooth structures on biodegradable polymer after the revitalization/revascularization procedure of platelet-rich plasma: tissue-engineered bone regeneration, scaffolds, J Dent Res 81:695, 2002. immature dog teeth with apical periodontitis, J Endod Tissue Eng 10:955, 2004. 262. Zanini M, Sautier JM, Berdal A, Simon S: Biodentine 36:56, 2010. 253. Yamagishi VT, Torneck CD, Friedman S, et al: Blockade of induces immortalized murine pulp cell differentiation into 244. Wei X, Ling J, Wu L, et al: Expression of mineralization TLR2 inhibits porphyromonas gingivalis suppression of odontoblast-like cells and stimulates biomineralization, markers in dental pulp cells, J Endod 33:703, 2007. mineralized matrix formation by human dental pulp stem J Endod 38:1220, 2012. 245. Wiesmann HP, Joos U, Meyer U: Biological and cells, J Endod 37:812, 2011. 263. Zhang CZ, Li H, Young WG, et al: Evidence for a local biophysical principles in extracorporal bone tissue 254. Yamamura T: Differentiation of pulpal cells and action of growth hormone in embryonic tooth development engineering: part II, Int J Oral Maxillofac Surg 33:523, inductive influences of various matrices with reference in the rat, Growth Factors 14:131, 1997. 2004. to pulpal wound healing, J Dent Res 64 Spec No:530, 264. Zhang J, An Y, Gao LN, et al: The effect of aging on the 246. Windley W 3rd, Teixeira F, Levin L, et al: Disinfection of 1985. pluripotential capacity and regenerative potential of human immature teeth with a triple antibiotic paste, J Endod 255. Yang B, Chen G, Li J, et al: Tooth root regeneration periodontal ligament stem cells, Biomaterials 33:6974, 31:439, 2005. using dental follicle cell sheets in combination with a 2012. 247. Witherspoon DE, Small JC, Regan JD, Nunn M: dentin matrix-based scaffold, Biomaterials 33:2449, 265. Zhang T, Kurita-Ochiai T, Hashizume T, et al: Retrospective analysis of open apex teeth obturated with 2012. Aggregatibacter actinomycetemcomitans accelerates mineral trioxide aggregate, J Endod 34:1171, 2008. 256. Yang X, Yang F, Walboomers XF, et al: The performance atherosclerosis with an increase in atherogenic factors in 248. Wysocki GP, Daley TD, Ulan RA: Predentin changes in of dental pulp stem cells on nanofibrous PCL/gelatin/nHA spontaneously hyperlipidemic mice, FEMS Immunol Med patients with chronic renal failure, Oral Surg Oral Med scaffolds, J Biomed Mater Res A 93:247, 2010. Microbiol 59:143, 2010. Oral Pathol 56:167, 1983. 257. Yassen GH, Platt JA: The effect of nonsetting calcium 266. Zhang W, Walboomers XF, Jansen JA: The formation of 249. Xu L, Tang L, Jin F, et al: The apical region of developing hydroxide on root fracture and mechanical properties of tertiary dentin after pulp capping with a calcium tooth root constitutes a complex and maintains the ability radicular dentine: a systematic review, Int Endod J phosphate cement, loaded with PLGA microparticles to generate root and periodontium-like tissues, J 46:112, 2013. containing TGF-beta1, J Biomed Mater Res A 85:439, Periodontal Res 44:275, 2009. 258. Yassen GH, Vail MM, Chu TG, Platt JA: The effect of 2008. 250. Xu X, Jha AK, Harrington DA, et al: Hyaluronic acid-based medicaments used in endodontic regeneration on root 267. Zhang X, Tamasi J, Lu X, et al: Epidermal growth factor hydrogels: from a natural polysaccharide to complex fracture and microhardness of radicular dentine, Int Endod receptor plays an anabolic role in bone metabolism in networks, Soft Matter 8:3280, 2012. J 46:688, 2013. vivo, J Bone Miner Res 26:1022, 2011. 251. Yamada Y, Ueda M, Hibi H, Nagasaka T: Translational 259. Yildirim S, Can A, Arican M, et al: Characterization of 268. Zhao S, Sloan AJ, Murray PE, et al: Ultrastructural research for injectable tissue-engineered bone dental pulp defect and repair in a canine model, Am J localisation of TGF-beta exposure in dentine by chemical regeneration using mesenchymal stem cells and Dent 24:331, 2011. treatment, Histochem J 32:489, 2000. platelet-rich plasma: from basic research to clinical case 260. Yip WK: The prevalence of dens evaginatus, Oral Surg study, Cell Transplant 13:343, 2004. Oral Med Oral Pathol 38:80, 1974. CHAPTER 11

Evaluation of Outcomes

YUAN-LING NG | KISHOR GULABIVALA

CHAPTER OUTLINE Context of Evaluating Endodontic Outcomes Pulpotomy Types of Disease and Their Treatment Summary of Prognostic Factors for Vital Pulp Therapy What Are Surrogate Outcome Measures? Outcomes of Nonsurgical Root Canal Treatment Types of Outcome Measures Factors Affecting Periapical Health or Healing Following Root What Is the Purpose of Evaluating Outcomes? Canal Treatment Effectiveness of Procedures Factors Affecting Tooth Survival Following Root Factors Affecting Outcomes Canal Treatment Value for Prognostication Impact of Root Canal Treatment on Quality of Life Outcome Measures for Endodontic Treatment Outcome of Nonsurgical Retreatment Outcome Measures for Vital Pulp Therapy Procedures Outcome of Surgical Retreatment Outcome Measures for Nonsurgical Root Canal Treatment Factors Affecting Periapical Health or Healing Following and Retreatment Periapical Surgery and Root-End Filling Outcome Measures for Periapical Surgery Factors Affecting Periodontal Incisional Wound Healing Outcomes of Vital Pulp Therapy Procedures Factors Affecting Tooth Survival Following Periapical Surgery Indirect Pulp Capping (One-Step versus and Root-End Filling Stepwise Excavation) Impact of Periapical Surgery on Quality of Life Direct Pulp Capping Concluding Remarks

CONTEXT OF EVALUATING Toward the 1990s, the improvements in general health ENDODONTIC OUTCOMES and longer survival of Western populations as well as their teeth, coupled with the rising costs of health care for longer- The development of medical and dental practices has been living individuals, prompted a host of reevaluations of the guided by prevailing philosophies and consensus of expert altered economic burdens on society. Among them, the cost- opinions. The foundations of modern endodontics were shaken effectiveness of treatment procedures for management of diseases when Billings97 brought the apparent relationship between oral loomed large. Thus began the era of evidence-based medicine sepsis and bacterial endocarditis to the attention of dentistry and dentistry, with its emphasis on costs, benefits, and out- and medicine. He had strengthened the concept further from comes of treatment. Attempts to pool outcome data for greater Miller134 who had coined the term focus of infection to highlight power brought with it the realization that studies originating a possible link between “mouth germs” and systemic disease. from different centers varied in diverse ways, leading to very The disastrous consequences of the focal infection era were heterogeneous data that challenged formal attempts at drawing sealed when Hunter96 delivered his famous address at McGill definitive conclusions. Characterization of data types and its University. Fears of fatal oral sepsis from deficient root canal quality prompted calls for standardization of approaches in treatments led to widespread extraction of pulpless teeth. End- measuring outcomes to enable more meaningful pooling. odontics virtually disappeared from many dental schools, Because of the better established science of evidence assess- whereas in some areas, treatment was restricted to anterior ment, a modern revisitation of the “focal infection era”94 did teeth. The focal infection theory reigned for about 50 years83 not lead to the same threat to the endodontic discipline that until around 1940. was evident at the beginning of the 20th century. In fact, it was The discipline of endodontics was rescued by individual, suggested as an opportunity to secure research funding for diligent practitioners in Europe and USA who meticulously assessing and managing the importance of dental care on sys- recorded their treatments, as well as their outcomes, to dem- temic health. onstrate the effectiveness of the procedures in controlling root Interestingly, the modern threat to endodontics was posed canal infection. It was through these individual endeavors of by the cost-economic pressures exerted on treatment planning merit that the reputation of endodontic procedures was restored decisions centering on the question of whether to “save the and the discipline granted its specialist status in 1952 in the tooth” or extract it with replacement by an implant-supported United States.59 crown.238 Once again, the science of evidence-based practice

474 CHAPTER 11 Evaluation of Outcomes 475 has helped to avert irrational treatment recommendations “viewable” through various imaging techniques (such as radi- leaning toward extraction of savable teeth.54,100,103 ography). The process therefore calls upon the clinician to assimilate various sources of information to form a judgment Types of Disease and Their Treatment about the presence or absence of disease. The statement, “Endodontists provide endodontic treatment to manage endodontic disease” is a gross oversimplification that Types of Outcome Measures masks important subtleties in recognition of the nature of the In its broadest sense, an outcome measure for a treatment diseases and how best to address them. Endodontists manage intervention may constitute any consistently anticipated and inflammation of the specialized connective tissues within and measurable consequence of the treatment. The prepared shape surrounding the teeth; more specifically, they deal with inflam- of the root canal system, bacterial load reduction, and technical mation that generally commences in the pulp tissue and pro- quality of the root filling may all be regarded as outcome mea- gresses to the periradicular tissues via portals of communication sures by this definition. But the ultimate clinical measure of a that convey the neurovascular supply. Incipient or established treatment outcome is assessing the prevention and resolution pulpitis may be managed by vital pulp therapy when judged of disease. reversible, largely because a sufficient body of pulp tissue The outcome of endodontic treatment may be assessed in remains healthy and unaffected. More advanced pulpitis that four dimensions as in other medical disciplines.9 The first approaches the foramina into the periodontal ligament may dimension is physical/physiologic and related to presence or require root canal treatment. When pulpal inflammation, absence of pulpal/periapical health/disease, pain, and function. necrosis, or infection encroaches on the apical periodontal The second dimension assesses longevity or tooth survival. ligament, root canal treatment is required. Apical periodontitis The third dimension relates to economics and assesses direct that persists despite technically adequate root canal treatment and indirect costs. Finally, the fourth dimension examines psy- may require root canal retreatment, periradicular surgery, or chologic aspects involving perceptions of oral health–related extraction to manage the source of the persistent disease. End- quality of life (OHRQoL) and aesthetics. odontic treatment is therefore a collective and nonspecific term for a range of procedures directed at managing the WHAT IS THE PURPOSE OF spread of pulpal inflammation or infection. Endodontic treat- ment encompasses the following procedures: EVALUATING OUTCOMES? 1. Vital pulp therapy (indirect pulp therapy, direct pulp Apart from the importance of developing a solid foundation of capping, pulpotomy, regenerative pulp therapy) evidence-based practice, it is important to evaluate treatment 2. Nonsurgical root canal treatment outcomes for a number of reasons. 3. Nonsurgical root canal retreatment 4. Surgical retreatment Effectiveness of Procedures The ideal outcome for endodontic treatment consists of First, treatment procedures must be effective. Otherwise, there controlled reduction of inflammation, accompanied by healing is no reason to recommend them to patients as a treatment through regeneration, although sometimes repair may follow option. The patient must be properly informed as to the risks, instead. Unfortunately, none of the involved tissues are within benefits, and potential outcomes of the offered treatment. The the direct sight of the clinician, hidden as they are by their availability of pooled outcome data and consensus guidelines housings of hard tooth structure or alveolar bone and their offers both patients and endodontists reassurance and confi- gingival/mucosal coverings. Consequently, surrogate measures dence in the validity and predictability of the offered proce- must be employed to evaluate the presence or absence of the dure. However, the pooled average outcome data may not disease process and its resolution. The evaluation process is pertain if the clinician does not have the requisite experience, further complicated by the lack of direct correlation between skill, and personal outcomes matching at least these average measures of the disease process and its clinical manifestation. figures of performance. Personal outcome data offer the patient more precise measures for comparison and expectation. It also WHAT ARE SURROGATE aids practitioners in refining their technique and knowledge to further improve their outcomes and, ultimately, to help improve OUTCOME MEASURES? the overall pooled data.34 The treating clinician must assess his Signs of acute inflammation are classically described in the or her own personal outcome data and personal expectation of “triple response” exhibited by mechanically injured skin, success. If the clinician is uncertain of a result or feels that which includes altered color (redness), texture/contour (swell- the outcome could be enhanced by treatment from another ing), and sensation (pain), which are directly accessible and clinician, then referral to someone else more qualified is viewable. These changes have a direct correlation with histo- paramount. pathologic and molecular alterations. Chronic inflammation does not necessarily exhibit the same highly visible signs and Factors Affecting Outcomes symptoms of its histopathologic character. When hidden from Pooling data (that is preferably homogeneous) offers the view, as are pulpal and periradicular tissues, the task of recog- potential to evaluate and prioritize the factors that exert a nizing the presence or absence of chronic inflammation is even dominant influence on outcomes. In this way, protocols for more challenging. The clinician is therefore left with indirect treatment may be improved in a progressive fashion, bringing or associated (proxy) changes by which to judge the presence about the perfect meld of technical, clinical, and biologic or absence of disease; these are called “surrogate” measures. insight necessary for the highest performance and most pre- Some of these may have to be inferred through associations, dictable outcome. Evaluation of factors affecting treatment some may be directly visualized, and others are only indirectly outcomes arguably provides the strongest influence for making 476 PART II The Advanced Science of Endodontics changes that will improve the clinical outcomes. Weighing the a goal will require a diligent and conscientious collection of relative importance of individual factors should help to identify data from many sources. Analyses of such powerful datasets the key biologic factors at play and how best to manage them will ultimately yield proper insight into outcomes of the pro- from a clinical or technical perspective. cedures we use. Coupled with biologic insight of the problem, it will become possible to derive new therapeutic solutions Value for Prognostication necessary to deliver them. Prognostication, which could be defined as the prediction, projection, prophesizing, or foretelling the likely outcome of OUTCOME MEASURES FOR treatment, is not often well defined in endodontics. The overall prognosis of a tooth depends on the interaction among three ENDODONTIC TREATMENT individual and often independent variables, including end- Before determining the success or failure of an endodontic odontic, periodontal, and restorative prognoses. Each has a set procedure, the parameters as to what is considered a success of subsidiary factors that must be considered to derive an or a failure have to be discussed. These are termed the outcome overall prognosis. Finally, the tooth in question must be con- measures of the procedures. sidered from a strategic perspective, relative to its position in the dental arch and the contribution it makes in the dynamic occlusion. Outcome Measures for Vital Pulp A deeper analysis of the factors affecting outcomes as prog- Therapy Procedures nostic indicators is merited in order to clarify the degree of The techniques used to maintain the vitality and health of complexity inherent in the problem. A comparison may be pulps in teeth with extensive caries or those with traumatic/ made with the randomized controlled trial of a drug as a treat- mechanical exposures include (1) indirect pulp capping with ment intervention. The drug therapy is delivered as a clearly one-step or stepwise caries excavation; (2) direct pulp capping prescribed and standardized dose regimen delivered by speci- of exposed pulps; and (3) partial/full pulpotomy procedures fied timings to effect an anticipated blood or target tissue for more extensively involved pulps. The surrogate outcome concentration for a specified duration. Data recording is measures adopted in studies include (1) clinical success confined to compliance with the prescription and possibly (pulp sensitivity to cold test and absence of pain, soft-tissue checking of actual blood levels achieved as well as the final swelling, sinus tract, periradicular radiolucency, or pathologic outcome effect. root resorption), (2) patient satisfaction, (3) adverse events In stark contrast, a surgical intervention, regardless of any (pain, swelling, tooth fracture), and (4) tooth extraction.89,136 standardization of the described procedural protocol, is subject Although not providing a specific follow-up strategy, the to enormous variation in its delivery dependent on interpreta- quality guidelines of the European Society of Endodontology61 tion and execution of the protocol by each operator. To add to suggests “initial review at no longer than 6 months and there- the levels of complexity and variation, surgical protocols are after at further regular intervals.” They also provide the criteria often multistep, sequential procedures, each prospective step for judging the favorable outcome of vital pulp therapy (Table dependent on the previous for its efficacy. Even characterizing 11-1). There are substantial differences in the criteria adopted and accurately recording any variations in treatment protocols becomes a challenge because many aspects and steps must be documented. This leads to the next challenge, that of demon- strating compliance with the accuracy of such complex data TABLE 11-1 gathering. Furthermore, it becomes important from an analytic Criteria for Assessing the Outcome of Pulp Therapy perspective to not only consider the effect of individual steps (factors) alone but also any interactive effect between the mul- Quality Guidelines for tiple steps inherent in the procedure. Even when such compre- Endodontic Treatment: Guidelines on Pulp Therapy for hensive prospective outcome data are available, utilizing these Consensus Report of the Primary and Immature Permanent data to individually calculate the prognosis in a particular European Society of Teeth (The American Academy of scenario may be challenging. Clinically, there may be two dis- Endodontology (2006) Paediatric Dentistry 2014) tinct ways to estimate prognosis: (1) to apply heuristic prin- 1. Normal response to pulp 1. Tooth vitality maintained ciples and intuitively gauge the effect of dominant factors or, sensitivity tests (when 2. Absence of posttreatment at the other end of the extreme, (2) to mathematically input feasible) signs or symptoms such as comprehensive data into an algorithmic model to calculate the 2. Absence of pain and sensitivity, pain, or swelling estimated outcome. An even more sophisticated approach may other symptoms 3. Occurrence of pulp healing and be to use mathematical modeling to calculate iterations on 3. Radiologic evidence of reparative dentin formation variations within the system. At present, the evidence base is dentinal bridge formation 4. Absence of radiographic insufficient to allow such a sophisticated approach. 4. Radiologic evidence of evidence of internal or external It therefore follows that evaluating outcomes and the factors continued root formation root resorption, periapical that affect them are fundamental to the creation of a suitable in immature teeth radiolucency, abnormal data foundation for future application to endodontic prognos- 5. Absence of clinical and calcification, or other pathologic tication. The endodontic treatment option is in competition radiographic signs of changes with other alternative therapies and therefore, for the disci- internal root resorption 5. Teeth with immature roots pline to survive and thrive, a suitable blend of efficacy, effi- and apical periodontitis should show continued root ciency, utility, predictability, and cost-effectiveness data must development and apexogenesis be generated for its principal therapies. Achievement of such CHAPTER 11 Evaluation of Outcomes 477 in published clinical studies on outcomes of vital pulp of the disease process depends on preventing or terminating therapy89,136 with few studies adopting all of the criteria. The this interaction. frequency of radiologic review adopted in published studies Prevention of apical periodontitis applies to the clinical also varies substantially, with some recommending the first situation where it is judged that the pulp is irreversibly review at 1 month followed by subsequent 3 monthly inflamed, necrotic, or infected to the extent that vital pulp reviews.172,242 The relative benefit of acquired information therapy would not resolve the problem, which therefore versus unnecessary radiation to patients has been questioned.66 requires pulpectomy. The implication is that the procedure An initial assessment at 6 to 12 weeks, followed by a review 6 demands asepsis as a prime requirement. The precise technical and 12 months after treatment, seems to have been accepted details of the protocol may not be so important, as long as they and is recommended. Some studies reported up to 10 years are focused on effective and aseptic removal of the pulp tissue. follow-up on case series demonstrating lower success rates, This is validated by the high chance of retaining periapical inferring the possibility of slow spread of pulpal inflammation health (judged by conventional radiography), regardless of the and late failures. The conclusion is that longer follow-up clinical protocol used.153 periods are merited. Once the periapical lesion has become established, the chal- The process at each review consists of obtaining a history lenge is a different one because the purpose now is to remove of symptoms, coupled with an examination to determine the the bacterial biofilm and effect switching-off the periapical host presence or absence of tenderness to palpation of adjacent soft response. The challenge seems to be greater still if the periapi- tissues, tenderness to pressure and percussion of the tooth, cal lesion is larger as it is associated with a more diverse infec- signs of radiographic pulpal, and periapical changes, and tion. A number of approaches (protocols) have been used to responses to pulp tests. The accuracy of pulp tests may be achieve this general aim. The periapical healing process that limited in pulpotomized teeth because of the distance of the occurs after root canal treatment is less clear. Nevertheless, remaining pulp tissue from the tooth’s surface. In the case of pulp capping and pulpotomy, additional tests include radio- graphic verification of the presence of the calcific barrier (Fig. 11-1) and its integrity by removal of the dressing and direct probing. Although an initial examination at 6 weeks has been suggested, this can be modified for the radiographic assess- ment. If there is no evidence of complete bridge formation, the treatment is considered failed and root canal treatment should be considered. In addition, in the case of incompletely formed roots there should be radiographic evidence of continued root development (Fig. 11-2).

Outcome Measures for Nonsurgical Root Canal Treatment and Retreatment Root canal treatment may be employed either to prevent or resolve periapical disease. Given that periapical lesions develop as a result of interaction between bacteria (and their by-products) FIG. 11-1 Histologic view of intact calcific bridge formation following and the host defenses, it is clear that prevention or resolution pulpotomy.

A B

FIG. 11-2 Immature maxillary right lateral and central incisors (A) showing continued root development following pulpotomies (B). 478 PART II The Advanced Science of Endodontics ideal healing would eventually result in regeneration and the that residual infection in the apical anatomy is typical follow- formation of cementum over the apical termini, isolating the ing completion of root canal treatment and that an ongoing root canal system from the periapex (Fig. 11-3); but this is not interaction continues between the residual infection, root an inevitable result. Incomplete removal of the infection will filling material, and host defenses, which plays a definitive role reduce but not eliminate the periradicular inflammatory reac- in determining the final healing outcome. tion, and in fact this is generally what happens.147 This implies The culture test has been used during root canal treatment as an interim measure of the efficacy of the chemomechanical procedure; however, it has fallen out of favor in contemporary practice for a variety of reasons.139 The outcome measures that quantitate healing subsequent to root canal treatment are the absence of clinical signs and symptoms of persistent periapical disease.17,18 The definitive outcome measure (in conjunction with the absence of signs and symptoms), however, is periapi- cal healing, because the treatment is aimed at resolution of periapical disease (Fig. 11-4).152 Clinical judgment of the outcome of treatment is based on the absence of signs of infec- tion and inflammation, such as pain, tenderness to pressure/ percussion of the tooth, tenderness to palpation of the related soft tissues, absence of swelling and sinus tract, and radio- graphic demonstration of reduction in the size of the periapical lesion (if sufficient time has lapsed), with a completely normal development of the periodontal ligament space. Although the majority of periapical lesions heal within 1 year, healing may continue for up to 4 years or longer.229 Absence of signs and symptoms of periapical disease with radiographic evidence of a persistent periapical radiolucency may indicate either fibrous repair (Fig. 11-5) or persistent FIG. 11-3 Low-power view of healing by cementum formation when chronic inflammation or infection. Only time and acute exac- Sealpex is used (black particles are residual Sealapex and root filling material). erbation would identify the latter, whereas the former should (Courtesy Prof. M. Tagger.) remain asymptomatic.

A B

C

FIG. 11-4 Resolution of periapical disease after root canal treatment of the mandibular left molar with preop- erative periapical radiolucency associated with mesial and distal roots (A), delayed healing caused by extruded filling material from the distal canal (B), and nearing complete healing as extruded material is resorbed (C). CHAPTER 11 Evaluation of Outcomes 479

variety of simulated defects created in bone in pig226 or human165,221 jaws. Although the validity of clinical outcome data derived using conventional radiographic techniques has been questioned,256 the routine use of CBCT is not recom- mended62,91,197 owing to its higher radiation dosage.5,91 The use of CBCT, which may be more sensitive for detection of periapi- cal healing,114,167 may give lower healing rates and longer dura- tions to complete healing. Many studies consider the treatment successful only when both radiographic and clinical criteria are satisfied.69 It has been documented that a small proportion of cases present with persistent symptoms despite complete radiographic resolution of the periapical lesion.176 Comparison of success rates esti- mated with or without clinical examination revealed no93 or only a very small difference (1%).150 FIG. 11-5 Histologic view of fibrous periapical healing. The widely accepted definition for endodontic success and failure by Strindberg229 embraces both radiographic and clinical findings (Table 11-2). Friedman and Mor69 pre- Longevity measures include survival of the root canal fill- ferred the terms healed, healing, and diseased instead of success ings or treatment118,228,236 and tooth retention or survival.111,149,190 and failure because of the potential of the latter to confuse The term functional retention was coined by Friedman in 200469 patients. The “healed” category corresponds to “success” as to mean retention of the tooth in the absence of signs and defined by Strindberg,229 whereas “healing” corresponds to symptoms regardless of the radiographic presence of a lesion. “success” as defined by Bender and colleagues17,18 (see The term functionality should further and more specifically Table 11-2). cover the functional utility of the tooth—that is, some patients The long duration of the periapical healing process coupled may complain that despite the absence of specific signs and with the reduced recall rates at longer follow-up periods has symptoms of infection or inflammation, they find it impossible provided opportunity for setting the thresholds of success at to use the tooth because it “feels” weak. either complete healing or partial healing (reduced lesion size). Modern definitions of health embrace a broader spectrum The success criteria used for complete resolution have been of measures including psychological well-being.116 Instruments described either as “strict”152 or “stringent,”69 whereas the cri- to assess these aspects have been developed in general medi- teria established for reduction in periapical radiolucency size cine and adapted for application to dentistry, such as the Oral has been described as either “loose”152 or “lenient.”69 The fre- Health Related Quality of Life instrument.55,87,115 There is no quency of adoption of these two thresholds has been similar definitive instrument for measuring these aspects in endodon- in previous studies; the expected success rates using “strict” tics as yet. Studies published so far have mainly adapted ver- criteria would be expected to be lower than those based on sions of the Oral Health Impact Profile (OHIP). “loose” criteria. The literature finds the difference to vary from The periapical status of root-treated teeth has traditionally 4% to 48%.153 been assessed using two-dimensional conventional radio- A periapical index (PAI) consisting of five points on the graphic imaging. The use and limitations of two-dimensional scale was devised for measuring the periapical status and radiographic images for assessing treatment outcome has been used in some studies.157,159 However, the studies only reported reviewed thoroughly and reported to suffer from low sensitiv- the increase or decrease in mean scores for the clinical factors ity, particularly in the posterior parts of the mouth.98 The under investigation coupled with the proportion of successful development of digital imaging technology80 brought the cases. This approach precluded direct comparison of their possibility of image manipulation, including digital subtrac- data with studies reporting conventional dichotomized out- tion,50,158,243,259 densitometric analysis,156 correction of gray comes. Other studies using the index dichotomized the scores values,32 and the manipulation of brightness and contrast.75 into “healthy” (PAI 1 or 2) or “diseased” (PAI 3 to 5) catego- However, none of these approaches has addressed the main ries,160 thus allowing the data to be compared directly with deficiencies of accurately elucidating and quantitating the the more traditionally used binary outcomes of success or actual existence or progression of periapical bone loss. failure. In this system of designation, given that the PAI score Cone-beam computed tomography (CBCT), a new three- 2 represents periodontal ligament widening, it effectively dimensional imaging technique requiring only 8% of the effec- signals adoption of the “loose” threshold. The longitudinal tive dose of conventional computed tomography,119 has been follow-up86 of 14 cases presenting with widened apical peri- proposed as a means of overcoming the problem of superim- odontal ligament (PAI score at 2) for 10 years revealed unfa- position of tissue layers and structures. The diagnostic values vorable future healing in only a small proportion of the cases of periapical radiography and cone-beam computed tomogra- (28%, 4/14). phy were compared on dogs’ teeth using histology as the com- parative method of determining apical periodontitis 180 days Outcome Measures for Periapical Surgery after root canal treatment.44 CBCT was found to be significantly Nonsurgical root canal treatment alone may fail to resolve more accurate in detecting minor bone lesions compared with apical periodontitis in a small proportion of cases because two-dimensional radiology.44,174 Other evidence to support the it may not allow access to the infection or resolution of the superior sensitivity of CBCT is available from studies using a cause of the infection (i.e., root fracture or crestal bone 480 PART II The Advanced Science of Endodontics

TABLE 11-2 Criteria for Determination of Periapical Status Strindberg (1956) Bender et al. (1966, a and b) Friedman & Mor (2004) Success Success Healed Clinical: No symptoms Clinical: Absence of pain/swelling; Clinical: Normal presentation Radiographic: The contours, width, and structure disappearance of fistula; no loss of Radiographic: Normal presentation of the periodontal margin were normal function; no evidence of tissue destruction OR Radiographic: An eliminated or arrested area The periodontal contours were widened mainly of rarefaction after a posttreatment around the excess filling interval of 6 months to 2 years Failure Diseased Clinical: Presence of symptoms Radiolucency has emerged or Radiographic: A decrease in the periradicular persisted without change, even rarefaction when the clinical presentation is OR normal Unchanged periradicular rarefaction OR OR Clinical signs or symptoms are An appearance of new rarefaction or an increase present, even if the radiographic in the initial rarefaction presentation is normal Uncertain Healing Radiographic: There were ambiguous or Clinical: Normal presentation technically unsatisfactory control radiographs Radiographic: Reduced radiolucency that could not, for some reason, be repeated OR The tooth was extracted prior to the 3-year follow-up owing to the unsuccessful treatment of another root of the tooth

communication to the periapical infection). Where the cause Indirect Pulp Capping (One-Step versus is microbial persistence, the infection may be localized intra- radicularly (in the apical canal anatomy [Fig. 11-6] or in the Stepwise Excavation) apical dentinal tubules [Fig. 11-7] or extraradicularly [Fig. The most conservative management for extensive caries is indi- 11-8]). In these instances, a surgical approach to the periapex rect pulp capping using either the one-step or the stepwise may be required in addition to the nonsurgical approach to excavation approach. The stepwise excavation approach with removing such microbes (see Fig. 11-7). initial partial caries removal was advocated to reduce the risk The success of periapical surgery has been assessed with the of pulpal exposure and is generally considered to be associated same clinical and radiographic criteria as for nonsurgical root with poorer outcomes. The clinical outcomes of the one- canal treatment. However, the radiographic criteria for success- step or stepwise approaches have been compared in three ful periapical healing are different from those for nonsurgical randomized controlled trials on permanent teeth with root canal treatment140,186 (Table 11-3; Figs. 11-9, 11-10, and deep caries.19,113,121,122 Stepwise excavation was found to be 11-11). Moreover, periodontal attachment loss in the form of associated with a lower rate of pulp exposure and higher marginal gingival recession is an additional criterion for mea- chance of long-term clinical success than the one-step excava- suring the outcome of periapical surgery. tion approach19,113 (Table 11-4). Maltz and colleagues,121,122 Now that the parameters have been discussed as to however, found the contrary, and they attributed the low long- the variables that need to be considered when assessing term clinical success rate of the stepwise approach to patients healing or nonhealing outcomes, the following discussion being noncompliant in returning for scheduled completion of will describe the expected outcomes of the various endodon- treatment. Interestingly, comparison of the cost-effectiveness tic procedures. of the one-step versus the stepwise excavation techniques using health-economic modeling involving simulated treat- OUTCOMES OF VITAL PULP ment of a molar tooth with deep caries in a 15-year-old patient revealed the one-step procedure to accrue lower long-term THERAPY PROCEDURES costs as well as longer tooth retention and pulp vitality.200 The vital pulp therapy procedures discussed in this chapter Meta-analyses of data from studies19,20,65,81,89,113,120-122,141 include those for managing extensive caries with a high risk listed in Table 11-4 revealed that the weighted pooled of pulpal exposure or pulps exposed through caries or trau- success rate for indirect pulp capping using the one-step matic injuries. approach (81.7%; 95% confidence interval [CI]: 72.7%, 90.6%) CHAPTER 11 Evaluation of Outcomes 481

D D D D

BA BA

AC

AC

B C

BA

BA BA

AC GR AC

A D E

FIG. 11-6 Axial sections through the surgically removed apical portion of the root with a therapy-resistant periapical lesion (GR). Note the cluster of bacteria (BA) visible in the root canal. Parts B–E show serial semithin sections taken at varying distances from the section plane of (A) to reveal the emerging (B) and gradually widening (C–E) profiles of an accessory root canal (AC). Note that the accessory canal is clogged with bacteria (BA). Original magnification: A ×52; B–E ×62. (From Nair PN, Sjogren U, Krey G, Sundqvist G: Therapy-resistant foreign body giant cell granuloma at the periapex of a root-filled human tooth, J Endod 16:589, 1990.) 482 PART II The Advanced Science of Endodontics

TABLE 11-3 Classification of Periapical Healing Following Apical Surgery Rud et al. (1972) and Molven et al. (1987) Complete Healing Reformation of a Periodontal Space with Normal width and lamina dura to be followed around the apex Slight increase in width of apical periodontal space but less than twice the width of noninvolved parts of the root Tiny defect in the lamina dura (maximum 1 mm) adjacent to the root filling Complete Bone Repair with A B Bone bordering the apical area does not have the same density as surrounding noninvolved bone No apical periodontal space can be discerned Incomplete Healing (Scar Tissue) The Rarefaction Has Decreased in Size or Remained Stationary with Bone structure recognized within the rarefaction The irregular periphery of the rarefaction and demarcation by a compact bone border The rarefaction located asymmetrically around the apex Angular connection between the rarefaction and the periodontal space An Isolated Scar Tissue in the Bone with Findings Above Uncertain Healing The Rarefaction Has Decreased in Size with The size larger than twice the width of the periodontal space C Lamina-dura like bone structures around the border A circular or semicircular periphery Symmetric location around the apex as a funnel-shaped extension of the periodontal space Bone structure discernible within the bony cavity A collar-shaped increase in width of lamina dura coronal to the radiolucency Unsatisfactory Healing (Failures) The rarefaction has enlarged or is unchanged If a case still demonstrated “uncertain healing” 4 years postoperatively, the treatment should be considered a failure186

D

FIG. 11-7 A, Adequate root filling demonstrated on radiograph. B, View of (Fig. 11-12) was similar to that for the stepwise approach the periapical surgery and of the tooth shown in (A) shows (81.9%; 95% CI: 72.1%, 91.7%) (Fig. 11-13). stained root dentine. C, Resected root showing stained/infected dentine. Calcium hydroxide cement was the preferred lining mate- D, Histologic view of the root end shown in (C) showing infected dentinal rial for the pulpal surface, whereas zinc oxide–eugenol–based tubules (S). cement was the preferred base material in most studies. Recently, resin-modified-glass-ionomer liner has also been used, but the type of lining material did not influence the outcome (see Table 11-4). The age of patients, presence of preoperative pain, and pulpal exposure during excavation were significant negative prognostic factors.19 CHAPTER 11 Evaluation of Outcomes 483

D

AC

AC

CW A B

NG FI NG NG

C

FIG. 11-8 Actinomyces in the body of a human periapical granuloma. The colony (AC in B) is magnified in B. The rectangular area demarcated in B is magnified in C. Note the starburst appearance of the colony with needle-like peripheral filaments surrounded by few layers of neutrophilic granulocytes (NG), some of which contain phagocytosed bacteria. A dividing peripheral filament (FI) is magnified in the inset. Note the typical Gram-positive wall (CW): D = dentine. Original magnification: A ×60; B ×430; C ×1680; inset ×6700. (From Nair PR, Schroeder H: Periapical actinomycosis, J Endod 10:567, 1984.)

tooth location and type; pulp exposure type, size, and its loca- Direct Pulp Capping tion; and the restoration type, size, and quality did not have a Direct pulp capping is performed on teeth with pulp exposures significant influence on success. Although the outcome of caused by caries, caries excavation, or traumatic injuries. Most direct pulp capping of teeth with immature versus mature studies investigating the clinical outcome of direct pulp apices has not been systematically compared in individual capping on permanent teeth had excluded teeth with signs studies, indirect comparison of pooled data from different and symptoms of irreversible pulpitis and apical pathosis studies1 revealed that teeth with immature roots were associ- (Table 11-5). Saline, sodium hypochlorite, and chlorhexidine ated with significantly more successful outcomes. have been reportedly used to irrigate the exposed pulp and to The type of capping material was another significant prog- achieve hemostasis. Calcium hydroxide paste and mineral tri- nostic factor,90,197,207 with MTA performing superiorly to oxide aggregate (MTA) were the commonly used capping mate- calcium hydroxide in a randomized controlled trial90 and in a rials (see Table 11-5). systematic review.1 A meta-analysis of data from studies* listed in Table 11-5 revealed the weighted pooled success rate to be 70.1% (95% Pulpotomy CI: 59.9%, 80.2%) (Fig. 11-14). The patient’s age and sex; Earlier studies on the outcome of partial pulpotomy (specifying varying degrees of coronal pulp removal) had only included teeth with vital traumatic pulp exposures; however, more *Please see the following references: 12, 19, 21, 64, 65, 72, 88, 90, 92, 128, 132, recent studies have also included teeth with carious pulp expo- 133, 193, 207, and 254. sures. Teeth with spontaneous or severe pain and signs and 484 PART II The Advanced Science of Endodontics

A B

C D

FIG. 11-9 A, A maxillary right central incisor having undergone periapical surgery. B, Incomplete periapical healing at 1 year postoperatively. Complete healing at 3 (C) and 4 years (D) postoperatively.

symptoms of apical pathosis were excluded (Table 11-6). On the other hand, the outcomes of full pulpotomies (com- pletely removing the coronal pulp and retaining the radicular pulp) had only been investigated in teeth with carious expo- sures (Table 11-7). Saline irrigation has been preferred for achieving hemostasis, whereas calcium hydroxide and more recently MTA were the preferred pulp capping materials (see Table 11-6). A meta-analysis of data from studies listed in Tables 11-610,11,19,40,71,125,131,177 and 11-76,30,31,52,58,126,187,192,235,251,255 revealed the weighted pooled success rate to be 79.3% (95% CI: 66.7%, 91.8%) for partial pulpotomies (Fig. 11-15) and 82.4% (95% CI: 69.3%, 95.4%) for full pulpotomies (Fig. 11-16). The effects of potential prognostic factors for pulpotomy have not been explored systematically except for the pulp capping material. Randomized controlled trials revealed that MTA achieved similar outcomes in partial177 or full pulpoto- mies58 when compared with calcium hydroxide. FIG. 11-10 Example of incomplete healing with isolated scar tissue. Text continued on p. 495 A B C

FIG. 11-11 Example of uncertain healing. Periapical radiographs of a maxillary left central incisor taken immediately after periapical surgery (A), at 2 years postoperatively (B), and at 3 years postoperatively (C).

Jordan et al. 1971 and 1978

Haskell et al. 1978

Fitzgerald and Heys 1991

Leksell 1996

Gruythuysen et al. 2010 FIG. 11-12 Forest plot showing individual study and weighted pooled Bjorndal et al. 2010 probabilities of success for one-step indirect pulp capping. Maltz et al. 2011

Maltz et al. 2012a and b

Combined 0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 Success_probability

Nagamine 1993

Leksell 1996

Bjorndal and Thylstrup 1998

FIG. 11-13 Forest plot showing individual Bjorndal et al. 2010 study and weighted pooled probabilities of success for stepwise indirect pulp capping. Maltz et al. 2012a and b

Combined

0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 Success_probability 486 PART II The Advanced Science of Endodontics

of for

after have

not

up

vital reduce

success

success was influence not

of

was dressing low did

duration

than completion patients higher followed exposure; exposure exposure CH patients

with

of the effect CH

preoperative capping to

remained stepwise, for

of of evidence

significant

stepwise better

11 were

older pulp no than of no

without without without

yr,

exposure

brand

teeth 11 with pathosis significant duration had on complete; pain, success associated significantly rate regardless treatment; rate attributed returning treatment

Notes 24 The 47/57 42/70 88/94 Stepwise Indirect

and yr

=

mo yr 3

mo)

36 11 given

yr mo, (mean 43 (clinical radiographic outcome) to mo yr mo to yr yr yr

Duration after Treatment 6 Not 6 1 3 1 1 1 3 10 18 PDL PDL PDL of PDL PDL PDL PDL/no

asymptomatic/ intact intact intact perforation, asymptomatic intact resorption asymptomatic/ asymptomatic/ Criteria for Success Vital/ Clinical Asymptomatic Asymptomatic/ Absence Vital/intact Vital/intact Vital/intact Vital/ Vital Survive/ Vital

Life)

+ filling

or sealing CH/GIC GIC/

or Dycal)/

with versus versus ZnPO4 cresatin,

hydraulic CH/Amal

ZOE.

then then then +

or

ZOE/ZOE

visit: CH visit:

(Dycal (Dycal

or amalgam improved ZOE cement tannin-fluoride versus temporary material/GIC composite composite amalgam composite Capping Material CH/GIC 1st 2nd CH, CH CH Polycarboxylate CH/ZOE CH/temporary GIC CH/ZOE GIC/amalgam CH/ZOE pain pain

2/3) 1/2) pulpitis pulpitis pulpitis

pathosis pathosis

> > ( ( pathosis

caries caries caries caries caries caries caries provoked

given spontaneous, apical spontaneous, apical

without without without pain persistent persistent Pretreatment Status Carious Vital Apical Deep Deep Deep Vital Deep Not Deep Deep Only Vital Carious No No Vital Deep Vital Carious No No 18 18 60 46

37 16 given

to to or to to (25-38) younger

6 to to

Age 11 8 14 20 17 6 Not 29 Up ≥ mo) mo) yr) yr) (74.1%) (18 (97.1%) (100%) (100%) (84.8%) (91.3%) (82.5%) (60.0%) (92.6%) (62.4%) (97.1%) (61.5%) (18 (69.3%) (91.7%)

8 (3 (3 48 39 21 47 42 87 93 33 16 87 70 236 106 111 Success (%) 102 8 46 70 94 34 26 48 23 57 149 101 243 112 143 No. of Teeth 1-step Stepwise Stepwise 1-step 1-step 1-step Stepwise 1-step 1-step Stepwise Stepwise Stepwise 1-step Type of Indirect Pulp Capping Stepwise series series series series series series

(multicenter) Case Case CCT Case CCT RCT Case Case Case RCT Study Design RCT

al.,

2011 2012

al., 1993 et

& 1982 al., b)

and 2011) 2011) 2010 & et 1991 al., al., (in

1996

et

1967 et et Hayashi Hayashi

al., al., al., and

1971 Hayashi 2011) 1978 (in et Heys, (in et Thylstrup, 1998 2010 et (a TABLE TABLE 11-4 Studies Investigating the Clinical/Radiographic Outcome of Indirect Pulp Capping of Permanent Teeth Study More, Jordan Sawusch, Fitzgerald Nagamine, Leksell, Bjorndal Bjorndal Gruythuysen Maltz Maltz CHAPTER 11 Evaluation of Outcomes 487

of for

after have

not up

vital reduce success

success was influence

not of was

dressing low did

duration than completion

patients

higher

followed exposure; exposure exposure

CH patients with

of the effect CH

preoperative capping

to remained stepwise,

for

of of evidence significant stepwise better 11 were

older pulp no

than of no without without without yr, exposure brand teeth 11 with pathosis significant duration had on complete; pain, success associated significantly rate regardless treatment; rate attributed returning treatment Notes 24 The 47/57 42/70 88/94 Stepwise Indirect

and

yr = mo yr 3 mo) 36 11 given yr mo, (mean 43 (clinical radiographic outcome) to mo yr mo to yr yr yr

Duration after Treatment 6 Not 6 1 3 1 1 1 3 10 18

PDL PDL PDL

of PDL PDL PDL PDL/no

asymptomatic/ intact intact intact perforation, asymptomatic intact resorption asymptomatic/ asymptomatic/ Criteria for Success Vital/ Clinical Asymptomatic Asymptomatic/ Absence Vital/intact Vital/intact Vital/intact Vital/ Vital Survive/ Vital Life)

+

filling or sealing GIC/ CH/GIC

or Dycal)/ with versus versus ZnPO4 cresatin, hydraulic CH/Amal

ZOE. then then then + or ZOE/ZOE visit: CH visit: (Dycal (Dycal or amalgam improved ZOE cement tannin-fluoride versus temporary material/GIC composite composite amalgam composite Capping Material CH/GIC 1st 2nd CH, CH CH Polycarboxylate CH/ZOE CH/temporary GIC CH/ZOE GIC/amalgam CH/ZOE

pain pain

2/3) 1/2) pulpitis pulpitis pulpitis

pathosis pathosis

> > ( ( pathosis caries caries caries caries caries caries caries provoked

given spontaneous, apical spontaneous, apical without without without pain persistent persistent Pretreatment Status Carious Vital Apical Deep Deep Deep Vital Deep Not Deep Deep Only Vital Carious No No Vital Deep Vital Carious No No 18 18 60 46

37 16 given to to or to to (25-38) younger

6 to to

Age 11 8 14 20 17 6 Not 29 Up ≥

mo) mo)

yr) yr) (97.1%) (74.1%) (18 (100%) (100%) (84.8%) (91.3%) (82.5%) (60.0%) (92.6%) (62.4%) (97.1%) (61.5%) (18 (69.3%) (91.7%)

8 (3 (3 48 39 21 47 42 87 93 33 16 87 70 236 106 111 Success (%) 102 8 46 70 94 34 26 48 23 57 149 101 243 112 143 No. of Teeth 1-step Stepwise Stepwise 1-step 1-step 1-step Stepwise 1-step 1-step Type of Indirect Pulp Capping Stepwise Stepwise Stepwise 1-step Stepwise series series series series series series

(multicenter) Study Design Case Case CCT Case CCT RCT Case Case Case RCT RCT

al., 2011 2012

al., 1993 et

&

1982 al., b) and 2011) 2011) 2010

& et 1991 al., al., (in 1996

et 1967 et et Hayashi Hayashi al., al., al., and

1971 Hayashi 2011) 1978 (in et Heys, (in et Thylstrup, 1998 2010 et (a TABLE TABLE 11-4 Studies Investigating the Clinical/Radiographic Outcome of Indirect Pulp Capping of Permanent Teeth Study More, Jordan Sawusch, Fitzgerald Nagamine, Leksell, Bjorndal Bjorndal Gruythuysen Maltz Maltz 488 PART II The Advanced Science of Endodontics

no

not

and no

site not

higher had lower age survival various

had rates

was effects effects setting had

type

did

by rate materials placement no type, of

had the

rate type

exposure significant

tooth was

paste age 22-year

of

exposure permanent

success tooth

brand

to significant difference in among capping affected and tooth significant survival CH have effect of significant of restoration significantly success

There 5- Success Type Older The Age, Immediate Notes mo yr

yr 10 9 12 yr yr

mo mo

5 yr to yr to yr yr to yr

3 24 > 6 5 12 3 4.5 5 2 1 1 Duration after Treatment root PDL PDL PDL PDL PDL PDL PDL PDL PDL/ PDL Vital/

intact intact intact intact intact intact asymptomatic/ intact intact continuous development bridge/ asymptomatic/ root development/ intact asymptomatic/ asymptomatic/ asymptomatic/ asymptomatic/ asymptomatic/ asymptomatic/ Vital/ Dentine Vital/intact Vital/ Vital/ Vital/ Asymptomatic/ Asymptomatic Asymptomatic Vital/ Vital/ Vital/ Criteria for Success

or or

or gold given/ /

material 4

given not amalgam crown amalgam composite cast restoration ZOE ZOE/amalgam ZOE Base Composite Not GIC ZOE ZOE/GIC Composite ZnPO ZOE/composite Base/ Restorative Material

+

CH or

4 or +

penicillin cresatin

or +

antibiotic, glycerrhetinic acid antibiotics, ZOE, other materials CH CH Corticosteroid CH CH CH CH CH CH/ZnPO MTA CH MTA Capping Material

and CHX

2 NaOCl O 2 2 cotton H O

2 0.2% NaOCl

given given given given chloramine H

or pellets 3% Saline Saline Not Not Not 2% Sterile 10% Not 3% Saline 5.25/6% Hemostasis

or to

its cold with

pain pain mm) pulpitis pulpitis

or during during due during to removal removal removal exposure

or exposure exposure exposure exposure exposure

2.5 PDL other caries sweat provoked to given periapical pain intense periapical

traumatic exposure cavity preparation caries pathosis caries caries, removal or no pathosis caries pain Not Carious Vital Asymptomatic Molar/premolar Carious Asymptomatic Carious Exposure No No Vital Exposure Carious No Exposure Sensitive No Carious Deep Potential Reversible Intact Carious (.25 Reversible Exposure Only Vital Pretreatment Status 67 44 60 69 70 38

12 45 74 9 given given

to to to to to to

to to to to

16 15 8 6 Not 20 20 Not 10 9 7 25 Age (year) (88.0%) (74.7%) (88.0%) (95.1%) (64.7%) (76.4%) (81.2%) (51.7%) (23.6%) (93.3%) (98.0%) (75.0%) (31.8%)

6 7 33 13 36 15 29 28 48 141 115 117 485 Success (%)

8 17 44 29 30 49 22 53 160 133 510 123 154 (1-step) (2-step) No. of Teeth

series series series series series series series series series series Case Case Case Case Case Case Case Case Case Case Study Design RCT RCT

&

al.,

al.,

1966

et &

et al., al., al., al., al., al.,

et 1971 et 1978 Schuman, 1985 et 1985 Heys, 1991 et 1996 1999 et 2000 2006 2008 et 2010 TABLE TABLE 11-5 Studies Investigating the Clinical/Radiographic Outcome of Direct Pulp Capping of Permanent Teeth Study Weiss, Shovelton Haskell Gillien Horsted Fitzgerald Matsuo Santucci, Barthel Farsi Bogen Bjorndal CHAPTER 11 Evaluation of Outcomes 489

no not and no

site not

higher had lower age

survival Continued various had rates was effects effects setting had

type did by rate materials placement no type, of

had the rate type exposure significant tooth was paste age 22-year of exposure permanent success tooth brand to significant difference in among capping affected and tooth significant survival CH have effect of significant of restoration significantly success There 5- Success Type Older The Age, Immediate Notes mo yr

yr

12 10 9 yr yr mo mo

5 yr to yr yr to yr to yr

3 24 > 6 5 12 3 4.5 5 2 1 1 Duration after Treatment

root

PDL

PDL PDL PDL PDL PDL PDL PDL PDL/ PDL Vital/

intact intact intact intact intact intact asymptomatic/ intact intact continuous development bridge/ asymptomatic/ root development/ intact asymptomatic/ asymptomatic/ asymptomatic/ asymptomatic/ asymptomatic/ asymptomatic/ Vital/ Dentine Vital/intact Vital/ Vital/ Vital/ Asymptomatic/ Asymptomatic Asymptomatic Vital/ Vital/ Vital/ Criteria for Success or or

or

gold given/ / material 4 given not amalgam crown amalgam composite cast restoration ZOE ZOE/amalgam ZOE Base Composite Not GIC ZOE ZOE/GIC Composite ZnPO ZOE/composite Base/ Restorative Material

+

CH or

4 or

+ penicillin cresatin or + antibiotic, glycerrhetinic acid antibiotics, ZOE, other materials CH CH Corticosteroid CH CH CH CH CH CH/ZnPO MTA CH MTA Capping Material

and

CHX 2 NaOCl O

2 2 cotton H O

2 0.2% NaOCl given given given given chloramine H

or pellets 3% Saline Saline Not Not Not 2% Sterile 10% Not 3% Saline 5.25/6% Hemostasis

or to

its cold with

pain

pain

mm) pulpitis pulpitis

or during during due during to removal removal removal exposure

or exposure exposure exposure exposure exposure

2.5

PDL other caries

sweat provoked

to given periapical pain intense periapical traumatic exposure cavity preparation caries pathosis caries caries, removal or no pathosis caries pain Not Carious Vital Asymptomatic Molar/premolar Carious Asymptomatic Carious Exposure No No Vital Exposure Carious No Exposure Sensitive No Carious Deep Potential Reversible Intact Carious (.25 Reversible Exposure Only Vital Pretreatment Status 67 44 60 69 70 38

74 9 12 45 given given

to to to to to to to to to to

16 15 8 6 Not 20 20 Not 10 9 7 25 Age (year) (88.0%) (74.7%) (88.0%) (95.1%) (64.7%) (76.4%) (81.2%) (51.7%) (23.6%) (93.3%) (98.0%) (75.0%) (31.8%)

6 7 33 13 36 15 29 28 48 141 115 117 485 Success (%) 8 17 44 29 30 49 22 53 160 133 510 123 154 (1-step) (2-step) No. of Teeth series series series series series series series series series series Study Design Case Case Case Case Case Case Case Case Case Case RCT RCT

& al.,

al.,

1966

et &

et al., al., al., al., al., al.,

et 1971 et 1978 Schuman, 1985 et 1985 Heys, 1991 et 1996 1999 et 2000 2006 2008 et 2010 TABLE TABLE 11-5 Studies Investigating the Clinical/Radiographic Outcome of Direct Pulp Capping of Permanent Teeth Study Weiss, Shovelton Haskell Gillien Horsted Fitzgerald Matsuo Santucci, Barthel Farsi Bogen Bjorndal 490 PART II The Advanced Science of Endodontics

and

and tooth,

the type;

higher

had of not and

on site CH for

rate and and

have associated did tooth success

restoration size, dentist, significant exposure, capping significantly

than MTA

not

was sex;

of

capping immediate placement permanent restoration significantly success location exposure type; type, quality have effects with higher rate pulp pulp characteristics did significant influence results Use Age; MTA Patient, Notes mo mo

18 27

to to

yr

12 12 2 Duration after Treatment

or

or

of

PDL/

pathosis PDL

clinical

canal requiring

resorption/

radiographic evidence apical asymptomatic/ intact no not extraction root treatment Vital/no Vital/ Vital/intact Criteria for Success

crown amalgam

or or GIC/composite GIC/composite GIC Base/ Restorative Material CH

or

MTA MTA MTA CH Capping Material CHX NaOCl

NaOCl

0.12% 2.5% 5.25% Hemostasis

or exposure

mechanical exposure traumatic, mechanical exposure Carious Carious Carious, Pretreatment Status 85

78 90 89

to

to to to

8 21 9 8 Age (year) (80.6%) (70.5%) (45.1%) (64.3%)

86 23 81 116 Success (%) 51 122 126 144 No. of Teeth

series series Case Case Study Design RCT

al., al.,

al.,

et et

et

2010 2010 2013 TABLE11-5 Studies Investigating the Clinical/Radiographic Outcome of Direct Pulp Capping of Permanent Teeth—cont’d Study Mente Miles Hilton CHAPTER 11 Evaluation of Outcomes 491

Weiss 1966 Shovelton et al. 1971 Shovelton et al. 1971 Horsted et al. 1985 Gillien and Schuman 1985 Fitzgerald and Heys 1991 Matsuo et al. 1996 Santucci 1999 Barthel et al. 2000 FIG. 11-14 Forest plot showing Farsi et al. 2006 individual study and weighted pooled Bogen et al. 2008 probabilities of success for direct Bjorndal et al. 2010 pulp capping. Miles et al. 2010 Mente et al. 2010 Hilton et al. 2013 Hilton et al. 2013

Combined 0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 Success_probability

Cvek 1978

Mejare and Cvek 1993

Fuks et al. 1993

Mejare and Cvek 1993

Mass and Zilberman 1993 FIG. 11-15 Forest plot showing individual study and weighted pooled Barrieshi-Nusair and Qudeimat 2006 probabilities of success for partial pulpotomy. Qudeimat et al. 2007

Bjorndal et al. 2010

Combined

0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 Success_probability

Masterton 1966

Russo et al. 1982

Santini 1983

Caliskan 1993

Caliskan 1995

Waly 1995 FIG. 11-16 Forest plot showing indi- Teizeirz et al. 2001 vidual study and weighted pooled prob- abilitiesf o success for full pulpotomy. Witherspoon et al. 2006

DeRosa 2006

El Meligy and Avery 2006

Combined

0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 Success_probability 492 PART II The Advanced Science of Endodontics

in during CH no affect

duration

maturity MTA

not was

exposure, and exposure

of root did outcome significant difference outcome between and with Size There Notes mo teeth mo yr

mo

for yr yr yr 4 60 140

to

2 2 2 to

to to

45.6

to yr to to

14 1 0.5 1 24 1 24.5 1 Duration after Treatment pulpotomy

partial

root

signs PDL of PDL/ PDL/root PDL/root PCL/root PDL/root

versus tissue

symptoms/ intact continued development/ hard barrier bridge/root development/ vital intact development intact development intact development and intact development capping Asymptomatic/ Vital/asymptomatic/ Absence Vital/intact Asymptomatic/ EPT/asymptomatic/ Asymptomatic/vital Asymptomatic/ Criteria for Success pulp

ZOE

direct crown crown crown oxide

visit:

visit:

and Composite cement amalgam or or or ZOE GIC 1st 2nd Zinc GIC/amalgam GIC/amalgam ZOE ZOE/ Base/ Restorative Material

exposure,

CH

hard

CH

visit: without visit:

powder

nonsetting CH setting then set cement CH 1st 2nd CH MTA CH CH CH CH MTA Pulp Dressing Material teeth

for

capping hydroxide solution Saline Calcium Saline Saline Saline Saline Saline Hemostasis material. pulp

in

indirect

dressing mm mm mm

than to mm to mm 4 4

given given given to

pulp 4.0 1 2 diameter; 2 3 deep deep deep to to as N/A 0.5 Not Not Less 2 2 Not Exposure Size stepwise

MTA to

versus

due or wound wound removal

versus of

exposure exposure exposure

during 1-step caries caries provoked

tissue of

apical apical apical exposure caries caries bleeding without signs degeneration exposure pathosis pathosis pulpitis pathosis pain removal hydroxide Traumatic Vital Bleeding Exposure Pulp Traumatic Vital Bleeding Molar Deep Asymptomatic No Carious Asymptomatic No Carious Molar Reversible No Carious Molar Deep Only Vital Expose Pretreatment Status outcomes calcium the 25 13.1 13.3 44 38

*.

given given given to to to

to to

with Not 12 Not 7.5 Not 7.2 6.8 25 Age comparing comparing trial labeled trial those (96.7%) (100%) (79.5%) (91.4%) (93.5%) (75.0%) (91.3%) (92.9%) (34.5%)

(66.7%) controlled 58 26 35 32 29 4 21 21 26 10 Success (%) controlled except series randomized randomized a (2-step) case a (1-step) is is 60 26 31 6 28 No. of Teeth 44 35 28 23 29 were

(2007)

(2010) & al. 1989 1993

al., al. studies &

et 1978 & et

et al., al., al.,

the

removal. et 1993 Zilberman, 1993 Cvek, Nusair Qudeimat, 2006 et 2007 * et 2010 * All TABLE TABLE 11-6 Studies Investigating the Clinical/Radiographic Outcome of Partial Pulpotomy of Permanent Teeth Author Cvek, Baratieri Fuks Mass Mejare Barrieshi- Qudeimat Bjorndal NB: Qudeimat Bjørndal caries CHAPTER 11 Evaluation of Outcomes 493

in during

CH

no

affect duration maturity MTA not was

exposure, and exposure of root did outcome significant difference outcome between and with Size There Notes

mo teeth

mo yr mo for

yr yr yr 4 60 140 to

2 2 2 to to to 45.6 to to to yr

14 1 0.5 1 24 1 24.5 1 Duration after Treatment pulpotomy

partial root signs PDL

of PDL/ PDL/root PDL/root PCL/root PDL/root

versus tissue

symptoms/ intact continued development/ hard barrier bridge/root development/ vital intact development intact development intact development and intact development capping Asymptomatic/ Vital/asymptomatic/ Absence Vital/intact Asymptomatic/ EPT/asymptomatic/ Asymptomatic/vital Asymptomatic/ Criteria for Success

pulp

ZOE direct crown crown crown oxide visit: visit: and Composite cement amalgam or or or

ZOE GIC 1st 2nd Zinc GIC/amalgam GIC/amalgam ZOE ZOE/ Base/ Restorative Material exposure,

CH hard CH visit: without visit: powder nonsetting CH setting then set cement CH 1st 2nd CH MTA CH CH CH CH MTA Pulp Dressing Material teeth

for

capping hydroxide solution Saline Calcium Saline Saline Saline Saline Saline Hemostasis material. pulp

in indirect

dressing

mm mm mm

than to mm to mm 4 4 given given given to

pulp 4.0 1 2 diameter; 2 3 deep deep deep to to

as N/A 0.5 Not Not Less 2 2 Not Exposure Size stepwise

MTA to

versus

due or wound wound removal

versus of

exposure exposure exposure during

1-step caries caries provoked tissue of

apical apical apical exposure caries caries bleeding without signs degeneration exposure pathosis pathosis pulpitis pathosis pain removal hydroxide Traumatic Vital Bleeding Exposure Pulp Traumatic Vital Bleeding Molar Deep Asymptomatic No Carious Asymptomatic No Carious Molar Reversible No Carious Molar Deep Only Vital Expose Pretreatment Status

outcomes

calcium

the 25 13.1 13.3

44 38

*.

given given given to to to to to

with

Not 12 Not 7.5 Not 7.2 6.8 25 Age comparing

comparing

trial labeled

trial

those

(96.7%) (100%) (79.5%) (91.4%) (93.5%) (75.0%) (91.3%) (92.9%) (34.5%)

(66.7%)

controlled

58 26 35 32 29 4 21 21 26 10 Success (%) controlled

except

series

randomized

randomized a

(2-step)

case a

(1-step)

is

is No. of Teeth 60 26 31 6 28 44 35 28 23 29

were

(2007)

(2010)

&

al. 1989

1993

al., al. studies &

et

1978 & et et al., al., al.,

the

removal. et 1993 Zilberman, 1993 Cvek, Nusair Qudeimat, 2006 et 2007* et 2010* All

TABLE TABLE 11-6 Studies Investigating the Clinical/Radiographic Outcome of Partial Pulpotomy of Permanent Teeth Author Cvek, Baratieri Fuks Mass Mejare Barrieshi- Qudeimat Bjorndal NB: Qudeimat Bjørndal caries 494 PART II The Advanced Science of Endodontics

with

was

significant

7.5 medicament

no <

healing

and

had effect associated age Sex Poor Notes mo w mo mo

mo

yr 72 32 88 20

70 4

to to to to

to w mo to yr yr yr

1 8 6 1 16 5 24 14 1 1 13 Duration after Treatment

bridge/ bridge/ resorption/

PDL PDL PDL/root

development/ development

PDL

material. given

dentine intact root vital dentine intact PDL/no root intact development PDL asymptomatic Criteria for Success Vital/asymptomatic Intact Not Asymptomatic/intact Vital/asymptomatic/ Asymptomatic/intact Vital/asymptomatic/ Asymptomatic Vital/bridge/ Vital/asymptomatic/ Asymptomatic/bridge/

dressing

or or

pulp

rest

as

MTA

given given given given

composite composite and

Base/Restorative Base/Restorative Material GIC Amalgam Not Not ZOE ZOE/Amalgam Not ZOE/Amalgam Not Permanent ZOE hydroxide

calcium Ledermix

or +

Capping Material CH CH CH CH CH CH MTA MTA CH CH NEC CH-glutaraldehyde CH CH comparing

trial

wool

given given given NaOCl

pellet controlled

Hemostasis Not Not Cotton Saline Saline Not 6% Saline teeth

or

apical randomized

a pulpitis

or

pulpitis pulpitis pulp

traumatized traumatic

apex

pathosis pathosis was

exposure exposure exposure exposure or or without exposure

pathosis

caries or

given given which apical apical

near exposed pathosis exposure Pretreatment Status Not Carious No Carious Symptomatic Hyperplastic Carious Asymptomatic Apical Carious Molar Deep With Not Carious Immature No Saline Carious Irreversible Carious Irreversible (2006),

22 24 62

39 28 16 12 16 given

Avery

to to to

to to to to to &

Age 6 9 Not 10 10 6 6 7 14 Meligy

El

(83.3%) (93.3%) (51.4%) (91.7%) (92.3%) (90.0%) (82.9%) (65.4%) (86.7%) (100%) (79.0%) (100%)

except

25 28 22 24 18 34 17 13 15 15 12 192 Success (%) series

24 26 20 26 15 15 19 12 30 30 41 373 case

No. of Teeth were

al.,

2009

& 1993 1995 2006 al., 2006

2006

et

1983

studies

&

et

1995

al., the

1966 1982 2001 Avery, et Ehsani, Meligy All

TABLE TABLE 11-7 Studies Investigating the Clinical/Radiographic Outcome of Full Pulpotomy of Permanent Teeth Author Masterson, Russo Santini, Caliskan, Caliskan, Waly, Teizeira DeRosa, El Witherspoon Asgary NB: CHAPTER 11 Evaluation of Outcomes 495

Grahnen and Hansen 1961 Engstrom and Lundberg 1965 Heling and Tamshe 1970 Adenubi and Rule 1976 Jokinen et al. 1978 Barbakow et al. 1980 Pekruhn 1986 Sjogren et al. 1990 Friedman et al. 1995 Cheung 2002 Chugal et al. 2001 Hoskinson et al. 2002 Moshonov et al. 2005 Gesi et al. 2006 Liang et al. 2011 Ng et al. 2011 Ricucci et al. 2011

Combined 0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 VITALSS_pr

FIG. 11-17 Forest plot showing results of pooled and individual study’s probability of maintained periapical health for preoperatively vital teeth undergoing root canal treatment (pooled probability = 0.83; 95% confidence interval: 0.77, 0.89).

A systematic review and meta-analysis of the factors affect- Summary of Prognostic Factors for Vital ing primary root canal treatment outcome conducted by the Pulp Therapy authors revealed the following: the mean success rate was 83% In summary, vital pulp therapy performed under guideline when vital pulpectomy was performed (Fig. 11-17), which standards with optimal coronal seal achieved promising long- reduced to 72% when the root canal treatment procedure was term success in teeth with carious, mechanical, or traumatic used to eradicate the established infection associated with a exposures of healthy pulps. periapical lesion (Fig. 11-18). The most important factors affecting the outcome of vital pulp therapy are preexisting health of the pulp, adequate removal of infected hard or soft tissues, careful operative tech- Factors Affecting Periapical Health or nique to avoid damage to residual tissues, and elimination of Healing Following Root Canal Treatment microbial leakage around the final restoration. It can be diffi- The factors influencing the maintenance of periapical health cult to gauge the health of the residual pulp as it is a matter or periapical healing of preexisting lesions following root canal of subjective assessment and relies on experience in pulp diag- treatment may be broadly classified into patient factors (age, nosis. The degree of pulp bleeding upon exposure is a more sex, general health, tooth anatomy, preoperative pulpal and reliable tool to judge the status of the pulp than the preopera- periapical status), treatment factors (operator variables, canal tive clinical signs and symptoms. Continued bleeding after 10 enlargement, irrigation, medication, culture test, and obtura- minutes, even after rinsing with sodium hypochlorite solution, tion), and restorative factors. Some factors had a profound may suggest that the residual pulp was still heavily inflamed impact on success rates, whereas others showed a negligible and a complete pulpectomy may be a more effective treatment effect. Patient factors characterizing the nature of the disease modality. Removal of infected tissue is a matter of subjective showed the most significant effect (periapical status), whereas experience but may be aided by various dyes. The final factor most of the treatment factors were found to exert a less signifi- relies on the correct choice of restorative material and its ade- cant effect; the exceptions were the apical extent of root canal quate manipulation to prevent leakage. treatment relative to the root canal terminus. In addition, the Factors such as age and health of the patient, size and quality of the postoperative restorative care also exerted a nature (carious or traumatic) of pulp exposure, and its dura- profound influence on outcome of treatment. tion of exposure to the oral environment (up to 48 hours) do not in themselves compromise outcomes of vital pulp therapy. Patient Factors Patient’s age and sex consistently had no significant effect on OUTCOMES OF NONSURGICAL ROOT outcome, whereas some specific health conditions (diabe- tes,54,67 compromised immune response123) apparently had a CANAL TREATMENT significant influence. The evidence for the effect of host In contrast to other areas of endodontics, the number of studies immune response characterized by the general health of the and extent of investigation of nonsurgical root canal treatment patient is, however, weak. Emerging evidence indicates that the is more comprehensive, yielding a much greater insight even host response measured by polymorphisms of various genes though the quality and scope of the research does not always involved in periapical healing may have an effect on out- reach the highest levels. comes142,183,212,213 (Table 11-8). The importance of the host 496 PART II The Advanced Science of Endodontics

Auerbach 1938 Buchbinder 1941 Castagnola and Orlay 1952 Heling and Tamshe 1970 Cvek 1972 Werts 1975 Adenubi and Rule 1976 Heling and Shapira 1978 Jokinen et al. 1978 Barbakow et al. 1980 Cvek et al. 1982 Boggia 1983 Pekruhn 1986 Bystrom et al. 1987 Halse and Molven 1987 Safavi et al. 1987 Akerblom and Hasselgren 1988 Sjogren et al. 1990 Murphy et al. 1991 Friedman et al. 1995 Calisken and Sen 1996 Peretz et al. 1997 Sjogren et al. 1997 Lilly et al. 1998 Weiger et al. 2000 Cheung 2002 Hoskinson et al. 2002 Peters and Wesselink 2002 Chugal et al. 2003 Huumonen et al. 2003 Khedmat 2004 Chu et al. 2005 Aqrabawi 2006 Doyle et al. 2006 Conner et al. 2007 Molander et al. 2007 Sari and Duruturk 2007 Chevigny et al. 2008 Cotton et al. 2008 Penesis et al. 2008 Siqueira et al. 2008 Hsiao et al. 2009 Mente et al. 2009 Tervit et al. 2009 Ng et al. 2011 Ricucci et al. 2011 Liang et al. 2012 Combined

0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 NVPASS_pr

FIG. 11-18 Forest plot showing results of pooled and individual study’s probability of periapical health for teeth associated with nonvital pulps and periapical radiolucencies undergoing root canal treatment (pooled probability = 0.72; 95% confidence interval: 0.67, 0.78).

TABLE 11-8 Type of Genes Investigated in Studies on SNPs and Periapical Healing Study Gene Function Genes Findings Siqueira et al., 2009 Interleukin 1 IL-1α, IL-1β No significant association Fcγ receptor FcγRIIa, FcγRIIIb Significant association Morsani et al., 2011 Interleukin IL-1β Significant association Siqueira et al., 2011 Fcγ receptor FcγRIIIa No significant association Rôças et al., 2014 Pattern recognition receptors CD14, TLR4 No significant association

response to maintenance of periapical health or periapical role than other complexities such as the number and curvature healing was also supported by the statistically significant clus- of canals. tering effect of multiple teeth within the same patient in a The presence and size of a periapical lesion seem to have prospective study.150 the most negative effect on periapical health/healing; it there- The widespread perception that single-rooted teeth with fore follows that these factors must be accounted for when less complicated anatomy should benefit with more predictable analyzing the influence of any other factor. The profound and favorable outcomes proves to be untrue. Having accounted change in success rates once a periapical lesion becomes estab- for potential confounding factors such as the presence of peri- lished is interesting, as it is correlated with the establishment apical disease, tooth type does not seem to exert a strong influ- of infection in the apical canal anatomy. This seems to suggest ence on success rates. This would appear to be counterintuitive that once the apical canal complexities become infected, it may but may be explained by the logical inference that canal com- be much more difficult to eradicate the infection. The negative plexities in the apical anatomy probably play a more dominant influence of large periapical lesions has a reasonable biologic CHAPTER 11 Evaluation of Outcomes 497 explanation: the diversity of bacteria (by number of species on medicolegal implications of root canal instrument ingestion and their relative abundance) is greater in teeth with larger or aspiration by the patient.61 periapical lesions.230 An endodontic infection is more likely to persist in canals with a higher number of bacteria present Magnification and Illumination preoperatively.27 In addition, larger lesions may represent Endodontists have repeatedly reinforced the value of magnifi- longer-standing root canal infections that may have penetrated cation and illumination during root canal treatment,166 but a deeper into dentinal tubules and accessory anatomy in the systematic review failed to draw any objective conclusions complex canal system206 where mechanical and chemical on their influence as no article was identified in the current decontamination procedures may not be so effective. Larger literature that satisfied their inclusion criteria.46 A prospective lesions may also represent cystic transformation.145 Finally, the study investigated this factor,150 but researchers found only an host response may also play a part, as patients with larger insignificant influence on the final outcome. Use of a micro- lesions may innately respond less favorably to residual bacte- scope may sometimes assist location of the second mesiobuccal ria.147 This speculation may crystallize into distinct questions canal in maxillary molars, but this only made a small difference for further biologic research into the nature of interactions to the success rates associated with mesiobuccal roots, when a among host, bacterial infection, and treatment intervention. periapical lesion was present.150 The true benefit of a micro- Most of the other investigated preoperative factors (pain, scope can only be verified through a randomized controlled tooth tenderness to percussion, soft tissue tenderness to palpa- trial. However, canal negotiation with less tooth structure tion, soft tissue swelling and sinus tract, periodontal probing removal and fewer procedural accidents is favorable and seems defect of endodontic origin, root resorption) are in fact differ- intuitively more consistent with the use of superior magnifica- ent clinical manifestations of periapical disease.254 They may tion and illumination. therefore act as surrogate measures or complement “presence and size of periapical lesion” in measuring the effect of severity Mechanical Preparation: Size, Taper, Extent, of periapical disease within a broad continuous spectrum. and Procedural Errors Of these, only presence of preoperative pain,68 sinus tract,150 The root canal system may be mechanically prepared to a req- swelling,150 and apical resorption229 have been found to be uisite size and taper199 using a variety of instruments of differ- significant prognostic factors that have been associated with ent cutting designs, tips, tapers, and materials of construction. significantly reduced success rates in root canal treatment. Their efficacy is often tested in laboratory studies, and the The biologic explanation for the negative impact of sinus instruments and their utility may have well-characterized tract and swelling, either in the acute or chronic form, on properties.95 Investigation of the influence of type of instru- periapical healing is interesting, as both represent suppuration ment used for canal enlargement has been undertaken in one and the proliferation of microbiota into the periapical tissues, nonrandomized prospective study, but the outcome is likely with the inference being that the host tissues must have subjective because of many factors, including the protocol become locally overwhelmed. The precise reasons for reduced adopted for teaching technical skills.150 In this study, the better success rates under these conditions remain unclear but success rates for hand or rotary NiTi instruments compared must somehow be related to the nature of the host-microbial with stainless steel instruments150 were attributable to the fact interaction. that tactile skills training was achieved through a preliminary focus on the use of stainless steel files to develop tactile sensi- Treatment Factors tivity and consistency. Only on demonstration of this compe- Operator tency did the trainees progress to NiTi instruments. More Although the impact of operator qualification and skill has not important, such senior students may also have had a better been specifically investigated, systematic review has shown understanding of the biologic rationale for root canal treat- that the involved clinicians may be grouped into undergradu- ment. The ability to gain and maintain apical patency as well ate students, general practitioners, postgraduate students, and as to avoid procedural errors was better instilled in the senior specialists. Studies show a clear trend in superior outcomes by students, whereas in selected cases, NiTi instruments appear greater experience and training. Clearly technical skills play an capable of achieving the same in primary root canal treatment important role, but this is difficult and often impossible to undertaken by undergraduates.175 quantify. In addition, the technical abilities must be augmented A key tenet of the European Society of Endodontology by the overall understanding of the biologic issues and the (ESE)61 guidelines is that root canal debridement must be quest for superior treatment by the operator. If clinicians do extended to the terminus of the canal system, which is expressed not feel they can do optimal treatment, it is incumbent upon variously as extension to the “apical constriction,” or to “0.5 to them to refer the patient to a more qualified practitioner. 2 mm from the radiographic apex,” or to the “cementodentinal junction.” This guideline is broadly supported by the fact that Isolation outcome of treatment is compromised by canal obstruction or The use of rubber dams in modern root canal treatment is failure to achieve patency to the canal terminus.150,216,229 Ng and widely accepted, and the justification seems almost empirical. colleagues150 reported a twofold reduction in the success of One study on retreatment244 analyzed the influence of rubber treatment when the patency to the canal terminus was not dam use compared to cotton roll isolation and found signifi- achieved. It could be speculated that the lack of mechanical cantly higher success rates with the former approach. Another negotiability of canals may be due to the presence of obstruc- study reported a significantly higher success rate of root canal– tions caused by “denticles,” tertiary dentine, acute branching treated teeth76 when a rubber dam was employed during post or a fine plexus of apical canals, or dentine/organic debris. placement compared to when it was not. Perhaps as a conse- The continued debate on the optimal size of apical prepara- quence, the principal justification for rubber dam use is based tion remains topical in the absence of definitive evidence; the 498 PART II The Advanced Science of Endodontics

findings from relevant in vitro and clinical studies have been could be influenced by the initial size of the canal, the type of previously reviewed.15 So far, four clinical outcome studies instrument used, and operator experience. have considered this issue or have systematically investigated Triangulation of the data on the effects of canal preparation the effect of apical size of canal preparation on treatment size and taper on treatment outcome may intuitively lead to outcome.93,102,150,188,223,229 One randomized controlled trial the conclusion that as far as current best evidence indicates, it revealed that enlargement of the canal to three sizes larger is not necessary to over-enlarge the canal to achieve periapical than the first apical binding file was adequate188 (the mean healing. An apical preparation size of ISO 30 with a .05 taper final size was ISO #30). The observational studies95,150,229 had for stainless steel instrumentation or .06 taper for NiTi instru- not designed their investigation with apical canal size as their mentation is sufficient. Precisely what biologic and hydrody- principal focus and neither had they found a statistically sig- namic mechanisms underpin such sufficiency is more difficult nificant influence from this factor; nevertheless, they all to define based on available evidence. Although a number reported the same inverse trend of decreasing success rates of laboratory studies4,84,112 have investigated the interaction with an increase in size of apical preparation. It was specu- between canal dimensions and irrigation or obturation dynam- lated that canal preparation to larger apical sizes may compro- ics, the precise physical, chemical, or biologic mechanisms that mise treatment success by generation of more apical dentine ultimately enable periapical healing remain unknown, although debris, which in the absence of an adequate irrigation regimen collaborations with fluid dynamics specialists84 and (micro) serves to block apical canal exits that may still be contami- biologists82 may ultimately yield a clearer picture. nated with bacteria. Continued generation of dentine debris, Procedural errors during root canal preparation include in the absence of sufficient irrigation, may lead to what is canal blockage, ledge formation, apical zipping and transporta- termed dentine mud, which ultimately creates a blockage. The tion, straightening of canal curvature, tooth or root perforation impatient or neophyte clinician fails to resist the temptation at the pulp chamber or radicular level, and separation of instru- to force the instrument back to length, resulting in the classi- ments. Instrument separation during treatment has been found cally described procedural errors of apical transportation, to reduce the success rate significantly150,229; however, the canal straightening, and perforation. An alternative mecha- reported prevalence of instrument separation was low (0.5% nism is required to explain the higher failures in initially large to 0.9%) in these studies, precluding an analysis of causative canals; it is likely that immature roots present a different factors. A case-control study225 revealed no significant differ- debridement challenge, where the canal shape is not amenable ence in success rates between periapically involved teeth with to planing of the main portions of the canal by conventional or without retained separated instruments. The stage of canal instruments. Perhaps an intracanal brush may be a more suit- debridement at which instrument separation occurred and the able cleaning device in such teeth. The findings from these justification for their retention may have implications on the studies therefore do not concur with views that more effective outcome. The coronoapical location of a separated instrument bacterial debridement may be achieved with larger apical and whether the instrument was successfully bypassed were preparations.33,164,184 found to have no effect on treatment outcome. The issue of apical preparation size should be considered together with that of the size and taper of the rest of the canal Irrigant preparation. Again, there is a paucity of sufficient direct evi- Different chemical agents have been used as irrigants for root dence for the influence of degree of canal taper on root canal canal treatment, singly or in various combinations, both in treatment outcome. The ESE guidelines63 recommend only that clinical practice and in the studies reviewed. They have canal preparation should be tapered from crown to apex included water, saline and solutions of , sodium without stipulating any particular degree of taper. Three studies hypochlorite, iodine, chloramine, sulfuric acid, EDTA, hydro- have analyzed the influence of canal preparation taper on gen peroxide, organic acid, Savlon, urea peroxide, and Biosept primary treatment and retreatment outcome, although again, (a quaternary ammonium compound).153 Most of the studies none had focused their investigation primarily on this had used sodium hypochlorite as an irrigant153 regardless of factor.93,150,220 Smith and coworkers,220 using loose criteria for whether it was primary treatment or retreatment. This is con- determination of success, found that a “flared” preparation sistent with the ESE guidelines61 for irrigation, which recom- (wide taper) resulted in a significantly higher success rate mend a solution possessing disinfectant and tissue-dissolving compared with a “conical” preparation (narrow taper); the properties. exact degree of taper was not reported and the effects of other One prospective study150 systematically investigated the treatment and nontreatment parameters were not controlled. effect of the irrigant on the success rates of root canal retreat- In contrast, Hoskinson and associates93 and Ng and col- ment, which, although not a randomized controlled trial, leagues,150 using strict criteria, did not find any significant revealed interesting new findings on the effects of irrigants. difference in treatment outcome between narrow (.05) and Even though a higher concentration of sodium hypochlorite wide (.10) canal tapers. The controlled use of stainless steel made negligible difference to treatment outcome, the additional instruments in a step-back technique may create .05 (1 mm use of other specific irrigants had a significant influence on step-back) or .10 (0.5 mm step-back) tapers, although, of success rates.150 The finding of a lack of improvement in peri- course, uncontrolled use of such instruments may generate a apical healing with the use of a higher concentration NaOCl variety of shapes. Ng and colleagues150 also compared these solution is consistent with previous clinical/microbiologic find- (.05 and .10) preparation tapers with .02, .04, .06, and .08 ings.29,41 Comparing 0.5% to 5.0% NaOCl solution for irriga- tapers (generally achieved by using greater taper nickel- tion, it was found that the concentration of solution, per se, did titanium instruments) and found no significant effect on treat- not appear to increase the proportion of teeth rendered culture- ment outcome. They cautioned that their investigation of the negative29 or associated with greater periapical healing.41 As influence of canal preparation taper without randomization iodine and sodium hypochlorite are both halogen-releasing CHAPTER 11 Evaluation of Outcomes 499 agents and attack common key protein groups,129 the finding The use of a mixture of calcium hydroxide and chlorhexi- that the additional use of 10% povidone-iodine for irrigation dine has been tested based on the speculation that the mixture had no additional influence on treatment success was as would be more effective against E. faecalis.13,78,198 expected. Surprisingly, however, the additional use of 0.2% chlorhexidine solution for irrigation was found to reduce the Root Canal Bacterial Culture Results Prior success of treatment significantly.150 This finding was in com- to Obturation plete contrast to previous reports211,252 on its equivalent or In the past, in various centers of endodontic excellence, com- superior in vivo antibacterial efficacy when compared with pletion of root canal treatment by obturation would only be sodium hypochlorite solution. The use of chlorhexidine as a acceptable after a negative culture test was obtained from the final irrigant following sodium hypochlorite irrigation had canal, confirming the absence of bacteria in the part of the root been recommended107 and was justified on several grounds, canal system that could be sampled.23,70,143 This practice has including its substantivity in root dentin (i.e., prolonged anti- fallen out of clinical favor because of the perceived predict- bacterial effect),185 relative lack of toxicity,117 and broad- ability and good prognosis of root canal treatment without spectrum efficacy.130 Not until recently has alternate irrigation microbiologic sampling. Sampling procedures are considered with sodium hypochlorite and chlorhexidine solution raised lengthy, difficult, often inaccurate, requiring laboratory support serious concerns because of their interaction product. The and having low benefit-to-cost ratio.138,139 A preobturation interaction product is thought to be an insoluble precipitate negative culture result may increase treatment success containing para-chloroaniline, which is cytotoxic and carcino- twofold (Fig. 11-19). One large study201 helped contribute to genic.14,24 Apart from mutually depleting the active moiety in the demise of the canal-culture test; however, even this study the two solutions for bacterial inactivation, the precipitate may showed a 10% difference in success in favor of the negative cause persistent irritation to the periapical tissue and block culture test when periapical disease was present. The outcome dentinal tubules and accessory anatomy, possibly explaining is even worse when a positive culture test result combines with the observed lower success rate when chlorhexidine was used the presence of a periapical lesion. as an additional irrigant. Numerous studies* have evaluated the effect of different Ng and associates150 also found that the additional use of stages of root canal treatment on the intraradicular microbiota, EDTA had a profound effect on improving radiographically both qualitatively and quantitatively (Table 11-9). Some studies observed periapical healing associated with root canal treat- merely report positive culture tests, whereas others have iden- ment (OR = 1.5 [1.1, 2.0]). In contrast, the observed synergis- tified and quantified intraradicular microbiota before and after tic effect of sodium hypochlorite and EDTA had been previously various stages of treatment. demonstrated in terms of bacterial load reduction26 but not The effect of the “mechanical preparation” of the canal(s) periapical healing. The long-term (≥ 2 years) outcome of their on the microbiota has been tested using only water or saline cases stratified by canal disinfection protocols25 did not support as the irrigant. Taken collectively, the studies show that nega- their microbiologic findings. Their reported success rate for tive cultures were achieved on a weighted pooled average in alternate irrigation with sodium hypochlorite and EDTA solu- 31% of the cases (range 0 to 79%). When sodium hypochlorite tions (67%) was low when compared to the success rate for (concentration range 0.5% to 5%) irrigation supplemented the irrigation using saline (91%), 0.5% sodium hypochlorite “mechanical preparation,” the frequency of negative cultures (92%), or 5% sodium hypochlorite (86%) solutions.162 The immediately increased to a weighted pooled average of 52% reported outcome data were unexpected, as preobturation (range 13% to 95%) (see Table 11-9). negative bacterial culture was achieved in all cases. Given the Most studies report culture reversals during the interap- complexity of their study design (clinical and microbiologic), pointment period when active antibacterial dressing is not used their sample size was restricted to 11 to 15 teeth per group, in the root canal system between appointments. The reversals limiting their outcome data. The synergistic effect of the two are due to regrowth of residual bacteria or recontamination by disinfectants has been attributed to the chelating properties of bacteria from coronal restoration leakage. When active interap- the sodium salts of EDTA, and their roles have been reviewed pointment antibacterial dressing is used, negative cultures in by Zehnder.261 EDTA solution assists negotiation of narrow or the subsequent visit were achieved on average in 71% of cases sclerosed canals by demineralization of root dentine and helps (range 25% to 100%) (see Table 11-9). in the removal of compacted debris from noninstrumented canal anatomy. It may also facilitate deeper penetration of Effect of Persistent Bacteria on Root Canal sodium hypochlorite solution into dentine by opening dentinal Treatment Outcome tubules and removing the smear layer from the instrumented The bacteria present in preobturation cultures have included surface. Lastly, it may help detach or break up biofilms adher- Enterococcus, Streptococcus, Staphylococcus, Lactobacillus, Veil- ing to root canal walls.85 lonella, Pseudomonas, Fusobacterium species, and yeasts. Studies have been variable as to the relationship between individual Medicament species and treatment failure. Although the overall failure rate Most previous treatment outcome studies have not standard- for cases with positive cultures was 31%, teeth testing positive ized the type of root canal medicament used in the interap- for Enterococcus species had a failure rate of 55%, and teeth pointment period, but the use of several different medicaments with positive cultures for Streptococcus species had 90% has been reported. The list was consistent with that recom- Text continued on p. 504 mended in the ESE guidelines for a medicament with disinfec- tant properties and included calcium hydroxide, creosote, and *Please see the following references: 2, 7, 10, 16, 26-29, 33, 37, 41, 42, 60, 77, 79, 153 iodine solutions. However, there is an absence of studies 99, 104, 108, 110, 124, 137, 154, 161, 163, 168, 169, 173, 179, 181, 208, 209, investigating the influence of this factor on treatment outcome. 210, 214, 215, 217, 227, 248, 252, 257, and 258. 500 PART II The Advanced Science of Endodontics

Rhein et al. (1926)

Appleton (1932)

Buchbinder (1941)

Forstell (1963)

Seltzer et al. (1963)

Bender et al. (1964)

Engstrom et al. (1964)

Engstrom and Lundberg (1965)

Oliet and Sorin (1969)

Storms (1969)

Heling and Shapira (1978)

Matsumoto et al. (1987)

Sjogren et al. (1997)

Peters and Wesselink (2002)

Molander et al. (2007)

Combined

.022142 20 40 60 Logor_negvspos_culture

FIG. 11-19 Forest plot showing pooled and individual study’s odds ratio (OR) for periapical health of teeth undergoing root canal treatment with preobturation negative versus positive culture test results (pooled OR = 2.1; 95% CI: 1.5, 2.9).

TABLE 11-9 Summary of Studies Evaluating the Effect of Root Canal Treatment Procedures on Bacterial Presence by Culture Percentage of Samples with Bacterial Presence Study Year Sample Size At Baseline After Preparation ± Irrigation Next Visit (after Dressing+/−) Auerbach 1953 60 teeth 93% (56/60) Chlorinated soda (double strength): — 22% (12/56)

Ingle & Zeldow 1958 89 teeth 73% (65/89) H2O: 70% (62/89) — Some initially −ve became +ve after treatment Stewart et al. 1961 77 teeth 100% (77/77) 0.5% NaOCl + Gly-oxide: 2% (1/44) No dressing: 0.5% NaOCl + 3% H2O2: 9% (3/33) 0.5% NaOCl + Gly-oxide: 34% (15/44)

0.5% NaOCl + 3% H2O2: 39% (17/33) Nicholls 1962 155 teeth 100% (155/155) Alkaline chloramine: 53% (39/74) —

H2O2 and 2% NaOCl: 50% (30/60)

H2O and 2% NaOCl: 71% (15/21) Grahnén & Krasse 1963 97 teeth 77% (75/97) NaCl: 72% (23/32) No dressing: Biocept: 66% (21/32) NaCl: 47% (15/32) Nebacin: 36% (12/33) Biocept: 47% (15/32) Some initially −ve became +ve Nebacin: 18% (6/33) after Tx CHAPTER 11 Evaluation of Outcomes 501

TABLE 11-9 Summary of Studies Evaluating the Effect of Root Canal Treatment Procedures on Bacterial Presence by Culture—cont’d Percentage of Samples with Bacterial Presence Study Year Sample Size At Baseline After Preparation ± Irrigation Next Visit (after Dressing+/−)

Engström 1964 223 teeth 60% (134/223) Biosept or Iodophor, plus alcohol, 5% I2 in 10% IKI: 2nd visit: 43% (untreated or chloroform, and 0.5% NaOCl: No (58/134); 3rd visit: 22% retreated) data (29/134); 4th visit: 8% (9/134); 5th visit: 3% (4/134); 6th visit: 2% (3/134); 7th visit: 16% (22/134)

Olgart 1969 207 teeth 72% (149/207) H2O2 and 0.5% NaOCl or H2O2 and No dressing: 34% (70/207) 1% NaOCl: 43% (88/207) Bence et al. 1973 33 teeth 100% (33/33) Pre-irritation: No dressing: 1st file: 93%, enlargement with #3: 8% dentine, 12% pp samples of 14%, #4: 11%, #5: 21% (32% of teeth with negative culture instruments showed +ve culture, after irrigation regardless of size) 5.25% NaOCl: 48-hr culture: 4% dentine, 10% pp 5-day culture: 8% dentine, 26% pp Akpata 1976 20 extracted 100% (20/20) NaCl: 65% (13/20) 38% CMCP: 20% (2/10) teeth When PP sample −ve, crushed tooth yielded −ve culture; When PP +ve, crushed teeth yielded +ve or −ve cultures Cvek et al. 1976 108 teeth NaCl group: 53% NaCl: 83% (15/18) — (18/34) 0.5% NaOCl: 59% (17/29) 0.5% NaOCl 5% NaOCl: 68% (15/22) group: 63% (29/46) 5% NaOCl group: 79% (22/28) Byström & 1981 15 teeth 100% (15/15) Saline: 100% (15/15) No dressing: 47% (7/15) Sundqvist (5th visit) Where initial bacteria load high, difficult to eliminate Byström & 1983 15 teeth 100% (15/15) 0.5% NaOCl: 87% (13/15) No dressing: 20% (3/15) Sundqvist (5th visit) Byström & 1985 60 teeth 100% (60/60) 0.5% NaOCl: No data No dressing: Sundqvist 5% NaOCl: No data 0.5% NaOCl: 12/20 (2nd visit); 5% NaOCl + 15% EDTA: No data 8/20 (3rd visit) 5% NaOCl: 10/20 (2nd visit); 6/20 (3rd visit) 5% NaOCl + 15% EDTA: 11/20 (2nd visit); 3/20 (3rd visit) Byström et al. 1985 65 teeth 100% (65/65) 0.5% NaOCl: No data CH: 0/35 (1 month), 1/35 5.0% NaOCl: No data (2-4 days) CP/CMCP (2 wks): 10/30 Sjögren & 1987 31 teeth 100% (31/31) 0.5% NaOCl plus ultrasonic No dressing: 29% (9/31) at 2nd Sundqvist debridement: No data visit; 23% (7/31) at 3rd visit Continued 502 PART II The Advanced Science of Endodontics

TABLE 11-9 Summary of Studies Evaluating the Effect of Root Canal Treatment Procedures on Bacterial Presence by Culture—cont’d Percentage of Samples with Bacterial Presence Study Year Sample Size At Baseline After Preparation ± Irrigation Next Visit (after Dressing+/−)

Koontongkaew 1988 15 teeth 100% (15/15) 3% H2O2/5.25% NaOCl: No data CMCP: 1-day dressing: 40% et al. (2/5); 3-day dressing: 20% (1/5); 7-day dressing 10% (1/10) No dressing: 60% (3/5) after 1 day, 20% (1/5) after 3 or 7 days Reit & Dahlén 1988 35 teeth 91% (32/35) 0.5% NaOCl: No data CH: After 14 days: 23% (8/35); after 21 days: 26% (9/35) Molander et al. 1990 25 teeth 96% (24/25) 0.04% iodine: No data Clindamycin: After 14 days: 16% (4/25); after 21 days: 24% (6/25) Sjögren et al. 1991 30 teeth 100% (30/30) 0.5% NaOCl: 50% (15/30) CH: 10 min: 50% (6/12) at 1 wk later 7 day: 0% (0/18) (none after 1-5 wks later without dressing) Ørstavik et al. 1991 23 teeth 96% (22/23) NaCl irrigation and enlarged to: CH: 34% (8/23); #20-25: 87% (20/23) #35/40: 40% (6/15) further to #35-80: No data #>40: 25% (2/8) Yared & Bou 1994 60 teeth 100% (60/60) 1% NaOCl: CH: 0% (0/60) Dagher Enlarged to #25: 73% (22/30) Enlarged to #40: 23% (7/30) Gomes et al.. 1996 42 root canals: 95% (40/42) 2.5% NaOCl: No data Empty canal (7-10 days): 73% Untreated (29/40) (n = 15) Retreated (n = 27) Sjögren et al. 1997 55 teeth (single 100% (55/55) 0.5% NaOCl: 40% (22/55) — canal) Dalton et al. 1998 46 teeth 100% (46/46) NaCl + NiTi files: 68% (15/22); — NaCl + K-files: 75% (18/24) Reit et al. 1999 50 teeth 84% (42/50) Enlarged to #35 (curved) or 5% IKI (5-7 days): 44% (22/50) #50 (straight) with 0.5% NaOCl: No Empty (7 days): 44% (22/50) data Peciuliene et al. 2000 25 teeth 80% (20/25); 2.5% NaOCl and 17% EDTA: No data Medication unknown: 28% (7/25) Shuping et al. 2000 42 teeth 98% (41/42) 1.25% NaOCl: 38% (16/42) CH: 8% (3/40) Lana et al. 2001 31 teeth 87% (27/31) 2.5% NaOCl: No data CH: 13% (4/31) Empty for 7 days: 23% (7/31) Peciuliene et al. 2001 40 teeth 83% (33/40) 2.5% NaOCl and 17% EDTA: 30% CH (10-14 days): 25% (5/20)

(10/33) IKI: 2% I2 in 4% KI (10 min): 5% (1/20) Peters et al. 2002 42 teeth Instrumentation to Enlarged to #35 with 2% NaOCl: CH (4 weeks): 71% (15/21); #20: 100% 23% (10/42) further irrigation: 43% (9/21) (42/42) CHAPTER 11 Evaluation of Outcomes 503

TABLE 11-9 Summary of Studies Evaluating the Effect of Root Canal Treatment Procedures on Bacterial Presence by Culture—cont’d Percentage of Samples with Bacterial Presence Study Year Sample Size At Baseline After Preparation ± Irrigation Next Visit (after Dressing+/−) Card et al. 2002 40 mandibular 95% (38/40) 1% NaOCl No data teeth/canals Profile instrumentation (.04 taper): 0/13 of cuspids and bicuspids, 5/27 of mesiobuccal canals Further LightSpeed instrumentation to size 57.5-65: 3/27 mesiobuccal canals of molars Only 1/16 of those mesiobuccal canals with detectable communication with the mesiolingual canals had +ve culture after the first preparation using ProFile instruments Kvist et al. 2004 96 teeth 98% (94/96) 0.5% NaOCl: 63% (60/96) CH (7 days): 36% (16/44) IPI (10 min): 29% (15/52) Chu et al. 2006 88 canals 99% (87/88) 0.5% NaOCl: No data CH, Septomixine forte, or Ledermix: 36% (32/88) Exposure of pulp, tooth type, acute versus chronic condition, size of lesion, and type of medication had no significant effect Paquette et al. 2007 22 teeth (single 100% (22/22) 2.5% NaOCl: 68% (15/22) 2% CHX: 45% (10/22) canal) Siqueira et al. 2007a 11 teeth (single 100% (11/11) 2.5% NaOCl: 55% (6/11) CH/CPMC: 9% (1/11) root) Siqueira et al. 2007b 11 teeth (single 100% (11/11) 2.5% NaOCl: 45% (5/11) CH: 18% (2/11) root) Vianna et al. 2007 24 teeth (single 100% (24/24) Saline + 2% CHX gel: 33% (8/24) 2% CHX, CH or mixture: 54% root) (13/24) Type of medication had no significant effect Wang et al. 2007 43 canals 91% (39/43) Saline + 2% CHX gel: 8% (4/39) 2% CHX + CH: 8% (3/36) Size of apical preparation (40 versus 60) had no significant effect. Markvart et al. 2012 24 teeth 88% (21/24) 2.5% NaOCl: 63% (15/24) 17% EDTA irrigation and 10 min 5% IKI medication: 50% (12/24) Box preparation (#60): 67% (8/12) Cone preparation (#25-30): 33% (4/12) Xavier et al. 2013 48 teeth (single 100% (40/40) 1% NaOCl: 75% (9/12) CH: 75% (18/24) canal) 2% CHX: 75% (9/12) NaOCl, sodium hypochlorite; CH, calcium hydroxide; CP, camphorated phenol; CMCP, camphorated monochlorophenol; PP, paper point sample; r culture, culture test before obturation. 504 PART II The Advanced Science of Endodontics

failures.70 In another study, good-quality root canal treatment canal filling material has been the most frequently and thor- on 54 teeth with asymptomatic periapical disease gave an oughly investigated. In these previous studies, the apical extent overall success rate of 74%, but teeth with positive cultures for of root fillings has been classified into three categories for Enterococcus faecalis achieved a success rate of only 66%.231 statistical analyses: more than 2 mm short of radiographic apex The success rate for teeth with no bacteria was 80%, whereas (short), 0 to 2 mm within the radiographic apex (flush), and that for teeth with bacteria in the canal before obturation was extended beyond the radiographic apex (long).153 The apical 33%. These associations cannot be regarded as direct cause- extent of root filling was found to have a significant influence and-effect associations, but they further emphasize the need to on the success rates of treatment, regardless of the periapical determine a relationship between microbial diversity and treat- status.150,153 Flush root fillings were associated with the highest ment outcome. success rates, whereas long root fillings were associated with A monkey-model study63 used a four- or five-strain infection the lowest success rates. to test the effect of debridement and obturation procedures on Most previous retrospective studies could not distinguish outcome. When bacteria remained after chemomechanical between the effects of apical extent of instrumentation versus debridement, 79% of the root canals were associated with non- the apical extent of obturation; however, the London Eastman healed periapical lesions, compared with 28% when no bacte- study150 was able to separate the effect of these two factors and ria were found to remain. Combinations of several residual found them both to independently and significantly affect peri- bacterial species were more frequently related to nonhealed apical healing. The factors correlated with each other, consis- lesions than were single strains. When no bacteria remained at tent with the fact that canals are normally filled to the same the end of chemomechanical debridement, healing occurred extent as canal preparation. independently of the quality of the root filling. In contrast, Extrusion of cleaning, medication, or filling materials when bacteria remained in the canal system, there was a greater beyond the apical terminus into the surrounding tissues may correlation with nonhealing associated with poor-quality root result in delayed healing or even treatment failure due to a fillings than in technically well-performed fillings. In root foreign body reaction.105,146,218,260 Magnesium and silicon from canals where bacteria were found after removal of the root the talc-contaminated extruded gutta-percha were found to filling, 97% had not healed, compared with 18% for those root induce a foreign body reaction, resulting in treatment failure.146 canal systems with no bacteria detected upon removal of root An animal study has shown that large pieces of subcutaneously filling. The study emphasizes the importance of reducing bac- implanted gutta-percha in guinea pigs were well encapsulated teria below detection limits before permanent root filling in in collagenous capsules, but fine particles of gutta-percha order to achieve optimal healing conditions for the periapical induced an intense, localized tissue response.218 The inference tissues. It also reinforces the view that obturation does indeed that perhaps extrusion of large pieces of gutta-percha may play a role when residual infection is present. not impact on periapical healing was not supported by data Regardless of the technique used for obtaining a sample for from previous studies.150,153 The discrepancy may possibly be a canal culture, the presence of a negative culture seems to accounted for by bacterial contamination of the extruded have a positive impact on treatment outcome. The association gutta-percha in the clinical data. of specific species with treatment failure is not well established The radiographic evidence of “sealer puffs” extruding but the identity of the small group of species isolated from through the main apical foramina and lateral/accessory canals positive cultures is relatively constant and may hold answers has been pursued with vigor by some endodontists based on to treatment resistance and failure. However, it is important to the undaunted belief of its value as “good practice.” Their understand that there are many other factors that can influence perception is that this represents a measure of root canal system root canal treatment outcome. cleanliness, and they ardently argue that healing would follow, albeit with some delay. The published evidence on the effects Root Filling Material and Technique of sealer extrusion into the periapical tissues has been contra- The interrelationship among the core root filling material, dictory. Friedman and colleagues68 found that extrusion of a sealer (for filling the gaps between the core material and canal glass ionomer-based sealer significantly reduced success rates. surface), and technique for their placement complicates the In contrast, Ng and associates150 reported that extrusion of a investigation of the effect of obturation and technique on treat- zinc oxide–eugenol based–sealer had no significant effect on ment outcome. In previous studies on treatment outcome, the periapical healing. The discrepancy may be attributed to the most commonly used core root filling material was gutta- difference in sealer type and the duration of treatment percha with various types of sealer or gutta-percha softened in follow-up. The radiographic assessment of the presence or chloroform (chloropercha).153 The sealers used may be classi- resorption of sealer may be complicated by the radiolucent fied into zinc oxide–eugenol–based, glass ionomer–based, and property of its basic components and the insufficient sensitivity resin-based types.153 Materials such as Resilon, SmartSeal, and of the radiographic method used to detect small traces of it.150 MTA have been adopted but have not significantly penetrated It is possible that, in some cases, the radiographic disappear- the market, except for the use of MTA in surgical repairs or ance of extruded sealer may simply be due to resorption of the repairs for immature apices. In any case, there is no evidence radio opaque additive, barium sulfate, or its uptake by macro- to show that the nature of root filling material and the tech- phages, still resident in the vicinity.146 nique used for placement has any significant influence on Extruded glass ionomer–based68 zinc oxide–eugenol– treatment outcome. based,98 silicone-based98 sealers or endomethasone22 were not found to be resorbed/absorbed by periapical tissues after 1 year. Apical Extent of Root Filling Traces of calcium hydroxide–based sealer (Sealapex) could still Of the many intraoperative factors associated with success and be detected after 3 years.194 In the latter study, treatments were failure of root canal treatment, the apical extent of the root carried out on primary molar teeth and the canals were CHAPTER 11 Evaluation of Outcomes 505 obturated with Sealapex without gutta-percha. With longer centers around better patient acceptability and cost-effectiveness duration of follow-up, complete resorption of extruded versus the preference of multiple-visit treatments based on zinc oxide–eugenol–based sealers (Procosol, Roth Elite)8 and biologic rationale.224 The premise for multiple-visit treatments a resin-based sealer (AH Plus, Dentsply/DeTrey, Konstanz, has been that primary debridement is not completely effective Germany)194 was demonstrated in 69% and 45% of the cases in eliminating all the adherent bacterial biofilm147 and the after 4 and 5 years, respectively. Ng and associates150 advanced residual bacteria may multiply and recolonize the canal two explanations for the difference between the effect of system.25,29 Therefore, the proponents consider it desirable to extruded core gutta-percha and the zinc oxide–eugenol sealer: use the interappointment period to dress the canal with a long- the latter is antibacterial and may kill residual microorganisms, lasting or slow-release antibacterial agent capable of destroying whereas it is also more soluble and readily removed by host or incapacitating residual bacteria, as well as to take the oppor- cells compared to gutta-percha. tunity to gauge the initial periapical response before root filling. Calcium hydroxide has served in this capacity for many Quality of Root Filling years because of its ability to dissolve organic tissue, kill bac- Another much investigated parameter of obturation in retro- teria, detoxify antigenic material, and act as a slow-release spective studies has been the radiographic measure of the agent because of its low solubility-product in an aqueous envi- “quality of root filling.” The rationale for complete obturation ronment. However, its antibacterial ability has come under of the root canal system is to prevent recontamination by colo- close scrutiny, with advocates suggesting that the material is nization from the residual infection or newly invading bacteria. not suitable for this purpose.195 A final resolution to this debate Both are supposedly prevented by a “tight” seal with the canal is awaited based on robust clinical evidence. Most of the pub- wall and an absence of voids within the body of the material. lished randomized controlled trials found no significant influ- Quality of root filling may therefore be regarded either as poor ence of healing attributable to number of treatment visits, but root-filling technique or as a surrogate measure of the quality they all lacked robust statistical power. of the entire root canal treatment, because good obturation The debate about the merits of single- or multiple-visit relies on properly executed preliminary steps in canal prepara- treatments will continue unabated given the respective tion. A systematic review153 reported that the criteria for strengths of the motivational drivers among the opposing judging the quality of root fillings have not been well defined groups. The issue may only be resolved by properly docu- in previous studies.43,93,216 An unsatisfactory root filling has mented, large randomized controlled trials (which are cur- been defined as “inadequate seal,” “poor apical seal,” or “radio- rently unavailable) because undocumented variables (i.e., graphic presence of voids.” Nevertheless, satisfactory root fill- operator skill, biologic or technical case complexity, and ings were found to be associated with significantly higher patient compliance) would continue to bias the outcome. success rates than unsatisfactory root fillings.153 Post Root Canal Treatment Restorative Factors Acute Exacerbation During Treatment Effect of Quality and Type of Restoration The causes for interappointment “flare-up” or pain have not The placement of a coronal restoration after root canal obtura- been precisely determined, and several hypothetical mecha- tion is the final step in the management of teeth undergoing nisms involving chemical, mechanical, or microbial injury to root canal treatment. It has been shown to have a major influ- the periradicular tissues, as well as psychological influences, ence on endodontic outcomes. Teeth with satisfactory coronal have been suggested as contributory to postpreparation pain restorations were found to have significantly better periapical or swelling.202,203 Although these factors have not been specifi- healing compared with those with unsatisfactory restorations cally studied in the context of periapical healing, acute “flare- (OR = 3.31; 95% CI: 1.07, 10.30).153 The term satisfactory ups” during treatment were not found to be significantly restorations has been defined as a restoration with no evidence associated with periapical healing in two studies.102,216 In con- of marginal discrepancy, discoloration, or recurrent caries with trast, the London Eastman study150 found that pain or swelling absence of a history of decementation.93,182 occurred in 15% of cases after chemomechanical debridement Given that one of the roles of coronal restorations is to and was found to significantly reduce success as measured by prevent postoperative root canal reinfection via coronal leakage, periapical healing. This interesting finding may be explained the criteria for unsatisfactory restoration given by Hoskinson by the hypothesis that “flare-ups” were caused by extrusion of and colleagues93 could not infer coronal leakage when the contaminated material during canal preparation. Such material inner core was still intact. Consequently, the London Eastman may elicit a foreign body reaction or (transient) extraradicular study150 adopted a different classification and definition for infection, resulting in treatment failure in a proportion of unsatisfactory restorations in order to illustrate obvious and such cases. Alternatively, acute symptoms may be the result of potential coronal leakage more accurately. The two groups of incomplete chemomechanical debridement at the first appoint- unsatisfactory restorations were defined as those with (1) ment, leading to a shift in canal microbial ecology favoring the obvious signs of exposed root filling and (2) potential leakage growth of more virulent microorganisms, leading to further indicated by marginal defects and history of de-cementation. postpreparation pain and treatment failure. The exact biologic It is perhaps this strategy that contributed to the finding of a mechanisms of failure in these cases remain obscure and profound effect (OR = 10.7; 95% CI: 3.7, 31.5) of coronal warrant further investigation. leakage on the endodontic outcome. A number of investigations have been performed based on Number of Treatment Visits comparisons between the types of post root canal treatment The number of treatment visits for completing root canal treat- restorations, including permanent versus temporary restora- ment and its effect on periapical healing remains an ongoing tions,43,68,150 crown versus acrylic restorations,43,68,150,216 pres- controversy. Generally, the argument for single-visit treatment ence versus absence of posts,68,150 and nonabutment versus 506 PART II The Advanced Science of Endodontics

abutment.150,216 Teeth that had been permanently restored were functioning as bridge abutments compared with those restored associated with significantly higher success rates than their as individual units following root canal treatment.216 temporarily restored counterparts in some studies43,68 but not in others.38,150 The type of permanent restoration43,68,150,216 was Summary of Factors Influencing found to have no significant influence on the outcome of Periapical Healing Following Nonsurgical treatment. Root Canal Treatment It has often been recommended that it would be wise to The following factors are considered as having a major impact provide a subseal over the root filling in case of loss of a per- on periapical health subsequent to root canal treatment: manent or temporary restoration; the subseal would be glass ♦ Presence (Fig. 11-20) and size (Fig. 11-21) of periapical ionomer (GIC) or zinc oxide–eugenol cement.196 The place- lesion ment of a GIC or zinc oxide–eugenol (IRM) cement lining ♦ Patency at the canal terminus (achieving patency signifi- coronal to the gutta-percha filling and underneath the perma- cantly increased the chance of success twofold)150 nent core in order to provide an additional antibacterial coronal ♦ Apical extent of chemomechanical preparation in relation seal was found in a prospective study to have no beneficial to the radiographic apex (Fig. 11-22) 150 effect on treatment success. ♦ Outcome of intraoperative culture test (see Fig. 11-19) In summary, the preceding findings overall support the ESE ♦ Iatrogenic perforation (if present, reduces the odds of guidelines61 that an adequate restoration should be placed after success by 30%)150 root canal treatment to prevent subsequent bacterial recontami- ♦ Quality of root canal treatment judged by the radiographic nation. Therefore, the provision of a good-quality coronal resto- appearance of the root filling (Fig. 11-23) ration, regardless of type, should be considered the final part of ♦ Quality of the final coronal restoration (Fig. 11-24) the root canal treatment procedure following obturation. The following factors are considered as having minimal impact on root canal treatment outcome: Use of Root Treated Teeth as Abutments for Prostheses ♦ Age of patient and Occlusal Contacts ♦ Gender of patient Mechanical stress on restorations is a function of the role of ♦ Tooth morphologic type individual teeth in the occlusal scheme. The pattern of occlusal ♦ Specific root canal treatment protocol and technique (prep- loading both in static and dynamic occlusion is dictated by aration, irrigation, and obturation material and technique) whether the teeth are involved as single units or abutments Contemporary improvements in mechanical and chemical (bridge/denture) and whether they have holding or guiding canal preparation have not resulted in an increase in the contacts. It is reasonable to expect that bridge and denture success rate for root canal treatment over the past century (Fig. abutments may be placed under unfavorable loads, as may 11-25). This observation may be explained by the currently last-standing teeth in the dental arch.127 These teeth may there- available techniques not being effective in eliminating the fore be expected to have lower success rates because of a infection in the apical canal anatomy. potential increase in the development of cracks and fractures It is notable that all factors that have a strong influence on due to fatigue. This observation has been confirmed for teeth periapical health after root canal treatment are associated

Castagnola and Orlay 1952

Heling and Tamshe 1970

Adenubi and Rule 1976

Jokinen et al. 1978

Barbakow et al. 1980

Friedman et al. 1995

Lilly et al. 1998

Hoskinson et al. 2002

Ng et al. 2011

Combined

.038591 10 20 30 LogOR_nonvital_nopa_pa

FIG. 11-20 Forest plot showing pooled and individual study’s odds ratio (OR) for periapical health of teeth undergoing root canal treatment with preoperative nonvital pulps and absence of periapical radiolucencies versus teeth with nonvital pulps and presence of periapical radiolucencies (pooled OR = 2.4; 95% CI: 1.7, 3.5). CHAPTER 11 Evaluation of Outcomes 507

Selden 1974

Matsumoto et al. 1987

Sjogren et al. 1990

Calisken and Sen 1996

Hoskinson et al. 2002

Siqueira et al. 2008

Ng et al. 2011

Ricucci et al. 2011

Combined

.447755 5 10 15 20 LogOR_small_largelesion_all

FIG. 11-21 Forest plot showing pooled and individual study’s odds ratio (OR) for periapical health of teeth undergoing root canal treatment with preoperative large (> 5 mm) versus small (< 5 mm) periapical radiolucencies (pooled OR = 2.2; 95% CI: 1.3, 3.7).

Harty et al. 1970 Heling and Tamshe 1970 Adenubi and Rule 1976 Heling and Shapira 1978 Jokinen et al. 1978 Kerekes 1978 Kerekes 1978 Heling and Kischinovsky 1979 Barbakow et al. 1980 Klevant and Eggink 1983 Halse and Molven 1987 Peak 1994 Hoskinson et al. 2002 Aqrabawi 2006 Doyle et al. 2006 Liang et al. 2011 Ng et al. 2011 Ricucci et al. 2011

Combined

0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 A UNDERFSS_pr

FIG. 11-22 Forest plot showing pooled and individual study’s probability of periapical health for teeth undergo- ing root canal treatment with underextended (0.76 [0.71, 0.82]) (A), flush (0.81 [0.76, 0.86]) (B), or overextended (0.66 [0.56, 0.75]) (C) root fillings. Continued 508 PART II The Advanced Science of Endodontics

Harty et al. 1970 Heling and Tamshe 1970 Adenubi and Rule 1976 Heling and Shapira 1978 Jokinen et al. 1978 Kerekes 1978 Kerekes 1978 Heling and Kischinovsky 1979 Barbakow et al. 1980 Klevant and Eggink 1983 Halse and Molven 1987 Peak 1994 Hoskinson et al. 2002 Aqrabawi 2006 Doyle et al. 2006 Liang et al. 2011 Ng et al. 2011 Ricucci et al. 2011

Combined

0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 B FLUSHSS_pr

Harty et al.1970 Heling & Tamshe 1970 Adenubi & Rule 1976 Heling & Shapira 1978 Jokinen et al. 1978 Kerekes 1978 Kerekes 1978 Heling & Kischinovsky 1979 Barbakow et al. 1980 Boggia 1983 Klevant & Eggink 1983 Halse & Molven 1987 Sjogren et al. 1990 Cvek 1992 Peak 1994 Hoskinson et al. 2002 Aqrabawi 2006 Doyle et al. 2006 Liang et al. 2011 Ng et al. 2011 Ricucci et al. 2011

Combined

0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 C OVERFSS_pr FIG. 11-22, cont’d CHAPTER 11 Evaluation of Outcomes 509

Harty et al. 1970

Adenubi and Rule 1976

Kerekes 1978

Kerekes 1978

Nelson 1982

Sjogren et al. 1990

Heling et al. 2001

Liang et al. 2011

Ng et al. 2011

Liang et al. 2012

Combined

.165487 10 20 30 Logor_satRF_unsatRF

FIG. 11-23 Forest plot showing pooled and individual study’s odds ratio (OR) of periapical health for teeth undergoing root canal treatment with good quality versus suboptimal quality root filling (pooled OR = 3.9; 95% CI: 2.5, 6.2).

Heling & Shapira 1978 Heling & Kischinovsky 1979 Swartz et al. 1983 Safavi et al. 1987 Ricucci et al. 2000 Heling et al. 2001 Hoskinson et al. 2002 Chugal et al. 2003 Siqueira et al. 2008 Liang et al. 2011 Ng et al. 2011 Ricucci et al. 2011 Liang et al. 2012

Combined

.526221 5 10 15 Logor_satrest_unsatrest

FIG. 11-24 Forest plot showing pooled and individual study’s odds ratio (OR) of periapical health for teeth undergoing root canal treatment with satisfactory versus unsatisfactory coronal restoration at follow-up (pooled OR = 1.9; 95% CI: 1.5, 2.5). in some way with a persistent root canal infection. Further treatment (Fig. 11-26). The most common reasons for such improvements in root canal treatment outcomes may therefore tooth loss were due to problems of endodontic origin, tooth/ be achieved by understanding the nature of the root canal infec- root fracture, or restoration failure.149,151 Consistent with the tion (especially apically) and the manner in which the micro- studies on periapical healing, the considerations affecting the biota is altered or eliminated during root canal treatment. survival of endodontically treated teeth may be divided into patient, intraoperative, and restorative factors.

Factors Affecting Tooth Survival Following Patient Factors Root Canal Treatment Ng and colleagues151 found that teeth in patients suffering from A systematic review and meta-analysis has shown that 93% of diabetes or receiving systemic steroid therapy had a higher endodontically treated teeth survive at 2 years postoperatively; chance of being extracted after root canal treatment. The nega- but this survival reduced to 88% at 10 years following tive influence of diabetes on tooth survival is consistent with 510 PART II The Advanced Science of Endodontics

Buchbinder 1941

Castagnola and Orlay 1952

Grahnen and Hansen 1961

Engstrom and Lundberg 1965

Combined

0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 A Probability of success—strict

Blayney 1922

Auerbach 1938

Morse and Yates 1941

Castagnola and Orlay 1952

Seltzer et al.. 1963

Zeldow and Ingle 1963

Bender et al.. 1964

Combined 0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 B Probability of success—loose

Harty et al. 1970

Heling and Tamshe 1970

Cvek 1972

Werts 1975

Adenubi and Rule 1976

Heling and Shapira 1978

Jokinen et al. 1978

Kerekes 1978

Kerekes 1978

Combined 0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 C Probability of success—strict

FIG. 11-25 Forest plot showing pooled and individual study’s probability of periapical health for teeth undergo- ing root canal treatment by “decade of publication” and “criteria for success.” CHAPTER 11 Evaluation of Outcomes 511

Selden 1974

Adenubi and Rule 1976

Jokinen et al. 1978

Soltanof 1978

Heling and Kischinovsky 1979

Combined 0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 D Probability of success—loose

Barbakow et al. 1980

Cvek et al. 1982

Boggia 1983

Klevant and Eggink 1983

Pekruhn 1986

Bystrom et al. 1987

Halse and Molven 1987

Safavi et al. 1987

Akerblom and Hasselgren 1988

Combined 0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 E Probability of success—strict

Barbakow et al. 1980

Nelson 1982

Boggia 1983

Morse et al. 1983

Oliet 1983

Swartz et al. 1983

Bystrom et al. 1987

Matsumoto et al. 1987

Shah 1988

Combined 0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 F Probability of success—loose FIG. 11-25, cont’d Continued 512 PART II The Advanced Science of Endodontics

Sjogren et al. 1990

Murphy et al. 1991

Cvek 1992

Reid et al. 1992

Peak 1994

Friedman et al. 1995

Calisken and Sen 1996

Peretz et al. 1997

Sjogren et al. 1997

Lilly et al. 1998

Combined

0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 G Probability of success—strict

Murphy et al. 1991

Jurcak et al. 1993

Smith et al. 1993

Peak 1994

Friedman et al. 1995

Calisken and Sen 1996

Orstavik 1996

Trope et al. 1999

Combined 0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 H Probability of success—loose FIG. 11-25, cont’d

the report by Mindiola and associates,135 whereas the influence reason for tooth loss after endodontic treatment. Ng and of steroid therapy had never been reported previously. It may coworkers149 found that tooth type had a significant influence be argued that patients with diabetes are more susceptible to on survival. Maxillary premolars and mandibular molars were periodontal disease74 or have a lower success rate of root canal found to have the highest frequency of extraction, with tooth treatment67 because of being immunologically compromised. fracture being the most common reason. The observation is However, the researchers reported that over 50% of such teeth consistent with previous reports on fracture incidence of max- were extracted due to persistent pain. Some of these observa- illary premolars and mandibular molars.56,109 The factors tions may be explained by the presence of neuropathy, a debili- “proximal contacts” and “terminal (last standing) teeth” were tating painful complication of diabetes.57 It is interesting to found to affect tooth survival considerably,149,151 but they were note that systemic steroid therapy is often prescribed to control significantly correlated with molar teeth. Most of the extrac- such chronic pain.39,51,101 tions of terminal teeth or teeth with one or fewer proximal contacts were due to tooth fracture.151 The observation may be Tooth Morphologic Type and Location explained by the unfavorable distribution of occlusal forces Tooth types (i.e., location within the arch) may vary with and higher nonaxial stress on terminal teeth and those with respect to their susceptibility to tooth fracture, a common fewer than two proximal contacts. Other possible reasons for CHAPTER 11 Evaluation of Outcomes 513

Ricucci et al. 2000 Weiger et al. 2000 Chugal et al. 2001 Peak et al. 2001 Benenati and Khajotia 2002 Cheung 2002 Hoskinson et al. 2002 Peters and Wesselink 2002 Chugal et al. 2003 Huumonen et al. 2003 Field et al. 2004 Khedmat 2004 Chu et al. 2005 Moshonov et al. 2005 Aqrabawi 2006 Doyle et al. 2006 Gesi et al. 2006 Conner et al. 2007 Molander et al. 2007 Sari and Duruturk 2007 Chevigny et al. 2008 Cotton et al. 2008 Penesis et al. 2008 Siqueira et al. 2008 Witherspoon et al. 2008 Hsiao et al. 2009 Mente et al. 2009 Tervit et al. 2009 Liang et al. 2011 Ng et al. 2011 Ricucci et al. 2011 Liang et al. 2012 Combined

0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 I Probability of success—strict

Weiger et al. 2000 Deutsch et al. 2001 Heling et al. 2001 Peak et al. 2001 Pettiett et al. 2001 Benenati and Khajotia 2002 Peters and Wesselink 2002 Cheung and Chan 2003 Huumonen et al. 2003 Negishi et al. 2005 Conner et al. 2007 Molander et al. 2007 Sari and Duruturk 2007 Zmener and Pameijer 2007 Penesis et al. 2008 Siqueira et al. 2008 Witherspoon et al. 2008 Ng et al. 2011

Combined

0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 J Probability of success—loose FIG. 11-25, cont’d their higher rate of loss are (1) failure of root canal treatment Preoperative Conditions of Teeth in a terminal tooth may be accepted more willingly because of The presence of preoperative periapical lesions, which is the little perceived aesthetic value and (2) clinicians may be less most significant prognostic factor for periapical healing, was likely to offer further treatment on terminal molar teeth due found to have no significant influence on tooth survival.151 On to difficult access. Therefore, when restoring molar teeth, the the other hand, the presence of preoperative periodontal favorable distribution of occlusal forces must be considered, probing defects of endodontic origin, preoperative pain, and especially on teeth with one or fewer adjacent teeth or on preoperative sinus tracts were found to reduce tooth sur- terminal teeth. vival.151 These observations are consistent with a previous 514 PART II The Advanced Science of Endodontics

Lazarski et al. (2001)

Lynch et al. (2004)

Nagasiri and Chitmongkolsuk (2005)

Tan et al. (2006)

Wang et al. (2011)

Combined 0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 A Rate_2 yr

Makie et al. (1993) Alley et al. (2004) Tilashaski et al. (2004) Caplan et al. (2005) Nagasiri and Chitmongkolsuk (2005) Chen et al. (2007) Salvi et al. (2007) Lumley et al. (2008) Chen et al. (2008) Salebrabi and Rotstein (2010) Fleming et al. (2010) Ng et al. (2011) Setzer et al. (2011) Ferrari et al. (2012) Skupien et al. (2013)

Combined 0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 B Rate_5 yr

Makie et al. (1993)

Aquilino and Caplan (2002)

Caplan et al. (2002)

Dammaschke et al. (2003)

Salehrabi and Rotstein (2004)

Caplan et al. (2005)

Stoll et al. (2005)

Fonzar et al. (2009)

Combined 0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 C Rate_10 yr

FIG. 11-26 Forest plot showing pooled and individual study’s probability of 2-year (pooled probability = 0.93; 95% CI: 0.90, 0.95) (A), 5-year (pooled probability = 0.92; 95% CI: 0.91, 0.93) (B), and 10-year (pooled probabil- ity = 0.88; 95% CI: 0.84, 0.92) (C) survival of teeth undergoing root canal treatment. CHAPTER 11 Evaluation of Outcomes 515 report that the mere presence of a persistent periapical lesion were incisors or canines. Therefore, the inference is that the was not a sufficient reason for dentists and patients to opt use of such retention should be particularly avoided in premo- for further treatment, either retreatment or extraction.180 The lar and molar teeth. Alternative treatment options should be negative impact of preoperative pain on survival outcome considered for molar or premolar teeth lacking sufficient tooth highlights the importance of accurate pain diagnosis. In some structure. instances, the pain may have been of nonendodontic origin and Ng and colleagues151 observed that teeth functioning as therefore would persist after treatment.176 In other instances, prosthetic abutments had poorer survival rates; however, the preoperative pain of endodontic origin may persist following number of teeth (n = 94) in the study that functioned as abut- treatment, as a result of peripheral or central sensitization. ments was too small a sample to be considered statistically Therefore, effective pain diagnosis and management for patients significant. As previously described, the explanation may presenting with preoperative pain are crucial. reside in the excessive and unfavorable distribution of occlusal The presence of preoperative cervical resorption and perfo- stresses on abutment teeth. If possible, root-treated teeth ration was also found to significantly reduce tooth survival.151 should be avoided as abutments for prostheses or in provision This was as expected because tooth fracture and reinfection of occlusal guidance in excursive movements. due to leakage through the resorption and perforating defects are likely sequelae in such cases. In the presence of reinfection, clinicians are more inclined to suggest extraction due to the Summary of Factors Influencing the Survival of intuitive perception of poor long-term prognosis of such teeth. Teeth Following Root Canal Treatment The following conditions have been found to significantly Treatment Factors improve tooth survival following root canal treatment: Considering all of the intraoperative factors, the “lack of ♦ Nonmolar teeth (Fig. 11-27) patency at apical foramen” and the “extrusion of gutta-percha ♦ Teeth with both mesial and distal adjacent teeth (Fig. 11-28) 151 root filling” were found to be the most significant intraopera- ♦ Teeth not located as the distal-most tooth in the arch 151 tive factors in reducing tooth survival. In the presence of ♦ Teeth (molar) with cast restorations after treatment persistent problems and knowing that the treatment objective (Fig. 11-29) of cleaning to the canal terminus could not be achieved, ♦ Teeth not requiring cast post and core for support and patients and dentists may be more likely to opt for extraction retention of restoration111,151 sooner than later. ♦ Teeth not functioning as abutments for fixed prosthesis3,111,191,151 Restorative Factors ♦ Absence of preoperative deep periodontal probing defects, Protection of teeth with crowns or cast restorations has not pain, sinus tract, or perforation151 been shown to influence periapical healing; however, the ♦ Achievement of patency at canal terminus and absence of placement of good cores had a positive effect on endodontic root-filling extrusion during treatment151 outcome. In contrast, placement of crowns or cast restorations In addition, it is important to ensure favorable distribution of was found to improve tooth survival.149,151,189 This suggests that occlusal forces when designing restorations for endodontically crowns and cast restorations help prevent tooth fracture, treated teeth. whereas the mere placement of a satisfactory core is sufficient to prevent reinfection after endodontic treatment. Unfortu- nately, the study was not able to investigate the interrelation- Impact of Root Canal Treatment on ship between tooth morphologic type, the extent of tooth Quality of Life tissue loss after treatment, or the type of final restoration. The impact of root canal treatment on the oral health–related Although the clinical inference from these findings is that cast quality of life of patients has been evaluated using the short restorations should preferably be placed on all teeth after root form (OHIP-14) or modified version (OHIP-17) of the Oral canal treatment, this is probably a gross exaggeration of the Health Impact Profile (OHIP-14)219 (Table 11-10). The dis- true need. On the basis of laboratory178 and clinical findings,144 tinctly positive impact of root canal treatment was apparent, posterior teeth with compromised marginal ridges (mesial regardless of cultural background of the patient group or the or distal), together with heavy occlusal loading evidenced measure used.55,73,87,115 As expected, physical pain, psycholo­ by faceting, may benefit from full coverage restorations. The gical discomfort (feeling tense), and disability (difficulty restoration design should attempt to preserve as much remain- in relaxing) were the most improved domains following ing tooth tissue as possible; the implication is that the so-called treatment. nonaesthetic but technically demanding partial veneer onlays and partial coverage crowns would be the restorations of OUTCOME OF NONSURGICAL choice for root-treated teeth. In anterior teeth, the missing tooth tissue may often be adequately replaced with composite RETREATMENT restorative materials. A crown is only indicated when tooth When root canal treatment fails to resolve periapical disease, structure or aesthetics become compromised. it is often considered appropriate to retreat the tooth using The use of cast post and cores for retention of restorations conventional approaches first, especially when the previous have also been found to reduce tooth survival.149,151 It may be treatment is technically deficient (Fig. 11-30). This requires speculated that the presence of a post has different effects on removal of the previous root-filling material and any other anterior versus posterior teeth as they are subjected to different material placed for restorative reasons. Correction of any pro- directions and amount of occlusal force. It has been reported cedural errors may also be required, if possible. All materials that only 12% of the extracted teeth with cast post and cores must be removed in their entirety to ensure delivery of 516 PART II The Advanced Science of Endodontics

Lazarski et al. (2001)

Aquilino and Caplan (2002)

Alley et al. (2004)

Salehrabi and Rotstein (2004)

FIG. 11-27 Forest plot showing Chen et al. (2007) pooled and individual study’s odds Salvi et al. (2007) ratio f (OR) o survival probability for nonmolar versus molar teeth (pooled Salebrabi and Rotstein (2010) OR = 1.4; 95% CI: 1.1, 1.6). Ng et al. (2011)

Wang et al. (2011)

Combined .238017 5 10 Logor_nonmolar_molar

Aquilino and Caplan (2002)

Alley et al. (2004) FIG. 11-28 Forest plot showing pooled and individual study’s odds ratio (OR) of survival probability for teeth with both mesial and distal con- Ng et al. (2011) tacts present versus absent (pooled OR = 2.6; 95% CI: 1.8, 3.7).

Combined .149639 10 20 30 40 Logor_proximal2_01

Lazarski et al. (2001)

Aquilino and Caplan (2002)

FIG. 11-29 Forest plot showing Alley et al. (2004) pooled and individual study’s odds ratio (OR) of survival probability for Lynch et al. (2004) teeth restored with cast versus plastic restoration (pooled OR = 3.5; 95% CI: 2.6, 4.7). Ng et al. (2011)

Combined .516111 10 20 30 Logor_crown_nocrown CHAPTER 11 Evaluation of Outcomes 517

identical to those affecting primary root canal treatment. Of TABLE 11-10 the potential prognostic factors unique to retreatment cases, The OHIP-14 and Modified Oral Health Impact the most significant factor influencing the outcome of treat- Profile Instrument (OHIP-17) (Dugas et al., 2002) ment is the ability to remove or bypass preexisting root-filling material or separated instruments during retreatment. This is OHIP # Item Question understandable because it would have a direct impact on the OHIP1 Have you had trouble pronouncing words because of ability to achieve canal patency and canal disinfection at the your teeth and mouth? apical terminus.150 OHIP2 Have you felt that your sense of taste has worsened because of your teeth or mouth? OUTCOME OF SURGICAL RETREATMENT OHIP3 Have you had painful aching in your mouth? OHIP4 Have you found it uncomfortable to eat any foods Factors Affecting Periapical Health because of your teeth or mouth? or Healing Following Periapical Surgery OHIP5* Have you had to alter the temperature of the foods that and Root-End Filling you eat because of your teeth or mouth? There are a number of published systematic reviews on OHIP6 Have you been self-conscious because of your teeth or the prognostic factors for periapical surgery with apical 45,47,48,155,204,205,239-241,250 mouth? filling. The major limitations of this pooled data include the variable length of time for evaluating OHIP7 Have you felt tense because of your teeth or mouth? success following treatment as well as the radiographic criteria OHIP8 Has your diet been unsatisfactory because of your teeth used to assess healing. An unpublished meta-analysis130 of data or mouth? from the studies listed in Table 11-11 reveals that the weighted OHIP9 Have you had to interrupt meals because of your teeth pooled probability of success from periapical surgery with ret- or mouth? rograde restorations, based on complete radiographic healing, OHIP10 Have you found it difficult to relax because of your teeth is 67.5% (95% CI: 62.9%, 72.0%) (Fig. 11-33). The trend shows or mouth? higher success rates reported in more recent studies. This observation is consistent with the much higher pooled success OHIP11* Have you found it difficult to fall asleep because of your rate of 92% (95% CI: 86%, 95%) revealed in the meta-analysis teeth or mouth? of prospective outcome data of surgical endodontic treatments OHIP12* Have you ever been awakened by problems with your performed by a “modern” technique (using magnification, root- teeth or mouth? end resection with minimal or no bevel, retrograde cavity prep- OHIP13 Have you been embarrassed because of your teeth or aration with ultrasonic tips, and modern retrograde root canal 240,241 mouth? filling materials). In congruence with these findings, Setzer and colleagues reported that the pooled success rate of OHIP14 Have you been irritable with other people because of treatment using a microsurgical approach (94%; 95% CI: 89%, your teeth or mouth? 98%) was more favorable than traditional root-end surgery OHIP15 Have you had difficulty doing your usual jobs because (59%; 95% CI: 55%, 63%).205 However, the studies included in of problems with your teeth or mouth? the latter meta-analysis differed in design, case selection, dura- OHIP16 Have you felt that life in general was less satisfying tion after treatment, and the provision of preoperative nonsur- because of your teeth or mouth? gical treatment, which may bias the higher success rate of the OHIP17 Have you been totally unable to function because of contemporary treatment. 130 your teeth or mouth? The unpublished meta-analysis stratified by duration fol- lowing treatment reveals that the pooled probability of success OHIP-14 contains all the items except those with an asterisk (*). based on complete healing plateaued after 2 years postopera- tively, being 51% (95% CI: 42%, 60%) after 6 months, 68% (95% CI: 63%, 73%) after 12 months, 76% (95% CI: 67%, 84%) antibacterial agents to all surfaces of the root canal dentin (see after 24 months, 74% (95% CI: 52%, 95%) after 48 months, Fig. 11-30). The periapical healing rates of root canal retreat- and 74% (95% CI: 66%, 82%) after more than 48 months. It ment are generally perceived to be lower compared to primary is therefore advisable to follow up on cases that have under- treatment for the following reasons: gone periapical surgery for a minimum period of 2 years and 61 ♦ Obstructed access to the apical infection up to 4 years as suggested in quality guidelines. ♦ A potentially more resistant microbiota The factors having a major impact on outcome of periapical The outcomes from a range of studies show that the mean surgery with retrograde cavities and fillings in teeth with pre- weighted success rate of nonsurgical root canal retreatment is operative periapical radiolucencies revealed in the (as yet) 66% (Figs. 11-31 and 11-32), which is about 6% lower than unpublished meta-analyses130 of data from studies listed in in the case of primary treatment on teeth with apical peri- Table 11-11 are as follows: 148,150 odontitis. However, it has also been shown that the sur- ♦ Small (≤ 5 mm) versus large (> 5 mm) periapical lesion vival rate of teeth having undergone nonsurgical root canal (risk ratio = 1.2; 95% CI: 1.1, 1.3) retreatment may be similar to that for primary root canal ♦ Periapical lesion involving one versus both cortical plates treatment.151 (risk ratio = 1.2; 95% CI: 1.0, 1.5) The factors affecting outcomes of periapical health and ♦ Absence versus presence of previous surgery (risk ratio = tooth survival following root canal retreatment are otherwise 1.2; 95% CI: 1.1, 1.3) 518 PART II The Advanced Science of Endodontics

A B

C

FIG. 11-30 Tooth with technically deficient root canal treatment (A); having undergone (B and C) root canal retreatment.

Grahen and Hansson 1961 Engstrom et al. 1964 Selden 1974 Bergenholtz et al. 1979 Molven and Halse 1988 Sjogren et al. 1990 Friedman et al. 1995 Danin et al. 1996 Sundqvist et al. 1998 Hoskinson et al. 2002 Gorni and Gagliani 2004 Caliskan 2005 Chevigny et al. 2008

Combined 0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 pr_success_pa

FIG. 11-31 Forest plot showing pooled and individual study’s probability of complete periapical healing follow- ing root canal retreatment.

♦ Using magnification versus without the use of magnifica- intermediate restorative material (IRM) versus amalgam; tion during surgery (risk ratio = 1.5; 95% CI: 1.3, 1.8) the use of MTA resulted in a similar outcome to SuperEBA ♦ Root-end resection with minimum versus obvious bevel (risk ratio = 1.0; 95% CI: 0.99, 1.1) or IRM (risk ratio = 1.1; (risk ratio = 1.3; 95% CI: 1.2, 1.4) 95% CI: 0.98, 1.1); SuperEBA and IRM were associated with ♦ Use of ultrasonic tip versus bur for retro-cavity preparation significantly higher chances of success than amalgam (risk (risk ratio = 1.3; 95% CI: 1.2, 1.4) ratio = 1.2; 95% CI: 1.1, 1.3) as the retro-filling material ♦ Use of retro-filling material with mineral trioxide aggregate The preoperative presence of signs or symptoms (risk ratio (MTA) cement, super ethoxybenzoic acid (EBA) cement, or = 1.2; 95% CI: 1.1, 1.3),249 periodontal status (risk ratio = 2.1; CHAPTER 11 Evaluation of Outcomes 519

TABLE 11-11 Studies Investigating Periapical Healing Following Apical Surgery Sample Size Duration after Type of Radiographic Criteria Treatment Author Design Examination Radiograph for Success Patients Teeth Roots (months) Harty et al., 1970 Retrospective C & R Pa Other 169 6-60 Nordendram, 1970 Prospective C & R Pa Rud et al., 1972 66 66 6-24 Rud et al., 1972 Prospective R Pa Rud et al., 1972 237 12-180 Finne et al., 1977 Prospective C & R Pa Persson, 1973 156 218 36 Hirsch et al., 1979 Prospective C & R Pa Rud et al., 1972 467 467 6-36 Ioannides & Borstlap, 1983 Retrospective C & R Pa Other 50 50 45 6-60 Allen et al., 1989 Retrospective C & R Pa Rud et al., 1972 175 12-60 Amagasa et al., 1989 Prospective C & R Pa Other 42 64 12-90 Dorn & Gartner, 1990 Retrospective R Pa Other 488 6-120 Grung et al., 1990 Prospective C & R Pa Rud et al., 1972 161 12-96 Lustmann et al., 1991 Retrospective C & R Pa Rud et al., 1972 123 6-96 Rapp et al., 1991 Retrospective R Pa Rud et al., 1972 331 226 6-24 Rud et al., 1991 Retrospective C & R Pa Rud et al., 1972 388 388 12 Waikakul et al., 1991 Prospective C & R Pa Other 34 62 6-24 Pantschev et al., 1994 Prospective C & R Pa Persson, 1973 79 103 36 Jesselen et al., 1995 Prospective C & R Pa Other 67 82 12-60 Rud et al., 1996 Prospective R Pa Rud et al., 1972 347 12-48 Sumi et al., 1996 Retrospective C & R Pa Other 86 157 6-36 Jansson et al., 1997 Retrospective C & R Pa Other 59 59 11-16 Testori et al., 1999 Retrospective R Pa Rud et al., 1972 130 181 302 12-72 Von Arx & Kurt, 1999 Prospective C & R Pa Von Arx & Kurt, 1999 38 43 12 Zuolo et al., 2000 Prospective C & R Pa Molven et al., 1987 106 102 12-48 Pecora et al., 2001 RCT R Pa Rud et al., 1972 20 20 6 Penarrocha et al., 2001 Retrospective R Pa Von Arx & Kurt, 1999 30 31 71 12 Rahbaran et al., 2001 Retrospective C & R Pa Other 154 154 48-108 Rud et al., 2001 Prospective C & R Pa Rud et al., 1972 520 834 6-150 Von Arx et al., 2001 Prospective C & R Pa Other 24 25 39 12 Jensen et al., 2002 RCT C & R Pa Rud et al., 1972 122 Rubinstein & Kim, 2002 Prospective C & R Pa Rud et al., 1972 52 59 59 68 Tobon et al., 2002 RCT R Pa Rud et al., 1972 25 26 12 Vallecillo et al., 2002 Prospective R Pa Other 29 29 12 Chong et al., 2003 RCT C & R Pa Molven et al., 1987 86 86 12-24 Maddalone & Gagliani, 2003 Prospective C & R Pa Molven et al., 1987 79 120 3-36 Schwartz-Arad et al., 2003 Prospective R Pa Other 101 122 6-45 Platt et al., 2004 RCT C & R Pa Molven et al., 1987 28 34 12 Gagliani et al., 2005 Prospective C & R Pa Rud et al., 1972 164 168 231 60 Lindeboom et al., 2005 RCT C & R Pa Rud et al., 1972 100 100 12 Marti-Bowen et al., 2005 Retrospective C & R DPT Von Arx & Kurt, 1999 52 71 95 6-12 Taschieri et al., 2005 Prospective C & R Pa Rud et al., 1972 32 46 12 Taschieri et al., 2007 Prospective C & R Pa Molven et al., 1987 28 28 12 Tsesis et al., 2006 Retrospective C & R Pa Rud et al., 1972 71 88 6-48 Marin-Botero et al., 2006 RCT C & R Pa Rud et al., 1972 30 30 12 Continued 520 PART II The Advanced Science of Endodontics

TABLE 11-11 Studies Investigating Periapical Healing Following Apical Surgery—cont’d Sample Size Duration after Type of Radiographic Criteria Treatment Author Design Examination Radiograph for Success Patients Teeth Roots (months) Taschieri et al., 2006 RCT C & R Pa Molven et al., 1987 53 71 12 De Lange et al., 2007 RCT C & R Pa Rud et al., 1972 290 290 12 Leco-Berrocal et al., 2007 Prospective R Pa and DPT Other 45 45 6-24 Penarrocha et al., 2007 Prospective C & R Pa Von Arx & Kurt, 1999 235 333 384 6-144 Taschieri et al., 2007a Prospective C & R Pa Molven et al., 1987 17 27 12 Taschieri et al., 2007b Prospective C & R Pa Molven et al., 1987 41 59 12 Von Arx et al., 2007 Prospective C & R Pa Rud et al., 1972 200 177 12 Walivaara et al., 2007 Prospective C & R Pa Rud et al., 1972 54 55 12 Garcia et al., 2008 Prospective C & R DPT Von Arx & Kurt, 1999 92 106 129 6-12 Kim et al., 2008 Prospective C & R Pa Molven et al., 1987 148 24 Penarrocha et al., 2008 Prospective C & R DPT Von Arx & Kurt, 1999 278 278 12 Taschieri et al., 2008b Prospective C & R Pa Molven et al., 1987 27 31 12 Taschieri et al., 2008a RCT C & R Pa Molven et al., 1987 61 100 25 Christiansen et al., 2009 RCT C & R Pa Molven et al., 1987 25 12 Dominiak et al., 2009 Retrospective C & R Pa Other 106 106 12 Ortega-Sanchez et al., 2009 Retrospective C & R DPT Von Arx & Kurt, 1999 30 30 37 Pantschev et al., 2009 Retrospective C & R Pa Other 147 12 Waalivaara et al., 2009 RCT C & R Pa Molven et al., 1987 131 147 12 Barone et al., 2010 Prospective C & R Pa PAI 129 48-120 Garcia-Mira et al., 2010 Retrospective C & R DPT Von Arx & Kurt, 1999 75 87 12 Taschieri et al., 2010 Retrospective C & R Pa Molven et al., 1987 76 112 48 Von Arx et al., 2010 Prospective C & R Pa Rud et al., 1972 339 12 Goyal et al., 2011 RCT C & R Pa Rud et al., 1972 25 25 12 Penarrocha et al., 2011 Retrospective C & R DPT Von Arx & Kurt, 1999 150 178 178 12 Song et al., 2011a Retrospective C & R Pa Molven et al., 1987 441 3-12 Song et al., 2011b Prospective C & R Pa Molven et al., 1987 42 42 12 Taschieri et al., 2011 Retrospective C & R Pa Molven et al., 1987 33 43 12-48 Waalivaara et al., 2011 RCT C & R Pa Molven et al., 1987 153 194 12-21 Penarrocha et al., 2012 Prospective C & R DPT Von Arx & Kurt, 1999 23 31 12-19 Song & Kim, 2012 RCT C & R Pa Molven et al., 1987 192 192 12 Von Arx et al., 2012 Prospective C & R Pa Rud et al., 1972 170 12-60 Kreisler et al., 2013 Prospective C & R Pa Rud et al., 1972 255 281 6-12 Penarrocha et al., 2013 Retrospective C & R Pa and DPT Von Arx & Kurt 96 139 6-12 Song et al., 2013a Prospective C & R Pa Molven et al., 1987 344 12-120 Song et al., 2013b Prospective C & R Pa Molven et al., 1987 135 12 Song et al., 2014 Retrospective C & R Pa Rud et al., 1972 115 12-96 Taschieri et al., 2013 Retrospective C & R Pa Molven et al., 1987 86 6-12 Villa-Machado et al., 2013 Retrospective C & R Pa PAI 154 171 12-192 Li et al., 2014 Retrospective C & R Pa Molven et al., 1987 82 101 48 RCT, randomized controlled trial; C, clinical; R, radiographic; Pa, periapical; DPT, dental pantomogram; PAI, periapical index. CHAPTER 11 Evaluation of Outcomes 521

Grahen and Hansson 1961

Selden 1974

Sjogren et al. 1990

Hoskinson et al. 2002

Chevigny et al. 2008

Hsiao et al. 2009

Mente et al. 2009

Ng et al. 2011

Ricucci et al. 2011

Combined

.359988 5 10 Logor_pa1st_pa2nd

FIG. 11-32 Forest plot showing pooled and individual study’s odds ratio of periapical health for teeth with preoperative periapical radiolucency undergoing primary root canal treatment versus root canal retreatment (pooled OR = 1.5; 95% CI: 1.0, 2.1).

Harty et al. 1970 Nordendram 1970 Rud et al. 1972 Finne et al. 1977 Hirsch et al. 1979 Ioannides and Borstlap 1983 Allen et al. 1989 Dorn and Gartner 1990 Grung et al. 1990 Lustmann et al.1991 Rapp et al.1991 Rud et al. 1991 Waikakul et al. 1991 Pantschev et al. 1994 Jesseln et al. 1995 Rud et al. (mod rp) 1996 Jansson et al. 1997 Testori et al. 1999 Von Arx and Kurt 1999 Rud et al. 2001 Pecora et al. 2001 Penarrocha et al. 2001 Rahbaran et al. 2001 Von Arx et al. 2001 Rubinstein and Kim 2002 Tobon et al. 2002 Vallecillo et al. 2002 Chong et al. 2003 Maddalone and Gagliani 2003 Schwartz-Arad et al. 2003 Platt et al. 2004 Gagliani et al. 2005 Marin-Botero et al. 2006 De Lange et al. 2007 Leco-Berrocal et al. 2007 Taschieri et al. 2007 Garcia et al. 2008 Kim et al. 2008 Taschieri et al. 2008 Pantschev et al. 2009 Christiansen et al. 2009 Dominiak et al. 2009 Ortega-Sanchez et al. 2009 Von Arx et al. 2010 Taschieri et al. 2010 Taschieri et al. 2011 Goyal et al. 2011 Song et al. 2011b Penarrocha et al. 2012 Song and Kim 2012 Penarrocha et al. 2013 Villa-Machado et al. 2013 Song et al. 2013c Li et al. 2014 Combined

0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 Strict_s_pr

FIG. 11-33 Forest plot showing pooled and individual study’s probability of complete periapical healing follow- ing apical surgery. 522 PART II The Advanced Science of Endodontics

95% CI: 1.1, 3.8),222 and quality of the coronal restoration (risk surgical cases at the time of this writing. The reported median ratio = 1.6; 95% CI: 1.2, 2.1)249 have also been revealed as survival time for first-time surgery was 92.1 months (95% CI: significant prognostic factors by individual studies. 40.9, 143.4) and that for resurgery was 39.1 months (95% CI: The following factors have minimal effect on surgical 6.1, 72.1).253 The failure events, however, included tooth retreatment43: extraction and clinical and radiographic signs of periapical ♦ Age of patient disease after treatment. ♦ Gender of patient ♦ General health of patient Impact of Periapical Surgery ♦ Tooth type ♦ Quality of the preexisting root canal filling, as judged on Quality of Life radiographically The impact of periapical surgery on the patient’s quality of life ♦ Histologic diagnosis of the biopsied periapical lesion (cyst has only been evaluated using a questionnaire including three or granuloma) domains: physical function (chewing, talking, sleeping, daily The use of guided periodontal tissue regenerative routine, and work), physical pain, and other physical symp- membrane or grafting material has been advocated for cases toms (swelling, bleeding, nausea, bad taste/breath).49 It was with through-and-through defects (missing buccal and palatal concluded that the papilla-base incision flap design resulted in cortical plates), but there are conflicting reports as to their a lower impact on physical pain and other symptoms within benefits.53,162,170,232-234,237,249 The unpublished meta-analyses130 of the first week postoperation. The impact on physical pain has data from these studies did not reveal a significant influence of also been explored by two other studies35,36; both reported the such approaches on the periapical healing outcome. postoperative pain was of relatively short duration, with the intensity peaking at 3 to 5 hours postoperatively and progres- sively decreasing with time with no significant influencing Factors Affecting Periodontal Incisional factors identified. Wound Healing Periodontal attachment level after periapical surgery is an addi- tional physical outcome measure compared to nonsurgical root canal treatment. Studies have compared the effect of different CONCLUDING REMARKS soft tissue incision techniques (intrasulcular with or without The procedures used to maintain pulp vitality and for preven- involving interproximal papilla, submarginal, papilla-base, tion and treatment of periapical disease are able to achieve etc.).106,245-247 As expected, all concluded that negligible mar- excellent outcomes. The outcome data and potential prognos- ginal recession could be achieved by adopting a flap design that tic factors should be considered during treatment options avoided reflection of the interproximal papilla. appraisal and planning. Despite the fact that most important The improvement in outcomes of periapical surgery has prognostic factors are beyond the control of clinicians, optimal been attributed to modern surgical techniques plus greater outcomes for individual cases may still be achieved by per- biologic awareness of clinicians.205,250 In addition, and perhaps forming the procedure to guideline standards. From a health even more important, case selection may be more critical in and economic perspective, conventional root canal treatment excluding potential failures, inferring that prognostication may is a highly cost-effective treatment as a first-line intervention have improved. to extend the life of the tooth with a preoperative periapical lesion.103,171 If a root canal treatment subsequently fails, non- surgical and surgical retreatments are also more cost-effective Factors Affecting Tooth Survival Following than replacement with a prosthesis.103,171 Ultimately, all sources Periapical Surgery and Root-End Filling of evidence must be assessed for biasing influences based Unlike nonsurgical root canal treatment, the outcome measure on the local cultures, expertise, treatment predilection, and of tooth survival had only been adopted by one study253 on funding sources.

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Int Endod J 40:209, 2007. J Endod 39:1335, 2013. Taschieri S, Del Fabbro M, Testori T, Weinstein R: Microscope Santini A: Assessment of the pulpotomy technique in human Smith CS, Setchell DJ, Harty FJ: Factors influencing the success versus endoscope in root-end management: a randomized first permanent mandibular molars: use of two direct of conventional root canal therapy—a five-year retrospective controlled study, Int J Oral Maxillofac Surg 37:1022, 2008a, inspection criteria, Br Dent J 155:151, 1983. study, Int Endod J 26:321, 1993. doi:10.1016/j.ijom.2008.07.001. Epub Aug 20, 2008. Santucci PJ: Dycal versus Nd:YAG laser and Vitrebond for direct Soltanoff W: A comparative study of the single-visit and the Taschieri S, Machtou P, Rosano G, et al: The influence of pulp capping in permanent teeth, J Clin Laser Med Surg multiple-visit endodontic procedure, J Endod 4:278, 1978. previous non-surgical re-treatment on the outcome of 17:69, 1999. Song M, Jung IY, Lee SJ, et al: Prognostic factors for clinical endodontic surgery, Minerva Stomatol 59:625, 2010. Sari S, Durutűrk L: Radiographic evaluation of periapical healing outcomes in endodontic microsurgery: a retrospective study, Taschieri S, Weinstein T, Tsesis I, et al: Magnifying loupes of permanent teeth with periapical lesions after extrusion of J Endod 37:927, 2011. Erratum in J Endod 37:1595, versus surgical microscope in endodontic surgery: AH Plus sealer, Oral Surg Oral Med Oral Pathol Oral Radiol 2011a. a four-year retrospective study, Aust Endod J 39:78, Endod 104:e54, 2007. Song M, Kim E: A prospective randomized controlled study of 2013. Sawusch RH: Direct and indirect pulp capping with two new mineral trioxide aggregate and super ethoxy–benzoic acid as Teixeira LS, Demarco FF, Coppola MC, Bonow ML: Clinical and products, J Am Dent Assoc 104:459, 1982. root-end filling materials in endodontic microsurgery, J Endod radiographic evaluation of pulpotomies performed under Schwartz-Arad D, Yarom N, Lustig JP, Kaffe I: A retrospective 38:875, 2012. intrapulpal injection of anaesthetic solution, Int Endod J radiographic study of root-end surgery with amalgam and Song M, Kim SG, Lee SJ, et al: Prognostic factors of clinical 34:440, 2001. intermediate restorative material, Oral Surg Oral Med Oral outcomes in endodontic microsurgery: a prospective study, Tervit C, Paquette L, Torneck CD, et al: Proportion of healed Pathol Oral Radiol Endod 96:472, 2003. J Endod 39:1491, 2013a. teeth with apical periodontitis medicated with two percent Selden HS: Pulpoperiapical disease: diagnosis and healing: a Song M, Kim SG, Shin SJ, et al: The influence of bone tissue chlorhexidine gluconate liquid: a case-series study, J Endod clinical endodontic study, Oral Surg 27:271, 1974. deficiency on the outcome of endodontic microsurgery: a 35:1182, 2009. Seltzer S, Bender IB, Turkenkopf S: Factors affecting successful prospective study, J Endod 39:1341, 2013b. Testori T, Capelli M, Milani S, Weinstein RL: Success and failure repair after root canal therapy, J Am Dent Assoc 67:651, Song M, Nam T, Shin S-J, Kim E: Comparison of clinical in periradicular surgery: a longitudinal retrospective analysis, 1963. outcomes of endodontic microsurgery: 1 year versus Oral Surg Oral Med Oral Pathol Oral Radiol Endod 87:493, Setzer FC, Boyer KR, Jeppson JR, et al: Long-term prognosis of long-term follow-up, J Endod 40:490, 2014. 1999. endodontically treated teeth: a retrospective analysis of Song M, Shin SJ, Kim E: Outcomes of endodontic micro- Tilashalski KR, Gilbert GH, Boykin MJ, Shelton BJ: Root preoperative factors in molars, J Endod 37:21, 2011. resurgery: a prospective clinical study, J Endod 37:316, canal treatment in a population-based adult sample: Shah N: Non-surgical management of periapical lesions: a 2011b. status of teeth after endodontic treatment, J Endod 30:577, prospective study, Oral Surg Oral Med Oral Pathol 66:365, Stewart GG, Cobe H, Rappaport H: Study of new medicament in 2004. 1988. chemomechanical preparation of infected root canals, J Am Tobón SI, Arismendi JA, Marín ML, et al: Comparison between Shovelton DS, Friend LA, Kirk EE, Rowe AH: The efficacy of pulp Dent Assoc 63:33, 1961. a conventional technique and two bone regeneration capping materials: a comparative trial, Br Dent J 130:385, Stoll R, Betke K, Stachniss V: The influence of different factors techniques in periradicular surgery, Int Endod J 35:635, 1971. on the survival of root canal fillings: a 10-year retrospective 2002. Shuping GB, Orstavik D, Sigurdsson A, Trope M: Reduction of study, J Endod 31:783, 2005. Trope M, Delano EO, Orstavik D: Endodontic treatment of intracanal bacteria using nickel-titanium rotary Storms JL: Factors that influence the success of endodontic teeth with apical periodontitis: single vs. multivisit treatment, instrumentation and various medications, J Endod 26:751, treatment, J Can Dent Assoc (Tor) 35:83, 1969. J Endod 25:345, 1999. 2000. Strindberg LZ: The dependence of the results of pulp therapy on Tsesis I, Rosen E, Schwartz-Arad D, Fuss Z: Retrospective Siqueira JF Jr, Guimaraes-Pinto T, Rocas IN: Effects of certain factors: an analytic study based on radiographic and evaluation of surgical endodontic treatment: traditional versus chemomechanical preparation with 2.5% sodium hypochlorite clinical follow-up examinations, 1956, Mauritzon. modern technique, J Endod 32:412, 2006. and intracanal medication with calcium hydroxide on Sumi Y, Hattori H, Hayashi K, Ueda M: Ultrasonic root-end Vallecillo Capilla M, Muñoz Soto E, Reyes Botella C, et al: cultivable bacteria in infected root canals, J Endod 33:800, preparation: clinical and radiographic evaluation of results, Periapical surgery of 29 teeth: a comparison of conventional 2007. J Oral Maxillofac Surg 54:590, 1996. technique, microsaw and ultrasound, Med Oral 7:46, 50, Siqueira JF Jr, Magalhaes KM, Rocas IN: Bacterial reduction Sundqvist G, Figdor D, Persson S, Sjogren U: Microbiologic 2002. in infected root canals treated with 2.5% NaOCl as an analysis of teeth with failed endodontic treatment and the Vianna ME, Horz HP, Conrads G, et al: Effect of root canal irrigant and calcium hydroxide/camphorated outcome of conservative re-treatment, Oral Surg Oral Med procedures on endotoxins and endodontic pathogens, paramonochlorophenol paste as an intracanal dressing, Oral Pathol Oral Radiol Endod 85:86, 1998. Oral Microbiol Immunol 22:411, 2007. J Endod 33:667, 2007. Swartz DB, Skidmore AE, Griffin JA: Twenty years of endodontic Villa-Machado PA, Botero-Ramírez X, Tobón-Arroyave SI: Siqueira JF Jr, Rocas IN, Paiva SS, et al: Bacteriologic success and failure, J Endod 9:198, 1983. 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Taschieri S, Corbella S, Tsesis I, et al: Effect of guided tissue prospective clinical study with a one-year follow-up, Int Siqueira JF Jr, Rocas IN, Provenzano JC, et al: Relationship regeneration on the outcome of surgical endodontic Endod J 34:520, 2001. between Fcgamma receptor and interleukin-1 gene treatment of through-and-through lesions: a retrospective von Arx T, Jensen SS, Hanni S: Clinical and radiographic polymorphisms and post-treatment apical periodontitis, study at 4-year follow-up, Oral Maxillofac Surg 15:153, assessment of various predictors for healing outcome 1 year J Endod 35:1186, 2009. 2011. after periapical surgery, J Endod 33:123, 2007. 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Siqueira JF Jr, Rôças IN, Riche FN, Provenzano JC: Clinical Taschieri S, Del Fabbro M, Testori T, et al: Endodontic surgery von Arx T, Kurt B: Root-end cavity preparation after apicoectomy outcome of the endodontic treatment of teeth with apical using 2 different magnification devices: preliminary results of using a new type of sonic and diamond-surfaced retrotip: periodontitis using an antimicrobial protocol, Oral Surg Oral a randomized controlled study, J Oral Maxillofac Surg a 1-year follow-up study, J Oral Maxillofac Surg 57:656, Med Oral Pathol Oral Radiol Endod 106:757, 2008. 64:235, 2006. 1999. CHAPTER 11 Evaluation of Outcomes 531

Von Arx T, Peñarrocha M, Jensen S: Prognostic factors in apical Oral Med Oral Pathol Oral Radiol Endod 108:784, Witherspoon DE, Small JC, Harris GZ: Mineral trioxide aggregate surgery with root-end filling: a meta-analysis, J Endod 2009. pulpotomies: a case series outcomes assessment, J Am Dent 36:957, 2010. Waly NG: A five-year comparative study of calcium Assoc 137:610, 2006. Waikakul A, Punwutikorn J: Clinical study of retrograde filling hydroxide-glutaraldehyde pulpotomies versus calcium Witherspoon DE, Small JC, Regan JD, Nunn M: Retrospective with gold leaf: comparison with amalgam, Oral Surg Oral hydroxide pulpotomies in young permanent molars, Egypt analysis of open apex teeth obturated with mineral trioxide Med Oral Pathol 71:228, 1991. Dent J 41:993, 1995. aggregate, J Endod 34:1171, 2008. Wälivaara DÅ, Abrahamsson P, Fogelin M, Isaksson S: Wang CH, Chueh LH, Chen SC, et al: Impact of diabetes Xavier AC, Martinho FC, Chung A. et al: One-visit versus Super-EBA and IRM as root-end fillings in periapical surgery mellitus, hypertension, and coronary artery disease on tooth two-visit root canal treatment: effectiveness in the removal with ultrasonic preparation: a prospective randomized clinical extraction after nonsurgical endodontic treatment, J Endod of endotoxins and cultivable bacteria, J Endod 39:959, study of 206 consecutive teeth, Oral Surg Oral Med Oral 37:1, 2011. 2013. Pathol Oral Radiol Endod 112:258, 2011. Wang CS, Arnold RR, Trope M, Teixeira FB: Clinical efficiency of Yared GM, Dagher FE: Influence of apical enlargement on Wälivaara DA, Abrahamsson P, Isaksson S, et al: Prospective 2% chlorhexidine gel in reducing intracanal bacteria, J Endod bacterial infection during treatment of apical periodontitis, study of periapically infected teeth treated with periapical 33:1283, 2007. J Endod 20:535, 1994. surgery including ultrasonic preparation and retrograde Weiger R, Rosendahl R, Löst C: Influence of calcium hydroxide Zeldow BI, Ingle JI: Correlation of the positive culture to the intermediate restorative material root-end fillings, J Oral intracanal dressings on the prognosis of teeth with prognosis of endodontically treated teeth: a clinical study, Maxillofac Surg 65:931, 2007. endodontically induced periapical lesions, Int Endod J J Am Dent Assoc 66:9, 1963. Wälivaara DA, Abrahamsson P, Sämfors KA, Isaksson S: 33:219, 2000. Zmener O, Pameijer CH: Clinical and radiographical evaluation Periapical surgery using ultrasonic preparation and Weiss M: Pulp capping in older patients, N Y State Dent J of a resin-based root canal sealer: a 5-year follow-up, thermoplasticized gutta-percha with AH Plus sealer 32:451, 1966. J Endod 33:676, 2007. or IRM as retrograde root-end fillings in 160 consecutive Werts R: Endodontic treatment: a five-year follow-up, Dent Zuolo ML, Ferreira MO, Gutmann JL: Prognosis in periradicular teeth: a prospective randomized clinical study, Oral Surg Survey 51:29, 1975. surgery: a clinical prospective study, Int Endod J 33:91, 2000. CHAPTER 12

Structure and Functions of the Dentin-Pulp Complex

INGE FRISTAD | ELLEN BERGGREEN1

CHAPTER OUTLINE Morphologic Zones of the Pulp Innervation The Pulp-Dentin Complex Steps and Mechanisms in Pain Perception Odontoblast Layer Detection: The First Step in Pain Perception Cell-Poor Zone Neuropeptides Cell-Rich Zone Pulp Testing Pulp Proper Sensitivity of Dentin Cells of the Pulp Peripheral Sensitization Odontoblast Hyperalgesia and Allodynia Odontoblast Process Inflammatory Mediators Relationship of Odontoblast Structure to Secretory Function Painful Pulpitis Pulp Fibroblast Plasticity of Intradental Nerve Fibers Macrophage Tissue Injury and Deafferentation Dendritic Cell Processing: The Second Step in Pain Perception Lymphocyte Perception: Thalamus to Cortex Mast Cell Vascular Supply Metabolism Regulation of Pulpal Blood Flow The Pulpal Interstitium And Ground Substance Fluid Drainage Hyaluronan Circulation in the Inflamed Pulp Elastic Fibers Pulpal Repair The Inflamed Interstitium Pulpal Calcifications Connective Tissue Fibers of the Pulp Age Changes The Trigeminal System

MORPHOLOGIC ZONES OF THE PULP Odontoblast Layer The outermost stratum of cells of the healthy pulp is the odon- The Pulp-Dentin Complex toblast layer (Figs. 12-1 and 12-2). This layer is located imme- The dental pulp and dentin function as a unit, and the odon- diately subjacent to the predentin. The odontoblast processes, toblasts represent a crucial element in this system. The odon- however, pass on through the predentin into the inner part of toblasts are located in the periphery of the pulp tissue, with dentin. Consequently, the odontoblast layer is actually com- extensions into the inner part of dentin. Dentin would not exist posed of the cell bodies of odontoblasts. In addition, capillar- unless produced by odontoblasts, and the dental pulp is depen- ies, nerve fibers, and dendritic cells may be found among the dent on the protection provided by the dentin and enamel. odontoblasts. Likewise, integrated dynamics of the pulp-dentin complex In the coronal portion of a young pulp that is actively secret- imply that impacts on dentin may affect the pulpal components ing collagen, the odontoblasts assume a tall columnar form.62 and that disturbances in the dental pulp will in turn affect the The odontoblasts vary in height; consequently, their nuclei are quantity and quality of the dentin produced. not all at the same level and are aligned in a staggered array, often described as a palisade appearance. This organization 1The authors acknowledge the outstanding work of Drs. Henry Trowbridge, Syngcuk makes the layers appear to be three to five cells in thickness Kim, Hideaki Suda, David H. Pashley, and Fredrick R. Liewehr in previous editions of even though there is only one actual layer of odontoblasts. this text. The present chapter is built on their foundational work. Between adjacent odontoblasts there are small intercellular

532 CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 533

Dentin

Predentin Odontoblast layer Cell-poor zone Cell-rich zone

Pulp proper

FIG. 12-1 Morphologic zones of the mature pulp.

Dentin

Odontoblast process

Predentin

Desmosome-like junction Odontoblast

Capillary

Nerve fiber

Cell-poor Fibroblast Zone

Nerve fiber Cell-rich Venule zone Arteriole

FIG. 12-2 Diagrammatic representation of the odontoblast layer and subodontoblastic region of the pulp. spaces approximately 30 to 40 nm in width. Odontoblast cell are able to spread out laterally.240 During maturation and aging, bodies are connected by tight and gap junctional com- there is a continued ongoing crowding in the odontoblast layer, plexes.29,62,160 Gap junctions are formed by connexin proteins113 particularly in the coronal pulp, due to narrowing of the pulp that permit cell-to-cell passage of signal molecules. space. Apoptosis of odontoblasts seems to adjust for this The odontoblast layer in the coronal pulp contains more limited space during development.252 cells per unit area than in the radicular pulp.240 Whereas the There is a series of specialized cell-to-cell junctions (i.e., odontoblasts of the mature coronal pulp are usually columnar, junctional complexes), including desmosomes (i.e., zonula those in the midportion of the radicular pulp are more cuboidal adherens), gap junctions (i.e., nexuses), and tight junctions (Fig. 12-3).62 Near the apical foramen, the odontoblasts appear (i.e., zonula occludens) that connect adjacent odontoblasts. as a squamous layer of flattened cells. Because fewer dentinal Spot desmosomes located in the apical part of odontoblast cell tubules per unit area are present in the root than in the crown bodies mechanically join odontoblasts together. The numerous of the tooth, the odontoblast cell bodies are less crowded and gap junctions provide permeable pathways through which 534 PART II The Advanced Science of Endodontics

FIG. 12-3 Low columnar odontoblasts of the radicular pulp. The cell-rich zone is inconspicuous.

M

FIG. 12-4 A, Electron micrograph of a mouse molar odontoblast M demonstrating gap junctions (arrows), nucleus (N), mitochondria (M), Golgi complex (G), and rough endoplasmic reticulum (RER). N B, High magnification of a section fixed and stained with lanthanum nitrate to demonstrate a typical gap junction. (Courtesy Dr. Charles F. Cox, School of Dentistry, University of Alabama.)

G

B

RER A

signal molecules can pass between cells (Fig. 12-4) to synchro- width that is relatively free of cells (see Fig. 12-1) and hence nize secretory activity that produces relatively uniform preden- is called the cell-free layer of Weil. It is traversed by blood capil- tin layers (see Fig. 12-2). These junctions are most numerous laries, unmyelinated nerve fibers and the slender cytoplasmic during the formation of primary dentin. Gap junctions and processes of fibroblasts (seeFig. 12-2). The presence or absence desmosomes have also been observed joining odontoblasts to of the cell-poor zone depends on the functional status of the the processes of fibroblasts in the subodontoblastic area. Tight pulp.62 It may not be apparent in young pulps, where dentin junctions are found mainly in the apical part of odontoblasts forms rapidly, or in older pulps, where reparative dentin is in young teeth. These structures consist of linear ridges and being produced. grooves that close off the intercellular space. However, tracer studies suggest direct passage of small elements from subodon- Cell-Rich Zone toblastic capillaries to predentin and dentin between the In the subodontoblastic area, there is a stratum containing a odontoblasts.352 It appears that tight junctions determine relatively high proportion of fibroblasts compared with the the permeability of the odontoblast layer when dentin is more central region of the pulp (see Fig. 12-1). It is much more covered by enamel or cementum by restricting the passage of prominent in the coronal pulp than in the radicular pulp. molecules, ions, and fluid between the extracellular compart- Besides fibroblasts, the cell-rich zone may include a variable ments of the pulp and predentin.29 During cavity preparation, number of immune cells like macrophages and dendritic cells, these junctions are disrupted, thereby increasing dentin but also undifferentiated mesenchymal stem cells. permeability.375,376 On the basis of evidence obtained in rat molar teeth, it has been suggested126 that the cell-rich zone forms as a result of Cell-Poor Zone peripheral migration of cells populating the central regions of Immediately subjacent to the odontoblast layer in the coronal the pulp, commencing at about the time of tooth eruption. pulp, there is often a narrow zone approximately 40 µm in Migration of immunocompetent cells out of and into the CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 535 cell-rich zone has been demonstrated as a result of antigenic cytoplasm, consists of an assembly of smooth-walled vesicles challenge.421 Although cell division within the cell-rich zone is and cisternae. Numerous mitochondria are evenly distributed rare in normal pulps, the death of the odontoblasts triggers a throughout the cell body. RER is particularly prominent, con- great increase in the rate of mitosis. Because odontoblasts are sisting of closely stacked cisternae forming parallel arrays that postmitotic cells, irreversibly injured odontoblasts are replaced are dispersed diffusely within the cytoplasm. Numerous ribo- by cells that migrate from the cell-rich zone onto the inner somes closely associated with the membranes of the cisternae surface of the dentin.98 This mitotic activity is probably the first mark the sites of protein synthesis. Within the lumen of the step in the formation of a new odontoblast layer.73,256,258,339 cisternae, filamentous material (probably representing newly Studies implicate stem cells as a source for these replacement synthesized protein) can be observed. odontoblasts.346 The odontoblast appears to synthesize mainly type I colla- gen,208,401 although small amounts of type V collagen have been Pulp Proper found in the extracellular matrix (ECM). In addition to pro- The pulp proper is the central mass of the pulp (see Fig. 12-1). teoglycans38,83,123 and collagen,208,220 the odontoblast secretes It consists of loose connective tissue and contains the larger dentin sialoprotein40 and phosphophoryn,40,69 a highly phos- blood vessels and nerves. The most prominent cell in this zone phorylated phosphoprotein involved in extracellular mineral- is the fibroblast. ization.40,77 Phosphophoryn is unique to dentin and is not found in any other mesenchymal cell types.77 The odontoblast CELLS OF THE PULP also secretes both acid phosphatase and alkaline phosphatase. The latter enzyme is closely linked to mineralization, but the Odontoblast precise role of alkaline phosphatase in dentinogenesis is not Because odontoblasts are responsible for dentinogenesis, both completely understood. Acid phosphatase, a lysosomal enzyme, during tooth development and aging, the odontoblast is the may be involved in digesting material that has been resorbed most characteristic and specialized cell of the dentin-pulp from predentin matrix.87 complex. During dentinogenesis, the odontoblasts form dentin In contrast to the active odontoblast, the resting or inactive and the dentinal tubules, and their presence within the tubules odontoblast has a decreased number of organelles and may makes dentin a living responsive tissue. become progressively shorter.62,240 These changes can begin Dentinogenesis, osteogenesis, and cementogenesis are in with the completion of root development and eruption when many respects quite similar, and odontoblasts, osteoblasts, and dentin production shifts from primary to secondary dentin. cementoblasts have many characteristics in common. Each of Direct actions of odontoblasts on dental nerves and vice these cells produces a matrix composed of collagen fibrils, versa have been proposed based on the excitability of odonto- noncollagenous proteins, and proteoglycans that are capable of blasts, the differential expression of receptors for neuropep- undergoing mineralization. The ultrastructural characteri­ tides on odontoblasts (Fig. 12-6), the demonstration of the stics of odontoblasts, osteoblasts, and cementoblasts are thermosensitive transient receptor potential (TRP) ion chan- likewise similar in that each exhibits a highly ordered rough nels, and the finding that all nine voltage-gated sodium chan- endoplasmic reticulum (RER), a prominent Golgi complex, nels are variably expressed on odontoblasts in developing, secretory granules, and numerous mitochondria. In addition, mature, and aging rat teeth.52,71,107,221,231,232 In addition, a pos- these cells are rich in RNA, and their nuclei contain one or sible function of odontoblast in immune regulation has been more prominent nucleoli. These are the general characteristics proposed by the finding of innate immune components in the of protein-secreting cells. odontoblast layer.382 Hence, the odontoblasts should be capable The most significant differences among odontoblasts, osteo- of recognizing and differentially responding to bacterial com- blasts, and cementoblasts are their morphologic characteristics ponents, thereby serving immune and pulp-dentine barrier and the anatomic relationship between the cells and the functions. mineralized structures they produce. Whereas osteoblasts and cementoblasts are polygonal to cuboidal in form, the fully developed odontoblast of the coronal pulp is a tall columnar Odontoblast Process cell.62,240 In bone and cementum, some of the osteoblasts and A dentinal tubule forms around each of the major odontoblas- cementoblasts become entrapped in the matrix as osteocytes tic processes. The odontoblast process occupies most of the or cementocytes, respectively. The odontoblast, on the other space within the tubule and coordinates the formation of peri- hand, leaves behind a cellular process to form the dentinal tubular dentin. tubule, and the cell body resides outside the mineralized tissue. Microtubules and microfilaments are the principal ultra- Lateral branches between the major odontoblast processes structural components of the odontoblast process and its interconnect178,253 through canaliculi, just as osteocytes and lateral branches.110,158 Microtubules extend from the cell body cementocytes are linked through the canaliculi in bone and out into the process.110,151 These straight structures follow a cementum. This provides a pathway for intercellular commu- course that is parallel with the long axis of the cell and impart nication and circulation of fluid and metabolites through the the impression of rigidity. Although their precise role is mineralized matrix. unknown, theories as to their functional significance suggest The ultrastructural features of the odontoblast have been the that they may be involved in cytoplasmic extension, transport subject of numerous investigations. The cell body of the active of materials, or the provision of a structural framework. Occa- odontoblast has a large nucleus that may contain up to four sionally, mitochondria can be found in the process where it nucleoli (Fig. 12-5). The nucleus is situated at the basal end of passes through the predentin. the cell and is contained within a nuclear envelope. A well- The plasma membrane of the odontoblast process closely developed Golgi complex, centrally located in the supranuclear approximates the wall of the dentinal tubule. Localized 536 PART II The Advanced Science of Endodontics

Predentin

Microtubule Secretory vesicle Lysosome

Mitochondria

Centriole Golgi complex

RER Cilia

Nucleus

Nucleolus

FIG. 12-5 Diagram of a fully differentiated odontoblast. RER, Rough endoplasmic reticulum.

electron-dense limiting membrane called the lamina limi- tans.260,354,398 A narrow space separates the limiting membrane from the plasma membrane of the odontoblast process, except for the areas where the process is constricted. Dentin In restoring a tooth, the removal of enamel and dentin often Odontoblast disrupts odontoblasts.41,45,73,80,202,257 It would be of considerable Odontoblast process Nucleus clinical importance to establish the extent of the odontoblast processes in human teeth, because with this knowledge, the clinician would be in a better position to estimate the impact of the restorative procedure on the underlying odontoblasts. However, the extent to which the process penetrates into dentin has been a matter of considerable controversy. It has long been thought that the process is present throughout the full thickness of dentin. Although ultrastructural studies using transmission electron microscopy have described the process FIG. 12-6 Confocal microscopic image showing an odontoblast with its as being limited to the inner third of the dentin,48,111,354,398 it process expressing neurokinin 2 receptor. Neurokinin 2 has affinity to all the should be noted that this could possibly be the result of shrink- neuropeptides of the neurokinin family. The dotted line represents the border age occurring during fixation and dehydration. Other studies of the predentin. (From Fristad I, Vandevska-Radunovic V, Fjeld K, et al: NK1, employing scanning electron microscopy have described the NK2, NK3 and CGRP1 receptors identified in rat oral soft tissues, and in bone process extending further into the tubule, often as far as the and dental hard tissue cells, Cell Tissue Res 311:383-391, 2003.) dentoenamel junction (DEJ),130,179,337,412 but it has been sug- gested that what has been observed in scanning electron micro- constrictions in the process occasionally produce relatively graphs is actually the lamina limitans.354,355,398 large spaces between the tubule wall and the process. Such In an attempt to resolve this issue, monoclonal antibodies spaces may contain collagen fibrils and fine granular material directed against microtubules were used to demonstrate that presumably represents ground substance (see also The tubulin in the microtubules of the process. Immunoreactivity Pulpal Interstitium and Ground Substance in this chapter). was observed throughout the dentinal tubule, suggesting that The peritubular dentin matrix within the tubule is lined by an the process extends throughout the thickness of dentin.337 CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 537

However, a study employing confocal microscopy found that fibrils at the base of the odontoblast process are approximately odontoblast processes in rat molars do not extend to the outer 15 nm in diameter, fibrils in the region of the calcification front dentin or DEJ, except during the early stages of tooth develop- have attained a diameter of about 50 nm. ment.48 It is likely that the walls of tubules contain many Similar tracer studies400 have elucidated the pathway of syn- proteins originally derived from odontoblasts that no longer thesis, transport, and secretion of the predentin proteoglycans. remain at that site. Because dentin matrix has no turnover, The protein moiety of these molecules is synthesized by the these antigens remain fixed in place. From a clinical perspec- RER of the odontoblast, whereas sulfation and addition of the tive, it is important to remember that these processes in the glycosaminoglycan (GAG) moieties to the protein molecules tubules represent appendages from living odontoblasts in the take place in the Golgi complex. Secretory vesicles then trans- pulp, which explains why the dentin must be considered a vital port the proteoglycans to the base of the odontoblast process, tissue, the destruction of which will affect the pulp. where they are secreted into the predentin matrix. Proteogly- The odontoblast is considered to be a fixed postmitotic cell cans, principally chondroitin sulfate, accumulate near the cal- in that once it has fully differentiated, it apparently cannot cification front. The role of the proteoglycans is speculative, undergo further cell division. If this is indeed the case, the life but mounting evidence suggests that they act as inhibitors of span of the odontoblast coincides with the life span of the calcification by binding calcium. It appears that just before viable pulp. However, its metabolic activity can be dynamically calcification, the proteoglycans are removed, probably by lyso- altered (described under the heading Pulpal Repair). somal enzymes secreted by the odontoblasts.84 Pulp Fibroblast Relationship of Odontoblast Structure to Fibroblasts are the most numerous cells of the pulp. They Secretory Function appear to be tissue-specific cells capable of giving rise to cells Studies using radiolabeled chemicals have shed a great deal that are committed to differentiation (e.g., odontoblast-like of light on the functional significance of the cytoplasmic cells) if given the proper signal. These cells synthesize types organelles of the active odontoblast.401,400 In experimental I and III collagen, as well as proteoglycans and GAGs. animals, intraperitoneal injection of a collagen precursor Thus, they produce and maintain the matrix proteins of the (e.g., 3H-proline) is followed by autoradiographic labeling of ECM. Because they are also able to phagocytose and digest the odontoblasts and predentin matrix401 (Fig. 12-7). Rapid collagen, fibroblasts are responsible for collagen turnover in incorporation of the isotope in the RER soon leads to labeling the pulp. of the Golgi complex in the area where the procollagen is Although distributed throughout the pulp, fibroblasts are packed and concentrated into secretory vesicles. Radiolabeled particularly abundant in the cell-rich zone. The early differen- vesicles can then be followed along their migration pathway tiating fibroblasts are polygonal and appear to be widely sepa- until they reach the base of the odontoblast process. Here they rated and evenly distributed within the ground substance. fuse with the cell membrane and release their tropocollagen Cell-to-cell contacts are established between the multiple pro- molecules into the predentin matrix by the process of cesses that extend out from each of the cells. Many of these exocytosis. contacts take the form of gap junctions that provide for elec- It is now known that collagen fibrils precipitate from a solu- tronic coupling or chemical signaling from one cell to another. tion of secreted tropocollagen and that the aggregation of In terms of ultrastructure, the organelles of the immature fibro- fibrils occurs on the outer surface of the odontoblast plasma blasts are generally in a rudimentary stage of development, membrane. Fibrils are released into the predentin and increase with an inconspicuous Golgi complex, numerous free ribo- in thickness as they approach the mineralized matrix. Whereas somes, and sparse RER. As they mature, the cells become stellate in form, and the Golgi complex enlarges, the RER proliferates, secretory vesicles appear, and the fibroblasts take on the characteristic appearance of protein-secreting cells. In addition, collagen fibrils accumulate along the outer surface of the cell body. With an increase in the number of blood vessels, nerves, and collagen fibers, there is a relative decrease in the number of fibroblasts in the pulp. Many fibroblasts of the pulp are characterized by being rela- tively undifferentiated. A more modern term for undifferenti- ated cells is stem cells. Many pulpal cells do seem to remain in a relatively undifferentiated modality, compared with fibro- blasts of most other connective tissues.136 This perception has been supported by the observation of large numbers of reticulin-like fibers in the pulp. Reticulin fibers have an affinity for silver stains and are similar to the argyrophilic fibers of the pulp. However, in a careful review, it appears that actual reticu- lin fibers may not be present in the pulp; instead the previously described fibers are actually argyrophilic collagen fibers.15 The fibers apparently acquire a GAG sheath, and it is this sheath that is impregnated by silver stains. In the young pulp, the FIG. 12-7 Autoradiograph demonstrating odontoblasts and predentin in a nonargyrophilic collagen fibers are sparse, but they progres- developing rat molar 1 hour after intraperitoneal injection of 3H-proline. sively increase in number as the pulp ages. 538 PART II The Advanced Science of Endodontics

Many experimental models have been developed to study Dendritic Cell wound healing in the pulp, particularly dentinal bridge forma- Dendritic cells are accessory cells of the immune system. tion after pulp exposure or pulpotomy. One study98 demon- Similar cells are found in the epidermis and mucous mem- strated that mitotic activity preceding the differentiation of branes, where they are called Langerhans cells.173,280 Dendritic replacement odontoblasts appears to occur primarily among cells are primarily found in lymphoid tissues, but they are also perivascular fibroblasts. widely distributed in connective tissues, including the pulp319 Pulpal fibroblasts seem to take active part in signaling path- (Fig. 12-9). These cells are termed antigen-presenting cells and ways in the dental pulp. For example, fibroblast growth and are characterized by dendritic cytoplasmic processes and the synthesis are stimulated by neuropeptides; in turn, fibroblasts presence of class II MHC complexes on their cell surface produce nerve growth factor (NGF) and proinflammatory (Fig. 12-10). In the normal pulp they are mostly located in the cytokines during inflammation.31,408,413 NGF plays an impor- periphery of the coronal pulp close to the predentin, but they tant role not only in tooth development but also in regulating migrate centrally in the pulp after antigenic challenge.421 They neuronal and possibly odontoblast responses to injury via acti- are known to play a central role in the induction of T cell– vation of similar neurotrophin receptors expressed on both dependent immunity. Like antigen-presenting macrophages, cell types (see also Plasticity of Intradental Nerve Fibers in dendritic cells engulf protein antigens and then present an this chapter).408 assembly of peptide fragments of the antigens and MHC class II molecules. It is this assembly that T cells can recognize. Then Macrophage the assembly binds to a T-cell receptor and T-cell activation Macrophages are monocytes that have left the bloodstream, entered the tissues, and differentiated into various subpopula- tions. The different subpopulations can be studied by their anti- genic properties in immunohistochemical studies. Many are found in close proximity to blood vessels. A major subpopulation of macrophages is active in endocytosis and phagocytosis (Fig. 12-8). Because of their mobility and phagocytic activity, they are able to act as scavengers, removing extravasated red blood cells, dead cells, and foreign bodies from the tissue. Ingested material M is destroyed by the action of lysosomal enzymes. Another subset of macrophages participates in immune reactions by processing antigen and presenting it to memory T cells.281 The processed antigen is bound to class II major histocompatibility complex LY (MHC) molecules on the macrophage, where it can interact with P specific receptors present on naive or memory T cells.133 Such interactions are essential for T cell–dependent immunity. Similar to fibroblasts, macrophages take an active part in the signaling pathways in the pulp. When activated by the appropriate inflam- matory stimuli, macrophages are capable of producing a large variety of soluble factors, including interleukin 1, tumor necrosis factor, growth factors, and other cytokines. One study showed that a subset of macrophages express lymphatic markers, indicat- ing a link between macrophages and lymphatic function and FIG. 12-8 Immunoelectron micrograph of an HLA-DR+ matured macro- development.19 phage (M) in the human pulp, showing a phagosome (P). Ly, lymphocyte.

D OB

FIG. 12-9 Class II antigen-expressing dendritic cells in the pulp and dentin border zone in normal human pulp, as demonstrated by immunocytochemistry. D, dentin; OB, odontoblastic layer. CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 539

FIG. 12-10 Immunoelectron micrograph of a dendritic-like cell (DC) in the human pulp, showing a dendritic profile with a relatively small number of lysosomal structures. DC

T cell Helper T cell receptor MHC class II (self) Activation

Antigen peptide Antigen-presenting cell (not self) Antigen

FIG. 12-11 Function of MHC class II molecule-expressing cells. They act as antigen-presenting cells that are essential for the induction of helper T cell–dependent immune responses. occurs (Fig. 12-11). Fig. 12-12 shows a cell-to-cell contact an important inflammatory mediator, as well as many other between a dendritic-like cell and a lymphocyte. chemical factors. Lymphocyte Hahn and colleagues133 reported finding T lymphocytes in METABOLISM normal pulps from human teeth. T8 (suppressor) lymphocytes The metabolic activity of the pulp has been studied by measur- were the predominant T-lymphocyte subset present in these ing the rate of oxygen consumption and the production of samples. Lymphocytes have also been observed in the pulps of carbon dioxide or lactic acid.26,95,96,97,135,320 An investigation impacted teeth.201 The presence of macrophages, dendritic employed an oxygen-sensitive microelectrode inserted into a cells, and T lymphocytes indicates that the pulp is well rat incisor pulp with a micromanipulator.418 The authors equipped with cells required for the initiation of immune reported that odontoblasts consumed O2 at the rate of responses.173,319 B lymphocytes are scarcely found in the normal 3.2 ± 0.2 ml/min/100 g of pulp tissue.418 uninflamed pulp. Because of the relatively sparse cellular composition of the pulp, the rate of oxygen consumption is low in comparison Mast Cell with that of most other tissues. During active dentinogenesis, Mast cells are widely distributed in connective tissues, where metabolic activity is much higher than after the crown is com- they occur in small groups in relation to blood vessels. Mast pleted. As would be anticipated, the greatest metabolic activity cells are seldom found in the normal pulp tissue, although they is found in the region of the odontoblast layer, and the lowest are routinely found in chronically inflamed pulps.319 The mast is found in the central pulp, where most of the nerves and cell has been the subject of considerable attention because of blood vessels are located.25 its dramatic role in inflammatory reactions. The granules of In addition to the usual glycolytic pathway, the pulp has the mast cells contain heparin, an anticoagulant, and histamine, ability to produce energy through a phosphogluconate (i.e., 540 PART II The Advanced Science of Endodontics

FIG. 12-12 Immunoelectron micrograph of a cell resembling a dendritic cell and a lymphocyte. They show cell-to-cell contact.

pentose phosphate) shunt type of carbohydrate metabolism,97 a metabolic pathway that permits tissues to function under varying degrees of ischemia. This could explain how the pulp manages to withstand periods of low perfusion resulting from induced by epinephrine-containing infiltra- tion anesthesia.190 Several commonly used dental materials (e.g., eugenol, zinc oxide and eugenol, calcium hydroxide, silver amalgam) inhibit oxygen consumption by pulp tissue, indicating that these agents may be capable of depressing the metabolic activity of pulpal cells.96,172 One study135 found that application of orth- odontic force to human premolars for 3 days resulted in a 27% reduction in respiratory activity in the pulp. This study utilized carbon-14-labeled succinic acid in the medium. As cells metab- 14 olize succinic acid, they produce CO2 that can be trapped and quantitated by a liquid scintillation counter.135 This technique FIG. 12-13 Delicate network of pulpal collagen fibers as demonstrated by requires only a few milligrams of tissue. the Pearson silver impregnation method.

THE PULPAL INTERSTITIUM AND GROUND SUBSTANCE connective tissue fibers also synthesize the major constituents The interstitium consists of the interstitial fluid and the inter- of the ECM. Whereas the fibers and cells have recognizable stitial (extracellular) matrix and occupies the extracellular and shapes, the ECM is described as being amorphous. It is gener- extravascular space. It is amorphous and generally regarded as ally regarded as a gel rather than a solid. Because of its content a gel rather than a solid. Its constituents are similar in all of polyanionic polysaccharides, the ECM is responsible for the tissues, but their relative amount varies. The major structural water-holding properties of connective tissues.404 component of the interstitium is collagen (Fig. 12-13). The Nearly all proteins of the ECM are glycoproteins.237 Proteo- network of collagen fibers also supports the other components glycans are an important subclass of glycoproteins.123 These of the interstitium, the proteoglycans, hyaluronan, and elastic molecules support cells, provide tissue turgor, and mediate a fibers. The two former components represent the glycosami- variety of cell interactions. They have in common the presence noglycans of the interstitial matrix. of GAG chains and a protein core to which the chains are Because of its content of polyanionic polysaccharides, the linked. Except for heparan sulfate and heparin, the chains are interstitium is responsible for the water-holding properties of composed of disaccharides. The primary function of GAG connective tissues and acts as a molecular sieve in regulating chains is to act as adhesive molecules that can bond to cell the diffusion of substances through this space. The magnitude surfaces and other matrix molecules. of the excluded volume has important consequences because Fibronectin is a major surface glycoprotein that, together the effective protein concentration in the interstitium is higher with collagen, forms an integrated fibrillary network that influ- than the value that would be estimated from fluid volume ences adhesion, motility, growth, and differentiation of cells. per se.404 Laminin, an important component of basement membranes, Connective tissue consists of cells and fibers, both binds to type IV collagen and cell surface receptors.118 Tenascin embedded in ground substance or ECM. Cells that produce is another substrate adhesion glycoprotein. CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 541

In the pulp, the principal proteoglycans include hyaluronic The Inflamed Interstitium acid dermatan sulfate, heparan sulfate, and chondroitin Hyaluronidases, and chondroitin sulfatases of lysosomal and sulfate.130 The proteoglycan content of pulp tissue decreases bacterial origin, are examples of the hydrolytic enzymes that approximately 50% with tooth eruption.220 During active den- can attack components of the interstitium. During infection tinogenesis, chondroitin sulfate is the principal proteoglycan, and inflammation, the physical properties of the pulp tissue particularly in the odontoblast and predentin layer, where it is may then be altered due to production of such degrading somehow involved with mineralization; with tooth eruption, enzymes.145,318 In addition to their own damaging effect, they hyaluronic acid and dermatan sulfate increase, and chondroitin may also pave the way for the deleterious effects of bacterial sulfate decreases greatly. toxins, increasing the magnitude of the damage.177 The consistency of a connective tissue (e.g., the pulp) is The pathways of inflammation and infection are strongly largely determined by the proteoglycan components of the influenced by the particular composition of the interstitium in ground substance. The long GAG chains of the proteoglycan every tissue and its degradation by either host or microbial molecules form relatively rigid coils constituting a network enzymes. that holds water, thus forming a characteristic gel. Hyaluronic acid in particular has a strong affinity for water and is a major component of ground substance in tissues with a large fluid CONNECTIVE TISSUE FIBERS OF THE PULP content, such as Wharton’s jelly of the umbilical cord. The Two types of structural proteins are found in the pulp: collagen water content of the young pulp is very high (approximately and elastin. Elastin fibers are confined to the walls of arterioles 90%), so the ground substance forms a cushion capable of and, unlike collagen, are not a part of the ECM. protecting cells and vascular components of the tooth. A single collagen molecule, referred to as tropocollagen, Ground substance also acts as a molecular sieve in that consists of three polypeptide chains, designated as either it excludes large proteins. Cell metabolites, nutrients, and alpha-1 or alpha-2 depending on their amino acid composition wastes pass through the ground substance between cells and and sequence. In the human pulp, the amount of collagen is blood vessels. In some ways, ground substance can be likened reported to be 26% to 32% of dry weight in premolars and to an ion exchange resin, because the polyanionic chains of molars.378 Type I and type III collagen represent the major the GAGs bind cations. In addition, osmotic pressures can be subtypes of collagen in the pulp, and type I is found in thick altered by excluding osmotically active molecules. Thus, pro- striated fibrils throughout the pulp tissue.206,336 The different teoglycans can regulate the dispersion of interstitial matrix combinations and linkages of chains making up the tropocol- solutes, colloids, and water, and (in large measure) they lagen molecule have allowed collagen fibers and fibrils to be determine the physical characteristics of a tissue, such as classified into a number of types: the pulp. ◆ Type I collagen is found in skin, tendon, bone, dentin, Degradation of ground substance can occur in certain and pulp. inflammatory lesions that have a high concentration of macro- ◆ Type II collagen is found in cartilage. phage lysosomal enzymes. Proteolytic enzymes, hyaluroni- ◆ Type III collagen is found in most unmineralized connective dases, and chondroitin sulfatases of lysosomal and bacterial tissues. It is a fetal form found in the dental papilla and the origin are examples of the hydrolytic enzymes that can attack mature pulp. In bovine pulp, it constitutes 45% of the total components of the ground substance. The pathways of inflam- pulp collagen during all stages of development.378 mation and infection are strongly influenced by the state of ◆ Types IV and VII collagen are components of basement polymerization of the ground substance components. membranes. ◆ Type V collagen is a constituent of interstitial tissues. Hyaluronan ◆ Type VI collagen is a heterotrimer of three distinct chains, Another major structural component of the interstitial matrix alpha 2 (VI) and alpha 3 (VI), and is widely distributed is hyaluronan. It is an unbranched, random-coil molecule from in low concentrations in soft tissues at interfibrillar repeating nonsulfated disaccharide units and is found in the filaments. interstitium as free molecules or bound to cells, possibly via Type I collagen is synthesized by odontoblasts and osteoblasts; the connection to fibronectin.205 Its large molecular weight fibroblasts synthesize types I, III, V, and VII collagen. together with its protein structure accounts for its unique In collagen synthesis, the protein portion of the molecule is properties. It has a high viscosity even at low concentration, formed by the polyribosomes of the RER of connective tissue exhibits exclusion properties, and has a strong affinity cells. The proline and lysine residues of the polypeptide chains for water. are hydroxylated in the cisternae of the RER, and the chains are Hyaluronan is one of several types of GAGs in the pulp.219,237 assembled into a triple-helix configuration in the smooth endo- The hyaluronan receptor-1 is expressed on lymphatic vessels plasmic reticulum. The product of this assembly is termed and also on immune cells in the dental pulp.19 Hyaluronan is procollagen, and it has a terminal unit of amino acids known as removed from the tissue by the lymphatics and metabolized in the telopeptide of the procollagen molecule. When these mole- the lymph nodes101 and by endothelial cells in the liver.134,298 cules reach the Golgi complex, they are glycosylated and pack- aged in secretory vesicles. The vesicles are transported to the Elastic Fibers plasma membrane and secreted by way of exocytosis into the Elastic fibers constitute an elastin core and a surrounding extracellular milieu, thus releasing the procollagen. Here microfibrillar network and provide elasticity to the tissue.289 the terminal telopeptide is cleaved by a hydrolytic enzyme, and The amount of elastin in the interstitial matrix in most tissue the tropocollagen molecules begin aggregating to form collagen is small. There is no evidence for elastic fibers in the matrix in fibrils. It is believed that the GAGs somehow mediate aggrega- the pulp.134,298 tion of tropocollagen. The conversion of soluble collagen into 542 PART II The Advanced Science of Endodontics

Cortex Dura Thalamus Trigeminal ganglion

Eye

CF Sinus

TMJ

Tooth Spinal tract nucleus Muscle Skin

FIG. 12-14 Dense bundles of collagen fibers (CF) in the apical pulp.

insoluble fibers occurs as a result of cross-linking of tropocol- lagen molecules. The presence of collagen fibers passing from FIG. 12-15 Schematic drawing illustrating the convergence of sensory the dentin matrix between odontoblasts into the dental pulp information from teeth to higher brain centers. has been reported in fully erupted teeth.28 Larger collagen fiber bundles are much more numerous in the radicular pulp than experimental conditions, a nonpainful sensation (i.e., prepain) in the coronal pulp. The highest concentration of these larger has been reported.247 Thus, not all afferent neurons that inner- fiber bundles is usually found near the apex (Fig. 12-14). Pulp- vate the pulp are nociceptors. The innervation of the pulp ectomy procedures should engage the pulp with a barbed includes both afferent neurons, which conduct sensory impulses, broach in the region of the apex, as it generally affords the best and autonomic or efferent neurons,163 which provide neurogenic opportunity to remove this tissue intact.366 modulation of the microcirculation, inflammatory reactions,148 and perhaps regulate dentinogenesis.46 THE TRIGEMINAL SYSTEM The sympathetic innervation of teeth derives from the supe- rior cervical ganglion (SCG).8,301 Postganglionic sympathetic Innervation nerves travel with the internal carotid nerve, join the trigemi- Pain is a subjective phenomenon involving not only sensory nal nerve at the ganglion, and supply teeth and supporting physiologic responses, but also emotional, conceptual, and structures via the maxillary and mandibular division of the motivational aspects of behavior. The existence of peripheral trigeminal nerve.239 Sympathetic fibers appear with blood “nociceptive” (pain-detecting) sensory neurons forms the basis vessels at the time the vascular system is established in the for pain, and pain sensations of varying qualities and intensi- dental papilla.104 In the adult tooth pulp, sympathetic fibers ties are evoked by activation of the intradental nerves innervat- form plexuses, usually around pulpal arterioles (Fig. 12-16). ing teeth. Noxious stimuli in teeth are transmitted in primary Stimulation of these fibers results in constriction of the arteri- afferent neurons located in the trigeminal ganglion via second- oles and a decrease in blood flow.1,82 The sympathetic neuron order neurons in the brain stem to the brain (Fig. 12-15; see terminals contain the classic neurotransmitter, norepinephrine also Chapter 17 and later in this chapter). Transmission of (NE), and neuropeptide Y (NPY) (see Neuropeptides later in sensory information consists of a cascade of events involving this chapter). NPY is synthesized in sympathetic neurons and input, processing, and sensing,329 so the control of dental pain supplied to terminals by axonal transport. By contrast, NE is should be based on an understanding of the origin of pain mainly produced locally in the terminals. Compared with the signals and the complex modulation that may take place locally sensory nerves, these fibers are most often located in deeper and at higher levels. The sensory system of the pulp appears parts of the pulp proper, but fibers have also been found in to be well suited for signaling potential damage to the tooth. close relation to odontoblasts.1,164 The tooth is innervated by a large number of myelinated and The presence of parasympathetic cholinergic nerves in unmyelinated axons. The number of axons entering a human dental tissues has been and is still controversial, although it premolar may reach 2000 or more, and each axon can arborize has been concluded that there is absence of parasympathetic to form multiple points of innervation.90,170,169 vasodilation in the cat dental pulp.286,323 It has been reported Regardless of the nature of the sensory stimulus (i.e., that the neuropeptide vasoactive intestinal polypeptide (VIP) thermal, mechanical, chemical, electric [e.g., pulp tester]), is localized in the parasympathetic neurons.225,226 The origin of almost all afferent impulses generated from pulp tissue result VIP-containing fibers in the pulp is uncertain insofar asno in the sensation of pain. However, when the pulp is weakly form of surgical denervation has resulted in complete loss of stimulated by an electric pulp tester under carefully controlled these fibers from the dental pulp.390 CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 543

Sensory nerve fibers are usually classified according to their diameter, conduction velocity, and function as shown in Table 12-1. The pulp contains two types of sensory nerve fibers: myelinated (A fibers) and unmyelinated (C fibers). It has been shown that there is some functional overlap between pulpal A and C fibers, as both fiber types can be nocicep- tors.163,169,170,246,267 The A fibers include both A-beta and A-delta fibers. The A-beta fibers may be slightly more sensitive to stimulation than the A-delta fibers, but functionally these fibers are grouped together in the dental pulp, because both innervate the dentinal tubules, and both are stimulated by dentinal fluid movement (Fig. 12-17). Approximately 90% of the A fibers in dental pulp are A-delta 246fibers. Table 12-2 summarizes the principal characteristics of the main sensory fibers. During the bell stage of tooth development, “pioneer” nerve fibers enter the dental papilla following the path of blood vessels.104 Although only unmyelinated fibers are observed in the dental papilla, a proportion of these fibers are probably A fibers that have lost or not developed their myelin sheath. Myelinated fibers are the last major structures to appear in the developing human dental pulp.11 The number of nerve fibers gradually increases, and branching occurs as the fibers approach dentin. During the bell stage, very few fibers enter FIG. 12-16 Histologic section, immunohistologically stained for neuropep- the predentin.104 tide Y (NPY), shows the distribution of sympathetic nerves in the root pulp of The sensory nerves of the pulp arise from the trigeminal a rat molar. NPY fibers are seen associated with blood vessels. (Courtesy Dr. nerve and pass into the radicular pulp in bundles by way of Inge Fristad, Department of Clinical Dentistry, University of Bergen.)

TABLE 12-1 Location of Intradental Classification of Nerve Fibers Sensory Nerves Conduction Type of Fiber Function Diameter (µm) Velocity (m/sec) A-alpha Motor, 12-20 70-120 proprioception A-beta Pressure, touch 5-12 30-70 A-gamma Motor, to muscle 3-6 15-30 spindles A-delta Pain, 1-5 6-30 temperature, touch A-fibers, myelinated C-fibers, unmyelinated B Preganglionic < 3 3-15 autonomic FIG. 12-17 Schematic drawing illustrating the location of A and C fibers C dorsal root Pain 0.4-1 0.5-2 in the dental pulp. Myelinated A fibers are located in the periphery of the pulp, penetrating the inner part of dentin. Unmyelinated C fibers are located in the Sympathetic Postganglionic 0.3-1.3 0.7-2.3 deeper part of the pulp proper. sympathetic

TABLE 12-2 Characteristics of Sensory Fibers Fiber Myelination Location of Terminals Pain Characteristics Stimulation Threshold A-delta Yes Principally in region of Sharp, pricking Relatively low pulp-dentin junction C No Probably distributed Burning, aching, less bearable Relatively high, usually throughout pulp than A-delta fiber sensations associated with tissue injury 544 PART II The Advanced Science of Endodontics

FIG. 12-18 Histologic section, immunohistologically stained for calcitonin FIG. 12-20 Histologic section, immunohistologically stained for calcitonin gene-related peptide (CGRP), shows distribution of sensory nerves in the gene-related peptide (CGRP), shows the distribution of sensory nerves in a apical area of a rat molar. Nerve fibers are seen associated with blood vessels rat molar. Nerves enter the coronal pulp in bundles and ramify in a network and enter the dental pulp in nerve bundles. (Courtesy Dr. Inge Fristad, Depart- beneath the odontoblasts (i.e., plexus of Raschkow), before entering between ment of Clinical Dentistry, University of Bergen.) the odontoblasts and the inner part of dentin.

patients have difficulty identifying the inflamed tooth until the inflammation reaches the periradicular tissue, which is highly innervated with proprioceptors. This is discussed in greater detail in Chapter 17. M In the human premolar, the number of unmyelinated axons entering the tooth at the apex reached a maximal number M shortly after tooth eruption.170 At this stage, an average of 1800 unmyelinated axons and more than 400 myelinated axons were found, although in some teeth fewer than 100 myelinated axons were present. Five years after eruption, the number of M A fibers gradually increased to more than 700. The relatively U late appearance of A fibers in the pulp may help to explain why the electric pulp test tends to be unreliable in young teeth, as A fibers are more easily electrically stimulated than C fibers.108 M M A quantitative study of axons 1 to 2 mm coronal to the root apex of fully developed human canine and incisor teeth170 FIG. 12-19 Electron micrograph of the apical pulp of a young canine tooth, reported a mean of about 360 myelinated axons in canines and showing in cross section myelinated nerve axons (M) within Schwann cells. incisors, whereas there were 1600 to 2200 unmyelinated axons. Smaller, unmyelinated axons (U) are enclosed singly and in groups by Schwann However, this does not reflect the actual number of neurons cells. (Courtesy Dr. David C. Johnsen, School of Dentistry, Case Western supporting a single tooth, because multiple branching of the Reserve University.) axons may occur in the peripheral tissues. Overall, approxi- mately 80% of the axons were unmyelinated fibers.169,170 The nerve bundles pass upward through the radicular pulp together with blood vessels (see Fig. 12-18). Once they reach the foramen in close association with arterioles and venules the coronal pulp, they fan out beneath the cell-rich zone, (Fig. 12-18). Each of the nerves entering the pulp is invested branch into smaller bundles, and finally ramify into a plexus within Schwann cells, and the A fibers acquire their myelin of single-nerve axons known as the plexus of Raschkow (Fig. sheath from these cells. With the completion of root develop- 12-20). Full development of this plexus does not occur until ment, the myelinated fibers appear grouped in bundles in the the final stages of root formation.90 It has been estimated that central region of the pulp (Fig. 12-19). Most of the unmyelin- each axon entering the pulp sends at least eight branches to ated C fibers entering the pulp are located within these fiber the plexus of Raschkow. There is prolific branching of the bundles; the remainder is situated toward the periphery of the fibers in the plexus, producing a tremendous overlap of recep- pulp (see Fig. 12-17).307 It should be noted that single neurons tor fields.143,267,268,269,273 It is in the plexus that the A fibers have been reported to innervate the pulps of multiple teeth in emerge from their encircling Schwann cells and branch repeat- animal studies.155 Assuming a similar innervation pattern in edly to form the subodontoblastic plexus. Finally, terminal humans, this finding may partially explain why patients often axons pass between the odontoblasts as free nerve endings have difficulty localizing pulpal pain to a specific tooth. An (Figs. 12-21 and 12-22). The extent to which dentin is alternative explanation to this clinical observation is that the innervated has been the subject of numerous investiga- pulp has a relatively low density of proprioceptors, and thus tions.42,45,46,49,90,215 With the exception of the innervation of CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 545

D PD OP OL Fb

A B

FIG. 12-21 Detailed histologic section, immunohistologically stained for calcitonin gene-related peptide (CGRP), shows the distribution of sensory nerves in the odontoblast layer of a rat molar. (Courtesy Dr. Inge Fristad, C D Department of Clinical Dentistry, University of Bergen.) FIG. 12-23 Schematic drawing showing distribution of nerve fibers in the dentin-pulp border zone. A, Fibers running from the subodontoblastic plexus to the odontoblast layer. D, dentin; Fb, fibroblast; OL, odontoblast layer; OP, NF odontoblast process; PD, predentin. B, Fibers extending into the dentinal tubules in the predentin. C, Complex fibers that branch extensively in the predentin. D, Intratubular fibers extending into the dentin.

PD O into the dentinal tubules for only a few micrometers, but a few may penetrate as far as 100 µm (see Fig. 12-20). The area covered by a single such terminal complex often reaches thou- sands of square micrometers.131,268 O Intratubular nerve endings are most numerous in the area of the pulp horns, where as many as 40% of the tubules may contain fibers.46,215 The number of intratubular fibers decreases in other parts of the dentin, and in root dentin FIG. 12-22 Unmyelinated nerve fiber (NF) without a Schwann cell covering only about 1% of dentinal tubules contain fibers. This notion located between adjacent odontoblasts (O) overlying pulp horn of a mouse has been challenged in a study that stained pulps for protein molar tooth. Predentin (PD) can be seen at upper right. Within the nerve, there gene-product 9.5, a specific marker for nerves.230 In that study, are longitudinally oriented fine neurofilaments, microvesicles, and mitochon- root dentin appeared to be as well innervated as coronal dentin. dria. (From Corpron RE, Avery JK: The ultrastructure of intradental nerves in The anatomic relationship between the odontoblast processes developing mouse molars, Anat Rec 175:585, 1973.) and sensory nerve endings has led to much speculation as to the functional relationships between these structures, if any.45 When present, nerve fibers lie in a groove or gutter along dentinal tubules, discussed later in this chapter, the bulk of the surface of the odontoblast process, and toward their termi- dentin is devoid of sensory nerve fibers. This offers an explana- nal ends, they twist around the process like a corkscrew. The tion as to why pain-producing agents (e.g., potassium chlo- cell membranes of the odontoblast process and the nerve ride) do not always elicit pain when applied to exposed dentin. fiber are closely approximated and run closely parallel for Similarly, application of topical anesthetic solutions to dentin the length of their proximity, but they are not synaptically does not decrease its sensitivity. A high concentration of lido- linked.160 caine solution is needed to block the response of intradental Although it may be tempting to speculate that the odonto- nerves to mechanical stimulation of the dentin.4 blasts and their associated nerve axons are functionally inter- One investigator131 studied the distribution and organiza- related and that together they play a role in dentin sensitivity, tion of nerve fibers in the dentin-pulp border zone of human there is a paucity of evidence supporting this hypothesis. If the teeth. On the basis of their location and pattern of branching, odontoblast were acting as a classic receptor cell,* it would several types of nerve endings were described (Fig. 12-23). have chemical, electric, or mechanical communication with Some fibers were found running from the subodontoblastic the adjacent nerve fiber. However, researchers have been nerve plexus toward the odontoblast layer. However, these unable to find classical anatomic structures (e.g., synaptic fibers do not reach the predentin; they terminate in extracel- junctions) that could functionally couple odontoblasts and lular spaces in the cell-rich zone, the cell-poor zone, or the odontoblast layer. Other fibers extend into the predentin and run through a dentinal tubule in close association with an *A receptor cell is a non–nerve cell capable of exciting adjacent afferent nerve fibers. odontoblast process. Most of these intratubular fibers extend Synaptic junctions connect receptor cells to afferent nerves. 546 PART II The Advanced Science of Endodontics

nerve fibers together. With regard to the membrane properties of odontoblasts, it has been reported that the membrane poten- Cerebral cortex tial of the odontoblast is low (−24 to −30 mV),198,227 and that the cell does not respond to electric stimulation.198,406 It would appear that the odontoblast does not possess the properties of Thalamus an excitable cell. Further, the sensitivity of dentin is not dimin- ished after disruption of the odontoblast layer.37,216 It is still “Perception” possible that odontoblasts could modulate neuronal function via alteration in sodium channel activity or the release of para- Trigeminal Trigemino- crine factors that diffuse to the closely approximated nerve ganglion thalamic terminal. Main sensory tract nucleus Another study showed that a reduction in pulpal blood flow, induced by stimulation of sympathetic fibers leading to the 82 pulp, results in depressed excitability of pulpal A fibers. The Oralis excitability of C fibers is less affected than that of A fibers by Motor 365 nuclei a reduction in blood flow. Interpolaris Of clinical interest is the evidence that nerve fibers of the RF pulp may be resistant to necrosis85,255 because their cell bodies “Detection” are found in ganglia outside the pulp. Because nerve bundles in general are more resistant to autolysis than other tissue ele- Caudalis ments, even in degenerating pulps, C fibers might still be able to respond to noxious stimulation. It may be that C fibers remain excitable even after blood flow has been compromised Medullary dorsal horn in the diseased pulp, as C fibers are often able to function in “Processing” the presence of hypoxia.365 This may explain why instrumenta- tion of the root canals of apparently nonvital teeth sometimes FIG. 12-24 Schematic diagram of the pathway of transmission of nocicep- elicits pain. On the other hand, histologic studies on nonvital tive information from the orofacial region. The trigeminal pain system is a teeth failed to demonstrate high levels of innervation, leading complex multilevel system that begins with the detection of tissue damaging to the suggestion that pain may be due to the transfer of stimuli in the periphery, the processing of that input at the level of the medul- noxious chemicals to terminals located in periapical tissues.255 lary spinal cord, and the final perception of what is felt as pain in the cerebral cortex. There is growing appreciation for the concept that once a noxious Steps and Mechanisms in Pain Perception stimulus is detected in the periphery, there is the opportunity for a great deal When activated by a stimulus sufficient to cause tissue damage of modification of the message prior to ultimate perception. or release of inflammatory mediators, nerve endings in the pulp and periradicular tissues begin to send bursts of mes- sages to the central nervous system (CNS) that may eventu- smaller, less myelinated Aδ and yet smaller and unmyelinated ally be perceived as pain. The anatomic pathway for this C fibers that conduct information likely to be perceived as pain. transmission of information has been fairly well established, These two classes of pain-sensing nerve fibers, or nociceptors, and it is tempting to view the perception of pain of orofacial are both found in the tooth pulp, but there are three to eight origin as a simple graded response to the intensity of the times more unmyelinated C fibers than δA fibers.41,45,170,371 It stimulus. However, researchers have come to realize that the should be noted that this classification system is based purely pain system is a complex, multilevel system that begins with on the size and myelination of the neurons and does not neces- the detection of tissue-damaging stimuli in the periphery, the sarily indicate function. For example, another class of pulpal C processing of that input at the level of the medullary spinal fibers are the postganglionic sympathetic efferents found in cord, and the perception of what is felt as pain in higher brain association with blood vessels, where they regulate pulpal regions such as the cerebral cortex (Fig. 12-24). After a blood flow2,187,299,300 and may also influence the activity of noxious stimulus is detected in the periphery, there is ample peripheral nociceptors (for reviews, see Perl296 or Hargreaves137). opportunity for a great deal of endogenous and possibly exog- Because most pulpal sensory fibers are nociceptive, their termi- enous modification of the message prior to its ultimate- per nal branches are free nerve endings, and physiologic stimula- ception. The clinician deals with all three levels of the pain tion by any modality (temperature, hyperosmotic fluids) results system in diagnosing and treating odontalgia, and a practitio- in the perception of pure pain, which can be difficult for ner with a basic understanding of each level will be able to patients to localize. Under experimental conditions, electrical recognize therapeutic opportunities and apply effective pain stimulation can result in a prepain sensation that is also difficult control methods. to localize. Once inflammation has extended to the periodontal ligament, which is well endowed with Aβ discriminative touch receptors, localization of pain is more predictable with light Detection: The First Step mechanical stimuli such as the percussion test. in Pain Perception In the normal uninflamed pulp and periradicular tissues, a Various types of peripheral neurons are found in the trigeminal noxious stimulus causes depolarization of nociceptors suffi- system, including large-diameter, heavily myelinated Aα, Aβ, cient to generate action potentials by means of the opening of and Aγ fibers associated with motor, proprioception, touch, voltage-gated sodium channels (Nav). After generation of an pressure, and muscle spindle stretch functions. But it is the action potential, not only is information sent to the CNS, but CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 547 also in an antidromic fashion (i.e., in the reverse direction of sensory A fibers at the dentin-pulp border zone. Smaller C the impulse) in which proinflammatory neuropeptides such fibers of the pulp do not respond to the conventional pulp as substance P (SP), calcitonin gene-related peptide (CGRP), tester because significantly more current is needed to stimulate neurokinins, and the classic neurotransmitter, glutamate, are them.267 Bender and associates17 found that in anterior teeth, released from afferent terminals in the pulp and periradicular the optimal placement site of the electrode is the incisal tissues. edge, as the response threshold is lowest at that location and increases as the electrode is moved toward the cervical region Neuropeptides of the tooth.

Of immense importance in pulp biology is the presence of Cold tests using carbon dioxide (CO2) snow or liquid refrig- neuropeptides in pulpal nerves.42,46,49,349 Pulpal nerve fibers erants and heat tests employing heated gutta-percha or hot contain neuropeptides such as calcitonin gene–related peptide water activate hydrodynamic forces within the dentinal tubules, (CGRP) (see Figs. 12-18, 12-20, and 12-21),41 substance P which in turn excite the intradental A fibers. C fibers are gener- (SP),268,285 neuropeptide Y (see Fig. 12-16), neurokinin A ally not activated by these tests unless they produce injury to (NKA),12 and VIP.228,389 In rat molars, the largest group of intra- the pulp. It has been shown that cold tests do not injure the dental sensory fibers contains CGRP. Some of these fibers also pulp.108 Heat tests have a greater potential to produce injury, contain other peptides, such as SP and NKA.42,45 Release of but if the tests are used properly, injury is not likely. these peptides can be triggered by numerous stimuli, including tissue injury,12 complement activation, antigen-antibody reac- Sensitivity of Dentin tions,280 or antidromic stimulation of the inferior alveolar The mechanisms underlying dentin sensitivity have been a nerve.285,282 Once released, vasoactive peptides produce vascu- subject of interest for many years. How are stimuli relayed from lar changes that are similar to those evoked by histamine and the peripheral dentin to the sensory terminals located in the bradykinin (i.e., vasodilation).282 In addition to their neurovas- region of the dentin-pulp border zone? Converging evidence cular properties, SP and CGRP contribute to inflammation and indicates that movement of fluid in the dentinal tubules is the promote wound healing.31,369 The release of CGRP can be mod- basic event in the arousal of dentinal pain.35,37,263,385,387 It now ified by sympathetic agonists and antagonists,115,138 offering the appears that pain-producing stimuli, such as heat, cold, air promise of using such agonists to treat dental pain. This latter blasts, and probing with the tip of an explorer, have in common point is important because clinicians use sympathetic agonists the ability to displace fluid in the tubules.35,246 This is referred every day—the vasoconstrictors present in local anesthetic to as the hydrodynamic mechanism of dentin sensitivity. The solutions may have direct effects on inhibiting dental nerve hydrodynamic theory suggests that dentinal pain associated activity. Local anesthetic reduction in pain may be due to the with stimulation of a sensitive tooth ultimately involves actions of both the local anesthetic and the vasoconstric- mechanotransduction. Classical mechanotransducers have tor.137,138 In cats, capsaicin acutely activates and chronically been recognized on pulpal afferents, providing a mechanistic blocks the TRPV1-expressing classes of C and A-delta nocicep- support to this theory.149 Thus, fluid movement in the dentinal tors in the pulp.163 In addition, the chronic application of tubules is translated into electric signals by receptors capsaicin ointment to skin has been shown to relieve pain in located in the axon terminals innervating dentinal tubules. patients, suggesting that clinical trials evaluating chronic appli- Using single-fiber recording techniques, a positive correlation cation of capsaicin for treating pulpal or periradicular pain may was found between the degree of pressure change and the be of value. number of nerve impulses leaving the pulp (Figs. 12-25 and Antidromic stimulation of nerves (i.e., toward the periph- 12-26).246,266,384 Thus, the outward fluid movements (negative eral terminals) simply means that the afferent barrage goes in pressure) produce a much stronger nerve response than inward the opposite direction of the orthograde stimulation (toward movements.246,388 the CNS). Normally, sensory nerves are stimulated at their In experiments on humans, brief application of heat or cold peripheral terminations, and their action potentials then travel to the outer surface of premolar teeth evoked a painful response toward the brain. In antidromic nerve stimulation, the sensory before the heat or cold could have produced temperature nerve is usually cut. The peripheral end of the nerve is then changes capable of activating sensory receptors in the underly- electrically stimulated. This causes an action potential to travel ing pulp.266,372 The evoked pain was of short duration: 1 or backward toward the periphery, which causes release of neu- 2 seconds. The thermal diffusivity of dentin is relatively low, ropeptides in the pulp.285 All branches of the nerve also depo- yet the response of the tooth to thermal stimulation is rapid, larize and release neuropeptides (the so-called axon reflex).322 often less than 1 second. Evidence suggests that thermal stimu- It has been reported246,285 that mechanical stimulation of lation of the tooth results in a rapid movement of fluid into dentin produces vasodilation within the pulp, presumably by the dentinal tubules. This results in activation of the sensory causing the release of neuropeptides from intradental sensory nerve terminal in the underlying pulp. Presumably, heat fibers (neurogenic inflammation).285 Electric stimulation of expands the fluid within the tubules faster than it expands the tooth has a similar effect.152 The pulpal concentrations of dentin, causing the fluid to flow toward the pulp, whereas cold CGRP, SP, and NKA are elevated in painful human teeth over causes the fluid to contract more rapidly than dentin, produc- healthy control teeth extracted for orthodontic reasons.12 ing an outward flow. It is speculated that the rapid movement These peptides are also elevated in pulps beneath advancing of fluid across the cell membrane of the axon terminal activates carious lesions.310,311 a mechanosensitive receptor, similar to how fluid movement activates hair cells in the cochlea of the ear. All axon terminals Pulp Testing have membrane channels through which charged ions pass, The electric pulp tester delivers a current sufficient to over- and this initial receptor current, if sufficient, can trigger come the resistance of enamel and dentin and stimulate the voltage-gated sodium channels to depolarize the cell, leading 548 PART II The Advanced Science of Endodontics

FIG. 12-25 Response of a single dog pulp nerve fiber to repeated hydrostatic pressure stimulation pulses. Lower solid wavy line of each recording indicates the stimulation pressure applied to the pulp. Upper line (kPa) is the femoral artery blood pressure curve recorded to indicate the relative changes in the pulse pressure and the heart cycle. (Modified from Närhi M: Activation of dental pulp nerves of the cat and the dog with hydrostatic pressure, Proc Finn Dent Soc 74[suppl 5]:1, 1978.)

400

300 Desiccation

200

100 Fluid movement No. of impulses in 5 seconds resulting from capillary force 0

–400 –200 0 200 400 Pressure (mm Hg) FIG. 12-26 The average number of impulses recorded from dentine after application of pressure stimuli to the dentine. More impulses are recorded after application of negative pressure (outward fluid flow) than after positive Odontoblast pressure (inward fluid flow). (From Vongsavan N, Matthews B: The relationship between the discharge of intradental nerves and the rate of fluid flow through Nerve fiber dentine in the cat, Arch Oral Biol. 52:643, 2007.) to a barrage of impulses to the brain. Some ion channels are activated by voltage, some by chemicals, and some by mechani- FIG. 12-27 Diagram illustrating movement of fluid in the dentinal tubules cal pressure.246,263,268 In the case of pulpal nerve fibers that are resulting from the dehydrating effect of a blast of air from an air syringe. activated by hydrodynamic forces, pressure would be trans- duced, gating mechanosensitive ion channels. reduced. If fluid is removed from the outer end of exposed The dentinal tubule is a capillary tube with an exceedingly dentinal tubules by dehydrating the dentinal surface with an small diameter.*111 The physical properties of capillarity are air blast or absorbent paper, capillary forces produce a rapid significant because fluid force increases as the diameter is outward movement of fluid in the tubule (Fig. 12-27). According to Brännström,35 desiccation of dentin can theoreti- cally cause dentinal fluid to flow outward at a rate of 2 to 3 mm/ *To appreciate fully the dimensions of dentin tubules, understand that the diameter of the tubules (about 1 µm) is much smaller than that of red blood cells (about sec. In addition to air blasts, dehydrating solutions containing 7 µm). The thickness of coronal dentin is about 3 mm, so each tubule is 3000 µm hyperosmotic concentrations of sucrose or calcium chloride can long but only 1 µm in diameter. Thus, each tubule is 3000 diameters long. produce pain if applied to exposed dentin. CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 549

Investigators have shown that it is the A fibers rather than the C fibers that are activated by hydrodynamic stimuli (e.g., heat, cold, air blasts) applied to exposed dentin.265,273 However, if heat is applied long enough to increase the temperature of the dentin-pulp border by several degrees Celsius, then C fibers may respond, particularly if the heat produces injury. It seems that the A fibers are mainly activated by a rapid displace- ment of the tubular contents.263 Slow heating of the tooth produced no response until the temperature reached 111° F (43.8° C), at which time C fibers were activated, presumably because of heat-induced injury to the pulp. These C fibers are called polymodal nociceptors because they contain numerous receptors that confer the ability to detect and respond to many different types of stimuli.268,269 Capsaicin, the pungent active ingredient in hot peppers, is known to simulate C fibers and a Scanning electron micrograph of a shallow groove (between subset of A-delta fibers.163 Capsaicin activates a receptor termed FIG. 12-28 white arrowheads) created in polished dentin by a dental explorer tine under the “transient receptor potential, subtype vanilloid 1” or a force of 30 g (30 cN). Note the partial occlusion of the tubules by smeared TRPV1.58 The TRPV1 receptor is expressed primarily on a matrix. (From Camps J, Salomon JP, Meerbeek BV, et al: Dentin deformation major subclass of nociceptors and responds to heat (> 110° F after scratching with clinically relevant forces, Arch Oral Biol 48:527, 2003.) [> 43° C]), certain inflammatory mediators, and acid (pH< 6). Thus TRPV1 has been considered a molecular integrator of polymodal noxious stimuli.277 The ability of the TRPV1 antago- nist, capsazepine, to inhibit acid-, heat-, and capsaicin-activated trigeminal neurons222 has led to the development of new drugs (i.e., TRPV1 antagonists) for the treatment of pulpal pain. Eugenol is known to activate and ultimately desensitize TRPV1, and this may explain the anodyne action of zinc oxide eugenol temporary restorations.415 It has also been shown that pain-producing stimuli are more readily transmitted from the dentin surface when the exposed tubule apertures are open157,171 and the fluid within the tubules is free to flow outward.171,246,387 For example, acid treatment of exposed dentin to remove the smear layer opens the tubule orifices and makes the dentin much more responsive to stimuli such as air blasts and probing.157,269 Perhaps the most difficult phenomenon to explain is den- FIG. 12-29 Odontoblasts (arrows) displaced upward into the dentinal tinal pain associated with light probing of dentin. Even light tubules. pressure of an explorer tip can produce strong forces.* These forces have been shown to mechanically compress dentin and close open tubule orifices with a smear layer that causes suf- bodies of odontoblasts are displaced upward in the dentinal ficient displacement of fluid to excite the sensory receptors in tubules, presumably by a rapid movement of fluid in the tubules the underlying pulp (Fig. 12-28).54,55 Considering the density produced when exposed dentin is desiccated, as with the use of the tubules in which hydrodynamic forces would be gener- of an air syringe or cavity-drying agents (Fig. 12-29).79,202 ated by probing, multiple nerve endings would be simultane- Such cellular displacement results in the destruction of odon- ously stimulated when a dental explorer is scratched across toblasts, because cells thus affected soon undergo autolysis and dentin. Another newly suggested explanation is that the nerves disappear from the tubules. Displaced odontoblasts may even- innervating teeth are special in nature and that low-threshold tually be replaced by stem cells that migrate from the cell-rich mechanoreceptors are signaling nociceptive input in teeth.103 zone of the pulp, as discussed later in the chapter. Similar low-threshold mechanoreceptors convey tactile sensa- The hydrodynamic theory can also be applied to an under- tion in skin. The authors used the term low-threshold “algo- standing of the mechanism responsible for hypersensitive neurons” for these nerves. This theory is not in conflict with dentin.36,37 There is controversy regarding whether exposed the hydrodynamic theory but may help to explain the sensa- dentin is simply sensitive or becomes truly hypersensi- tion of pain felt after weak mechanical stimuli like air puffs tive.268,269,273 Growing evidence indicates that new sodium and water spray. channels responsible for activating nerves are expressed in Another example of the effect of strong hydraulic forces that nerve tissue exposed to inflammation.119,137,308 An increase in are created within the dentinal tubules is the phenomenon of the density of sodium channels or their sensitivity may con- odontoblast displacement. In this reaction, the nuclei and cell tribute to dentinal hypersensitivity. Hypersensitive dentin is also associated with the exposure of dentin normally covered by cementum or enamel. The thin layer of cementum is fre- *A force of 44 cM (44 g) applied to an explorer having a tip 40 µm in diameter would produce a pressure of 2437 MPa on the dentin.54 This is far in excess of the compres- quently lost as gingival recession exposes cementum to the oral sive strength of dentin, listed as 245MPa, as evidenced by the shallow grooves lined environment. Cementum is subsequently worn away by brush- by smear layers created in dentin using this force.55 ing, flossing, or using toothpicks. Once exposed, the dentin 550 PART II The Advanced Science of Endodontics

may respond to the same stimuli to which any exposed dentin BOX 12-1 surface responds (e.g., mechanical pressure, dehydrating Peripheral Mechanisms Contributing to agents). Although the dentin may at first be very sensitive, Hyperalgesia and Allodynia within a few weeks the sensitivity usually subsides. This desen- sitization is thought to occur as a result of gradual occlusion Mechanism of the tubules by mineral deposits, thus reducing hydrody- 142 ♦ Composition and concentration of inflammatory mediators namic forces. In addition, deposition of reparative dentin over 199,243,324 ♦ Changes in afferent fiber: activation and sensitization the pulpal ends of the exposed tubules probably also reduces 51 ♦ Changes in afferent fiber: sprouting sensitivity, because reparative dentin is less innervated by Changes in afferent fiber: proteins51,119,409 44 ♦ sensory nerve fibers. However, some hypersensitive dentin 264,265 ♦ Tissue pressure does not spontaneously desensitize, so hypersensitivity may be 250 ♦ Tissue temperature caused by either inflammatory changes in the pulp or mechani- 166,210,295 ♦ Sympathetic primary afferent fiber interactions cal changes in the patency of dentinal tubules. 272 ♦ Aβ fiber plasticity Currently, the treatment of hypersensitive teeth is directed toward reducing the functional diameter of the dentinal tubules Modified from Hargreaves KM, Swift JQ, Roszkowski MT, et al: Pharmacology of periph- eral neuropeptide and inflammatory mediator release, Oral Surg Oral Med Oral Pathol to limit fluid movement. Four possible treatment modali- 78:503, 1994. ties287,373,389 can accomplish this objective: 1. Formation of a smear layer on the sensitive dentin by burnishing the exposed root surface157,268,269 2. Application of agents, such as oxalate compounds, that form insoluble precipitates within the tubules157,292 TABLE 12-3 3. Application of agents such as hydroxyethyl methacrylate (HEMA) with or without glutaraldehyde that are thought Effect of Inflammatory Mediators on Nociceptive to occlude tubules with precipitated plasma proteins in Afferent Fibers dentinal fluid325 Effect on Effect on Human 4. Application of dentin bonding agents to seal off the tubules303 Mediator Nociceptors Volunteers Dentin sensitivity can be modified by laser irradiation, but clinicians must be concerned about its effect on the pulp.346,357 Potassium174 Activate ++ Protons218,343 Activate Peripheral Sensitization ++ Serotonin16,174 Activate ++ Following repeated noxious stimuli, both A and polymodal C 16,174,211 fiber nociceptors undergo a process of sensitization manifested Bradykinin Activate +++ by three obvious changes in response patterns. First, firing Histamine174 Activate + thresholds may decrease, so that previously non-noxious Tumor necrosis factor-α Activate ? stimuli may trigger discharges, contributing to the sensation Prostaglandins27 Sensitize of pain (allodynia). Second, after-discharges may occur, so that ± noxious stimuli may produce an even greater increase in the Leukotrienes27,229 Sensitize ± perceived intensity of pain (hyperalgesia). And third, firing Nerve growth factor213,297 Sensitize ++ may occur spontaneously, contributing to the development of 132 Substance P Sensitize ± spontaneous pain. These changes are often seen in endodontic 99 pain patients and may be explained in part by the effects of Interleukin 1 Sensitize (?) ? chemical mediators released into inflamed pulp and perira- +, Positive; ++, very positive; +++, extremely positive; ±, equivalent; ?, unknown. dicular tissues. Such mediators include substances produced Modified from Fields H: Pain, New York, 1987, McGraw-Hill. from damaged tissues, agents of vascular origin, and peptides released from the nerve fibers themselves (Table 12-3). Other mechanisms of peripheral sensitization are listed in Box 12-1.

Hyperalgesia and Allodynia TABLE 12-4 Three characteristics of hyperalgesia are (1) spontaneous pain, Signs of Hyperalgesia and Allodynia and (2) a decreased pain threshold, and (3) an increased response Endodontic Diagnostic Tests to a painful stimulus.137 The peripheral mechanisms for these symptoms include a decrease in firing threshold, an increase Related Diagnostic Tests in responsiveness to noxious stimuli, and development of Signs of Hyperalgesia or Symptoms spontaneous discharges of nociceptors. All three of these char- Spontaneous pain Spontaneous pain acteristics can be seen in patients experiencing inflammatory pain of pulpal origin (Table 12-4). It is recognized that hyper- Reduced pain threshold Percussion test, palpation test, algesia can be produced by sustained inflammation, as in the throbbing pain case of sunburned skin. Clinical observation has shown that Increased response to painful Increased response to pulp test the sensitivity of dentin often increases when the underlying stimuli (electric or thermal test) pulp becomes acutely inflamed, and the tooth may be more From Hargreaves KM, Swift JQ, Roszkowski MT, et al: Pharmacology of peripheral difficult to anesthetize. This is due in part to the upregulation neuropeptide and inflammatory mediator release, Oral Surg Oral Med Oral Pathol of tetrodotoxin-resistant (TTX-resistant) sodium channels in 78:503, 1994. CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 551 inflamed neural tissue.119,137 NGF seems to play an important found predominantly on nociceptors—channels thought to be role in hyperalgesia. NGF regulates chronic inflammatory relatively resistant to .9,120 When administered to rats hyperalgesia by controlling gene expression in sensory before an inflammatory insult, ibuprofen, a nonselective COX neurons,274 including genes involved in inflammatory hyperal- inhibitor, has been shown to block the increased expression 74 127,128 gesia in the dental pulp. Although a precise explanation for of Nav1.7 and Nav1.8. Therefore, if the concentrations of hyperalgesia is lacking, apparently localized elevations in PGs in inflamed pulp and periradicular tissues can be tissue pressure and inflammatory mediators that accompany decreased with nonsteroidal antiinflammatory drugs (NSAIDs) acute inflammation play an important role.151,154,344,362,379 Clini- or corticosteroids, postoperative pain may be relieved; in cally, we know that when the pulp chamber of a painful tooth addition, more profound local anesthesia may be achieved in with an abscessed pulp is opened, drainage of exudate soon patients with hyperalgesia of pulpal origin.140,161,254 It is inter- produces a reduction in the level of pain. This suggests that esting to note that sensory neurons themselves are a source mechanical stimuli may contribute substantially to pain during of PGs; during inflammation, the levels of PGE2 appear to inflammatory hyperalgesia. increase in dorsal root ganglia and spinal cord, suggesting From a clinical point of view, thermal allodynia is the term that NSAIDs also have a central site of action (discussed in that best describes a patient whose chief complaint is “I have Chapter 4).235,405 pain when I drink cold beverages.” Mechanical allodynia is Bradykinin (BK) is a proinflammatory mediator derived involved when the chief complaint is “It now hurts when I bite from circulating plasma proteins and also causes direct activa- on this tooth.” These previously non-noxious stimuli now tion of nociceptive neurons, resulting in pain. Increased levels cause the perception of pain. Hyperalgesia is manifested in of BK have been demonstrated in the inflamed dental pulp,207 endodontic pain patients when noxious stimuli (e.g., refriger- and the presence of growth factors associated with inflamma- ant sprays or carbon dioxide snow used in the cold test) tion (e.g., nerve growth factor) have been reported to cause an produce much more pain than they would in teeth with normal increase in the expression of mRNA encoding B1 and B2 recep- pulp tissues. Spontaneous pain involves episodes of pain that tors in primary cultures of rat trigeminal ganglia,363 as well as seem to be unprovoked. All these changes can be partly other receptors such as TRPV1 and TRPA1.74,168 The transient explained by sensitization of peripheral nerve endings in the receptor potential subtype V1 (TRPV1) is the “capsaicin recep- pulp and periradicular tissues. tor”; it plays a key role in mediating inflammatory pain. TRPA1 Many silent nerve fibers are present in the normal pulp268,269 is expressed on capsaicin-sensitive neurons74 and interacts and are termed silent because they are not excited by with TRPV1.314 Bradykinin likely increases the excitability of ordinary external stimuli. Once they are sensitized through nociceptive neurons through its action on TRPV1 and TRPA1 pulpal inflammation, they begin to respond to hydrodynamic (for review, see Tominaga et al358). stimuli.46,268,269,273 This phenomenon may provide an additional Cytokines are a diverse group of regulatory proteins synthe- mechanism for dentin hypersensitivity. The molecular mecha- sized and secreted by a variety of cell types, such as leukocytes, nisms of this activation are not known in detail but involve neurons, and glia. In particular, tumor necrosis factor α (TNF- upregulation of numerous genes and their products.12,45,119 α) and the interleukins IL-1β, IL-6, and IL-8 are thought to play a role in the neuroplastic changes that occur in nocicep- Inflammatory Mediators tors innervating inflamed tissues, leading to hyperalgesia.197 Among the best characterized of the inflammatory mediators Application of TNF-α rapidly sensitizes TRPV1,183 contribut- are the prostaglandins (PGs), which are derived from arachi- ing to activation of the capsaicin-sensitive class of nociceptors. donic acid via the action of the cyclooxygenase (COX) enzyme All of the above are thought to exist in the inflamed pulp (for systems. The human COX enzyme is known to exist in at review, see Fouad100) and are thought to act at least in part by least two forms, COX-1 and COX-2. COX-1 is constitutively causing increased release of prostanoids.347 expressed and produces PGs that are involved in basic house- keeping functions such as cytoprotection in the stomach, Painful Pulpitis regulation of blood flow in the kidneys, and the formation of From the foregoing, it is apparent that pain associated with the thromboxane A2. The formation of thromboxane A2 can ulti- stimulation of the A fibers does not necessarily signify that the mately lead to platelet aggregation; therefore, inhibition of pulp is inflamed or that tissue injury has occurred. Clinically, thromboxane A2 should decrease platelet aggregation. COX-2 pain produced by A fibers in response to the hydrodynamic is inducible, synthesized in inflamed tissues (including dental mechanism has a sharp or bright quality as contrasted with the pulp),262 and is important in the production of the proinflam- dull, boring, or throbbing pain associated with C fibers. A matory PGs as well as the vasodilating prostacyclin (PGI2). fibers have a relatively low threshold of excitability to external Although they do not produce pain if applied alone, PGs are stimuli,246,267 and painful pulpitis is more likely to be associated known to sensitize peripheral nociceptors, which increases with nociceptive C fiber activity indicative of pulpal tissue the algogenic (pain-producing) properties of serotonin and injury.267-269,273 The clinician should carefully examine symp- bradykinin.76,124 The exact mechanism by which PGs increase tomatic teeth to rule out the possibility of hypersensitive neuronal excitability is not clear, but there is a growing body dentin, cracked or leaky fillings, or fracture lines—each of of evidence to suggest that they activate the PG E receptor which may initiate hydrodynamic forces—before establishing subtypes EP2 and EP3 in the trigeminal system293 and exert a diagnosis of reversible or irreversible pulpitis (see also their effects by regulating the activity of certain ion chan- Chapters 1 and 21). nels,394 including voltage-gated sodium channels (for a review, Pain associated with an inflamed or degenerating pulp may 86 see England ). For example, application of prostaglandin E2 be either provoked or spontaneous. The hyperalgesic pulp may (PGE2) to isolated dorsal root ganglion neuronal somata more demonstrate a lowered threshold of pain by responding to than doubles the responsiveness of certain sodium channels stimuli that usually do not evoke pain (allodynia), or the 552 PART II The Advanced Science of Endodontics

pain may be exaggerated and persist longer than normal (GDNF); the smaller trigeminal neurons remain dependent on (hyperalgesia).6 On the other hand, the tooth may commence NGF.200,305 This suggests that GDNF may function as a neuro- to ache spontaneously in the absence of any external stimu- trophic factor for the subset of larger neurons supporting the lus.137 Spontaneous, unprovoked pain generally indicates a tooth, which apparently mediate mechanosensitive stimuli, pulp that is seriously damaged and generally will not respond whereas NGF is suggested to support neurons responsible for to noninvasive therapy. nociception.259 NGF is the most extensively investigated among the trophic factors.209 Binding of target-derived NGF is depen- Plasticity of Intradental Nerve Fibers dent on specific TrkA receptors located on the axonal surface, It has become apparent that the innervation of the tooth is a with subsequent internalization and transport to the cell body, dynamic process in which the number, size, and cytochemistry where the effects are mediated. of nerve fibers can change because of 102,224,349aging, tooth Regulation of neural changes during inflammation seems to injury,42,45,46,49 and dental caries.310 For example, in rats, nerve be a function of NGF expression.47,53 NGF receptors are found fibers sprout into inflamed tissue surrounding sites of pulpal on intradental sensory fibers and Schwann cells.45 Evidence injury, and the content of CGRP and SP increases in these indicates that NGF is synthesized by fibroblasts in the coronal sprouting fibers.42,45,46,49,53 When inflammation subsides, the subodontoblastic zone (i.e., cell-rich zone), particularly in the number of sprouts decreases. Fig. 12-30 compares the normal tip of the pulp horn.45 Maximal sprouting of CGRP- and distribution of CGRP-immunoreactive sensory fibers in an SP-containing nerve fibers corresponds to areas of the pulp adult rat molar with those beneath a shallow cavity prepara- where there is increased production of NGF.53 Fig. 12-31 shows tion. The innervational pattern in normal and inflamed teeth the expression of NGF-mRNA in a pulp horn subjacent to is governed by neuronal growth factors. Neurotrophic and cavity preparation. target-derived factors regulate neuronal structure, survival, and It has been suggested that neuroimmune interactions take function and are important for the maintenance of neuronal place in the dental pulp, because a coordinated increase of phenotype characteristics. During development, all dental pulpal nerves and immune cells has been demonstrated.173,319 fibers appear to require nerve growth factor (NGF) and express In addition, recruitment of immunocompetent cells has been its receptor, TrkA, at some stages,244 whereas in adult teeth the demonstrated in the dental pulp after electrical tooth stimula- large trigeminal neurons are potentially dependent only on tion.66,106 Similar responses have been seen in periapical bone dental pulp–derived, glial cell line–derived neurotrophic factor in rats with radicular pulpitis.195 Neurogenic inflammation is generally thought to enhance healing, because denervated teeth show poorer healing following pulp exposures than innervated teeth.42,50 Another consideration in the neural response to inflamma- tion is the possibility of a change in the distribution and activ- ity of voltage-gated sodium channels. In particular, mice

lacking the gene for Nav1.7 show reduced painlike behaviors D when treated with a variety of proinflammatory agents.270 Also rd implicated in the altered firing characteristics of nociceptors O innervating inflamed tissues are the sodium channels that are resistant to tetrodotoxin (TTX), the biotoxin found in the C tetraodon pufferfish. The two main TTX-R sodium channels

are Nav1.8 and Nav1.9, and both have been shown to be increased two- to fourfold in inflamed dental pulp collected

ir * A R B FIG. 12-30 A, Normal distribution of calcitonin gene–related peptide (CGRP)-immunoreactive sensory fibers in adult rat molar. Nerve fibers typically are unbranched in the root (R), avoid interradicular dentin (ir), and form many branches in coronal pulp (C) and dentin (D). Nerve distribution is often asym- metric, with endings concentrated near the most columnar odontoblasts (in this case on the left side of the crown). When reparative dentin (rd) forms, it alters conditions so that dentinal innervation is reduced (magnification ×75). B, Shallow class I cavity preparation on the cervical root of a rat molar was made 4 days earlier. Primary odontoblast (O) layer survived, and many new CGRP-immunoreactive terminal branches spread beneath and into the injured pulp and dentin. Terminal arbor can be seen branching (arrowhead) from a larger axon and growing into the injury site. Scale bar: 0.1 mm (magnification. FIG. 12-31 NGF-mRNA is upregulated in the mesial pulp horn 6 hours after A, ×45). (From Taylor PE, Byers MR and Redd PE: Sprouting of CGRP nerve cavity preparation. (From Byers MR, Wheeler EF, and Bothwell M: Altered fibers in response to dentin injury in rat molars. Brain Res 461:371-376, expression of NGF and p75 NGF-Receptor mRNA by fibroblasts of injured teeth 1988.) precedes sensory nerve sprouting. Growth Factors 6:41-45, 1992.) CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 553 from patients with a diagnosis of irreversible pulpitis.396,402

When exposed to PGE2, neurons isolated from dorsal root ganglia cells have been shown to increase TTX-resistant sodium channel currents within minutes,120 indicating an increased activation of existing channels, rather than de novo protein synthesis. These sodium channels are relatively resis- tant to lidocaine,313 and this may explain the difficulty in achieving profound anesthesia in inflamed tissues (see also Chapter 4).140 Tissue Injury and Deafferentation When a peripheral nerve is cut or crushed, an interruption of the afferent input to the CNS occurs, which is called deaffer- entation. It would be logical to assume that the result of deaf- ferentation would be anesthesia of the formerly innervated FIG. 12-32 The interstitial cells of the trigeminal tract of medullary dorsal area, but occasionally other symptoms may occur, which may horn express c-fos during chronic tooth pulp inflammation in rat. (Adapted surprisingly include pain. Following nerve injury, a dramatic from Byers MR, Chudler EH, Ladarola MJ: Chronic tooth pulp inflammation shift in transcription of neuropeptides, receptors, and sodium causes transient and persistent expression of Fos in dynorphin-rich regions channels has been documented. The bidirectional contact of rat brainstem. Brain Res 861:191-207, 2000.) between the nerve cell and the peripheral target tissue is lost, and the neurons change into a state of either regeneration or neuronal cell death. The impact on neurons in the trigeminal ganglion is dependent on the injury site. A peripheral injury If such changes also occur in humans, they may help to has less effect than a more centrally located one. However, even explain why certain patients may complain of vague, poorly a small pulp exposure induces neuronal changes, both in the described pain for months following endodontic treatment. If trigeminal ganglion and at the second-order neuronal level in their pulpitis caused sprouting of periapical nerves,49,50,53,195 the brain stem.43,391 Because each single-rooted tooth contains these nerves may have taken part in transport of peripheral about 2000 nerve fibers,169,170 extirpation of the pulp is shown signaling molecules to the cell body by way of retrograde to cause both neurochemical and degenerative changes of their axoplasmic flow.42,53 This could induce changes in the expres- cell bodies in the gasserian (trigeminal) ganglion.129,186,334 The sion of many genes, resulting in central sensitization6,41 that central projection of these nerves to the spinal nucleus of the may require many months to correct.351 Reports of nerve trigeminal nerve is also affected,367 and there is evidence for sprouting in human inflamed pulps have been confirmed in transsynaptic changes330,334 that are reflected in the sensory different studies.310,312,319 Such reactions might contribute to cortex. Even larger responses would be expected from tooth increases in dentin sensitivity as well as expansion of receptive extraction, where both periodontal ligament and pulpal inner- fields.268,269,273 Sprouting of sympathetic fibers has also been vation are destroyed. reported,147 but the timing appears to be different. The func- When an axon is severed peripherally, a complete degenera- tional implications and how this relates to pain mechanisms tion of the cell bodies may not always occur.392 Attempted are unknown, but it has been suggested that these reactions regeneration by axonal sprouting may result in altered expres- are involved in healing and nociception following pulpal sion of various receptors, resulting in sensitivity to norepi- inflammation.115,147,148 nephrine (via increased activity)278 or acetylcholine (via increased cholinergic receptor activity),72 Processing: The Second Step sensitizing sensory neurons to autonomic activity. In addition, dorsal horn neurons, deprived of their normal sensory input, in Pain Perception may begin to respond to other nearby afferents. Thus, normal The Medullary Dorsal Horn inhibitory influences are reduced, and a widening of the After activation of peripheral nociceptors, nerve impulses in sensory receptive field is produced, which can produce central the form of action potentials convey information about the sensitization (see Central Sensitization later in this chapter). intensity (encoded by firing frequency), quality (encoded by Phantom tooth pain is another term often used synonymously type of neuron activated), and temporal features (encoded by with pain following deafferentation. Different reports suggest onset, duration, and offset of depolarization) of the peripheral an incidence of persistent pain following pulpectomy to be in stimuli to the CNS. In the trigeminal pain system, these action the range of 3% to 6%.238,383 potentials arrive at the trigeminal spinal tract nuclear complex Following tissue injury or tissue inflammation, extensive located in the medulla.141,214,329 Three distinct subnuclei can be changes occur in the gene expression of sensory ganglion found in this complex. Named for their anatomic position, neurons and, by way of transsynaptic mechanisms, in their they are the subnuclei oralis, interpolaris, and caudalis (see central projections.45,268,269,334 One example is the upregulation Fig. 12-24). Although the more rostral subnuclei (oralis and of inducible gene transcription factors such as c-fos43 and dif- interpolaris) receive some nociceptive input from oral tissues,70 ferent subsets of sodium channels.119 This is thought to result most such input is received at the level of the subnucleus in alterations in threshold properties and the size of receptor caudalis.89,233,329 Because of its organizational similarity to the fields. C-fos is not normally expressed in neurons in the brain dorsal horn of the spinal cord (which receives nociceptive stem, but chronic pulpitis causes prolonged increases in c-fos input from the somatosensory system), the subnucleus cauda- expression is some brain stem neurons (Fig. 12-32).43 lis has been termed the medullary dorsal horn. 554 PART II The Advanced Science of Endodontics

Components of the Medullary Dorsal Horn interneurons release the amino acid, glycine, or gamma amino The medullary dorsal horn relays information to higher centers butyric acid (GABA).214,331 in the brain and serves as the site of much potential processing The terminals of neurons that descend from brain structures of the signals from primary afferent sensory nerve fibers. such as the locus coeruleus and nucleus raphe magnus tend to Output from this region can be increased (hyperalgesia), inhibit nociceptive transmission at the level of the medullary decreased (analgesia), or misinterpreted (referred pain). dorsal horn.14 These terminals release a variety of neuroeffec- Understanding the functional components involved in such tive agents, including the endogenous opioid peptides (EOPs). processing not only helps explain some of these clinical phe- The EOPs, similar in three-dimensional structure to many of nomena but also allows evaluation of potential therapeutic the exogenous opiates from which their name derives, are modalities currently under investigation. Functional compo- released in response to nociceptive input and act to suppress nents include the central terminals of primary nociceptors the pain system. The EOPs likely are partly responsible for the (Aδ and C fiber afferents), the second-order projecting neurons, placebo effect seen in pain control studies, because this effect interneurons, the terminals of descending neurons, and glial241 can be reversed by administration of the opioid antagonist, cells (for review, see Hargreaves137). naloxone.139,212 Primary afferent fibers (whose cell bodies are located in the The final component of the medullary dorsal horn complex trigeminal ganglion) transmit signals to projection neurons via to be considered is the glial cell population. Historically con- the release of transmitters such as the excitatory amino acid, sidered to be solely supportive in function, they are now rec- glutamate, and the neuropeptide, substance P. Receptors for ognized to play an important role in the pain processing these neurotransmitters are found on postsynaptic membranes system.302,397 Following nociceptive input from primary affer- and include the N-methyl-D-aspartate receptor (NMDA) ents, glia release cytokines such as TNF-α and IL-1, as well as and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid certain PGs that may facilitate the activity of projection receptor (AMPA) classes of glutamate receptors and the neu- neurons. Glial modulating agents have been shown to be effec- rokinin 1 (NK1) class of substance P receptors. Antagonists to tive in experimental models of neuropathic pain,348 and NSAIDs these receptors have been shown to reduce hyperalgesia in potentially could exert part of their mechanism by animal studies.59 In a human clinical trial using an oral surgery acting at this level. model, the AMPA/kainate antagonist, LY293558, was shown to be antihyperalgesic.117 NK1 antagonists have shown promising Central Sensitization results in animal studies, but in general, they have displayed Central sensitization can be defined as an increased responsive- limited analgesic efficacy in humans.156 ness of central nociceptive neurons to peripheral stimulation The cell bodies of the second-order (projection) neurons in that occurs in addition to peripheral sensitization of the primary the trigeminal pain system are found in the medullary dorsal afferent nociceptors. Central sensitization is thought to be a horn; their processes cross the midline and project rostrally major cause of hyperalgesia and allodynia.204 Clinical trials to the thalamus via the trigeminothalamic tract (Fig. 12-33). implicate central sensitization in patients reporting pain due to From the thalamus, third-order neurons relay information to irreversible pulpitis. In one survey of nearly 1000 patients, 57% the cerebral cortex via a thalamocortical tract. Once signals of patients with irreversible pulpitis reported mechanical have reached the cortex, the input may be perceived as pain. allodynia (pain due to percussion).288 This appears to be due at Evidence exists that referred pain is caused by convergence of least in part to central sensitization, as both the ipsilateral (pul- afferent input from different areas onto the same projection pitis) tooth and a contralateral (normal) tooth demonstrated neurons. mechanical allodynia to a force transducer.185 Thus, central Approximately 50% of subnucleus caudalis neurons are sensitization contributes to a spread of endodontic pain, and the estimated to receive convergence of sensory input from cutane- clinical application of bite force transducers may provide a novel ous and deep structures.329 In one study of a cat, a single method for diagnosing pain mechanisms.184,185 nucleus caudalis neuron received input from sensory neurons Different studies shed light on the molecular mechanisms innervating the cornea, the skin overlying the maxilla, a maxil- involved in central sensitization (for review, see Cousins and lary premolar tooth, and a mandibular canine and premolar Power63 and Hargreaves137), but the process is generally initi- tooth on one side.333 Subnuclei oralis and interpolaris also ated by a barrage of nociceptive impulses from peripheral C receive converging input from orofacial and muscle afferents.332 fibers. The level and duration of pain prior to endodontic This would explain the clinical observation of patients who intervention have been cited in several studies as predictors of perceive pain in a particular tooth that actually originates from postoperative endodontic pain,364,393 and this may be due to either a different tooth or structure (see also Chapter 17). In such a prolonged and intense input from C nociceptors. Any such cases, anesthetizing the tooth suspected by the patient reduction of such a barrage should limit the occurrence of would afford no relief. However, if an anesthetic is delivered central sensitization and the development of pain of longer selectively to the suspected primary source of pain, the patient’s duration after tissue injury (including surgical and nonsurgical discomfort should greatly diminish.279 Likewise, if the source endodontic procedures). The use of long-acting local anesthet- of a perceived were located in a muscle of mastica- ics following tonsillectomies and third molar extractions has tion, palpation of that muscle should aggravate the pain.410 been shown to provide pain relief far beyond the duration of In the medullary dorsal horn, local circuit interneurons the peripheral tissue anesthesia.125,167 have the potential to affect transmission of nociceptive input A reduction in the chemical mediators of inflammation at from primary afferents to projection neurons. Depending on the level of the medullary dorsal horn also should reduce sen- the transmitter released, these neurons have the ability to sitization of second-order neurons. Decreasing the synthesis of enhance or diminish the signal. Typically, excitatory interneu- proinflammatory PGs, cytokines, nitric oxide, or the useof rons release glutamate or substance P, whereas inhibitory drugs that block the receptors of such agents probably will CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 555

From From To LC NRM thalamus Thalamus

Glia C fibers TNFα IL-1β PGs TGG SP SP Glu Glu Periaqueductal gray NE SP Glu Trigeminal Locus ganglion Projection coeruleus 5HT neuron Aβ

M-ENK NOS COX GABA glycine Nucleus GABA raphe magnus glycine NO Medullary dorsal horn Interneuron PGs

AMPA GABAB 5HT1A/D Subnucleus caudalis NMDA α2 NK-1 FIG. 12-33 Schematic diagram of the perception and modulation of orofacial pain. Activation of primary afferent fibers (in this example from an inflamed maxillary molar) leads to the entry of a nociceptive signal that is conveyed across a synapse in the subnucleus caudalis of the trigeminal spinal nucleus. The second-order neuron projects to the thalamus; the information is then relayed to the cortex. A great deal of processing of nociceptive input can occur at the level of the medullary dorsal horn (MDH). The inset depicts a typical wide dynamic range (WDR) projection neuron and its relationship with other components of the MDH. Primary afferent fibers release the excitatory amino acid, glutamate—which binds and activates either AMPA or NMDA receptors—and substance P—which activates NK-1 receptors on the WDR neuron or excitatory interneurons. Descending fibers from the locus coeruleus (LN) and nucleus raphe magnus (NRM) secrete serotonin (5HT) and norepinephrine (NE), respec- tively, which inhibit transmission. Release of γ amino butyric acid (GABA), the amino acid, glycine, and endogenous opioid peptides such as met-enkephalin (M-ENK) also inhibit transmission of nociceptive information. Projection neurons may have autocrine or paracrine effects by the synthesis and release of prostaglandins (PGs) and nitric oxide (NO) via the action of cyclooxygenase (COX) and nitric oxide synthase (NOS), respectively. Glial cells can modulate nociceptive processing by the release of cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β). The + sign indicates an excitatory action, whereas the − sign denotes an inhibitory action.

become accepted pharmacotherapy in the future. For example, clinician) that a disproportionately large portion of the application of an inflammatory agent to the tooth pulp sensory cortex in humans is devoted to input from orofacial of rat maxillary molars results in an increased receptive field regions.294 of Aβ touch receptors on the face. This can be blocked by It is becoming increasingly obvious that higher-order (i.e., pretreatment with a glutamate NMDA receptor antagonist, cortical) perceptual processes have a profound effect on the indicating that such centrally acting drugs may offer highly ultimate state of pain the patient experiences (for a review, see efficacious means of treating odontogenic 59 pain. A similar Yaksh411). Memories of previous pain experiences provide a investigation implicated nitric oxide synthesis at the level of framework by which similar new experiences are judged and the subnucleus caudalis in the development of tactile hyper- serve to shape the patient’s response to a given stimulus. In the sensitivity following dental injury.416 Reduction in nitric oxide field of dentistry, the anxiety level of the patient at the time of synthase levels may also provide protection from central treatment has been shown to affect not only the patient’s sensitization.234,248 response to pain experienced during treatment,78,407 but also the tendency of the patient to recall the experience as painful Perception: Thalamus to Cortex or unpleasant even 18 months after treatment.112 The clinician The final anatomic step in the trigeminal pain pathway should do everything possible to control a patient’s anxiety relies on neurons that leave the thalamus and extend to level prior to endodontic treatment (see also Chapter 28). One the cerebral cortex (see Fig. 12-24). The patient actually simple pretreatment method is to provide patients with posi- perceives a stimulus as painful at the cortical level. It is inter- tive written information regarding the control of pain during esting to note (but likely of no surprise to the experienced their endodontic treatment. In a placebo-controlled clinical 556 PART II The Advanced Science of Endodontics

trial, 437 endodontic patients were given one of five informa- Capillary blood flow in the coronal portion of the pulp is tive paragraphs to read prior to treatment. One of the para- nearly twice that in the root portion.192 Moreover, blood flow graphs contained positive information about pain during in the region of the pulp horns is greater than in all other areas treatment. Patients completed questionnaires following treat- of the pulp.249 In young teeth, capillaries commonly extend ment that evaluated their dental anxiety and dental fear. Sub- into the odontoblast layer, thus ensuring an adequate supply jects given positive information were shown to be less fearful of nutrients for the metabolically active odontoblasts (Fig. of pain during endodontic therapy.381 Along with a positive and 12-37). In the subodontoblastic capillaries, fenestrations are caring attitude, pharmacologic intervention may help reduce observed in the vessel wall.306 These fenestrations are thought anxiety. Nitrous oxide has been shown to be effective in a to promote rapid transport of fluid and metabolites from the dental setting,75 but it may interfere with radiography proce- capillaries to the adjacent odontoblasts. The average capillary dures during endodontic therapy. In a placebo-controlled clini- density is about 1400/mm2, which is greater than in most other cal trial in patients undergoing the extraction of impacted third tissues of the body.386 molars, 0.25 mg of oral triazolam (a benzodiazepine) provided comparable anxiolysis to intravenous diazepam titrated to a typical clinical endpoint.176 Of course, the patient so medicated must be provided transportation to and from the dental office, and the potential drug-drug interactions with other centrally acting agents such as opioids, barbiturates, and alcohol must AL be considered. One interaction that should be considered is the capacity of grapefruit juice to prolong the half-life of tri- azolam.217 It has been shown that furanocoumarins in grape- fruit juice inhibit cytochrome P450 3A4,290 which is the enzyme VL responsible for the metabolism of triazolam in the liver. Patients should be told not to take oral triazolam with grapefruit juice.

VASCULAR SUPPLY TCN Blood from the dental artery enters the tooth by way of arteri- oles having diameters of 100 µm or less. These vessels pass through the apical foramen or foramina with nerve bundles. Smaller vessels may enter the pulp by way of lateral or acces- sory canals. They are richly innervated by autonomic and sensory nerves, and the regulation of blood flow seems to be dominated by neuronal control2,20,188,283,359 (Fig. 12-34). The arterioles course up through the central portion of the FIG. 12-35 High-power scanning electron micrograph of vascular network radicular pulp and give off branches that spread laterally in the radicular pulp of a dog molar showing the configuration of the subodon- toward the odontoblast layer, beneath which they ramify to toblastic terminal capillary network (TCN). Venules (VL) and arterioles (AL) are form a capillary plexus196 (Fig. 12-35). As the arterioles pass indicated. (Courtesy Dr. Y. Kishi, Kanagawa Dental College, Kanagawa, into the coronal pulp, they fan out toward the dentin, diminish Japan.) in size, and give rise to a capillary network in the subodonto- blastic region351 (Fig. 12-36). This network provides the odon- toblasts with a rich source of metabolites.

TCN VL

VL AL 100 VL

FIG. 12-36 Subodontoblastic terminal capillary network (TCN), arterioles 20 ␮m (AL), and venules (VL) of young canine pulp. Dentin would be to the far left and the central pulp to the right. Scale bar: 100 µm. (From Takahashi K, Kishi FIG. 12-34 Substance P–positive nerve fibers in the wall of pulpal blood Y, Kim S: A scanning electron microscopic study of the blood vessels of dog vessels. (Courtesy Dr. K.J. Heyeraas.) pulp using corrosion resin casts, J Endod 8:131, 1982.) CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 557

Blood passes from the capillary plexus, first into postcapil- Doppler flowmetry can be used to detect revascularization of lary venules (see Figs. 12-36 and 12-38) and then into larger traumatized teeth.79,88 Although measurement of pulpal blood venules.196 Venules in the pulp have unusually thin walls, and flow would be an ideal tool for determining pulp vitality, the the muscular layer is discontinuous,68 which may facilitate the use of laser Doppler and other techniques is limited due to movement of fluid in or out of the vessel. The collecting sensitivity, specificity, reproducibility, and costs. venules become progressively larger as they course to the central region of the pulp. The largest venules have a diameter Regulation of Pulpal Blood Flow that may reach a maximum of 200 µm, considerably larger Under normal physiologic conditions, pulpal vascular tone is than the arterioles of the pulp. controlled by neuronal, paracrine, and endocrine mechanisms The resting pulpal blood flow is relatively high, averaging that keep the blood vessels in a state of partial constriction. 0.15 to 0.60 ml/min/g tissue,245,360 and blood volume represents The pulpal blood flow is also influenced by vascular tone in about 3% of pulpal wet weight,30 approximately the same as in neighboring tissues. Vasodilatation in these tissues has been mammary tumor tissue.403 As would be anticipated, pulpal shown to cause a drop in pulpal blood flow due to reduction blood flow is greater in the peripheral layer of the pulp in local arterial pressure of the teeth and thereby reduced (i.e., the subodontoblastic capillary plexus)196 where the pulpal perfusion pressure.361 The “stealing” of dental perfusion oxygen consumption has been shown to be higher than in the pressure makes the dental pulp vulnerable in clinical situations central pulp.26 with inflammatory processes in the adjacent tissues, as in gin- Changes in pulpal blood flow can be measured through givitis and periodontitis. dentin using laser Doppler flowmeters. Sensitivity to move- Neuronal regulation of blood flow is extensive in the pulp. ment requires that they are stabilized in an occlusal stent or a There is little or no vasoconstrictor tone of sympathetic origin modified rubber dam clamp.93,321 Because up to 80% of the in the dental pulp during resting conditions,165,359 but a neuro- Doppler signal originates from periodontal tissue, it is helpful nal vasodilator tone caused by release of sensory neuropeptides to cover periodontal tissues with a black rubber dam.144 Laser has been demonstrated (Fig. 12-39).21,20 There are α-adrenergic receptors in the pulp,162 and stimula- tion of the cervical sympathetic trunk causes vasoconstriction and fall in pulpal blood flow that can be partially reversed by α-receptor blockade.188,359 NPY, colocalized with norepineph- rine in pulpal sympathetic nerve fibers, contributes also to vasoconstriction in the pulp.81,194 Increase in pulpal blood flow is observed after electrical tooth stimulation and is caused by the release of sensory neu- ropeptides followed by vasodilatation.20,153,181 CGRP released from sensory nerve fibers is mainly responsible for the observed vasodilatation.21,20 Glutamate, present in CGRP negative sensory afferent nerve fibers in the pulp, also has a vasodilatory effect when applied in the pulp during experimental conditions.419 There is evidence for sympathetic modulation of sensory neuropeptide release in the dental pulp138; presynaptic adreno- ceptors are found on the sensory nerve terminals and attenuate the release of vasodilators from the sensory nerves.34,180 FIG. 12-37 Blood vessels in the pulp horn fan out into the odontoblast Muscarinic receptors have been identified in the pulp,32 layer. (Courtesy Dr. S.R. Haug.) and the parasympathetic neurotransmitter acetylcholine (ACh)

FIG. 12-38 Postcapillary venule draining blood from subodonto- blastic capillary plexus. 558 PART II The Advanced Science of Endodontics

0 Similarly to the effect of norepinephrine released from sym- Ϫ5 pathetic nerve fibers in the pulp, epinephrine released from the Ϫ10 adrenal medulla will cause vasoconstriction due to activation of α-adrenergic receptors in the pulp. Another catecholamine, Ϫ15 dihydroxyphenylalanine (DOPA), also induces vasoconstric- Ϫ20 tion in pulpal arterioles when applied intraarterially.417 Ϫ25 * * Ϫ30 * Fluid Drainage * Ϫ35 Interstitial fluid, which accumulates in the tissue during normal conditions through net filtration out of the blood Percent of control Ϫ40 * vessels or during inflammation where the net filtration is Ϫ45 * h-CGRP(8-37) increased, must be removed in order to maintain normal fluid Ϫ50 * SR 140.33 balance. In most tissues in the body, the lymphatic vessels Ϫ55 drain excess fluid from the peripheral tissue and return it into PBF GBF MAP the blood vessel system. In addition, the lymphatic system is FIG. 12-39 Effect of antagonist infusion of h-CGRP(8-37) (calcitonin gene– important because it transports captured antigen and presents related peptide inhibitor) and SR 140.33 (substance P inhibitor) on basal it in the lymph nodes. The existence of lymphatics in the pulp pulpal blood flow (PBF) and gingival blood flow (GBF). (From Berggreen E, has been a matter of debate because it is difficult to distinguish Heyeraas KJ: Effect of the sensory neuropeptide antagonists h-CGRP[8-37] between blood and lymphatic vessels by ordinary microscopic and SR 140.33 on pulpal and gingival blood flow in ferrets, Arch Oral Biol techniques without specific lymphatic markers. 45:537, 2000.) Several specific lymphatic markers have now been applied, and contradictory conclusions have been drawn. One of the markers tested is vascular endothelial growth factor receptor (VEGFR-3) known to be expressed by lymphatic endothelial causes vasodilatation and increases blood flow in the tissue.417 cells in adult tissue.114,242 The receptor expression was reported The vasodilation evoked by acetylcholine has been demon- in human and mouse pulp tissue, but to identify lymphatic strated to be partly dependent on nitric oxide (NO) production. vessels, use of more than one lymphatic marker is recom- VIP, which coexists with ACh in postganglionic neurons, is mended. However, other studies have failed to demonstrate found in the dental pulp105,377 and has been demonstrated to lymphatic markers in the pulp 114,242 and one study showed cause vasodilatation and increase in pulpal blood flow in cats.283 lymphatic vessel endothelium receptor-1 (LYVE-1) staining in On the other hand, Sasano and coworkers323 failed to dem- immune cells in human pulp, but not in vascular structures onstrate parasympathetic nerve-evoked vasodilatation in the (Fig. 12-40). Taken together it seems that the pulp is not cat dental pulp, leaving pulpal vascular responses to parasym- supplied with draining lymphatic vessels. pathetic neurotransmitters with some uncertainty. Transcapillary Fluid Exchange Local Control of Blood Flow In all tissues in the body, the fluid transport between the blood The microvascular bed in the dental pulp has the ability to vessels and the interstitial space is regulated by differences in regulate hemodynamics in response to local tissue demands. colloid osmotic and hydrostatic pressures in the plasma and Endothelin-1 is located in the endothelium of pulpal vascula- the interstitium, and by properties in the capillary membrane ture,57 and close intraarterial infusions of endothelin-1 reduce (Fig. 12-41). From the interstitium excess fluid is transported pulpal blood flow.22,116,417 However, endothelin-1 does not seem back to the blood circulation through the lymphatic system. to influence blood vessel vascular tone under basal, resting The pulp seems to be an exception along with, for example, conditions.22 the brain and bone marrow, as lymphatic vessels are not The endothelium in pulpal blood vessels modulates vascu- detected in the tissue (see the previous section). During normal lar tone by release of vasodilators such as prostacyclin and NO. conditions, a steady state is achieved as the fluid filtered into A basal synthesis of NO provides a vasodilator tone on pulpal the interstitial space equals the amount of fluid transported out vessels.20,223 The shear forces that blood flow exert on endothe- of the same compartment. Using radioisotopes, the interstitial lial cells seem to regulate the release of NO.73 fluid volume in the pulp was measured and averaged 0.6 ± Adenosine is released from ischemic and hypoxic tissue and 0.03 ml/g wet weight,30 demonstrating that as much as 60% of is probably important in the metabolic regulation of blood flow the extracellular fluid in dental pulp is located outside the in periods of low pulpal oxygen tension. When applied from vascular system. Measurements of interstitial fluid pressure in the extraluminal side of the vessel wall, adenosine mediates the pulp with the micropuncture method have given values vasodilatation in pulpal vessels.417 that range from 6 to 10 mm Hg,20,150 but higher values mea- sured with different methods have also been reported.39,380,385 Humoral Control of Blood Flow Colloid osmotic pressure (COP) measurements in intersti- Evidence for humoral control of pulpal blood flow exists and tial fluid isolated from rat incisors have shown a relatively high takes place when vasoactive substances transported by the pulpal COP, reaching 83% of plasma COP.30 The high value bloodstream reach the receptors in the pulp tissue. Angiotensin may imply that the normal permeability of pulpal vessels to II is produced by activation of the renin/angiotensin system plasma proteins is relatively high or the drainage of plasma and exerts a vasoconstrictive basal tone on pulpal blood proteins is ineffective. vessels.22 The angiotensin II receptors, AT1 and AT2, have been Because lymphatic vessels are lacking inside the pulp, identified in the rat pulp.341 excess interstitial fluid and proteins must be transported CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 559

LYVE-1 CD68 Merged

FIG. 12-40 Immune cells in normal human pulp are immunopositive to LYVE-1, known as a lymphatic vessel marker. The CD68+/LYVE-1+ cells derive from the monocytic lineage of cells. Immunostaining demonstrated the lack of LYVE-1+ lymphatic vessels in the pulp. (Courtesy Dr. A Virtej.)

Capillary Proteoglycan Kf, ␴ Capillary hydrostatic pressure Pc Interstitial fluid P Lymph flow if pressure Plasma colloid COPc osmotic pressure

COPif Interstitial fluid Cell-collagen colloid osmotic pressure attachment integrin ␣2␤1 Net capillary filtration Jv Collagen bundles Hyaluronan

FIG. 12-41 Interstitial structure and pressures that govern transcapillary fluid transport. Kf, Capillary filtration coefficient;σ , capillary reflection coefficient for plasma proteins. (From Wiig H, Rubin K, Reed RK: New and active role of the interstitium in control of interstitial fluid pressure: potential therapeutic consequences,Acta Anaesthesiol Scand 47:111, 2003.) out of the pulp by other transport routes in order to achieve a steady-state situation. Two possibilities exists: (1) transport of fluid in the interstitial compartment toward the apical part of 50 the pulp and furthermore out of the apex, and (2) a combina- tion of fluid reabsorption into pulpal blood vessels in addition 0 LDF percent to transport of protein-rich fluid toward the apex. ᭝ Circulation in the Inflamed Pulp 30 Minute Inflammation in the pulp takes place in a low-compliance environment composed of rigid dentinal walls. Compliance is 0 defined as the relationship between volume (V) and interstitial mm Hg IFP pressure (P) changes: C = Δ V/ Δ P. Consequently, in the low- compliant pulp, an increase in blood or interstitial volume will 150 lead to a relatively large increase in the hydrostatic pressure in mm Hg the pulp. The acute vascular reactions to an inflammatory A

P 0 stimulus are vasodilatation and increased vascular permeabil- ity, both of which will increase pulp interstitial fluid pres- FIG. 12-42 Original simultaneous recordings of percent change in pulpal sure151,154,359,379 and may tend to compress blood vessels and blood flow Δ( LDF%), interstitial fluid pressure (IFP), and systemic blood pres- counteract a beneficial blood flow increase (Fig. 12-42). sure (PA mm Hg) in a cat during electrical tooth stimulation. Note that when Classical studies have demonstrated that an increase in IFP is first decreasing after an initial rise, the pulpal blood flow reaches its intrapulpal tissue pressure promoted absorption of tissue fluid maximal level (arrows), demonstrating compression of vessels in the first back into the circulation, thereby reducing the pressure.151,154 phase. (Courtesy Dr. K.J. Heyeraas.) This observation can explain why pulpal tissue pressure in inflamed pulps may persist in local regions for long observa- tion periods,362 contradicting the old concept of a wide, 560 PART II The Advanced Science of Endodontics generalized collapse of pulpal venules and cessation of blood Clinical Aspects flow (pulpal strangulation theory). The influence of posture on pulpal blood flow has been The delivery of dental restorative procedures may lead to observed in humans.60 Significantly greater pulpal blood flow substantial increases or decreases in pulpal blood flow, depend- was measured when subjects changed from an upright to a ing on the precise procedure and time point sampled.189 Vaso- supine position. The supine position increases venous return active mediators are locally released upon an inflammatory from all tissues below the level of the heart, thereby increasing insult, and in the pulp, prostaglandin E2, bradykinin, SP, and cardiac output and producing a transient increase in systemic histamine have all been demonstrated to increase pulpal blood blood pressure. The increase in blood pressure stimulates flow after application.191,284 In contrast, serotonin (5-HT) is baroreceptors that reflexively decrease sympathetic vasocon- released primarily from the platelets, and given intraarterially, striction to all vascular beds, thereby increasing peripheral it has been shown to reduce pulpal blood flow.193,417 blood flow. Acute inflammation in the dental pulp induces an immedi- Patients with pulpitis often report an inability to sleep at ate rise in blood flow and can reach a magnitude of upto night because they are disturbed by throbbing tooth pain. In nearly 200% of control flow followed by increased vascular addition to the lack of distractions normally present during the permeability.152,154 day, the following mechanism may be operative in patients A common outcome of pulpal inflammation is development with inflamed pulps. When these patients lie down at the end of tissue necrosis. One study found circulatory dysfunction of the day, their pulpal blood flow probably increases due developed in the pulp after exposure to lipopolysaccharide to the cardiovascular postural responses described earlier. (LPS) from gram-negative bacteria.30 This may increase their already elevated pulpal tissue pres- In addition, the inflammatory cytokines IL-1 and TNF-α sure,151,154,344,362,379 which is then sufficient to activate sensitized are elevated in the inflamed pulp. When the endothelium is pulpal nociceptors and initiate spontaneous pulpal pain. Thus, exposed to endotoxin, it expresses cytokines, chemokines, the “throbbing” sensation of toothache was previously consid- and thromboxane A2. The latter has been demonstrated to be ered as an effect of the pulsation in the pulp that follows heart produced in the pulp exposed to LPS281 and induces vasocon- contractions (systole), but a study investigating the rhythm of striction. The set of changes in endothelial function have toothache and the patient pulse showed lack of synchrony been called endothelial perturbation and were first described in between the two parameters.251 The authors raised an alterna- endothelial cells exposed to endotoxin or to cytokines such tive hypothesis: that the throbbing quality is not a primary as IL-1, TNF-α, and IL-6.24,271 The activated endothelium also sensation but rather an emergent property, or perception, participates in procoagulant reactions that promote fibrin clot whose “pacemaker” lies within the CNS causing intermittent formation.345 A reduced pulpal perfusion due to endothelial increases in pulpal tissue pressure. perturbation might be the consequence of bacterial infection impairing pulpal defense mechanisms and promoting necro- sis. Downregulation of vascular endothelial growth factor PULPAL REPAIR (VEGF) expression in stromal cells and reduced microvessel The inherent healing potential of the dental pulp is well rec- density have been observed in human dental pulps with irre- ognized. As in all other connective tissues, repair of tissue versible pulpitis.10 VEGF is an essential proangiogenic factor, injury commences with débridement by macrophages, fol- and the reduced microvessel density might also lead to lowed by proliferation of fibroblasts, capillary buds, and the reduced pulpal perfusion and contribute to development of formation of collagen. Local circulation is of critical impor- pulpal necrosis. tance in wound healing and repair. An adequate supply of blood is essential to transport immune cells into the area of Vascular Permeability pulpal injury and to dilute and remove deleterious agents from Increased vascular permeability takes place as a result of acute the area. It is also important to provide fibroblasts with nutri- inflammation, and vascular leakage has been demonstrated in ents from which to synthesize collagen. Unlike most tissues, the pulp after release of inflammatory mediators such as the pulp has essentially no collateral circulation; for this prostaglandin, histamine, bradykinin, and the sensory neuro- reason, it is theoretically more vulnerable than most other peptide, SP.182,191,236 tissues. In the case of severe injury, healing would be impaired LPS and lipoteichoic acid () from gram-negative and gram- in teeth with a limited blood supply. It seems reasonable to positive bacteria, respectively, cause upregulation of VEGF assume that the highly cellular pulp of a young tooth, with a in activated pulpal cells.33,353 VEGF increases vascular perme- wide-open apical foramen and rich blood supply, has a much ability,91,328 and it is likely that it also causes leakage in pulpal better healing potential than an older tooth with a narrow vessels. It is a potent agent, because its ability to enhance foramen and a restricted blood supply. microvascular permeability is estimated to be 50,000 times Dentin can be classified as primary, secondary, or tertiary, higher than that of histamine.335 Cytokines such as IL-1 and depending on when it was formed. Primary dentin is the TNF-α are released into the pulp interstitial fluid during regular tubular dentin formed before eruption, including inflammation30 and upregulate VEGF mRNA gene expression mantle dentin. Secondary dentin is the regular circumferential in pulpal fibroblasts.60 dentin formed after tooth eruption, whose tubules remain The resulting increased vascular permeability allows continuous with that of primary dentin. Tertiary dentin is the increased transport of proteins through the capillary vessel wall irregular dentin that is formed in response to abnormal and results in increased COP in the tissue. In acute pulpitis stimuli, such as excess tooth wear, cavity preparation, restor- induced by LPS, it has been shown that COP in the pulp can ative materials, and caries.64,65 In the past, tertiary dentin reach the level of plasma COP, meaning that a protein transport has been called irregular dentin, irritation dentin, reparative barrier between plasma and interstitium can be eliminated.30 dentin, and replacement dentin. Much of the confusion was CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 561 caused by a lack of understanding of how tertiary dentin is formed. If the original odontoblasts that made secondary dentin are responsible for focal tertiary dentin formation, that particular type of tertiary dentin is termed reactionary dentin.339 Generally, the rate of formation of dentin is increased, but the tubules remain continuous with the secondary dentin.342 However, if the provoking stimulus caused the destruction of the original odontoblasts, the new, less tubular, more irregular dentin formed by newly differentiated odontoblast-like cells is called reparative dentin. In this dentin the tubules are usually not continuous with those of secondary dentin. Initially, the newly RD formed cells tend to be cuboidal in shape, without the odon- toblast process that is necessary to form dentinal tubules. They seem to form in response to the release of a host of growth factors that were bound to collagen during the formation of secondary dentin.92,309,339 The loss of the continuous layer of odontoblasts exposes unmineralized predentin that is thought to contain both soluble and insoluble forms of TGF-β, insulin- FIG. 12-43 Reparative dentin (RD) deposited in response to a carious lesion like growth factor (IGF)-1 and IGF-2, bone morphogenetic in the dentin. (From Trowbridge HO: Pathogenesis of pulpitis resulting from proteins (BMPs), VEGF, and other growth factors that attract dental caries, J Endod 7:52, 1981.) and cause proliferation and differentiation of mesenchymal stem cells to form reparative dentin and new blood vessels. During caries progression, bacterial acids may solubilize these growth factors from mineralized dentin, liberating them to diffuse to the pulp, where they could stimulate reactionary dentin formation. This is also thought to be the mechanism of action of calcium hydroxide during apexification treatment. Despite its high pH, calcium hydroxide has a slight demineral- izing effect on dentin and has been shown to cause the release of TGF-β.338 TGF-β and other growth factors stimulate and accelerate reparative dentinogenesis. Other researchers have attempted to apply growth factors to dentin to allow it to diffuse through the tubules to the pulp.316,317,340 Although this has been successful, the remaining dentin thickness must be so thin that this approach may not be practical from a thera- peutic perspective. Others have inserted deoxyribonucleic acid (DNA)-sequenced BMP-7 into retroviruses to transfect ferret pulpal fibroblasts to stimulate increased BMP-7 production. Although this was successful in normal pulps,316 it was unsuc- FIG. 12-44 Layer of cells forming reparative dentin. Note the decreased cessful in inflamed pulps.315 Specific amelogenin gene splice tubularity of reparative dentin compared with the developmental dentin products, A+4 and A-4, adsorbed onto agarose beads and above it. applied to pulp exposures, induced complete closure and min- eralization of the root canal in rat molars.122 The regulation of It will be recalled that secondary dentin is deposited cir- peritubular dentin formation is not well understood. Some cumpulpally at a slow rate throughout the life of the vital have claimed that this is a passive process resulting in occlu- tooth.342 In contrast, when a carious lesion has invaded dentin, sion of the tubules over time, but it has also been claimed that the pulp usually responds by depositing a layer of tertiary this is a mechanism under odontoblast control. If odontoblasts dentin over the dentinal tubules of the primary or secondary could be stimulated to form excessive peritubular dentin by dentin that communicate with the carious lesion (Fig. 12-43). the application of an appropriate biologic signaling molecule Similarly, when occlusal wear removes the overlying enamel to the floor of cavity preparations, then the tubules ofthe and exposes the dentin to the oral environment, tertiary dentin remaining dentin could be occluded, rendering this dentin is deposited on the pulpal surface of the exposed dentin. Thus, impermeable and protecting the pulp from the inward diffu- the formation of tertiary dentin allows the pulp to retreat sion of noxious substances that might leak around restora- behind a barrier of mineralized tissue.370 tions.291 These are examples of how molecular biology may be Compared with primary or secondary dentin, tertiary dentin used in future restorative dentistry. tends to be less tubular, and the tubules tend to be more irregu- The term most commonly applied to irregularly formed lar with larger lumina. In some cases, particularly when the dentin is reparative dentin, presumably because it frequently original odontoblasts are destroyed, no tubules are formed. The forms in response to injury and appears to be a component of cells that form reparative dentin are often cuboidal and not as the reparative process. It must be recognized, however, that columnar as the primary odontoblasts of the coronal pulp this type of dentin has also been observed in the pulps of (Fig. 12-44). The quality of tertiary dentin (i.e., the extent to normal, unerupted teeth without any obvious injury.276 which it resembles primary or secondary dentin) is quite 562 PART II The Advanced Science of Endodontics

A PD

F

FIG. 12-45 Swiss-cheese type of reparative dentin. Note the numerous F areas of soft-tissue inclusion and infiltration of inflammatory cells in the pulp.

variable. If irritation to the pulp is relatively mild, as in the case of a superficial carious lesion, then the tertiary dentin formed may resemble primary dentin in terms of tubularity and degree of mineralization. On the other hand, dentin deposited in response to a deep carious lesion may be relatively atubular and poorly mineralized, with many areas of interglobular B C dentin. The degree of irregularity of this dentin is probably determined by numerous factors, such as the amount of inflam- FIG. 12-46 Autoradiographs from dog molars illustrating uptake of mation present, the extent of cellular injury, and the state of 3H-thymidine by pulp cells preparing to undergo cell division after pulpotomy differentiation of the replacement odontoblasts. and pulp capping with calcium hydroxide. A, Two days after pulp capping. The poorest quality of reparative dentin is usually observed Fibroblasts, endothelial cells, and pericytes beneath the exposure site are in association with marked pulpal inflammation.64,370 In fact, labeled. B, By the fourth day, fibroblasts (F) and preodontoblasts adjacent to the dentin may be so poorly organized that areas of soft tissue the predentin (PD) are labeled, which suggests that differentiation of pre­ are entrapped within the dentinal matrix. In histologic sec- odontoblasts occurred within 2 days. C, Six days after pulp capping, new tions, these areas of soft-tissue entrapment impart a Swiss- odontoblasts are labeled, and tubular dentin is being formed. (Titrated thymi- cheese appearance to the dentin (Fig. 12-45). As the entrapped dine was injected 2 days after the pulp capping procedures in B and C.) (From soft tissue degenerates, products of tissue degeneration are Yamamura T, Shimono M, Koike H, et al: Differentiation and induction of released that further contribute to the inflammatory stimuli undifferentiated mesenchymal cells in tooth and periodontal tissue during assailing the pulp.370 wound healing and regeneration, Bull Tokyo Dent Coll 21:181, 1980.) It has been reported that trauma caused by cavity prepara- tion that is too mild to result in the loss of primary odontoblasts does not lead to reparative dentin formation, even if the cavity dentin synthesize type I (but not type III) collagen, and they preparation is relatively deep.73 This has been confirmed both are immunopositive for dentin sialoprotein. in rat teeth258 and human teeth.256 However, chronic pulpal Destruction of primary odontoblasts can occur from cutting inflammation associated with deep caries produces reparative cavity preparations dry,80,202 from bacterial products such as dentin. This reparative dentin is formed by new odontoblast- endotoxins shed from deep carious lesions,18,395 or from like cells. For many years, it has been recognized that destruc- mechanical exposure of pulps.257 Such pulpal wounds do not tion of primary odontoblasts is soon followed by increased heal if the tissue is inflamed.64 Local fibroblast-like cells divide, mitotic activity within fibroblasts of the subjacent cell-rich and the new cells then redifferentiate in a new direction to zone. It has been shown that the progeny of these dividing cells become odontoblasts. Recalling the migratory potential of differentiate into functioning odontoblasts.98 Investigators414 ectomesenchymal cells from which the pulpal fibroblasts are have studied dentin bridge formation in healthy teeth of dogs derived, it is not difficult to envision the differentiating odon- and found that pulpal fibroblasts appeared to undergo dedif- toblasts moving from the subodontoblastic zone to the area of ferentiation and revert to undifferentiated mesenchymal stem injury to constitute a new odontoblast layer. Activation of cells (Fig. 12-46). The similarity of primary odontoblasts to antigen-presenting dendritic cells by mild inflammatory pro- replacement odontoblasts was established by D’Souza and col- cesses may also promote osteoblast/odontoblast-like differen- leagues.77 They were able to show that cells forming reparative tiation and expression of molecules implicated in mineralization. CHAPTER 12 Structure and Functions of the Dentin-Pulp Complex 563

Recognition of bacteria by specific odontoblast and fibroblast membrane receptors triggers an inflammatory and immune response within the pulp tissue that would also modulate the repair process.121 Although many animal studies have shown dentin bridge formation in healthy pulps following pulp capping with adhe- sive resins,64 such procedures fail in normal human teeth.61 When small mechanical pulp exposures are inadvertently made in healthy teeth, the recommendation has been to place a small, calcium hydroxide–containing dressing on the wound. After setting, the surrounding dentin can be bonded using a no-rinse, self-etching primer adhesive.175 Calcium silicate cements like mineral trioxide aggregate (MTA) have also been recognized to promote hard-tissue formation, and available information indicates that the dentin bridge formed under these cements is more dense and has fewer defects compared with calcium hydroxide–containing dressings.3,5,261,368 The formation of atubular “fibrodentin” is another potential FIG. 12-47 Diffusion of dye from the pulp into reparative dentin. Note product of newly differentiated odontoblasts, provided that a atubular zone between reparative dentin (RD) and primary dentin on the left. 15 capillary plexus develops beneath the fibrodentin. This is (From Fish EW: Experimental investigation of the enamel, dentin, and dental 64,98 consistent with the observation made by other researchers pulp, London, 1932, John Bale Sons & Danielson.) that the newly formed dentin bridge is composed first of a thin layer of atubular dentin on which a relatively thick layer of tubular dentin is deposited. The fibrodentin was lined by cells case. It is well known that reparative dentin can be deposited resembling mesenchymal cells, whereas the tubular dentin was in a pulp that is irreversibly injured and that its presence does associated with cells closely resembling odontoblasts. not necessarily signify a favorable prognosis (see Fig. 12-45). Other researchers342 studied reparative dentin formed in The quality of the dentin formed, and hence its ability to response to relatively traumatic experimental class V cavity protect the pulp, to a large extent reflects the environment of preparations in human teeth. They found that seldom was the cells producing the matrix. The presence of a single tunnel reparative dentin formed until about the 30th postoperative defect64 through reparative dentin would circumvent the pro- day. The rate of dentin formation was 3.5 µm/day for the first tective effect of atubular reparative dentin. Therefore, any clini- 3 weeks after the onset of dentinogenesis, after which it cal attempt at pulp therapy must include sealing dentin with decreased markedly. By postoperative day 132, dentin forma- bonding agent. tion had nearly ceased. Assuming that most of the odontoblasts Periodontally diseased teeth have smaller root canal diam- were destroyed during traumatic cavity preparation, as was eters than teeth that are periodontally healthy.203 The root likely in this experiment, the 30-day delay between cavity canals of such teeth are narrowed by the deposition of large preparation and the onset of reparative dentin formation is quantities of reactionary dentin along the dentinal walls.327 The thought to reflect the time required for the proliferation, migra- decrease in root canal diameter with increasing age, in the tion, and differentiation of new replacement odontoblasts. absence of periodontal disease, is more likely to be the result Does reparative dentin protect the pulp, or is it simply a of secondary dentin formation. form of scar tissue? To serve a protective function, it would One study showed that in a rat model, frequent scaling and have to provide a relatively impermeable barrier that would root planing resulted in reparative dentin formation along the exclude irritants from the pulp and compensate for the loss of pulpal wall subjacent to the instrumented root surface.146 developmental dentin. The junction between developmental However, given that normal rat root dentin is only 100 µm and reparative dentin has been studied using a dye diffusion thick, these procedures are probably more traumatic to the technique, which demonstrated the presence of an atubular pulp in the rat model than in humans, where normal root zone situated between secondary dentin and reparative dentin dentin is more than 2000 µm thick. (Fig. 12-47).94 In addition to a dramatic reduction in the Not uncommonly, the cellular elements of the pulp are number of tubules, the walls of the tubules along the junction largely replaced by fibrous connective tissue over a span of 5 were often thickened and occluded with material similar to decades. It appears that in some cases, the pulp responds to peritubular matrix.326 Taken together, these observations would noxious stimuli by accumulating large fiber bundles of colla- indicate that the junctional zone between developmental and gen, rather than by elaborating reparative dentin (Fig. 12-48). reparative dentin is an atubular zone of low permeability. However, fibrosis and reparative dentin formation often go Moreover, the accumulation of pulpal dendritic cells was hand in hand, indicating that both are expressions of a repara- reduced after reparative dentin formation, which may indicate tive potential. In periodontally diseased teeth, the pulp tissue the reduction of incoming bacterial antigens.319 is found to be the site of an enhanced process of collagenous One group356 studied the effect of gold foil placement on fibrosis associated with an inflammatory infiltrate.56 human pulp and found that this was better tolerated in teeth With the expanding knowledge of tooth regeneration and in which reparative dentin had previously been deposited biologic mechanisms of functional dental tissue repair, current beneath the cavity than in teeth that lacked this deposit. It treatment strategies are beginning to give way to evolving fields would thus appear that reparative dentin can protect the such as tissue engineering and biomimetics. Pulpal stem cells pulp,18 but it must be emphasized that this is not always the in scaffolds have been shown to produce pulplike tissues with 564 PART II The Advanced Science of Endodontics

CB

FIG. 12-48 Fibrosis of dental pulp showing replacement of pulp tissue by FIG. 12-50 with a smooth surface and concentric laminations large collagen bundles (CB). in the pulp of a newly erupted premolar extracted in the course of orthodontic treatment.

Pre-op 7 Month recall

FIG. 12-49 Immature tooth with a necrotic infected canal with apical periodontitis. The canal is disinfected with copious irrigation with sodium hypochlorite and an antibiotic paste. Seven months after treatment, the patient is asymptomatic, and the apex shows healing of the apical periodontitis and some closure of the apex. (From Banchs F, Trope M: Revascularization of immature permanent teeth with apical periodontitis: new treatment protocol? J Endod 30:196, 2004.) FIG. 12-51 Diffuse calcification near the apical foramen.

tubular-like dentin,87 and in animal models, root perforations have been treated with scaffolds of collagen, pulpal stem cells, in at least 50% of all teeth. In the coronal pulp, calcification and dentin matrix protein 1, resulting in organized matrix usually takes the form of discrete, concentric pulp stones (Fig. similar to that of pulpal tissue.304 12-50), whereas in the radicular pulp, calcification tends to be Studies investigating new possibilities for regeneration of diffuse (Fig. 12-51).374 There is no clear evidence as to whether the pulp/dentin complex are now frequently reported and are pulp calcification is a pathologic process related to various described in detail in Chapter 10. A first interesting case report, forms of injury or a natural phenomenon. The clinical signifi- which has been followed up by others, has led to new strategies cance of pulp calcification is that it may hinder root canal for treatment of necrotic immature roots (Fig. 12-49; see also treatment. Chapter 10).13 In the future, the field of pulpal repair will prob- Pulp stones (denticles) range in size from small, micro- ably develop rapidly, and new treatment strategies will appear. scopic particles often seen in association with the wall of arte- rioles to accretions that occupy almost the entire pulp chamber (Fig. 12-52). The mineral phase of pulp calcifications has been PULPAL CALCIFICATIONS shown to consist of typical carbonated hydroxyapatite.374 His- Calcification of pulp tissue is a common occurrence. Although tologically, two types of stones are recognized: (1) those that estimates of the incidence of this phenomenon vary widely, it are round or ovoid, with smooth surfaces and concentric is safe to say that one or more pulp calcifications are present laminations (see Fig. 12-50), and (2) those that assume no