The International Journal of Periodontics & Restorative Dentistry

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Connective Tissue–Cementum Regeneration: A New Histologic Regeneration Following the Use of Enamel Matrix Derivative in Dehiscence- Type Defects. A Dog Model

Khalid Alhezaimi, BDS, MSc, FRCD(C)1/Thakib Al-Shalan, BDS, MS, PhD1 Total periodontal healing that occurs Rory O’Neill, DMD, MS2/Tanazi Shapurian, DMD, MS3 through regeneration has been Jafar Naghshbandi, DDS, MS4/Paul Levi Jr, DMD5 defined as the reconstitution of a new 5,6 Terrence Griffin, DMD periodontium, which involves the formation of alveolar bone, new connec- The goal of this investigation was to evaluate histologically and histometrically the tive tissue attachment (NA), and new healing process in dehiscence-type defects treated by enamel matrix derivative cementum (NC).1 Alternatively, repair (EMD). Five adult female beagle dogs were used. Buccal osseous dehiscences were is defined as healing by replacement surgically created on the maxillary canines and the second and fourth premolars. with epithelium, connective tissue, or Thirty defect sites were randomly assigned to treatment and control groups both that matures into various non- (15 defect sites for each group). The treatment group received EMD application, functional types of scar tissue and is while the control groups received no EMD. After 4 months of healing, the dogs termed new attachment.1 Histolo- were sacrificed and tissue blocks were prepared. Histometric parameters were gically, patterns of repair include a long employed to evaluate the type of periodontal tissues that formed in the defects. junctional epithelium, adhesion of new All created dehiscence defects in the test sites treated by EMD had formed func- 2 tional connective tissue fibers inserted into regenerated cellular cementum. The connective tissue, and ankylosis. The mean amount of apicocoronal regenerated cementum was 3.74 ± 0.43 mm in EMD- guided tissue regeneration (GTR) tech- treated sites, whereas the control sites had not formed cementum in the created nique employs a barrier membrane to dehiscence defects (P < .000). Statistically significant differences were found be- allow selective colonization of the tween treatment and control sites. Within the limits of this study, it can be conclud- wound area by periodontal ligament ed that EMD alone effectively promoted new cementum and functionally oriented cells, space making, stabilization of connective tissue formation. (Int J Periodontics Restorative Dent 2009;29:425–433.) the wound, and protection of the blood clot.3–6 1Assistant Professor, College of Dentistry, King Saud University, Riyadh, Saudi Arabia. 2Clinical Associate Professor, School of Dental Medicine, Tufts University, Boston, Previously, in a series of experi- Massachusetts. mental studies in dogs, the periodon- 3 Clinical Assistant Professor, School of Dental Medicine, Tufts University, Boston, tal tissues formed after GTR were char- Massachusetts. 4Private Practice, Pheonix, Arizona. acterized and compared with pristine 5Associate Professor, School of Dental Medicine, Tufts University, Boston, Massachusetts. (untreated) periodontal tissues in 6Chairman and Director of Periodontology Department, School of Dental Medicine, Tufts mandibular premolars. The NC dif- University, Boston, Massachusetts. fered on the test side from the pristine

Correspondence to: Khalid Al-Hezaimi, PO Box 103851, Riyadh 11616, Saudi Arabia; side, and it was suggested that the email: [email protected]. healing results following GTR showed

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Fig 1a (left) Normal anatomy of the canine maxilla.

Fig 1b (right) Dehiscence-type defects were created with reference notches at 8 Buccal mm and 5 mm coronal to the cemento- dehiscence CEJ enamel junction (CEJ) on the canine and second premolar (P2), respectively. 5 mm 8 mm Notch (landmark for measurements) CEJ

the characteristics of repair rather than Emdogain (Biora), an EMD, possesses Periodontal surgery regeneration.7–9 Another study evalu- the potential to stimulate new con- ated periodontal healing following nective tissue attachment formation in Supragingival scaling was done using GTR therapy of dehiscence-type human intrabony defects.19 an ultrasonic scaler twice a week dur- defects in monkeys. The healing in that The aim of the present study was ing a 3-week housing period prior to study suggested that the periodontal to evaluate the type of periodontal tis- the surgery. Buccal full-thickness structure resembled that of the pristine sues that will form following EMD mucoperiosteal flaps were raised, and tissues.10 application in dehiscence-type defects all soft tissue adherent to the teeth Biologic mediators have recently in a dog model. and alveolar bone was removed. received considerable attention, with Under irrigation with sterile saline, the a variety of growth and differentiation buccal alveolar bone, the exposed factors locally applied to periodontal Method and materials periodontal ligament, and the cemen- wounds to promote regenera- tum of the canine, second premolar tion.11–13 Among these, the efficacy of Five adult female beagle dogs were (P2), and fourth premolar (P4) were enamel matrix protein (EMD) as an used (mean weight 13.4 kg, mean age removed by means of a no. 6 round adjunct to periodontal regeneration 15 months). The Institutional carbide bur and hand curettes. The therapy has been demonstrated in Committee on Animal Research at distances between the cemento- both in vitro and in vivo studies.14–17 King Saud University, Riyadh, Saudi enamel junction and the apical end of Application of an acid extract of Arabia, approved the study. The non- the defect were standardized to 8 mm porcine enamel matrix on pathologi- surgical and surgical procedures were and 5 mm for the canine and the pre- cally exposed root surfaces has been performed under general anesthesia molars, respectively (Fig 1). shown to result in periodontal con- using Ketalar (10 mg/kg body weight; The buccal half of the interradicu- nective tissue attachment formation in Pfizer Inc) and local anesthesia using lar parts of the alveolar bone was both animal and clinical trials.18 A Xylocaine (Astra) with epinephrine removed to eliminate the possibility human histologic study indicated that 5 mg/mL. of accelerated healing from the adja-

The International Journal of Periodontics & Restorative Dentistry

cent bone. A notch was made on the washed in running water, dehydrated, root surface to serve as a landmark for and embedded in paraffin. Bucco- future measurements of histologic sec- lingual sections of 7 µm were obtained tions and was prepared at the apical using a microtome with a diamond level of the surgically reduced bone. blade and stained with Retic and Three teeth were used per side, with Masson trichrome. 15 teeth per treatment variable. With the study’s split-mouth design, 30 teeth were assigned ran- Histomorphometric analysis domly to 2 groups: the control group, which did not receive EMD (n = 15 The measurements were performed sites); and the test group, which was with a light microscope linked to a treated with placement of etching eth- video camera, computer, and software ylenediaminetetraacetic acid 24% for (Buehler). The descriptive analysis of 2 minutes, followed by EMD (Emdo- each histomorphometric parameter in gain) (n = 15). The flaps were reposi- each dog was done by a single exam- tioned to their presurgical level and iner, who evaluated the type of wound sutured using Vicryl 5-0 (Ethicon). healing following EMD application and All animals received intramuscu- the presence of periodontal ligament larly injected Medicycline vet (5 mg/kg (PDL), cementum, and bone. body weight once a day for 3 days; A comparison was made of the Norbrook Lab Ltd). Sutures were tissue formed in the defect compart- removed after 2 weeks. Plaque control ments in the control and test sites. procedures, which included topical The analyses were confined to the application of a 0.2% chlorhexidine following: digluconate solution, were performed twice weekly for 4 months after surgery. • Position 1: The level of the crestal bone on the pristine side was compared with the corresponding area Histologic preparation and on the test side in both control and analysis test groups. • Position 2: The base of the notch on After 4 months, the animals were sac- the test side was compared with the rificed with an overdose of sodium corresponding area on the pristine pentobarbital 3%. The segments of side in both control and test groups. the jaws containing the teeth with the buccal dehiscence were removed en bloc along with the adjacent teeth and Data analysis alveolar bone. The blocks were fixed in 10% neutral formalin solution for 1 The standardized paired t test was week. They were decalcified in a solu- used to analyze the results. tion of equal parts of 50% formic acid and 20% sodium citrate for 10 weeks. The decalcified specimens were

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Fig 2 Test site showing 1.9 mm of regenerated bone coronal to the notch after EMD application. The new bone is associated with newly formed cementum and functionally oriented fibers (modified Masson trichrome; ϫ20).

Notch

Results cementum, and the tissue was similar to that on the corresponding pristine Test defects side (Figs 3 and 4). The coronal extent of this NA was 3.74 ± 0.43 mm, corre- NA including NC, PDL, and bone had sponding to 58% ± 4% of the defect formed within the defect compart- height from the base of the notch. ments in 3 of the 15 teeth that received EMD, and the mean new bone height from the base of the notch was 1.9 Control defects mm (Fig 2). The bone height in the other teeth in the test group was at the No NA formed, and the bone height same level as the base of the notch or at each site was apical to the base of had resorbed apically (0.4 ± 0.5 mm). the notch (0.7 ± 0.4 mm) (Fig 5). In Although there was no bone coronal to addition, there was no NC in any of the the notch in 13 of the test sites, there control sites. Twelve teeth had a long was evidence of functionally aligned junctional epithelial attachment (Figs 6 connective tissue fiber attachment and 7), and three teeth had connective associated with the newly formed tissue adhesion.

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Notch

Fig 3a (top left) Histologic section of a test site showing regeneration of cementum and PDL covering the entire defect without bone (Retic; ϫ10). NC = newly formed cementum; OC = old cementum.

Figs 3b (top center) and 3c (top right) Perpendicular connective tissue fibers inserted into newly formed cellular cementum (Retic; ϫ60). NC = newly formed cementum. NC Fig 3d (right) Continuity of the newly formed cementum (NC), which overlaps the old cementum (OC), apical to the notch is evident (Retic; ϫ40). OC

Fig 4a (left) Test site shows the connective attachment to the newly formed cementum (NC) and shows cementum artifact (lower arrow) (Retic; ϫ10). Notch Fig 4b (right) Pristine site (palatal side) shows bone, cementum, and PDL (Retic; ϫ10).

NC Palatal

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Notch

Fig 5 Control site shows bone below the Fig 6a Control site histologic section at Fig 6b Higher-power view (ϫ40). notch level, associated with absence of the notch area showing a long junctional cementum (modified Masson trichrome; epithelium with no evidence of cementum ϫ20). or bone formation (modified Masson trichrome; ϫ20).

Fig 7a (left) Control site shows epithelial downgrowth that resulted in a long junc- Notch tional epithelium (LJE) (modified Masson trichrome; ϫ10).

Fig 7b (right) Pristine site (palatal side) shows bone, cementum, and PDL (Retic; Palatal ϫ10).

LJE

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Discussion with EMD, with or without membranes, showed cementum formation when When Melcher first presented the basic compared with open flap debride- concept of periodontal wound heal- ment, which showed a slight or no ing, he suggested that there are two cementum formation in most cases.24 types of healing: regeneration (bone, Hammerström et al reported regener- PDL, and cementum) and repair (long ation (ie, bone, PDL, and cementum) junctional epithelium, connective tissue following the application of EMD in a adhesion, and ankylosis).2 New attach- buccal dehiscence model in mon- ment is defined as the union of con- keys15; however, the authors used no nective tissue or epithelium with the histologic landmark (ie, notch at the root surface that has been deprived of base of the defect), which might have its original attachment apparatus; this led to more variation in measurements. attachment may be epithelial attach- In the present study, there was a clear ment or connective tissue adaptation attachment between the connective and may include cementum.2 The new tissue and NC without the presence of attachment does not have a functional bone. The authors speculate that the connective tissue fiber but rather is an lack of complete regeneration with adaptation of the connective tissue to EMD could be attributed to the lack of the dentin or cementum. In the present space maintenance, which was one of study, 80% of the control group had a Melcher’s key prerequisites. long junctional epithelium and 20% Periodontal tissue modes of heal- had connective tissue adhesion, which ing have been classified into two is in agreement with previous types. Regeneration is defined as a reports.20–22 These earlier findings biologic process by which the archi- showed that after detachment of gin- tecture and function of lost tissue are gival fibers and the removal of cemen- completely renewed. Repair, on the tum following a flap procedure, much other hand, is a biologic process by of the root becomes covered by which continuity of disrupted tissue is epithelium and not by cementum. In restored by new tissues that do not the present test group, following EMD replicate the structure and function of application, NC was identified within the lost tissues.15 The present find- most of the new attachment, and it ings exhibit an absence of bone; how- appeared to be firmly anchored to the ever, the formation of functionally ori- root dentin, with functionally oriented ented connective tissue fibers inserted connective tissue fibers inserted into into a newly formed cellular cemen- cementum similar to the tissue arrange- tum was demonstrated in the surgi- ment observed on the pristine side. cally induced dehiscence-type One experimental study using a defects. This is called connective tis- monkey model provided histologic sue–cementum regeneration for two evidence of periodontal regeneration reasons: (1) in cases of dehiscence with enamel matrix proteins alone and where bone did not exist originally enamel matrix protein plus GTR.23 and regeneration of the original tissue Another study found that sites treated that has been deprived as a result of

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periodontal disease, cementum and 8. Araújo MG, Berglundh T, Lindhe J. On the connective tissue fibers become dynamics of periodontal tissue formation in degree III furcation defects. An experi- imperative; and (2) to the authors’ mental study in dogs. J Clin Periodontol knowledge, this is the first study to 1997;24:738–746. demonstrate histologically a functional 9. Araújo MG, Berglundh T, Lindhe J. GTR connective tissue fiber insertion into treatment of degree III furcation defects regenerated cellular cementum. The with 2 different resorbable barriers. An experimental study in dogs. J Clin Perio- results of the present study show that dontol 1998;25:253–259. a new type of regeneration with con- 10. Graziani F, Laurell L, Tonetti M, Gottlow J, nective tissue and newly formed Berglundh T. Periodontal wound healing cementum can occur in the dog following GTR therapy of dehiscence-type model following the use of biomimicry defects in the monkey: Short-, medium- and long-term healing. J Clin Periodontol mediators in areas of dehiscence. 2005;32:905–914. 11. Howell TH, Martuscelli G, Oringer J. Polypeptide growth factors for periodon- References tal regeneration. Curr Opin Periodontol 1996;3:149–156. 1. Ten Cate AR. Glossary of terms. J Perio- 12. King GN, Cochran DL. Factors that mod- dontol 1977;48:611–612. ulate the effects of bone morphogenetic 2. Melcher AH. On the repair potential of protein-induced periodontal regeneration: periodontal tissues. J Periodontol 1976;47: A critical review. J Periodontol 2002;73: 256–260. 925–936. 3. Gottlow J, Nyman S, Karring T, Lindhe J. 13. Takayama S, Murakami S, Shimabukuro Y, New attachment formation as the result of Kitamura M, Okada H. Periodontal regen- controlled tissue regeneration. J Clin Perio- eration by FGF-2 (bFGF) in primate mod- dontol 1984;11:494–503. els. J Dent Res 2001;80:2075–2079. 4. Karring T, Nyman S, Gottlow J, Laurell L. 14. Hammarström L. Enamel matrix, cemen- Development of the biological concept of tum development and regeneration. J Clin guided tissue regeneration—Animal and Periodontol 1997;24:658–668. human studies. Periodontol 2000 15. Hammarström L, Heijl L, Gestrelius S. 1993;1:26–35. Periodontal regeneration in a buccal dehis- 5. Lindhe J, Pontoriero R, Berglundh T, Araujo cence model in monkeys after application M. The effect of flap management and of enamel matrix proteins. J Clin Perio- bioresorbable occlusive devices in GTR dontol 1997;24:669–677. treatment of degree III furcation defects. 16. Heijl L. Periodontal regeneration with An experimental study in dogs. J Clin enamel matrix derivative in one human Periodontol 1995;22:276–283. experimental defect. A case report. J Clin 6. Nyman S, Karring T, Lindhe J, Plantén S. Periodontol 1997;24:693–696. Healing following implantation of peri- 17. Slavkin HC. What is the role of the host in odontitis-affected roots into gingival con- periodontal disease? Periodontal Abstr nective tissue. J Clin Periodontol 1980;7: 1975;23:101–103. 394–401. 18. Venezia E, Goldstein M, Boyan BD, 7. Araújo M, Berglundh T, Lindhe J. The peri- Schwartz Z. The use of enamel matrix odontal tissues in healed degree III furca- derivative in the treatment of periodontal tion defects. An experimental study in defects: A literature review and meta- dogs. J Clin Periodontol 1996;23:532–541. analysis. Crit Rev Oral Biol Med 2004;15: 382–402.

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19. Sculean A, Chiantella GC, Windisch P, Donos N. Clinical and histologic evaluation of human intrabony defects treated with an enamel matrix protein derivative (Emdo- gain). Int J Periodontics Restorative Dent 2000;20:374–381. 20. Levine HL, Stahl SS. Repair following periodontal flap surgery with the retention of gingival fibers. J Periodontol 1972;43: 99–103. 21. Listgarten MA. Electron microscopic study of the junction between surgically denud- ed root surfaces and regenerated periodontal tissues. J Periodontal Res 1972;7: 68–90. 22. Stahl SS, Slavkin HC, Yamada L, Levine S. Speculations about gingival repair. J Perio- dontol 1972;43:395–402. 23. Sculean A, Donos N, Brecx M, Reich E, Karring T. Treatment of intrabony defects with guided tissue regeneration and enamel-matrix-proteins. An experimental study in monkeys. J Clin Periodontol 2000; 27:466–472. 24. Lekovic V, Camargo PM, Weinlaender M, Kenney EB, Vasilic N. Combination use of bovine porous bone mineral, enamel matrix proteins, and a bioabsorbable membrane in intrabony periodontal defects in humans. J Periodontol 2001;72:583–589.

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