REVISTA DE ODONTOLOGIA DA UNESP Rev Odontol UNESP, Araraquara. nov./dez., 2011; 40(6): 310-316 © 2011 - ISSN 1807-2577 ORIGINAL ARTICLE Effect of platelet-rich plasma on healing of class III furcation defects treated with autogenous bone grafting and guided tissue regeneration: a histomorphometric study in dogs José Marcos Alves FERNANDESa, Emilio BARBOSA E SILVAa, Rodrigo Otávio Citó César RÊGOa, Rafael Scaf de MOLONa, Daniela Leal ZANDIM-BARCELOSa, Luis Carlos SPOLIDÓRIOb, Joni Augusto CIRELLIa aDepartamento de Diagnóstico e Cirurgia, Faculdade de Odontologia de Araraquara, UNESP – Univ Estadual Paulista, 14801-903 Araraquara - SP, Brazil bDepartamento de Fisiologia e Patologia, Faculdade de Odontologia de Araraquara, UNESP – Univ Estadual Paulista, 14801-903 Araraquara - SP, Brazil Fernandes JMA, Barbosa e Silva E, Rêgo ROCC, Molon RS, Zandim-Barcelos DL, Spolidório LC, Cirelli JA. Efeito do plasma rico em plaquetas na reparação de lesões de furca classe III tratadas com enxerto ósseo autógeno e regeneração tecidual guiada: estudo histomorfométrico em cães. Rev Odontol UNESP. 2011; 40(6): 310-316. Resumo Objetivo: O objetivo deste estudo foi avaliar histologicamente os efeitos do plasma rico em plaquetas (PRP), quando usado em combinação com enxerto ósseo autógeno e membrana bioabsorvível (Resolut®) no tratamento de defeitos de furca Classe III em cães. Material e método: Cinco cães foram usados neste estudo. Defeitos de furca classe III (5  mm de altura e de profundidade) foram criados cirurgicamente no terceiro pré-molar inferior de ambos os lados. Nove semanas após a primeira cirurgia, os terceiros pré-molares foram tratados com raspagem e alisamento radicular e cada defeito recebeu um dos seguintes tratamentos: Enxerto ósseo autógeno + membrana (grupo C) ou PRP + enxerto ósseo autógeno + membrana (grupo T). Após um período de cicatrização de 90 dias, os animais foram sacrificados. Processamento histológico de rotina e coloração com hematoxilina e eosina e tricrômico de Masson foram realizados para determinar o efeito dos tratamentos na regeneração dos tecidos periodontais. Os dados foram analisados pelo teste T2 de Hotelling (p  <  0,05). Resultado: A análise histomorfométrica da área de furca não mostrou nenhuma diferença estatisticamente significativa entre os grupos C e T. Os dois grupos de tratamento demonstraram resultados regenerativos semelhantes, com os defeitos de furca parcialmente preenchidos e a regeneração periodontal foi limitada à marca experimental apical das lesões. (p > 0,05). Conclusão: Dentro dos limites deste estudo, concluiu-se que o uso de PRP não melhorou a regeneração periodontal em defeitos de furca classe III tratados com enxerto ósseo autógeno e membrana bioabsorvível. Palavras-chave: Defeitos de furca; plasma rico em plaquetas; regeneração tecidual guiada; membrana bioabsorvível Abstract Objective: The purpose of this study was to evaluate the effects of the platelet-rich plasma (PRP) when used in combination with autogenous bone graft and bioabsorbable membrane (Resolut®) in the treatment of Class  III furcation defects in dogs. Material and method: Class III furcation defects (5 mm in height and in depth) were surgically created in the mandibular third premolars of five mongrel dogs. After nine weeks, the lesions were treated with scaling and root planning and each defect received one of the following treatments: autogenous bone graft + membrane (group C) or PRP + autogenous bone graft + membrane (group T). After a healing period of 90 days, the animals were sacrificed. Routine histological processing and staining with hematoxilyn and eosin and Masson trichrome were performed and a histomorphometric analysis determined the effect of the treatments on periodontal tissue regereneration. Data were analyzed by Hotelling’s T-squared (p < 0.05). Result: No statistically significant difference between C and T groups was observed by the histomorphometric analysis of the furcation area. Both treatment groups demonstrated similar regenerative results with the furcation defects partially filled and periodontal regeneration limited to the experimental notches of the lesions. (p > 0.05). Conclusion: According to the present results, PRP does not enhance the periodontal regeneration in class III furcation defects treated with autogenous bone graft and bioabsorbable membrane. Keywords: Furcation defects; platelets-rich plasma; guided tissue regeneration; absorbable membrane. Rev Odontol UNESP. 2011; 40(6): 310-316 Effect of platelet-rich plasma on healing of class III furcation defects… 311 INTRODUCTION The ultimate goal of the periodontal therapy is the restitution of the architecture and function of the original support structures (i.e. root cementum, periodontal ligament and alveolar bone) which have been destroyed during the course of periodontal disease1. The use of bone grafts and bone substitutes, guided tissue regeneration (GTR) and, more recently, the application of polypeptide growth factors (PGFs) to the surgical wound are some of the commonly employed techniques used to promote periodontal regeneration2-7. GTR therapy was introduced in 1980s and consists in the placement of a physical barrier over a periodontal osseous defect. This technique can prevent the faster proliferation of the gingival connective tissue and epithelial oral cells from growing into the bone defect, allowing the cells of the periodontal ligament to colonize the blood clot and regenerate the periodontal lost tissue8. However, the results obtained with this therapy in the treatment of large-size defects such as Class  III furcation defects are very limited9-11. The amount of regenerated tissue and the course of soft tissue healing are dependent on the individual healing potential12,13, which is significantly influenced by the presence and amount of polypeptide growth factors (PGFs) naturally available in the wound13,14. Based on this knowledge, the application of certain growth factors has been used to promote periodontal regeneration. Findings from in vitro experiments15,16, animals17,18 and clinical studies19,20 have indicated a potential influence of different growth factors on the regeneration of periodontal tissues. These proteins were used alone21, in combination with bone substitutes19,20, or in addition to GTR therapy22. Platelet-rich plasma (PRP), which is an autologous volume of plasma with a four to fivefold-increased platelet concentration above baseline, is a proven source of growth factors23. The use of PRP is based on the potential of the plasma to release multiple wound-healing growth factors and cytokines23 which are responsible for increasing collagen production, recruiting other cells to the site of injury, increasing cell mitosis, inducing cell differentiation and initiating vascular in-growth24. It has also been verified the application of the PRP in addition to other regenerative procedures in intra-bony periodontal defects25,26 and class II furcation defects27. At present, it is still unknown an effective therapy that result in periodontal regeneration of Class III furcation defects. Thus, the aim of the present study was to evaluate histologically and histometrically the additional effect of PRP on the treatment of Class III furcation defects comparing the use of PRP associated to bone graft + GTR to bone graft + GTR only. MATERIAL AND METHOD 1. Sample Experimental group comprised five male mongrel dogs, weighing approximately 15  kg each and estimated ages of 1.5 to 3 years old, maintained in the animal facilities of the School of Dentistry at Araraquara - UNESP. The study protocol was conducted according to the recommendations of the National Council for the Control of Animal Experimentation (CONCEA) and the protocol was approved by the local Institutional Experimentation Committee for Animal Care and Use (protocol 05/2003). These animals were systemically healthy and showed no clinical and radiographic signs of destructive periodontal disease. A total of 10 third lower premolars were used and the teeth of the same animal were randomly distributed into 2 groups of treatment. 2. Creation of Class III Furcation Defects The creation of class  III furcation defects was performed as previously described7. Initially, the anesthetic procedure used throughout the study included a previous sedation of the animals with IM injection of cloridrate of chlorpromazine, 0.2  mL.kg–1 (Bayer S.A. Saúde Animal, São Paulo, SP, Brasil) followed by induction of general anesthesia with IV sodium thiopental, 0.5  mL.kg–1 (Abbott Laboratórios do Brasil Ltda., São Paulo, SP, Brasil). To control bleeding and ensure deep anesthesia, 2% lidocaine (Cristália Produtos Químicos Farmacêuticos Ltda., Itirapina, SP, Brasil) containing noradrenaline (1:100,000) was infiltrated into the mucosa. After, a full-thickness mucoperiosteal flap was reflected and the interradicular bone of each third premolar was removed with rotatory # 2 round burr (KG Sorensen, São Paulo, SP, Brasil) and hand micro Ochsenbein chisels (Neumar, São Paulo, SP, Brasil) to create class III furcation defects. These defects had 5 mm of vertical height from cementoenamel junction (CEJ) to the marginal bone and 5  mm of width on vestibular and lingual aspects (Figure 1). The roots were scaled with Gracey curettes (Neumar, São Paulo, SP, Brasil) to remove all periodontal ligament fibers, and filled with heat-softened gutta-percha (Odahcam, Herpe Produtos Dentários Ltda., Rio de Janeiro, RJ, Brasil) to avoid spontaneous regeneration. Sutures (Ethicon 4.0 Atraloc, Johnson & Johnson, São Paulo, SP, Brasil) were removed after 7 days and the animals were maintained during 8 weeks with water-softened food to increase plaque accumulation, leading to root contamination as well as the development of a chronic inflammatory reaction. After this period, the dogs were anesthetized again to remove the gutta- percha with curettes and check the cronification of the defects. The animals also received supragingival scaling and dental prophylaxis. Chemical plaque control with daily (5 times/week) topical application of 0.2% chlorhexidine solution was initiated. 3. PRP Preparation PRP was prepared according to Anitua28 (1999). One 5 mL tube containing 0.5  mL of 3.2% sodium citrate solution as an anticoagulant (VacutaineTM, Becton Dickson UK Ltd., Belliver Industrial, State Plymouth, England) was drawn from each dog. The tubes were centrifuged (SIN – Sistema de Implante Nacional Ltda – Brasil) at 1200 rpm for 8 minutes at room temperature. The blood was then separated into three basic parts: red blood cells (at the bottom of the tube), PRP (a discrete gray line in the middle 312 Fernandes et al. Rev Odontol UNESP. 2011; 40(6): 310-316 of the tube) and platelet-poor plasma (at the top of the tube). The portion corresponding to the PRP was pipetted out from each tube and stored in a sterile glass recipient. Subsequently, 10% calcium chloride was added to the preparation to activate platelets and to form the platelet gel. The autogenous bone graft was associated with the platelet gel and used to fill in the furcation defects. 4. Regenerative Treatment The treatment of the defects was performed as previously described7. Briefly, after one week of the gutta-percha removal, full-thickness mucoperiosteal flaps were elevated, granulation tissue was removed and the root surfaces were thoroughly scaled with manual instruments (Gracey curettes, Neumar, São Paulo, SP, Brasil). Reference notches were done on the root surface at the marginal bone level both on mesial and distal aspect. After these procedures, the treatment varied according to the group in which the teeth were randomly assigned: • Test group (group T): The defects of this group were filled with autogenous bone graft + PRP and bioabsorbable membranes Gore Resolut XT (W.L. Gore & Associates, Inc. Arizona) were trimmed according to the GRT principles and fitted to the vestibular and lingual surfaces (Figure 2). Resorbable sutures (Vycril Ethicon 6.0, Johnson & Johnson, São Paulo, SP, Brasil) placed around the cervical third of the tooth stabilized the membranes and flaps were coronally repositioned and secured with suspended and interrupted sutures; • Control group (group C): The defects received the same treatment described for the test group, except the PRP application. At the end of surgical procedures, the dogs received IM injections of antibiotics (penicilin G benzatine, 40,000 mL.kg–1) and analgesics (dipirone, 2 mL.10 kg–1) as post-operative medications. The animals were then introduced into a reparative period of 90 days. During the first 4 weeks, the wounds were protected by feeding the dogs a soft diet. Chemical plaque control with daily (5 times/week) topical application of 0.2% chlorhexidine solution was maintained. 5. Sacrifice and Histological Processing The animals were sacrificed by an overdose of sodium thiopental (Abbott Laboratórios do Brasil Ltda., São Paulo, SP, Brasil) 3 months after the regenerative treatment. The jaws were removed, dissected and the blocks containing the experimental specimens were fixed in formalin 10% and decalcified in Morse solution. After decalcification, routine histological processing and paraffin embedding were done, and 5 μm thick tissue slices were obtained throughout the blocks on the mesiodistal plane. For each tooth, five sections were selected, including the first and last sections that showed the references notches on both mesial and distal roots and three sections equally spaced between those two representing the vestibular, central and lingual portions of the furcations. These selected sections were stained with hematoxylin and eosin (H/E) and Masson Trichrome. 6. Histometric Analysis The histometric analysis was performed as previously described7 and was done by another examiner that was also blind to the treatment groups. A computerized image analysis system (Diastar - Cambridge Instruments, Buffalo, NY, USA) consisting of Figure 1. Class III furcation defect. Figure 2. Absorbable membrane positioned according to GTR protocol. Rev Odontol UNESP. 2011; 40(6): 310-316 Effect of platelet-rich plasma on healing of class III furcation defects… 313 a light microscope coupled to a video camera DXC-107AP/107AP - (Sony Eletronics Inc., Japan) and connected to a microcomputer with image analysis software Jandel Sigma Scan Pro, Version 2.0 (Jandel Corporation-San Rafael, CA, USA).was used to obtain linear and area measurements. The following variables were analyzed: 1) Free: linear extension of root surface not covered by any tissue and in contact with dental plaque; 2) EP (epithelium): linear root surface extension covered by epithelial tissue plus the extension of epithelial tissue present within the lesion that did not contact dental plaque; 3) CE (cementum): root surface extension with newly formed cementum; 4)  CT (connective tissue): root surface extension with attached connective tissue fibers without any cementum; 5) LPR (Linear periodontal regeneration): linear root surface extension covered with new cementum and new alveolar bone; 6)  ES (empty space): furcation area without any tissue; 7) STA (soft tissue area): defect area filled by connective and epithelial tissues; 8)  MTA (mineralized tissue area): defect area filled by mineralized tissue (Figure 3). 7. Descriptive Analyses An experienced pathologist (LCS) that was blind to the treatment groups performed the histological descriptive analysis. The type and quality of tissues formed within the defect as well as the presence of any unusual tissue reactions such as resorption and anquilosis were evaluated. 8. Statistical Analysis To analyze differences between T and C groups in all area and linear histometric measurements, T2 Hotelling multivariate statistical test with 0.05 of significant level and Bonferroni test were used. RESULT 1. Descriptive Analysis The healing was uneventful on the experiment and control sides in all dogs; no suppuration or abscess formation was observed. The results of the descriptive histological analysis for both treatment groups were similar. The furcation lesions were predominantly filled out by a dense connective tissue covered by an epithelium of variable thickness. This epithelium demonstrated normal characteristics with pronounced projections for the subjacent connective tissue which showed an inflammatory infiltrate with different intensity, probably in response to the presence of dental plaque in the furcation roof. Newly formed cementum was observed at the root notch and eventually extending coronally until the furcation roof (Figures  4 and 5). This new cellular cementum had variable thickness without the presence of incremental lines, denoting disorganized apposition. In some areas of the notches, suggestive image of fibers collagen from the connective tissue imbedded inside the cementum was observed. Bone regeneration was negligible, extending only to the base of the furcation defect. Figure 3. Schematic representation of linear and area variables evaluated in the histometric analysis. EP CT CE MTA STA ES FREE Figure 4. a) Panoramic view of the class III furcation in the Test group, showing poor bone regeneration within the defect (Hematoxylin and Eosin, original magnification ×20); b) Masson Trichrome, original magnification ×20). D D M MO O M M CJ CJ E E CE CE a b Figure 5. a) Panoramic view of the class III furcation in the Control group, showing poor bone regeneration within the defect (Hematoxylin and Eosin, original magnification ×20); b) Masson Trichrome, original magnification ×20). E E D D M M M O O CJ CJ CE CE a b 2. Histometric Analysis Histometric results are shows in Table 1. No connective tissue was found in contact with the root surface in all specimens. 314 Fernandes et al. Rev Odontol UNESP. 2011; 40(6): 310-316 DISCUSSION The present study evaluated the effect of platelet-rich plama (PRP) associated to GTR and autogenous bone graft in bone regeneration of class  III experimental furcation defects created in dogs. After 3 months of healing, no additional benefits were observed in that type of defect when PRP was added to the regenerative treatment. The PRP is derived from a preparation of an autologous platelet concentrate from the patient blood serum. It is activated by calcium chloride, and the result of this activation is the release of a cascade of growth factors present in the granules of platelets29. The mechanism of this technique is based on the release of growth factors from the clot fibrin, rich in these components, during a natural healing event. The PRP is also known by its osteoinductive properties. However, in our results, those properties were not demonstrated and no additional benefits on the periodontal regeneration were observed when PRP was associated to autogenous bone graft and absorbable membrane. In addition, bone regeneration in the furcation of both groups was poorly achieved. This result can be attributed to the critical size of the defects. In the present study, the class III furcation lesions had a cervical to apical height and width of 5 mm and a limited amount of periodontal tissue was left only at the apical area of the defect, reducing its regenerative potential. According to Park  et  al.30 (1995), periodontal regeneration of class III furcation requires longer healing period (more than 11 weeks) and the result seems to be more variable and unpredictable. These defects tend to be more vulnerable to bacterial infection and bone healing is less predictable and reliable on them. Previous studies7,22,30,31 reported in the literature have described positive results for class III furcation defects in dogs after regenerative treatments with growth factors. Methodological variation may explain divergent results observed in the present study in comparison to those, e.g. defect size (as previously described) and the use of mongrel dogs. This fact can result in significant genetic variability that leads alteration in the metabolism and physiology of the tissues, such as inflammatory reaction on the defect. Also, adequate biofilm control play an important role in the regeneration treatment success, since the presence of biofilm in the defect area during the healing process is described as one of the failure causes of periodontal regeneration therapies. In our study, even though daily chlorhexidine application was performed, biofilm was observed attached to the root surface of the furcation roof in various histological cuts. This may be justified by the difficulty of adequate biofilm control in the class III furcation after gingival tissue recession and furcation exposure, which usually happens at the beginning of the healing process. Some of the previously cited studies used specific growth factors instead of PRP. The PRP is a pool of different growth factors with unknown concentration, which may have significant variability and, consequently, unpredictable effects. Additional studies are necessary to better understand the real regenerative potential of PCR, especially in critical size defects. According to Mellonig32 (1999), it is important to note that the type of regenerative material to be used is only one of the determinants for successful outcome. Other important issues to be considered is: patient selection, the morphology and size of the defect, occlusal considerations, tooth mobility, root preparation, suture closure of the flap, antibiotic coverage, plaque control, wound stabilization. All these variables are considered important when seeking a true periodontal regeneration. CONCLUSION The association of platelet-rich plasma with autogenous bone graft and absorbable membrane did not show additional benefits on the periodontal regeneration in class III furcation in dogs when compared to autogenous bone graft and resorbable membrane only. Table 1. Histometric parameters: mean, standard deviation and range (mm for linear or mm2 for area measurements) for each regenerative treatment C group (n = 5) (range) T group (n = 5) (range) p EP 3.16 ± 1.34 (2.09 – 5.51) 3.70 ± 1.64 (2.25 - 6.53) >0.05 CE 2.97 ± 1.19 (1.41 – 4.04) 2.23 ± 1.01 (0.72 – 3.13) >0.05 LPR 0.74 ± 0.84 (0.00 – 1.63) 0.51 ± 0.60 (0.00 – 1.43) >0.05 ES 1.62 ± 0.52 (1.01 – 2.41) 1.88 ± 0.47 (1.38 – 2.45) >0.05 STA 7.71 ± 1.09 (6.45 – 8.88) 7.39 ± 1.00 (6.31 – 8.90) >0.05 MTA 0.44 ± 0.33 (0.00 – 0.77) 0.09 ± 0.1 (0.00 – 0.22) >0.05 REFERENCES 1. Karring T, Lindhe J, Cortellini P. Regenerative periodontal therapy. In: Lindhe J, Karring T, Lang NP. 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