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Evaluation of collagen-based membranes for guided bone regeneration, by three-dimensional computerized microtomography
Vol. 114 No. 4 October 2012
Evaluation of collagen-based membranes for guided bone regeneration, by three-dimensional computerized microtomography Paulo Guilherme Coelho, DDS, MS, BS, MsMtE, PhD,a Gabriela Giro, DDS, MS, PhD,b Wanki Kim, DDS,a Rodrigo Granato, DDS, MS, PhD,c Charles Marin, DDS, MS, PhD,c Estevam Augusto Bonfante, DDS, MS, PhD,c Samara Bonfante, DDS, MS, PhD,d Thomas Lilin, DMV,e and Marcelo Suzuki, DDS,f New York, NY, USA; Araraquara, São Paulo, Brazil; Rio de Janeiro, Brazil; Maisons Alfort, France; and Boston, MA, USA ˜ O PAULO, UNIGRANRIO UNIVERSITY; ÉCOLE New YORK UNIVERSITY; STATE UNIVERSITY OF SA NATIONALE VETERINAIRE ALFORT; AND TUFTS UNIVERSITY SCHOOL OF DENTAL MEDICINE Objective. The aim of this study was to evaluate the use of a collagen-based membrane compared with no treatment on guided bone regeneration by 3-dimensional computerized microtomography (CT). Study Design. Defects were created between the mesial and distal premolar roots of the second and third premolars (beagle dogs; n ⫽ 8). A collagen-based membrane (Vitala; Osteogenics Biomedical Inc., TX, USA) was placed in one of the defects (membrane group; n ⫽ 16), and the other was left untreated (no-membrane group; n ⫽ 16). Left and right sides provided healing samples for 2 and 16 weeks. Three-dimensional bone architecture was acquired by CT and categorized as fully regenerated (F, bone height and width) or nonregenerated (N). Results. Chi-square tests (95% level of significance) showed that tooth did not have an effect on outcome (P ⫽ .5). Significantly higher F outcomes were observed at 16 weeks than 2 weeks (P ⫽ .008) and in membrane group than in nomembrane group (P ⫽ .008). Conclusions. The collagen-based membrane influenced bone regeneration at the furcation. (Oral Surg Oral Med Oral Pathol Oral Radiol 2012;114:437-443)
Bone is a natural porous complex composite with unique properties of remodeling to adapt its microstructure to external mechanical stress. Bone is also one of the tissues with high demand for reconstruction or replacement.1,2 Bone defects may result from trauma, surgery, and disease.3 The predictable and full regeneration of bone tissue defects in the maxillofacial system has represented a Supported in part by Osteogenics (Lubbock, Texas), the Oral and Maxillofacial Surgery division at the Federal University of Santa Catarina (Brazil), and the New York University Summer Research Program (Wanki Kim). a Department of Biomaterials and Biomimetics, New York University; and Director for Research, Department of Periodontology and Implant Dentistry New York University College of Dentistry. b Department of Oral Diagnosis and Surgery, Araraquara Dental School, State University of São Paulo. c Postgraduate Program in Dentistry, School of Oral Sciences, UNIGRANRIO University. d Private Practice, Bauru, São Paulo, Brazil. e Department of Experimental Research, École Nationale Veterinaire Alfort. f Department of Operative Dentistry and Prosthodontics, Tufts University School of Dental Medicine. Received for publication Jun 29, 2011; returned for revision Oct 28, 2011; accepted for publication Nov 27, 2011. © 2012 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter doi:10.1016/j.oooo.2011.11.032
challenge to the dental profession since its early days. Examples of techniques currently used in an attempt to regenerate bone defects include the use of various classes of synthetic biomaterials, such as biopolymers and bioceramics, animal-derived grafting materials, bone autografts, the use of osteogenic biomolecules, such as growth factors,4 or a combination of these techniques. Although all of these methods have presented varied levels of success in bone regeneration, they have been limited by the materials’ capability to appropriately fill the physical dimensions of the defect over time (eventually allowing soft tissue migration into the defect region) and/or their unmatched degradation rate relative to bone’s growth ability. Such difficulties have been especially demonstrated in periodontal defects, owing to their complex geometry and
Statement of Clinical Relevance Three-wall defects may substantially benefit from the use of collagen-based membranes compared with no treatment. Bone width and height was restablished in vivo in most defects of the membrane group as observed by microscopic computerized tomography.
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ORAL AND MAXILLOFACIAL SURGERY 438 Coelho et al.
boundaries represented by various tissues (i.e., soft tissue, bone, and cementum). To improve this circumstance, the use of temporary or permanent physical barriers, called guided tissue regeneration (GTR), has been used. Such a technique has been shown to be effective in restoring complex periodontal defects, because it may allow the maintenance of the defect physical dimensions over time while providing a separation between tissue with different healing kinetics, such as soft tissue and bone. The use of physical barriers (membranes) prevents the migration of epithelial cells into the defect and ensures that cells from periodontal ligament and the alveolar bone have time for regeneration of the injured area.5-7 Several nonabsorbable or bioabsorbable biocompatible materials have been used as membranes in GTR. Although clinical and histologic studies have demonstrated that similar results can be achieved using either material,8,9 several authors have shown the advantages of collagen-based resorbable membranes over nonresorbable, because resorbable membranes exclude the need of a new intervention.10,11 However, the collagenbased membrane’s ability to achieve and subsequently retain its position and dimensions for sufficient time to allow appropriate posttreatment bone morphology has raised concerns.10,11 Although clinical and histologic studies have demonstrated the ability of GTR in the regeneration of periodontal tissues,10,11 these methodologies have not quantified the technique’s ability to restore bone physical dimensions. The present study used microscopic computerized tomography (microCT) to evaluate the effectiveness of a resorbable collagen-based membrane on guided bone regeneration at the furcation region of beagle dogs’ maxillary premolars compared with no treatment.
MATERIALS AND METHODS This study consisted of 8 female beagle dogs aged ⬃1.5 years and in good health. This study was performed under approval of the committee for animal experimentation at the École Nationale Veterinaire Alfort, Maisons-Alfort, France. The dogs were acquired 2 weeks before the first surgical intervention and were under experimentation for 16 weeks. Sixteen weeks before killing the dogs, we carried out the first surgical procedure on the right side of the maxilla (n ⫽ 8 each group), and 2 weeks (n ⫽ 8 each group) before killing them, we carried out the second surgical procedure on the left side. Figure 1, A, demonstrates the presurgical aspect of the beagle dog posterior maxilla. All surgical procedures were performed under general anesthesia. The preanesthetic procedure comprised an intramuscular (IM) administration of atropine sulfate (0.044 mg/kg) and xylazine hydrochloride (8 mg/kg).
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General anesthesia was then obtained after an IM injection of ketamine chlorate (15 mg/kg). After an intrasulcular incision, mucoperiosteal flaps were made and the bone tissue was exposed (Figure 1, B). The standardized defect created at the furcation region of the second and third upper premolars was 4 mm in height from the bone crest level by 2.5 mm of horizontal width and 3 mm in depth (Figure 1, C). The defects were created by means of a 2-mm diameter cylindric bur. After the defects were created, a collagen-based (collagen derived from decellularized porcine pericardium) membrane (Vitala, Osteogenics Biomedical, Lubbock, TX) was placed in one of the defects (membrane group; n ⫽ 16) and the other one was left uncovered (nomembrane group; n ⫽ 16; Figure 1, D). The site of the membrane placement was changed for each dog and resulted in a balanced study design presenting the same number in membrane and no-membrane groups per tooth and experimental time in vivo. The flaps were coronally repositioned with resorbable sutures (4-0 Vicryl; Ethicon; Johnson and Johnson, Miami, FL; Figure 1, E). After surgery, the animals were subjected to a soft diet for a period of 7 days. Postsurgical medication included antibiotics (penicillin, 20,000 UI/kg) and analgesics (ketoprophen, 1 mL/5 kg) for a period of 48 hours after surgery. We killed the dogs with anesthesia overdose. After the dogs were killed, the samples were placed in 10% buffered formalin. The evaluation concerning bone filling the defects was performed by microCT (microCT 40; Scanco Medical, Basserdorf, Switzerland) with a resolution of 18 m per slice. The X-ray energy level used was 70 kVp, and the current level 114 A. Integration time was 150 ms with a total scanning time of ⬃30 minutes (78 mA). The 3-dimensional (3D) construction of the maxilla was made using the system proprietary software (microCT 40). After 3D reconstruction, the defects were electronically sectioned by computer software and assigned to one of the following categories depending of the defect regeneration: fully regenerated (F), when both bone height and width were completely regenerated, or nonregenerated (N). Statistical analysis was performed by multiple 2 tests considering the effects of tooth, time in vivo, and treatment on outcome (SPSS Statistics version 20; SPSS, Chicago, IL). Statistical significance was set to 95%.
RESULTS The surgical procedures and follow-up demonstrated no complications regarding procedural conditions or postoperative infection. No clinical concerns or adverse events, such as membrane exposure, were detected, and a clinically healthy soft tissue aspect was observed throughout the course of the study.
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Fig. 1. Surgical procedure sequence. A, Presurgical aspect of the beagle dog posterior maxilla; B, total flap reflection; C, creation of standardized defects at the furcation region; D, collagen-based membrane placement on one premolar and no-treatment on the other; and E, suture.
The 3D reconstructions were created successfully and allowed the determination of whether bone height and width were regenerated for the different experimental groups (Figure 2). For accurate assessment as to whether height or width was regained, the 3D reconstructions were sectioned through the defect thickness (Figures 3 and 4). The distribution of treatment outcomes showed that bone height and width was reestablished in 1 membranecovered defect at 2 weeks (Figure 5). At 16 weeks, all membrane-covered defects except 1 presented bone height and width gain, reflecting a substantial change in treatment outcome distribution compared with 2 weeks (Figure 5). In contrast, no no-membrane defects except 1 presented bone height and width reestablishment after 16 weeks in vivo (Figure 5). The 2 analysis considering treatment group and outcome showed that the membrane group presented a significantly higher number of fully regenerated defects com-
pared with the no membrane group (P ⫽ .008). When time in vivo and treatment outcome were considered, a significantly higher number of fully regenerated sites were observed at 16 weeks compared with 2 weeks. Finally, 2 analysis showed no effect of defect site (second or third premolar; P ⫽ .5; Figure 6).
DISCUSSION Over the years, the composition of the physical barriers used for GTR has ranged from polymeric materials to different organic materials (mainly collagen). Although the polymer-based barriers provide adequate physical property stability over time, their main drawbacks lie in the need for a second surgical intervention and the higher likelihood of infection due to its exposure. On the other hand, although the exposure and subsequent likelihood for collagen-based membrane is practically eliminated, concerns regarding their adequate physical integrity and degradation kinetics have been raised.5-7
ORAL AND MAXILLOFACIAL SURGERY 440 Coelho et al.
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Fig. 2. Three-dimensional reconstruction of (A) 2 weeks’ and (B) 16 weeks’ membrane (M) and nonmembrane (NM) treated defects. For this particular animal, full height and width were regenerated after 16 weeks. After 2 weeks, only the membrane group presented reestablishment of the height and width. The yellow dotted line demonstrates the lack of bone width gain at the NM group.
Fig. 3. Representative microCT reconstruction for no-membrane group at (A, B) 2 weeks and (C, D) 16 weeks. The reconstruction in A represents the full 3D morphology of the defect and in B a detailed evaluation through the partial thickness of the defect at 2 weeks (no width and height were reestablished). The reconstruction in C represents the full 3D morphology of the defect and in D a detailed evaluation through the partial thickness of the defect at 16 weeks (where only height was reestablished but no width).
Some clinical and histologic findings have rationalized that the expansion of GTR as tissue reattachment in periodontally compromised teeth is highly desirable for their longevity.8-11 However, although in vivo stud-
ies have been concerned with the presence and kinetics of tissue regeneration in periodontal defects, quantifiable assessment of the reestablishment of defect dimensions is not possible through 2-dimensional histologic
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Fig. 4. Representative microCT reconstruction for the membrane group at (A, B) 2 weeks and (C, D) 16 weeks. The reconstruction in A represents the full 3D morphology of the defect and in B a detailed evaluation through the partial thickness of the defect at 2 weeks. The reconstruction in C represents the full 3D morphology of the defect and in D a detailed evaluation through the partial thickness of the defect at 16 weeks. For this representative subject, both height and thickness were reestablished at both 2 and 16 weeks in vivo.
sections. Such quantification is especially crucial when one is evaluating resorbable barriers that may lose structural integrity over time in vivo, potentially being subjected to compression/dislodgement and thereby only partial instead of full defect regeneration. From a clinical perspective, full restoration of the defect form and function facilitates subsequent surgical and prosthetic treatment in both anterior and posterior regions. MicroCT has been successfully used for the study of hard tissues, because of the high linear attenuation coefficient of the calcified bone and dental matrices.12 The technique has been used to study the 3D morphology of root canals,13 finite element modeling for biomechanical assessment of bone strength,14 bone microarchitecture and morphology,15 and alveolar bone loss induced by different systemic and local condi-
tions,16-18 but to date no study has used this imaging tool to investigate bone regeneration using GTR. In a general fashion, our results are in agreement with earlier clinical6,9,19-24 and laboratory in vivo25-30 investigations that have shown the predictability of this therapy in restoring periodontal tissues. The periods of 2 and 16 weeks in vivo were selected in an attempt to demonstrate the early and long-term periods of periodontal tissue healing.25-30 Although the presence of bone at different levels and physical distribution was expected at 16 weeks,29 the present study demonstrated that woven bone was already present in both membrane and no-membrane groups as early as 2 weeks in vivo in the 3-wall defect created in the furcation region of dog premolars. These results are in contrast to those found by Araujo et al.,29 who reported that at 2 weeks the furcation
ORAL AND MAXILLOFACIAL SURGERY 442 Coelho et al.
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Fig. 5. Distribution of treatment outcomes as a function of time and experimental group.
GTR therapy,5,22,32,33 several studies have used GTR in tandem with grafting materials. Because all but 1 no-membrane defect resulted in the absence of bone regeneration it can be suggested that the dimensions of the defect created was effective in simulating a critical-size defect, considering that it did not regenerate within the observed follow-up without adjunctive measures.34,35 Although the present methodology allows 3D assessment of the bone architecture at the created defects, clinical and histologic evaluation to confirm to what extent new cellular cementum31,36 and reattachment degree37-41 effectively occurred are yet to be performed. REFERENCES Fig. 6. Distribution of treatment outcomes as a function of tooth.
defect should be filled with granulation tissue and that woven bone was present only after 4 weeks of healing. This discrepancy is likely accounted for by the nature of the defect created by Araujo et al.,29 which did not allow the presence of 3 walls for bone healing and thereby changed the healing kinetics compared with the present study. Along with the suitable structural integrity of the membrane used in the present study, the high incidence of fully regenerated sites after 16 weeks was due the presence of 3-walled defects. As demonstrated by Christgau et al.,31 a potential advantage of placing a physical barrier on a defect created in the furcation region relates to the presence of the cells of residual periodontium that may improve new bone formation. Because the space provided by the physical barrier is one of the most important factors for
1. Service RF. Tissue engineers build new bone. Science 2000;289:1498-500. 2. Hench LL, Wilson J. An introduction to bioceramics. Advanced series in ceramics. Singapore: World Scientific 1993. 3. Chaput C, Selmani A, Rivard CH. Artificial scaffolding materials for tissue extracellular matrix repair. Curr Opin Orthop 1996;7:62-8. 4. Coimbra ME, Elias CN, Coelho PG. In vitro degradation of poly-L-D-lactic acid (PLDLA) pellets and powder used as synthetic alloplasts for bone grafting. J Mater Sci Mater Med 2008;19:3227-34. 5. Polimeni G, Xiropaidis AV, Wikesjö UM. Biology and principles of periodontal wound healing/regeneration. Periodontol2000 2006;41:30-47. 6. Needleman IG, Worthington HV, Giedrys-Leeper E, Tucker RJ. Guided tissue regeneration for periodontal infra-bony defects. Cochrane Database Syst Rev 2006;(2):CD001724. 7. Siciliano VI, Andreuccetti G, Siciliano AI, Blasi A, Sculean A, Salvi GE. Clinical outcomes following treatment of noncontained intrabony defects with enamel matrix derivative (EMD) or guided tissue regeneration (GTR). A 12-month randomized controlled clinical trial. J Periodontol 2011;82:62-71. 8. de Andrade PF, de Souza SL, de Oliveira Macedo G, Novaes AB Jr, de Moraes Grisi MF, Taba M Jr, Palioto DB. Acellular dermal
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and DFDBA: a histometric study in dogs. Braz Oral Res 2009;23:307-12. Araújo MG, Berglundh T, Lindhe J. GTR treatment of degree III furcation defects with 2 different resorbable barriers. An experimental study in dogs. J Clin Periodontol 1998;25:253-9. Pontoriero R, Nyman S, Ericsson I, Lindhe J. Guided tissue regeneration in surgically-produced furcation defects. An experimental study in the beagle dog. J Clin Periodontol 1992;19:159-63. Trombelli L, Lee MB, Promsudthi A, Guglielmoni PG, Wikesjö UM. Periodontal repair in dogs: histologic observations of guided tissue regeneration with a prostaglandin E1 analog/methacrylate composite. J Clin Periodontol 1999;26:381-7. Araújo MG, Berglundh T, Albrekstsson T, Lindhe J. Bone formation in furcation defects. An experimental study in the dog. J Clin Periodontol 1999;26:643-52. Caffesse RG, Dominguez LE, Nasjleti CE, Castelli WA, Morrison EC, Smith BA. Furcation defects in dogs treated by guided tissue regeneration (GTR). J Periodontol 1990;61:45-50. Christgau M, Caffesse RG, Schmalz G, D’Souza RN. Extracellular matrix expression and periodontal wound-healing dynamics following guided tissue regeneration therapy in canine furcation defects. J Clin Periodontol 2007;34:691-708. Tonetti MS, Prato GP, Cortellini P. Factors affecting the healing response of intrabony defects following guided tissue regeneration and access flap surgery. J Clin Periodontol 1996;23:548-56. Haney JM, Nilvéus RE, McMillan PJ, Wikesjö UM. Periodontal repair in dogs: expanded polytetrafluoroethylene barrier membranes support wound stabilization and enhance bone regeneration. J Periodontol 1993;64:883-90. Schmitz JP, Hollinger JO. The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop Relat Res 1986;205:299-308. Wikesjö UM, Selvig KA. Periodontal wound healing and regeneration. Periodontol 2000 1999;19:21–39. Simsek SB, Keles GC, Baris S, Cetinkaya BO. Comparison of mesenchymal stem cells and autogenous cortical bone graft in the treatment of class II furcation defects in dogs. Clin Oral Investig Published online Ahead of Print. Cortellini P, Carnevale G, Sanz M, Tonetti MS. Treatment of deep and shallow intrabony defects. A multicenter randomized controlled clinical trial. J Clin Periodontol 1998;25:981-7. Zucchelli G, Brini C, De Sanctis M. GTR treatment of intrabony defects in patients with early-onset and chronic adult periodontitis. Int J Periodontics Restorative Dent 2002;22:323-33. Cortellini P, Tonetti MS, Lang NP, Suvan JE, Zucchelli G, Vangsted T, et al. The simplified papilla preservation flap in the regenerative treatment of deep intrabony defects: clinical outcomes and postoperative morbidity. J Periodontol 2001;72:1702-12. Silvestri M, Sartori S, Rasperini G, Ricci G, Rota C, Cattaneo V. Comparison of infrabony defects treated with enamel matrix derivative versus guided tissue regeneration with a nonresorbable membrane. J Clin Periodontol 2003;30:386-93. 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;72:583-9.
Reprint requests: Samara Bonfante Rua Henrique Savi 5-75 Bauru, São Paulo Brazil, 17012-590 [email protected]
Evaluation of collagen-based membranes for guided bone regeneration, by three-dimensional computerized microtomography Paulo Guilherme Coelho, DDS, MS, BS, MsMtE, PhD,a Gabriela Giro, DDS, MS, PhD,b Wanki Kim, DDS,a Rodrigo Granato, DDS, MS, PhD,c Charles Marin, DDS, MS, PhD,c Estevam Augusto Bonfante, DDS, MS, PhD,c Samara Bonfante, DDS, MS, PhD,d Thomas Lilin, DMV,e and Marcelo Suzuki, DDS,f New York, NY, USA; Araraquara, São Paulo, Brazil; Rio de Janeiro, Brazil; Maisons Alfort, France; and Boston, MA, USA ˜ O PAULO, UNIGRANRIO UNIVERSITY; ÉCOLE New YORK UNIVERSITY; STATE UNIVERSITY OF SA NATIONALE VETERINAIRE ALFORT; AND TUFTS UNIVERSITY SCHOOL OF DENTAL MEDICINE Objective. The aim of this study was to evaluate the use of a collagen-based membrane compared with no treatment on guided bone regeneration by 3-dimensional computerized microtomography (CT). Study Design. Defects were created between the mesial and distal premolar roots of the second and third premolars (beagle dogs; n ⫽ 8). A collagen-based membrane (Vitala; Osteogenics Biomedical Inc., TX, USA) was placed in one of the defects (membrane group; n ⫽ 16), and the other was left untreated (no-membrane group; n ⫽ 16). Left and right sides provided healing samples for 2 and 16 weeks. Three-dimensional bone architecture was acquired by CT and categorized as fully regenerated (F, bone height and width) or nonregenerated (N). Results. Chi-square tests (95% level of significance) showed that tooth did not have an effect on outcome (P ⫽ .5). Significantly higher F outcomes were observed at 16 weeks than 2 weeks (P ⫽ .008) and in membrane group than in nomembrane group (P ⫽ .008). Conclusions. The collagen-based membrane influenced bone regeneration at the furcation. (Oral Surg Oral Med Oral Pathol Oral Radiol 2012;114:437-443)
Bone is a natural porous complex composite with unique properties of remodeling to adapt its microstructure to external mechanical stress. Bone is also one of the tissues with high demand for reconstruction or replacement.1,2 Bone defects may result from trauma, surgery, and disease.3 The predictable and full regeneration of bone tissue defects in the maxillofacial system has represented a Supported in part by Osteogenics (Lubbock, Texas), the Oral and Maxillofacial Surgery division at the Federal University of Santa Catarina (Brazil), and the New York University Summer Research Program (Wanki Kim). a Department of Biomaterials and Biomimetics, New York University; and Director for Research, Department of Periodontology and Implant Dentistry New York University College of Dentistry. b Department of Oral Diagnosis and Surgery, Araraquara Dental School, State University of São Paulo. c Postgraduate Program in Dentistry, School of Oral Sciences, UNIGRANRIO University. d Private Practice, Bauru, São Paulo, Brazil. e Department of Experimental Research, École Nationale Veterinaire Alfort. f Department of Operative Dentistry and Prosthodontics, Tufts University School of Dental Medicine. Received for publication Jun 29, 2011; returned for revision Oct 28, 2011; accepted for publication Nov 27, 2011. © 2012 Elsevier Inc. All rights reserved. 2212-4403/$ - see front matter doi:10.1016/j.oooo.2011.11.032
challenge to the dental profession since its early days. Examples of techniques currently used in an attempt to regenerate bone defects include the use of various classes of synthetic biomaterials, such as biopolymers and bioceramics, animal-derived grafting materials, bone autografts, the use of osteogenic biomolecules, such as growth factors,4 or a combination of these techniques. Although all of these methods have presented varied levels of success in bone regeneration, they have been limited by the materials’ capability to appropriately fill the physical dimensions of the defect over time (eventually allowing soft tissue migration into the defect region) and/or their unmatched degradation rate relative to bone’s growth ability. Such difficulties have been especially demonstrated in periodontal defects, owing to their complex geometry and
Statement of Clinical Relevance Three-wall defects may substantially benefit from the use of collagen-based membranes compared with no treatment. Bone width and height was restablished in vivo in most defects of the membrane group as observed by microscopic computerized tomography.
437
ORAL AND MAXILLOFACIAL SURGERY 438 Coelho et al.
boundaries represented by various tissues (i.e., soft tissue, bone, and cementum). To improve this circumstance, the use of temporary or permanent physical barriers, called guided tissue regeneration (GTR), has been used. Such a technique has been shown to be effective in restoring complex periodontal defects, because it may allow the maintenance of the defect physical dimensions over time while providing a separation between tissue with different healing kinetics, such as soft tissue and bone. The use of physical barriers (membranes) prevents the migration of epithelial cells into the defect and ensures that cells from periodontal ligament and the alveolar bone have time for regeneration of the injured area.5-7 Several nonabsorbable or bioabsorbable biocompatible materials have been used as membranes in GTR. Although clinical and histologic studies have demonstrated that similar results can be achieved using either material,8,9 several authors have shown the advantages of collagen-based resorbable membranes over nonresorbable, because resorbable membranes exclude the need of a new intervention.10,11 However, the collagenbased membrane’s ability to achieve and subsequently retain its position and dimensions for sufficient time to allow appropriate posttreatment bone morphology has raised concerns.10,11 Although clinical and histologic studies have demonstrated the ability of GTR in the regeneration of periodontal tissues,10,11 these methodologies have not quantified the technique’s ability to restore bone physical dimensions. The present study used microscopic computerized tomography (microCT) to evaluate the effectiveness of a resorbable collagen-based membrane on guided bone regeneration at the furcation region of beagle dogs’ maxillary premolars compared with no treatment.
MATERIALS AND METHODS This study consisted of 8 female beagle dogs aged ⬃1.5 years and in good health. This study was performed under approval of the committee for animal experimentation at the École Nationale Veterinaire Alfort, Maisons-Alfort, France. The dogs were acquired 2 weeks before the first surgical intervention and were under experimentation for 16 weeks. Sixteen weeks before killing the dogs, we carried out the first surgical procedure on the right side of the maxilla (n ⫽ 8 each group), and 2 weeks (n ⫽ 8 each group) before killing them, we carried out the second surgical procedure on the left side. Figure 1, A, demonstrates the presurgical aspect of the beagle dog posterior maxilla. All surgical procedures were performed under general anesthesia. The preanesthetic procedure comprised an intramuscular (IM) administration of atropine sulfate (0.044 mg/kg) and xylazine hydrochloride (8 mg/kg).
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General anesthesia was then obtained after an IM injection of ketamine chlorate (15 mg/kg). After an intrasulcular incision, mucoperiosteal flaps were made and the bone tissue was exposed (Figure 1, B). The standardized defect created at the furcation region of the second and third upper premolars was 4 mm in height from the bone crest level by 2.5 mm of horizontal width and 3 mm in depth (Figure 1, C). The defects were created by means of a 2-mm diameter cylindric bur. After the defects were created, a collagen-based (collagen derived from decellularized porcine pericardium) membrane (Vitala, Osteogenics Biomedical, Lubbock, TX) was placed in one of the defects (membrane group; n ⫽ 16) and the other one was left uncovered (nomembrane group; n ⫽ 16; Figure 1, D). The site of the membrane placement was changed for each dog and resulted in a balanced study design presenting the same number in membrane and no-membrane groups per tooth and experimental time in vivo. The flaps were coronally repositioned with resorbable sutures (4-0 Vicryl; Ethicon; Johnson and Johnson, Miami, FL; Figure 1, E). After surgery, the animals were subjected to a soft diet for a period of 7 days. Postsurgical medication included antibiotics (penicillin, 20,000 UI/kg) and analgesics (ketoprophen, 1 mL/5 kg) for a period of 48 hours after surgery. We killed the dogs with anesthesia overdose. After the dogs were killed, the samples were placed in 10% buffered formalin. The evaluation concerning bone filling the defects was performed by microCT (microCT 40; Scanco Medical, Basserdorf, Switzerland) with a resolution of 18 m per slice. The X-ray energy level used was 70 kVp, and the current level 114 A. Integration time was 150 ms with a total scanning time of ⬃30 minutes (78 mA). The 3-dimensional (3D) construction of the maxilla was made using the system proprietary software (microCT 40). After 3D reconstruction, the defects were electronically sectioned by computer software and assigned to one of the following categories depending of the defect regeneration: fully regenerated (F), when both bone height and width were completely regenerated, or nonregenerated (N). Statistical analysis was performed by multiple 2 tests considering the effects of tooth, time in vivo, and treatment on outcome (SPSS Statistics version 20; SPSS, Chicago, IL). Statistical significance was set to 95%.
RESULTS The surgical procedures and follow-up demonstrated no complications regarding procedural conditions or postoperative infection. No clinical concerns or adverse events, such as membrane exposure, were detected, and a clinically healthy soft tissue aspect was observed throughout the course of the study.
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Fig. 1. Surgical procedure sequence. A, Presurgical aspect of the beagle dog posterior maxilla; B, total flap reflection; C, creation of standardized defects at the furcation region; D, collagen-based membrane placement on one premolar and no-treatment on the other; and E, suture.
The 3D reconstructions were created successfully and allowed the determination of whether bone height and width were regenerated for the different experimental groups (Figure 2). For accurate assessment as to whether height or width was regained, the 3D reconstructions were sectioned through the defect thickness (Figures 3 and 4). The distribution of treatment outcomes showed that bone height and width was reestablished in 1 membranecovered defect at 2 weeks (Figure 5). At 16 weeks, all membrane-covered defects except 1 presented bone height and width gain, reflecting a substantial change in treatment outcome distribution compared with 2 weeks (Figure 5). In contrast, no no-membrane defects except 1 presented bone height and width reestablishment after 16 weeks in vivo (Figure 5). The 2 analysis considering treatment group and outcome showed that the membrane group presented a significantly higher number of fully regenerated defects com-
pared with the no membrane group (P ⫽ .008). When time in vivo and treatment outcome were considered, a significantly higher number of fully regenerated sites were observed at 16 weeks compared with 2 weeks. Finally, 2 analysis showed no effect of defect site (second or third premolar; P ⫽ .5; Figure 6).
DISCUSSION Over the years, the composition of the physical barriers used for GTR has ranged from polymeric materials to different organic materials (mainly collagen). Although the polymer-based barriers provide adequate physical property stability over time, their main drawbacks lie in the need for a second surgical intervention and the higher likelihood of infection due to its exposure. On the other hand, although the exposure and subsequent likelihood for collagen-based membrane is practically eliminated, concerns regarding their adequate physical integrity and degradation kinetics have been raised.5-7
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Fig. 2. Three-dimensional reconstruction of (A) 2 weeks’ and (B) 16 weeks’ membrane (M) and nonmembrane (NM) treated defects. For this particular animal, full height and width were regenerated after 16 weeks. After 2 weeks, only the membrane group presented reestablishment of the height and width. The yellow dotted line demonstrates the lack of bone width gain at the NM group.
Fig. 3. Representative microCT reconstruction for no-membrane group at (A, B) 2 weeks and (C, D) 16 weeks. The reconstruction in A represents the full 3D morphology of the defect and in B a detailed evaluation through the partial thickness of the defect at 2 weeks (no width and height were reestablished). The reconstruction in C represents the full 3D morphology of the defect and in D a detailed evaluation through the partial thickness of the defect at 16 weeks (where only height was reestablished but no width).
Some clinical and histologic findings have rationalized that the expansion of GTR as tissue reattachment in periodontally compromised teeth is highly desirable for their longevity.8-11 However, although in vivo stud-
ies have been concerned with the presence and kinetics of tissue regeneration in periodontal defects, quantifiable assessment of the reestablishment of defect dimensions is not possible through 2-dimensional histologic
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Fig. 4. Representative microCT reconstruction for the membrane group at (A, B) 2 weeks and (C, D) 16 weeks. The reconstruction in A represents the full 3D morphology of the defect and in B a detailed evaluation through the partial thickness of the defect at 2 weeks. The reconstruction in C represents the full 3D morphology of the defect and in D a detailed evaluation through the partial thickness of the defect at 16 weeks. For this representative subject, both height and thickness were reestablished at both 2 and 16 weeks in vivo.
sections. Such quantification is especially crucial when one is evaluating resorbable barriers that may lose structural integrity over time in vivo, potentially being subjected to compression/dislodgement and thereby only partial instead of full defect regeneration. From a clinical perspective, full restoration of the defect form and function facilitates subsequent surgical and prosthetic treatment in both anterior and posterior regions. MicroCT has been successfully used for the study of hard tissues, because of the high linear attenuation coefficient of the calcified bone and dental matrices.12 The technique has been used to study the 3D morphology of root canals,13 finite element modeling for biomechanical assessment of bone strength,14 bone microarchitecture and morphology,15 and alveolar bone loss induced by different systemic and local condi-
tions,16-18 but to date no study has used this imaging tool to investigate bone regeneration using GTR. In a general fashion, our results are in agreement with earlier clinical6,9,19-24 and laboratory in vivo25-30 investigations that have shown the predictability of this therapy in restoring periodontal tissues. The periods of 2 and 16 weeks in vivo were selected in an attempt to demonstrate the early and long-term periods of periodontal tissue healing.25-30 Although the presence of bone at different levels and physical distribution was expected at 16 weeks,29 the present study demonstrated that woven bone was already present in both membrane and no-membrane groups as early as 2 weeks in vivo in the 3-wall defect created in the furcation region of dog premolars. These results are in contrast to those found by Araujo et al.,29 who reported that at 2 weeks the furcation
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Fig. 5. Distribution of treatment outcomes as a function of time and experimental group.
GTR therapy,5,22,32,33 several studies have used GTR in tandem with grafting materials. Because all but 1 no-membrane defect resulted in the absence of bone regeneration it can be suggested that the dimensions of the defect created was effective in simulating a critical-size defect, considering that it did not regenerate within the observed follow-up without adjunctive measures.34,35 Although the present methodology allows 3D assessment of the bone architecture at the created defects, clinical and histologic evaluation to confirm to what extent new cellular cementum31,36 and reattachment degree37-41 effectively occurred are yet to be performed. REFERENCES Fig. 6. Distribution of treatment outcomes as a function of tooth.
defect should be filled with granulation tissue and that woven bone was present only after 4 weeks of healing. This discrepancy is likely accounted for by the nature of the defect created by Araujo et al.,29 which did not allow the presence of 3 walls for bone healing and thereby changed the healing kinetics compared with the present study. Along with the suitable structural integrity of the membrane used in the present study, the high incidence of fully regenerated sites after 16 weeks was due the presence of 3-walled defects. As demonstrated by Christgau et al.,31 a potential advantage of placing a physical barrier on a defect created in the furcation region relates to the presence of the cells of residual periodontium that may improve new bone formation. Because the space provided by the physical barrier is one of the most important factors for
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