Sérgio Miguel Andrade de Matos. Aplicação de matrizes enriquecidas com moduladores biológicos na regeneração de tecidos periodontais e tecidos ósseos

Transcrição

1 Sérgio Miguel Andrade de Matos Aplicação de matrizes enriquecidas com moduladores biológicos na regeneração de tecidos periodontais e tecidos ósseos Coimbra 2008

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3 Dissertação de candidatura ao Grau de Doutor apresentada à Faculdade de Medicina da Universidade de Coimbra. Orientadores: Professor Doutor João Luís Maló de Abreu Professor Doutor Mariano Sanz Alonso A elaboração deste trabalho decorreu no Departamento de Medicina Dentária, Estomatologia e Cirurgia Maxilo-Facial, no seu Laboratório de Histologia de Tecidos Duros e no Laboratório de Investigação Experimental dos Hospitais da Universidade de Coimbra. A Faculdade de Medicina da Universidade de Coimbra não aceita qualquer responsabilidade em relação à doutrina e à forma desta dissertação. 3

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5 I. RESUMO Os defeitos ósseos do complexo maxilo-facial constituem uma das problemáticas mais prementes em Medicina Dentária. Estes defeitos, resultantes de doença ou traumatismos, podem acarretar graves problemas funcionais e estéticos, principalmente quando associados a estados de desdentação ou que ponham em risco a dentição natural. No âmbito da Peridontologia, os defeitos periodontais infra-ósseos representam um desafio clínico de grande complexidade microbiológica e morfológica. A sua permanência implica a persistência de um nicho ecológico desfavorável, com elevada probabilidade de contínua perda de inserção ou recidiva. A terapêutica ideal a aplicar nestas situações seria aquela que possibilitasse a reabilitação da estrutura anatomo-morfológica e funcional do periodonto. As técnicas regenerativas apresentam-se como a escolha de eleição, uma vez que possibilitam a reconstituição dos tecidos danificados, ou perdidos, como resultado da doença periodontal. Nas últimas duas décadas tem sido reunida uma considerável evidência clínica e histológica que comprova a possibilidade de alcançar regeneração periodontal em humanos. Vários materiais de enxerto ósseo (auto-enxertos, alo-enxertos, xenoenxertos e materiais aloplásticos) têm sido utilizados como matrizes na regeneração de defeitos infra-ósseos periodontais, geralmente com resultados clínicos positivos. Recentes revisões sistemáticas indicam que estes materiais são significativamente mais eficazes que o desbridamento cirúrgico simples na melhoria dos níveis de inserção clínica e do preenchimento ósseo. Adicionalmente, também têm sido aplicados noutras indicações como na regeneração óssea de defeitos do rebordo alveolar, com eficácia clínica comprovada. Contudo, resultados heterogéneos e estudos insuficientes com um desenho experimental comparável impediram a formulação de conclusões definitivas sobre a utilização específica dos vários materiais de enxerto. Com o objectivo de promover modalidades biológicas que possam estimular a regeneração de tecidos, desenvolveram-se matrizes bioactivas com afinidades para a adesão celular. Desta abordagem, resultou a combinação do composto ABM/P-15, constituído por um mineral anorgânico derivado bovino (ABM) ao qual está ligado irreversivelmente um peptídeo sintético do domínio da ligação celular P-15. Este último, representa uma réplica de uma sequência de 15 aminoácidos derivados da molécula de colagénio tipo I, que está especificamente envolvida na estimulação da migração, 5

6 adesão e proliferação celular de osteoblastos e fibroblastos. Estudos recentes demonstraram resultados clínicos positivos da sua aplicação no tratamento de defeitos periodontais infra-ósseos, bem como efeitos benéficos a longo prazo e a comprovação histológica de regeneração periodontal em humanos. Numa tentativa de melhorar o manuseamento clínico, controlar a migração das partículas e optimizar a eficácia clínica, foi desenvolvida uma nova formulação deste material granulado, combinado com um veículo transportador das partículas de ABM/P- 15 constituído por um hidrogel de carboximetilcelulose e glicerol. A possibilidade de injectar o produto no defeito, mantendo-o no local desejado, sem necessidade de hidratação e compactação, representa um salto qualitativo importante nas propriedades de manuseamento de um material de enxerto ósseo. Foi, igualmente, sugerido que a criação de um espaçamento mais homogéneo entre partículas, condição sine qua non para uma adequada colonização celular e invasão de neovasos, poderia promover uma regeneração óssea mais acelerada e uma remodelação mais rápida, com expectáveis benefícios clínicos quantitativos e qualitativos. Este material, de acordo com o nosso conhecimento até à data, apresenta um nível de evidência científica muito escasso relativamente à avaliação das suas potencialidades regenerativas. Assim, o objectivo deste trabalho consistiu em avaliar o ABM/P-15 como material de enxerto, comparando a formulação de partículas isoladas sem qualquer veículo de transporte (ABM/P-15 granulado) com a formulação de partículas transportadas num hidrogel (ABM/P-15 hidrogel) e identificar alguma eventual reacção estranha ou complicações com estes materiais. Com o intuito de alcançar este propósito, definiramse dois níveis de avaliação através dos seguintes estudos experimentais: 1) avaliação da eficácia clínica no tratamento de defeitos peridontais infra-ósseos, através de um ensaio clínico aleatorizado; e 2) avaliação histológica do desempenho biológico, através de estudos de experimentação animal em modelos de regeneração óssea. No ensaio clínico prospectivo de boca dividida, controlado e com alocação aleatória, foram seleccionados dezanove pacientes diagnosticados com periodontite crónica moderada a severa, a partir da consulta de Periodontologia do Departamento de Estomatologia, Medicina Dentária e Cirurgia Maxilo-Facial da Faculdade de Medicina da Universidade de Coimbra, para participar voluntariamente neste ensaio. Cada paciente deveria ter pelo menos dois defeitos periodontais infra-ósseos proximais não adjacentes e em dentes separados, com uma profundidade superior ou igual a 3 mm após efectuada a terapia periodontal causal. A terapia cirúrgica consistiu na reflexão de retalhos de espessura total muco-periósteos para acesso à instrumentação radicular dos dentes envolvidos e preenchimento dos defeitos com o material teste (ABM/P-15 hidrogel) e o material controlo (ABM/P-15 granulado), utilizando uma distribuição 6

7 aleatorizada. Aos 6 meses do pós-operatório, procedeu-se a uma cirurgia de reentrada para obtenção de dados documentais sobre os resultados dos tecidos duros. As alterações das medições dos tecidos moles e duros foram avaliadas em todos os defeitos entre o período inicial de base e os 6 meses. A cicatrização pós-operatória decorreu normalmente sem registo de qualquer tipo de complicação e revelou uma excelente resposta dos tecidos moles em ambos os grupos de tratamento. Não se verificaram, igualmente, efeitos adversos ou queixas dos pacientes relacionadas com os dois materiais de enxerto. Após o período final de avaliação, não se identificaram diferenças significativas entre ambos os tratamentos para qualquer uma das medições clínicas. O grupo teste demonstrou um preenchimento ósseo médio de 3,10 ± 0,85 mm (75,0%) versus 3,09 ± 1,11 mm (73,7%) do grupo controlo e uma percentagem de resolução média do defeito de 85,8% versus 81,9%, respectivamente. Foram obtidos dados semelhantes para as percentagens médias da resolução do defeito (85,8 ± 10,7% para o grupo teste versus 81,9 ± 13,3% para o grupo controlo). Relativamente aos parâmetros clínicos primários dos tecidos moles, registaram-se para o grupo teste e para o grupo controlo, respectivamente, ganhos de CAL de 2,89 ± 1,58 mm versus 3,41 ± 1,95 mm, reduções de PPD de 4,02 ± 1,19 versus 4,19 ± 1,55 e aumentos na recessão gengival de 1,13 ± 0,96 mm e 0,75 ± 1,08 mm. No acto da reentrada cirúrgica, identificámos diferenças macroscópicas na área da crista óssea dos defeitos tratados com as duas formulações dos materiais de enxerto. Designadamente, era notória no grupo controlo, de uma forma consistente, a presença de um maior número de partículas embebidas no tecido neoformado, comparativamente com o verificado no grupo teste. Esta constatação deixava antever possíveis diferenças qualitativas nos tecidos regenerados. Com o objectivo de clarificar o comportamento biológico dos materiais em questão e a influência das distintas formulações no processo de cicatrização, efectuaram-se dois estudos de experimentação animal com modelos de regeneração óssea em coelhos albinos da estirpe da Nova Zelândia. O primeiro estudo preliminar, utilizou um modelo retardado de cicatrização óssea, com defeitos cranianos de reduzida contenção física. Foram executados cirurgicamente defeitos circulares, bilaterais e transcorticais de 8 mm de diâmetro nos ossos parietais. A amostra constituída por 10 animais foi dividida equitativamente em dois grupos, cujos animais foram sacrificados, respectivamente, às duas e às quatro semanas do pósoperatório. O segundo estudo consistiu num modelo de cicatrização óssea de dimensão crítica, com defeitos de maior contenção física. Foram executados defeitos cilíndricos nos membros opostos na face distal do fémur, com 5 mm de diâmetro e 10 mm de 7

8 profundidade, através da cortical lateral e sem perfuração da cortical oposta. A amostra constituída por 21 animais foi dividida equitativamente em três grupos, cujos animais foram sacrificados, respectivamente, às duas, quatro e oito semanas do pós-operatório. Todas as amostras foram preparadas para avaliação histológica em material não descalcificado, com uma técnica de coloração com azul de Toluidina. Efectuou-se uma análise histológica qualitativa e histomorfométrica, em que se avaliaram os seguintes parâmetros: percentagem de novo osso, percentagem de partículas e resolução do defeito. Em ambos os modelos animais, o ABM/P-15 granulado teve um desempenho biológico superior quando comparado com o ABM/P-15 na formulação em hidrogel, tanto a nível de uma maior maturação do tecido ósseo formado, como a nível de uma quantidade significativamente superior de novo osso, presença de partículas e área total de matriz mineralizada (novo osso e partículas). Como conclusão deste trabalho experimental pode afirmar-se que o tratamento de defeitos periodontais infra-ósseos, tanto com o ABM/P-15 granulado como com o ABM/P-15 na formulação em hidrogel, resultou em melhorias significativas em termos de ganhos de CAL, reduções de PPD e preenchimento ósseo comparado com os valores iniciais de base prévios ao tratamento. Além disso, o ensaio clínico não demonstrou superioridade de nenhuma formulação do material de enxerto. Complementarmente, a evidência histológica qualitativa e quantitativa, providenciada pelos dois modelos animais de regeneração óssea, revelou que o ABM/P-15 granulado funcionou como um excelente suporte osteocondutor, possibilitando a síntese de uma matriz mineralizada, constituída inicialmente por uma rede trabecular de osso imaturo, que evoluiu para um tecido ósseo organizado com características de maturação funcional numa tentativa de reposição da arquitectura óssea original. Por sua vez, o ABM/P-15 hidrogel evidenciou uma migração anómala das partículas que comprometeu a formação de uma matriz mineralizada robusta. O carácter aleatório da distribuição das partículas transportadas no veículo tipo hidrogel determinou uma osteocondução imprevisível e, consequentemente, uma menor quantidade e qualidade do tecido ósseo desenvolvido, comprometendo a evolução do processo de maturação da matriz óssea. Assim, revelase fundamental desenvolver estudos no sentido de melhorar as características de transporte dos veículos dos materiais de enxerto granulados com o intuito de optimizar o seu desempenho em defeitos ósseos/periodontais, que apresentem factores de potencial regenerativo mais exigentes. 8

9 II. SUMMARY 1. Introduction Periodontal disease is one of the most prevalent pathologies worldwide, and lost of periodontal tissue support is one of its most deleterious consequences. The destruction of alveolar bone is a characteristic anatomical sequela due to the apical spread of periodontitis. The extent, severity and type of infrabony lesions can compromise the dentition and affect dental prognosis in a decisive way. It is conjectured that these lesions are often associated with ecological adverse niches, such as deep pockets, and might represent site-specific risk indicators for disease progression (Papapanou & Tonetti 2000). To achieve the primary goal of periodontal treatment, defined as the maintenance of the natural dentition in health and comfortable function (Zander et al. 1976), one should ideally seek for the restitutium ad integrum of the damaged periodontal apparatus. Regenerative procedures are indicated where the endpoint achieved would improve the local anatomy and/or function and prognosis of the tooth/teeth or jaw region (AAP 1996b). Considerable histological and clinical evidence has been gathered over the last two decades indicating that regeneration of periodontal tissues lost as a result of periodontitis can be achieved in humans (Sculean et al. 2003; Cortellini & Tonetti 2000). Two recent systematic reviews (Trombelli et al. 2002; Reynolds et al. 2003), summarizing the clinical outcomes following application of specific biomaterials to the treatment of deep infrabony defects, indicated that bone grafts and bone substitutes were significantly more effective than open flap debridement in improving attachment levels and in reducing probing depths. However, differences in clinical attachment level gains varied greatly with respect to the different biomaterials used and due to this heterogeneity in the results between studies, the authors were unable to draw conclusions on the use of specific graft biomaterials (Trombelli et al. 2002). With the goal of achieving a bioactive graft, an anorganic bovine-derived matrix (ABM) enhanced with the P-15 peptide was developed. P-15 is a synthetic peptide composed of a defined sequence of 15 amino acids, identical to a potent domain of the cell alfa-1 chain receptor of type I collagen that is uniquely involved in the binding of cells, particularly fibroblasts and osteoblasts (Bhatnagar et al. 1999). The ABM is a 9

10 natural microporous hydroxyapatite. Multicenter clinical evaluations of the ABM/P-15 have demonstrated clinical superiority in infrabony defect fill when compared to DFDBA, open flap debridement, and the ABM graft material alone (Yukna et al. 1998, 2000). A thirty-six month follow-up study has also showed a beneficial effect on the long-term clinical management of periodontal infrabony defects (Yukna et al. 2002a). Moreover, the evaluation of a human biopsy has evidenced periodontal regeneration in a case treated with ABM/P-15 (Yukna et al. 2002b). Recently, a new clinical presentation of this material has been developed with the purpose of improving its clinical handling, controlling particle migration and optimizing its clinical efficacy. It was suggested that creating a more homogenous interparticle spacing, which is a sine qua non condition for a proper cellular and vascular colonization, could promote a faster bone regeneration with expectable quantitative and qualitative clinical benefits. This has been accomplished by combining the ABM/P-15 particulate graft with a biocompatible hydrogel carrier consisting of carboxymethylcellulose and glycerol. To date the level of cientific evidence regarding the evaluation of the regenerative potential of this graft material is very low. Therefore, the purpose of the present work was to compare the standard ABM/P- 15 particulate with the new ABM/P-15 hydrogel and to identify any unusual clinical findings or complications seen with its application. To achieve this goal, two levels of evaluation of the grafts were defined: a) clinical efficacy in the treatment of periodontal infrabony defects, using a randomized controlled clinical trial; and b) histological evaluation of the biological performance, using bone regeneration animal models. 2. Randomized clinical trial 2.1 Materials and methods Experimental Design Two different forms of the bone graft material (ABM/P-15) were evaluated for the treatment of infrabony defects in a prospective, randomized, controlled, split-mouth clinical trial. At least two infrabony periodontal defects in each patient were treated randomly by the hydrogel form of the ABM/P-15 graft (test group), or the particulate form of the ABM/P-15 graft (control group). Patients were followed for 6 months. 10

11 Study Population The patients were selected from those attending the Periodontal Clinic at the University of Coimbra Dental School. They were informed in detail about the objectives and possible risks associated with their participation in the study by signing an informed consent. The Ethical Committee from the University Hospital of Coimbra approved the study protocol, the patient information sheet and the informed consent. Nineteen patients with a diagnosis of moderate to severe chronic periodontitis were entered into the study. Participation criteria included at least two non-adjacent periodontal infrabony defects with an intraosseous component 3 mm and a probing depth 6 mm (the defect depth was evaluated clinically and radiographically at screening but had to be confirmed intrasurgically); 30 years of age; non-smokers at screening; free of systemic diseases or systemic medication that could interfere with the post-surgical healing process; no endodontic involvement of study teeth and no mobility superior to grade II. Teeth were excluded when their prognosis was bad (mobility grade III or presenting fremitus) or if the defect extended to the furcation. Materials The anorganic bone matrix used in this clinical investigation is a totally deproteinated bovine-derived natural hydroxyapatite with a particle size ranging from 250 to 420 µm. This graft material incorporates the 15-amino acid sequence (P-15), which is a synthetic replica of the cell-binding domain of type I collagen, in a proportion of 200 ng P-15 for each gram of ABM. Two different formulations were compared: the test formulation where the graft is vehicled in a hydrogel (putty-like form) and the control formulation, which is the standard particulate ABM/P-15 graft. The hydrogel formulation is ABM/P-15 suspended in a water-based carrier containing sodium carboxymethylcellulose and glycerol, widely used biocompatible components. Both products are commercially available in the United States and the European Union, were supplied by the manufacturer, and were used per the manufacturer s instructions. Pre-surgical Phase Prior to surgery, all patients received a thorough periodontal examination, including motivation and instructions in oral hygiene, and multiple sessions of scaling and root planing until reaching acceptable levels of plaque control (plaque index 15%) and inflammation control (bleeding index 15%). The baseline examination was carried out 4 to 6 weeks after the completion of this basic periodontal therapy. 11

12 Surgical Procedure The surgical procedure consisted of elevation of full-thickness buccal and lingual mucoperiosteal flaps at the affected teeth. Once the flaps were reflected and the granulation tissue was eliminated completely, the selected defects were debrided meticulously, and the affected roots were scaled and planed with ultrasonic and manual instruments. The intrasurgical measurements were recorded, and the test or control graft materials were selected randomly. The randomization was done by rolling a die and assigning the odd numbers to ABM/P-15 particulate and even numbers to ABM/P-15 hydrogel. Because some patients harbored more than two defects, the number of selected defects was not identical for each treatment modality. Forty-seven defects were treated: 26 with the control material and 21 with the test material. For consistency in the measurements, the site at each selected defect with the deepest infrabony component was identified and measured, and it was used for the follow-up examinations. The selected replacement graft was packed carefully in the interior of the defect until it was filled completely and level with the alveolar crest. The flaps were repositioned, aiming primary closure without any tension, and sutured. All surgical procedures were performed by well-trained periodontists. Post-surgical instructions and infection control Patients were put on systemic doxycycline for 10 days: 200 mg on the day of surgery and then 100 mg per day for the following 10 days. All patients were instructed to rinse with a 0,12% chlorhexidine twice a day for 6 weeks and were not allowed to perform any interdental hygiene procedures during this period. Sutures were removed 2 weeks post-surgery. All patients were placed on a strict recall schedule following surgery (2, 4 and 6 weeks and 3 and 6 months). At every recall appointment, the healing was evaluated and patients were asked about any post-surgical complications. At these visits, a professional supragingival polishing was carried out together with oral hygiene reinforcement. The study concluded at the 6-month visit, when the reentry procedure was scheduled and then all patients were placed on routine recall. Reentry flap surgeries were performed to visualize the defects under investigation and obtain documentation on the hard tissue outcome measurements. 12

13 Outcome Measurements All clinical recordings were made by a single calibrated examiner, who was not one of the periodontists who performed the regenerative surgical procedures. The evaluator was masked to the treatment provided for each bony defect. Intraexaminer calibration was determined by taking repeated measurements of the same pockets until reaching a high degree of repeatability (90% agreement within 1 mm). Data analysis We onsidered the split-mouth design as a specialized form of a randomized block design, where the subject serves as the block. Therefore, the subject is the unit of analysis, with the difference between treatments within each subject consisting of the outcome to be analyzed. Based on previous clinical trials using the same bone replacement graft, we designed a study with a similar sample size (Yukna et al. 1998, 2000). The t test or Wilcoxon signed-rank test for paired data (depending on the normality of the distribution) were used to compare the response between the test and control replacement grafts for the soft and hard tissue main outcome variables. Intragroup changes between baseline and 6 months of the same variables also were assessed using the paired t test. The Fischer exact test was used to compare the percentage of predominant walls between groups at baseline. A significance level of 5% was established. 2.2 Results Patient follow-up All 19 patients (13 males and 6 females with a mean age of 49 years and ranging in age from 36 to 64 years) completed the study. Twenty-one defects were evaluated in the test group (ABM/P-15 hydrogel), and 26 defects in the control group (ABM/P-15 particulate); all soft and hard tissue outcome measurements were recorded at baseline and 6 months. When comparing both treatment groups, no differences were encountered for any of the studied variables. Oral hygiene Plaque control and inflammation around the affected teeth did not show any difference between the groups at baseline. During the 6 months of the study, both 13

14 indexes maintained stable (between 0,5 and 0,6 for plaque index and 0,2 and 0,3 for modified gingival index), which demonstrated that the population had a high degree of oral hygiene and inflammation control. Outcome measurements Postoperative healing was uneventful in all cases and revealed excellent soft tissue response in both treatment groups. There were no untoward effects or patient complaints related to the particulate or hydrogel bone graft substitutes. There were no significant differences between both treatments at 6 months for any of the outcome measurements, regarding both hard and soft tissue parameters. The test group demonstrated a mean bone fill of 3,10 ± 0,85 mm versus 3,09 ± 1,11 mm in the control group, which represents a mean defect fill of 75,0% and 73,7%, respectively. Very similar data was obtained for the mean percentages of defect resolution (85,8% ± 10,7% for the test group versus 81,9% ± 13,3% for the control group). In both groups, gains in CAL were about 3 mm (2,89 ± 1,58 in the test group versus 3,41 ± 1,95 in the control group), PPD reductions about 4 mm (4,02 ± 1,19 in the test group versus 4,19 ± 1,55 in the control group), and changes in the gingival recession were -1,13 ± 0,96 mm in the test group and -0,75 ± 1,08 mm in the control group. The frequency distribution of CAL gains and bone defect fill was also evaluated. Clinically significant CAL gains ( 4 mm) were obtained in 21,7% of the defects treated with ABM/P-15 in hydrogel versus 46,9 % of the defects treated with ABM/P-15 particulate. The mean percentage of clinically significant defect bone fill ( 80%) was similar with both grafts (39,1% in the test group versus 34,4% in the control group). When intragroup differences were evaluated, gains in CAL and PPD reductions between baseline and 6 months were statistically significant in both groups (P<0,0001). In both groups, gingival recession increased between baseline and 6 months; these differences were significant for both treatment groups, specially the test group. Upon reentry, macroscopic differences were noted between the two graft materials. While the particulate material in the control group was clearly present on the crestal area of the defect, in the test group, the bone crest at the defect site was more homogeneous, although the presence of small particulate material was evident. To evaluate the biological behaviour of the materials and to clarify whether the different physical properties of the two formulations had any influence on the process of bone cicatrization, further studies on experimental animal models were performed. 14

15 3. Experimental animal models The experimental protocols used in the present studies were approved by the National Authoraties - DGV (Direccção Geral de Veterinária) and the animals were housed and manipulated according to the National Legislation (Portaria nº: 1005/92 de 23 de Outubro; Portaria nº: 1131/97 de 7 de Novembro). 3.1 Materials and methods Preliminary rabbit cranial bone model - experimental protocol A total of 10 adult male New Zealand White rabbits (weight 3,5-4,5 Kg) were used, divided in two groups. Each group was composed by 5 animals, which were sacrificed at 2 and 4 weeks postoperative. The ABM/P-15 hydrogel form (test material) and the ABM/P-15 particulate form (control material) were compared to each other. Full-thickness circular 8 mm defects were created, establishing a so-called delayed healing model (Kramer et al. 1968b; Damien et al. 1991), which precludes spontaneous bone regeneration during the experimental period. Two defects were created per rabbit and each one received randomly one of the materials or remained empty as a negative control. Only 2 negative controls per group were used, since historical empty defects with limited bone regeneration are well established in this model. General anesthesia was obtained by intramuscular injection of ketamine and xylazine at a dose of 35 mg/kg and 2 mg/kg, respectively, and maintained during the intervention through an intravenous administration of ketamine (50 mg/ml) as needed. Both skulls were shaved, prepped with povidone-iodine solution and draped. A paramedian incision was made approximately five centimeters in length extending from the parietal to the frontal area to gain access to the underlying calvaria. A full-thickness flap was raised using blunt and sharp dissection. Two bilateral defects were created through the parietal bone using pilot and depth trephines mounted on a powered handpiece, leaving the dura mater intact. After copious irrigation with physiologic saline, to remove bone debris, the materials were carefully placed to completely fill the defects. Soft-tissues were meticulously closed in layers with resorbable sutures for the periosteum and non-resorbable for the skin sutures. 15

16 Rabbit cancellous bone model experimental protocol A total of 21 adult male New Zealand White rabbits (weight 3,5-4,5 Kg) were used, divided in three groups. Each group was composed by 7 animals, which were sacrificed at 2, 4 and 8 weeks postoperative. The ABM/P-15 hydrogel form (test material) and the ABM/P-15 particulate form (control material) were compared to each other. Two defects were created per rabbit in the femur of opposite limbs, establishing the so-called critical size defect (Oonishi et al. 1994; Chan et al. 2002). Each defect received randomly one of the materials or remained empty as a negative control. Only 2 negative controls per group were used, since previous studies using this model have demonstrated limited bone regeneration in empty defects. General anesthesia was obtained by intramuscular injection of ketamine and xylazine at a dose of 35 mg/kg and 2 mg/kg, respectively, and maintained during the intervention through an intravenous administration of ketamine (50 mg/ml) as needed. Both hind limbs were shaved, prepped with povidone-iodine solution and draped. An incision of approximately 1.5 centimeter was made over the distal femur in the lateral aspect of the knee joint. Blunt dissection through the underlying muscles exposed the periosteum which was elevated to identify the epiphysis. Care was taken to avoid creating communication with the knee joint cavity. A cylindrical defect was drilled with irrigation using increasing size drills to a final 5 mm diameter by 10 mm depth through the lateral cortex of the distal femur and extending to, but not through, the opposite medial cortex. The defect was placed within the epiphysis, which mainly contains cancellous bone, and avoiding the epiphyseal plate and bone marrow cavity of the diaphysis. Following meticulous rinse with sterile saline for bone debris, one of the materials was placed into the defect site (typical volume of graft material placed in defect was approximately 0,2 cc) or left untreated. The wound was meticulously closed in layers with resorbable sutures for the periosteum and non-resorbable for the skin sutures. Pos-operative management and sacrifice Butorphanel tartrate (0,2 mg/kg body weight, s.c., 2 days) was administered for immediate post-surgery pain control and a long acting amoxycilin was given as a single dose antibiotic (1 mg/kg, s.c.). At the end of the study periods, animals were sacrificed with an overdose of intravenous barbiturate. Bone harvest was done en bloc with a power saw and soft tissue carefully dissected away from the defect site for histological preparation. 16

17 Histologic evaluation All specimens were prepared for histologic evaluation, according to a routine protocol for undecalcified sections (Donath 1995). Individual specimens were fixed by immersion in 10% formalin solution immediately after collection. Following fixation, the specimens were dehydrated in graduated ethyl alcohol from 60 to 100 % and were submitted to resin infiltration in a continuous agitation unit. The specimens were then embedded in a light-cured methylmethacrylate polymer and allowed to polymerize. The ground sections were obtained by cutting the specimens on high-speed, water cooled Exakt System sectioning saw. Two ground sections were obtained from the cranial defect: one coronal in the middle of the defect and other transversal in the remaining half. Three transversal ground sections were obtained from the femur defect, in its outer, middle and inner third. Final samples were mounted on acrylic slides, ground to approximately 50 microns and stained with toluidine blue. A qualitative and quantitative histological evaluation was performed, analyzing the inflammatory response, the bone cicatrisation process and the following histomorphometric parameters: percentage of new bone formation, percentage of graft particles, percentage of new bone related to the graft particles and defect resolution (new bone plus graft). The histomorphometry was carried out using an optical system associated with the histometry software package Bioquant Nova with image-capturing capabilities. Evaluations were related to the total area of the defects obtained in each sample. Statistical analysis Means and standard deviations were calculated for intra and intergroup comparison, using a Mann-Whitney test at a 95% level of significance (P 0.05). 3.2 Results There were no adverse tissue reactions to any of the implanted graft materials. In both experimental models during the various evaluation periods, the ABM/P-15 particulate demonstrated a superior biological behaviour compared with the ABM/P-15 hydrogel, not only promoting a more advanced bone tissue maturation, but also showing a statistically significant higher amount of new bone formation, presence of graft particles and total area of mineralized matrix (defect resolution). 17

18 4. Discussion The clinical study used a split-mouth design as a specialized form of a randomized block design where the subject is the unit of analysis. This design has been considered adequate for evaluating periodontal regenerative procedures in a recent systematic review (Needleman et al. 2005) evaluating the efficacy of guided tissue regeneration procedures, where half of the selected controlled randomized clinical trials (eight of 16 selected studies) used this split-mouth design. The sample size calculation used in this study was not designed for an equivalence clinical trial (Gunsolley et al. 1998) because this study aimed to carry out a preliminary comparison of the clinical efficacy of a new bone replacement graft with a new presentation for clinical use. However, this sample size is consistent with other clinical trials (Richardson et al. 1999; Camargo et al. 2000; Lekovic et al. 2001; Scheyer et al. 2002) using xenogeneic bone grafts (ranging from 17 to 23 patients); we did not compare the experimental replacement graft to the open flap debridement (negative control) with this sample because many studies (Camargo et al. 2000; Schultz et al. 2000; Sculean et al. 2003) already demonstrated statistical superiority of natural hydroxyapatite bone grafts over access flap surgery. This fact also was documented recently by two systematic reviews (Trombelli et al. 2002; Reynolds et al. 2003) on the efficacy of bone replacement grafts in the treatment of infrabony periodontal defects. This study failed to demonstrate superiority of the experimental replacement graft over the control. Six months after surgery, both treatments resulted in similar improvements in the studied outcome measurements (clinical attachment gains, probing depth reductions and defect bone fill). The magnitude of the CAL gain (3.41 mm) and defect bone fill (3.09 mm) observed in the control treatment group (ABM/P- 15 particulate) was comparable to the best results obtained (ranging from 1.3 to 4.0 mm for CAL gain and from 2.2 to 3.0 mm for bone fill) in recently published studies (Richardson et al. 1999; Lekovic et al. 2001; Scheyer et al. 2002; Sculean et al. 2003) using bovine-derived xenogenic grafts in the treatment of infrabony defects. These results are clearly comparable to those reported by Yukna et al. (1998, 2000) using the same material (ABM/P-15 particulate). In this study we obtained a mean defect bone fill of 73.7% (3.09 mm) compared to 72.3% (2.8 mm) and 72.9% (2.9 mm) reported in those two previous studies. Similarly, in this study we obtained a defect resolution of 81.9% versus 79.9% and 78.4% reported in those studies. These results also are comparable to those obtained in a clinical trial, using the same experimental design (split-mouth, controlled clinical trial with reentry at 6 months), that also evaluated the 18

19 efficacy of a bovine-derived xenograft in combination with a collagen membrane (Camargo et al. 2000). The investigators reported a mean defect bone fill of 3.8 mm and a mean alveolar crest resorption of 0.67 mm. Most of the defects in both treatment groups shared a similar morphology (between 85 and 90% were a combination of 2- and 3-wall defects); therefore, we believe that the presence of at least two bony walls also influenced the magnitude of the CAL gains and defect fill obtained in both groups. The consistency in the results obtained with this replacement graft gives support to its predictable use in the treatment of periodontal infrabony defects. The differences in CAL gain reported in the different studies can be explained by the known prognostic factors in the outcome of any periodontal regenerative surgery, such as the patient s plaque and infection control, the patient s smoking status, the surgeon s variability, and the surgical technique. In this study, we tried to control all of these factors, and under these circumstances, it is more likely that higher CAL gains and a higher percentage of defect bone fill can be obtained. The magnitude of the obtained results is dependent on the initial depth of the defect (Cortellini et al. 1998), which also may explain the slight differences in the results among the previous studies. This is probably the case when comparing our results to the study by Sculean et al. (2003), in which the initial periodontal defects were significantly deeper than in this study. The experimental material that we used (ABM/P-15 hydrogel) demonstrated similar outcomes, compared to the control (ABM/P-15 particulate), in soft tissue variables (mean CAL gain of 2.89 mm and mean PPD reduction of 4.02 mm) and in hard tissue variables [mean bone defect fill of 3.1 mm (75.0%) and mean defect resolution of 85.8%]. The clinical handling of this new form of hydrogel was very good. The material is supplied in prefilled syringes, which facilitates its application because it does not need prehydration. When evaluating the outcomes obtained with this experimental product, the results showed high variability, and there was a tendency not to see significant differences between the groups. The lower crestal resorption and the resulting lower recession shown in the control group may have been be due to a better space-maintaining capacity of the particulate graft that facilitated the preservation of the supracrestal connective tissue and, with this, the regenerative results (Cortellini & Tonetti 2000; Zucchelli et al. 2003; Tonetti et al. 2004). However, the limited sample size of the present study did not allow us to clarify whether the different physical properties of the products had any influence on the regenerative results. In the search for an ideal bone replacement graft in the treatment of periodontal infrabony defects, its physical properties must allow for the stabilization of the initial blood clot; serve as a scaffolding for the invasion of cells with regenerative capacity, 19

20 together with blood vessels; and allow for the maintenance of space under the flap. Under these principles, because the hydrogel formulation contains less mineral particles per volume and has a wider space in its matrix, it should allow for a greater invasion of adjacent tissues through these spaces. When evaluating the macroscopic outlook during the reentry surgery, the experimental group showed less granular material than the control grup; however, this did not translate into a greater defect bone fill or defect resolution. The only available information on the different biological behavior of both products in humans, comes from a histological case report study (Hahn et al. 2003), in which the particulate ABM/P-15 graft was compared to the hydrogel form in two fresh extraction sockets filled with both materials. At 13 weeks, the socket filled with the hydrogel form was void of particles and was filled with tissue with histologic characteristics of woven bone. Compared to the socket filled with the particulate form, the hydrogel-filled socket, besides the absence of granules, demonstrated almost twice the amount of vital bone. A comparative study in dogs (Vastardis et al. 2005), although using a different formulation (ABM/P-15 putty), also resulted in significantly greater bone formation in the defects treated with the putty form (49.3%) compared to the particulate form (14.8%). Conversly, a preclinical study (Scarano et al. 2003) performed in cortical bone defects in rabbits demonstrated that the defects treated with the ABM/P-15 particulate graft had a statistically significant greater regeneration of new bone, with a more mature type, whereas the defects treated with the ABM/P-15 hydrogel had a greater resorption of the particles. Nevertheless, the positive effect of the hydrogel form on bone formation also was established in vitro (Nguyen et al. 2003) by comparing different carriers for the ABM/P-15 particles and for the ABM particles without the P-15 sequence. The carboxymethylcellulose hydrogel carrier containing ABM/P-15 enhanced cell adhesion, enhanced osteoblastic activity with increased osteogenic gene expression for alkaline phosphatase and bone morphogenetic proteins, and promoted matrix mineralization. However, in the present clinical trial, the possible differences between the two distinct formulations were not detected. Therefore, we have used animal models with experimental bone defects to discern wether the hydrogel form was more advantageous than the standard ABM/P-15 bone replacement graft. The qualitative and quantitative histological evidence, provided by the two animal studies, with different types of bone defect containment, revealed that the ABM/P-15 particulate graft worked as an excellent osteoconductive scaffold, promoting the formation of a mature mineralized matrix. The initial immature new trabecular bone evolved to a functional organized tissue in an attempt to restore the original bony architecture. On the other hand, the ABM/P-15 hydrogel showed an abnormal particle 20