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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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.

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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, xeno-

enxertos 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,

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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, definiram-

se 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

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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 pós-

operatório. 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

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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, revela-

se 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.

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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

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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.

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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.

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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.

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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

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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.

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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.

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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.

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).

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

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,

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

21

migration that compromised the formation of a well-established mineralized matrix. The

random distribution of the particles, vehicled by the hydrogel, determined an

unpredictable osteoconduction and, consequently, a lower quantity and quality of new

bone compromising the maturation process of the mineralized tissue.

5. Conclusions

Based on the results from the clinical trial and the experimental animal models, it

can be concluded the following:

1. Both the graft materials, evaluated in the various experimental models,

demonstrated an adequate biocompatibility, which was confirmed by the absence of

adverse tissue reactions and anatomo-pathological alterations of the surrounding

tissues.

2. The treatment of periodontal infrabony defects with the ABM/P-15 particulate

and ABM/P-15 hydrogel bone replacement grafts resulted in significant improvements in

terms of CAL gains, PPD reductions and bone defect fill, when compared to baseline.

3. The controlled randomized clinical trial failed to demonstrate the superiority of

one form of the graft material over the other.

4. On the bone defects experimental models, both graft materials achieved a

superior new bone formation comparing with the negative unfilled defects.

5. Both bone regeneration animal models, provided qualitative and quantitative

histological evidence about the superior biological performance of the ABM/P-15

particulate graft over the ABM/P-15 hydrogel graft. The control graft material

demonstrated, not only, a more advanced maturation of the bony tissue, but also, a

significantly higher formation of new bone, presence of particles and total area of

mineralized matrix (new bone plus particles).

6. The delayed healing cranial bone defect model, demonstrated that the ABM/P-

15 particulate was the only graft that allowed for space provision, proper particle spatial

distribution, preservation of the original defect tickness and absence of soft tissue

collapse.

22

7. The ABM/P-15 particulate graft demonstrated, on both animal models, an

excellent osteoconductive behaviour and allowed the synthesis of a mineralized matrix,

initially formed by trabecular woven bone, which has evolved to an organized osseous

tissue, with functional maturation characteristics in an attempt to re-establish the

original bony architecture.

8. The ABM/P-15 hydrogel graft demonstrated, on both animal models, an

anomalous particle migration, which has compromised the formation of a robust

extracelular mineral matrix. The random distribution pattern of the particles, delivered

by the hydrogel based-vehicle, determined an unpredictable osteoconduction and,

consequently, a decreased quality and quantity of the developed mineralized tissue,

compromising the evolution of the bone maturatin process.

9. The critical size bone defect model in the femur, suggested the existence of a

minimum maturation threshold, in the early periods, that has influenced the

cicatrization evolution towards a regenerative or reparative pathway. In this context,

only the control graft material has favoured a predictable implementation of the iniciatic

regenerative cascade with a sufficient magnitude for the formation of a mature bony

tissue in the latter experimental periods, due to the fulfillment of an osteopromotive

matrix.

10. Active particle resorption of both graft materials was identified on the animal

studies during the various periods of time. However, in the latter stage of evolution (8

weeks), on the femur model, a stabilization of this process was observed without

significant modifications of the global particle surface area.

11. It is important to develop further studies in order to optimize the performance

of the delivery systems of graft materials in bone/periodontal defects with more

demanding regenerative potential factors.

23

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