UNIVERSIDADE FEDERAL FLUMINENSE FACULDADE DE ODONTOLOGIA

AVALIAÇÃO HISTOMORFOMÉTRICA COMPARATIVA DE MÉTODOS DE HIDRATAÇÃO DE UM FOSFATO DE CÁLCIO BIFASICO

Niterói 2017

UNIVERSIDADE FEDERAL FLUMINENSE FACULDADE DE ODONTOLOGIA

AVALIAÇÃO HISTOMORFOMÉTRICA COMPARATIVA DE MÉTODOS DE HIDRATAÇÃO DE UM FOSFATO DE CÁLCIO BIFASICO

IGOR GUIMARÃES BARROS PAULINELLI SANTOS Dissertação apresentada à Faculdade de Odontologia da Universidade Federal Fluminense, como parte dos requisitos para obtenção do título de Mestre, pelo Programa de Pós- Graduação em Odontologia.

Área de Concentração: Clínica Odontológica

Orientador: Profa. Dra. Carolina Miller de Mattos Santana.

Niterói 2017

FICHA CATALOGRÁFICA

S237 Santos, Igor Guimarães Barros Paulinelli

Avaliação histomorfométrica comparativa de métodos de hidratação de um fosfato de cálcio bifásico / Igor Guimarães Barros Paulinelli Santos; orientadora: Prof.ª Dr.ª Carolina Miller de Mattos Santana – Niterói: [s.n.], 2018.

54 f.: il.

Inclui tabelas

Dissertação (Mestrado em Clínica Odontológica) – Universidade Federal Fluminense, 2018.

Bibliografia: f.50- 54

1.Biocompatibilidade 2.Cicatrização 3.Alvéolos dentários 4.Ratos I.Santana, Carolina Miller de Mattos [orien.] II.Título

CDD 617.695

BANCA EXAMINADORA

Profa. Dra. Carolina Miller Mattos de Santana Instituição: Universidade Federal Fluminense

Decisão: ________________Assinatura:______________________________

Prof. Dr. Ronaldo Barcelos de Santana Instituição: Universidade Federal Fluminense

Decisão: ________________Assinatura:______________________________

Profa. Dra. Mônica Diuana Calasans Maia Instituição: Universidade Federal Fluminense

Decisão: ________________Assinatura:______________________________

Prof. Dr. Eduardo Sanches Gonçales Instituição: Universidade de São Paulo

Decisão: ________________Assinatura:______________________________

DEDICATÓRIA

Aos meus pais Grace e Eduardo por todo apoio, carinho e dedicação que sempre tiveram. Apesar da distância, sempre se fizeram presentes em meu desenvolvimento

pessoal e profissional;

Ao meu irmão Lucas pelo carinho e amizade imensurável;

Aos meus familiares e amigos pelo apoio e confiança de sempre.

À minha namorada Crislaine por todo carinho, apoio e paciência.

AGRADECIMENTOS

À Deus pela vida e por ser a razão de tudo;

Aos meus orientadores Carolina Miller e Ronaldo Barcellos pelos ensinamentos, incentivo e confiança depositada ao longo da minha formação;

Ao meu grande amigo e incentivador Raphael Monte Alto por todo apoio pessoal e profissional;

Aos professores, Mônica Diuana Calasans Maia, Adriana Therezinha Neves Novelino Alves, Rodrigo Resende e Marcelo Uzeda pelos ensinamentos, atenção e

ajuda na realização deste trabalho;

Aos colegas Jonatan, Lívia, Giovana e Fernanda por toda ajuda na realização do trabalho;

Aos meus professores e amigos Gustavo Oliveira dos Santos, Edgard Mello Fonseca , Mario Groisman e Raul Feres Monte Alto por todos os ensinamentos e

generosidade;

Aos meus amigos Guilherme Diaz , Edgard Belladonna , Antônio Costa Neto , Rodrigo Modena, Pedro Paulo Albuquerque, Marcus Bertolo, Leandro Machado,

Renan Arruda e Eduardo Mendes pelo companheirismo de sempre;

À Universidade Federal Fluminense, por ter me acolhido durante minha formação e a todos colaboram com ela;

Á CAPES, pela cessão de bolsa de estudos no ano de 2015.

Muito obrigado.

RESUMO

Santos IGBP. Avaliação histomorfométrica comparativa de métodos de hidratação de um fosfato de cálcio bifásico. [dissertação]. Niterói: Universidade Federal Fluminense, Faculdade de Odontologia; 2017.

Após a exodontia de um elemento dental, as mudanças volumétricas são inerentes a este processo. O preenchimento alveolar pós-extração com biomateriais pode reduzir essas mudanças volumétricas e proporcionar um ambiente mais favorável para o futuro restabelecimento de próteses com implantes. Entre os biomateriais disponíveis, os compostos cerâmicos bifásicos demonstraram ter bons resultados clínicos. No entanto, poucos estudos avaliaram histologicamente sua resposta tecidual em termos de seu método de hidratação e incorporação. O objetivo deste estudo foi avaliar in vivo e, de forma comparativa, o potencial osteocondutor do substituto ósseo cerâmico bifásico composto por fosfato beta-tricálcico e hidroxiapatita (Bone Ceramic®, Straumann) após diferentes metodologias de hidratação em alvéolos dentários de ratos. Os ratos Wistar (n = 20) foram distribuídos aleatoriamente em dois subgrupos (G1, G2) de acordo com os métodos de hidratação utilizados. Após a realização dos procedimentos de anestesia, antisepsia e extração do incisivo central superior direito, os alvéolos foram preenchidos com biomaterial cerâmico bifásico hidratado por dois métodos diferentes. Grupo 1: alvéolo preenchido com biomaterial cerâmico bifásico hidratado no sangue; Grupo 2: alvéolo preenchido com biomaterial cerâmico bifásico hidratado em solução salina fisiológica, então a região foi suturada em ambos os grupos. Os animais foram eutanasiados após 1 e 6 semanas para a remoção dos blocos ósseos contendo o biomaterial e foram submetidos ao processamento histológico. Secções de cinco μm de espessura das amostras desmineralizadas foram coradas com Hematoxilina e Eosina (HE) e sujeitas a análise histomorfométrica. A formação de osso foi limitada 7 dias após o procedimento de extração e aumentou em ambos os grupos entre 7 e 42 dias a partir da cirurgia, demonstrando um aumento do volume ósseo, dependente do tempo, durante todo o período experimental (p <0,05). A hidratação do SBC com solução salina aumentou significativamente a nova formação óssea e reduziu o volume do tecido conjuntivo após 42 dias, demonstrando que o método de hidratação pode influenciar significativamente a cicatrização óssea em tais defeitos e, portanto, deve ser cuidadosamente realizado.

Palavras-chave: Biocompatibilidade, Cicatrização, Alvéolos dentários, Ratos.

ABSTRACT

Santos IGBP. Comparative histomorphometric evaluation of methods of hydratations of a bifasic calcium fosfate.[dissertation]. Niterói: Universidade Federal Fluminense, Faculdade de Odontologia; 2017.

After the exodontia of a dental element volumetric changes are inherent to this process. The filling of the post-extraction well with biomaterials can reduce these volumetric changes and provide a more favorable environment for the future

prosthetic reestablishment with implants. Among the biomaterials available, biphasic ceramic compounds have been shown to have good clinical results. However, few studies have histologically evaluated their tissue response in terms of their hydration and incorporation method. The objective of this study was to evaluate in vivo, and in a comparative way, the osteoconductive potential of the biphasic ceramic bone substitute composed of beta-tricalcium phosphate and hydroxyapatite (Bone Ceramic®, Straumann) after different hydration methodologies in rat dental alveoli.

Wistar rats (n = 20) were randomly distributed in two subgroups (G1, G2) according to the hydration methods used. After performing the procedures of anesthesia, antisepsis and extraction of the right upper central incisor the alveoli were filled with biphasic ceramic biomaterial hydrated by two different methods. Group 1: alveolus filled with biphasic ceramic biomaterial hydrated in blood; Group 2: alveolus filled with biphasic ceramic biomaterial hydrated in physiological saline, then the region was sutured. The animals were euthanized after 1 and 6 weeks for removal of the bone blocks containing the biomaterial and were submitted to histological processing. Five μm thick sections of the demineralized samples were stained with Hematoxylin and Eosin (HE) and subjected to histomorphometric analysis. Bone formation was limited 7 days after the extraction procedure and increased in both groups between 7 and 42 days from surgery, demonstrating a time dependent increase of bone volume

throughtout the experimental period (p<0.05). The hydration of SBC with saline significantly increased new bone formation and reduced connective tissue volume after 42 days demonstrating that hydration method may significantly influence bone healing in such defects, and, thus should be carefully performed.

Keywords: Biocompatibility, Healing, Dental alveoli, Rats.

1 - INTRODUÇÃO

Inúmeros fatores podem levar à exodontia de um elemento dentário, dentre eles podemos destacar a doença periodontal, cárie, reabsorção radicular e fraturas.

Essa condição leva a diversas alterações biológicas que podem repercutir na manutenção volumétrica do sítio pós-exodontia.^1,2

Após a remoção de um elemento dentário, um coágulo sanguíneo preenche o alvéolo e estimula o processo de formação óssea. No período de 72 horas pode-se observar a presença de tecido de granulação. Após 7 dias o alvéolo já está preenchido por tecido conjuntivo imaturo , com a formação de osteóide.O processo de maturação do tecido conjuntivo e mineralização do osteóide ocorre com 20 dias após a exodontia e um osso trabecular pode ser observado com seis semanas.^3-5

Entretanto, esse processo não resulta na reconstituição total do volume ósseo alveolar. As alterações volumétricas inerentes a esse processo cirúrgico já foram amplamente discutidas na literatura. Van der Weijden et al. (2009)⁶, em uma revisão sistemática da literatura, encontraram que, durante o período de cicatrização pós- extração, as médias ponderadas das mudanças mostraram a perda clínica em espessura (3,87 mm) como sendo maior do que a perda em altura, avaliada tanto clinicamente (1,67–2,03 mm) como radiograficamente (1,53 mm).

Sabe-se que essa transformação é da ordem de 40 % no sentido vertical e 60

% no sentido horizontal, durante os dois primeiros anos pós-exodontia^7,8. Essa remodelação tecidual pode comprometer o reestabelecimento protético com o uso de implantes, sobretudo na tentativa de se buscar um posicionamento tridimensional ideal.⁹

O avanço no desenvolvimento da ciência dos biomateriais e a evolução das técnicas cirúrgicas têm proporcionado resultados favoráveis no sentido de minimizar as alterações dimensionais decorrentes da extração de um elemento dentário, visando à manutenção e recuperação da arquitetura alveolar prévia.

Algumas metodologias podem ser empregadas para atenuar essas alterações dimensionais e viabilizar a futura instalação de implantes, dentre elas o uso de substitutos ósseos, juntamente com o uso de membranas, têm apresentado bons resultados clínicos e histológicos, no preenchimento de defeitos ósseos^10,11.

Dentre os materiais usados para esse preenchimento destacam-se os enxertos ósseos autógenos, alógenos, xenógenos e aloplásticos¹².

O enxerto de osso autógeno é considerado o padrão ouro na regeneração óssea em função do seu potencial osteogênico, de osteoindução e de osteocondução. Entretanto, o procedimento de remoção do enxerto requer um segundo sítio cirúrgico, o que aumenta a complexidade e morbidade do procedimento, sobretudo quando há necessidade de grande volume de material, além de vários relatos de complicações pós-cirugicas^13,14.

Já o enxerto de origem alógena é obtido de doadores humanos vivos ou de cadáveres e são processados e armazenados antes do uso. Essa origem apresenta uma importante desvantagem que é a possibilidade da haver a ocorrência exacerbada da resposta imunológica¹⁵, além da pouca disponibilidade de banco de ossos. Por outro lado, os xenoenxertos, sejam de origem bovina, suína ou equina, também conhecidos como osso mineral natural, são amplamente utilizados, consistindo em um arcabouço ósseo inerte de estrutura tridimensional semelhante à matriz óssea mineralizada¹⁶. Apesar dos excelentes resultados clínicos e histológicos^17,18, esses materiais também podem apresentar o risco de antigenicidade,por se tratar de uma origem diferente da espécie humana^19,20.

Na tentativa de se encontrar um substituto ósseo ideal, diversos materiais aloplásticos têm sido pesquisados. Essa classe de materiais têm trazido benefícios nos campos das pesquisas e tratamentos, uma vez que reduz a morbidade de procedimentos cirúrgicos e tem a opção de diferentes formas de apresentação, tamanho, textura, grau de porosidade, grau de cristalinidade e solubilidade²¹.

Estes devem apresentar como propriedades físico-químicas e biológicas:

biocompatibilidade, degradação controlada e substituição simultânea por novo osso formado, osteocondução e integridade mecânica, a fim de suportar a cicatrização após os procedimentos de regeneração óssea guiada²².

Dentre os materiais sintéticos utilizados na regeneração óssea as cerâmicas de fosfato de cálcio sintéticas tem sido amplamente utilizadas, apresentando bons resultados clínicos e histológicos^23,24. As cerâmicas de fosfato de cálcio são biocompatíveis e possuem propriedades bioativas. A sua constituição química inorgânica é semelhante à do osso natural, o que as torna substitutos ósseos promissores nos campos ortopédicos e maxilofacial ²⁵.

Esses biomateriais apresentam propriedade de osteocondução, ou seja, funcionam como arcabouço para a implantação de osteoblastos e deposição de matriz óssea, favorecendo a formação de trabéculas ósseas em meio às partículas

do material ²⁶. O processo de neoformação óssea começa na periferia da área de enxerto, por meio da diferenciação de células mesenquimais indiferenciadas em osteoblastos e sua migração em direção ao biomaterial. As partículas dos biomateriais podem ser dissolvidas pela ação de macrófagos através de fagocitose e redução do pH nos fagolisossomos. Os íons cálcio e fosfato liberados estimulam a nucleação secundária de hidroxiapatita favorecendo a mineralização da matriz óssea

27. Assim, o tecido ósseo é formado e sofre processo de remodelação com participação de osteoblastos e osteoclastos.

A hidroxiapatita (HA) [Ca10(PO4 )6(OH)2] e o tricálcio fosfato (TCP) [Ca3(PO 4)2] são cerâmicas fosfato de cálcio amplamente utilizadas. Estes biomateriais são atóxicos e apresentam ótima atividade osteocondutiva. Eles apresentam diferenças em relação a sua composição e em relação ao seu metabolismo, apresentando tempos diferentes de degradação²⁸. A hidroxiapatita apresenta uma desvantagem importante do ponto de vista biológico que é sua baixa solubilidade, o que interfere na sua degradação e substituição por osso neoformado²⁹.

O TCP está disponível em 2 formas: uma modificação a alta temperatura, α-TCP (α-Ca3(PO4)2), que é produzido a temperaturas superiores a 1125ºC e uma modificação a baixa temperatura, β-TCP (β-Ca3(PO4)2), produzida a temperaturas inferiores a 1125ºC. Em contraste com α-TCP, β-TCP é termodinamicamente estável num meio ambiente biológico e dentro de um intervalo de temperatura normal.

Apesar de um grau semelhante de solubilidade, a biodegradação do β-TCP é mais rápido do que o da α-TCP, porque esta última forma hidrolisa parcialmente ou completamente em hidroxiapatita Ca5[OH(PO4)3]. Os cristais resultantes têm uma morfologia não fisiológica e não são reabsorvidos, devido ao seu baixo nível de solubilidade e podem entrar no sistema linfático por fagocitose³⁰.

No entanto, a porosidade dos biomateriais à base de TCP é inversamente proporcional à sua estabilidade. Esta perda de estabilidade é frequentemente citada como uma limitação no uso de cerâmicas de TCP na prática clínica. Uma alternativa para superar este problema é associar cerâmicas à base de TCP com outros tipos de cerâmicas que apresentam maior estabilidade, como a hidroxiapatita, o que pode manter a sua resistência mecânica até a reabsorção ser alcançada.^31,32

Diversos substitutos ósseos cerâmicos foram formulados com diferentes proporções de TCP e HA . Dentre essas formulações, o Bone Ceramic® - Straumann (BC) é um substituto ósseo 100% sintético, com morfologia para

estimular a formação de osso vital. De acordo com o fabricante, possui índice de porosidade de 90%, com poros interconectados de diâmetro entre 100 a 500 mícrons. É composto por fosfato de cálcio bifásico, uma combinação de hidroxiapatita (HA) a 60% em peso e 40% em peso de beta- tricálcio fosfato.³³ Em alguns estudos , o BC mostrou ser um substituto ósseo com propriedades osteocondutoras favoráveis para o reparo em defeitos ósseos, com resultados semelhantes, no tocante a quantidade de osso mineralizado, quando o mesmo foi comparado com osso bovino anorgânico (Bio- Oss®). Entretanto, houve maior quantidade dos componentes de tecido mole e diferentes características de reabsorção para o grupo BC.³⁴

Segundo orientações de fabricantes, e diversos outros estudos utilizando biomateriais cerâmicos como substituto ósseo, sua hidratação deveria ser feita com sangue do paciente³⁵. No entanto a disponibilidade de material sanguíneo na região a ser abordada pode não ser suficiente para suprir a necessidade, principalmente quando grande quantidade de material se faz necessário. Nesse caso, fontes periféricas de coleta sanguínea podem ser necessárias para a correta hidratação do enxerto.

Outros materiais permitem o uso de soro fisiológico estéril para a incorporação e hidratação das partículas de enxerto. Essa fonte de hidratação facilita o uso dos substitutos ósseos em função da sua fácil disponibilidade e menor morbidade para o paciente, principalmente quando fontes periféricas são necessárias para a coleta sanguínea.

No entanto, a literatura carece de trabalhos que apresentem os eventos fisiológicos desses diferentes métodos de incorporação das partículas de enxerto, bem como seu potencial osteocondutivo. Nesse contexto, este estudo in vivo se propõe a avaliar histologicamente e histomorfometricamente as reações teciduais a um substituto ósseo constituído de Beta-tricalcio Fosfato e Hidroxiapatita, com diferentes metodologias de hidratação, em alvéolos de ratos.

2 - METODOLOGIA

2.1 - Material

Este estudo experimental in vivo foi aprovado pela Comissão de Ética no Uso de Animais (CEUA/NAL).

Biomaterial

Neste estudo foi utilizado um substituto ósseo cerâmico bifásico de beta-tricálcio fosfato e hidroxiapatita, contendo 60% e 40 % de cada componente, respectivamente (Straumann Bone Ceramic ® - Institute Straumann, Switzerland).

Possui índice de porosidade de 90%, com poros interconectados de diâmetro entre 100 a 500 mícrons. Foram utilizados 2 embalagens de 0,5 g do biomaterial e foram divididos entre os objetos de estudo.

2.1.1 - Caracterização dos animais

Nesta pesquisa foram utilizados ratos Wistar (n=20) machos ou fêmeas, com peso médio de 300 gramas, que foram divididos aleatoriamente em 4 caixas contendo 5 animais e distribuídos em 2 subgrupos de acordo com os períodos experimentais de 1 Semana (T1) e 6 semanas (T2) após os procedimentos cirúrgicos. Cada grupo de caixas foi dividido em 2 grupos de acordo com a metodologia que foi empregada ao alvéolo.Sendo:

- Grupo 1 : alvéolo preenchido com substituto ósseo cerâmico bifásico composto por beta tricálcio fosfato e hidroxiapatita hidratado em sangue;

-Grupo 2: alvéolo preenchido com substituto ósseo cerâmico bifásico composto por beta tricálcio fosfato e hidroxiapatita hidratado em soro fisiológico;

O biomaterial do grupo 1 foi coletado do próprio alvéolo do animal.Com uma seringa estéril, com uma agulha de 1,2 x 25 mm, foi coletado do alvéolo do animal uma quantidade de sangue suficiente para a hidratação completa do biomaterial que seria utilizado para o preenchimento alveolar pós-exodontia.

Todos os animais foram mantidos em caixas com ração e água sem restrições no laboratório de experimentação animal (LEA) do Núcleo de Animais de Laboratório (NAL) ambos da Universidade Federal Fluminense (UFF).

Figura 1. A) Alvéolo pós exodontia do incisivo central superior direito. B) Alvéolo preenchido com biomaterial.

2.2 - Método

Procedimentos cirúrgicos: Os animais receberam anestesia geral sob injeção intramuscular de 75mg/kg de Ketamina (cloridrato de cetamina, Veltbrands, Brasil) e 1,5ml/kg de Rompun (xilazina, Veltbrands, Brasil). Após anestesia geral foi realizada a sindesmotomia, luxação e extração do incisivo central superior direito com uma sonda milimetrada nº5, em seguida foi realizado o preenchimento dos alvéolos dentários com os biomateriais e seguindo-se a sutura com fio de seda 5.0 (Ethicon®, Johnson & Johnson, Brasil) e anti-sepsia com gaze e solução alcoólica de clorexidina 2% para proteção da ferida cirúrgica e evitar contaminação secundária.

Procedimentos e cuidados pós-operatórios: Todos os procedimentos cirúrgicos foram realizados em dias da semana que permitissem a realização do protocolo de analgesia nos dois dias seguintes à cirurgia, de acordo com as orientações do Prof.

Fabio O. Ascoli. Foi ministrado antiinflamatório (Meloxicam) 1mg/kg, via subcutânea a cada 24 horas, que foi iniciado no dia da cirurgia e mantido por mais 2 dias.

Obtenção das amostras: Decorridos os períodos experimentais de 1 e 6 semanas, 5 animais de cada grupo experimental receberam dose letal de anestésico geral para coleta das amostras e tecidos circunjacentes.

(A) (B)

Figura 2. Amostras prontas para inclusão em parafina

Processamento das amostras: As amostras provenientes de 5 animais por período experimental foram fixadas, descalcificadas, classificadas e processadas para inclusão em parafina e posterior cortes de 5μm foram obtidos e corados com Hematoxilina e Eosina (HE) conforme Tabela 1.

Análise dos resultados: As lâminas foram observadas em um microscópio de luz de campo claro (Olympus Bx 43). As imagens selecionadas foram capturadas por uma câmera digital (Olympus SC 100) utilizando um sistema de captura (CellSens Standard). Foram feitas aleatoriamente entre 8 e 10 imagens por corte histológico correspondente a cada animal sem que houvesse sobreposição das mesmas. Cada imagem foi aberta no software ImagePro Plus® e sobre cada imagem foi sobreposta uma grade com 247 pontos com espaçamento igual entre si. A cada ponto foi atribuída uma definição entre cinco possíveis: osso nativo, tecido conjuntivo, osso neoformado, biomaterial e outros. Os dados foram tratados estatisticamente (ProStat, New York, USA).

A análise descritiva da resposta tecidual aos biomateriais foram avaliadas em função da presença de células gigantes, de tecido fibroso, de neoformação vascular e óssea. No decorrer do estudo não ocorreram complicações cirúrgicas ou infecção em nenhum animal.

2.3. Local

A pesquisa foi desenvolvida no Laboratório de Biotecnologia Aplicada (LABA) e Laboratório de Experimentação Animal (LEA) da Universidade Federal Fluminense (UFF).

2.4. Tratamento Estatístico

A estatística descritiva foi expressa por meio +/- de desvio padrão (DP). A análise de variância (ANOVA) foi utilizada para comparar os resultados inter-grupo. Então, as comparações inter-grupo e intra-grupo foram analisadas por teste Wilcoxon (variáveis qualitativas ordinais) ou teste Chi-quadrado (variáveis qualitativas ordinais). O valor alfa menor que 0,05 foi utilizado para declarar significância estatística.

3 - ARTIGO PRODUZIDO

HISTOLOGICAL AND HISTOMORPHOMETRIC COMPARATIVE ANALYSIS OF METHODS OF HYDRATION OF A BIPHASIC CERAMIC BONE SUBSTITUTE COMPOSED BY BETA TRICALCIUM PHOSPHATE AND HYDROXYAPATITE

Igor Guimarães Barros Paulinelli Santos¹ Carolina Miler Mattos de Santana¹

Mônica Diuana Calasans-Maia²

Adriana Therezinha Neves Novelino Alves³ Ronaldo Barcellos de Santana¹ *

1. Department of Periodontology. School of Dentistry, Federal Fluminense University, Niterói, Rio de Janeiro, Brazil.

2. Department of Oral Surgery. School of Dentistry, Federal Fluminense University, Niterói, Rio de Janeiro, Brazil.

3. Department of Stomatology. School of Dentistry, Federal Fluminense University, Niterói, Rio de Janeiro, Brazil.

*Corresponding author

Key words: Biocompatibility, healing, hydroxyapatite, Dental alveoli, Osteogenesis, Rats.

ABSTRACT

After the exodontia of a dental element volumetric changes are inherent to this process. However, some measures can be taken to minimize these effects. The filling of the post-extraction well with biomaterials can reduce these volumetric changes and provide a more favorable environment for the future prosthetic

reestablishment with implants. Among the biomaterials available, biphasic ceramic compounds have been shown to have good clinical results. However, few studies have histologically evaluated their tissue response in terms of their hydration and incorporation method. The objective of this study was to evaluate in vivo, and in a comparative way, the osteoconductive potential of the biphasic ceramic bone substitute composed of beta-tricalcium phosphate and hydroxyapatite (Bone Ceramic®, Straumann) after different hydration methodologies in rat dental alveoli.

Wistar rats (n = 20) were randomly distributed in two subgroups (G1, G2) according to the hydration methods used. After performing the procedures of anesthesia, antisepsis and extraction of the right upper central incisor the alveoli were filled with biphasic ceramic biomaterial hydrated by two different methods. Group 1: alveolus filled with biphasic ceramic biomaterial hydrated in blood; Group 2: alveolus filled with biphasic ceramic biomaterial hydrated in physiological saline, then the region was sutured. The animals were euthanized after 1 and 6 weeks for removal of the bone blocks containing the biomaterial and were submitted to histological processing. Five μm thick sections of the demineralized samples were stained with Hematoxylin and Eosin (HE) and subjected to histomorphometric analysis. Bone formation was limited 7 days after the extraction procedure and increased in both groups between 7 and 42 days from surgery, demonstrating a time dependent increase of bone volume

throughtout the experimental period (p<0.05). The hydration of SBC with saline significantly increased new bone formation and reduced connective tissue volume after 42 days demonstrating that hydration method may significantly influence bone healing in such defects, and, thus should be carefully performed.

INTRODUCTION

Numerous factors can lead to the extraction of a dental element, among them we can highlight periodontal disease, caries, root resorption and fractures. This condition leads to several biological alterations that can affect the volumetric maintenance of the post-extraction site ^1,2.

After removal of a dental element, a blood clot fills the alveolus and stimulates the bone formation process. After 7 days the alveolus is already filled with immature connective tissue, with the formation of osteoid. The process of maturation of the connective tissue and mineralization of the osteoid occurs with 20 days after the extraction and a trabecular bone can be observed at six weeks^3-5.

However, this process does not result in total reconstitution of alveolar bone volume. The volumetric changes inherent to this surgical process have already been widely discussed in the literature. Van der Weijden et al. (2009)⁶, in a systematic review of the literature, found that, during the post-extraction healing period, the weighted averages of the changes showed clinical loss in thickness (3.87 mm) as being greater than loss in height, evaluated both clinically (1.67-2.03 mm) and radiographically (1.53 mm).

It is known that this transformation is of the order of 40% in the vertical direction and 60% in the horizontal sense, during the first two years post-extraction^7,8. This tissue remodeling may compromise prosthetic reestablishment with the use of implants, especially in an attempt to achieve an ideal three-dimensional positioning⁹. Advances in the development of biomaterials science and the evolution of surgical techniques have provided favorable results in order to minimize the dimensional changes resulting from the extraction of a dental element, aiming the maintenance and recovery of the previous alveolar architecture.

Some methodologies may be used to attenuate these dimensional changes and to make possible the future implantation of implants, among them the use of bone substitutes, together with the use of membranes, have presented good clinical and histological results in the filling of bone defects^10,11.

Among the materials used for this filling, autogenous, allogenic, xenogeneic and alloplastic bone grafts are highlighted¹².

The graft of the autogenous bone is considered the gold standard in the bone regeneration in function of its osteogenic potential of osteoinduction and

osteoconduction. However, the graft removal procedure requires a second surgical site, which increases the complexity and morbidity of the procedure, especially when there is a need for a large volume of material, besides several reports of postoperative complications^13,14.

Allogeneic origin grafts are obtained from living human donors or cadavers and are processed and stored before use. This origin has a significant disadvantage, which is the possibility of an exacerbated occurrence of the immune response¹⁵, in addition to the low availability of bone banks. On the other hand, xenografts, whether of bovine, porcine or equine origin, also known as natural mineral bone, are widely used, consisting of an inert bone structure of three-dimensional structure similar to the mineralized bone matrix¹⁶. Despite the excellent clinical and histological results^17,18, these materials may also present the risk of antigenicity, since it is a different origin of the human species^{19 , 20}.

In an attempt to find an ideal bone substitute, several alloplastic materials have been researched. This class of materials has brought benefits in the fields of research and treatment, since it reduces the morbidity of surgical procedures and has the option of different forms of presentation, size, texture, degree of porosity, degree of crystallinity and solubility²¹.

These should present as physical-chemical properties: biocompatibility, controlled degradation and simultaneous replacement by new bone formation, osteoconduction and mechanical integrity, in order to support healing after guided bone regeneration procedures²².

Among the synthetic materials used in bone regeneration, synthetic calcium phosphate ceramics have been widely used, presenting good clinical and histological results^23,24. Calcium phosphate ceramics are biocompatible and have bioactive properties. Their inorganic chemical constitution is similar to that of natural bone, which makes them promising bone substitutes in the orthopedic and maxillofacial fields ²⁵.

These biomaterials have properties of osteoconduction, that is, they act as a framework for the implantation of osteoblasts and deposition of bone matrix, favoring the formation of bone trabeculae in the midst of the particles of material ²⁶. The process of new bone formation begins at the periphery of the graft area , through the differentiation of undifferentiated mesenchymal cells into osteoblasts and their migration towards the biomaterial. The biomaterial particles can be dissolved by the

action of macrophages through phagocytosis and pH reduction in the phagolysosomes. The released calcium and phosphate ions stimulate the secondary nucleation of hydroxyapatite favoring mineralization of the bone matrix ²⁷. Thus, the bone tissue is formed and undergoes a process of remodeling with the participation of osteoblasts and osteoclasts.

Hydroxyapatite (HA) [Ca10 (PO4) 6 (OH) 2] and tricalcium phosphate (TCP) [Ca3 (PO4) 2] are widely used calcium phosphate ceramics. These biomaterials are non- toxic and have excellent osteoconductive activity. They differ in their composition and in relation to their metabolism, presenting different times of degradation²⁸. Hydroxyapatite has a biologically significant drawback which is its low solubility, which interferes with its degradation and replacement by neoformed bone²⁹.

TCP is available in two forms: a high temperature modification, α-TCP (α-Ca 3 (PO 4 2)), which is produced at temperatures above 1125 ° C and a modification at low temperature, b-TCP (b-Ca 3 (PO4) 2), produced at temperatures below 1125 ° C.

In contrast to α-TCP, b-TCP is thermodynamically stable in a biological environment and within a normal temperature range. Although a similar degree of solubility, the biodegradation of B-TCP is faster than that of α-TCP, because the latter form partially or completely hydrolyzes in hydroxyapatite Ca5 [OH (PO 4) 3]. The resulting crystals have a non-physiological morphology and are not reabsorbed because of their low solubility and can enter the lymphatic system by phagocytosis.

However, the porosity of the TCP-based biomaterials is inversely proportional to their stability. This loss of stability is often cited as a limitation in the use of TCP ceramics in clinical practice. An alternative to overcome this problem is to associate ceramics with TCP with other types of ceramics that present greater stability, such as hydroxyapatite, which can maintain its mechanical resistance until the resorption is achieved.^31,32

Several ceramic bone substitutes have been formulated with different proportions of TCP and HA. Among these formulations, Bone Ceramic® - Straumann (BC) is a 100% synthetic bone substitute, with morphology to stimulate the formation of vital bone. According to the manufacturer, it has a porosity index of 90%, with interconnected pores with a diameter between 100 and 500 microns. It is composed of two-phase calcium phosphate, a combination of hydroxyapatite (HA) at 60% by weight and 40% by weight of beta-tricalcium phosphate.³³ In some studies, BC has been shown to be a bone substitute with favorable osteoconductive properties for

repair In bone defects, with similar results, regarding the amount of mineralized bone, when it was compared with anorganic bovine bone (Bio-Oss®). However, there were more soft tissue components and different resorption characteristics for the BC group

34.

According to manufacturer guidelines, and several other studies using ceramic biomaterials as a bone substitute, their hydration should be done with the patient's blood.³⁵ However, the availability of blood material in the region to be addressed may not be sufficient to meet the need, especially when large quantities of material is required. In this case, peripheral sources of blood collection may be necessary for the correct hydration of the graft.

Other materials allow the use of sterile saline for the incorporation and hydration of the graft particles. This source of hydration facilitates the use of bone substitutes due to their easy availability and lower morbidity for the patient, especially when peripheral sources are necessary for blood collection.

However, the literature lacks papers that present the physiological events of these different methods of incorporation of graft particles, as well as their osteoconductive potential. In this context, this in vivo study aims to evaluate histologically the tissue reactions to a bone substitute constituted of Beta-tricalcium Phosphate and Hydroxyapatite, with different hydration methodologies, in alveoli of rats.

MATERIALS AND METHODS

Biomaterial

In this study, a biphasic beta-tricalcium phosphate and hydroxyapatite ceramic replacement was used, containing 60% and 40% of each component, respectively. It has a porosity index of 90%, with interconnected pores with a diameter of between 100 and 500 microns.

Animals and surgical procedures

All procedures were carried out in accordance with conventional guidelines in the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health 85-23, revised 1996) and were approved by the local Institutional Animal Care and Use Committee of Federal Fluminense University, Niteroi, Brazil. In this study, male or female Wistar rats (n = 20), weighing 300 grams, were randomly divided into 4 boxes containing 5 animals and distributed in 2 groups according to the experimental periods of 1 week (T1) and 6 Weeks (T2) after the surgical procedures. Each group of boxes will be divided into 2 sub-groups according to the methodology that will be used in the alveolus:

- Group SBC-B: alveolus filled with biphasic ceramic bone substitute composed of beta tricalcium phosphate and hydroxyapatite hydrated in blood;

-Group SBC-S: alveolus filled with biphasic ceramic bone substitute composed of beta tricalcium phosphate and hydroxyapatite hydrated in physiological saline;

All animals were anesthetized with ketamine (20 mg/kg) (Virbac, Jurubatuba, SP, Brazil) and xylazine (1 mg/Kg) (FortDodge, São Cristovão, RJ, Brazil). The surgical area was scrubbed with sterile gauze soaked with 4 % alcoholic chlorhexidine, rinsed with sterile water and then draped. Subsequently, syndesmotomy of periodontal tissue was performed using a syndesmotome (Duflex®, Rio de Janeiro, Rio de Janeiro, Brazil), and the upper-right incisor was extracted with a clinical probe adapted to this tooth (Figure 3A). The dental alveolar sockets were grafted with the biomaterials, and sutured with a 5–0 nylon interrupted suture. (Johnson & Johnson Medical Ltd., Blue Ash, Ohio, United States).

At 7 and 42 days after implantation, the animals were anesthetized and euthanized with an overdose of the anesthetic solution and samples containing the biomaterials were removed and fixed in 4 % buffered formalin at pH 7.0. The specimens were decalcified in fast bone demineralization solution (Allkimia Ltda, Campinas, Brazil) for 48 h, washed for 1 h, dehydrated in ethanol (Vetec Química Fina Ltda., Duque de Caxias, Brazil), clarified in xylol (Vetec Ltda.), and embedded in paraffin (Vetec Ltda.).

Histological analysis

Decalcified paraffin sections (5 um thick) were stained with hematoxylin-eosin (HE) and examined by an experienced pathologist in the field of biomaterials biocompatibility without knowing the tested animal groups (blind analysis). A descriptive analysis comparing the intra and inter-group biological responses was based on the type and intensity of the inflammatory alterations and repair processes (fibrosis, new blood vessels and osteogenesis). Photomicrographs were obtained with a digital camera (Cybershot DsC-W300, Sony, Manaus, Brazil) connected to an optical microscope (FWl-1000, Feldman Wild Leitz, Manaus, Brazil).

Histomorphometric analysis

For histomorphometric analysis, a light microscope (Olympus BX43, Tokyo, Kanto, Japan) with 10x of magnfication was used. The microscope was connected to a computer and each HE-stained histological slice corresponding to the alveolar region was captured by scanning by Image acquisition software (Cellsens® 1.9 Digital, Tokyo, Kanto, Japan). One expert observer analysed ten non-consecutive images of each section. With the Image-Pro Plus® 6.0 (Media Cybernetics, Silver Spring,

Maryland, USA), a grid of 247 points were superimposed on captured field, permitting the determination of newly formed bone and the residual biomaterial. The bone volume density (BV/ TV%) was calculated by bone volume over total volume, indicating the fraction of volume of interest that was occupied by bone. For biomaterial volume density (BiomatV/TV%) and connective tissue volume density (CT/TV%), the same calculation method was applied. The areas were expressed in percentage.

RESULTS

Histological analyses

Morphological analysis was performed at light microscope after hematoxylin- eosin staining. The Figures 3-15 contain representative photomicrographs of alveolar socket from each implanted biomaterials at magnification of 400X. After seven days, both groups exhibited a mild inflammatory response, which mainly consisted of mononuclear cells, blood vessels of different calibers, and loose connective tissue around the particles, with collagen fibers randomly dispersed in the area.

Hemorrhagic exudate, inflammatory cells and a few multinucleated inflammatory giant cells were also observed. Few trabeculae of newly formed bone with osteoblasts pavement on periphery, interspersed by connective tissue containing serum hemorrhagic exudate were noted in both groups. New bone formation occurred only in contact with the residual bone walls. Both biomaterial groups presented the dental socket filled by connective tissue characterized by granulation reaction permeating a small fraction of biomaterial spheres.

Figure 3- . Photomicrograph of BoneCeramic and Saline solution after 7

days of implantaion. Presence of connective tissue (CT) permeating

biomaterial particles (BC) and preexisting bone tissue (PB).40x

magnification Stain: Hematoxillin and Eosin.

Figure 4 -. Photomicrograph of BoneCeramic and Saline solution after 7

days of implantaion. Presence of connective tissue (CT) permeating

biomaterial particles (BC).40x magnification Stain: Hematoxillin and

Eosin.

Figure 5 . Photomicrograph of BoneCeramic and Saline solution after 7

days of implantaion. Presence of connective tissue (CT) permeating

neoformed bone islands (NB) adjacent to preexisting bone tissue

(PB).400x magnification Stain: Hematoxillin and Eosin.

Figure 6. Photomicrograph of BoneCeramic and Saline solution after 7

days of implantation. Presence of biomaterial (BC), surrounded by

connective tissue (CT). Inflammatory activity adjacent to the material

(black arrows). 400X magnification. Stain: Hematoxillin and Eosin

Figure 7. . Photomicrograph of BoneCeramic and Saline solution after 7

days of implantation. Presence of richly cellularized connective tissue

(CT), particles of biomaterial (BC), neoformed bone projections and

preexisting bone tissue (PB). The black arrow indicating osteoblastic

paving. 400X magnification. Stain: Hematoxillin and Eosin

Figure 8. Photomicrograph of BoneCeramic and Blood after 7 days of

implantation. Presence of preexisting bone tissue (PB) and biomaterial

particles (BC). It is possible to observe a small area of early formation of

immature bone tissue (NB). . 40X magnification. Stain: Hematoxillin and

Eosin

Figure 9- . Photomicrograph of BoneCeramic and Blood after 7 days of implantation. Presence of biomaterial particle (BC), connective tissue (CT) and presence of preexisting bone tissue (PB).40X

magnification.Stain: Hematoxillin and Eosin

Figure 10.. Photomicrograph of BoneCeramic and Blood after 7 days of implantation. Presence of preexisting bone tissue (PB), connective tissue (CT) rich in inflammatory cells around biomaterial particles (BC). It is possible to observe a small area of early formation of immature bone tissue (NB). . 400X magnification. Stain: Hematoxillin and Eosin

CT

Figure 11. . Photomicrograph of BoneCeramic and Blood after 7 days of

implantation. Presence of biomaterial particle (BC), connective tissue

(CT) and early formation of immature bone tissue (NB). 400X

magnification. . 400X magnification. Stain: Hematoxillin and Eosin

Figure 12. . Photomicrograph of BoneCeramic and Blood after 7 days of implantation. Presence of biomaterial particle (BC), area of connective tissue with inflammatory infiltrate (CT). It is possible to observe a area of preexisting bone tissue (PB) and early formation of immature bone tissue (NB). 400X magnification. Stain: Hematoxillin and Eosin

After 42 days, the alveolar socket of both groups was almost filled by newly formed bone interspersed by connective tissue. Inflammatory cells and multinucleated giant cells were rare and localized to the periphery of the SBC particles, and new bone was present around the particles. At high magnification, it was observed that the trabeculae periphery was surrounded by a large osteoblasts pavement. Both groups of biomaterials presented a significant reduction of biomaterial amounts compared with seven-day period. A considerable amount of newly formed bone was observed surrounding the biomaterials particles

CT

Figure 13. Photomicrograph of BoneCeramic and Saline solution after 42

days of implantation. Presence of neoformed bone tissue (NB) and

conective tissue (CT).40X magnification. Stain: Hematoxillin and Eosin

Figure 14. Photomicrograph of BoneCeramic and Saline solution after 42

days of implantation. Presence of neoformed bone tissue (NB) .40X

magnification. Stain: Hematoxillin and Eosin

Figure 15. Photomicrograph of BoneCeramic and Saline solution after 42

days of implantation. Presence of biomaterial (BC), neoformed bone

tissue (NB). 400X magnification. Stain: Hematoxillin and Eosin

Figure 16: Photomicrograph of BoneCeramic and Saline solution after 42 days of implantation.Presence of neoformed bone tissue (NB) and small espaces of conective tissue (CT). 400X magnification. Stain:

Hematoxillin and Eosin

Figure 17: Photomicrograph of BoneCeramic and Saline solution after 42 days of implantation. Presence of biomaterial (BC), neoformed bone tissue (NB) and preexisting bone tissue (PB). 400X magnification. Stain:

Hematoxillin and Eosin

Figure 18. Photomicrograph of BoneCeramic and Blood after 42 days of

implantation.Presence of neoformed bone tissue (NB) and island of

connective tissue (CT). 40x magnification. Stain: Hematoxillin and Eosin

Figure 19. Photomicrograph of BoneCeramic and Blood after 42 days of

implantation.Presence of neoformed bone tissue (NB) and island of

biometerial particles (BC). 40x magnification. Stain: Hematoxillin and

Eosin

Figure 20. Photomicrograph of BoneCeramic and Blood after 42 days of

implantation.Presence of neoformed bone tissue (NB) and island of

connective tissue (CT). 400x magnification. Stain: Hematoxillin and

Eosin

Figure 21. Photomicrograph of BoneCeramic and Blood after 42 days of implantation. Presence of neoformed bone (NB). 400x magnification.

Stain: Hematoxillin and Eosin

Figure 22. Photomicrograph of BoneCeramic and Blood after 42 days of implantation Area with neoformed bone tissue (NB). 400x magnification.

Stain: Hematoxillin and Eosin

Histomorphometric analysis

Data from histomorphometric evaluation of extraction sites following implantation of biphasic ceramic material are presented in frame 1 and 2.

Frame 1 – Histomorphometric evaluation of extraction sites following implantation of biphasic ceramic material – 7 days (mean ± SD).

Frame 2 – Histomorphometric evaluation of extraction sites following implantation of biphasic ceramic material – 42 days (mean ± SD).

SBC - Blood SBC - Saline 7 days 7 days Native Bone ^9,61 ± ± 7,75 3,77 ± 2,12 Connective

Tissue ^50,59 ± 32,04 76,50 ± 12,01 New Bone _8,22 ± 7,06 9,99 ± 12,47 Residual

Particles ^27,66 ± 34,90 4,04 ± 7,23 Other _3,90 ± 5,82 1,74 ± 7.09

SBC - Blood SBC - saline 42 days 42 days Native Bone 0.0 ± 0.0 2,30 ± 3,52 Connective

Tissue ^40,31 ± 16,81 27,81 ± 9,08 New Bone _52,51 ±13,42 61,35 ± 12,48 Residual

Particles ^7,98 ± 3,56 5,82 ± 7,79 Other _3,12 ± 2,81 2,70 ± 3,71

BV/TV% (p<0.05)

Bone formation was limited 7 days after the extraction procedure and diferences between SBC-B and SBC-S were not significant (P=0.17). Histomorphometric analysis showed an increase of BV/TV% in both groups between 7 and 42 days from surgery (table 1), demonstrating a time dependent increase of BV/TV% throughtout the experimental period. (p<0.05). However, this increase was significantly more robust for the SBC-S group, which exhibited significantly (p<0.001) more new bone after 42 days in comparison with 7 days after extraction. After 42 days SBC-S group exhibited significantly more new bone than SBC-B group (P<0.001). The increased new bone formation in CHA group after 42 days, in comparison with 7 days after extraction, did not reach statistical significance (P= 0.07).

BiomatV/TV%

As expected, both groups exhibited residual particles of grafted material 7 days after surgery. A time-dependent biosorption was observed for both groups as documented by a significant decrease in BiomatV/TV% of SBC-B at 42 days after surgery compared with 7 days (P=0.02). Similar results were found for SBC-S group (P=0.01). No difference was found between the groups for residual BiomatV/TV%

after 42 days of healing (P>0.99).

CT/TV%

Both groups exhibited verying amounts of connective tissue 7 days after surgery, albeit aproximatey 50% more in the SBC-S group (77% versus 51%, respectively). A time-dependent reduction in connective tissue volume was observed for both groups

as documented by a significant decrease in CT/TV% at 42 days after surgery compared with 7 days. Interestingly, despite the higher CT/TV% at 7 days, the SBC- S group exibited significantly less CT volume than SBC-B after 42 days (28% versus 40%, respectively).

DISCUSSION

The present study demonstrated that a biphasic ceramic material may be an adequate biomaterial for bone replacement since it exhibited biocompatibility, biosesorbability and osteoconduction. Data showed that the method of hydration of the biomaterial, saline or blood, may influence osteogenesis in extraction socket defects. Interestingly, contrary to expectations, hydration of the biomaterial with saline solution previously to implantation positively enhanced bone regeneration.

A time dependent increase of bone volume was documented throughout the experimental period. Surprisingly, bone regeneration was significantly enhanced in SBC-S group by 20% (61 versus 52%, respectively) in comparison with SBC-B after 42 days of healing. To the best of our knowledge, no previous study has evaluated the effects of hydration method on the behavior of biomaterials on bone healing and regeneration. Moreover, despite the higher amounts of connective tissue at 7 days, the SBC-S group exibited significantly less CT volume than SBC-B after 42 days (28% versus 40%, respectively). Thus, by simply hydrating the SBC with saline, rather than blood, before grafting, it is possible to significantly improve the regenerative potential of this biomaterial by augmenting the amount of bone regeneration and reducing connective tissue formation, ultimately resulting in denser bone ³⁶ at the treted site.

It is important to notice, however, that blood was used as the hydration method in most of previous clinical studies with SBC in humans ^37,38,39 and, despite the adequate clinical results reported, based on the present findings, it may be anticipated that osteogenesis could have been improved even further. Unfortunately, several studies did not clearly reported the method of hydration used ^40,-45 hampering the possibility to compare the results with other studies. Thus, the present findings also support the notion that future studies must clearly state the method employed for the hydration of the biomaterial in order to better document the experimental procedures and clarify the analytic methods.

It is concluded that SBC is biocompatible, bioresorbable and osteoconductive when grafted in alveolar dental sockets. The hydration of SBC with saline significantly increased new bone formation and reduced connective tissue volume after 42 days demonstrating that hydration method may significantly influence bone healing in such defects, and, thus should be carefully performed. These findings may present clinical relevance and must be carefully taken under consideration by the clinician for the management and grafting of biomaterials.

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Revista de Odontologia da UNESP, p. 119-123, 2014. Disponível em:

<http://www.scielo.br/pdf/rounesp/v43n2/1807-2577-rounesp-43-02-00119.pdf>. Acesso em 30 Out. 2014.