UNESP - Universidade Estadual Paulista “Júlio de Mesquita Filho” Faculdade de Odontologia de Araraquara Amanda de Carvalho Silva Leocádio Osseointegração de implantes com diferentes macro e microestruturas instalados em áreas sem enxertia ou com osso bovino desproteinizado associado ou não à medula óssea fresca: estudo pré-clínico em coelhos Araraquara 2020 UNESP - Universidade Estadual Paulista “Júlio de Mesquita Filho” Faculdade de Odontologia de Araraquara Amanda de Carvalho Silva Leocádio Osseointegração de implantes com diferentes macro e microestruturas instalados em áreas sem enxertia ou com osso bovino desproteinizado associado ou não à medula óssea fresca: estudo pré-clínico em coelhos Tese apresentada ao programa de Pós- Graduação em Odontologia, área de concentração - Implantodontia, da Faculdade de Odontologia de Araraquara, da Universidade Estadual Paulista para obtenção do título de Doutora em Odontologia. Orientador: Elcio Marcantonio Júnior Co-orientador: Guilherme José Pimental de Oliveira Araraquara 2020 Leocádio, Amanda de Carvalho Silva Osseointegração de implantes com diferentes macro e microestruturas instalados em áreas sem enxertia ou com osso bovino desproteinizado associado ou não à medula óssea fresca: estudo pré-clínico em coelhos / Amanda de Carvalho Silva Leocádio.-- Araraquara: [s.n.], 2020 149 f.; 30 cm. Te Tese (Doutorado em Odontologia) – Universidade Estadual Paulista, Faculdade de Odontologia Orientador: Prof. Dr. Elcio Marcantonio Junior Coorientador: Prof. Dr. Guilherme José Pimentel de Oliveira 1. Osseointegração 2. Implantes dentários 3. Transplante ósseo 4. Medula óssea 5. Torque 6. Materiais biocompatíveis I. Título Ficha catalográfica elaborada pela Bibliotecária Marley C. Chiusoli Montagnoli, CRB/5646 Universidade Estadual Paulista (Unesp), Faculdade de Odontologia, Araraquara Diretoria Técnica de Biblioteca e Documentação Amanda de Carvalho Silva Leocádio Osseointegração de implantes com diferentes macro e microestruturas instalados em áreas sem enxertia ou com osso bovino desproteinizado associado ou não à medula óssea fresca: estudo pré-clínico em coelhos Comissão julgadora Tese para obtenção do grau de Doutor Presidente e orientador: Professor Dr. Elcio Marcantonio Júnior 2º Examinador: Professor Dr. Ronaldo Célio Mariano 3º Examinador: Professor Dr. Rafael Silveira Faeda 4º Examinador: Professora Dra. Luana Carla Pires Verzola 5º Examinador: Professora Dra. Pâmela Letícia dos Santos Araraquara, 30 de março de 2020. DADOS CURRICULARES Amanda de Carvalho Silva Leocádio NASCIMENTO: 08 de novembro de 1989 – Poços de Caldas – MG FILIAÇÃO: Moisés Pereira da Silva Maria Betânia de Carvalho Silva Graduação em Odontologia 2008-2013 Faculdade de Odontologia de Alfenas Universidade Federal de Alfenas-MG Pós- Graduação em Ciências Odontológicas – Nível Mestrado 2013-2015 Faculdade de Odontologia de Alfenas Universidade Federal de Alfenas-MG Pós-graduação em Implantodontia – Nível de Especialização 2016-2018 Associação Paulista de Cirurgiões-Dentistas APCD Araraquara-SP Pós-graduação em Implantodontia – Nível de Doutorado 2016-2020 Faculdade de Odontologia de Araraquara Universidade Estadual Paulista - UNESP À Deus, meu Senhor fiel e verdadeiro amigo, dedico primeiramente este trabalho, que por seu amor e cuidado para comigo, me concedeu forças e oportunidade para a sua realização. A minha família amada, em especial, aos meus pais, que estiveram sempre presentes, me apoiando, incentivando aos meus objetivos e muito trabalharam para que eu pudesse alcançá-los. Ao meu esposo, Everton, por toda a sua paciência, amor e compreensão. AGRADECIMENTOS Deus, Agradeço-te por estar no meu caminho e conduzi-lo, pois sem ti nada seria possível. “Lâmpada para os meus pés é tua palavra, e luz para o meu caminho.” (Salmos, cap. 119, v. 105) Família Em especial aos meus pais, Moisés e Betânia, Papai e mamãe, obrigada por todo apoio e amor incondicionais. Obrigada por me ampararem em todos os momentos da minha vida, por me ensinarem a cultivar a paciência, o bem, a honestidade; por se esforçarem exaustivamente para eu ser e ter o melhor em tudo! Aos meus irmãos Moisés Júnior, Márcio e Osmanda e aos meus sobrinhos Isabelle, Nicolas e Heitor Sem vocês, com certeza, grande parte dos meus momentos de alegria não estaria presente. Ao meu sogro Jesiel e a minha sogra Esdra, A vocês todo meu amor e gratidão. “Amor de família é a coisa mais inexplicável do mundo, nem um pai consegue dizer para um filho o quanto o ama, nem o filho sabe dizer ao pai, então simplesmente demonstram.” Esposo Ao Everton, Amor, obrigada por estar sempre ao meu lado, me incentivando a buscar meus sonhos e por compreender minha ausência. “A frase mais sincera que existe, aquela que faz a diferença todos os dias, não importa a hora em que seja dita, não importa o tempo que resta, as dificuldades que venha, ela sempre vencerá obstáculos: EU TE AMO!” Amigos Matusalém Silva Júnior, A sua dedicação e empenho no desenvolvimento deste trabalho foi fundamental para que nós chegássemos até aqui. As longas horas de árduo trabalho durante os procedimentos cirúrgicos se tornaram bem mais leves e divertidas com você e sua esposa, Kelli Silveira, uma doce mulher. Não me deixaram na mão em um só momento. Não mediram distância, deixaram seus filhos e trabalho, mas estavam aqui. Sou muito, mas muito grata mesmo, por tudo que fizeram. Anjos, verdadeiros anjos que Deus mandou na minha vida! Ao querido Profº Dr. Ronaldo Célio Mariano (UNIFAL-MG), O senhor com certeza foi minha fonte de inspiração na graduação para seguir o mestrado e doutorado e também a Implantodontia como especialidade. Um professor extremamente dedicado e exigente, e ao mesmo tempo afetuoso. Tenho muita admiração e respeito pelo senhor. “Talvez eu não tenha tantos amigos. Mas os que eu tenho são os melhores que alguém poderia ter...” Agradecimentos Especiais Ao meu amigo e orientador Prof. Dr. Élcio Marcantonio Júnior: Professor, obrigada pelo privilégio de ser sua orientada e por todas as oportunidades me concedidas. Agradeço a sua amizade e o seu carinho, estando sempre disposto a me ajudar de maneira compreensiva e paciente. Gostaria de ter- lhe retribuído com muito mais dedicação, mas os caminhos sinuosos da vida muitas vezes nos obrigam a caminhar um pouco distantes. Ao meu Co- Orientador Profº Dr. Guilherme José Pimentel Lopes de Oliveira, Gui você é especial, além da paciência e dedicação, você tem o dom de transferir conhecimento. É um grande professor! Você já foi longe e irá ainda mais! Agradeço pela amizade e por toda ajuda para a realização deste trabalho. Sempre acessível e disponível em tudo que precisei. Sempre me tratou com muito carinho e atenção! “Ensinar não é transferir conhecimentos, mas criar as possibilidades para a sua produção ou a sua construção. Quem ensina aprende ao ensinar e quem aprende ensina ao aprender.” Paulo Freire Ao Programa de Pós-graduação em Odontologia da Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP/ARARAQUARA), sob coordenação dos professores Dr. Joni Augusto Cirelli e Dr. Paulo Sergio Cerri e todo o corpo docente, pela formação e exemplo. Ao professor Dr. Rafael Faeda e ao colega Gustavo da Col Santos Pinto agradeço pela paciência, disponibilidade e ajuda na realização das cirurgias deste trabalho. Ao Felipe Pinotti, doutorando em Odontologia pela Faculdade de Odontologia da Unesp (Araraquara) e da Técnica de laboratório Claudinha pela disponibilidade em realizar o processamento histológico dos cortes não descalcificados das peças desta pesquisa. Ao Pedrinho, pela ajuda preciosa e dedicação na confecção dos cortes histológicos descalcificados apresentados neste trabalho. Ao funcionário do biotério de coelhos Celso Luis Borsato. À Luana do laboratório de microCT. A todos os funcionários da Disciplina de Periodontia e aos demais funcionários e colegas do Departamento de Diagnóstico e Cirurgia. Aos funcionários da Seção de Pós-Graduação pela paciência e admirável interesse em nos ajudar. À NEODENT, pela concessão dos materiais utilizados nesta pesquisa (Curitiba, Paraná, Brasil / PAP 0107.16 e 2508.17). A todos que, direta ou indiretamente, contribuíram para a elaboração deste trabalho, minha mais sincera gratidão. “O primeiro dever da inteligência é desconfiar dela mesma.” Albert Einstein “Poderia ser pior!” Per-Ingvar Brånemark Leocádio ACS. Osseointegração de implantes com diferentes macro e microestruturas instalados em áreas sem enxertia ou com osso bovino desproteinizado associado ou não à medula óssea fresca – Estudo pré-clínico em coelhos. [Tese de doutorado]. Araraquara: Faculdade de Odontologia da UNESP; 2020. RESUMO Implantes com diferentes macro (CI-Implante Cilíndrico e HCI-Implante Cônico Híbrido) e microestruturas (NP-Jateamento+ataque ácido e AQ-Jateamento+ataque ácido+imersão em solução isotônica de cloreto de sódio 0,9 %) foram testados em áreas de osso nativo ou enxertadas prévia ou imediatamente com osso bovino desproteinizado associado ou não à medula óssea fresca (DBB e DBB/BM). Na primeira hipótese foi testado a estabilidade primária e o processo de osseointegração em implantes com diferentes macroestruturas (CI vs. HCI) na metáfise tibial de coelhos. 24 coelhos foram divididos em 3 períodos (2, 4 e 8 semanas). Cada animal recebeu bilateralmente 2 implantes de cada grupo. Todos os implantes foram avaliados quanto ao torque de inserção. Um dos implantes foi submetido ao torque de remoção e análise histológica e o outro foi utilizado para análise microtomográfica e histométrica (%BIC-Contato Osso-Implante). Os HCI apresentaram maior torque de inserção (32.93±10.61 Ncm vs. 27.99± 7.80Ncm) e maior %BIC no período de 8 semanas (79.08±11.31% vs. 59.72±11.29%) que CI. CI apresentaram maiores valores de torque de remoção que HCI no período de 8 semanas (91.05 ± 9.32 Ncm vs. 68.62 ± 13.70 Ncm). Não houve diferenças em relação aos dados microtomográficos. Na segunda e na terceira hipóteses, foi avaliado a influência de diferentes macros (CI vs. HCI) e microestruturas de implantes (NP vs. AQ) no processo de osseointegração em áreas previamente (metáfise tibial e seio maxilar) ou imediatamente (metáfise tibial) enxertadas (DBB vs. DBB/BM).16 coelhos foram avaliados em um período de 180 dias. Cada animal foi submetido à criação de um defeito ósseo bilateral na metáfise tibial e preenchidos com DBB ou DBB/BM. Foi executado acesso bilateral à membrana do seio maxilar e enxerto. Após 90 dias, os defeitos da metáfise tibial foram trefinados e submetidos a instalação de implantes (CI vs. HCI). No seio maxilar implantes foram instalados (NP vs. AQ). Um segundo defeito foi confeccionado na metáfise tibial, preenchido e seguido da instalação imediata de implantes (CI vs. HCI). 90 dias após os animais foram eutanasiados. Foi verificado nas biópsias ósseas (DBB vs. DBB /BM) que não houve diferenças estatísticas quanto à porcentagem de tecidos mineralizados (75.42±10.11 vs. 77.42±9.57) e em relação a porcentagem de tecido ósseo (23.08±9.95 vs. 27.37±5.83), biomaterial (20.81±11.34 vs. 26.40±7.16) e tecido conjuntivo (51.81±10.53 vs. 50.52±14.31). BV/TV% dos implantes com diferentes macroestruturas, diferença estatística foi verificada no DBB independente do momento de instalação dos implantes (Imediato vs. Tardio) nos HCI (83.34±5.87 e 85.69±7.91, respectivamente) comparado aos CI (DBB-CI: 70.39±15.18 e 77.60±8.31, respectivamente). BIC%, diferença estatística foi verificada nos CI imediatos comparado ao tardio em áreas enxertadas com DBB somente. Já nas diferentes microestruturas foi verificado que não houve diferenças na quantidade no BV/TV% (DBB-NP:33.25±19.67 ; DBB-AQ:35.15±22.17; DBB/BM-NP:39.71±24.21; DBB/BM- AQ: 36.40±23.07) e na BIC% (DBB-NP:58.94±24.37; DBB-AQ:52.52±24.36; DBB/BM- NP: 61.66±14.60; DBB/BM-AQ:64.06±23.30). HCI apresentaram maior estabilidade primária e melhor padrão de osseointegração que os CI ao final de 8 semanas de avaliação em osso nativo tipo I e a adição de BM e o tipo de macro ou microestrutura não influenciaram na osseointegração dos implantes instalados em áreas com DBB. Palavras chave: Osseointegração. Implantes dentários. Transplante ósseo. Medula óssea. Torque. Materiais biocompatíveis. Leocádio ACS. Osseointegration of implants with different macro and microstructures installed in areas without grafting or with deproteinized bovine bone associated or not with fresh bone marrow - Pre-clinical study in rabbits. [Tese de doutorado]. Araraquara: Faculdade de Odontologia da UNESP; 2020. ABSTRACT Implants with different macro (CI-Cylindrical Implant and HCI-Hybrid Conical Implant) and microstructures (NP-Sandblasting + acid attack and AQ-Sandblasting + acid attack + immersion in 0.9% sodium chloride isotonic solution) were tested in areas of native bone or grafted previously or immediately with deproteinized bovine bone associated or not with fresh bone marrow (DBB and DBB / BM). In the first hypothesis, primary stability and the osseointegration process in implants with different macrostructures (CI vs. HCI) in the tibial metaphysis of rabbits were tested. 24 rabbits were divided into 3 periods (2, 4 and 8 weeks). Each animal received two implants bilaterally from each group. All implants were evaluated for insertion torque. One of the implants was submitted to removal torque and histological analysis and the other was used for microtomographic and histometric analysis (% BIC-Bone Contact-Implant). The HCI showed a higher insertion torque (32.93 ± 10.61 Ncm vs. 27.99 ± 7.80Ncm) and a higher BIC% in the 8-week period (79.08 ± 11.31% vs. 59.72 ± 11.29%) than CI. CI showed higher removal torque values than HCI in the 8 week period (91.05 ± 9.32 Ncm vs. 68.62 ± 13.70 Ncm). There were no differences in relation to microtomographic data. In the second and third hypotheses, the influence of different macros (CI vs. HCI) and implant microstructures (NP vs. AQ) in the process of osseointegration in areas previously (tibial metaphysis and maxillary sinus) or immediately (tibial metaphysis) was evaluated grafted (DBB vs. DBB / BM) .16 rabbits were evaluated over a period of 180 days. Each animal was submitted to the creation of a bilateral bone defect in the tibial metaphysis and filled with DBB or DBB / BM. Bilateral access to the maxillary sinus membrane and graft was performed. After 90 days, defects in the tibial metaphysis were redefined and implanted (CI vs. HCI). Implants were installed in the maxillary sinus (PN vs. AQ). A second defect was made in the tibial metaphysis, filled in and followed by the immediate installation of implants (CI vs. HCI). 90 days after the animals were euthanized. It was verified in bone biopsies (DBB vs. DBB / BM) that there were no statistical differences regarding the percentage of mineralized tissues (75.42 ± 10.11 vs. 77.42 ± 9.57) and in relation to the percentage of bone tissue (23.08 ± 9.95 vs. 27.37 ± 5.83), biomaterial (20.81 ± 11.34 vs. 26.40 ± 7.16) and connective tissue (51.81 ± 10.53 vs. 50.52 ± 14.31). BV / TV% of implants with different macrostructures, statistical difference was verified in DBB regardless of the moment of implant implantation (Immediate vs. Late) in HCI (83.34 ± 5.87 and 85.69 ± 7.91, respectively) compared to CI (DBB-CI: 70.39 ± 15.18 and 77.60 ± 8.31, respectively). BIC%, statistical difference was found in the immediate IC compared to the late IC in areas grafted with DBB only. In the different microstructures, it was verified that there were no differences in the quantity in BV / TV% (DBB-NP: 33.25 ± 19.67; DBB-AQ: 35.15 ± 22.17; DBB / BM-NP: 39.71 ± 24.21; DBB / BM-AQ: 36.40 ± 23.07) and in BIC% (DBB-NP: 58.94 ± 24.37; DBB-AQ: 52.52 ± 24.36; DBB / BM-NP: 61.66 ± 14.60; DBB / BM-AQ: 64.06 ± 23.30). HCI showed greater primary stability and better pattern of osseointegration than CIs after 8 weeks of evaluation in native bone type I and the addition of BM and the type of macro or microstructure did not influence the osseointegration of implants installed in areas with DBB. Keywords: Osseointegration. Dental implants. Bone transplantation. Bone marrow. Torque. Biocompatible materials. Lista de Abreviaturas Abreviaturas usadas no texto: AQ: Superfície Acqua® %BIC: Contato osso implante expressa em porcentagem BM: Medula Óssea %BV/TV: Relação entre volume ósseo e volume total da amostra expressa em porcentagem CEUA: Comitê de Ética no uso de animais CI: Implante Cilíndrico COBEA: Colégio Brasileiro de Experimentação Animal DBB: Osso bovino desproteinizado HA: Hidroxiapatita HCI: Implante Cônico Híbrido HE: Hematoxilina Eosina NP: Superfície Neoporos® ROI: Região de interess SUMÁRIO 1 INTRODUÇÃO ................................................................................ 16 2 PROPOSIÇÃO ................................................................................. 21 3 Publicações .................................................................................... 22 3.1 Publicação 1 ................................................................................ 22 3.2 Publicação 2 ................................................................................ 37 3.3 Publicação 3 ................................................................................ 72 4 DISCUSSÃO .................................................................................... 101 5 CONCLUSÃO ................................................................................. 110 REFERÊNCIAS ............................................................................. 111 APÊNDICE ..................................................................................... 119 ANEXO A ...................................................................................... 148 ANEXO B ....................................................................................... 149 16 1 INTRODUÇÃO A Odontologia, na era da Implantodontia, tem possibilitado a utilização de implantes dentais osseointegráveis no tratamento do edentulismo 1,2. As reabilitações orais tradicionais, com próteses fixas ou removíveis, apoiadas sobre dentes e/ou mucosa, deixaram de ser o tratamento de escolha dos pacientes totais ou parcialmente edêntulos, uma vez que, esses pacientes tomaram conhecimento e vivenciam a possibilidade de reabilitação do sistema estomatognático com próteses implanto-suportadas, as quais, hoje, representam o tratamento que mais se aproxima da dentição natural 3. Apesar das altas taxas de sobrevivência clínica dos sistemas de implantes dentais contemporâneos, falhas ainda ocorrem, podendo estar associadas a diversos fatores mecânicos ou biológicos 4. A falta de formação óssea adequada ou volume ósseo de apoio para facilitar a osseointegração têm sido relatados como principais influências sobre a previsibilidade e falha do implante 5,6. As taxas de sobrevivência também variam com o local do implante na cavidade bucal e de outros fatores, tais como design do implante, biocompatibilidade, carregamento, densidade óssea, técnica cirúrgica e outros 6, 7. Portanto, uma série de modificações no projeto de implante e técnicas cirúrgicas tem sido introduzida para melhorar o contato osso-implante, ancoragem e distribuição do estresse 5, 8, 9, 10. Muitas dessas mudanças levaram a uma bem- sucedida terapia com implantes, mesmo nas mais difíceis situações clínicas. Devido a isso, a busca por protocolos que acelerem o processo de osseointegração, e consequentemente o carregamento protético, tem sido o foco de várias pesquisas na área da Implantodontia 1, 2. 17 Quanto às modificações estruturais dos implantes, elas podem ser executadas ao nível da macro ou da microestrutura 11, 12. Essas modificações alteram estágios distintos na estabilidade dos implantes, sendo que alterações na macroestrutura têm sido relacionadas com a obtenção da estabilidade primária dos implantes enquanto que as alterações de microestrutura afetariam o processo de osseointegração, e, consequentemente, a estabilidade secundária dos implantes 13, 14, 15,16. Neste contexto, a macroestrutura exerce fundamental importância na obtenção da estabilidade primária dos implantes em áreas com osso de pobre qualidade, como na região posterior da maxila, ou em condições de pós extrações com instalação imediata do implante e possível carregamento em áreas estéticas17. Estudos com implantes cônicos e com implantes com roscas com ausência de bordas cortantes e com câmeras de cicatrização tem demonstrado não apenas melhorar a estabilidade primária, como também acelerar o processo de osseointegração18, 19. Com relação às modificações de microestrutura, têm sido verificado que diversas alterações nas superfícies dos implantes que visam modificar suas propriedades físico-químicas tais como a rugosidade de superfícies, a molhabilidade e a sua composição química demonstram serem capazes de acelerar e melhorar a qualidade da osseointegração, resultando em maior deposição óssea e redução do período de reparo, principalmente em regiões de má qualidade óssea 20, 21, 22. Mas, apesar de um dos mecanismos mais fascinantes no organismo ser a alta capacidade do tecido ósseo se regenerar, a doença periodontal, tumores, traumas, exodontias, anomalias de desenvolvimento, patologias, ressecções oncológicas e perda fisiológica de massa óssea, podem levar a defeitos ósseos perenes, os quais não possuem a capacidade de se regenerar espontaneamente. 18 Especialmente na região bucomaxilofacial, a reabsorção severa de mandíbula e maxila, associada à perda dos dentes, pode levar a defeitos anatômicos significativos comprometendo a função e a estética e, em muitos casos inviabilizando a reabilitação oral com implantes 23. E, uma vez que as cirurgias ósseas reconstrutivas, antes da reabilitação oral com próteses implanto-suportadas, são muitas vezes necessárias para evitar ou até mesmo recuperar a perda óssea vertical e horizontal e, principalmente, obter uma quantidade e qualidade ósseas adequadas, garantindo a estabilidade primária de inserção do implante, tornou-se constante a busca por novos biomateriais que possam modular, alterar ou estimular a atividade osteogênica nos defeitos teciduais24, 25. Atualmente, como possíveis soluções utilizam-se enxertos autógenos, alógenos, aloplásticos, xenogênicos e fatores de crescimento. Como o recrutamento de células, a modulação do processo inflamatório e a promoção do reparo, por cicatrização ou regeneração, são influenciados pelas suas características físicas, químicas e biológicas 26, cada material apresenta vantagens e limitações, justificando o grande número de estudos realizados para verificar o efeito de novos biomateriais, associados ou não, à outros biomateriais utilizados rotineiramente. Dentre os biomateriais alternativos a utilização dos enxertos autógenos, podemos citar os enxertos homólogos, xenógenos, ou aloplásticos sendo que cada material apresenta vantagens e limitações27, 28, 29, 30, 31. Os xenoenxertos têm ganhado destaque e o mais utilizado e pesquisado até o momento tem sido o osso bovino desproteinizado. Esse tipo de enxerto tem sido utilizado com sucesso em situações clínicas diversas tais como em elevação do assoalho do seio maxilar 32, 33 e manutenção de alvéolo pós-extração 34. Mas, assim como os demais, esses substitutos ósseos apresentam como único mecanismo de ação a osteocontutividade 19 35, 36, 37 o que causa um retardo na cicatrização dos defeitos ósseos, que pode ter como consequência o retardo na instalação dos implantes 38. Considerando que a presença de um pequeno subconjunto de células- tronco, células progenitoras e osteoblastos são transferidos juntamente com o enxerto autógeno, em grandes defeitos ósseos sua presença torna a reconstrução mais previsível, mesmo sendo o nível de celularidade baixo 39. A adição de células ou fatores de crescimento a um biomaterial essencialmente ostecondutor poderia resultar em um biomaterial potencialmente comparável ao enxerto autógeno40, 41, 42. Neste contexto, a associação do osso bovino desproteinizado com o osso autógeno e fatores de crescimento tais como o plasma rico em plaquetas41 e a rhBMP242 foram testados com intuito de melhorar a formação óssea. Outra alternativa que tem sido investigada é a utilização da medula óssea fresca associado a biomateriais osteocondutores, pois teoricamente, as células tronco e os fatores de crescimento presentes nesse adjuvante biológico, quando associadas a um arcabouço, podem adicionar ao mesmo um potencial de osteogênese e osteoindução para formação óssea 39, 43, 44, 45. Não há um consenso sobre a melhor metodologia para o uso de células estromais da medula óssea. Três alternativas principais têm sido propostas: (1) Uso do enxerto de medula óssea autóloga fresca ("in natura"); (2) Uso de concentrado de células da medula óssea autóloga (por centrifugação); e, (3) Uso de cultivo de células estromais da medula óssea autóloga 39, 40, 41, 42. Algumas desvantagens foram associadas com a utilização da metodologia de cultura celular: custo, tempo (requer um período de algumas semanas entre a coleta de células, obtenção da cultura e transplante), maior risco de contaminação, e a aplicabilidade clínica rotineira da cultura celular pode esbarrar na necessidade de 20 dois procedimentos cirúrgicos separados. Outro questionamento sobre a necessidade ou não de se cultivar as células é a falta de informação sobre o número mínimo de células requeridas para promoção do reparo ósseo 39, 46, 47, 49. Dessa forma, a utilização do enxerto de medula de forma fresca seria uma abordagem mais simples do que outros que já foram propostos47. O efeito de áreas enxertadas com biomateriais osteocondutores associados ao aspirado de medula óssea sobre a osseointegração dos implantes foi pouco avaliado até o momento. Portanto, o objetivo desse estudo foi verificar a influência de um implante experimental com macroestrutura compactante perfurante (HCI - Implante Cônico Híbrido) comparada a um implante controle (CI - Implante Cilíndrico) no processo de osseointegração em osso nativo (Osso tipo I) (Publicação 1) ou em defeitos cirúrgicos prévio e imediatamente enxertados com osso bovino desproteinizado isolado (DBB) ou associado com medula óssea fresca (DBB/BM) na metáfise tibial de coelhos; e avaliar a neoformação óssea das biópsias colhidas da região previamente enxertada (Publicação 2). Além disso, avaliar o efeito da uma superfície de implante altamente hidrofílica (AQ - Implante Acqua: jateamento de óxido, a subtração ácida e manutenção do implante em solução isotônica de cloreto de sódio 0,9 %) comparada a uma hidrofóbica (NP - Implante NeoPoros: jateamento de óxido e subtração ácida) sobre a osseointegração em seios maxilares de coelhos enxertados com DBB vs. DBB/BM (Publicação 3). 21 2 PROPOSIÇÃO Hipóteses A) A macroestrutura compactante perfurante dos implantes Cônicos Híbridos (HCI) favorece o processo de osseointegração comparado a um implante cilíndrico (CI) em osso nativo, prévio ou imediatamente enxertado com osso bovino desproteinizado associado ou não à medula óssea fresca (DBB vs. DBB/BM) na metáfise tibial de coelhos. B) A superfície hidrofílica (AQ) favorece o processo de osseointegração comparado a uma superfície hidrofóbica (NP) de implantes instalados na região do seio maxilar previamente enxertado (DBB vs. DBB/BM). C) O enxerto xenogênico de origem bovina, constituído de hidroxiapatita pura (HA) e sem componentes orgânicos, sinterizado à altas temperaturas (DBB), apresenta neoformação óssea e osseointegração melhorados quando associado à medula óssea fresca (BM). Objetivos específicos Para avaliar a hipótese esse projeto foi dividido nos seguintes objetivos específicos: 1) Avaliar o efeito da modificação da macroestrutura de implantes (HCI vs. CI) com superfície hidrofílica modificada por jateamento e ataque ácido sobre a osseointegração em osso tipo I (metáfise tibial) de coelhos (Publicação 1). 2) Avaliar o efeito da modificação da macroestrutura de implantes (HCI vs. CI) com superfície hidrofílica modificada por jateamento e ataque ácido sobre a osseointegração em defeitos ósseos criados na metáfise tibial prévio ou imediatamente enxertados com osso bovino desproteinizado associado ou não à medula óssea fresca (DBB vs. DBB/BM). Além de comparar a neoformação óssea e osseointegração do enxerto xenogênico, constituído de hidroxiapatita pura (HA) e sem componentes orgânicos nos diferentes grupos (DBB vs. DBB/BM) (Publicação 2). 3) Avaliar o efeito de uma superfície hidrofílica e modificada por ataque ácido e por jateamento (AQ) sobre a osseointegração em comparação a uma superfície hidrofóbica com o mesmo tipo de tratamento para modificação de superfície (NP) instalados na região do seio maxilar previamente submetido ao levantamento do assoalho e enxertia (DBB vs. DBB/BM) (Publicação 3). 22 3 PUBLICAÇÕES 3.1 Publicação 1 Original Reseach: Evaluation of implants with different macrostructures in type I bone - Pre-clinical study in rabbits Amanda de Carvalho Silva Leocádio (Leocádio ACS) - PhD in Implantology1 Matusalém Silva Júnior (Silva Jr. M) - Master in Implantology2 Guilherme José Pimentel Lopes de Oliveira (Oliveira GJPL) - Professor in Periodontology3 Gustavo da Col Santos Pinto (Pinto GCS) - PhD in Implantology1 Rafael Silveira Faeda (Faeda RS) - Professor in Periodontology4 Luis Eduardo Marques Padovan (Padovan LEM) - Professor in Implantology2 Élcio Marcantonio Júnior (Marcantonio Jr E) - Professor in Periodontology1 1 Department of Diagnosis and Surgery, School of Dentistry at Araraquara, Univ. Est. Paulista / UNESP, Araraquara, Brazil 2 Post Graduation Course in Implantology, Instituto Latino-americano de pesquisa odontológica (ILAPEO), Curitiba, Brazil. 3 Department of Periodontology, School of Dentistry at Uberlândia, Federal University of Uberlândia, Uberlândia, Brazil 4 Post Graduation Course in Odontology, Universidade de Araraquara/ UNIARA, Araraquara, Brazil Corresponding author: Élcio Marcantonio Júnior Humaitá St., 1680. Zip code: 14801-130, Araraquara, Brazil. Phone: +55 (16) 33016378 e-mail: junior.elcio@gmail.com Conflicts of Interest: Professor Doctor Élcio Marcantonio Junior is a consultant for the company Neodent. The other authors have no conflict of interest in this study. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. * Artigo publicado no periódico Materials 23 Article Evaluation of Implants with Different Macrostructures in Type I Bone—Pre-Clinical Study in Rabbits Amanda de Carvalho Silva Leocádio 1,*, Matusalém Silva Júnior 2, Guilherme José Pimentel Lopes de Oliveira 3, Gustavo da Col Santos Pinto 1, Rafael Silveira Faeda 4, Luis Eduardo Marques Padovan 2 and Élcio Marcantonio Júnior 1,* 1 Department of Diagnosis and Surgery, School of Dentistry at Araraquara, Sao Paulo State University (UNESP), Araraquara 14801-385, Brazil; gustavo.dcsp@gmail.com 2 Post Graduation Course in Implantology, Latin American Institute of Dental Research (ILAPEO), Curitiba 80710-150, Brazil; jr@matusaodontologia.com (M.S.J.); padovan@iocp.com.br (L.E.M.P.) 3 Department of Periodontology, School of Dentistry at Uberlândia, Federal University of Uberlândia, Uberlândia 38408-160, Brazil; guioliveiraodonto@hotmail.com 4 Post Graduation Course in Odontology, University of Araraquara/UNIARA, Araraquara 14801-320, Brazil; rafaelfaeda@gmail.com * Correspondence: a.carvalhos@hotmail.com (A.d.C.S.L.); elcio.marcantonio@unesp.br (E.M.J.); Tel.: +55-(35)- 99138-5571 (A.d.C.S.L.); +55-(16)-33016378 (E.M.J.) Received: 23 February 2020; Accepted: 22 March 2020; Published: 26 March 2020 Abstract: The objective of this study was to assess the primary stability and the osseointegration process in implants with different macrostructures (Cylindrical vs. Hybrid Conical) in rabbit tibiae. Twenty-four (24) rabbits were used, divided into 3 experimental periods (2, 4 and 8 weeks) with 8 animals each. Each animal bilaterally received 2 implants from each group in the tibial metaphysis: Cylindrical Implant (CI) and Hybrid Conical Implant (HCI). All implants were assessed for insertion torque. After the experimental periods, one of the implants in each group was submitted to the removal counter-torque test and descriptive histological analysis while the other implant was used for microtomographic and histometric analysis (%Bone-Implant Contact). HCI implants showed higher insertion torque (32.93 ± 10.61 Ncm vs. 27.99 ± 7.80 Ncm) and higher % of bone-implant contact in the 8-week period (79.08 ± 11.31% vs. 59.72 ± 11.29%) than CI implants. However, CI implants showed higher values of removal counter-torque than HCI implants in the 8-week period (91.05 ± 9.32 Ncm vs. 68.62 ± 13.70 Ncm). There were no differences between groups regarding microtomographic data. It can be concluded that HCI implants showed greater insertion torque and bone- implant contact in relation to CI implants in the period of 8 weeks when installed in cortical bone of rabbits. Keywords: dental implants; macrostructure; osseointegration 1 . Introduction The use of osseointegrated dental implants in the treatment of partial or total edentulism has been a widely used procedure in recent years [1,2]. However, despite the high rates of the clinical survival of contemporary dental implant systems, failures still occur, which may be associated with several mechanical or biological factors [3]. The lack of adequate bone formation or bone support volume to facilitate osseointegration has been reported as the main influence on the predictability and failure of the implant [4,5]. Survival rates also vary with the location of the implant in the oral cavity and other factors, such as implant design, biocompatibility, loading, bone density, surgical technique, and others [5,6]. Materials 2020, 13, 1521; doi:10.3390/ma13071521 www.mdpi.com/journal/materials 24 In this context, a series of changes in the implant design and surgical techniques have been introduced to improve bone-implant contact, anchorage and stress distribution [7–9]. Many of these changes have led to successful implant therapy, even in the most difficult clinical situations. Because of this, the search for protocols that accelerate the osseointegration process, and consequently the prosthetic loading, has been the focus of several research papers in the area of Implantology [1,2]. Therefore, these structural modifications of the implants can be performed at the nano- or microstructure level [10–12], and they would alter different stages in the stability of the implants, as changes in the macrostructure have been related to the achievement of the primary stability of the implants, while the changes in microstructure would affect the osseointegration process and, consequently, the secondary stability [11–14]. Considering that changes in the implant macrostructure have been reported as important for obtaining primary stability of implants in areas with poor quality bone, such as in the posterior region of the maxilla [15], studies with conical implants and threaded implants with no sharp edges and with healing chambers have been shown not only to improve primary stability but also to accelerate the osseointegration process [11]. In this context, the emergence of implants with a conical structure and with changes in the conformation of the threads, in order to make them more compressive, allows the installation of implants with good primary stability [16–18], as well as the use of implants with smaller sizes, since they can be used in areas with limited bone availability [19]. Consequently, that the macrostructure is an extremely important characteristic for obtaining primary stability, a parameter related to the success of the implant osseointegration, changes in the implant macrostructure have been proposed in order to improve the initial locking of the implants. Thus, the objective of this study was to verify the influence of an experimental implant with perforating compacting macrostructure (HCI—Hybrid Conical Implant: Helix Acqua, Neodent®, Grand Morse, Curitiba, Brazil) compared to a control implant (CI—Cylindrical Implant: Titamax Acqua, Neodent®, Cone Morse, Curitiba, Brazil) in the process of osseointegration in cortical bone (tibial metaphysis) of rabbits. 2. Materials and Methods This project was carried out in accordance with the Ethical Principles for Animal Experimentation after approval by the Ethics Committee on Animal Use (ECAU) of the Faculty of Dentistry of Araraquara (FOAr-UNESP) (11/2016). For the present research, 24 male New Zealand albino rabbits (~5 months old and 4–5 kg) were used. The animals were kept in an environment with a temperature of 22–24 °C, with a controlled light cycle (12 h light and 12 h dark) and consumption of solid food and water ad libitum throughout the experimental period. The study was conducted according to the ARRIVE protocol. 25 2.1. Experimental Outline The 24 rabbits were randomly divided into 3 experimental periods (2, 4 and 8 weeks). Each animal bilaterally received two implants from each group in the cortical bone of the animals’ tibia, with the side selected at random. Two different macrostructures were assessed: Hybrid Conical Implant (HCI) and Cylindrical Implant (CI). The CI implant used in this study is characterized by a cervical diameter equal to the diameter of the implant body (3.75 mm in diameter × 11 mm in height). Presence of triangular and double threads that facilitate the quick implant insertion, with minimal trauma, and apex morphology with the presence of self-cutting chambers (Figure 1A,C,E). The HCI implant is characterized by an increased cervical diameter in relation to the implant body (3.75 mm in diameter × 11.5 mm in height). In addition, these implants have compacting and double trapezoidal threads, with a conical apex containing helical chambers designed to optimize secondary stability (Figure 1B,D,F). Figure 1. Macrostructure of implants installed in the cortical bone of rabbits. Body (A), threads (C) and apex (E) of the Cylindrical Implant; Body (B), threads (D) and apex (F) of the Hybrid Conical Implant. 2.2. Surgical Procedure The animals were initially weighed and anesthetized intramuscularly, with a combination of ketamine (Quetamina Agener®, Agener União Ltd.a, São Paulo, SP, Brazil—0.35 mg/kg) and xylazine (Rompum, Bayer AS, São Paulo, SP, Brazil—0.5 mg/kg). Subsequently, trichotomy and antisepsis were performed with 10% iodinated polyvinylpyrrolidone (IPVP) with 1% active iodine in the right and left portions of the tibial metaphysis of the animal (Figure 2A). Local anesthesia (Mepivacaine Hydrochloride 2% + Adrenaline 1:100,000) was also applied in the region, to allow peripheral vasoconstriction, reducing local bleeding and optimizing the surgical procedure (Figure 2B). Then, using a No. 15 scalpel blade, a dermo-periosteal incision of approximately 5 cm in length was performed (Figure 2C,D). This allowed for a 26 delicate dissection so that the bone surface of the tibial metaphysis was exposed, and the surgical beds were prepared according to the manufacturers’ recommendations (Neodent®—Curitiba, Brazil), using metal drills under abundant refrigeration with sterile saline solution (Figure 2E,F). Two implants of each type were installed in the tibiae, and the side was selected at random. The upper implants had a distance of 3 cm in relation to the lower ones (Figure 2G–I). Both implants had the same type of surface (Sandblasting + acid attack + immersion in 0.9% sodium chloride isotonic solution) and were manufacture with the same titanium alloy (Ti-6Al4V). The cover screws were installed (Figure 2J) and then the soft tissues were repositioned and sutured plane by plane with resorbable thread (Vicryl®, ETHICON, Sao Paulo, Brazil) and nonresorbable thread (Shalon®-Nylon 3-0, Shalon surgical wires Ltda., Goias, Brazil) (Figure 2K,L). After surgery, all animals received a single intramuscular dose of antibiotic (Pentabiótico®, WyethWhitehall Ltd.a, São Paulo, Brazil—0.1 mL/kg) and analgesic (Tramadol Hydrochloride 50 mg/mL, Tramadol®, Medley, São Paulo, Brazil—5 mg/kg IM). After the periods of 2, 4 and 8 weeks, the animals were euthanized through anesthetic overdose. Figure 2. Surgical procedure for installing implants in the tibial metaphysis of rabbits. Schematic drawing to define the tibial metaphysis and local anesthesia (A); Incision in layers (B–D); Detachment and exposure of the tibia, and perforation according to the manufacturer’s recommendations under abundant sterile saline irrigation (E,F); Installation of the implants (G,H); Implants and cover screws installed (I,J); Internal suture of the muscular fascia with resorbable thread and external suture with non-resorbable thread (K,L). 2.3. Biomechanical Assessment (Insertion Torque and Removal Torque) All installed implants were submitted to the assessment of the insertion torque, and for that, the torque was noted after the installation of the implants (at the bone level). After euthanasia, in each analysis period (2, 4 and 8 weeks), the medial portions of the samples obtained from the tibia were reopened to expose the implants and perform the reverse torque. The samples were stabilized in a small vise, and a hexagonal wrench was connected to both the implant and the torque wrench 27 (Tohnichi, model ATG24CN-S, Tokyo, Japan-with a graduated scale of 0.05 Ncm, measuring the strength from 3 to 24 Ncm), and an anti-clockwise movement was performed to remove the implants, increasing the torque until the rotation of the implant occurred inside the bone tissue, completely disrupting the bone-implant interface, when the torque wrench registered the maximum torque peak necessary for this disruption. This maximum peak required to move the implant was noted as the removal torque value. The remnants of the tibia related to the upper implants that were previously removed for the removal torque analysis were then reduced and immersed in 10% formaldehyde for 48 h, washed in running water for 4 h and then decalcified in Ethylenediaminetetraacetic Acid (E.D.T.A. 7%) for two months. 2.4. Descriptive Histology of Decalcified Sections After the decalcification period, the parts from the sites where the implants were removed for analysis of the removal torque were embedded in paraffin, cut into a microtome (6 µm-thick) and stained using the hematoxylin-eosin (HE) technique. Five slides were obtained with 3 sections each in the central region of the site where the implant was inserted. Three sections were assessed that were 36 µm apart, and the first section for assessment was selected randomly. The sections were assessed by means of a DIASTAR optical microscope (Leica Reichert & Jung products, Wetzlar, Germany) with 5× and 10× magnifications, and the quality of bone tissue in the vicinity of the implant bed was assessed. This analysis was performed by an experienced trained examiner, blind for the type of implant used. 2.5. Microtomographic Analysis (µCT) The parts from the sites where the implants were kept (lower tibial implants), after the fixation process with 4% formaldehyde for 48 h and washing with running water for 4 h, were dehydrated in an alcohol solution. Subsequently, they were subjected to the scanning process through the µCT and the following parameters were used: the size of the pixel image was 2000 × 1336 (18 µm), the thickness of the sections was 12 µm, the magnification of the image was 10×, the voltage of the X-ray tube was 50 kV, the beam was 496 µA, and the electrical current was adjusted to 0.1 mA. The three- dimensional images were reconstructed using a reconstruction software (NRecon 1.6.1.5, SkyScan N. V., Belgium). The parameters for reconstruction were: Beam Hardening Correction = 4%, Ring Artifact Correction = 3, Smoothing = 1, Postalignment = 1.00. Subsequently, the scanned images were reoriented in three planes (coronal, axial and sagittal) to standardize the position before performing the volumetric analysis. The measurements for the Volumetric analysis (3D) were carried out using specific software (CT Analyzer 1.10.1.0, SkyScan N. V., Belgium), following the selection of an area of interest (ROI— region of interest) in a cylindrical shape that circumscribed in 0.5 cm the diameter of the implants. Although the implants received a cover screw, there was bone formation inside the cover screw in some cases. In order to prevent this bone formation from interfering with the analysis of the volume of 28 mineralized tissue around the implant, a second ROI was established to remove the volume of mineralized tissues that could have been formed in this region. With the results obtained in the two ROI’s, it was possible to define the volume of mineralized tissues using the following formula: Total bone volume of mineralized tissues in the main ROI—Volume of mineralized tissues within the cover screw = Volume of mineralized tissues (BV/TV). The grayscale threshold used was 25–90, and the values of the volume of mineralized tissue around the implant were obtained as a percentage. The entire analysis was performed by a single trained examiner, who was blind for the type of training performed [20]. 2.6. Histometric Analysis (BIC) The biopsies with the lower implants that were submitted to the fixation process and microtomographic analysis were subsequently dehydrated in an alcohol solution in a series of increasing concentrations. The plastic infiltration was performed with mixtures of glycolmethacrylate (Technovit 7200 VLC) and ethyl alcohol, following gradual variations, ending with two infiltrations of pure glycol methacrylate. After plastic infiltration, the specimens were embedded in resin and polymerized. Therefore, the specimens were sectioned longitudinally, along the main axis of the implant, using a high precision diamond disk. The blocks were mounted on an acrylic slide with the help of Tecnovit 4000 resin (Kulzer, Wehrheim, Germany). Using a cutting and micro-wear system (Exact-Cutting, System, Apparatebau Gmbh, Hamburg, Germany), the blades were processed with a section of approximately 50– 70 µm thick. The pieces were stained with Stevenel’s Blue for histomorphometric analysis. Histometry assessed the percentage of mineralized bone in direct contact with the implant surface (BIC—bone to implant contact) in the extension of the cervical third of the implant. The measurements were made using a DIASTAR optical microscope (Leica Reichert & Jung products, Wetzlar, Germany), with a 10-fold magnification objective lens, through which the images were captured and sent to a PC, with the aid of a video camera (Leica Reichert & Jung products, Wetzlar, Germany). The values were determined using image analysis software (Image J, Jandel Scientific, San Rafael, CA, USA), by a blind examiner, calibrated and trained for this analysis [21]. %BIC = Direct contact of the bone with the implant surface × 100/Extension of the cervical third of the implant 2.7. Statistical Analysis The data from the biomechanical (insertion and removal torque), microtomographic (BV/TV) and histometric (%BIC) analyses were submitted to the Shapiro-Wilk normality test which confirmed that the data were distributed according to the central distribution theorem. The parametric paired ttest was used for the inferential analysis of the data comparing the different groups of macrostructures (CI vs. HCI). The One- way ANOVA test was applied to compare the different assessment periods within 29 each group. The GraphPad Prism 6 software (San Diego, CA, USA) was employed in the statistical tests that were applied at the significance level of 5%. Sample calculation was performed using the paired t-test based on the histometric data of bone-implant contact from the study by Faeda et al. 2012 [21], which assessed the effect of different implant surfaces on osseointegration in rabbits. It was found that the difference between the BIC averages among different implant surfaces in order to promote a statistically significant difference was 25.95% (SD = 8.34). Therefore, the use of 8 rabbits per group in each period would be sufficient to obtain a power β and α of the study greater than 0.9 and equal to 0.05, respectively 3. Results The animals tolerated well the surgical procedure and remained healthy throughout the experimental period. 3.1. Biomechanical Analysis It was found that the HCI implants had a higher insertion torque than the CI implants. There was a progressive increase in implant removal torques in all groups, regardless of the type of macrostructure assessed. It was found that the CIs presented higher removal torque than HCIs in the period of 8 weeks. Figures 3 and 4 and Table 1 show the data on the mean and standard deviation of the biomechanical analysis. Figure 3. Mean and standard deviation of implant insertion torque with different macrostructures. **p < 0.01—Differences between groups of implants with different macrostructures—Paired t-test. 30 Figure 4. Mean and standard deviation of implant removal torque with different macrostructures. **p < 0.01 - Differences between groups of implants with different macrostructures—Paired t-test. Different letters represent different levels of statistically significant differences among the periods within each group (p < 0.05). One-way ANOVA complemented by the Tukey test. Table 1. Data on the mean and standard deviation for the insertion and removal torque of all groups. Insertion Torque Removal Torque Implant type/Period 2 weeks 4 weeks 8 weeks CI 27.99 ± 7.80 * 38.01 ± 17.09 c 68.01 ± 8.46 b 91.05 ± 9.32 **,a HCI 32.93 ± 10.61 * 41.69 ± 5.98 b 53.88 ± 18.50 a,b 68.62 ± 13.70 **,a *p < 0.01—Differences between groups of implants with different macrostructures—Paired t-test. **p < 0.01— Differences between groups of implants with different macrostructures—Paired t-test. Different letters (a, b and c) represent different levels of statistically significant differences among the periods within each group (p < 0.05). One- way ANOVA complemented by the Tukey test. 3.2. Microtomographic Analysis There were no differences between groups regarding the BV/TV data. However, there was a progressive increase in this parameter with the increase in the time of the experimental periods in both groups. Figure 5 and Table 2 show the data on the mean and standard deviation from the BV/TV data obtained through microtomographic analysis of all groups. Figure 6 shows the microtomographic images of the implants with the different macrostructures. 31 Figure 5. Mean and standard deviation for BV/TV data obtained through microtomographic analysis of all groups. Different letters represent different levels of statistically significant differences among the periods within each group (p < 0.05). One-way ANOVA complemented by the Tukey test. Table 2. Data on the mean and standard deviation for BV/TV data obtained through microtomographic analysis of all groups. Implant Type/Period 2 weeks 4 weeks 8 weeks CI 29.68 ± 6.77 b 46.06 ± 5.68 a 49.95 ± 7.36 a HCI 29.58 ± 5.78 b 45.80 ± 7.78 a 52.19 ± 10.77 a Different letters (a and b) represent different levels of statistically significant differences among the periods within each group (p < 0.05). One-way ANOVA complemented by the Tukey test. Figure 6. Microtomographic images representative of implants with different macrostructures CI: Cylindrical Implant (1) and HCI: hybrid conical implant (2). Axial slice (A); Sagittal slice (B); and Coronal slice (C). 3.3. Descriptive and Histometric Histological Analysis The histological description was performed in decalcified sections in the region associated with the first thread of the implants. There were no differences in the histological aspects of bone tissue associated with the different macrostructures of the implants, thus, this description was performed together, varying only the description of the different experimental periods. At two weeks, it was possible to observe the formation of new bone in the region of the threads that presented a trabecular aspect with rounded osteocytes, immature mineralized matrix without organization in the form of concentric lamellae, active osteoblasts with intense organization of the formation of Haversian channels and presence of medullary tissue. At 4 weeks, it was observed an increase in the formation of the mineralized matrix associated with a change in the appearance of the matrix to a more organized condition with the formation of concentric lamellae and a reduction in the number and diameter of the Haversian channels. There was also a reduction in medullary tissue, presence of rounded osteocytes in large numbers and a lower 32 number of osteoblasts. The appearance of peri-implant bone tissue has changed little over the 8-week period compared to the 4-week period. The presence of a mineralized matrix was observed in an advanced stage of mineralization, with the presence of well-defined Haversian channels, presence of rounded osteocytes, organization of the mineralized matrix in concentric lamellae and reduced amount of medullary tissue (Figure 7). It was found that HCI implants (79.08 ± 11.31%) had a higher %BIC in the 8-week period compared to CI implants (59.72 ± 11.29%). Figure 8 and Table 3 show the data of the mean and standard deviation from the %BIC data obtained through the histometric analysis of all groups. Figure 9 shows images representative of the non- decalcified sections of the implants with different macrostructures and in the different experimental periods. Figure 7. Histological images representative of decalcified sections of implants with different macrostructures and in different experimental periods. CI: Cylindrical Implant and HCI: Hybrid Conical Implant. Both in the periods of 2, 4 and 8 weeks. 5× and 10× magnification. Figure 8. Mean and standard deviation for %BIC data obtained through histometric analysis of all groups. **p < 0.01— Differences between groups of implants with different macrostructures—Paired t-test. Table 3. Data on the mean and standard deviation for %BIC data obtained through histometric analysis of all groups. Implant Type/Period 2 weeks 4 weeks 8 weeks CI 69.64 ± 13.22 62.21 ± 11.19 59.72 ± 11.29 * HCI 68.62 ± 11.97 63.49 ± 16.77 79.08 ± 11.31 * 33 *p < 0.01—Differences between groups of implants with different macrostructures—Paired t-test. Figure 9. Histological images representative of the non-decalcified sections of the implants with different macrostructures and in different experimental periods. CI: Cylindrical Implant and HCI: Hybrid Conical Implant. Both in the periods of 2, 4 and 8 weeks. 5× and 10× magnification. 4. Discussion In general, in this study, it was found that HCI implants showed better primary stability, which consequently resulted in greater bone-implant contact at the end of the 8 weeks of assessment, which demonstrates that this type of macrostructure can clinically receive occlusal loads earlier, be used in bones of lower density and accelerate the osseointegration process when compared to CI implants. It has been determined that the implant macrostructure promotes changes in terms of obtaining primary stability [22,23]. The results of this study support studies that demonstrated that conical implants have superior primary stability when compared to cylindrical implants. However, despite the statistically significant difference obtained in this study, cylindrical implants also showed good results for primary stability [18,24]. This finding may be related to the fact that the experiment was carried out on the tibial bone, which has the characteristics of a type I bone, due to the presence of a thick cortical region that allowed the implants to lock properly [25,26]. It is likely that, in a bone of poorer quality, the differences in primary stability for HCI implants are even greater when compared to CI implants, a hypothesis that needs to be tested in the future. Conflicting data in this study was that the removal torque of CI implants was higher than that of HCI implants in the period of 8 weeks. A factor that could have interfered with this aspect would be that the higher insertion torque of the HCI implants would 34 have induced greater necrosis of the cortical bone tissue and delayed the osseointegration process [27,28]. However, the insertion torque values of the HCI implants did not exceed 45 Ncm, which is not related to these adverse events [28]. In addition, the histological assessment of this study proved the similarity between implants with different macrostructures in the same experimental period assessed. The implants in this study had bicortical locking, where their apexes were locked to the posterior cortical bone of the tibia of the rabbits. Despite the fact that the surface area of the implants was not measured, it is possible to observe that the apexes of HCI implants are smaller than those of CI implants, it is possible that this increase in the contact area of the surface of the CI implants may have benefited the increased removal torque of these implants [29]. HCI implants are indicated for all types of bone densities. In the case of type I and II bones, an overrun drill should be used. However, during the installation of these implants, manual torque and counter-torque are often necessary to avoid damage to adjacent tissues. For this reason, the apex of HCI implants (Figure 1F) has already been developed with low intensity helical cameras to facilitate counter torque during manual installation. This fact can also explain the removal torque result of this study. It has also been reported in the literature that primary stability is essential for the osseointegration process to occur in a predictable way and that the degree of osseointegration is correlated with the quality of this stability [30–32], a fact reinforced by the findings of this study that demonstrated a higher degree of %BIC associated with HCI implants when compared to what was observed in the CI implants in the 8- week period, however, without altering the quality of the newly formed bone. This finding demonstrates that the HCI macrostructure benefits the acceleration of the osseointegration process and that perhaps this difference is even more relevant in more challenging clinical conditions (e.g., low-density native bone, grafted areas), however, the animal model used does not allow to infer on the real impact of the HCI macrostructure on osseointegration in these clinical conditions. The results of this study should be interpreted with caution because, in addition to the limitations mentioned above, this model cannot extrapolate clinical conditions such as the application of immediate or early loading, which could alter the osseointegration process in these implants. In addition, the bicortical locking offered by the tibia of rabbits, which in fact helps in the primary stability of implants, is not a common event to be expected in daily clinical practice, where implants usually lock in their most coronal portion in the cortical bone of the maxilla or the mandible. Thus, the effect of HCI on osseointegration still requires further investigation. 5. Conclusions According to the results from and within the limits of this study, it is observed that HCI Implants showed greater insertion torque and bone-implant contact in relation to CI implants in the period of 8 weeks when installed in cortical bone of rabbits. However, the removal torque of CI implants was higher than that of HCI implants in the period of 8 weeks. 35 Author Contributions: Conceptualization, A.d.C.S.L. and M.S.J.; data curation, G.J.P.L.d.O.; formal analysis, A.d.C.S.L. and G.J.P.L.d.O.; funding acquisition, E.M.J.; methodology, A.d.C.S.L., M.S.J., G.d.C.S.P. and R.S.F.; project administration, A.d.C.S.L., L.E.M.P. and E.M.J.; software, G.J.P.L.d.O.; supervision, L.E.M.P. and E.M.J.; validation, G.J.P.L.d.O.; writing—original draft, A.d.C.S.L., M.S.J., G.J.P.L.d.O. and E.M.J.; writing—review & editing, A.d.C.S.L., G.J.P.L.d.O. and E.M.J. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the company Neodent (Curitiba, Paraná, Brazil/PAP 0107.16). Acknowledgments: We do hereby thank the Department of Diagnosis and Surgery and the Graduate Program in Dentistry at the Faculty of Dentistry of Araraquara-UNESP, Araraquara, São Paulo, Brazil the CAPES (Coordination for the Improvement of Higher Education Personnel) and the company Neodent (Curitiba, Paraná, Brazil). Conflicts of Interest: Professor Doctor Élcio Marcantonio Junior is a consultant for the company Neodent. The other authors have no conflict of interest in this study. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. Papaspyridakos, P.; Mokti, M.; Chen, C.-J.; Benic, G.I.; Gallucci, G.O.; Chronopoulos, V. Implant and Prosthodontic Survival Rates with Implant Fixed Complete Dental Prostheses in the Edentulous Mandible after at Least 5 Years: A Systematic Review. Clin. Implant. Dent. Relat. Res. 2013, 16, 705–717. 2. Åstrand, P.; Ahlqvist, J.; Gunne, J.; Nilson, H. Implant Treatment of Patients with Edentulous Jaws: A 20Year Follow-Up. Clin. Implant. Dent. Relat. Res. 2008, 10, 207–217. 3. Lee, J.-T.; Cho, S.A. Biomechanical evaluation of laser-etched Ti implant surfaces vs. chemically modified SLA Ti implant surfaces: Removal torque and resonance frequency analysis in rabbit tibias. J. Mech. Behav. Biomed. Mater. 2016, 61, 299–307. 4. El-Askary, A.S.; Meffert, R.M.; Griffin, T. Why do dental implants fail? Part I. Implant. Dent. 1999, 8, 173– 185. 5. Battula, S.; Lee, J.; Wen, H.; Papanicolaou, S.; Collins, M.; Romanos, G. Evaluation of Different Implant Designs in a Ligature-Induced Peri-implantitis Model: A Canine Study. Int. J. Oral Maxillofac. Implant. 2015, 30, 534–545. 6. Cameron, H.U.; Pilliar, R.M.; Macnab, I. The effect of movement on the bonding of porous metal to bone. J. Biomed. Mater. Res. 1973, 7, 301–311. 7. Steigenga, J.T.; Al-Shammari, K.F.; Nociti, F.H.; Misch, C.E.; Wang, H.-L. Dental implant design and its relationship to long- term implant success. Implant. Dent. 2003, 12, 306–317. 8. Junker, R.; Dimakis, A.; Thoneick, M.; Jansen, J.A. Effects of implant surfasse coatings and composition on bone integration: A systematic review. Clin. Oral Implant. Res. 2009, 20, 185–206. 9. Pilliar, R.M. Overview of surface variability of metallic endosseous dental implants: Textured and porous surface-structured designs. Implant. Dent. 1998, 7, 305–314. 10. Castilho, G.A.A.; Martins, M.; Macedo, W.A.A. Surface characterization of titanium Based dental implants. Braz. J. Phys. 2006, 36, 1004–1008. 11. Javed, F.; Romanos, G.E. The role of primary stability for successful immediate loading of dental implants. A literature review. J. Dent. 2010, 38, 612–620. 12. Salerno, M.; Itri, A.; Frezzato, M.; Rebaudi, A. Surface Microstructure of Dental Implants Before and After Insertion. Implant. Dent. 2015, 24, 1–255. 13. Chiapasco, M.; Gatti, C.; Rossi, E.; Haefliger, W.; Markwalder, T.H. Implant-retained mandibular overdentures with immediate loading. A retrospective multicenter study on 226 consecutive cases. Clin. Oral Implant. Res. 1997, 8, 48–57. 14. Chong, L.; Khocht, A.; Suzuki, J.B.; Gaughan, J. Effect of Implant Design on Initial Stability of Tapered Implants. J. Oral Implant. 2009, 35, 130–135. 15. Wennerberg, A.; Albrektsson, T.; Andersson, B.; Krol, J.J. A histomorghometric study of screw-shaped and removal torque titanium implants with three different surface topographies. Clin. Oral Implant. Res. 1995, 6, 24–30. 16. Negri, B.; Calvo-Guirado, J.L.; Maté Sánchez de Val, J.E.; Delgado Ruiz, R.A.; Ramirez Fernandez, M.P.; Gomez Moreno, G.; Aguilar Salvatierra, A.; Guardia, J.; Munoz Guzon, F. Biomechanical and Bone Histomorphological Evaluation of Two Surfaces on Tapered and Cylindrical Root Form Implants: An Experimental Study in Dogs. Clin. Implant. Dent. Relat. Res. 2012, 15, 799–808. 17. Calvo-Guirado, J.L.; Gomez Moreno, G.; Aguilar-Salvatierra, A.; Mate Sanchez de Val, J.E.; Abboud, M.; Nemcovsky, C.E. Bone remodeling at implants with different configurations and placed immediately at different depth into extraction sockets. Experimental study in dogs. Clin. Oral Implant. Res. 2014, 26, 507– 515. 18. Gehrke, S.A.; Martínez, C.P.-A.; Piattelli, A.; Shibli, J.A.; Markovic, A.; Guirado, J.L.C. The influence of three different apical implant designs at stability and osseointegration process: Experimental study in rabbits. Clin. Oral Implant. Res. 2016, 28, 355– 361. 36 19. Calvo-Guirado, J.L.; Torres, J.A.L.; Dard, M.; Javed, F.; Martínez, C.P.-A.; De Val, J.E.M.S. Evaluation of extrashort 4-mm implants in mandibular edentulous patients with reduced bone height in comparison with standard implants: A 12-month results. Clin. Oral Implant. Res. 2015, 27, 867–874. 20. Pinotti, F.E.; De Oliveira, G.J.P.L.; Aroni, M.A.T.; Marcantonio, R.A.C.; Marcantonio, E. Analysis of osseointegration of implants with hydrophilic surfaces in grafted areas: A Preclinical study. Clin. Oral Implant. Res. 2018, 29, 963–972. 21. Faeda, R.S.; Spin-Neto, R.; Marcantonio, E.; Guastaldi, A.C. Laser ablation in titanium implants followed by biomimetic hydroxyapatite coating: Histomorphometric study in rabbits. Microsc. Res. Tech. 2012, 75, 940–948. 22. Akkocaoglu, M.; Uysal, S.; Tekdemir, I.; Akca, K.; Cehreli, M.C. Implant design and intraosseous stability of immediately placed implants: A human cadaver study. Clin. Oral Implant. Res. 2005, 16, 202–209. 23. De Oliveira, G.J.P.L.; Barros-Filho, L.A.B.; Queiroz, T.; Marcantonio, Élcio; Barros, L.A.B. In Vitro Evaluation of the Primary Stability of Short and Conventional Implants. J. Oral Implant. 2016, 42, 458–463. 24. Sakoh, J.; Wahlmann, U.; Stender, E.; Nat, R.; Al-Nawas, B.; Wagner, W. Primary stability of a conical implant and a hybrid, cylindric screw-type implant in vitro. Int. J. Oral Maxillofac. Implant. 2006, 21, 560– 566. 25. Turkyilmaz, I.; Sennerby, L.; McGlumphy, E.A.; Tozum, T. Biomechanical Aspects of Primary Implant Stability: A Human Cadaver Study. Clin. Implant. Dent. Relat. Res. 2009, 11, 113–119. 26. Marquezan, M.; Osório, A.; Sant’Anna, E.; Souza, M.M.; Maia, L.C. Does bone mineral density influence the primary stability of dental implants? A systematic review. Clin. Oral Implant. Res. 2011, 23, 767–774. 27. Markovic, A.; Mišić, T.; Milicic, B.; Calvo-Guirado, J.L.; Aleksić, Z.; Đinić, A. Heat generation during implant placement in low-density bone:effect of surgical technique, insertion torque and implant macro design. Clin. Oral Implant. Res. 2012, 24, 798–805. 28. Duyck, J.; Roesems, R.; Cardoso, M.V.; Ogawa, T.; Camargos, G.D.V.; Vandamme, K. Effect of insertion torque on titanium implant osseointegration: An animal experimental study. Clin. Oral Implant. Res. 2013, 26, 191–196. 29. Lee, J.; Frias, V.; Lee, K.; Wright, R.F. Effect of implant size and shape on implant success rates: A literature review. J. Prosthet. Dent. 2005, 94, 377–381. 30. Montes, C.C.; Pereira, F.A.; Thomé, G.; Alves, E.D.M.; Acedo, R.V.; De Souza, J.R.; Melo, A.C.M.; Trevilatto, P. Failing Factors Associated with Osseointegrated Dental Implant Loss. Implant. Dent. 2007, 16, 404–412. 31. Gill, A.; Rao, P. Primary stability: The password of implant integration. J. Dent. Implant. 2012, 2, 103. 32. Bataineh, A.; Al-Dakes, A.M. The influence of length of implant on primary stability: An in vitro study using resonance frequency analysis. J. Clin. Exp. Dent. 2017, 9, e1–e6. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). 37 3.2 Publicação 2 Original Reseach: Analysis osseointegration of implant of diffrent macrostructures in areas previously or immediately grafted with deproteinized bovine bone associated or not with fresh bone marrow Amanda de Carvalho Silva Leocádio (Leocádio ACS) - PhD in Implantology1 Matusalém Silva Júnior (Silva Jr. M) - Master in Implantology2 Guilherme José Pimentel Lopes de Oliveira (Oliveira GJPL) - Professor in Periodontology3 Élcio Marcantonio Júnior (Marcantonio Jr E) - Professor in Periodontology1 1 Department of Diagnosis and Surgery, School of Dentistry at Araraquara, Univ. Est. Paulista / UNESP, Araraquara, Brazil 2 Post Graduation Course in Implantology, Instituto Latino-americano de pesquisa odontológica (ILAPEO), Curitiba, Brazil. 3 Department of Periodontology, School of Dentistry at Uberlândia, Federal University of Uberlândia, Uberlândia, Brazil Corresponding author: Élcio Marcantonio Júnior Humaitá St., 1680. Zip code: 14801-130, Araraquara, Brazil. Phone: +55 (16) 33016378 e-mail: junior.elcio@gmail.com Conflicts of Interest: Professor Doctor Élcio Marcantonio Junior is a consultant for the company Neodent. The other authors have no conflict of interest in this study. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. * Artigo submetido no periódico Journal of Periodontology 38 Abstract Background: To assess the osseointegration of implants of different macrostructures (CI - Cylindrical and HCI - Hybrid Conical) in areas grafted with deproteinized bovine bone (DBB) alone or associated with fresh bone marrow (DBB/BM), with variation in the time of implant placement (Immediate or Delayed). Material and methods: Sixteen (16) rabbits were randomly divided into two groups according to the association of biomaterials used to fill the bone defect created in the animals' tibia: DBB vs. DBB/BM. After 90 days, bone biopsies from this region were collected and implants with different macrostructures were installed: CI and HCI. At the same time, a second bilateral defect was created in the tibial metaphysis and filled with the same materials, followed by the immediate implant placement. Euthanasia was performed 90 days after the second surgical procedure. The microtomographic analysis was performed to assess the number of mineralized tissues over the entire length of the bone biopsy and the number of implants over their entire length (%BV/TV). In addition, the histomorphometric analysis was performed to assess the composition of biopsies (%Bone; %Biomaterial and %Connective Tissue), and bone-implant contact (%BIC). Results: There were no differences in the number of mineralized tissues and the composition of biopsies between the DBB and DBB/BM groups. HCI and CI showed no differences for %BIC, however, HCI showed higher values of %BV/TV in areas grafted with DBB when compared to CI. Conclusions: Osseointegration in areas grafted with DBB was not affected by the addition of BM or the macrostructure of the implants. 39 Keywords: Dental implants; Bone marrow; Osseointegration; Bone substitute; Macrostructure. Introduction The treatment of edentulism with implant-supported prostheses has been applied in a predictable way in a wide variety of clinical conditions1. However, in some cases, it is not possible to install implants due to the limited availability of bone tissue, and guided bone regeneration procedures are necessary before or at the same time as the implant placement2. Osteoconductive bone substitutes of xenogeneic origin have been used in bone grafting procedures as an alternative to the use of autogenous bone graft as they promote good clinical results that allow rehabilitation on implants with high success and survival rates in these areas. However, due to the absence of osteogenic and osteoinductive potential for bone tissue formation, these biomaterials have a delayed repair process compared to the autogenous graft3. Due to such limitation, it has been proposed the association of biomaterials of xenogeneic origin with other bone substitutes or growth factors that improve their biological potential for bone tissue formation4-6. The use of fresh bone marrow (BM) infiltrate can be useful to accelerate bone repair in areas grafted with osteoconductive bone substitutes because it is a store of osteochondral progenitor stem cells that has the potential to differentiate into osteoblasts7,8. Obtaining good primary stability of the implants (that is necessary for the osseointegration process to occur) is another problem related to areas grafted with osteoconductive biomaterials9. Bone tissue of poor density, such as grafted 40 bone, makes it difficult to obtain the minimum insertion torque necessary for predictable osseointegration. In order to solve this clinical problem, structural changes in the macrostructure of the implants (e.g. Implant shape, shape and size of threads) have been assessed in order to improve the stability of implants in low-density bone10, 11. The emergence of implants with a conical structure and compressive threads allows the installation of implants with good primary stability, as well as the use of implants with smaller sizes since they can be used in areas with limited bone availability 12,13. However, conical implants can also excessively increase the insertion torque, which can exacerbate the bone remodeling process and consequently impair the osseointegration process 14,15. Therefore, this preclinical study aimed to verify the influence of an experimental implant of hybrid macrostructure with compacting and perforating threads (HCI - Hybrid Conical Implant), compared to a control implant (CI - Cylindrical Implant), on the osseointegration in areas previously or immediately grafted with deproteinized bovine bone (DBB) alone or associated with fresh bone marrow (BM) in surgical defects created in rabbits’ tibiae. Material and methods This research was carried out in accordance with the ARRIVE protocol for conducting preclinical studies. The protocol was submitted and approved by the Ethics Committee on Animal Use (ECAU) of the State University of São Paulo (Unesp), Faculty of Dentistry of Araraquara, Brazil (n°15/2017). For this research, 16 male rabbits (Albinos, New Zealand) were used (4-5 kg and 9-12 months of age). These animals were kept in an environment with a temperature of 22-24ºC, 41 with a controlled light cycle (12 hours light and 12 hours dark) and consumption of solid food and water ad libitum throughout the experimental period. Experimental Outline To assess the influence of different macrostructures of titanium implants in areas previously or immediately grafted with xenogeneic grafts of bovine origin (Straumann® Cerabone®, Switzerland, Germany/0.5-1.0mm granules) associated or not with fresh bone marrow, 16 rabbits were used in a period of 180 days. The animals were randomly assigned to two experimental groups: Control Group (DBB: Deproteinized bovine bone graft) and Experimental Group (DBB/BM: Deproteinized bovine bone graft associated with fresh autologous bone marrow). Initially, a bilateral bone defect (5 mm in diameter) was created in the tibial metaphysis of each animal. The defects were filled with DBB or DBB/BM. After 90 days, a bone biopsy was collected and implants of different macrostructures were placed in these previously grafted areas: Hybrid Conical Implant - HCI (Cylindrical body and helical conical apex) and Cylindrical Implant - CI. Each grafted region (right and left side) received an implant with different macrostructures (HCI or CI). In addition, in this surgical procedure, a second bilateral defect (5 mm in diameter) in the tibial metaphysis, located 4 mm from the first defect, was filled with the same materials described for the first defects (DBB or DBB/BM), but with immediate installation of implants of different macrostructures (HCI or CI). Ninety days after the second surgical procedure, all animals were euthanized by anesthetic overdose. Implants 42 The CI implant used in this study is characterized by a cervical diameter equal to the diameter of the implant body (3.75mm in diameter x 11mm in height). Presence of triangular and double threads that facilitate the quick implant insertion, with minimal trauma, and apex morphology with the presence of self- cutting chambers (Titamax Acqua, Neodent®, Cone Morse, Curitiba, Brazil) (Figure 1A, 1C, and 1E). The HCI implant is characterized by an increased cervical diameter in relation to the implant body (3.75mm in diameter x 11.5mm in height). In addition, these implants have compacting and double trapezoidal threads, with a conical apex containing helical chambers designed to optimize secondary stability (Helix Acqua, Neodent®, Grand Morse, Curitiba, Brazil) (Figure 1B, 1D, and 1F). Surgical procedure The animals were weighed and anesthetized intramuscularly, with a combination of ketamine (Quetamina Agener®; Agener União Ltda, São Paulo, SP, Brazil) - 0.35 mg/kg) and xylazine (Rompum, Bayer AS, São Paulo, SP, Brazil - 0.5 mg/kg). Subsequently, bilateral trichotomy in the region of the tibial metaphysis and antisepsis with 10% polyvinylpyrrolidone (PVPI) with 1% active iodine were performed. Local anesthesia with Mepivacaine Hydrochloride 2% and Adrenaline 1:100,000 was also applied in the region, to allow peripheral vasoconstriction, reducing local bleeding and optimizing the surgical procedure. Then, using a No. 15 scalpel blade, a dermo-periosteal incision of approximately 5 cm in length was performed on the inside of the rear leg, just below the knee (Figure 2A). This allowed for delicate dissection so that the bone surface was exposed. The surgical defect was made using a 5mm diameter trephine drill (3i, Neodent, Brazil), coupled in a counter-angle and micromotor, under abundant 43 refrigeration with 0.9% sterile saline (Figure 2B, 2C and 2D). Circular markings were also made 2 mm anterior and 2 mm posterior to the margins of the surgical defect and filled with gutta-percha. These markings were located on an imaginary longitudinal line dividing the surgical defect in half. The markings were made with a carbide truncated conical drill (Carbide Truncated Conical Drill for High Rotation Speed no. 16 702, Dentsply, Brazil) coupled in high rotation speed also under continuous irrigation with 0.9% sterile saline. These markings were useful to identify the center of the original surgical defect during implant installation and laboratory processing, allowing to locate the original bone margins of the defect during histological analysis. After filling the defects with the respective materials (DBB or DBB/BM according to the experimental group) (Figure 2E, 2F, 2G, 2H and 2I), a collagen membrane was placed on the surface of the defect (Jason® membrane, Straumann®, Curitiba, Brazil) (Figure 2J). The fresh autologous bone marrow used was obtained from the tibial metaphysis with the aid of a Lucas 86 curette (Figure 2E). Finally, the soft tissues were repositioned and sutured per plane by using simple interrupted stitches with resorbable thread (Vicryl® Ethicon, 4-0, Johnson & Johnson, Brazil) and non-resorbable thread (Nylon 3-0, Shalon®, Brazil) (Figure 2K and 2L). After surgery, all animals received a single dose of antibiotic (Pentabiótico®, Wyeth-Whitehall Ltda, São Paulo, Brazil - 0.1 ml/kgl) and analgesic (Tramadol Hydrochloride 50mg/ml, Tramadol®, Medley, São Paulo, Brazil - 5 mg/Kg IM). Ninety days after the first surgical procedure, the animals were submitted to the second surgical procedure in which the entire protocol of anesthesia, surgical access, and postoperative care was repeated. When accessing the previously grafted defects, a bone biopsy of the defect was 44 collected using a 3.3mm trephine drill (Figure 3A, 3B and 3C). The preparation for installing the implants was carried out according to the recommendations of the manufacturers of the implant system (Neodent®, Curitiba, Paraná, Brazil) so that the bone was milled with metal drills under abundant refrigeration with saline. The implants were inserted to the bone level and the cover screws were threaded. One implant was installed on the right side and one on the left side according to the different groups (CI or HCI) (Figure 3D). After the installation of the implants in the previously grafted areas, the second defect (5mm diameter) in the tibial metaphysis was made and grafted (DBB or DBB/BM according to the experimental group) (Figure 3E, 3F, 3G, 3H, 3I and 3J). The defect was made and filled in the same way as described in the first surgical procedure, however, this defect was performed 2 mm from the distal marking (with gutta-percha) of the previously grafted area and received an implant immediately according to the different groups (CI or HCI) (Figure 3K, 3L and 3M). The soft tissues were repositioned again and sutured per plane as previously described (Figure 3N, 3O, 3P and 3Q). Microtomographic Analysis (µCT) The parts from the sites where the bone biopsies were collected and the implants were installed, fixed in 4% formaldehyde for 48 hours and kept in alcohol at 70°. The samples underwent a µCT scan (Skyscan, Aatselaar, Belgium), with the following scanning parameters: 18 μm3 voxel, 10x image magnification, X- ray tube voltage of 50 kV, 496 uA beam and the electrical current adjusted to 0.1 mA. The three-dimensional images were reconstructed, reoriented and analyzed by specific software (NRecon, Data Viewer, Ctan, SkyScan, Belgium). 45 The biopsies without implants were assessed for the volume of mineralized tissues over their entire length, while the samples with implants were assessed for the volume of mineralized tissues around the implants, and a region of interest was determined covering their entire length and exceeding its diameter by 0.5 mm. The threshold used for both analyses was 25 – 90 16. Histomorphometric Analysis After the decalcification period, the bone biopsies collected from previously grafted areas were embedded in paraffin, cut into a microtome (6µm-thick) and stained using the hematoxylin-eosin (HE) technique. Five slides with 3 sections each were obtained in the central region of the biopsy. Three sections were assessed that were 36 µm apart, and the first section for assessment was selected randomly. The sections were assessed using a DIASTAR optical microscope (Leica Reichert & Jung Products, Germany) with 5x and 10x magnifications and the amount of bone tissue, biomaterial, and connective tissue was assessed, expressed as the average of the percentages of the 3 sections assessed in each sample. The biopsies with implants were subjected to dehydration in alcohol with increasing concentration and infiltrated later in solutions of glycolmethacrylate (Technovit 7200 VLC) and ethyl alcohol, following gradual increases in the concentration of glycolmethacrylate until infiltration in this pure solution. The specimens were embedded in resin, polymerized and sectioned longitudinally along the main axis of the implant with a high precision diamond disk. The blocks were assembled on an acrylic slide with the help of the Tecnovit 4000 resin (Kulzer, Wehrheim, Germany), and using a cutting and micro-wear system (Exact-Cutting, System, Apparatebau Gmbh, Hamburg, Germany). The slides 46 were processed so they had a 50-70 μm thick section (approximately). The parts were stained with Stevenel’s Blue and fuchsin and were analyzed by histometric analysis that assessed the amount of direct contact between the bone and the surface throughout the implant (%BIC). Measurements were made using photomicrographs obtained by an optical microscope (DIASTAR, Leica Reichert & Jung products, Germany), with 10x magnification, with the aid of a video camera (Leica Reichert & Jung products, Germany). The values were determined using image analysis software (Image J, Jandel Scientific, San Rafael, CA, USA). All analyses in this study were performed by trained examiners, blind for the experimental groups. Statistical Analysis The Shapiro-Wilk normality test was applied, which demonstrated that the data were distributed according to the theorem of the central data distribution. The parametric paired t-test was used for the inferential analysis of the data comparing the different groups of macrostructures (CI vs. HCI), to compare the results of the grafted areas (DBB vs. DBB/BM) and the shape of the installed implants (Immediate vs. Delayed). The GraphPad Prism 6 software (San Diego, CA, USA) was employed in the statistical tests that were applied at the significance level of 5%. Results The animals tolerated well the surgical procedure and remained healthy throughout the experimental period. µCT Analysis 47 There were no statistically significant differences between groups for %BV/TV of biopsies without implants, although the group of DBB associated with BM had higher valuesthan biopsies where DBB was used alone (75.42 ± 10.11 vs. 77.42 ± 9.57). Table 1 show the data from the microtomographic analysis of the groups in this study. Figure 4 shows images representative of the microtomographic analysis of all groups. It was observed that, regardless of the time of implant placement (Immediate vs. Delayed), HCI implants showed higher %BV/TV values in the areas grafted with deproteinized bovine bone alone (DBB-HCI: 83.34 ± 5.87 and 85.69 ± 7.91, respectively) compared to CI implants (DBB-CI: 70.39 ± 15.18 and 77.60 ± 8.31, respectively). The other groups assessed did not show statistically significant differences. Table 3 show the data on mean and standard deviation from the microtomographic analysis of the areas grafted with deproteinized bovine bone alone or associated with bone marrow. Figure 6 shows images representative of the microtomographic analysis of all groups. Histomorphometric Analysis Although the DBB/BM group had higher values regarding the percentage of bone and connective tissue (27.37 ± 5.83 and 51.81 ± 10.53, respectively) compared to the DBB group (23.08 ± 9.95 and 50.52 ± 14.31, respectively) and although the DBB group (26.40 ± 7.16) had higher values regarding the percentage of biomaterial compared to the DBB/BM group (20.81 ± 11.34), no statistical difference was detected between the grafted areas concerning the percentage of bone, biomaterial, and connective tissue. Table 2 show the data on mean and standard deviation for the percentage of bone (2A), 48 biomaterial (2B) and connective tissue (2C) of the different groups (DBB vs. DBB/BM). In biopsies of non-decalcified sections, it was found that CIs installed immediately in areas grafted with deproteinized bovine bone alone showed higher %BIC than the same implants installed later in areas grafted with deproteinized bovine bone alone (36.22 ± 6.07 vs. 23.32 ± 5.29). The other groups assessed did not show statistically significant differences. Table 4 show the data on mean and standard deviation from the histometric analysis of %BIC of the areas grafted with deproteinized bovine bone alone or associated with bone marrow. Figure 7 shows the histological images representative of the non- decalcified sections of all groups assessed. Discussion In this study, it was proposed to assess the osseointegration of two different implant macrostructures (HCI vs. CI) installed immediately or later in rabbits’ tibiae grafted with DBB associated or not with BM. As a general finding, the areas grafted with BM showed a better pattern of osseointegration and new bone formation between the biomaterial particles, however, no statistically significant differences were detected in the analyses employed. As for the different macrostructures assessed, in the grafted areas with DBB, regardless of the time of implant placement (Immediate vs. Delayed), HCI had higher valuesfor the percentage of mineralized tissues around the implant compared to CI. On the other hand, regarding the percentage of bone-implant contact, only in the areas grafted with DBB, CIs showed statistical differences when comparing the 49 Immediate vs. Delayed implant installation (36.22 ± 6.07 and 23.32 ± 5.29, respectively). Deproteinized bovine bone (DBB) is widely used and researched as a bone substitute due to its physicochemical properties similar to those of human bone, its osteoconductive potential, and availability. The sintering temperature of bone substitutes is an important parameter that can affect their properties17. When sintered at low temperatures (up to 300°C), the mineral crystals of the bone matrix are preserved (Bio Oss®, Geistlich Pharma, Wolhusen, Switzerland, for example)18. In contrast, when sintered at high temperatures (Straumann® Cerabone®, Switzerland, Germany) (initial oxidative combustion at temperatures around 800°C followed by a second heat treatment at 1,250°C), this process results in an increase in the crystal size (500-1000%) and crystal density, which makes it comparable to a ceramic-based material17-19. The present study used DBB sintered at high temperatures, and bone biopsies collected from previously grafted areas (after 90 days of healing) showed particles of the biomaterial incorporated into the newly formed bone. This indicates that this DBB, in fact, worked as a scaffold for bone apposition. The use of bone substitutes capable of offering an adequate framework, with emphasis on bovine xenogeneic grafts, has been a good option. But the osteoconduction of these materials, when combined with other mechanisms of action, such as osteoinduction and osteogenesis, seems to have even better results 20. Therefore, a favorable alternative would be the association of this (essentially osteoconductive) filling material with the fresh bone marrow that has osteogenic and osteoinductive properties because it contains osteochondral progenitor stem cells and growth factors that would induce greater differentiation 50 and osteoblastic activity. In addition, the use of this biological adjuvant could work as a biological connection between the graft particles, further favoring tissue regeneration 21-25. There is no consensus on the best methodology for the use of bone marrow stromal cells. Three main alternatives were proposed: (1) Use of fresh autologous bone marrow graft ("in natura"); (2) Use of autologous bone marrow cell concentrate (by centrifugation); and, (3) Use of autologous bone marrow stromal cell culture 21-26. The use of bone marrow enriched with mononuclear fraction obtained by centrifugation showed better results of bone formation associated with DBB (28.17 ± 3.19%) than the use of fresh bone marrow associated with the same bone substitute (21.14 ± 7.38%) according to a study by Pelegrine et al. (2014)27. However, some disadvantages have been associated with the use of the cell culture methodology: cost, time (requires a period of a few weeks between cell collection, obtaining the culture and transplantation), increased risk of contamination, and the routine clinical applicability of the cell culture may require two separate surgical procedures. Another question about the need (or not) to cultivate the cells is the lack of information on the minimum number of cells required to promote bone repair26-30. Therefore, the use of fresh bone marrow made in the present study would be a simpler approach than other uses that have already been proposed. In a study of critical defects in rabbit calvaria, the use of fresh bone marrow associated with DBB showed a greater formation of bone tissue (21.14 ± 7.38%) compared to the use of DBB alone (13.06 ± 5.24 %) 8 weeks after the grafting procedure26. As in a study of vertical bone reconstruction in rabbit calvaria, the stem cells derived from the marrow associated with a block of DBB resulted in 51 increased bone formation when compared to the association of the same block with stem cells from adipose tissue (11.9 ± 7.5% vs. 7.6 ± 5.6%) 8 weeks after the surgical procedure 8. In the present study, no statistical differences were identified as observed in the studies mentioned in which BM was added to DBB. This may have occurred due to the long experimental period in our study (90 days for the healing of the previous graft and for the post-installation of the implants) compared to the aforementioned studies (60 days). In addition, other forms of application of BM should be tested with regard to the possibility of positively influencing the osseointegration process in areas grafted with DBB, because when BM was used (DBB vs. DBB/BM) the quality of the connection between the particles was evidently higher. The implant macrostructure directly influences the primary stability of the implants and, consequently, the success of their osseointegration. As for the macrostructure of the implants used (CI vs. HCI), several studies in the literature have shown that implants with conical structure and with changes in the conformation of threads, in order to make them more compressive, allow the installation of implants with good primary stability. Therefore, they are indicated in areas with limited bone availability or in low-density regions 31-33. The results of this study showed that the percentage of mineralized tissue around the implants was statistically higher in the HCI implant compared to the CI implant when using DBB alone, regardless of the time of implant placement (Immediate or Delayed). In the bone-implant contact, although both tested implants showed good results, the statistical difference in osseointegration was only observed in the CI implant when installed in areas previously or immediately grafted with DBB alone, with better results when immediately installed (36.22 ± 6.07 and 23.32 ± 52 5.29, immediate and delayed respectively). These results support studies in the literature since the decision to perform previous or immediate grafting will be defined by the type of bone defect found at the beginning of the treatment plan and by the possibility of primary implant locking. After tooth extraction, the alveolus has an intrinsic cone shape. When it receives immediate implants, the primary locking occurs only in the apical or apical-middle portion of the implant. Therefore, the macrostructure of the selected implant is extremely important. If such locking does not occur, bone regeneration or repair of the alveolus should first be achieved with or without prior grafting depending on the type of defect obtained after tooth loss. And, the alveolus has often dimensions larger than the implant diameter, forming a space between the cervical region of the implant and the bone tissue. Implant locking in these clinical situations occurs basically in the apical portion of the alveolus, which requires the filling of the space between the implant and the alveolus wall with biomaterials34. In this study, the second defect produced in the tibial metaphysis of rabbits with a cervical diameter (in the upper cortical area of the tibia) 1.25mm larger than the implant diameter (5mm vs. 3.75mm, respectively), and implant locking only in the apical portion of the (lower cortical) defect simulated a clinical situation of immediate implant after tooth extraction35. As the bone width in the cervical portion directly influences the %BIC, it is important to use biomaterials to fill these defects, so we use a bovine xenogeneic graft. The surfaces modified by sandblasting with abrasive particles followed by acid subtraction have demonstrated good clinical results, success, and survival of the implants. However, they have a low degree of wettability. For this reason, this surface treatment has been associated with the procedure of 53 maintaining the implant in a 0.9% sodium chloride isotonic solution, resulting in a surface that stands out for being exceptionally hydrophilic, with a contact angle of about 0° aga