UNESP - Universidade Estadual Paulista “Júlio de Mesquita Filho” Faculdade de Odontologia de Araraquara Camila Soares Lopes Propriedades biológicas, físicas e químicas de uma medicação intracanal biocerâmica Araraquara 2022 UNESP - Universidade Estadual Paulista “Júlio de Mesquita Filho” Faculdade de Odontologia de Araraquara Camila Soares Lopes Propriedades biológicas, físicas e químicas de uma medicação intracanal biocerâmica Tese apresentada à Universidade Estadual Paulista (Unesp) - Faculdade de Odontologia, Araraquara, para obtenção do título de Doutor em Odontologia, na Área de Endodontia. Orientador: Prof. Dr. Paulo Sérgio Cerri Coorientadora: Profª Drª Juliane Maria Guerreiro-Tanomaru Araraquara 2022 Camila Soares Lopes Propriedades biológicas, físicas e químicas de uma medicação intracanal biocerâmica Comissão julgadora Tese como requisito para obtenção do grau de doutor em Odontologia Presidente e Orientador: Prof. Dr. Paulo Sérgio Cerri 2º Examinador: Prof. Dr. Gustavo Sivieri de Araújo 3º Examinador: Profª. Drª. Naiana Viana Viola 4º Examinador: Profª. Drª. Raquel Assed Bezerra Segato Araraquara, 20 de maio de 2022. DADOS CURRICULARES Camila Soares Lopes NASCIMENTO: 05 de março de 1989 – Alfenas - Minas Gerais FILIAÇÃO: Ercílio Lopes e Eveline Soares Lapa Lopes 2011- 2015: Graduação em Odontologia pela Universidade Federal de Alfenas, UNIFAL/MG, Alfenas-Minas Gerais. 2016-2018: Mestre em Ciências Odontológicas, área de concentração em Odontologia, pela Universidade Federal de Alfenas, UNIFAL/MG, Alfenas-Minas Gerais. 2019-2021: Especialização em Endodontia pela Faculdade Herrero - FAEPO, Araraquara-SP. 2018-Atual: Doutoranda em Odontologia, área de concentração em Endodontia, pela Universidade Estadual Paulista “Júlio de Mesquita Filho”, Faculdade de Odontologia de Araraquara – FOAr/UNESP, Araraquara-SP. Dedico este trabalho à minha avó Zilda Teodoro Miranda (in memorian) e toda minha ancestralidade, que de alguma forma me permitiram estar aqui hoje lutando pelos meus ideais e mudando nossa história. AGRADECIMENTOS Agradeço a Deus, pela sua infinita misericórdia, por ter abençoado esta jornada me dando força e coragem para enfrentar todos os obstáculos e por ter colocado pessoas que me ajudaram e me guiaram por toda trajetória. Aos amigos de luz que me acompanham nessa caminhada me protegendo e iluminando os meus passos, que nunca me desamparam. Aos meus pais que me ensinaram que não há vitórias sem batalhas, sem esforço e gratidão. Que é preciso força e determinação para ir atrás dos sonhos. Obrigada por terem me dando o dom da vida e pela chance de trilharmos juntos está caminhada. A minha família, tios e primos, por todo apoio e torcida. Em especial ao meu primo Guilherme que, mais que um primo, é parte de mim e me ensina os mais nobres dos sentimentos. À minha madrinha Maria de Lourdes que me ajudou e financiou em todos os meus sonhos. Obrigada por tudo, por ser modelo de coragem, pelo apoio e por todas as orações e intenções emanadas. Ao meu tio José Maria que sempre frisou a importância do estudo e conhecimento na vida de uma pessoa (in memorian). Aos meus avôs e minhas avós que de onde estiverem vibram pela minha felicidade (in memorian). À minha amiga de luz Cristina por todo amparo, conselhos e ajuda espiritual. Às minhas amigas de infância Cínthia por ter me apoiando na decisão de trilhar essa nova trajetória e ter me acompanhado nos primeiros dias aqui em Araraquara. A Marília por ser inspiração e me ensinar a lidar com os desafios da pós-graduação. À Marcella, Karine e Luiza pelo apoio e torcida de sempre. À Profª. Drª. Naiana Viana Viola, não foi sorte, foi Deus. Essa vitória não seria possível sem você que foi minha inspiração desde a graduação e que foi meu maior apoio durante toda caminhada. Obrigada por ter reconhecido em mim o potencial que eu mesma nunca reconheci e por ser em minha vida o modelo de determinação, potência feminina, bondade e fé. Gratidão por ser mais que uma professora, uma eterna amiga. Ao meu orientador Prof. Dr. Paulo Sérgio Cerri. Obrigada por ter aceitado me orientar, pelos ensinamentos e conhecimentos compartilhados, pela sua dedicação e competência, pelas vitórias e derrotas que passamos juntos durante essa trajetória, me ensinando a levantar a cabeça e não desistir. Pela oportunidade de ter sido bolsista FAPESP, pois essa conquista não foi só minha. Minha eterna gratidão pelas experiências que passamos juntos. À minha coorientadora Profª. Drª. Juliane Maria Guerreiro Tanomaru por todos os ensinamentos, tanto os profissionais quanto os pessoais. Por ser essa referência feminina e profissional que enaltece conhecimento e determinação. Ao Prof. Dr. Mário Tanomaru Filho por todo acolhimento, carinho, oportunidades que me deu desde minha chegada em Araraquara. Obrigada professores pela colaboração neste trabalho e minha eterna gratidão por todo apoio e incentivo na minha vida acadêmica, foi uma honra poder conviver, trabalhar e aprender com vocês. À Prof.ª Dr.ª Estela Sasso Cerri pela colaboração neste trabalho, pelos ensinamentos na parte experimental com animais mostrando conhecimento e, ao mesmo tempo, amor pelos animais. À Prof.ª Dr.ª Gisele Faria pela sua disposição e carinho em sempre ajudar e dividir seus conhecimentos e pela colaboração na banca de qualificação deste trabalho. Ao Prof. Dr. Guilherme Ferreira da Silva muito obrigado pelas considerações e sugestões para o crescimento deste trabalho durante a banca de qualificação Aos amigos e companheiros da pós-graduação Fernanda, Mateus, Karina, Airton e Járder que, além de todo apoio e amizade, ajudaram e dividiram seus conhecimentos para que eu pudesse realizar este trabalho e que muitas vezes me estimularam a continuar trabalhando com seriedade em busca dos meus objetivos. À doutoranda Jéssica Arielli Pradelli, sou tão grata a Deus por ter te conhecido no meu primeiro dia em Araraquara. Obrigada por estar sempre do meu lado me apoiando, me acompanhando e sendo luz em meus dias. Sem você essa e tantas outras vitórias não seriam possíveis. Gratidão pela parceria nos momentos felizes e turbulentos. À doutoranda Rafaela Inada, obrigada pela sua amizade e disposição em sempre me ajudar, por me acolher na sua casa e ser minha parceira de viagens, proporcionado grandes memórias registradas nas melhores fotos. Obrigada por me ensinar a ter paciência e calma no dia a dia. À Mariana Mena Barreto Pivoto-João, você e sua família foram uns dos maiores presentes em Araraquara. Gratidão por essa amizade linda, saudades amiga! Aos alunos da pós-graduação Giselle, Evelin, Marcela, Cris, Hernan, Vitor e Lívia por fazerem parte desta trajetória, obrigada pela convivência e aprendizado durante esses anos e por estarem sempre dispostos a me ajudar. Aos alunos das disciplinas de Endodontia que sempre me acolheram e me respeitaram durante os estágios docentes. Aos funcionários da Faculdade de Odontologia de Araraquara – UNESP por todo apoio e suporte, em especial ao Pedro, Sandra, Alexandre, Cristiano e Renan. Muito obrigada por tudo! Aos professores que aceitaram o convite para compor a banca de defesa desta tese de doutorado, Prof. Dr. Gustavo Sivieri de Araújo, Profª. Drª Raquel Assed Bezerra Segato e Profª. Drª Naiana Viana Viola pela disponibilidade em melhorar nossos trabalhos. Em especial aos professores e alunos da Universidade Federal de Alfenas, vocês foram a base de toda essa história incentivando, ajudando e sempre torcendo por mim. À Faculdade de Odontologia de Araraquara - FOAr, Universidade Estadual Paulista – UNESP, representada pelo Prof. Dr. Edson Alves de Campos e pela Prof.ª Dr.ª Patrícia P. Nordi Sasso Garcia. Pela infraestrutura desta instituição que se tornou minha segunda casa, na qual, tenho muito orgulho de ter feito parte. Ao Programa de Pós-Graduação em Odontologia, FOAr/UNESP, na pessoa do Coordenador Prof. Dr. Paulo Sérgio Cerri, pela oportunidade de se obter uma formação acadêmica de excelência e apoio. À FAPESP – Fundação de Amparo à Pesquisa do Estado de São Paulo (Processo nº 2018/16848-2) pelo apoio financeiro essencial para realização dessa pesquisa. A todos aqueles que direta ou indiretamente contribuíram para a execução desta tese. “Que nada nos defina, que nada nos sujeite. Que a liberdade seja a nossa própria substância, já que viver é ser livre.” Simone de Beauvoir Disponível em:https://veja.abril.com.br/coluna/pilulas-de-sabedoria/frase-do-dia-simone-de-beauvoir- 2/. Lopes CS. Propriedades biológicas, físicas e químicas de uma medicação intracanal biocerâmica. [tese de doutorado]. Araraquara: Faculdade de Odontologia da UNESP; 2022. RESUMO O objetivo do estudo foi avaliar a biocompatibilidade e o potencial bioativo de uma medicação intracanal biocerâmica, Bio-C Temp (BIO; Angelus, Londrina, Brasil), em comparação a medicação à base de hidróxido de cálcio, Calen (CAL; SS White, Rio de Janeiro, Brasil) implantados nos tecidos do subcutâneo de 60 ratos, durante 7, 15 30 e 60 dias. Análise de propriedades físico-químicas, incluindo radiopacidade, pH, solubilidade, escoamento, alteração volumétrica (%) e capacidade de preenchimento foram conduzidas com as medicações BIO e o CAL. Publicação 1: A espessura das cápsulas, o número de células inflamatórias (CI), células imunopositivas à interleucina-6 (IL-6) e IL-10 e conteúdo de colágeno foram mensurados. Os níveis séricos da transaminase glutâmico-oxalacética (TGO) e transaminase glutâmico- pirúvica (TGP), foram avaliados 7 e 60 dias. Os dados foram submetidos à ANOVA e teste Tukey (p≤0,05). Os níveis séricos de TGO e TGP foram semelhantes (p > 0,05). BIO e CAL apresentaram maior espessura das cápsulas aos 30 dias reduzindo aos 60 dias. Em todos os períodos, o número de CI e células IL-6- imunopositivas foram maiores em CAL. O número de células IL-10-imunopositivas aumentou significantemente no decorrer do tempo em BIO E CAL. Aos 60 dias, BIO mostrou maior conteúdo de colágeno em comparação ao CAL (p < 0,0001). Os danos teciduais inicialmente provocados por BIO foram suprimidos mais rapidamente em comparação àqueles provocados por CAL, favorecendo a reparação do tecido conjuntivo. Publicação 2: Neste estudo, os níveis séricos de cálcio (Ca+2), fósforo (P-) e fosfatase alcalina (ALP), a imunoexpressão de osteocalcina (OCN) e osteopontina (OPN), o número de fibroblastos e a deposição de calcita amorfa foram avaliados. Os dados foram submetidos à ANOVA/Tukey (p≤0,05). Aos 7 e 15 dias, os níveis de Ca+2 foram maiores nas amostras do CAL enquanto que o nível de P- foi maior aos 7 dias em BIO e CAL. O nível de ALP foi alto em BIO e CAL até os 30 dias, reduzindo significantemente aos 60 dias. Em BIO e CAL, um aumento significante no número de fibroblastos, na imunoexpressão de IL-10, OCN e OPN foram detectados ao longo do tempo. BIO e CAL apresentaram estruturas von Kossa-positivas e birrefringentes enquanto que, no grupo controle (tubos vazios), estas estruturas não foram observadas. BIO, assim como CAL, causou aumento dos níveis séricos de ALP, enzima essencial na biomineralização, e induziu células do tecido subcutâneo a sintetizar OCN e OPN, além da deposição de calcita, indicando que BIO tem potencial bioativo. Publicação 3: No presente estudo, o pH, radiopacidade, escoamento, solubilidade (após 7 e 14 dias imersão em agua destilada e PBS), alteração volumétrica e capacidade de preenchimento (por micro- CT) foram avaliadas. Foram aplicados os testes estatísticos ANOVA/Tukey e teste T (p≤0,05). CAL mostrou maior pH, escoamento, alteração volumétrica e solubilidade após 14 dias de imersão em água destilada e 7 e 14 dias em PBS. BIO apresentou maior radiopacidade e capacidade de preenchimento (p<0,05). Apesar de a medicação biocerâmica apresentar menor perda volumétrica e capacidade de preenchimento satisfatória, evitando vazios e falhas, o Bio-C Temp mostrou menor alcalinidade do meio em comparação ao Calen. Palavras chave: Endodontia. Imuno-histoquímica. Materiais dentários. Propriedades físicas. Propriedades químicas. Lopes CS. Biological, physical and chemical properties of a bioceramic intracanal medication [tese de doutorado]. Araraquara: Faculdade de Odontologia da UNESP; 2022. ABSTRACT The aim of the study was to evaluate the biocompatibility and bioactive potential of a bioceramic intracanal medication, Bio-C Temp (BIO; Angelus, Londrina, Brazil), in comparison to a calcium hydroxide-based medication, Calen (CAL; SS White, Rio de Janeiro, Brazil) implanted into the subcutaneous tissues of 60 rats for 7, 15, 30 and 60 days. Analyses of physicochemical properties, including radiopacity, pH, solubility, flowability, volumetric change (%) and filling capacity were conducted with BIO and CAL medications. Publication 1: The thickness of the capsules, the number of inflammatory cells (IC), immunopositive cells for interleukin-6 (IL-6) and IL-10, and collagen content were measured. Serum glutamic-oxalacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) levels were evaluated at 7 and 60 days. The data were submitted ANOVA and Tukey’s test (p ≤ 0.05). Significant differences in the serum GOT and GPT levels were not detected (p > 0.05). The capsules around the BIO and CAL specimens were thicker at 30 days than at 60 days. In all periods, the number of IC and IL-6-immunopositive cells was significantly lower in BIO than in CAL specimens. In BIO and CAL specimens, the number of IL-10-immunopositive cells increased significantly over time. At 60 days, the capsules around the BIO contained greater collagen content than in CAL specimens (p < 0.0001). Tissue damage initially caused by BIO was suppressed more quickly compared to that caused by CAL, favoring connective tissue repair. Publication 2: In this study, the concentration of calcium (Ca+2), phosphorus (P-) and alkaline phosphatase (ALP) in the serum, the immunoexpression of osteocalcin (OCN) and osteopontin (OPN), the number of fibroblasts and the deposition of amorphous calcite were evaluated. Data were submitted to ANOVA and post-hoc Tukey test (p≤0.05). At 7 and 15 days, serum Ca+2 levels were higher in the CAL than in BIO samples while the P- concentration was highest in BIO and CAL in comparison to control group (CG; empty tubes) at 7 days. The ALP level was high in BIO and CAL up to 30 days, decreasing significantly at 60 days. In BIO and CAL, a significant increase in the number of fibroblasts, in the immunoexpression of IL-10, OCN and OPN was detected over time. In constrast, OCN- and OPN-immunolabelled cells were not seen in the CG specimens. BIO and CAL specimens showed von Kossa-positive structures and birefringent deposits, while in the CG, these structures were not observed. BIO, as well as CAL, caused an increase in the serum ALP levels, an essential enzyme in biomineralization, and induced subcutaneous tissue cells to synthesize OCN and OPN, in addition to calcite deposition, indicating that BIO has bioactive potential. Publication 3: In the present study, pH, radiopacity, flowability, solubility (after 7 and 14 days immersion in distilled water and PBS), volumetric change and filling capacity (by micro-CT) were evaluated. ANOVA and Tukey’s test and T test (p≤0.05) were applied. CAL showed higher pH, flow, volumetric change and solubility after 14 days of immersion in distilled water, and at 7 and 14 days in PBS. BIO showed greater radiopacity and filling capacity (p<0.05). Despite the bioceramic medication presenting less volumetric loss and satisfactory filling capacity, avoiding voids and failures, Bio-C Temp showed lower alkalinity of the medium compared to Calen. Keywords: Endodontics. Immunohistochemistry. Dental Materials. Physical properties. Chemical properties. SUMÁRIO 1 INTRODUÇÃO .................................................................................12 2 PROPOSIÇÃO ..................................................................................18 3 PUBLICAÇÕES ................................................................................19 3.1 Publicação 1 .................................................................................20 3.2 Publicação 2 .................................................................................49 3.3 Publicação 3 .................................................................................75 4 DISCUSSÃO ....................................................................................94 5 CONCLUSÃO ..................................................................................102 REFERÊNCIAS ...............................................................................103 APÊNDICE A ...................................................................................113 APÊNDICE B ...................................................................................127 ANEXO A ........................................................................................138 ANEXO B ........................................................................................139 12 1 INTRODUÇÃO A complexidade da anatomia do sistema de canais radiculares dificulta a completa limpeza e desinfecção1. Dessa maneira, mesmo após o preparo biomecânico os micro-organismos podem permanecer no seu interior2, sendo essa a principal causa de fracasso do tratamento endodôntico3. O uso da medicação intracanal é necessária para os dentes com necrose pulpar pois a microbiota já se difundiu por todo o sistema de canais e no periápice. Assim, a medicação intracanal tem o papel de complementar a desinfecção dos canais radiculares, cemento apical e túbulos dentinários1, se difundido pela dentina em áreas onde o preparo biomecânico não pode ser realizado, estimulando a reparação tecidual e o prognóstico endodôntico4. Além disso, durante o intervalo entre as sessões a medicação intracanal também atua como barreira física prevenindo a reinfecção e reduzindo o risco de proliferação dos remanescentes bacterianos5. As pastas à base de hidróxido de cálcio têm sido amplamente utilizadas como medicação intracanal em diferentes condições endodônticas6, além de serem utilizadas no intuito de otimizar a desinfecção do canais radiculares, trocas periódicas são preconizadas em casos que há necessidade de formação e reparo de tecido mineralizado7, após trauma dentário6, no controle de reabsorções radiculares, no tratamento de lesões periapicais extensas, apicificações, perfurações radiculares8, na endodontia regenerativa6 e na pulpotomia9. O hidróxido de cálcio destaca-se devido ao fato de apresentar ação antimicrobiana e excelentes propriedades biológicas4. As pastas de hidróxido de cálcio possuem a capacidade de inativar a endotoxina bacteriana10, dissolver tecido necrótico11, estimular a diferenciação de osteoblastos12, inibir a reabsorção dentária13 e possui efeito biológico mineralizador8,13. A ação do hidróxido de cálcio é decorrente da dissociação iônica dos íons cálcio e hidroxila7,8,14, pois proporciona um ambiente alcalino que favorece sua ação antimicrobiana e efeito mineralizante14. O efeito antimicrobiano do hidróxido de cálcio é atribuído principalmente à liberação de seus íons hidroxila em meio aquoso9, que se difundem através de túbulos dentinários até a superfície externa da raiz15. Estes íons são radicais livres com alto poder de reatividade16 e capacidade de destruir componentes estruturais da membrana celular das bactérias e diminuir sua atividade celular17. Além disso, os íons cálcio tem função na remoção de gás carbônico que constitui fonte respiratória 13 de bactérias anaeróbias18. O pH alcalino proporcionado pelo hidróxido de cálcio ao microambiente favorece o reparo e a calcificação ativa8,14,19, uma vez que, neutraliza o ácido lático dos osteoclastos e ativa fosfatase alcalina (ALP), enzima que desempenha um papel crucial na formação do tecido duro9. Os íons cálcio também desempenham um papel essencial na mineralização, estimulando a expressão do gene da fibronectina13. Entretanto, a difusão de íons é dependente do veículo e das associações utilizadas na pasta à base de hidróxido de cálcio19. Características como a velocidade da dissociação iônica20, solubilidade21 e difusão22 estão relacionadas ao tipo do veículo associado à medicação. Quando o hidróxido de cálcio é associado a um veículo viscoso, como propilenoglicol 400 e polietilenoglicol 400, a liberação desses íons é lenta e progressiva23,24. A pasta Calen (SS White Artigos Dentários, Rio de Janeiro, RJ, Brasil) é uma medicação intracanal à base de hidróxido de cálcio associada a um veículo viscoso, o polietilenoglicol 400. Esta pasta fornece ao microambiente um pH alcalino, possui ação antimicrobiana21 apresentando capacidade de inativar a endotoxina bacteriana10. Além disso, a pasta Calen é biocompatível25,26, com capacidade de induzir a deposição de nódulos mineralizados27, bem como, a formação de tecido mineralizado19, proporcionando reparo apical satisfatório14,19,28. Embora a medicação à base de hidróxido de cálcio apresente adequadas propriedades biológicas, a permanência da pasta no interior do canal radicular por longos períodos pode afetar as propriedades mecânicas da dentina radicular por degradação do colágeno6. A alcalinidade do hidróxido de cálcio pode também enfraquecer a raiz29 aumentando a suscetibilidade à fratura30, além da medicação à base de hidróxido de cálcio ser permeável aos fluídos dos tecidos e solúvel no canal radicular31. Por outro lado, o hidróxido de cálcio não é efetivo contra todas as bactérias9, apresentando baixa efetividade frente à espécie Enterococcus faecalis32. Estudos têm avaliado a associação do hidróxido de cálcio com outras substâncias com o intuito de melhorar seu espectro de ação33-35. Materiais endodônticos biocerâmicos, ou seja, à base de silicatos de cálcio, têm se destacado por serem bioativos36, devido a interação do material com a dentina formando uma zona intermediária mineralizada na presença de umidade37. Os materiais biocerâmicos são biocompatíveis38-44, não citotóxicos45-47, mostram potencial biotivo45,47e capacidade de induzir a regeneração óssea48, devido pelo 14 menos em parte ao pH alcalino proporcionado ao meio e a liberação de íons cálcio49. Os materiais biocerâmicos também apresentam ação antibacteriana45,47,50 e tomam presa em ambiente úmido51. Os materiais à base de silicato de cálcio são rotineiramente utilizados na Endodontia em procedimentos envolvendo a regeneração pulpar e o reparo de tecidos duros, como material reparador durante o capeamento pulpar, pulpotomia, apexificação, reparo de perfuração e retrobrutação, bem como, na obturação dos canais radiculares52. Atualmente existe um interesse crescente no desenvolvimento de novos materiais endodônticos à base de silicato de cálcio53. Deste modo, a Angelus® Indústria de Produtos Odontológicos S/A (Londrina, Brasil)54 desenvolveu um novo medicamento endodôntico biocerâmico, o Bio-C Temp. Esta medicação é uma pasta, pronta para o uso, à base de silicatos de cálcio associada aos radiopacificadores tungstato de cálcio e óxido de titânio; esse medicamento biocerâmico também apresenta na sua composição o aluminato de cálcio, óxido de cálcio e resina base como veículo. O Bio-C Temp é indicado como um curativo intracanal no tratamento endodôntico, retratamento, em dentes com rizogênese incompleta, perfurações e reabsorções externas e internas54. De acordo com o fabricante, o Bio-C Temp dispensa e/ou elimina a necessidade de novas aplicações, constituindo-se numa vantagem em relação às medicações à base hidróxido de cálcio que exigem trocas frequentes54. O Bio-C Temp deve ser removido do canal radicular após algum tempo por meio de protocolos semelhantes aos usados para as pastas à base de hidróxido de cálcio54. As partículas de silicato de cálcio, presente na pasta biocerâmica, em ambiente úmido hidratam formando o silicato de cálcio hidratado na superfície da partícula. No entanto, as cadeias poliméricas presentes no material dificultam a ligação das partículas hidratadas e, em consequência, impossibilita que a medicação tome presa53. Este novo medicamento à base de silicato de cálcio mostrou efeito não citotóxico em extratos mais diluídos associados à cultura de fibroblastos53, células da polpa dental humana (hDPCs)55,56 e células osteoblásticas humanas (Saos-2)27. O Bio-C Temp induziu atividade osteogênica em ensaio in vitro em células da linhagem Saos-2, demonstrada pela deposição de nódulos mineralizados e atividade de ALP semelhante às medicações à base de hidróxido de cálcio, UltraCal XS (UltraDent 15 Product, Inc., Indaiatuba, SP, Brasil) e Calen. No entanto, com relação à atividade antimicrobiana, o Bio-C Temp apresentou menor efetividade contra E. faecalis na forma planctônica e de biofilme por meio do teste de contato direto e de cristal violeta em comparação as medicações à base de hidróxido27. Embora o meio aquoso contendo o Bio- C Temp tenha apresentado pH alcalino, o valor deste foi inferior ao Ultracal XS, uma medicação à base de hidróxido de cálcio associada a um veículo aquoso, nos períodos de 1, 24 e 72 horas. No entanto, diferenças significantes nos valores do pH das soluções contendo Bio-C Temp e Ultracal XS não foram detectadas aos 7 dias. A liberação de cálcio do material biocerâmico para o microambiente foi menor do que a pasta de hidróxido de cálcio apenas no período de 24 horas. Baseado nas normas International Organization for Standardization (ISO 6876:2016), o Bio-C Temp apresentou maior radiopacidade (cerca de 7 mm/Al) e ambos os materiais não promoveram penetração nos túbulos dentinários como foi demonstrado pela análise com microscopia confocal de varredura a laser (CLSM)53. Quando comparado aos materiais reparadores biocerâmicos, Bio-C Repair (Angelus, Londrina - PR, Brasil) e Biodentine (Septodont, Saint Maur des Fossés, France), o Bio-C Temp forneceu soluções com alcalinidade semelhante, porém maior liberação de cálcio após 24 horas de imersão. Utilizando dentes bovinos, os autores observaram que os três materiais foram associandos com a descolaração dental e a medicação biocerâmica mostrou menor radiopacidade que o Bio-C Repair que apresenta na composição óxido de zircônia como radiopacificador22. Estudo similar em dentes bovinos demonstrou que o Bio-C Temp apresentou maior radiopacidade e promoveu menor alteração de cor dentinária em comparação ao Ultracal XS e ao material reparador MTA Flow (UltraDent Product, Inc., Indaiatuba, SP, Brasil)55. Uma vez que a medicação entra em íntimo contato com tecidos perirradiculares, sua composição química e os compostos tóxicos liberados podem interferir na resposta inflamatória e, consequentemente, no processo de reparo57. O implante de materiais dentários no tecido conjuntivo subcutâneo de ratos é um método recomendado pela ISO 1099358 que permite avaliar a reação inflamatória em resposta aos materiais bem como permite avaliar as cascatas de eventos que culminam na regressão do processo inflamatório e a sua substituição por tecido conjuntivo denso, contendo fibroblastos e fibras colágenas40,42. Assim, a análise em 16 diferentes períodos permite avaliar a intensidade do processo inflamatório, os principais tipos celulares e se o mesmo persiste ao longo do tempo, constituindo-se num parâmetro de avaliação das propriedades biológicas dos materiais dentários59. Os materiais endodônticos podem liberar substâncias que estimulam a migração e a diferenciação das células inflamatórias, induzindo também a produção de citocinas moduladoras da inflamação, tais como a interleucina 6 (IL-6)40-44,60,61. Esta citocina tem se constituído num parâmetro de análise do processo inflamatório, pois apresenta uma correlação direta com a regressão do processo inflamatório nas cápsulas formadas em resposta aos implantes de materiais endodônticos39. Em contrapartida a IL-10 apresenta uma marcante função anti-inflamatória mostrando a capacidade de inibir a síntese e liberação de citocinas pró-inflamatórias, como a IL-1, IL-6 e IL-862,63. Portanto, a detecção da IL-10 possibilita uma melhor compreensão dos mecanismos relacionados na regressão da resposta inflamatória induzida pelos biomateriais endodônticos64. Além disso, a neoformação de constituintes da matriz dos tecidos mineralizados pode ser avaliada por imuno-histoquímica para detectar as diversas proteínas existentes, como osteocalcina e osteopontina65. Estudos relataram que materiais contendo silicato de cálcio induziram a imunoexpressão de osteopontina66,67 e osteocalcina38,43,66,67 nas células do tecido conjuntivo do subcutâneo de ratos. Adicionalmente, estudos reportaram que materiais à base de silicato de cálcio induzem a precipitação de cálcio nas cápsulas formadas adjacentes aos implantes no subcutâneo de ratos, evidenciado pelo método histoquímico de von Kossa38,41-44,60. A implantação no subcutâneo de ratos também permite investigar se os materiais odontológicos podem promover alterações sistêmicas, uma vez que estes materiais liberam substâncias que podem por via sanguínea podem atingir órgãos intimamente associados à manutenção do metabolismo do organismo44. A dosagem dos níveis séricos das enzimas transaminase glutâmico-oxalacética (TGO) e transaminase glutâmico-pirúvica (TGP) podem ser utilizadas como parâmetros bioquímicos da função hepática68. Além disso, constituintes inorgânicos de tecidos mineralizados, incluindo cálcio (Ca+2) e fósforo (P-), e ALP, uma enzima envolvida na mineralização, podem ser mesurados por meio da coleta de sangue no intuito de investigar se os materiais endodônticos causam alterações nesses parâmetros associados à biomineralização66. 17 Por ouro lado, a reação tecidual (biocompatibilidade e potencial bioativo) de um material odontológico, incluindo a medicação intracanal, está intimamente associada as suas propriedades fisico-químicas. Deste modo, a investigação destas propriedades, dentre elas o pH, liberação de íons cálcio e solubilidade são relevantes para prever o comportamento clínico das medicações21. É importante que a medicação se difunda pelos túbulos dentinários e ramificações laterais do sistema de canais radiculares, preenchendo todo o canal e agindo por contato direto ou indireto nos túbulos dentinários69. A radiopacidade da medicação intracanal é importante para avaliar se o preenchimento dos canais radiculares ocorreu satisfatoriamente, além de permitir o acompanhamento clínico do tratamento21. A microtomografia computadorizada (micro-CT) é uma ferramenta de análise tridimensional não destrutiva70 que complementa o teste convencional de análise de solubilidade permitindo uma análise volumétrica (em mm³) dos materiais e a capacidade de preenchimento dos materiais71 por meio da análise da interface dentina e material, uma vez que, a alta resolução, permite observar a presença de defeitos e espaços vazios72. Essas análises são importantes, pois a perda volumétrica do material, bem como, a presença de espaços vazios após o preenchimento do canal impede o contato direto do material com as bactérias prejudicando a ação antimicrobiana, além de permitir a reinfecção bacterina no interior do canal radicular21. Portanto, a total eliminação de micro-organismos resistentes continua sendo um grande desafio na terapia endodôntica, sendo fundamental a busca por medicamentos eficazes73. Considerando que é essencial o conhecimento das propriedades fisico-químicas para a escolha de uma de medicação intracanal23 e o possível contato de um material endodôntico com os tecidos periapicais, é importante que as propriedades físico-químicas e biológicas de novos materiais, ou modificações na composição de materiais já conhecidos, sejam avaliadas por diferentes metodologias74,75. 18 2 PROPOSIÇÃO Avaliar a resposta sistêmica, local e o potencial bioativo de uma medicação intracanal biocerâmica, Bio-C Temp (Angelus, Londrina, Brasil), bem como avaliar algumas propriedades físicas e químicas desta medicação. Os resultados obtidos desta pasta biocerâmica foram comparados ao Calen (SS. White Art. Dent. Ltda, RJ, Brasil), uma pasta à base de hidróxido de cálcio, usada rotineiramente na clínica endodôntica. Publicação 1: Avaliação da biocompatibilidade da medicação intracanal, Bio- C Temp, em comparação a pasta Calen. Para atingir o objetivo foram realizadas as análises morfológicas, morfométricas e reações imuno-histoquímicas para detecção da interleucina-6 e interleucina-10 nas cápsulas formadas ao redor dos implantes. Adicionalmente, mensurações dos níveis séricos de TGO e TGP foram realizadas para verificar se as medicações induzem alterações nas enzimas hepáticas. Publicação 2: Avaliar se o Bio-C Temp promove alterações nas concentrações de cálcio, fósforo e fosfatase alcalina no soro após a implantação no tecido conjuntivo do subcutâneo de ratos. Além disso, o potencial bioativo do Bio-C Temp foi avaliado nas cápsulas formadas ao redor dos implantes. Esta hipótese foi avaliada por meio da detecção imuno-histoquímica de osteocalcina e de osteopontina, reação de von Kossa e da análise de cortes não corados sob luz polarizada. Publicação 3: Avaliação do pH, radiopacidade, escoamento, solubilidade, bem como, alteração volumétrica e capacidade de preenchimento da medicação intracanal Bio-C Temp em comparação ao Calen. 19 3 PUBLICAÇÕES O presente trabalho resultou em três artigos. O artigo 1 intitulado “Hepatic enzymes and immunoinflammatory response to Bio-C Temp bioceramic intracanal medication implanted into the subcutaneous tissue of rats”, publicado no “Scientific Reports”. Lopes CS, Delfino MM, Tanomaru-Filho M, Sasso-Cerri E, Guerreiro-Tanomaru JM, Cerri PS. Hepatic enzymes and immunoinflammatory response to Bio-C Temp bioceramic intracanal medication implanted into the subcutaneous tissue of rats. Sci Rep. 2022; 12(1): 2788. O artigo 2 intitulado “Bioactive potential of Bio-C Temp demonstrated by systemic markers of mineralization and immunoexpression of bone proteins after implantation in rat subcutaneous tissues” foi redigido para submissão no “Journal of Biomedical Materials Research Part B”. (Em revisão pelos autores para submissão). O artigo 3 intitulado “Physicochemical properties of a calcium silicate intracanal medication: Bio-C Temp” será submetido a publicação no “International Endodontic Journal”. 20 3.1 Publicação 1  Hepatic enzymes and immunoinflammatory response to Bio-C Temp bioceramic intracanal medication implanted into the subcutaneous tissue of rats Camila Soares Lopes 1 , Mateus Machado Delfino 1 , Mário Tanomaru-Filho 1 , Estela Sasso- Cerri 2 , Juliane Maria Guerreiro-Tanomaru 1 , Paulo Sérgio Cerri 2* 1 Department of Restorative Dentistry, Dental School – São Paulo State University (UNESP), Araraquara, SP, Brazil. 2 Laboratory of Histology and Embryology - Department of Morphology, Genetics, Orthodontics and Pediatric Dentistry - Dental School – São Paulo State University (UNESP), Araraquara, SP, Brazil. Running title: In vivo evaluation of Bio-C Temp * Address for all correspondence: Prof. Dr. Paulo Sérgio Cerri Laboratory of Histology and Embryology – Dental School São Paulo State University (UNESP), Araraquara, SP, Brazil. Phone: +55 16 33016497 Fax: +55 16 33016433 e-mail: paulo.cerri@unesp.br  Artigo escrito segundo normas do periódico Scientific Reports. Artigo aceito em 5 de janeiro de 2022. 21 ABSTRACT This study evaluated the tissue reaction and serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) levels of a new bioceramic intracanal medicament, Bio-C Temp (BIO). The biological properties of BIO were compared with calcium hydroxide- based intracanal medicament (Calen; CAL), used as gold pattern. Polyethylene tubes filled with BIO or CAL, and empty tubes (control group, CG) were implanted into subcutaneous tissue of rats. After 7, 15, 30 and 60 days, the samples were embedded in paraffin for morphological, quantitative and immunohistochemistry analyses. At 7 and 60 days, blood samples were collected for analysis of serum GOT and GPT levels. The data were submitted to two-way ANOVA and Tukey’s test (p ≤ 0.05). No significant difference was detected in serum GOT and GPT levels among BIO, CAL and CG specimens. In all periods, BIO specimens exhibited lower number of inflammatory cells and immunoexpression of IL-6, a pro-inflammatory cytokine, than CAL specimens. The reduction of these parameters was accompanied by significant increase in the collagen content and in the immunoexpression of IL-10, a cytokine involved in the tissue repair, over time. Biochemical analysis suggests that Bio-C Temp had no hepatotoxicity effect. Moreover, histological findings indicate that Bio-C Temp is biocompatible. INTRODUCTION The complexity of the root canal system can interfere with its complete cleaning and disinfection and even after biomechanical preparation, microorganisms can remain in the root canal system, leading to endodontic treatment failure 1 . Therefore, the use of intracanal medication may enhance the disinfection of root canal system, apical cementum and dentinal tubules 2 . Moreover, intracanal medication acts as a physical barrier preventing reinfection and reducing the risk of bacteria proliferation 3 . The calcium hydroxide pastes have been widely used as an intracanal medication acting as a complement to biomechanical preparation due to its antimicrobial potential 2 , ability to dissolve organic tissue 4 , stimulate the osteoblast proliferation 5 , and induction of mineralized tissue formation 6 . There is the search for new medications or associations with the materials routinely used in order to enhance the action of the intracanal dressing 7 . The bioceramic material has been used due to excellent biocompatibility and bioactive potential of the calcium silicate 8 , which promotes the deposition of hydroxyapatite on its 22 surface 9 . The bioactivity of bioceramic materials favor the survival and differentiation of osteoblasts, cells actively involved in the periapical repair 10 enabling the formation of mineralized tissue 11 . Thus, a bioceramic intracanal medication, Bio-C Temp, has been developed by Angelus (Angelus, Londrina, PR, Brazil), ready to use. The Bio-C Temp is indicated for conventional endodontic treatment, endodontic retreatment, apexification and endodontic regeneration 12 . According to the manufacturer, Bio-C Temp contains calcium silicates associated with calcium tungstate and titanium oxide radiopacifiers, besides calcium aluminate, calcium oxide and base resin. This new intracanal medication has shown acceptable cell viability 12-14 and osteogenic activity in vitro 12 , however, there are no in vivo studies on the biological properties of this material. The implantation into subcutaneous tissues of rats is a method, which has been widely used to analyze the tissue response to the materials as well as the type and extent of the inflammatory reaction 15 . The aim of this study was evaluate whether Bio-C Temp induces changes in the serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) levels and the tissue reaction induced by this bioceramic intracanal medication in the subcutaneous connective tissue in different time points. The biological response induced by Bio-C Temp was compared with a calcium hydroxide-based paste (Calen, SS.White, Rio de Janeiro, RJ, Brazil), used as gold pattern. The null hypothesis was that Bio-C Temp does not present better biological properties when compared with the calcium hydroxide paste. RESULTS Serum hepatic enzymes level As shown in the Figs. 1A and 1B, significant differences in serum TGO and TGP concentrations among BIO, CAL and CG specimens (p  0.05) were not observed, at 7 and 60 days. Moreover, from 7 to 60 days, no significant difference was also seen in serum TGO and TGP levels in all groups (p  0.05). 23 Figure 1 (A-B) – The serum levels of glutamic-oxaloacetic transaminase (GOT - Fig. A) and glutamic-pyruvic transaminase (GPT – Fig. B) in Bio-C Temp, Calen and Control groups at 7 and 60 days. In rats with intracanal medication, the GOT and GPT levels were similar to CG rats. From 7 to 60 days, significant differences are not seen in the serum GOT and GPT levels. Values expressed as mean ± SD. Tukey’s test (p≤0.05). In each period, superscript letters indicate the analysis among groups; same letters = no significant difference. The superscript numbers indicate the analysis of each group over time; same numbers = no significant difference. 24 Morphological findings, capsule thickness and numerical density of ICs In all groups, well-defined capsules were around the implants (Fig. 2A-L). In BIO and CAL there was an increase in the thickness of the capsules over time. Moreover, material particles were often observed dispersed by the capsules (Fig. 2A, B, D, E, G, H, J and K). At 7 days, necrotic areas were observed in the surface of capsules of BIO and CAL (Fig. 2A-B); in the CAL, necrotic areas were seen at all periods. The CG specimens were surrounded by thin capsules (Fig. 2 C, F, I and L). According to Figure 2M, no significant difference was found in the capsule thickness between BIO and CAL specimens at 7 days (p = 0.98). The capsules of BIO specimens were significantly thicker than in CAL (p < 0.0001) at 15, 30 e 60 days. In BIO and CAL specimens, a significant increase in the capsule thickness was verified at 30 days, but a significant reduction was noticed at 60 days (p < 0.0001). At all periods, GC specimens exhibited the lowest values in the capsule thickness and a significant reduction was detected over time (p < 0.0001). At 7 days, the capsules of all groups contained several ICs, mainly macrophages and lymphocytes, and few fibroblasts (Figs. 3A-C). However, significant differences were observed among the groups (p < 0.0001); the lowest values of ICs were obtained in CG capsules while the highest values were found in CAL specimens (Figs. 3A-M). In BIO and CG, a significant reduction in the number of ICs was observed over time (p < 0.0001). Although the number of ICs reduced significantly in CAL specimens from 7 to 15 days (p < 0.0001), no significant difference (p = 0.61) was detected between the periods of 15 and 30 days. At 60 days, the number of ICs in all groups was lower (p < 0.0001) than other time points (Fig. 3M). 25 26 Figure 2 (A-L) – Light micrographs of sections showing an overview of capsules adjacent to the opening of the implanted tubes (T), at 7 (Figs. A-C), 15 (Figs. D-F), 30 (Figs. G-I) and 60 (Figs. J-L) days. In all periods, well-defined capsules (C) are seen. At 15, 30 and 60 days, thick capsules are adjacent to the BIO (D, G and J) and CAL (E, H and K) groups. In BIO specimens, several particles (brown/black colour) are dispersed throughout the capsules (C). In all periods, thin capsules are observed in the CG. N, necrosis area. HE. Bars: 152 µm. Figure 2M – The graphic shows the values (expressed as mean ± standard deviation) of capsule thickness (in µm) of Bio-C Temp (BIO), Calen (CAL) and Control (GC) groups at 7, 15, 30 and 60 days. In each period, the comparison among the groups is indicated by superscript letters; different letters = significant difference. The superscript numbers indicate the analysis of each group over time; different numbers = significant difference. Tukey’s test (p≤0.05). 27 28 Figure 3 (A-L) – Light micrographs showing the innermost portion of capsules. Inflammatory cells (arrows), fibroblasts (Fb), collagen fibers (CF) and material particles (arrowheads) are seen in the capsules. At 15, 30 and 60 days, the capsules of the control group exhibit few inflammatory cells (arrows) and a gradual increase in fibroblasts (Fb) and collagen fibers (CF). BV, blood vessels; HE. Bars: 18 µm. Figure 3M – The graphic shows the values (expressed as mean ± standard deviation) of the numerical density of inflammatory cells in the capsules. In each period, the comparison among the groups is indicated by superscript letters; different letters = significant difference. The superscript numbers indicate the analysis of each group over time; different numbers = significant difference. Tukey’s test (p≤0.05). 29 Immunohistochemical detection of IL-6 The capsules of all groups showed ICs, particularly neutrophils and macrophages, containing strong immunolabelling in their cytoplasm (brown-yellow colour) in all periods. Moreover, some immunolabelled fibroblasts were also observed (Figs. 4A-L). A reduced immunoexpression was observed in the capsules of CG, particularly at 30 and 60 days (Figs. 4 C, F, I and L). The quantitative analysis (Fig. 4M) revealed that the number of IL-6-immmunostained cells decreased significantly from 7 to 60 days (p < 0.0001), in all groups. In all periods, the number of immunolabelled cells was significantly lower around the BIO specimens than in CAL (p < 0.0001) while the lowest values were observed in the capsules of CG. Immunohistochemical detection of IL-10 In all periods, the immunohistochemistry reaction for detection of IL-10 revealed immunolabelled cells (brown-yellow colour) in the capsules of all groups. Although some ICs and fibroblasts exhibited immunolabelling for IL-10, strong immunolabelling in the cytoplasm of mast cells was often seen in the capsules of all groups (Figs. 5A-L). According to Figure 5M, the lowest values of IL-10-immunolabelled cells were found in all groups at 7 days. From 7 to 60 days, the immunoexpression of IL-10 increased significantly in all groups (p < 0.0001). At 7 days, no significant difference among the groups was detected (p > 0.05). At 15, 30 and 60 days, the number of IL-10-immunolabelled cells was significantly lower in CG specimens than in BIO and CAL groups (p < 0.0001). On 15 th day, the highest values were found in BIO specimens while, at 30 and 60 days, the highest values were observed in the CAL specimens (p < 0.0001). 30 31 Figure 4 (A-L) - Light micrographs showing portions of capsule of sections submitted to immunohistochemistry reaction to detection of IL-6 (brown/yellow colour) and counterstained with haematoxylin. In all periods, inflammatory cells with strong immunolabelling in their cytoplasm (arrows) are present in the capsules of all groups. Immunolabelled fibroblasts (Fb) are also observed. BV, blood vessels; material particles (arrowheads). Bars: 18 µm. Figure 4M – The graphic shows the values (expressed as mean ± standard deviation) of the numerical density of IL-6-immunolabelled cells in the capsules around Bio-C Temp (BIO), Calen (CAL) and Control (GC) specimens at 7, 15, 30 and 60 days. In each period, the comparison among the groups is indicated by superscript letters; different letters = significant difference. The superscript numbers indicate the analysis of each group over time; different numbers = significant difference. Tukey’s test (p≤0.05). 32 33 Figure 5 (A-L) - Light micrographs showing portions of capsule of sections submitted to immunohistochemistry reaction to detect IL-10 (brown/yellow colour) and counterstained with haematoxylin. Note that few immunostained cells are present in the capsules of all groups, particularly at 7 days (Figs. A-C). In all groups, an evident immunolabelling is present in the mast cells (MC). Inflammatory cells (arrows); BV, blood vessels; Arrowhead: material particles. Bars: 18 µm. Figure 5M – The graphic shows the values (expressed as mean ± standard deviation) of the numerical density of IL-10-immunolabelled cells in the capsules around Bio-C Temp (BIO), Calen (CAL) and Control (GC) specimens at 7, 15, 30 and 60 days. In each period, the comparison among the groups is indicated by superscript letters; different letters = significant difference. The superscript numbers indicate the analysis of each group over time; different numbers = significant difference. Tukey’s test (p≤0.05). 34 Birefringent collagen content in capsules At 7 days, all groups exhibited capsules with few and thin birefringent collagen fibres (Figs. 6A-C). On 60 th day, an evident increase in the birefringence was observed in the capsules of all groups (Figs. 6D-F). The quantitative analysis (Fig. 6G) showed that the amount of birefringent collagen was similar among the groups (p ˃ 0.05) at 7 and 15 days. Although no significant difference between BIO and CAL was found at 30 days, the amount of birefringent collagen was significantly greater in BIO than CAL specimens at 60 days (p < 0.0001). After 30 and 60 days, the collagen content was significantly greater in CG than in BIO and CAL groups (p < 0.0001). The analysis revealed a significant increase in the collagen amount in BIO group at 60 days (p < 0.0001). In contrast, no significant difference was detected in the CAL group over time (p > 0.05). In the CG, the amount of collagen was significantly greater at 30 and 60 days than in the periods of 7 and 15 days. 35 36 Figure 6 (A–F) – Light micrographs showing portions of capsule from sections subjected to picrosirius-red and analyzed under polarized illumination. At 7 days, thin birefringent collagen fibres (in red-orange) are dispersed in the capsules. At 60 days, the capsules exhibit thick bundles of birefringent collagen; note an accentuated birefringence in capsules of BIO (Fig. D) and CG (Fig. F) groups compared to the CAL group (Fig. E). Bars: 18 µm. Figure 6M – The graphic shows the values (expressed as mean ± standard deviation) of the amount of birefringent collagen (in percentage) in the capsules around Bio-C Temp (BIO), Calen (CAL) and Control (GC) specimens at 7, 15, 30 and 60 days. In each period, the comparison among the groups is indicated by superscript letters; different letters = significant difference. The superscript numbers indicate the analysis of each group over time; different numbers = significant difference. Tukey’s test (p≤0.05). 37 DISCUSSION This is the first study that evaluated the serum GOT and GPT levels and biocompatibility in vivo of Bio-C Temp bioceramic intracanal medicament after implantation into the subcutaneous tissues of rats. Morphological and quantitative analyses of capsules around intracanal medications at different time points allows evaluating the injured tissue as well as the complex cascade of cytokines caused by these medicaments, which may lead to chronic inflammatory reaction or tissue repair 16-18 . Furthermore, this methodology allows us to investigate whether dental materials could promote systemic changes 18 . The null hypothesis was rejected since Bio-C Temp favouring the connective tissue repair quickly in comparison with the calcium hydroxide paste. In the present study, both the bioceramic medication and calcium hydroxide-based paste did not promote changes in serum GOT and GPT levels in comparison with CG after 7 and 60 days of implantation. Therefore, the intracanal medications may not be hepatotoxic, since the measurement in serum of GOT and GPT levels can be used as biochemical parameters of liver function 19 . Usually, hepatic enzymatic changes are associated with structural liver damage 20 . Low cellular viability has been demonstrated when extracts of Bio-C Temp is added to human dental pulp cells (hDPCs) 14 and osteoblast-like cell line (Saos-2) 12 . The low cytocompatibility of this bioceramic intracanal medication may be explained by presence of titanium dioxide 14 , which promotes cell damage culminating in cell death by apoptosis 21 . According to the manufacturer Bio-C Temp also contains base resin in its composition. It is known that resins can release substances, which may promote local and systemic adverse reactions 22 . Here, serum GOT and GPT levels suggest that Bio-C Temp and calcium hydroxide-based pastes may not promote liver injury. However, further studies are need to evaluate whether these intracanal medications promote structural changes in the liver. The morphological and quantitative analyses revealed that, in all groups, the greatest values of inflammatory cells were observed at 7 days. The inflammatory reaction induced by empty polyethylene tubes suggest that surgical trauma may stimulate the recruitment of inflammatory cells and, may be responsible, at least the part, for highest numerical density of inflammatory cells initially found, as reported in other studies 16,17,23-25 . However, the number of ICs in the BIO and CAL specimens was around two folds greater than in CG, which may be attributed to the alkaline pH and 38 chemical composition of these intracanal medications. The Calen paste provides an alkaline pH around 12.4 to microenvironment 26 and, Bio-C Temp provides pH around 10.79 after 7 days 13 . The alkaline pH induces the recruitment of ICs to the microenvironment 16,27,28 and formation of a coagulation necrosis zone 27 in the tissue surface in contact with the material 15,29,30 . At all periods, necrotic areas remained until 60 days and the greatest number of ICs were observed in the capsules around calcium hydroxide-based paste specimens, indicating, therefore, that the release of hydroxyl ions may be maintained for a long time in the in calcium hydroxide-based pastes associated with a viscous vehicle. Furthermore, this group exhibited greater IL-6-immunostaining and lower amount of collagen than Bio-C Temp. IL-6 is a multifunctional pro-inflammatory cytokine, being an indicative parameter of the inflammatory reaction intensity 31,32 . Studies have shown a direct correlation between the reduction in the number of ICs and IL-6 immunoexpression in the capsules adjacent to the calcium silicate-based materials implanted in rat subcutaneous 17,31,33,34 . In the present study, the reduction in the immunoexpression of IL-6 was also accompanied by decrease in the number of ICs reinforcing the concept that this interleukin may modulate the inflammatory reaction in response to the endodontic materials. The irritating potential of Calen can also be justified by the harmful activity of zinc ions from the radiopacifying agent zinc oxide present in this paste 35 . However, this irritant potential decreases over time corroborating with other studies 15,29,30 . The thickness of capsules around BIO and CAL specimens increased significantly until on 30 th day. The thickening of connective tissue around intracanal medications was accompanied by increase of material particles dispersed throughout the capsules. The massive presence of material particles may be due to the flow and solubility of these pastes, particularly, of Bio-C Temp. However, these properties are needed to the filling of the root canals and penetration into the dentinal tubules and periapical region 3 , acting in areas which are not achieved by the channel instrumentation 2 . The Calen has as vehicle the polyethylene glycol 400, which improves the dissolution of calcium hydroxide and release of hydroxyl ions 26 . Bio-C Temp contains in its composition the base resin as a vehicle, which allows better insertion of the material in the root canals 36 . From 30 to 60 days, the thickness of capsules around intracanal medications reduced significantly suggesting a remodelling process of the connective tissue of these capsules. 39 There is evidence showing a strong positive correlation between IL-6 and matrix metalloproteinases (MMP), such as MMP-1 and MMP-9, enzymes responsible for extracellular matrix degradation 37 . Here, it is possible to suggest that the IL-6 may exert a control on the degradation of extracellular matrix components since the highest values of this interleukin were parallel to lowest collagen content in the CAL specimens while the lowest immunoexpression of IL-6 was concomitant with accentuated amount of collagen in CG specimens. These findings point to a participation of IL-6 in the breakdown of extracellular matrix components in the capsules around implants in the subcutaneous tissue. Moreover, the reduction in the immunoexpression of IL-6 was also accompanied by gradual increase of IL-10-immunostaining over time. In the present study, several mast cells exhibited an accentuated immunolabelling for IL-10. Although IL-10-immunopositve mast cells were observed in all periods, the marked presence of immunostained mast cells at 60 days may be associated with the high formation of collagen, suggesting tissue repair. One of the anti-inflammatory actions of IL-10 is the ability to modulate the production of inflammatory cytokines by mast cells. Mast cells contain IL- 10 receptor, and, via IL-10, release several inflammatory mediators, which mediate the immune and inflammatory responses [Blank] and participate in the tissue repair and remodeling 32,38 . The accentuated number of mast cells in the capsules around a reparative calcium silicate-based biomaterial, Biodentine (Septodont, Saint-Maur-des-Fossèes, France), was associated with the proliferation of fibroblasts and, consequently, with the collagen formation supporting the concept that this cell is involved in the connective tissue repair 32 . Moreover, there is evidence that IL-10 inhibits IL-6 production by mast cells in response to inflammation and bacterial infection 39 . Thus, our findings taken together indicate that the increase in the IL-10 may inhibit the immunoexpression of IL-6 in the capsules mitigating the inflammatory reaction promoted by bioceramic and calcium hydroxide intracanal medications implanted in the subcutaneous tissues. No study evaluating the biological in vivo behavior of the Bio-C Temp was found in the literature. Our findings indicate that, in the initial period, bioceramic medication induced an intense inflammatory reaction, which gradually reduced over time. Thus, at 60 days, the capsules around Bio- C Temp specimens showed bundles of collagen fibres surrounding the material particles, pointing to a 40 reorganization of the connective tissue. The biocompatibility of tricalcium silicate-based materials 16,23,25 seems to be due to the stable chemical comportment of the calcium silicate in biological environment 40 . Therefore, it is expected that Bio-C Temp, when in contact with periapical tissues, may induce the tissue repair. However, further studies are necessary to confirm the safety and viability of this new intracanal medication in clinical practice. In conclusion Serum GOT and GPT levels suggest that bioceramic and calcium hydroxide- based intracanal medications had no hepatotoxicitiy effect. Bio-C Temp caused initial tissue damage that was quickly suppressed in comparison to those caused by calcium hydroxide-based paste, favouring the repair of connective tissue indicating, therefore, that the new bioceramic material is biocompatible. MATERIALS AND METHODS Experimental procedures The present research protocol was approved by the Ethical Committee for Animal Research. Sixty adult male Holtzman rats (Rattus norvegicus albinus) weighing 220-250 g were used. The rats were maintained in polyethylene cages under 12h light/12h dark cycle at controlled temperature (23±2°C) and humidity (55±10%), with water and food provided ad libitum. The study was carried out in accordance with the US National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications nº 80-23, 1996). Sixty rats were randomly distributed into three groups containing 20 animals each: BIO (BIO- C TEMP Group, Angelus, Londrina, Brazil); CAL (Calen Group, SS. White Art. Dent. Ltda, RJ, Brazil) and CG (control group, empty polyethylene tubes). Each cage was identified according to the group and period. The sample size for this study was calculated based in previous studies [16-19]. The size sample was calculated considering an alpha error of 0.05 to recognize a significant difference and 90% test power for detection of 50% difference among experimental groups and CG. Thus, a sample of 5 rats per group in each time point was required, totalling twenty animals per group. The animals were anaesthetized with ketamine hydrochloride (80 mg/kg of body weight) and xylazine hydrochloride (8 mg/kg of body weight) by intraperitoneal route. After shaved and 41 disinfection with 5% iodine solution, a 2.0 cm-long incision was made in a head-to-tail orientation using a nº 15 scalpel (Fibra Cirúrgica, Joinvile,SC, Brazil). The polyethylene tube (Embramed Indústria e Comércio Ltda, São Paulo, São Paulo, Brazil) with 10.0 mm length and 1.6 mm diameter, previously sterilized with ethylene oxide, were implanted into the subcutaneous tissue. In each animal, two polyethylene tubes of the same group were implanted for 7, 15, 30 and 60 days. Serum hepatic enzymes level To evaluate whether intracanal medicantions cause changes in the hepatic enzymes (GOT and GPT), the blood samples were collected through cardiac puncture with BD Vacutainer® Blood Collection Tubes (SSTII Plus, BD Biosciences) at 7 and 60 days. After clot formation, the blood was centrifuged (Excelsa® II 206 BL; Fanem Ltda., Guarulhos, SP, Brazil) at 3500 rpm for 10 minutes and the serum was stored at -20° C. The GOT and GPT serum concentrations were determined using the AST (aspartate aminotransferase) and ALT (Ianine Aminotransferase), respectively, by chemiluminescence immunoassay kits (Beckman Coulter, CAR, USA). Concentrations were measured using a multiparametric automatic analyzer (Cobas ÍNTEGRA® 400 Plus; Roche Diagnóstica Brasil Ltda., São Paulo, SP, Brazil). The analyses were performed at São Lucas Clinical and Microbiological Laboratory. The experiments were carried out in duplicate and the averages were calculated. Procedures for paraffin embedding The tissue reaction promoted by intracanal medications was carried out in all groups at 7, 15, 30, and 60 days. The animals were euthanized with overdose of ketamine and xylazine and, subsequently, the specimens containing the implants surrounded by tissues were removed and immersed in 4% formaldehyde (freshly prepared from paraformaldehyde) buffered at pH 7.2 with 0.1 M sodium phosphate. After 48 hours, the specimens were dehydrated with ethanol graded concentrations, treated with xylene, and embedded in paraffin. In each specimen, forty serial longitudinal sections (6 µm thick) were obtained and adhered to glass slides. Three non-serial sections were stained with Carazzi’s haematoxylin and eosin (HE) for morphological analysis of the capsules, and to estimate the capsule thickness and the numerical density of inflammatory cells. Three non-serial sections were subjected to the picrosirius-red to estimate the collagen content. Other non-serial 42 sections were adhered to slides previously treated with silane 4% (Sigma-Aldrich) and submitted to the immunohistochemistry reactions for detection of interleukin-6 (IL-6) and IL-10. The histological description and quantitative data were obtained using a digital camera (DP- 71, Olympus, Tokyo, Japan) attached to a light microscope (Olympus BX-51, Tokyo, Japan) and an image analysis system (Image-Pro Express 6.0, Olympus). The analyses were conducted by one calibrated and blinded examiner. Numerical density of inflammatory cells The ICs was obtained from three non-serial sections of each implant. In each section, an image of the central portion of capsule in close juxtaposition to the opening of implanted tube was captured at x695 magnification. In this standardized field (0.09 mm 2 ), the number of inflammatory cells (neutrophils, lymphocytes, plasma cells, and macrophages) was computed using the image analysis system. Thus, the number of inflammatory cells per millimetre square of capsule was obtained dividing the total number of inflammatory cells by the total standardized field of capsule 16,17,23,31,34 . Capsule thickness For each specimen, three HE-stained non-serial sections were used, totalling15 sections per group in each period. To estimate the capsule thickness, an image at x65 magnification was captured; the measurement was made from the capsule surface to the adjacent tissues (in micrometers). In each specimen, the mean value of capsule thickness was calculated from the three sections analyzed 31,33,34 . Immunohistochemical detection of IL-6 and IL-10 For the detection of interleukin-6 and interleukin-10, mouse monoclonal anti-IL-6 antibody (Abcam Inc., Cambridge, MA, USA; code: ab 9324) and mouse monoclonal anti-IL-10 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; code SC-8438) were used. After deparaffinization and hydration, the slides were immersed in 0.001M sodium citrate buffer (pH 6.0) and subjected to microwave two cycles for 20 minutes at 96-98ºC. After cooling-off, the slides were washed in 0.01 M sodium phosphate buffer (PBS) for 15 minutes. For inactivation of endogenous peroxidase, the sections were immersed for 30 minutes in 5% aqueous hydrogen peroxide. After washing in PBS, the 43 sections were incubated for 20 minutes with 2% bovine serum albumin (Sigma-Aldrich Co., Saint Louis, Missouri, USA) at room temperature. Afterwards, the sections were incubated with anti-IL-6 antibody (diluted at 1:400) or anti-IL-10 antibody (diluted at 1:100) in a humid chamber at 4º C for 16 hours. After washings with PBS, the sections were incubated with the Labeled StreptAvidin-Biotin kit (Universal Dako LSAB, Dako Inc., Carpinteria, CA, USA; K0675) for 60 minutes at room temperature. Peroxidase activity was revealed by 3,3'-diaminobenzidine chromogen (DAB substrate, Vector Laboratories, Burlingame, CA, USA) for 3 min. The sections were stained with Carazzi's haematoxylin. As a negative control, the sections were incubated with non-immune serum. Subsequently, the quantification of immunostained cells was performed. The number of IL6- and IL10-immunolabelled cells was estimated in the capsules of five specimens of each group/period. In each section, a standardized area (0.09 mm 2 ) was captured at x695 magnification. Using the image analysis system, the number of immunolabelled cells (brown-yellow colour) was computed 17,23,31,34 . Measurement of birefringent collagen The amount of collagen was estimated in three non-serial sections stained with picrosirius-red and analyzed under polarized illumination. In each section, the birefringent collagen was measured in a standardized field (0.09 mm 2 ) of the capsule at x695 magnification. All images were captured with standardized light intensity, field diaphragm aperture, condenser diaphragm and exposure time. The amount of birefringent collagen was performed using the ImageJ image processor, which provided the number of pixels of each colour frequency (red, orange, yellow and green) occupied in the total pixel value of the captured images 25,33 . 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C. & Gavini, G. Evaluation of radiopacity, pH, release of calcium ions, and flow of a bioceramic root canal sealer. J. Endod. 38, 842- 845, DOI: http://dx.doi.org/10.1016/j.joen.2012.02.029 (2012). Acknowledgments This work was carried out with the financial support from the São Paulo State Research Foundation (FAPESP: 2018/16848-2), National Council for Scientific and Technological Development (CNPq) and CAPES (code 001). Author contributions C.S.L. contributed to the conceptualization, experiment, methodology, analysis, writing, editing, and critical review of the article. M.M.D. contributed to the experiment, methodology, analysis, writing, and critical review of the article. M.T.-F. contributed to the conceptualization, and critical review of the article. E.S.-C. contributed to the experiment, methodology, analysis, writing, and critical review of the article. J.M.G.-T. contributed to the experiment, analysis, writing, and critical review of the article. P.S.C. contributed to the conceptualization, funding acquisition, methodology, supervision, project administration, writing, and critical review of the article. All authors reviewed the manuscript. Competing interests The authors declare no competing interests. http://dx.doi.org/10.3389/fimmu.2014.00453 http://dx.doi.org/10.1172/JCI118506 http://dx.doi.org/10.1016/j.joen.2012.02.029 49 3.2 Publicação 2 Bioactive potential of Bio-C Temp demonstrated by systemic markers of mineralization and immunoexpression of bone proteins after implantation in rat subcutaneous tissues ABSTRACT To evaluate in vivo the bioactive potential of bioceramic intracanal medication Bio-C Temp (BIO) in comparison to a calcium hydroxide-based intracanal medication (Calen; CAL). Polyethylene tubes filled with medications, and empty tubes (control group, CG,) were implanted into the subcutaneous tissue of rats. After 7, 15, 30 and 60 days, the blood was collected for calcium (Ca +2 ), phosphorous (P - ) and alkaline phosphatase (ALP) measurement, and the implants with surrounding tissues were removed. The number of fibroblasts (FB), immunohistochemistry for detection of osteocalcin (OCN) and osteopontin (OPN), von Kossa method and analysis of unstained sections under polarized light were performed. The data were submitted to two-way ANOVA and Tukey test (p<0.05). No significant difference was observed in the serum Ca+2 level between BIO and CG samples, in all periods. At 7 days, the serum P- levels were greater in BIO and CAL samples than in CG (p<0.0001), but no difference was seen among the groups at 15, 30 and 60 days. After 7, 15 and 30 days, greater ALP levels were observed in BIO and CAL specimens than in CG (p<0.0001) whereas no significant difference was observed among the groups at 60 days. From 7 to 60 days, an increase in the number of FB, OCN- and OPN-immunolabelled cells was observed in BIO and CAL groups (p<0.0001). At 60 days, no significant difference in the number of FB was seen between BIO and CAL. BIO and CAL specimens showed von Kossa-positive and birefringent structures while, in the CG specimens, these structures and OCN- and OPN-immunolabelled cells were not seen. Bio-C Temp caused an increase in the levels of ALP, an essential enzyme for biomineralization process, and induced the production of osteocalcin and osteopontin by connective tissue cells as well as the deposition of calcite, suggesting bioactive potential. Keywords: bioactive material; calcium silicate; immunohistochemistry; osteocalcin; osteopontin.  Artigo escrito segundo normas do periódico Journal of Biomedical Materials Research Part B Applied Biomaterials. 50 INTRODUCTION Calcium hydroxide is recommended during endodontic treatment due to its antimicrobial action and biological properties, such as its ability to induce deposition of mineralized tissue. 1 It has been demonstrated that calcium hydroxide induces mineralization due to its ionic dissociation giving rise to calcium and hydroxyl ions in an aqueous medium 3 providing an alkaline environment that favors repair tissue. 4 Alkaline environment favors the calcification, 3 since neutralizes lactic acid from osteoclasts and activates alkaline phosphatase (ALP), an enzyme that has an important role in the formation of hard tissue. 1 Calcium ions also play an essential role in mineralization, stimulating the expression of the fibronectin gene 4 . Previously to the use of a repairing material or filling periodic changes of calcium hydroxide-based intracanal medicament are used 5 to induce the formation and repair of mineralized tissue. 6 Among the indications, these intracanal medications are used for treatment of dental trauma, 5,7 root perforations 6 , root resorption, in pulpotomy and to induce apexification. 2 However, it has been suggested that long-term exposure to calcium hydroxide-based medications induces collagen degradation 5 and, hence, weakening the root dentine. 8 Moreover, the calcium hydroxide-based pastes have low radiopacity and flow capacity, which difficult their insertion into the root canal. 9 Bioceramic endodontic materials have stood out for being bioactive, 10 due to the interaction of the material with dentine, forming a mineralized intermediate zone in the presence of moisture. 11 Furthermore, the bioceramic materials modulate the response of host cells favoring the bone repair, since favors the osteoblasts survival and differentiation, cells actively involved in the periapical repair 12 enabling the formation of mineralized tissue. 13 Bioceramic endodontic materials are biocompatible, 14,15 non-cytotoxic 16 and have satisfactory physico-chemical properties. 16-18 There is currently a growing interest in the development of new materials based on bioactive calcium silicate. 19 Bio-C Temp (Angelus Dental Products Industry, Londrina, PR, Brazil) is a ready-to- use bioceramic paste, which contains calcium silicates associated with calcium tungstate and titanium oxide radiopacifiers, calcium aluminate, calcium oxide and base resin as a vehicle that does not allow hydration and setting of the material. According to the manufacturer, this material can be used in teeth with treatment of incomplete root formation, perforations, external and internal root resorption, previously to the use of repair and filling materials. Bio- C Temp eliminates the need for new applications, constituting an advantage over calcium hydroxide-based medications that require frequent changes. 20 Moreover, Bio-C Temp has 51 been suggested in pulpotomy cases for the induction of a calcified barrier 21 and treatment of and endodontic regeneration. 22,23 In vitro studies have demonstrated alkaline phosphatase activity and deposition of mineralized nodules in cultures of Saos-2 cells containing diluted extracts of Bio-C Temp, suggesting that this medication is cytocompatible. 19,21-23 Bio-C Temp is biocompatible when implanted into subcutaneous tissues as well as does not cause changes in liver enzymes. 24 In addition, this bioceramic medication provides an alkaline pH to the microenvironment, releases calcium ions when immersed in solution 19,21 and exhibits radiopacity about 7 mm/Al. 19 No in vivo study evaluating the bioactive potential of calcium silicate medication Bio- C Temp was found in the literature. Materials that promote increase in osteogenic response are of great value for endodontic therapy. 12 Extracellular matrix proteins of mineralized tissues, such as osteocalcin (OCN) and osteopontin (OPN) can be evaluated by immunohistochemistry reactions. 14,25-27 In addition, inorganic constituents of mineralized tissues including calcium (Ca +2 ) and phosphorus (P - ), and ALP, an enzyme involved in the mineralization, can be measured in the serum in order to investigate whether the endodontic materials cause changes in these parameters associated with the 26,28 . Therefore, in the present study, we evaluated the bioactive potential in vivo of an intracanal medication of calcium silicate, Bio-C Temp, in comparison with calcium hydroxide-based intracanal medication (Calen, SS. White Art. Dent. Ltda, RJ, Brazil). For this purpose, the concentrations of Ca +2 , P - and ALP in the serum were measured at 7, 15, 30 and 60 days after implantation of Bio-C Temp in the subcutaneous tissues of rats. Moreover, immunoexpression of OCN and OPN, typical proteins of mineralized tissues, and deposition of calcite in the capsules around the implants were also evaluated. MATERIAL AND METHODS Experimental procedures This research was approved by the Ethical Committee for Animal Research of XXXX XXX (# 22/2018). Sixty Holtzman (Rattus norvegicus albinus) adult male rats, weighing 250- 280 g were randomly distributed into three groups (n=20 per group): BIO (BIO-C TEMP Group, Angelus, Londrina, Brazil), CAL (Calen Group, SS. White Art. Dent. Ltda, RJ, Brazil) and CG (control group, empty polyethylene tubes). Calen intracanal medication, a calcium hydroxide-based paste. 52 The polyethylene tubes (Embramed Indústria Comércio, São Paulo, SP, Brazil) with 10.0 mm length and 1.6 mm diameter, previously sterilized with ethylene oxide, were filled with the materials or kept empty (CG) and implanted into the subcutaneous connective tissue. Previously, the animals were anaesthetized with an intraperitoneal injection of ketamine hydrochloride (80 mg/kg of body weight) combined with xylazine hydrochloride (8 mg/kg of body weight). After trichotomy and disinfection with 5% iodine solution, a 2.0 cm-long incision was made in a head-to-tail orientation using a nº 15 scalpel (Fibra Cirúrgica, Joinvile, SC, Brazil), and the polyethylene tube was placed into the subcutaneous pocket. After 7, 15, 30 and 60 days of implantation, the animals were anaesthetized, as previously described, and cardiac puncture of the left ventricle was performed with a vacuum tube (Vacuette®, Greiner Bio-One Brasil Produtos Médicos Hospitalares Ltda., Americana, SP, Brazil) with a needle (Med Goldman Indústria e Comércio Ltda., Manaus, AM, Brazil) and connected to a BD Vacutainer® adapter (Becton Dickinson Indústrias Cirúrgicas Ltda.,Curitiba, PR, Brazil). After blood collection, the animals were killed with anesthetic overdose and the implants and surrounding tissues were removed. The specimens were fixed for 48 hours in 4% formaldehyde buffered with 0.1 M sodium phosphate at pH 7.2, and the specimens were processed for paraffin embedding. Non-serial sections (6 µm thick) were adhered to slides previously treated with 4% silane (Sigma-Aldrich). The sections were subjected to immunohistochemistry reactions for detection of OCN and OPN. Other non- serial sections were submitted to the von Kossa histochemical reaction to evaluate the calcium deposits in the capsules and Masson’s trichrome staining was used for the quantification of fibroblasts. 29 Concentration of Ca +2 , P - and ALP in the serum After blood collection (as described above) and clot formation, the blood was centrifuged (Excelsa® II 206 BL; Fanem Ltda., Guarulhos, SP, Brazil) at 3500 rpm for 10 minutes and the serum was stored at -20 ° C. The calcium, phosphorus and ALP concentrations were determined using the Calcium Arsenazo III assay kit (Beckman Coulter, Indianapolis, Indiana, USA), Inorganic Phosphorus (Beckman Coulter) and Alkaline Phosphatase (Beckman Coulter), respectively. The sample absorbance was read on a spectrophotometer (Hitachi, model U 1100, Tokyo, Japan). The absorbance of calcium was determined at 660/700nm, phosphorus at 340nm and ALP at 410/480 nm. The experiments were carried out in triplicate and the averages were calculated. 53 Numerical density of fibroblasts The number of fibroblasts was estimated from three non-serial sections stained with Masson’s trichrome. The images of the capsules adjacent to the implants were captured with a camera (DP-71, Olympus - Japan) attached to a light microscope (Olympus, model BX-51), using the x40 objective (x695 final magnification). The fibroblasts, identified as fusiform or elliptical shape cells were estimated in a standardized field (0.09 mm 2 ) using an image analysis system (Image-Pro Express Olympus). In each specimen, the fibroblasts were counted in a total standardized area of 0.27 mm 2 , and the number of fibroblasts per mm 2 was estimated. 29, Immunohistochemical detection of OCN and OPN Deparaffinized sections were immersed in 0.001 M sodium citrate buffer at pH 6.0 and heated in a microwave oven at 96-98ºC for 20 minutes. After cooling, the slides were washed in 0.01 M sodium phosphate buffer (PBS) for 15 minutes and immersed for 30 min in 5% hydrogen peroxide to inactive the endogenous peroxidase. The sections were incubated for 20 minutes with 2% bovine serum albumin (Sigma-Aldrich Co., Saint Louis, Missouri, USA) at room temperature. Sections were then incubated for 16 hours with mouse anti- osteocalcin monoclonal antibody (Sigma-Aldrich Co., Saint Louis, Missouri, USA; code SAB1306277; diluted at 1:300) or mouse anti-osteopontin monoclonal antibody (Abcam Inc., Cambridge, MA, USA; code: ab 166709; diluted at 1:100) in a humid chamber at 4ºC. After washings, the sections were incubated for 1 hour with labelled polymer-HRP (EnVision + Dual Link System-HRP, Dako Inc., Carpinteria, CA, USA; K4061) at room temperature. The peroxidase activity was revealed by 3,3'-diaminobenzidine chromogen (ImmPACTTM DAB substrate, Vector Laboratories, Burlingame, CA, USA) for 3 min. The slides were washed with distilled water and, subsequently, the sections were counterstained with haematoxylin. In negative controls, the sections were incubated with non-immune serum. The number of OCN- and OPN-immunolabelled cells was quantified in the capsules of all the animals. Using a light microscope (Olympus, BX-51) and an image analysis system (Image-Pro Express Olympus), the number of immunolabelled cells was estimated in a standardized area (0.09 mm 2 ) of the capsule in close juxtaposition to the opening of implanted tube. 14 von Kossa histochemical reaction and analysis under polarized light The deparaffinized sections were incubated in an aqueous solution containing 5% silver nitrate, under the action of an incandescent light (100 watts) for 1 hour. After 54 incubation, the sections were washed quickly in distilled water and, subsequently, the sections were immersed in 5% sodium hyposulfite for 5 minutes. Then, the sections were washed in running water and for 5 minutes in distilled water. After washing, the sections were submitted to the picrosirius-red method, washed, dehydrated and mounted in resinous medium (Permount®, Fisher Scientific, New Jersey, USA) for analysis under a light microscope. Dewaxed sections of all specimens were mounted in resinous medium (Permount®, Fisher Scientific, New Jersey, USA). These unstained sections were analyzed under polarized light to assess the presence of birefringent structures in the capsules. 15,30,31 Statistical analysis The statistical analyses were performed using the GraphPad Prism 6.01 program (GraphPad Software, Inc., La Jolla, CA, United States). The data were submitted to two-way ANOVA analysis of variance, followed by the Tukey test (p<0.05). All data were presented as mean ± standard deviation. RESULTS Concentration of calcium, phosphorus and ALP in the serum In all periods, the serum Ca +2 concentration in BIO group was similar to that of CG (p>0.05). In contrast, the serum Ca +2 levels were greater in CAL group than in BIO and CG groups at 7 and 15 days. At 30 and 60 days, no significant difference among BIO, CAL and CG specimens was observed in the Ca +2 concentration (p<0.0001). From 7 to 60 days, the Ca +2 level reduced significantly in all groups (p<0.0001) (Figure 1 A). As shown in Figure 1B, the serum P - levels in BIO and CAL groups were greater than in CG specimens at 7 days. Moreover, significant difference was not found between BIO and CAL groups (p=0.0537). At 15, 30 and 60 days, no significant difference was seen among groups (p>0.05). From 7 to 60 days, significant reduction in the P - level was observed in all groups (p<0.0001). According to the Figure 1C, the serum ALP levels in the BIO and CAL groups were significantly higher than in the CG specimens at 7, 15 and 30 days (p<0.0001). Moreover, there was no significant difference in the ALP concentration between BIO and CAL medications (p>0.05). At 60 days, no significant difference was found among the BIO, CAL and CG groups (p>0.05). From 30 to 60 days, the serum ALP levels decreased significantly in 55 the BIO and CAL groups (p<0.0001) while no significant difference in the ALP concentration was observed in the CG specimens over time (p>0.05). Histological features and number of fibroblasts in the capsules The analysis of sections stained with Masson’s trichrome (2A- 2L) showed some changes in the cellular population and extracellular matrix components from capsules adjacent implants over time. After 7 days, the capsules contained few fibroblasts among many inflammatory cells. Over the course of 15 and 30 days, a decrease in the number of inflammatory cells in parallel to an increase in the number of fibroblasts and bundles of collagen fibres, stained in blue, were observed. At 60 days, the capsules exhibited mainly fibroblasts intermingled with an enhanced content of thick collagen fibre bundles compared to those seen at 7 days. According to Figure 2M, the quantitative analysis of the capsules of all groups rev