Índia Olinta de Azevedo Queiroz Tese de Doutorado Análise de propriedades biológicas do MTA em condição normal e hiperglicêmica Orientador: Prof. Titular João Eduardo Gomes Filho Araçatuba – SP 2017 Índia Olinta de Azevedo Queiroz Análise de propriedades biológicas do MTA em condição normal e hiperglicêmica Tese apresentada à Faculdade de Odontologia de Araçatuba, Universidade Estadual Paulista “Júlio de Mesquita Filho” - UNESP como parte dos requisitos para obtenção do título de Doutor em Endodontia. Orientador: Prof. Titular João Eduardo Gomes Filho Araçatuba – SP 2017 Catalogação na Publicação (CIP) Diretoria Técnica de Biblioteca e Documentação – FOA / UNESP Queiroz, Índia Olinta de Azevedo. Q3a Análise de propriedades biológicas do MTA em condição normal e hiperglicêmica : influência do diabetes na biomera- lização do MTA / Índia Olinta de Azevedo Queiroz. – Araça- tuba, 2017 184 f. : il. ; tab. Tese (Doutorado) – Universidade Estadual Paulista, Faculdade de Odontologia de Araçatuba Orientador: Prof. João Eduardo Gomes Filho 1. Diabetes mellitus 2. Inflamação 3. Cimentos dentários 4. Calcificação fisiológica I. T. Black D24 CDD 617.67 Dados Curriculares Índia Olinta de Azevedo Queiroz Nascimento 19/04/1986 - Caetité/BA Filiação Maria Suelly de Souza Azevedo Queiroz João Queiroz Pinto 2004- 2008: Curso de Graduação em Odontologia Universidade Estadual de Montes Claros – UNIMONTES 2010- 2012: Curso de Especialização em Endodontia Associação Brasileira de Odontologia (ABO/MG) 2011- 2013: Mestrado em Ciências Odontológicas, área de concentração em Endodontia na Faculdade de Odontologia de Araçatuba, Universidade Estadual Paulista “Júlio de Mesquita Filho” FOA/UNESP 2013- 2016: Doutorado Ciências Odontológicas, área de concentração em Endodontia na Faculdade de Odontologia de Araçatuba Universidade Estadual Paulista “Júlio de Mesquita Filho” FOA/UNESP Associações: Associação Brasileira de Odontologia Associação Mineira de Cirurgiões Dentistas Sociedade Brasileira de Pesquisa Odontológica International Association of Dental Research American Association of Dental Research Dedicatória Dedicatória Dedico este trabalho... A Deus Pela bênção de viver..... "A vontade de Deus nunca irá leva-lo aonde a Graça de Deus não possa protegê-lo.” Francisco Cândido Xavier Dedicatória A minha família... À minha mãe, Maria Suelly de Souza Azevedo Queiroz, “Uma mãe é capaz de ensinar mais do que cem professores.” A minha inspiração, meu alicerce e meu exemplo de vida. Simplesmente a pessoa que não mede esforços para me ver feliz. Obrigado por todos os momentos dedicados a mim, pelos conselhos, pelo amor, pela honestidade e pelo afeto. Mãe, sem você isso não seria possível!!!Amo você!!! Ao meu pai, João Queiroz Pinto, “Na longa jornada da vida muitos mestres encontramos, alguns seguimos, outros abandonamos, dentre todos, um deles é o que mais amamos”. Ao meu herói e meu maior exemplo de simplicidade e bondade. Obrigada por todos os ensinamentos passados, pelo amor, carinho, respeito e por sempre cuidar de mim...Amo você!!! À minha irmã, Indira Augusta de Azevedo Queiroz, “A felicidade está em nossas mãos”. Sua simplicidade, sensibilidade e o jeito como você consegue abrir mão das coisas em função dos outros me fazem enxergar outro lado da vida. Obrigada pelo carinho, atenção e amor...Amo você!!! À minha irmã, Indiane Souza de Azevedo Queiroz. “Pessoas grandes são aquelas que lutam por ideais”. Sua força de vontade, determinação e coragem me inspiram a cada dia. Obrigada por compartilhar comigo sua vida e sempre acreditar em mim. Tenho muito orgulho de ser sua irmã!!! Amo você!!! Agradecimentos Especiais Agradecimentos Especiais Aos professores responsáveis diretos pelo desenvolvimento deste trabalho... Prof. Dr. João Eduardo Gomes Filho Por simplesmente “ser você”. Por ser não somente um orientador e sim um mentor. Pela preocupação, paciência, respeito, calma e atenção que teve comigo durante toda a minha formação (mestrado e doutorado). Por acreditar em mim mesmo com toda a minha inexperiência. Pela liberdade que sempre me proporcionou para que fizesse minhas próprias escolhas e desenvolvesse minhas atividades. Por todas as vezes que dividir com você meus medos e anseios estando sempre disposto a me ajudar e ouvir. Obrigada por me fazer querer ser uma pessoa melhor. Ter você como meu orientador por todos esses anos foi uma honra e privilégio. Muito obrigada por tudo!!! Prof. Dr. Edilson Ervolino “Meu coorientador”, pela forma responsável e atenciosa com que me acolheu desde o meu mestrado, pela maneira como me ajudou, conduzi e orientou em todas as etapas deste trabalho. Muito obrigado pelos ensinamentos em biologia óssea, seu entusiasmo foi um dos motivos que me fizeram procurar um laboratório de biologia óssea nos EUA. Por todas as vezes que em que pode me escutar e me ajudar. Obrigada pela sua amizade e por dividir comigo parte do seu conhecimento. Prof. Dra. Sandra Helena Penha de Oliveira Por ser uma inspiração para mim. Pela forma carinhosa como sempre me acolheu e me ensinou, bem como nos momentos onde fui “desabafar” e estava disposta a me ouvir. Pela prontidão em me atender em todos os momentos que precisei. Por confiar e acreditar em mim e por me oferecer uma oportunidade que jamais vou esquecer. Muito obrigada pela amizade, carinho e respeito. Admiro muito você!!! Prof. Dr. Ivo Kalajzic Rigor, exigência e competência te definem. Pela maneira como compartilhou comigo seus conhecimentos e experiências em biologia óssea através das aulas, reuniões e experimentos. Pela forma acolhedora como me recebeu Agradecimentos Especiais durante meu doutorado sanduíche, tentando do seu jeito fazer com que minha experiência em outro país fosse a melhor possível. Pelos “happy hour” no The Half Door, pelas conversas, conselhos e risadas no “lab time” (especialmente nas sextas-feiras). Muito obrigada por tudo!!! Agradecimentos Especiais Aos os grandes responsáveis por “hold me” todos esses anos, sem vocês não seria nada... Thiago Machado, “Algumas vezes na vida, você encontra uma pessoa especial; a que muda sua vida simplesmente por estar nela; a que te faz rir até você não poder mais parar; a que te faz acreditar que realmente tem algo bom no mundo.” Pelo companheirismo, amizade, atenção, carinho, paciência e amor. Por estar sempre ao meu lado, principalmente nos momentos em que mais precisei. E por nunca desistir de mim...Muito obrigada por fazer parte da minha vida!!! Loiane Massunari, “Ninguém cruza nosso caminho por acaso e nós não entramos na vida de alguém sem nenhuma razão. ”Fiote”, às vezes eu me pergunto o que seria de mim sem você na minha vida para “puxar meu freio” rsrsrsrs.. Loi, sua amizade foi um dos melhores presentes que a pós-graduação me ofereceu. Muito obrigada, pela amizade, confiança, apoio, carinho e atenção que sempre teve comigo. Pelos nossas comilanças nos jantares, almoços e gelatos.. hehehe.. Adoro você “fiote”!!! Renata Oliveira Samuel, “Amigo é coisa para se guardar do lado esquerdo do peito...” pelos momentos de carinho e amizade que divididos desde o primeiro dia do mestrado. Pelo tempo que passamos juntas, dividindo experiências, dificuldades, sonhos, anseio, desejos e aflições. Pelo seu jeito corajoso de ir atrás de seus sonhos. Por ser a melhor “roommate”. Pelas gargalhadas e lágrimas. Pelas noites regadas a vinho e pizzas. Pelas nossas noites solteiras. Pelas nossas viagens. Pelo carinho com que sempre teve comigo, mesmo com meus defeitos. Obrigada por todos esses anos!!! Nelci Vieira, “Minha segunda mãe”. A mãezona, a que me escuta, me ajuda, me ensina, me aconselha. Nel, eu não tenho palavras para te descrever, ou descrever o que você representou e representa na minha vida. Obrigada por tudo minha mãezona!!! Amo você!!! Agradecimentos Especiais Simone Watanabe, minha eterna amiga/supervisora/orientadora. Japinha, eu devo a você parte da minha formação. Muito obrigada por me ensinar, me apoiar e estar sempre ao meu lado. Serei eternamente grata... Gabrielly Cristinni Rezende, pela amizade, compreensão e ajuda. Muito obrigada pelo carinho com que sempre me acolheu, principalmente quando estava em Santa Fé. Por me escutar, pelas viagens, pelos tempos alegres/malucos na micro, pelas festas no rancho e pelas nossas comilanças nos jantares, almoços e gelatos...hehehe... Adoro você Gaby!!! Ludmila Santos, “A vida é marcada pela presença de pessoas queridas e que apesar da distância, ficam sempre em nossos corações”. Lud, esse tempo que esteve em Araçatuba, você foi capaz de me ajudar a viver novamente e a lembrar das minhas origens. Muito obrigada por tudo, pela amizade, alegria e carinho. Sinto sua falta... Gustavo Sivieri de Araújo, “o amigo”, uma pessoa de uma bondade e natureza simples que poucos conhecem, escondido atrás do Professor Sivieri...hehehe...Muito obrigada por ter me deixado conhecer esse seu outro lado e por dividir uma parte da sua vida comigo nos trabalhos, festas, happy hours, drinks e viagens... Agradecimentos Agradecimentos À Faculdade de Odontologia de Araçatuba, da Universidade Estadual Paulista “Júlio de Mesquita filho” – UNESP, na pessoa seu Diretor Prof. Titular Wilson Roberto Poi e Vice-Diretor Prof. Titular João Eduardo Gomes Filho, pelo empenho e dedicação com que o conduz. Ao programa de Pós-Graduação em Ciência Odontológica da Faculdade de Odontologia de Araçatuba – UNESP representado pelo seu coordenador Prof. Dr. Luciano Tavares Ângelo Cintra, pela competência e afinco na condução do programa de pós-graduação. À Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP, pelo total apoio financeiro para a realização deste trabalho através da concessão da Bolsa de Doutorado (processo nº 2013/06641-8) e da Bolsa de Doutorado sanduíche (BEPE) (processo nº 2014/13750-0). Aos docentes da disciplina de Endodontia da Faculdade de Odontologia de Araçatuba – UNESP, Prof. Prof. Dr. Luciano Tavares Ângelo Cintra, Dr. Rogério de Castilho Jacinto, Prof. Dr. Gustavo Sivieri de Araújo, Prof. Dr. Elói Dezan Júnior, Prof. Dr. José Arlindo Otoboni Filho, Prof. Dr. Mauro Juvenal Nery, e novamente ao meu orientador Prof. Dr. João Eduardo Gomes Filho pelo aprendizado, apoio e contribuição durante minha formação. Ao Departamento de Ciências Básicas da Faculdade de Odontologia de Araçatuba da Universidade Estadual Paulista “Júlio de Mesquita Filho” - UNESP, representado pelos Prof. Dr. Edilson Ervolino, pela oportunidade de realizar o processamento laboratorial imunoistoquímico; pela Profa. Dr. Sandra Helena Penha de Oliveira, por me proporcionar realizar todo o meu experimento in vitro; e pela Profa. Dr. Rita Cássia Menegati Dornelles pela disponibilidade de ceder todo material necessário para análises bioquímicas. Ao Departamento de Odontologia Infantil e Social da Faculdade de Odontologia de Araçatuba da Universidade Estadual Paulista “Júlio de Mesquita Filho” - UNESP, representado pelo Prof. Dr. Alberto Carlos Botazzo Delbem, por Agradecimentos disponibilizar o laboratório e equipamentos necessários para realizar para análises bioquímicas. Ao Prof. Dr. Luciano Tavares Ângelo Cintra, pelas conversas, conselhos e por sempre disposto a ensinar/ajudar. Por dividir seus conhecimentos, experiências e sempre buscar tirar o melhor de cada pessoa. Luciano, obrigada pelos ensinamentos/lições e por ter sido esse exemplo de professor/conselheiro/orientador durante minha formação; e ao Prof. Dr. Eloi Dezan Júnior, pela simplicidade, generosidade e paciência em dividir seus conhecimentos clínicos. Pelo seu jeito extrovertido, atencioso e por sempre “pensar” nos seus alunos, buscando ferramentas/meios de nos incentivar a sermos professionais melhores. Aos amigos Wagner Garcez de Melo, você realmente é o que podemos chamar de um excelente Professional/Professor. Muito obrigada pela inestimável ajuda durante todos os meus experimentos nos finais de semana, na redação do texto, nas análises estatísticas e no mais importante em dividir comigo seus conhecimentos de forma tão gentil e espontânea; e Luis Gustavo Narciso, pela preciosa ajuda durante as coletas sanguíneas. Aos amigos Marcos Frozoni e Guilherme Bonduki, pela confiança e por me ajudarem na realização do meu doutorado sanduíche, me proporcionando um crescimento pessoal e profissional. Serei eternamente grata a vocês pelo que fizeram e pela amizade dispensada à minha pessoa. Aos amigos do programa de pós-graduação Ciência Odontológica da Faculdade de Odontologia de Araçatuba – UNESP, Área de Concentração de Endodontia, Diego Valentim, Ludmila Santos, Loiane Massunari, Gabrielly Rezende, Paulo Tobias, Renata Samuel, Marcelo Wayama, Mariane Azuma, Luciana Louzada, Annelise Katrine, Francine Beneti, Carlos Bueno, Christine Mem Martins, Renan Dal Fabro, Leticia, Camila, Amanda e Vanessa, e da iniciação científica Luanna Gonçalves, Larissa Gonçalves e Aline Ávila, pela amizade e pelos momentos de alegria, conversas, brincadeiras e descontrações proporcionadas. Agradecimentos Aos amigos do Laboratório de Farmacologia do Departamento de Ciências Básicas da Faculdade de Odontologia de Araçatuba da Universidade Estadual Paulista “Júlio de Mesquita Filho” - UNESP, Aline Takamiya, Victor Balera, Dayane Queiroz, Leticia, Carluci Beltran, Maria Fernanda Lopes, Fernanda Demarqui, pela paciência, apoio, ensinamentos e por sempre estarem dispostos a me ajudar e ensinar. Vou sentir saudades da “Farmaco B” que com certeza é a melhor!!! Aos funcionários do Departamento de Odontologia Restauradora da Faculdade de Odontologia de Araçatuba da Universidade Estadual Paulista “Júlio de Mesquita Filho” - UNESP, Nelci Vieira, Cláudia Neves Corrêa, Elaine Cristina Francischini Ferreira e Peterson Moura, pela amizade, paciência e colaboração, apoio e incentivo. O que seria desse departamento sem a presença de vocês!!! Aos funcionários do Departamento de Cirurgia e Clínica Integrada, Odair, Dirce e Paulo da Faculdade de Odontologia de Araçatuba da Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP, pelo carinho, dedicação e simpatia com que sempre me atenderam todas às vezes. Muito obrigada por me ensinarem e me ajudarem durante as etapas desse trabalho. Aos funcionários da Seção Técnica de Graduação e Pós-Graduação da Faculdade de Odontologia de Araçatuba - UNESP, Valéria Queiroz Marcondes Zagatto, Lílian Sayuri Mada e Cristiane Regina Lui Matos, pela eficiência e presteza de sempre. Obrigada pela ajuda durante minha representação discente e por tudo o que fizeram por mim durante esses anos. À Alice e família, pela amizade e pela forma como me acolheram em Araçatuba; Aos meus amigos de “Moc”, Amanda Normanha, Geraldo Edson, Rafael Santiago, Paulo Sergio, Lara Mota, Mariana Silveira e Swed, pela amizade, apoio e carinho sempre. A todos aqueles que, direta ou indiretamente, contribuíram para a realização deste trabalho!!! Agradecimentos To the responsible for my USA life experience... To University of Connecticut Health Center – UCHC, specially to Center of Regenerative Medicine and Skeletal Development, Department of Reconstructive Sciences of School of Dental Medicine for received and given me the opportunity to develop my PhD and improve my knowledge. To Professor Dr. Ivo Kalajzic, Center of Regenerative Medicine and Skeletal Development, for open his lab and share with me your knowledge and experiences, to be a mentor that gave me the opportunity to know more about bone biology. Thank you for everything!! To Professor Dr. Mina Mina and Ivana Vidovic, Department of Craniofacial sciences of School of Dental Medicine, for always be receptive, for the gentleness in share hers knowledge and for all the help in the in vivo experiment. To my co-workers, Emilie Roeder, Xi Wang, Paola Vizzarri, Brya Matthews, Devin Shaheen and Mara for friendship, affection, dedication and sympathy that always had with me. Thanks for sharing yours expertise with me and help me all time and for the fun times together. I hope to see you all again. To friends from Department of Reconstructive Sciences and Department of Craniofacial sciences of School of Dental Medicine, Lipin, Yalin, Zhihua Wu (Lisa), Bharbara and Anu for friendship and attention that always had with me. To Dipika Gupta, Nidhi Gupta, Tulika Sharma, Marian George and Bandita Adhikari, my “american/indian family”, thank you for taking care of me during my USA life, to make my life more happy, to teach me another culture and for the drinks and party that we shared. I miss our house, girls!! To Dipika Gupta, roommate and friend, the girl that loves her job, the inspiration for everyone close to her and an example of researcher. A friend that listened me when I was crying and happy, holding me when I was drunk (hehe) Agradecimentos and capable to do everything to see a friend happy. Dipiii, I miss you so much, thanks for everything and as I said I will see you again soon. To Emilie Roeder, for make my life in the lab easier, better and unforgettable, for sharing and teach me in the lab. For the trips together, for your patience, friendship and be an amazing friend…. To Mariana Quezado, for help and take care of me when I arrived, for the New York days, for all the happy hours together and for teach me chemistry and mathematics. See you soon in BH, Mari. To the three Musketeers, Fani Memi and Debargha Bassuli, for the friendship, for listen me in the bad and good times, for trying to speaking and learning Portuguese, for all the drinks, talking, happy hours, for the new year’s day/eve and for be the two most incredible friend that I knew… To all my other USA/UCHC/World friend, Fabiana Saoki, Martinna Bertolini, Alexandro Lima, Ivana Vidovic, Nilse dos Santos, Melissa Car, Hank Hrdlicka, Yulia Pustovalova, Alexandra Pozhidaeva, Anushree Vk, Guilia Vigone, Igor Matic, Ryan Russell, Sara Acevedo, Guivini Gomes, Alberto Ortega, Scotti Danger Adamson, Tosin Quadri, Nicole Glidden and Anilei Hoare, Luciana Arraes, Anthar Darwish, Jelena Vidas and Candace Reeve, for friendship and all the good/funny times that we spend together. Epígrafe "Cada sonho que você deixa para trás é um pedaço do seu futuro que deixa de existir.” Steve Jobs Resumo Resumo 22 Queiroz, IOA. Análise de propriedades biológicas do MTA em condição normal e hiperglicêmica. [Tese]. Araçatuba: UNESP – Univ. Estadual Paulista; 2017. O objetivo deste estudo foi analisar as propriedades biológicas do MTA em condição normal e hiperglicêmica. Para tanto, esse trabalho foi dividido em duas partes, sendo que a primeira teve como objetivo avaliar o efeito do MTA no processo de reparo do Ligamento Periodontal (PDL) e na diferenciação de células mesenquimais progenitoras do PDL (PDSCs) e da Medula Óssea (BMSCc) após injuria dental. Uma perfuração na região de furca do primeiro molar superior de camundongos transgênicos (αSMACreERT2/Ai9/Col2.3GFP) foi realizada e os efeitos do MTA após 2, 17 e 30 dias de lesão, foram examinados e comparados com resina composta (AS) utilizando análise histológica e epifluorescência. Além disso, BMSCs e PDSCs desses camundongos foram isoladas, cultivadas e os efeitos do MTA na proliferação celular e diferenciação osteogênica foram avaliados. Os resultados indicaram que o MTA promoveu a regeneração do PDL e do osso alveolar na área da injuria dental. No entanto, demonstrou efeitos negativos na diferenciação osteogênica de PDSCs e BMSCc. A segunda parte, teve como objetivo avaliar a influência da Diabetes Mellitus na proliferação celular, produção de citocinas, resposta tecidual, capacidade de mineralização e na expressão local e sistêmica de marcadores ósseos. Para alcançar esses objetivos, células de linhagem fibroblásticas L929 foram cultivadas em alta concentração de glicose e a influência do MTA na proliferação celular e na produção de citocinas das IL-1β, IL-6 e TNF-α foram observados às 6, 24, 48 e 72 horas; tubos de polietileno foram implantados no tecido subcutâneo de ratos normais e diabéticos (induzidos pelo Aloxano) e a influência do MTA na resposta tecidual, produção de citocinas e na capacidade de mineralização em condição diabética foram observadas através de técnicas histológicas e imunoistoquímicas aos 07 e 30 dias; analises bioquímicas para Cálcio, Fósforo e Fosfatase Alcalina e imunoistoquímica para osteocalcina e osteopontina, aos 07 e 30 dias, também foram realizadas com a finalidade de verificar a influência do MTA na expressão local e sistêmica de marcadores ósseos. O quadro hiperglicêmico promoveu, in vitro, um aumento da produção Resumo 23 de IL-6 e comprometeu a proliferação celular após 72hs. Independente da condição diabética, a resposta tecidual e a capacidade de produção de IL-1β, IL-6 e TNF-α de ambos MTA não foi alterada, embora uma redução na intensidade de fluorescência do MTA Branco foi observada aos 14 dias em animais diabéticos. Por outro lado, o quadro hiperglicêmico inibiu a produção local de osteocalcina e osteopontina na presença dos dois MTA e aumentou os níveis séricos de Fósforo e Fosfatase Alcalina. Assim, concluiu-se que, o MTA promoveu a regeneração do PDL e do osso alveolar na área da injuria dental, contudo, apresentou um efeito negativo com relação à diferenciação osteogênica e, que em condições hiperglicêmicas, o MTA Cinza melhores resultados biológicos quando comparado ao MTA Branco. Palavras-chaves: Diabetes Mellitus, Inflamação, Cimentos Dentários, Calcificação Fisiológica. Abstract Abstract 25 Queiroz, IOA. Analysis of biological properties of MTA in normal and hyperglycemic conditions. [Thesis]. Araçatuba: UNESP – Univ. Estadual Paulista; 2017. The aim of this study was to analyze the biological properties of MTA in normal and hyperglycemic conditions. Therefore, this study were divided into two parts; the first part aim was to evaluate MTA effect on healing of periodontal ligament (PDL) and differentiation of mesenchymal progenitor cells in PDL (PDSCs) and bone marrow stromal cells (BMSCc) following dental injury. Perforation on the pulp floor in the furcation area in the first maxillary molars of transgenic mice (αSMACreERT2/Ai9/Col2.3GFP) were performed and the effects of MTA after 2, 17, 30 days of injury, were examined and compared to AS using histological and epifluorescence analysis. Additionally, BMSCs and PDSCs from these mice were isolated, cultured and the effects of MTA on cell proliferation and osteogenic differentiation were evaluated. The results indicated that MTA promoted regeneration of injured PDL and alveolar bone in the area of dental injury. However, it has demonstrated negative effects on the osteogenic differentiation of PDSCs and BMSCs. The aim of second part was to evaluate the influence of Diabetes Mellitus on cell proliferation, cytokine production, tissue response, mineralization ability and local and systemic expression of bone markers. To achieve these goals, L929 fibroblasts cell line were cultured under high glucose concentration and the influence of MTA on cell proliferation and production of cytokine IL-1β, IL-6 and TNF-α were observed at 6, 24, 48 and 72 hours; polyethylene tubes were implanted in the subcutaneous tissue of normal and diabetic rats (induced by Alloxan) and the influence of MTA on tissue response, cytokines production and mineralization ability in diabetic condition were observed by histological and immunohistochemical techniques at 07 and 30 days; biochemical analysis for Calcium, Phosphorus and Alkaline Phosphatase and immunohistochemistry for osteocalcin and osteopontin were also performed, at 07 and 30 days, in order to verify the influence of MTA on the local and systemic expression of bone markers. The hyperglycemic state promoted an increase on IL-6 production and impaired L929 proliferation after 72hs. Independent of the diabetic condition, the tissue response and ability to produces IL-1β, IL-6 and TNF-α by both MTA was not change, although a Abstract 26 reduction on fluorescence intensity of White MTA was observed after 14 days in diabetic animals. Moreover, hyperglycemia state inhibited the local production of osteocalcin and osteopontin in the presence of both MTA and increased serum levels of Phosphorus and Alkaline Phosphatase. Thus, it was concluded that MTA promoted regeneration of PDL and alveolar bone in the area of dental injury, moreover, it had a negative effect in relation to osteogenic differentiation; and under hyperglycemic condition, Gray MTA showed better biological results when compared with White MTA. Keywords: Diabetes Mellitus, Inflammation, Dental Cements, Physiological Calcification. Lista de Abreviaturas Lista de Abreviaturas 28 LISTA DE ABREVIATURAS ADA – Associação Americana de Diabetes Al2O3 – Óxido de Alumínio ALP – Fosfatase Alcalina ANOVA – Análise de Variância AS – Compósito resinoso autoadesivo BMSCs – Células Mesenquimais da Medula Óssea. BSP – Sialoproteína Óssea Ca – Cálcio CaP – Cálcio Fosfato cDNA – Ácido Desoxirribonucleico complementar CEUA – Comissão de Ética no Uso Animal CO2 – Gás carbônico DM – Diabetes Mellitus DMEM – Meio Essencial Mínimo de Dulbecco DNA – Ácido Desoxirribonucleico EDTA – Ácido Etilenodiaminotetracético ELISA – Ensaio de Imunoabsorção Enzimática FAPESP = Fundação de Amparo à Pesquisa do Estado de São Paulo FBS – Soro Fetal Bovino FeO – Óxido de Ferro Fe2O3 – Óxido Férrico Fig. – Figura g – Gramas GAPDH – Gliceraldeído-3-fosfato desidrogenase GFP - Proteína Verde Fluorescente GMA – Glicol metacrilato g/mL– Gramas por Mililitros GMTA – Mineral Trióxido Agregado Cinza h – Horas H&E – Hematoxilina e Eosina IDF – Federação Internacional de Diabetes IL1-β – Interleucina 1 beta Lista de Abreviaturas 29 IL-6 – Interleucina 6 IP – Intraperitoneal IR – Imunorreatividade Kg – Quilogramas L–929 – Células de Linhagem Fibroblástica L–929 M – Molar Mg – Magnésio mg – Miligramas mg/dL– Microgramas por Decilitros mg/kg – Miligramas por Quilogramas mg/ml – Miligramas por Mililitros MgO – Óxido de Magnésio min – minutos mL – Mililitros mm – Milímetro mM – Milimolar MSCs – Células mesenquimais indiferenciadas MTA – Mineral Trióxido Agregado MTA-CM – Meio de cultura condicionado com Mineral Trióxido Agregado nm – Nanômetro OC – Osteocalcina OCN – Osteocalcina OH- - Hidroxila OPN – Osteopontina OZE – Óxido de Zinco e Eugenol P – Fósforo PBS – Tampão fosfato-salino PDL – Ligamento Periodontal PDSCs – Células Progenitoras do Ligamento Periodontal. pH – potencial Hidrogeniônico RNA – Ácido Ribonucleico rpm – Rotação por minuto RT-qPCR – Reação em Cadeia de Polimerase com transcriptase reserva em tempo real Lista de Abreviaturas 30 Runx-2 – Fator de transcrição relacionado ao Runt 2 s – Segundos Si – Silício TM – Tamoxifeno TNF-α – Fator de necrose tumoral alfa UV- Luz Ultravioleta U/L – Unidades por Litro U/mL – Unidades por Mililitros VH – Veículo VK – Von Kossa vs – versus WMTA – Mineral Trióxido Agregado Branco ZOE – Óxido de Zinco e Eugenol % – Por cento °C – Graus Célsius ® – Marca registrada α – alfa β – Beta x – Vezes n – Tamanho da amostra α-MEM – Meio Essencial Mínimo alfa α-SMA – Actina de Músculo Liso alfa α-SMACreERT2/Ai9/Col2.3GFP – Animal Triplo Transgênico µg – Microgramas μm – Micrômetros µm2 – Micrometros quadrados µg/g – Microgramas por gramas µg/ml – Microgramas por Mililitros Lista de Figuras e Tabelas Lista de Tabelas e Figuras 32 Artigo 1: Figure 1: Effects of experimental perforation of the integrity of PDL and alveolar bone_________________________________________________________ 61 Figure 2: Effect of MTA on regeneration of PDL and the underlying alveolar bone_________________________________________________________ 62 Figure 3: Effect of MTA on apical region ____________________________ 63 Figure 4: Effect of MTA-CM on cell viability and osteogenesis of PDLCs___ 64 Figure 5: Effect of MTA-CM on cell viability and osteogenesis of BMSC cultures______________________________________________________ 65 Figure 6: Effects of MTA-CM on the SMA9+ progenitors and their osteogenic differentiation in vitro___________________________________________ 66 Supplemental Figure 1: Schematic representation of experimental PDL injury________________________________________________________ 67 Artigo 2: Figure 1: Effect of both Gray MTA and White MTA extract on L929 proliferation under high or normal glucose concentration after 6, 24, 48, and 72 hs______ 80 Figure 2: Influence of hyperglycemic condition on IL-6 production by fibroblasts upon MTA treatment ____________________________________________ 81 Figure 3: Graph showing immunostaining patterns for IL-1β and IL-6 observed in normal and diabetic groups_____________________________________ 82 Figure 4: Immunostaining patterns for TNF-α observed in the normal and diabetic groups_________________________________________________ 83 Artigo 3: Table 1: Inflammatory scores specimens stained with hematoxylin-eosin___ 95 Table 2: Medium of samples in Each Group categorized necrosis, presence of mineralization and Fluorescence intensity____________________________ 95 Figure 1: Response found in normal group at 30 days__________________ 96 Figure 2: Response found in diabetic group at 30 days_________________ 98 Lista de Tabelas e Figuras 33 Artigo 4: Figure 1: Graph showing the serum levels of calcium, phosphorus and alkaline phosphatase for healthy group (a, b, c) and diabetic group (d, e, f) on days 7 and 30______________________________________________________ 118 Figure 2: Graph showing comparison between the calcium, phosphorus and alkaline phosphatase serum levels in healthy and diabetic groups on day 7 (a, b, c) and 30 (d, e, f)____________________________________________ 119 Figure 3: Photomicrographs showing the histological appearance of immunolabelling for OCN and OPN found in healthy and diabetic groups on day 30__________________________________________________________ 120 Sumário Sumário 40 Sumário Introdução .................................................................................... 42 Proposição ................................................................................... 46 Artigo 1: Mineral Trioxide Aggregate improves healing response of periodontal tissue to injury ............................................................. 48 Artigo 2: Hyperglycemic condition interferes on cell proliferation and IL-6 production stimulated by Gray MTA ........................................ 69 Artigo 3: Diabetes mellitus affects mineralization ability of white mineral trioxide aggregate ............................................................. 85 Artigo 4: Effect of Diabetes Mellitus on local and systemic bone marker expression induced by Gray versus White Mineral Trioxide Aggregate .................................................................................... 101 Conclusão .................................................................................. 121 Referências ................................................................................ 123 Anexos ....................................................................................... 130 Anexo 1 – Comitê de Ética ....................................................... 130 Anexo 2 – Protocolos experimentais – In vitro .......................... 131 Anexo 3 – Protocolos experimentais – In vivo .......................... 137 Anexo 4 - Diretrizes para publicação dos trabalhos .................. 150 Introdução Introdução 42 Introdução A "Medicina Endodôntica" visa estudar a relação e/ou associação entre doenças sistêmicas e as de origem endodônticas (1-4). Entre elas, a Diabetes Mellitus (DM) que é uma doença complexa, progressiva e debilitante de origem metabólica caracterizada por um quadro de hiperglicemia crônica que promove alterações no metabolismo dos carboidratos, lipídios, proteínas, água e eletrólitos resultantes da insuficiente secreção/ação do hormônio insulina (5). DM é considerada como um fator modulador das infecções endodônticas (6). No entanto, esta relação ainda não está completamente elucidada, estudos mostram que as alterações na reposta imune e a persistência do estado inflamatório associadas a DM podem interferir e comprometer o reparo dos tecidos periapicais (7-9). A hiperglicemia crônica decorrente da DM promove a ativação de vias que aumentam a inflamação (3,5). Assim, a elevação dos níveis inflamatórios sistêmicos altera diversas funções do sistema imune (10,11) como o comprometimento da resposta leucocitária e o aumento da expressão de citocinas pró-inflamatórias, promovendo uma redução da capacidade de defesa celular e aumentando a susceptibilidade à infecção e inflamação, afetando diretamente a integridade dos tecidos pulpares e periapicais e interferindo no processo de reparo (3, 12-14). DM também tem sido associada com alterações no processo de reparo ósseo (15,16), onde mecanismos fisiopatológicos relacionados à perda óssea como a redução da atividade osteoblástica, diminuição da síntese de colágeno e alterações no metabolismo do cálcio e fosforo e na expressão de marcadores de formação óssea tem sido observados em indivíduos diabéticos (17,18). Entretanto, os mecanismos pelos quais a DM interfere no metabolismo ósseo e, portanto, no processo de reparo/cicatrização ainda precisam ser esclarecidos, sabe-se, que controle da inflamação é essencial para que o processo de reparo ocorra, uma vez que, na presença de um quadro hiperglicêmico, a persistência da inflamação leva uma estimulação, pelos neutrófilos, da condrogênese e inibição da osteogênese (19,20). Introdução 43 O osso é um tecido dinâmico que está em constante remodelação e a diferenciação osteoblástica é regulada por uma série de hormônios, citocinas e múltiplos fatores de transcrição (21-23) e que podem ser inibidos e/ou alterados pelo quadro hiperglicêmico (24). Deste modo, a estimulação da reabsorção óssea, através da inibição da osteogênese, acarreta no aumento da reabsorção óssea periapical (25,26). Além disso, em indivíduos diabéticos, a redução da capacidade de reparo também está associada a diminuição da resistência à infecção bacteriana e maior susceptibilidade as infecções endodônticas (6, 27, 28). A infecção endodôntica é tratada através da eliminação dos micro- organismos patogênicos e o restabelecimento da normalidade dos tecidos apicais e periapicais afetados, bem como da utilização de materiais capazes de promover reações de teciduais favoráveis, apresentarem adequadas propriedades físicas e químicas, que sejam indutores de mineralização e que possam favorecer e contribuir para a reparo periapical (29,30). Uma vez que, os cimentos reparadores e obturadores estão em intimo contato com tecidos perirradiculares, sua composição química, bem como, compostos tóxicos liberados pelos mesmos podem interferir na resposta inflamatória e, consequentemente, no processo de reparo (31-33). Assim, materiais com as mais variadas bases: óxido de zinco e eugenol, resina epóxica, ionômero de vidro, hidróxido de cálcio e Agregado Trióxido Mineral (MTA), podem ser encontrados. Óxido de Zinco e Eugenol (ZOE) é um cimento composto de um pó de Óxido de Zinco e um líquido o Eugenol, utilizado que nos mais diversos procedimentos endodônticos: proteção pulpar direta, em selamento provisório, como obturador endodôntico, em revestimento de cavidades profundas. É um cimento que apresenta ação antimicrobiana (34), bom selamento marginal (35), ação anestésica e anti-inflamatória local (36). No entanto, devido ao seu componente líquido: Eugenol quando aplicado diretamente sobre os tecidos pode desencadear uma resposta inflamatória crônica dos tecidos periapicais (37) e danos sobre o tecido pulpar (38). MTA é um cimento reparador à base de silicato de cálcio que foi introduzido em 1993 por Torabinejad (39) com a finalidade de proporcionar o Introdução 44 selamento de comunicações patológicas ou iatrogênicas entre o dente e sua superfície externa (49,41). Entretanto, devido às suas excelentes propriedades físicas, químicas e biológicas (42,43) este passou a ser rotineiramente utilizado nas mais diversas situações clínicas (pulpotomias, capeamentos pulpares, apicogêneses, apicificações e obturação dos canais radiculares) (41). Estudos, in vitro e in vivo, mostram que o MTA é um material bioativo (44); biocompatível (45); promove a proliferação e diferenciação de células mesenquimais/progenitoras da polpa dentária (46) e ligamento periodontal (47), além de induz dentinogênese (48), cementogênese (49) e osteogênese (50). MTA também é capaz de estimular a produção de citocinas (51,52) e a expressão marcadores ósseos (49, 53). Inclusive em condições hiperglicêmicas o MTA mostrou-se biocompatível, promoveu mineralização (54) e foi capaz de induzir a formação de ponte de dentina (55,56). MTA encontra-se atualmente disponível sob duas formas MTA Cinza e MTA Branco, onde a principal diferença entre ambos está na redução das concentrações de Al2O3, MgO, e FeO encontras no MTA Branco (57). Apenas poucos estudos comparando MTA Cinza e MTA foram realizados, alguns mostrando semelhanças; são biocompatíveis (58), capazes de induzir a proliferação celular (59) e de estimular a formação de ponte de dentina (60), e outros diferenças; MTA Cinza favorece a adesão osteoblástica (61), porém cementoblastos e queratinócitos crescem melhor na superfície do MTA Branco (62). Embora, o MTA Branco tenho sido introduzido como uma alternativa para evitar o pigmentação dental produzida pelo MTA Cinza, estudos in vitro e em in vivo, também verificaram pigmentação dental causada pelo MTA Branco (63,64). Recentemente, o MTA também começou a ser empregado em procedimentos endodônticos regenerativos (65,66), uma vez que os tecidos dentários (polpa dentária, ligamento periodontal e osso alveolar) são fonte rica e acessível de células mesenquimais/progenitoras (67,68), e tais procedimentos envolvem a interação e diferenciação das células mesenquimais/progenitoras, bem como a utilização de biomateriais (69). No entanto, os mecanismos envolvidos nessa interação ainda não estão totalmente explicados. Introdução 45 A diferenciação de células osteoprogenitoras é um principais processos responsáveis pela formação e remodelação óssea, com isso, torna-se um pré- requisito compreender e analisar as vias envolvidas no desenvolvimento ósseo (70). Em função disso, investigações tem utilizando animais transgênicos e marcadores visuais (GFP - proteína verde fluorescente) sob o controle da actina de músculo liso (α-SMA) (promoter) e do colágeno tipo I 2.3kb (promoter) expressado por osteoblastos maduros com a finalidade de identificar a subpopulação de células progenitoras que expressam α-SMA e exibem um potencial osteogênico (71-73). Deste modo, como uma das propriedades do MTA é induzir a mineralização e promover o reparo nos tecidos onde é aplicado, torna-se relevante compreender e verificar a influência do MTA no processo de diferenciação osteogênica de células mesenquimais, bem como, no processo de reparo dos tecidos periodontais após injuria dental, através da utilização de animais transgênicos. Ao mesmo tempo, como a DM é uma desordem metabólica que altera a resposta inflamatória e, portanto, afeta o processo de mineralização, justifica-se o estudo da influência dos MTA Cinza e MTA Branco na viabilidade celular, resposta tecidual, produção de citocinas, capacidade de mineralização e na expressão local e sistêmica de marcadores ósseos em condição diabética. Proposição Proposição 46 Proposição O presente trabalho teve o intuito de avaliar as propriedades biológicas do MTA em condição normal e hiperglicêmica. Os objetivos específicos foram:  Avaliar, in vivo, os efeitos do MTA na reparação dos tecidos periodontais e ósseo após injuria dental (perfuração) usando animais transgênicos (αSMACreERT2/Ai9/Col2.3GFP);  Avaliar, in vitro, a influência do MTA na proliferação e diferenciação de células progenitoras da Medula Óssea e do Ligamento Periodontal utilizando linhagem de animais transgênicos (αSMACreERT2/Ai9/Col2.3GFP);  Avaliar, in vitro, a influência do MTA na viabilidade celular e na produção de citocinas IL-1β, IL-6 e TNF-α em condição hiperglicêmica;  Avaliar, in vivo, a influência do MTA na resposta tecidual, na produção de citocinas IL-1β, IL-6 e TNF-α e capacidade de mineralização em condição diabética;  Avaliar, in vivo, a influência do MTA na produção local (osteocalcina e osteopontina) e sistêmica (Cálcio, Fósforo e Fosfatase Alcalina) de marcadores de formação óssea em condição diabética. Artigo 1 Artigo 1 Artigo 1 48 Artigo 1: Mineral Trioxide Aggregate improves healing response of periodontal tissue to injury Abstract Objectives and Background: Mineral Trioxide Aggregate (MTA) a biomaterial used in endodontic procedures as it exerts beneficial effects on regenerative processes. In this study we evaluate MTA effect on healing of PDL and differentiation of mesenchymal progenitor cells in PDL and bone marrow stromal cells following periodontal ligament and alveolar bone injury. Materials and Methods: We used an inducible Cre-loxP in vivo fate mapping approach to examine the effects of MTA on the contributions of descendants of cells expressing αSMA-CreERT2 transgene to the PDL and alveolar bone after experimental injury to the root furcation on the maxillary first molars. The effects of MTA after 2, 17, 30 days of injury, were examined and compared to AS using histological and epifluorescence analysis. The effects of two dilutions of MTA (MTA 1:5 and MTA 1:50) on proliferation and differentiation of mesenchymal progenitor cells derived from bone marrow (BMSC) and periodontal ligament (PDSCs) from αSMACreERT2;Ai9/Col2.3GFP were examined using presto blue assay, alkaline phosphatase and Von Kossa staining. The expression of markers of differentiation were assessed by real time PCR Results: Histological and epifluorescence analyses showed better repair of injury in teeth restored with MTA as shown by greater expansion of SMA9+ and 2.3GFP+ cells as compared to AS. We also observed positive effect on alveolar bones and apical region on distant from the site of injury. The in vitro data showed that MTA supported viability of the PDL fibroblasts but not their differentiation. MTA did not exert effect on BMSCs viability during the 9 days in cultures, but resulted in significant decreases in von Kossa staining and levels of expression of OC and Bsp as compared to OM and control media. In BMSCs and PDL cells grown in presence of MTA there were marked decrease in SMA9-stained and 2.3GFP-stained areas as compared to OM indicating the reduced levels of expression of markers of osteogenesis. Conclusion: MTA promotes regeneration of injured PDL and alveolar bone reflected as contribution of progenitors (SMA9+ cells) into osteoblasts (Col2.3+ Artigo 1 49 cells). In vitro effects of MTA are supportive to viability of the PDL progenitor but have negative effects on osteogenic differentiation of both PDL and BMSCc. Keywords: MTA, injury, periodontal ligament, progenitor cells, differentiation Introduction Mineral Trioxide Aggregate (MTA), a calcium silicate–based cement, is a bioactive biomaterial used extensively in almost all endodontic therapies including root perforation repair, apexification, apexogenesis, pulpotomy and root-end filling (1). MTA has been used extensively in regenerative endodontic procedures (2,3). MTA has been reported to have low cytotoxicity, and well as the ability to promote proliferation and differentiation of stem/progenitor cells resulting in cementogenesis, dentinogenesis and osteogenesis (4-9). Dental tissues are a rich source of mesenchymal stem cells (MSCs) that participate in healing and regeneration following injury or infection (10-12). Previous in vivo linage tracing studies in our laboratory showed that alpha- smooth muscle actin (αSMA) expressing cells residing in perivascular areas within a number of tissues including PDL, dental pulp, bone marrow and periosteum represent a population of mesenchymal progenitor cells (12-16). Following periodontal injury, αSMA+ cells expand and differentiate into osteoblasts in the alveolar bone, fibroblasts in the PDL and cementoblasts (16). Our studies also showed that this population is capable of giving rise to a second generation of odontoblasts during reparative dentinogenesis (15). Despite numerous studies on the effects of MTA on various dental tissues, the underlying mechanisms of the effects of MTA on regeneration of periodontal tissues and surrounding alveolar bone and its effects on differentiation of stem/progenitor cells are not fully understood. We designed the present study to gain insight into the effects of MTA on perivascular cells expressing αSMA during repair of the periodontium and surrounding alveolar bone using cell lineage-tracing experiments in developing mouse molars. We utilized the previously characterized αSMACreERT2;Ai9/Col2.3GFP transgenic animal in which αSMA serves as a marker of progenitor cells in PDL. In these transgenic animals Col2.3GFP transgene serves as a marker for identification of PDL cells, mature osteoblasts and cementoblasts. Artigo 1 50 Materials and Methods Transgenic mice The αSMACreERT2;Ai9/Col2.3GFP mice have been previously described (13). For in vivo and in vitro lineage tracing experiments αSMACreERT2;Ai9 (cross between αSMACreERT2 Cre reporter mice with Ai9 mice from Jackson Labs, Bar Harbor, ME, USA) and αSMACreERT2;Ai9/Col2.3GFP (cross between αSMACreERT2;Ai9 with Col2.3GFP mice) were used. Animal protocols were approved by the Institutional Animal Care Committee. Tooth injury in vivo Four to six weeks old transgenic mice were injected with corn oil (vehicle, VH) or tamoxifen (TM) (75 ug/g body weight) twice in 24h intervals. Two days later, mice were anesthetized with an intraperitoneal injection of ketamine (87 mg/kg) and xylazine (13 mg/kg) and experimental pulp perforations on maxillary first molars were performed as previously described (17). Briefly, class I cavity was prepared with a carbide round burr (diameter 0.40 mm) on the occlusal surface of first maxillary molars. Pulp chambers were opened, and coronal pulp tissues were removed with pulp extractor (VDW® STERILE Barbed Broaches, VDW GmbH, Munich, GE) up to the root canal orifices. A perforation was created in the center of the floor of the pulp chamber using an endodontic hand file number #15 (Dentsply, Tulsa, OK, USA) (Supplemental Figure 1). The perforation area was filled with one-step self-etching Adhesive System (AS) (Clearfill SE Bond; Kuraray, Okayama, JP) (AS, controls) or White ProRoot MTA (Dentsply), prepared according to the manufacturer recommendations. The cavities were then sealed with a light-cured composite resin (SDI wave restorative system, SDI Wave, SDI Inc, Itasca, IL, USA) in both groups. Animals were sacrificed by intra-cardiac perfusion with 4% paraformaldehyde in PBS (17) at various time points (2, 17 and 30 days). The maxillary arches were isolated, cleaned of soft tissue, fixed in 4% paraformaldehyde solution for additional 24h and then, decalcified with 14% EDTA for 7 days. Decalcified tissues were placed in 30% sucrose solution overnight and embedded in cryomatrix (Thermo Shandon, Pittsburg, PA, USA). Seven-micrometer sections were obtained using the Leica cryostat and Artigo 1 51 mounted using a CryoJane tape transfer system (Instrumedics, St Louis, MO, USA). Sections were imaged using a AxioScan.Z1 (Carl Zeiss). Adjacent sections were processed for hematoxylin/eosin staining and analyzed by light microscopy. Cell isolation and culture Primary bone marrow stromal cells (BMSCs) were prepared from the hind limbs of 4-6 week old αSMACreERT2;Ai9/Col2.3GFP mice as previously described (18, 19). The cells were plated in 12 well culture plates at a density of 106/cm2 for 7 days in basal medium: α-modified essential medium (α-MEM), 10% fetal bovine serum (FBS, Life Technologies, Carlsbad, CA, USA) and 100 U/ml of penicillin, 100 mg/ml of streptomycin (1% PS). Cre activity was induced by 1µM of 4‐OH‐Tamoxifen added at days 2 and 4 of culture. At day 7, cells were grown in 4 different conditions including control basal medium, dilutions of MTA-CM in basal medium and osteogenic media (OM) (α- MEM 10% FBS + 50ug/ml ascorbic acid + 8mM β–glycerophosphate). Medium was changed every two days. PDL cells were isolated from 4-6 week old αSMACreERT2;Ai9/Col2.3GFP mice as previously described (12). Briefly, the mandibles and maxilla were dissected from the surrounding tissues, rinsed in 0.12% chlorhexidine digluconate (Clorhexidina, Villevie, Joinville, SC, BRA), for 30 secs, and washed in PBS. Molars with the adherent PDL were removed from the surrounding alveolar bone and digested in Dulbecco's modified Eagle's medium (DMEM) with 2 mg/ml Collagenase P (Sigma-Aldrich, Saint Louis, MO, USA) and 0.25% trypsin (Life Technologies), and digested at least for 2 h at 37°C. Following washing, PDL cells were seeded in DMEM 20% FBS + 1% PS and cultured in 5% oxygen for 7 days. The medium was changed every 2 days and the Cre activity was induced by 1µM of 4‐OH‐Tamoxifen added at days 3 and 5 of culture. At day 7, the cells were transferred to normoxic conditions until confluence (around day 11) and then trypsinized and plated in 24 well plates at a density 105/cm2. Medium was changed to different treatments the following day. Artigo 1 52 MTA Conditioned Medium White ProRoot MTA was mixed with sterile water according to the manufacturer’s instructions. MTA discs were prepared under aseptic conditions as described previously with minor modifications (20). Briefly, the discs were created by using a sterile cylindrical polyethylene tube (diameter: 6mm; height: 3mm). The MTA discs were kept a 5% CO2 incubator at 37°C for 6 hours for setting. After 6 hours, the discs were sterilized by ultraviolet (UV) light for 1 hour. The discs were incubated in α-MEM 10% FBS at 37oC in a humidified atmosphere containing 5% CO2 for 3 days (1 mL of α-MEM 10% FBS for each disc). After 3 days, the supernatants were collected, filtered through a sterile 0.22µm filter (Sigma-Aldrich, Saint Louis, MO, USA). The supernatant collected was referred as MTA conditioned media (MTA-CM). Two different dilutions of MTA-CM were used (high dilution, 1:50 and low dilution, 1:5). Cell viability assay Cell viability was determined using a Presto Blue assay (Thermo Fisher Scientific, Waltham, MA, USA). At various time points the Presto blue reagent was added to the cell medium, incubated for 2 hours and the fluorescence intensity was measured (560nm excitation/590nm emission). The experiments were performed in triplicate. Histochemical analysis of cell cultures The histochemical staining for alkaline phosphatase (ALP) was performed on cultures fixed in 10% formalin for 5 minutes using 86-R Alkaline Phosphatase kit (Sigma Aldrich) according to the manufacturer instructions. The number of ALP positive colonies per well was counted. Mineralization was assessed after 21 days of culture using von Kossa staining as described previously (21). Plates were imaged on a flat bed scanner and mineralized area was quantified using ImageJ. Detection of epifluorescence Expression of GFP and tdTomato was imaged on an inverted Observer Z1 microscope (Carl Zeiss). The region scanned covered approximately half the Artigo 1 53 well area. Fluorescent area proportion for each channel was quantified using ImageJ. RNA extraction and gene expression RNA was extracted using Trizol reagent (Life Technologies) (19). Reverse transcription was performed using iScript™ cDNA Synthesis Kit (Bio- Rad, Hercules, CA, USA). The expression of osteogenic genes osteocalcin (Oc, Mm03413826_mH), bone sialoprotein (Bsp, Mm00492555_m1) was assessed by RT-qPCR (14). The gene expression was normalized by the expression of a housekeeping gene (GAPDH). Statistics Data were subjected to statistical analysis by using the GraphPad Prism (version 5.0) software. For all parametric data, ANOVA followed by Tukey´s test was used. The p value was considered significant at 0.05. Results Effects of MTA on periodontal tissue regeneration In the present study we used the previously characterized αSMACreERT2;Ai9/Col2.3GFP mouse to examine the effects of MTA on regeneration of periodontal tissue following injury. Adhesive system (AS) without MTA has been used as a control for the effects of MTA on healing. In these experiments perforation on the pulp floor in the furcation area in the first maxillary molars was performed on 4-6 weeks old TM- injected αSMACreERT2;Ai9/Col2.3GFP mice. In uninjured tissue, Col2.3GFP expression (referred to as 2.3GFP) was detected in osteoblasts, osteocytes within the alveolar bone, cementoblasts on the root surface, odontoblasts lining the pulp chamber and roots and in PDL fibroblasts surrounding the roots in the remaining areas of the teeth (Figure 1B-E). Histological analysis of the injured area showed that perforation at the pulpal floor resulted in local destruction of dentin, odontoblasts, PDL in the furcation area and the underlying alveolar bone as evident by the lack of 2.3GFP expression in these locations (Figure 1). Examination of the area underneath the injury showed presence of a very few αSMA9-tdTomato+ (referred as SMA9) and 2.3GFP+ cells in teeth filled with AS Artigo 1 54 and MTA (Figure 1). At this time the number of SMA9+ in bone marrow of the alveolar bone and in dental pulp were increased as compared to uninjured controls (Figure 1). Examination of the area of injury, 17 to 30 days following injury showed increase in expression of SMA9+, 2.3GFP+ and cells co- expressing SMA9+ and 2.3GFP+ at the site of injury in teeth filled with AS and MTA (Figure 2). Histological analysis in teeth filled with AS showed that the area underneath the injury contained necrotic tissue that was separated from alveolar bone with a relatively large fibrous area filled with small and elongated 2.3GFP+ fibroblasts, some of which were also SMA9+, oriented parallel to the bone surface (Figure 2A). A few cells co-expressing both transgenes were detected at day 17 but not at day 30 after injury (Figure 2B). Histological examination of teeth filled with MTA showed that the area underneath the injury contained dentin chips, PDL-like fibroblasts and well- organized alveolar bone. SMA9+ and 2.3GFP+ cells were detected in the PDL- like fibroblasts and the alveolar bone in the area of repair. A few cells co- expressing both transgenes (SMA9+/2.3GFP+) were detected in the area of repair and underlying alveolar bone at day 17 (Figure 2A). The number of SMA9+/2.3GFP+ cells in PDL-like cells and alveolar bones increased at day 30 (Figure 2B). These observations together indicated contribution of SMA9+ cells in the organized PDL-like fibroblasts and underlying alveolar bone in teeth filled with MTA, but limited differentiation of SMA9+ cells to mature lineages in injuries filled with AS (Figure 2). Further examination of these teeth showed that injury at the pulp floor and restorative materials also had a significant effect on the expression of these transgenes in the apical regions (Figure 3). Examination at day 30, showed significant expansion in the SMA9+ cells in the apical regions in injured teeth as compared to uninjured teeth (Figure 3). The apical region of teeth filled with MTA showed organized structure containing SMA9+ cells and 2.3GFP+ osteoblasts and cementoblasts. There were numerous double labeled cells in this area indicating the contribution of SMA9+ cells to osteoblasts and cementoblasts. Unlike the periapical region of teeth filled with MTA, the periapical regions of teeth filled with AS was very disorganized with significantly lower numbers of SMA9+ and 2.3GFP+ and no double labeled cells. These data Artigo 1 55 indicate that application of MTA resulted in better repair of the PDL in the area under the injury as well as in the apical region. Effect of MTA-CM on PDLC In vitro To gain a better understanding of the underlying process mediated by MTA, we examined the effects of media conditioned with MTA (MTA-CM) on the cell viability, presence of SMA9+ cells and differentiation of PDL progenitors. In these experiments the effects of two different concentrations of MTA-CM were compared to osteogenic media (OM) and control media. Presto Blue assay showed that OM media increased the viability of the PDL cells as compared to controls at day 7. However, MTA at both concentrations had negative effects on cell viability at day 7, although this effect was greater at the lower concentration (Figure 4A). We also examined the effect of MTA-CM on osteogenic differentiation, by ALP staining and gene expression. In PDL cells, OM did not affect the ALP staining (Figure 4B) but resulted in increased levels of expression of OC and BSP as compared to controls. Both high and low concentrations of MTA-CM decreased the number of ALP+ colonies compared to both control and OM. MTA-CM also failed to induce expression of OC and BSP compared to control (Figure 4C). These observations showed that MTA-CM had negative effects on both viability and differentiation of PDL fibroblasts. Effect of MTA-CM on Bone marrow stromal cells (BMSC) In vitro We observed significant effects of MTA application on the alveolar bone underneath the site of injury as well as in the apical region. Therefore, we also examined the effects of MTA-CM on BMSCs. Our results showed that OM increased viability or cell number in the cultures while MTA-CM had a slight but significant negative effect on viability at both concentrations (Figure 5A). Treatment with OM increased von Kossa staining and increased levels of OC and BSP expression in BMSCs as compared to controls. Both concentrations of MTA-CM resulted in significant decreases in von Kossa staining and levels of expression of OC and BSP as compared to OM and control media (Figure 5C- D). Artigo 1 56 Effects of MTA-CM on the SMA9+ progenitors and their osteogenic differentiation in vitro BMSCs and PDL cells grown in OM showed marked increases in SMA9+ and 2.3GFP+ areas as compared to controls indicating the positive roles of osteogenic media on progenitor cells and their differentiation (Figure 6A-B). In BMSCs and PDL cells grown in both concentrations of MTA-CM SMA9+ and 2.3GFP+ areas were significantly lower compared to OM, and 2.3GFP+ area was reduced compared to control (Figure 6A-B). Reduced levels of expression of markers of osteogenesis in these cultures were therefore related to reduced number of progenitor cells giving rise to osteoblasts. Discussion Despite the progress made in understanding the biological effects of MTA, the mechanism of its effects on wound healing and the nature of hard- tissue formation remain unclear. Therefore, our study focused on the effect of MTA on mesenchymal progenitor cells during repair of PDL and surrounding tissues. We showed that placement of MTA in site of injury at the furcation area can significantly improve the healing process. Compared to AS-filled teeth, where the injured area contained necrotic tissue and a large fibrous layer typical of scarring, in MTA filled teeth PDL-like fibroblasts and well-organized tissue were present. These observations are consistent with previous observations that have shown bacteria invasion and necrosis of tissues in teeth filled with AS due to its inadequate sealing properties (22,23). On the other hand, it is well documented that MTA is a commonly used restorative material because of its biological and sealing properties that reduce bacterial invasion (24). Previous studies showed contribution of SMA9+ cells and their ability to differentiate into mature cell types, including PDL fibroblasts, osteoblasts and cementoblasts during growth and in repair after PDL injury (16). Our lineage tracing study showed significant expansion of SMA9+ cells, 2.3GFP+ cells and cells co-expressing SMA9/2.3GFP at the perforation site, confirming a biological response and healing process promoted by MTA. Furthermore, MTA induced contribution of SMA9+ cells in repair of alveolar bone underlying injured PDL, indicating that MTA promotes bone repair. Following PDL regeneration and alveolar bone healing, MTA showed a positive effect on distant root periodontal Artigo 1 57 complex. Unlike the disorganized apical region in AS capped teeth, in teeth filled with MTA this area showed organized structure containing differentiated SMA9+/2.3GFP+ osteoblasts and cementoblasts. These observations suggest that MTA mediates regeneration through interactions with periodontal ligament and alveolar bone progenitor/stem cells. In contrast to our in vivo observations, in vitro results showed that MTA conditioned media does not support differentiation of BMSCs and PDLCs. The lack of differentiation in our in vitro studies is also different from previously reported positive effects of MTA on the formation of mineralized nodules and expression of cemento/osteoblastic marker genes in PDLCs and BMSCs (9,25,26). Explanation for these differences can be the amount of calcium, aluminum, bismuth and silicon ions released by MTA into media and its variation depending of concentration utilized, which might inhibit or suppress cell growth and functions resulting in changes in the cell response, such as proliferation and differentiation (27-29). It has been also reported that MTA enhances proliferation of human dental pulp cells through sustained release of calcium ions (30). In contrast, others have shown that rate of calcium ion release from MTA were higher during the first three hours with subsequent decreases thereafter (31). Furthermore, high concentrations of MTA have been shown to exert cytotoxic effects on human PDL fibroblasts (32). Taken together, these findings may explain decreased in viability when MTA-CM was used in PDLC and BMSC cultures. Although the outcomes of the in vitro experiments may depend on the cell type, different culture systems and concentrations of MTA, the most likely explanation for these differences is the lack of mineralization inducing reagents such as ascorbic acid and β–glycerophosphate in media used to examine the effects of MTA on differentiation. Although cell studies results are relevant, it is not possible to assess the complex interactions between materials and host. Therefore, we primarily evaluated the in vivo effects of the MTA on the progenitor lineages. Collectively, our findings showed contribution of SMA9+ cells in soft tissue repair and newly calcified bone matrix formation as well as positive effect of MTA on PDL and alveolar bone injury. Artigo 1 58 Acknowledgments We would like to thank all individuals who provided reagents, valuable input and technical assistance in various aspects of this study, including Drs. Emilie Roeder and Xi Wang. This work was supported by R01-DE016689 (MM), R01-AR055607 (IK) & R90-DE022526 grants from National Institute of Health (NIDCR); by #2014/13750-0 scholarship from São Paulo Research Foundation (FAPESP). BGM is supported by Connecticut Stem Cell grant 14-SCA-UCHC- 02. The authors declare no conflicts of interest. References 1. Torabinejad M, Parirokh M. Mineral trioxide aggregate: a comprehensive literature review—part II: leakage and biocompatibility investigations. J Endod 2010;36:190–202. 2. Chueh LH, Ho YC, Kuo TC, et al. Regenerative endodontic treatment for necrotic immature permanent teeth. J Endod 2009;35:160-4. 3. Paryani K, Kim SG. Regenerative endodontic treatment of permanent teeth after completion of root development: a report of 2 cases. J Endod 2013;39:929-34. 4. Maroto M, Barbería E, Vera V, García-Godoy F. Dentin bridge formation after white mineral trioxide aggregate (white MTA) pulpotomies in primary molars. Am J Dent 2006;19:75-9. 5. Parirokh M, Torabinejad M. 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Sealing effectiveness of materials used in furcation perforation in vitro. Int Dent J 2005;55:133-41. 23. Lodiene G, Kleivmyr M, Bruzell E, Ørstavik D. Sealing ability of mineral trioxide aggregate, glass ionomer cement and composite resin when repairing large furcal perforations. Br Dent J 2011;12:210(5):E7. 24. Torabinejad M, Watson TF, Pitt Ford TR. Sealing ability of a mineral trioxide aggregate when used as a root end filling material. J Endod. 1993;19:591-5. 25. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 2004;364:149–155. 26. Hakki SS, Bozkurt SB, Hakki EE, Belli S. Effects of mineral trioxide aggregate on cell survival, gene expression associated with mineralized tissues, and biomineralization of cementoblasts. J Endod 2009;35:513-9. 27. Hoppe A, Güldal NS, Boccaccini AR. A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics. Biomaterials 2011;32:2757–2774 28. Wu BC, Kao CT, Huang TH, et al. Effect of verapamil, a calcium channel blocker, on the odontogenic activity of human dental pulp cells cultured with silicate-based materials. J Endod 2014;40:1105-11. 29. Chen I, Salhab I, Setzer FC, et al. A New Calcium Silicate-based Bioceramic Material Promotes Human Osteo- and Odontogenic Stem Cell Proliferation and Survival via the Extracellular Signal-regulated Kinase Signaling Pathway. J Endod 2016;42:480-6. 30. Takita T, Hayashi M, Takeichi O, et al. Effect of mineral trioxide aggregate on proliferation of cultured human dental pulp cells. Int Endod J 2006;39:415- 22. 31. Duarte MA, Demarchi AC, Yamashita JC, Kuga MC, Fraga Sde C. pH and calcium ion release of 2 root-end filling materials. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;95:345-7. 32. Keiser K, Johnson CC, Tipton DA. Cytotoxicity of mineral trioxide aggregate using human periodontal ligament fibroblasts. J Endod 2000;26:288- 91. Artigo 1 61 Figure 1: Effects of experimental perforation of the integrity of PDL and alveolar bone. Images of sagittal sections through maxillary molars from αSMACreERT2;Ai9/Col2.3GFP mice are shown. In all images the dental pulp is denoted by dashed lines, the site of injury by an arrow and the injury to the PDL and underlying alveolar bone by dotted lines. (A – E) Bright field (A) and epifluorescence (B) images of intact molars isolated 14 days post TM injection. C - E are higher magnification of boxed area shown in B. Note the expression of SMA9 + cells (red) and 2.3GFP + cells (green) in dental pulp, periodontal ligament (PDL) and alveolar bone (ab). Also note a few cells co-expressing SMA9/Col2.3-GFP (yellow) detected in PDL indicating the differentiation of SMA9 into PDL fibroblasts. (F – O) Bright field (F, K) and epifluorescence (G, L) images of sections through injured maxillary molars filled with AS (F-J) and MTA (K-O) isolated 2 days post PDL injury. H – J are higher magnification of boxed area shown in G. M – O are higher magnification of boxed area shown in L. Note the lack of detectable SMA9 + and 2.3GFP + cells at the sites of injury. Also note expansion of SMA9 + and 2.3GFP + cells in bone marrow of the alveolar bone surrounding injury in molars restored with MTA (L – O). P is an H&E stained section of a maxillary molar isolated 2 days after injury (indicated by arrow). Note the destruction of dentin in the pulpal floor, PDL and alveolar bone at the site of injury. Also note residual dentin chips (*). Scale bars=100µm. Artigo 1 62 Figure 2. Effect of MTA on regeneration of PDL and the underlying alveolar bone. Representative bright field and epifluorescence images of sagittal sections through injured maxillary molars from αSMACreERT2;Ai9/Col2.3GFP animals. In all images the dental pulp is denoted by dashed lines, the site of injury by an arrow and the sites of repair by dotted lines. (A) Histology of injured molars 17 days after injury and restoration with AS (a – f) and MTA (g – l). c – f are higher magnification of the boxed area outlined in b and i – l are higher magnification of boxed area in h. SMA9 + cells are present in PDL region (outlined by dotted lines) and alveolar bone (ab) in both groups (c and i). Thick layer of cells expressing 2.3GFP are evident in AS restored molars (d). Co-expression of SMA9 and 2.3GFP (yellow, arrowheads) is indicated. (B). Images of molars 30 days after injury and restoration with AS (a – f) and MTA (g – l). c – f are higher magnification of the boxed area outlined in b and i – l are higher magnification of boxed area in h. Note increase in number of SMA9 + cells in repaired PDL and alveolar bone in molars restored with MTA (i) as compared to molars restored with AS (c). Also note increase in co-expression of SMA9 + and 2.3GFP + cells (arrowheads) in repaired PDL of molars restored with MTA (f) as compared to molars restored with AS (l). Scale bar=50µm, (f and l) in A and B=25µm. (m - p) are images of H&E stained section of maxillary molars 30 days after injury (indicated by arrow). n and p are higher magnifications of boxed areas in m and o, respectively. Repaired PDL and PDL region are outlined with dotted line. Note the lack of alveolar bone repair and large fibrous area containing fibroblastic cells in PLD region in molars restored with AS (m and n). Also note well-organized repaired alveolar bone(ab), PDL with PDL- like fibroblasts, and partial dentin repair (arrowheads) in molars restored with MTA (n and o). Scale bar=100µm. Artigo 1 63 Figure 3. Effect of MTA on apical region. A – D are representative bright field and fluorescent images of a sagittal section through the root of an uninjured maxillary molar at day 30 post TM injection showing expression of SMA9+ (B) and 2.3GFP+ (C) cells in dental pulp (outlined with dotted line), alveolar bone (ab), periodontal ligament (PDL), cementoblasts (cb) and osteoblasts (ob) in apical area. Note co-expressing SMA9/Col2.3-GFP (yellow) detected in cementoblasts and osteoblasts. Scale bar=100µm. E – L are representative images of the apical root region at day 30 post injury restored with AS (E – H) and MTA (I – L). Note significant expansion of SMA9+ (J) and 2.3GFP+ (K) cells in apical area of MTA restored molars as compared to AS restored molars (F-G). Also note lack of co-expressing cells in apical cementoblasts and osteoblasts in molars restored with AS (H) as compared to MTA restored molars where co-expression of SMA9 and 2.3GFP is present in both cementoblasts and osteoblasts (L). Note increase in co-expressing cells in apical region of molars restored with MTA as compared to control without injury. Scale bar=50µm. Artigo 1 64 Figure 4. Effect of MTA-CM on cell viability and osteogenesis of PDLCs. PDLC were cultured in control medium, osteogenic medium (OM) or MTA-CM diluted 1:50 or 1:5. (A) Cell viability was determined by using Presto blue reagent at days 2, 4 and 7 after treatment initiation. (B) ALP staining was performed on day 7 of treatment and positive colonies were counted. (C) Expression of OC and BSP was determined at day 7 of treatment, normalized to GAPDH expression. Results represent mean ± SEM values from three independent experiments. #: p≤0.05 (vs. Osteogenic media); *: p<0.05 (vs. Control); 0: p<0.05 (vs. MTA 1:50). Artigo 1 65 Figure 5. Effect of MTA-CM on cell viability and osteogenesis of BMSC cultures. BMSC were cultured in control medium, osteogenic medium (OM) or MTA-CM diluted 1:50 or 1:5. (A) Cell viability was determined by using Presto blue reagent at days 7 (before MTA-CM), 9, 12 and 15. (B) Mineralization by von Kossa staining was assessed on day 21 and expressed as area stained. (C) Expression of OC and BSP was determined at day 21 of culture, normalized to GAPDH expression. Results represent mean ± SEM values from three independent experiments. #: p≤0.05 (vs. Osteogenic media); *: p<0.05 (vs. Control); 0: p<0.05 (vs. MTA 1:50) Artigo 1 66 Figure 6. Effects of MTA-CM on the SMA9+ progenitors and their osteogenic differentiation in vitro. (A-B) Images of scanned PDLC and BMSC cultures analyzed at the end point (day 7 for PDLC and day 21 for BMSC). Increase of the SMA9 and 2.3GFP positive areas in OM group as compared to control in both PDLC and BMSC cultures was observed. Also, note significant decreases in SMA9+ and 2.3GFP+ areas in both concentrations of MTA-CM as compared to OM in both cultures. Image analysis was competed on whole cell culture wells and area of fluorescence was measured. The images are generated from one representative of three biological replicates. Artigo 1 67 Supplemental Figure 1. Schematic representation of experimental PDL injury. A. Scheme of lineage tracing experiments where 4-6 weeks old αSMACreERT2/Ai9 transgenic mice were injected with tamoxifen (TM) twice in 24- hour interval. PDL injury was performed 48 hours after second injection and animals were chased at indicated time points after PDL injury. B. Schematic representation of experimental PDL injury where access cavity preparation was performed using dental carbide round bur. Access through the floor of dental pulp chamber to PDL was created using endodontic K file size 15 and PDL with surrounding alveolar bone was injured with the same instrument. In experimental group injury was capped with MTA and composite associated with adhesive system (AS). In control group injury was capped with AS and tooth was restored with composite. Artigo 2 Artigo 2 69 Artigo 2: Hyperglycemic condition interferes on cell proliferation and IL-6 production stimulated by Gray MTA Abstract Introduction: Diabetes mellitus (DM) affects inflammatory and immune responses and impairs healing processes. We investigated DM influence on cell proliferation and cytokine production induced by Gray Mineral Trioxide Aggregate (GMTA) and White MTA (WMTA). Methods: L929 fibroblasts were cultured under high or normal glucose concentration. Effects of GMTA and WMTA on cell proliferation and IL-1β, IL-6, and TNF-α production were investigated using Alamar Blue assay and ELISA, respectively, at 6, 24, 48, and 72h. Moreover, polyethylene tubes containing GMTA, WMTA, and empty tubes (control) were implanted into dorsal connective tissues of Wistar rats previously assigned normal and diabetic groups (Alloxan induced). After 7 and 30 days, the tubes with surrounding tissues were removed, fixed, and subjected to immunohistochemical analysis of IL-1β, IL-6, and TNF-α. Nonparametric and parametric data were statistically analyzed (p<0.05). Results: In vitro assays showed no detectable production of IL-1β and TNF-α. The hyperglycemic condition promoted IL-6 up-regulation production (p<0.05) and impaired cell proliferation at 72h (p<0.05). Under high glucose condition, GMTA induced greater cytotoxicity and IL-6 production than WMTA did. In vivo assay showed no differences in IL-1β, IL-6, and TNF-α production between both systemic conditions in presence of GMTA and WMTA at all time points. Conclusion: Hyperglycemic conditions interfered on cell proliferation and IL-6 production stimulated by GMTA. Moreover, no up-regulation of inflammatory cytokines in diabetic animal tissues was observed in the presence of GMTA and WMTA. Keywords: Diabetes Mellitus; Cytokines; Gray MTA; White MTA Artigo 2 70 Introduction Diabetes mellitus (DM) is a metabolic disease considered as a modulator of endodontic infections (1). The hyperglycemic state promoted by DM can alter immune functions through impairment of leucocyte responses and cellular defense capacity reduction, leading to increased susceptibility to infection and inflammation and a direct effect on dental pulp integrity and periapical healing process (2-7). During periapical healing, proinflammatory cytokines IL-1β, IL-6, and tumor necrosis factor alpha (TNF-α) play an essential role in inflammatory response development (8,9). Moreover, tissue response to biomaterials depends on innate and nonspecific immune responses (5,8,10). The root end filling material is set in close contact with periapical tissue and its chemical composition can interfere on inflammatory response and consequently affect repair process (9,11). Mineral Trioxide Aggregate (MTA) has been widely studied as an endodontic material since it was introduced (12). Both in vivo and in vitro investigations showed that MTA is biocompatible, shows low cytotoxicity, and stimulates hard tissue formation (13,14). Besides, MTA stimulates the release of IL-1β, IL-6, TNF-α, and growth factors from cells (15-17). Even in diabetic conditions, MTA does not alter tissue response, promotes mineralization and induces dentin bridge formation (6,14,18). Despite these investigations, the biological mechanism of MTA, especially its relationship with systemic conditions, is still not completely elucidated. DM induces alterations in immune cell function and stimulates expression of proinflammatory cytokines (3), thus compromising dental pulp and periapical response. In this study, we aimed to evaluate in vitro and in vivo effects of both Gray and White MTA on cell viability and cytokine production under diabetic conditions. Materials and Methods In vitro study Cell Culture L929 mouse fibroblasts were grown in Dulbecco Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, GIBCO Artigo 2 71 BRL, Gaithersburg, MD) streptomycin (50 g/mL), and 1% antibiotic/antimycotic cocktail (300 U/mL penicillin, 300 µg/mL streptomycin, 5 µg/mL amphotericin B) (GIBCO BRL, Gaithersburg, MD) under standard cell culture conditions (37°C, 100% humidity, 95% air, and 5% CO2). The hyperglycemic condition was simulated with cell culture media supplemented with a high concentration of glucose (25 mM glucose). MTA conditioned media Gray and White MTA Angelus® (Angelus Indústria de Produtos Odontológicos S/A, Londrina, PR, Brazil) were mixed according manufacturer’s instructions. MTA discs were prepared as previously described (19) with some modifications. Briefly, the discs were formed using a sterile polyethylene tube (5 mm in diameter and 3 mm in height) and kept in a 5% CO2 incubator at 37°C to setting for 6h. Then, the discs were removed from the mold and sterilized in ultraviolet (UV) light for 1h. Each disc was immersed in 1 mL of DMEM with 10% FBS and incubated in a humidified atmosphere containing 5% CO2. After 3 days, the discs were discarded and supernatants (extract) were collected and filtered through a sterile 0.22 µm filter (Sigma-Aldrich, Saint Louis, MO, USA). The collected supernatants were referred as the GMTA extract or WMTA extract. An extract dilution of 1:50 was used in this study. Cell proliferation assay Cell proliferation was determined using Alamar Blue reduction assay (Alamar Blue® Cell Viability Reagent, Thermo Fisher Scientific, Waltham, MA, USA) following manufacturer’s instructions. L929 fibroblasts were seeded into 24-well plates (104 cells/mL medium per well) and incubated for 24h in a humidified air atmosphere of 5% CO2 at 37oC. After, both MTA extract (1:50) and Alamar Blue reagent (1:10) was added to the culture medium and after 6h, 24h, 48h, and 72h, 200 µl of medium was transferred to a 96 well plate. Optical density (OD) was measured at 570 nm and 600 nm. Alamar blue reduction was calculated with a manufacturer provided formula. The percentage of reduction level reflects the cell proliferation. The controls were cultured in media without MTA extracts. The experiments were performed in triplicate. Artigo 2 72 Inflammatory cytokine production assay For cytokine assay, L929 fibroblasts were seeded into 24-well plates (104 cells/mL medium per well) and incubated for 24h in a humidified atmosphere of 5% CO2 at 37°C. After incubation, both MTA extracts were added to cells at the dilution of 1:50. After 6, 24, 48, and 72h of extracts addition, the culture media were collected and levels of IL-1β, IL-6, and TNF-α were evaluated using DuoSet ELISA kits according to manufacturer’s recommendations (R&D Systems, Minneapolis, MN, USA). Cells cultured without MTA extracts served as controls. In vivo study Animals Twenty male Wistar albino rats weighing 250–280 grams and within 3 to 4 months-old were used in this study. The animals were divided in two main groups: normal and diabetic. The diabetic condition was induced as described previously (14). The study was approved and performed according to the guidelines of Ethical Committee (protocol number 00557-2013). Surgical procedure and immunohistochemical analyses Polyethylene tubes (Abbott Labs of Brazil, São Paulo, SP, Brazil) filled with Gray and White MTA Angelus or empty tubes were implanted in the dorsal connective tissue of rats (14). On 7 and 30 days after implantation, six animals from each group were euthanized and the implanted tubes along with the surrounding tissues were excised, fixed, processed and embedded in paraffin. The tissues were then sliced into 5μm semi-serial sections and submitted to immunohistochemistry using an indirect immunoperoxidase technique for detecting IL-1β (Rabbit anti-IL-1β SC 7884), TNF-α (Goat anti-TNF-α SC 1348), and IL–6 (Rabbit anti-IL-6 SC 1265). Cytokine production near the tube opening was analyzed at 400x magnification (Leica Microsystems, Wetzlar, Germany). A semi-quantitative immunostaining analysis was performed and the criteria for establishing immunoreactivity patterns were adapted from Garcia et al. (20): score 0: total absence of immunoreactivity (IR); score 1 (low IR pattern): IR in approximately 25% of cells per field; score 2 (moderate IR pattern): IR in approximately 50% of Artigo 2 73 cells per field; score 3 (high IR pattern): IR in approximately 75% of cells per field. Statistical analysis Data were subjected to statistical analysis by using the GraphPad Prism (version 5.0) software. For nonparametric data, Kruskal-Wallis test was used followed by Dunn test, and for parametric data, ANOVA followed by Bonferroni’s correction. The p value was considered significant at 5%. Results Effects of MTA on cell proliferation under high glucose concentration The effects of MTA on proliferation of L929 fibroblasts grown at high or normal glucose concentration are shown in Figure 1. In normal conditions, L929 proliferation was not affected by GMTA and WMTA. In contrast, under hyperglycemic conditions, GMTA treatment showed reduced cell proliferation at 24h (p<0.05). Comparison between all groups in both conditions indicated that hyperglycemic conditions impaired the cell proliferation at 72h (p<0.05). Influence of DM on cytokine production upon MTA treatment ELISA was used to assess IL-1β, TNF-α, and IL-6 production by L929 fibroblasts grown under high or normal glucose concentration after MTA treatment. Irrespective of hyperglycemic condition, no production of IL-1β and TNF-α was observed in the presence of both MTAs. However, significant effects on IL-6 production were observed as shown in Figure 2. In normal conditions, Control group released more IL-6 than that by the GMTA-treated group at 6 and 48h (p<0.05). At 48h, IL-6 production upon WMTA treatment significantly reduced compared to that in Control group (p<0.05). Under hyperglycemic condition, at 6 h, Control group secreted more IL-6 than GMTA-treated group (p<0.05). However, at 48h, WMTA-treated group showed more IL-6 production than Control group (p<0.05). Differences between both MTAs were observed only at 24h, when GMTA-treated cells showed more IL-6 production than WMTA-treated cells (p<0.05). Additionally, hyperglycemic conditions promoted up-regulation of IL-6 production in all groups (p<0.05). Artigo 2 74 In contrast, in vivo experiment revealed the presence of IL-1β, TNF-α, and IL-6 positive cells in all groups evaluated on day 7 and 30. However, no significant difference was detected among all groups in both systemic conditions. Cells stained positively for IL-1β, TNF-α, and IL-6 are shown in Figures 3 and 4. Discussion Determination of relationship between systemic diseases and their oral manifestation, especially with regard to tissue response to endodontic materials is challenging, and few studies have emphasized this relationship (6,14,18). DM is a systemic disease associated with impaired cell proliferation and raise on proinflammatory cytokine production (3, 21). In this study, we aimed to evaluate the effect of both Gray and White MTA on cell proliferation and cytokine production under hyperglycemic conditions. Additionally, to simulate hyperglycemic condition, we used glucose concentrations corresponding to those observed in patients with poorly controlled diabetes (25mM) (22). It has been previously reported that glucose induces alteration of gene regulation, differentiation, and cell proliferation (21). Cell growth is dependent on glucose concentration, and high glucose concentration is known to impair cell proliferation (21,22). In our study, cell proliferation was affected by high glucose concentration only at 72h. This result is in agreement with the report by Li et al. (21) that evaluated effect of high glucose concentrations (25mM) in two different types of cell and demonstrated that cell proliferation varies according to evaluated cell type and the glucose exposure time, where increasing exposure time results on decreasing cell proliferation. L929 proliferation was not inhibited in normal conditions by both MTAs, thus confirming low cytotoxicity of these materials as reported previously (16,23). In contrast, Haglund et al. (24) reported that MTA caused inhibits growth of L929 fibroblasts. However, under hyperglycemic conditions, GMTA showed increased cytotoxicity only at 24h. DM is associated with altered calcium homeostasis (25) and the difference in chemical composition of both MTAs (26) results in release of more Ca+2 ions with GMTA treatment in initial periods (27), which could increase intracellular calcium levels and alter cell functions. These facts in relation to impaired cell proliferation induced by Artigo 2 75 hyperglycemic state and observation that related cell cultures show better growth upon treatment with WMTA (28), could explain the obtained results. Moreover, no difference in cytotoxicity and cell proliferation between GMTA and WMTA has been described in literature (23). Although studies have shown release of cytokines IL-1β, TNF-α, and IL-6 by cells grown in presence of both MTAs (15-17), no expression of IL-1β and TNF-α was observed with both tested materials, irrespective of diabetic condition. However, IL-6 production was observed at all time points evaluated. IL-6 is a pro- and anti-inflammatory cytokine released by several cell types in response to irritants (29) and can be correlated with suppression of IL-1β and TNF-α transcription (30). Besides, IL-1β and IL-6 were not previously detected in presence of MTA probably owing to cell type and time of exposure to MTA (24). In addition, the effect of MTA on cytokine production depends of cell culture type, MTA composition, and specific cytokines investigated (13). In this study, MTA extract was used to avoid direct contact between the test material and cells, which could change both properties of material and cell response (19,31). IL-6 expression has significant anti-inflammatory effects in modulating infection-stimulated bone destruction (32). In normal conditions, ELISA revealed IL-6 production in the presence of both MTAs corroborating with previous studies (15,16). In addition, Control group produced more IL-6 compared to that with GMTA at 6h and 48h, and with WMTA at 48h. These findings can be explained by studies that reported an anti-inflammatory effect for MTA resulting in decreased production of some proinflammatory cytokines (8,33). Meanwhile, the average IL-6 production was higher in hyperglycemic condition than that under normal conditions. Glucose intake is reported to cause oxidative stress and inflammatory changes at cellular and molecular levels, which promotes upregulation of IL-6 and other cytokines (34), thus corroborating our findings. Furthermore, at 24h, GMTA-treated cells showed more IL-6 production than WMTA-treated cells did, and at 48h, WMTA-treated cells showed more IL-6 production than Control group did. The ionic dissociation of GMTA may be changed by its high concentration of Fe2O3 (26), resulting in release of more Ca+2 ions at the initial time compared to that by WMTA (27). Because an increase in Ca+2 ions may act as a tissue irritant and Artigo 2 76 IL-6 being a cytokine released in response to irritants, we considered that these facts might explain our results in the diabetic conditions. Although cell culture studies are widely used to investigate the cytotoxicity of a biomaterial, they cannot be used to examine host-biomaterial interaction. Thus, to better understanding the DM influence on cytokine production of both MTAs, a subcutaneous implantation study was performed. Irrespective of diabetic condition, IL-1β, IL-6, and TNF-α immune-reactive cells were identified at all time points evaluated in presence of both MTAs. In fact, previous studies reported IL-1β, IL-6, and TNF-α production in presence of MTA (8,33). However, no difference was observed between both MTAs and systemic conditions. Although DM induced an inflammatory response and cytokines release (3,34), no evidence of a direct correlation between DM and production of IL-1β, IL-6, and TNF-α upon MTA treatment was established. In addition, no reports evaluating such cytokine production were found in existing literature. The ability of MTA to precipitate apatite during acute phase of inflammation, together with its alkalinity, might induce modifications in gene expression and signaling pathways (35). These facts together with anti- inflammatory effect of MTA (8,33) and its capacity to promote no significant tissue inflammation (14) could affect these cytokines production and explain ours findings. Besides, earlier reports have shown no difference in inflammatory response to MTA in diabetic animals (14,18). Therefore, it is clear that further investigations are necessary to clarify the correlation between DM and inflammatory and immune response to endodontic materials. On basis of these results, we concluded that hyperglycemic condition interfered on cell proliferation and IL-6 production observed with GMTA treatment at 24hs. However, IL-1β, IL-6, and TNF-α production in tissue of diabetic animals was not altered in presence of both MTAs. Conflicts of interest The authors deny any conflicts of interest related to this study. Artigo 2 77 Acknowledgments