AMANDA CASELATO ANDOLFATTO SOUZA Ação antimicrobiana e antibiofilme de flavonóis isolados ou incorporados em hidrogéis termossensíveis e sua influência sobre marcadores de mineralização em células odontoblastóides para aplicação em Endodontia Araçatuba 2022 AMANDA CASELATO ANDOLFATTO SOUZA Ação antimicrobiana e antibiofilme de flavonóis isolados ou incorporados em hidrogéis termossensíveis e sua influência sobre marcadores de mineralização em células odontoblastóides para aplicação em Endodontia Tese apresentada à Faculdade de Odontologia da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de Araçatuba, para obtenção do título de Doutor em Ciência Odontológica, área de concentração Endodontia. Orientadora: Prof. Assoc. Cristiane Duque Araçatuba 2022 Catalogação na Publicação (CIP) Diretoria Técnica de Biblioteca e Documentação – FOA / UNESP Souza, Amanda Caselato Andolfatto. S729a Ação antimicrobiana/antibiofilme de flavonóis isolados ou incorporados em hidrogéis termossensíveis e sua influência sobre marcadores de mineralização em células odontoblastói- des para aplicação em endodontia / Amanda Caselato Andolfatto Souza. - Araçatuba, 2022 149 f.: il. ; tab. Tese (Doutorado) – Universidade Estadual Paulista, Faculdade de Odontologia de Araçatuba Orientadora: Profa. Cristiane Duque 1. Flavonóis 2. Anti-infecciosos 3. Biofilmes 4. Hidrogéis 5. Citotoxicidade 6. Odontoblastos I. T. Black D24 CDD 617.67 Claudio Hideo Matsumoto – CRB-8/5550 Dados Curriculares Amanda Caselato Andolfatto Souza Nascimento 20.07.1990 – Araçatuba - SP Filiação Antônio Roberto Andolfatto de Souza Sirlene de Fátima Caselato Souza 2009/2012 Curso de Graduação em Odontologia pela Faculdade de Odontologia da Ribeirão Preto – UNAERP. 2014/2016 Curso de Especialização em Endodontia, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Faculdade de Odontologia de Araçatuba - FOA 2016/2018 2018/2022 Mestrado em Ciência Odontológica, área de concentração Endodontia, Universidade Estadual Paulista "Júlio de Mesquita Filho", Faculdade de Odontologia – FOA, com bolsa CAPES. Doutorado em Ciência Odontológica, área de concentração Endodontia, Universidade Estadual Paulista "Júlio de Mesquita Filho", Faculdade de Odontologia – FOA, com bolsa CAPES. Associações CROSP - Conselho Regional de Odontologia de São Paulo. SBPqO - Sociedade Brasileira de Pesquisa Odontológica. COMISSÃO EXAMINADORA TESE PARA OBTENÇÃO DO GRAU DE DOUTOR Profa. Dra. Cristiane Duque - Orientadora. Professora Associada do Departamento de Odontologia Preventiva e Restauradora, Disciplina de Odontopediatria, Faculdade de Odontologia, FOA-UNESP, Araçatuba, São Paulo, Brasil. Profa. Dra. Simone Nataly Busato de Freria – Pesquisadora colaboradora do Departamento de Diagnóstico Oral, Disciplina de Microbiologia e Imunologia, Faculdade de Odontologia de Piracicaba, FOP-Unicamp, Piracicaba, São Paulo, Brasil. Prof. Dr. Leopoldo Cosme - Professor Doutor da Disciplina de Endodontia, Faculdade de Odontologia, Universidade Federal de Alagoas, UFAL, Maceió, Brasil. Profa. Dra. Aimée Maria Guiotti - Professora Doutora do Departamento de Materiais Odontológicos e Prótese, Disciplina de Materiais Dentários, Faculdade de Odontologia de Araçatuba, FOA –UNESP, Araçatuba, São Paulo, Brasil. Prof. Dr. Gustavo Sivieri de Araújo - Professor Doutor do Departamento de Odontologia Preventiva e Restauradora, Disciplina de Endodontia da Faculdade de Odontologia de Araçatuba, FOA – UNESP, Araçatuba, São Paulo, Brasil. Dedicatória Dedico este trabalho à minha família, meus pais Sirlene e Antônio, meu irmão Vitor Hugo, meus tios Marina e José Roberto, e à minha orientadora Cristiane Duque. Por todo suporte, compreensão, exemplos de vida e carinho que recebi nesses anos de pós-graduação. Sou muito grata. Agradecimentos AGRADECIMENTOS À Deus, primeiramente, por toda sua grandeza!! Obrigada por me proporcionar a família em que nasci, o ambiente em que vivo e as pessoas da minha vida. Assim tenho a oportunidade de crescer e evoluir. Obrigada pela vida privilegiada. Obrigada por todo discernimento, felicidade, caráter e luz no meu caminho! Sou eternamente grata pela Sua companhia! Aos meus amados pais, Sirlene de Fátima Caselato Souza e Antônio Roberto Andolfatto de Souza. Obrigada por tanto amor, carinho, dedicação, educação, respeito e alegria! Por mostrar a importância disso isso a mim e ao meu irmão! Isto é, com toda certeza, fundamental para formação do nosso caráter. Obrigada por todos os bons exemplos sempre transmitidos a nós, como generosidade, amor ao próximo e humildade. Obrigada por serem a minha base! Me sinto privilegiada diante de tanto amor e acolhimento! E por ter tido uma infância, a melhor fase da minha vida, tão alegre! Obrigada por sempre estarem ao meu lado em todos os momentos! Obrigada por me incentivarem a seguir meus sonhos e batalhar junto comigo para que eles se tornem realidade. Obrigada por, na maioria das vezes, passarem por cima de suas vontades para que as minhas sejam feitas! Sou eternamente grata por me ensinarem que sem a perseverança não há nada que possamos conseguir e sem ela não chegaremos a lugar algum. Obrigada por serem tão humanos, atenciosos e amorosos comigo! Poderia tentar explicar o amor que sinto por vocês com palavras, mas estas são pequenas perto do meu sentimento! A conquista desse trabalho é nossa. Muito obrigada por tudo! Amo vocês incondicionalmente! Peço desculpas pelas minhas falhas. Ao meu querido irmão, Vitor Hugo Caselato Andolfatto Souza, pela companhia, pela força, pelos momentos felizes, por tantos ensinamentos e pelo seu jeito de ser. Obrigada por ser um dos meus alicerces. Eu te amo incondicionalmente!! Conte comigo para o que você precisar! À minha querida cunhada, Eduarda Sayeg Corbucci, pela delicadeza, pela educação e pelas conversas! Obrigada por estar ao lado do meu irmão, fazê-lo feliz e amado. Por ajudá-lo tanto! Amo você! Aos meus queridos tios, Marina Aparecida Colaferro e José Roberto de Souza Andolfatto, por me acolherem em sua residência como uma verdadeira filha, por tamanho zelo e proteção desde a época da especialização que se iniciou em 2014, aliás por sempre receber a mim e minha família. Muito obrigada por serem tão atenciosos comigo. O amor que sinto por vocês é imensurável! Contem sempre comigo. Obrigada por não me deixarem cair nos momentos de dificuldade durante esse tempo, quando meus pais estavam em São José do Rio Preto, e por toda ajuda, carinho e união! A conquista desse trabalho é nossa! Essa homenagem se estende à minha prima Camila Colaferro. Obrigada pelas conversas, pelos conselhos e pelos momentos de risadas. Muito obrigada, Mi, por ser esse presente na minha vida! Amo muito vocês! Às minhas queridas famílias Caselato e Andolfatto, por apoiarem meus sonhos, mesmo sem entender muito o porquê de algumas coisas, por estarem sempre presentes, por me fazerem rir mesmo em tempos de agonia, por me ensinarem a ver a beleza da vida. Muito obrigada pelas orações, pelo tempo e pelo amor dedicado a mim!! A cada um de vocês o meu muito obrigada! Amo muito vocês! À minha querida orientadora, Cristiane Duque! Professora, sempre admirei o jeito carinhoso com que trata as pessoas, a admiração que tenho por você vai além de simples palavras. Pois a senhora é muito mais que um exemplo profissional, é exemplo de caráter, de humildade, de bondade, de perseverança, de dedicação e de ser humano. Serei eternamente grata a todos os ensinamentos transmitidos a mim. Obrigada pelo cuidado e delicadeza nesses anos de doutorado. Desculpa minhas falhas, sei que posso não ser a pessoa mais fácil de lidar em certos momentos. Muito obrigada por tudo! Agradeço aos meus queridos companheiros de equipe que se tornaram amigos a quem sou muito grata! Jesse Augusto Pereira, obrigada por me ensinar com seu jeito tranquilo, que energia boa a sua. Rafaela Laruzo Rabelo, obrigada pelo seu jeito descontraído e amigo. Karina Sampaio Caiaffa, obrigada pela sua disposição e pelos ensinamentos, pelas conversas no laboratório, sempre que precisei você esteve ao meu lado. Vanessa Rodrigues dos Santos, obrigada por me ensinar calmamente os procedimentos que fizemos juntas e pelo seu senso de humor ímpar. Gabriela Braga Pacheco, um agradecimento especial para você, por me ajudar e me acompanhar cuidadosamente nesses últimos meses do doutorado, que foram bem intensos, sem você não teria dado certo, muito obrigada! Obrigada por ser tão prestativa e amorosa. Contem sempre comigo! Agradeço aos meus amigos do laboratório de odontopediatria, Letícia Capalbo, Heitor Ceolin, Igor Zen, Francyenne Castro, Priscila Toniatto, Caio Sampaio, Gabriel Nunes e Leonardo Morais, por proporcionarem um ambiente harmonioso e leve mesmo com todas as responsabilidades, às vezes tensas, da pós-graduação. Além disso, por toda ajuda que eu precisei. Obrigada por serem tão prestativos e amigos dentro ou fora do ambiente de trabalho! Contem sempre comigo! Aos meus queridos amigos da época do mestrado, quando ainda frequentava o laboratório de endodontia, Cristiane Cantiga e Pedro Henrique Chaves, obrigada por serem bons amigos, me darem tanto suporte e compartilhar momentos alegres comigo. Contem sempre comigo! À minha grande e querida amiga, Carolina Cardoso de Moraes Barros, que esteve ao meu lado em todos os momentos da pós-graduação, que se tornou uma irmã. Obrigada pelos conselhos, pelas conversas, pelo acolhimento em horas difíceis, pela amizade e por se fazer tão presente na minha vida! Amo você! À grande e querida amiga Carina dos Santos Souza, pelo seu jeito sossegado e amigo de ser! Muito obrigada por deixar meus dias mais leves, com nossas conversas depois de um longo dia ou durante os nossos treinos funcionais na sua casa ou na academia. Obrigada pelo companheirismo e por ser tão presente!!! Amo você! À professora Thayse Hosida e Marcelle Danelon, pelo aceite para fazer parte da banca examinadora do meu exame geral de qualificação tão prontamente. Tive a oportunidade de conhece-las dentro do laboratório de odontopediatria quando comecei o mestrado, e desde então as admiro muito pelo trabalho e dedicação de vocês. Obrigada! O agradecimento se estende aos suplentes Natalia Leal Vizoto e Gustavo Sivieri de Araújo, obrigada pela disponibilidade e atenção. À Profa. Dra. Simone Nataly Busato de Freiria, pela disponibilidade e aceite ao convite de participar da banca examinadora. Ao Prof. Dr. Gustavo Sivieri de Araújo, obrigada pela disponibilidade e aceite do convite para participar da banca examinadora da defesa da tese do meu doutorado. Tive o prazer de conhecer o senhor durante o meu mestrado, o senhor foi sempre muito solícito, dando dicas nos seminários e encorajando os alunos a buscarem sua melhor versão. Admiro sua trajetória e seu trabalho, professor. À Profa. Dra. Aimée Maria Guiotti, com quem tive o prazer de conviver em algumas disciplinas durante a pós. Obrigada por ter aceito o convite para participar da banca examinadora, professora. Sempre admirei seu trabalho, seu jeito atencioso e dócil foi bem marcante para mim durante as disciplinas, professora. Ao Prof. Dr. Leopoldo Cosme, com quem tive o prazer de conviver por um período no departamento de endodontia da FOA. Aprendi muito com você, obrigada pelos momentos alegres no departamento e por me ajudar com alguns trabalhos. Obrigada por compartilhar essa energia boa que você tem e por ter aceitado o convite da banca examinadora na defesa da tese. Aos professores José Antonio Santos Souza, Thiago Cruvinel da Silva e Ana Claudia Okamoto, pela disponibilidade e aceite do convite para participar da minha banca examinadora de defesa da minha tese de doutorado. A todos os professores da Pós-Graduação pelos ensinamentos transmitidos nas disciplinas da pós-graduação. Vocês contribuíram muito para meu crescimento, agradeço por me ajudarem a chegar até aqui. Foi uma honra ter a oportunidade de conhecê-los. Meus sinceros agradecimentos! Aos Funcionários da FOA-UNESP, por serem acolhedores, por desejarem bom-dia, até amanhã, bom trabalho. Agradeço pelo cafezinho e as bolachinhas no departamento. Pelos momentos de descontração pelas risadas. Por sempre se disporem a nos ajudar, muito obrigada. Aos vigilantes, por cuidar da instituição com tanta responsabilidade. As funcionárias da seção de pós-graduação, Cris, Lilian e Valéria, por trabalharem para que toda a burocracia seja executada corretamente, pela paciência de sempre esclarecendo todas as minhas dúvidas e pelos e-mails informando o que era importante. À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Capes – Código de Financiamento 001 – que apoiou a realização do presente trabalho. Agradeço por me concederem recursos fundamentais para minha manutenção na pós-graduação e para a conclusão do doutorado. À Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP, processo: #2017/10940-1, pela concessão da bolsa, que permitiu a realização deste estudo. Agradeço pelo auxílio- pesquisa que foi fundamental para o desenvolvimento desta tese. Epígrafe “Um dia, quando olhar para trás, os dias de luta serão os mais bonitos” Sigmund Freud Resumo Geral SOUZA, A.C.A. Ação antimicrobiana/antibiofilme de flavonóis isolados ou incorporados em hidrogéis termossensíveis e sua influência sobre marcadores de mineralização em células odontoblastóides para aplicação em Endodontia. 2022. 149f. Tese (Doutorado) em Ciência Odontológica - área Endodontia, Faculdade de Odontologia, Universidade Estadual Paulista, Araçatuba, 2022. RESUMO Ainda não foi encontrado um medicamento capaz de desinfetar os canais radiculares e permitir a recuperação celular e a regeneração tecidual em dentes permanentes jovens com comprometimento endodôntico. Dois importantes flavonóis detectados no vinho tinto, morina (MO) e miricetina (MY), são atualmente estudados por suas amplas propriedades biológicas, incluindo atividade antimicrobiana. No entanto, o desenvolvimento de sistemas de liberação controlada pode ser útil para a liberação desses flavonóis para fins de terapia endodôntica. Este estudo avaliou a citocompatibilidade e os efeitos antimicrobianos/antibiofilme de MO e MY, isolados ou incorporados em hidrogéis termorreversíveis de quitosana-poloxamer-β-glicerofosfato de sódio (CPG), além dos efeitos de MO e MY, isolados e combinados sobre a viabilidade, atividade de ALP e produção de nódulos de mineralização em células MDPC-23. A atividade antimicrobiana dos compostos foi avaliada em Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii e Fusobacterium nucleatum em condições planctônicas, em biofilmes dual-espécies e multiespécies e analisadas por contagem bacteriana e microscopia de varredura. Os hidrogéis CPG foram caracterizados por reometria de fluxo e oscilatória, temperatura de gelificação, perfil de textura e análise de bioadesão em espécimes de dentina. MO, MY e controles (hidróxido de cálcio – CH e clorexidina – CHX) foram incorporados em hidrogéis de CPG e o efeito do antibiofilme sobre biofilmes multiespécies formados em amostras de dentina radicular foi avaliado por microscopia confocal. O efeito de toxicidade dos compostos isolados ou incorporados em hidrogéis de CGP foi determinado em cultura de fibroblastos por ensaios de resazurina. Os dados foram analisados estatisticamente pelos testes ANOVA e Tukey considerando p < 0,05. A combinação de MO e MY foi sinérgica ou aditiva contra bactérias endodônticas testadas a partir de concentrações de 0,03 mg/mL MO + 0,06 mg/mL MY e não foram tóxicas para fibroblastos até 0,125mg/mL. MO + MY apresentou melhor efeito sobre biofilmes dual-espécies e multiespécies considerando suas menores concentrações quando comparados com os flavonóis isolados. Os hidrogéis CPG foram caracterizados como termorreversíveis e com propriedades mecânicas e bioadesivas adequadas. Hidrogéis de CPG carregados com MO+MY, CH e CHX apresentaram efeitos inibitórios semelhantes quando aplicados em biofilmes multiespécies formados no interior dos túbulos dentinários radiculares por 48h e seus extratos apresentaram citotoxicidade acima de 50% de diluição. As células semelhantes a odontoblastos (MDPC-23) foram expostas a diferentes concentrações de MO, MY, isoladamente ou em combinação e CH como controle positivo por 24h e 48h, e troca contínua de meio osteogênico por 8 dias e 14 dias. As combinações de MO+MY ou CH também foram incorporadas em hidrogéis de quitosana-poloxamer-β-glicerofosfato e seus extratos em meio de cultura celular foram coletados após 48h e 7 dias. Viabilidade celular, atividade de fosfatase alcalina (ALP) e ensaios de deposição de nódulos mineralizados (MN) foram realizados pelo método de resazurina, ensaios de monofosfato de timolftaleína e coloração com vermelho de alizarina, respectivamente. Todos os compostos não causaram citotoxicidade nas concentrações testadas em 24h e 8 dias e 0,5 mg/mL MO e MY isolados reduziram a viabilidade celular em 48h. A atividade de ALP e a deposição de MN foram aumentadas para a combinação MO+MY e CH em células MDPC-23. Extratos de hidrogel de 7 dias contendo ou não MO+MY não foram citotóxicos até diluição de 25% em 48h e em baixas concentrações estimularam a atividade de ALP e deposição de MN aos 8 e 14 dias de avaliação. Em conclusão, a combinação de morina e miricetina incorporada ou não em hidrogéis de CPG apresentou efeito antibiofilme sobre patógenos orais e baixa toxicidade sobre fibroblastos. Morina e miricetina em baixas concentrações, isoladas, em combinação ou em hidrogéis CPG não foram citotóxicas e foram eficazes na indução de marcadores de mineralização em células semelhantes a odontoblastos. Palavras-chave: flavonóis, atividade antimicrobiana, biofilmes, hidrogel, citotoxicidade, odontoblastos, mineralização dentinária. General Abstract SOUZA, A.C.A. Antimicrobial/antibiofilm action of flavonols alone or incorporated in thermosensitive hydrogels and their influence on mineralization markers in odontoblast-like cells for endodontic applications. 2022. 149f. Tese (Doutorado) em Ciência Odontológica - área Endodontia, Faculdade de Odontologia, Universidade Estadual Paulista, Araçatuba, 2022. ABSTRACT A drug capable of disinfecting the root canals and allow cell recovery and tissue regeneration in permanent young teeth with endodontic problems has not been found yet. Two important flavonols detected in red wine, morin (MO) and myricetin (MY), are currently studied for their wide biological properties including antimicrobial activity. However, the development of controlled release systems could be useful for the delivery of these flavonols for endodontic therapies. This study evaluated the cytocompatibility and antimicrobial/antibiofilm effects of MO and MY, alone or incorporated in thermoreversible chitosan- poloxamer hydrogels containing sodium β-glycerophosphate (CPG), in addition to the effects of isolated and combined morin and myricetin flavonols on viability, ALP activity and production of mineralization nodules in MDPC-23 cells. Antimicrobial activity of the compounds was evaluated on Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii, and Fusobacterium nucleatum under planktonic conditions, on dual-species and multispecies biofilms and analyzed by bacterial counts and scanning microscopy. CPG hydrogels were characterized by flow and oscillatory rheometry, gelation temperature, texture profile and bioadhesion analysis in dentin specimens. MO, MY and controls (calcium hydroxide – CH and chlorhexidine – CHX) were incorporated in CPG hydrogels and antibiofilm effect on multispecies biofilms formed in radicular dentin samples were evaluated by confocal microscopy. Cytotoxicity of the compounds alone or incorporated in CGP hydrogels was determined on fibroblasts culture by resazurin assays. Data were statistically analyzed by ANOVA and Tukey considering p < 0.05. The combination of MO and MY had synergistic or additive against oral bacteria tested starting at concentrations of 0.03 mg/mL MO + 0.06 mg/mL MY and they were not toxic to fibroblasts up to 0.125mg/mL. MO + MY had better effect on dual-species and multispecies biofilms considering their lower concentrations when compared with the flavonols alone. CPG hydrogels were characterized as thermoreversible and with adequate mechanical and bioadhesive properties. CPG hydrogels loaded with MO+MY, CH and CHX have similar inhibitory effects when applied on multispecies biofilms formed inside root dentin tubules for 48h and their extracts were cytotoxicity above 50% dilution. Furthermore, the effects of morin, myricetin, alone or in combination or incorporated in chitosan-based hydrogels on cytotoxicity and expression of mineralization markers in odontoblast-like cells. The MDPC-23 cells were exposed to different concentrations of morin (MO), myricetin (MY), alone or in combination and calcium hydroxide (CH) as a positive control for 24h and 48h, and continuous osteogenic medium changing for 8 days and 14 days. The combinations of MO+MY or CH were also incorporated in chitosan-poloxamer-β- glycerophosphate hydrogels and their extracts in cell culture media were collected after 48h and 7 days. Cell viability, alkaline phosphatase (ALP) activity and assays mineralized nodules (MN) deposition were performed using resazurin method, thymolphthalein monophosphate assays and alizarin red staining, respectively. Data were statistically analyzed considering p<0.05. All compounds were non-toxic at the concentrations tested at 24h and 8 days and 0.5 mg/mL MO and MY alone reduced cell viability at 48h. ALP activity and deposition of MN were increased for MO+MY combination and CH in MDPC-23 cells. 7 days hydrogel extracts containing or not MO+MY were not cytotoxic up to 25% dilution at 48h and at low concentrations stimulated ALP activity and MN deposition at 8 and 14 days of evaluation. In conclusion, the combination of morin and myricetin incorporated or not in CPG hydrogels presented antibiofilm effect on oral pathogens and low cytotoxicity on fibroblasts. Morin myricetin at low concentrations, alone, in combination or in CPG hydrogels were not cytotoxic and were effective in inducing mineralization markers in odontoblast-like cells. Key words: flavonols, antimicrobial activity, biofilms, hydrogel, cytotoxicity, odontoblasts, dentin mineralization. Lista de figuras LISTA DE FIGURAS Capítulo 1 Figure 1 Effect of the flavonoids morin (MO) and myricetin (MY) alone or in combination (MO + MY) and controls calcium hydroxide (CH) and chlorhexidine (CHX) on the viability of fibroblasts (3T3) after 24h of exposure, using staining with resazurin. Concentrations in mg/mL. 51 Figure 2 Effect of flavonoids and controls on dual-species biofilms (48-72h) of oral bacteria after 36h of treatment. Graphics on the left side represent microbial total counts and graphics on the right side represent E. faecalis counts. 52 Figure 3 Representative scanning electron microscopy images of 14-day multispecies biofilms under 1000x magnification. Biofilms were treated for 48 hours with A: Morin (MO) - 5 mg/mL; B - Myricetin (MY) – 5 mg/mL, C - Morin + Myricetin (MO+MY) – 2,5 mg/mL each one, D - Calcium hydroxide (CH) – 1 mg/mL, E - CHX - 0.5 mg/mL, and F - Control – Bacterial growth without antimicrobial agents. G. Mean (SD) of the bacterial counts detected after 48h of the biofilm treatment with flavonoids and controls. 54 Figure 4 Continuous rheological behavior of hydrogels composed of chitosan, poloxamer, and β-glycerophosphate (CPG). Empty symbols indicate an upward curve, and full symbols, a downward curve. 55 Figure 5 Frequency sweep profile of storage modulus G’ (opened symbols) and loss modulus G’’ (closed symbols) of hydrogels composed of chitosan, poloxamer and β-glycerophosphate (CPG). (A) Analyzes performed at 25℃, (B) Analyzes performed at 37 ℃. 56 Figure 6 Temperature sweep analyses demonstrating of storage modulus G’ (blue symbol) and loss modulus G’’ (green symbol) and loss tangent (Tan d) (red symbol) of hydrogels composed of chitosan, poloxamer and β- glycerophosphate (CPG). 57 Figure 7 Representative confocal microscopy images of bovine root dentin specimens contaminated for 14 days with multispecies biofilms and treated for 48 hours with the following groups with A: Hydrogel (CPG) with Morin + Myricetin (MO+MY) – 2,5 mg/mL each one, B – CPG 58 with calcium hydroxide (CH) – 1 mg/mL, C – CPG with CHX - 0.5 mg/mL, D – CPG alone, E - Control – Bacterial growth without antimicrobial agents. F. Mean (SD) of the percentages of dead cells of multispecies biofilms after 48h of treatment with flavonoids and controls. Bacterial counts were obtained by Image J analysis. Figure 8 Effect of the 48h (A) and 7 days (B) extracts of chitosan-poloxamer hydrogels with glycerophosphate (CPG) containing morin and myricetin in combination (MO+MY) and controls calcium hydroxide (CH) and chlorhexidine (CHX) on the viability of fibroblasts (3T3) after 24h of exposure, using staining with resazurin. Concentrations in mg/mL. 59 Capítulo 2 Figure 1 Experimental design of the study. A. MDPC-23 treatments with MO, MY, combination of MO+MY and CH. B. MDPC-23 treatments with 48h and 7 days extracts of hydrogels containing MO+MY and CH 78 Figure 2 Percentage of MDPC-23 viability after 24 and 48h of treatment with morin (MO), myricetin (MY), combinations of MO + MY and calcium hydroxide (CH). 82 Figure 3 Percentage of MDPC-23 viability at 8 and 14 days of evaluation after 48h treatment with morin (MO), myricetin (MY), combinations of MO + MY and calcium hydroxide (CH). 83 Figure 4 ALP activity of MDPC-23 cells at 8 days of evaluation after 48h of the treatment with morin (MO), myricetin (MY), combinations of MO + MY and calcium hydroxide (CH). 84 Figure 5 Mineralized nodules deposition of MDPC-23 cells at 14 days of evaluation after 48h of the treatment with morin (MO), myricetin (MY), combinations of MO + MY and calcium hydroxide (CH). 85 Figure 6 Percentage of MDPC-23 viability after 48 h treatments with 48h and 7 days hydrogels extracts containing or not morin (MO) + myricetin (MY) or calcium hydroxide (CH). 86 Figure 7 Percentage of MDPC-23 viability after 48 h treatments with 48h and 7 days hydrogels extracts containing or not morin (MO) + myricetin (MY) or calcium hydroxide (CH). 87 Figure 8 Percentage of MDPC-23 viability at 14 days of evaluation after 48h treatments with 48h and 7 days hydrogels extracts containing or not morin (MO) + myricetin (MY) or calcium hydroxide (CH 88 Figure 9 ALP activity of MDPC-23 cells at 8 days of evaluation after 48h treatments with 48h and 7 days hydrogels extracts containing or not morin (MO) + myricetin (MY) or calcium hydroxide (CH). 89 Figure 10 Mineralized nodules deposition by MDPC-23 cells at 14 days of evaluation after 48h treatments with 48h and 7 days hydrogels extracts containing or not morin (MO) + myricetin (MY) or calcium hydroxide (CH). The values are expressed in means/standard deviations. CPG - Quitosan, poloxamer and b-glicerophosphate hydrogels. 90 Suppl. 1 Suppl.1. Images of alizarin red staining obtained by inverted light microscope, indicating the bioactive effect of flavonols on the MDPC- 23 cells relative to mineralized nodules formations. The greatest nodules deposition can be seen in the groups MY, MO+MY and CH, compared to the control (DMEM). 99 Suppl. 2 Images of alizarin red staining obtained by inverted light microscope, indicating the bioactive effect of 7 days-CPG hydrogels extracts containing the flavonols and controls on the MDPC-23 cells relative to mineralized nodules formations. All groups (CPG + MOMY, CPG CH and CPG presented greater nodules deposition when compared to the control (DMEM). 100 Lista de tabelas LISTA DE TABELAS Capítulo 1 Table 1 Flavonols and controls used in the present study 41 Table 2 Minimal inhibitory concentration (MIC), minimal bactericidal/fungicidal concentration (MBC) and fractional inhibitory concentration (FIC) in mg/mL for the flavonoids and control chlorhexidine against the oral microorganisms tested 50 Sumário SUMÁRIO Introdução Geral 29 Capítulo 1 36 Abstract 38 Introduction 39 Material and Methods 41 Results 50 Discussion 60 Conclusions 63 References 63 Capítulo 2 71 Highlights 72 Abstract 72 Introduction 76 Material and Methods 77 Results 82 Discussion 91 References 95 Conclusão 102 Anexos 103 Anexo A 104 Anexo B 109 Anexo C 110 29 Introdução Geral 30 INTRODUÇÃO GERAL Após a erupção de um dente permanente na cavidade bucal, ainda são necessários por volta de quatro anos para o desenvolvimento completo dos canais e o fechamento dos ápices radiculares. Dessa forma, o tratamento de dentes permanentes jovens que sofreram danos pulpares, por vezes irreversíveis, antes do fechamento fisiológico normal do ápice radicular é um desafio em Endodontia, pois além de eliminar a infecção, o medicamento deve permitir ou até estimular o fechamento do ápice e a estimulação da mineralização do local da lesão periapical (Iglesias-Linares et al., 2013; Andreasen & Kahler, 2015). Quando ocorre uma infecção endodôntica, as bactérias no canal radicular ficam aderidas às paredes dentinárias formando biofilmes e certos fatores como ambiente anaeróbio, interações entre microrganismos e disponibilidade de nutrientes estabelecem a composição dessa microbiota residente (Ricucci e Siqueira, 2010 a,b; Swimberghe et al., 2019). Devido às irregularidades anatômicas do sistema de canais radiculares, as bactérias podem se proliferar para as ramificações apicais, canais laterais, istmos, túbulos dentinários o que dificulta o desbridamento e a desinfecção durante o tratamento endodôntico (Ricucci e Siqueira, 2010 a,b; Swimberghe et al. , 2019). Consequentemente, uma alta frequência e abundância das mesmas espécies de bactérias comumente encontradas em infecções primárias, como estreptococos e actinomyces, foram encontradas em infecções pós-tratamento. Uma das espécies mais prevalentes encontradas nessas condições é Enterococcus faecalis, um coco Gram-positivo aeróbio, considerado um patógeno oportunista associado à cavidade oral, trato gastrointestinal e vagina humanos conhecido por sua capacidade de se estabelecer em ambientes hostis com baixos níveis de oxigênio e complexas comunidades microbianas (Lee e Tan, 2014; Alghamdi e Shakir 2020). A capacidade de E. faecalis de se proliferar rapidamente e formar biofilme nas paredes do canal radicular e a grande resistência a agentes antimicrobianos, torna esse patógeno um dos mais difíceis de ser eliminado durante o tratamento endodôntico (Estrela et al., 2008; Murad et al. 2012). A pasta de hidróxido de cálcio é largamente utilizada para o tratamento de dentes permanentes imaturos, seja em situações de pulpite reversível, em procedimentos de terapia pulpar vital (como capeamento pulpar direto ou pulpotomia) em que se busca a apicigênese ou completa formação da raiz, seja em casos de dentes com pulpite irreversível/necrose pulpar em que se tradicionalmente realiza-se procedimentos de 31 apicificação para induzir a formação de barreira apical de tecido duro (Rafter, 2005; AAE, 2022). As desvantagens da apicificação residem nas múltiplas sessões de troca da medicação ao longo de um extenso período, a citotoxicidade do medicamento que impede a proliferação de células remanescentes e a incompleta formação das raízes que consequentemente aumentam o risco de reinfecções e de fraturas dentárias (Rafter et al., 2005; Andreasen et al; 2002; Khoshkhounejad et al., 2019). A busca por tratamentos capazes de bioestimular as células remanescentes (fibroblastos, odontoblastos, entre outras) ou induzir a diferenciação de células tronco da polpa dentária (Asgary et al., 2014; Chen et al., 2015; Huang et al., 2008; Yuan et al., 2011), promover uma desinfecção eficaz sem causar citotoxicidade (Bottino et al., 2013; Kim et al., 2008; Park et al., 2008), ou ainda criar um arcabouço capaz de manter a liberação adequada de biomoléculas e também ser suporte para proliferação e diferenciação celular (Bottino et al., 2015) tem sido alvo de diversos estudos no campo da terapia endodôntica regenerativa (RET). Diferentes abordagens da RET estão sendo estudadas como alternativas aos procedimentos de apicificação, comumente realizados em dentes permanentes jovens, pois visam continuar a deposição de tecido duro nas paredes dentinárias e dessa forma, permitir o adequado comprimento e resistência à raiz evitando fraturas (Banchs e Trope, 2004; Pulyodan et al., 2020). A análise de marcadores de mineralização em células diferenciadas ou indiferenciadas tem sido amplamente realizada com o intuito de analisar a capacidade bioestimulatória de diferentes materiais, visando induzir o processo de reparo ou regeneração (Da Silva et al., 2010; Wang et al., 2017). Dentre esses marcadores de mineralização estão a enzima fosfatase alcalina (ALP) e as proteínas não-colagenosas denominadas de proteína da matriz dentinária (DMP-1) e sialofosfoproteína dentinária (DSPP), que participam da mineralização da dentina e maturação das fibras colágenas durante o processo de dentinogênese. Essas proteínas permanecem no interior do substrato dentinário, sendo liberadas em resposta às injúrias teciduais, para estimular odontoblastos primários a produzirem dentina terciária reacional, ou ainda para estimular a diferenciação de células pulpares em células semelhantes a odontoblastos que irão produzir dentina terciária reparadora (De Souza et al., 2014; Douglas et al., 2014). Compostos bioativos derivados de plantas como os flavonoides, têm recebido destaque na literatura devido à sua amplitude terapêutica. Flavonoides constituem as classes mais importantes de polifenóis, com mais de 5.000 compostos já descritos na literatura (Shi et al., 2006; Ponce-Vargas et al., 2013). Estão presentes em pequenas 32 quantidades em frutas e vegetais, geralmente como metabólitos secundários e apresentam papel importante para defesa das plantas contra agentes externos. Estudos tem apontado diversas propriedades farmacológicas dos polifenóis, como ação antimicrobiana, antioxidante, anti-inflamatória, osteogênica e anti-osteoclastogênica (Rafter et al., 2005; Shi et al., 2006; Domitrovic et al., 2011; Ponce-Vargas et al., 2013). Eles apresentam uma estrutura comum (flavona), sendo constituídos por um núcleo 2-phenyl-1,4- benzopyrone consistindo de dois anéis benzênicos ou aromáticos (A e B) unidos por três carbonos que formam um anel heterocíclico, denominado de anel C. De acordo com as variações no anel C, os flavonoides são divididos nas seguintes classes: flavonas, flavonóis, flavanonas (3-hidroxi-flavona), flavanonol (2,3-dihydroflavonol), flavonóis (ou catequinas), antocianidinas e chalconas. Dois importantes flavonóis detectados no vinho tinto, a morina e a miricetina, têm sido estudado atualmente por sua biodisponibilidade e propriedades biológicas (Fang et al., 2007). A morina (3,5,7,2',4'-pentahidroxiflavona) é um flavonol encontrado em Morus alba L (amoreira branca), vinho tinto, amêndoa (Prunus dulcis, família Rosaceae), castanha (Castanea sativa), Acridocarpus orientalis, e outras frutas (Gopal, 2013). A miricetina (3,5,7-Trihidroxi-2-(3,4,5-trihidroxifenil)-4-cromenona) é um flavonol presente em muitos vegetais, bagas e frutas, chás e vinho tinto, principalmente na forma de glicosídeos, ao invés de agliconas livres (Song et al., 2021). Estudos demonstraram o potencial in vitro da morina e seus complexos na diferenciação de osteoblastos e no aumento dos níveis de marcadores gênicos osteogênicos (Vimalraja et al., 2019), bem como a prevenção in vivo de defeitos ósseos (Wan et al., 2020). A miricetina também estimula a diferenciação osteoblástica de células mesenquimais da medula óssea com aumento na atividade de ALP, na deposição de colágeno e mineralização óssea (Hsu et al., 2007) e também aumentou a proliferação e expressão de DMP-1 em células odontoblastóides (Baldion et al., 2021). Polímeros naturais ou sintéticos e materiais inorgânicos têm sido utilizados para o desenvolvimento de sistemas de liberação controlada para engenharia endodôntica (Rahmanian-Devin et al., 2021). Esses carreadores poliméricos podem ser utilizados em diferentes formas: scaffolds, hidrogéis e nanopartículas ou combinações dessas formas (Yao et al., 2016; Tentor et al., 2017; Serna et al., 2021). Os hidrogéis são um tipo de polímero poroso tridimensional com alta flexibilidade, baixa toxicidade, adequada biocompatibilidade e biodegradabilidade e podem ser usados como scaffolds ou sistemas injetáveis de liberação de moléculas (Dhivya et al 2015; Rahmanian-Devin et al., 2021). 33 Um dos polímeros naturais mais utilizados é a quitosana, uma mistura de d-glucosamina ligada a β-1,4 N-acetilglucosamina, adquirida pela desacetilação da quitina. A quitosana tem sido aplicada em curativos de lesões, implantes ósseos e sistemas de carreamento de moléculas (Ito & Hidaka, 1997; Felt et al., 1998; Jarry et al., 2001). Possui amplo espectro de aplicação e atividade biológica, como biocompatibilidade, biodegradação a bioprodutos não tóxicos, atividade de reparo tecidual, funcionalidade e propriedades antimicrobianas (Peers et al., 2021). A quitosana é um dos polímeros mais naturais usados para o desenvolvimento de sistemas de liberação lenta de fármacos. Outro polímero, o poloxamer 407(mistura de óxido de etileno e óxido de propileno), também foi adicionado ao hidrogel à base de quitosana, para promover propriedades termorreversíveis e otimizar a formulação do medicamento (Dumortier et al., 2006; Giuliano et al., 2018). Outras vantagens desse copolímero são a baixa toxicidade, biocompatibilidade, facilidade de preparação de gel, boa compatibilidade com uma ampla gama de biomoléculas, incluindo flavonóides, aumentando sua solubilização e prolongando seu perfil de liberação para muitos tipos de aplicações (Dumortier et al., 2006; Giuliano et al., 2018; Yu et al., 2018). Hidrogéis à base de quitosana contendo flavonóides foram desenvolvidos como curativos no tratamento de feridas (Soares et al., 2020), como hidrogéis injetáveis para atividade antibacteriana e regeneração óssea (Lišková et al., 2015; Arpornmaeklong et al., 2021) e outros aplicações médicas e são considerados uma fonte sustentada para liberação desses compostos. Esta combinação de flavonóis foi incorporada em hidrogéis de quitosana-poloxamer 407-beta-glicerofosfato e foi avaliada sua citocompatibilidade e efeitos antibiofilme em canais radiculares. Este estudo avaliou os efeitos da morina, miricetina, isoladamente ou em combinação ou incorporada em hidrogéis à base de quitosana na citotoxicidade e expressão de marcadores de mineralização em células semelhantes a odontoblastos 34 Objetivos Gerais 35 OBJETIVOS Os objetivos deste estudo foram avaliar: 1) O efeito dos flavonóis morina e miricetina, isolados e combinados, sobre bactérias associadas com infecção endodôntica, em condições planctônicas e em biofilmes mistos, bem como sua toxicidade em fibroblastos; 2) O efeito de combinações dos flavonóis morina e miricetina incorporados em hidrogéis de quitosana-poloxamer-β-glicerofosfato sobre biofilmes multiespécies intracanais, bem como sua toxicidade em fibroblastos; 3) O efeito dos flavonóis morina e miricetina, isolados e combinados sobre a viabilidade, sobre a atividade de ALP e produção de nódulos de mineralização em células MDPC-23; 4) O efeito de combinações dos flavonóis morina e miricetina incorporados em hidrogéis de quitosana-poloxamer-β-glicerofosfato sobre a viabilidade, atividade de ALP e produção de nódulos de mineralização em células MDPC-23. 36 Capítulo 1 37 Cytotoxicity and antimicrobial/antibiofilm action of flavonols alone or incorporated in chitosan-poloxamer-glycerophosphate hydrogels for endodontic regenerative therapy Amanda C A Souza1, Gabriela Pacheco de Almeida Braga1, Vanessa Rodrigues dos Santos1, Tais de Cassia Ribeiro2, Gabriel Flores Abuna3, Marlus Chorilli2, Cristiane Duque1 1 Department of Preventive and Restorative Dentistry, Araçatuba Dental School, São Paulo State University (UNESP), Araçatuba, Brazil 2Department of Drugs and Medicines, School of Pharmaceutics Sciences, São Paulo State University, Araraquara, Brazil 3 Department of Foundational Science East Carolina University, Greenville NC. *Corresponding author: Cristiane Duque Department of Preventive and Restorative Dentistry, São Paulo State University – (UNESP), Araçatuba Dental School, Address: R. José Bonifácio, 1193, CEP: 16015-050, Araçatuba-SP, Brazil Tel: (+55) 1836363315 E-mail: cristianeduque@yahoo.com.br, cristiane.duque@unesp.br The authors declare that have no conflicts of interest. *The manuscript is according to the guidelines for authors of Biomaterials 38 Abstract The treatment of infected young permanent teeth that have suffered irreversible pulp damage before the normal physiological closure of the root apex needs a medicament capable of disinfecting the root canals and allow cell recovery and tissue regeneration has not yet been found. Two important flavonoids detected in red wine, morin (MO) and myricetin (MY), are currently studied for their wide biological properties including antimicrobial activity. This study evaluated the cytocompatibility and antimicrobial/antibiofilm effects of MO and MY, alone or incorporated in thermoreversible chitosan-poloxamer hydrogels containing sodium β-glycerophosphate (CPG). Antimicrobial activity of the compounds was evaluated on Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii, and Fusobacterium nucleatum under planktonic conditions, on dual-species and multispecies biofilms and analyzed by bacterial counts and scanning microscopy. CPG hydrogels were characterized by flow and oscillatory rheometry, gelation temperature, texture profile and bioadhesion analysis in dentin specimens. MO, MY and controls and controls (calcium hydroxide – CH and chlorhexidine – CHX) were incorporated in CPG hydrogels and antibiofilm effect on multispecies biofilms formed in radicular dentin samples were evaluated by confocal microscopy. Toxicity effect of the compounds alone or incorporated in CGP hydrogels was determined on fibroblasts culture by resazurin assays. Data were statistically analyzed considering p < 0.05. The combination of MO and MY had synergistic or additive against oral bacteria tested starting at concentrations of 0.03 mg/mL MO + 0.06 mg/mL MY and they were not toxic to fibroblasts up to 0.125mg/mL. MO + MY had better effect on dual-species and multispecies biofilms considering their lower concentrations when compared with the flavonols alone. CPG hydrogels were characterized as thermoreversible and with adequate mechanical and bioadhesive properties. CPG hydrogels loaded with MO+MY, CH and CHX have similar inhibitory effects when applied on multispecies biofilms formed inside root dentin tubules for 48h and their extracts were cytotoxicity above 50% dilution. In conclusion, the combination of morin and myricetin incorporated or not in CPG hydrogels has antibiofilm effect on oral pathogens and low cytotoxicity. Key words: morin, myricetin, antimicrobial, biofilms, hydrogel, cytotoxicity 39 1. Introduction Molecular studies indicated that approximately 500 different species, predominantly bacteria, harbor endodontic infections including primary, secondary, persistent, and extra radicular infections and most prevalent phylotypes belong to five phyla: Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria and Fusobacteria [1-3]. The core microbiota most frequently found in primary intraradicular infections is composed predominantly by anaerobic bacteria, including Gram-negative bacteria, among them Fusobacterium nucleatum, Porphyromonas spp., Treponema species and Gram-positive bacteria, specially Actinomyces, Streptococcus, Propionibacterium species [4-8]. Due to the anatomical irregularities of the root canal system, bacteria can spread to the apical ramifications, lateral canals, isthmuses, dentinal tubules and the effective debridement and disinfection become a quite challenge. The ability of E. faecalis for growing as a biofilm on root canal walls and as a mono-infection in treated canals without synergistic support from other bacteria makes high resistance to antimicrobial agents a very resistance pathogen to root canal treatment [9-11]. Considering immature permanent teeth with pulp necrosis and apical periodontitis, high bacterial load and the presence of virulent species causes inflammation which leads to incomplete healing and root development. Calcium hydroxide paste is widely used for the treatment teeth with irreversible pulpitis/pulp necrosis in which apexification procedures are traditionally performed to induce the formation of an apical hard tissue barrier [12-14]. The disadvantages of apexification reside in the multiple sessions of medication change over an extended period, the cytotoxicity of the medication that prevents the proliferation of remaining cells and the incomplete formation of the roots that consequently increase the risk of reinfections and dental fractures [15,16]. Current studies have evaluated some procedures of regenerative endodontic therapy (RET), a tissue engineering concept based on the use of stem cells and growth factors in scaffolds to regenerate the pulp-dentin complex, for the endodontic treatment of young permanent teeth [15,17]. These procedures are considered an alternative to apexification because they aim to continue deposition of hard tissue in dentinal walls promoting adequate length and strength to the root avoiding further fractures [15,17]. One of the challenges of RET procedures is to achieve a level of disinfection in root canals which do not interfere with survival of cellular and molecular sources essentials to promote mineralization and root development [18]. Since RET recommend minimal or 40 no mechanical instrumentation, an inter-visit medicament is indicated to reduce substantially microbial load and allow cell recovery and tissue regeneration [16,18]. Flavonoids constitute a major group of polyphenolic compounds which are directly associated with the health-promoting properties of red wine [19,20]. Two important flavonoids detected in red wine, morin and myricetin, have been currently studied for their bioavailability and biological properties [20]. Morin (3,5,7,2',4'- pentahydroxyflavone) is a flavonol found in Morus alba L (white mulberry), red wine, almond (Prunus dulcis, family Rosaceae), sweet chestnut (Castanea sativa), Acridocarpus orientalis, and other fruits [21]. It exerts antioxidant, anti-inflammatory and anticarcinogenic effects [22-24]. Myricetin (3,5,7-Trihydroxy-2-(3,4,5-trihydroxyphenyl)-4-chromenone) is a flavonol present in many vegetables, berries and fruits, teas, and red wine, mainly in the form of glycosides, instead of free aglycones [25-27]. Antimicrobial, antithrombotic, and antiatherosclerotic activities as well as antioxidant and anti-inflammatory effects have been reported for myricetin [25-27]. Natural or synthetic polymers and inorganic materials have been used for the development of controlled release systems for endodontic engineering [28]. These polymeric carriers can be used in different forms: scaffolds, hydrogels and nanoparticles or combinations of these forms [29-31]. Hydrogels are a type of three-dimensional porous polymer with high flexibility, low toxicity, biocompatibility, and biodegradability and can be used as scaffold or injectable drug delivery system [28,32]. One of the most used natural polymers is the chitosan, a mixture of β-1,4-linked d-glucosamine and N-acetylglucosamine, acquired by deacetylation of chitin [32,33]. Chitosan have been applied in injury dressings, bone implants, and delivery systems [33-35]. It has a wide application spectrum and biological activity, such as biocompatibility, biodegradation to non-toxic bioproducts, tissue repair activity, functionality, and antimicrobial properties [36]. As this polymer is usually soluble only in acidic media, β-glycerophosphate (GP) is used to neutralize chitosan solutions and obtain a pH close to 7 without making the polymer precipitates [35,37]. In addition, phosphate salts promoted soluble chitosan solutions to become gel at body temperature [28]. One limitation of chitosan is the absence of thermoreversible properties, which is interesting for optimizing drug formulation (fluid state at the administration and sol-gel transition temperature at body temperature promoting prolonged release of pharmacological agents). Poloxamer 407 copolymer (ethylene oxide 41 and propylene oxide blocks) have been largely used in various drug delivery systems including with chitosan due to their advantages such as thermoreversibility, low toxicity, biocompatibility, easy gel preparation methods, good compatibility with a wide range of biomolecules, including flavonoids, enhancing their solubilization and prolonging their release profile for many types of applications [38-40]. This study firstly aimed to evaluate the antimicrobial and antibiofilm properties of morin and myricetin alone or in combination and their cytocompatibility. This combination of flavonols was incorporated in poloxamer 407-chitosan hydrogels containing beta-glycerophosphate and evaluated its cytocompatibility and antibiofilm effects as drug delivery system for endodontic purposes. 2. Material and Methods 2.1.Flavonols and controls The following flavonols were evaluated: morin (MO, # M4008) and myricetin (MY, #70050) – 30 mg/mL. The compounds were all solids, of analytical standard, >90% (TLC). Flavonol stock solutions were prepared and frozen in dimethylsulfoxide - DMSO [41]. Chlorhexidine gluconate (2 mg/mL) (#C9394) and calcium hydroxide (1 mg/mL) (CH #232932) diluted in sterile water were used as positive control [42]. All compounds were filtered on a 0.22 µm membrane filter. The products were obtained from Sigma- Aldrich (St. Louis, MO/USA). Table 1 shows the flavonols and controls used in this study, their codes, synonyms, chemical structure, empirical formula, and molecular weight. Table 1. Flavonols and controls used in the present study Compound Code* Synomyms Chemical structure Empirical formula Molecular weight Morin (MO) #M4008 2′,3,4′,5,7- Pentahydroxyflavone C15H10O7 302.24 42 Myricetin (MY) #70050 3,3′,4′,5,5′,7- Hexahydroxyflavone, Cannabiscetin, Myricetol C15H10O8 318.24 Chlorexidine digluconate (CHX) #C9394 1,6-Bis(N5-[p- chlorophenyl]-N1- biguanido)hexane; 1,1′- Hexamethylenebis(5-[p- chlorophenyl]biguanide) C22H30Cl2N10 505.44 Calcium hydroxide (CH) #232932 calcium hydrate, lime, hydrated lime, caustic lime, lime hydrate, slaked lime Ca(OH)2 74.09 *Codes and information taken from the Sigma-Aldrich company website (https://www.sigmaaldrich.com). 2.2.Microbial strains and growing conditions The following standard strains were used in the present study: Enterococcus faecalis (ATCC 51299), Actinomyces israelii (ATCC 12102), Streptococcus mutans (ATCC 25175), and Fusobacterium nucleatum (ATCC 25586), gently donated by the Oswaldo Cruz Foundation – FIOCRUZ, Rio de Janeiro. The culture media used for each bacterial species were Mitis Agar Salivarius Agar (Difco Laboratories, Kansas City, MO, USA) with 0.2U/mL bacitracin for S. mutans, Brain Heart Infusion Agar – BHIA (Difco) for A. israelii and E. faecalis. For F. nucleatum, the medium used was BHIA containing 5mg/mL of hemin, 5mg/mL of menadione, 0.5% of yeast extract, and 5% of defibrinated sheep blood. The microorganisms were incubated at 37 °C in an atmosphere of 5% CO2, except for F. nucleatum which was grown in jars with an anaerobic system (Anaerogen, Oxoid, Thermo Scientific, Waltham, MA, USA) containing 5% CO2, 10% H2, and 85% N2. All assays were performed in triplicate on three independent days. 43 2.3.Evaluation of the antimicrobial activity of the compounds 2.3.1. Determination of Minimal Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) The MIC assays were measured by the microdilution method, based on the CLSI (Clinical and Laboratory Standards Institute - M7-A9) standards (CLSI, 2012), with some modifications [42-43]. Cultures of the microbial species mentioned in the previous item were subcultured in Miller-Hinton (MH) medium (Difco) for bacteria. Cells were centrifuged and washed with sodium phosphate buffer - PBS (10 mM, pH 6.8). The number of cells was adjusted to obtain 1-5x 105 cells/ml in MH medium for bacteria. Counts were validated by counting on MH agar. Serial dilution of the flavonoids [morin (MO) and myricetin (MY) and chlorhexidine (CHX)] were prepared in water and incubated with an inoculum of each culture (1 to 0.0001 mg/mL) for 24h (and 48h for F. nucleatum) at 37o C under the atmospheric conditions described for each species. Microbial suspensions were incubated in sterile water or chlorhexidine gluconate as negative and positive controls, respectively. After this period, the microtiter plates were stained with resazurin (#R7017, Sigma-Aldrich) incubated for 4h and analyzed at 570 and 600nm in a spectrophotometer (Biotek, Winooski, VT). MIC was defined as the lowest concentration of each compound in which there was no detectable growth by the colorimetric method. The media containing this MIC concentration and two consecutive higher concentrations were serially diluted and plated on MH agar or SDA medium and incubated at 37o C for 48 h. The colony forming units (CFU) were counted using a binocular stereomicroscope and the MBC determined when the antimicrobial agent reduced more than 99.9% of the microorganisms compared to the negative control. 2.3.2. Determination of Fractional Inhibitory Concentration (FIC) To analyze the synergistic effect between morin and myricetin, the microdilution method was conducted on a checkerboard [44,45]. The same concentrations of morin (from 0.25 to 1 mg/mL) were pipetted into all lines (x-axis) of the 96-well microplates. In the columns (y-axis), myricetin was added at concentrations from 0.125 to 1 mg/mL. Thereafter, bacterial cultures were adjusted to the final concentration of 1×5× 106 CFU/mL. The plates were subsequently inoculated at 37 °C for 24 h. The samples were plated into MH agar plates to obtain the rates of cell survival. The combination values were derived from the highest dilution of the antimicrobial combination that did not show bacterial growth. The FIC index (FICI) was calculated using the following 44 formula: FICI = (MIC of antimicrobial A in combination/MIC of A alone) + (MIC of antimicrobial B in combination/MIC of B alone). Synergy was interpreted for FICI≤0.5, addictive for 0.54. 2.4.Cytotoxicity assays The cytotoxicity of flavonoids was determined on culture of fibroblasts L3T3-L1 (CL- 173TM). Cells were grown in Dulbecco's modified Eagle medium (DMEM, Dulbecco's Modified Eagle's, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and 100 IU/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L glutamine (Gibco) in a humidified oven containing 5% CO2 and 95% air at 37°C (Isotemp Fisher Scientific, Pittsburgh, PA, USA). Cultures were subcultured every 2 days until reaching 80% confluence. After treatment with trypsin-EDTA (0.25% Trypsin-EDTA1x, Gibco) for 5 min at 37oC, cells were stained with Trypan Blue (Sigma-Aldrich) and counted in a Neubauer chamber using an inverted microscope. Next, they were planted at a cell density of 5x 104 cells/well in 96-well plates and incubated for 24 h. The cells were treated with morin, myricetin, morin + myricetin, CHX and CH (from 0.06 to 0.015 mg/mL) for 24 h. Cells were washed with PBS and resazurin 70µM in DMEM was added for 4h. After that, the plates were analyzed in a spectrophotometer (Biotek, Winooski, VT) at 570 and 600nm. The final values were obtained by the subtractions between the absorbance values at both wavelengths, and they were converted into percentage of cell viability considering the growth in DMEM medium as 100% and the means determined for each group [46,47]. 2.5.Evaluation of the antibiofilm activity of the compounds 2.5.1. Effect of the compounds on dual-species biofilms Flavonols and controls (Morin 5 mg/mL, Myricetin 5 mg/mL, Morin 2.5 mg/mL + Myricetin 2.5 mg/mL, CHX 0.05 and 0.5 mg/mL and CH 1mg/mL) were evaluated in dual-species biofilms associating E. faecalis with A. israelii, E. faecalis with S. mutans, and E. faecalis with F. nucleatum, following the methodology proposed by Gao et al. 2016 [48] and modified by Dos Santos et al. 2021 [49]. Compounds were used in 10x MIC or 10x FIC. Briefly, bacterial cultures, pre-adjusted to 1-5x 103 CFU/mL were mixed in equal proportions in BHI broth medium containing 1% glucose. After 48h of biofilm formation (or 72h for E. faecalis with F. nucleatum) the same treatments described for mono-species biofilms were carried out and the plates were incubated for 36h. After 45 scraping of biofilms, aliquots from all wells were resuspended, serially diluted, and plated in BHI for total bacterial count and in BHI containing 0.1mg/mL (E. faecalis + S. mutans) and 0.5 mg/mL (other biofilms) of cefuroxime to allow only the growth of E. faecalis for 48h. The count of species that were inoculated with E. faecalis was calculated by subtracting the number of total bacteria on BHI agar from those counted on the antibiotic containing BHI [48]. 2.5.2. Effect of the compounds on multispecies biofilms and scanning electron microscopy The multispecies biofilm assays were conducted following the same steps described previously for dual-species biofilms, however, all bacteria (E. faecalis, A. israelii, S. mutans and F. nucleatum) were mixed in equal aliquots at the same concentration (1-5x 103 CFU/mL) in BHI broth containing 1% glucose. After 1 week of growth in anaerobic conditions, biofilms were washed twice with sterile saline solution and flavonols (morin 5 mg/mL, myricetin 5 mg/mL, morin 2.5 mg/mL + myricetin 2.5 mg/mL) and controls (CHX 0.5 mg/mL and CH 1mg/mL) were inserted into each well. The plates were incubated for 48h at 37°C in anaerobic conditions. After scraping of biofilms, aliquots from all wells were resuspended, serially diluted, and plated on BHI agar and the plates incubated for 48h for further counting of CFU/mL. The same experiments were conducted in parallel in coverslips for microscopic analysis. The samples were dehydrated by washing in a series of ethanol (70% for 10min, 95% for 10 min, and 100% for 20 min) and air-dried in a desiccator. Afterwards, coverslips were mounted into aluminum stubs, sputter coated with gold, and analyzed in a scanning electron microscope (Leo, Cambridge, MA, USA) [50]. 2.6.Preparation of chitosan poloxamer hydrogel with β-glycerophosphate (CPG) Chitosan low molecular weight (50-190 Da; 75–85% deacetylated, #448869) Poloxamer 407 (#16758); Acetic acid (Synth, Labsynth®, Brazil) and ultrapure water (#W4502). Hydrogels were prepared according to the cold method first described by Schmolka et al. (1972) with some modifications [51]. First, chitosan (1% wt/vol) was dissolved in a solution of acetic acid (1% wt/vol) using mechanical stirrer (100 rpm) for about 3h. Following complete dissolution, the chitosan dispersion was then refrigerated (4oC) and used as a solvent for the poloxamer 407 (P-407) dispersion. After, P-407 (18% wt/vol) was added to the chitosan dispersion and stored at 4o C for about 24h. 46 For the preparation of P-407/chitosan hydrogels containing sodium β- glycerophosphate (CPG), a β-glycerophosphate solution was prepared by dissolving the β-glycerophosphate powder (#G9422) (0.5 g) in 1 mL of distilled water. Then, this solution was carefully dropped with the aid of a Pasteur pipette into the CH dispersion before adding the P-407, which was left under stirring for 30 minutes and then the P-407 was added to the dispersion. The hydrogels were sterilized in an autoclave at 121 ℃ for 15 minutes. 2.7.Characterization of CPG hydrogel 2.7.1. Flow rheometry Rheometry was analyzed using an AR2000 pressure-controlled rheometer (TA Instruments, New Castle, DE, USA) with cone/plate geometry (40 mm diameter at a 52 μm gap). Continuous shear analysis of the CPG hydrogel was performed at 37 ±0.1 °C. The formulations were carefully placed to the inferior plate, and allowed to equilibrate for at least 1 min prior to analysis [51]. Flow curves were measured over shear rates ranged from 0 to 100 s−1 (increased over a period of 120 s, held at the upper limit for 10 s, and then decreased over a period of 120 s). The consistency index and the flow index were determined using eq. [52] for a quantitative analysis of flow behavior (1) 𝜏 = 𝐾. 𝛾! where τ is the shear stress (Pa), k is the consistency index [(Pa s) n], γ is the rate of shear (s−1), and n is the flow behavior index (dimensionless). 2.7.2. Oscillatory Rheometry Oscillatory analysis of hydrogels was carried out after the linear viscoelastic region in which stress is directly proportional to strain and the storage modulus remains constant was identified. Frequency sweep analysis was carried out at the frequency range of 1–10 Hz at a constant stress of 1 Pa [52]. The CPG hydrogel was carefully applied to the plate as described previously. After determination of the linear viscoelastic region of hydrogels, frequency sweep analysis was evaluated from 1 to 10.0 Hz Oscillatory analysis of hydrogels was performed at 25 and 37 ± 0.1 °C. 2.7.3. Sol–gel transition temperature The determination of sol-gel transition temperature (Tsol-gel) of the CPG hydrogel was performed in oscillatory mode with temperature ramp, using cone-plate as 47 described above. The temperature sweep analysis was performed over the temperature range of 18-50°C at a defined frequency (1.0 Hz) and rate of heating 3 °C/min using a controlled stress [52]. 2.7.4. Texture profile analysis Texture profile analysis (TPA) was performed by using a TA-XT plus texture analyzer (Stable Micro Systems, Surrey, UK) as described by Calixto et al. 2016 [51]. The CPG hydrogel were weighed (7 g), placed in 50 mL centrifuge tubes, and centrifuged to remove the air bubbles present. Then, the hydrogels were allowed to stand for 12 hours until analysis. Then, tubes were placed below the 10 mm analytical probe, which was lowered at a constant speed of 1 mm/s 1 until it reached the hydrogel surface. Contact was detected by a triggering force of 2 mN, and then the probe continued down to 10 mm depth in the hydrogel. Then, the probe returned to the surface (0.5 mm/s), and after 5 seconds, a second compression was initiated. The test results were used to plot a force– time curve, from which the mechanical parameters were calculated such as hardness, compressibility, adhesiveness, and cohesion. This process was repeated seven times at 37° C± 0.5°C [52]. 2.7.5. Bioadhesion assay A TA-XT plus texture analyzer (Stable Micro Systems) was used to measure the tensile strength according to the methodology described by Calixto et al. [52]. The dentin model (4mm diameter x 2mm depth) was fastened to the cylindrical probe (diameter, 10 mm). A 50 mL centrifuge tube containing the CPG hydrogel was placed below the probe. The probe was lowered at a constant speed of 1 mm/s until the teeth model and hydrogels surface were made to be in contact with each other, as detected by a triggering force of 2 mN. The teeth model and hydrogels were kept in contact for 60 seconds, and no external force was applied during this time interval. After 60 seconds, the teeth model was drawn upward (0.5 mm/s) until the contact between the surfaces was broken. During this experiment, a force–time curve was plotted, and the work of adhesion and peak adhesion were calculated from this plot. Seven replicates were analyzed at 37 ± 0.5 °C. 2.8.Preparation of CPG hydrogels with flavonols and controls CPG hydrogels containing flavanols were prepared following the protocol previous described: 18% 407-poloxamer, 1% chitosan, 1% hypochlorite acid, 10% sodium β- 48 glycerophosphate and added 2.5mg/mL of morin and 2.5 mg/mL of myricetin or 1mg/mL of CH and 0.5 mg/mL of CHX as controls. CPG hydrogels were kept under agitation for 1 h and after that they were incubated for 24h at 5 °C for total solubilization before use. 2.9.Effect of CPG hydrogels on multispecies biofilms and confocal microscopy analysis This study was approved by the Animal Committee of Araçatuba Dental School, UNESP, Brazil (Protocol: 711/2019) and conducted in accordance with Ma et al. 2011 [53]. Roots of bovine incisors were separated from crowns using a diamond disc (KG Sorensen D91, Barueri, SP, Brazil) at 1mm below the cement-enamel junction. Then, 4mm cylinder specimens were obtained from the roots horizontally sectioned with two diamond discs of 0.6 mm (Isomet 5000, BuehlerLtd, LakeBluff, IL, USA) and their root canals were enlarged with a # 6 drill. The cylindrical specimens were again sectioned in two half cylindrical halves and the dentin specimens (n=4/group) were washed with distilled water and ultrasonically cleaned with 17% EDTA for 3 min and deionized water for 5 min. After autoclaving for 15 minutes at 121 ° C, they were then inserted into a microtube with the canal side (pulp) up and fixed with composite resin. Cultures of E. faecalis, A. israelii, S. mutans and F. nucleatum were mixed in equal aliquots at the same concentration (1-5x 103 CFU/mL), centrifuged and resuspended in BHI broth at the same initial volume. In the microtubes with the dentin specimens, 0.5 mL of the bacterial culture were inoculated and centrifuged at 1400 x g, 2000 x g, 3600 x g, and 5600 x g, twice each, for 5 minutes to promote dentin infection. A fresh aliquot of bacteria was added between each centrifugation and the old discarded. All microtubes were incubated at 37°C in BHI supplemented with 1% glucose broth for 2 weeks in an atmosphere of 5% CO2. The culture medium was changed every 72h. After this period, dentin samples were washed with sterile water and treated with 1mL P-407/chitosan hydrogels with β- glycerophosphate (CPG) containing flavonols (MO and MY) and controls (CHX and CH) in the following groups: 1) CPG + MOMY, 2) CPG + CH, 3) CPG + CHX and 4) CPG and 5) sterile water. Each sample was immersed in 350 μL of each solution for 48h and was then washed with sterile water for 1 min at 37 °C to avoid residual effects of the treatments. Subsequently, the samples were cut into two new halves, for observation of the surface longitudinally to the dentinal canals visible by Confocal Laser Scanning Microscopy - CLSM [53]. The analysis was performed at the Bauru School of Dentistry – USP. The samples were stained with 100 µL of fluorescent LIVE/DEAD BacLight 49 Bacterial Viability stain (L13152, Molecular Probes, Eugene, OR), according to the manufacturer’s instructions. CLSM images were acquired using software (LAS AF Leica Microsystems) at a resolution by 1024 pixels. Ten-micrometer-deep scans were obtained with the CLSM from two randomly selected places for each dentin specimen (total of 8 images/group). Quantification of the red fluorescence ratio in relation to green-and-red fluorescence was determined by software denominated Image J 1.48 (NIH, Bethesda, MA, USA), indicating the proportion of dead cells for each antimicrobial agent tested [43]. 2.10. Cytotoxicity of CPG hydrogels After preparation, CPG hydrogels loaded with MO+MY, CH and CHX were kept at 5 °C for 24h. Then, 1mL of hydrogels at the liquid state were incubated in 24 wells microplates at 37 °C overnight to acquire the gel state. Then, the DMEM culture medium was added over the hydrogel and incubated for 48 h and 7 days. The supernatant was harvested after 48 h and 7 days for sequential treatment. The viability rate was evaluated by colorimetry resazurin assay (Sigma-Aldrich), as previously described. Briefly, fibroblastic 3T3 cells were seeded into the 96 well plates (1-5x 104 cells/well) and incubated for 24 h under standard cell culture conditions. After incubation, DMEM was removed, and the dilutions of the hydrogel’s extracts were added to the cells (from 50% to 6.25%). 3T3 cultured in DMEM without any extract was used as the control. After the 24h treatment, the extracts were removed and 200 μL of resazurin added (70μM) for incubation at 37o C for 4 h. At each time point, 100 μL of the medium was transferred to another 96 well plate to absorbance reading at 570 and 600 nm in spectrophotometer (Biotek, Winooski, VT). 2.11. Statistical analysis In virtue of amplitude of CFU/mL counts, microbial data were transformed in Log (CFU+1/mL). The constant +1 was added because some specimens had a zero count. Data from cytocompatibility and microbiological assays were expressed in means/standard deviation and submitted to ANOVA and Tukey tests. SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) was used to run the statistical analysis. 50 3. Results 3.1.Antimicrobial activity of the flavonoids Table 2 shows the MIC, MBC and FIC values obtained for the flavonols against the oral bacteria tested compared with the control chlorhexidine (CHX). The MIC/MBC values of morin (MO) and myricetin (MY) ranged between 0.125 and 1 mg/mL and F. nucleatum was the most sensitive bacteria. The combination of MO and MY showed effect against all bacterial species tested with FICI below 0.7, showing synergistic or addictive effect. F. nucleatum was the most sensitive bacteria and E. faecalis was the most resistant bacteria to the effect of the flavonols. CHX presented the best inhibitory effects on bacteria tested. Table 2. Minimal inhibitory concentration (MIC), minimal bactericidal/fungicidal concentration (MBC) and fractional inhibitory concentration (FIC) in mg/mL for the flavonoids and control chlorhexidine against the oral microorganisms tested. E. faecalis A. israelii S. mutans F. nucleatum Morin (MO) 0.5 (1) 0.25 (0.5) 0.25 (0.5) 0.125 (0.25) Myricetin (MY) 0.5 (1) 0.25 (0.5) 0.5 (0.5) 0.125 (0.25) Morin (MO) + Myricetin (MY) 0.25+ 0.25 FICI= 0.75 0.125+0.015 FICI = 0.37 0.06+0.125 FICI = 0.5 0.03 +0.06 FICI = 0.75 Chlorhexidine (CHX) 0.004 (0.009) 0.0001 (0.004) 0.0001 (0.001) 0.0003 (0.0006) MIC results were based on resazurin staining and MBC results were based on CFU/mL count in Miller Hinton Agar (MHA) medium. MBC = >99.9% bacterial reduction what is in parentheses. The growth of microorganisms without antimicrobials in MHA was considered 100%. 3.2.Effect of flavonoids on fibroblast viability The effect of MO, MY, their combinations and controls calcium hydroxide (CH) and CHX on fibroblasts L3T3 after 24h can be seen in Figure 1. Flavonols or their 51 combination were not cytotoxic below 0.125 mg/mL or 0.125 + 0.125 mg/mL for each flavonoid in combination. Cell viability was higher than 70% at these concentrations. CH was not cytotoxic at any concentration tested. On the contrary, CHX was cytotoxic at all concentrations evaluated. Figure 1. Effect of the flavonoids morin (MO) and myricetin (MY) alone or in combination (MO + MY) and controls calcium hydroxide (CH) and chlorhexidine (CHX) on the viability of fibroblasts (3T3) after 24h of exposure, using staining with resazurin. Concentrations in mg/mL. a Different letters show statistical difference among the groups and concentrations, according to ANOVA and Tukey test (p<0.05). 3.4.Antibiofilm activity of the compounds on microplates Figure 2 shows the effect of flavonols on dual-species biofilms of E. faecalis combined with S. mutans, A. israelli or F. nucleatum compared to controls. No E. faecalis growth was observed when the dual-species biofilms were treated with MO or MO+MY combination. The same was observed for the total counts of microorganisms presented in dual-species biofilms with S. mutans or A. israelii, when they were treated with MO and MO + MY. Although low count for total microorganisms was detected after MO treatment in dual species biofilms with E. faecalis and F. nucleatum, no difference was found between MO and MO+MY. MY significantly reduced the growth of all dual- species biofilms tested, similar or superior to CHX at the highest MIC value observed in 52 this study. CH and CHX 10x MIC was not effective against biofilms of E. faecalis and S. mutans and biofilms of E. faecalis and A. israelli and reduced the growth of E. faecalis and F. nucleatum biofilms. Figure 2. Effect of flavonoids and controls on dual-species biofilms (48-72h) of oral bacteria after 36h of treatment. Graphics on the left side represent microbial total counts and graphics on the right side represent E. faecalis counts. Values are presented as means and standard deviations. Morin (MO) - 5 mg/mL (10x the highest MIC value); Myricetin (MY) – 5 mg/mL (10x the highest MIC value), Morin + Myricetin (MO+MY) – 2,5 mg/mL each one (10x the highest FIC value), Calcium hydroxide (CH) – 1 mg/mL, CHX - 0.5 mg/mL (100x the highest MIC value), and Control – Bacterial growth without antimicrobial agents. a Different letters show statistical difference between the groups of antimicrobials for each double species tested, according to ANOVA and Tukey's test (p<0.05). 3.5.Effect of the compounds on multispecies biofilms and scanning electron microscopy Figure 3 (A-G) shows representative images of scanning electron microscopy of multispecies biofilms with E. faecalis, S. mutans, A. israelli and F. nucleatum after 53 treatment with the flavonols and controls. Evident biofilm disorganization and areas with “crack” areas and a substantial reduction in bacterial presence and in extracellular matrix are observed in Figures 3A, C and E when multispecies biofilms were treated with MO, MO+MY and CHX 100x MIC. Figures 3B and 3D show little or no biofilms disorganization when they were treated with MY and CH, respectively. Figure 3E and Fshows the complex and organized structure of multispecies biofilms with no influence of antimicrobial agents. Bacteria co-aggregated and incorporated in an extracellular polymeric matrix can be seen in this Figure. Figure 3G presents the effect of all compounds on bacterial counts (in CFU/mL) in the multispecies biofilms confirming that MO, MO+MY and CHX were the most effective treatments, followed by MY and CH. 54 Figure 3. Representative scanning electron microscopy images of 14-day multispecies biofilms under 1000x magnification. Biofilms were treated for 48 hours with A: Morin (MO) - 5 mg/mL; B - Myricetin (MY) – 5 mg/mL, C - Morin + Myricetin (MO+MY) – 2,5 mg/mL each one, D - Calcium hydroxide (CH) – 1 mg/mL, E - CHX - 0.5 mg/mL, and F - Control – Bacterial growth without antimicrobial agents. G. Mean (SD) of the bacterial counts detected after 48h of the biofilm treatment with flavonoids and controls. a Different letters show statistical difference between the antimicrobial groups, according to ANOVA and Tukey test (p<0.05). 55 3.6.Characterization of CPG hydrogels 3.6.1. Flow Rheometry Figures 4 demonstrates the flow curves of CPG hydrogel. The hydrogel exhibited behavior non-Newtonian of the type pseudoplastic once the relation between the shear rate (Pa) and shear stress (1/s) is not linear. In addition, according to downward curve that overlapped upward curve the hydrogel showed thixotropic flow behavior characterized by the decrease in viscosity during the increase in the shear rate, and when this rate is decreased, the viscosity increases again. According to the equation 1 (See Material and Methods) for a quantitative analysis of flow behavior, the consistency index (K) was 0.6120 and the flow index (n) was 0.9715 confirming that CPG hydrogel exhibited shear thinning behavior (n<1) being characterized as pseudoplastic fluid. Figure 4. Continuous rheological behavior of hydrogels composed of chitosan, poloxamer, and β-glycerophosphate (CPG). Empty symbols indicate an upward curve, and full symbols, a downward curve. 3.6.2. Oscillatory Rheometry The storage modulus and loss modulus were analyzed from the frequency sweep analysis. Figure 5 shows results of the oscillatory rheometry of CPG hydrogel. According to the analysis performed, CPG hydrogel exhibited viscoelastic characteristics at 25oC where storage modulus > storage modulus. In addition, frequency dependence is observed (Figure 5A). At this temperature, the loss modulus is predominant, revealing that the poloxamer micelles are not orderly packed and that the featuring a less-structured system. However, at 37o C (Figure 5B), the rapid increase in the storage modulus reflects the 56 formation of a strong gel network with storage modulus > loss modulus, independent of the oscillatory frequency. Figure 5. Frequency sweep profile of storage modulus G’ (opened symbols) and loss modulus G’’ (closed symbols) of hydrogels composed of chitosan, poloxamer and β- glycerophosphate (CPG). (A) Analyzes performed at 25℃, (B) Analyzes performed at 37 ℃. 3.6.3. Sol–gel transition temperature The Tsol-gel of CPG hydrogel was investigated by oscillatory analysis, monitoring the variation of storage modulus and loss modulus as a function of the temperature in the range between 10 and 60o C. According to Figure 6 can be observed that the Tsol-gel occurs in three phases. First, before the gelation process, the loss module presents values lower than the storage modulus demonstrating viscoelastic characteristics, as shown in oscillatory rheology. The second phase corresponds to the beginning of the gelation process, which starts approximately 24 °C, temperature at which the storage modulus exceeds the loss modulus. In addition, the phase transition can be observed by abrupt change in Tan d is observed with values of <1 indicating a greater storage modulus compared with loss modulus. Rheological analyzes confirmed that CPG hydrogel was thermosensitive and had thermoreversible properties. 57 Figure 6. Temperature sweep analyses demonstrating of storage modulus G’ (blue symbol) and loss modulus G’’ (green symbol) and loss tangent (Tan d) (red symbol) of hydrogels composed of chitosan, poloxamer and β-glycerophosphate (CPG). 3.6.4. Texture profile and bioadhesion analysis Texture profile results are divided in hardness (measured by the peak of force applied by the probe on the hydrogels), compression (the work required to deform the product during compression), adhesiveness (the work required to overcome the attractive forces between the surfaces of the hydrogels and probe) and cohesiveness (the force that binds molecules) (Friedman et al., 1963; Jones et al., 2009). The results obtained by the CPG hydrogel were as follows: hardness - 10.38 mN; compressibility - 76.05 mN/s, adhesiveness – 68.24 mN/s and cohesiveness – 0.73 mN. Bioadhesion results in teeth model when the CPG hydrogel was kept in contact for 60 seconds were as follows: peak of adhesion – 0.034 N and area of adhesion – 0.102 N/s. 3.7. Antibiofilm activity of the compounds on root dentin specimens Figure 7A-F shows the results observed when 14 days multispecies biofilms (with E. faecalis, S. mutans, A. israelli and F. nucleatum) formed inside root canals were exposed to the CPG hydrogels containing the antimicrobial agents for 48h. Representative confocal images of CPG hydrogels are observed in Figures 7A-E comparing the dead (red spots) and live (green spots) patterns among the groups. Similar results were observed for CPG+MOMY, CPG+CH and CPG+ CHX. CPG hydrogel had little antibiofilm effect and did not differ from the control. These results were confirmed in Figure 7F. 58 Figure7.Representative confocal microscopy images of bovine root dentin specimens contaminated for 14 days with multispecies biofilms and treated for 48 hours with the following groups with A: Hydrogel (CPG) with Morin + Myricetin (MO+MY) – 2,5 mg/mL each one, B – CPG with calcium hydroxide (CH) – 1 mg/mL, C – CPG with CHX - 0.5 mg/mL, D – CPG alone, E - Control – Bacterial growth without antimicrobial agents. F. Mean (SD) of the percentages of dead cells of multispecies biofilms after 48h of treatment with flavonoids and controls. Bacterial counts were obtained by Image J analysis. a Different letters show statistical difference between the antimicrobial groups, according to ANOVA and Tukey test (p<0.05). 59 3.8.Effect of CPG hydrogel extracts containing flavonols on fibroblast viability Figure 8A-B shows the effect of the 48h and 7 days extracts of CPG hydrogels containing MO+MY, CH and CHX, or without antimicrobials agents on fibroblast cells after 24h of exposure. All CPG hydrogels (containing or not the antimicrobials) diluted 50% had cytotoxic effect reducing the cell viability below 70%, for both 48h and 7 days extracts. Dilutions below 50% had no cytotoxic effect after 24h of extracts exposure. Figure 8. Effect of the 48h (A) and 7 days (B) extracts of chitosan-poloxamer hydrogels with glycerophosphate (CPG) containing morin and myricetin in combination (MO+MY) and controls calcium hydroxide (CH) and chlorhexidine (CHX) on the viability of fibroblasts (3T3) after 24h of exposure, using staining with resazurin. Concentrations in mg/mL. a Different letters show statistical difference among the groups and concentrations, according to ANOVA and Tukey test (p<0.05). 60 4. Discussion Regarding the challenge of developing a material capable of treating the pulpar/periapical infection and allowing the apex closure in young permanent teeth, this study proposed evaluated the antimicrobial activity and cell viability of flavonols as potential agents for endodontic engineering. Both flavonols had antimicrobial effect against oral bacteria tested at the concentrations above 0.125 mg/mL, and the combination of morin and myricetin affected the bacterial growth from 0.03 mg/mL morin with 0.06 mg/mL myricetin, confirming the synergistic or addictive effect of these flavonols. They were also not toxic to fibroblasts up to 0.125mg/mL, alone or in combination. MO + MY had better effect on dual-species and multispecies biofilms considering their lower concentrations when compared with the flavonols alone. Morin have showed superior antimicrobial effect in comparison with other flavonols such as myricetin, naringin, rutin and quercetin against A. actinomycetemcomitans, A. naeslundii, A. viscosus, E. faecalis, E. coli, L. casei, S. aureus and Candida albicans in planktonic conditions (54). Another study compared the antibacterial activity of morin and quercetin and their sulfonic derivatives and morin was the most effective growth inhibitor of the six bacterial strains: E. coli and carbapenem- resistant E. coli, S. aureus, and methicillin-resistant S. aureus (MRSA). The structure- activity relationship revealed that 2',4'-dihydroxylation of the B ring in the flavonoid structure is important for significant antibacterial activity of morin [55]. Morin was also combined with other flavonoids such as rutin and quercetin and had better effect against several clinical isolates of MRSA and S. aureus ATCC than when tested alone. This combination (morin+rutin+quercetin) with the antibiotics amoxicillin, ampicillin, cephradine, ceftriaxone, imipenem, and methicillin showed synergism. Potassium leakage, which represented the effect on cytoplasmic membrane of bacteria, was highest for morin + rutin + quercetin that improved further in combination with imipenem [56]. Other study observed a decrease by 3- to 16-fold on the MIC of antibiotics (ciprofloxacin, tetracycline, and erythromycin) when combined with quercetin and morin against S. aureus strains. Similar to our study, these authors also evaluated the cytotoxicity and fibroblast IC 50 of 41.8 and 67.5 was observed for quercetin and morin [57]. The effect of new antimicrobial agents on biofilms have been considered in the medical studies, since biofilms are implicated in the pathogenesis and chronicity of several bacterial infections, including endodontic infections, that are difficult to 61 successfully eradiate with conventional antimicrobial therapies in virtue of mechanisms of antibiotic resistance and tolerance [58,59]. These mechanisms are related to composition of biofilm ECM, including polysaccharides, antibiotic-modifying enzymes and cell wall-modifying enzymes, extracellular DNA, oxygen and nutritional gradient, oxidative stress responses, efflux pumps, quorum sensing and genetic diversity [59]. The interactions between different species increases the antimicrobial tolerance in polymicrobial infections by exchange of antibiotic resistant genes or components of ECM, becoming relevant the study of mixed-species microbial biofilms instead of single- species biofilms [59-61]. The concentration of compounds (10x the highest MIC value) was chosen based on previous studies which have shown that lower concentrations of antimicrobials have no significant effect on polymicrobial biofilms [62,63] considering that biofilm cells are at least hundreds of times more resistant to antibacterial agents than planktonic cells (up to 1,000-fold increase) [64,65]. In the present study, MO and MO+MY (at the half concentration tested for the flavonols alone) were the most effective compounds against both dual-species and multispecies biofilms. MY also reduced significantly bacterial counts (3.39-8.78 log in total bacterial counts and 3.35-11.02 log in E. faecalis counts) in biofilms superior to the control CHX at the highest concentration (1.25-4.72 log in total bacterial counts and 1.43- 5.59 log in E. faecalis counts). MEV images showed the formation of cracks or gaps mainly in the biofilms treated with MO or MO+MY evidencing the biofilm disorganization. Morin at concentrations exceeding 225 µM (68µg/mL) reduced up to 65% biofilm biomass of Streptococcus pyogenes [66] and inhibited the biofilm adhesin and disrupted the established biofilm, beside to reduce motility, spreading and exopolysaccharides production of S. aureus resistant strains [67]. Synergism between antibiotics and morin was effective in reducing biofilm of S. aureus strains, decreasing antimicrobial resistance to antibiotics [68]. Both morin and myricetin have demonstrated to reduce adhesion and biofilm formation of S. mutans by the inhibition of sortase A, a transpeptidase that attaches protein Pac to the cell surface of S. mutans during adhesion phase [69,70]. The combination of myricetin with tt-farnesol and fluoride also significantly reduced water-soluble exopolysaccharides in the ECM [71]. Poloxamer 407 have been incorporated in chitosan hydrogels to allow thermoreversibility and optimize drug formulations. For endodontic purposes, for example, could be useful as intracanal medicament for delivery of antimicrobial agents 62 reducing the microbial load and allowing tissue recovery and regeneration. Hydrogels with chitosan, poloxamer, and b-glycerophosphate (CPG) have not studied yet. CPG hydrogel was characterized as pseudoplastic fluid with the ability to form a strong gel network with storage modulus superior to loss modulus at 37o C. In addition, oscillatory rheology showed that hydrogels are thermoresponsive, since the change in temperature promotes an increase in viscosity and consequently induces the in situ gelification process at 37o C resulting in well-structured systems. Texture profile and bioadhesion analysis revealed adequate hardness, resistance to compression, adhesiveness, and cohesiveness, besides the ability to adhere in dentin. Pseudoplasticity refers to a reduction in viscosity when the shear rate increases, which is a desirable feature of pharmaceutical formulations since the reduction in viscosity facilitates the administration of the formulation [52,72]. Texture properties are important for the formulations, contributing to patient acceptance, clinical efficacy, and final product approval. They improve the product spreadability on the biological substrate such as the teeth, skin, or mucosa; the bioadhesion which ensures the retention at the site of application, and viscosity that allows efficient releasing and absorbing of the drug [52,72]. The advantages of using bioadhesive systems as drug carriers include prolongation of drug residence time at the absorption site, intensified contact with the epithelial barrier, decreased frequency of drug application, and improved patient compliance with therapy [72]. In this study, CPG hydrogels were loaded with MO+MY and controls CH and CHX and they have similar inhibitory effects when applied on multispecies biofilms formed inside root dentin tubules for 48h. Dilutions below 50% of CPG hydrogels extracts obtained after 48h and 7 days in solution had no cytotoxic effect after 24h of extracts exposure. Antimicrobial and antibiofilm effect of morin were observed when incorporated in controlled release films and tables based on different natural polymers, gellan gum or sodium alginate [73,74]. Morin incorporated in tablets or films formed with gellan gum and sodium alginate reduced bacterial viability, acidogenicity and insoluble extracellular polysaccharide (iEPS) in A. naeslundii and S. mutans single biofilms [73]. The same authors evaluated the effect of morin incorporated in gellan gum system on the establishment of polymicrobial biofilms from human saliva for 48h and observed lower bacterial viability mainly aciduric bacteria in tablets compared to films. Acidogenicity and iEPS were also affected by the systems containing morin. Myricetin or the combination of myricetin and miconazole were incorporated in poloxamer hydrogels and 63 observed good stability, excellent sensitivity, and controlled drug release. MY reduced MIC50 and MIC80 of miconazole against C. albicans in planktonic conditions [75]. 5. Conclusions In summary, the combination of morin and myricetin have antimicrobial/antibiofilm effect on oral pathogens and low cytotoxicity. This combination was successfully incorporated in poloxamer 407-chitosan hydrogels containing beta- glycerophosphate, characterized as thermoreversible and with adequate mechanical and bioadhesive properties, and affected multispecies biofilms formed in dentin tubules, as wells as, affected minimally fibroblast cells. The findings of the present study corroborate that combination therapy is preferred over monotherapy in treating multispecies biofilm- related diseases, such as endodontic infections. Acknowledgments This work was supported by FAPESP (#2017/10940-1) and CAPES (financial code #001). Conflict interest There is not conflict of interest. 6. References 1. J.M. Shin, T. Luo, K.H. 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