UNESP - Universidade Estadual Paulista “Júlio de Mesquita Filho” Faculdade de Odontologia de Araraquara Eran Nair Mesquita de Almeida Avaliação in vitro dos efeitos do clareamento dentário com fotossensibilização em dentes não vitais Araraquara 2023 UNESP - Universidade Estadual Paulista “Júlio de Mesquita Filho” Faculdade de Odontologia de Araraquara Eran Nair Mesquita de Almeida Avaliação in vitro dos efeitos do clareamento dentário com fotossensibilização em dentes não vitais Tese apresentada à Universidade Estadual Paulista (Unesp), Faculdade de Odontologia de Araraquara para obtenção do título de Doutora em Ciências Odontológicas, na Área de Dentística Restauradora Orientador: Prof. Dr. Marcelo Ferrarezi de Andrade Araraquara 2023 A447a Almeida, Eran Nair Mesquita de Avaliação in vitro dos efeitos do clareamento dentário com fotossensibilização em dentes não vitais / Eran Nair Mesquita de Almeida. -- Araraquara, 2023 71 p. : il., tabs. Tese (doutorado) - Universidade Estadual Paulista (Unesp), Faculdade de Odontologia, Araraquara Orientador: Marcelo Ferrarezi de Andrade 1. Clareamento dental. 2. Dente não vital. 3. Peróxido de hidrogênio. I. Título. Sistema de geração automática de fichas catalográficas da Unesp. Biblioteca da Faculdade de Odontologia, Araraquara. Dados fornecidos pelo autor(a). Essa ficha não pode ser modificada. Eran Nair Mesquita de Almeida Avaliação in vitro dos efeitos do clareamento dentário com fotossensibilização em dentes não vitais Comissão julgadora Tese para obtenção do grau de Doutora em Ciências Odontológicas Presidente e orientador: Prof. Dr. Marcelo Ferrarezi de Andrade 2º Examinador: Prof. Dr. Milton Carlos Kuga 3º Examinador: Prof. Dr. Andrea Abi Rached Dantas 4º Examinador: Profa. Dra. Camila Cruz Lorenzetti 5º Examinador: Prof. Dr. João Felipe Besegato Araraquara, 24 de Fevereiro de 2023. DADOS CURRICULARES Eran Nair Mesquita de Almeida NASCIMENTO: 26 de Novembro de 1990 – São Luís – MA FILIAÇÃO: Mãe: Maria de Fátima Mesquita Almeida Pai: Edvan de Almeida Junior 2019 – 2023 Doutorado em Ciências Odontológicas área de Dentística Restauradora. Universidade Estadual Paulista Júlio de Mesquita Filho, UNESP, Brasil. 2017 – 2019 Mestrado em Ciências Odontológicas área de Dentística Restauradora. Universidade Estadual Paulista Júlio de Mesquita Filho, UNESP, Brasil. 2018 – 2019 Capacitação em Reabilitação Oral: Próteses implantosuportadas e sobre dentes. Associação Paulista de Cirurgiões Dentista, APCD, Brasil. 2016 – 2018 Especialização em Dentística Restauradora. Núcleo de Pós-Graduação e Pesquisa em Odontologia, FAEPO, Brasil. 2016 – 2016 Aperfeiçoamento Profissionalizante. Universidade Estadual Paulista Júlio de Mesquita Filho, UNESP, Brasil. 2015 – 2015 Habilitação em Laserterapia. Instituto Pós-Saúde, PÓS-SAÚDE, Brasil. 2015 – 2015 Aperfeiçoamento em Estética Branca e Vermelha. Centro Integrado de Educação Continuada, CIEC, Brasil. 2009 – 2014 Graduação em Odontologia. Universidade Ceuma, UNICEUMA, Brasil. 2006 – 2008 Ensino Médio (2º grau). Centro de Ensino Upaon-Açu, UPAON, Brasil. Dedico este trabalho aos meus país e em especial à minha avó, Iracema Figueiredo de Almeida, pelo amor e apoio incondicional. AGRADECIMENTOS À Deus, que sempre esteve ao meu lado, com ele fui capaz de ultrapassar limites e ir além. Aos meus pais Fátima Mesquita e Edvan Junior, que me deram a vida. Muito obrigada! Amo vocês! Aos meus familiares e em especial, meus tios Guaracy Figueiredo, Emanuel Mesquita e Tadeu Mesquita e à minha prima Samantha Mesquita, que mesmo distantes me deram através do exemplo o apoio e a força necessária para superar os desafios. Aos amigos de pós-graduação e os da minha amada São Luís do Maranhão, por tornarem a trajetória mais tranquila. À Faculdade de Odontologia de Araraquara por me receber tão bem e pela oportunidade de cursar o doutorado. Agradeço ao corpo docente do programa de Pós-graduação em Ciências Odontológicas, coordenado pela Profa. Dra. Andreia Bufalino, em especial ao meu orientador e membro do corpo docente da Disciplina de Dentística Restauradora, Prof. Dr. Marcelo Ferrarezi de Andrade, pela confiança e incentivo. Agradeço ao corpo docente da Disciplina de Dentística Restauradora, Profa. Dra. Andréa Abi Rached Dantas, Profa. Dra. Alessandra Nara de Souza Rastelli e Prof. Dr. Osmir Batista de Oliveira Junior. Ao Prof. Dr. José Roberto Cury Saad, Prof. Dr. Carlos Milton Kuga e Prof. Dr. Edson Alves de Campos, que desde o início se dispuseram a participar da minha banca examinadora durante o exame de pré-qualificação e qualificação do doutorado, meu sincero agradecimento. Ao Magnífico Reitor Prof. Dr. Pasqual Barretti e Vice-Reitora Profa. Dra. Maysa Furlan da Universidade Estadual Paulista “Júlio de Mesquita Filho”. À Faculdade de Odontologia de Araraquara, representados pelo Diretor Prof. Dr. Edson Alves de Campos, à Vice-Diretora Profa. Dra. Patrícia Petromilli Nordi Sasso Garcia. Agradeço a todos os funcionários do Departamento de Odontologia Restauradora. Agradeço à seção técnica de pós-graduação pela gentileza e competência, gratidão Cristiano e Alexandre. À toda equipe de professores da Especialização em Dentística Restauradora da Fundação Araraquarense de Ensino e Pesquisa- FAEPO ano de 2018, aqui representados pela Profa. Dra. Anna Thereza, muito obrigada, vocês foram essenciais. O sonho de passar no seletivo para o doutorado tornou-se possível devido aos aprendizados que adquiri com vocês. Aos laboratórios de Dentística da FORP-USP e Laboratório Multiusuários da ESALQ-USP e aos respectivos técnicos Sra. Patríca Marchi e Sr. Renato Salaroli pelo auxilio e disponibilidade durante todo o período das análises. À CAPES: o presente trabalho foi realizado com o apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Código de financiamento 001. “Que a força e benção de cada geração alcancem sempre e inundem a geração seguinte.” Bert Hellinger  Hellinger B. Oração aos antepassados. Almeida ENM. Avaliação in vitro dos efeitos do clareamento dentário com fotossensibilização em dentes não vitais [tese de doutorado]. Araraquara: Faculdade de Odontologia da UNESP; 2023. RESUMO O objetivo deste estudo in vitro foi avaliar os efeitos da técnica de clareamento utilizando Led Violeta sobre a resistência à fratura, formação da camada híbrida, microdureza e estabilidade de cor em diferentes períodos de tempo (T1: 7 dias, T2: 14 dias, T3: 21 dias, T30: 30 dias e T9M: 9 meses após a última sessão clareadora). Para avaliação da resistência a fratura e formação da camada híbrida os grupos foram: C: Sem tratamento (grupo controle); HP: Whiteness HP Maxx 35% (FGM); HP-BL: Whiteness HP Maxx 35% (FGM) + Led Azul Twin Flex Evolution (MMOptics); HP-VL: Whiteness HP Maxx 35% (FGM) + Led Violeta – Bright Max Whitening (MMOptics). Para o teste de microdureza e cor, diferentes protocolos foram testados: C: Sem tratamento (grupo controle); HP: Whiteness HP 35% (FGM); HP-BL: Whiteness HP 35% (FGM) + Led Azul Twin Flex Evolution (MMOptics); HP-VL: Whiteness HP 35% + Led Violeta – Bright Max Whitening (MMOptics); VL: Led Violeta – Bright Max Whitening (MMOptics), que foi adicionado para avaliação de cor. Na resistência a fratura foram utilizados (N = 40) espécimes, formação da camada híbrida (N = 20), microdureza (N = 40) e estabilidade de cor (N = 50). Os protocolos clareadores foram aplicados de acordo com a recomendação dos fabricantes dos produtos e dos equipamentos, totalizando 3 sessões de clareamento, com intervalo de 7 dias entre elas. Os espécimes permaneceram em saliva artificial a 37°C durante todo o período referente aos experimentos, sendo substituída a cada 7 dias. Os dados obtidos foram analisados por análise de variância (ANOVA one-way, teste de kruskal-Wallis e ANOVA two-way, respectivamente) e pós teste ao nível de 5% de significância. Não foram encontradas diferenças significativas entre os protocolos de clareamento quanto ao efeito sobre a resistência à fratura (p > 0,05). Na avaliação da formação camada híbrida, todos os grupos com HP interferiram em algum nível. Na microdureza HP e C demonstraram respectivamente a maior e menor redução da microdureza dentinária após o uso dos protocolos de clareamento dental (p < 0.05). No entanto, não houve diferença entre os protocolos HP-BL e HP-VL (p > 0.05). Na avaliação de cor os valores médios (DP) de ∆E e ∆WID, os grupos HP, HP BL e HP VL apresentam maior alteração de cor quando comparados a C e VL. HP, HP BL e HP VL não apresentam diferenças significativas entre si após 9 meses da realização do protocolo de clareamento. Palavras chave: Clareamento dental. Dente não vital. Peróxido de hidrogênio. Almeida ENM. In vitro evaluation of the effects of tooth whitening with photosensitization on non-vital teeth [tese de doutorado]. Araraquara: Faculdade de Odontologia da UNESP; 2023. ABSTRACT The objective of this in vitro study was to evaluate the effects of the bleaching technique using Led Violet on the resistance to fracture, formation of the hybrid layer, microhardness and color stability in different periods of time (T1: 7 days, T2: 14 days, T3: 21 days, T30: 30 days and T9M: 9 months after the last whitening session). For the evaluation of resistance to fracture and formation of the hybrid layer, the groups were: C: No treatment (control group); HP: Whiteness HP Maxx 35% (FGM); HP-BL: Whiteness HP Maxx 35% (FGM) + Twin Flex Evolution Blue Led (MMOptics); HP-VL: Whiteness HP Maxx 35% (FGM) + Violet Led – Bright Max Whitening (MMOptics). For the microhardness and color test, different protocols were tested: C: No treatment (control group); HP: Whiteness HP 35% (FGM); HP-BL: Whiteness HP 35% (FGM) + Twin Flex Evolution Blue Led (MMOptics); HP-VL: Whiteness HP 35% + Violet Led – Bright Max Whitening (MMOptics); VL: Led Violet – Bright Max Whitening (MMOptics), which was added for color evaluation. The fracture resistance (N = 40) specimens, hybrid layer formation (N = 20), microhardness (N = 40) and color stability (N = 50) were used. The bleaching protocols were applied according to the recommendations of the product and equipment manufacturers, totaling 3 bleaching sessions, with an interval of 7 days between them. The specimens remained in artificial saliva at 37°C throughout the period referring to the experiments, being replaced every 7 days. The data obtained were analyzed by analysis of variance (one-way ANOVA, Kruskal-Wallis test and two-way ANOVA, respectively) and post-test at a 5% significance level. No significant differences were found between bleaching protocols regarding the effect on fracture toughness (p > 0.05). In evaluating the hybrid layer formation, all groups with HP interfered at some level. In terms of microhardness, HP and C showed, respectively, the greatest and smallest reduction in dentin microhardness after the use of dental bleaching protocols (p < 0.05). However, there was no difference between the HP-BL and HP-VL protocols (p > 0.05). In the color evaluation, the mean values (SD) of ∆E and ∆WID, the HP, HP BL and HP VL groups present greater color change when compared to C and VL. HP, HP BL and HP VL do not present significant differences among themselves after 9 months of the bleaching protocol. Keywords: Tooth bleaching. Tooth, nonvital. Hydrogen peroxide. SUMÁRIO 1 INTRODUÇÃO .................................................................................11 2 PROPOSIÇÃO ..................................................................................13 2.1 Objetivos Específicos ..................................................................13 3 PUBLICAÇÕES ................................................................................14 3.1 Publicação 1 .................................................................................14 3.2 Publicação 2 .................................................................................29 3.3 Publicação 3 .................................................................................47 4 CONCLUSÃO ..................................................................................68 REFERÊNCIAS ...............................................................................69 ANEXO ...................................................................................................... 71 11 1 INTRODUÇÃO A alteração da cor dos dentes é uma preocupação constante entre os pacientes, podendo afetar a autoestima e o convívio social. O processo de escurecimento dentário em dentes não vitais ocorre geralmente devido a trauma na coroa dentária e a necrose pulpar, onde o extravasamento do sangue proveniente da polpa é responsável pelo manchamento progressivo da estrutura. Assim, quanto menor o tempo de exposição ao agente pigmentante e menor grau de escurecimento, maior a probabilidade de sucesso no tratamento clareador1– 4. Para o clareamento de dentes não vitais preconiza-se a técnica imediata. Essa técnica é realizada apenas em consultório, e consiste na aplicação de géis clareadores em altas concentrações como o peróxido de hidrogênio (PH) 35% que é posicionado na câmara pulpar e face vestibular do dente a ser clareado. Pode-se associar a utilização de uma fonte de energia LED azul e laser, que promove a aceleração na reação clareadora, proporcionando assim ganho no tempo de realização do procedimento5,6. O (PH) tem a capacidade de liberar substâncias altamente reativas com o poder de penetrar no esmalte e na dentina, possibilitando que cadeias moleculares longas de pigmento sejam segmentação, gerando cadeias moleculares menores e cromaticamente menos escurecidas7. Esta reação química que ocorre quando utilizado um gel clareador é conhecida como oxirredução8,9. No que se refere aos riscos associados a técnica clareadora em dentes não vitais, o mais preocupante é a reabsorção radicular externa. Este tipo de reabsorção ocorre na região cervical da coroa dentária. Para evitar este dano é essencial a realização do tampão com material ionômerico na região cervical intracoronal10. Embora a técnica de clareamento dentário seja uma alternativa conservadora, menos dispendiosa e eficaz, relados na literatura associam os protocolos utilizados a possíveis alterações no dente, como redução da microdureza o que pode influenciar na resistência à fratura. Ainda neste contexto, é importante ressaltar que o oxigênio residual proveniente do gel clareador continua na estrutura dentária após a última sessão, o que causa a diminuição na resistência adesiva imediatamente após aos protocolos clareadores. Desta forma, preconiza-se aguardar de 7 a 14 dias para realização de futuras restaurações11–14. 12 Recentemente, surgiu o clareamento com fonte de luz violeta, tratando- se de uma luz visível e não ionizante. Sua ação ocorre por mecanismos físicos, possibilitando a quebra das moléculas de pigmento dos dentes15–17. A técnica para dentes não vitais deve ser realizada em consultório, podendo associar à utilização de gel clareador à base de peróxido de hidrogênio (PH), dessa maneira buscando a longevidade na estabilidade da cor. O grande diferencial é que a luz pode ser utilizada isoladamente, mesmo com ausência do gel clareador, o que a torna uma tecnologia inovadora18,19. Considerando a ausência de estudos sobre a técnica com a utilização de luz violeta como alternativa clareadora em dentes não vitais, torna- se importante a realização de pesquisas que avaliem seu potencial clareador e seu efeito sobre diferentes características da estrutura dentária. 13 2 PROPOSIÇÃO Comparar o efeito e a longevidade de diferentes protocolos de clareamento dental para dentes despolpados. 2.1 Objetivos Específicos Avaliação da resistência à fratura, após término dos protocolos para as diferentes técnicas de clareamento; Avaliação da formação da camada híbrida, após término dos protocolos para as diferentes técnicas de clareamento; Avaliação da microdureza dentinária, após término dos protocolos para as diferentes técnicas de clareamento; Avaliação da efetividade na alteração de cor, 7 dias após cada sessão de clareamento dentário; Avaliação da longevidade na alteração de cor, 30 dias e 9 meses após término dos protocolos de clareamento dentário. 14 3 PUBLICAÇÕES A partir desta pesquisa de doutorado produzimos três artigos que são apresentados a seguir. 3.1 Publicação 1 Efeito do protocolo de clareamento com LED violeta sobre a resistência a fratura e formação da camada híbrida de dentes tratados endodonticamente Resumo Objetivo: Avaliar in vitro o efeito do protocolo de clareamento com LED violeta associado a alta concentração de peróxido de hidrogênio (HP) sobre a resistência a fratura e formação da camada híbrida. Métodos: Quarenta dentes bovinos foram tratados endodonticamente e aleatorizados em quatro grupos (n=10): C - Controle, HP - Peróxido de hidrigênio 35%, HP-BL - Peróxido de hidrigênio 35%, + blue LED, HP-VL - Peróxido de hidrigênio 35%, + violet LED. Foram realizadas 3 sessões de clareamento dental com intervalo de 7 dias entre elas. As coroas foram restauradas 10 dias após a última sessão e submetidas ao teste de resistência a fratura. Cinco espécimes de cada grupo foram preparados para avaliação da formação da camada híbrida por meio de imagens de microscopia eletrônica de varredura (MEV). Os resultados de resistência a fratua foram obtidos a partir dos testes de ANOVA one-way e os de MEV a partir do teste Kruskal- Wallis considerando o nível de significância de 5%. Resultados: Não houve diferença significativa no teste de resistência a fratura (p > 0,05). HP e HP-BL apresentaram maior alteração na formação da camada hibrida que o grupo C (p < 0,05). HP-VL foi similar aos grupos C, HP e HP-BL (p > 0,05).  O artigo segue as normas do periódico Photodiagnosis and Photodynamic Therapy ao qual será submetido. 15 Conclusão: Não foram observados efeitos na redução da resistência a fratura para todos os grupos avaliados. Independente do protocolo realizado, a presença de HP interferiu em algum nível na formação da camada hibrida quando realizada restauração 10 dias após a última sessão de clareamento. Palavras-Chave: Clareamento dental; Peróxido de hidrogênio; Endodontia; Fratura; LED violeta. Introdução O clareamento dental é um tratamento estético conservador, eficaz e que demanda menor tempo clínico [1]. Em dentes tratados endodonticamente, pode ser utilizada a técnica interna e externa, onde o componente mais utilizado é o gel de peróxido de hidrogênio (HP)[2– 4]. O HP atua através de mecanismos químicos, onde há liberação de substâncias altamente reativas chamadas de radicais livres, que são capazes de penetrar na estrutura dental e quebrar as moléculas de pigmento dos dentes [1,5]. A técnica de clareamento com HP pode ser associada a utilização de fontes de luz. O LED violeta (405 ± 10 nm) surgiu como uma tecnologia inovadora que promove o clareamento através de mecanismos físicos, diferentemente do LED azul (450 ± 10 nm), que é capaz de acelerar o processo clareador apenas na presença de um gel clareador [6]. Segundo Panhoca et al. [7], a luz violeta utilizada isoladamente pode produzir energia suficiente para quebrar os pigmentos no esmalte. Klaric et al. [8], afirma que ela tem a capacidade de quebrar as moléculas de pigmento da dentina, que por serem fotorreceptoras, são reduzidas sem causar prejuízos térmicos ao dente. Em revisão sistemática realizada por Rossi et al. [9] os autores relatam que o LED violeta utilizado sozinho parece ter potencial para clarear os dentes, embora com efeito menor 16 quando comparado a utilização de peróxidos. O HP em alta concentração associado a luz violeta parece potencializar o efeito do clareamento. Infelizmente, a utilização de altas concentrações de HP sozinho já foi associada a alterações na estrutura dental, como alteração da microdureza, morfologia da superfície, composição química e formação da camada híbrida [8,10]. Com base na literatura correlata, segue-se em busca de novos protocolos que mantenham a eficácia clareadora dos géis de alta concentração (padrão ouro) e reduzam possíveis efeitos adversos. Sabe-se que a dentina é a maior parte da estrutura dentária e qualquer alteração em suas propriedades biomecânicas causa impacto na resistência dental [11–13]. Além disso, a formação de uma camada híbrida íntegra e estável é fundamental para propiciar uma adesão satisfatória ao longo do tempo [14,15]. No entanto, ainda não se sabe como o LED violeta associado ao HP age sobre a resistência e formação da camada híbrida. Portanto, o presente estudo teve como objetivo avaliar os efeitos de protocolos de clareamento dental utilizando HP 35% associado ou não a irradiação com LED violeta ou LED azul sobre a resistência à fratura e formação da camada híbrida de dentes tratados endodonticamente. A hipótese nula testada foi que os protocolos clareadores não influenciam na resistência à fratura e formação da camada híbrida. Materiais e Metódos Preparação dos Espécimes Inicialmente o projeto foi submetido à Comissão de Ética em Pesquisa em Animais. Devido à perda de dentes no momento da seleção dos espécimes que fariam parte da pesquisa, foram solicitados (n = 80) dentes bovinos. Os dentes foram armazenados em solução de timol 1%, a 4°C. No momento do uso, foram lavados em água corrente por 12 horas. Primeiro os dentes foram polidos com pasta de pedra-pomes mais água com auxílio de escova Robinson em 17 baixa rotação. Imediatamente após foram avaliados com lupa estereoscópica, e somente fizeram parte aqueles que não apresentaram trincas, fraturas ou qualquer desgaste na face vestibular. Avaliação da resistência a fratura Foi realizado acesso coronário com ponta diamantada esférica 1014 e 1016 (KG Sorensen, São Paulo, Brasil) em quarenta dentes incisivos bovinos e preparo químico e mecânico para obturação do canal radicular e restauração provisória segundo técnica descrita por Garrido et al. [16]. As raízes de todos os espécimes foram incluídas em um plástico matriz (largura interna de 16,5 mm x comprimento de 20,0 mm) com resina acrílica autopolimerizável (Classic Jet, São Paulo, SP, Brasil) até a junção cemento- esmalte, de acordo com adaptação da técnica proposta por Kuga et al [12]. Os espécimes foram deixados intocados por 24 horas até a polimerização da resina estar completa. Então, foi realizada a remoção de 3 mm de guta-percha e confeccionado o tampão cervical com um ionômero de vidro autopolimerizavel (Vidrion R; SS White, Rio de Janeiro, RJ, Brasil) que foi colocado como uma barreira cervical até a junção cemento - esmalte. Em seguida, um pellet de algodão foi colocado na câmara pulpar e a cavidade foi vedada com material restaurador temporário (RT) (Villevie; Dentalville do Brasil Ltda., Joinvile, SC, Brasil). Na sessão seguinte, os dentes então foram aleatorizados em quatro grupos (n= 10). Os materiais utilizados estão descritos na tabela 1 e os protocolos clareadores dos grupos seguem abaixo: 18 Tabela 1. Grupos investigados, protocolos de clareamento e marcas comerciais utilizadas neste estudo. Groups Protocolo de clareamento (marca registrada, fabricante, endereço) C Grupo controle HP Peróxido de hidrigênio 35% (Whiteness HP MAXX- FGM, Joinville, SC, Brazil) HP-BL Peróxido de hidrigênio 35% (Whiteness HP MAXX- FGM, Joinville, SC, Brazil) + blue LED (Twin Flex Evolution - MMOptics, São Carlos, SP, Brazil) HP-VL Peróxido de hidrigênio 35% (Whiteness HP MAXX - FGM, Joinville, SC, Brazil) + violet LED (Bright Max Whitening - MMOptics, São Carlos, SP, Brazil)  C: Sem protocolo de clareamento. Os espécimes foram armazenados em água destilada a 37ºC. O meio foi trocado a cada 7 dias.  HP: A manipulação do gel Whiteness HP MAXX (FGM, Joinville, SC, Brasil) foi realizada de acordo com as recomendações do fabricante. A proporção 3:1 de HP e espessante foi manipulada por 10 segundos até sua homogeneização. Em seguida, uma camada de aproximadamente 1 mm de espessura do gel foi aplicada. Foram realizadas duas aplicações de 15 min, totalizando 30 min.  HP-BL: A manipulação do gel Whiteness HP MAXX (FGM, Joinville, SC, Brasil) foi realizada como descrita anteriormente e o gel foi fotoativado por LED azul (Twin Flex Evolution - MMOptics, São Carlos, SP, Brasil) por 9 minutos, de acordo com as instruções do fabricante. Duas aplicações de gel clareador e irradiação com LED azul foram realizadas em cada sessão. Cada sessão teve 18 min de duração.  HP-VL: A manipulação do gel Whiteness HP MAXX (FGM, Joinville, SC, Brasil) foi 19 realizada como descrita anteriormente. O gel foi aplicado e o LED violeta foi ativadologo em seguida. A irradiação consistiu em vinte ciclos de 60 segundos, com pausa de 30 segundos, de acordo com as instruções do fabricante. Foram realizadas três sessões em cada grupo, com intervalo de 7 dias entre cada uma. Nos grupos HP, HP-BL e HP-VL, o gel clareador foi aplicado na superfície vestibular e dentro da câmara pulpar. Os espécimes foram imersos em saliva artificial durante todo o período experimental e foi realizada a troca da solução a cada 7 dias. Após cada sessão de clareamento, todo o gel clareador foi aspirado e a coroa dentária irrigada com água destilada. O acesso coronário foi temporariamente protegido com algodão e RT (Villevie; Dentalville do Brasil Ltda., Joinvile, SC, Brasil). No C (controle), após o tratamento endodôntico, a coroa dentária foi restaurada com resina composta. As coroas dentárias dos grupos foram restauradas após 10 dias da última sessão de clareamento. A dentina foi condicionada com ácido fósforico a 37% (Codac 37, FGM, Joinville, SC, Brasil), o sistema adesivo etch-and-rinse foi aplicado (Adper Scotchbond Multipurpose, 3M ESPE, Sumaré, SP, Brazil) e a restauração foi realizada com resina (Charisma, Heraeus Kulzer, São Paulo, SP, Brazil). Os dentes foram mantidos em saliva artificial a 37°C e após 7 dias foram submetidos ao teste de resistência à fratura em uma máquina de teste eletromecânica (EMIC DL 2000; São José dos Pinhais, PR, Brasil). Foi utilizada carga de compressão a uma velocidade constante de 0,5 mm/minuto, com célula de carga de 5 kN. Para adaptação da amostra ao aparelho de ensaio, foi utilizado um dispositivo cilíndrico com ponta cônica [12]. A carga final necessária para fraturar a coroa foi registrada. Os dados obtidos na resistência a fratura foram submetidos a 20 análise de Variância ANOVA one-way e pós teste de Tukey, considerando o nível de significância de 5%. Análise de microscopia eletrônica de varredura Os espécimes para a avaliação da formação da camada híbrida (N = 24) entre o sistema adesivo e a dentina coronária foram obtidos a partir de corte transversal, realizado em espécimes que passaram pelo teste de resistência a fratura. Foi utilizado disco diamantado montado em máquina de cortes Isomet 1000 (Buhler, Lake Bluff Illinois USA), separando a coroa da raiz na região cervical. Após, foi realizado seccionamento no longo eixo da coroa dental, assim obtendo duas secções. A secção inicialmente foi polida com lixa na granulação #1200 montada em politriz DP-10 (Panambra Struers) e lavada em ultrassom por 3 minutos, para a remoção de eventuais resíduos. Na sequência, foram submersas em ácido clorídrico 6N, por 30 segundos, lavadas em água destilada e imersas em solução de hipoclorito de sódio a 2,5%, por 10 minutos e finalmente lavadas também em água destilada por outros 10 minutos. Após os espécimes estarem secos, foram obtidas impressões individualizadas com vinil polissiloxano (Express XT, 3M ESPE, St. Paul, MN, USA), da superfície preparada e obtidas réplicas em resina epóxi (Buehler, São Paulo, SP, Brasil). Estas foram metalizadas e preparadas para análise em microscopio eletrônico de varredura (JMS 5310, JEOL, Tóquio, Japão), utilizando 20kV. Foi obtida uma imagem de cada espécime (500x de magnificação) Fig. 1. Posteriormente, as imagens foram avaliadas na região na interface dente – restauração, foi utilizada uma medida descritiva de 0 a 3 para avaliar a continuidade da camada híbrida. Assim, "0" representando a continuidade da camada híbrida em todos os terços da imagem capturada, ou seja, ausência de fissuras em todos os terços; "1" representando uma continuidade em dois terços; "2" representando uma continuidade em pelo menos um terço; e "3" indicando que todos os terços 21 apresentaram uma descontinuidade na camada ou fissuras híbridas [17]. Os dados obtidos foram submetidos ao teste Kruskal-Wallis considerando o nível de significância de 5%. Resultados Resistência à fratura Não foram encontradas diferenças significativas entre os protocolos de clareamento (p > 0,05). Tabela 2. Média, desvio padrão e intervalo de confiança da resistência à fratura de dentes tratados endodonticamente de acordo com diferentes protocolos de clareamento dentário. Grupos Média (DP) Intervalo de Confiança (IC) C 926,89 (149,56) a (834,19-1019,58) HP 690,35 (232,64) a (546,16-834,54) HP-BL 722,35 (221,05) a (585,35-859,36) HP-VL 778,65 (206,14) a (650,88-906,42) a Letras iguais indicam que não houve diferença estatisticamente significativa entre os grupos (p > 0,05). C: Controle; HP: Peróxido de hidrogênio 35%; HP-BL: Peróxido de hidrogênio 35% e luz LED azul; HP-VL: Peróxido de hidrogênio 35% e luz LED violeta. Microscopia eletrônica de varredura HP e HP-BL demonstraram maior alteração na formação da camara híbrida quando comparados ao Controle (P > 0.05). Por outro lado, não houve diferença entre HP-VL e os protocolos C, HP e HP-BL (P < 0.05). A tabela 3 demonstra a média e desvio padrão da continuidade na formação da camada hibrida após os protocolos de clareamento dental. 22 Figura 1. (A) - C: Controle; (B) - HP: Peróxido de hidrogênio 35%; (C) - HP-BL: Peróxido de hidrogênio 35% e luz LED azul; (D) - HP-VL: Peróxido de hidrogênio 35% e luz LED violeta. Tabela 3. Valores médios (desvio padrão) de acordo com os grupos e tempo de avaliação em relação ao número de lacunas e a continuidade da camada híbrida. Pontuações de medida descritiva de 0 a 3. Groups Avaliação da camada hibrida Mediana 1Q – 3Q C 0 a 0 – 0.5 HP 3 b 2,5 - 3 HP-BL 3 b 3 - 3 HP-VL 2 ab 2 - 3 ab Letras diferentes indicam diferença estatisticamente significativa entre os grupos (Teste de Kruskal-Wallis p<0.05). C: Controle; HP: Peróxido de hidrogênio 35%; HP-BL: Peróxido de hidrogênio 35% e luz LED azul; HP-VL: Peróxido de hidrogênio 35% e luz LED violeta. 23 Discussão O protocolo de clareamento em dentes tratados endodonticamente utilizando LED violeta associado HP 35% não reduziu significativamente a resistência a fratura, o mesmo foi observado nos grupos C, HP e HP-BL. Em relação a formação da camada híbrida, HP-VL foi similar aos demais grupos avaliados. No entanto, HP e HP-BL apresentaram alteração significativa em comparação ao grupo C. Portanto, a hipótese nula foi parcialmente rejeitada. Dentes sem vitalidade possuem características diferentes de dentes vitais. Essa diferença morfológica pode ser atribuída à cárie, fraturas e menor quantidade de remanescente dental, podendo apresentar ainda um maior grau de pigmentação dos dentes [11,18]. Devido à essa maior pigmentação, o HP em alta concentração é normalmente utilizado [2,4,19]. No entanto, seu uso está associado a efeitos deletérios sobre os componentes orgânicos e inorgânicos da estrutura dental [8]. Considerando os últimos achados acerca da luz violeta no clareamento de dentes tratados endodonticamente [2,4], tornou-se pertinente a realização de estudos para avaliar seu efeito e segurança sobre a resistência a fratura e a formação da camada hibrida. Segundo Ribeiro et al. [20] e Manzoli et al. [21] o protocolo HP associado ao LED violeta apresenta efeito similar ao HP utilizado sozinho. Em nosso estudo avaliamos os efeitos do LED violeta com o HP, considerando a capacidade de diminuir o tempo de realização da técnica de clareamento sem causar efeitos deletérios a estrutura dental [22]. A realização de três sessões de clareamento não afetou a resistência a fratura nos grupos experimentais. Os resultados observados neste estudo diferiram dos achados por Galloza et al. [3], que verificou maior redução da resistência a fratura após três sessões de clareamento. No entanto, nesse estudo os autores utilizaram o peróxido de hidrogênio em uma proporção diferente e associado a pigmento, o que pode ter gerado uma maior catálise de HP e ocasionado redução da resistência a fratura. 24 Outra questão a ser considerada, é que mesmo não existindo diferença significativa entre o grupo HP-VL e o C, ainda há a possibilidade de interferência na formação da camada híbrida quando a restauração é realizada 10 dias após a última sessão clareadora. Acredita-se que a presença do gel HP 35%, mesmo que por um menor período de tempo possa afetar a formação da camada híbrida [23]. A literatura mostra que o HP causa alterações no substrato e a presença de radicais oxidativos (ROS) promove dano a adesão das restaurações quando realizadas logo após o clareamento, pois afeta a polimerização dos materiais resinosos [23]. Somado a isso, o uso da luz gera calor, aumentando a liberação de ROS e acelerando a reação clareadora [24]. Segundo Manzoli et al. [21], o LED violeta que atua em 405 nm associado ao HP não afeta negativamente a temperatura, o que pode justificar os resultados encontrados neste estudo. Apesar disso, vale considerar que algum aterfato técnico durante a manipulação dos espécimes excepcionalmente pode ter desencadeado fissuras na interface de adesão dos dentes clareados, sendo interessante a realização de estudos complementares por meio da desmineralização e desproteinização do substrato dentinário para confirmar o efeito durante procedimentos adesivos. Conclusão O uso de HP 35% associado à irradiação com LED violeta ou LED azul não interferiu na resistência a fratura de dentes tratados endodonticamente. No entanto, alterações na formação da camada híbrida de dentes restaurados após 10 dias da última sessão de clareamento foram observadas para os grupos HP e HP-BL em comparação ao grupo controle, diferentemente do grupo HP-VL. 25 Referências [1] B.M. Maran, A. Burey, T. de Paris Matos, A.D. Loguercio, A. Reis, In-office dental bleaching with light vs. without light: A systematic review and meta-analysis, J. Dent. 70 (2018) 1–13. https://doi.org/10.1016/j.jdent.2017.11.007. [2] E.N.M. de Almeida, J.F. Bessegato, D.D.L. dos Santos, A.N. de Souza Rastelli, V.S. Bagnato, Violet LED for non-vital tooth bleaching as a new approach, Photodiagnosis Photodyn. Ther. 28 (2019) 234–237. https://doi.org/10.1016/j.pdpdt.2019.08.024. [3] M.O.G. Galloza, K.C.F. Jordão-Basso, M.C. Bandeca, S.O. Costa, A.H. Borges, M.R. Tonetto, F.C. Tirintan, K.C. Keine, M.C. Kuga, Effects of the ratio between pigment and bleaching gel on the fracture resistance and dentin microhardness of endodontically treated teeth, J. Contemp. Dent. Pract. 18 (2017) 1051–1055. https://doi.org/10.5005/jp-journals-10024-2174. [4] L.M. Teodosio, L. Gambarini, A.L. Faria-e-Silva, F. de C.P. Pires-de-Souza, A.E. de Souza-Gabriel, J.F. Mazzi-Chaves, M.D. Sousa-Neto, F.C. Lopes-Olhê, Bleaching effect of violet LED of 405–410 nm on stained endodontically treated teeth, Photodiagnosis Photodyn. Ther. 39 (2022) 16–18. https://doi.org/10.1016/j.pdpdt.2022.102929. [5] K. Luk, L. Tam, M. Hubert, Effect of light energy on peroxide tooth bleaching, J. Am. Dent. Assoc. 135 (2004) 194–201. https://doi.org/10.14219/jada.archive.2004.0151. [6] B.P. de Oliveira, A.N. Souza Rastelli, V. Salvador Bagnato, V. Hugo Panhoca, Dental Bleaching Using Violet Light Alone: Clinical Case Report, Dentistry. 7 (2017). https://doi.org/10.4172/2161-1122.1000459. [7] V.H. Panhóca, B.P. de Oliveira, V.S. Bagnato, Dental bleaching efficacy with light application: In vitro study, Photodiagnosis Photodyn. Ther. 12 (2015) 357. https://doi.org/10.1016/j.pdpdt.2015.07.128. 26 [8] E. Klaric, M. Rakic, I. Sever, O. Milat, M. Par, Z. Tarle, Enamel and dentin microhardness and chemical composition after experimental light-activated bleaching, Oper. Dent. 40 (2015) E132–E141. https://doi.org/10.2341/14-148-L. [9] B. Rossi, S. Morimoto, T.K. Tedesco, S.R. Cunha, A.C.R.T. Horliana, K.M. Ramalho, Effectiveness of Violet LED alone or in association with bleaching gel during dental photobleaching: A Systematic Review, Photodiagnosis Photodyn. Ther. 38 (2022) 102813. https://doi.org/10.1016/j.pdpdt.2022.102813. [10] F. Benetti, C.A.A. Lemos, M. de Oliveira Gallinari, A.M. Terayama, A.L.F. Briso, R. de Castilho Jacinto, G. Sivieri-Araújo, L.T.A. Cintra, Influence of different types of light on the response of the pulp tissue in dental bleaching: a systematic review, Clin. Oral Investig. 22 (2018) 1825–1837. https://doi.org/10.1007/s00784-017-2278-9. [11] R. Krishan, F. Paqué, A. Ossareh, A. Kishen, T. Dao, S. Friedman, Impacts of conservative endodontic cavity on root canal instrumentation efficacy and resistance to fracture assessed in incisors, premolars, and molars, J. Endod. 40 (2014) 1160–1166. https://doi.org/10.1016/j.joen.2013.12.012. [12] M.C. Kuga, J.M. dos Santos Nunes Reis, S. Fabrício, I. Bonetti-Filho, E.A. de Campos, G. Faria, Fracture strength of incisor crowns after intracoronal bleaching with sodium percarbonate, Dent. Traumatol. 28 (2012) 238–242. https://doi.org/10.1111/j.1600- 9657.2011.01077.x. [13] R.D.T. Leonardo, M.C. Kuga, F.A. Guiotti, C. Andolfatto, N.B. De Faria-Júnior, E.A. De Campos, K.C. Keine, A.A. Rached Dantas, Fracture resistance of teeth submitted to several internal bleaching protoLEONARDO, R. D. T. et al. Fracture resistance of teeth submitted to several internal bleaching protocols. Journal of Contemporary Dental Practice, v. 15, n. 2, p. 186–189, 2015. col, J. Contemp. Dent. Pract. 15 (2015) 186– 189. https://doi.org/10.5005/jp-journals-10024-1512. 27 [14] D.H. Pashley, R.M. Carvalho, Dentine permeability and dentine adhesion, J. Dent. 25 (1997) 355–372. https://doi.org/10.1016/S0300-5712(96)00057-7. [15] A.C.S. Barboza, P.H. dos Santos, L.R. do Vale, M. de Oliveira Gallinari, A. Assmann, C.M.P. Vidal, T.C. Fagundes, A.L.F. Briso, Dental bleaching with violet LED: Effects on dentin color change, resin-dentin bond strength, hybrid layer nanohardness and dentinal collagen biostability, Photodiagnosis Photodyn. Ther. 33 (2021). https://doi.org/10.1016/j.pdpdt.2020.102141. [16] L.D.M.A. Garrido, M.C. Kuga, N.G. Kalatzis-Sousa, A.A.R. Dantas, M.R. Tonetto, R.D.T. Leonardo, K.C.F. Jordão-Basso, S.L. Lima, A.H. Borges, M.C. Bandeca, Influence of the number of bleaching sessions on fracture resistance and dentin microhardness of endodontically treated teeth, World J. Dent. 8 (2017) 5–9. https://doi.org/10.5005/jp-journals-10015-1402. [17] L.R. Abou-Id, F.L.S.A. Morgan, G.A.B. Silva, L.T. de A. Poletto, L.D. Lanza, R. de C. Albuquerque, Ultrastructural evaluation of the hybrid layer after cementation of fiber posts using adhesive systems with different curing modes, Braz. Dent. J. 23 (2012) 116–121. https://doi.org/10.1590/S0103-64402012000200005. [18] M. Hönn, K. Dietz, A. Godt, G. Göz, Attraktivität von Gesichtsprofilen unterschiedlicher skelettaler Anomalieausprägungen, J. Orofac. Orthop. 66 (2005) 187–196. https://doi.org/10.1007/s00056-005-0445-0. [19] P.D. Marin, P.M. Bartold, G.S. Heithersay, Tooth discoloration by blood: An in vitro histochemical study, Endod. Dent. Traumatol. 13 (1997) 132–138. https://doi.org/10.1111/j.1600-9657.1997.tb00026.x. [20] R.A.O. Ribeiro, V. Peruchi, L. de O. Fernandes, C. Anselmi, I.P.M. Soares, J. Hebling, C.A. de S. Costa, The influence of violet LED application time on the esthetic efficacy and cytotoxicity of a 35% H2O2 bleaching gel, Photodiagnosis Photodyn. Ther. 40 28 (2022) 103069. https://doi.org/10.1016/j.pdpdt.2022.103069. [21] T.M. Manzoli, J. Lucas, D.S. Gomes, F. Besegato, M.B. Gelio, L.D. Galvani, E. Alves, F. Bordini, Violet LED applied on dental enamel associated with high concentration of hydrogen peroxide: impacts on bleaching, pH and temperature, Photodiagnosis Photodyn. Ther. (2022) 103133. https://doi.org/10.1016/j.pdpdt.2022.103133. [22] J.L.S.G. Costa, J.F. Besegato, J.F. Zaniboni, L.D. Galvani, M.C. Kuga, Effects of tooth bleaching protocols with violet LED and hydrogen peroxide on enamel properties, Photodiagnosis Photodyn. Ther. 38 (2022). https://doi.org/10.1016/j.pdpdt.2022.102733. [23] V. Cavalli, M. Sebold, M.S. Shinohara, P.N.R. Pereira, M. Giannini, Dentin bond strength and nanoleakage of the adhesive interface after intracoronal bleaching, Microsc. Res. Tech. 81 (2018) 428–436. https://doi.org/10.1002/jemt.22995. [24] R.S. Gonçalves, C.A.S. Costa, D.G.S. Soares, P.H. Dos Santos, L.T.A. Cintra, A.L.F. Briso, Effect of different light sources and enamel preconditioning on color change, H2O2 penetration, and cytotoxicity in bleached teeth, Oper. Dent. 41 (2016) 83–92. https://doi.org/10.2341/14-364-L. 29 3.2 Publicação 2 Effect of non-vital tooth bleaching photoactivated with blue or violet LED on color and microhardness Graphical Abstract  O artigo segue as normas do periódico Photodiagnosis and Photodynamic Therapy ao qual foi submetido. 30 Abstract Background: To evaluate the efficacy of non-vital dental bleaching protocols using 35% hydrogen peroxide photoactivated with violet LED on color and microhardness of endodontically treated teeth. Methods: Forty specimens were selected and randomized into 4 groups (n = 10): C - Control, HP - 35% hydrogen peroxide, HP + BL - 35% hydrogen peroxide + blue LED, HP + VL - 35% hydrogen peroxide + violet LED. Three bleaching sessions were performed for each group. Color analysis was performed 7 days after each bleaching session. Two-way repeated measure ANOVA and Bonferroni test were used to evaluate the effect of different bleaching protocols and evaluation times on the dependent variables (∆E and ∆L). Dentin microhardness was measured 24 hours after the third bleaching session. Data were evaluated by ANOVA and Tukey’s test at a significance level of 5%. Results: Differences on ∆E and ∆L were verified after the first and second bleaching sessions (p < 0.05) and showed stability after the third one, for all the groups. No differences were observed among HP, HP + BL, and HP + VL groups, regardless of the evaluation time (p > 0.05). HP and C showed the greatest and smallest reduction in dentin microhardness (p < 0.05), respectively. No difference between HP + BL and HP + VL protocols (P > 0.05) was observed. Conclusions: High concentration hydrogen peroxide (35%) photoactivated with violet LED bleached endodontically treated teeth effectively. However, the same protocol negatively affected the dentin microhardness, but not in the same level of 35% HP solely used. Keywords: Endodontics, Dental bleaching, Hydrogen peroxide, LED, Microhardness. 31 1. Introduction Dental bleaching is a safe, minimally invasive, and effective aesthetic treatment, showing satisfactory outcomes when well indicated [1–4]. To achieve adequate color change in a short time, high concentration hydrogen peroxide (HP) gels combined with longer exposure time have been proposed [5]. However, the biological and morphological safety of this procedure may be compromised since self-reported tooth sensitivity, and negative effects on the elastic modulus, microhardness, and toughness of the enamel and dentin can be observed [5–7]. The bleaching process occurs when free oxygen radicals are released during the decomposition of HP. These free radicals diffuse through the enamel and dentin breaking the pigmented macromolecules that give yellowish appearance to teeth. However, this process is slow, and the use of light sources such as halogen light, light emitting diodes (LEDs), diode lasers, argon lasers and plasma arc lamps have been proposed to accelerate the decomposition of HP [2,8]. The combined use of HP and violet LED has been emerged since the wavelength between 405 – 410 nm coincides with the wavelength of pigmented molecules, which allows that the photons break long carbon chains into smaller ones and increase the generation of free radicals from the HP [9–13]. Previous studies have shown positive effects when combining violet LED irradiation and concentration hydrogen peroxide on vital teeth [12,14]. Teodosio et al. [20], in vitro study were one of the first to report in the literature on the efficacy of bleaching non-vital tooth using the violet LED associated with 35% hydrogen peroxide, however, there is only one other case report study where the whitening effect on non- vital teeth was evidenced [4]. Despite these studies, there are no comparative evaluations on bleaching with violet LED and blue LED protocol or and the effects on the morphological 32 alterations. More studies are needed to establish the appropriate irradiation time and bleaching protocols [20]. This in vitro study aimed to evaluate the efficacy of bleaching protocols using high concentration hydrogen peroxide photoactivated or not with violet LED and blue LED on the color change and dentin microhardness of non-vital tooth. The null hypothesis tested was the absence of effects on color change and dentin microhardness. 2. Materials and methods 2.1. Specimens’ preparation Bovine teeth were obtained and stored in 0.1% thymol solution at 4ºC. At the moment of use, the teeth were washed for 12 hours to remove residues. After, the teeth were cleaned using pumice stone and Robinson brush (KG Sorensen Ind. E Com. Ltd, São Paulo, Brazil) at low-speed rotation. All teeth were evaluated in a stereomicroscope (20x magnification) and only those without enamel cracks or defects were included. 2.2. Experimental groups C: Control group; HP: 35% Hydrogen peroxide (Whiteness HP - FGM, Joinville, SC, Brazil); HP + BL: 35% Hydrogen peroxide (Whiteness HP - FGM, Joinville, SC, Brazil) + blue LED (Twin Flex Evolution - MMOptics, São Carlos, SP, Brazil); HP + VL: Hydrogen peroxide 35% (Whiteness HP - FGM, Joinville, SC, Brazil) + Violet LED (Bright Max Whitening - MMOptics, São Carlos, SP, Brazil). 33 2.3. Color change evaluation The color was evaluated at: T0 – initial color assessment (after staining); T1 – 7 days after the first bleaching session; T2 – 7 days after the second bleaching session; T3 – 7 days after the third bleaching session. Seventy-five bovine teeth were selected and transversally sectioned at the cervical region using a diamond-coated disk coupled in a precision cutting machine (IsoMet 1000, Buhler, Lake Bluff, IL, USA) to separate the crown from the root. After that, enamel/dentin specimens were obtained from the buccal surface of the crown (5 x 5 x 2 mm). The specimens were planned with silicon carbide sandpapers (#600, #1200, #1500) coupled in a politrix (DP- 10, Panambra Struers) and then polished with diamond paste and felt discs. After that, the specimens were submitted to ultrasonic bath in distilled water for 5 min. The color assessment was performed using a digital spectrophotometer (CM-2600d, Konica Minolta) in the same room and under similar illumination conditions. Three color readings enamel for each specimen were performed using the CIELab color system (L* a* e b*). After, the specimens were stained with defibrinated sheep blood (NewProv; Pinhais, PR, Brazil) according to Freccia et al. [15]. After the blood staining, the specimens were washed in running water and stored in a 24-well plate with only the buccal surface in contact with artificial saliva. The specimens were stored for 30 days, with the artificial saliva being systematically changed every 7 days. After staining, the simulation of the endodontic treatment was performed according to the adaptation of the technique described by Janiboni et al. [17]. To select the included specimens, a new color assessment was performed after the blood staining and the ΔE values were calculated. The forty included specimens were randomized into the experimental groups (Table 1). This selection was performed to ensure the initial color standardization of the specimens. 34 Table 1. Distribution of groups according to the bleaching protocol used. Groups Bleaching protocols C No bleaching protocol. Buccal face immersed in distilled water, changing the distilled water every 7 days. HP HP 35% hydrogen peroxide applied to the buccal surface (enamel) and pulp chamber (dentin) of the specimens. Two 15-minute applications of the whitening gel in the same session. Three sessions were performed at 7-day intervals. HP + BL 35% hydrogen peroxide applied to the buccal surface (enamel) and pulp chamber (dentin) of the specimens and activation of the blue LED for 9 minutes. Two applications of whitening gel and use of the blue LED in the same session. Three sessions were performed at 7-day intervals. HP + VL 35% hydrogen peroxide was applied to the buccal surface (enamel) and pulp chamber (dentin) of the specimens. The gel was applied and the violet light was allowed to activate for 2 minutes (5 cycles of 60 seconds, with a 30- second pause). Two applications of whitening gel and activation of the violet led in the same session. In total 10 light activation cycles. Three sessions were performed at 7-day intervals. 35 Table 1 shows the bleaching protocols performed. A 1-mm thin layer of the bleaching gel was applied over the buccal and palatal surfaces of the specimens. To change the bleaching gel during the session, a sterilized gauze was used. At the end of the session, the gel was removed with air/water jets for 30 seconds and the teeth were stored at 37ºC with the buccal surface submerged in water, changing the medium every 7 days. To determine the color change after the treatment protocols at different evaluation times, ΔE (according to the CIELab system) was calculated [16]. Data normality and sphericity were verified by Shapiro-Wilk (p ≥ 0.059) and Mauchly (p < 0.001) tests, respectively. The Greenhouse-Geisser correction was used due to the sphericity assumption was not proven. Data were analyzed using the PASW Statistics software (version 22.0; SPSS Inc., Chicago, IL, USA), with a significance level of 5%. Two-way ANOVA for repeated measures and Bonferroni test were used to evaluate the effect of different treatments (C, HP, HP + BL, HP + VL) and evaluation times (T1, T2, T3) on the dependent variables (∆Eab, and ∆L). 2.4. Microhardness evaluation To evaluate the microhardness, enamel/dentin specimens (n = 75) were obtained from the buccal surface (4 x 4 x 2 mm). The simulation of the endodontic treatment was performed [17]. Then, the pulp chamber dentin surface was prepared for the microhardness test according to the protocol performed by Aranda-Garcia et al. [18]. Surface planning was performed with #300 to #1200 sandpaper (Norton, Lorena, SP, Brazil) and polishing with aluminum oxide (Profill; SS White, Rio de Janeiro, Brazil). Afterwards, the specimens were washed with distilled water. Microhardness measurements were obtained with a Hardness Testing Machine (Buehler, Japan) and a Knoop indenter with a load of 10 g for 20 sec. Initial measurements of 36 dentin microhardness were performed in the palatal surface of the dental crown 7 days after the endodontic treatment. Three indentations were performed for each specimen with a distance of 200 µm between them. The mean microhardness was calculated for each specimen and only those that presented values close to the general mean were included (n = 40). After that, the included specimens were randomized (n = 10) into the four experimental groups. The final microhardness evaluation was performed 24 hours after the 3rd bleaching session. The difference between the initial and final values was interpreted as the effect on dentin microhardness. The value was expressed as percentages. Data were evaluated using ANOVA and Tukey's test at a significance level of 5%. 3. Results 3.1. Color change evaluation Table 2 shows the mean and standard deviation of ∆E values. Color changes after the first and second bleaching sessions in the HP, HP + BL and HP + VL groups (P < 0.05) were observed. However, no differences were observed between the protocols (p > 0.05) regardless of the evaluation time. Group C showed no color change. ∆L results behaved similarly. The HP, HP + BL and HP + VL groups showed color change until the second bleaching session and remained stable after the third one. The inter- group comparisons showed that HP + VL was similar to HP and HP + BL groups (p > 0.05) (Table 3). 37 Table 2. Mean (standard deviation) of ∆E values according to the experimental groups and evaluation times. Group T1 T2 T3 C 3.04 (1.41)Aa 4.17 (1.67)Aab 3.71 (1.46)Aa HP 9.64 (1.68)Ba 11.63 (1.36)Bb 11.96 (0.95)Bb HP + BL 10.15 (3.07)Ba 12.75 (2.48)Ba 12.81 (3.62)Bab HP + VL 8.69 (2.52)Ba 11.96 (2.93)Bb 11.56 (2.09)Bb A-a Different uppercase letters at the same column (p ≤ 0.001) or different lowercase letter at the same row (p ≤ 0.021) denote statistically significant difference within the same evaluation time or group, respectively. C, control; HP, Whiteness HP 35%; HP + BL, Whiteness HP 35% (FGM) + Blue Twin Flex Evolution LED; HP + VL, Whiteness HP 35% + Violet Led – Bright Max Whitening. 38 Table 3. Mean (standard deviation) of ∆L values according to the experimental groups and evaluation times. Groups T1 T2 T3 C - 0.18 (2.32)Aa 0.71 (2.82)Aab - 0.07 (2.88)Aa HP 8.94 (1.68)Ba 11.02 (1.41)Bb 11.13 (1.13)Bb HP + BL 9.48 (2.87)Ba 12.18 (2.23)Bb 12.02 (3.88)Bab HP + VL 7.82 (2.79)Ba 11.39 (2.73)Bb 10.87 (1.99)Bb A-a Different uppercase letters at the same column (p ≤ 0.001) or different lowercase letters at the same row (p ≤ 0.044) denote statistically significant difference within the same evaluation time or group, respectively. C, control; HP, Whiteness HP 35%; HP + BL, Whiteness HP 35% (FGM) + Blue Twin Flex Evolution LED; HP + VL, Whiteness HP 35% + Violet Led – Bright Max Whitening. 3.2. Microhardness evaluation HP and C showed the greatest and smallest reduction in dentin microhardness after the tooth whitening, respectively (P < 0.05) (Fig.1). On the other hand, no differences between the HP + BL and HP + VL were observed (P > 0.05). Table 4 shows the arithmetic mean and standard deviation of the percentage of dentin microhardness reduction after the tooth whitening protocols. 39 Figure 1. Mean and confidence interval of the reduction in microhardness (%) according to the experimental groups. Table 4. Arithmetic mean, standard deviation and percentage of dentin microhardness reduction (in Knoop) according to the experimental groups. Groups Mean (SD) Percentage reduction C 35.63 (0.0082)a 0.01 HP 30.55 (1.84)c 13.31 HP + BL 32.53 (3.73)b 8.40 HP + VL 32.45 ( 3.12)b 8.73 abc Different letters indicate significant differences between the evaluated groups (P < 0.05). C, control; HP, Whiteness HP 35%; HP + BL, Whiteness HP 35% (FGM) + Blue Twin Flex Evolution LED; HP + VL, Whiteness HP 35% + Violet Led – Bright Max Whitening. 4. Discussion This study evaluated bleaching efficacy and the microstructural effect of dental bleaching using 35% HP and violet LED irradiation in non-vital tooth. The values of ΔE and 40 ΔL in HP + Violet LED were significantly higher than the control group (no treatment) and were the same as in the HP and HP + Blue LED groups. These data show that the dental bleaching was effective. Our results indicate that the whitening protocol with two applications per session of the gel (HP 35%) for 2 min, followed by 5 cycles of 60 sec of light activation and pause time of 30 seconds, contributed positively to the color change. On the other hand, Gallinari et al. [7] showed that the greatest whitening effect with HP 35% occurs regardless of the use of violet light after three cycles with 7 light activations each, totaling 21 light activation cycles. However, in our study, 10 light activation cycles were performed per session, so it is believed that the main benefit of using the violet LED may be the reduction in the time of the whitening session. Kury et al. [11] verified through a randomized controlled clinical trial that the violet LED associated with HP 35% resulted in a greater color change compared to the group with HP 35% gel alone. In this context, it is important to emphasize that the duration of the application of the bleaching gel may interfere with the effectiveness of dental bleaching. In our study, the HP group consisted of two 15-minute applications of the whitening gel, totalizing 30 minutes. This time was estimated so that there was better standardization between the groups evaluated. The HP group showed no significant differences in the values of ΔE and ΔL compared to HP+BL and HP+VL. Despite recent findings about the effectiveness of violet light when associated with different concentrations of peroxides [19–22], there are still few studies reporting its effect on the mechanical properties of dentin. Therefore, this study aimed to evaluate the effect of the bleaching protocols with violet LED and HP on the dentin microhardness. Regarding microhardness, HP + VL showed less decrease compared to HP group. However, in all the bleaching protocols that used high concentration hydrogen peroxide some 41 level of alteration in dentin microhardness was observed. Thus, our null hypothesis was rejected. HP + BL was used in this study to compare their effect with violet LED. Group C, without treatment, was chosen as a negative control, to allow comparison with the other groups. HP, on the other hand, represents the gold standard within whitening procedures, therefore, comparison is essential when new whitening protocols are tested [2,7,9,17,23,24]. Microhardness analysis can evaluate after the bleaching protocols the structural damage in dentin [25], and the greater reduction of microhardness can lead to a lower resistance to tooth fracture. For this reason, the evaluated area of dentin was standardized, since the different diameters of the dentinal tubules in the region could result in bias [26,27]. Possibly, the shorter contact time of the gel with the enamel/dentin contributed to the lower reduction of microhardness in HP + BL and HP + VL (Fig.1). Therefore, it is suggested that shorter contact of HP time promoted by the use of LED resulted in a smaller reduction of the organic components of the dentin and alterations in the morphology of the dental structure that influence the mechanical properties [28]. Our study describes the color and microstructural effect of combining violet LED or blue LED photoactivation with 35% hydrogen peroxide as an innovative alternative protocol for non-vital tooth bleaching. Based on the limitations of this in vitro study, it can be concluded that the photoactivation with violet LED combined with 35% hydrogen peroxide is an interesting conservative approach. However, further studies must be performed to evaluate this protocols under a clinical setting. 5. Conclusion Within the limitations of this in vitro study, the bleaching of non-vital tooth using high concentration hydrogen peroxide and violet LED photoactivation was effective in color change. All the bleaching protocols with HP decreased the dentin surface of the specimens microhardness; however, the solely use of hydrogen peroxide showed the greatest reduction. 42 Acknowledgments The authors would like to thank MMOptics equipments for materials’ donation. References [1] R.S. Kobayashi, M.Z.D. Picolo, M. Kury, B. de A. Resende, F.L. Esteban Florez, V. Cavalli, Effects of dental bleaching protocols with violet radiation on the color and chemical composition of stained bovine enamel, Photodiagnosis Photodyn. Ther. 34 (2021) 102194. https://doi.org/10.1016/j.pdpdt.2021.102194. [2] B.M. Maran, A. Burey, T. de Paris Matos, A.D. Loguercio, A. Reis, In-office dental bleaching with light vs. without light: A systematic review and meta-analysis, J. Dent. 70 (2018) 1–13. https://doi.org/10.1016/j.jdent.2017.11.007. [3] S. Kossatz, A. Dalanhol, T. Cunha, A. Loguercio, A. Reis, Effect of light activation on tooth sensitivity after in-office bleaching, Oper. Dent. 36 (2011) 251–257. https://doi.org/10.2341/10-289-C. [4] E.N.M. de Almeida, J.F. Bessegato, D.D.L. dos Santos, A.N. de Souza Rastelli, V.S. Bagnato, Violet LED for non-vital tooth bleaching as a new approach, Photodiagnosis Photodyn. Ther. 28 (2019) 234–237. https://doi.org/10.1016/j.pdpdt.2019.08.024. [5] F. Benetti, C.A.A. Lemos, M.O. Gallinari, A.M. Terayama, A.L.F. Briso, R. de Castilho Jacinto, G. Sivieri-Araújo, L.T.A. Cintra, Influence of different types of light on the response of the pulp tissue in dental bleaching: a systematic review, Clin. Oral Investig. 22 (2018) 1825–1837. https://doi.org/10.1007/s00784-017-2278-9. [6] A.E.C.G. Dos Santos, S.K. Bussadori, M.M. Pinto, D.A. Pantano Junior, A. Brugnera, F.A.A. Zanin, M.F.S.D. Rodrigues, L.J. Motta, A.C.R.T. Horliana, Evaluation of in- 43 office tooth whitening treatment with violet LED: Protocol for a randomised controlled clinical trial, BMJ Open. 8 (2018) 1–9. https://doi.org/10.1136/bmjopen-2017-021414. [7] M.O. Gallinari, T.C. Fagundes, L.M.A.V. Da Silva, M.M.B. De Almeida Souza, A.C. De Souza Barboza, A.L.F. Briso, A new approach for dental bleaching using violet light with or without the use of whitening gel: Study of bleaching effectiveness, Oper. Dent. 44 (2019) 521–529. https://doi.org/10.2341/17-257-L. [8] K. Luk, L. Tam, M. Hubert, Effect of light energy on peroxide tooth bleaching, J. Am. Dent. Assoc. 135 (2004) 194–201. https://doi.org/10.14219/jada.archive.2004.0151. [9] T.W.S Daltro, S.A.G de Almeida, M.F. Dias, P.C. Lins-Filho, C.H.V. da Silva, R.P. Guimarães, The influence of violet LED light on tooth bleaching protocols: In vitro study of bleaching effectiveness, Photodiagnosis Photodyn. Ther. 32 (2020) 4–7. https://doi.org/10.1016/j.pdpdt.2020.102052. [10] J.L.S.G. Costa, J.F. Besegato, J.F. Zaniboni, M.C. Kuga, LED/laser photoactivation enhances the whitening efficacy of low concentration hydrogen peroxide without microstructural enamel changes, Photodiagnosis Photodyn. Ther. 36 (2021) 102511. https://doi.org/10.1016/j.pdpdt.2021.102511. [11] M. Kury, E.E. Wada, S.S Palandi, M.Z.D. Picolo, M. Giannini, V. Cavalli, Colorimetric evaluation after in-office tooth bleaching with violet LED: 6- and 12- month follow-ups of a randomized clinical trial, Clin. Oral Investig. 26 (2022) 837– 847. https://doi.org/10.1007/s00784-021-04062-9. [12] A.N. de S. Rastelli, H.B. Dias, E.T. Carrera, A.C.P. de Barros, D.D.L. dos Santos, V.H. Panhóca, V.S. Bagnato, Violet LED with low concentration carbamide peroxide for dental bleaching: A case report, Photodiagnosis Photodyn. Ther. 23 (2018) 270–272. 44 https://doi.org/10.1016/j.pdpdt.2018.06.021. [13] B.M. Fernandes, M.H. Tanaka, A.L.B.M. De Oliveira, R.S. Scatolin, Color stability of dental enamel bleached with violet LED associated with or without Low concentration peroxide gels, Photodiagnosis Photodyn. Ther. 33 (2021) 102101. https://doi.org/10.1016/j.pdpdt.2020.102101. [14] A.D.N. Lago, W.D.R. Ferreira, G.S. Furtado, Dental bleaching with the use of violet light only: Reality or Future?, Photodiagnosis Photodyn. Ther. 17 (2017) 124–126. https://doi.org/10.1016/j.pdpdt.2016.11.014. [15] W.F. Freccia, D.D. Peters, A technique for staining extracted teeth: a research and teaching aid for bleaching, J. Endod. 8 (1982) 67–69. https://doi.org/10.1016/S0099- 2399(82)80260-4. [16] T.W.S. Daltro, S.A.G. de Almeida, M.F. Dias, P.C. Lins-Filho, C.H.V. da Silva, R.P. Guimarães, The influence of violet LED light on tooth bleaching protocols: In vitro study of bleaching effectiveness, Photodiagnosis Photodyn. Ther. 32 (2020) 4–7. https://doi.org/10.1016/j.pdpdt.2020.102052. [17] J.F. Zaniboni, V. de Souza, W.G. Escalante-Otárola, T.P. Leandrin, E. Fernández Godoy, J.F. Besegato, M.C. Kuga, Cleaning and microstructural effects of amyl acetate on pulp chamber dentin impregnated with epoxy resin-based endodontic sealer, J. Esthet. Restor. Dent. (2022) 1–8. https://doi.org/10.1111/jerd.12966. [18] A.J. Aranda-Garcia, M.C. Kuga, G.M. Chavéz-Andrade, N.G. Kalatzis-Sousa, M.A. Hungaro Duarte, G. Faria, M.V. Reis Só, N.B. Faria-Junior, Effect of final irrigation protocols on microhardness and erosion of root canal dentin, Microsc. Res. Tech. 76 (2013) 1079–1083. https://doi.org/10.1002/jemt.22268. 45 [19] B. Rossi, S. Morimoto, T.K. Tedesco, S.R. Cunha, A.C.R.T. Horliana, K.M. Ramalho, Effectiveness of Violet LED alone or in association with bleaching gel during dental photobleaching: A Systematic Review, Photodiagnosis Photodyn. Ther. 38 (2022) 102813. https://doi.org/10.1016/j.pdpdt.2022.102813. [20] L.M. Teodosio, L. Gambarini, A.L. Faria-e-Silva, F. de C.P. Pires-de-Souza, A.E. de Souza-Gabriel, J.F. Mazzi-Chaves, M.D. Sousa-Neto, F.C. Lopes-Olhê, Bleaching effect of violet LED of 405–410 nm on stained endodontically treated teeth, Photodiagnosis Photodyn. Ther. 39 (2022) 16–18. https://doi.org/10.1016/j.pdpdt.2022.102929. [21] C.F.S. Menezes, G.S. Furtado, G. Sarra, M.M. Marques, V.P. Rodrigues, A.D.N. Lago, Violet led dental whitening: Effectiveness and biological safety: An in vitro study, Photodiagnosis Photodyn. Ther. 39 (2022). https://doi.org/10.1016/j.pdpdt.2022.102965. [22] T.M. Manzoli, J.L.S.G. Costa, J.F. Besegato, M.B. Gelio, L.D. Galvani, E.A.F Bordini, M.C. Kuga, A.A.R Dantas, Violet LED applied on dental enamel associated with high concentration of hydrogen peroxide: impacts on bleaching, pH and temperature, Photodiagnosis Photodyn. Ther. (2022) 103133. https://doi.org/10.1016/j.pdpdt.2022.103133. [23] R.S. Gonçalves, C.A.S. Costa, D.G.S. Soares, P.H. Dos Santos, L.T.A. Cintra, A.L.F. Briso, Effect of different light sources and enamel preconditioning on color change, H2O2 penetration, and cytotoxicity in bleached teeth, Oper. Dent. 41 (2016) 83–92. https://doi.org/10.2341/14-364-L. [24] M.O.G. Galloza, K.C.F. Jordão-Basso, M.C. Bandeca, S.O. Costa, A.H. Borges, M.R. 46 Tonetto, F.C. Tirintan, K.C. Keine, M.C. Kuga, Effects of the ratio between pigment and bleaching gel on the fracture resistance and dentin microhardness of endodontically treated teeth, J. Contemp. Dent. Pract. 18 (2017) 1051–1055. https://doi.org/10.5005/jp-journals-10024-2174. [25] M. Srivastava, R. Yeluri, on the Microhardness and Shear Bond Strength To Bleached Dentin, (2021) 1–7. [26] D. Pashley, A. Okabe, P. Parham, The relationship between dentin microhardness and tubule density, Dent. Traumatol. 1 (1985) 176–179. https://doi.org/10.1111/j.1600- 9657.1985.tb00653.x. [27] M. Kazemipoor, S. Azad, F. Farahat, Concurrent effects of bleaching materials and the size of root canal preparation on cervical dentin microhardness, Iran. Endod. J. 12 (2017) 298–302. https://doi.org/10.22037/iej.v12i3.15774. [28] E. Klaric, M. Rakic, I. Sever, O. Milat, M. Par, Z. Tarle, Enamel and Dentin Microhardness and Chemical Composition After Experimental Light-activated Bleaching, Oper. Dent. 40 (2015) E132–E141. https://doi.org/10.2341/14-148L. 47 3.3 Publicação 3 Effectiveness and color stability of dental bleaching photoactivated by violet LED on blood-stained teeth Graphical Abstract  O artigo segue as normas do periódico Photodiagnosis and Photodynamic Therapy ao qual foi submetido. 48 Abstract Background: Few studies have investigated the effect of violet LED irradiation associated or not with bleaching agents on blood-stained teeth. This in vitro study aimed to evaluate the whitening efficacy and color stability of non-vital dental bleaching using 35% hydrogen peroxide (HP) photoactivated with violet LED (VL) compared to 35% HP alone and 35% HP photoactivated with blue LED (BL). Methods: Fifty bovine dental crowns were used to obtain specimens of 5x5x2 mm. After selection based on a previous colorimetric analysis, the specimens were blood-stained and randomly assigned into five groups (n = 10): control (no treatment); 35% HP, 35% HP/BL; 35% HP/VL; and VL. Three bleaching sessions were performed and the colorimetric analysis (∆Eab, ∆L, and ∆WID) was recorded after 7 days, 30 days, and 9 months of the last bleaching session. Two-way repeated measures ANOVA followed by Bonferroni post-hoc test was used at a significance level of 5%. Results: 35% HP, 35% HP/BL, and 35% HP/VL showed the higher values of ∆Eab, ∆L, e ∆WID (P < 0.05), without intra- and intergroup differences (P > 0.05). C and VL were similar in all the evaluation times (P > 0.05), showing lower values of ∆Eab, ∆L, and ∆WID (P < 0.05). Conclusions: 35% HP/VL can be a viable alternative for dental bleaching in endodontically- treated teeth, showing whitening efficacy similar to 35% HP solely used, even after a 9-moth follow-up. VL used alone was not effective to bleach blood-stained teeth. Keywords: Dental bleaching; Hydrogen peroxide; Color change; Blue LED; Violet LED. 49 1. Introduction Violet light-emitting diode (LED) is an innovative light source in dental bleaching working by physical mechanisms [1]. Its wavelength (405 – 410 nm) is capable to bleach the dental structure, even when solely used [2]. The conventional bleaching protocols are based on chemical mechanisms in which a hydrogen peroxide(HP)-based agent can be associated or not with a light source [3–6] . HP is responsible to bleach the dental structure by the release of highly reactive molecules that penetrate into the enamel and dentin and break complex pigmented molecules (chromophores). This chemical reaction is so-called oxi-reduction in which long carbon chains are transformed into smaller ones, making the appearance of teeth visually lighter [7–9]. Besides, light sources such as diode lasers, LED units, and halogen lamps can be used as photocatalysts to accelerate the bleaching reaction by the generated heat, improving the bleaching efficacy [10]. However, Maran et al. [11], in a systematic review, pointed out that the use of light sources did not improve the bleaching efficacy of the in-office technique using HP. With the upcoming of violet LED, novel studies have been conducted to validate its bleaching efficacy when associated with HP, or even alone, in teeth stained with various pigments [12–20]. In terms of endodontically-treated teeth, the literature is scarce regarding the clinical viability of violet LED photoactivation [21,22]. Color change in endodontically-treated teeth can occur due to the presence of sealer residues in the coronal region, necrosis, or blood leakage after trauma. The tooth discoloration progressively occurs; thus, the exposure to the pigment must be reduced or eliminated to achieve a better prognosis of color change after bleaching [23]. In addition, tooth discoloration may impair the self-esteem and social living of the patient, as well as the quality of life. It is 50 important to emphasize that dental bleaching is a conservative, effective, and time-saving approach compared to prosthetic rehabilitation [10,24]. The use of violet LED has demonstrated promising results when associated with HP- based agents [18,25]. Bearing in mind the few studies in endodontically-treated teeth, this study aimed to long-term evaluate the bleaching efficacy of in-office dental bleaching using violet LED associated or not with 35% HP agents compared to 35% HP alone or associated with blue LED. The null hypothesis tested was that the different bleaching protocols have the same efficacy in terms of color change over the evaluation times. 2. Materials and methods 2.1. Specimens’ preparation The sample size was determined using the G*Power software [26] considering ANOVA for repeated measures, an effect size of 0.6, [12,16,27] a significance level of 5% (α = 0.05), and a power test of 95% (1 – β error). After the calculation, the sample size was defined as 8 specimens per group. Eighty-five bovine incisors without enamel cracks or defects were previously selected. All teeth were stored in 0.1% thymol solution at 4ºC until the beginning of the experiments. Specimens of 5x5x2 mm were obtained from the central region of the buccal surface of the crown using a precision cutting machine (IsoMet 1000; Buehler Inc., Lake Bluff, IL, USA). After that, the enamel surface of the specimens was planned with silicon carbide sandpapers in decreasing granulation (#600, #1200, #1500) and polished with diamond past and felt discs. After polishing, the specimens were submitted to ultrasonic bath in distilled water for 5 minutes (Cristófoli, Campo Mourão, PR, Brazil), and then stored at 37ºC (± 1°C) until the first colorimetric analysis. 51 Tooth staining was performed after the first colorimetric assessment. The staining protocol used defibrinated sheep blood (NewProv; Pinhais, PR, Brazil), according to an adaptation of the staining technique proposed by Freccia et al. [28] and Guimarães et al. [29]. This protocol consisted of three days of blood immersion. After that, the specimens were cleaned in running water, and then dried and stored in a 24-well culture cell plate containing cotton and distilled water to create a humid environment for 4 weeks. After staining, the simulation of the endodontic treatment was performed according to the adaptation of the technique described by Janiboni et al. [30,31] and Del Carpio-Perochena et al. [32] by immersing the specimens in a 2.5% sodium hypochlorite solution (Bioquímica, São José do Rio Preto, SP, Brazil) for 30 minutes, refreshing the solution every 3 minutes. 17% EDTA (Asfer, São Caetano do Sul, SP, Brazil) was applied for 3 minutes and then an epoxy resin-based cement containing calcium hydroxide (Sealer Plus; MK Life, Porto Alegre, RS, Brazil) was impregnated on the dentin surface using a microbrush (KG Sorensen, São Paulo, SP, Brazil). After 15 min, the cement was removed from the surface with a sterilized gauze and 95% ethanol. Then, the specimens were strategically placed to guarantee that only the enamel surface was in contact with the artificial saliva, in order to simulate the oral conditions. 2.2. Specimens’ allocation A blinded operator randomized the specimens into the five experimental groups only after the staining protocol. To standardize and avoid sample bias, all the specimens were submitted to colorimetric analysis using a digital spectrophotometer (CM 2600d - Konica Minolta, Petrópolis, RJ, Brazil) to ensure similar ∆Eab values among them. 52 2.3. Experimental groups The materials evaluated are shown in Table 1. The bleaching protocols were performed as follows: Table 1. Investigated groups, bleaching protocols, and trademarks used in this study. Groups Bleaching protocol (Trademark, manufacturer, address) C Control group 35% HP 35% Hydrogen peroxide (Whiteness HP - FGM, Joinville, SC, Brazil) 35% HP/BL 35% Hydrogen peroxide (Whiteness HP - FGM, Joinville, SC, Brazil) + blue LED (Twin Flex Evolution - MMOptics, São Carlos, SP, Brazil) 35% HP/VL Hydrogen peroxide 35% (Whiteness HP - FGM, Joinville, SC, Brazil) + violet LED (Bright Max Whitening - MMOptics, São Carlos, SP, Brazil) VL Violet LED (Bright Max Whitening - MMOptics, São Carlos, SP, Brazil)  C: No bleaching protocol. Specimens were stored with the buccal surface immersed in distilled water at 37ºC. The medium was changed every 7 days.  35% HP: Three bleaching sessions of 30 min using the Whiteness HP gel (FGM, Joinville, SC, Brazil) were performed with an interval of 7 days. The handling of the gel was performed according to the manufacturer recommendations. The proportion 3:1 of HP and thickener was handled for 10 sec until its homogenization. After that, an approximately 1-mm thickness layer of the gel was applied over the buccal surface (enamel) and pulp chamber of the specimens with two applications of 15 min, totalizing 30 min.  35% HP/BL: Three bleaching sessions using the Whiteness HP gel (FGM, Joinville, SC, Brazil) photoactivated by blue LED (Twin Flex Evolution - MMOptics, São 53 Carlos, SP, Brazil) were performed with an interval of 7 days. Each session had 18 min of duration. The gel was handled as previously described above. The gel was applied over the buccal surface (enamel) and pulp chamber (dentin) of the specimens and immediately photoactivated by blue LED irradiation for 9 min, according to the manufacturer’s instructions. Two applications of bleaching gel and blue LED irradiation was performed in each session.  35% HP/VL: Three bleaching sessions using the Whiteness HP gel (FGM, Joinville, SC, Brazil) photoactivated by violet LED (Twin Flex Evolution - MMOptics, São Carlos, SP, Brazil) were performed with an interval of 7 days. Each session had 18 min of duration. The gel was handled as previously described above. The gel was applied over the buccal surface (enamel) and pulp chamber (dentin) of the specimens and after 2 min they were photoactivated by violet LED. The irradiation consisted of five cycles of 60 seconds, with 30-sec pause, according to the manufacturer’s instructions. Two applications of bleaching gel and violet LED irradiation was performed in each session.  VL: Three bleaching sessions using only violet LED irradiation were performed with an interval of 7 days. Each session had 29 min and 30 sec of duration. The violet LED source was applied on the buccal surface (enamel) and pulp chamber (dentin) of the specimens. The irradiation consisted of twenty cycles of 60 sec with a 30-sec pause, according to the manufacturer’s instructions. After the end of each bleaching session, the gel was removed with air/water-jet for 30 sec and the specimens were stored in a 24-well culture cell plate with the buccal surface immersed in artificial saliva at 37ºC. 54 2.4 Colorimetric analysis To evaluate the color change, a digital spectrophotometer was used (CM 2600d - Konica Minolta, Petrópolis, RJ, Brazil). The colorimetric analysis was performed before and after the staining protocols, and 7 days after the final bleaching session. To long-term evaluate the bleaching efficacy, the colorimetric analysis was also performed after 30 days and 9 months after the final bleaching session. All the colorimetric analysis was performed in the same room with similar illumination conditions by the same operator. Three color readings were performed for each specimen in each period of evaluation. The color change was evaluated by the colorimetric systems: CIELab (∆E* and ΔL*) [16] and ∆WID (CIELAB-based Whiteness Index for Dentistry) [15,33]. 2.5 Statistical analysis After verifying the normality and sphericity of the data by Shapiro-Wilk (p ≥ 0.059) and Mauchly tests (p < 0.001), respectively, the Greenhouse-Geisser correction was used. Two-way repeated measures ANOVA followed by Bonferroni post-test was used for multiple comparisons. The PASW statistical software (version 22.0; SPSS Inc., Chicago, IL, USA) was used at a significance level of 5%. 3. Results Table 2 shows the mean and standard deviation values for ∆E. 35% HP, 35% HP/BL and 35% HP/VL showed higher values than C and VL, regardless of the evaluation time (p < 0.05). No differences among the groups that used 35% HP were observed after 30 days and 9 months of the final bleaching session (p > 0.05). 55 Table 2. Mean (standard deviation) values for ∆Eab according to the groups and evaluation time. Groups Evaluation time T1 T2 T3 C 3,59 (1,42) Aa 3,62 (1,49) Aab 6,03 (2,72) Aab 35% HP 11,87 (0,89) Ba 11,32 (3,06) Ba 12,66 (2,88) Ba 35% HP/BL 12,76 (3,58) Ba 12,64 (3,73) Ba 14,06 (2,94) Ba 35% HP/VL 11,47 (2,11) Ba 10,83 (3,91) Ba 13,23 (3,03) Ba VL 3,31 (1,47) Aa 3,94 (2,16) Aab 6,24 (1,53) Ab A-a Different uppercase letters in the same column (p ≤ 0.001) or lowercase letter in the same row (p ≤ 0.021) denote statistcally significant difference within the same evaluation time and group, respectively. C, no treatment (negative control); 35% HP, Whiteness HP 35% (positive control); 35% HP/BL, Whiteness HP 35% + Blue LED; 35% HP/VL, Whiteness HP 35% + Violet LED; VL, Violet LED. T1, 7 days after the 3rd bleaching session; T2, 30 days after the 3rd bleaching session; T3, 9 months after the 3rd bleaching session. Similarly, the ΔL results showed higher values for 35% HP, 35% HP/BL, and 35% HP/VL compared to C and VL, regardless of the evaluation time (p > 0.05) (Table 3). 56 Table 3. Mean (standard deviation) values for ∆L according to the groups and evaluation time. A-a Different uppercase letters in the same column (p ≤ 0.001) or lowercase letter in the same row (p ≤ 0.021) denote statistcally significant difference within the same evaluation time and group, respectively. C, no treatment (negative control); 35% HP, Whiteness HP 35% (positive control); 35% HP/BL, Whiteness HP 35% + Blue LED; 35% HP/VL, Whiteness HP 35% + Violet LED; VL, Violet LED. T1, 7 days after the 3rd bleaching session; T2, 30 days after the 3rd bleaching session; T3, 9 months after the 3rd bleaching session. Regarding the ∆WID, the groups 35% HP, 35% HP/BL, and 35% HP/VL showed higher values than C and VL (p < 0.05) in T1, T2, and T3. For 35% HP/BL and 35% HP/VL a decrease in the color change was observed after 30 days. No differences were observed among the groups that used hydrogen peroxide (35% HP, 35% HP/BL, and 35% HP/VL), regardless of the evaluation time (p > 0.05). VL showed lower ∆WID values, similar to the control group in all evaluation periods. Group Evaluation time T1 T2 T3 C - 0,05 (2,79) Aa 0,36 (1,66) Aa 4,67 (2,64) Ab 35% HP 11,10 (1,18) Ba 10,32 (3,66) Ba 12,07 (2,83) Ba 35% HP/BL 12,00 (3,76) Ba 11,79 (3,82) Ba 13,43 (2,67) Ba 35% HP/VL 10,84 (1,96) Ba 9,85 (3,06) Ba 12,55 (2,86) Ba VL 0,55 (2,24) Aa - 0,36 (1,44) Aab 4,97 (1,75) Ac 57 Table 4. Mean (standard deviation) values for ∆WID according to the groups and evaluation time. Group Evaluation time T1 T2 T3 C 4,26 (3,31) Aa 4,05 (2,25) Aab 5,71 (3,65) Ab 35% HP 15,76 (2,96) Ba 14,61 (2,89) Ba 12,75 (3,93) Ba 35% HP/BL 14,21 (5,52) Ba 12,79 (5,63) Bab 12,49 (5,37) Ba 35% HP/VL 12,99 (5,57) Ba 11,61 (6,14) Bb 11,22 (5,82) ABb VL 6,46 (2,83) Aab 5,92 (2,47) Aab 7,07 (2,87) Aac A-a Different uppercase letters in the same column (p ≤ 0.001) or lowercase letter in the same row (p ≤ 0.021) denote statistcally significant difference within the same evaluation time and group, respectively. C, no treatment (negative control); 35% HP, Whiteness HP 35% (positive control); 35% HP/BL, Whiteness HP 35% + Blue LED; 35% HP/VL, Whiteness HP 35% + Violet LED; VL, Violet LED. T1, 7 days after the 3rd bleaching session; T2, 30 days after the 3rd bleaching session; T3, 9 months after the 3rd bleaching session. 4. Discussion This study evaluated the effectiveness of non-vital dental bleaching with or without 35% HP associated or not with violet LED as an alternative protocol for blood-stained teeth compared to the conventional 35% HP and 35% HP + Blue LED. The null hypothesis was rejected, since the groups behaved differently after 7 days, 30 days and 9 months of the last bleaching session. The dimension of the specimens was standardized in 5 x 5 x 2 mm to allow the initial color reading in a Minolta Spectrophotometer. Our methodology was chosen to reproduce the tooth discoloration classified as an intrinsic post-eruptive stain and simulate the dental bleaching in a non-vital tooth that suffers dental trauma, culminating in blood overflowing into 58 the dentin tubules and progressive staining of the tooth [34]. Thus, we performed the staining of the specimens by blood immersion and the endodontic treatment after 4 weeks in order to reproduce the clinical scenario in a more reliable manner. As described by Teodósio et al. [22], first, we performed the tooth staining and then the endodontic treatment. The staining protocol was performed according to the technique proposed by Freccia et al. [28], and Guimarães et al. [29], which has been used to stain extracted teeth after blood pigmentation. The existence of different bleaching and staining protocols increases the risk of bias [11,12,18,22,35,36]. Because of this, we chosen to perform three sessions using the HP for a maximum of 30 minutes [17], ensuring a more accurate intergroup comparison by the standardization of the bleaching protocols. The HP/BL and HP/VL groups consisted of sessions of 18 minutes, while the duration of the sessions for VL group was 29 minutes and 30 sec. The HP/BL and HP/VL protocols followed manufacturer's recommendations. Regarding the protocol using violet LED alone, we used the same protocol indicated for vital teeth (twenty cycles of 60 sec with a 30-sec pause). Although the bleaching technique with violet light alone can bleach the tooth, the color change is not comparable to that promoted by HP [18]. Considering the dimension of the specimens, it was easily possible to irradiate the enamel and dentin simultaneously, by positioning the LED perpendicular to the surface of the specimen. Moreover, we ensure that the distance between the acrylic tip of the violet LED source and the dental surface not exceed 2.0 cm, otherwise, in this case, a large portion of the delivered light would be lost, reducing the necessary light intensity for the photobleaching, according to the manufacturer’s instructions. The colorimetric analysis was carried out only 7 days after the last bleaching session, preventing misinterpretation of the results due to dehydration of the dental surface, and the presence of residual free radicals in the structure that could change the tooth color [35]. To 59 ensure greater accuracy in color readings, the coordinates of the CIELab system (∆E and ΔL) and Whiteness Index for Dentistry (ΔWID), which represents the evaluation of whiteness in dentistry [33], were used. Our results showed that blue and violet LEDs associated with HP 35%, in high concentration, can achieve the same bleaching efficacy of HP used alone, which is in accordance with a systematic review carried out by Maran. et al. [11], that stated that the bleaching efficacy is not influenced by the use of light. However, it is important to emphasize the possibility of reducing the clinical time without affecting the long-term efficacy of the bleaching when high concentration HP is photoactivated by LED units. In our study ∆Eab, ∆L, and ∆WID showed similar behavior for the groups that used HP with or without light irradiation regardless of the evaluation time (T1, T2 and T3). On the other hand, the violet led used alone did not change the tooth color significantly in all evaluation times (Tables 2, 3, and 4). VL showed no statistically significant differences compared to the control group. Despite the differences within the same group according to the evaluation time, we can observe that there was a pattern for all the values. It is possible to infer that the use of high concentration hydrogen peroxide associated with light or not, directly influences the effectiveness and potential of whitening in non-vital teeth. An interesting outcome we found was to verify that the efficacy and color stability of bleaching using violet LED and HP was similar to the gold standard protocol (35% HP). In a previous study carried out by Ribeiro et al. [37] the authors claim that violet LED photoactivation increased the color change of enamel and dentin when associated with 35% HP, regardless of the irradiation time (15 min, 30 min or 45 min). Galinnari et al. [12] showed that 35% HP used alone on teeth stained with black tea, has a greater whitening effect, regardless of the use of violet light. Teodósio et al. [22], observed that the HP photoactivated 60 or not with a violet LED presents greater color change when compared to the violet LED alone in endodontically treated teeth. It is claimed that the violet LED photoactivation appears to enhance the bleaching efficacy of peroxides [20,36]. However, a systematic review highlighted that the clinical efficacy of violet LED photoactivation are still questionable due to a high heterogeneity of related studies, limited number of randomized clinical trials, lack of standardization of the bleaching protocols, and the quality of in vitro studies [18]. The violet LED irradiation can be a peroxide-free alternative in dental bleaching, but with lower bleaching efficacy than the association with peroxides [38,39]. Therefore, it justifies the need for longer periods of time and even a possible increase in the number of sessions in order to achieve a satisfactory whitening result. In our study, to allow comparison of groups, we standardized the performance of 3 sessions for each protocol. It has been demonstrated that violet light has limited penetration into the dental structure. Thus, it can be inferred that dental bleaching using only violet LED irradiation would be restricted to enamel since the photobleaching is hindered by the poor penetration of the light [40]. Moreover, the light may not achieve the pigmented molecules located in deeper areas close to the enamel/dentin junction. Particularly in non-vital teeth after dental trauma, the dentin tends to be high pigmented due to the blood overflow into the dentin tubules [23,34]. In this context, the use of violet LED irradiation alone may not achieve successful bleaching, and longer exposure times can be used as an attempt to overcome these limitations. To the best of knowledge, the main purpose of this in vitro study was to evaluate novel bleaching protocols using violet LED photoactivation in blood-stained teeth. Despite the promising results of using violet LED associated with high concentration hydrogen peroxide, it must be considered that in vitro studies cannot accurately reproduce the in vivo conditions. 61 Thus, in vivo studies are crucial to evaluate the clinical efficacy of bleaching protocols using violet LED. 5. Conclusion Within the limitations of this in vitro study, it can be concluded that 35% hydrogen peroxide photoactivated by violet LED is effective to bleach blood-stained non-vital teeth, showing a similar color change to hydrogen peroxide used alone or photoactivated by blue LED after a 9-month follow-up. However, the sole use of violet LED irradiation did not promote successful bleaching, being comparable to the group with no treatment. Acknowledgments: The authors would like to thank MMOptics equipments for materials’ donation. References [1] V.H. Panhóca, B.P. de Oliveira, V.S. Bagnato, Dental bleaching efficacy with light application: In vitro study, Photodiagnosis Photodyn. Ther. 12 (2015) 357. https://doi.org/10.1016/J.PDPDT.2015.07.128. [2] B.P. de Oliveira, A.N.S. Rastelli, V.S. Bagnato, V.H. Panhoca, Dental Bleaching Using Violet Light Alone: Clinical Case Report, Dentistry. 7 (2017). https://doi.org/10.4172/2161-1122.1000459. [3] A.N.S. Rastelli, H.B. Dias, E.T. Carrera, A.C.P. de Barros, D.D.L. dos Santos, V.H. Panhóca, V.S. Bagnato, Violet LED with low concentration carbamide peroxide for dental bleaching: A case report, Photodiagnosis Photodyn. Ther. 23 (2018) 270–272. https://doi.org/10.1016/j.pdpdt.2018.06.021. [4] A.D.N. Lago, W.D.R. Ferreira, G.S. Furtado, Dental bleaching with the use of violet 62 light only: Reality or Future?, Photodiagnosis Photodyn. Ther. 17 (2017) 124–126. https://doi.org/10.1016/j.pdpdt.2016.11.014. [5] F. Zanin, Recent Advances in Dental Bleaching with Laser and LEDs., Photomed. Laser Surg. 34 (2016) 135–6. https://doi.org/10.1089/pho.2016.4111. [6] A.D.N. Lago, P.M. de Freitas, E.M. dos S. Araújo, A.B. Matos, N. Garone-Netto, Is It Necessary to Prepare the Enamel before Dental Bleaching?, Int. J. Dent. 2017 (2017) 1–6. https://doi.org/10.1155/2017/5063521. [7] S. Al Shethri, B.A. Matis, M.A. Cochran, R. Zekonis, M. Stropes, A clinical evaluation of two in-office bleaching products., Oper. Dent. 28 (n.d.) 488–95. http://www.ncbi.nlm.nih.gov/pubmed/14531592 (accessed March 19, 2019). [8] V.B. Haywood, History, safety, and effectiveness of current bleaching techniques and applications of the nightguard vital bleaching technique., Quintessence Int. 23 (1992) 471–88. http://www.ncbi.nlm.nih.gov/pubmed/1410249 (accessed March 19, 2019). [9] R.A. Feinman, G. Madray, D. Yarborough, Chemical, optical, and physiologic mechanisms of bleaching products: a review., Pract. Periodontics Aesthet. Dent. 3 (1991) 32–6. http://www.ncbi.nlm.nih.gov/pubmed/1888902 (accessed March 19, 2019). [10] S.R. Kwon, P.W. Wertz, Review of the Mechanism of Tooth Whitening, J. Esthet. Restor. Dent. 27 (2015) 240–257. https://doi.org/10.1111/jerd.12152. [11] B.M. Maran, A. Burey, T.P. Matos, A.D. Loguercio, A. Reis, In-office dental bleaching with light vs. without light: A systematic review and meta-analysis, J. Dent. 70 (2018) 1–13. https://doi.org/10.1016/j.jdent.2017.11.007. [12] M.O. Gallinari, T.C. Fagundes, L.M. da Silva, M.B. de Almeida Souza, A.C.S. Barboza, A.L.F. Briso, A new approach for dental bleaching using violet light with or without the use of whitening gel: Study of bleaching effectiveness, Oper. Dent. 44 63 (2019) 521–529. https://doi.org/10.2341/17-257-L. [13] M.O. Gallinari, L.T.A. Cintra, M.B. de Almeida Souza, A.C.S. Barboza, L.M.B. Esteves, T.C. Fagundes, A.L.F. Briso, Clinical analysis of color change and tooth sensitivity to violet LED during bleaching treatment: A case series with split-mouth design, Photodiagnosis Photodyn. Ther. 27 (2019) 59–65. https://doi.org/10.1016/j.pdpdt.2019.05.016. [14] L.E.S. Soares, D.A. da Rosa, A.A. Martins, V. Cavalli, P.C.S. Liporoni, D.P. da Silva, M. Kury, Effects of experimental bleaching agents on the mineral content of sound and demineralized enamels, J. Appl. Oral Sci. 26 (2018) 1–11. https://doi.org/10.1590/1678-7757-2017-0589. [15] J.L.S.G. Costa, J.F. Besegato, M.C. Kuga, Bleaching and microstructural effects of low concentration hydrogen peroxide photoactivated with LED/laser system on bovine enamel, Photodiagnosis Photodyn. Ther. 35 (2021). https://doi.org/10.1016/j.pdpdt.2021.102352. [16] T.W.S. Daltro, S.A.G. de Almeida, M.F. Dias, P.C. Lins-Filho, C.H.V. da Silva, R.P. Guimarães, The influence of violet LED light on tooth bleaching protocols: In vitro study of bleaching effectiveness, Photodiagnosis Photodyn. Ther. 32 (2020) 4–7. https://doi.org/10.1016/j.pdpdt.2020.102052. [17] C.F.S. Menezes, G.S. Furtado, G. Sarra, M.M. Marques, V.P. Rodrigues, A.D.N. Lago, Violet led dental whitening: Effectiveness and biological safety: An in vitro study, Photodiagnosis Photodyn. Ther. 39 (2022). https://doi.org/10.1016/j.pdpdt.2022.102965. [18] B. Rossi, S. Morimoto, T.K. Tedesco, S.R. Cunha, A.C.R.T. Horliana, K.M. Ramalho, Effectiveness of Violet LED alone or in association with bleaching gel during dental photobleaching: A Systematic Review, Photodiagnosis Photodyn. Ther. 38 (2022) 64 102813. https://doi.org/10.1016/j.pdpdt.2022.102813. [19] B.R. de Souza, A.D.N. Lago, L.S. Ferreira, E. Mayer-Santos, P.M. de Freitas, S. Morimoto, K.M. Ramalho, In-office bleaching protocols using violet LED: A split mouth case report, Photodiagnosis Photodyn. Ther. 36 (2021) 102497. https://doi.org/10.1016/j.pdpdt.2021.102497. [20] J.L.S.G. Costa, J.F. Besegato, J.F. Zaniboni, M.C. Kuga, LED/laser photoactivation enhances the whitening efficacy of low concentration hydrogen peroxide without microstructural enamel changes, Photodiagnosis Photodyn. Ther. 36 (2021) 102511. https://doi.org/10.1016/j.pdpdt.2021.102511. [21] E.N.M. de Almeida, J.F. Bessegato, D.D.L. dos Santos, A.N.S. Rastelli, V.S. Bagnato, Violet LED for non-vital tooth bleaching as a new approach, Photodiagnosis Photodyn. Ther. 28 (2019) 234–237. https://doi.org/10.1016/j.pdpdt.2019.08.024. [22] L.M. Teodosio, L. Gambarini, A.L. Faria-e-Silva, F. de C.P. Pires-de-Souza, A.E. de Souza-Gabriel, J.F. Mazzi-Chaves, M.D. Sousa-Neto, F.C. Lopes-Olhê, Bleaching effect of violet LED of 405–410 nm on stained endodontically treated teeth, Photodiagnosis Photodyn. Ther. 39 (2022) 16–18. https://doi.org/10.1016/j.pdpdt.2022.102929. [23] S.A. Nathoo, The chemistry and mechanisms of extrinsic and intrinsic discoloration., J. Am. Dent. Assoc. 128 Suppl (1997) 6S-10S. http://www.ncbi.nlm.nih.gov/pubmed/9120149 (accessed March 19, 2019). [24] M.Q. Alqahtani, Tooth-bleaching procedures and their controversial effects: A literature review., Saudi Dent. J. 26 (2014) 33–46. https://doi.org/10.1016/j.sdentj.2014.02.002. [25] J.L.S.G. Costa, B.R. Nogueira, O.B.O. Junior, H. Prete