Faculdade de Odontologia de Piracicaba
JOYCE FIGUEIREDO MACEDO DE LIMA
EFEITO DE MATERIAIS BIOMIMÉTICOS NA
REMINERALIZAÇÃO DENTINÁRIA
EFFECT OF BIOMIMETIC MATERIALS ON DENTIN
REMINERALIZATION
Piracicaba 2019
JOYCE FIGUEIREDO MACEDO DE LIMA
EFEITO DE MATERIAIS BIOMIMÉTICOS NA
REMINERALIZAÇÃO DENTINÁRIA
EFFECT OF BIOMIMETIC MATERIALS ON DENTIN
REMINERALIZATION
Dissertação apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para obtenção do título de Mestra em Clínica Odontológica, na Área de Dentística.
Dissertation presented to the Piracicaba Dental School of the University of Campinas in partial fulfilment of the requirements for the Degree of Master in Dental Clinic in Operative Dentistry Area.
Orientador: Prof. Dr. Flavio Henrique Baggio Aguiar Coorientador: Prof. Dr. Klaus Heinz Rischka
ESTE EXEMPLAR CORRESPONDE A VERSÃO FINAL DA DISSERTAÇÃO DEFENDIDA PELA ALUNA JOYCE FIGUEIREDO MACEDO DE LIMA ORIENTADA PELO PROF. DR. FLAVIO HENRIQUE BAGGIO AGUIAR E COORIENTADA PELO PROF. DR. KLAUS HEINZ RISCHKA.
Piracicaba 2019
DEDICATÓRIA
AGRADECIMENTOS
O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) - Código de Financiamento 001 e do Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), processo no 132749/2018-1.
Agradeço ao criador de todas as coisas e Mestre dos mestres, Deus, por dar sentido a minha vida, por ter guiado os meus passos e aberto todas as portas necessárias para que eu concluísse mais esse desafio. Tudo que tenho e sou é Teu!
À Universidade Estadual de Campinas, nas pessoas do Magnífico Reitor Prof. Dr. Marcelo Knobel e Coordenadora Geral da Universidade Profa. Dra. Teresa Dib Zambon. À Direção da Faculdade de Odontologia de Piracicaba, nas pessoas do Diretor Prof. Dr. Francisco Haiter Neto e Diretor Associado Prof. Dr. Flavio Henrique Baggio Aguiar. À Profa. Dra. Karina Gonzales Silvero Ruiz, coordenadora geral dos cursos de Pós-Graduação e ao Prof. Dr. Valentim Adelino Ricardo Barão, coordenador do curso de Pós-Graduação em Clínica Odontológica.
Ao meu orientador Prof. Dr. Flavio Henrique Baggio Aguiar pela paciência, pelas oportunidades, pelo apoio e ensinamentos durante todo o mestrado, por fazer tudo que estava ao seu alcance para tornar a pesquisa possível, e principalmente por sempre acreditar em mim. Seu incentivo foi essencial durante todo o processo. Meu muito obrigada professor! Independente de para onde eu for, o senhor sempre terá o meu respeito e admiração.
Ao meu coorientador Prof. Dr. Klaus Heinz Rischka, pela síntese do peptídeo DOPA-Ahx-(Gly)3-(Glu)5, pelo envio de parte dos materiais e por todos os ensinamentos presenciais e por e-mail. Suas contribuições foram de muita importância para a execução da pesquisa.
À professora Dra. Regina Puppin Rontani por todas as contribuições, ensinamentos e ajuda constante, principalmente ao manter as portas do laboratório de Odontopediatria da FOP-UNICAMP e de sua sala sempre abertas para mim. Admiro muito sua dedicação como profissional e não tenho dúvidas de que a senhora foi a melhor co-orientadora não formal que eu poderia ter!
Ao professor Dr. Ubirajara Pereira Rodrigues Filho e a professora Dra. Priscila Christiane Suzy Liporini por todos os ensinamentos e por permitir a realização das análises de
difração de raios-X na USP-São Carlos e de µ-EDX na UNIVAP, respectivamente.
À professora Dra.Maria do Carmo Aguiar Jordão Mainardi, pela ajuda nos primeiros passos da pesquisa e por ter sempre uma palavra de encorajamento.
Ao técnico do Laboratório de Odontopediatria da FOP-UNICAMP, Marcelo Côrrea Maistro, pelo auxílio no preparo das soluções, e ao técnico do Laboratório de Patologia da FOP – UNICAMP, Adriano Luís Martins, pelo auxílio no uso dos equipamentos de ponto crítico e de microscopia eletrônica de varredura.
Aos Profs. Drs. Lúcia Trazzi Prieto, Carolina Bosso André e Waldemir Francisco Vieira Júnior pelas valiosas contribuições no exame de qualificação.
À professora Dra. Ana Carolina Botta, pela amizade e por todos os conselhos e ensinamentos que ultrapassam a vida acadêmica. Desde o estágio de iniciação científica na SBU-EUA onde tive o prazer de conhecê-la, seu exemplo como profissional despertou em mim o interesse pelo ensino e pela pesquisa, e eu serei sempre grata à Deus por ter cruzado os nossos caminhos!
Ao professor Dr. Boniek Castillo Dutra Borges por ter acredito no meu potencial e ter dado todo incentivo para que eu viesse fazer pós-graduação na FOP-UNICAMP.
A todos os Profs. Drs. que compõe o corpo docente do Departamento de Dentística da FOP-UNICAMP: Giselle Marchi Baron, José Roberto Lovadino, Vanessa Cavalli Gobbo, Marcelo Giannini, Débora Alves Nunes Leite Lima, Luís Alexandre Maffei Sartini Paulillo por toda dedicação e ensino.
Aos queridos colegas de turma Priscila Regis Matos Pedreira, Janaína Emanuela Damasceno dos Santos, Enrico Angelo, Marco Túlio de Oliveira Filho, Maria Del Carmen Choque Yaya, Matheus Kury Rodrigues, Marcela Alvarez Ferreti, Larissa Daniela Orlando e Giovana Masiero Fontanetti, Beatriz Curvello de Mendonça, Jorge Rodrigo Soto Monteiro pela amizade e boa convivência. Em especial, à amiga Danielle Ferreira Sobral de Souza, que desde a primeira vez que nos falamos no dia da seleção, eu senti que era uma pessoa que valia a pena ter por perto! Obrigada por sua sinceridade, por toda ajuda e principalmente por ter compartilhado comigo tanto os momentos bons como os ruins. Sua amizade foi um grande
presente que recebi durante o mestrado e quero mantê-la por toda a vida.
Aos demais colegas de pós-graduação, Mariana Dias Flor Ribeiro, Laura Nobre Ferraz, Bruna Guerra Silva, Daylana Pacheco da Silva, que apesar de não serem da mesma turma de mestrado, também alegraram os meus dias com suas companhias. Em especial, ao amigo Josué Júnior Araújo Pierote palavras me faltam para agradecer por tanto! Obrigada por sempre estar à disposição, pela parceria e por todo conhecimento transmitido. Admiro muito sua versatilidade e competência como profissional, e tenho certeza que seu futuro na Odontologia será brilhante! À amiga Renata Pereira, por me ensinar o valor da persistência na pesquisa e por ser uma das pessoas mais amáveis e gentis que já conheci. Sua presença trouxe leveza aos meus dias e eu só tenho motivos para agradecer por tê-la conhecido. Também gostaria de agradecer ao amigo Rodrigo Barros Esteves Lins, pela ajuda com a análise estatística e por ser sempre tão prestativo.
Aos alunos de Graduação, pela boa convivência e troca de experiências durante e após o estágio docência. Em especial, a aluna Bruna Scarcello Strini pela amizade, e por ter aceitado o desafio de no seu primeiro ano de graduação, ser a minha primeira orientanda de iniciação científica. Sinto que aprendi muito mais do que ensinei; obrigada por me proporcionar essa experiência e por toda sua dedicação. Conte sempre comigo!
A Aliança Bíblia Universitária (ABU-FOP/UNICAMP), especialmente nas pessoas de Mônica Viviane Freire, Ariane Bezerra Santos, Thayná Melo de Lima e Andréa Oliveira pela amizade, por todos os momentos preciosos que compartilhamos juntas e por me aproximarem mais de Deus. Meus dias não na FOP não teriam sido os mesmos sem vocês!
Agradeço também a FAEPEX – UNICAMP pelo incentivo financeiro para compra dos materiais que foram utilizados.
Aos meus pais Samuel Renovato de Lima e Janice Figueiredo Macedo de Lima, pelo amor que ultrapassa as fronteiras geográficas e me alcança com todo apoio emocional, financeiro e espiritual de que preciso. Não tenho palavras para agradecer por tudo o que vocês já fizeram por mim, mas quero honrá-los enquanto viver! Agradeço também ao meu irmão Jonathan Figueiredo Macedo de Lima por todo incentivo, e à minha avó Edna Figueiredo de Macedo (em memória), que por mais que não tenha tido a alegria de me ver conquistar o
título de mestra, sempre me apoiou e teve papel importante na minha formação. Sinto muito a sua falta vovó, mas jamais a esquecerei!
Ao meu melhor amigo e noivo, Kayo Victor Santos Marques, por mais uma vez compreender a minha ausência e ser o meu maior incentivador. Seu amor foi meu refúgio em muitos dias difíceis, e sua firmeza exemplo para eu seguir em frente mesmo diante de tantos obstáculos. Essa vitória é tão minha quanto sua! Aproveito para estender os meus agradecimentos ao meu sogro Kitaçuá Pinheiro Marques (em memória) e a minha sogra Vanicelly de Lourdes Santos Marques por todo amor, apoio e orações constantes.
Aos queridos amigos Ana Camila Batista Medeiros de Assis, Diego Figueiredo Nóbrega, Letícia Fagundes Marinho e Luide Michael Rodrigues França Marinho por terem me acolhido como família e por todo suporte em Piracicaba. Jamais esquecerei o que fizeram por mim e dos bons momentos que tivemos juntos.
Enfim, a todos que, direta ou indiretamente, contribuíram para a realização desse trabalho, muito obrigada!
“Ó profundidade das riquezas, tanto da sabedoria, como da ciência de Deus! Quão insondáveis são os Seus juízos, e quão inescrutáveis os Seus caminhos! Por que quem compreendeu a mente do Senhor? Ou quem foi Seu conselheiro?
Ou quem lhe deu primeiro a Ele, para que lhe seja recompensado? Porque dEle e por Ele, e para Ele, são todas as coisas;
Glória, pois, a Ele eternamente. Amém”.
RESUMO
A remineralização de dentina é de considerável interesse clínico para a Hipersensibilidade Dentinária e o desenvolvimento de análogos biomiméticos capazes de regular a nucleação e o crescimento da hidroxiapatita (HAp) ainda é um desafio. O objetivo deste trabalho foi avaliar in vitro o potencial de remineralização dentinária utilizando os seguintes poli-catecóis: Polidopamina, Poli-DOPA, Poli-Ácido Cafeico e o peptídeo DOPA-RP ((DOPA-Ahx-(Gly)3-(Glu)5)contendo tanto domínio de ligação ao colágeno como ao cálcio que foi sintetizado por química em fase sólida. Amostras de dentina foram obtidas de terceiros molares humanos e condicionadas com ácido fosfórico 37% por 2 minutos para desmineralização. Em seguida, foram divididas aleatoriamente em 4 grupos experimentais (n=10) e imersas em solução tampão fosfato (PBS) (pH 6) recém preparada contendo 1mg/ 10mL do respectivo análogo biomimético e 1mg/ 10mL de Lacase mantida por 12 h em estufa a 37 °C. Após o tratamento biomimético, as amostras foram imersas em 10 ml de solução remineralizadora a 37 °C e a solução foi trocada todos os dias por 10 dias. Amostras de dentina intacta e desmineralizadas foram utilizadas como grupos controle (n = 10) e mantidas em água deionizada nas mesmas condições que os grupos experimentais. A dentina remineralizada foi caracterizada por microscopia eletrônica de varredura (MEV), espectroscopia de raios X por dispersão de energia (μEDX) e difração de raios-X (DRX). A aplicação de diferentes poli-catecóis e do peptídeo DOPA-RP promoveu a nucleação de cristais e a formação de HAp, similar à dentina biológica intacta, tanto na superfície dentinária como nas paredes dos túbulos dentinários. Ao imitar o papel das proteínas não-colagenosas carregadas in vivo, os polímeros consistindo de grupos catecol mostraram a capacidade de modificar as propriedades da superfície da dentina desmineralizada, promovendo a formação de minerais. O uso de poli-catecóis pode ser, portanto, incentivado para o desenvolvimento de uma técnica terapêutica para hipersensibilidade dentinária.
ABSTRACT
Remineralization of dentin is of considerable clinical interest for Dentin Hypersensitivity and the development of biomimetic analogs able to regulate hydroxyapatite (HAp) nucleation and growth is still a challenge. This study aimed to evaluate in vitro the potential for dentin remineralization using the following prepared poly(catechols): poly(dopamine), poly(DOPA), poly(caffeic acid) and the peptide DOPA-RP ((DOPA-Ahx-(Gly)3 -(Glu)5)containing both collagen and calcium-binding domains, which was synthesized by solid-phase chemistry. Dentin samples were prepared from sound human molars and etched with 37% of phosphoric acid for 2 minutes. The samples were randomly divided into 4 experimental groups (n = 10) and immersed in a 1mg/ 10mL freshly prepared solution of the respective biomimetic analog on phosphate buffer solution (PBS) (pH 6) with 1mg/ 10mL of Laccase for 12 h at 37 °C. After the treatment reaction, they were immersed in 10mL of the calcium and phosphate remineralization solution at 37 °C and the solution was changed every day for 10 days. Samples of intact and demineralized dentin were used as control groups (n = 10) and kept in deionized water under the same conditions of the experimental ones. The remineralized dentin was characterized by scanning electron microscopy (SEM), energy dispersion X-ray spectroscopy (μEDX) and X-ray diffraction (DRX). The application of different poly(catechols) and DOPA-peptide promoted crystal nucleation and the formation of HAp similar to biological intact dentin, which covered both the dentin surface and dentinal tubules walls. By mimicking the role of charged non-collagenous proteins in vivo, polymers consisting of catechol groups showed the ability to modify demineralized dentin surface properties, promoting mineral formation. The use of poly(catechols) may be encouraged for the development of a therapeutic technique for dentin hypersensitivity.
Key-words: Biomimetic mineralization, Dentin Collagen, Hydroxyapatite, Dentin Hypersensitivity
SUMÁRIO
1 INTRODUÇÃO...14
2 ARTIGO: Bioinspired Catechol Chemistry for Dentin Remineralization: A new approach for the treatment of Dentin Hypersensitivity………...19
3 CONCLUSÃO...40
REFERÊNCIAS...41
APENDICE 1 – Delineamento experimental e metodologia ilustrada...44
ANEXOS Anexo 1 – Certificado do Comitê de Ética em Pesquisa...55
Anexo 2 – Relatório de verificação de originalidade e prevenção de plágio...56
1 INTRODUÇÃO
A hipersensibilidade dentinária (HD) é definida como uma dor derivada de exposição de dentina em resposta a estímulos químico, térmico, tátil e osmótico, que não pode ser atribuído a nenhuma outra forma de doença (Holland et al., 1997). De acordo com uma revisão sistemática e meta-análise recente, é uma manifestação clínica que atinge grande parte da população mundial (11,5%) e tende a um crescimento contínuo na prática odontológica (Zeola et al. 2019).
Existem muitas teorias que tentam explicar o mecanismo patogênico da dor, mas a mais aceita é a teoria hidrodinâmica de Brännström (Brännström et al., 1963). De acordo com essa teoria, a HD é causada pelo deslocamento rápido do fluído localizado dentro dos túbulos dentinários frente aos diferentes estímulos. Esse movimento provoca deformação das fibras nervosas do tecido pulpar (A-δ, A-β ou C), e como resultado, o paciente experimenta uma dor aguda, de curta duração e bem localizada (Amarassena et al., 2010). De acordo com Boiko et al. (2010), a experiência da dor pode afetar a qualidade de vida, desde as atividades diárias mais simples como comer, beber, falar e escovar os dentes, bem como na capacidade de interação social e emocional de um indivíduo.
Muitos fatores etiológicos e predisponentes têm sido identificados, porém todos eles estão associados com mudanças na estrutura dentária que resultam em exposição dos túbulos dentinários ao meio ambiente oral (Molina et al., 2016). Uma vez expostos, fatores como pobre controle do biofilme, desgaste, erosão cervical, escovação incorreta, refluxo do suco gástrico e exposição excessiva a bebidas ácidas resultam em desmineralização progressiva da dentina exposta (Oliveira et al., 2012) que pode levar ao agravamento da sintomatologia dolorosa. Em virtude de seus hábitos alimentares e estilo de vida, os adultos jovens (18-35 anos) são os mais vulneráveis ao desenvolvimento da HD (West et al., 2013 e Zeola et al., 2019).
Apesar do extenso número de agentes terapêuticos fabricados para uso próprio ou de administração profissional (West et al., 2015), todos eles se baseiam em dois objetivos: impedir a movimentação do fluído no interior dos túbulos dentinários através da obliteração dos mesmos, ou no bloqueio neural dos receptores pulpares, ou em ambos simultaneamente (Oliveira et al., 2012). Contudo, o bloqueio neural dos receptores é um processo reversível;
para induzir a despolarização dos nervos e promover significante alívio da dor, a concentração iônica nas terminações nervosas no interior dos túbulos precisa ser mantida. Uma vez que o uso de produtos a base de potássio é cessado, os íons no local de ação são difundidos e a sensação de alívio é perdida, obrigando o paciente a realizar o tratamento constantemente (Cummins, 2010). A oclusão dos túbulos por sua vez, pode ser obtida através da deposição de pequenas partículas, como o fluoreto de estanho e o cloreto de estrôncio, que formam uma barreira física na abertura dos túbulos dentinários e na superfície de dentina exposta (West et al., 2015). Contudo, o desafio ácido diário afeta negativamente a manutenção dos plugs formados, e também obriga o paciente a realizar o tratamento constantemente para alívio da dor (Trushkowsky et al., 2011). Desta forma, ainda não existe um tratamento eficaz a longo prazo e por isso o tratamento da HD é considerado desafiador (West et al., 2015).
Markowitz e Pasheley (2007) propuseram que os novos tratamentos para hipersensibilidade dentinária precisam tornar a dentina mais resistente tanto ao ataque mecânico como ao químico e isso pode ser obtido através de duas formas. Primeiro, ao aumentar a densidade da superfície de dentina exposta por si só, é possível melhorar a resistência ao desgaste. Segundo, através da formação de plugs com minerais de conteúdo similar ao da dentina, ou seja, que contenham cálcio e fosfato, é possível bloquear a difusão dos ácidos através dos túbulos até a dentina sub-superficial, aumentando assim sua resistência ao ataque ácido. Além disso, eles sugeriram que o tratamento ideal deveria mimetizar o processo dessensibilizante natural que leva a oclusão espontânea dos túbulos dentinários com o tempo; um tratamento ideal iria, portanto, produzir uma dentina não sensível e esclerótica. Os autores concluíram que qualquer tratamento que consiga selar completamente os túbulos dentinários irá restaurar a superfície de dentina exposta a um estado de saúde (Cummins, 2010).
Dentro desse contexto, um método promissor é a indução da formação de minerais através de métodos de remineralização dentinária. Por definição, é um processo que consiste em repor os minerais da dentina desmineralizada. Porém, em virtude de sua estrutura hierarquizada em forma de túbulos e seu alto conteúdo de matéria orgânica (20%), o processo de remineralização da dentina é muito complexo e difícil de ser obtido (Cao et al., 2015). Existem duas vias de cristalização que podem estar envolvidas nesse processo. A primeira, conhecida como teoria clássica, baseia-se em uma abordagem descendente de
remineralização, em que a formação dos cristais se dá através da adição de íons sobre cristais pré-existentes (Zhou et al., 2012). Entretanto, essa via de cristalização não é capaz de remineralizar completamente a dentina (Niu et al., 2014), pois há poucos cristais residuais na dentina desmineralizada, e mesmo quando sua formação consegue ser induzida por meio de métodos de remineralização, os cristais formados são muito grandes para caber entre as fibras colágenas, e portanto, só induzem formação mineral extra-fibrilar (Zhou et al., 2012). A teoria não-clássica de cristalização, por sua vez, baseia-se em uma abordagem ascendente, em que a formação dos cristais depende do uso de precursores amorfos minerais (Zhou et al., 2012). Devido as suas propriedades líquidas, eles conseguem se difundir nas matrizes de colágeno, onde passam por uma transformação de fase até eventualmente formarem cristais intimamente mineralizados às fibras colágenas (Olszta et al., 2003).
Assim como a remineralização extra-fibrilar é importante para obliteração dos túbulos e redução da HD (Zhou et al., 2012), a remineralização intra-fibrilar é também de fundamental importância para a recuperação das propriedades mecânicas da dentina (Cao et al., 2013) e, portanto, para a manutenção da integridade desse tecido. Desta forma, a melhor forma de remineralizar a dentina de modo duradouro, prevenindo os sintomas da HD é através de um método que induza as duas vias de cristalização. Muitos métodos de remineralização da dentina tem sido desenvolvidos, porém nenhum deles foi capaz de remineralizar completamente a dentina (Zhou et al., 2012).
No processo de biomineralização, ou seja, no processo cujos organismos vivos secretam minerais orgânicos a partir dos quais estruturas rígidas como ossos e dentes são formados (Hajir et al., 2014), as proteínas não-colágenas (PNCs) desempenham papéis muito importantes (Cao et al., 2015). Embora representem menos de 10% do conteúdo orgânico de dentina, essas proteínas possuem grande afinidade pelas fibras colágenas e pelos íons cálcio (Cao et al., 2015) e através dos seus domínios de aminoácidos (AA), são responsáveis por regular os processos de nucleação e crescimento dos cristais de hidroxiapatita (HAp) (Tavafoghi et al., 2016). Considerando a importância de suas propriedades, muitos pesquisadores estão tentando encontrar na natureza ou sintetizar análogos capazes de mimetizar o papel das PNCs no processo de remineralização dentinária.
Um desses possíveis análogos é encontrado nas proteínas dos mexilhões. Essas proteínas são ricas em 3,4-diidroxifenilalanina (DOPA), um aminoácido responsável pelas propriedades adesivas que mantêm os mexilhões ligados aos mais variados tipos de superfícies (Liu et al., 2016). Messersmith et al. (2007) foi o primeiro a explorar o potencial químico dessas proteínas. Ele usou a dopamina para mimetizar a composição das proteínas adesivas dos mexilhões, e observou que quando exposta a soluções aquosas, ela sofre uma reação de auto-polimerização (Polidopamina) formando uma fina película aderente. Essa capacidade de adesão foi atribuída aos grupos catecol, presente tanto na DOPA quando na dopamina, que tem demonstrado possuir grande afinidade por ambos materiais orgânicos e inorgânicos (Liu et al. 2016). Além disso, foi demonstrado que a película formada pode funcionar como uma plataforma para reações secundárias (Ye et al., 2011). Diante disso, muitos esforços têm sido feitos em diversas áreas do conhecimento para explorar o potencial de modificação de superfícies inspirado na química dos mexilhões.
Zhou et al. (2012) avaliaram o potencial de remineralização através do uso da Polidopamina sobre a dentina desmineralizada e demonstraram que a película formada promoveu notável remineralização da dentina, na qual todos os túbulos foram ocluídos por cristais de HAp densamente compactados. Apesar do potencial teórico, apenas esse estudo foi encontrado avaliando o potencial de remineralização através do uso de moléculas que possuem grupos catecol sobre a dentina desmineralizada, e por isso decidimos estender o conhecimento e também avaliar a própria DOPA e o Ácido Cafeico.
Considerando ainda que ambas PNCs carregadas positiva e negativamente são necessárias para a precipitação de HAp (Tavafoghi et al. 2016), decidimos sintetizar um peptídeo com base no potencial de carga. Para isso, um peptídeo simples com um domínio de ácido glutâmico para ligação ao cálcio e um espaçador rígido curto foi sintetizado e denominado peptídeo de remineralização (RP). Para a adesão a dentina, um DOPA-aminoácido foi ligado ao peptídeo por um ácido amino-hexanóico de ligação flexível (Ahx)-linker. O peptídeo DOPA-Ahx-(Gly)3-(Glu)5 foi projetado para interagir com o colágeno através de uma ligação entre os grupos catecol (DOPA) e a superfície orgânica (Ye et al., 2011), enquanto os múltiplos ácidos glutâmicos residuais (L-Glu) atraem os íons cálcio (II) para o processo de remineralização da dentina (Tavafoghi et al., 2016). As três glicinas residuais foram
usadas como um outro espaçador na estrutura do peptídeo. Além disso, a combinação de glicina com ácido glutâmico aumenta a afinidade de ligação ao cálcio (II) (Tang et al., 2016).
Desta forma, o objetivo deste trabalho foi avaliar o potencial remineralizador da dentina in vitro através do uso de Polidopamina, Poli-DOPA, Poli-Ácido cafeico e do peptídeo sintético DOPA-RP.
2 ARTIGO: Bioinspired Surface Chemistry for Dentin Remineralization: A new approach for the treatment of Dentin Hypersensitivity
Artigo submetido ao periódico Dental Materials (Anexo 3)
Joyce Lima, Maria do Carmo Aguiar Jordão Mainardi, Regina Maria Puppin Rontani, Ubirajara Rodrigues-Filho, Priscila Christiane Suzy Liporoni, Marcelo Calegaro, Klaus Rischka, Flavio Henrique Baggio Aguiar
ABSTRACT
Objective: Dentin remineralization is of considerable clinical interest for dentin hypersensitivity and developing biomimetic analogs that can regulate hydroxyapatite (HAp) nucleation and growth remains a challenge. This study aimed to evaluate in vitro the potential for dentin remineralization using the following biomimetic in situ prepared poly(catechols): poly(dopamine), poly(DOPA), poly(caffeic acid) and a synthesized DOPA-peptide possessing collagen and calcium-binding domains (DOPA-Ahx-(Gly)3-(Glu)5). Methods: Dentin samples were immersed in a freshly prepared phosphate-buffered saline (PBS) containing the respective catechol and laccase. After the reaction, they were immersed in calcium and phosphate remineralization solution, which was changed every day for 10 days. Samples of intact and demineralized dentin were used as control groups and kept in deionized water under the same experimental conditions. The remineralized dentin was characterized by scanning electron microscopy (SEM), energy dispersion X-ray spectroscopy (μEDX) and X-ray diffraction (DRX). Results: The application of different poly(catechols) and DOPA-peptide promoted crystal nucleation and the formation of HAp similar to biological intact dentin, which covered both the dentin surface and dentinal tubules walls. Significance: By mimicking the role of charged non-collagenous proteins in vivo, polymers consisting of catechol groups showed the ability to modify demineralized dentin surface properties, promoting mineral formation. The use of poly(catechols) may be encouraged for the development of a therapeutic technique for dentin hypersensitivity.
Key-words: Bioinspired Mineralization, Dentin Collagen, Hydroxyapatite, Dentin Hypersensitivity
1. Introduction
Dentin hypersensitivity (DH) is an increasing clinical manifestation that affects a large part of the world’s population (11.5%) [1]. Many etiological and predisposing factors have been identified, but all of these are related to changes in dental structure that result in the exposure of the dentinal tubules to the oral environment [2]. Once exposed, the rapid displacement of the fluid located inside the dentinal tubules over different stimuli causes a deformation of the pulp nerve fibers (type A-δ, A-β or C), resulting in painful symptomatology [3] that can affect quality of life [4].
Despite the extensive number of therapeutic agents manufactured for self or professional administration [5], all are based on two objectives: obliterating the dentinal tubule to prevent movement of fluid, or a neural blockade of the pulp receptors, or both simultaneously [6]. However, the pulp receptors’ neural blockade is reversible, and as the daily acid challenge negatively affects the maintenance of the plugs that obliterate the dentinal tubules, the pain relief is compromised and leads the patient to require constant treatment in both situations [7]. Thus, there is currently no effective long-term treatment, and for that reason, DH is still considered challenging [5]. Markowitz and Pasheley (2007) [8] proposed that new treatments for DH should make dentin more resistant to both mechanical and chemical attack and should mimic the natural desensitizing process, leading to a spontaneous occlusion of the dentinal tubules over time; an ideal treatment would, therefore, produce non-sensitive and sclerotic dentin.
Within this context, a promising method is the induction of mineral formation by dentin remineralization methods. By definition, it is a process that replaces the minerals of demineralized dentin. However, due to its hierarchical structure in the form of tubules and its high organic matter content (20%), the dentin remineralization process is very complex and difficult to obtain [9]. Although they represent less than 10% of the organic content of dentin, studying the structures and functions of noncollagenous proteins (NCPs) becomes a general strategy because these proteins are responsible for regulating the processes of nucleation and growth of HAp crystals through their amino acid domains (AA) on natural biomineralization [10]. Considering their properties, researchers try to synthesize or find in nature analogs capable of mimicking the role of NCPs in dentin remineralization.
Inspired by the chemistry of mussel adhesive proteins, catechol-containing compounds have shown the ability to self-assemble over both organic and inorganic materials, serving as
a template for surface biomodification [11]. Zhou et al. (2012) [12] evaluated the potential of remineralization through the use of poly(dopamine) on demineralized dentin and demonstrated that the coating formed over the dentin promoted remarkable remineralization, in which all the tubules were occluded by densely compacted HAp crystals. Despite the theoretical potential, their study was only found to be evaluating the remineralization potential through the use of catechol containing compounds on demineralized dentin, and for this reason, we decided to extend the knowledge and also evaluate poly(DOPA) (DOPA = 3,4-dihydroxyphenylalanine) and poly(caffeic acid). Moreover, Zhou et al. (2012) [12] kept the dentin samples on the biomimetic solution for 24 h. Although poly(catechols) are able to self-polymerize under oxygen exposure [13], it is necessary to accelerate this reaction for clinical applications. Therefore, we included the Trametes versicolor laccase on the solution due to its ability to catalyze the oxidation reaction of various aromatic compounds [14] and to achieve the highest redox potential among laccases [15].
Considering that both positively and negatively charged NCPs are necessary for HA precipitation [10], we decided to synthesize a peptide based on charge potential. For this purpose, a simple peptide with a domain of glutamic acid for calcium binding and a short rigid spacer was synthesized, herein called the remineralization peptide. For the immobilization on the dentin surface, a DOPA-amino acid was attached to the designed peptide by a flexible aminohexanoic acid (Ahx)-linker. The peptide DOPA-Ahx-(Gly)3-(Glu)5 was designed to interact with the collagen through a bond between the catechol groups (DOPA) and the organic surface [11], while the multiple glutamic acid (L-Glu) residues attract calcium(II) ions for the dentin remineralization process [10] and even di-glutamate possesses a strong binding for calcium(II) [16]. The three glycine residues are used as a further spacer in the peptide. Furthermore, the combination of glycine with glutamic acid increases the calcium(II)-binding affinity [17].
Hence, the aim of this study was to evaluate the dentin remineralization potential in vitro of poly(dopamine), poly(DOPA), poly(caffeic acid) and a DOPA containing remineralization peptide (DOPA-RP). The null hypothesis tested is that the proposed remineralization methods would not be able to produce a remineralization of dentin.
2. Materials and Methods
2.1 Dentin Samples Preparation
This study was reviewed and approved by an Ethics Committee in Human Research under protocol #80645317.9.0000.5418. Thirty extracted sound human third molars were collected, and the soft tissue attached to them was removed. The teeth were stored in a 0.5% thymol solution for at least 1 month prior to the experiment. Each tooth was sliced perpendicular to its longitudinal axis using an IsoMet Low Speed diamond saw (Buehler, Lake Bluff, IL, USA). The dentin surface exposed was polished on 600-, 1200- and 2000-grit silicon carbide papers, respectively, under running water (Buehler EcoMet 5, Lake Bluff, IL, USA) and ultrasonically cleaned for 10 min between each grit (Ultrasound Ultrason 1440 D-Odontobrás Ind. E Com. Med. Odont. Ltda, Rio Preto, SP, Brazil). A second slice, also perpendicular to the longitudinal axis, was made in order to obtain a 1.5 mm dentin slice. Each slice was then sectioned in half using a surgical chisel and hammer. Sixty dentine samples without cracks or hypomineralization were selected for use in this study. They were stored in deionized water in a refrigerator at 4 °C prior to their use.
2.2 Peptide Synthesis
The peptide DOPA-Ahx-(Gly)3-(Glu)5 (DOPA-RP) was obtained by solid phase peptide synthesis (SPPS) on a 433A peptide synthesizer (Applied Biosystems, Foster City, CA, USA). All applied amino acids were Fmoc-protected at the α-amino group. In the case of DOPA, the orthogonal protecting group was acetonide (Merck Bioscience, Darmstadt, Germany) and for glutamic acid tert.-butyl (Iris Biotech GmbH, Marktredwitz, Germany). A glutamic acid preloaded TCP resin was applied with a size of 200-400 mesh (Intavis Bioanalytical Instruments AG, Tübingen, Germany). The synthesis was performed in NMP using the FastMocTM protocol (HBTU (2-(1Hbenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) in a solution of HOBt (1-hydroxybenzotriazole) in DMF (dimethylformamide) in the presence of DIEA (diisopropylethylamine) for coupling and a 20% solution of Piperidine in NMP for the Fmoc removal before coupling (all Iris Biotech GmbH, Marktredwitz, Germany). The peptide was cleaved from the resin by a mixture of 95% TFA (trifluoroacetic acid)/2.5%water/2.5% triisopropylsilane. After the cleavage, the peptide was obtained by precipitation in ice-cold methyl tert.-butyl ether. The precipitated peptide was centrifuged, and the supernatant was
discarded. The remaining peptide was solubilized in UHQ water and freeze-dried in a lyophilisator (Martin Christ Alpha 1-4, Osterode am Harz, Germany). The obtained peptide was characterized by MALDI-ToF MS (Autoflex speed, Bruker Daltonik, Bremen, Germany) using α-CHCA ( -cyano-4-hydroxycinnamic acid) as a matrix.
2.3 Dentin Samples Demineralization
With the exception of the intact dentin group, all dentin samples were acid etched with 37% phosphoric acid for 2 min to promote demineralization and exposition of the collagen fibrils. After being abundantly rinsed with deionized water, they were randomly divided into the following groups: poly(dopamine, poly(DOPA), poly(caffeic acid), DOPA-RP (experimental groups), intact dentin and demineralized dentin (control groups).
2.4 Dentin Samples Biomimetic Treatment
All dentin samples from the experimental groups were immersed in a freshly prepared solution containing 1 mg/mL of the respective biomimetic analog (dopamine, L-DOPA, caffeic acid [all from Sigma-Aldrich, Schnelldorf, Germany] and the synthesized peptide, DOPA-Ahx-(Gly)3-(Glu)5 (DOPA-RP)) in phosphate-buffered saline (PBS) (pH 6) with 1 mg/10mL of laccase (ASA Spezialenzyme GmbH, Wolfenbüttel, Germany) for 12 h at 37 °C. After that, the dentin samples were rinsed with PBS solution (pH 6) to remove the unbound biomimetic analog. Then, the dentine samples were placed at the bottom of sealed falcon tubes filled with 10 mL of the remineralizing solution, which was prepared according to the procedure described by Fan et al. (2009) [18]. The solution contained 2.58 mM calcium (CaCl2·2H2O) (Dinâmica, Indaiatuba, SP, Brazil), 1.55 M phosphate (KH2PO4) (Dinâmica, Indaiatuba, SP, Brazil), 1 mg/L fluoride (NaF) (Thermo Scientific Orion, Waltham, Massachusetts, USA), and 180 mM NaCl (Dinâmica, Indaiatuba, SP, Brazil) and was buffered by 50 mM of tris(hydroxymethyl)-aminomethane (Tris)-hydrochloric acid (Dinâmica, Indaiatuba, SP, Brazil). The remineralizing solution pH was adjusted using 0.1 M HCl and 0.1 M NaOH to 7.6 and was stored at 4 °C prior to use. The remineralizing solution was replaced every day for 10 days.
For the control groups, the dentin samples were stored under the same conditions as the experimental ones but immersed in deionized water.
2.5 Characterization of the remineralization precipitates
The surface morphology of the samples was observed by SEM (JEM 1400, JEOL, Tokyo, Japan) with a beam voltage at 15 kV and a working distance of 10 mm. Four samples were selected from each group. They were fixed with 2.5% glutaraldehyde for 12 h, gradually dehydrated using ethanol (at a gradient concentration of 25%, 50%, 75%, 95% and 100%) until a critical point (Critical Point Drying, CPD 030, Balzers, Liechtenstein) as the final dehydration step, and then sputter-coated with gold (Sputter Coater, SCD 050, Balzers Union Aktiengesellschaft, Liechtenstein).
The quantitative element analysis of Ca, P, and OH as well as the Ca/P ratio was conducted by energy-dispersive X-ray analysis (μEDX-1300, Shimadzu, Japan) at an accelerating voltage of 15 kV and a working distance of 25 mm. Five samples from each group were used, and three spot measurements (spot size 2 nm) were made on every sample. For the statistical analysis, Bartlett’s test and the Shapiro-Wilk test were used to verify the homogeneity of the data. The Kruskal-Wallis and Dunn post hoc tests were used to assess and compare the statistical differences between the groups, respectively (p<0.05).
The mineral phase and crystallinity were carried out by X-ray diffraction (DRX) using a diffractometer (D8 Advance, Bruker, Germany) with Cu K radiation. One sample was selected from each group. The data were collected in the 2θ range of 10–60o at a scan rate of 2o per min. The diffractogram’s software treatment (OriginLab, Northampton, USA) consisted of a subtraction of the baseline and normalization. The Rietveld method was performed in order to measure the size of the crystallites formed.
3. Results
3.1 SEM analysis
Prior to the experiment, we performed a pilot study to evaluate the surface of intact and demineralized dentin after immersion in the remineralizing solution for 10 days. In the group with intact dentin, it was not possible to observe the dentin tubules because the mineral deposited on its surface covered all of them (Fig. 1A and B). The group with demineralized dentin, on the other hand, presented surfaces with completely opened dentinal tubules, with little mineral content deposited on its surface, and with an absence of peritubular and intertubular dentin (Fig. 1C and D). The SEM images show that even after immersion in a
supersaturated calcium and phosphate solution for 10 days, the collagen fibrils, in the absence of the biomimetic analogs, could not induce dentin remineralization.
Regarding the control groups, which were stored in deionized water for 10 days during the experiment, the control group of intact dentin presented surfaces with opened dentinal tubules, with no mineral content deposited on the surface, and with peritubular and intertubular dentin present (Fig. 2A and B). In contrast, the control group that had been acid-etched with 37% phosphoric acid for 2 min presented surfaces with completely opened dentinal tubules, with no mineral content deposited on the surface, and with the presence of a demineralized collagen fiber matrix (Fig. 2C and D).
1A 1B
Figure 1. A and B: Surface of intact dentin after immersion in the remineralizing solution for 10 days. Visualization of completely covered dentinal tubules (1000x and 5000x). C and D: Surface of demineralized dentin after acid-etching with 37% phosphoric acid for 2 min and immersion in the remineralizing solution for 10 days. Visualization of opened dentinal tubules with little mineral deposition on the surface (1000x) and the presence of a demineralized collagen fiber matrix (5000x).
In the experimental groups, it was possible to observe that the different treatments of dentin were responsible for different changes in these surfaces when compared to the control groups. The SEM images show that after immersion in the remineralizing solution for 10 days, each biomimetic analog induced mineral formation and almost all dentin tubules were covered by a Ca and P layer. Spherical agglomerates had precipitated over the surface and were massively interconnected to each other (Figures 3A, 3C, 3E, 3G).
On the transverse sections, it was possible to observe that the treatment with the poly(catechols) also changed the surface properties of the dentinal tubules, allowing homogenous mineral formation (Figs. 4E, 4F, 4G, 4H). On the other hand, in the demineralized dentin group of the pilot study, there was a heterogenous crystal deposition on the lumen of the dentinal tubules that was only observed for this group (Fig. 4B). This is probably due to the mineral layer formed over the surface, which covered the dentinal tubules’ entrance of the intact dentin group after 10 days of immersion on the remineralizing solution (Fig. 4A).
2B 2A
Figure 2. A and B: Surface of intact dentin. Visualization of the opened dentinal tubules (1000x) and the presence of peritubular and intertubular dentin (5000X). C and D: Dentin surface after acid-etching with 37% phosphoric acid for 2 min. Visualization of the opened dentinal tubules (1000x) and the presence of a demineralized collagen fiber matrix (5000x).
Po ly (d op am in e) 3D Po ly (D O PA ) 3E Po ly (c af fe ic a ci d) 3G DO PA R P 3C 3F 3H
Figure 3. A and B: Surface of dentin after treatment with polydopamine. Visualization of the surface partially covered by a Ca and P layer (1000x) and the shrinkage of collagen fibrils (5000x). C and D: Surface of dentin after treatment with poly(DOPA). Visualization of the surface partially covered by a Ca and P layer and the dentin tubules partially occluded (1000x). In the 5000x magnification image, it is also possible to observe the presence of intertubular dentin. E and F: Surface of dentin after treatment with poly(caffeic acid). Visualization of the surface partially covered by a Ca and P layer (1000x) and the shrinkage of collagen fibrils (5000x). G and H: Surface of dentin after treatment with DOPA-RP. Visualization of the surface partially covered by a Ca and P layer (1000x) and the shrinkage of collagen fibrils (5000x). Visualization of the surface partially covered by a Ca and P layer (1000x) and the shrinkage of collagen fibrils (5000x).
Figure 4. A: Transverse section of intact dentin after immersion in the remineralizing solution for 10 days. Visualization of a mineral layer formed on the top of the dentinal tubules (asterisk). B: Transverse section of demineralized dentin after immersion in the remineralizing solution for 10
days. Visualization of the mineral crystal deposited on the surface walls of the dentinal tubules (arrow). C: Transverse section of intact dentin after immersion in deionized water for 10 days. Visualization of the peritubular (a) and intertubular (b) dentin. D: Transverse section of demineralized dentin after immersion in deionized water for 10 days. Visualization of the demineralized collagen matrix (arrow), absence of the peritubular dentin, and the presence of accessory channels. E, F, G and H: Transverse sections of demineralized dentin after treatment with poly(dopamine), poly(DOPA), poly(caffeic acid) and DOPA-RP, respectively, and immersion on the remineralizing solution for 10 days. Visualization of homogenous mineral formation on the walls of the dentinal tubules (arrows).
3.2 Energy-dispersive X-ray (μEDX) analysis
The Bartlett test revealed that there was no homoscedasticity between the groups for Ca (p = 0.0133), P (p = 0.0002), OH (p = 0.0037) and Ca/P (p <0.0001). In addition, data from at least one of the groups did not present normality (Shapiro-Wilk test) for all variables. Thus, the data were submitted to the Kruskal-Wallis and Dunn tests (adjusted for multiple comparisons).
For the Ca and OH content and the Ca/P ratio, no statistically significant differences were observed between groups, except for the group of demineralized dentin, which presented lower values of Ca and higher values of OH and Ca/P compared to the other groups (p<0.05).
For the P content, the group of demineralized dentin presented the lowest statistically significant value, which was different from all other groups (p<0.05). Even though the caffeic acid group presented values that were statistically significantly different when compared to the control ones, the experimental groups did not differ between them (p<0.05), as shown in Table 1.
Table 1. Median of mineral content and Ca/P ratio in wt % (1st and 3rd quartiles) of the controls and experimental groups Ca P OH Ca/P Intact Dentin 27.32 (26.81-28.89) a 11.72 (11.43-12.22) a 60.96 (58.89-61.76) b 2.359 (2.34-2.37) b Demineralized Dentin 21.72 (20.98-23.42) b 8.056 (7.42-9.05) c 70.2 (67.59-71.44) a 2.699 (2.58-2.81) a Poly(dopamine) 26.94 (25.87-27.55) a 11.38 (10.67-12.08) ab 61.72 (60.27-63.47) b 2.379 (2.36-2.42) b Poly(DOPA) 27.09 (26.26-28.48) a 11.2 (10.87-12.15) ab 61.53 (59.19-62.95) b 2.401 (2.34-2.49) b Poly(caffeic acid) 26.6 (24.90-27.24) a 11.11 (10.06-11.68) b 62.26 (61.02-65.04) b 2.391 (2.34-2.47) b DOPA-RP 26.02 (24.40-27.85) a 10.99 (9.77-12.12) ab 63.03 (60.03-66.12) b 2.365 (2.27-2.53) b
3.3 X-Ray Diffraction (XRD) Analysis
In order to access the crystallinity of the remineralization treatments, an XRD of the cross-section of the dentin samples was performed. The demineralization treatment induced an amorphization of the dentin, as can be observed in the increase at the amorphous halo, a region marked with AmH in Fig. 5. All treatments seem to have induced recovery of the crystallinity of the dentin based on the decrease of the amorphous halo and the presence of narrow diffraction hydroxyapatite, marked as HA1 to HA4 [Powder Diffraction Database, file PDF 09-0432] on Fig. 5. HA1 is assigned to the (002) plane, and the HA2 multiplet is assigned to the (211), (112), (300) and (202) planes from left to right. Multiplet HA3 is assigned to the planes (212), (310) and (221), while multiplet HA4 is assigned to (222), (312), (213), (321), (410), (402), (004), (104), (322) and (313). The difference graph between the diffractogram of the demineralized samples and the experimental groups does show a slight shift in peak position, thus indicating the induction of a slight isotropic change in the cell parameters of hydroxyapatite (Supplemental Data). The Rietveld method was used to measure the shape and size of the crystallites produced. The results show that the crystallites formed by all biomimetic analogs were hexagonal in shape, similar to biological apatite. Regarding the size, the crystallites were bigger than biological apatite but smaller than 30 nm (Table 2), which means that they are able to fit in the gaps between the collagen fibrils (67 nm). Overall, the XRD results prove the remineralization and even the formation of larger crystallites.
Figure 5. XRD spectra of the control groups after storage in deionized water for 10 days and of the experimental groups after remineralization for 10 days
Table 2. Structural parameters of dental samples obtained by the refinement of structures using the Rietveld method
Groups Rwp / GOF Type Space Group Network Parameters / Å Size of the crystallites
/nm
a c
Intact Dentin 6.076 / 1.10 Hexagonal P63/m 9.4391 6.8871 10.3 Polydopamine 4.166 / 1.24 Hexagonal P63/m 9.4361 6.8834 21.528
Poly-dopa 5.682 / 1.11 Hexagonal P63/m 9.4472 6.8904 27.542
Poly-caffeic acid 5.147 / 1.05 Hexagonal P63/m 9.4453 6.8906 23.552
DOPA-RP 5.223 / 1.05 Hexagonal P63/m 9.4488 6.8852 20.679
4. Discussion
NCPs with a high affinity for calcium ions and collagen fibrils, such as dentine matrix protein (DMP1) and dentine phosphophoryn (DPP, DMP2), are responsible for regulating the processes of nucleation and growth of HAp crystals in the natural biomineralization of dentin [9]. In this study, we used polydopamine, poly(DOPA), poly(caffeic acid) and DOPA-RP as analogs to mimic the functions of the NCPs on dentin remineralization. Based on the results,
the null hypothesis that the proposed remineralization methods would not be able to produce dentin remineralization was rejected.
According to the XRD analysis, all experimental groups induced mineral formation since there was a decrease of the amorphous halo on the spectra of all groups as well as a presence of narrow diffraction hydroxyapatite peaks at 002, 211, 112, 300 and 200 (Fig. 5). This finding was confirmed by SEM micrographs, in which it is possible to observe the presence of mineral agglomerates over the dentin surface that almost covered it entirely after immersion in the remineralizing solution for 10 days (Figs. 3A, 3C, 3E and 3G). To the best of our knowledge, our study is the first to use DOPA and caffeic acid in an attempt to induce dentin remineralization, beyond the synthetized peptide DOPA-RP. However, all of these have catechol groups (Fig. 6), and it is already known that dopamine [11], DOPA [19] and caffeic acid [20] are susceptible to self-polymerization or enzyme-catalyzed polymerization. This is possible because molecules bearing catechol are easily oxidized and converted into reactive
o-quinones, which self-polymerizes, resulting in an adherent coating on virtually any substrate
[21]. In this study, we used Trametes versicolor laccase to catalyze the oxidation reaction under immersion of the demineralized dentin samples in PBS solution at 37 °C for 12 h in order to simulate the oral cavity conditions. The reason for choosing this particular enzyme is its ability to catalyze the oxidation reaction of various aromatic compounds [14] and to achieve the highest redox potential among laccases [15]. Kurniawati and Nicell (2008) [15] thoroughly characterized Trametes versicolor laccase in terms of its catalytic stability and its effectiveness as a biocatalyst and observed that the oxidation reaction was optimal at pH 6. However, they observed significant transformation over a pH range of 4 to 7 when sufficient laccase was present in the reacting solution. In our study, even though the dentin samples were kept in PBS solution for 12, after only 10 min it was possible to visually observe a reddish-brown color change in the solutions containing dopamine, DOPA and caffeic acid, suggesting poly(catechol) formation.
Our pilot study demonstrates that collagen fibrils do not have the capacity to initiate HAp mineralization, even under supersaturated solutions (Fig. 1), which is in agreement with studies of Tavafoghi et al. (2016) [10] and Gajjeraman et al. (2007) [22]. Therefore, we assume that the surface biomodification of the collagen fibrils was a direct result of the catechol oxidation followed by the polymerization reaction of the catechol-containing molecules used. The binding mechanism of the polymer-like layer to the collagen is not fully understood.
Nevertheless, considering that o-quinone functional groups are capable of covalent coupling to thiol and amine-containing molecules [23], there might be covalent or strong non-covalent interactions taking place between the functional o-quinone groups of the polymer-like layers formed and the exposed collagen.
Furthermore, these layers could act as a chelating layer for calcium, phosphate, fluoride, and hydroxyl ions close to the exposed collagen. Such a polymer-like layer may increase the local activity as well as provide a template for the nucleation of HAp. In this study, we used a remineralizing solution as the source of the ions necessary to promote the remineralization of dentin. However, in future clinical applications, these ions could be easily addressed by the saliva, which is also a supersaturated calcium and phosphate solution and is the source of the natural process that leads to the spontaneous occlusion of the dentinal tubules of people who suffer from DH over time [24].
Regarding the μEDX analysis, the minerals formed using all the proposed biomimetic analogs were identified to have a statistically similar Ca/P ratio to the intact dentin group (p<0.05), and a statistically different Ca/P ratio to the demineralized dentin group (p>0.05). These results confirm the content of mineral agglomerates observed over the dentin surface of all experimental groups and are in agreement with the proposed ability of the biomimetic analogs to induce Ca and P nucleation.
Even though it was not possible to visualize each crystal unit through the SEM analysis used, the Rietveld method demonstrates that all biomimetic analogs were able to induce the formation of crystallites that were hexagonal in shape, similar to biological apatite [25], with a size suitable for filling the interfibrillar spaces (67nm) [26] (Table 2). Beyond changing the surface properties of the demineralized dentin, it is fundamental for the biomimetic analogs to induce the formation of crystals in between the collagen fibrils in order to recover the mechanical properties of dentin [27] and maintain its integrity as a tissue against the challenges of daily habits [28]. Different crystallite sizes among the experimental groups could be related to different chelating abilities, e.g., the stability constants of the ion with the functional group in the polymer-like layer as well as the presence of cavity or channels inside this layer.
Another interesting finding was the difference observed on the SEM transverse sections regarding the dentinal tubules. While on the demineralized dentin group immersed remineralization solution for 10 days of the pilot study showed an heterogenous crystal
deposition on the lumen of the dentinal tubules due to the local ion supersaturation (Fig. 4B), in all experimental groups there was a homogenous mineral formation on the surface of the dentinal walls, resembling the peritubular dentin that was lost during the demineralization process (Figures 4E, 4F, 4G, 4H). Many treatments of HD have been able to occlude the dentinal tubules through a deposition of small particles, such as tin fluoride and strontium chloride, which form a physical barrier preventing the movement of the dentinal fluid [5]. However, the daily acid challenge adversely affects the maintenance of the plugs formed and obliges the patient to constantly repeat the treatment to ensure pain relief [7]. For this reason, the treatment of HD is still considered challenging [5]. Nevertheless, our method has shown the ability of the catechol-containing molecules to induce mineral formation not only on the exposed dentin surface but also on the walls of the dentinal tubules. This mechanism resembles what happens in vivo [24] through the formation of sclerotic dentin, which reduces the dentin permeability for patients suffering from HD, but in a faster way. However, it remains necessary to prove whether the mineral formation observed using the catechol-containing molecules will make dentin more resistant to both mechanical and chemical attack, although since it has the same content of HAp, this is expected.
Recently, Sheng et al. (2015) [29] demonstrated that the functionalization of surfaces using poly(dopamine) thin films could be performed in a one-step photoreaction under “sunlight” irradiation. Compared with other polymerization methods, light-initiated/controlled polymerization methods have specific advantages, which include mild reaction conditions, fast polymerization rates, low toxicity and biocompatibility [30], making all of them interesting for dental clinical applications. This finding expands the possibility to use poly(dopamine), and perchance the other catechol-containing molecules, not only as self-administered agents but also as a professionally applied agent for the treatment of DH.
In the developed world, the changes in nutritional habits and lifestyle have had a large impact on the physiologic remineralization-demineralization process, and the consequence is the increasing rate of dental hypersensitivity and its progressive state, erosion [31]. The challenge in the next decade is to develop smart materials that may counter the demineralization process [25]. According to Cummins (2010) [24], it seems that the best way to treat DH in the long term is mimicking the natural remineralizing process that happens over time for patients who suffer from this pathology. Our methods have shown the ability of catechol-containing molecules to bind to demineralized dentin, modifying its surface
properties and inducing mineral formation. However, more studies are needed to assess the Ca and P layer’s stability against the challenges of daily habits as well as the cytotoxicity of unpolymerized molecules prior to their development into a therapeutic technique.
Figure 6. Chemical structure of Dopamine, L-DOPA, Caffeic Acid and DOPA-RP
5. Conclusion
Molecules containing catechol groups have demonstrated the ability to bound to the demineralized dentin surface, thereby changing its properties and inducing the formation of HAp, similar to biological intact dentin, on both the dentin surface and on the dentinal tubule walls. Although the resistance of this new layer still needs to be validated, we believe that poly(catechols) offer great potential for use in the treatment of dentin hypersensitivity.
6. Acknowledgments
This work was supported by the Coordination for the Improvement of Higher Education Personnel (CAPES) and the National Council for Scientific and Technological Development (CNPq: 132749/2018-1). The authors cordially thank Dr. Arno Cordes from “ASA Spezialenzyme GmbH”, Wolfenbüttel, Germany for providing laccase. The authors declare no conflicts of interest with respect to the authorship and/or publication of this article.
7. References
[1] Zeola LF, Soares PV, Cruz-Cunha J. Prevalence of dentin hypersensitivity: Systematic review and meta-analysis. J Dent 2019;81:1-6.
[2] Molina A, García-Gargallo M, Montero E, Tobías A, Sanz M, Martín C; Clinical efficacy of desensitizing mouthwashes for the control of dentin hypersensitivity and root sensitivity: a systematic review and meta-analysis. Int J Dent Hyg 2016;15:84–94.
[3] Brännström M, ed. A hydrodynamic mechanism in the transmission of painproducing stimuli through dentine. Sensory Mechanisms in Dentine: Proceedings of a Symposium; 1962; London, England: Pergamon Press; 1963.
[4] Boiko O, Baker S, Gibson B, Locker D, Sufi F, Barlow A, Robinson P. Construction and validation of the quality of life measure for dentine hypersensitivity (DHEQ). J Clin Periodontol 2010;37:973–980.
[5] West N, Seong J, Davies M. Management of dentine hypersensitivity: efficacy of professionally and self-administered agents. J Clin Periodontol 2015;42:256–302.
[6] Oliveira J, Oliveira M, Santos A, Vadillo J, Campos C, Chaves M; Hipersensibilidade dentinária: considerações para o sucesso em seu manejo clínico. HU Rev 2012;15:13-20. [7] Trushkowsky R, Oquendo A. Treatment of dentin hypersensitivity. Dent Clin North Am 2011;55:599–608.
[8] Markowitz K, Pashley DH; Discovering new treatments for sensitive teeth: The long path from biology to therapy. J Oral Rehabil 2007;35:300–315.
[9] Cao C, Mei M, Li Q, Lo E, Chu C. Methods for Biomimetic Remineralization of Human Dentine: A Systematic Review. Int J Mol Sci 2015;16:4615–4627.
[10] Tavafoghi M, Cerruti M. The role of amino acids in hydroxyapatite mineralization. J R Soc Interface 2016;13:20160462.
[11] Ye Q, Zhou F, Liu W. Bioinspired catecholic chemistry for surface modification. Chem Soc Rev 2011;40:4244–4258
[12] Zhou YZ, Cao Y, Liu W, Chu CH, Li QL. Polydopamine-Induced Tooth Remineralization. ACS Appl Mater Interfaces 2012;4:6901-6910.
[13] Faure E, Falentin-Daudré C, Jéröme C, Lyskawa J, Fournier D, Woisel P, Detrembleur C. Catechols as versatile platforms in polymer chemistry. Prog Polym Sci 2012;236-270.
[14] Su J, Fu J, Wang Q, Silva C, Cavaco-Paulo A. Laccase: a green catalyst for the biosynthesis of poly-phenols. Crit Rev Biotechnol 2017;1549-7801.
[15] Kurniawatti S, Nicell JA. Characterization of Trametes Versicolor laccase for the transformation of aqueous phenol. Bioresour Technol 2008;99:7825-7834.
[16] Vavrusova M, Skibstedt LH. Calcium binding to dipeptides of aspartate and glutamate in comparison with orthophosphoserine. J Agric Food Chem 2013;61:5380-5384.
[17] Tang N, Skibstedt LH. Calcium Binding to Amino Acids and Small Glycine Peptides in Aqueous Solution: Toward Peptide Design for Better Calcium Bioavailability. J Agric Food Chem 2016;64:4376-4389.
[18] Fan Y, Sun, Z, Moradian-Oldak J. Controlled remineralization of enamel in the presence of amelogenin and fluoride. Biomaterials 2009;30:478−483.
[19] Moulay S. Dopa/Catechol-Tethered Polymers: Bioadhesives and Biomimetic Adhesive Materials. Polym Rev 2014;54:436-513.
[20] Xu P, Uyama H, Whitten JE, Kobayashi S, Kaplan DL. Peroxidase-Catalyzed in Situ Polymerization of Surface Orientated Caffeic Acid. J Am Chem Soc 2005;127:11745-11753. DOI: 10.1021/ja051637r
[21] Duan L, Yuan Q, Xiang H, Yang X, Liu L, Li J. Fabrication and characterization of a novel collagen-catechol hydrogel. J Biomater Appl 2018;32:862-70.
[22] Gajjeraman S, Narayanan K, Hao J, Qin C, George A. Matrix macromolecules in hard tissues control the nucleation and hierarchical assembly of hydroxyapatite. J Biol Chem 2007;282:1193–1204.
[23] Wei H, Ren J, Han B, Xu L, Han L, Jia L. Stability of polydopamine and poly(DOPA) melanin-like films on the surface of polymer membranes under strongly acidic and alkaline conditions. Colloids Surf B Biointerfaces 2013;110:22-28.
[24] Cummins D. Recent advances in dentin hypersensitivity: clinically proven treatments for instant and lasting sensitivity relief. Am J Dent 2010;23:3-13.
[25] Abou NEA, Aljabo A, Strange A, Ibrahim S, Coatchup M, Young AM, Bozec L, Mudera V. Demineralization-remineralization dynamics in teeth and bone. Int J Nanomedicine 2016;11:4743-4763.
[26] Breschi L, Maravic T, Cunha SR, Comba A, Cadenaro M, Tjaderhane L, Pashley DH, Tay FR, Mazzoni A. Dentin bonding systems: From dentin collagen structure to bond preservation and clinical applications. Dent Mater 2018;34:78-96.
[27] Cao Y, Mei M, Xu J, Lo E, Li Q, Chu C. Biomimetic mineralisation of phosphorylated dentine by CPP-ACP. J Dent 2013;41:818–825.
[28] Niu L, Zhang W, Pashley D, Breschi L, Mao J, Chen J, Tay F. Biomimetic remineralization of dentin. Dent Mater 2014;30:77–96.
[29] Sheng W, Li B, Wang X, Dai B, Yu B, Zhou F. Brushing up from “anywhere” under sunlight: a universal surface-initiated polymerization from polydopamine-coated surfaces. Chem Sci 2015;6:2068-073.
[30] Liu M, Zeng G, Wang K, Wan Q, Tao L, Zhang X et al. Recent developments in polydopamine: an emerging soft matter for surface modification and biomedical applications. Nanoscale 2016;8:16819-840.
[31] Gambon DL, Brand HS, Veerman EC. Dental erosion in the 21st century: what is happening to nutritional habits and lifestyle in our society? Br Dent J 2012;213(2):55-57.
8. Supplemental Data * * * * * * * * * * * * * * * * * * * * * * * *
XRD spectra comparing the difference between the control group of demineralized dentin and the intact dentin group, as well as the experimental groups, respectively. HAp peaks marked as asterisks and the amorphous halo with an arrow.
3 CONCLUSÃO
Pôde-se concluir que polímeros consistindo de grupos catecóis demonstraram a capacidade de se ligar à superfície da dentina desmineralizada, alterando suas propriedades e induzindo a formação de HAp tanto na superfície dentinária quanto na parede dos túbulos dentinários. Embora a resistência dessa nova camada formada ainda precise ser validada, acreditamos que os poli-catecóis apresentam grande potencial para o desenvolvimento de uma técnica terapêutica para hipersensibilidade dentinária.
REFERÊNCIAS*
Amarasena N, Spencer J, Ou Y, Brennan D. Dentine hypersensitivity: australian dentists’ perspective. Aust Dent J. 2010; 55:181–187.
Boiko O, Baker S, Gibson B, Locker D, Sufi F, Barlow A, et al. Construction and validation of the quality of life measure for dentine hypersensitivity (DHEQ). J Clin Periodontol. 2010; 37:973–980.
Brännström M, ed. A hydrodinamic mechanism in the transmission of pain-producing stimuli thorugh dentine. In: Sensory Mechanisms in Dentine: Proceedings of a Symposium; 1962; London, England: Pergamon Press; 1963.
Cao C, Mei M, Li Q, Lo E, Chu C. Methods for Biomimetic Remineralization of Human Dentine: A Systematic Review. Int. J. Mol. Sci. 2015; 16:4615–4627.
Cao Y, Mei M, Xu J, Lo E, Li Q, Chu C. Biomimetic mineralisation of phosphorylated dentine by CPP-ACP. J Dent. 2013; 41:818–825.
Cummins D. Recent advances in dentin hypersensitivity: clinically proven treatments for instant and lasting sensitivity relief. Am J Dent. 2010; 23:23:3-13.
Hajir M, Graf R, Tremel Wolfgang. Stable amorphous calcium oxalate: synthesis and potential intermediate in biomineralization. Chem Commun. 2014; 50:6534-536.
Holland G, Narhi M, Addy M, Gangarosa L, Orchardson R. Guidelines for the design and conduct of clinical trials on dentine hypersensitivity. J Clin Periodontol. 1997; 24:808–813 Markowitz K, Pashley DH; Discovering new treatments for sensitive teeth: The long path from biology to therapy. J Oral Rehabil. 2007;35: 300–315.
Lee H, Dellatore SM, Miller WM, Messersmith PB; Mussel-inspired surface chemistry for multifunctional coatings. Science. 2007; 318:426-430.
* De acordo com as normas da UNICAMP/FOP, baseadas na padronização do International Committee of Medical
Liu M, Zeng G, Wang K, Wan Q, Tao L, Zhang X et al. Recent developments in polydopamine: an emerging soft matter for surface modification and biomedical applications. Nanoscale. 2016; 8:16819-40.
Molina A, García-Gargallo M, Montero E, Tobías A, Sanz M, Martín C; Clinical efficacy of desensitizing mouthwashes for the control of dentin hypersensitivity and root sensitivity: a systematic review and meta-analysis. Int J Dent Hyg. 2016; 15: 84–94.
Niu L, Zhang W, Pashley D, Breschi L, Mao J, Chen J, et al. Biomimetic remineralization of dentin. Dent Mater. 2014; 30:77–96.
Oliveira J, Oliveira M, Santos A, Vadillo J, Campos C, Chaves M; Hipersensibilidade dentinária: considerações para o sucesso em seu manejo clínico. HU Rev. 2012; 15:13-20.
Olszta M, Odom D, Douglas E, Gower L. A new paradigma for biomineral formation:
mineralization via an amorphous liquid-phase precursor. Connect Tissue Res. 2003; 44:326– 34.
Povimonsky AG, Rapaport H. Peptide coating applied on the spot improves osseointegration of titanium implants. J Mater Chem B. 2017; 5:2096-2105.
Tang N, Skibstedt LH. Calcium Binding to Amino Acids and Small Glycine Peptides in Aqueous Solution: Toward Peptide Design for Better Calcium Bioavailability. J Agric Food Chem
2016;64:4376-4389.
Tavafoghi M, Cerruti M. The role of amino acids in hydroxyapatite mineralization. J R Soc Interface. 2016; 13:20160462.
Trushkowsky R, Oquendo A. Treatment of dentin hypersensitivity. Dent Clin North Am. 2011; 55:599–608.
West N, Sanz M, Lussi A, Bartlett D, Bouchard Philipe, Bourgeois D. Prevalence of dentine hypersensitivity and study of associated factors: A European population-based cross-sectional study. J Dent. 2013; 41:841-851.
West N, Seong J, Davies M. Management of dentine hypersensitivity: efficacy of professionally and self-administered agents. J Clin Periodontol. 2015; 42:S256–S302.
Ye Q, Zhou F, Liu W. Bioinspired catecholic chemistry for surface modification. Chem Soc Rev. 2011; 40:4244–4258
Zeola LF, Soares PV, Cruz-Cunha J. Prevalence of dentin hypersensitivy: Systematic review and meta-analysis. J Dent. 2019.
Zhou YZ, Cao Y, Liu W, Chu CH, Li QL. Polydopamine-Induced Tooth Remineralization. ACS Appl Mater Interfaces. 2012; 4:6901-6910.
