UNIVERSIDADE ESTADUAL DE CAMPINAS Faculdade de Odontologia de Piracicaba
GABRIELA DE ALENCAR PINTO MAGALHÃES
EFEITO DO PEPTÍDEO DE AUTO-MONTAGEM (P11-4) E DA PROTEÍNA DA
MATRIZ DENTINÁRIA 1(DMP-1) NA RESISTÊNCIA DA UNIÃO À MICROTRAÇÃO DA RESINA COMPOSTA À DENTINA AFETADA POR
CÁRIE.
Piracicaba 2020
GABRIELA DE ALENCAR PINTO MAGALHÃES
EFEITO DO PEPTÍDEO DE AUTO-MONTAGEM (P11-4) E DA PROTEÍNA DA
MATRIZ DENTINÁRIA 1(DMP-1) NA RESISTENCIA DA UNIÃO À
MICROTRAÇÃO DA RESINA COMPOSTA À DENTINA AFETADA POR CÁRIE.
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 Materiais Dentários.
Orientadora: Profa. Dra. Regina Maria Puppin Rontani
Este trabalho corresponde à versão final da dissertação defendida pela aluna Gabriela de Alencar Pinto Magalhães, e orientada pela Profa. Dra. Regina Maria Puppin Rontani.
Piracicaba 2020
Ficha catalográfica
Universidade Estadual de Campinas Biblioteca da Faculdade de Odontologia de Piracicaba
Marilene Girello - CRB 8/6159
Magalhães, Gabriela de Alencar Pinto,
M27e MagEfeito do peptídeo de automontagem (P11-4) e da Proteína da Matriz
Dentinária 1 (DMP-1) na resistência da união à microtração da resina
composta à dentina afetada por cárie / Gabriela de Alencar Pinto Magalhães. – Piracicaba, SP : [s.n.], 2020.
MagOrientador: Regina Maria Puppin Rontani.
MagDissertação (mestrado) – Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba.
Mag1. Dentina. 2. Cárie dentária. 3. Biomimética. I. Puppin-Rontani, Regina Maria, 1959-. II. Universidade Estadual de Campinas. Faculdade de Odontologia de Piracicaba. III. Título.
Informações para Biblioteca Digital
Título em outro idioma: Effect of a self-assembly peptide (P11-4) and Dentin Matrix Protein
1 (DMP-1) on the bond strength of caries affected dentin to composite resin
Palavras-chave em inglês:
Dentin Dental caries Biomimetics
Área de concentração: Materiais Dentários Titulação: Mestra em Materiais Dentários Banca examinadora:
Regina Maria Puppin Rontani [Orientador] Mário Alexandre Coelho Sinhoreti Rafael Pino Vitti
Data de defesa: 27-02-2020
Programa de Pós-Graduação: Materiais Dentários
Identificação e informações acadêmicas do(a) aluno(a)
- ORCID do autor: https://orcid.org/0000-0002-7001-8850 - Currículo Lattes do autor: http://lattes.cnpq.br/8814403569329048
DEDICATÓRIA
Dedico este trabalho aos meus pais Ciro Gomes Magalhães e Mariêta de Alencar Pinto Magalhães e aos meus amados irmãos Antônio Moreira Magalhães Neto, Ana Beatriz de Alencar Pinto Magalhães e Ciro Gomes Magalhães Filho.
AGRADECIMENTOS ESPECIAIS
A Deus porque sempre esteve no controle da minha vida me fazendo chegar a lugares inimagináveis. Agradeço pelo Senhor ter permitido cada dificuldade, pois somente assim consegui ser melhor que antes.
Ao meu pai, pelo apoio emocional que me ajudou a permanecer nessa jornada e a não desistir pois eu sabia que "eu poderia segurar a sua mão para atravessar a avenida". Obrigado pelo apoio financeiro e pelos extras, que me animaram bastante nos dias mais difícies aqui em Piracicaba.
À minha orientadora, Profa. Dra. Regina Maria Puppin Rontani, que é dona de um sorriso encantador, de um olhar forte e brilhante capaz de entusiasmar quem está ao seu redor. Muito obrigada pelo seu otimismo, sua atenção, sua confiança, sua paciência e todos os ensinamentos passados até então, gratidão professora!
AGRADECIMENTOS
À Universidade Estadual de Campinas, por meio do seu Magnífico Reitor, Prof. Dr. Marcelo Knobel, e à Faculdade de Odontologia de Piracicaba (FOP/UNICAMP), nas pessoa do diretor Prof. Dr.Francisco Haiter Neto.
Às amigas que a casa do cavalo me proporcionou Talita Mallini Carletti, Rafaela de Holanda Costa, May Anny Alves Fraga e Carolina Garfias Yamashita. Nada é por acaso, dia após dia somos chamados a ser melhores do que fomos ontem, por isso conviver com vocês me fez crescer, diante das diferenças fomos nos tornando parecidas.
Ao Rafael Rocha Pacheco, pelo companheirismo e carinho nessa reta final. A sua companhia me estimula diariamente na vida acadêmica. Admiro a sua fé em Deus, o seu caráter, a sua inteligência e a sua incrível habilidade de tornar as coisas mais simples e belas.
Agradeço ao nosso grupo de estudo, "Team Puppin-Rontani”, nossas reuniões semanais com direito a bolo e muita discussão científica, muito contribuíram para eu chegar até aqui.
Aos professores da Área de Materiais Dentários:
Ao Prof. Dr. Mário Fernando de Goes, pelas conversas, puxões de orelha, ensinamentos.
Ao Prof. Dr Américo Bortolazzo Correr, pelas suas piadas que fazem rir porque são muito boas. Admiro sua paciência, cordialidade, inteligência e bondade.
Ao Prof. Dr Lourenço Correr Sobrinho, primeiramente, por ter tirado meu carro do prego, mas também, pela simpatia e carisma incomparáveis, proporcionando bons momentos de confraternização, o que une as pessoas do laboratório.
Ao Prof. Dr Simonides Consani por transmitir de forma única o seu conhecimento, nos ensinando a pensar, estimulando a busca profunda pelos porquês, o que refletiu na minha vida pessoal, não me contentando mais o campo da superficialidade. Ao Prof.Dr. Mário Alexandre Coelho Sinhoreti, que para mim é sinônimo de serenidade, obrigada pela paciência e tranquilidade em transmitir conhecimento, obrigada pelo seu olhar fraterno.
Ao Sr. Marcos Cangiani, "Marcão", por estar sempre a disposição e me assustando no laboratório, isso me lembrou que eu deveria viver com mais leveza
À Sra. Selma Aparecida Barbosa Segalla, por estar sempre de prontidão para resolver burocracias, executando com muita seriedade seu ofício
Às professoras do Departamento de Ciências Odontológicas e Odontologia Infantil, Área de Odontopediatria, e ao técnico do laboratório de odontopediatria Sr. Marcelo Correa Maistro, que sempre me ajudou com a preparação soluções, explicando o funcionamento dos equipamentos e calculando as concentrações pacientemente.
Às meninas da limpeza que sempre sorriam para mim nos corredores e mantinham o nosso ambiente de trabalho e estudo limpos.
A todos que contribuíram direta ou indiretamente para a realização deste trabalho.
O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) - Código de Financiamento 001
RESUMO
Nesse estudo foi avaliado o potencial efeito do peptídeo de automontagem (P11-4), e da
associação deste à Proteína da Matriz Dentinária 1 (DMP-1) na resistência da união à microtração (µTBS) de um compósito resinoso à dentina afetada por cárie (DAC). Trata-se de um estudo in vitro, que utilizou 48 terceiros molares humanos hígidos. A porção radicular e oclusal dos terceiros molares foram removidas, formando fatias de dentina (~4mm). Em seguida, foram divididos aleatoriamente em 6 grupos (n=8): C+ (Dentina Hígida, sem tratamento); C- (DAC, sem tratamento); DMP-1 (DAC tratada com 1µg/ml de DMP-1); P11-4
(DAC tratada com 1 µg/ml de P11-4); DMP-1: P11-4(DAC tratada com 1:0,5 µg/ml de DMP-1:
P11-4); 1P11-4 (DAC tratada com 1mg/ml de P11-4). As amostras de dentina dos grupos DAC
foram submetidos ao método biológico de formação de cárie (S.mutans–UA159). Então, os tratamentos foram realizados sobre a dentina seguindo-se os protocolos de cada grupo, brevemente: 50µL de cada agente biomineralizador foi aplicado sobre a superfície da DAC por 5min, removido o excesso com papel absorvente, e 50µL de solução supersaturada em cálcio e fosfato foi aplicada por 1min. Foi realizado o procedimento adesivo utilizando-se o sistema Adper Single Bond 2 e em seguida foram construídos blocos de resina sobre a dentina. Então os conjuntos resina/dentina foram armazenados por 24h a 37°C em solução de Fluido Corporal Simulado (Body Fluid Solution), e sob pressão pulpar simulada. Em seguida os blocos foram seccionados perpendicularmente à área de união e os palitos obtidos (área de secção transversal-1mm2) foram submetidos ao teste de µTBS, (MPa). Os dados foram submetidos aos testes Shapiro–Wilk e Levene e em seguida à ANOVA one-way e teste de Tukey e a diferença entre os grupos experimentais e os grupos controles foi verificada pelo teste de Dunnett, considerando α =0,05. Os valores obtidos para a µTBS para o grupo 1P11-4 (33,1 ±4,7 MPa), seguido do
grupo da associação DMP-1: P11-4(19,9 ±5,8 MPa) foram significativamente maiores que o
C-(12,1 ±7,3 Mpa). Os valores de µTBS para o grupo P11-4 (17,3 ±3,2 MPa) não diferiram dos
obtidos para o grupo DMP-1(12,2 ±3,6 MPa) e do C-. O grupo da dentina hígida apresentou significativamente maior µTBS (57,2 ±12.9 MPa). O padrão de falha foi, predominantemente, do tipo mista para todos os grupos. Em conclusão, o presente estudo verificou que o 1P11
-4[1mg/ml] e a associação entre a DMP-1 e o P11-4[1:0,5 µg/ml] melhora a resistência união
imediata à dentina afetada por cárie.
ABSTRACT
This study aimed to evaluate the effect of the association between Dentin Matrix Protein 1
(DMP-1) and the P11-4, a self-assembly peptide, on the microtensile bond strength (µTBS) of
the Caries-Affected Dentin (CAD). Middle dentin slices (+/-4mm thick) were obtained from 48 healthy human third molars. They were randomly divided into 6 groups (n = 8): C+ (sound
dentin); C: CAD; DMP-1: CAD + DMP-1 (1µg/ml); P11-4: CAD/P11-4 (1 µg/ml); 1P11-4: CAD+
P11-4 (1mg/ml), and DMP-1/P11-4: CAD/ DMP-1(1 µg/ml): P11-4(0.5 µg/ml). Artificial caries
lesions were performed by a biological method (S.mutans - UA159) on dentin surfaces on the CAD groups. After selective caries removal, CAD was reached by selective caries removal and treated following the groups. It was applied 50µL of each biomineralization agent on the DAC to 5 min, dry the excess, and 50µL of supersaturated calcium and phosphate solution was applied to 1 min. Then, bonding procedure using Adper Single Bond 2 was accomplished over the dentin and a composite resin block was built. All the resin/dentin sets were stored in Body Fluid Solution (BFS) for 24h, under simulating pulpal pressure, at 37°C. The beams (1mm2- cross-sectional area), were submitted to the µTBS (MPa). Data was verified by Shapiro-Wilk and Levene` tests. One-way ANOVA and Tukey post hoc tests were used to determine the significant differences between groups and Dunnett's test to determine the difference between the experimental and the control groups, all tests were performed considering α = 0.05. 1P11-4
group and DMP-1 showed, respectively, the highest and the lowest µTBS (33.1 ±4.7 MPa and
12.2 ±3.6 MPa), followed by association DMP-1: P11-4 (19.9 ±5.8 MPa). The groups 1P11-4
and DMP-1: P11- 4 showed significantly higher µTBS than C-(12.1 ±7.3 MPa). There are no
significant differences between P11-4(17.3 ±3.2 MPa) and DMP-1: P11-4 groups. DMP-1 and
P11-4 did not differ from C-. Sound dentin (C+) showed the highest µTBS values (57.2 ±12.9
MPa). Premature failure decreased when used these biomimetics agents and mixed failure mode
was predominantly for all groups. In conclusion 1P11-4 [1mg/ml] and the association of
DMP-1 and P11-4 (DMP-1: P11- 4) improves immediate caries-affected dentin bond strength. Thus,
the effect of P11- 4 on µTBS is concentration-dependent, and the association between P11- 4,
even in the lowest concentration with DMP-1 provides similar results for CAD, improving the adhesion of resin materials and also allowing diminish the tooth damage.
SUMÁRIO
1 INTRODUÇÃO ... 12
2 ARTIGO:"Effect of a self-assembling peptide (P11-4) and Dentin Matrix Protein (DMP-1) on the bond strength of caries-affected dentin to composite resin” ... 16
3 CONCLUSÃO ... 31
REFERÊNCIAS* ... 32
APÊNDICE ... 35
Apêndice 1- Sequência metodológica para remoção da dentina infectada e aplicação dos diferentes tratamentos. ... 35
ANEXOS ... 37
Anexo 1 – Certificado de aprovação do Comitê de Ética em Pesquisa da Faculdade de Odontologia de Piracicaba. ... 37
Anexo 2 – Comprovante de submissão do artigo. ... 38
12
1 INTRODUÇÃO
O manejo das lesões cariosas tem mudado nos últimos anos, migrando de uma abordagem na qual o tecido cariado é completamente removido e substituído por um material restaurador, para uma abordagem voltada à mínima intervenção, como a técnica de remoção seletiva de cárie. Nessa técnica, o tecido cariado é removido seletivamente, sendo a sensibilidade tátil o principal parâmetro de definição para o limite de remoção do tecido, uma vez que este apresenta diferentes zonas, como a dentina infectada e a dentina afetada, que se fundem, o que dificulta a distinção pelo clínico (Schwendike, 2018; Frencken & Innes, 2018). As zonas da lesão cariosa profunda apresentam características particulares quanto aos aspectos morfológicos, bacteriológicos, histológicos e, consequentemente, diferente resposta à capacidade de remineralização (Banerjee et al, 2017; Schwendike, 2018).
Apesar da dificuldade de distinção entre as camadas, a dentina infectada, caracterizada pela consistência amolecida, com alto conteúdo bacteriano, deve ser removida da cavidade, uma vez que representa um tecido necrótico, não passível de remineralização. Por outro lado, a dentina afetada por cárie (DAC) deve ser mantida na cavidade, visto que é passível de remineralização. Isso porque esse tecido apresenta prolongamentos odontoblásticos, fibras colágenas parcialmente degradadas, dada a presença de resíduos cristalinos em sua estrutura, sendo de consistência firme e resistente ao corte (Schwendike, 2018)
A estrutura dentinária é composta por: 50% de minerais, 30% de componentes orgânicos e 20% de água, em volume (Betancourt, Baldion & Castellanos, 2019). O colágeno tipo I representa 90% da matriz orgânica dentinária e 10% de proteínas não-colágenas (NCP), principalmente fosfoproteínas e proteoglicanas (Bedran-Russo, Ravindran & George, 2012). O colágeno tem importância fundamental sobre o módulo de elasticidade, resistência à tensões (tração, flexão, cisalhamento) e propriedades bioquímicas da dentina devido às ligações cruzadas presentes em sua organização hierárquica (dos Santos, Karol & Bedran-Russo, 2011).
Devido ao alto teor de matéria orgânica e água, o procedimento adesivo no substrato dentinário apresenta os seguintes agravantes: degradação hidrolítica da camada adesiva, pela sorção de água na camada híbrida; e, infiltração incompleta dos monômeros resinosos na rede de colágeno desmineralizada, deixando o colágeno exposto e suscetível à ação colagenolítica das metaloproteinases da matriz (MMPs) e cisteínas catepsinas (CTs) (Pashley et al, 2004),o que contribui para a instabilidade na interface adesiva. Na DAC, a adesão é ainda mais desafiadora, pois, além desses agravantes, as propriedades mecânicas da DAC são consideravelmente inferiores às da dentina hígida (Balooch et al, 1998, Costa et al. 2017), o que pode ser refletido na qualidade da adesão e consequentemente na longevidade das
13 restaurações adesivas neste substrato. A adesão em DAC pode se apresentar de 20-50% inferior à da dentina hígida. No entanto, estudos mostram que é possível, após tratamento biomineralizador, estabelecer a interação desse substrato com os materiais adesivos e atingir valores de resistência da união similares ou superiores aos da dentina hígida (Costa et al. 2017, Barbosa-Martins et al 2018a e 2018b, de Sousa et al 2019).
No intuito de diminuir ou impedir uma das principais fontes de degradação da interface de união resina/dentina é amplamente pesquisado o uso de inibidores de MMPs, tais como luz UV, cross-linkers de colágeno naturais e sintéticos, e agentes de reforço na abordagem remineralizadora, como forma de se estabilizar o colágeno (Nair, Gautieri, Chang & Buehler , 2013; Toledano & Osorio, 2015). Para melhorar as propriedades mecânicas da DAC é imprescindível reestabelecer a integridade do colágeno, por meio da mineralização extra e intrafibrilar. No entanto, essas estratégias parecem não ter alcançado a mineralização intrafibrilar, que é mais complexa e pode ser conseguida a partir da biomineralização (Bertassoni,2017).
A abordagem remineralizadora e a biomineralização podem ser diferenciadas por dois mecanismos distintos. A primeira, também conhecida como “teoria da nucleação” ou modelo clássico, é mediada por íons flúor, cálcio e fosfato, e caracterizada pelo crescimento cristalográfico desordenado. A biomineralização ocorre por meio da via de cristalização não-clássica, mediada por análogos biomiméticos, envolvendo a auto-montagem de materiais metaestáveis ou partículas precursoras amorfas, isto é, a transformação de nanopartículas fosfato de cálcio amorfo (ACP) em cristalitos de hidroxiapatita (HAP), reproduzindo o que de fato ocorre na biomineralização. Enquanto que o modelo clássico não atinge a hierarquia estrutural intrafibrilar, portanto, não há deposição de HAP dentro da matriz colágena (Sear, 2012).
No processo de formação da dentina, a mineralização do colágeno é iniciada quando as vesículas da matriz extracelular disponibilizam os minerais para o meio intracelular e são armazenados nas mitocôndrias, até serem liberados para o meio extracelular em formato de ACP, preenchendo toda a rede colágena. As subsequentes transformações de fases do mineral permitem que o mineral amorfo adote uma forma mais cristalina e morfologia estável da HAP, promovendo o que se conhece como mineralização intrafibrilar. Assim, a fase mineral torna-se o primeiro constituinte da dentina (hidroxiapatita carbonatada deficiente em cálcio). Aceita-se que a nucleação de nanocristais de HAP se iniciam nas áreas com espaços “gap zones” das fibrilas de colágeno, progridem via crescimento do cristal, e seguem a fibra colágena longitudinalmente e por entre os espaços intermoleculares separando a tripla-hélice sendo denominados cristais intrafibrilares. Esses cristais também se encontram entre as fibrilas e são
14 chamados cristais extrafibrilares. É importante considerar que o tamanho, espessura e alinhamento dos cristais intrafibrilares são guiados, de forma fisicamente restrita, pelas moléculas de colágeno adjacentes, enquanto o mineral extrafibrilar assume formas aleatórias e tamanhos maiores (Bertassoni, 2017).
Avanços na engenharia tecidual têm sido implementados por meio de peptídeos de automontagem, que promovem a biomodificação na superfície dentária, regenerando o esmalte dental. Não obstante, no tecido dentinário, provavelmente, propicia a mineralização intrafibrilar, aumentando a espessura na fibra colágena e inibindo as MMPs, melhorando as propriedades mecânicas da dentina (de Sousa et al, 2019). Isso é possível, pois, a dentina contém moléculas bioativas, enzimas e fatores de crescimento que são ativados durante o reparo tecidual., ainda que esta seja caracterizada como um tecido acelular. Dessa forma, apesar de desmineralizada, a DAC apresenta fibras colágenas parcialmente degradadas e prolongamentos odontoblásticos, sendo possível a interação com esses agentes biomodificadores (Linde & Goldberg, 1993).
É crescente a ênfase na importância da fase orgânica, que opera em nano escala, contribuindo com as propriedades mecânicas da dentina e do esmalte. Por meio da prática da Odontologia de mínima intervenção com ênfase na engenharia tecidual, acredita-se que ao melhorar a qualidade da DAC pela indução da biomineralização ou do processo "de novo
biomineralization", previamente ao procedimento adesivo, a remineralização tecidual pode ser
favorecida e contribuir para a longevidade das restaurações adesivas, atrasando o ciclo restaurador destrutivo.
A DMP-1 é uma fosfoproteína ácida não colágena expressa pelos osteócitos e osteoblastos durante a maturação dentinária. Essa fosfoproteína é composta pelos aminoácidos: Ser (22%), Glu (15%) e Asp (13%) que, apresentam muitos sítios de fosforilação, tornando-a altamente carregada (-15,8), hidrófilo, com alta afinidade pelos íons Ca2+. Devido a alta carga negativa, a DMP-1 estabiliza o ACP, induzindo a nucleação mineral principalmente nos terminais N e C, correspondentes às gap zones do colágeno e também extrafibrilar, protegendo mecanicamente a fibra colágena de forma indireta (George et al, 1993; Narayanan et al, 2003; Beniash et al, 2011). Dessa forma, a DMP-1 desempenha função regulatória sobre a mineralização da matriz óssea e dentinária. Não obstante, essa fosfoproteína é capaz de organizar os cristais minerais em arranjos paralelos com seu eixo, o que caracteriza uma estrutura mineralizada (Beniash et al, 2011).
O P11-4, disponível comercialmente no Curodont™ Repair (Credentis, Windisch,
Suíça), é um peptídeo sintético, self-assembly, derivado da amelogenina que mimetiza o processo conhecido com “de novo biomineralization”. Inicialmente, esse peptídio foi indicado
15 para o tratamento de lesões de cárie no esmalte (Kirkham et al. 2007, Kind et al 2017). Ainda neste substrato, o P11-4 entra no corpo da lesão, onde o potencial hidrogênio é baixo, passando
do estado líquido para uma consistência viscosa como um gel, devido a formação das β-sheets. Com isso, forma uma matriz 3D, que serve como um arcabouço para a deposição mineral e suporte para o crescimento de cristais, e é capaz de promover a nucleação de hidroxiapatita, devido a alta afinidade por íons Ca2+. Além disso, o peptídeo mostrou boa interação com a fibra de colágeno, uma vez que forma uma rede polimérica em torno da fibra, como uma barreira física, dificultando a difusão das enzimas colagenolíticas, e reduzindo a degradação do colágeno (de Sousa et al, 2018).
Além de saber da importância da regulação da deposição mineral, pela DMP-1, supõe-se que a presupõe-sença de um arcabouço proteico formado pelo P11-4 na dentina desestruturada
poderia auxiliar na mineralização dessa superfície, a partir de íons Ca2+ e PO4 do fluido
dentinário. Esse processo poderia mimetizar a biomineralização decorrente da dentino ou osteogênese.Sabendo ainda que a biomineralização é facilitada na presença do peptídeo, de forma similar à apresentada pela DMP-1, poderia haver uma interação positiva entre a DMP-1 e o P11-4, uma vez que a DMP-1 induz a deposição de matriz extracelular e o P11-4 acelera e
organiza o processo de nucleação e cristalização da hidroxiapatita. Logo, o presente estudo teve como objetivo investigar o efeito do P11-4 e DMP-1 na resistência de união da DAC
16 2 ARTIGO:"Effect of a self-assembling peptide (P11-4) and Dentin Matrix Protein
(DMP-1) on the bond strength of caries-affected dentin to composite resin” Abstract
This study aimed to evaluate the effect of the association between Dentin Matrix Protein 1
(DMP-1) and the P11-4, a self-assembly peptide, on the microtensile bond strength (µTBS) of
the Caries-Affected Dentin (CAD). Middle dentin slices (+/-4mm thick) were obtained from 48 healthy human third molars. They were randomly divided into 6 groups (n = 8): C+ (sound
dentin); C: CAD; DMP-1: CAD + DMP-1 (1µg/ml); P11-4: CAD/P11-4 (1 µg/ml); 1P11-4: CAD+
P11-4 (1mg/ml), and DMP-1/P11-4: CAD/ DMP-1(1 µg/ml): P11-4(0.5 µg/ml). Artificial caries
lesions were performed by a biological method (S.mutans - UA159) on dentin surfaces on the CAD groups. After selective caries removal, CAD was reached by selective caries removal and treated following the groups. It was applied 50µL of each biomineralization agent on the DAC to 5 min, dry the excess, and 50µL of supersaturated calcium and phosphate solution was applied to 1 min. Then, bonding procedure using Adper Single Bond 2 was accomplished over the dentin and a composite resin block was built. All the resin/dentin sets were stored in Body Fluid Solution (BFS) for 24h, under simulating pulpal pressure, at 37°C. The beams (1mm2- cross-sectional area), were submitted to the µTBS (MPa). Data was verified by Shapiro-Wilk and Levene` tests. One-way ANOVA and Tukey post hoc tests were used to determine the significant differences between groups and Dunnett's test to determine the difference between the experimental and the control groups, all tests were performed considering α = 0.05. 1P11-4
group and DMP-1 showed, respectively, the highest and the lowest µTBS (33.1 ±4.7 MPa and
12.2 ±3.6 MPa), followed by association DMP-1: P11-4 (19.9 ±5.8 MPa). The groups 1P11-4
and DMP-1: P11- 4 showed significantly higher µTBS than C-(12.1 ±7.3 MPa). There are no
significant differences between P11-4(17.3 ±3.2 MPa) and DMP-1: P11-4 groups. DMP-1 and
P11-4 did not differ from C-. Sound dentin (C+) showed the highest µTBS values (57.2 ±12.9
MPa). Premature failure decreased when used these biomimetics agents and mixed failure mode
was predominantly for all groups. In conclusion 1P11-4 [1mg/ml] and the association of
DMP-1 and P11-4 (DMP-1: P11- 4) improves immediate caries-affected dentin bond strength. Thus,
the effect of P11- 4 on µTBS is concentration-dependent, and the association between P11- 4,
even in the lowest concentration with DMP-1 provides similar results for CAD, improving the adhesion of resin materials and also allowing diminish the tooth damage.
17 Introduction
The bonding longevity on the dentin surface is challenging due to its high content of the organic phase, and water. Then, the hydrolytic degradation is inherent to the bonding procedure on dentin, as well as water sorption in the hybrid layer, and incomplete infiltration of resin monomers, which leaves collagen fibers exposed, and susceptible to the collagenolytic action of matrix metalloproteinases (MMPs) and cysteines-cathepsin (CTs) enzymes [1,2]. Additionally, it is exacerbated on demineralized dentin [3], inducing a significant instability on the bonding interfaces, due to lower mechanical properties of caries-affected dentin (CAD) than sound dentin [4].
Mineralized dentin structures reveal a mineral accumulation in intra and extrafibrillar compartments of type I collagen, with a substantial amount of the mineral being situated in the extrafibrillar space. These features promote and are responsible for the dentin mechanical properties, such as elastic modulus and hardness [5]. The elastic modulus of sound dentin is about 15-20 MPa, while demineralized dentin is about 0.1-10 MPa [4]. Then, the mineral loss observed on CAD can be replaced by water and the partially degraded collagen being the last one. Despite this, CAD is partially demineralized due to the remained apatite crystals adhered to the collagen fibrils and preservation of odontoblastic extensions, which can remineralize [6]. Rather than its physiopathological aspect, CAD provides a substrate capable of interacting with adhesive materials, demonstrating resin/dentin bonding values reach up only 50% of those of sound dentin [7,8,9].
Concerning the improvement of CAD as a substrate for dentin bonding, some approaches can be found as the use of bioglass, MMPs inhibitors, collagen cross-linkers, and ordinary remineralizing agents, acting on mineral deposition onto collagen fibers [10]. These approaches, however, fail to mimic the dentin biomineralization, concerning CAD bond strength as showed by Barbosa-Martins et al (2018), who found that CAD treated with a 2% NaF provided significantly lower micro tensile bond strength (µ-TBS) than biomineralization approaches using CPP-ACP and P11-4, a self-assembly peptide present in Curodont Repair [7].
The proposed action for the biomineralization mechanism differs from remineralization, once biomineralization cannot only aggregate inorganic components, as the remineralization process does, but it is a complex process, that involves non-collagenous proteins (NCPs), providing hydroxyapatite crystals nucleation and growth. Mineralization initiates when minerals are available from extracellular matrix vesicles to the intracellular medium. Then, they are released as amorphous calcium phosphate (ACP) format to the extracellular medium and are deposited into the collagen network, following transformation phases until getting a more
18 crystalline form and a stable morphology of hydroxyapatite, which represents the interfibrillar mineralization [11].
Moreover, over the past decades, there has been an increasing emphasis on the importance of the organic matrix, which operates at the nanoscale to contribute to the properties of dentin. Briefly, among NCP’s, especially the dentin matrix phosphoprotein 1 (DMP-1), a SIBLING family protein (small integrin-binding ligand N-linked glycoproteins), acts in the mineralization process stabilizing the calcium amorphous phase onto intrafibrillar collagen [12]. Furthermore, due to a high negative charge, DMP-1 acts as a glue, protecting collagen fibers as an indirect physical barrier [13,14].
Recent works have been proved that biomimetic agents like the synthetic self-assembly peptide, P11-4, available from Curodont Repair®, increases the thickness of collagen, protecting
it from enzymatic degradation and improves the µ-TBS on CAD [9]. Considering the needs for
improvement of CAD substrate for a bonding procedure, and knowing the participation of DMP-1 on biomineralization process, and also that DMP-1 cannot act on mature dentin, applying DMP-1 on deep dentin would act onto odontoblast and could activate a new mineralization process. Also, P11-4, a synthetic peptide, and DMP-1, applied before the bonding
procedure would impact positively on bond strength. Therefore, the present study aimed to investigate the effect of a self-assembly peptide P11-4 and DMP-1 on the bonding interface to
artificial CAD produced by biological methods (S.mutans).
The hypotheses tested were: P11-4 improves the CAD bond strength showing values
similar to DMP-1(H1); The association between DMP-1 and P11-4 increases the CAD bond
strength (H2); and, the DMP-1 and P11-4 present similar µ-TBS values than sound dentin (H3).
Materials and Methods
Study design
This study was based on a single randomized model, consisting of forty-eight teeth assigned into 6 groups (n=8) according to the dentin surface treatment: Positive Control: C+; Negative Control: C-; DMP1:caries-affected dentin treated by DMP1 [1µg/ml]; P11-4:
caries-affected dentin treated by P11-4[1µg/ml];DMP-1/P11-4: caries-affected dentin treated by
DMP-1/P11-4[1:0.5µg/ml];1P11-4: caries-affected dentin treated by P11-4[1mg/ml].
Specimen obtaining
Forty-eight sound human third molars were collected after patients informed consent, approved by the Ethics Committee (Protocol number 12319919.0.0000.5418). Teeth were stored in a 0.1% thymol solution at 4o C for no longer than 2 months after extraction. A 4.0 mm
19 coronal dentin slice from each tooth was obtained by sectioning 2.0 mm below cement-enamel junction (CEJ), and 2.0 mm above CEJ, using a slow-speed water-cooled diamond saw (Isomet 1000, Buehler Ltd., Lake Bluff, IL, USA). The dentin surface of each specimen was wet polished with a 600-grit SiC paper (Arotec, São Paulo, Brazil) (30 s) to create a standardized smear layer. Eight dentin slices were used for sound dentin (control group C+), and the others were immediately subjected to the production of caries lesions in vitro.
Groups Assignments
Positive Control - Sound Dentin (C+) - without treatment.
The dentin slices were stored in deionized water for 24h. Then, the bonding procedure was conducted. All dentin slices were etched with 37% phosphoric acid (All Prime Dental Products, SC, Brazil) by 15s and the surfaces were washed with deionized water (1min). The water excess was removed with absorbent paper until the surface getting moist. A single operator applied the adhesive according to the manufacturer’s instruction. Two layers of Adaper Single Bond 2 (3M Dental Products, St. Paul, Brazil) were actively applied with microbrush and soft air jet was applied (10s, 10cm distance) to volatilize the solvents onto the dentin surface. LED curing unit (Bluephase, Ivoclar Vivadent; Schaan, Liechtenstein) was set to the low power mode with an irradiance of 650 mW/cm2 (15 s). A 4 mm height block composite resin was built-up on dentin surface using universal Filtek Z250 XT- A3 (3M ESPE- Sumaré-SP, Brazil), in 2 mm height increments, individually, and light-cured (30 s) using the same LED curing unit, but set by a radiometer (Ophir, Laser Mensurament Group, Israel) to the high power mode with a light intensity of 1200 mW/cm2 [7,8,15].
Negative Control - Caries-affected Dentin (C-) without treatment.
Artificial Caries Formation- Biological Model and Selective Caries Removal
Forty specimens’ surfaces were coated with red acid-resistant nail varnish (Colorama, São Paulo, SP, Brazil). Sequentially, they were fixed with orthodontic wire on the lids of glass vials containing 400 mL of sterile deionized water and were sterilized with gamma radiation (14.5 kGy doses) at constant room temperature (27 °C) [18]. Then, the specimens from caries-affected dentin groups were transferred to another glass vial containing 400 mL of sterile broth brain-heart infusion (BHI) culture medium (LabCenter, São Paulo, Brazil), supplemented with 0.5% yeast extract (LabCenter, São Paulo, Brazil), 1% glucose (LabCenter, São Paulo, Brazil), 2% sucrose (LabCenter, São Paulo, Brazil), and 2% of S. mutans (UA159, FOP-UNICAMP,
Piracicaba, SP, Brazil) incubated at 37oC and supplemented with 10% CO
2, pH 4.0. The optical
20
Inoculation occurred only on the first day of the experiment, but the medium was renewed every 48 h for the 14 days. The broth was Gram stained daily to monitor contamination. The resulting biofilm formed over the dentin slices was removed with sterile gauze with deionized water and the softer dentin layer was removed using a low-speed rotary instrument and tungsten carbide spherical #8 drill (JET; Beavers Dental, Morrisburg, Canada). The threshold for caries removal was used based on the surface dentin hardness like caries-affected dentin, as well as leathery dentin and hard to be cut [16,17]. A single operator was previously trained to perform the selective caries removal.
DMP-1—caries-affected dentin treated by DMP-1 [1µg/ml];
rDMP1— Recombinant Mouse Dentin Matrix Protein 1 (R&D Systems, Minneapolis, MN, USA) was diluted in PBS 1X, until the concentration 1µg/ml. The dentin slices (n=8) were washed with deionized water (10s), water excess was removed with absorbent paper, and 50µL of DMP1 [1µg/ml] was applied onto the surface (5 min). The excess was removed with absorbent paper and 50µL of a supersaturated solution of Ca2+ and PO4 (1.5 mM calcium, 0.9 mM phosphate, 150 mM KCl, 20 mM cacodylic buffer, pH 7.0) was applied for 1min this way ions it is available enough to biomimetics agents action. Solution excess was removed with absorbent paper.
P11-4 — caries-affected dentin treated by P11-4[1µg/ml];
A water-based P11-4 solution [1µg/ml] was made from a stock solution (10 mg/mL)
prepared by dissolving the lyophilized powder in deionized water at 25°C, as recommended by the manufacturer (Credentis, Windisch, Switzerland). The dentin slices (n=8) were washed with deionized water (10s) and water excess was removed with absorbent paper. Then, 50µL of P11
-4 was left onto the surface by 5 min. After that, the excess was removed with absorbent paper and a 50µL of supersaturated solution Ca2+ and PO4 was applied for 1min [18].
1P11-4 — caries-affected dentin treated by P11-4[1mg/ml];
A water-based P11-4 solution [1mg/ml] was made from a stock solution (10 mg/mL)
prepared by dissolving the lyophilized powder in deionized water at 25°C, as recommended by the manufacturer. The dentin slices (n=8) were treated similar to P11-4[1µg/ml] group.
21
DMP-1/P11-4—caries-affected dentin treated by DMP1/P11-4[1:0.5µg/ml];
The proportion of 1DMP-1:0.5 P11-4 [µg/ml] as a result of the solution of 985 µL of PBS 1x
added to 10 µL of DMP1 [1µg/ml] and 5 µL of P11-4 [1µg/ml]. The dentin slices (n=8) were
washed with deionized water (10s) and water excess was removed with absorbent paper. Then, 50µL of DMP-1/P11-4[1:0.5µg/ml] was applied onto the surface (5 min) and the excess was
removed with absorbent paper. After, a 50µL of supersaturated solution Ca2+ and PO4 was
applied for 1min. Finally, the solution excess was removed with absorbent paper and the etching and bonding procedures were performed similarly to the C+ group.
All the slices were submitted to the bonding procedure similarly to described for the C+ group
Pulpal Pressure Simulation
All resin/dentin sets were stored in Body Fluid Solution (BFS) (136.8 mM NaCl; 4.2 mM NaHCO3; 3.0 mM KCl; 1.0 mM K2HPO4.3H2O; 1.5 mM MgCl2.6H2O; 2.5 mM CaCl2; 0.5
mM Na2SO4; 3.08 mM Na3N), at simulated pulp pressure, at 37oC for 24 hours prior to
µ-TBS.
Pulpal pressure was simulated [19] with a BFS which presents a similar ionic composition to human plasma and kept at 37ºC in the stove. All resin/dentin sets from all groups were positioned laterally exposing only the pulpal chamber to facilitate removal without forcing the interface, and they were fixed to the lid of cylindrical containers with dental wax, according to the distribution of the respective groups, for 24 hours. Subsequently, the lid was sealed to the cylindrical container, that was previously filled with BFS at 20 cm height, and the system was turned upside down to induce a simulated pulpal pressure.
Micro-tensile Bond Strength Test (µ-TBS)
After storage in BFS, the specimens were sectioned perpendicularly to the resin/dentin interface
to produce dentin–resin beams with 1 mm2 cross-sectional area, using a low-speed diamond
saw (ISOMET 1000, Buehler Ltd., Lake Buff, IL, USA). A total of thirteen to fifteen beams were obtained per tooth. Each beam was measured previously with a digital caliper (Mitutoyo; Kawasaki, Japan) to determine the cross-sectional area. For µ-TBS measurements, each beam was fixed to a microtensile device with a cyanoacrylate glue (Super Bonder Power Flex-Gel Control, Loctite, Henkel Ltda, Itapevi, São Paulo, Brazil) and tested in universal testing machine (EZ-test; Shimadzu, Kyoto, Japan) with a 500-N load cell (cross-head speed: 1.0
22
mm/min) until failure. The µ-TBS values were calculated and expressed in MPa, according to the formula: R = F (kgF) x 0.098/A, where A = bonding surface area (mm2), F = value of force
obtained at the failure, and R = bond strength value (MPa). The value (MPa) attained from the beams of the same resin-bonded tooth was averaged and the tooth was used as an experimental unit for statistical analysis.
Analysis of Failure Mode
The failure modes of all fractured specimens from the µ-TBS analysis were evaluated under a stereomicroscope at 40x magnification and scored as follows: adhesive, mixed (involving resin composite, adhesive and/or dentin), cohesive in resin composite and cohesive in dentin. Then, 20% of the specimens of each group were examined with a scanning electron microscope (SEM) (JSM-5600LV, JEOL; Tokyo, Japan), with an 80x magnification, operated at 15 kV. The fractured surfaces of the beams were paired, air dried, mounted on aluminum stubs, gold-coated, and examined [8,16,20].
Statistical analysis
The µ-TBS values were submitted to the Shapiro–Wilk test to assess data normality and Levene’s test to show homoscedasticity of variances. one-way ANOVA and Tukey post hoc test, considering 5% as significant level, were used to determine statistically significant differences between different dentin treatments in four levels: DMP-1 [1ug/ml], P11-4 [1µg/ml],
DMP-1/P11-4 [1:0.5µg/ml] and 1P11-4 [1mg/ml] on dentin/resin bond strength. Besides,
Dunnett’s test was used to determine statistically significant differences between experimental groups and the control groups (sound dentin and caries-affected dentin). All statistical analyses were carried out using SPSS software (IBM SPSS Statistics 21).
23 Results
Table 1 shows the average and standard deviations of µ-TBS (MPa) of the experimental groups and control groups (C+ and C-).
Table 1. Average and standard deviation of µ-TBS (MPa) of the experimental groups and control groups (C- and C+)
GROUPS µ-TBS (MPA) C+ (SOUND DENTIN) 57.2 ±12.9* C- (DAC) 12.1 ±7.3& DMP-1 12.2 ±3.6 C& P11-4 17.3 ±3.2 BC& 1P11-4 33.1 ± 4.7 A DMP-1/P11-4 19.9 ±5.8 B
Average followed by * means the significant difference between the positive control group (C+) and experimental group, by Dunnet test (p<0.05). Average followed by & means the significant difference between the negative control group (C-) and experimental group, by Dunnet test (p<0.05). Average followed different uppercase letters means a significant difference between experimental groups by one-way ANOVA and Tukey tests (p<0.05).
Considering C+, sound dentin, although none of the affected dentin treatments reach the µ-TBS of the sound dentin, 1P11-4 group achieved around 60% of the sound dentin µ-TBS
values. However, considering C-, the caries-affected dentin, the µ-TBS values reached the highest value when treated as 1P11-4 group (p=0.000), with a high concentration of P11-4,
followed by DMP-1/P11-4 (p=0.019).
Comparing the experimental groups, the highest values were found for 1P11-4 and
DMP-1/P11-4 (p=0.000 and p=0.000, respectively) groups and the lowest values were found to
DMP-1(p=0.115). Intermediary µ-TBS (MPa) values were found to P11-4 group with no significant
difference from DMP-1/P11-4 (p=0.672) and DMP-1(p=0.115).
The specimen's failure modes are shown in Figure 1. The predominant failure modes of all groups were mixed failure. The mixed failure mode was found in the same proportion for DMP-1/P11-4 (78%), DMP-1 (77%), P11-4 (77%) groups, and it was decreased for 1P11-4 (66%),
C-(65%) and C+ (53%) groups. The lowest percentage of adhesive failure was found for DMP-1/P11-4 (5%), followed by P11-4 (14%) and 1P11-4 (16%), DMP-1(20%), C- (27%) and C+
(33%) groups. Cohesive failure in composite resin was observed in the DMP-1 specimens (2%), C- (4%), P11-4 (6%), C+ (7%), 1P11-4 (7%) and the DMP-1/P11-4 (10%). Cohesive failure in
dentin was observed in the 1P11-4 (12%), C+ (7%), DMP-1/P11-4(7%), C- (4%), P11-4(3%) and
DMP-1 (1%) groups.
It can be observed in Figure 1 that premature failures or debonding previously tested were showed at the highest percentage on C-, on CAD dentin, potentially decreasing after treatment with DMP-1 and P11-4. On the opposite side, the mixed failures increased at the same
24
Figure 1 - Distribution of failure modes and premature failures. C+; C-; DMP-1 (caries-affected dentin treated by DMP-1 [1µg/ml]); P11-4 (caries-affected dentin treated by P11
-4[1µg/ml]); DMP-1/P11-4(caries-affected dentin treated by DMP-1/P11-4[1:0.5µg/ml]) and
1P11-4 (caries-affected dentin treated by P11-4[1mg ml]).
Discussion
In this study, we highlighted the association of amelogenin-derived self-assembly peptide (P11-4), presented in Curodont Repair and Dentin Matrix Protein 1 (DMP-1) on the
improvement of demineralized dentin substrate by bond strength between Caries Affected Dentin (CAD)/composite resin.
This is the first study of DMP-1 and P11-4 association on the bond strength of CAD. Our
first hypothesis that P11-4 improves the CAD bond strength showing similar values of DMP-1
was rejected. It was observed that 1P11-4, exhibited significantly higher µ-TBS (MPa). It can
be explained by the different concentrations between them (1,000x higher). However, P11-4 in
low concentration showed no significant difference between DMP-1 and C- (Table 1). Therefore, in the same concentration, P11-4 and DMP-1 present similar effects on CAD bond
strength.
P11-4 (Ac-QQRFEWEFEQQ-NH2) is a self-assembly peptide that is used as an
injectable scaffold for treating bone defects, dental hypersensitivity and dental decay [21]. The self-assembly property is possible due to β-sheet binding formation, but, in lower concentration it is diminished [22] The β-sheet is characterized by a periodic repetition of hydrophilic and hydrophobic amino acids, which subsequently arrange via intermolecular hydrophobic and electrostatic interactions, resulting in a highly stable 3D matrix of nanofibers scaffolds [23]. The self-assembling peptide scaffold induces ''de novo'' nucleation of hydroxyapatite, which means new nucleation from these peptides, further increases enamel remineralization and on collagen fibers [9] and delay demineralization [24]. When used on demineralized dentin, the bond strength
was higher than the other remineralize agent as CPP-ACP [7]. P11-4, in lower pH, passes from a
25 framework for mineral deposition, by Ca2+ e PO
4 from saliva, and crystal growth support [26].
Also, hydroxyapatite formation appears to be faster and produces larger and better-organized crystals in the presence of P11-4 [9]. We suggest that for the 1P11-4 group, the β-sheet formation
was high due to the 1mg/ml concentration, and it was not jeopardizing the self-assembly property, consequently, the improvement on CAD substrate was better than P11-4 group.
Because of the lower concentration of P11-4 group, [1µg/ml], probably there was a disadvantage
in its self-assembly property, due to a lower β-sheet formation, which was reflected on the lower bond strength values found.
The similarity between P11-4 and DMP-1 on CAD bond strength can be explained by
DMP-1 and P11-4 interaction with type I collagen. DMP-1 presents a common collagen-binding
domain, —DSESSEEDR—, which displays a strong affinity for the N-telopeptide located in the gap zones region of type I collagen [27]. The P11-4 presents 3 glutamic acid residues
(Ace-QQRFEWEFEQQ-NH2) [28], which is a similar acidic amino acid cluster being the major interaction domain with collagen [9]. We hypothesize that probably both agents compete for the same spot on the collagen surface.
As seen in Table 1, the bond strength of DMP-1/P11-4 was significantly higher than C-.
The second hypothesis was that the association between 1µg/ml of DMP-1 and 0.5µg/ml of P11
-4 (this concentration promotes higher mineralization nucleus in odontoblastoids cells- unpublished results) increase the caries-affected dentin bond strengths were accepted. Probably not only the concentration but also the DMP-1 mechanism, has improved caries-affected dentin substrate. DMP-1 is an acidic non-collagen phosphoprotein, which is important to the bone and dentin biomineralization matrix process The biomineralization activity of DMP-1 is due to the phosphorylation sites, which makes them highly negatively charged, assisting in the recruitment of calcium ions and the nucleation and growth of hydroxyapatite crystals [29]. It is crucial to preserve the mechanical properties of dental tissues [30]. However, DMP-1 cannot be produced by mature dentin [31], but when deposited in the dentin matrix can interact with old odontoblasts and type I collagen, presenting mineralization activity [32].
In this way, DMP-1 and P11-4 mineralization ability may have improved CAD bond
strength. As seen in figure 1, it is important to mention that was found a high percentage of premature failure on C- group (59%), probably were adhesive failure, compared with C+(3%). Only when DAC was treated by biomimetics agents the premature failure was diminished, for example, 1P11-4 group showed 16% premature failure, and mixed failure has been increasing.
The sound dentin composition is hydroxyapatite crystals, type I collagen fiber and non-collagen macromolecules carefully hierarchical organized. The presence of mineral content is directly related to the dentin mechanical properties. The collagen fibers play an important role in
26 the mechanical and structural properties of dentin substrate, due to crystal growth, initially at interfibrillar levels, specifically, in gap zones. This growth happens longitudinally, follow the collagen molecular structure, as a guide to crystallographic alignment. The extrafibrillar crystals do not follow a scaffold and may form crystals large and disorganized. When fibers lose minerals, the fiber becomes susceptible to the action of water, hydrolytic degradation, MMPs and CTs enzymatic degradation, in the gap zones [33, 34]. As known, CAD is a hypomineralized and porous surface, leading to a replacement of minerals by water in equal volume, ranging from 14% to 53% compared to the 10% found in sound dentin [35,6,36]. As a consequence, the strength and ductility, nanoindentation hardness and elastic modulus decrease in the demineralized dentin. When using adhesive materials, it is also a challenge to occurs the satisfactory monomeric infiltration, once hydrophobic monomers harmfully penetrate [11], due to the high water content and the low mechanical properties characteristics of this substrate, as mentioned before. This way, the bonding procedure on demineralized dentin could be harmed, getting a lower bond strength, as it was found in this study. CAD group showed the lowest micro tensile bond strength (Table 1).
The third hypothesis that biomineralization using the DMP-1 and P11-4 agents present
similar µ-TBS values than sound dentin, was not proved. This study found that none treatments on CAD achieved sound dentin µ-TBS values. The sound dentin group preparation involved SiC ground, while the other groups, caries was removed with tungsten burn. According to surface preparation, it can observe clear differences in irregularities, roughness and smear layer thickness
[37]. The smear layer created with tungsten burn is more irregular, thick and compact, as a result
of porous and weak mechanical properties Also, demineralized dentin presents less HAP content. It may compromise bonding effectiveness to dentin [38]. On another hand, the smear layer at Sic ground dentin shows more regular, relatively flat and smooth [39].
These results do not corroborate with recent studies with Curodont™ Repair on CAD. Barbosa-Martins et al,2018b and De Sousa et al 2019, used Curodont™ Repair on caries-affected dentin, produced by the biological model, and, achieve a similar bond strength of the sound dentin, in 24h [8,9]. We assume that the difference between our findings and those could be explained due to the different concentrations of P11-4 used. In our study was used 1/10
Barbosa-Martins' concentration and 1/5 De Sousa's concentration. As previously commented, the P11-4
concentration is important to form high β-sheet bands, consequently high mineralization potential of CAD, improving the properties of the dentin substrate. Besides, P11-4 can improve
the effect on the bond strength as shown by this study (Table 1). Even in a lower concentration than the recommended by the manufacturer, the 1P11-4 still gets around 60% µ-TBS of the
27 sound dentin. Nonetheless, our sound dentin bond strength values were similar or higher than found in previous studies [7,8,9]. Moreover, all findings can be based basically on the
concentration impacts on the β-sheet formation, primordially do self-assembly property, and on the DMP-1 phosphorylation sites.
This in vitro study presents limitations as lower concentrations used and a short-term evaluation, besides being a micro-mechanical model. Due to the DMP-1 high cost and knowing that its effect cannot be found on mature dentin, P11-4 can be an alternative for reestablishing
the mechanical properties of CAD substrate. Further, studies are being conducted to evaluate the effect of long-term and using higher concentrations of P11-4 and also for evaluating the
molecular and nanostructural modifications happening in the dentin substrate focusing on the increasing of adhesive dental restorations longevity.
Conclusion
The association between DMP-1 and P11-4 for treatment of affected dentin and promotes
a significant enhancement on µ-TBS as compared with untreated caries-affected dentin. The effect of the P11-4 peptide seems to be depending on its concentration since the higher
concentration the higher µ-TBS values are provided. The treatment with 1mg/ml of P11-4 reached
around 60% of sound dentin µ-TBS. The use of P11-4 for caries-affected dentin treatment before
the restorative procedure shows to be a promising approach for restorative treatment.
Acknowledgments
This research was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES, AUX CAPES-PROEX 0878/2018).
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30 28. Aggeli, A.; Bell, M.; Boden, N.; Carrick, L.M.; Strong, A.E. Self-assembling peptide
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31 3 CONCLUSÃO
A associação entre os agentes biomiméticos, DMP-1 e P11-4, aumentou
significativamente µ-TBS da DAC, em comparação com a dentina afetada por cárie não tratada.
O efeito do P11-4 sobre a DAC, em relação a µ-TBS, quanto maior a concentração, maior
a resistência de união resina/dentina. O tratamento da DAC com 1 mg/ml de P11-4,
conseguiu recuperar em torno de 60% da µ-TBS da dentina hígida.
O uso de P11-4 no tratamento da dentina afetada por cárie, antes do procedimento
30
*De acordo com as normas da UNICAMP/FOP, baseadas na padronização do International Committee of Medica Journal Editors - Vancouver Group. Abreviatura dos periódicos em conformidade com o PubMed.
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35 APÊNDICE
Apêndice 1- Sequência metodológica para remoção da dentina infectada e aplicação dos diferentes tratamentos.
Figura 1- Remoção da dentina infectada por cárie.
(A) Aspecto da dentina cariada artificialmente pelo método biológico. (B) Dentina cariada após remoção do biofilme com gaze e água deionizada. (C) Remoção da camada superficial, similar a dentina infectada por cárie, com broca carbide n°8. (D) Aspecto após remoção da dentina infectada. (E) Limpeza com água deionizada por 10 seg. (F) Remoção do excesso de água com papel absorvente.
A B
C
F E
36 Figura 2- Diferentes tratamentos aplicados nos grupos experimentais.
Figura 3- Tratamento da dentina. (A, B) Aplicação de 50 µl da solução (diferentes tratamentos) sobre a superfície da DAC por 5 min. (C) Remoção do excesso da solução. (D, E) Aplicação de 50 µl da solução supersaturada em cálcio e fosfato por 1 min.
Figura 4- Procedimento de Adesão.(A) Condicionamento ácido (15s). (B, C)Lavagem com água deionizada (60s). (D)Remoção o excesso de água com papel absorvente.(E) Aspecto de dentina úmida, após o condicionamento.(F) Aplicação do adesivo com microbrush, 2 camadas, intercalando leve jato de ar entra elas.(G) Fotoativação do adesivo 15s (650mW/cm2). (H) Construção do bloco de resina composta, técnica incremental. (I) Fotoativação do compósito resinoso por 30s (1200mW/cm2). (J) Amostra do bloco dentina/resina composta, aproximadamente 8mm.
A B C D E
F G H I J
37
ANEXOS
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A self-assembly peptide (P11-4) mimics Dentin Matrix Protein 1 (DMP-1) improving µTBS of caries affected dentin to composite resin. Puppin-Rontani, Regina
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