UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE ODONTOLOGIA DE PIRACICABA
LUIZ FILIPE BARBOSA MARTINS
INFLUÊNCIA DE AGENTES REMINERALIZADORES NA
LONGEVIDADE DA UNIÃO RESINA/DENTINA
INFLUENCE OF REMINERALIZING AGENTS ON THE
DENTIN/RESIN BOND DURABILITY
PIRACICABA 2018
LUIZ FILIPE BARBOSA MARTINS
INFLUÊNCIA DE AGENTES REMINERALIZADORES NA LONGEVIDADE DA UNIÃO RESINA/DENTINA
INFLUENCE OF REMINERALIZING AGENTS IN THE DENTIN/RESIN BOND DURABILITY
Tese apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para obtenção do título de Doutor em Odontologia, área de concentração em Odontopediatria.
Thesis presented to the Piracicaba Dental School of the University of Campinas in partial fulfillment of the requirements for the degree of Doctor in Dentistry, in Pediatric Dentistry area.
Orientador: Profa. Dra. Regina Maria Puppin Rontani ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA PELO ALUNO
LUIZ FILIPE BARBOSA MARTINS E
ORIENTADA PELA PROFA. DRA. REGINA MARIA PUPPIN RONTANI.
PIRACICABA 2018
DEDICATÓRIA
Este trabalho é dedicado aos meus pais, Joaquim
Viana Martins e Lúcia Maria Barbosa Martins.
Por serem meus maiores mestres e sempre me incentivarem a lutar pelos meus sonhos.
AGRADECIMENTO ESPECIAL
“Cada coisa tem sua hora e cada hora o seu cuidado”. Rachel de Queiroz
À minha orientadora, Profa. Dra. Regina Maria Puppin Rontani, pelo exemplo de pessoa e pesquisadora, pelo carinho e pela dedicação extraordinária em todas as fases do trabalho. Agradeço por ter acreditado que no final tudo daria certo. Obrigado pelo incentivo constante e por contribuir com sua grande experiência na área da odontopediatria para o meu crescimento profissional.
AGRADECIMENTOS
A Deus, pelas bênçãos concedidas, por todas experiencias e por nunca ter me abandonado. À Universidade Estadual de Campinas – UNICAMP, na pessoa de seu Magnífico Reitor
Prof. Dr. Marcelo Knobel.
À Faculdade de Odontologia de Piracicaba - FOP, na pessoa de seu diretor, Prof. Dr.
Guilherme Elias Pessanha Henriques e diretor associado Prof. Dr. Francisco Haiter Neto.
A Coordenadora dos Programas de Pós-Graduação da Faculdade de Odontologia de Piracicaba – UNICAMP, Prof. Dra. Cinthia Machado Tabchoury.
À coordenação do Programa de Pós-Graduação em Odontologia (PPGO) da FOP-UNICAMP, por meio do Coordenador Prof. Dr. Marcelo C. Meneghin.
Ao PPGO da FOP-UNICAMP e a todos os professores envolvidos, pela generosidade, bem como pelo estímulo, apoio e ensinamentos.
Aos funcionários da FOP-UNICAMP sempre dispostos a ajudar e solucionar os problemas surgidos.
Às maravilindas da 5ª turma de doutorado em Odontologia-Odontopediatria da FOP-UNICAMP, Darlle dos Santos Araújo, Lenita Marongoni Lopes, Micaela Cardoso e
Jossaria Pereira de Sousa, pela convivência harmoniosa e troca de experiências, bem como
por todos os momentos em que nos apoiamos mutuamente e pelo apoio e compreensão durante esse tempo todo.
À maravilhosa, Jossaria Pereira de Sousa, os desafios e as dificuldades de cada momento contribuíram para que nos tornássemos amigos e companheiros até o final. Agradeço pela amizade e companheirismo fortalecios durante a saudável convivência.
À gata garota, Lívia Alves Araújo, por toda a ajuda, companhia e carinho para comigo em vários momentos durante a minha vida.
Aos colegas de pós-graduação da FOP-UNICAMP, pela ajuda e atenção nos momentos em que precisei, nas minhas várias temporadas em Piracicaba – SP. Meu agradecimento.
À minha banca avaliadora da qualificação Profa. Dra. Fernanda Pascon, Profa. Dra. Kamila
Kantovitz e Prof. Dr. Américo Correr, pelas excelentes contribuições.
A todos os meus familiares, em especial a Vovó Fransquinha (in memoriam), aos meus tios, primos e primas, presentes em todos os momentos da minha vida, que, apesar da distância física, estão sempre ao meu lado.
Aos meus irmãos Igor Martins, Marêssa Martins e Alyne Martins por todo amor e carinho. Aos meus padrinhos Luciana Martins e Marcelo Aragão obrigado por todo apoio e carinho durante esse tempo.
À Coordenação do Curso de Odontologia do Centro Universitário Católica de Quixadá -
UNICATÓLICA, em especial aos professores Mardonio Rodrigues e Diego Lima.
Aos amigos professores Prof. Me. Helder Ferreira e Profa. Me Sofia Vasconcelos, por todo apoio e compreensão.
Ao amigo Juscelino Freitas, obrigado por todo o apoio e carinho duante todo esse tempo. Às amigas Thayla Hellen e Rafaela Costa, alunas de pós-graduações da Faculdade de Odontologia de Piracicaba, por sempre estarem na torcida, amizade, companheirismo e por sempre acreditarem que nosso sonho se tornaria realidade.
Aos meus amigos, que aproveito para me desculpar pela ausência em muitos momentos importantes na vida de vocês, quando não foi possível estar presente para me dedicar a esta tese. Sei que me compreenderam, pela importância da causa.
À Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), pelo apoio financeiro para esta pesquisa.
À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), pela concessão de bolsa em determinado período deste Doutorado.
E a todos que, direta ou indiretamente, contribuíram para a realização deste trabalho, o meu sincero agradecimento.
“É melhor escrever errado a coisa certa do que escrever certo a coisa errada...”
RESUMO
Os objetivos deste trabalho foram avaliar diferentes métodos de produção de cárie (químico-DDC e biológico-DDB) sobre a resistência da união (μTBS) imediata da dentina desmineralizada e tratada por agentes remineralizadores (Fluoreto de Sódio (NaF), MI Paste™ (MP) e Curodont™ Repair (CR), molhabilidade simulando os procedimentos adesivos, μTBS, nanoinfiltração (NL) e quantificação mineral (EDS) nos tempos de armazenamento (24h, 6 meses e 18 meses). 268 terceiros molares hígidos foram aleatorizados em: 1º estudo (N=63) e 2º estudo (N=205) que foram seccionados o terço oclusal para expor a superfície dentinária. 1º estudo: blocos de dentina foram distribuídos para μTBS (n=6) e microscopia por Luz Polarizada (PLM) (n=3), de acordo com controle: G1-Dentina Hígida-DH; e grupos experimentais: G2-DDC; G3-DDC/NaF; G4-DDC/MP; G5-DDC/CR; G6-DDB; G7-DDB/NaF; G8-DDB/MP; G9-DDB/CR. Para PLM duas secções de 0,15 μm de cada dente foram utilizadas dos grupos G1, G2 e G6. Foi realizado o teste de μTBS a 1 mm/min/500N. Os dados de μTBS foram submetidos à ANOVA fatorial/Tukey (α=5%). Os dados dos sítios de fratura foram submetidos ao teste Kruskal-Wallis (α=5%). Os maiores valores de μTBS foram encontrados com a utilização de MP e CR, independentemente do método de produção de cárie (p<0,05). O padrão de fratura mista e adesiva foi o mais encontrado, não apresentando diferença significativa entre os métodos de produção de cárie (p=0,9967). 2º estudo: blocos de dentina foram divididos em cinco grupos (n=8) para análise de molhabilidade, em 15 grupos para μTBS (n=8), NL/EDS (n=8), de acordo com os grupos controles e experimentais/tempos de armazenamento. Para análise de molhabilidade, a dentina amolecida foi removida por polimento e tratada como descrito acima, e avaliada usando o método da gota séssil. μTBS foram testados a 1 mm/min/500N. O padrão e fratura foi avaliado de forma descritiva em MEV. Para análise de NL a deposição de prata foi avaliada por MEV/EDS. As mesmas amostras de NL foram utilizadas para avaliar EDS quanto ao teor de minerais e a relação Ca/P. Os dados de molhabilidade foram submetidos ao teste ANOVA one-way/teste t, os dados de μTBS, NL e EDS foram submetidos ao teste ANOVA fatorial/teste t/Tukey (α=5%). DD e NaF apresentaram menor molhabilidade (p<0,01), enquanto MP apresentou o maior molhabilidade (p<0,01). CR exibiu valores similares a DH (p>0,05). Houve interação significativa entre o os fatores em estudo considerando μTBS e NL (p<0,001). MP e CR apresentou os maiores valores de μTBS, independentemente do tempo de armazenamento (p<0,001). MP não apresentaram diferença entre 24h e 6 m de tempo de armazenamento em NL (p>0,05). Todos os outros grupos mostraram aumento de Ag na camada híbrida após 6 m de armazenamento (p<0,001). Houve
um aumento do teor dos minerais e da relação Ca/P para os grupos de MP e CR (p<0,001). Conclui-se que o uso dos modelos de produção de cárie artificial produz diferenças na μTBS considerando o material utilizado. A abordagem de remineralização favoreceu as propriedades físicas, a μTBS, NL e propriedades químicas, reduzindo a degradação a longo prazo, restaurando condições semelhantes ou superiores às encontradas para a DH.
ABSTRACT
The aims of this work was to evaluate different methods of caries (chemical-DDC and biological-DDB) on immediate bond strength (μTBS) of the demineralized dentin and treated with remineralizing agents (Sodium Fluoride (NaF), MI Paste™ (MP) and Curodont™ Repair (CR), wettability simulating adhesive procedures, μTBS, nanoinfiltration (NL) and mineral quantification (EDS) at storage times (24h, 6-month and 18-month). 268 sound third molars were randomized in: 1st study (N=63) and 2nd study (N=205) that the occlusal third was sectioned to expose the dentin surface. 1st study: dentin blocks were distributed to μTBS (n=6) and Polarized Light Microscopy (PLM) (n=3), according to control: G1-Sound Dentin-SD; and experimental groups: G2-DDC; G3-DDC/NaF; G4-DDC/MP; G5-DDC/CR; G6-DDB; G7-DDB/NaF; G8-DDB/MP; G9-DDB/CR. For PLM, two sections of each tooth (0.15 μm) were used from groups G1, G2 and G6. The μTBS test was performed at 1 mm/min/500N. The μTBS data were submitted to factorial/Tukey ANOVA (α=5%). The data of the fracture pattern were submitted to the Kruskal-Wallis test (α=5%). The highest values of μTBS were found with MP and CR, independently of the caries production method (p<0.05). The mixed and adhesive fracture pattern was the most commonly found, with no significant difference between caries production methods (p=0.99). 2nd study: dentin blocks were divided into five groups (n=8) for analysis of wettability, in 15 groups for μTBS (n=8), NL/EDS (n=8), according to control and experimental groups/storage times. For wettability analysis, the softened dentin was removed by polishing and treated as described above, and evaluated using the sessile drop method. μTBS were tested at 1 mm/min/500N. The fracture pattern was evaluated descriptively in SEM. For analysis of NL the deposition of silver was evaluated by MEV/EDS. The same NL samples were used to evaluate EDS for mineral content and Ca/P ratio. The wettability data were submitted to the one-way ANOVA/t test, the μTBS, NL and EDS data were submitted to the ANOVA factorial/t and Tukey test (α=5%). DD and NaF presented lower wettability (p<0.01), while MP presented the highest wettability (p<0.01). CR showed values like DH (p>0.05). There was a significant interaction between the factors under study considering μTBS and NL (p<0.001). MP and CR presented the highest values of μTBS, regardless of storage time (p<0.001). MP presented no difference between 24h and 6m of storage time in NL (p>0.05). All other groups showed increase of Ag in the hybrid layer after 6 m of storage (p<0.001). There was an increase in the mineral content and the Ca/P ratio for the MP and CR groups (p<0.001). It is concluded that the use of artificial caries production models produces differences in μTBS considering the material used. The remineralization approach favored the physical properties,
μTBS, NL and chemical properties, reducing long-term degradation, restoring conditions similar or superior to those found for SD.
SUMÁRIO
1 INTRODUÇÃO ... 15
2 ARTIGOS………...19
2.1 Artigo 1: Remineralizing agents recovered the micro tensile bond strength of demineralized dentin...19
2.2 Artigo 2: Biomimetic agents can slow down caries-affected dentin/resin interface degradation………39
3 DISCUSSÃO ... 66
4 CONCLUSÃO ... 71
REFERÊNCIAS ... 72
ANEXOS ... 77
ANEXO 1 - COMPROVANTE CEP FOP/UNICAMP ... 77
ANEXO 2 - COMPROVANTE DE SUBMISSÃO DE ARTIGO ... 78
1 INTRODUÇÃO
Considerada como um compósito natural, poroso, úmido e heterogêneo, a dentina é constituída por cristais de apatita (70%) e água (10%) envolvendo uma matriz proteica (20%), apresentando como um dos principais constituintes o colágeno Tipo I (Perdigão et al., 2013). Entretanto, com a evolução dos materiais adesivos, aplicando as novas tecnologias, surge uma tendência de preservação da estrutura de dentina afetada pelo processo carioso (Yoshida et al., 2004; Joves et al., 2014; Barbosa-Martins et al., 2018)
As importantes contribuições obtidas por meio dos trabalhos de Fusayama, (1979), sugeriram que a dentina afetada pelo processo carioso apresentava condições para preservação, sendo passível de remineralização. Ainda, mostraram que a estrutura colágena não estava desnaturada e se apresentava desprotegida pela perda mineral. A destruição da matriz do colágeno na dentina requer uma combinação da desmineralização, proveniente da liberação de ácidos pelos microorganismos como subprodutos da metabolização de carboidratos, e, a ativação de proteases (colagenases/gelatinases), que são classes de enzimas capazes de clivar ligações peptídicas dentro da estrutura de colágeno em condições fisiológicas de pH e temperatura. As metaloproteinases da matriz (MMPs) intrínsecas ao organismo são endopeptidases zinco/cálcio dependentes, que foram aprisionadas na matriz dentinária mineralizada durante o processo de biomineralização do dente (Tjaderhane et al., 2013a). A liberação e consequente ação de degradação destas enzimas são fatores também responsáveis pela longevidade das interfaces adesivas. O restabelecimento das condições normais do conteúdo mineral destas estruturas afetadas poderia favorecer positivamente a durabilidade da união, impedindo a ação destas enzimas (Tjaderhane et al., 2013a).
Clinicamente, de acordo com o conceito de mínima intervenção, o uso de restaurações que utilizam materiais resinosos tem sido aceito como tratamento eletivo e conservador para lesões de cárie (Joves et al., 2014). Em cavidades que apresentem envolvimento da dentina cariada, a remoção seletiva do tecido cariado é preconizada, contribuindo para a preservação da estrutura sadia e em cavidades profundas (Joves et al., 2014).
A dentina afetada pelo processo carioso apresenta diferenças (bioquímica, morfológica) (Kidd, 2004) e histológica (Kidd&Fejerskov, 2004), em relação à dentina sadia (Tjaderhane et al., 2013b). A perda da porção mineral que protege as fibrilas colágenas na dentina torna a estrutura de colágeno vulnerável, aumentando a suscetibilidade à degradação enzimática (Tjaderhane et al., 2013b).
Na tentativa de mimetizar lesões de cárie em dentina, modelos in vitro (método químico: ciclagem de pH (Ten Cate, 1982), soluções desmineralizantes (Bertassoni et al., 2010), desmineralização com gel ácido e modelo biológico: desmineralização por biofilme de S.
Mutans (Pacheco et al., 2013)), de produção artificial foram desenvolvidos para fins de
simulação da condição clínica em modelos controlados e avaliação da resistência da união (Clarkson et al., 1984; Arends et al., 1997; de Carvalho et al., 2008; Pacheco et al., 2013). Cada método de produção de cárie apresenta um protocolo especifico, e é de grande relevância que estes protocolos sejam comparados para um melhor embasamento na indicação do protocolo experiemental mais adequado, uma vez que a desmineralização produzida pode ter influencia nos procedimentos adesivos a longo prazo (Ten Cate, 1982; Bertassoni et al., 2010).
Baseando-se na longevidade dos procedimentos adesivos, eleva-se a preocupação de favorecer os mecanismos que envolvem a união aos substratos dentinários e, de desenvolver técnicas e biomateriais capazes de mimetizar, reforçar e reestruturar adequadamente a estrutura dentinária afetada, que foi debilitada pelo processo carioso por meio da remineralização biomimética (Perdigão et al., 2013; Tjaderhane et al., 2013b; Niu et al., 2014; Barbosa-Martins et al., 2018).
A remineralização biomimética baseia-se em replicar o que ocorre no processo de biomineralização, cuja proposta, permitiria que os espaços extras e intrafibrilares da matriz de colágeno ocupado anteriormente pela água sejam substituídos por cristais de nano-apatitas (Veis et al., 2012; Perdigão et al., 2013; Niu et al., 2014), por meio de agentes remineralizantes, deixando menor quantidade de colágeno exposto, evitando maior degradação da camada híbrida, sem contudo interferir na penetração do sistema adesivo, uma vez que o processo de recristalização seria ordenado (Tay&Pashley, 2009). Esta remineralização promoveria a reposição mineral perdida pelo processo carioso, de forma ordenada, envolvendo e protegendo as fibrilas colágenas, reestruturando e promovendo um reforço na estrutura do tecido afetado. Dessa forma, a remineralização biomimética pode ser considerada uma estratégia interessante para melhorar a estabilidade da união resina/dentina, bem como controlar a cárie primária e secundária (Liu et al., 2011).
Historicamente, a eficácia do fluoreto de sódio (NaF) como agente remineralizador e cariostárico tem sido bem estabelecida, e a ação tópica deste agente tem sido utilizada por profissionais para deter a progressão de lesões de cárie ativas. O mecanismo de ação do fluoreto é decorrente da ligação aos íons Ca2+, que promoveram a formação de fluoreto de cálcio (Ca2F). Os fluoretos agem promovendo a deposição mineral tanto na superfície quanto em profundidade em áreas desmineralizadas, decorrentes da difusão desordenada dos íons em direção ao interior
do tecido dentinário afetado (Hamba et al., 2011; Prabhakar et al., 2013). Entretanto, a maior deposição de ions minerais relaciona-se ao Fluoreto de Cálcio, localizando-se na superfície da área afetada, seja esmalte ou dentina. Essa deposição descontrolada poderia interferir na permeabilidade do tecido aos monômeros resinosos e produzir uma camada híbrida não homogênea, produzindo uma união menos estável entre a resina e a dentina (Arends et al., 1989; Arends et al., 1990; Mukai et al., 2002).
Um agente que se baseia em um nanocomplexo da proteína do leite, o fosfopeptídeo de caseína (CPP) é derivado de um grupo de peptídeos da caseína, que juntamente com fosfato de cálcio amorfo (ACP) tem sido pesquisado com o objetivo de liberação lenta de elementos químicos (Ca2+ e PO43-), contribuindo para o processo de remineralização (Reynolds, 1998; Reynolds, 2008; Rahiotis&Vougiouklakis, 2007). Teoricamente, o CPP pode servir como um análogo biomimético de proteínas fosforiladas, não colagenosas, da dentina, pelo recrutamento de nanoprecursores de ACP nos espaços das áreas das fibrilas colágenas (Poggio et al., 2013). Utilizando um modelo animal para comparar a atividade do CPP com o flúor na redução de lesões de cáries, Reynolds et al. (1998) constataram que a aplicação de CPP-ACP, duas vezes ao dia sobre os dentes seria equivalente ao uso de 500 ppm de flúor. Então, o CPP-ACP foi
incorporado a uma pasta disponível no mercado (MI PasteTM - GC International, Itabashi-ku,
Tóquio, Japão), e tem sido considerado um ótimo agente remineralizador tanto do esmalte quanto da dentina, e rotineiramente indicado para o tratamento da sensibilidade dentinária (Yamaguchi et al., 2006; Adebayo et al., 2010; Comar et al., 2013).
Além disso, na tentativa de fortalecer a unidade resina/dentina, Borges et al. (2013) avaliaram a resistência da união na interface resina/dentina após a aplicação de CPP-ACP e observaram que essa aplicação na superfície dentinária hígida produziram maiores médias de resistência de união quando comparado a dentina hígida sem tratamento, sugerindo que a aplicação de MI Paste™ contendo CPP-ACP, poderia proporcionar um efeito preventivo ao processo de desmineralização. Barbosa-Martins et al. (2018), aplicando o CPP-ACP em dentina desmineralizada observaram que esta associação promoveu maiores médias de resistência da união mesmo quando comparado a dentina hígida. Dessa forma, levantou-se a hipótese de que a remineralização das fibras colágenas da camada híbrida pode representar uma abordagem atrativa e inovadora para a união resina/dentina desmineralizada uma vez que com a remineralização ocorreria o aumento mineral nas áreas desmineralizadas e o possível reestabelecimento das propriedades mecânicas, aprimorando o substrato para a adesão.
Outro agente que apresenta um mecanismo de remineralização biomimética dos tecidos
contato com a superfície porosa, por exemplo, de uma lesão de cárie em esmalte, modifica-se de um estado líquido isotrópico para o de hidrogel elastomérico com aspecto fibrilar. O peptídeo P11-4 tem a capacidade de atrair metabólitos minerais, como o cálcio, induzindo a precipitação de fosfato de cálcio proveniente de soluções supersaturadas, os quais posteriormente se unem e transformam-se em cristais de hidroxiapatita (Kirkham et al., 2007; Kind et al., 2017). Kirkham et al. (2007) e Brunton et al. (2013), sugeriram que o peptídeo P11-4 poderia ser útil na modulação do aumento do ganho mineral, sendo capaz de iniciar um processo de remineralização biomimética.
Desta forma, uma melhor compreensão da ação de agentes remineralizadores sobre o substrato dentinário afetado (desmineralziado), pode representar implicações positivas na longevidade dos procedimentos adesivos. Baseado nas evidências científicas encontradas sobre
a ação dos agentes NaF, CPP-ACP e o peptídeo P11-4 contido no Curodont™ Repair,
transportando a ação destes agentes remineralizadores para o substrato dentinário, principalmente na dentina cariada e na camada híbrida, sua implicação na longevidade da união resina/dentina, parece justificável a investigação de agentes remineralizadores que possam reduzir a degradação hidrolítica e atividade de enzimas proteolítica, decorrente da mineralização intrafibrilar do colágeno dentinário.
Assim, as hipóteses desta tese são:
1. Diferentes métodos de produção de cárie (método químico e biológico)
produzem diferença nos valores de resistência da união entre o adesivo e a dentina desmineralizada;
2. A dentina desmineralizada e tratada por agentes remineralizadores recupera os
valores da resistência da união;
3. A molhabilidade da dentina afetada por cárie produzida pelo método biológico
e tratada por diferentes agentes remineralizadores é maior do que a obtida para a dentina hígida;
4. O tratamento da dentina afetada por cárie produzida pelo método biológico e
tratada por diferentes agentes remineralizadores aumenta a resistência da união, reduz a nanoinfiltração e aumenta a quantidade de mineral na camada híbrida comparada a dentina hígida;
5. O tempo é fator determinante na estabilidade da resistência da união,
nanoinfiltração e quantidade de mineral observado na camada híbrida ao longo do tempo.
Esta tese será apresetada no formato alternativo de acordo com o Art. 2º da Informação CCPG/001/2015, § 2°.
2 ARTIGOS
2.1 Artigo 1: Remineralizing agents recovered the micro tensile bond strength of demineralized dentin
Artigo submetido à revista Materials
Luiz Filipe Barbosa-Martins1, DDS, MSc
Jossaria Pereira de Sousa1, DDS, MSc
Lívia Araujo Alves2, DDS, MSc, PhD
Robert Philip Wynn Davies3 BSc, Mres, PhD
Regina Maria Puppin-Rontanti4* DDS, MSc, PhD
1 Department of Pediatric Dentistry, Piracicaba Dental School, State University of Campinas,
Piracicaba, São Paulo, Brazil.
2 Department of Oral Diagnosis, Piracicaba Dental School, State University of Campinas, Piracicaba, SP, Brazil.
3 Researcher of Division of Oral Biology, School of Dentistry, Faculty of Medicine & Health, University of Leeds, Leeds, United Kingdom
4 Professor and Researcher Senior, Departments of Pediatric Dentistry and Restorative Dentistry, Piracicaba Dental School, University of Campinas, Piracicaba, São Paulo, Brazil.
Corresponding author:
Profa. Dra. Regina Maria Puppin Rontani
Departments of Pediatric Dentistry and Restorative Dentistry, Piracicaba Dental School, Univdersity of Campinas.
Limeira Avenue, Zip Code: 13414-903. Areião, Piracicaba, São Paulo E-mail address: rmpuppin@unicamp.br
ABSTRACT
Background: The aim this study was to investigate the influence of Dentin Caries-like Lesions
Producing Model (chemical and biological models of caries induction) on microtensile bond strength-μTBS of etch-and-rinse adhesive system and the effect of remineralizing agents such as Sodium Fluoride (NaF), CPP-ACP (casein phospopeptide amorphous calcium phosphate)
contained in Curodont™ Repair (CR) (Credentis AG, Dorgstrasse, 69CH-5210, Windisch, Switzerland) on caries-affected dentin. In addition, in order to confirm the caries lesion depths, sound and demineralized dentin (DDC and DDB) were evaluated using polarized light microscopy-PLM (n=3). Materials and Methods: Sound dentin blocks obtained from third molars were distributed in groups (μTBS-n=6/PLM-n=3) according to dentin conditions: G1-Sound Dentin; G2-Demineralized Dentin by Chemical model-DDC; G3-DDC/NaF; G4-DDC/MP; G5-DDC/CR; G6-Demineralized Dentin by Biological Model)-DDB; G7:DDB/NaF; G8:DDB/MP and G9:DDB/CR. G2-G5 groups were demineralized with 6% carboxymethylcellulose gel and 0.1 M lactic acid in a KOH solution at pH 5.0 for 48h. G6-G9 groups were submitted to the biological model using S. mutans biofilm for 7 days. Tooth sections of 0.15 µm were cut longitudinally of the occlusion surface, and 2 sections per tooth were obtained. Sections were soaked in water and viewed with a PLM. Then all dentin blocks were submitted to bonding procedure with Adper™ Single Bond 2 adhesive system followed by building of a Filtek Z350XT 4mm high block. All sets were immersed in deionized water
for 24h and then sectioned with ≅ 1mm2 (cross-section) beams. The beams were submitted to
the μTBS test at 1.0mm/min and 500N loading. Failure sites were evaluated by SEM (scanning electron microscopy (150x). The μTBS data were submitted to factorial ANOVA and Tukey's test (p<0.05). Results: The μTBS values obtained to dentin caries-like lesions producing model were lower compared with sound dentin and remineralizing agents, but were not affected by method of caries induction and caries lesion depth (p<0.05). The highest values were found when demineralized dentin was treated with MP and CR, regardless caries lesion depth (p<0.05). There was a predominance of adhesive, mixed in the present study. Conclusion: It was concluded that the use of the artificial dentin caries production models does not produce differences in the strength of the resin/dentin dentin bond. However, MP and CR remineralizing agents were able to enhance adhesive procedures even at different models of caries lesion.
Keywords: Dentin; Desmineralization; Microtensile bond strength
INTRODUCTION
During routine dental restorations the hybrid structure formed during the dental bonding procedure occurs by the infiltration and subsequent polymerization of monomers around the demineralized collagenous matrix [1]. The oral cavity is a severe environment for the resin-dental bond to survive for a reasonable length of time, with thermomechanical changes, chemical attacks by acids and enzymes and other factors posing routine daily challenge.
Therefore, to achieve effective and stable bonding, the preservation of dentin collagen is critical, since collagen represents the major organic component of the dentin matrix [2].
Caries is the most common disease worldwide [3], and the immediate bond strengths to caries-affected dentin are commonly 20-50% lower than to sound dentin [4-6]. The restoration of the normal conditions of the mineral content of the caries-affected dentin, prevents the action of enzymes in addition to providing a increased bond durability [7].
Biomimetic remineralization mimics the process of natural biomineralization by replacing demineralized collagen matrix water with apatite crystallites [7]. Caries-affected dentin is comprised of about 14% to 53% of water when compared by comparison sound dentin exhibits a much lower value ca. 10% to 12% [8]. Therefore, by replacing water with minerals at the dentine-resin interface, this would increase the mechanical properties and inhibit water-related hydrolysis [9].
It has recently been demonstrated that the use of remineralizing agents in dentin could recover the mechanical properties of the substrate [10]. In addition to sodium floride (NaF) and sodium phosphate (Na3PO4), casein phosphopeptide amorphous calcium phosphate (CPP-ACP), which is derived from milk protein, promotes the release of calcium phosphate assisting in enamel and dentin remineralization [11]. Mainly by inhibiting demineralization and enzymatic degradation [12]. Furthermore, recent studies have shown that the use CPP-ACP has no negative effect on bond strength [13,14]. The peptidic biomimetic matrix ‘P11-4’, which has been incorporated into a clinical product (Curodont™ Repair) has shown encouraging results in early clinical trials. It has been shown to improve the visual appearance of carious lesions and increases the opacity on X-rays after treatment of proximal caries [15,16]. Additionally, following the application of P11-4 and subsequent bonding procedures an increase in the resin-dentin bond strength has been observed [17].
The restructure of the demineralized collagen matrix found in caries-affected dentin process by interventions such as Sodium Fluoride (NaF), CPP-ACP contained in MI Paste™
(GC International) and P11-4 peptide contained in Curodont™ Repair (Credentis AG), prior to
adhesive procedures by an etch-and-rinse adhesive system (Adper ™ Single Bond 2 (3M ESPE) in the demineralized dentin could be a promising proposal for adhesive clinical procedures.
Simulating adhesive procedures via mechanical methods (i.e. µTBS) use demineralized dentin and the lack of standardization of the caries lesions creates technical difficulties for evaluation [18]. To date no information is available regarding the implication of induced caries-like lesions and the action of remineralizing agents on bonding procedures.
In vitro models have been used to produce demineralized dentin under controlled conditions [19-22]. Chemical methods provide a superficial dentin demineralization, resulting in a substrate with similar hardness compared to natural caries-affected dentin [19]. Conversely, the microbiological method promotes an excessive softening of dentin, but with a more comparable morphological pattern of collagen degradation to natural caries lesions [19-22].
To ascertain the potential efficacy of this clinical procedure we aim to evaluate number of remineralization treatments and investigate to what extent the method used in producing the simulated dentin-like caries lesions has on the micro tensile bond strength after treatment. The hypothesis was that there are no significant differences between µTBS of etch-and-rinse adhesive system and demineralized dentin treatment with 0.2% NaF, MI Paste™ and Curodont™ Repair, regardless of the artificial caries development model used.
MATERIALS AND METHODS Specimen Preparation
Sixty-three noncarious human third molars were collected after patients' informed consent, as approved by the Ethics Committee of Piracicaba Dental School, University of Campinas, (Protocol: 37634814.5.0000.5418). The teeth were stored in 0.1% thymol solution at 4oC for no longer than 2 months after extraction. Three teeth were used in a polarized light microscopy in order to check the caries dentin depth and the others to be used for a µTBS tests. A 4.0 mm 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). Six dentin slices were used for sound dentin (control group-CG), and the others were randomly assigned into 2 groups (n=24), according to the caries method production. The dentin surface of each specimen was wet polished with a 600-grit SiC paper (Arotec, São Paulo, Brazil) for 30 s to create a standardized smear layer. The dentin surfaces were carefully examined under a stereomicroscope at ×50 magnification to confirm the absence of enamel islets. The specimens were immediately subjected to production of caries in vitro. The group distribution can be seen on Flowchart.
Flowchart - Experimental design. DDC - demineralized dentin provided by chemical model; DDC/NaF -
DDC+ 2% NaF; DDC/MP - DDC+ MI Paste™; DDC/CR - DDC+ Curodont™ Repair; DDB- demineralized dentin provided by biological model; DDB/NaF - DDB+2% NaF; DDB/MP - DDB+ MI Paste™; DDB/CR - DDB+ Curodont™ Repair.
Dentin Caries-like Lesions Producing Model
Sixty dentin slices (54 teeth for µTBS and 6 for polarized light microscopy) were randomly assigned into 2 groups according to dentin-like caries lesions producing models: chemical (carboxymethylcellulose acid gel) and biological (Streptococcus mutans - UA159 biofilm).
Chemical model
The specimens were submerged in vials containing 5 mL of 6% carboxymethylcellulose acid gel (0.1 M lactic acid titrated to pH 5.0 in a KOH solution) at pH 5.0 and 37ºC. The specimens remained in the gel for 48 hours without renewal (Pacheco et al., 2013). This model supposedly provided a demineralized dentin similar to caries affected dentin.
Biological model
The specimens were fixed with orthodontic wire on the lids of glass vials containing 250 mL of sterile deionized water and were sterilized with gamma radiation (14.5 kGy dose) – for 60 hours (Pacheco et al., 2013). Then, they were transferred to another glass vial containing 250 mL of sterile brain-heart infusion (BHI) broth (LabCenter, São Paulo, Brazil) supplemented
with 0.5% yeast extract (LabCenter, São Paulo, Brazil), 0.5% glucose (LabCenter, São Paulo, Brazil), 1% sucrose (LabCenter, São Paulo, Brazil) and 2% S. mutans (UA159) incubated at
37ºC and supplemented with 10% CO2, pH around of 4.0. Starter culture was transferred into
250 mL of fresh BHI and grown for 4 h at 37°C under aerobic conditions. Optical density at 550 nm (A550) of all bacterial suspensions was adjusted to 0.05 prior to inoculation. Inoculation occurred only in the first day of the experiment, but the broth was renewed every 48 h during 7 days. Contamination of the broth was checked every day using Gram staining. The biofilm over the teeth were removed with gauze and the softer dentin layer was accomplished using # 6 carbide drills; the removal was stopped when it was observed the appearance of dentin similar to caries-affected dentin (Pacheco et al., 2013).
Polarized Light Microscopy
After providing dentin caries-like lesions, three specimens of each dentin caries-like lesions producing models and control group were sectioned perpendicular to the occlusal surface to obtain slices. The two more central slices of each tooth was selected and polished with #800, #1200, #2400 and #4000-grit silicon carbide (SiC) paper (Buehler, Lake Buff, IL, USA), obtaining a 0.15 µm dentin thickness. Dentin slices were analyzed for the depth of demineralization on PLM (Leica DMLP, Leica microsystems, Germany). The dentin slices were stored in water and kept humid throughout the investigation. Measurements were carried out with a PLM (Leica DMLP, Leica microsystems) caries area was visualized using 20x/0.4 (corr) objective to analyze carious dentin morphology. Standard settings for contrast, brightness and light were used for all images. Four measurements were made in different parts of the same lesion from the lesion border to the deepest part of the lesion, for each dentin caries-like lesion model. An average depth for each specimen was calculated from the individual values based on depth difference between dentin caries-like lesions and sound dentin. Dentin caries-like lesions observed in the specimens submitted to Chemical Model presented x=12.69 µm average depth and Biological Model x=148.55 µm, measured using PLM.
Dentin Surface Treatment
Thirty-six teeth were assigned to 6 groups according to the remineralization treatment: demineralized dentin by chemical model (DDC) + treated with 0.2% NaF Solution (1 min)-DDC/NaF; DDC + treated with MI Paste™ (1 min)-DDC/MP; and DDC + treated with
Curodont™ Repair-DDC/CR applied (5 min) plus Ca++ and PO
43-Solution (1 min); demineralized dentin by biological model (DDB) + 0.2% NaF Solution-DDB/NaF; DDB +
treated with MI Paste™ applied (1 min)-DDB/MP; and DDB + treated with Curodont™ Repair - DDB/CR applied (5 min) plus Ca++ and PO43-Solution (1 min).
The specimens of groups DDC/NaF and DDB/NaF were remineralized using 0.1 mL of the 0.2% NaF 0.2% NaF solution was applied on demineralized dentin surface and left dry for 1 min at room temperature for the groups DDC/NaF and DDB/NaF. 0.1 mL of the MI Paste™ was applied onto the surface of the specimens from DDC/MP and DDB/MP groups with microbrush for 1 min at room temperature, the excess paste was removed by washing with deionized water. For DDC/CR and DDB/CR groups, 50 µL of the Curodont™ Repair was applied and left for 5 min, then, a Ca++ and PO
43- solution was applied and left onto surface for 1 min. For all treatments, the solution excess was removed by with absorbent paper (Barbosa-Martins et al., 2018).
Bonding Procedures
A single operator applied the adhesive according to the manufacturer's instruction (Table 1). An LED light-curing unit (Bluephase, Ivoclar Vivadent; Schaan, Liechtenstein) was
set to the low power mode with a light intensity of 650 mW/cm2. A nanohybrid resin composite
(Filtek Z350 XT, A2, 3M ESPE) was used to create resin composite buildups in four layers of 1 mm each. Each layer was light cured for 20 s, followed by a final polymerization of 60 s. The specimens were then stored at 100% humidity at 37ºC for 24 h.
Microtensile Bond Strength Test (µTBS)
After storage time, the specimens were sectioned perpendicularly to the interface to
produce dentin-resin beams with 1 mm2 at cross-sectional area, using a low speed diamond saw
(ISOMET 1000, Buehler Ltd., Lake Buff, IL, USA). From six to eight beams were obtained per tooth, each beam was measured with a digital caliper (Mitutoyo; Kawasaki, Japan) to determine the cross-sectional area. All beams were kept in deionized water for 24 h.
For µTBS measurement, each beam was fixed to a microtensile device with cyanocrylate glue (Super Bonder (#1883519), Loctite, Henkel Corp., Rocky Hill, CT, USA), and tested in a universal testing machine (DL 2000, EMIC, Equipment and Systems Ltda., São José dos Pinhais, PR, Brazil). The test was carried out with a load cell of 500 N at 1.0 mm/min cross speed until failure. The µTBS values were expressed in MPa.
Table 1. Materials, manufactures, components, batch numbers and application mode of tested
materials.
Materials
(manufactures) Main components
Batch
number Application mode
0.2% NaF Solution 0.2g of NaF in 100 ml deionized water Made in the Lab* 1. Apply 1.0 mL of 0.2% NaF solution, pH 7.0.
Ca++ and PO4
3-Solution
Saturated solution of Ca++ and PO43- (1.5 mmol/L calcium, 0.9 mmol/L phosphate, and 150 mmol/L KCl in 20 mmol/L cacodylic buffer, pH 7.0.) (Zancopé et al., 2016) [23]. Made in the Lab 1. Apply 0.1mL of Ca++ and PO43- solution, pH 7.0. MI™ Paste - GC Internacional, Tóquio, Japão
Glycerol, CPP-ACP, D-Sorbitol, Propylene glycol, Titanium dioxide and silicon N2347319 1. Apply 0.1mL of MI™ Paste Curodont™ Repair - Credentis AG, Dorfstrasse, Windisch, Switzerland
P11-4 peptide – amino acid sequence -
(Ace-Gln-Gln-Arg-Phe-Glu-Trp-Glu-Phe-Glu-Gln-Gln-NH2)
N342x
1. Apply 50µL of Curodont™ Repair for 5 min, pH 7.0. 2. Apply 0.1 mL of Ca++ and PO43- solution, pH 7.0. Scotchbond™ Universal Etchant - 3M ESPE; St Paul, MN, USA
32 % phosphoric acid N345 1. Apply etchant for 15 s 2. Rinse for 10 s
Adper Single Bond 2.0 – 3M ESPE; St
Paul, MN, USA
HEMA, water, ethanol, Bis-GMA, dimethacrylates, amines, metacrylate functional copolymer of polyacrylic and polyitaconic acids, 10% by weight of 5 nanometer-diameter spherical sílica particles
N42912
3. Blot water excess 4. Apply 2 consecutive
coats of adhesive for 15 s with gentle agitation 5. Gently air dry for 5 s 6. Light-cure for 10 s
Filtek™ Z350 XT -
3M ESPE; St Paul, MN, USA
BIS-GMA, Bis-EMA, UDMA, TEG-DMA, camphorquinone, non-agglomerated silica nanoparticles
N98354 1. Incremental insertion 2mm 2. Light-cure for 20 s
*Pediatric Dentistry Laboratory
Analysis of Failure Mode
Fractured beams were evaluated by scanning electron microscopy (SEM) (JEOL, JSM – 5600LV, Tokyo, Japan), at x50 and x150 magnification to determine the failure mode, which was classified as follows: cohesive into dentin (CD), cohesive into composite (CC), adhesive (A) or mixed (M).
Statistical Analysis
Bond strength values for each group were analyzed by Shapiro–Wilk test in order to assess the normality of the data distribution. Factorial ANOVA and post hoc Tukey test were
used to determine statistically significant differences between factors: the dentin-like caries lesions model (two levels – chemical and biological models) and dentin remineralization
treatment (four levels – SD, DD, NaF+DD, CPP-ACP+DD and P11-4+DD) on dentin/resin bond
strength, and additional Dunnett test to determine statistically significant differences between the experimental groups and the control group (sound dentin). The kruskal-wallis test was used to evaluate the failure mode. The R Software version 3.4.3 (The R Foundation for Statistical Computing, Vienna, Áustria), was used to perform the tests. Statistical difference was set at α=5%.
RESULTS
Factorial ANOVA revealed a significant interaction between studied factors: dentin caries-like lesion model and remineralization treatment (p<0.001). In addition, there was a statistically significant difference concerning artificial dentin caries-like lesion model (p<0.001), and also between treatments (p<0.001).
As shown in Table 2, a Tukey test revealed that µTBS of NaF to demineralized dentin for both, chemical and biological dentin caries-like lesion models, were significantly lower than other remineralizing agents (p<0.001). Biological DCLL model significantly reduced the microtensile bond strength when the dentin was treated by NaF and Curodont™ Repair (p<0.001). Chemical DCLL model provided higher μTBS than biological one, when demineralized dentin was treated by NaF and Curodont Repair (p<0.05). In addition, dentin demineralized by the chemical DCLL treated with Curodont™ Repair provided the highest bond strength, and there was no significant difference from MI Paste™ for the same dentin condition, and they were significant higher than sound dentin (p<0.05). However, when DCLL was treated with MI Paste™, there was no influence of the DCLL model (p>0.05) and they were significant higher than sound dentin (p<0.05). However, only when NaF was used can be observed lower μTBS than sound dentin (p<0.05). For both DCLL models, demineralized dentin treated with NaF, MI Paste™ and Curodont™ Repair showed significant higher μTBS than demineralized dentin (p<0.01). Demineralized dentin group showed the lowest bond strength for all groups (p<0.01) (Table 2).
Table 2. Average and standard deviation of μTBS of demineralized dentin considering the
Artificial Caries Development Models.
Experimental Groups Artificial Caries Development Models Chemical Model Biological Model
Sound Dentin 43.32±4.35
Demineralized Dentin 21.96±5.92 Ca * 22.89±2.68 Da *
Demineralized Dentin + NaF 33.43±10.42 Ba * 26.94±6.70 Cb *
Demineralized Dentin + MI Paste™ (CPP-ACP) 45.25±8.83 Aa* 47.95±6.69 Aa *
Demineralized Dentin + Curodont™ Repair (P11-4) 46.42±12.03 Aa* 42.07±7.83 Bb
Uppercase letters represent statistically significant difference in the column (p<0.001). Lowercase letters represent no statistically significant difference in the row (p>0.05). * indicates statistically significant difference with the control group (sound dentin) (p<0.05) by additional Dunnett’s test.
The failure modes of specimens are shown in Figure 2. The failure modes of DDC (76%) and DDB (85%) specimens were predominantly adhesive failure; Mixed failure were found for DDC – NaF (84%), DDB – NaF (61%), DDC - MI Paste™ (54%), DDC - Curodont™ Repair (68%) and DDB - Curodont™ Repair (85%); cohesive failure in composite resin were found for Sound Dentin (56%), DDB - MI Paste™ (65%) and DDC – Curodont™ Repair (24%); cohesive failure in dentin was observed in the DDC - MI™ Paste (38%) and DDB - MI Paste™ (12%) groups. The often failure patterns were adhesive and mixed for all groups, except for DDB-MI Paste™. There was no statistically significant difference between the fracture type by Kruskal-Wallis's test, concerning DCLL model (p=0.9967).
Figure 3 shows representative SEMs for the fracture patterns observed for the differents groups. Figure 3A - Cohesive failure on composite; 3B - Adhesive failure; 3C Mixed failure; and 3D - cohesive failure on dentin.
Figure 3. SEM image of failure modes. A. Cohesive failure in Resin Composite; B. Adhesive failure;
C. Mixed failure and D. Cohesive failure in Dentin. Abbreviations shows areas of Co. Composite; Ad.
Adhesive; De. Dentin. 0 10 20 30 40 50 60 70 80 90 100 Sound
Dentin DDC DDB DDC + NaF DDB + NaF DDC + MI Paste™ DDB + MI Paste™ Curodont™ DDC + Repair
DDB + Curodont™
Repair Adhesive Mixed Cohesive in Composite Cohesive in Dentin
Figure 2. Distribution of failure modes. DDC – Demineralized Dentin by chemical model; DDB –
Demineralized Dentin by biological model; NaF – Sodium Floride; MI Paste™ - CPP-ACP – Casein
DISCUSSION
To date, no information is available regarding the implication of the Dentin Caries-like Lesions on bond strength to adhesive systems. In addition, no information has been found concerning the using of remineralizing agents in permanent teeth. In the present study evaluated the influence of artificial caries development models (chemical and biological) and substrate conditions on resin/dentin bond strength. In this study, the null hypothesis was rejected, since there was a significant influence of the DCLL model and demineralized dentin treatment on μTBS of a etch & rinse adhesive system. Therefore, the highest µTBS values were found for demineralized dentin treated with MI Paste™, and for Curodont™ Repair treatment (p<0.001), considering the chemical DCLL model.
The caries lesion provided by a biological model, which uses S. mutans biofilm, seems to be quite similar to the natural ones, based on molecular and structural evaluations [23]. Another model used for providing dentin caries-like lesions is the chemical one, as it can be used to simulate caries-affected dentin [5]. It has been recently demonstrated that immediate µTBS of adhesive systems to dentin of primary teeth was not affected by the different DCLL [25]. However, there were no implications with regard to the performance of different caries models on DCLL bond strength of permanent teeth.
This study corroborates previous investigations demonstrating that the bonding procedures on demineralized dentin [26, 27] present lower µTBS to demineralized dentin when compared to a sound one, regardless of the artificial caries development model (Table 2). Morphological changes in the substrate provided by caries production process can induce a decreased µTBS [20, 28]. This reduction can be associated with changes in physical and chemical properties of the demineralized substrate when compared to sound dentin [29]. Demineralized dentin provides a high porosity in the inter-tubular dentin, exposure of collagen fibers along with decrease in mineral content [30] and partial penetration of resin monomers and a non-homogeneous hybrid layer [31]. In a porous hybrid layer, over time, mineral and organic matrix would be degraded giving rise to gaps which may be visible using a SEM which show a higher rate of degradation [31]. In addition, the demineralization of dentin surface results in a more hydrophobic surface, avoiding the wettability of the adhesive [17].
It is desirable that bonding between mineralized tooth tissues, as dentin, and the restorative materials must be sufficiently effective to resist at varied challenges, such as biofilm attack, hydrolytic and enzymatic degradation, thermal and mechanical stress from repeated loading over many months or years [32, 33]. The reinforcement of demineralized collagen matrix can be achieved by remineralizing agents [10, 34]. The current biomimetic
remineralization approach provides a proof-of-concept that utilizes nanotechnological principles to mimic natural biomineralization, extending the longevity of resin-dentin bonds [10]. The mineral reinforcement of collagen matrix found in demineralized dentin appears to be a strategy that restores conditions found on sound dentin, as seen in the present study. In the present study the biomimetic remineralization strategy provided a higher or similar μTBS to demineralized dentin than sound one, while the NaF remineralization provided higher μTBS values than demineralized dentin, but lower than sound one.
We have observed that a single active application MI Paste™ in demineralized dentin for 1 minute before the bonding procedures can significantly increase the μTBS values than those found for sound dentin. In contrast, to the present study, different application protocols are described in the literature, showing that the neither 3 min application of MI Paste™, on sound dentin [13], 5 minutes [35] or 60 minutes per day for 7 days [36] did not show significant differences from control groups. Those studies evaluated the effect of CPP-ACP on bond strength to sound dentin before bonding procedure, but did not show any improved effect on bond strength.
Concerning affected dentin, Bahari et al., (2014) [37] showed that 5 consecutive days of CPP-ACP application for 15 minutes did not have any significant effect on μTBS of SB to demineralized dentin. However, it has been considered that the methodology used in that study was quite different from that used in the present study. Firstly, according to the methodology description, even sound dentin was submitted to the CPP-ACP action, since each tooth, with both sound and caries-affected dentin, was submitted to CPP-ACP application. The casein phosphopeptides (CPP) have been described to bind amorphous calcium phosphate, forming nano-complexes of casein phosphopeptide–amorphous calcium phosphate (CPP–ACP), thereby stabilizing in calcium phosphates [38, 39]. Calcium and phosphate ions can easily diffuse into the porous lesion and deposits in the partially demineralized crystals and rebuild hydroxy-apatite crystals [40]. This further substantiates the theory that CPP-ACP is considered a biomaterial [41]. The presence of bioavailable calcium and phosphate present the MI Paste™ can maintain a supersaturated state in dental substrate [11]. Studies have demonstrated that CPP-ACP could reduce demineralization and increase remineralization of dentin [41, 42]. It is possible, that in this study the CPP-ACP could have been impregnated onto caries-affected dentin, and sound dentin [13, 35] in the same way.
It is well established that the collagen matrix serves as a scaffold for crystal deposition but does not provide a mechanism for orderly nucleation of hydroxyapatite [34]. The results of the present study can be attributed to the ability of CPP-ACP to increase deposition of crystals
on the dentin surface [36]. Furthermore, the CPP also has the capacity to stabilize nano-ACP [43]. Therefore, the deposition and stabilization may result in a restructuring of the characteristics found in sound dentin, showing highest the µTBS. This reinforcement approach of demineralized collagen matrix structure may favor the bonding procedure. Further studies should be carried out to verify the stability of the bonding strength when CPP-ACP is used.
It is generally believed that extracellular matrix proteins, which play an important role in controlling apatite nucleation and growth in the dentin remineralization process [44], mediate a biomineralization process. Biomimetic remineralization represents a different approach to this by attempting to backfill the demineralized dentin collagen with liquid-like ACP nanoprecursor particles that are stabilized by biomimetic analogs of noncollagenous proteins [10, 45]. In this way, maybe this particular nucleation would provide a regular and feasible restructuration of the demineralized dentin and also would provide a favorable substrate for bonding, due to the more hydrophyllic nature of the substrate.
Another interesting finding of the present study was the fact that the µTBS means of MI Paste™ group was higher than those found in sound dentin, and did not show a significant difference between either artificial caries development models.
The remineralization process and artificial caries development model, results from this study showed that the artificial caries development model only affects the μTBS of
demineralized dentin treated with NaF and the remineralized P11-4. Only the P11-4 approach
using the DCLL chemical model provided significantly higher μTBS than sound dentin. The chemical model provided a significantly higher μTBS when dentin was treated with NaF and
P11-4. Although it did not affect the adhesion to demineralized dentin per se (Demineralized
Dentin groups). The chemical model of DCLL provided a lower content of type I collagen and higher content of calcium and phosphate ions, than Biological one [23]. The collagen is the precursor for mineralization, acting as a scaffold for mineral aggregation. However, when NaF is used, only a deposition of ions and CaF formation occurs on dentin surface. Possibly the high content of mineral ions decreased the surface energy of the demineralized dentin. The opposite
can be observed when biomimetic remineralization happens, using CPP-ACP or P11-4. In this
case, the mineralization occurs by organized crystal formation guided by the scaffold. This kind of surface can experience a high surface energy and a high level of wettability by resin monomers providing the highest µTBS.
The results of the study indicated that the treatment with P11-4, with a single application showed significant improvement on µTBS. Despite the fact that there was significant difference between the DCLL models, the µTBS values still show higher values. For the biological model
when the peptide P11-4 was used no significant difference from sound dentin was observed. It is suggested that the use of the P11-4 is able to nucleate hydroxyapatite and to promote repair of caries-like lesions in vitro. We have no knowledge in the literature of any report in which the treatment with P11-4 has been conducted in demineralized dentin, associating its effects with bonding procedures. However, research groups using other peptides, observed that this strategy mimics the functions of non-collagenous proteins (NCPs) [46, 47]. This suggests that the action of the P11-4 peptide reflects the reinforcing of demineralized collagen matrix.
The potential for enamel lesion repair of P11-4 gel may mimic the functions of NPCs
[15, 48, 49]. Several studies indicate that P11-4 forms three dimensional fibrillar hierarchical structures resulting in gels in response to specific environmental triggers [48, 49]. Assembled P11-4 forms scaffold-like structures with negative charge domains, mirroring biological macromolecules in mineralised tissue extracellular matrices (ECM) [49].
Fluoride is well-known for its proved anticariogenic and antimicrobial capacity. Its ability to prevent demineralization and promot remineralization by calcium phosphate precipitation on dental surface by reducing the dissolution of hydroxyapatite [50]. Thus, the effect of demineralized dentin treated with NaF on the µTBS, improved the µTBS compared to demineralized dentin, but did not reach the sound dentin µTBS. It has to be considered that the dentin etching with 35% phosphoric acid increases the bonding efficacy of dental adhesives and removes the smear layer and the superficial part of the dentin, opening dentin tubules, demineralising the dentin surface and increasing the microporosity of the intertubular dentin [51]. Despite the phosphoric acid used in the etching procedure removing part of the mineral deposits, maybe the reinforcement provided by NaF on demineralized dentin structure would improve the µTBS compared to demineralized dentin [52, 53]. However, the differences found with µTBS of Chemical and Biological models can be explained by the deeper demineralization provided by biological model than the chemical one. Therefore, it is known that the mineral deposition of fluorides occurs on surface and generally causes hyper mineralization of the dentin and in dentin tubules [10, 54-56]. The disorganized precipitation and deposit of mineral on dentin may mechanically obliterate the tubules reducing the performance of the restorative material [56], providing less efficiency of NaF treatment.
Another important aspect is that the NaF and Curodont Repair treatments showed statistically significant difference between the DCLL. With regard to the treatment with NaF, a previous study [57] showed that this treatment is able to reduce the subsurface dentin demineralization compared with the control from 30 to 50 µm depth. At the other depths (60-220 µm) NaF showed no positive effect. Such results may be attributed to a possible reaction
between NaF and demineralized dentin, producing soluble fluoride. In the present study, the demineralized dentin associated with NaF treatment exhibit mixed failure type. This result may be associated with a surface reinforcement, which was partially removed by the etching with the phosphoric acid during bonding procedures.
Except of with the treatment with NaF, Curodont Repair has shown no negative influence on the bond strength, as the biological DCLL it did not differ from sound dentin. This behavior can be attributed to different interactions with the demineralized substrate, since the depth produced by the biological model DCLL was higher than chemical one. It has been reported that the P11-4 scaffold can act as nucleator for hydroxyapatite, infiltrating into the porous lesions and increase the mineral diffusion within the lesion, restructuring the affected tissue [15].
Moreover, regardless the artificial caries development model, this study results suggest that the use of, remineralizing agents can reinforce the mechanical properties of demineralized dentin and would favor the durability of resin-dentin bonds, since the demineralized substrate treated provides an organized mineral surface. However, the degree of improvement in bonding strength is dependent on the artificial caries development model and dentin treatment. Futher studies have to be carried out in order to observe the long-term efficacy of the remineralized dentin bonded to adhesive systems.
CONCLUSION
Based on the results of this study it can be concluded that:
MI Paste™ and Curodont™ Repair recovered higher μTBS values when compared to sound dentin. Each agent shows a different interaction in each evaluated condition. The remineralizing treatment of demineralized dentin is a potential approach for increasing bond strength of etch & rinse adhesive system.
Competing interests
The authors declare that they have no competing interests.
Funding
This work was supported by grants of the Cordination for the Improvement of Higher Education Personnel (CAPES) and São Paulo Research Foundation (FAPESP) (2011/16634-3, 2015/12660-0) by the research financial support and to Credentis (AG, Dorfstrasse, Windisch, Switzerland) for providing one of the materials tested.
Ethical Approval
This work was approved by the Ethics Committee of Piracicaba Dental School, University of Campinas (Protocol number 123/2014).
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