JOSSARIA PEREIRA DE SOUSA
USO DE UM PEPTÍDEO SELF-ASSEMBLING NA
BIOMODIFICAÇÃO DA DENTINA AFETADA POR CÁRIE:
ANÁLISE QUÍMICA E BIOMECÂNICA
USE OF A SELF-ASSEMBLING PEPTIDE ON THE
BIOMODIFICATION OF CARIES AFFECTED DENTIN:
CHEMICAL AND BIOMECHANICAL ANALYSES
Piracicaba 2018
USO DE UM PEPTÍDEO SELF-ASSEMBLING NA
BIOMODIFICAÇÃO DA DENTINA AFETADA POR CÁRIE:
ANÁLISE QUÍMICA E BIOMECÂNICA
USE OF A SELF-ASSEMBLING PEPTIDE ON THE
BIOMODIFICATION OF CARIES AFFECTED DENTIN:
CHEMICAL AND BIOMECHANICAL ANALYSES
Tese apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutora em Odontologia, na Área de 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.
Orientadora: Profa. Dra. Regina Maria Puppin Rontani
Este exemplar corresponde à versão final da Tese defendida pela aluna Jossaria Pereira de Sousa e orientada pela Profa. Dra. Regina Maria Puppin Rontani.
Piracicaba 2018
Ficha catalográfica
Universidade Estadual de Campinas
Biblioteca da Faculdade de Odontologia de Piracicaba Marilene Girello - CRB 8/6159
Sousa, Jossaria Pereira de, 1989-
So85u Uso de um peptídeo self-assemblingna biomodificação da dentina afetada por cárie : análise química e biomecânica / Jossaria Pereira de Sousa. – Piracicaba, SP : [s.n.], 2018.
Orientador: Regina Maria Puppin Rontani.
Tese (doutorado) – Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba.
Sou1. Dentina. 2. Cárie dentária. 3. Remineralização dentária. 4. Peptídeos. 5. Inibidores de proteases. 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: Use of a self-assembling peptide on the biomodification of caries-affected dentin : chemical and biomechanical analyses
Palavras-chave em inglês: Dentin Dental caries Tooth remineralization Peptides Protease inhibitors
Área de concentração: Odontopediatria
Titulação: Doutora em Odontologia
Banca examinadora:
Regina Maria Puppin Rontani [Orientador] Kamila Rosamilia Kantovitz
Fabíola Galbiatti de Carvalho Alan Roger dos Santos Silva Sérgio Luiz Pinheiro
Data de defesa: 03-08-2018
A Comissão Julgadora dos trabalhos de Defesa de Tese de Doutorado, em sessão pública realizada em 03 de Agosto de 2018, considerou a candidata JOSSARIA PEREIRA DE SOUSA aprovada.
PROFª. DRª. REGINA MARIA PUPPIN RONTANI
PROF. DR. SÉRGIO LUIZ PINHEIRO
PROFª. DRª. FABÍOLA GALBIATTI DE CARVALHO
PROFª. DRª. KAMILA ROSAMILIA KANTOVITZ
PROF. DR. ALAN ROGER DOS SANTOS SILVA
A Ata da defesa com as respectivas assinaturas dos membros encontra-se no processo de vida acadêmica do aluno.
Dedico este trabalho aos meus queridos pais João
Everaldo Freitas de Sousa e Elba Pereira de Sousa,
que foram a base forte da nossa família, que não
mediram esforços para prover as melhores oportunidades
aos seus três filhos, e que sempre estiveram ao meu lado
me apoiando durante esse longo caminho na vida
acadêmica.
A Deus, em primeiro lugar, que me deu o dom da vida, que sempre esteve ao meu lado nos
momentos felizes e difíceis dessa caminhada, que iluminou os meus passos, que me fez forte,
determinada a alcançar os meus sonhos e nunca permitiu que eu desistisse.
À minha orientadora Profa. Dra. Regina Maria Puppin Rontani, por ter me acolhido de
forma tão carinhosa há 4 anos na FOP/UNICAMP. Por ter depositado em mim tanta
confiança para execução deste trabalho. Pela dedicação, paciência, disponibilidade, e
serenidade ao conduzir os seus alunos na carreira acadêmica. Obrigada pelos ensinamentos
em Odontopediatria, pelas conversas, pelo apoio e por acreditar no meu potencial. Obrigada
de coração.
Aos Profs. Fábio Dupart Nascimento, Ivarne Tersariol, e Rafael Guzzela pela importante
participação no desenvolvimento da tese, por abrirem as portas dos laboratórios da
UNIFESP e do Centro Interdisciplinar de Mogi das Cruzes para execução de vários
experimentos. Obrigada pela oportunidade de aprender novas metodologias, e por nunca
pouparem esforços para me ajudar no processo de execução e redação do manuscrito.
À Profa. Dra. Ana Karina Bedran-Russo, orientadora durante o estágio no exterior pelo
College of Dentistry da University of Illinois at Chicago (UIC), por abrir as portas do
seu laboratório e me receber tão bem. Obrigada pelos ensinamentos, pela oportunidade de
conhecer novas metodologias, por contribuir para o meu enriquecimento intelectual. Obrigada
pela atenção, por ser um apoio e conforto a alguém que está tão distante de seu país, amigos
e família. Esse período foi importantíssimo para o meu crescimento pessoal e profissional.
Aos meus pais Elba e Freitas, mais uma vez, que tanto torcem pelo meu sucesso, amam-me
acima de tudo, estão ao meu lado em pensamento e dentro do meu coração, que sempre
prezaram pela educação de seus filhos, e me ensinaram princípios dos quais eu nunca
esquecerei, Obrigada por tudo que fizeram e fazem por mim. Tudo o que sou devo a vocês.
carinho e amor a todo reencontro em João Pessoa, e pelas boas energias emanadas para
finalização dessa tese.
A Luiz Filipe Barbosa Martins, ou meu amigo Filipe, primeira pessoa que me acolheu na
área de Odontopediatria na FOP/UNICAMP. Você foi o melhor amigo que eu poderia ter
aqui. Nós compartilhamos a mesma área de atuação, mesma orientadora, e até a mesma
casa, e durante todo esse tempo nunca nos desentendemos. Obrigada por ser meu segundo
orientador, por me mostrar o funcionamento do laboratório, por ensinar tudo o que sabia de
pesquisa e Odontopediatria. Obrigada pelas conversas, pelos desabafos, pela companhia
diária, pelos almoços no bandeijão, Obrigada por ser quem é, e por sempre prezar pela
amizade em detrimento à competição, Nós somos um time e uma família. Sentirei muito a sua
falta.
A Kelly Scudine e Lívia Alves, por serem as melhores roommates que eu poderia ter. Lívia,
pela nossa amizade desde a graduação na UFPB, por me acolher em Piracicaba, dar-me
todo o suporte inicial e ser minha família também. Vivemos momentos memoráveis que eu
nunca esquecerei. Obrigada pela torcida mútua em busca das realizações profissional e
pessoal. Kelly, por seu jeitinho alegre e saltitante, obrigada pela companhia diária na
Pediatria e em casa. Obrigada por ser firme quando tem que ser, e por acreditar em mim.
Vocês duas sabem que os últimos meses foram bem esgotantes, mas a amizade de vocês me
fortaleceu. Amo até o infinito.
À Faculdade de Odontologia de Piracicaba – UNICAMP, na pessoa do seu Diretor
Prof. Dr. Guilherme Elias Pessanha Henriques e Diretor Associado Prof. Dr. Francisco
Haiter Neto.
À Profa. Dra. Karina Gonzales Silveiro Ruiz, coordenadora geral dos cursos de
Pós-Graduação e ao Prof. Dr. Francisco Carlos Groppo, coordenador do curso de Pós
Graduação em Odontologia.
À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes) pela concessão de
bolsa de doutorado no primeiro ano do curso.
À Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp #2014/22899-8,
#2017/08164-3 e 2015/12660-0), pelo bolsa de Doutorado concedida nos demais anos,
pela concessão de Bolsa BEPE para estágio no exterior e pelo apoio financeiro para
execução da pesquisa.
Ao Departamento de Odontologia Restauradora da University of Illinois at Chicago –
UIC (Chicago, IL, EUA), por todo apoio e solicitude para o desenvolvimento do estágio
no exterior no laboratório da Profa. Dra. Ana Karina Bedran Russo. À Yvette Allania,
Lívia Trevelin, Camila Zamperini, Mariana Reis e Alyne Simões pelo acolhimento no
laboratório, pela amizade criada, pelo suporte, e por todo o conhecimento compartilhado e
por toda generosidade comigo. Meu muito obrigada.
Às professoras do Departamento de Odontologia Infantil da área de Odontopediatria da
Faculdade de Odontologia de Piracicaba, professoras Dra. Marinês Nobre dos Santos
Uchôa e Dra. Fernanda Miori Pascon pela coordenação da área de Odontopediatria
durante os quatros anos de Doutorado. Às demais professoras, Profa. Dra. Maria Beatriz
Duarte Gavião, Profa. Dra. Regina Maria Puppin Rontani, e Profa. Dra. Carolina Steiner
profissionais e professoras.
Às professoras colaboradoras do Departamento de Odontologia Infantil da área de
Odontopediatria da Faculdade de Odontologia de Piracicaba, Profa. Dra. Paula Midori
Castelo Ferrua, Profa. Dra. Taís de Souza Barbosa e Profa. Dra. Kamila Rosamilia
Kantovitz, pela convivência e troca de ensinamentos ao longo desses anos.
Aos professores do Departamento de Odontologia Infantil da área de Ortodontia da
Faculdade de Odontologia de Piracicaba, Profa. Dra. Maria Beatriz Araújo Magnani,
Profa. Dra. Vânia Célia Vieira de Siqueira, Prof. Dr. João Sarmento Pereira Neto e
Prof. Dr. Eduardo César Almada Santos, pela convivência diária, pelos ensinamentos e
transmissão de conhecimentos.
À secretária do Departamento de Odontologia Infantil, Shirley Rosana Sbravatti Moreto,
pela disponibilidade e prontidão, e ao técnico de laboratório da Odontopediatria, Marcelo
Corrêa Maistro, pela disposição em ajudar os alunos na preparação de soluções e no
manuseio dos equipamentos. Obrigada pelo seu bom-humor diário.
Aos colegas de turma de Doutorado da Área de Odontopediatria, Darlle Araújo, Lenita
Lopes, Luiz Filipe Martins e Micaela Cardoso pela amizade, troca de experiências, pela
convivência diária e por inúmeros momentos vivenciados.
Aos colegas do Programa de Pós-Graduação em Odontologia, Área de Odontopediatria,
Aline Laignier, Aline Pedroni, Aline Tavares, Andréia Cardoso, Camila Nobre, Carlos
Velazco, Cynthia Arias, Daniela Cibim, Daniela Prado, Emerson Tavares, Fernanda
Mazoni, Gabriela Borghi, Juana Huamani, Karina Sousa, Kelly Moreira, Lívia
Nazaretti, Luciana Solera, Maria Carolina Salomé, Pedro Rebouças, Priscila Giovani,
Rafaela Costa, , Raquel Kobayashi, Rosa Abuhadba e Samuel Chaves, por todos esses
Aos amigos da Paraíba que também ingressaram na FOP/UNICAMP, Elis Lira,
Rodrigo Lins, Louise Dornelas, Mayara Abreu, Jaíza Araújo, Renally Wanderley,
Marina Moreno, Larissa Fernandes, Bruno Mariz, que juntos tornaram-se uma grande
família e me fizeram sentir mais perto do aconchego de casa. Amo vocês.
Aos amigos que fiz ao longo dessa jornada na FOP-UNICAMP, nas áreas de Cariologia,
Saúde Coletiva, Farmacologia, Fisiologia, Microbiologia, Prótese Dentária, Dentística,
Periodontia, Cirurgia, Endodontia, Materiais Dentários, Patologia e Radiologia.
Aos alunos de graduação, os quais foram essenciais para o desenvolvimento das minhas
habilidades como docente.
A todos que direta ou indiretamente contribuíram para a realização deste trabalho.
Recentemente um peptídeo do tipo self-assembling P11-4 foi desenvolvido com o objetivo de
melhorar a remineralização de lesões inicias de cárie, entretanto, sua aplicabilidade no tecido dentinário não foi aventada. O presente estudo investigou a interação do P11-4 com
componentes inorgânicos e orgânicos da matriz dentinária; e posteriormente, verificou a influência do P11-4 na interface de união à dentina artificialmente afetada por cárie (DaC). Os
experimentos foram divididos em três assuntos: 1-nucleação de hidroxiapatita (HAP), 2-interação com colágeno tipo I, e 3-influência na interface da união resina/DaC. Alteração na fluorescência intrínseca do P11-4 foi avaliada a fim de verificar a afinidade deste peptídeo com
íons cálcio. Análise de espalhamento de luz dinâmico (DLS) e monitoramento do pH mensuraram a razão de formação de HAP, enquanto a Microscopia de Força Atômica (AFM) avaliou o tamanho dessas partículas formadas. Ressonância de Plasma de Superfície (SPR) e AFM foram aplicadas para avaliar a interação do peptídeo com fibras colágenas. A inibição do P11-4 sobre a degradação do colágeno foi observada por meio de ensaios fluorimétricos e
SDS-PAGE. Por fim, avaliou-se a influência do peptídeo P11-4 na interface de união
resina/DaC, onde oitenta e quatro terceiros molares humanos livres de cárie foram requeridos. Os dentes foram randomizados em seis grupos, de acordo com o substrato (DH:dentina hígida, DaC:dentina afetada por cárie, e DaC+P11-4:DaC tratada com P11-4), e tempo de
armazenamento (24 h e 6 meses). Procedimento adesivo foi realizado com sistema adesivo convencional AdperTM Single Bond 2, e posteriormente bloco de resina composta com 4 mm de espessura foi construído. As amostras foram armazenadas em água deionizada a 37° C por 24 h, sendo então realocadas nos três ensaios: resistência de união à microtração-µTBS (n=8), nanoinfiltração (n=3) e zimografia in situ (n=3). A normalidade dos dados foi verificada pelo teste Kolmogorov Sminorv, e testes estatísticos apropriados foram utilizados para avaliar diferença entre os grupos, considerando p<0.05. P11-4 teve sua fluorescência diminuída com o
aumento da concentração de Ca2+ (Kd=0.63±0.07 mM). DLS e monitoramento do pH
mostraram que o P11-4 aumentou significantemente a razão de conversão de partículas de
fosfato de cálcio amorfo (ACP) para fosfato octacálcico (OCP), enquanto que a velocidade de conversão de OCP para HAP decresceu. AFM demonstrou que o P11-4 reduziu a variabilidade
no tamanho das partículas de HAP formadas. SPR e AFM mostraram que o P11-4 se liga às
fibras de colágeno tipo I, aumentando o seu diâmetro de 214±4 nm para 308±5 nm (p<0.0001). SDS-PAGE evidenciou diminuição na degradação do colágeno com o aumento da concentração do P11-4. Tratamento da DaC com P11-4 melhorou, imediatamente, a
atividade proteolítica na camada híbrida foi observada (p<0.05). Tais efeitos reduziram ao longo do tempo, porém mantiveram-se superior ao grupo DaC. Em conclusão, o presente estudo verificou que o P11-4 contribui para a nucleação de HAP, inibe a degradação do
colágeno tipo I, e melhora a união imediata à DaC.
Palavras-chave: dentina; cárie dentária; remineralização dentária; peptídeos; inibidores de
Recently, a rationally designed self-assembling peptide P11-4 has been developed to enhance
remineralization on initial caries lesions; yet, its applicability on dentin tissue remains unclear. Thus, the present study investigated the interaction of P11-4 with the inorganic and
organic dentin compounds. Furthermore, the effect of the self-assembling peptide on the bonding interface to artificial caries-affected dentin (CaD) was studied. Experiments were divided in three main arms: 1- nucleation of hydroxyapatite, 2- interaction with collagen type-I, and 3- influence on the resin/CaD bonding interface. Fluorescence changes in the P11-4
tryptophan was measured to verify the affinity of P11-4 to calcium ions. Dynamical light
scattering (DLS) and pH monitoring were used to evaluate the rate of HAP formation in the presence of P11-4, while Atomic Force Microscopy (AFM) screened the size of HAP particles
formed. Surface Plasmon Resonance (SPR) and AFM were used to measure the interaction of the peptide P11-4 with collagen fibers. Fluorometric and SDS-PAGE assays were performed to
evaluate collagen degradation inhibition by P11-4. Finally, the influence of P11-4 on bonding
interface to CaD was verified. Eighty-four extracted human third molars were randomized in six different groups (n=11), according to dentin substrate (SD:sound dentin, CaD:caries-affected dentin, and CaD + P11-4:CaD treated with P11-4), and storage-time (24 h and 6
months). Adhesion to substrates were done with the two-step etch-and-rinse adhesive system AdperTM Single Bond 2, and a 4-mm-thick resin composite block was built. Samples were stored in deionized water at 37°C for 24 h. Afterwards, samples of each group were allocated into to three different analysis: microtensile bond strength -µTBS (n=8), nanoleakage (n=3) and in situ zymography (n=3). Kolmogorov Sminorv test was applied to evaluate data normality, and appropriate tests were chosen to verify difference between groups, p< 0.05. P11-4 had its intrinsic fluorescence decayed in function of the Ca2+ concentration (Kd=
0.63±0.07 mM). Dynamical light scattering and pH monitoring showed P11-4 increased
significantly the rate of conversion of amorphous calcium phosphate (ACP) into octacalcium phosphate (OCP), while the velocity of conversion of OCP into HAP phase was decreased. AFM analysis demonstrated P11-4 standardized the HAP particle growth by reducing the
particle size variability. SPR and AFM explorations showed P11-4 bind to immobilized
collagen I fibers, increasing its diameter from 214±4 nm to 308±5 nm (p<0.0001). Complementary, SDS-PAGE demonstrated the increase of P11-4 concentrations impaired the
collagen degradation from bacterial collagenase. Regarding bonding analysis, the immediate treatment of CaD with P11-4 enhanced the mechanical performance of bonding interface
but still higher compared to CaD group. In conclusion, the present study observed that P11-4
induces the nucleation of hydroxyapatite and protects Collagen Type I against bacterial collagenase degradation. In addition, P11-4 improves the stability of the hybrid layer formed
by artificial caries-affected dentin.
SUMÁRIO
1. INTRODUÇÃO ... 16 2. ARTIGO: Prevention of collagen type I proteolytic degradation using self-assembling peptide P11-4: improving the biomechanical properties of the resin/dentin interface …… 21
3. CONCLUSÃO ... 53 REFERÊNCIAS ... 54 APÊNDICE 1 - Sequência da metodologia empregada para o tópico: Influência do peptídeo P11-4 na estabilidade de união resina/dentina afetada por cárie... 57
ANEXOS ... 64 Anexo 1 - Comprovante de aprovação no Comitê de Ética em Pesquisa da FOP/UNICAMP... 64 Anexo 2 - Comprovante de submissão de artigo científico... 65
1 INTRODUÇÃO
A cárie dentária é uma doença biofilme-mediada, açúcar-dependente, multifatorial e dinâmica, resultado de um desequilíbrio nos ciclos de desmineralização e remineralização dos tecidos dentais mineralizados (Bedran-Russo e Zamperini, 2017; Pitts et al., 2017; Weber et al., 2018). Localmente, a lesão de cárie se caracteriza pela gradual desmineralização do esmalte e/ou cemento, podendo atingir tecidos dentários mais profundos, como o complexo dentino-pulpar (Featherstone 1996; Bertassoni et al., 2009). Diferentemente do esmalte, a lesão de cárie em dentina progride rapidamente, uma vez que se trata de um tecido estruturalmente complexo e hidratado, com 30% de seu volume constituído por matéria orgânica (Bertassoni et al. 2009; Tjaderhane et al., 2012). Assim, a lesão de cárie em dentina caracteriza-se pela perda progressiva de mineral extra e intrafibrilar, com exposição do componente orgânico e, por último, a degradação permanente da matriz colagenosa (Tjäderhane et al. 2013; Bedran-Russo e Zamperini, 2017).
Fusayama e Terachima (1972) foram os pioneiros a caracterizarem estruturalmente o tecido dentinário cariado, diferenciando-o em duas zonas: a dentina infectada e a dentina afetada pela cárie. A primeira e mais superficial trata-se de uma zona de destruição tecidual, com invasão bacteriana, extensa desmineralização e desnaturação das fibrilas colágenas, enquanto que a segunda e mais profunda apresenta-se como um substrato parcialmente desmineralizado, com fibrilas colágenas levemente alteradas, porém passíveis de reestruturação. Tais características fazem da dentina afetada por cárie o substrato de escolha nas abordagens restauradoras atuais, as quais tem se baseado em um conceito de Odontologia Minimamente Invasiva e máxima preservação tecidual (Pinna et al., 2015; Toledano et al., 2017; Giacomini et al., 2017).
Apesar disso, a adesão à dentina afetada por cárie apresenta certas peculiaridades, tendo em vista que alterações morfológicas são identificadas nesse substrato. A porosidade da dentina intertubular, por exemplo, resulta em um condicionamento ácido mais agressivo (Nakajima et al. 2011). Além disso, o maior conteúdo aquoso, a presença de uma smear layer espessa, e a formação de depósitos minerais ácido-resistentes na superfície dentinária são fatores que interferem no embricamento dos monômeros resinosos ao longo das fibrilas colágenas (Yoshiyama et al, 2002; Innoue et al., 2006). Desta forma, uma adesão inadequada é alcançada entre compósito e dentina, culminando na diminuição das propriedades mecânicas e longevidade dessa interface e(Pinna et al., 2015; Giacomini et al., 2017; Costa et al., 2017).
Paralelamente, recentes investigações têm destacado o papel de proteases intrínsecas da dentina na progressão da lesão de cárie e na longevidade das restaurações (Mazzoni et al., 2012; Tjaderhane et al., 2013; Vidal et al., 2014; Mazzoni et al., 2015). Tais enzimas, conhecidas como metaloproteinases da matriz (MMPs) e cisteíno-catepsinas (CCs), são expostas e ativadas pela desmineralização das fibrilas colágenas, decorrente da ação dos ácidos derivados do metabolismo bacteriano, e/ou pelo condicionamento ácido inerente aos procedimentos adesivos (Tezvergil-Mutluay et al., 2017). Assim, as MMPs e CCs atuam de maneira sinérgica em diferentes porções do colágeno, causando a solubilização permanente dessa molécula, e afetando por último a estabilidade da camada híbrida (Giacomini et al., 2017; Turco et al., 2017).
Estudos atuais têm sido categóricos ao considerarem a remineralização da dentina afetada por cárie o ponto chave para a longevidade das restaurações adesivas (Zhong et al., 2013; Niu et al., 2014; Tezvergil-Mutluay et al., 2017). A dentina afetada por cárie, apesar de desmineralizada, apresenta algumas regiões intrafibrilares intactas com presença de mineral
(Bahari et al., 2014). Tais regiões, conhecidas como gap zones, são consideradas o ponto para
nucleação dos cristais de hidroxiapatita (Bertassoni et al., 2009). Além disso, a presença de mineral intrafibrilar nesse substrato mantém a estrutura secundária das moléculas de colágeno, funcionando como arcabouço para a remineralização extrafibrilar da dentina e recuperação de suas propriedades mecânicas (Bedran-Russo e Zamperini, 2017).
Ao longo dos anos, diferentes estratégias vêm sendo aplicadas em busca da completa remineralização da dentina afetada por cárie (Bertassoni et al., 2009). Tais abordagens incluem a aplicação de soluções fontes de fluoreto, cálcio e fosfato (Niu et al., 2014). Entretanto, essas estratégias têm falhado em seu objetivo pelo fato de promoverem a formação e deposição heterogênea de cristais de fosfato de cálcio na superfície dentinária, porém sem alcançar as regiões mais profundas da matriz dentinária (Zhong et al., 2013). Baseando-se nisso, novos esforços têm sido direcionados ao desenvolvimento de materiais que possam induzir a formação mineral em uma escala nanométrica, mimetizando a biomineralização do tecido dentinário pelas proteínas da matriz dentinária (DMP-1).
Tay e Pashley (2008) foram os primeiros a transpor os conceitos da biomineralização da dentina, identificando possíveis análogos sintéticos das DMP-1. Agentes como o ácido poliacrílico, trimetafosfato de sódio e ácido polivinilfosfônico teriam a função de estabilizar íons de cálcio e fosfato em solução, promovendo assim a formação de nanoprecursores de fosfato de cálcio amorfo metaestáveis nos espaços intrafibrilares do colágeno. Sequencialmente, tais análogos promoveriam a transformação dos nanoprecursores em
cristais de hidroxiapatita, ordenando o crescimento desses cristais ao longo dos espaços intra e extrafibrilares (Niu et al., 2014; Padovano et al., 2015). Portanto, diversos estudos têm avaliado e comprovado a efetividade de tais agentes na remineralização das interfaces adesivas, seja como pré-tratamento da dentina ou incorporados aos sistemas adesivos contemporâneos (Niu et al., 2014; Abuna et al., 2016).
De forma alternativa uma segunda fonte de agentes biomiméticos remineralizadores vem sendo destacada, os peptídeos do tipo self-assembling. A inovação dessa abordagem se apresenta pelo fato de uma simples molécula com uma sequência de aminoácidos predefinida ter a capacidade de se transformar em uma superestrutura funcionalmente complexa (Whitesides e Grzybowski, 2002). Portanto, peptídeos do tipo self-assembling poderiam servir como promissores arcabouços para a deposição e crescimento ordenado de minerais, além de apresentarem-se como materiais biocompatíveis aos tecidos dentais, fazendo dessas moléculas interessantes alvos para aplicação clínica (Zhong et al., 2013).
Particularmente, um peptídeo de auto-organização, o P11-4, tem ganhado bastante
notoriedade no que diz respeito às suas propriedades remineralizadoras. O P11-4 trata-se de um
peptídeo contido no Curodont Repair®, composto por uma cadeia de 11 aminoácidos (CH3CO-Gln-Gln-Arg-Phe-Glu-Trp-Glu-Phe-Glu-Gln-Gln-NH2) que, ao ser submetido a
específicos estímulos (queda de pH), passa de um estado líquido isotrópico para o de gel elastomérico (Aggeli et al., 2003). Tal propriedade permite a formação de um complexo tridimensional do tipo folha-beta usado como arcabouço para a deposição mineral (Brunton et al., 2013).
Estudos in vitro têm reportado o efeito remineralizador do P11-4 sobre lesões de cárie
não cavitadas em esmalte (Kirkham et al., 2007; Jablonski-Momeni et al., 2014; Schmidlin et al., 2016). Estudo clínico randomizado demonstrou que a aplicação do P11-4 combinado ao
flúor facilitou o processo de remineralização de lesões iniciais de cárie em esmalte, bem como a inativação dessas lesões após 6 meses de avaliação, em comparação a aplicação de verniz fluoretado, não produzindo quaisquer efeitos adversos relacionados ao tratamento (Alkilzy et al., 2018). Baseando-se nesses achados e nas características químicas e estruturais desse peptídeo, sugere-se que o P11-4penetre nas porosidades da lesão do esmalte, formando um
arcabouço tridimensional. Consecutivamente, os radicais aniônicos do P11-4 passariam a atrair
íons cálcio provenientes da saliva, e estes íons fosfato, resultando na progressiva e ordenada precipitação de cristais de fosfato de cálcio dentro da lesão de esmalte (Kirkham et al., 2007; Kyle et al., 2010).
Apesar de as evidências científicas demonstrarem que o P11-4 possivelmente controla a
deposição e crescimento de cristais de hidroxiapatita no esmalte (Silvertown et al., 2017; Kind et al., 2017), o comportamento desse peptídeo na dentina permanece desconhecido. Deste modo, o presente estudo apresentou como objetivos verificar o potencial do P11-4 em
nuclear hidroxiapatita, bem como avaliar a interação desse peptídeo com importante componentes da matriz dentinária, colágeno e proteases da matriz. E em segundo momento, a presente investigação almejou avaliar o efeito do tratamento da dentina artificialmente afetada por cárie com o peptídeo P11-4 na estabilidade das restaurações adesivas, verificando
alterações estruturais e mecânicas dessa interface.
Desta forma, o presente estudo foi conduzido em três principais tópicos: nucleação de hidroxiapatita, interação com colágeno tipo I, e influência na interface da união resina/dentina afetada por cárie; os quais encontram-se descritos no Quadro 1, e será apresentado em capítulo único: (1) Prevention of collagen type I proteolytic degradation using self-assembling peptide P11-4: Improving the biomechanical properties of the resin/dentin interface.
Quadro 1. Delineamento experimental do estudo
2 ARTIGO:
Prevention of collagen type I proteolytic degradation using self-assembling peptide P11
-4: Improving the biomechanical properties of the resin/dentin interface
Jossaria P. de Sousa1, Rafael G. Carvalho2, Luiz F. Barbosa-Martins1, Ricardo J. S. Torquato2, Kátia C. U. Mugnol3, Fábio D. Nascimento3, Ivarne L. S. Tersariol2* and Regina M. Puppin-Rontani1*
1Department of Pediatric Dentistry, Piracicaba Dental School, University of Campinas -
UNICAMP, Campinas, SP, Piracicaba, São Paulo, 13414-903, Brazil.
2Department of Biochemistry, Federal University of São Paulo, São Paulo, SP, 04021-001,
Brazil.
3Interdisciplinary Center of Biochemistry Investigation, University of Mogi das Cruzes, Mogi
das Cruzes, SP, 08780-911, Brazil.
Financial Support: FAPESP (Grants 2013/05822-9, 2014//22899-8 and 2015/03964-6), CNPq and CAPES
*Corresponding Author: Fábio D. Nascimento
Interdisciplinary Center of Biochemistry Investigation, University of Mogi das Cruzes Address: 200, Dr. Cândido Xavier de Almeida e SouzaAvenue, Mogi das Cruzes, SP, Brazil, Zip Code: 08780-911 / Fax: +55 11 4799-5233 / e-mail: [email protected]
ABSTRACT
The major goal in operative dentistry is to develop a true regenerative approach that totally recovers hydroxyapatite crystals within the caries lesion. Recently, a rationally designed self-assembling peptide (SAP) P11-4 (Ace-QQRFEWEFEQQ-NH2) has been developed to
enhance remineralization on initial caries lesions; yet, its applicability on dentin tissues remains unclear. Thus, the present study investigated the interaction of P11-4 with the
inorganic and organic dentin components. Furthermore, the effect of the self-assembling peptide on the proteolytic activity and mechanical properties of the bonding interface to artificial caries-affected dentin was studied. Intrinsic fluorescence analysis, dynamic light scattering with pH monitoring and Atomic Force Microscopy (AFM) showed SAP P11-4
induces hydroxyapatite (HAP) crystal nucleation process improving the structural match among HAP crystallite aggregates. Surface Plasmon Resonance (SPR) and AFM indicated that SAP P11-4 binds to Collagen Type I fibers, increasing their diameter from 214 ± 4 nm to
308 ± 5 nm (P< 0.0001). SAP P11-4 also increased the resistance of Collagen Type I fibers
against the proteolytic activity of collagenases. The immediate treatment of artificial caries-affected dentin with SAP P11-4 enhanced the µTBs of the bonding interface (P< 0.0001)
reaching values of sound dentin, and decreasing the proteolytic activity at the hybrid layer (HL); however, such effects impaired after 6 months of water storage (P< 0.05). In conclusion, the present study observed that SAP P11-4 induces the nucleation of
hydroxyapatite and interacts with Collagen Type I. In addition, SAP P11-4 improves the
stability of the hybrid layer formed by artificial caries-affected dentin.
Key-words: dentin, tooth remineralization, protease inhibitors, self-assembling peptide,
1. Introduction
Dental caries is a biofilm-mediated, sugar-driven, multifactorial, dynamic disease that results in the phasic demineralization and remineralization of dental hard tissues [1]. These tissues have poor regeneration capability because of the lack of regenerative cells and vascularization [2]. In the complex caries progression process involving dietary sugars, bacterial metabolism and demineralization, the collagenous organic matrix becomes exposed and destroyed by resident [3] and bacterial proteases [4], allowing the lesion to expand. It is possible to find numerous non-, or minimally invasive therapies for caries, such as hygiene education [5], fluorides [6], phosphopeptide compounds [7], xylitol [5], and infiltrative resins [8]; however, when caries progresses to the point of breaking down the dental tissues, composite restorations are imperative to preserve the tooth functionality [9].
According to the current philosophy of minimal intervention, the outer, infected, and highly disorganized layer of dentin should be removed, while the inner layer, called caries-affected, can be remineralized and maintained in bonding procedures [10-14]. In fact, the remineralization of the partially demineralized dentin underlying the hybrid layer would enhance the mechanical properties of this interface, ultimately improving the durability of resin composite restorations [15-17]. Additionally, remineralization of dentin seems to have a critical role upon the collagen degradation, the presence of mineral within the intrafibrilar collagen spaces blocks the mobility of endogenous enzymes within the extracellular matrix, thereby inhibiting their proteolytic activity [16]. Thus, a true regenerative approach that enables de novo hydroxyapatite crystals formation within the carious lesion remains one of the major goal in the operative dentistry field.
Based on today’s understanding of the dental biomineralization process, new efforts have been made to develop synthetic analogs of non-collagenous proteins (NCPs), which are involved in the events of nucleation and growth of hydroxyapatite (HAP) crystals in hard tissues [18]. Such analogs have been designed mirroring the amphiphilic characteristics of NCPs, with polar groups complexing inorganic ions, and non-polar sidechains governing the matrix-matrix interactions [19]. The harnessing of peptides in remineralization approaches, arises due to their biocompatibility, the ease of rationally designing specific motifs in order to engineer particular properties [16,20]. A relatively simple peptide with a molecular recognition motif can self-assemble into much more complex and functional superstructures [21]. Moreover, a relatively short peptide sequences that mimics natural proteins may allow for better, ordered growth of mineral crystals [19].
Recently a rationally designed self-assembling peptide (SAP) P11-4
(Ace-QQRFEWEFEQQ-NH2) was introduced onto the market. Designed to enhance
remineralization and inhibit demineralization on enamel subsurface lesions [22]. P11-4 is an
eleven-amino acid peptide that undergoes well-characterized hierarchical self-assembly into three-dimensional fibrillar scaffolds in response to specific environmental triggers [23,24], offering a new generation of well-defined biopolymers with a range of potential applications [25]. Assembled P11-4 forms scaffold-like structures with negative charge domains available
on its surface, these domains attract Ca2+ and other related ions, inducing surface mediated de
novo precipitation of HAP [22,26]. Based on this, P11-4 would act on dentin as an analog to
NCPs, initiating dentine mineralization through hydroxyapatite nucleation [15,16].
Therefore, the present study aimed to investigate the interaction of fibrillar P11-4 with
inorganic and organic components of dentin as well as the effect of P11-4 on the proteolytic
activity and mechanical properties of the bonding interface to artificial caries-affected dentin. The null hypothesize tested was that P11-4 peptide does not interact with components of
dentin, and has no influence on the adhesion to caries-affected dentin.
2. Material and methods
2.1 Hydroxyapatite formation and interaction with components of dentin matrix
2.1.1 Mineralization reaction
The mineralization reaction was performed in ultrapure water (18.2 mMillipore, USA). A 10 mM calcium and a 10 mM phosphate stock solution were prepared by dissolving CaCl2*2H2O (Sigma) and K2HPO4 (Merck) in deionized water at 25 ± 1 ºC. CaCl2 and
K2HPO4 solutions of different concentrations were prepared by dilution from the
afore-mentioned stock solutions. The supersaturated solutions were made by adding an equal volume of CaCl2 solution to K2HPO4 solution, the calcium stock was stirred gently at 200 rpm
before adding phosphate stock (Ca/P=1.67). The reaction was followed under stirring for typically 0–2 h; these conditions were chosen to achieve initial phase separation of amorphous calcium phosphate (ACP) within 15–20 min. [27,28].
2.1.2 Intrinsic Fluorescence of the Peptide P11-4 Assays
The interaction of Ca2+ with P11-4 was analyzed by measuring the change in the
(0-3.45 mM). P11-4 has a tryptophan residue which is sensitive to Ca2+ binding [29]. The intrinsic
fluorescence of the tryptophan residue on P11-4 was monitored in 50 mM Tris-HCl buffer (pH
7.4) at 25 °C by measuring the emission of fluorescence (5 nm slit) between at 300-400 nm after excitation at λex = 295 nm (5 nm slit) in a RF-6000 spectrofluorometer (Shimadzu, São Paulo, Brazil), in the absence or in the presence of different concentrations of CaCl2 (0-3.45
mM). The 1 cm2 path-length quartz cuvette containing 2 ml of buffered P11-4 solution (5 μM)
was placed in a thermostatically controlled cell compartment under constant magnetic stirring in the spectrofluorometer for 5 min prior to the addition of small aliquots of a highly concentrated CaCl2 solution with minimal dilution (less than 5%) and the decrease in
fluorescence signal was recorded. The dependence of the relative fluorescence change, i.e., ΔF = (Fobs – F0) where F0 is the initial P11-4 solution fluorescence value and Fobs is the
observed fluorescence value after each addition of Ca2+ was analyzed by nonlinear least-squares data fitting by the binding Equation 1 according to Judice et al. [30], using GraFit 5.0 Software.
Where P is the total P11-4 concentration, Ca2+ represents the added CaCl2 concentration, n the
stoichiometry, Kd the dissociation constant and ΔFmax is the maximum fluorescence change.
The same procedure described above was used to analyze the effect of hydroxyapatite (HAP) supersaturated solutions upon the decay of the intrinsic fluorescence of P11-4. The
dependence of the relative fluorescence change was also analyzed in function of time to measure the adsorption rate (kA) of P11-4 on the surface of crystalline particles according to:
Where Fobs and F∞ are the fluorescence of SAP P11-4 at a given time t and at infinite time,
respectively, and kA is the adsorption rate.
2.1.3 Dynamic Light Scattering and pH Analysis
The formation of crystalline material ACP/OCP (octacalcium phosphate) was monitored using a light-scattering detector; by measuring the radial size of the crystalline particles as function of the reaction time. This assay investigated the nucleation rate (nm/s) of
calcium phosphate crystals in the absence or in the presence of 5 μM of P11-4 at different
concentrations of supersaturated solutions, CaCl2 (0.25 - 10 mM) and K2HPO4 (0.15 - 6 mM)
at Ca/P=1.67. The kinetics of calcium phosphate crystal formation was monitored for 120 min using a dynamic light-scattering equipment Zetasizer Nano ZS90 (Malvern Instruments Ltd, Malvern, United Kingdom) at 25 ºC. The rate of crystalline material formation can be described by a first-order law [31] and be fitted according to the first-order relationship:
Where R and Rmax are the radius (nm) of the crystalline particles at a given time t and at
infinite time, respectively, and kobs is the observed first-order rate.
The conversion of the ACP to OCP intermediate is acid mediated and conversely the conversion of OCP to apatite is base mediated [28,32]. Therefore, the time course of formation of HAP (Ca/P= 1.67) was monitored as a function of the pH variation throughout the mineralization reaction medium. This media contained a mixture of 2.5 mM CaCl2 and 1.5
mM K2HPO4 solutions, in absence or in the presence of 5 μM P11-4 at 25 ºC using the MPT-2
accessory in order to automate the measurement of nanoparticle size as a function of pH (Malvern Instruments Ltd, Malvern, United Kingdom).
2.1.4 Atomic Force Microscopy Analysis
Images of the HAP nanocrystallites were obtained by Atomic Force Microscopy (AFM) in a SPM 9600 scanning probe microscope (Shimadzu Corporation, Kyoto, Japan) by measurement of the interaction forces between the tip and the sample surface containing the samples. The samples were analyzed using a scanner of 125 nm in the contact mode by using a triangular silicon tip (OMCL–TR800PSA-1, Olympus®), and the resonant frequencies of the cantilever were found to be 210–230 kHz. To prepare the samples, 20 μL of the solution containing the mixture of 2.5 mM CaCl2 and 1.5 mM K2HPO4 in the absence or in the
presence of SAP P11-4 (0.2% p/v) was deposited after 30 min of reaction on 0.5 cm2 mica
plates and the analyses were performed at 25 ºC after complete material drying. The diameter (nm) of the HAP nanocrystallites was measured from the scanning images in the absence or in the presence of P11-4.
The procedure described above was used to analyze the effect of the P11-4 upon the
self-assembling properties of collagen type I fibrils. AFM images of the Collagen Type I fibrils (0.2% w/v) in water were obtained by AFM in the contact mode in the absence or in
the presence of P11-4 (0.2% w/v). Also, the diameter of Collagen Type I fibrils was measured
from the scanning images in the absence or in the presence of P11-4 at 25 ºC.
2.1.5 Surface Plasmon Resonance Analysis
The interaction of P11-4 with Collagen Type I was monitored by Surface Plasmon
Resonance (SPR) in a Biacore T200 (GE Healthcare Life Science). Collagen Type I conjugated with Biotin was immobilized on a CM5 chip sensor containing Streptavidin (Chip SA, GE Healthcare). In the interaction assays, different concentrations of SAP P11-4 (1-2000
μM) diluted in 50 mM Tris-HCl buffer (pH 7.4) containing 100 mM NaCl were applied at a flow rate of 30 μl/min for the total time of 360 s. The regeneration of the surface after the injection of each SAP P11-4 concentration was performed with 0.005% SDS solution followed
by 2 M NaCl for 30 s at flow rate of 30 μl/min. The blank in the absence of SAP P11-4 was
subtracted of each sensorgrams and the resulting sensorgrams were used to calculate the affinity constants, which were processed by the surface distribution model using the software EVILFIT [33].
2.1.6 MMP-2 and Bacterial Collagenase Assays
The influence of P11-4 upon recombinant human MMP-2 (ab198429) and bacterial
collagenase from Clostridium histolyticum (Endo Pharmaceuticals, Malvern, USA) endopeptidase activities were monitored fluorometrically with the aid of a synthetic FRET-peptide substrate analog of Collagen Type I, Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 [34]. The fluorescence of 7-methoxycoumarin-4-acetyl (Mca) is quenched by the Dpa group, N-3-(2,4-dinitrophenyl)-L-α, β-diaminopropionyl, until cleavage separates them, MMPs cleave this substrate between Gly-Leu [Neumann et al., 2004]. The fluorescence intensity was monitored on a thermostatic Shimadzu RF-6000 spectrofluorometer (Shimadzu Corporation, Kyoto, Japan). The steady-state kinetic assays with fluorogenic substrate were performed in 50 mM HEPES buffer (pH 7.4) containing 10 mM CaCl2 at 37 °C. The MMP-2 was
pre-activated by its pre-incubation in a solution containing 50 mM HEPES (pH 7.4), 10 mM CaCl2, 0.05% Brij-35, and 1 mM APMA (amino-phenyl mercuric acetate) for 2 h. All
reactions were done in 1×1 cm cross section quartz cuvette, the excitation and emission wavelengths were set at 328 and 400 nm, respectively. The initial rate of hydrolysis at 5 μM substrate concentration in the absence or in the presence of SAP P11-4 (0 - 100 μM) was
fluorescence versus Mca concentration were constructed. All kinetic experiments were performed in triplicates.
2.1.7 Influence of P11-4 on Collagenase Proteolysis of Collagen Type I
Collagen Type I (2 mg/ml) was incubated with Clostridium histolyticum collagenase in the absence or in the presence of SAP P11-4 (0, 6, 12 and 24 mM) for 30 min. The
enzymatic reaction was performed in 50 mM HEPES buffer (pH 7.4) containing 10 mM CaCl2 at 37 °C. Aliquots of reactions mixtures were collected at 30 min and the reaction was
stopped by addition of SDS-PAGE sample buffer consisting of 200 mM Tris-HCl (pH 7.0) 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.025% bromophenol blue (1∶1 v/v) and finally boiled for 5 min. Samples were submitted to 10% SDS-PAGE and the products of Collagen Type I hydrolysis were visualized by Coomassie blue staining. The efficiency of fibrillar Collagen Type I cleavage by bacterial collagenase in the presence or in the absence of P11-4 was indicated by the disappearance of the alpha-, and beta chains detected by scanning
densitometry.
2.2 Influence of P11-4 on the bonding interface to artificial caries-affected dentin
2.2.1 Preparation and Allocation of Specimens
Eighty-four extracted non-carious human third molars were obtained with approval by local Ethics Committee (CAAE: 3763.4814.5.0000.5418), forty-eight used on microtensile bond strength (n=8), eighteen for nanoleakage (n=3) and eighteen for in situ zymography (n=3) assays. Teeth were stored in 0.1% thymol solution at 4 ºC for no more than two months after extraction. The occlusal portion and the roots were removed using a water-cooled diamond saw (ISOMET 1000; Buehler, Lake Bluff, IL, USA) to obtain a 4-mm-thick flat dentin specimen. All the surfaces of the dentin specimens were coated with a red acid-resistant nail varnish (Colorama, CEIL; São Paulo, SP, Brazil), except the occlusal flat surface, which was ground with 600 grit silicon carbide sandpaper to create a smear layer.
Dentin specimens were allocated into six groups (n=14), according to the dentin substrate: sound dentin (SD), caries-affected dentin (CaD), and caries-affected dentin treated with P11-4 (CaD + P11-4); and the storage times: 24 h and 6 months. CaD and CaD + P11-4
groups were submitted to artificial caries development, and the last one (CaD + P11-4) to the
assigned to the following analysis: microtensile bond strength (n=8), and nanoleakage (n=3) and in situ zymography (n=3).
2.2.2 Artificial Carious Lesion Formation
The protocol for artificial carious lesion formation was adapted from Pacheco et al. [36]. In brief, all dentin specimens were previously sterilized with gamma radiation at 14.5 kGy for 60 h. Fifty-six dentin specimens were incubated in a cariogenic solution containing brain-heart infusion (BHI) broth (Bacto Brain Heart Infusion; Becton Dickinson and Company, Sparks, MD, USA), supplemented with 2% glucose (LabSynth; Synth, São Paulo, SP, Brazil), 1% glucose (LabSynth; Synth) and 0.5% yeast extract (Bacto Yeast Extract; Becton Dickinson and Company), This solution was inoculated with 2% of Streptococcus
mutans (UA159) at 106 CFU/ml on the first day of the experiment. The broth was renewed daily. The remaining twenty-eight dentin specimens were used as positive control (sound dentin).
After seven days of incubation, dentin specimens were removed from the cariogenic solution, rinsed and cleaned to remove the biofilm attached in the occlusal surfaces. Tactile inspection showed the formation of two layers: a superficial and softer layer resembling caries-infected dentin, and an inner and more cut-resistant layer, like caries-affected dentin. Specimens from CaD and CaD + P11-4 groups had the superficial layer removed with
cylindrical tungsten carbide burs #8 (JET; Beavers Dental, Morrisburg, ON Canada) in a slow speed handpiece (Dabi Atlante; Ribeirão Preto, SP, Brazil) to expose the artificial caries-affected dentin. The limit for removal of the softened carious tissue was considered when the dentin presented a surface darkened and slightly hard to an excavation with slight pressure [14]. A single experienced and trained operator performed this procedure.
2.2.3 Self-assembling peptide treatment
A water-based P11-4 solution (5 mg/ml) was prepared and applied on the occlusal
surface of caries-affected dentin specimens for 5 min. Afterward, a solution supersaturated of calcium and phosphate (1.5 mM calcium, 0.9 mM phosphate, 150 mM KCl, 20 mM cacodylic buffer, pH 7.0) was dispensed on dentin surface for more 1 min [37], with the excess removed using a tissue paper.
2.2.4 Bonding procedure
All dentin surfaces were bonded with the two-step etch-and-rinse adhesive system Adper Single Bond 2 (3M Dental Products, St. Paul, USA), according to the manufacturer’s instructions. Initially, the dentin surface was etched for 15 s with 35% phosphoric acid gel (Ultra Etch; Ultradent, Indaiatuba, SP, Brazil) and then rinsed. Excess water was removed using a cotton pellet and two consecutive coats of the adhesive were gently applied to the etched dentin surface with a microbrush, air-dried to evaporate the solvent, and light cured for 15 s using a Light emitting diode (LED) curing light (Ivoclar Vitadent, Schaan, Liechtenstein), with an intensity of 1200 mW/cm2. Then, a 4-mm-thick resin composite block (Filtek™ Z350 XT Flow, 3M ESPE, St. Paul, MN, USA) was built on the dentin surface in 1-mm increments. Afterward, the resin/dentin sets were stored in deionized water at 37 °C for 24 h [38].
2.2.5 Microtensile bond strength (µTBS) test
Bonded specimens (n=8) were sectioned into resin-dentin beams with a cross-sectional area of 1 mm2 to evaluate the microtensile bond strength to different dentin substrates (SD, CaD, and CaD + P11-4) and water storage times (24 h and 6 months). Specimens were
individually fixed with cyanoacrylate-based adhesive (Super Bonder Power Flex-Gel Control; Henkel Ltda, Itapevi, SP, Brazil) to a specially designed apparatus and then, to the grips of a universal testing machine (EMIC DL 500, EMIC - Equipamentos e Sistemas de Ensaio, São José dos Pinhais, PR, Brazil). Finally, they were tested under tension (50 N load) at a 1 mm∕min crosshead speed until failure. μTBS values were expressed in MPa [38].
2.2.6 Nanoleakage analysis
Three resin-dentin slices were selected from the middle portion of each resin/dentin set of the tested groups (n=3). Slices were immersed in a 50 wt% ammoniacal silver nitrate solution in darkness for 24 h, rinsed thoroughly in distilled water, and then immersed in photographic developing solution for 8 h under a fluorescent light to reduce silver ions into metallic silver grains within voids along the bonded interface [39].
Afterwards, the silver-impregnated slices were embedded in acrylic resin and polished using 600-, 1200-, 2000-grit SiC papers, felt discs and aluminum oxide paste (Buehler, Lake Bluff, IL, USA), with an ultrasonic cleaning bath of 20 min after each abrasive/polishing step. Slices were then demineralized with 37% phosphoric acid for 5 s, rinsed with deionized water for 30 s, and dried. Subsequently, they were deproteinized with 10% NaOCl for 5 min, rinsed
in an ultrasonic bath, and left to dry for 24 h at room temperature. Finally, slices were coated with carbon (Balzers-SCD 050 Sputter Coater), analyzed using SEM (JSM-5600LV; JEOL, Tokyo, Japan) and observed in the backscattered electron mode at 15 kV.
The amount of silver nitrate uptake in the hybrid layer was registered using an Energy-Dispersive X-ray spectroscopy (EDS) detector couplet to SEM. Three representative and different areas of each bonding interface slices were chosen for the measurements and the atomic percentage of silver (at % Ag) quantified by Easy Macro software (VANTAGE- digital microanalysis system) available in SEM [40].
2.2.7 In situ Zymography
Three slices of 150 μm-thick were obtained from longitudinal cuts of each resin/dentin sets using a water-cooled diamond saw (ISOMET 1000; Buehler, Lake Bluff, IL, USA). The slices were polished manually with a sequence of 600-, 800-, 1200-grit SiC papers. Each slice was glued to a microscope slide with a cyanoacrylate-based adhesive (Super Bonder Power Flex-Gel Control; Henkel Ltda, Itapevi, SP, Brazil). In situ zymography was performed with quenched fluorescein-conjugated gelatin as the MMP substrate (E-12055; Molecular Probes, Eugene, OR, USA). A 1.0 mg/ml stock solution of fluorescein-labeled gelatin was prepared by the addition of 1.0 ml water to the vial containing the lyophilized substrate that was stored at -20 °C until use. The gelatin stock solution was diluted 1:8 with the dilution buffer (NaCl 150 mM, CaCl2 5 mM, Tris-HCl 50 mM, pH 8.0), and an anti-fading agent was added
(Prolong Gold Mountant; Molecular Probes, Eugene, OR, USA). A 50-μL quantity of the fluorescent gelatin mixture was placed on top of each slab and covered with a coverslip. Slides were light protected and incubated in humidified chambers at 37 °C for 48 h [41]. Briefly, hydrolysis of quenched fluorescein-conjugated gelatin substrate, indicative of endogenous gelatinolytic enzyme activity, was assessed by examination under a multi-photon confocal microscope Leica TCS SP5 (Leica Microsystems, Mannheim, Germany). Optical sections were acquired from different focal planes, and the stacked images were analyzed, quantified, and processed with ImageJ Software (NIH).
2.3 Statistical Analysis
Data normality was verified by Kolmogorov Smirnov test. Two-way analysis of variance (ANOVA) followed by the Bonferroni test were carried out to analyze the diameter of HAP nanocrystalline particles by AFM, the remaining soluble Collagen Type I after collagenase degradation by SDS-PAGE, µTBS and atomic Ag% atomic HL. F test was used
to compare the variance between HAP particle size measured by AFM in the absence or in the presence of SAP P11-4. Pearson correlation coefficient as used to measure the strength of
association between the velocity of adsorption of P11-4 into a HAP particle and the nucleation
rate of the HAP in supersaturated solutions. Unpaired t-test was used to compare the difference between first-order rate of the HAP crystal growth, the time of conversion of ACP to OCP,the velocity of conversion of OCP into HAP and the diameter of the collagen type I fibrils in the absence or in the presence of P11-4. Kruskall-Wallis and Wilcoxon tests were
used to evaluate the intensity of fluorescence at HL. All tests were performed setting the significant level as =5%, using SPSS version 20 software and GraphPad Prism 6.0 software.
3. Results and Discussion
Work recently published by Habraken et al. [28] demonstrated that calcium triphosphate-associated complexes can aggregate in 3D branched polymeric nanometer structures termed pre-nucleation cluster for calcium phosphate; these pre-nucleation complexes bind calcium ions from solution continuously forming insoluble spherical particle of amorphous calcium phosphate (ACP). Continued calcium uptake converts ACP into octacalcium phosphate (OCP) and subsequently into hydroxyapatite (HAP). The results depicted in the Figs 1-3 show the effect P11-4 on the nucleation of HAP. The results depicted
in the Fig. 1A show that the peptide P11-4 could bind calcium ions as expected [22]. The
interaction of Ca2+ with P
11-4 was analyzed by measuring the change in the intrinsic
fluorescence emission spectra of 5 μM peptide P11-4 as function of Ca2+ concentration (0 -
3.45 mM). The peptide P11-4 has a tryptophan residue which is florescence sensitive to
calcium binding. The interaction of calcium with the peptide promoted the decrease of the emission of its intrinsic fluorescence in function of the Ca2+ concentration (Fig. 1A). The spectrofluorometric assays indicated that Ca2+ interact with a specific and saturable site of P11-4, showing a dissociation constant Kd = 0.63 ± 0.07 mM (Fig. 1B).
Calcium phosphate (Ca/P= 1.67) crystalline particles were prepared by the instantaneous addition of a phosphate solution to a gently stirred solution of calcium ions. The formation of crystalline material ACP/OCP was monitored by measuring the radial size of the crystalline particles in function of the reaction time with the aid of a light-scattering detector (Fig. 1C). Fig. 1C shows that the size of the particles grew with time and the formation rate of crystalline material can be described by first-order kinetics [31]. Light scattering analysis demonstrated that the presence of 5 µM P11-4 did not change the rate of ACP/OCP crystal
growth was Kobs = 0.54± 0.13 min-1 and in the presence of 5 μM SAP P11-4 the rate was Kobs =
0.52 ± 0.12 min-1 (P= 0.955). As well as, the presence of P11-4 did not alter the average size
of the crystalline particles, in the presence of peptide the size of the crystalline particles was 669 ± 61 nm and in the absence of P11-4 was 534 ± 54 nm (P= 0.0984).
Data from the literature show that the conversion of saturated solution of amorphous calcium phosphate (ACP) in hydroxyapatite (HAP) occurs in two distinct phases, the first phase is acid mediated and converts ACP into octacalcium phosphate (OCP), the second phase is based mediated and indicates the conversion of the OCP intermediary to hydroxyapatite [28,32]. During the mineralization reaction in deionized water, 2.5 mM CaCl2
and 1.5 mM K2HPO4 (Ca/P= 1,67), the pH was monitored as a function of time in the absence
or in the presence of 5 µM SAP P11-4 (Fig. 1D). The data presented in the Fig. 1D shows that
the presence of 5 µM P11-4 increased significantly the rate of conversion of ACP into OCP,
the time of conversion of ACP to OCP (first phase) had decreased significantly from 15 ± 1 min to 6 ± 1 min, while the kinetics of conversion of OCP into HAP also decreased significantly from 0.035 ± 0.02 min-1 to 0.013± 0.01 min-1 (Fig. 1D). Taken together, this data
strongly suggest that P11-4 can organize the nucleation of HAP crystals.
As showed above, the HAP nucleation induction time in the presence of P11-4 is
longer than in its absence, such as in a system with the usual random foreign particles. From kinetics, the increase observed upon nucleation induction time is mainly caused by the effect of P11-4 on the transport of growth units to the surface of the crystalline clusters because of
collision, adsorption, and desorption, although the presence of P11-4 can lower the nucleation
energy barrier [42]. Indeed, the adsorption rate (kA) of the peptide P11-4 on the surface of a
crystalline particle was a sigmoidal function of the HAP supersaturated solutions (Fig. 2A) as was nucleation rate of the crystalline material (Fig. 2B). As expected, the kinetics of adsorption of P11-4 on the surface of a crystalline particle strongly correlates positively with
the nucleation rate of the crystalline material on HAP supersaturated solutions, Pearson = 0.994, CI95% = 0.971 – 0.999, P< 0.0001 (Fig. 2C). The present results indicate that although the presence of P11-4 slows down the HAP nucleation process, P11-4 can improve the
structural match among HAP crystallite aggregates by templating Ca2+ for HAP crystallite formation [43]. This was verified by our experiments using Atomic Force Microscope as discussed below.
To investigate the influence of the P11-4 upon HAP nucleation-complex, we used in
situ AFM, which allows the direct monitoring of the mineralization processes on the surfaces with nanometer detail (Fig. 3). AFM images indicate that HAP nanocrystallites obtained from
the solution in the absence of P11-4 are in a disordered or random manner (Fig. 3A). Yet, the
HAP crystallite aggregates formed in the presence of P11-4 show a remarkably ordered
microstructure. AFM image in Fig. 3B reveals a compact and ordered microstructure of HAP assemblies. In each assembly, the HAP nanocrystallites mediated by P11-4 are aligned parallel
to each other. Also, the AFM analysis of HAP formation (Figs. 3A and 3B) demonstrated that the P11-4 standardized the HAP particle growth by reducing the particle size variability. In the
absence of P11-4 (Fig 3A), there is a population of HAP crystals of heterogeneous sizes, with
three distinct groups according to their diameter measured by AFM (Fig. 3C): Small Size, diameter = 76 ± 3 nm (a); Intermediate Size, diameter = 130 ± 6 nm (b) and Large Size, diameter = 266 ± 11 nm (c). In the presence of P11-4, only a single population with mean
diameter = 134 ± 2 nm was observed (d). Fig. 3D shows that P11-4 did not alter the mean
HAP particle size, as in the absence the mean of the HAP particle size was 144 ± 15 nm and in the presence P11-4 it was 134 ± 2 nm, corroborating the light scattering data showed in Fig.
1C. On the other hand, P11-4 drastically reduced the variance of the HAP particle size by
standardizing their growth, the F-test to compare the variances between the groups was strongly significant (P< 0.0001).
Recent literature has shown that P11-4 can diffuse into the subsurface of the enamel
carious lesion and supports nucleation of de novo hydroxyapatite nanocrystals [26,43]. The application of P11-4 monomers onto an enamel carious lesion causes a higher concentration of
P11-4 within the carious lesions [26]. These results strongly suggest that prior to initiation of
HAP crystal nucleation, the peptide P11-4 can interact with the proteins of the extracellular
matrix from enamel and dentin as evidenced on this study. The interaction of the peptide P11-4
monomers with collagen I was monitored by surface plasmon resonance (Figs. 4A and 4B). When increasing concentrations of P11-4 monomers were injected onto the collagen surface
the interaction between peptide P11-4 with Collagen Type I occurs in a saturable and very
stable way. The peptide P11-4 binds to collagen I in a dose-dependent and saturable manner,
showing a dissociation constant Kd = 405 ± 25 μM (Fig. 4B). SPR analyses also shows that the peptide P11-4 specifically binds to immobilized Collagen Type I.
Acid-solubilized Collagen Type I is known to self-assemble in vitro in a temperature-dependent manner affecting fibril and fiber dimensions [44]. The details of the Collagen Type I fibril structure in the presence of P11-4 was obtained by AFM analysis. AFM images of
0.19% Collagen Type I (w/v) in mica holder in the absence (Fig. 4C) or in the presence of 0.20% SAP P11-4 (Fig. 4D) at 25º C. AFM images reveals larger diameter fibers in the
20 fibers) and 290 - 330 nm in its presence (n= 20 fibers). SAP P11-4 increased the diameter
of the Collagen Type I fibers of 214 ± 4 nm to 308 ± 5 nm (Fig. 4E) (P< 0.0001). These results clearly demonstrated that SAP P11-4 can interact with Collagen Type I and this
interaction can increase the diameter of the resulting fibers.
We observed distinct microstructural changes induced by polymerizing acid-solubilized rat tail tendon collagen in the presence of SAP P11-4 while keeping the mass
content of collagen constant. Collagen Type I fiber diameters assessed by AFM images increased in the presence of the peptide P11-4. This observation is likely due to the
self-assembly properties of collagen and to polymerization kinetics, in which peptide P11-4 can
affect the balance of hydrophobic, electrostatic, and hydrogen bond forces between polymerizing collagen monomers and fibrils, thus favoring lateral fibril and fiber growth. Altering the collagen hydrogel microstructure allows one to specifically study how this microstructure affects tissue mechanical properties [44].
Since MMP-2 is one of the most abundant gelatinolytic enzymes present in dentin matrix and has a crucial role in the caries progression and hydrolytic degradation of hybrid layers [3,45,46], we verified whether P11-4 modified the proteolytic activity of MMP-2 on
dentin (Fig. 5A). The proteolytic activities of the human recombinant MMP-2 and bacterial collagenases were monitored spectrofluorometrically with the aid of a synthetic FRET-peptide substrate analog of collagen, Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 [35]. The results depicted in the Fig. 5A show that P11-4 (0 - 100 μM) did not directly inhibit the
proteolytic activities of MMP-2 and bacterial collagenase on dentin. Fig. 5B shows a SDS-PAGE image analyzing the proteolytic fragments of fibrillar Collagen Type I (2 mg/ml) by bacterial collagenase in the absence or in the presence of P11-4 at concentration of 0, 6, 12 and
24 mM. Scanning densitometry indicated that in the absence of P11-4 bacterial collagenase
completely degrades native fibrillar collagen and the presence of P11-4 partially inhibits the
enzymatic degradation of fibrillar Collagen Type I by bacterial collagenase.
Numerous literature reports have correlated increased collagen polymerization with increasing hydrogel linear tensile modulus [44,47,48]. Also, the stability of collagen-P11-4
complex based films was assessed in vitro collagenase digestion tests that are widely used as an accelerated model of degradation [48].We observed that the specific mechanism of action of bacterial collagenase was sensitive to the presence of P11-4, likely due to the slower
diffusion of the enzyme in increased collagen polymerization networks induced by peptide P11-4. Taken together, our results suggest that the presence of P11-4 can protect the dentine
