UNIVERSIDADE FEDERAL FLUMINENSE FACULDADE DE ODONTOLOGIA
INFLUÊNCIA DA MORFOLOGIA SUPERFICIAL DO ESMALTE NA AÇÃO DE SISTEMAS ADESIVOS AUTOCONDICIONANTES DE DIFERENTES GRAUS DE ACIDEZ APLICADOS EM UMA OU DUAS CAMADAS
Digite para introduzir texto
Niterói 2017
UNIVERSIDADE FEDERAL FLUMINENSE FACULDADE DE ODONTOLOGIA
INFLUÊNCIA DA MORFOLOGIA SUPERFICIAL DO ESMALTE NA AÇÃO DE SISTEMAS ADESIVOS AUTOCONDICIONANTES DE DIFERENTES GRAUS DE ACIDEZ APLICADOS EM UMA OU DUAS CAMADAS
JÉSSICA HORTA CARNEIRO
Dissertação apresentada à Faculdade de Odontologia da Universidade Federal Fluminense, como parte dos requisitos para obtenção do título de Mestre, pelo Programa de Pós-Graduação em Odontologia.
Área de Concentração: Dentística
Orientadora: Profa. Dra. Laiza Tatiana Poskus
Niterói 201
DEDICATÓRIA
Dedico este trabalho a minha mãe, que sempre esteve ao meu lado me incentivando e dando suporte pra que eu realizasse todos os meus sonhos, e
que se pudesse moveria céus e terras por mim.
AGRADECIMENTOS
Agradeço primeiramente a Deus por ter me guiado até aqui e por vencer tantos obstáculos que somente eu e ele sabemos.
A minha orientadora Laiza Poskus pela sua imensa sabedoria, ajuda e inspiração durante toda essa longa jornada que foi o mestrado.
Aos professores da banca que se disponibilizaram não só em comparecer mas também em estudar e contribuir de alguma forma com meu trabalho. Aos professoras da dentística UFF pelos ensinamentos e dedicação que me foi cedidos.
F D E z E pessoal da turma nova. Tantas risadas e tantos momentos bons fizeram com que tudo fosse mais fácil.
Aos amigos Fabiana e Andre Luiz por terem se tornado um presente da UFF pra mim, vocês fizeram que meus dias indo pra Niterói fossem mais leves e mais engraçados. Espero que essa amizade dure pra vida toda.
Ao senhor Zé Maria com toda sua paciência e seu tempo e dedicação gastos me ajudando sempre no laboratório.
Gostaria de agradecer imensamente a minha família por ser minha base e meu porto seguro diante todas as minha dificuldades.
Ao meu avô Décio que sempre foi meu ídolo e minha fonte de inspiração para viver.
A minha mãe Monique e minha irmã Andressa por estarem sempre do meu lado me ajudando em tudo que eu sempre precisei.
Ao meu noivo Silas por todo amor, carinho e compreensão com minhas ausências e mudanças de humor durante o mestrado. Eu te amo muito.
RESUMO
CARNEIRO JH. Influência da morfologia superficial do esmalte na ação de sistemas adesivos autocondicionantes de diferentes graus de acidez aplicados em uma ou duas camadas [dissertação]. Niterói: Universidade Federal Fluminense, Faculdade de Odontologia; 2017.
Objetivo: Verificar a espessura da camada de esfregaço (CE) e a rugosidade (Sa) em esmalte, utilizando instrumentos rotatórios de diferentes granulações e analisar a influência da morfologia superficial do esmalte na eficácia adesiva de sistemas autocondicionantes experimentais com diferentes graus de acidez, aplicados em uma ou duas camadas. Materiais e métodos: Para análise da Sa e CE (n=5), superfícies planificadas da face vestibular de dentes bovinos foram desgastadas com diferentes instrumentos: R (ponta diamantada de granulação regular 3101) e FF (ponta diamantada de granulação extra fina 3101FF). A medição da rugosidade (n=5) foi efetuada por meio de um perfilômetro tridimensional (Sa). Imagens da superfície clivada foram obtidas no microscópio eletrônico de varredura para medição da CE, utilizando o software Image J. Posteriormente, 80 dentes bovinos tiveram a superfície vestibular de esmalte planificada e distribuídas nos grupos de acordo com a superfície de esmalte tratada (R e FF), com os adesivos (Suave – M; Intermediário: - I; forte – S; Controle comercial Clearfil SE Bond - C) e com o número de camadas (Uma camada -1 e Duas camadas - 2), formando os grupos: MR1, MR2, MFF1, MFF2, IR1, IR2,IFF1,IFF2, SR1, SR2, SFF1, SFF2, CR1, CR2. CFF1, CFF2. A ê ã (μTBS) f z
microcisalhamento em máquina de ensaios universal. Resultados: O grupo R levou a maior Sa e CE em esmalte do que o FF (p<0.05). Somente para o sistema autocondicionante suave, a ponta diamantada R diminuiu os valores de μTBS (p<0.05). O número de camadas aplicadas não influenciou nos resultados.Conclusão: Instrumentos rotatórios de maior granulação produziram uma maior CE e Sa. Para adesivos suaves, em esmalte seria recomendada a utilização de pontas de granulação extra fina para melhorar a união.
PALAVRAS CHAVE: camada de esfregaço; rugosidade; acidez; autocondicionante; esmalte; cisalhamento
ABSTRACT
CARNEIRO JH. Enamel morphology and its influence on the performance of self-etching adhesive systems of different acidities applied in one or two coats. [Dissertation]. Niterói: Universidade Federal Fluminense, Faculdade de Odontologia; 2017.
Purpose: To analyze the surface roughness (Sa) and smear layer thickness (SL) of enamel ground with different grit-sized diamond burs, and to verify the influence of this morphology on the adhesive effectiveness of experimental self-etching systems of different acidities, applied in one or two coats. Materials and methods: The vestibular surface of bovine teeth was ground with the diamond burs: R (regular grit diamond bur) and FF (extra-fine grit diamond bur). The enamel surface roughness (n=5) was analyzed using a three-dimensional profilometer (Sa). For SL, after cleavage of the specimen, scanning electron microscope images (n=5) were obtained and the measurement was performed using the Image J software. For microshear bond strength (μSBS), 60 bovine teeth were distributed in the following groups, according to the diamond bur (R and FF), the experimental self-etching adhesives (Mild - M; Intermediate: - I; Strong – S) and the number of adhesive coats (One coat -1 and Two coats - 2): MR1, MR2, MFF1, MFF2, IR1, IR2, IFF1,IFF2, SR1, SR2, SFF1, SFF2. Clearfil SE Bond was used as commercial reference Microshear bond strength (μSBS) test was performed using a chisel in a universal testing machine. ANOVA and Student-N w ’ w f f y Results: The diamond
bur R led to the highest Sa and SL than the FF (p <0.05). Only for the mild experimental adhesive system, the regular diamond bur caused a decrease in the μTBS values (p <0.05). The number of adhesive coats applied to the surface did not influence the results. Conclusion: The coarser diamond bur produced higher Sa and SL, and this morphology was an important factor for the adhesion, being recommended the use of extra-fine grit burs on enamel to improve it, mainly when a mild self-etching adhesive is used.
Key words: smear layer; roughness; acidity; self-etching; enamel; shear bond strength
1 - INTRODUÇÃO
A utilização de sistemas autocondicionantes para confecção de restaurações diretas e indiretas têm sido bastante investigada, já que tornam a técnica restauradora mais simples, por envolver menos passos, e menos crítica, por não se necessitar controlar a umidade dentinária.49 Além disso, tais sistemas, por promoverem o condicionamento ácido e a infiltração de monômeros de maneira simultânea, evitariam a formação de uma camada de dentina desmineralizada e não infiltrada pelos adesivos, mais sujeita à degradação pelas metaloproteinases.53
Tais sistemas devem ser suficientemente ácidos, para promover a desmineralização e infiltração simultânea do substrato durante sua aplicação.26, 32 Dependendo do sistema, o pH de sua formulação pode ser classificado como super suave (pH por volta de 2.5) suave (pH ao redor de 2.0), intermediário (pH ao redor de 1.5), forte (pH menor do que 1.0).48
Quando aplicados em esmalte, os sistemas de maior pH, por terem mais baixo grau de acidez, não tem se mostrado tão eficazes, com um padrão de desmineralização aquém ao obtido com os sistemas do tipo condicione e lave, sendo preconizada a utilização de um condicionamento seletivo desta estrutura com ácido fosfórico.10, 21, 28, 47 Quando possuem um pH mais baixo, ou seja, um grau de acidez maior, a resistência de união em esmalte tem mostrado melhores resultados.24, 33, 34 No entanto, em dentina, parece que os sistemas autocondicionantes de maior pH, tem se mostrado mais eficazes. 7, 40 O ideal seria que pudéssemos contar com um sistema que condicionasse eficientemente tanto o esmalte como a dentina, ao mesmo tempo.
Como se sabe, diferentes instrumentos rotatórios são capazes de criar camadas de esfregaço e uma rugosidade superficial em esmalte e dentina com características próprias.4, 16, 23 Tem sido mostrado que instrumentos de maior granulação causam uma maior rugosidade superficial e uma camada de esfregaço mais espessa tanto em esmalte como em dentina. 4, 16, 23, 42
Os sistemas de menor acidez têm mostrado não remover totalmente a camada de esfregaço, principalmente quando esta camada é mais espessa.4,
23
No entanto, é controversa na literatura a afirmação de que a espessura dessa camada exerça uma influência direta na resistência de união à dentina, uma vez que certos estudos mostram que camadas de esfregaço mais espessas levam a menores valores de resistência de união para sistemas autocondicionantes15, 37, 41 e outros não observam nenhuma influência deste tipo. 18, 42, 44 Em se tratando de esmalte, pouco é abordado sobre a ação dos adesivos autocondicionantes em superfícies com diferentes espessuras de camada de esfregaço. Tem sido verificado que diferentes camadas de esfregaço formadas em esmaltes influenciam na interação de adesivos autocondicionantes. 4, 23 No entanto, em outro estudo38 a resistência de união de compósitos em superfícies de esmalte tratadas com diferentes instrumentos foi similar mesmo quando diferentes sistemas autocondicionantes foram utilizados.11, 45
Para aumentar os valores de resistência de união em dentina, tem sido proposta a aplicação de uma segunda camada do sistema autocondicionante principalmente quando sistemas suaves são empregados.3, 31
Contrariamente, em estudo prévio, a aplicação de duas camadas de adesivos autocondicionantes de passo único não aumentou os valores de resistência de união. 2
Sendo assim, primeiramente o objetivo deste estudo foi verificar a espessura da camada de esfregaço e a rugosidade superficial em esmalte, obtidas após desgaste com instrumentos rotatórios de diferentes granulações. Também foi objetivo, analisar a influência da morfologia superficial do esmalte na eficácia adesiva de sistemas autocondicionantes experimentais com diferentes graus de acidez, aplicados em uma ou duas camadas.
As hipóteses nulas do presente estudo são: 1) Instrumentos rotatórios de diferentes granulações não influenciarão na espessura da camada de esfregaço e na rugosidade superficial do esmalte; 2) Os valores de resistência de união não serão dependentes da morfologia superficial do esmalte e do grau de acidez do sistema autocondicionante empregado. 3) A aplicação de uma ou duas camadas do sistema autocondicionante no esmalte não influenciará nos valores de resistência de união, independentemente do sistema adesivo empregado e da qualidade do esmalte.
2.1-Preparo dos dentes
Após desinfecção com solução de cloramina 0,5% e estocagem em água destilada a 37°C (Estufa, Sociedade Fabbe Ltda / Indústria Brasileira, São Paulo, Brasil), a face vestibular de dentes bovinos foi planificada com lixa de carbeto de silício #600 durante 30s, utilizando uma polidora automática sob refrigeração com água (Politriz Dpu-10, Struers, Denmark). Posteriormente, as raízes foram removidas utilizando um disco diamantado em cortadeira metalográfica (Cortadeira metalográfica Isomet 1000 precision saw - Buehler, Lake Bluff, IL, USA), ao nível da linha cemento-esmalte. Para a rugosidade e camada de esfregaço, um segundo corte, paralelo ao primeiro, foi realizado aproximadamente na metade da coroa, obtendo-se dois fragmentos.
A seguir, a face vestibular planificada dos dentes foi desgastada de acordo com os seguintes grupos: R (Desgaste com a ponta diamantada 3101 de granulação regular (KG SORENSEN, São Paulo, Brasil); FF (Desgaste com a ponta diamantada 3101FF de granulação extra Fina (KG SORENSEN, São Paulo, Brasil). Para padronização, utilizou-se um dispositivo de preparos dentários onde uma caneta de alta rotação foi acoplada e o desgaste foi realizado com refrigeração, perfazendo 10 ciclos de vaivém. A broca foi trocada por uma nova a cada 5 superfícies de esmalte preparadas.
Um fragmento de cada dente (n=5) foi utilizado para fazer a leitura da rugosidade do esmalte (Sa), por meio de um perfilômetro tridimensional (Form Talysurf 60i, Taylor Hobson, Leicester, UK), sendo escaneada uma área de 1mm2 com uma resolução-z de 20 nm, 40,000 passos no eixo-X e ç 2μ x Y
Onde z é a altura do ponto medido nas coordenadas X e Y
2.3- Medição da Camada de Esfregaço
Inicialmente foi efetuada uma canaleta no sentido ocluso-cervical do dente, na porção palatina (n=5), para permitir a clivagem do espécime e a análise da superfície transversal ao desgaste. Após fixação com solução de karnovski (glutaraldeído a 2,5%, paraformaldeído a 2% em tampão fosfato de sódio a 0,1M e pH 7,4) por 12 horas a 4ºC, foi efetuada lavagem com cacodilato de Sódio (0,1M e pH 7,4) durante uma hora, sendo realizadas três trocas da solução a cada 15 minutos, sem agitação dos tubos de ensaio. Uma última lavagem com água destilada foi realizada por 1 min, com agitação dos tubos. Para desidratação, eles foram imersos em concentrações crescentes de etanol (50%, 70%, 90%, 100%). Após isso,os espécimes foram deixados por 24 horas em estufa a 37°C com sílica coloidal para secagem.
Os espécimes foram fixados em stubs e estes, a um porta-amostra de baixo vácuo. O conjunto foi levado ao microscópio eletrônico de varredura por (Phenom proX,Phenom-World BV, Eindhoven, Holland) a uma tensão de
15Kv, sendo obtidas três imagens com aumento de 3000 vezes para cada fragmento. Em cada imagem foram realizadas 3 medições da camada de esfregaço (µm) utilizando o software Image J (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA), totalizando 9 medições para cada fragmento, obtendo-se uma média para cada dente.
2.4- Manipulação dos adesivos experimentais
Tanto o sistema adesivo comercial como os experimentais utilizados no presente estudo foram autocondicionantes de dois passos. Primeiramente, um adesivo e três primers experimentais (suave-M, intermediário-I, e forte-S) foram manipulados de acordo com a formulação descrita no Quadro 1. Com o objetivo de modificar o grau de acidez dos primers, foi adicionado ácido clorídrico 37% p.a. O pH dos primers foi aferido utilizando o medidor de pH S220 SevenCompactTM pH/Ion (Mettler Toledo, Scwerzebacj, Switzerland, SWZ) em intervalos de 24 horas, até sua estabilização, quando foram então utilizados. O sistema adesivo autocondicionante Clearfil SE Bond (Kuraray Inc, Osaka, Japan) foi utilizado como controle comercial. Para permitir a fotoativação, foram incorporados, tanto nos primers experimentais como no adesivo, canforoquinona (0,5% p/p) e etil N,N-dimetil-4aminobenzoato (EDMAB) (0,5 %p/p) para atuarem, respectivamente, como fotoiniciador e agente de redução (Aldrich Chemical, Inc., Milwaukee, WI, EUA).
Tabela 1 – Composição dos materiais utilizados COMPOSIÇÃO TITULAÇÃO COM HCL pH Primer experimental Primer: 10-MDP (30%), TEGDMA, (30%), etanol (34%), água (5%), canforoquinona (0,5%), EDMAB (0,5%) 0 gotas 2,5 1 (0 20 μ ) 1,5 3 (0 60μ ) 0,8 Adesivo Experimental
Adesivo: Bis-GMA (50%), TEGDMA (29%), HEMA (20%), canforoquinona (0,5%), EDMAB (0,5%) Primer
comercial (Clearfil SE Bond)
Primer: 10-MDP, HEMA, DMA hidrófilo, água, amina terciária, canforoquinona
Clearfil SE Bond
Adesivo: 10-MDP, HEMA, Bis-GMA, DMA hidrófilo, água, silica coloidal silanizada, amina terciária, canforoquinona,
2,5
O grau de conversão monomérica foi avaliado através de espectroscopia infravermelha com transformada de Fourier – FT-IR. Misturas produzidas com 0,6 μL de primer e 0,6 μ de adesivo foram inseridas em uma matriz de teflon (Ø= 4,0 mm e h = 1,0 mm) posicionada sobre o cristal de ATR do espectrômetro (ALPHA-P FT-IR, Bruker Optics). Para cada sistema adesivo, foram obtidos espectros das misturas não polimerizadas e após a fotoativação por 40 s com irradiância de 600 mW/cm2, (n = 5). Foram aplicadas 20 varreduras com resolução de 4 cm-1, com temperatura controlada de 25 ± 1ºC. Na análise dos espectros FT-IR, foi considerado o intervalo entre 1700 e 1600 cm-1, para a observação dos sinais em 1609 e 1639 cm-1, correspondentes, respectivamente, as ligações vinílicas aromáticas do bisfenol A e alifáticas do grupamento funcional metacrilato.
O GC% foi calculado utilizando a razão entre as integrais das bandas em 1639 cm-1 e em 1609 cm-1 das misturas polimerizadas e não polimerizadas, de acordo com a seguinte equação:
onde R = Integral da área da banda em 1639 cm-1 / Integral da área banda em 1609 cm-1
2.6- Microcisalhamento
Após preparo da superfície do esmalte de 80 dentes bovinos como já descrito anteriormente, os dentes foram distribuídos aleatoriamente nos grupos experimentais (n=5) descritos na Tabela 2:
Tabela 2 – Grupos experimentais PONTA DIAMANTADA SISTEMA ADESIVO AUTOCONDICIONANTE CAMADAS GRUPOS REGULAR (R) SUAVE (M) 1 CAMADA RM1 2 CAMADAS RM2
INTERMEDIÁRIO (I) 1 CAMADA RI1
2 CAMADAS RI2
FORTE (S) 1 CAMADA RS1
2 CAMADAS RS2
CLEARFIL SE BOND (C) 1 CAMADA RC1
2 CAMADAS RC2
EXTRA-FINA (FF)
SUAVE (M) 1 CAMADA
FFM1
2 CAMADAS FFM2
INTERMEDIÁRIO (I) 1 CAMADA FFI1
2 CAMADAS FFI2
2 CAMADAS
FFS2
CLEARFIL SE BOND (C) 1 CAMADA FFC1
2 CAMADAS FFC2
Primeiramente, os dentes foram embutidos em tubos de PVC utilizando resina acrílica incolor, ficando exposta apenas a superfície planificada de esmalte vestibular. A seguir, uma camada do primer de todos os grupos foi aplicada na superfície de esmalte ativamente utilizando uma microescova por 20 segundos. Após aplicação de um leve jato de ar durante 5 segundos, outra camada de primer foi aplicada da mesma maneira, somente para o grupo de 2 camadas. O adesivo foi então aplicado ativamente com uma microescova para todos os grupos e tubos de PVC de 0,6mm de diâmetro interno e 1,0 mm de altura (Mark Med, Bragança Paulista, SP, BR) foram posicionados na superfície. Em seguida, foi realizada a fotoativação durante 40 segundos, utilizando irradiância de 600 mW/cm2 (Radii-cal, Demetron Inc. Danburry, USA), sendo esta monitorada periodicamente por um radiômetro (modelo 100, Demetron Inc., Danburry, USA). O compósito Filtek Bulk Fill Flow (3MESPE, St Paul, MN, EUA) foi inserido no interior do tubo em incremento único e fotoativado por 40 segundos. Após 1 hora, os tubos foram removidos cuidadosamente com auxílio de uma lâ ˚ 11 (W x v CO TD Xishan, Jiangsu, China), e as restaurações foram avaliadas em um estereomicroscópio (SZ40, Olympus, Tóquio, Japão) com aumento de 40x.
para verificar ausência de bolhas e fendas. Após armazenagem em água 37˚C 24 f metidas ao teste de microcisalhamento na máquina de ensaios universal (DL 2000, EMIC, São José dos Pinhais, São Paulo), utilizando um cinzel a uma velocidade de 1,0 mm/min. (Figura 1)
Após ruptura, todas as superfícies de dentina foram analisadas em estereomicroscópio com aumento de 40x, classificando as falhas como: Adesivas – superfície de esmalte íntegra livre de compósito; Coesivas – superfície de esmalte fraturada, ou com compósito em toda extensão; Mistas – falha coesiva em esmalte e/ou compósito e adesiva.
Figura 1. Esquema da preparação dos espécimes para microcisalhamento.
Os dados foram submetidos ao teste de homocedasticidade de Levene usando o software Statgraphics Centurion XVI (StatPoint Technologies Inc, Warrenton, VA, EUA) . Sendo os dados homogêneos para todos os testes, a análise de variância de um fator foi aplicada para a rugosidade superficial, espessura da camada de esfregaço e grau de conversão, e a de três fatores, foi aplicada para a resistência de união. O teste de Student-Newman-Keuls (5%) foi aplicado para contraste entre as médias. O teste de correlação de Pearson foi aplicado para verificar a correlação entre rugosidade superficial e espessura da camada de esfregaço.
CONCLUSÕES
Dentro das limitações do presente estudo, pode-se concluir que:
1. A fresa de diamante mais espessa produziu uma maior rugosidade superficial do esmalte e espessura da camada de esfregaço.
2. A influência da morfologia do esmalte sobre os valores de resistência de união foi dependente do material adesivo, isto é, apenas para o sistema autocondicionante suave, obtiveram-se maiores valores de resistência da união com a ponta diamantada mais fina.
3. A aplicação de uma camada adicional de primer dos adesivos autocondicionantes no esmalte não trouxe benefícios à resistência de união, independentemente das suas acidez, mas também não a danificou.
ARTIGO ELABORADO
O presente estudo será enviado para a revista The Journal of Adhesive Dentistry.
Enamel morphology and its influence on the performance of self-etching adhesive systems of different acidities applied in one or two coats
Jéssica Horta Carneiroa/Laiza Tatiana Poskusb/ Eduardo Moreira da Silvac/ José Guilherme Antunes Guimarãesd
a
DDS, MSc student,Analytical Laboratory of Restorative Biomaterials, School of Dentistry, Federal Fluminense University, Niterói, Rio de Janeiro, Brazil. Idea, performed the experiments in partial fulfillment of requirements for a degree, wrote and proofread manuscript.
b
DDS, MSc, PhD, Associate Professor,Analytical Laboratory of Restorative Biomaterials, School of Dentistry, Federal Fluminense University, Niterói, Rio de Janeiro, Brazil.Idea, hypothesis, experimental design, wrote and proofread manuscript.
c
DDS, MSc, PhD, Associate Professor,Analytical Laboratory of Restorative Biomaterials, School of Dentistry, Federal Fluminense University, Niterói, Rio de Janeiro, Brazil.Idea, hypothesis, experimental design, wrote and proofread manuscript.
d
DDS, MSc, PhD, Associate Professor,Analytical Laboratory of Restorative Biomaterials, School of Dentistry, Federal Fluminense University, Niterói, Rio de Janeiro, Brazil.Idea, hypothesis, experimental design, wrote and proofread manuscript.
Corresponding author: Dra. Laiza Tatiana Pos – U v F F F O – S B 30 - C V C N B z - CEP 24020-140 - Phone: 55 21 2629-9832 - Fax: 55 21 2622-5739 - e-mail: poskus@vm.uff.br
ABSTRACT
CARNEIRO JH. Enamel morphology and its influence on the performance of self-etching adhesive systems of different acidities applied in one or two coats [Dissertation]. Niterói: Universidade Federal Fluminense, Faculdade de Odontologia; 2017.
Purpose: To analyze the surface roughness (Sa) and smear layer thickness (SL) of enamel ground with different grit-sized diamond burs, and to verify the influence of this morphology on the adhesive effectiveness of experimental self-etching systems of different acidities, applied in one or two coats. Materials and methods: The vestibular surface of bovine teeth was ground with the diamond burs: R (regular grit diamond bur) and FF (extra-fine grit diamond bur). The enamel surface roughness (n=5) was analyzed using a three-dimensional profilometer (Sa). For SL, after cleavage of the specimen, scanning electron microscope images (n=5) were obtained and the measurement was performed using the Image J software. For microshear bond strength (μSBS), 60 bovine teeth were distributed in the following
groups, according to the diamond bur (R and FF), the experimental self-etching adhesives (Mild - M; Intermediate: - I; Strong – S) and the number of adhesive coats (One coat -1 and Two coats - 2): MR1, MR2, MFF1, MFF2, IR1, IR2, IFF1,IFF2, SR1, SR2, SFF1, SFF2. Clearfil SE Bond was used as commercial reference Microshear bond strength (μSBS) test was performed using a chisel in a universal testing machine. ANOVA and Student-N w ’ w f f y Results: The diamond bur R led to the highest Sa and SL than the FF (p <0.05). Only for the mild experimental adhesive system, the regular diamond bur caused a decrease in the μTBS values (p <0.05). The number of adhesive coats applied to the surface did not influence the results. Conclusion: The coarser diamond bur produced higher Sa and SL, and this morphology was an important factor for the adhesion, being recommended the use of extra-fine grit burs on enamel to improve it, mainly when a mild self-etching adhesive is used.
Key words: smear layer; roughness; acidity; self-etching; enamel; shear bond strength
1- INTRODUCTION
Nowadays, self-etching adhesive systems have been extensively investigated for adhesive dentistry, as they are user friendly, with less clinical steps and technical sensitive than etch-and-rinse ones. 49 Also, it is expected that they did not leave any exposed collagen subject to degradation by metalloproteinases,53 since they are able to promote demineralization and monomer infiltration simultaneously, providing that they are acid enough.26, 32
The acidity of the self-etching adhesive systems can be classified as ultra-mild (pH ≈ 2.5), mild (pH ≈ 2.0), intermediate (pH ≈ 1.5) or strong (pH ≈ 1.0).48 On enamel, the milder systems have shown a lower demineralization pattern and bond strength values than that obtained with etch-and-rinse ones, being recommended an enamel selective conditioning.10, 21, 28, 36, 46, 47 For stronger systems better results have been found for bond strength to enamel.24, 33, 34 However, it seems that milder self-etching systems are more effective on dentin.7, 40 The ideal clinical condition would be if we can count on a self-etching adhesive that could treat efficiently both dentin and enamel simultaneously, resulting in high and long-lasting bond strength.
As is known, different rotary instruments are able to create smear layers and surface roughness on enamel or dentin with their own characteristics.4, 16, 23 Studies have shown that courser instruments can create a surface more roughness and a thicker smear layer, not only on enamel, but also on dentin.4, 16, 23, 42 In this respect, milder self-etching adhesive systems have not shown to remove completely the smear layer, especially when this is thicker.4, 19, 23 However, it is controversial in the literature to assert that the
thickness of this layer do influence on the dentin bond strength for self-etching systems, since some studies15, 37, 41 showed that thicker smear layers led to lower bond strength values, and others, did not observed any influence.18, 42,
44
With regards to enamel, little is addressed about the action of self-etching adhesives on different smear layer thickness or roughness. In this respect, the interaction between self-etching adhesives and enamel has shown to be smear layer thickness dependent4, 23 However, the bond strength of composites to enamel surfaces treated with different rotary instruments was similar, even when self-etching systems of different acidities were used.38 In spite of this, it is difficult to draw accurate conclusions concerning the effect of the acidity of self-etching adhesives on enamel hybridization, as these studies used commercial systems, which may have a greater variation in composition, making difficult the evaluation about the acidity as an isolated factor to obtaining an effective bonding. 11, 45
Given the difficulty to reaching higher bonding strength values with self-etching adhesive systems, it has been proposed a second application of them on dentin, especially when milder systems are used.3, 31 With respect to enamel, no significant improvement was obtained with this procedure, but the enamel was ground with a fine SiC paper.2
Based on the above considerations, the aim of this study was first to verify the surface roughness and thickness of the smear layer of enamel ground with different grit-sized diamond burs. Secondly, it was objective to analyze the influence of the enamel morphology on the adhesive effectiveness of experimental self-etching adhesive systems with different acidities applied in one or two coats.
The null hypotheses of the present study are: 1) Different grit-sized diamond burs do not influence the enamel surface roughness and smear layer thickness; 2) Different enamel morphology do not affect the adhesive performance, despite the acidity of the experimental self-etching systems. 3) The number of adhesive coats applied to the enamel do not influence the bond strength values, regardless of the adhesive acidity and the enamel morphology.
MATERIALS AND METHODS
Tooth preparation
After disinfection in 0.5% chloramine solution and storing in distilled water at 37°C, the enamel vestibular surface of 90 bovine teeth was planned with silicon carbide sandpaper # 600 for 30s, under water cooling. Subsequently, the roots were removed using a diamond saw at the level of the enamel-cement junction. For roughness and smear layer measurement, a parallel section was performed approximately at the middle of the crown, obtaining two fragments. For smear layer measurement, a groove at the palatal side of one fragment, for each tooth, was created to make possible a posterior cleavage.
The vestibular surface of the teeth was ground according to the following groups: R - regular grit-sized diamond bur 3101 (KG SORENSEN, São Paulo, Brazil); FF - extra-fine grit-sized diamond bur (KG SORENSEN, São Paulo, Brazil). For standardization of the applied pressure, a high-speed hand piece attached to a device for dental preparations was used under air
water spray, making 10 passes by each bur. This was replaced every each five prepared enamel surfaces.
Surface roughness
A fragment of each tooth (n=5) was used for measurement of the enamel surface roughness. The topographic analysis was performed using a 3D profilometer (Form Talysurf 60i, Taylor Hobson, Leicester, UK), scanning an area of 1mm2 (1 X 1mm), with a z-resolution of 20 nm, employing 40,000 steps in the X axis and a 2μm space in the Y axis. The roughness of the reconstructed 3D images was obtained using the 3D parameter Sa (average absolute deviation of the surface), applying the following equation:
Where z is the height of the point measured at the X and Y coordinates
Smear Layer thickness
After careful cleavage of the fragment through palatal groove, the specimens (n=5) were immersed in Karnovski solution (2.5% glutaraldehyde, 2% paraformaldehyde in 0.1M sodium phosphate buffer; pH=7.4) for 12 hours at 4°C. Then, they were immersed in sodium cacodylate (0.1M; pH=7.4) for one hour, in three different baths and were rinsed with distilled water for 1 minute. For dehydration, they were immersed in increasing concentrations of
ethanol (50%, 70%, 90%, 100%) and left for 24 hours in a heating chamber at 37°C with colloidal silica for drying.
The specimens were fixed in stubs and these, to a sample holder of low vacuum. Three imagens for each specimen were obtained by means a scanning electron microscope (Phenom proX, Phenom-World BV, Eindhoven, Holland) at a voltage of 15Kv and 3000X of magnification. For each image, three measurements of the smear layer (μm) were performed using the software Image J(Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA), performing 9 measurements for each specimen. An average of them was calculated for statistical analysis.
Experimental adhesives
A model experimental self-etching adhesives were manipulated according to the formulation described in Table 1. For modifying the acidity of the primers, hydrochloric acid (37%) was added. The pH of the primers was controlled using a pH meter (S220 SevenCompactTM pH/Ion, Mettler Toledo, Scwerzebacj, SWZ), each every 24 hour until stabilization, when they were used. The self-etching adhesive system Clearfil SE Bond (Kuraray Inc, Osaka, Japan) was used as a commercial reference. Camphorquinone (0.5% w/w) and ethyl N, N-dimethyl-4-aminobenzoate (EDMAB) (0.5% w/w) were incorporated to act as photoinitiator and reducing agent, respectively (Aldrich Chemical, Inc., Milwaukee, WI, USA).
Composition Titulation Hydrochloric Acid pH Experimental Primer Primer: 10-MDP (30%), TEGDMA, (30%), ethanol (34%), water (5%), camphorquinone (0,5%), EDMAB (0,5%) 0 drop 2,5 1 (0 20 μ ) 1,5 3 (0 60μ ) 0,8 Experimental Adhesive
Adhesive: Bis-GMA (50%), TEGDMA (29%), HEMA (20%), camphorquinone (0,5%), EDMAB (0,5%)
Commercial
Reference (Clearfil SE Bond)
Primer: 10-MDP, HEMA, Hydrophilic DMA, water, tertiary amine, camphorquinone
Adhesive: 10-MDP, HEMA, Bis-GMA, hydrophilic DMA, water, silanized colloidal silica, tertiary amine, camphorquinone,
2,5
Degree of conversion
This property was evaluated by Fourier Transform Infrared Spectroscopy (ALPHA-P FT-IR Spectrometer, Bruker Optics, Ettlingen, Germany), using attenuated total reflectance (ATR) technique (BrukerOptics, Ettlingen, Germany). 0.6μl of adhesive and 0.6μl of primer were mixed (n=5) and deposited on the ATR crystal, where spectra were obtained in the range of 1700-1600cm, for the observation of the signals at 1609-1cm and 1639-1cm corresponding, respectively, to the aromatic vinylic bonds of bisphenol A and aliphatics of the functional group methacrylate. The spectra were obtained with 40 scans and 4cm resolution. Subsequently, the adhesive systems were photoactivated for 40 seconds at 600 mW/cm2 (DEMI, Kerr Corporation,
Middleton, WI, USA), and the spectra were again obtained under the same conditions. The irradiance of the photoactivator was checked prior to the photoactivation of each adhesive through a radiometer (Model 100, Demetron Inc. Danburry, CT, USA). The degree of conversion (GC%) of each adhesive was calculated using the rate between the height of the signal at 1639-1cm and at 1609-1cm of the polymerized and unpolymerized films, according to the following equation:
where R =height of the band at 1639 cm-1 / band height at 1609 cm-1
Microshear bond strength
After preparing 80 bovine as previously described, they were randomly assigned to the groups described in Table 2 (n=5):
Table 2 – Experimental groups
Diamond Burs Self-etching adhesive Number of coats Experimental Groups
Regular (R)
Mild (M) 1 RM1
2 RM2
Intermediate (I) 1 RI1
2 RI2 Strong (S) 1 RS1 2 RS2 Clearfil SE Bond (C) 1 RC1 2 RC2 Extra-Fine (FF) Mild (M) 1 FFM1 2 FFM2
Intermediate (I) 1 FFI1
2 FFI2
Strong (S) 1 FFS1
2 FFS2
Clearfil SE Bond (C) 1 FFC1
Microshear bond strength
Firstly, the primer was applied actively on the enamel surface for 20 seconds, followed by a gentle air spray. A double application, in the same way, was performed in accordance with the table 2. Then, the adhesive was also applied actively and Small tubes (0.6mm in internal diameter and 1.0mm in high) obtained from a tracheal suction catheter (Mark Med, Bragança Paulista, SP, Br) were positioned on the enamel surface, and the photoactivation was realized for 40 seconds using an irradiance of 600 mW/cm2 (Radii-cal, Demetron Inc. Danburry, USA). The irradiance was monitored periodically using a radiometer (model 100, Demetron Inc., Danburry, USA). The Filtek Bulk Fill Flow composite (3MESPE, St Paul, MN, USA) was inserted into the tube in a single increment and photoactivated for 40 seconds. After 1 hour, the tubes were carefully removed with a scalpel blade (n°11, Wuxi Still Medical device CO.LTD, Xishan, Jiangsu, China), and the resin cylinders were evaluated using a steromicroscope (40X, SZ61TR Stereoscopic Microscope, Olympus, USA) to discard specimens with bubbles or failures. After storing in distilled water at 37 °C for 24 hours, the specimens were attached in a metal device and the microshear test was runned in a universal testing machine (DL 2000, EMIC, São José dos Pinhais, PR, Brazil), using a chisel at a speed of 1,0 mm/min. (Figure 1)
Figure 1. Schematic illustration of the μSBS test.
Failure mode analysis
After rupture, each dentin surface was evaluated with the stereomicroscope at 40X of magnification, and the failure mode was classified as adhesive (failures at the adhesive interface), cohesive (failures occurring within dentin or resin composite), or mixed (both adhesive and cohesive failures for the same fractured surface).
Statistical treatment
The data obtained were analyzed using Statgraphics Centurion XVI software (StatPoint Technologies Inc., Warrenton, VA, USA). After verification of the sample y f v ( v ’ ) y f variance of one factor was applied for surface roughness, smear layer thickness and degree of conversion, and of three factors, for microshear bond
strength. The Student-Newman-K ’ (5%) was applied for post-hoc T P ’ w f between the surface roughness and the smear layer thickness.
RESULTS
The means, standard deviation, and statistical differences are shown in Tables 3, 4, 5 and 6. For surface roughness, smear layer thickness and degree of conversion, there was a high statistical significance (p <0.001). For the bond strength test, only the "Adhesive" factor and the double interaction "diamond bur and self-etching adhesive" were statistically significant (p <0.001).
From Table 3, it can be observed that the regular grit diamond bur led to higher roughness and smear layer thickness than the extra-fine grit diamond bur (p <0.05). T P ’ w y strong positive correlation (R = 0.55) between the roughness and the smear layer. The Figure 2 shows the simple regression graph. It can be observed in Table 4 that the mild experimental adhesive (M) showed higher values for degree of conversion (p <0.05) than intermediate and strong adhesives. Table 3 – Means and SD of roughness (m) and smear layer thickness (m) after enamel grinding with different diamond burs
Figure 2 – Linear regression line of smear layer thickness plotted against roughness.
Table 4- Means and standard deviation (SD) of degree of conversion (%) for the adhesive systems studied
Adhesive Systems
M I S
96,23 ± 2,56a 79,73 ± 9,60b 77,34 ± 8,24b
*different letters indicate statistical significant differences (DC CSE=86,31)
From table 5, the intermediate self-etching adhesive obtained higher values of bond strength than the strong, and this, higher values than the mild (p <0.05). According to table 6, it can be observed that for mild self etching
SL = 18,89 + 2,19 * Sa Sa 0 2 4 6 8 10 0 10 20 30 40 50 S L t h ic k n e s s m) m) Properties Diamond bur (*) R FF Roughness 7,15 ± 1,28a 2,02 ± 0,59b
adhesives, the bond strength values lower when the coarser diamond bur was used (p <0.05). For the other adhesives, the diamond bur used did not influence the results. In accordance with Figure 3, the most of failures after shear bond strength test were adhesive. There were not failures completely cohesive.
Table 5 – Means and SD of microshear bond strength (MPa) for the adhesive systems
Adhesive Systems
M I S
12,62 ± 4,35c 27,04 ± 4,38a 16,59 ± 3,24b
*different letters indicate statistical significant differences (µSBS CSE=25,72)
Table 6 – Means and SD of microshear bond strength (MPa) to enamel ground with two different burs (R and FF)
M I S
R 10,72 ± 3,15d 28,73 ± 4,67a 16,45 ± 3,41b
FF 14,51 ± 4,69c 25,35 ± 3,52a 16,72 ± 3,25b
*different letters indicate statistical significant differences (µSBS CSER=25,74; CSEFF=25,69)
Figure 3– Failure modes DISCUSSION
Most of the laboratory tests about adhesion between the composites and the dental structure use silicon carbide papers (SiC) to prepare the specimens, in order to make easier to obtain and standardize flat dental surfaces.2, 16, 43 However, it was demonstrated in a previous study23 that the use of silicon carbide paper produces a different enamel substrate than that obtained with diamond burs, which could influence on the behavior and, consequently, on the adhesive effectiveness of the self-etching adhesives. Thus, in the present study, diamond burs of different granulations, instead of silicon carbide papers, were chosen to prepare the enamel surface, in order to mimic the clinical situation.
It is worth to mention that factors such as viscosity of the self-etching adhesive systems, their surface tension and the bonding potential between the different functional monomers and the residual hydroxyapatite, not only
0 10 20 30 40 50 60 70 80 90 100 RM FFM RI FFI RS FFS MISTA ADESIVA
can determine the bond effectiveness, but also the stability of it24, 51 The presentation of these systems in single or two bottles is another factor that could influence the bond results10, 35 Thus, in the present study, we tested experimental adhesives with similar composition, varying only the acidity, in order to allow more accurate comparisons between the experimental conditions. It was chosen the acidic functional monomer 10-MDP because it has been shown a high potential of chemical bonds to the hydroxyapatite, improving the performance and the longevity of the bond to the substrate51, 52
The degree of conversion of the experimental self-etching adhesive systems ranged from 96.23% to 77.34%, similar to previous studies,27, 50, 54 rendering the proposed formulation more reliable. This analysis was performed because it is a fundamental parameter to predict bond stability, and the degree of conversion may be related to the adhesion provided by the adhesive systems.6 In spite of the mild self-etching adhesive had shown higher degree of conversion than intermediate and strong ones, it did not led to higher values of µSBS. It has been shown that more viscous solutions are capable to reduce the polymer mobility. So, the free radical propagation can increase, improving the vinyl conversion and the polymerization rate.30 In spite of we had not verified the viscosity of the primers, the addition of the hydrochloric acid could have diminished the viscosity of them, impairing their degree of conversion.In adition, a lower pH could affect the polymerization initiating process by amines, reducing the degree of conversion.24 However, the buffering capacity induced by the hydroxyapatite of the enamel, could had increased the pH, making possible a more complete monomer conversion, so
that an higher bond strength values were found for the experimental stronger adhesives.
The first null hypothesis of this study was rejected since the use of diamond burs of different grit size influenced both the surface roughness and the smear layer thickness created on the enamel, i.e. the coarser diamond bur showed the greater roughness and smear layer thickness, which is in accordance with previous studies.4, 16, 23 It was also found a strong positive correlation between the thickness of smear layer and surface roughness, that is, the greater the roughness, the greater the smear layer thickness on the enamel.
When etch-and-rinse adhesive systems are used, longer resin tags are obtained due to the penetration of the monomers into the enamel microporosities12 which are responsible for enamel bond.5 For self-etching adhesive systems, shorter resin tags can be observed by means scanning electron microscopy (SEM) images. When evaluated by transmission electron microscopy (TEM), these tags are not purely resinous, being found resin-encapsulated crystallites, suggesting that the bond between enamel and resin achieved with self-etching systems is based on inter and intra-crystallite hybridization of the enamel surface, better than on the formation of resinous tags.12, 13
The acidity of the self-etching adhesive systems has been associated with its ability to produce an effective demineralization of the enamel surface, being found defined hybrid layers of 1 to 2μm for the strongest systems.45
However, the demineralization capacity of self-etching systems has been shown to be highly dependent on the nature of the enamel smear layer
present.23, 25 The removal of the smear layer with phosphoric acid and polishing the surface prepared with coarser diamond burs could improve the action of self-etching adhesives on the enamel surface and, consequently, the bond results, mainly when mild ones are employed.23
On the other hand, the surface roughness of a solid is a factor under the control of the clinician that influences its wettability by a liquid. It is expected that, the rougher the surface of the solid, the larger the surface area and the greater the wettability, improving the bond strength values.9 Thus, this study aimed to verify if the enamel morphology obtained with different grit-sized diamond burs, and the acidity of the self-etching adhesives could influence the enamel bond results.
According to the data obtained, the second hypothesis of this study was partially accepted, since the bond strength values depended on the acidity of the self-etching adhesive systems used (table 5), but the enamel morphology influenced the results only for the mild experimental adhesive. When the enamel surface was ground with the regular diamond bur, which in turn implied greater surface roughness and smear layer thickness (Table 3), the µSBS values were lower for the mild experimental adhesive. Other studies also showed for mild self-etching adhesives, an influence of different grit-size burs on the bond strength results. 4, 19, 23 It may be speculated that the low acidity of the mild adhesive primers was not able to etch successfully the enamel substrate when a thick smear layer was present. Bortoloto et al4 pointed out that the enamel smear layer has a high amount of hydroxyapatite compared to dentin. So, in thicker smear layers, higher amounts of H+ ions would be required to overcome the inherent buffering capacity caused by their
high hydroxyapatite content, that is, the acidity of the self-etching adhesive system would be fastly buffered by hydroxyapatite. Such authors even recommended that clinicians use a more acidic self-etching system or rotating instruments that would lead to the formation of a thinner enamel smear layer.
However, in a previous study38 the use of regular or super-fine diamond burs on enamel did not influence the bond strength values for self-etching adhesives with different acidities, although there had been a tendency of better results for the superfine grit bur. It should be emphasized that the authors used commercial adhesives of different composition, making more difficult the comparisons.
It could be expected that the more roughness enamel surface, and, consequently, the larger surface area for the mechanical interlocking9 would increase bond strength values. However, this did not occur. In fact, it was shown in a previous study1 that although different rotating instruments caused different surface roughness on the enamel surface, surface wettability was not significantly affected. Therefore, the authors of the present study could suppose that the thickness of the smear layer formed by the different rotary instruments was more decisive in obtaining the results of the bond strength than the surface roughness.
In general, intermediate and strong self-etching experimental adhesives showed higher values of enamel bond strength than the mild adhesive (table 5), corroborating the results found by Sevigcan et al.38 However, previous studies17, 20, 25 produced results which go against the presented here, despite having shown a less retentive enamel demineralization pattern for mild adhesives, with reduced potential
micromechanical interlocking.8, 25 These authors speculated that the commercial self-etching adhesive systems studied had different compositions, such as the presence of the functional monomer 10-MDP in the mild adhesive, which has a potential for chemical bonds with the hydroxyapatite, or even the differences between the wettability characteristics of the monomers. These factors could have influenced the better bond results for the mild self-etching adhesive, regardless of its lower acidity.
Also, in a previous study22 no difference was found for the enamel bond strength using a mild or strong self-etching adhesive. In that study, a medium-grit diamond bur was used, which could have created a thinner smear layer than a regular bur, influencing the results obtained. In fact, in the present study, when using the finer bur, the bond strength values obtained for mild or strong experimental self-etching adhesives were similar.
In the present study, it was unexpected that the stronger experimental adhesive had lower bond strength values than the intermediate one, since its potential for demineralization and micromechanical interlocking would be greater. It is known that under more acidic conditions, the hydrolytic stability of the methacrylates is impaired, that is, the ester portion of functional monomers can be hydrolyzed in the presence of an aqueous solution and low pH.24, 29 So, it may be speculated that, in the more acidic condition of the strong self-etching adhesive system, although the functional monomer 10-MDP be more stable, it may have undergone certain hydrolysis of the monomers, impairing its bond strength values.
It should be noted that most of the failures occurred were adhesive. When etch-and-rinse systems are employed, the entire demineralized area
created by the phosphoric acid is not filled by the resinous monomers,39 providing a non-infiltrated zone, less resistant to mechanical loads by the friability of the non-supported enamel, so that a cohesive failure in enamel could be the precursor of the failure.14 For self-etchings adhesives, the hydroxyapatite crystals are not removed, being involved by the resinous monomers, acting as an inorganic reinforcement in the region of the mechanical interlocking, which would make the rupture more difficult. Thus, there would be a predisposition for the adhesive failures.14
The third hypothesis of this study was accepted, since the number of coats of the self-etching primers applied on the enamel, irrespective of their acidities, did not influence the bond strength values, which is in accordance to a previous study.2 In dentin, previous studies have shown that the application of a double layer could benefit the bond strength of certain self-etching systems.2, 31 This has been associated with, among other factors, the occurrence of a faster buffering of the self-etching adhesive system in this substrate due to moisture, thus reducing its action. So, the application of two coats for the mild self-etching adhesive systems on dentin could increase the bond strength. This buffering may not have been as effective on enamel, a more mineralized and low moisture substrate, allowing a longer action of the adhesives even for the mild system, so that the application of a second layer did not improve the adhesion.
Within the limitations of the present study, it can be concluded that:
1. The coarser diamond bur produced a greater enamel superficial roughness and smear layer thickness.
2. The influence of the enamel morphology on the bond strength values was adhesive-material dependent, that is, only for mild self-etching system, higher bond strength values were obtained with the finer diamond bur.
3. The application of an additional coat of the primer self-etching adhesives on enamel did not bring benefits to the bond strength, irrespective their acidities, but did not also damage it.
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