UNIVERSIDADE ESTADUAL DE CAMPINAS Faculdade de Odontologia de Piracicaba
MARIANA DIAS FLOR RIBEIRO
DESEMPENHO FÍSICO-QUÍMICO DE INFILTRANTES
EXPERIMENTAIS E COMERCIAL: UM ESTUDO IN VITRO E
IN SITU
PHYSICOCHEMICAL PERFORMANCE OF EXPERIMENTAL
AND COMMERCIAL INFILTRANTS: AN IN VITRO AND IN
SITU STUDY
PIRACICABA 2019
MARIANA DIAS FLOR RIBEIRO
DESEMPENHO FÍSICO-QUÍMICO DE INFILTRANTES
EXPERIMENTAIS E COMERCIAL: UM ESTUDO IN VITRO E IN SITU
PHYSICOCHEMICAL PERFORMANCE OF EXPERIMENTAL AND
COMMERCIAL INFILTRANTS: AN IN VITRO AND IN SITU STUDY
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 Clínica Odontológica, na Área de Dentística.
Thesis presented to the Piracicaba Dental School of the University of Campinas in partial fulfillment of the requirements for the degree of Doctor in Dental Clinic, in Dentistry area.
Orientadora: Profª Drª Giselle Maria Marchi Baron
ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA TESE APRESENTADA PELA ALUNA MARIANA DIAS FLOR RIBEIRO, E ORIENTADA PELA PROFA. DRA. GISELLE MARIA MARCHI BARON.
PIRACICABA 2019
Dedico esse trabalho a Deus e a minha família, que são minha base de sustentação nessa vida terrena.
Agradeço primeiramente a Deus, que me sustentou, apoiou nessa árdua jornada e me capacitou para que esse dia chegasse. A Ele minha gratidão eterna pelo sopro da vida e por ter me permitido chegar muito além do que eu sonhei.
Agradeço à professora Giselle, que foi uma mãe para mim nesses quase 5 anos de estudo em Piracicaba. Obrigada por cada orientação, cada conselho, cada palavra e por acreditar em mim e me permitir viver essa experiência de pós-graduação na FOP/Unicamp.
Agradeço ao Roney, que nesse período de doutorado se tornou meu marido. Obrigada por nunca me deixar esquecer de que o amor existe e por escolher dividir a vida comigo. Obrigada por me apoiar em cada novo sonho e compartilhar tanto amor comigo.
Agradeço aos meus pais, Pedro e Ana, que nunca mediram esforços para que eu e minhas irmãs pudéssemos realizar nossos sonhos. Obrigada por sempre apoiarem nossos estudos e serem nossas referências de amor e companheirismo.
Agradeço às minhas irmãs, Marília e Ana Clara, por todo apoio e carinho nesse período que estive ausente. Sabemos que sempre podemos contar umas com as outras e esse laço é eterno. Gratidão por vocês existirem!
O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) - Código de Financiamento 001 e da Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), processo nº 2017/14378-6.
Agradeço à direção da Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas, na pessoa do Diretor Prof. Dr. Francisco Haiter Neto e vice-diretor Prof. Dr. Flávio Henrique Baggio Aguiar.
À Profa. Dra. Karina Gonzales Silverio Ruiz, coordenadora dos cursos de Pós--‐Graduação e ao Prof. Dr. Valentim Adelino Ricardo Barão, coordenadora do Programa de Pós-Graduação em Clínica Odontológica.
À Professora Giselle Maria Marchi Baron, que me acompanhou, orientou e apoiou nessa jornada de mestrado e doutorado.
Aos professores da banca de qualificação: Thatiana de Vicente Leite, Diogo Bonazzi Dressano e Flávio Henrique Baggio Aguiar pelas valiosas contribuições nesse trabalho.
Aos Professores da Área de Dentística, Giselle Maria Marchi; Vanessa Cavalli Gobbo; Débora Alves Nunes Leite Lima; Lúcia Trazzi Prieto; Flávio H. Baggio Aguiar; Luis Alexandre M. S. Paullilo; Luís Roberto M. Martins e Marcelo Giannini pelos ensinamentos compartilhados ao longo desse ciclo de mestrado e doutorado.
Aos companheiros Giselletes: Thati, Alan, Diogo, Carol, Priscila, Janaína, Marco Túlio, Ana e Gabi por compartilharem conhecimentos e apoiarem nos momentos em que precisei.
Aos professores da UFES, minha primeira casa universitária, que me apoiaram na decisão de ingressar no mestrado e seguir no doutorado.
Ao Rodrigo que além de amigo se tornou meu irmão de vida. Obrigada por ser minha inspiração de dedicação e conduta, sem nunca esquecer os princípios e valores humanos.
À Renata, que é minha referência na busca de evoluções pessoais, profissionais e espirituais. Obrigada por ser luz e compartilhar toda essa amizade comigo.
À equipe de microbiologia da FOP-Unicamp. Em especial à professora Renata Graner a à pós doutoranda Lívia Alves, por nos darem suporte à realização do teste de Unidades Formadoras de Colônias, descrito nessa tese.
Ao professor Roberto Braga, da USP São Paulo, que permitiu a realização do teste de Grau de Conversão, descrito nessa tese.
À equipe da área da bioquímica, professora Cínthia que permitiu o uso do laboratório da bioquímica para a realização das soluções utilizadas nessa tese e a Mayara Noronha, que me auxiliou em todo o preparo dessas.
Ao técnico Marcelo de Assumpcão, da USP São Carlos, que deu suporte à realização do teste de Microscopia de Força Atômica descrito nessa tese.
Ao técnico do laboratório Wanderlei Francisco por toda a dedicação na manutenção do laboratório e bem-estar comum.
À Andréia, por sempre dar suporte aos alunos e professores do Departamento de Clínica Odontológica.
Aos técnicos do microscópio confocal, Flávia Sammartino e Adriano Lima, por permitirem a realização do teste de Profundidade de Penetração descrito nessa tese.
A todas as funcionárias da limpeza, em especial à Magda e Selma por sempre serem receptivas, amáveis e zelarem pelo bem-estar de todos.
A todos que direta ou indiretamente contribuíram para a realização desse trabalho: muito obrigada!
“Se cheguei até aqui foi porque me apoiei no ombro dos gigantes”. (Isaac Newton)
Objetivos: Desenvolver infiltrantes experimentais contendo sal de difeniliodônio (DFI)
e quitosana em diferentes concentrações e avaliar seu desempenho físico-químico in
vitro (Artigo 1) e in situ (Artigo 2) em comparação com o infiltrante comercial. Materiais
e métodos: Foram desenvolvidos 9 infiltrantes experimentais contendo TEGDMA e
BisEMA, variando a concentração do DFI em 0; 0,5; 1 mol% e quitosana em 0; 0,12 e 0,25% - em peso, que foram comparados com o infiltrante comercial Icon® (controle positivo). A formulação foi realizada em ambiente com iluminação e temperatura controlados. Para o estudo in vitro, foram utilizados corpos de prova de infiltrantes polimerizados e realizados testes de Módulo de Elasticidade (n=10), Resistência à Flexão (n=10), Grau de Conversão (n=5), Sorção (n=5) e Solubilidade (n=5); e para o teste de Ângulo de contato (n=6) foram utilizados blocos de esmalte humano dental polido e infiltrantes não polimerizados. Para o estudo in situ, 132 blocos de esmalte humano dental (n=12/grupo) foram confeccionados e submetidos à simulação de lesão inicial de cárie. As amostras foram aleatorizadas com base na microdureza inicial do esmalte e divididas em 11 grupos de tratamento (9 infiltrantes experimentais, infiltrante comercial como controle positivo e grupo sem tratamento como controle negativo) e foram realizados os testes de Rugosidade (n=3), Área (n=3), Morfologia e Projeção Superficial com Microscópio de Força Atômica (n=3), Análise microbiológica por Teste de Unidades Formadoras de Colônias (UFC/mL; n=5) e Profundidade de Penetração (n=3) com Microscópio Confocal de Varredura à Laser. Os dados foram analisados por meio de modelos lineares generalizados e ANOVA one-way com post-hoc de Tukey e pelo teste de Dunnet (α=0.05). Resultados: In vitro - Grupos de infiltrantes contendo a mistura de DFI 1% e quitosana 0.12% e 0.25% tiveram melhores resultados de módulo de elasticidade, resistência à flexão e grau de conversão em comparação com o produto comercial Icon®. Porém, para sorção, solubilidade e ângulo de contato, o Icon® teve o melhor desempenho. In situ - Não houve diferença significativa entre os onze grupos quanto atividade microbiológica (UFC/mL), rugosidade e área (p<0.05). A profundidade de penetração foi significativamente maior no grupo sem tratamento do que no grupo com 0.5% de DFI e 0.25% de Quitosana (p<0.05). Conclusão: Os infiltrantes experimentais contendo sal de iodônio e quitosana testados apresentam similaridade em comparação com o infiltrante comercial Icon®.
Palavras-chave: Resinas, Sintéticas; Técnicas In Vitro; Hibridização in Situ
ABSTRACT
Objectives: To development experimental infiltrants containing diphenyliodonium salt
(DPI) and chitosan in different concentrations and to evaluate their physicochemical performance in vitro (Paper 1) and in situ (Paper 2) in comparison with the commercial infiltrant. Material and methods: Nine experimental infiltrants containing TEGDMA and BisEMA were prepared, varying the DPI concentration by 0; 0.5; 1 mol% and chitosan at 0; 0.12 and 0.25% - by weight, which were compared with the commercial Icon® infiltrant. The formulation was performed in environment with controlled lighting and temperature. For the in vitro analyses, polymerized infiltrants specimens were used and Elastic Modulus (n = 10), Flexural Strength (n = 10), Degree of Conversion (n = 5), Sorption (n = 5) and Solubility (n = 5) tests were performed; and for the Contact Angle test (n = 6), polished dental human enamel blocks and unpolymerized infiltrants were used. For the in situ study, 132 blocks of human dental enamel (n = 12 / group) were made and submitted to the simulation of initial caries lesion. Samples were randomized based on initial enamel microhardness and divided into 11 treatment groups (9 experimental infiltrants, commercial infiltrant as positive control and untreated group as negative control) and the Roughness (n = 3), Area (n=3), Morphology and Surface Projection (n = 3) with Atomic Force Microscope, Microbiological Analysis by Colony Forming Test (CFU / mL; n = 5) and Penetration Depth (n = 3) with Confocal Laser Scanning Microscope tests were performed. Data were analyzed using generalized linear models and one-way ANOVA with Tukey post-hoc and Dunnet's test (α = 0.05). Results: In vitro - Infiltrants groups containing 1% DPI and 0.12% and 0.25% chitosan in the blend had better results of elastic modulus, flexural strength and degree of conversion compared to the commercial Icon® infiltrant. In situ - There was no significant difference between the eleven groups regarding microbiological activity (CFU/mL), roughness and area (p <0.05). Penetration depth was significantly greater in the untreated group than in the 0.5% DPI and 0.25% Chitosan group (p <0.05). Conclusion: The experimental infiltrants containing iodonium salt and chitosan tested are similar when compared to the commercial infiltrant Icon®.
Key words: Resins, Synthetic; In Vitro Techniques; In Situ Hybridization,
SUMÁRIO
1 INTRODUÇÃO 13
2 ARTIGO 1: In vitro evaluation of physical properties of experimental infiltrants
containing iodonium salt and chitosan 16
3 ARTIGO 2: In situ analysis of physicochemical properties and penetration of experimental infiltrants containing iodonium salt and chitosan into artificial enamel
caries lesions 29
4 DISCUSSÃO 49
5 CONCLUSÃO 53
REFERÊNCIAS 54
ANEXOS
Anexo 1 – Aprovação no Comitê de Ética em Pesquisa 57 Anexo 2 – Comprovante de submissão dos artigos 58 Anexo 3 – Metodologia Ilustrada 59
1 INTRODUÇÃO
Apesar da constante evolução da Odontologia, a cárie dental continua sendo um dos distúrbios de saúde mais predominantes na sociedade moderna (Qu et al., 2015). Dessa maneira, torna-se importante o diagnóstico de lesões de carie em estágio inicial, o qual caracteriza-se principalmente pela perda mineral subjacente a uma superfície aparentemente intacta (Kidd et al., 2004). Essas lesões, também chamadas de lesões de mancha branca, podem ocorrer por vários fatores como a má higiene oral, hipofunção salivar, dieta cariogênica, etc. (Ceci et al., 2017).
Bem como a maioria das doenças humanas, a cárie pode ser controlada com facilidade e precisão nos estágios iniciais (Andrade Neto et al., 2016). Atualmente, a primeira opção de tratamento de lesões cariosas incipientes está relacionada aos procedimentos minimamente invasivos que promovem a remineralização da estrutura dental (Paris et al., 2007; Young, Featherstone, 2010; Ceci et al., 2017). Até a momento, o único material biocompatível capaz de impedir a progressão da cárie em estágios iniciais da doença é o infiltrante resinoso (Paris; Meyer-Lueckel, 2010), o qual consiste em uma mistura de monômeros de baixa viscosidade e sem carga que têm por objetivo o selamento e a paralisação da lesão cariosa em seus estágios iniciais (Doméjean et al, 2015; Andrade Neto et al., 2016).
Como mecanismo de ação, o infiltrante atua por capilaridade, ao penetrar no interior dos pequenos poros formados pela desmineralização da lesão de cárie; e, assim, impede que esses poros atuem como via de difusão de ácidos e minerais (Meyer-Lueckel & Paris, 2008; Paris S & Meyer-Lueckel, 2010). Consequentemente, há inibição do desenvolvimento da lesão cariosa, reforço da estrutura dentária; e paralisação e controle da lesão (Gurdogan et al., 2017). No entanto, como só há uma marca de infiltrante comercialmente disponível, dispendiosos esforços vêm sendo feitos no meio científico para encontrar um material com características semelhantes a esse produto (Araújo et al., 2013; Inagaki et al., 2016; Mathias et al., 2018; Flor-Ribeiro et al., 2019).
Entretanto, torna-se difícil estabelecer uma composição que seja capaz de unir fluidez à resistência, uma vez que baixa quantidade de monômeros hidrófobos,
relacionados às propriedades mecânicas, e alta quantidade de monômeros diluentes e solventes são necessários para diminuir a viscosidade do material (Paris et al., 2007).
Em geral, materiais resinosos utilizam em sua maioria o monômero bisfenol A glicidil dimetacrilato (BisGMA) devido ao seu alto peso molecular que confere um bom desempenho mecânico, porém, ele possui como desvantagem alta viscosidade. Para promover melhoria na sua fluidez, a mistura ou substituição por monômeros com menor peso molecular têm sido propostos, como por exemplo, o bisfenol A dimetacrilato etoxilado (BisEMA) que devido à ausência dos grupos hidroxila, confere menor viscosidade e também maior susceptibilidade à sorção de água (Gonçalves et al., 2009). Além da substituição do BisGMA, também pode-se optar pela utilização de co-monômeros, não tão resistentes à degradação, visando diminuir a viscosidade do produto final, sendo que o trietileno glicol dimetacrilato (TEGDMA) é o mais utilizado por possuir baixo peso molecular e baixa viscosidade (Floyd; Dickens, 2006), apresentando-se, inclusive, como principal componente do infiltrante comercial disponível, Icon®.
Adicionalmente, a inclusão de 2-hidroxi-etilmetacrilato (HEMA) aos infiltrantes pode ser utilizado com objetivo de aumentar a infiltração monomérica devido ao fato de esse monômero apresentar baixo peso molecular, e consequentemente baixa viscosidade (Gonçalves et al., 2009; Paris et al., 2012).
Em biomateriais dentais, o sal de difeniliodônio (DFI) tem mostrado influenciar positivamente as propriedades mecânicas dos materiais, bem como: reatividade e potencial de polimerização, resistência à flexão, módulo de elasticidade, grau de conversão (Ogliari et al., 2007; Gonçalves,2013), atividade antibacteriana (Flor-Ribeiro et al., 2019) e, por isso, passível de estudos mais aprofundados. Do mesmo modo, o polissacarídeo quitosana tem sido intensamente utilizado na área da odontologia, e possivelmente atua como um arcabouço de reforço aos metacrilatos (Diolosà et al., 2014), aumentando assim a resistência mecânica dos infiltrantes (Flor-Ribeiro et al., 2019).
De um modo geral, os infiltrantes são materiais recentes e, por isso, com limitados estudos nas bases de dados disponíveis atualmente, tanto in vitro como in situ. Dessa maneira, a avaliação comparativa entre infiltrantes experimentais contendo sal de difeniliodônio e quitosana ao infiltrante comercial, ainda não foi proposto. Por ser
evidente a necessidade de novos estudos que avaliem melhorias na composição dos infiltrantes, criação de novos materiais, além de investigar a influência de diferentes reagentes no material sobre suas propriedades químico-físicas, esse estudo objetivou desenvolver infiltrantes experimentais contendo sal de difeniliodônio e quitosana em diferentes concentrações e avaliar seu desempenho físico-químico in vitro (Artigo 1) e in
2. ARTIGO 1: IN VITRO EVALUATION OF PHYSICAL PROPERTIES OF
EXPERIMENTAL INFILTRANTS CONTAINING IODONIUM SALT AND CHITOSAN
Submitted in Journal of Applied Oral Science
Authors: Mariana Dias Flor Ribeiro, Rodrigo Barros Esteves Lins, Flávio Henrique
Baggio Aguiar, Giselle Maria Marchi
ABSTRACT
Objective: To development experimental infiltrants containing iodonium salt and chitosan
and evaluate their physical performance compared to commercial infiltrant. Material and
Methods: Nine experimental infiltrants containing TEGDMA and BisEMA were prepared
in a proportion of 75 and 25% by weight, respectively; 0.5 mol% camphorquinone and 1 mol% ethyl 4-dimethylaminobenzoate (EDAB) as photoinitiator system, varying iodonium salt (DPI) (0; 0.5; 1 mol%) and chitosan (0; 0.12 and 0.25 wt%) which were compared with the commercial Icon® infiltrant. The formulation was performed in environment with controlled lighting and temperature. Degree of Conversion (DC) test were performed with Fourier Transformer Infrared Spectroscopy FTIR (n = 5), Flexural Strength (FS), Elastic Modulus (EM) with three-point bending test in a universal testing machine EMIC (n = 10), Sorption (SO) and Solubility (SL) in water (n = 5) and Contact Angle (CA) (n = 6). Data were analyzed by one-way ANOVA with Tukey post-hoc and Dunnet's test. Results: Infiltrants groups containing the iodonium sal and chitosan in the blend had better results of modulus of elasticity, flexural strength and degree of conversion compared to the commercial Icon® infiltrant. However, for sorption, solubility and contact angle, Icon had the best performance. In the general, the infiltrant group containing 1% DPI and 0.12% chitosan showed the best results from the physical analyzes of this study. Conclusion: The addition of iodonium salt and chitosan in experimental infiltrants improve their physical properties.
INTRODUCTION
Restorative dentistry is an area that is constantly evolving, new materials and techniques are developed in order to improve and ensure longevity to the restorative treatment. In this context, in 2006 a new class of materials emerged in the market: that of resin infiltrants 1. It is a monomer blend with low viscosity, no filler, that aim to seal and paralyze the carious lesion in its early stages 2, 3.
In conjunction with modern Minimally Invasive Dentistry, infiltration technique has become increasingly common in recent years 2, 4. As an action mechanism, the infiltrant acts by capillarity, penetrating the small pores formed by the demineralization of the caries lesion; thus, they prevent these pores from acting as an acid and mineral diffusion pathway 5, 6. Consequently, there is inhibition of carious lesion development, strengthening of tooth structure, paralysis and lesion control 7, in addition to promoting increased enamel surface hardness 8.
However, as there is only one commercially available infiltrant brand, costly efforts have been made in the scientific community to formulate a material with improved characteristics relative to this product 9, 10, 11.
In past studies 11, 12, experimental infiltrants TEGDMA and BisEMA – based showed good physical, mechanical and antimicrobial behavior. With the progress of the studies, we aim to create an infiltrant with different monomeric compositions in relation to the commercial infiltrant, but with similar or superior physical-chemical behavior. In dental biomaterials, iodonium salt has shown excellent results in relation to the improvement of physical properties, with increased polymerization efficiency and increased mechanical strength when added to these materials 13, 14.
Similarly, there is evidence that chitosan, in addition to being known for its antimicrobial properties, also works by reinforcing the internal structure of methacrylate-based polymers 15, 12, which can intensify their mechanical strength and consequently increase the durability of these materials. To date no tests have been performed comparing these experimental infiltrants containing iodonium salt and chitosan to the commercial infiltrant. Thus, this study aimed to development experimental infiltrants
containing iodonium salt and chitosan in order to improve their physical characteristics regarding resistance, water behavior and contact angle and to compare them with the commercial Icon® infiltrant.
As a null hypothesis, we have that: Experimental infiltrants do not present statistical difference in relation to the commercial infiltrant in relation to the proposed physical tests.
MATERIALS AND METHODS
a. Experimental Infiltrant Formulation
Based on a previous study 12, nine experimental infiltrants were formulated in the
laboratory, and the control group consisted of the Icon® brand infiltrant - Table 1.
Table 1. Composition of infiltrating groups.
Groups Composition G1 Basic composition
G2 Basic composition + Chitosan (0,12%)
G3 Basic composition + Chitosan (0,25%)
G4 Basic composition + DPI (0,5%)
G5 Basic composition + Chitosan (0,12%) + DPI (0,5%)
G6 Basic composition + Chitosan (0,25%) + DPI (0,5%)
G7 Basic composition + DPI (1%)
G8 Basic composition + Chitosan (0,12%) + DPI (1%).
G9 Basic composition + Chitosan (0,25%) + DPI (1%).
G10 Commercial Infiltrant Icon®.
Base composition of experimental infiltrants: BisEma (25%), TEGDMA (75%), HEMA (10%), EDAB (1%), Camphorquinone (0,5%).
The basic composition of the experimental infiltrants was based on BisEma (Sigma Aldrich, St. Louis, USA) (25%) and TEGDMA (Sigma Aldrich, St. Louis, USA) (75%) as base monomers, HEMA (Sigma Aldrich, St. Louis, USA) (10%) as solvent, camphorquinone (Sigma Aldrich, St. Louis, USA) (0.5%) and EDAB amine (Sigma Aldrich, St. Louis, USA) (1%) as photoinitiation system. The variables used were chitosan (Sigma Aldrich, St. Louis, USA) (0; 0.12; 0.25 wt.%), and diphenyliodonium hexafluorophosphate salt - DPI (Sigma Aldrich, St. Louis, USA) (0; 0.5; 1 mol%). All materials used were weighed in high precision analytical balance (Chyo JEX-200, YMC Co, Tokyo, Japan) and handled in ambient with lighting and temperature control. The appliance used for the samples light curing was the VALO (Ultradent Products Inc; S. Jordan, UT, USA), irradiance: 1000 mW/cm2.
b. Elastic Modulus and Flexural Strength
Samples from each group (n = 10) were made in polyvinylsiloxane matrices (7 x 2 x 1 mm) (Express XT, 3M ESPE, St. Paul, USA) following ISO 4049: 2000 specifications except for sample size. The samples were photoactivated for 40 seconds and stored in an incubator at 37ºC for 24 hours. In a universal testing machine (Instron, Model 4111, Instron, Canton, USA) a three-point test was performed under load of 50N, 0.5mm / min, until the moment of specimen fracture. Through the Bluehill 2 software of the machine itself and based on the sample sizes, the elastic modulus in GPa and the flexural strength in MPa were calculated.
c. Degree of Conversion
This test was performed using a Fourier transformer infrared spectroscopy apparatus (FTIR, Vertex 70, Bruker Optik GmbH, Germany) with a wavelength range of 4000 cm -1 to 9840 cm -1 with a resolution of 6 cm -1. Unpolymerized samples (n = 5) were carefully placed into the reading hole (5mm x 1mm radius) of polyvinylsiloxane matrices (Express XT, 3M ESPE, St. Paul, USA) and read. Light curing was performed for 40 seconds. Each sample was subjected to 32 readings, both pre and post polymerization. Data were analyzed by Opus v.6 software (Bruker Optics, Germany). The degree of conversion was calculated as follows: 1- Residual double bonding rate (polymerized
sample / unpolymerized sample x 100); 2 – Degree of Conversion (%) = 100 - residual double bonds (%).
d. Water sorption and solubility
Circular shaped samples (n = 5, 5 x 1mm) were prepared in polyvinylsiloxane matrices (Express XT, 3M ESPE, St. Paul, USA) following ISO 4049: 2009 standard, except for the sample size, and the water volume used. After light curing, the samples were stored in a desiccator containing silica gel (Sigma Aldrich Chemical Co., St Louis, MO, USA) and kept in incubator at 37 °C. Every 24 hours, the samples were weighed on a precision analytical balance (Chyo JEX-200, YMC Co, Tokyo, Japan) and after 5 days with a variation less than 0.1mg, we obtained the constant initial mass (m1). Soon after, the samples were kept in eppendorfs with 2mL of water and kept in an oven at 37ºC for 7 days. After this period, the samples were washed in running water, dried with absorbent paper and weighed again to obtain the mass (m2). Every 24 hours, new weighing were performed until the mass had a variation less than 0.1mg to obtain the final mass (m3). The values were calculated using the formulas: So = (m2-m3) / V; SL = (m1-m3) / V.
e. Contact Angle
Upon approval by the Ethics Committee (No. 3.262.475), we used planned human enamel blocks (4 x 4mm) as the base where the infiltrant was dripped to assess surface contact between tooth-infiltrant. To measure the contact angle, we used the goniometer (Ramé hart-500f1 Succasunna, NJ, USA) attached to a camera. Each infiltrating group (n = 6) was dispensed into the respective enamel block by a high precision syringe (Ramé hart-500f1 Succasunna, NJ, USA). In total, 10 measurements were taken per sample, with a time interval of 0.05 s and 60 frames per second. At the end, the images were analyzed by DROPimage Advanced Software (Ramé hart-500f1 Succasunna, NJ, USA) which generated the results of the contact angles degrees (º).
f. Statistical analysis
Data were initially evaluated for sample normality and homogeneity and were approved by the Shapiro-Wilk and / or Kolmogorov-Smirnov tests.
For statistical analysis, one-way ANOVA and Tukey's post-hoc test was performed for comparison between experimental groups and Dunnet's test for
comparisons between groups (1-9) with control (Icon®), with significance of 95% (α = 0.05).
RESULTS
a. Elastic Modulus
The infiltrant groups containing 0.5% and 1% iodonium salt and 0.12% and 0.25% chitosan (Groups 5, 7, 8 and 9) had higher elastic modulus values, statistically significant in relation to Icon® commercial infiltrant (Group 10) - Table 2. Only Group 3 (0% DPI; 0.25% CH) was statistically lower than Icon®. All other groups were similar to the commercial infiltrant.
b. Flexural Strength
Similar to the elastic modulus, groups 5, 7, 8 and 9 also presented higher values of flexural strength, statistically significant in relation to Icon® infiltrant (Group 10). The other groups were equivalent to the commercial infiltrant.
c. Degree of Conversion
All experimental groups had superior results, statistically significant in relation to the Icon® infiltrant, and the highest values of degree of conversion were observed in Group 1 (0; 0%), Group 2 (0; 0,12%). Group 4 (0.5, 0%) and Group 5 (0.5, 0.12%).
d. Water sorption
For this test, the Icon® infiltrant presented the lowest water sorption values, being statistically similar only to Group 6 (0.5; 0.25%). However, such a group is statistically equivalent to Group 1 (0, 0%), 2 (0, 0.12%), 3 (0, 0.25%), 5 (0.5, 0.12%), 7 (1.0%) and 8 (1, 0.12%).
The Icon® infiltrant (Group 10) presented the lowest solubility value, being statically similar to Groups 1 (0; 0%), Group 2 (0; 0,12%), Group 3 (0; 0,25%), Group 4 (0.5, 0%) and Group 9 (1, 0.25%).
f. Contact angle
The lowest contact angle values were observed in Group 1 (0; 0%), Group 2 (0; 0.12%), Group 3 (0; 0.25%), Group 4 (0.5; 0%) and Group 8 (1; 0.12%). And, except for groups 5 (0.5%; 0.12%) and 9 (1; 0.25%) all other groups were statistically similar to Group 10 (Icon®).
Table 2. Mean (standard deviation) of elastic modulus (GPa), flexural strength (MPa), degree of conversion (%), water sorption
(g/mm3), solubility (g/mm3) and contact angle (º) of the experimental infiltrating groups and commercial group (Icon®).
Group Elastic modulus
Flexural Strength Degree of Conversion
Sorption Solubility Contact Angle
G1 (0% DPI; 0% CH) 1.22 (0.13) BC 93.91 (10.21) CD 83.43 (1.62)* A 46.62 (4.28)* ABC 16.20 (2.90) B 45.11 (6.24) D G2 (0% DPI; 0.12% CH) 1.02 (0.15) CD 78.88 (9.49) DE 81.71 (2.37)* AB 44.94 (1.89)* ABC 16.59 (3.43) B 51.05 (6.65) BCD G3 (0% DPI; 0.25% CH) 0.82 (0.17)* D 70.55 (10.83) E 78.97 (1.42)* CD 45.42 (3.46)* ABC 18.08 (10.53) B 52.09 (5.87) ABCD G4 (0.5% DPI; 0% CH) 1.07 (0.07) CD 93.72 (5.20) CD 81.13 (0.06)* ABC 49.81 (2.43)* A 20.29 (3.54) B 53.90 (7.12) ABCD G5 (0.5% DPI; 0.12% CH) 1.47 (0.24)* AB 135.35 (12.93)* A 80.81 (1.14)* ABCD 42.64 (3.56)* BC 40.22 (18.17)* A 61.02 (3.21)* AB G6 (0.5% DPI; 0.25% CH) 1.08 (0.23) C 95.64 (16.96) CD 79.70 (0.81)* BCD 41.29 (3.98) C 35.45 (15.12)* AB 56.88 (4.22) ABC G7 (1% DPI; 0% CH) 1.42 (0.18)* AB 126.19 (18.02)* A 80.11 (1.13)* BCD 44.34 (3.10)* ABC 26.59 (5.03)* AB 56.91 (4.06) ABC G8 (1% DPI; 0.12% CH) 1.40 (0.20)* B 107.11 (7.83)* BC 78.74 (0.59)* CD 44.01 (3.54)* ABC 29.75 (6.88)* AB 50.14 (4.57) CD G9 (1% DPI; 0.25% CH) 1.66 (0.12)* A 121.80 (13.55)* AB 78.29 (0.99)* D 49.30 (3.31)* AB 20.69 (2.17) B 61.36 (5.36)* A G10 (Icon®) 1.15 (0.15) 84.36 (17.58) 50.14 (2.34) 32.26 (13.63) 5.80 (19.75) 49.29 (1.93) Different letters indicate statistical difference (p <0.05).
DISCUSSION
Experimental infiltrants are of extreme clinical utility in controlling and stopping carious white spot lesions 6. However, there is still a gap regarding the ideal composition of infiltrants and their physical-mechanical behavior 11, 12.
According to the results of this study, the experimental infiltrants containing the addition of iodonium and chitosan salts showed statistically superior results compared to the commercial infiltrant for the flexural strength, elastic modulus and degree of conversion tests. However, we had experimental infiltrants similar to the commercial ones in the sorption, solubility and contact angle tests. Thus, the null hypothesis was partially rejected.
As resinous infiltrants are known to have no filler in their composition, we obtained low values of elastic modulus in general. We could evaluate that the groups had similar behaviors for both ME and RF, and group 5, 7, 8 and 9 even presented superior results in relation to the Icon® infiltrant (group 10) - Table 2. In this context, we believe that the higher concentration iodonium salt has been shown to increase the resistance of experimental infiltrants, which has been reported in other studies with other dental materials 13, 14. Chitosan, on the other hand, seems to act as a secondary factor in this increase in resistance, but it does show to improve the performance of DPI when used in certain concentrations, as already mentioned in the previous literature 12. This higher mechanical strength is interesting for commercial infiltrants, while they have the function of reinforcing the fragile structure of the enamel with early carious lesion 1.
Regarding the degree of conversion, we obtained extremely interesting findings. This is an important physical method for evaluating the polymeric chain of composites, and is known to indicate the formation of a polymer with good mechanical properties 16. The minimum degree of conversion percentage for composites is not precisely established in the literature; however, there is evidence that values below 65% are contraindicated 17. A high degree of conversion is desirable for resinous infiltrants, while favoring the formation of a more uniform and homogeneous polymer network of the material, reducing porosities and increasing acid penetration resistance 3. Nevertheless, in the present study, the Icon® infiltrant did not reach this minimum required conversion
average. There is a consensus that a high percentage of unreacted monomers indicate a sub polymerization of the composite and tends to compromise the mechanical properties of these materials 3. In contrast, all the experimental infiltrants we formulated presented values above 78% of conversion degree. It is known that the degree of conversion of dental materials can be influenced by the addition of particles, which can lead to an increase in the polymerization reaction of the material 18. Thus, we believe that, in the experimental infiltrants formulated in this study, the monomeric blend itself (or the polymerization agents - camphorquinone and amine EDAB), besides the presence of DPI may have led to a completer and more efficient polymerization reaction.
As for water sorption and solubility, in general, methacrylate monomers such as those addressed in this study are vulnerable to hydrolysis of their ester groups 19. Because of the hydrophilicity of these monomers, there is a high chance that the infiltrant will experience high sorption and solubility 20. For sorption, however, Group 6 (DPI 0.5% and Chitosan 0.12%) was the most similar in relation to the trademark, possibly due to the fact that DPI and chitosan at these concentrations acted as protectors of infiltrants by form a stiffer, better quality polymer, making it difficult for water molecules to enter the polymer chain 21. Regarding solubility, five of the nine experimental groups evaluated were similar to Icon®. We believe that despite the high concentration of TEGDMA in the composition of both experimental and commercial infiltrants, which is a hydrophilic and polar monomer 20 and recognized as the monomer that has the highest sorption capacity 22, the mixture of BisEMA as a monomeric base of the experimental infiltrants, in addition to DPI and chitosan may hinder/prevent the dissolution of the polymeric chain in general. Regarding the contact angle test, this is recognized as an important parameter for estimating the spreading of a liquid on a surface or the penetration of a liquid on a porous solid surface 23, as well as providing data about hydrophilia or hydrophobia of the material. As a rule, materials that form an angle less than 90º in relation to the surface are called hydrophilic and with an angle greater than 90º are hydrophobic 24.
In our study, all evaluated groups obtained contact angle data lower than 90º, which is a desirable factor for infiltrants. In addition, except for 2 experimental groups, the others were statistically similar to the commercially available brand, which reveals high
wettability of the experimental infiltrants in relation to the enamel surface. Taking into account all the benefits pointed out in relation to DPI and chitosan and the data we obtained in the other tests, as differential in relation to the contact angle test, we show that group 8 (DPI 1%; Chitosan 0.12%) presented the lowest statistical values. To date, only one study has evaluated the contact angle of experimental infiltrants and it has been shown that the hydrophilic nature of DPI has reduced the contact angle of experimental infiltrants 11. In relation to the chitosan, there are no studies showing this analysis.
In a general aspect, a previous study identified the mixture of 0.5% DPI with 0.12% chitosan to experimental infiltrants showed better mechanical performance among experimental infiltrants 12. However, in this new analysis and comparison with the Icon® infiltrant, it is suggested that in addition to the afore mentioned composition, blends containing 1% DPI and 0.12% or 0.25% chitosan indicate that they are statistically similar or even better compositions that the infiltrant trademark in relation to the physical-mechanical behavior of the infiltrants.
Further studies with experimental infiltrants are needed for this material to become popular and make its future commercialization viable; this, consequently, may result in an improvement in the population's quality of life, which would make it easier to access this innovative and atraumatic procedure to control and paralyze initial carious lesions.
CONCLUSION
- Experimental infiltrants containing iodonium salt and chitosan showed statistically superior results compared to Icon® for the Elastic Modulus, Flexural Strength and Degree of Conversion tests.
- Icon® commercial infiltrant showed better sorption, solubility and contact angle results, but was statistically similar to experimental infiltrants.
- Blends of experimental infiltrants containing 1% DPI and 0.12 and 0.25% chitosan showed the best results regarding physical properties.
REFERENCES
1. Paris S, Meyer-Lueckel H, Cölfen H, Kielbassa AM. Resin infiltration of artificial enamel caries lesions with experimental light curing resins. Dent Mater J. 2007 Jul;26(4):582-8.
2. Doméjean S, Ducamp R, Léger S, Holmgren C. Resin infiltration of noncavitated caries lesions: a systematic review. Med Princ Pract 2015;24: 216–221.
3. Andrade Neto DM, Carvalho EV, Rodrigues EA, Feitosa VP, Sauro S, Mele G, Carbone L, Mazzetto SE, Rodrigues LK, Fechine PB. Novel hydroxyapatite nanorods improve anti-caries efficacy of enamel infiltrants. Dent Mater. 2016 Jun;32(6):784-93.
4. Ceci M, Rattalino D, Viola M, Beltrami R, Chiesa M, Colombo M, Poggio C. Resin infiltrant for non-cavitated caries lesions: evaluation of color stability. J Clin Exp Dent. 2017 Feb 1;9(2):e231-e237.
5. Meyer-Lueckel H, Paris S. Progression of artificial enamel caries lesions after infiltration with experimental light curing resins. Caries Res. 2008; 42(2):117-24. doi: 10.1159/000118631.
6. Paris S, Meyer-Lueckel H. Inhibition of caries progression by resin infiltration in situ. Caries Res. 2010;44:47-54.
7. Gurdogan EB, Ozdemir-Ozenen D, Sandalli N. Evaluation of Surface Roughness Characteristics Using Atomic Force Microscopy and Inspection of Microhardness Following Resin Infiltration with Icon(®). J Esthet Restor Dent. 2017 May 6;29(3):201-208.
8. Arslan S, Zorba YO, Atalay MA, Özcan S, Demirbuğa S, Pala K, Perçin D, Ozer F. Effect of resin infiltrationon surface properties and Streprococcus mutans adhesion to artificial enamel lesions. Dent Mater J 2014; 34: 25-30.
9. Araújo GS, Sfalcin RA, Araújo TG, Alonso RC, Puppin-Rontani RM Evaluation of polymerization chacacteristics and penetration into enamel caries lesion of experimental infiltrants. J Dent. 2013 Nov;41(11):1014-9.
10. Inagaki LT, Dainezi VB, Alonso RC, Paula AB, Garcia-Godoy F, Puppin-Rontani RM, et al. Evaluation of sorption/solubility, softening, flexural strength and elastic modulus of experimental resin blends with chlorhexidine. J Dent. 2016 Jun;49:40-5. doi: 10.1016/j.jdent.2016.04.006.
11. Mathias C, Gomes RS, Dressano D, Braga RR, Aguiar FHB, Marchi GM. Effect of diphenyliodonium hexafluorophosphate salt on experimental infiltrants containing different diluents. Odontology. 2019 Apr;107(2):202-208.
12. Flor-Ribeiro, MD et al. Effect of iodonium salt and chitosan on the physical and antibacterial properties of experimental infiltrants Braz. Oral Res. 2019;33:e075.
13. Gonçalves LS, Moraes RR, Ogliari FA, Boaro L, Braga RR, Consani S. Improved polymerization efficiency of methacrylate-based cements containing an iodonium salt. Dent Mater. 2013 Dec;29(12):1251-5.
14. Dressano D, Palialol AR, Xavier TA, Braga RR, Oxman JD, Watts DC, et al. Effect of diphenyliodonium hexafluorophosphate on the physical and chemical properties of ethanolic solvated resins containing camphorquinone and 1-phenyl-1,2-propanedione sensitizers as initiators. Dent Mater. 2016 Jun;32(6):756-64.
15. Diolosà M, Donati I, Turco G, Cadenaro M, Di Lenarda R, Breschi L, Paoletti S. Use of methacrylate-modified chitosan to increase the durability of dentine bonding systems. Biomacromolecules. 2014 Dec 8;15(12):4606-13.
16. Aguiar TR, de Oliveira M, Arrais CA, Ambrosano GM, Rueggeberg F, Giannini M. The effect of photopolymerization on the degree of conversion, polymerization kinetic, biaxial flexure strength, and modulus of self-adhesive resin cements. J Prosthet Dent. 2015 Feb;113(2):128-34.
17. Galvão MR, Caldas SG, Bagnato VS, de Souza Rastelli AN, de Andrade MF. Evaluation of degree of conversion and hardness of dental composites photo-activated with different light guide tips. Eur J Dent. 2013 Jan;7(1):86-93.
18. Leitune VCB, Collares FM, Trommer RM, Andrioli DG,Bergmann CP, Samuel SMW. The addition of nanostructured hydroxyapatite to an experimental adhesive resin. J Dent 2013;41:321–7.
19. Van Landuyt KL, Snauwaert J, De Munck J, Peumans M, Yoshida Y, Poitevin A, et al. Systematic review of the chemical composition of contemporary dental adhesives. Biomater. 2007;28:3757-85.
20. Arslan S, Lipski L, Dubbs K, Elmali F, Ozer F. Effects of different resin sealing therapies on nanoleakage within artificial non-cavitated enamel lesions. Dent Mater J. 2018 Nov 30;37(6):981-987.
21. Ferreira P, Calvinho P, Cabrita AS, Schacht E, Gil MH. Synthesis and characterization of new methacrylate based hydrogels. Braz J Pharm Sci. 2006; 42(3): 419-427.
22. Sideridou I, Tserki V, Papanastasiou G. Study of water sorption, solubility and modulus of elasticity of light-cured dimethacrylate-based dental resins. Biomater. 2003;24:655-65.
23. Grundke K, Pöschel K, Synytska A, Frenzel R, Drechsler A, Nitschke M, Cordeiro AL, Uhlmann P, Welzel PB. Experimental studies of contact angle hysteresis phenomena on polymer surfaces – Toward the understanding and control of wettability for different applications. Adv Colloid Interface Sci. 2015 Aug;222:350-76.
24. Krasowska M, Terpilowski K, Chibowski E, Malysa K. Apparent contact angles and time of the three phase contact formation by the bubble colliding with Teflon surfaces of different roughness. Physicochemical Problems of Mineral Processing 2006; 40: 293-306.
3. ARTIGO 2: IN SITU ANALYSIS OF PHYSICOCHEMICAL PROPERTIES AND PENETRATION OF EXPERIMENTAL INFILTRANTS CONTAINING IODONIUM SALT AND CHITOSAN INTO ARTIFICIAL ENAMEL CARIES LESIONS
Submitted in Dental Materials
Authors: Mariana Dias Flor Ribeiro, Lívia Araújo Alves, Renata de Oliveira Mattos
Graner, Marcelo A. Pereira-da-Silva, Flávio Henrique Baggio Aguiar, Giselle Maria Marchi
ABSTRACT
Objectives: To evaluate in situ the physicochemical properties and depth of
penetration of experimental infiltrates containing iodonium and chitosan salt. Methods: 132 blocks of human dental enamel (n = 12 / group) were made and submitted to the simulation of initial caries lesion. Samples were randomized into 11 treatment groups: Nine groups of experimental infiltrants containing the triethylene glycol dimethacrylate monomeric base (TEGDMA) and bisphenol-A dimethacrylate ethoxylate (BisEMA) in proportion by 75 and 25% by weight and variations in concentration of iodonium salt (DPI) at 0; 0.5 and 1% and chitosan at 0; 0.12 and 0.25%; Icon® Commercial Infiltrant as Positive Control; No infiltrating treatment as negative control. The samples were exposed to the oral environment for 7 days in intraoral device. The following tests were performed: Penetration depth with Confocal Laser Scan Microscope; Roughness, Area, Projection and Surface Morphology with Atomic Force Microscope; and evaluation of microbiological activity by Colony Forming Units Test. For the microbiological activity, roughness and area data generalized linear models were used. Penetration depth data were analyzed by one-way analysis of variance (ANOVA) and Tukey test, with a significance level of 5%.
Results: There was no significant difference between the eleven groups regarding
microbiological activity (CFU), roughness and area (p <0.05). Penetration depth was significantly higher in the untreated group than in the 0.5% DPI and 0.25% Chitosan group (p <0.05). Significance: In situ, experimental infiltrants containing iodonium and chitosan salt are similar in comparison to commercial infiltrants.
Keywords: Resins, Synthetic; In Situ Hybridization Fluorescence; Onium
1. INTRODUCTION
Dental caries remains one of the most prevalent health disorders in modern society [1], and it is crucial to observe this disease in its early stage, which has as its main feature the mineral loss underlying an apparently intact surface [2] and its onset is usually associated with the adherence of a specific and complex biofilm on the enamel surface [1]. Early caries lesions, commonly referred to as white spot lesions, are common side effects of poor oral hygiene, salivary hypofunction, cariogenic diet etc. [3].
As with most human diseases, caries can be easily and precisely controlled in the early stages [4]. Currently, the first treatment option for incipient carious lesions is related to minimally invasive procedures that promote remineralization of the dental structure [3, 5, 6]. However, to date, the only biomaterial that can prevent the progression of caries in the early stages of the disease is the resinous infiltrant [7]. This material consists of low viscosity hydrophilic monomers based on dimethacrylates capable of penetrating the enamel (and even the external third) of demineralized dentin and paralyzing the progression of tooth decay [4, 7].
Its principle is based on perfusing the porous enamel with resin by capillary action, thus interrupting the demineralization process, which promotes lesion stabilization and inhibits the diffusion of bacteria, while preserving the healthy remaining structure [8]. Currently there is only one commercially available infiltrant (Icon®, DMG, Germany). According to the manufacturer, it is basically composed of triethylene glycol dimethacrylate (TEGDMA), initiators and additives; It is indicated to be applied in early white spot on smooth, proximal and occlusal surfaces [9, 10].
Several infiltrant formulations have been developed [5, 11, 12, 13], in which combinations of monomers, diluents and solvents are tested in order to obtain material with low viscosity, which presents rigid consistency after polymerization and high penetration speed. However, it is difficult to establish a composition that is capable of uniting flowability and strength, since a low amount of hydrophobic monomers related to mechanical properties and high amount of diluent monomers and solvents are necessary to decrease material viscosity [5].
The resinous materials mostly use the monomer bisphenol A glycidyl dimethacrylate (BisGMA) due to its high molecular weight that gives good mechanical performance, but its disadvantage is its high viscosity. To try to decrease this viscosity it has been replaced by another monomer, the ethoxylated bisphenol A dimethacrylate (BisEMA) which, due to the absence of hydroxyl groups, confers lower viscosity and susceptibility to water sorption [14]. In addition to replacing BisGMA with less viscous monomers, it is also possible to use co-monomers that are not as resistant to degradation as to decrease the viscosity of the final product, with triethylene glycol dimethacrylate (TEGDMA) being the most commonly used. have low molecular weight and consequently low viscosity [15], even presenting itself as the main component of the available commercial infiltrant, Icon®. Additionally, the inclusion of 2-hydroxyethyl methacrylate (HEMA) to the infiltrants may be used to increase monomeric infiltration due to the fact that this monomer has low viscosity and low molecular weight [14, 16].
In dental biomaterials, diphenyliodonium salt (DPI) has been shown to positively influence the mechanical properties of materials as well as: reactivity and polymerization potential, flexural strength, modulus of elasticity, degree of conversion [17, 18], antibacterial activity [13] and therefore subject to further study. In addition, in recent years chitosan polysaccharide has been extensively used in the field of dentistry and, in addition to its antimicrobial potential, possibly acts as a methacrylate reinforcement framework [19], increasing the mechanical resistance of infiltrants [13].
In general, infiltrants are recent materials and, therefore, with limited studies in the currently available databases, and contain mostly the limitations of studies that bring in vitro methodology. Thus, a clinical condition can be achieved through in situ studies, which are intermediate stage studies between laboratory experiments and clinical trials, which can reproduce clinical conditions and perform analyzes outside the oral cavity [20]. As it is evident the need for further studies that propose and evaluate improvements on the ideal composition of the infiltrants, besides investigating the influence of different material compositions on their chemical-physical properties, the purpose of this study was to evaluate in situ the properties of experimental infiltrants. compared to Icon® commercial infiltrant.
The hypotheses of this study are: 1) The DPI and Chitosan reduces roughness, area and reduces bacterial growth of experimental infiltrants compared to commercial ones. / 2) The DPI and chitosan increase penetration depth of experimental infiltrants.
2. MATERIALS AND METHODS
2.1. Ethics committee
This study was submitted and approved by the Ethics and Research Committee of the Piracicaba School of Dentistry (UNICAMP, Brazil), nº 3.262.475.
2.2. Teeth cleaning and preparation
Seventy human teeth were used. After their donation, they were stored in a buffered 0.1% thymol aqueous solution (Dinâmica, Piracicaba, São Paulo, Brazil). After disinfection, they were submitted to manual scraping with periodontal curette to remove organic debris and prophylaxis with rubber bowls, pumice paste (Maquira Dental Products, Maringá, PR, Brazil) and water. The teeth were examined under magnifying glass (Zeiss-Carl Zeiss from Brazil) with a four-fold magnification to check for cracks, cracks or staining, which could eventually influence the results of this study. Soon after, the teeth were stored in distilled water under refrigeration until the moment of their use. Sequential to tooth selection, the crown was separated from the root at 2 mm from the cementum-enamel junction using a double-sided diamond disk (KG Sorensen, Ind. Com., Barueri, SP, Brazil) coupled to the low rotation contra-angle. under constant irrigation.
2.3. Sample making
For this step, the coronary portion was fixed to the hot glue acrylic plate and from then on tooth blocks were obtained through a diamond cutting disc (Extec Dia. Wafer Blade 102 x 0.3 x 12.7 mm) coupled to a Metallographic Cutter (Isomet 1000, Buehler Ltda. Lake Buff, IL, USA). The blocks (n = 132) were obtained from the enamel portion of the free, buccal and lingual / palatine faces, with a dimension of approximately 4 x 4 x 2 mm – Figure 1.
Figure 1. Experimental design study. (1) Crown-root separation; (2) Cutting free enamel faces; (3) 4 x 4 x 2 mm blocks; (4) 132 randomized blocks in 11 groups.
To standardize the surfaces, the fragments were lightly planned in a polishing machine (Arotec S / A Industry and Commerce, Cotia - SP) with 600, 1200 and 2400 (Buehler) granulated sandpaper under refrigeration; and soon after polishing with felt discs and diamond solution (1 µm; Buehler) also coupled to the rotating polisher (Aropol E, Arotec, Cotia; São Paulo, SP, Brazil). Between each planning and polishing step, as well as at the end of this step, the specimens were washed with distilled water in an ultrasonic vat (Marconi, Piracicaba, SP, Brazil) to remove any debris present on the enamel surface. The samples were stored in ependorfs with distilled water and brought to the oven at 37ºC.
2.4. Sample esterilization
As this is an in situ study, the specimens need to be sterilized. This step was performed with gamma cell radiation at 25 kgy for 191 hours, by the Center for Nuclear Energy of Agriculture / USP, prior to the preparation of intraoral devices.
2.5. Volunteers selection
Twelve volunteers were selected to participate in this research. Inclusion criteria for volunteer selection were: presence of normal salivary flow, absence of caries and / or periodontal disease. Exclusion criteria were: patient using orthodontic devices, using drugs that interfere with salivary flow, smoking patients, and presence of fixed or removable prosthesis. All details about the study were clarified by the researchers prior to signing the Informed Consent Form.
2.6. Experimental infiltrant formulation
Based on previous studies (Araújo et al., 2013; Flor-Ribeiro et al., 2019), nine groups of experimental infiltrants were tested - Fig. 1.
The monomeric base was composed of 75% TEGDMA (Sigma Aldrich Chemical Co., St Louis, MO, USA) and 25% BisEMA (Sigma Aldrich Chemical Co., St Louis, MO, USA). The diluent used was 10% HEMA monomer (Sigma Aldrich Chemical Co., St Louis, MO, USA) and the photoinitiator system was composed of: 0.5% camphorquinone (Sigma Aldrich, St. Louis, USA) and 1% EDAB amine (Sigma Aldrich, St. Louis, USA). The variables used were chitosan (Sigma Aldrich, St. Louis, USA) (0; 0.12; 0.25 wt.%), and diphenyliodonium hexafluorophosphate salt - DPI (Sigma Aldrich, St. Louis, USA) (0; 0.5; 1 mol%). The materials used were weighed in high precision analytical balance (Chyo JEX-200, YMC Co, Tokyo, Japan) and handled in ambient with temperature and lighting control.
2.7. Making the intraoral device
Prior to the beginning of this in situ experimental stage, the volunteer was given prophylaxis and oral hygiene instruction and each one was given a toothbrush (Oral-B Indicator, Procter and Gamble, USA) and a toothpaste (Oral B, Procter and Gamble, USA), which were used throughout the experiment to standardize the oral hygiene of the volunteers.
The volunteers had the upper dental arch molded with alginate (Hydrogum - Zhermack, Badia Polesine, Italy) and the models were made of Type IV stone plaster (Asfer - Asfer Indústria Química Ltda., Sao Caetano do Sul, SP). The palatal intraoral devices were made of self-curing acrylic resin. During the making of the appliance, silicone blocks (4 mm x 4 mm x 2 mm) were positioned in the plaster model in order to provide the necessary places for the insertion of the dental blocks. Each plate had niches for 11 blocks, which corresponded to each infiltrating group that was submitted to salivary exposure. In an intraoral appliance, 11 loci that contained the samples were contained 9 with experimental infiltrants, 1 with Icon® (positive control), 1 untreated locus (negative control). The specimens were fixed with sticky wax (ASFER - Industria Química Ltda). The plates were adapted to each volunteer so that the surface of each specimen was in contact with the oral environment.
The device was used by patients throughout the day and night, removing it only during meals, fluid intake (except water) and oral hygiene. During this period, the devices were stored in a container with gauze soaked in water, which was provided to the volunteers. The volunteers were instructed not to subject the device with the specimens in solutions containing fluoride nor to brush the specimens with toothbrush and toothpaste. Devices were collected for analysis after 7 days.
2.8. Simulation of initial enamel caries lesion
Sequential to the test specimens, initial surface microhardness averages (346.28 ± 108.42 KHN) were obtained by means of a microdurometer (HMV-2000; Shimadzu Corporation, Tokyo, Japan) with a Knoop indentator 25 g diamond of load for 10 s [21]. Three indentations, 100 μm apart, were performed from the center of the surface. After this selection, the samples were randomized into 11 groups (n = 12), placed in individual support for pH cycling, with all faces of the samples protected with utility wax, except the polished enamel area (4mm x4mm). To develop sub-superficial artificial caries lesions, demineralization (DES) and remineralization (RE) solutions were used. Thus, with the adaptation of the methodologies used in the studies by Araújo et al., 2013 [22] and Andrade Neto et al., 2016 [4], the demineralizing solution was composed of 0.05 M
of acetate buffer solution with pH = 4.6, 50% saturated relative to hydroxyapatite. The remineralizing solution, which aims to simulate artificial saliva, was composed of 1.5 mM Calcium, 0.9 mM Phosphate in a 20.0 mM Tris (hydroxymethyl) aminomethane buffer solution with pH = 7.0.
To simulate the DES-RE cycle, the specimens were individually immersed in 20 ml DES solution for 4 h in an oven at 37 ° C, then washed with distilled water. They were then immersed in 50 ml of RE solution and stored in an oven at 37ºC for 20 h. These steps were repeated consecutively for 8 days, and on the 4th day, the solutions were replaced by new fresh solutions. At the end of the DES-RE cycle, the specimens were again washed with distilled water and kept at a relative humidity of 37ºC in an incubator.
2.9. Enamel Penetration Depth
The resin infiltration was screened and visualized based on the indirect dye marking technique [23]. The specimens were conditioned with 15% hydrochloric acid for 120s following the manufacturer's recommended protocol (Icon Etch, DMG, Hamburg, Germany). Soon after, this acid was flushed with a triple syringe for 30 sec. and then it was performed to the infiltration application [5]. To evaluate the infiltration depth, the specimens were impregnated with rhodamine B isothiocyanate ethanolic solution (RITC, Sigma Aldrich, Steinheim, Germany) at 0.1% for 12 h. Subsequently, the specimens were dried using air blasts for 10 s and a pure infiltrant was applied to the lesion surface. After 5 minutes, excess material was removed with a cotton swab and the material was photoactivated for 40 s (1000 mW / cm2, Valo, Ultradent Products Inc; S. Jordan, UT, USA). To remove excess red fluorophore, which was not incorporated by the infiltrant, the samples were stored in 30% hydrogen peroxide solution for 12 h at 37 °C. They were then washed with water for 60 s.
After, 0.5 mm thick tooth slices were obtained perpendicular to the lesion surface. These slices were polished with silicon carbide sandpaper (1200, 2400, Buehler, Lake Bluff, IL, USA). To evaluate the parts of the lesion where there was no infiltration, the specimens were immersed in 100 μM sodium fluoroscein ethanolic solution (NaFl; Sigma Aldrich) for 180 s and then washed with distilled water for 10 s.
Then, the specimens (n = 3) were evaluated by Confocal Laser Scanning Microscopy (Leica, TCS NT; Leica, Heidelberg, Germany) in dual fluorescence mode, using objective with magnifications of 20x and 63x, in dual fluorescence mode in which fluorescences were detected simultaneously (RITC: Ex 568 nm, At 590 nm; NaFl: Ex 488 nm, At 525/50 nm). Two-dimensional (XY-scan) images were obtained, with a resolution of 1024 x 1024 pixels. Then, the averages were calculated with the aid of ImageJ software (NIH, Bethesda, MD, USA), based on the defined points.
2.10. Atomic Force Microscope (AFM) Analysis
In this study, for qualitative surface analysis, three samples from each infiltrating group were randomly selected after each treatment. For this, the samples were washed in an ultrasonic bath and dried on absorbent paper. Then, the samples were fixed with double-sided tape on the metallic support and coupled under the atomic force microscope (Easy Scan 2, Nano Surf, Boston, MA, USA). Thus, 3D topographic images of size 15 µm x 15 µm were obtained for each group and from these, the following analyzes were performed: roughness, area, surface projection and surface morphology.
The analyzes were performed with a constant velocity of approximately 70N / m, with an oscillation frequency of 320KHz. It was operated in a tapping mode with a scan frequency of 0.7 Hz per line and a 2 μm scale.
2.11. Microbiological Analysis - Test of Colony Forming Units (CFU /
mL).
The samples (n = 5) were placed in ependorfs with 1 mL of sterile saline and shaken for 60 s with the aid of a vortex shaker (MX-S, Dlab, Riverside, CA, USA). From the liquid concentrate formed, serial dilutions were made up to 10-5. 25 μL of each dilution were plated in triplicate on specific microbial growth media: Agitis Mitis Salivarius Bacitracin to enumerate S. mutans; MRS agar to list species of lactobacillus.
For previously isolated and identified microorganisms, two suspensions were prepared on the Mac Farland Scale (barium sulphate suspension, scale 1.0).
From these samples, a 0.5 mL aliquot of S. mutans solution and 0.5 mL of L. acidophilus were collected and added into test tubes containing culture medium. All tubes were stored for 24 hours at 37º. After 24 hours dilution to 10-9 of S. mutans and L.
acidophillus solutions was performed. An aliquot of 0.1 mL was collected from each
dilution for cultivation (using the spread plate technique) on standard plate count agar (PCA) incubated at 37 °C for 24 hours. After this period the Colony Forming Units (CFU / mL) were counted.
From the cultures obtained and stored for the time and temperature described above, all plates that had CFU / mL less than or equal to 250 were counted and the result noted. In all experimental steps, without exception, the aseptic technique was valued and the tests performed in triplicates.
2.12. Statistical analysis
Data were initially evaluated through descriptive and exploratory analyzes. For the microbiological activity, roughness and area data generalized linear models were used. Penetration depth data were analyzed by one-way analysis of variance (ANOVA) and Tukey test. The analyzes were performed with the aid of the R and SAS programs, with a significance level of 5%.
3. RESULTS
3.1. Roughness and Area:
There was no significant difference between groups regarding roughness and area, p> 0.05. – Figure 2 - A and B.
Figure 2. Mean and standard deviation, depending on the treatment of: Roughness (A), Area (B), Microbiological analysis (C) and Penetration Depth (D).
Regarding the projection and surface morphology, the presence of more regular grains was observed in the groups 1 (0% DPI; 0% CH) and 8 (1% DPI; 0.12% CH), being visually similar to group 11 (without treatment). - Figure 3.
3.2. Microbiological analysis:
For lactobacillus acidophillus, no colony forming unit was present in any evaluated group.
Thus, CFU / mL data were evaluated only for S. mutans and there was no significant difference between the 11 groups analyzed, p> 0.05, Figure 2 - C.
3.3. Penetration depth:
Regarding the penetration depth, this was significantly higher in the untreated group than in the group with 0.5% DPI and 0.25% Chitosan (p <0.05), Figure 2 – D and Figure 4.
Figure 3. 3D images obtained by the Atomic Force Microscope. Observe greater homogeneity of samples in G1, G8 and No treatment.
Figure 4. Representative images of confocal laser scanning microscopy. Areas in red represent infiltrated areas and in green represents non-infiltrated enamel. The images confirm the resin infiltration in the evaluated specimens.
4. DISCUSSION
This study evaluates mechanical and microbiological properties of experimental infiltrants in an in situ model.
Under the conditions of the present study, there was no significant difference between the eleven groups regarding microbiological activity (CFU/mL), roughness and area (p <0.05), Fig. 2. Thus, the first hypothesis of the study was rejected.
In situ studies are considered intermediate between in vitro and in vivo studies because they allow the simulation of some clinical variables [24]. Thus, an in situ study model can simultaneously simulate intraoral events and standardize experimental conditions [25].
Most groups evaluated by Atomic Force Microscopy, including the commercial Icon® infiltrant, demonstrated a heterogeneous distribution of infiltrant grains on its surface (Fig. 3), similar to the study by Gurdogan et al., 2017 [26] and Taher et al., 2013 [27]. This heterogeneous appearance and irregular distribution of infiltrating grains may be caused by polymerization contraction, oxygen inhibition or incomplete solvent evaporation [5].
Evaporation of solvents during penetration of resin infiltrants can lead to unfilled pores within the resin layer [28]. In addition, Icon® (and similar experimental infiltrants may possibly lose the oxygen-inhibiting layer more easily because it is more easily worn by salivary flow or mechanical action [29].
However, group 1 (0% DPI; 0% CH) and 8 (1% DPI; 0.12% CH) were visually similar to the untreated group, and these had a more homogeneous surface. Such finding may be beneficial in relation to these two groups particularly, indicating the absence or lesser deposition of infiltrant on the enamel surface.
Clinically, white spot injury may or may not be associated with surface roughness [27]. In general, when the microporosities of demineralized enamel are with resin infiltrants, it is expected that surface roughness will decrease [30]. This roughness depends on the surface treatment and materials being used, as some materials promote a smoother surface (such as group 1 and 8), others may create a relatively rough surface. Rough surfaces have an increased risk of bacterial adhesion and plaque buildup compared to other surfaces, and as a result, rougher surfaces increase the risk of further demineralization [26].
