Bioestimulação transdentinária de células odontoblastóides através da aplicação de sinvastatina previamente ao uso do cimento de ionômero de vidro
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2017-02-23
Autores
Leite, Maria Luísa de Alencar e Silva [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O objetivo geral da presente pesquisa foi avaliar se o pré-tratamento da dentina com sinvastatina (SV) antes de aplicar o cimento de inômero de vidro (CIV) permitiria a difusão transdentinária desta droga em concentrações capazes de promover aumento da capacidade de células odontoblastóides em depositar e mineralizar matriz de dentina. No Estudo 1, foram determinadas concentrações de sinvastatina que fossem bioativas e citocompatíveis quando em contato com células odotoblastóides MDPC-23. Duas concentrações (0,01 e 0,1 μM) e três tempos de tratamento (24 h, 72 h e contínuo – até 14 dias) foram avaliados. No Estudo 2, diante da confirmação da hipótese da difusão transdentinária da SV por análise em cromatografia liquida em espectro de massa (LC-MS/MS), concentrações de 2,5 e 1,0 mg/mL foram selecionadas para avaliação do efeito bioativo transdentinário sobre células odontoblastóides MDPC-23 após pré-tratamento ou não da superfície dentinária com EDTA. Também nesse estudo foi avaliada a ação antimicrobiana (teste de disco-difusão) das concentrações de 2,5 e 1,0 mg/ml de SV sobre Streptococcus mutans e Lactobacillus acidophilus. No Estudo 3, a concentração que apresentou os melhores resultados no estudo anterior foi selecionada para avaliação do efeito bioativo transdentinário sobre células MDPC-23 associado ao protocolo de forramento cavitário com Ketac Molar (3M ESPE). Nos estudos que avaliaram o efeito bioativo da SV foram analisados os seguintes parâmetros celulares: viabilidade celular (MTT), atividade de ALP (timolftaleína monofosfato) e deposição de matriz mineralizada (alizarin red) (n = 6). A concentração de 2,5 mg/ml de SV também foi selecionada para avaliação da resistência de união ao microcisalhamento do CIV Ketac Molar à dentina, aplicando-a sobre a dentina pré-tratada ou não com EDTA. Os dados foram submetidos aos testes ANOVA e Tukey (p < 0,05). Os resultados do Estudo 1 demonstraram que baixas concentrações de SV em curto período de tratamento apresentaram efeito bioativo e citocompatível sobre células odontoblastóides MDPC-23. Em sequência, os estudos transdentinários demonstraram aumento na atividade de ALP e na deposição de matriz mineralizada quando a dentina foi tratada com SV; no entanto, apenas a SV 2,5 mg/mL, associada ou não ao EDTA, resultou em valores significativamente superiores ao controle negativo, para ambos os parâmetros testados. Desta forma, a concentração de 2,5 mg/mL de SV foi selecionada para realização dos experimentos subsequentes, onde foi possível observar que o forramento da dentina com CIV, realizado após tratamento do substrato com EDTA e SV apresentou a melhor resposta quanto a expressão dos marcadores fenotípicos testados. De modo complementar, observou-se ação antimicrobiana da SV nas concentrações testadas contra as linhagens avaliadas e que o pré-tramento da dentina com EDTA previamente a aplicação da SV resultou em valores de resistência de união significativamente superiores comparado ao tratamento isolado com SV. Assim, conclui-se que o tratamento da dentina com SV previamente ao forramento do substrato com CIV é capaz de aumentar a capacidade de células odontoblastóides em depositar matriz mineralizada rica em cálcio.
The aim of the present study was to determine whether the treatment of dentin with simvastatin (SV) before application of glass-ionomer cement (GIC), allows transdentinal diffusion of this drug at bioactive concentration capable of increasing the potential of odontoblast-like cells to deposit and mineralize dentin matrix. In the 1st study, the dose-response and time-course of SV were determined for the odontoblast-like MDPC-23 cells. Two concentrations (0.01 and 0.1 μM) of SV and three treatment times (24 h, 72 h and continuous – up to 14 days) were evaluated. In the 2nd study, the SV transdentinal diffusion hypothesis was confirmed by liquid chromatography mass spectrometry (LC-MS / MS). Then, concentrations of 2.5 and 1.0 mg/ml were selected to evaluate the transdentinal bioactivity of this drug with MDPC-23 cells, when dentin was treated or not with EDTA. Also in this study the antimicrobial activity (disc diffusion assay) of 2.5 and 1.0 mg/ml SV against Streptococcus mutans and Lactobacillus acidophilus was evaluated. In the 3rd study, the 2.5 mg/mL SV was selected to analyze the bioactivity of dentin treatment with SV followed by lining with Ketac Molar (3M ESPE). In all the studies, the following cell parameters were analyzed: cell viability (MTT), ALP activity (thymolphthalein monophosphate) and mineralized matrix deposition (alizarin red) (n=6). The concentration of 2.5 mg/ml SV was selected to evaluate dentin microshear-bond strength of GIC Ketac Molar when applied on dentin conditioned or not with EDTA. Data were submitted to ANOVA and Tukey test’s (p<0.05). The results of the 1st study demonstrated that exposing MDPC-23 to low concentrations of SV during short periods of time resulted in a bioactive effect associated with no toxicity to cultured cells. In the transdentinal study, it was detected an increase on ALP activity and mineralized matrix deposition when dentin was treated with SV; however, only SV 2.5 mg/ml, associated or not to EDTA, resulted in values significantly higher than negative control for both cell parameters. Finally, it was observed that lining dentin with GIC associated with pre-treatment with EDTA and SV 2.5 mg/ml resulted in the best cell responses regarding the expression of odontoblastic phenotypic markers. In addition, SV featured antimicrobial activity at both tested concentrations against the bacterial strains evaluated. Finally, pre-treatment of dentin with EDTA + SV resulted in significantly higher bond strength values compared to SV treatment. Therefore, based upon the methodology employed in the present in vitro, it was possible to concluded that the treatment of dentin substrate with a specific concentration of simvastatin before application of glass-ionomer cement enhances the capability of odontoblast-like cells to deposit calcium-rich mineralized matrix
The aim of the present study was to determine whether the treatment of dentin with simvastatin (SV) before application of glass-ionomer cement (GIC), allows transdentinal diffusion of this drug at bioactive concentration capable of increasing the potential of odontoblast-like cells to deposit and mineralize dentin matrix. In the 1st study, the dose-response and time-course of SV were determined for the odontoblast-like MDPC-23 cells. Two concentrations (0.01 and 0.1 μM) of SV and three treatment times (24 h, 72 h and continuous – up to 14 days) were evaluated. In the 2nd study, the SV transdentinal diffusion hypothesis was confirmed by liquid chromatography mass spectrometry (LC-MS / MS). Then, concentrations of 2.5 and 1.0 mg/ml were selected to evaluate the transdentinal bioactivity of this drug with MDPC-23 cells, when dentin was treated or not with EDTA. Also in this study the antimicrobial activity (disc diffusion assay) of 2.5 and 1.0 mg/ml SV against Streptococcus mutans and Lactobacillus acidophilus was evaluated. In the 3rd study, the 2.5 mg/mL SV was selected to analyze the bioactivity of dentin treatment with SV followed by lining with Ketac Molar (3M ESPE). In all the studies, the following cell parameters were analyzed: cell viability (MTT), ALP activity (thymolphthalein monophosphate) and mineralized matrix deposition (alizarin red) (n=6). The concentration of 2.5 mg/ml SV was selected to evaluate dentin microshear-bond strength of GIC Ketac Molar when applied on dentin conditioned or not with EDTA. Data were submitted to ANOVA and Tukey test’s (p<0.05). The results of the 1st study demonstrated that exposing MDPC-23 to low concentrations of SV during short periods of time resulted in a bioactive effect associated with no toxicity to cultured cells. In the transdentinal study, it was detected an increase on ALP activity and mineralized matrix deposition when dentin was treated with SV; however, only SV 2.5 mg/ml, associated or not to EDTA, resulted in values significantly higher than negative control for both cell parameters. Finally, it was observed that lining dentin with GIC associated with pre-treatment with EDTA and SV 2.5 mg/ml resulted in the best cell responses regarding the expression of odontoblastic phenotypic markers. In addition, SV featured antimicrobial activity at both tested concentrations against the bacterial strains evaluated. Finally, pre-treatment of dentin with EDTA + SV resulted in significantly higher bond strength values compared to SV treatment. Therefore, based upon the methodology employed in the present in vitro, it was possible to concluded that the treatment of dentin substrate with a specific concentration of simvastatin before application of glass-ionomer cement enhances the capability of odontoblast-like cells to deposit calcium-rich mineralized matrix
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Palavras-chave
Sinvastatina, Polpa dentária, Cimento de ionômero de vidro