Desenvolvimento e validação de um modelo de crescimento de biofilmes de S. mutans e estudo do efeito da sacarose na expressão de gtfBCD e dexA em biofilmes dentais formados in vitro e in situ / Development and validation of S. mutans biofilm growth model and study of effect of sucrose on gene expression of gtfBCD and dexA in dental biofilms formed in vitro and in situ

AUTOR(ES)
DATA DE PUBLICAÇÃO

2010

RESUMO

In vitro and in situ models have been developed to study dental biofilms. Protocols, that simulate the alternations of "feast or famine" episodes that happen in oral environment in the presence of dietary carbohydrates and allowing dental caries evaluation, are necessaries. On the other hand, few studies about gene expression of dental biofilm formed in situ were performed. Thus, the objectives of this research were: To develop and to validate a model of S. mutans biofilm formation simulating feast-famine episodes that allows biofilms and dental demineralization changes assessment. Also, to evaluate the effect of sucrose exposure on expression of gtfB, gtfC, gtfD and dexA of in vitro and in situ dental biofilms. For the first objectives, S. mutans UA159 biofilms were grown during 5 days on bovine enamel slabs at 37oC, 10% CO2 in ultra-purified culture media. To develop the model, the effect of sucrose concentration (1 - 20%) 8x/day and frequency (0 - 8x/day) exposure were evaluated and to validate the model, chlorhexidine (CHX) effect (0.012- 0.12%) 2x/day was tested. Bacterial viability, biofilm acidogenicity, biomass and polysaccharides were determined, and enamel demineralization was evaluated by surface hardness loss. To the last objective, for in vitro study, S. mutans UA159 biofilms were grown in the same conditions of previous experiments and were exposed to 1% sucrose constantly (control) or 10% sucrose 8x/day (intermittent group) and after 48, 72 and 120 h the biofilms were collected for analysis. For in situ experiment, a crossover study was conducted in two phases of 7 days each, with nine volunteers that wore intraoral palatal appliances containing 12 dental dentin slabs, which were extra orally submitted 8 times/day to sucrose solutions of 1%, 5%, 10% and 20% . On the 7th day, biofilms were collected for analysis. RNA from the in vitro and in situ biofilms were extracted and purified. Gene expression of gtfB, gtfC, gtfD and dexA were evaluated by real time PCR. In vitro development and validation results showed that 10% and 20% sucrose concentrations and frequency 8x/day provoked biofilm and enamel demineralization changes similar to control group. CHX showed dose-response effect decreasing biomass, bacterial viability and enamel demineralization (p<0.05). Also, 0.05% NaF did not show antimicrobial effect but had similar effect than 0.12% CHX decreasing enamel demineralization (p<0.05). With regard to gene expression of in vitro experiment, gene expression of intermittent group was constant along the time. In situ results showed that biofilm wet weight, lactobacillus, extracellular polysaccharides values of the biofilm increased according to the increase of sucrose exposure. No differences for gtfB gene expression between the groups were observed (p<0.05) and no levels of gtfC, gtfD and dexA were detected. In conclusion, the model developed and validated has potential to assess substances with antimicrobial effect on biofilm and dental demineralization. Quantification of gtfB, gtfC, gtfD and dexA gene expression levels were possible for S. mutans biofilms in vitro study but only gtfB gene expression of in situ study can be determined and was not regulated by sucrose concentration.

ASSUNTO(S)

expressão genica streptococcus mutans gene expression streptococcus mutans

ACESSO AO ARTIGO

Documentos Relacionados