Effect of concentration and frequency of sucrose exposure on glycosyltransferases and glucanases gene expression in dental biofilm

Abstract

Sucrose is the most cariogenic dietary carbohydrate because in addition to being fermented by oral bacteria, it is a substrate for the synthesis of extracellular (EPS) and intracellular polysaccharides (EPS). EPS, especially the insoluble ones, promote changes in the composition of the biofilms matrix enhancing its cariogenicity; these polysaccharides are considered critical virulence factors of mutans streptococci, the most cariogenic organisms in dental biofilms. The insoluble EPS are produced by glucosyltransferases (GTF) B and C as a result of the expression of gtfB and gtfC genes. On the other hand, the EPS can be degraded by glucanases, such as dextranases (products of dexA and dexB) which are responsible for remodelling the structure of the biofilms. Our objective is to examine whether the expression of glucosyltransferases and dextranases within biofilms are dependent on the frequency of sucrose exposure and its concentration. This study will be conducted in vitro and in situ. The in vitro study will will be performed using biofilms of S. mutans UA 159 formed on hydroxyapatite discs in batch cultures for 5 days. Biofilms will be exposed to sucrose at concentrations of 1%, 5%, 10% and 20% (w/v) 8 times a day. In a separate experiment, biofilms will be exposed to sucrose 2, 4, 8 times a day. Samples will be collected 30, 60 and 120 h after biofilm formation for gene expression analysis. In the in situ study, a pilot experiment will conducted to determine the number of days for dental biofilm formation. Fifteen volunteers will participate in this study with an experimental period of 14 days; they will use palatal intra-oral devices containing 12 bovine enamel blocks and will drip a solution of sucrose 20% 8 times daily. In the 3o, 7th and 14th days the dental biofilm formed will be collected for further analysis. This pilot study will provide the best experimental period for the in situ study per se. Fifteen volunteers using palatal intra-oral devices containing 8 bovine enamel blocks will participate in the in situ study, which will be performed in 4 experimental phases, In the first part, sucrose concentrations of 1, 5, 10 and 20% will be evaluated. In the second part, the frequency of 20 % sucrose exposure (2, 4 and 8 times/day) will be evaluated. In each experimental phase, one concentration or one frequency will be tested. The volunteers will brush their teeth 3 times/day with a fluoridated dentifrice of 1100 ppm of F. At the end of each experimental phase, dental biofilm formed on the enamel block will be collected for the following analysis: a) Gene expression using real-time PCR and b) chemical analyses of the polysaccharide, if sufficient sample is available, especially from in situ studies.