The aim of this work will be to compare the effect of Stevia (natural sweetener) to aspartame (synthetic sweetener), sucrose and xylitol on the development of dental caries under microcosm biofilm model in enamel and dentin. Two hundred and sixteen bovine enamel samples and 216 bovine root dentin samples (4 mm x 4 mm) will be prepared. In 24-well plates, each enamel or dentin sample will be exposed to 1.5 mL of inoculum (human saliva-glycerol + McBain saliva, 1:50) for 8 h. After the initial 8 h the inoculum will be removed, the samples will be washed with PBS (5 s), and receive 1.5 mL of fresh medium (McBain artificial saliva) for 16 h, completing the initial 24 h. From day 2 to day 5, the samples will be exposed to McBain saliva supplemented with 1% of the sugars/sweeteners (v = 1.5 mL, namely: Stevia, aspartame, sucrose or xylitol) for 8 h. There will be control samples exposed to McBain saliva without supplementation (n = 4/plate). Thereafter, the samples will be washed with PBS (5 s) and receive 1.5 ml of fresh medium (McBain artificial saliva without supplementation) for a further 16 h, completing the daily cycle. The culture will be carried out in a CO2 incubator at 5% and 37ºC. The biofilm culture will be performed in biological triplicate (n = 5 for each replicate, n final = 15). The response variable will be: 1) The percentages of live bacteria and extracellular polysaccharides (EPS) in the biofilm by fluorescence using the confocal microscope; 2) CFU counting for total microorganisms, total streptococci, mutans streptococci and total lactobacilli; 3) Quantification of lactic acid production (mmol/L) and 4) Quantification of enamel demineralization by using transverse microradiography. Data will be submitted to appropriate statistical analysis (parametric or non-parametric test), considering p <0.05.
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