The formation of a cariogenic biofilm is a complex process, which depends of factors related to the host and the environment. Hygiene habits, diet, salivary flow and antimicrobial agents may modulate the quantity and quality of the dental biofilm. One of the antimicrobial agents is fluoride, often applied as sodium fluoride (NaF). Recent studies have shown that titanium tetrafluoride (TiF4) has a better effect against enamel demineralization than NaF. However, there is no information if it might also have antimicrobial effect. Therefore, the present study complements the research field of the group that has focused on anti-caries effect of an experimental 4% TiF4 varnish and aims to evaluate the effect of this agent on the viability and acid production of microcosm biofilm and on enamel demineralization as well. The 4% TiF4 varnish will be compared to 5.42% NaF, 2% chlorexidine (positive control) and placebo varnishes/non treatment (negative control). For the microcosm biofilm formation, saliva will be collected from 10 healthy subjects, who must have not brush the teeth for the last 24 h and not drink or eat for the last 2h. The saliva will be diluted (70% saliva and 30% glycerol), and aliquots of 1ml will be stored at -80ºC. An average of 100 bovine enamel samples (2mm x 2mm) will be prepared and treated for 6h with the experimental varnishes containing: A) 4% TiF4 (pH 1.0, 2.45% F); B) 5.42% NaF (pH 5.0, 2.45% F); C) placebo; D) 2% Chlorexidine - Positive control or E) untreated (n=5x4 assays, 20 samples/group). These samples will be plated in 24-wells plates and exposed to the collected saliva for the biofilm formation. The live and dead bacteria will be evidenced by fluorescence using a confocal microscope (Assay 1) In the Assay 2, acidogenicity will be evaluated, by measuring the lactic acid using enzymatic methods. In the Assay 3, the microbial suspension removed from the biofilm will be diluted and spread in Agar plates for colony forming unit (CFU) counting for total microorganism, total streptococcus and mutans streptococcus. In the assay 4, the enamel demineralization will be measured using transverse microradiography. The data will be plotted and submitted to the statistical analyses (p<0.05).
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