This study aims to detect alterations in the protein profile of acquired pellicles formed on enamel and dentin in vitro after application of gels containing chlorhexidine or EGCG, as well as to evaluate if the modification of the pellicle by these treatments is able to change its protective potential against tooth demineralization. 135 bovine enamel blocks and 135 bovine dentin blocks (3x3 mm) will be used. Among them, 90 blocks per each substrate will be incubated in human saliva for the formation of acquired pellicle and 45 blocks per substrate will remain without acquired pellicle. The blocks with pellicle will be divided into 3 groups (n=30 per group) that will differ according to the type of gel that will be applied on them: placebo gel, EGCG gel or chlorhexidine gel. Among the 30 blocks of each group, half of them will be submitted to erosive challenge and analysis of calcium released and the remainder will be submitted to proteomic analysis of the pellicle. The 45 blocks without pellicle will be divided into three groups and submitted to erosive challenge and analysis of calcium released. The acquired pelliclee will be formed on the blocks by exposure to pool of human stimulated saliva (from 3 donors). After incubation of the blocks in saliva, the gels will be applied on them (blocks with and without acquired pellicle) in a thin layer for 1 min, followed by careful removal. The blocks with formation of pellicle will be incubated again in human saliva. The pellicles will be collected with filter paper soaked in 3 % citric acid and processed for analysis by nLC-MS/MS. Ninety blocks of each substrate [45 with pellicles formation (n = 15 per group) and 45 without acquired pellicle (n = 15 per group)] will be subjected to erosive challenge and subsequent analysis of calcium released in the citric acid solution. After the erosive challenge, the blocks with pellicle will be submitted to new collection of pellicle for proteomic analysis, in order that acid-resistant proteins can be identified. Proteomic analysis will be conducted employing nLC-ESI-MS/MS. The obtained MS/MS spectra will be searched against human protein databases (SWISS -PROT and TrEMBL) using the MASCOT algorithm. The proteomic data related to protein quantification will be analyzed using the PLGS software. Data regarding calcium released will be analyzed using the GraphPad InStat software. Initially the data will be checked for normality (Kolmogorov- Smirnov) and homogeneity (Bartlett's test) to select the appropriate statistical test. The significance level will be set at 5 %.
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