In dentistry, various diseases such as caries and tooth erosion have been studied. Recently, a new cystatin derived from sugarcane (CaneCPI-5) was produced in a heterologous expressions ystemand was shown to strongly bind to enamel, protecting against tooth erosion in vitro. However, the protective effect of CaneCPI-5 against dental caries, caused by acids produced by bacteria organized in a biofilm, after sugar consumption, has never been evaluated. In addition, matrix proteases are very important for the progression of dentin caries. Cystatins, includingCaneCPI-5, inhibit cysteine cathepsins, which can have a additional impact on the prevention of dentin caries. In addition, matrix proteases are very important for disease progression and cystatin, including CaneCPI-5, are cathepsin inhibitors, and may have an additional effect in preventing disease in dentin. The objective of this study is to evaluate the effect of different concentrations of CaneCPI-5 on the bacterial profile and viability of a microcosm biofilm as well as on the prevention of dentin demineralization. For the formation of a microcosm biofilm, saliva will be collected from 2 healthy subjects, who have not brushed their teeth in the last 24hours and have not ingested food or drinking liquids in the last 2 hours. Saliva will be diluted(70% saliva and 30% glycerol) and further mixed to McBain saliva (1:50) for the formation of acariogenic biofilm. Bovine enamel and dentin samples (4 mm x 4 mm; n=90) will be prepared for the formation of a microcosm biofilm for 5 days, which will be treated daily (1x60s/day),with 3concentrations of CaneCPI-5 (0.1, 0.5or 1.0 mg/ml), 0.12% chlorhexidine (positive control) or PBS (negative control).In Step 1, live and dead bacteria in the biofilm will be evidenced by fluorescence using confocal microscope. In Step 2, the demineralization caused by the biofilm in the studied conditions will be measured by transverse microradiography. The data will be subjected to appropriate statistical analysis (parametric or non-parametric test, p<0.05).
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