Effect of the water content on the collagenolytic/gelatinolytic activity of the dentin matrix

Abstract

Water has a paradoxical role in the process of dentin bonding. While it is important in the initial stages to keep the interfibrillar spaces open for the diffusion of the adhesive monomers, the presence of water negatively interferes with the infiltration of hydrophobic monomers, allowing phase separation. Residual water also negatively interferes with solvent evaporation and monomer-polymer degree of conversion. Additionally, water allows the activity of proteases residing in dentin, such as matrix metalloproteinases (MMPs) and cysteine cathepsins (CTAs), which can degrade collagen. To date, it is not known if the modulation of the amount of water within the dentin matrix could interfere with the activity of these proteases. Therefore, the main purpose of this study will be to evaluate if the amount of water in the demineralized dentin interferes with the proteolytic activity of this substrate. Human teeth will be collected, the roots will be cut and discarded, and the crowns will be pulverized to a fine powder. Enamel and dentin powders will be separated by density difference, and only the dentin will be collected. The dentin powder will be demineralized in 10% phosphoric acid. Ninety (n=90) eppendorf tubes will be each filled with 1.0 mg of the demineralized dentin powder. The tubes will be randomly and equitably distributed into 9 groups as a function of the water:ethanol ratio solutions. In weight percentage, the solutions will contain 0, 1, 5, 10, 20, 50, 70 or 100% water (positive control) in ethanol. Mineral oil will be used as negative control. The tubes will be stored in water bath at 37oC, under constant shaking, for 7 days. At the end of that period, total protein (Lowry's method), collagen concentration, hydroxyproline (HYP) release and ICTP fragments will be determined by colorimetric assays. The analyses will be made in duplicate. The independent variable of the study will be the "storage solution" (in 9 levels) while the response variables will be "total protein", "collagen concentration", "HYP", and "ICTP fragments". The number of replicates by group will be ten for each response variable (n=10). Data sets will be evaluated regarding adherence to the normal curve and homoscedasticity. Based on the results of these preliminary tests, the adequate statistical tests will be selected. The level of significance adopted will be 5%. (AU)