Study of the enzymatic profile and activity of the human dentinal fluid

Abstract

The destruction caused by the caries process can be either accelerated or limited by the responses of the pulp-dentin complex. The dentinal fluid is an important component in such mechanism, since it forms a communication path between pulp and different regions of dentin, allowing transportation of nutrients and molecules coming from the pulp. The dentin matrix degradation is observed in physiopathological processes, like in caries progression. It is believed that host derived proteolytic enzymes have a role in dentin organic matrix degradation. In this way, it is essential to better understand the mechanisms involved in collagen breakdown, the main component of dentin organic matrix. Therefore, the present proposal aims to verify the presence of different families of proteolytic enzymes in dentinal fluid, and after that, investigate a possible involvement of such enzymes in the progression of caries lesions. The hypothesis to be tested is that during the progression of dental caries, the proteolytic enzymes can be transported via the dentinal fluid, becoming able to degrade the collagen exposed in dentin. The objective of this study is to identify and map the families of proteases present in the dentinal fluid extracted from healthy and carious teeth, which will be initially analyzed by proteomics (protein profile). Then the endogenous proteolytic activity of dentinal fluid will be evaluated according to the enzymes previously identified. Liquid chromatography coupled to mass spectrometry (nLC-ESI-MS/MS) will be used for the identification and quantification of proteins extracted from the dentinal fluid. The main enzymes will be complimentary quantified by Western Blotting. The proteolytic activity of some previously detected enzymes will also be evaluated by spectrofluorimetric analysis using specific fluorogenic substrates, while the gelatinolytic activity will be investigated by zymography. (AU)