Evaluation of the pro-osteo/odontogenic effect of recombinant sugarcane cystatin, CaneCPI-5, on human pulp cells

Abstract

Cathepsins (a group of cysteine-proteases) play an important role in some pathological processes. It is known that an increase in their activity is related to the development of diseases such as osteoporosis and apical periodontitis (periapical lesion), as they stimulate the bone resorption process, through the proteolytic degradation of matrix components. Therefore, its inhibition could be therapeutic target for the control of these diseases. Cystatins are natural and reversible inhibitors of cysteine proteases. Phytocystatins are plant cystatins and some of them have already been produced in a recombinant way, such as CaneCPI-5 (derived from sugarcane), which will be the focus of this study because it presented pro-osteogenic potential observed in a preliminary study of our research group. The aim of this study will be to evaluate the cytocompatibility and the effect of CaneCPI-5 on proliferation, migration, osteogenic and odontogenic differentiation and the ability to stimulate mineralization of human dental pulp cells (hDPCs). HDPCs exposed to CaneCPI-5 and not exposed (control) will be evaluated for viability, by the methylthiazole tetrazolic (MTT) and neutral red assays; proliferation, by the bromodeoxyuridine incorporation assay (BrdU); migration, by transwell assay; gene expression of markers related to osteogenic and odontogenic differentiation, by quantitative real time polymerase chain reaction (qRT-PCR); protein production by Western Blotting, alkaline phosphatase activity, by calculating thymolphthalein release; and detection of inorganic precipitates, by alizarin red staining. If the pro-osteogenic and/or pro-odontogenic effect of CaneCPI-5 on hDPCs is confirmed, we will design trials to assess the effect of CaneCPI-5 on hDPCs in contact with dentin. If the results are promising, CaneCPI-5 may be a molecule with potential to be used in treatment techniques that aim the repair of the dentin-pulp complex and/or periapical. (AU)