Sugarcane-derived cysteine protease inhibitor: effect on osteoblastic and odontoblastic differentiation of human dental 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, especially cathepsin K, is related to the development of diseases, such as osteoporosis and apical periodontitis (periapical lesion), because they stimulate the bone resorption process. Therefore, its inhibition could be a therapeutic target for the control of that diseases. Cystatins are natural and reversible inhibitors of cysteine proteases. The cystatins, especially cathepsin K inhibitors, are part of an emerging class of drugs that are potent antagonists of osteoclastic activity, reducing bone loss. In addition to the inhibition of cysteine proteases, some studies have shown that cystatines may have a pro-osteogenic effect. The phytocystatins are plant cystatins, and some of them have already been produced recombinantly, such as CsinCPI-2 (derived from sweet orange) and CaneCPI-5 (derived from sugar cane). Study of our research group showed that CsinCPI-2 inhibited the cysteine proteases K and B, showed anti-inflammatory potential, and pro-osteogenic effect on human pulp cells. About CaneCPI-5, preliminary study, also from our research group, showed in pre-osteoblasts (MC3T3), that it present pro-ostegenic potential. The aim of the study will be to evaluate the effect of CaneCPI-5 on proliferation, migration, osteogenic and odontogenic differentiation of human dental pulp cells (hDPCs), in contact or not with the dentin. In a first step (without the presence of dentin), hDPCs exposed to CaneCPI-5 and not exposed (control) will be evaluated for viability using the alamar blue assay, proliferation by bromodeoxyuridine incorporation assay (BrdU), migration by transwell assay, gene expression of markers related to osteogenic and odontogenic differentiation through quantitative real-time polymerase chain reaction (qRT-PCR), protein production by Western Blotting, alkaline phosphatase activity by calculating thymophthalein release and detection of inorganic precipitates by alizarin red staining. In a second step, the effect of CaneCPI-5 on hDPCs in contact with dentin blocks will be evaluated. The viability of hDPCs will be evaluated by alamar blue and Live/Dead assays, adhesion and scattering using fluorescent labeling of F-actin filaments and cell differentiation through calcium-rich matrix deposition analysis using red staining of alizarin. If the results are promising, CaneCPI-5 could constitute a molecule with potential to be used in treatment techniques aimed the reparing of pulp-dentin complex and/or periapical, including endodontic regeneration techniques. (AU)