Pro-osteogenic effects of the phytocystatins CsinCPI-2 and CaneCPI-5 on bone repair - In vitro and in vivo study

Abstract

The present study aims to evaluate the pro-osteogenic effects of the phytocystatins CsinCPI-2 and CaneCPI-5, through in vitro and in vivo studies. Specifically, the in vitro study will assess the modulating impact of phytocystatins on the response of mouse bone marrow-derived macrophages (BMDMs) and on the osteoblastic differentiation of primary undifferentiated mouse bone marrow mesenchymal cells (BMSCs). The modulation of macrophages will be evaluated by analyses of gene expression (RT-qPCR) and protein production/secretion (Western blot / Cytometric Bead Array) of cytokines (pro- or anti-inflammatory) and other molecules that characterize a phenotypic profile within the spectrum between M1 or M2 polarization. To evaluate osteoblastic differentiation, gene expression and protein production of bone markers will be assessed, as well as alkaline phosphatase activity and mineralization nodule formation by the alizarin red method. Also, in the in vitro study, BMDM and BMSC will be grown in co-culture on poly-µ-caprolactone (PCL) scaffolds with phytocystatin incorporation. In this experiment, osteoblast differentiation under direct or indirect stimulation of phytocystatin will be evaluated. The in vivo study will be conducted after the in vitro results have been obtained, and will evaluate the bone repair of critical defects induced in mice's calvaria treated with 3D PCL scaffolds with the incorporation of phytocystatin that best show a favorable in vitro response to bone repair. Thirty-two BALB/C mice will be used, randomly divided into 4 experimental groups: Negative Control Group (untreated calvarial defect), Vehicle Group (calvarial defect + scaffold + vehicle gel), Positive Control Group (calvarial defect + scaffold + BMP2), and Treatment Group (calvarial defect + scaffold + selected phytocystatin). Microtomographic and histological analyses will evaluate bone formation in the defect areas. In addition, immunohistochemical analysis will be performed to identify and localize proteins related to osteoblast differentiation and bone formation. Appropriate statistical tests will be used for comparisons between the groups. In all procedures, a significance level of 5% will be adopted for the decision on the validity of the hypothesis tested.