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

• Grant number: 22/07424-0
• Support Opportunities: Regular Research Grants
• Start date: February 01, 2023
• End date: July 31, 2025
• Field of knowledge: Health Sciences - Dentistry - Periodontology
• Principal Investigator: Joni Augusto Cirelli
• Grantee: Joni Augusto Cirelli
• Host Institution: Faculdade de Odontologia (FOAr). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil
• Associated researchers: Andrea Soares da Costa Fuentes

Abstract

Intraoral bone defects comprise clinical conditions caused by different factors, which are pathological, traumatic and iatrogenic. The search for regenerative methods and biomaterials that promote the resolution of bone defects and the restoration of oral functional and aesthetic conditions are fundamental for the maintenance of the health and well-being of the population. 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 shows a favorable in vitro response to bone repair. Cell therapy with BMSCs in association with scaffolds and the cystatin will also be evaluated. Eighty-four BALB/C mice will be used, randomly divided into seven experimental groups: Negative Control Group/Coagulum (untreated calvarial defect), Vehicle Group (calvarial defect + scaffold + vehicle gel), Positive Control Group (calvarial defect + scaffold + BMP2), Treatment 1 Group (calvarial defect + scaffold + selected phytocystatin), Treatment 2 Group (same as Treatment Group in association with BMSC Cell Therapy), Treatment 3 Group (same as Vehicle Group in association with BMSC Cell Therapy) and Treatment 4 Group (same as Vehicle Group in association with Cell Therapy with BMSC previously treated with cystatin). The animals will be euthanized in 2 post-treatment periods: 05 days (n=6/group) and 42 days (n=6/group). The tissue collected in the defects after 05 days of repair will be analyzed by multiplex (protein analysis) and immunohistochemistry to evaluate proteins related to bone formation and osteoblastic differentiation/activation. Bone blocks with defects removed within 42 days will be analyzed by microtomography and histological analyses to assess bone formation in the defect areas. In addition, the 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 to decide the validity of the hypothesis tested. (AU)