Effect of BMP-9 overexpression on osteoblast differentiation of mesenchymal stem cell

• Grant number: 19/18221-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2020
• End date: March 31, 2021
• Field of knowledge: Health Sciences - Dentistry
• Principal Investigator: Helena Bacha Lopes
• Grantee: Georgia Kors Quiles
• Host Institution: Faculdade de Odontologia de Ribeirão Preto (FORP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

Previous studies of our research group have shown that the use of cell therapy has great potential to repair bone tissue; however, without yet inducing complete tissue regeneration. Based on this, we are developing a FAPESP-funded project (Thematic Project, Process No. 2017 / 12622-7) to establish a mesenchymal stem cell line (MSC) overexpressing bone morphogenetic protein 9 (BMP-9) using the technique of gene editing based on short interspersed, regularly interspersed Cas9 protein palindromes (CRISPR / Cas9) for use in this type of treatment. In this context, the complete in vitro characterization of these cells is necessary before the in vivo experiments. For this, cells already prepared expressing the VPR gene transcription activating protein (control group - CTMs-VPR) and with the guide RNA for the BMP-9 gene (CTMs-VPR-BMP-9) will be cultured in osteogenic culture media. non-osteogenic for periods of up to 21 days and the following parameters will be evaluated: (1) expression of the osteoblast marker genes, Runt-2 (Runx2), Osterix (Osx), alkaline phosphatase (Alp), osteocalcin (Oc ), osteopontin (Opn) and bone sialoprotein (Bsp) by real time PCR at 3, 7, 10 and 14 days; (2) in situ ALP activity at 3, 7, 10, and 14 days, (3) mineralized extracellular matrix formation by alizarin red staining at 14, 17, and 21 days, and (4) expression of genes related to BMP signaling pathway by PCR Array at 3 and 7 days. Data will be subjected to the normal curve adherence test to determine the appropriate statistical test and significance level will be 5% for the comparison between the cells groups.