Multi-User Equipment approved in grant 2017/21622-7: aparelho QuantStudio 7 Flex Real-Time PCR System

• Grant number: 19/05289-5
• Support type: Multi-user Equipment Program
• Duration: May 01, 2019 - April 30, 2026
• Field of knowledge: Health Sciences - Dentistry - Oral and Maxillofacial Surgery
• Principal researcher: Adalberto Luiz Rosa
• Grantee: Adalberto Luiz Rosa
• Home Institution: Faculdade de Odontologia de Ribeirão Preto (FORP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 17/12622-7 - Cell therapy: potential of mesenchymal stem cells, VEGF-A and BMP-9 to regenerate bone tissue, AP.TEM
• EMU web page: http://143.107.206.201/departamentos-mainmenu-28/803.html
• Type of equipment: Caracterização e Análises de Amostras - Proteínas/Ácidos nuclêicos - PCR
• Manufacturer: ThermoFisher Scientific
• Model: QuantStudio 7 Flex

Abstract

Bone tissue has high capacity of regeneration, but in several situations the extent of the injury overcomes its regenerative potential. In this scenario, therapies based on the use of mesenchymal stem cells (MSCs) have aroused the attention of many researchers for being a promising alternative compared with the available treatments. However, many molecular, cellular and tissue characteristics remain unmet in the literature to make cell therapy an effective treatment for bone repair both in healthy and systemic compromised patients by pathologies such as osteoporosis, diabetes and hypertension. In keeping with this, this research project consists of three subprojects that aim to: (1) evaluate the potential of MSCs harvested from either bone marrow (BM-MSCs) or adipose tissue (AT-MSCs) combined with vascular endothelial growth factor A (VEGF-A) and/or bone morphogenetic protein 9 (BMP-9) to repair bone defects; (2) evaluate the potential of these MSCs genetically edited to overexpress VEGF-A and/or BMP-9 to repair bone defects; and (3) evaluate the effect of BM-MSCs harvested from healthy rats on the osteoblastic differentiation of BM-MSCs harvested from osteoporotic, diabetic and hypertensive rats. To develop subprojects 1 and 2, BM-MSCs and AT-MSCs will be treated with VEGF-A and/or BMP-9 or genetically edited by clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) to overexpress VEGF-A and/or BMP-9. Those cells will be evaluated in vitro to assess their angiogenic and osteogenic potentials as well as their large scale genomic and proteomic profile. For bone repair, BM-MSCs and AT-MSCs either combined with VEGF-A and/or BMP-9 or edited to overexpress these factors will be directly injected into rat bone calvarial defects. To evaluate the presence of cells in the defects, Luc-expressing cells will be tracked by bioluminescent imaging. Up to 4 weeks, vasculogenesis and bone formation will be evaluated by in vivo micro-CT. After 4 weeks, the animals will be euthanized and the harvested calvaria evaluated by histological analysis. In the subproject 3, the effect of BM-MSCs harvested from healthy rats on the osteoblastic differentiation of BM-MSCs from osteoporotic, diabetic and hypertensive rats will be evaluated using an indirect co-culture model. These studies are the first step aiming to apply these cell therapies to regenerate bone defects in the presence of such pathologies. (AU)