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Mesenchymal stem cells and osteoblasts: evaluation of molecular signatures and their potential for bone regeneration

Grant number: 16/02556-4
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): May 01, 2016
Effective date (End): November 30, 2016
Field of knowledge:Health Sciences - Dentistry
Principal Investigator:Adalberto Luiz Rosa
Grantee:Victor Augusto Minari
Home Institution: Faculdade de Odontologia de Ribeirão Preto (FORP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

Bone has a high regenerative capacity, but in some cases the extension of the lesion prevents tissue repair. In this context, cell therapy has been the focus of several research groups as an alternative to the current treatments. However, several molecular, cellular and tissue aspects were not addressed by the literature and need to be investigated in order to make cell therapy an effective treatment for regenerating bone defects. Then, the aim of this study is to compare the molecular signatures and the potential of mesenchymal stem cells (MSCs) and osteoblasts to regenerate bone tissue. MSCs will be obtained from bone marrow (MSCs-BM) and adipose tissue (MSCs-AT) of rats, cultured in conditions to either keep MSCs features or to differentiate into osteoblasts (OBs-BM and OBs-AT, respectively). MSCs will be identified by the expression of CD29, CD31, CD34, CD45, CD90 and CD106 by flow cytometry. Gene expression of Runx2, alkaline phosphatase and osteocalcin will be evaluated by real-time PCR to confirm osteoblast differentiation. The molecular signatures of MSCs-BM, MSCs-AT, OBs-BM and OBs-AT will be evaluated by large-scale analyses of gene (microarray) and protein (proteomic) expression. For bone regeneration, cells will be directly injected (single injection x repeated injections) in bone defects created in rat calvarial two weeks before the first injection to mimic bone defect without self-regeneration capacity. Firstly, MSCs-BM, MSCs-AT, OBs-BM and OBs-AT transfected to express luciferase will be injected in bone defects to allow tracking cells to determine the interval between repeated injections. Then, the first cell injection will be carried out after two weeks of the bone defect creation and the additional injections will be done following the interval previously determined. Bone defects treated with a single cell injection and with the vehicle phosphate buffered saline without cells will also be evaluated. Six weeks after the first injection, the rats will be killed and the new-formed bone will be evaluated by microtomography, histological and real-time PCR analysis and elastic modulus and hardness analyses. The data will be submitted to normal distribution adherence to determine the appropriate statistical test. The results of this study may stablish new parameters regarding characteristics of candidate cells to be used in cell therapy and their injections to regenerate bone tissue as, up to now, there are no studies investigating the effect of repeated injections. (AU)