Bone has a high regenerative capacity, but in some situations the lesion extension prevents the tissue repair. In this context, cell therapy has been the focus of several research groups as an alternative to the current treatments. However, there are experimental aspects that need to be deeply investigated in order to make cell therapy an effective treatment for regenerating bone defects. Then, the aim of this study will be evaluate the potential of mesenchymal stem cells (MSCs) derived from either bone marrow or adipose tissue, as well as, osteoblasts differentiated from these cells to regenerate bone tissue when directly injected (repeated injections) into rat calvarial bone defects. MSCs will be obtained from bone marrow and inguinal adipose tissue of rats, cultured in conditions to either keep MSCs features or to differentiate into osteoblasts. MSCs will be characterized 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 in vivo experiments will be carried out with the four groups of cells, (1) MSCs derived from bone marrow, (2) MSCs derived from adipose tissue, (3) osteoblasts differentiated from bone marrow MSCs and (4) osteoblasts differentiated from adipose tissue MSCs, expressing luciferase. This will allow tracking these cells after direct injection into calvarial bone defects to determine the number of injections and its frequency. The first cell injection will be carried out after 2 weeks of the bone defect creation and at the end of 8 weeks the new-formed bone will be evaluated by microtomography, histological analysis and elastic modulus and hardness analyses. The data will be submitted to adherence to normal distribution to determine the appropriate statistical test. The results of this study may stablish new parameters regarding cell injections to regenerate bone tissue as, up to now, there are no studies investigating the effect of repeated injections.
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