Evaluation of the osteogenic growth factors effect in the differentiationof mesenchymal stem cells into osteoblasts

Abstract

The growth factors are biologically active polypeptides that regulate the recruitment and differentiation of bone cells during the bone regeneration process. In this context, the osteogenic growth peptide (OGP - osteogenic growth peptide) which is naturally present in the organism whose primary structure is identical to the C-terminal sequence from histone H4 has been investigated. Proteolytic cleavage of the OGP can generate the pentapeptide OGP (10-14). Both peptides stimulate the proliferation and differentiation of osteoprogenitor cells in vitro and increase bone formation in vivo. However, few studies have investigated the physiological mechanisms activated by OGP and OGP (10-14) peptides for bone regeneration. The aim of this study is to investigate in vitro which mechanism(s) are activated by OGP and OGP(10-14) for the differentiation of mesenchymal cells into osteoblasts. Mesenchymal stem cells (MSCs) will obtain and isolate from mice and after characterization, the cells will be stimulated by: BMP-2 (Positive Control - Group I); OGP (Group II), OGP(10-14) (Group III), and the negative control (only MSCs - Group IV). All groups will be evaluated for cell proliferation and differentiation. To identify intracellular mechanisms for MSCs to differentiate into osteoblasts through the OGP and OGP effect (10-14) will be realized the proteome and phosphoproteome. Thus we intend to identify the cellular mechanisms induced by these peptides, including secreted and intracellular proteins. In addition, the phosphoproteome allows evaluate the phosphorylated protein, which is essential to the understanding of signaling pathways. After collection of secreted proteins in the culture medium, the cell will be lysed to obtain phosphorylated and non-phosphorylated intracellular proteins. All samples will be submitted to proteome analysis, while only the phosphorylated proteins will also be submitted to phosphoproteome. After appropriate treatment of the Bioinformatics and statistical data, the most differentially expressed proteins in each group will be validated by flow cytometry or Western blot. In this study is expected to identify key cell signaling pathways including intracellular phosphorylation mechanisms during differentiation of osteoblastic process of mesenchymal cells induced by OGP and OGP (10-14) peptide. The results of this study will also promote greater security and reliability in the use of biomaterials functionalized with these peptides in for bone regeneration.