The Biological Effects Of Mechano-growth Factor On Human Bone Marrow-derived Mesenchymal Stem Cells

Bone marrow-derived mesenchymal stem cells(BMSCs)are a type of multipotent adult stem cells with that are capable of differentiate into multiple cell lineages, such as osteocytes, adipocytes, chondrocytes, nerve cells and muscle cells. BMSCs has generated a great amount of interest in fields of cell or gene therapy and tissue engineering due to their unique biological properties: easily obtained; fast proliferation; the ability to home to sites of tissue injury, to differentiate into various cell types, to secrete multiple bioactive molecules and to perform immunomodulatory functions.The differentiation of BMSCs is particularly important in fields of cell therapy and tissue engineering. BMSCs are subjected in a complicated fluid microenvironment. Many studies have shown that mechanical stimulation plays important roles in the differentiation of BMSCs. Mechano-growth factor(MGF), a newly discovered alternative splicing variant of insulin-like growth factor 1(IGF-1), is up-regulated in tissues following damage or mechanical stimuli. Several studies have demonstrated that the 24 amino acid peptide analog corresponding to the C-terminal E-domain of MGF(MGF-E) play important roles in protection of damaged tissues and cells and promoting repair of injury tissues. It was also found that MGF-E has effects in cell differentiation. However, the effects of MGF-E in differentiation of h BMSCs are unclear.In this article, we first studied the effects of MGF-E on proliferation and migration of h BMSCs. Next, we studied the effects of MGF on h BMSCs differentiation. In addition, we studied the underlying mechanism during differentiation. The main exprements and results of the study are as follows:① Effects of MGF-E on the proliferation and migration to hBMSCs Using the adjustable FX-4000 T cellular mechanical loading system, we treated the in vitro cultured h BMSCs with 12%, 1Hz equibiaxial cyclical mechanical stimulation. Then RT-PCR technology was used to detect the gene expression of MGF. The results showed that mechanical loading did not change the expression of MGF. Then, we studied the the proliferation and migration of h BMSCs by MGF-E peptides treatment. The results showed that MGF-E treatment significantly down regulated the proliferation rate of h BMSCs. Transwell and scratch experiments showed that 25 ng/m L and 50 ng/m L of MGF-E peptides treatment can significantly promote the cell migration but 100 ng/m L of MGF-E peptides did not change the cell migration compared with control.RT-PCR and western blot experiments showed that MGF-E could induce the expression of cyclooxygenase-2(COX-2). And NS-398(COX-2 inhibitor) treatmen could inhibit the MGF-E induced migration of h BMSCs. These results indicated that mechanical stimulation did not affect the expression of MGF in h BMSCs. MGF-E could promote the migration and down regulate the proliferation rate of h BMSCs.② Effects of MGF-E on osteogenic, adipogenic and chondrogenic differentiation of h BMSCs To investigate the effects of MGF-E on h BMSCs differentiation, we prepared osteogenic, adipogenic, chondrogenic induction medium as control group and 20 ng/m L MGF-E added induction medium as experimental group. The induction medium was replaced every three days. Due to low stability, MGF E peptide was added every day. Experimental results are as follows: 1) Alkaline phosphatase(ALP) activity assay, ALP staining and Alizarin Red S staining were performed to study the effects of MGF-E on osteogenic differentiation of h BMSCs. 2) Oil red O staining and Reverse Transcription–Polymerase Chain Reaction(RT-PCR) technique were performed to study the effects of MGF-E on adipogenic differentiation of h BMSCs. 3) Immunofluorescence staining and enzyme-linked immunosorbent assay(ELISA) technologies were performed to research the effects of MGF-E on chondrogenic differentiation of h BMSCs. Above studies demonstrated that MGF-E could promote h BMSCs differentiation in induction medium as it results in enhanced osteogenic, adipogenic and chondrogenic differentiation. This provides foundation basis for subsequent research.③ Effects of MGF overexpression on osteogenic and adipogenic differentiation of h BMSCs To characterize the roles that MGF played in h BMSCs differentiation, the MGF gene was cloned and carried by pc DNA3.1 vector, and then transfected into h BMSCs using a lipofectamine kit. The results showed that almost 50% of the cells can express MGF. Next, RT-PCR analysis was performed to verify the expression of MGF in h BMSCs. The MGF overexpressing h BMSCs were then cultured in induction medium to induce the differentiation into osteoblasts and adipocytes. Cells transfected with an empty vector were used as controls. In osteogenesis, Alizarin Red S staining showed that MGF overexpression enhanced calcium deposits compared with empty vector group under osteogenic induction medium after 7 days. Similarly, Oil Red O staining showed that MGF overexpression also increased the lipid droplets accumulation in adipogenic induction medium after 7 days. Quantification of calcium deposits and lipid droplets accumulation showed that levels of calcium deposits and lipid droplets accumulation in MGF overexpression group were increased compared with empty vector group, respectively. Our results indicated that MGF overexpression could induce h BMSCs differentiation under induction medium condition.④ MGF-E promote h BMSCs differentiation through delay the cell cycle In order to study the underlying mechanisms of MGF induced h BMSCs differentiation, flow cytometry assay was performed to study the effects of MGF-E on the cell cycle and RT-PCR assay was performed to study the gene expression of Cyclin E and cyclin-dependent kinase(CDK2). The results of flow cytometry assay showed that the cell numbers of h BMSCs in G1 phase were significantly increased after 24 hours MGF-E treatment in growth medium. It illustrated that MGF-E could induce the cell cycle arrested in G1 phase and the G1 phase were prolonged. RT-PCR results showed that the m RNA expression of cyclin E and CDK2 were significantly decreased after 24 hours treatment. These results indicated that the reason of MGF-E induced cell differentiation may be by reducing the expression of Cyclin E and CDK2 to arrest the cell cycle in G1 phase.In conclusion, our study demonstrated that MGF could promote h BMSCs migration and induce the differentiation of h BMSCs. It also provides a potential strategy of employing MGF for cell therapy and tissue engineering.