The Experimental Study On Differentiating Bone Marrow Mesenchymal Stem Cells Into Endothelial Cells In Vitro

Objective To detect the feasibility of bone marrow mesenchymal stem cells (BMSCs) differentiating into endothelial cell lineages in vitro, evaluate the biocompatibility of BMSCs-derived ECs with freeze-dried demineralized bone matrix (fdDBM), so as to provide the promising strategies for the establishment of vascular tissue engineered bone.Methods 1. BMSCs were isolated by bone marrow cells adherence, and nonadherent cells were removed carefully after 8h and fresh medium was replaced. MTT method was used to assess the third passage of subcultures.2. The third passage of subcultures were induced into endothelial cells with endothelial medium (10ng/ml VEGF , 2ng/ml bFGF and 2%FBS) for 3 weeks. Then the morphologic, ultrastructural and phenotypic characterizations of the endothelial cells were detected. MTT method was used to assess induced cells'growth and proliferation.3. Real-time Quantitative PCR (RT-qPCR) analysis indicated the relative gene content of genes (Flt-1, Flk-1, Tie-2 and eNOS) related to the cell differentiation of BMSCs. Vascular endothelial cells (VECs) was used as positive control. Relative gene expression data were analyzed using the 2^{-ΔΔCt}method.4. The production of nitric oxide (NO) by BMSCs-derived ECs was measured with an NO detection kit.5. A cell suspension of ECs was seeded and then cultured for 1, 3 and 7 days in freeze-dried demineralized bone matrix (fdDBM), then the biocompatibility and the interaction between the scaffolds and ECs were evaluated.Results 1. Isolation and characterization of BMSCs The morphology of BMSCs was spindle-like bearing round or oval-shape nuclei. Approximately 12 days, they reached confluence and became uniformly shaped. Growth curve of the third passage cells showed that there were differences of latency, of activity and flat periods.2. Characterization of BMSCs-derived ECsThe cells became into colony, ellipse and arranged like pebble 3 weeks later, and there were linear cord-like and tube-like struction. The cells also acquired important ultrastructural properties of endothelial cells as they contained Weibel-Palade bodies. CD31 and CD34, the specific markers of endothelial cell were positive in the cells. And the positive rate of CD34 increased significantly (p<0.05). The cells presented exponential growth and had no infinite senilism in short time.3. RT-qPCR Analysis①The expression of Flt-1mRNA in the BMSCs-derived-ECs group was lower than the VECs group (p<0.05), while BMSCs and BMSCs-derived-ECs was consistent with (p>0.05).②The expression of Flk-1mRNA increased remarkably in the BMSCs-derived-ECs group compared with the BMSCs group (p<0.05), and no differences were seen in the BMSCs-derived-ECs group and VECs group (p>0.05).③Both in the BMSCs-derived-ECs group and VECs group, the mRNA level of Tie-2 and eNOS showed no differences (p>0.05); while the BMSCs group didn't express these genes.4. Functional analysis of BMSCs-derived ECsThe BMSCs-derived ECs didn't release NO.5. The biocompatibility of fdDBM with BMSCs-derived ECsMany induced cells were adhered to the surface and inner walls of fdDBM network which appeared three dimensional structure, and spread extensively and proliferated well. Conclusion 1. The combination of VEGF (10ng/ml) and bFGF (2ng/ml), as well as low proliferating conditions (2%FBS), can induce BMSCs into endothelial cells,2. There is no significant gene expression difference between BMSCs -derived ECs and the vascular endothelial cells, but their biology function is inferior to mature vascular endothelial cells.3. BMSCs-derived endothelial cells have good biocompatibility with allogeneic fdDBM, and BMSCs will represent powerful alternatives to the construction of vascular engineered bone in vitro.