Study On The Effect That Umbilical Vein Endothelial Cells Produce On Bone Marrow Mesenchymal Stem Cells' Osteogenic Differentiation In Vitro Co-culture System And Its Mechanism

Bone defect is common diseases in plastic surgery, maxillofacial surgery and orthopedics. The rehabilitation of maxillary defects has been a difficult problem in clinical. The current method of constructing tissue engineered bone were more seeds with simple cells.Although bone can be, but there are problems of slow vascularization of bone tissue engineering, new bone growth retardation and other issues.Bone marrow Mesenchymal stem cells have the potential of multiline age differentiation and are the early development of the mesoderm cells. They can not only differentiate into mesoderm from the same Mesenchymal cells, but also break mesoderm boundaries, differentiate into mesodermal tissue, such as fat cells, bone cells, cartilage cells, cardiac cells, nerve cells, muscle cells, tendon cells and astrocytes, etc. Currently, we have make great progress in the study that induce pure BMSCs to osteoblastic differentiation.However, this way exist some problems, such as a long cycle into bone, low efficiency to formation bone, cells easy to aging and other shortcomings.In recent years, researchers found VECs have the ability of secreting bone morphogenetic protein, stimulating osteoblasts and their precursor cells to secrete vascular endothelial growth factor when it promotes osteoblast differentiation, and the vascular endothelial growth factor play a very important role in the process of angiogenesis and the formation of vascular. It can promote endothelial cells proliferation and angiogenesis. These studies have shown that endothelial cells can support bone marrow Mesenchymal stem cells to change into bone. It can also speed up the formation of blood vessels, provide the stem cells with nutrition during the process of changing into bone.So, this project focused on the human umbilical vein endothelial cells'effect on the human bone marrow Mesenchymal stem cells'Morphology, growth, differentiation and the express of Bmi-land Runx2 gene. In a word, I wish this project can provide the theoretical basis of bone tissue engineering, through the co-culture of umbilical vein endothelial cells and bone marrow Mesenchymal stem cells.[Method]1 We extracted a volunteer's bone marrow fluid and isolated the bone marrow mononuclear cells by the way of density gradient centrifugation. And we purified the MSCs by its characteristic of adhesion to the plastic bottom. In order to identify the MSCs, We cultured MSCs to passage to the third generation and then we detected CD34, CD29, and CD44's surface antigen expression by flow cytometry.2 We observed the morphological changes by phase contrast microscope. The co-culture of the third generation hMSCs and HUVECs was established in the rate of 1:1, and DMEM with 10 percent FBS were used as the medium of the joint culture system.3 The separate cultured hBMSCs and hUVECs as a negative control group.We observed the morphological changes by phase contrast microscope in 4th,6th,8th,10th day, and counted the number of each hBMSCs group by count plate; we detected alkaline phosphatase and osteocalin in three culture groups at 4th,6th,8th,10th day. And we make a statistical analysis about the test value with software SPSS17.0.4 We detected the expression of Bmil and Runx2 gene in hBMSCs group and co-culture group at 4th,6th,8th,10th day. At last, we make a statistical analysis about the test value with software SPSS17.0.[Result]1 We make an analysis and identification on third-generation hMSCs's cell phenotype By flow cytometry, the expression of CD34 is negative and the expression of CD29,CD44 are positive; We can get higher purity hBMSCs by the way of Ficoll density gradient centrifugation, which is used to isolate and purify hBMSCs.2 The hBMSCs,which are cultured with FBS, are elongated spindle and small. The Primary generation cells grow into groups at 4 to 5 days. The third generation of bone marrow Mesenchymal stem cells forms a single, and into the spindle, vortex-like distribution, there is no cell overlap. The hBMSCs grow in logarithmic at 4-6 days, and into platform at 8-10 days. HUVECs grow as monolayer, polygonal shape, cobblestone-like arrangement. The hBMSCs have clear boundary and rich cell slurry,nuclei were round or oval. Occasionally,they show dual-core and confluent at 5th. From first generation to fourth generation, the hBMSCs grow faster,2-3 days can be passage.3 The amount of alkaline phosphatase in each group gradually increased with time, and the co-culture group's ALP is the highest at all time; The ALP of Mesenchymal stem cells group and umbilical vein endothelial cells group did not change; The comparisons between co-culture group and other groups were statistically significant(P<0.01). The osteocalcin detected in each group was gradually increased with time. The highest osteocalcin of co-culture group is detected at 8th day. The comparisons between co-culture group and other groups were statistically significant(P<0.01).4 The volume of co-culture group's Bmi-1 and Runx2 gene was gradually increased with time, and the co-culture group's gene expression is the highest at all time; The expression of Bmi-1,Runx2 in Mesenchymal stem cells group and umbilical vein endothelial cells group did not change;The comparisons between co-culture group and other groups were statistically significant(P<0.01). The osteocalcin detected in each group was gradually increased with time. The highest osteocalcin of co-culture group is detected at 10th day. The comparisons between co-culture group and other groups were statistically significant(P<0.01).[Conclusion]1 We can get higher purity hBMSCs cultured with FBS by the way of Ficoll density gradient centrifugation, which is used to isolate and purify hBMSCs.2 It shows good compatibility about the co-culture of Mesenchymal stem cells and umbilical vein endothelial cells. Umbilical vein endothelial cells can promote bone marrow Mesenchymal stem cells proliferation.3 In vitro co-culture system, Umbilical vein endothelial cells can promote bone marrow Mesenchymal stem cells express Bmi-land Runx2 gene, and Induce bone marrow Mesenchymal stem cells differentiate into osteoblasts.