Experimental Studies On Biological Features Of Human Bone Marrow Mesenchymal Stem Cells For Tissue Engineering Application And Preparation Of It's Monoclonal Antibody

Bone defect is a very common surgical complication. By far, there are limited treatment for it, such as autologous or allograft bone transplation, artificial succedaneum of which none is suitable from all cases. With the development of bone tissue engineering, it seems to be the ideal solution.The bone tissue engineering is established on three footstones, they are seed cell, scaffold and growth factors in which the most important one is the seed cell. Now we can abtain the seed cell for bone, periosteum, bone marrow and embryo. For the absolutely advantage of mesenchymal stem cells (MSCs), many scholars have given great effort on it. With the hope to put MSCs into clinical applications,wo designed our experimental studies.PART I Cultivation and evaluation of the MSCs in vitroObjective: To find the most suitable culture condition of MSCs in vitro, and to study partial of it's biological characteristics.Methods: We isolation MSCs with density centriguging.after cultivation and proliferation, we observed the morphological changes with phase contrast microscope and trasmission elctron microscope. Also, we have drawn the growth curve and assayed the cell cycle and phenotype with flow cytometer. In the same time, we observed the effect put by the growth facter-bFGF.Results: The MSCs we cultured in vitro showed spindle cells in shape. They growed in fast proliferation and only taked 3-4days to get into logarithm growth phase; after passage,they show more uniformappearance;They displayed stem feature in electron microscope.And the bFGF can accelerate it's proliferation.Conclution: The best condition for isolation and culture of human bone marrow mesenchymal stem cells was successfully established and some biological characteristics were observed. It has found a base for further investigation and application of mesenchymal stem cells.PART II Experimental studies on osteogenic and chondrogenic differentiation of mesenchymal stem cells from human MSCsObjective: To explore osteogenic and chondrogenic potential of human bone marrow mesenchymal stem cells.Methords: MSCs which derived from human bone marrow with centriguging were induced ostergenic and chondrogenic differentiation under conditinal culture medium and were observed the change of morphology, alkaline phosphatase activity, matrix mineralization and expression of type I and type II collagen by immunohistochemical by assay and extracellular matrix.Results: MSCs became more poly-edged and round morphology after 14 days and induction in dexamethasone,P-glycerophoshpate and Vitamin C medium.Histological analysis of the cells revealed strong staining with ALP method, immunohistochemistry of type I collagens expression confirmed that the cells had osteogenic potential.Cultured cells also differentiated into chondrocytes after exposure to Vitman C and TGF-B1 in F12 medium,the cells uniformly spindle shape became round after 7 days induction,immunohistochemistry revealed expression of type II collagens and an abundance of glycosaminoglycans within the extracellular matrix.Conclusion: We successfully induce human mesenchymal stem cells derived from bone marrow differentiate into osteoblast like cells andchondrocytes.PART III Observation on growth of human osterblast induced from MSCs cultured with coral hydroxyapatite and the influence of bFGFObjective: To study the compatibility and growth of human osteoblast induced from MSCs cultured with coral hydroxyapatite.Methods: Osteoblast obtained from human MSCs induction were cultured with coral HA, cells were observed under phase contrast microscope and scanning elctron microscope.,and we also draw the growth curve and assayed the alkaline phosphatase activity both in experimental group ,bFGF group and control group.Results: Osteoblasts can attached and well growed on the three-demension suface of coral HA.The alkaline phosphatase activity is the same strong compared with control group. And the cell quantities in bFGF group is more than other two groups with t-test.