The Biological Characteristics Of Mesenchymal Stem Cells From Human Bone Marrow And Its Application For Bone Tissue Engineering

The biological characteristics of mesenchymal stem cells from human bone marrow and its application for bone tissue engineeringMesenchymal stem cells (MSCs) are a population of pluripotent cells which reside in the complex cellular arrangement in bone marrow and possess the highly proliferative capacities. In the definite condition,they can be coaxed to differentiate into osteoblasts, chondrocytes, myoblasts, tendon cells, hepatocytes, adipoccytes and even astrocytes. As a result of virtues,such as draw the materials easily,simple method of cultivation and highly proliferative capacities, MSCs has become the hot spot of the research of seed cells for tissue engineering, especially bone tissue engineering.In our study, the major aim is focused on the biological characterization of MSC, their theraeutic potentials in gene therapy and the application of tissue engineering.1. The biological characteristics of mesenchymal stem cells from human bone marrowMSCs were isolated by density gradient centrifugation with Percoll at a density of 1.073g/ml in order to eliminate unwanted cell types. The mononuclear cells were incubated in low Glucose Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum. MSCs were highly purified and culture-expanded . The morphology, phenotype, histology and biological properties of the cultured MSCs were investigated . Our research has demonstrated that MSCs possess some unique phenotypes. MSCs were fusiform shape,and the growth speed was swiffed in the earlier period. The amplification speed of cells would become slow after 10 generation. Flow-cytometry analysis showed that MSCs were positive for CD29,CD44,CD166, and negative for CD34 ,CD45. Cell cycle studies showed there were 83.11%,5.17%and 11.72% percentage of the cells in G₀/G₁, S and G₂/M respectively.Cytochemistry staining evaluation revealed that MSC were homogeneously positive for glycogen (periodic acid Schiff reaction,PAS), negative for Sudan black reaction (SB) and around 5%of them were positive for alkaline phosphatase (ALP).Our studies indicate that the MSCs own their peculiar biological characteristics of stem cells which differ the cells from clusters of HSCs.2.Differentiation of MSC into osteoblasts and adipocytes.In the specific condition, MSCs can be differentiated along multiple mesenchymal lineages. Firstly, The cultured human MSCs which exposed to 10-7M dexamethasone (Dex) , 50μg/ml ascorbic and 10mM β —glycerophosphate ( β -GP) in the presence of 10% FBS differentiate into osteoblastic lineage . Von Kossa's stain revealed heavy extracellular matrix deposition and calcium nodule formation. The positive stain of type I collagen was revealed by the immunohistochemical technique. MSCs were exposed in osteogenic induction for 1,2,3,4 weeks,and the cells increased their expression of alkaline phosphatase. The higher expression appeared during 2 to 3 weeks period, about 85%. Secondly, The cultured human MSCs differentiate into adipocytes lineages when 1μM dexamethasone and 10μ g/ml insulin were in the present of 10% FBS for 2 weeks. There was positive staining for Sudan black reaction (SB).3.Transfection of EGFP gene into MSCs and its expressionAs a marker gene, EGFP(enhanced green fluorescent protein) was widely applied to the investigation of scope of gene therapy. A variety of studies have demonstrated that MSCs may be an ideal target cell type in the gene therapy. To investigate the possibility, MSCs were transfectant by a retrovirus vector carrying the...