Experimental Study Of Chondrogenesis Differentiation Of MSCs In Vitro And In Vivo

Bone marrow mesenchymal stem cells(MSCs) are multipotential adult stem cells. MSCs share similar morphology of fibroblasts, reticular cells and endothelial cells. MSCs are present in a variety of tissues during individuall human body development, and in adults they are prevalent in bone marrow. From that readily available source, MSCs can be isolated, expanded in culture, and stimulated to differentiate into the main cells of bone, cartilage, muscle, marrow stroma, tendon, fat and a variety of other connective tissues. Because large numbers of MSCs can be generated in culture and be re-introduced into the in vivo setting, MSCs may be used in tissue-engineered constructs. This approach is now being explored to regenerate tissues that the body cannot naturally repair or regenerate when challenged. Moreover, MSCs can be transduced with retroviral and other vectors and is, thus, potential candidates to deliver somatic gene in local or systemic therapies. In order to separate MSCs severalmethods have been applied such as density gradient centrifugation, immunomagnetic beads, and static adhesive assay and flow cytometer. MSCs with phenotype similar to that of msenchymal cells,epithelial cells, fibroblast like cells and hemopoietic cells,MSCs often show the positive expression of CD29,CD 44H,CD71,CD 90,CD 106,CD 120 a ,CD124,CD166,SH1,SH2, SH3, SH4 SB 10, negative expression of CD1a, CD14, CD34, CD45, CD56 and ESA.MSCs could be induced into chondrocytes in vitro and formed cartilage in vivo. MSCs can be obtained by aspiration bone marrow and expanded fastly in vitro. MSCs may have stable phenotype after long time being cultured and subpopulated. MSCs may be induced into the chondrocytes phenotype When MSCs are expanded to the needs. Therefore,MSCs may be used in the tissue engeneering of cartilage.In vitro studies simulate the in vivo environment to induce MSCs by application of cytokines. In vivo studies, MSCs with scaffolds were implanted into animal to generate cartilage.But, it is very difficult to completely simulate the joint cavity environment in vivo by in vitro experiments. We investigated the chondrogenesis by implantation of MSCs/scaffolds constructs in joint cavity. Then, in order to discover the mechanism of chondrogenesis in joint cavity, we studied the effect of joint synovial cells and joint fluid on the differentiation of MSCs in vitro respectively. For gene sox9 is the main transcription factor that controls the chondrocyte differentiation of mesenchymal cells, we separated and identified human fetal bone marrow mesenchymal stem cells and transfected sox9 gene into the cells and observed the effects of sox9 onchondrocyte differentiation of MSCs.Part one, separation, identification and culture of MSCs with low plating density of rapid expansion MSCs. Methods: we separate the pure sheep MSCs by means of Percoll gradient centrifugation combinated with static adhesive assay. The separated cells have been demonstrated to be MSCs for most cells showed positive expression of CD44 and CD 166, negative expression of CD34 and CD45 by immunohistochemistry. Results: during the 12 days, after plating the cells at 10 to 1000 cells/cm2,the cultures underwent a time-dependent transition from early thin spindle shaped to wider spindle shaped cells.During the experiental time, the maximal width and area of the cells changed, achieving the peak rate in 7 days then decreasing at density of 10 cells/cm2 and 50 cells/cm2, achieving the peak rate at 5 day then decreasing at density of 100 cells/cm2 and 1000 cells/cm2.It may deduce that the small MSCs at low plating density could be expanded rapidly because the MSCs can keep small spindle shape at density of 10cells/cm2 and 50cells/cm2 for longer time than those at 100cells/cm2 and 1000cells/cm2.For colony forming efficiency and proliferation rate, it is significantly different between low density (10cells/cm2 ,50cells/cm2) and high density (100 cells /cm2 ,1000cells /cm2). The cells of passage 2 plated at densities of 10 cells/cm2 expanded approximately 650-fold in 1...