Adult Msc Induced Differentiation Of Bone And Cartilage Cells And The Differences Between The Fetal Msc Expressed Genes

Repairment of large bone defect and nonunion of fracture caused by all kinds of reasons are still an important problem waiting to be resolved so far. Tissue engineering provide a new method to resolve the above problem.The first problem that tissun engineering will resolve is to choose easily manufactured seed cells. Mesenchymal stem cell (MSC) are a population of pluripotent cells within the bone marrow microenvironment defined by their ability to differentiate into cells of osteogenic, chondrogenic, tendonogenic, adipogenic, and myogenic lineages. MSCs always remain the pluripotential during the long-term culture in vitro, they are perfect seed cells of tissue engineering. Because a very low abundance of MSCs is present in bone marrow (approximately one Per 1 O~- 106 nucleated celled), ex vivo expansion produces a significant increase in the number of MSCs that can be very important. The goals of the current study were to establish the techniques for the purification and culture-expansion of these adult human bone marrow-derived MSCs and develop a reproducible system for the vitro osteogenic and chondrogenic differentiation of adult human MSCs. Normal adult human bone marrow obtained from ribs, a small percentage of mesenchymal stem cells (MSCs) were isolated by using the density interface of 1 .O73gIml percoll. Adult 7 human MSCs were allowed to attach to the surface of the plates for three days, at which time the culture medium was replaced with fresh medium. The culture adherent hMSCs have been allowed to mitotically expand with complete medium changes occurring twice weekly. When adult human MSC in primary cultures expand to form colonies of several hundred cells that collectively cover 80% of the culture plate, the cells were passaged at 1:3 dilution after being detached from the culture substratum with trypsin. Then they were purified as well as expanded in Mesencult liquid culture system. In the osteoblast-inducible system, MSCs derived from variant passage were cultured in base media containing 1 0~ molIL dexamethasen, lOng/mi Na-~3-glycerophosphate (f3-GP) and 50 Li g /ml ascorbic acid, chondrogenesis was induced by culturing hMSC in micromass pellets in the presence of defined medium that include lOng/mi TGF-133. Optimal osteogenic and chondrogenic differentiation, as determined by osteoblastic and chondroblastic morphology, cytochemistiy and immunohistochemistry were used to detect the calcium deposition, alkaline phosphatase activity, cartilage cells and type I, II collagen. In our experiment, the adult hMSCs were expanded 15 passages and yielded 7.5 X 1012 MSCs. Some polygonal cells could be observed at about the 7~ day in culture system for inducing osteoblast differentiation and they were dyed AKP positve. By 21 days of induction, calcium deposition and type I collage can be observed. At the same time, 1 Onglml TGF43 in the chemically defined medium induced chondrogenic differentiation was evidenced by the appearance of cartilage cells and the immunohistochemical detection of type II collagen. Culture of adult human MSCs provide a useful model for evaluating the multiple factors responsible for step-wise progression of cells from undifferentiated precursors to secretory osteoblasts and chondroblasts, and eventually terminally differentiated osteocytes and cartilage cells, the different passages MSCs still remain the potential of osteogenic and chondrogenic differentiation.