Study On Differentiation Of Mesenchymal Stem Cells Derived From Rabbit Bone Marrow In Vitro

Objective To investigate characteristics of mesenchymal stem cells(MSCs) derived from rabbit bone marrow and valurate the feasibility of constructing tissue- engineering cell.Methods 1. By Percoll density gradient centrifuge and monolaryer culture in vitro MSCs were isolated from bone marrow blood and were purified. Furthermore, cytohistochemical characteristics of MSCs were determined.2. By Percoll density gradient centrifuge and monolaryer culture in vitro MSCs were isolated from rabbit bone marrow and were purified. Some MSCs were seeded into centrifuge tube culture and allowed to form three-dimensional aggregates in a chemically defined medium including transforming growth factor- 1(TGF-β,), dexamethasone, ascorbic acid, insulin, selenium and transferrin ,At same time, some MSCs were induced by monolaryer culture in same medium.3. By Percoll density gradient centrifuge and monolaryer culture in vitro MSCs were isolated from rabbit bone marrow and were purified. After that, some MSCs were induced into adipotic cell in adipogenesis supplement(AS) medium consisting of dexamethasone (10~8M) and insulin(10n g/ml), others were induced into osteoblast cell in the osteogenesis supplement(OS)medium consisting of dexamethasone (1(T8M) , VitaminC(50ii g/ml) and P -glycerophosphate(10mmol/L). Then, Specific markers of differentiation of both adipocytic and osteogenic cells were detected.Results 1. Isolated MSCs were homogeneous and their maintainal generation in LG-DMEM with 10%FBS have nearly same cell growth pattern. MSCs were positive for collegen I, glycogen periodic acid Schiff reaction (PAS), and were negative for acid phosphatase (ACP), alkaline phosphatase (ALP) and collegen II.2. After 7-21 days, MSCs in culture including TGF-p\, dexamethasone, ascorbic acid, insulin, selenium and transferrin progressed from fibroblastic-appearing cells to round —shaped cells. Their transformation toward chondrocytic phenotype were verified by a shift in express from collagen type I to collagen type II, and chondrogeness were evidenced by the positive appearance of toluiding blue merachromasia and the immunohistochemistry detection of type II collagen and in situhybridization detection of collagen type II mRNA.3.In adipogenesis culture, approximately 75% and 10% of MSCs appeared to be adipocytes and osteogenetic cells at day 21, respectively; while 10% and 10% in non-treated control group, respectively. In osteogenesis culture, approximately 20% and 40% of MSCs appeared to be adipocytes and osteogenetic cells at day 21, respectly; while 5% and 5% in non-treated controls, respectively.Conclusion MSCs have unique phenotype and growth patterns, and have the capacity to undergo chondrocytic differentiation, adipocytic differentiation and osteogenetic differentiation. Besides, these results also demonstrate an inverse relationship between the differentiation of adipocytes and the differentiation of osteogenic cells in this culture system and the regulation of adipogenesis and osteogenesis can occur at the level of a common precursor in vivo. In near future , MSCs may be used as functional cells in constructing tissue engineering.