An Experimental Study In Isolation, Culture And Chondrogenic Phenotype Differentiation Of Mesenchymal Stem Cells (MSCs) From The Bone Marrow Of Rats In Vitro

Objective: To explore a method of isolation, culture and chondrocytic phenotype differentiation of mesenchymal stem cells(MSCs) from the bone marrow of rats in vitro and to observe the biological characteristics of MSCs histomorphologically in order to offer experimental reference for the resources of seeding cells in cartilage tissue engineering.Methods: The bone marrow was aspirated from the bones of limbs of the rats and was isolated by gradient centrifugation in Percoll. The monouclear cells were collected and cultured in DMEM-LG with 15% fatal bovine serum(FBS). Cultures were maintained at 37 in humidified atmosphere chamber containing 5% C02 for10-14d. The higher purity of MSCs were obtained by the repeated removal of nonadherent cells. The passage cells were induced in DMEM-HG with 15% FBS, 10-7 mol/L dexamethasone, 50ug/ml VitC. The changes of morphology, growth and proliferation in vitro and expression of specific chondrogenic matrix after inducing were observed.Results: (1) MSCs, separated from the bone marrow of rats, were collected in higher purity by gradient centrifugation in Percoll and still kept the cells' activity. (2) Primary MSCs proliferated in visible symmetric colonies with long-spindle shape. The morphological characteristics of marrow-derived MSCs had no change during passage, and its homogeneity rose with passage. (3) The cells had negative staining of ALP during the primary and passage culture. Induced cells changed from a spindle-like fibroblastic appearance to a polygonal shape and showed strong heterochromatic to toluidine blue and positivestaining of collagen II.Conclusion: (1) It is a simple, effective and practical method to separate and obtain higher purity and activity of MSCs from the bone marrow of rats by gradient centrifugation in Percoll and fibronectin ashesion. (2) MSCs that are in low abundance can grow quickly when culturedin vitro. (3) MSCs from the bone marrow of rats can differentiate to be chondrogenic phenotype when cultured in a defined medium. MSCs can likely be served as optimal autogenous cell source for cartilage tissue engineering.