Basic Research of Human Mesenchymal Stem Cells was Differentiated to the Chondrocytes

Background:Cartilage defect is a tough problem in orthopaedics. There are many troubles in the repairs of the defects by using Micro-fractures, periosteal transplanted, Chondrocytestransplantation. In 1980s,the repairing have entered the era of tissue engineering. Growth factors, seed cells and scaffolds are three elements of tissue engineering. Today, the problem of seed cells is a bottleneck in this field. Recently, the bone marrow stromal cells(BMSCs) have caught scientists' eyes because of its plasticity and easy harvesting. But it is not clear about its isolation, purification, identification and conditions of multi-inducing. Moreover, we can't obtain enough cells for tissue engineering because of the aging of cells in vitro culture. With the developmentof tissue engineering, people are looking forward to obtaining BMSCs easily as standard cells in basic research and a cells bank in clinical tissue engineering. To satisfy the needs, the best way is to establish a cell line.Objectives:Exploring bone marrow mesenchymal stem cell culture conditions. Observe the capacity that the mesenchymal stem cell differentiated to cartilage for basic and clinical of orthopaedics useMethods:Firstly, the study explored the best condition to culture BMSCs, the materials are Hyclone fetal Bovine serum (FBS), bone marrows from five patients in operation with informed consents. Secondly, after obtaining the optimization way to culture BMSCs, we cultured the primary BMSCs. The bone was barrow obtained 5 volunteers, after digesting the primary cells, We identified the clone cells with flow cytometry. According to the needs of clinics, the study induced BMSCs into chondrocyte The conditional culture mediums,for inducing chondrocyte:TGF-β1, dexamethasone, vitamin C, the final concentration were 5ug/L, 10-8mmol/L,l0mmol/L; The BMSCs cells were cultured in the three conditional mediums. At 6, 12, 18, 24d, these cells were tested. The chondrocyte were stained with HE to observe the cells morphology, with immunocytochemical stain to observe the expression of collogen II .The study used the method In situ hybridization to observe the mRNAs expression of collogen II.Results:We identified the clone cells with flow cytometry, and found the cells expressed adhesion molecule CD29, CD71, CD 106, and, CD34, CD45were not detected. The result indicated the clone cells were not hematopoiesis cells. Indicated BMSCs have the character of plasticity and could be induced into chondrocytes. We can draw the conclusion that the BMSCs-2 were bone marrow stromal stem cells.Conclusions:Human bone marrow stromal stem cell's optimized culture condition: 10% FBS, L-DMEM medium, first exchange medium at 5-6 days, the isolation method of density gradient centrifugation is better than the full bone marrow in obtaining purified cells, the later is facilitating to the growth of BMSCs. We can obtain BMSCs by using limiting dilution assay. The cells have normal cell biological behaviors and characteristics of adult stem cells which can be used in tissue engineering field. MSC cell line had been applied in many laboratories. MSC are standard cells for laboratory study and cell bank for clinical tissue engineering.