To Study Chondrogenic Differentiation Of Rabbit Bone Marrow-derived Mesenchymal Stem Cells By Two Distinct Separation Methods

ObjectiveThe purpose of the present study was to contract the results of chondrogenic differentiation of rabbit bone marrow - dirived mesenchymal stem cells ( BM-SCs) by two distinct separation methods ,to set up an easier and more practical system of chondrogenic differentiation in vitro, and to provide preclinical base for the repair of articular cartilages injuries.Materials and MethodsAfter the vein anesthesia by 3% napental (1mg/kg) ,the bone marrow was obtained from the rabbit' s Greater trochanter of femur on the condition of asepsis. After aspiration and equation of rabbit bone marrow ,one share (A group) was isolated by the method of integral bone marrow and adhere culture , and another share ( B guoup) by density gradient centrifugation and adhere culture. The cells were cultured at 37℃/5% CO₂ in control medium ( LG - DMEM, 15% FBS,100units/ml of penicillin, 100mg/L of streptomycin). The cells' growth was observed in the inverted microscope and was drawn into the growth curve by the MTT method. After 3 passages, we changed to use another medium ( HG -DMEM,100units/ml of penicillin,100mg/L of streptomycin)to cuture the cells in the six - hole plate, and added the TGF - β1 (10ng/ml) in the medium. TGF - β 1 was used to induce chondrogenic differentiation of BMSCs. After 10 days of the differentiation of BMSCs, Collagen II and it' s mRNA in the cytolymph were detected by immunohistochemistry and in situ hybridization respectively.ResultObservation of Morphology of bone marrow - derived mesenchymal stem cell1) The group isolated by the method of integral bone marrow24 hours later, some cells began to adhere to the inner - surface of culture flask and the cell showed the morphous of dolicho - spindle or polygon. 48h later, some cell colonies were seen clearly, which composed of several cells or more. One week later, when the monolayer of adherent cells reached confluence, cells were trypsinized (0.25% trypsin) , resuspended in DMEM containing 15% FBS, and subcultured at a density of 1× 10⁶cells/ml.2) The group isolated by the method of density gradient centrifugation and adhere culture24 hours later, only some round cells adhered to the inner - surface, and 48hours later, the adherent cells began to show the morphous of polygon or dolicho- spindle. 72 hours later,a few of cell colonies were found in the medium. Twoweeks later, the monolayer of adherent cells reached confluence. After the futherpassages, the growth rate of cells was promoted.3 ) The differentiation of BMSCsSix days after the TGF - β1 was added into the medium, most cells appeared the morphous of polygon, especially in the region of high density of cells.BMSCs growth curveThe growth curve showed that the doubling rates of the cells in both groups were slow during 1st to 2nd day, and the cells'growth rate promoted obviously into the increased logarithmic phase during 3rd to 6th day. Afterwards, the cells growth entered the platform stage.The immunohistochemistry of Collagen IIAfter ten days in the medium in the six - pole plate, the positive rates of Collagen II immunohistochemistry of A group induced by TGF - β1 (experimental group) was 84.4 % ,and the positive rate of B experimental group was 85.2 %. The positive cells showed the dark - brown particles in the intracytoplasm, and the particles could be seen around the cells, too. We could not find the dark...