Study Of Exvivo Culture And Chondrogenesis Of Rabbit Mesenchymal Stem Cells

Objective: To study proliferation of mesenchymal stem cells(MSCs) affected by different concentration of bFGF ,and influenced by bFGF with TGF- β 1 in vitro. To evaluate chondrogenesis of MSCs affected by growth factors and time. To observe attachment of MSCs to the polymer graft surface.Method: MSCs were prepared from bone marrow aspirates obtained from the rabbit femur neck. After initial isolation and culture, observe the characterization of cells with progeneration and transfer of culture .(l)There are seven group cells of P4MSCs.Five group were cultured hi DMEM with different concentration of bFGF(0,10,50,80,100 ng/ml);other group pretreatment with TGF- β , followed by exposure of cells to bFGF, the other group treated with both bFGF and TGF- β1. The proliferation were detected by the method of MTT four days later. (2)Using semiquantitative reverse transcriptase- polymerase chain reaction (RT-PCR) to analysis chondrogenesis of MSCs of P3.P5, P7,P10, which cultured with bFGF and TGF- β1(both are 20ng/ml) four days. Culturing cells were stained with toluidine blue. The expression of type II collagen is detected by RT-PCR, indicate their chondrogenic potential. The expression dose of type II collagen of PS MSCs, which induced with bFGF is detected by RT-PCR, and the same way to chondrocytes. (3)P4 MSCs culturing with polymer were observed by scan electron microscopy after one month.Results: The proliferation of MSCs increased with high concentration of bFGF, l00ng/ml to the top(P<0.05).There are more cells proliferlation of MSCs sequentially exposed to TGF- β 1(10ng/ml) and bFGF (10ng/ml)than only with 80ng/ml bFGF(P<0.05). Toluidine blue stain has different colors. Type II collagen indicating chondrogenesis of MSCs was detected by RT-PCR. The gray level of type II collagenof cells with TGF- β1i and bFGF is larger than that only by bFGF. In the culture period ,the gray level of type II collagen expression increased with the time. P7 MSCs has the highest gray level(P<0.01). Scanning electron microscopy revealed layer of cells within the cell-polymer constructs. Around the cells or in polymer holes has some extracellular matrix.Conclusion: The concentration of growth factor is responsible to the proliferation of MSCs. Different factors has interaction each other. Using two factors can amplify the effects. Chondrogenesis vary with the time of culturing. There has a top of chondrogenesis of MSCs with time in vitro. MSCs can be well attached to polymer graft.