| Objective To explore transplanting autologous bone mesenchymal stem cells(BMSCs)induced by transforming growth factorβ1 composite with "two-phase" bone matrix gelatin(BMG) into rabbit intervertebral disc which has been removed by operation.To observe whether the BMSCs can differentiate into disc cells,and to observe its feasibility and efficiency of repairing disc.Method 1.48 experimental Japan rabbits were randomly divided into control group(n=24)and experimental group(n=24).By technique of cell culture in vitro, BMSCs were isolated from ilium in 24 experimental group rabbits,adding culture medium,mixing,centrifugaling,bloting upper fat and supematant,then cultivated them in culture bottle to culture primary cells.We gradually removed non-adherent cells by exchanging liquid,then we acquired purified BMSCs with the adhered property.2.We observed morphology of BMSCs,made the growth curve,in order to identify the BMSCs,we used immunohistochemical analysis after purification, passage,amplification,culture of BMSCs.3.When BMSCs were passaged to the third generation,in order to obtain seed cells that is cartilage precursor cells for tissue engineering,we added TGF-β1 and vitamin C on the forth days after culturing in the third generation.4.Acquiring the rabbits'iliums from experimental group at the same time, acquiring rabbits'iliums from control group,according the method of Urist,we modified and acquired the 'two-phase' BMG through degreasing,taking off the calium and getting out the protein,at last,the 'two-phase' BMG were cut into certain sizes and shapes which were needed.They were conserved in low temperature after sterilizing.5.Vaccinated induced autologous BMSCs from experimental group in the same autologous BMG.Set up a cell-carrier composite,observe it through the electric-microscope after culturing three days.6.Made the rabbit model of completely removing intervertebral disc,the experimental group were implanted the autologous cell-carrier composite.The control group were implanted autologous BMG only.The specimens were obtained in the eighth week and the twelfth week respectively for observing roughly.Then using HE staining,carbazole method,RT-PCR to detect proteoglycan,and using immunohistochemistry,RT-PCR to detect collagen II.At last,the data was analyzed with SPSS 11.5 statistical software.Results 1.BMSCs could be isolated,cultured and proliferated by marrow gentrifugalism and can adherence to plastic flask culture.The results of immunohistochemistry demonstrated that in vitro expanded BMSCs expressed mesenchymal cells'marker's CD44 not CD34.2.BMSCs can transformed to chondrocyte with the induction of TGF-β1 and VitaminC.We got a lot of cartilage precursor cells which were identified by immunohistochemical staining and toluidine blue positive.3.Through the observations by electron microscope.We can see rough surface,multiporosity,100um～800um size of pore in BMG,cells-carrier composite, and cells grow well in the surface and pore wall of BMG4.Intervertebral discs of experimental rabbits were repaired by cartilage endplate and fibrocartilage.,the disc's repair of control group were not as obvious as the experimental group.The content of proteoglycan and collagen II of cartilage endplate and fibrocartilage in experimental group were higher than control group significantly in both eight week and twelve week(P<0.05).There were significance in statistics.Conclusion BMSCs can differentiate into interverbral disc cells,proliferate,and repair disc's cartilage endplate and fibrocartilage.
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