Research On The Viability Of The MSCs Induced By The Nucleus Pulposus Cell Transplanted Into The Intervertebral Disc

Objective To study the viability of Mesenchymal stem cells promoted by co-cultured autologous nucleus pulposus cells of rabbit composite with "two-phase" bone matrix gelatin(BMG).Methods 1.DMEM/F12 was added into the bone marrow which was aspirated from the rabbit's shinbone.After blown,beaten and centrifuged,cells suspension was inoculated in the culture bottle to proceed to the primary culture,and by way of changing the culture medium gradually taking away the cells which didn't stick the wall to obtain the MSCs.2.In no aseptic conditions,taking out the entire spine from the killed rabbit which was aspirated the bone marrow before,and then taking the nucleus pulposus tissue carefully from the every intervertebral disk,and culture the nucleus pulposus cells by way of tissue culture.3.To obtain enough quantity of the MSCs and Nucleus Pulposus Cells through the passage and amplification in vitro.In the phase of culture,observing the cells changes of the morphology,and drawing their growth's curve.4.The NP cells were added into the six-well culture plate which culturing the third passage MSCs and in the meantime added the TGF-β₁ to promote the MSCs to differentiate,which should provide seed cells for tissue engineering of intervertebral disk.The effect of the induction was tested by RT-PCR technology.5.Getting out the part of the rabbit's ilium,according to the methed of modified Urist,the "two-phase" BMG was acquired through degreasing,taking off the calium and getting out the protein,and last it was cut as certain size and shape as needed.It could be conserved after sterilizing within a low temperature.6.Inoculated the MSCs induced by the Nucleus Pulposus cells into BMG to set up a composite of the cell-carrier,observed it through the electric-microscope after culturing three days.7.Removed the rabbit's nucleus pulposus tissue of the intervertebral disk by the operation,and divided its into two groups:the experimental group was implanted the composite and the experimental control group was implanted the BMG only.The specimens were observed roughly in the second,eighth and twelfth week respectively, at the same time were processed for HE staining and testting the content of the collagenâ…¡and proteoglycan.Results 1.After removing the upper adipose and supernatant by centrifuging,the bone marrow proceed to the primer culture in the F12/DMEM culture medium,with the medium changed regurally,it could be obtained large quantity of MSCs through culture and passage in vitro.2.By method of co-culture,the effect of the induced MSCs differentiate to the NP cells is well.3.Observed the BMG and the composite of the cell-carrier by the electric-microscope,the surface of the BMG was rough and had many small pores,the size of the small pores was 100μm-800μm,the cells were observed growing in the BMG well,and the cells were also growing inside the wall of the small pore.4.The graft of the experimental group could be convert to the NP tissue at the time of the eighth week and twelve week,comparatively,the experimental control group were fiber repair at the same time.By the way of collagen and sulfate-carbazole testing,the content of collagenâ…¡and proteoglycan were almost reach the normal level at the time of the twelfth week.