| Objective:Establish the rabbit disc degeneration model by the means of Nucleuspulposus aspiration. The disc imaging, histological and extracellular matrixexpression changes after the bone marrow mesenchymal stem cellstransplantation were observed at different times. To evaluate the effect ofbone marrow mesenchymal stem cells transplantation in the rabbitintervertebral disc degeneration.Method:(1) Construction of rabbit model of intervertebral disc degenerationEighteen healthy New Zealand white rabbits of either gender, aged sixmonths year and weighted approximately2.5kg, were divided into thenucleus pulposus aspiration group and the control group at random. Eachgroup had9rabbits. Using aseptic techniques, the spine was exposed to ananterior midline transperitoneal approach. The L3-4, L4-5, L5-6lumbarintervertelral discs were punctured by21-gauge needle and aspirated outabout5mg nucleus pulposus tissues. The control group just isolated theexposed intervertebral discs without any further processing. At4,8, and12weeks after the operation, three rabbits of each group were checked bymagnetic resonance imaging. The disc hight and changes in signal intensityon T2-weighted images were measured respectively. The histomorphology ofthe intervertebral disc was observed via gross specimen. (2) In-vitro isolation and culturation of bone marrow mesenchymal stemcells of rabbitRabbit bone marrow was collected by aspiration from the femur and tibiaunder sterile conditions. The bone marrow mesenchymal stem cells wereisolated and purified by the whole bone marrow culture method andadherence method. The cell morphology, anchorage-dependent rate and theproliferation ability in vitro were observed respectively.(3) Bone marrow mesenchymal stem cells transplantation treatment ofrabbit intervertebral disc degeneration36healthy New Zealand white rabbits, regardless of their gender, agedsix months year and weighing approximately2.5kg, were divided into thenormal control group, the cell transplantation group, and the intervertebraldisc degeneration group randomly (n=12). The L3-4, L4-5, and L5-6intervertebral discs were exposed through the median incision approach ofperitoneum. The normal control group was not given any treatment. Theintervertebral disc degeneration group just aspirated the nucleus pulposus. Forthe cell transplantation group, The L3-4, L4-5, and L5-6discs were injectedwith50μl of bone marrow mesenchymal stem cells (2.0×105cells/ml).Magnetic resonance imaging scans (MRI) on the3groups of rabbits wereperformed at4,8, and12weeks respectively after surgery, to measure theheight of the intervertebral disc and the change of signal intensity inT2-weighted images. HE dyeing and safranine and fast green dyeing wereconducted to observe the histomorphology of the intervertebral disc. Theexpression of extracellular matrix protein of nucleus pulposus were detectedby the collagen II immune histochemistry dyeing and Aggrecanimmunofluorescent staining. And animals were analyzed by real-time fluorescence quantitative polymerase chain reaction to measure the expressionlevel of mRNA in the two types of genes (Aggrecan and Collagen II) ofextracellular matrix in the nucleus pulposus.Result:(1) Observation of animal model: MRI on the rabbit lumbar spine revealsthat the height of the intervertebral disc is stable and T2-weighted imagesalways exhibits high signal intensity in the control group. In the nucleuspulposus aspiration group, the stabbed discs exhibit a progressive decrease ofsignal intensity in T2-weighted images; the intervertebral disc is darkened andthe height of the disc is decreased progressively at4weeks after surgery. Theheight is nearly lost at8weeks after surgery. The difference in the disc heightand the signal intensity in T2-weighted images between the two groupsthrough comparison has statistical significance (p<0.05) in each observationtime point. The anatomical morphology of isolated specimen reveals that theboundary between the annulus fibrosus and the nucleus pulposus is distinctand the jelly-like nucleus pulposus suffuses the whole intervertebral space inthe control group. In the nucleus pulposus aspiration group, the morphologyof the annulus fibrosus is basically complete, the nucleus pulposus isdwindled in size, and the disc height is decreased at4weeks after surgery.The nucleus pulposus is degenerated and shrunk and the annulus fibrosus isderanged or even broken at8and12weeks.(2) In-vitro isolation and culturation of bone marrow mesenchymal stemcells of rabbit: Adherence begins after culturation in vitro for24h. Celladherence is almost completed after5d. The cell fusion rate reaches morethan90%after15d around. Cells proliferate quickly and sufficient number of seed cells can be obtained within a short period of time. The cells are notdegenerated or differentiated after cell passage for12times.(3) Bone marrow mesenchymal stem cells transplantation treatment ofrabbit intervertebral disc degeneration: MRI reveals that the intervertebraldisc exhibits a decrease (to different degrees) in the disc height and the signalintensity in T2-weighted images (p<0.05) at4,8, and12weeks in theintervertebral disc degeneration group, compared with those in the normalcontrol group. They also decline over time. However, the aforesaid indicesturn out to rise (p<0.05) at4weeks in the cell transplantation group,compared with the intervertebral disc degeneration group. At8and12weeks,there is no obvious difference in the disc height and the signal intensity inT2-weighted images between the cell transplantation group and the normalcontrol group. The result of HE dyeing and safranine and fast green dyeingindicates that the intervertebral disc is deranged and the boundary between theperipheral annulus fibrosus and the nucleus pulposus is indistinct over time inthe intervertebral disc degeneration group. But in the cell transplantationgroup, the boundary of the histological structure is distinct and the nucleusand cytoplasm are stained obviously at4,8, and12weeks. Collagen IIimmune histochemistry dyeing reveals that the number of stained cells andthe secretion level of extracellular matrix are decreased progressively in theintervertebral disc degeneration group. The degeneration level of theintervertebral disc in the cell transplantation group is lower than that in thenormal control group. There is no obvious difference between the two groups(p>0.05) at4,8, and12weeks. Aggrecan immunofluorescent stainingindicates that the nucleus pulposus exhibits red or positive reaction and thewhole intervertebral disc is distinct in structure in the normal control group. In the cell transplantation group, the central nucleus pulposus exhibitspositive reaction at each time point, obvious red fluorescent can be seen in theintercellular substance, and the general structure remains complete. In theintervertebral disc degeneration group, the fluorescence intensity of thenucleus pulposus is weakened at4weeks and greatly reduced or evendistained and the morphology of the nucleus pulposus is indistinct at8and12weeks. Real-time fluorescence quantitative polymerase chain reaction revealsthat the expression level of mRNA in the two types of genes (Aggrecan andCollagen II) is decreased obviously at4,8, and12weeks in the intervertebraldisc degeneration group, different from that in the normal control group(p<0.05). In the cell transplantation group, the aforesaid index is decreasedtemporarily at4weeks but shortly it is found to approach the expression levelof mRNA in the two types of genes in the normal control group and there isno obvious difference between the two groups (p>0.05).Conclusion:1. Nucleus pulposus aspiration method can induce rabbit intervertebraldisc degeneration. Change of intervertebral disc in imageology in early stageof degeneration complies with Thompson Grade III. Such method facilitatesthe construction of model and achieves good repeatability.2. Whole bone marrow culture method and adherence method cansimplify the isolation process of bone marrow mesenchymal stem cells,strengthen the proliferation capability of cells cultured in vitro, and maintainthe stable consecutive passage phenotype. The bone marrow mesenchymalstem cell is a good source of seed cell for transplantation.3. The bone marrow mesenchymal stem cells for transplantation cansurvive and proliferate. The results suggest that BMSCs transplantation is effective in decelerating disc degeneration and repairing the degenerativedintervertebral discs. |
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