| Peripheral nerve injury is a common and serious disease exposed to physicalinjuries; the regeneration of injury nerve and its functional recovery are achallenge to current clinical. For short nerve gaps that there is no nerve tissue lossor possible approximation with minimal tension, we choose direct neurorrhaphyto suture the divided nerve through end-to-end or end-to-side coaptations. But forlong nerve gaps, we can't coaptate the divided nerve directly. Currently, we takeautologous nerve grafting as the gold standard treatment. But there are someinherent drawbacks limit autologous nerve grafting, such as limited availability ofdonor nerves, the need for a second surgery to obtain the donor nerve, donor sitemorbidity and secondary deformities, as well as mismatch between the injurednerve and the donor nerve, etc. So seeking promising alternatives to supplementor even substitute autologous nerve grafts is still a major challenge to peripheralnerve repair.In recent years, using tissue engineering grafts to bridge long gaps has produced promising results and gained extensive attention. With the deepeningresearch of tissue engineering, more and more high-simulation structure neuralscaffolds were reported. However, only few studies about compounds seed-cellswere reported.In this topic we choose collagen as the main material, and by freeze-dryingtechnology we successfully fabricated a collagen-chitosan scaffold which with aninterconnected porous structure and longitudinally oriented pore channels. Andwe choose bone marrow stem cells which were source wide, easy to cultivate andhave the ability to multiplex differentiation to combine with it. Finally, we use thecombined nerve scaffold to bridge a 15 mm long sciatic nerve defect in rats andevaluate its efficacy by using a combination of morphological and functionaltechniques. Specific content as follows:Part one: Culture and Differentiation of Bone marrow stem cells.[Objectives] To study the growth condition and training methods of rats bonemarrow stem cells for laying the foundation of combining with bone marrowstem cells and repairing rat peripheral nerve defects.[Methods] Taking weight around 70g SD rats to culture bone marrow stem cellsin vitro. Growth conditions and morphological characteristics of primary cells,passage cells and differentiated cells were monitored by an inverted phasecontrast microscope. And we also depict the growth curve.[Results] Cells grow in an adherent way and proliferate by the cell colony way.The 3rd generation cells are growing like a spindle, and can be induced intoneural cells and osteoblasts through simple methods.[Conclusion] The third generation of bone marrow stem cells have higher cellpurity and good stem cell activity, can be used as seed cells of tissue engineering. [Objectives] Developing a tissue engineering nerve scaffold, and observe thegrowth of bone marrow stem cells cultured in vitro on the scaffold, to provide thestage-experiment data for the transplantation of seed cells.[Methods] The scaffold was made of I-collagen and chitosan by freeze-dryingtechnology. Using scanning electronic microscope to observe there coursedirections and measure the size of the micropores and the factor of porosity. Thebone marrow stem cells were seeded on the collagen scaffolds and cultured for 3days, and then observe the growth of bone marrow stem cells cultured in thescaffold.[Results] All scaffolds were circular cylinder, the microscopic channels werearranged in parallel manners, and the pore sizes of the channels were uniform.The bone marrow stem cells were seeded in the scaffolds successfully.[Conclusion] I-collagen-chitosan scaffolds have good structure andbiocompatibility, and can be used for repairing the nerve injuries after combinedwith bone marrow stem cells.Part three: The effectiveness of combined collagen-chitosan scaffolds inbridging 15 mm long sciatic nerve defect in rats[Objectives] To investigate the effectiveness of combined collagen-chitosanscaffolds in bridging 15 mm long nerve gap in rats.[Methods] Using combination of morphological and functional techniques,including TEM, retrograde-labelling, immunohistology, electrophysiology andbehavioral tests, to investigate the effectiveness.[Results] After implantation, the nerve scaffold can maintained its microstructureintegrity for about four weeks. The results show that compound scaffold group and the pure scaffold group are close to autologous nerve grafts, compoundscaffold group little good, a large number of regenerative nerve fibers grow intothe remote. Fluorescence microscopy showed that scaffolds were almostcompletely degraded and were replaced by many regenerated nerve fibers(aftercross-linked by genipin,the scaffold spontaneous red fluorescence). TEM Results:there are many unmyelinated and myelinated nerve fibers co-exist. The newmyelin is thin but has integrity structure and well-structured lamellar. Functionresults: the compound scaffold group's motor nerve conduction velocity, latencyand amplitude are close to nerve autograft and no significant difference betweenthe two groups (p>0.05); FG labeled tracer shows that the number of positiveneurons which can be detected in the spinal anterior horn and dorsal root gangliais not far off auto-graft. The SFI results also confirm that the functional recoveryeffect of the compound nerve scaffold is close to nerve autograft, and better thanpure scaffold group.Bone marrow stem cells; Collagen; Scaffold... |
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