| Peripheral nerve injuries is common and results in incomplete or no functional recovery, particularly after a complete transection .The regeneration in the peripheral nervous system is incomplete and the treatment of severe lesions with nerve tissue loss is primarily aimed at recreating nerve continuity. Therapeutic approaches for the reconstruction of the peripheral nerve defects include end-to-end suturing, fascicular suturing, nerve grafts and so on. In some cases, however, the distance between the proximal and distal stumps is too large to allow direct approximation of transected nerves. Also,treatment of the nerve defect with autograft has inevitable disadvantages,such as limited supply of available nerve grafts and permanent loss of donor nerve function.The development of tissue engineering has provided a new strategy for the reparation of damaged nerves , the scaffold and seed cells are critical for the construction of tissue engineering nerve. Chitosan is a kind of high molecular compound produced from marine life with porosity, biodegradability and excellent biocompatibility. As the main composition of extracellular matrix, collagen protein determines cell migration, proliferation, and differentiation. Moerover collagen protein plays an important role in the process of axons regrowth and migration of Schwann cell. So the complex collagen-chitosan scaffolds we produced not only have excellent biocompatibility but also benefit the regeneration of axon.Residing in the peripheral nervous system and responsible for myelination , Schwann cells play a key role in promoting axonal regeneration. Athough they are the ideal seed cells in nerve regeneration, some difficulties are found in clinical application. First, it needs two times of surgery for autograft of SCs;Second,allograft of SCs may cause the immunological rejection in the host. Thus it is necessary to search other cells for nerve tissue engineering. Neural stem cells (NSCs) demonstrate the ability to self-renew, and they are capable of differentiating into neurons, astrocytes and oligodendrocytes. It was reported that neural stem cells could promote the nerve regeneration by differenting to specific neural cells or the Scs like cells. Thus the NSC may have a great potential in treating the damaged nerves.The neuroepithelial stem cells are derived from neural thbe and neual plate,which demonstrate vigorous ability to differentiate into neurons and glial cells. Thus these cells were employed as the seed cells in our experiment.Bsaed on the above, we design this experiment : complex collagen-chitosan scaffolds were made from chitosan and collagen protein by the method of freeze drying in proportion of 1 to 3. The neuroepithelial stem cells were isolated from the neural tube of Wistar rats of embryonic day 11.5(E11.5d day of gestation), cultivated in serum-free medium. To explore the biocompatibility of collagen-chitosan scaffold, the neuroepithelial stem cells were cultured with the collagen-chitosan scaffold, the growth of cells was observed by scanning electron microscope. 10mm sciatic nerve defects were bridged with collagen-chitosan conduits cultured with neuroepithelial stem cells, tubes without neuroepithelial stem cells served as controls. After grafting, the regenerated nerves were evaluated by electrophysiological examination, histological staining , retrograde tracing and electron microscope observe. Our purpose is to examine the effectiveness of collagen-chitosan conduits cultured with neuroepithelial stem cells for repairing a 10-mm defect in the rat sciatic and to provide the experimental evidence for repairing the damaged nerves.Results:The neruoepithelial stem cells grew suspended and form the neurospheres by division growth in the serum-free medium. With persistent culture, the amount of the cells in the neurosphere was increased rapidly and hundreds of cells constructed a sphere in the 5-6 days. The spheres were positive for anti-nestin antibody,thus the cells we cultured were NSCs ;A large number of neruoepithelial stem cells attached onto the surface of collagen-chitosan scaffold, and some could stretch out evections, so there was a good compatibility between collagen-chitosan scaffold and neruoepithelial stem cells; At 8 weeks after surgery,weak action potentials were found in the experiment group only. At 14 weeks after surgery,the conduction velocity and wave amplitude of experiment group was better than the control group,and the difference was evident (P<0.01) ; The histological features of regenerated nerves were observed by the toluidine blue stain and image anaiyzied : Density and average diameter of nerve fibre and area of myelin sheath in the neruoepithelial stem cells grafed group were better than the control group (P<0.01) ;TEM observing showed that axon formation was clearly richer in the neruoepithelial stem cells grafed group than the control group;Lumafluor labelled motoneuron cell bodies were found in the anterior horn of grey matter ipsilateral to the operated side in both groups,showing that nervous pathway had re-established;Also, cells positive for both anti-BrdU antibody and anti-S100 were observed in the regenerated tissue, and part of the grafted neural stem cells were shown to have differentiated into Schwann cells.To conclusion, The collage-chitosan conduits containing neuroepithelial stem cells have excellent biocompatibility, when transplanted to a site of peripheral nerve defect, neuroepithelial stem cells derived from neural tube could differentiate into Schwann cells and they contributed to the promotion of axonal regeneration. |
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