| It has been a difficult clinical problem to repair the nerve defects after the peripheral nerve injury. Suitable growth environment and pulpation sheath cells are essential for axon regeneration. It have been widely concerned for substrates and intervention methods that can induce nerve regeneration.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. The approaches for the reconstruction of the peripheral nerve defects include end-to-end suturing, fascicular suturing, nerve grafts and so on. 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.Schwann cell which derided from neural crest of early embryo is the mainly glial cell in the peripheral neural system and promotes nerve regeneration and recovery of function. It is seen as the candidate cell in the cell transplantation. While it is difficult to preserve the autoallergic Scs proliferate massively and steady characters, The neural crest cell is located in the neuroectoderm of early embryo and migrates from the neural tube to its destinations. It is regarded as the cell with the most differentiation potential for differentiating into neuron, glial and diverse noneural cells. The trunk neural crest stem cell can differentiate into neuron and glial which contribute to PNS. So it is considered as the primordium of PNS. The Neural crest stem cells are derived from neural tube 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.Chitosan is the chitin de-acetyl production whose suface have some positive charge in the natural world.It is the giant molecule polyoses which can degradative.Chitosan have good biocompatibility,plastically,bactericidal and cellular affinity.Its suface have some identity signals to the cells.Chitosan is widely used in the tissue engineering because it is benefit for the neural cell. The experiment proved that Chitosan is a kind of high molecular compound produced from marine life with porosity,biodegradability and excellent biocompatibility.We design this experiment:We isolate and culture the trunk neural crest stem cell using tissue culture of neural tube in the E10.5 Wistar rat embryo in defined medium, then observing its'biological properties. The trunk neural crest stem cells using tissure culture of neural tube in vitro were cultured on the chitosan fiber.Used immunofluoresecence to indentify the trunk neural crest cells.Observed the histocompatibility of them..the trunk neural stem cells were cultured with the chitosan-slilca sebific duct scaffold, the growth of cells was observed by scanning electron microscope.10mm sciatic nerve defects were bridged with chitosan-slilca sebific duct scaffold cultured with trunk neural stem cells, sebific gel tubes without trunk neural 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 chitosan-slilca sebific duct scaffold cultured with trunk neural stem cells for repairing a 10-mm defect in the rat sciatic and to provide the experimental evidence for repairing the damaged nerves.Results:Using tissure culture of neural tube, we can isolate and culture the trunk neural crest stem cells.The immuocytochemistry resulte showed that the cell were nestin positive and P75 positive.The trunk neural crest stem can grow adhered on the chitosan fiber after inoculation. Scanning elector microscope, the trunk neural crest stem cell were spindle.proliferated and migrated along fiber.The cell were arranged side by side or linked by head and tail of processes. The end of cells which S-100 positive were flat and expanded,adhered on the fiber as claw-shaped pseudopodium. There is good histocompatibibity between the chitosan fiber and the trunk neural crest stem cells, the trunk neural crest stem cell can differentiate into Schwann cell.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 analyzied:Density and average diameter of nerve fibre and area of myelin sheath in the trunk neural crest stem cells grafed group were better than the control group (P<0.01);TEM observing showed that axon formation was clearly richer in the trunk neural crest 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.To conclusion, There is good histocompatibibity between the chitosan fiber and the trunk neural crest stem cells, the trunk neural crest stem cell can differentiate into Schwann cell. They contributed to the promotion of axonal regeneration.
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