| The regeneration and function recovery of peripheral nerve is one of the hottest issues in the field of neuroscience. Because of the characteristics of peripheral nerve anatomically and functionally, anastomosis can not be achieved ideally in most cases. It is necessary to search some kinds of nerve substitute for the nerve repair. Over the past ten years, with the development of material science and molecular biology, more and more kinds of materials have come out. At present, the emphases of nerve repair research shift from nerve repair technology to nerve regeneration. There are four phrases in the repair of peripheral nerve defects: autologous nerve graft, homogeneous nerve graft, autologous non-nerve graft, and biomaterial substitute.Though autologous nerve graft is the golden standard in clinic for the peripheral nerve defects repair, this method can give rise to sense dysfunction in donor and misdirection regeneration of nerve fiber. Scientists have place focus on applying artificial nerve substitute to promote peripheral nerve regeneration, and to achieve the perfect objective of nerve regeneration quickly and function recovery completely. The peripheral nerve tissue engineering technology brings new hope to resolve the difficulties in peripheral neuroscience. The key point of tissue engineering nerve is to implant schwann cells cultured in vitro into a kind of tube which has a three-dimensional structure, bio- absorbabilty and half-penetrability, then to repair the peripheral nerve defects. It involves the culture of schwann cells in vitro, the construction of artificial nerve interior scaffold, and the processing of nerve conduit.In this experiment, tissue engineering nerve was constructed to repair peripheral nerve defects by applying the method of tissue engineering and exogenous growth factor. We have studied the culture of schwann cells in vitro, the complex of cells and biomaterials and the repair of sciatic nerve defects, and established a method to construct tissue engineering peripheral nerve. It is expectedfor a alternative method for repair peripheral nerve defects. There are three parts in this experiment as follows:1. Culture schwann cells(SCs) in vitro: the aim is to optimize culture condition of schwann cells(SCs) in vitro and obtain more purified SCs to repair peripheral nerve. Methods: five to seven-day-old SD rat's sciatic nerve were digested by trypsin and collagenase by many times. Cytosine arabinoside and geneticin were used to eliminate fibroblast, and forskolin can promote the proliferation of SCs. 0.125% typsin was used to purify SCs in the course of passage. Results large amount of SCs were harvested by this method, then these cells were identified with rabbit anti-S100 protein antibody by SABC immunohistochemistry(IHC) methods. The percentage of SCs purified was over 97%. Conclusion The method used in this paper is an optimal means to culture and purify SCs which can meet the needs of nerve repair by tissue engineering method.2. Study biological effect of TGF-β and rhBMP2 on the schwann cells in vitro: cultured SC was treated with 50ng/ml TGF-β and 50ng/ml rhBMP2 .SC proliferation activity was assessed by MTT and flow cytometry(FCM) mothed, and NGF synthesis was detected by ELISA mothed. Result TGF- and rhBMP2 can promote proliferation and NGF synthesis of SC significantly, and TGF-β is better than rhBMP23. Construct tissue engineering peripheral nerve: the aim is to establish a protocol of constructing tissue engineering peripheral nerve. The method is to mix the schwann cells which marked by BrdU, bovine acellular matrix (BAM), fetal bovine serum and DMEM on the special ratio and inject them into the PLGA tube, the compound change to the gel when incubated under 37℃ 5% CO2 for 30 min. After 24 hours, it was implanted the gluteal muscle of SD rat. After three weeks, the implanted Schwann cells were still survival by anti-BrdU staining. It's a perfect method to construct tissue engineering peripheral nerve on the basis of the thr... |
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