Study Of Using Chitosan Conduit Containing With Nerve Fragments Or Schwann Cells To Repair Rat's Peripheral Nerve Defect

Objective To investigate an effective technique for isolation, cultivation and purification of human Schwann cells of normal peripheral nerves cultured in vitro. To study the effect of using nerve conduits containing with nerve fragments or Schwann cells to repair rat's peripheral nerve defect, comparing with the effect of using solo-nerve conduits and nerve autochthonous graft and to provide theory foundation and experiment experience for further study on repair and regeneration of peripheral nerve defect.Method Part 1 of the experiment, normal peripheral nerves were obtained from sciatic nerves of 10 healthy female Wistar rats. Schwann cells were cultured with enzymatic digestion culture method and differential attachment method. The purity of Schwann cells was identified through S-100 protein immunohistochemistry staining, counted with the light microscope and the growth curve of Schwann cells was got. Then the nerve conduits and Schwann cells were cultured together.Based on the Part 1 of the experiment,32 Wistar rats with 10mm sciatic nerve defect of the right lower extremity were divided into four groups:the nerve defects of group A were repaired with autochthonous graft, group B with nerve conduits containing of Schwann cells, group C with nerve conduits containing of nerve fragments and group D with nerve conduits only. After 1 week,3 weeks,7 weeks,10 weeks and 13 weeks, morphology and functional rehabilitation were observed. And after 13 weeks, electrophysiology tests of rats were performed, including nerve conductive velocity (NCV) and nerve-muscle compound action potential amplitude (AMP). The samples of regeneration nerves were stained by toluidin blue and the density and area of myelinization axons were analyzed by computing image analysis system of image-pro plus 5.0 semi-qualitively.. Data were packed up by SPSS 14.0 statistical software with p<0.05 considered significant.Result More than 0.5×108/L Schwann cells were detected after four days under a microscope. Following the third passage, the number of Schwann cells was over 9-11×105/L. The purity of Schwann cell population was up to 85%. The right lower extremity of rats in group B and group C recovered well. The results of walking track analysis, electrophysiology tests and histology test of regeneration nerves on rats in group B and group C were significantly better than those in group D (P< 0.05), and similar with those in group A (P> 0.05). The results between group B and group C had no significant difference (P>0.05).Conclusion Results suggested that plenty and purified Schwann cells could be obtained by enzymatic digestion culture and differential attachment methods in a short time, which can be used for the source of peripheral nerve tissue engineering. The effect of using nerve conduits containing nerve fragments or Schwann cells were better than using nerve conduits only, especially for neurite regeneration and functional rehabilitation. In addition, nerve conduits containing nerve fragments or Schwann cells may have less immune reject reaction than nerve conduits only, although the effect may not be as well as autochthonous graft.