The Primary Culture Of Schwann Cells And The Research Of Peripheral Nerve Regeneration With RGD Composites Conduits

Schwann cells, a kind of glial cells in neural system, play an important role in the growth and regeneration of peripheral nerve. They can form the nerve sheath of myelin fibers and unmyelin fibers and also secret various nerve nutrition factors(NTFs) and cell adhesion molecules. Along with the development of tissue engineering, implanting nerve guide conduits(NGCs) containing with Schwann cells have been tested in pre-clinical studies to promote the regeneration of injured nerve. However, how to obtain a large amount of Schwann cells is the key of this technology. As artificial nerve guide conduits have been widely proved can enhance nerve regeneration, the study of the biomaterials which can improve cell affinity and constantly release nerve growth factors become crucial important in the research of peripheral neural tissue engineering.In this study, we developed an effective method of obtaining highly purified adult Schwann cells based on the differing rates of attachment of the various harvest cells, combining rapid trypsin digesting technique. After 7d culture, the purity of the Schwann cells obtained from predegenerated sciatic nerve could reach above 90% while the one of normal sciatic nerve was 86%, and the amount of the purified Schwann cells could reach above 10~6/ml which satisfied the clinical need in peripheral nerve repair.The successful regeneration of short nerve defect gaps of peripheral nerve by bridging with nerve guide conduits provide a new way for reparing long nerve defect gaps of peripheral nerve. In this experiment, we prepared a new type of nerve guide conduits-PRGD/PLA/TCP/NGF which have good degeneration rate and could release proteins constantly. In this type of conduits, PDLLA was chosen as the basic materials of nerve guide conduits, and were peptided with RGD, thenβ-TCP and NGF were mixed in the composites with a certain mixture ratio. During the following in vitro degeneration test, the changes of the weight and pH of PRGD/PLA/TCP/NGF film was investigated and was compared with PLA, PRGD/PLA and PRGD/PLA/TCP films. The results showed that PLA have the lowest degradation rate, the weight loss rate was only 25%, the PRGD/PLA,PRGD/PLA/TCP and PRGD/PLA/TCP/NGF film have the same degradation rate, the weight loss rate had been above 50% in 24th weeks. The pH of the supernant of the four materials appeared a declining trend during the first few days. The PRGD/PLA film had a higher decline rate of pH than PRGD/PLA/TCP and PRGD/PLA/TCP/NGF, the supernant of PRGD/PLA/TCP/NGF could reach as pH6.8. In addition, animal experiments were conducted to evaluate the biocompatibility of the RGD composites. The sciatic nerve defect model of adult rats was conducted in this experiment, then the defected nerves were bridged with PLA, PRGD/PLA/TCP and PRGD/PLA/TCP/NGF conduits respectively. After 3 months operation and 6 months operation, the rats were anaesthetized and conducted with function recovery evaluation, morphological evaluation, histological evaluation, electrophysiological assay to evaluate the recovery of the regenerated nerve. The results showed that: NGF could promote the regeneration of defected sciatic nerves effectively and the RGD composites have the good biocompatibility to enhance the regeneration of peripheral nerves.