| Summary: | 博士 === 國立臺灣大學 === 醫學工程學研究所 === 93 === In order to develop a nerve regenerated guidance channel, we used gelatin membrane to substitute for nerve autograft. The studies were divided into three parts, including the development of materials, biocompatibility tests (in vitro) of membranes immobilized with nerve growth factor (NGF) and in vivo tests of guidance channel. At the first, we used chitoson cross-linked with gelatin to compare with gelatin-tricalcium phosphate membranes by Fourier-transform infrared spectrophotometer (FTIR) analysis, differential scanning calorimetry (DSC) measurement, mechanical test and water contact angle evaluation. The results indicated that the gelatin-tricalcium phosphate membranes cross-linking with 0.05% glutaraldehyde solution (GTG) were suitable for nerve guidance channels. The ductility, mechanical strength and hydrophilic property of GTG were all good for nerve repair. Therefore, GTG membranes were used for the later experiments.
The results of in vitro study showed that the GTG composite, whether cross-linking with NGF or not, had no cytotoxic effect to Schwann cell culture. Comparing the GTG membrane immobilized with NGF (GEN) and GTG soaked in NGF solution without carbodiimide (GN composite) as cross-linking agent, the data confirmed more attachment of Schwann cells onto GEN composite. Although GTG cross-linking with NGF did not promote Schwann cell proliferation, the techniques we used in this study provided a method to fabricate a novel biomaterial incorporation with Schwann cells and covalently immobilized NGF. Besides, GEN membrane sustained released NGF at least 60 days, and the minimum amount of NGF released from GEN reached the concentration to stimulate PC12 cell differentiation. PC 12 cells showed significant neurite outgrowth on GEN membranes which was statistically higher than GTG without NGF immobilization. From these experiments, it can be concluded that the NGF immobilizing onto GTG membrane remaining the bioactivity.
The results of in vivo study indicated that the degradation rate of GTG and GEN membranes were moderate for nerve regeneration. The regenerated nerve fiber in GEN guidance channel cultured with Schwann cell (GENSc) showed denser nerve tissue, and the cross-section area of axon were larger than other groups. The histomorphometric, electrophysiologic, and functional assessments demonstrated that axon in GENSc recovered batter than other groups. Therefore, GEN guidance channel incorporated with Schwann cell can be a potential candidate for human peripheral nerve repair.
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