可持續釋放神經滋養因子之生物可分解性神經導管於周邊神經再生之研發與應用

Abstract

In this study, we focused on the development of artificial nerve guides that could offer sustained release of neurotrophic factors during the prolonged period of nerve regeneration. We used carbodiimide as a zero-length cross-linker to conjugate neurotrophic factors with gelatin-tricalcium phosphate composites (GTG). The bioactivity of neurotrophic factors after conjugation reaction, the release characteristics of neurotrophic factors grafted on GTG composites, and the in vitro biocompatibility of modified GTG composites were explored. With the immobilization of nerve growth factors (NGF) on GTG membranes, the releasing curve for NGF showing two distinctive parts with different slopes indicated that NGF was released from the composite in both diffusion-controlled mechanism and degradation-controlled mechanism. With the ELISA tests for β-NGF subunit, the biological activity of NGF molecules after the conjugation reaction could be confirmed. In addition, cultured PC12 cells also showed significant neurite outgrowth with NGF-grafted GTG membranes, which was statistically higher than GTG without NGF immobilization. Therefore, the technique used in the study is capable of immobilizing NGF on GTG membranes and retaining the bioactivity of NGF. Using the carbodiimide conjugation reaction, NGF, BDNF, and IGF-1 were then immobilized onto GTG membranes. Before the applications in animal studies, the in vitro biocompatibility of GTG membranes grafted with various neurotrophic factors was investigated. In PC12 cell culture, the total protein content and MTT assay indicated more cell attachment on the composites modified with growth factors. IGF-1 showed a higher survival promotion effect on PC12 cells than BDNF and NGF groups. On the other hand, NGF released from the composite showed the highest level of neurite outgrowth for PC12 cells. Cytotoxic effect was not induced by the conjugation reactions for the immobilization of neurotrophic factors. In animal studies, we first evaluated the in vivo applications of GTG conduits for peripheral nerve repair. Six months after sciatic nerve repair with GTG conduits, the biocompatibility and effects on nerve regeneration of GTG conduits were evaluated. The GTG conduits that we implanted were well tolerated by the host tissue, elicited a mild foreign body reaction, and had an optimal degradation over 24 weeks. No inflammation, infection, nor allergic reaction was noticed. Sections taken at the midconduits demonstrated typical regenerated nerve cables. Electrophysiological examinations and walking tract analysis also showed better recovery of functions for nerves repaired with GTG conduits when compared with nerves repaired with silicone tubes. When nerve repair was conducted with GTG conduits modified with various neurotrophic factors, the conduits were also well tolerated by the host tissue. In the regenerated nerves, the number of regenerated axons per unit area, the average axon size, the density of nerve fiber were even more improved by incorporating neurotrophic factors with GTG conduits. In the assessment of compound muscle action potential, reinnervation of gastrocnemic muscles, and sciatic function index, conduits modified with various neurotrophic factors showed a more favourable outcome in compound muscle action potential. Conduits with BDNF had a better recovery of gastrocnemic muscle weight. Therefore, GTG nerve guidance conduit incorporated with neurotrophic factors can be a potential candidate for peripheral nerve repair.