三鈣磷酸鹽強化之明膠基材表面固定神經生長因子及培養許旺氏細胞用於周邊神經再生導管之研究

本實驗以明膠為薄膜基材，發展出一個適用於神經修復的替代材料來取代現有的自體移植，並進行神經再生導管的研究。實驗分為三大部分：導管材料的發展、神經生長因子(NGF)的固定與體外生物相容性評估，以及導管植入坐骨神經之體內評估。首先在材料的研發上，我們比較了利用幾丁聚醣與明膠交聯的薄膜，以及添加三鈣磷酸鹽粉末以強化明膠機械強度的薄膜，分別進行紅外線光譜分析、微差掃描熱差分析、機械強度測試及水接觸角測試。結果顯示以低濃度戊二醛交聯及三鈣磷酸鹽粉末強化之明膠薄膜(GTG)為最適合用於神經再生導管的材料，其延性適中、機械強度佳且為較具親水性的材料。因此，在後續的實驗中將以GTG做為進行神經再生導管的材料。在體外生物相容性評估的結果顯示，GTG薄膜無論表面有無NGF的固定皆不會對許旺氏細胞(Schwann cell)造成細胞毒性的影響。此外，GTG薄膜表面固定NGF分子(GEN) 證實有較多的許旺氏細胞貼附，而使用物理性吸附的薄膜由於大部分NGF已被清洗掉，所以並沒有增加細胞貼附的作用，雖然GTG薄膜固定NGF分子並不能促進許旺氏細胞的增生，但GEN薄膜可以促進許旺氏細胞的貼附；而在NGF的釋放實驗中證實，GEN薄膜可以持續釋放NGF至少60天，且其最低釋放量亦達刺激PC12細胞的最小濃度。另外，在PC12細胞的培養中發現GEN組神經突的生長數量較GTG組高，顯示GEN薄膜釋放出的NGF具有刺激PC12細胞分化的生物活性。在神經導管植入大鼠坐骨神經的體內評估中顯示，GTG及GEN薄膜的降解速度適中，在神經修復完成前導管仍可提供一個管腔支持神經軸突生長。而在GEN導管內培養許旺氏細胞的GENSc組所再生的神經纖維有較緻密的軸突生長，組織間只有細小的裂隙，橫切面面積也較其他組的面積大。在電生理的實驗中也證實其再生的神經軸突之電傳導功能恢復指數較其他組為高，坐骨神經的運動功能也有相當程度的恢復。因此，本實驗使用之GEN導管結合許旺氏細胞的培養，具有相當好的潛力發展為用於人體的神經再生導管，以修復受損之周邊神經。

關鍵字

神經再生導管；神經生長因子；許旺氏細胞；生物相容性；體內評估

並列摘要

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.

並列關鍵字

nerve regenerated guidance channel ； nerve growth factor ； Schwann cell ； biocompatibility ； in vivo test

參考文獻

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三鈣磷酸鹽強化之明膠基材表面固定神經生長因子及培養許旺氏細胞用於周邊神經再生導管之研究

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