以非微影製程圖案化蠶絲明膠水膠應用於具備細胞排列、電刺激、及釋放生長因子功能之神經再生導管

Non-lithographically patterned silk-gelatin nerve conduit with functions of cell-alignment, electrical stimulation and NGF release to enhance neural regeneration

常晶晶(Jing-Jing Chang)

指導教授 ： 陳三元

國立交通大學/工學院/材料科學與工程系所/碩士(2018年)

摘要

周邊神經損傷是臨床常見的疾病，神經導管的植入式治療在修復受損神經時扮演了十分重要的角色。將神經導管植入斷裂神經處以橋接近端及遠端神經殘枝，除可降低因神經拉扯所造成的張力外，導管本身亦可協助引導軸突的再生，也可做為物理性支架，進而承載細胞、神經生長因子或其他生物性分子以促進受損神經的再生。另一方面，材料表面的微觀結構對細胞的生長、增殖、遷移和分化等多種行為有一定的影響。開發可降解仿神經基材並且具引導神經軸突有方向性生長功能是目前設計新一代神經導管的重點。
本研究以構建有序方向性表面結構之神經支架為目標，使用具有良好的生物相容性和生物可降解性的絲蛋白和明膠兩種天然高分子材料作為神經導管的支架材料，利用機械力拉伸絲蛋白和明膠所組成之彈性水膠，使用無光罩微影製程的物理機制，在施加預應變的狀態下，在該彈性水膠表面塗覆一層聚乳酸(PLA)薄膜，隨後釋放預應力，即可獲得具有一維有序排列波紋表面之可降解彈性基材。研究發現，由絲蛋白和明膠所組成之彈性水膠不僅機械強度接近周邊神經組織的機械強度，且降解性滿足神經軸突再生所需週期。使用無光罩微影製程的方法製備具有有序排列的波紋表面，依據其物理機制，通過改變上層PLA薄膜的厚度可以調控波紋的波長和振幅，即可得到一合適的波紋排列，使該雙層水膠可以引導神經軸突定向生長。
另一方面，研究引入氧化石墨烯及氧化鐵複合載體，因石墨烯具有豐富的官能基及良好的導電性，在近幾年已廣泛應用於生醫材料。載體製備利用化學共沉澱方式將氧化鐵修飾於石墨烯上，並利用正負電荷間的靜電力作用接上聚乙烯亞胺高分子(Polyethylenimine，PEI)，根據文獻，聚乙烯亞胺高分子具有高正電荷，可吸附帶有負電荷的神經生長因子（Nerve Growth Factor，NGF）。將該吸帶有NGF的載體滴於具有有序波紋排列的雙層水膠之上，透過磁鐵吸引磁性氧化鐵，使其沉積於波谷處，再利用氧化石墨烯的導電性，給予細胞一電流脈衝以刺激軸突的分化。體外實驗證實，具有波紋排列的水膠具有引導軸突定向生長的作用，且在電與化學的雙重效應影響下，分化的軸突長度顯著增長，最終達到可以刺激並引導神經軸突定向生長的目標。

關鍵字

周邊神經修復 ； 非微影製程 ； 圖案化表面 ； 細胞排列 ； 電刺激 ； 神經生長因子釋放

並列摘要

Peripheral nerve injury is a common clinical disease, implanted therapy with nerve conduits has played an important role in the field of damaged nerves repair. Implanted nerve conduits into the fractured nerve to bridge the proximal and the distal ends of nerve branch, in addition to reducing the tension caused by the nerve pulling, the catheter itself can also assist in guiding the regeneration of the axon, or as a physical scaffold which can carry cells, nerve growth factor (NGF) or other biological molecules to promote the regeneration of damaged nerves. On the other hand, the microstructure of the surface of the material has an obvious influence on various behaviors such as cell growth, proliferation, migration and differentiation. The development of degradable biomimetic substrates which can guide axons directional growth is currently the focus of designing a new generation of nerve conduits.
This study aims to construct a neural scaffold with a one-dimensional ordered surface structure, using two natural polymer materials silk fibroin and gelatin as scaffold materials, which have good biocompatibility and biodegradability. Take advantage of the non-lithography technique mechanism, using a mechanical force to stretch the elastic hydrogels composed of silk fibroin and gelatin, a polylactic acid (PLA) stiff thin film was coated on the surface of the hydrogel by solvent casting method under the pre-strain condition, then the pre-stress was released to obtain a biodegradable hydrogel having a one-dimensionally ordered corrugated surface. The experimental results showed that: the peripheral nervous system and the hydrogels composed of silk fibroin and gelatin had similar mechanical strength, moreover, the degradation time of the hydrogels satisfied the regeneration time of the nerve branch. The one-dimensional ordered corrugation pattern was prepared by non-lithography technique. According to its physical mechanism, the wavelength and amplitude of the corrugation can be adjusted by changing the thickness of the upper PLA film, so that a suitable corrugation arrangement can be obtained which can directionally guide the axon growth.
Subsequently, graphene oxide (GO) is widely explored in the field of biomedical materials in recent years, due to its rich of functional groups and have good electrical conductivity. In this study, nGO that is modified with Fe3O4 was prepared by chemical depositing, and coated branch 25k polyethyleneimine (bPEI) through electrostatic interaction to create a multifunctional carrier. According to previous researches, PEI is a positively charged polymer, which can be used to attract nerve growth factor (NGF) which has negative charged. The rGO-Fe3O4-PEI-NGF (GFPN) complexes would deposit at the groove of the corrugation pattern using the magnet attraction of iron oxide nanoparticles, and then the conductivity of the graphene oxide was utilized to give electrical pulse to the cells which would stimulate axonal differentiation. In vitro experiments confirmed that the SG4 hydrogel with corrugated pattern has the function of guiding the neurites directional growth, moreover, under the influence of the electrical and chemical dual effects, the length of the differentiated neurites were significantly increased, and finally it can achieve the goal of stimulating and guiding the axon directional growth.

並列關鍵字

Peripheral nerve repair ； Non-lithography ； Patterned surface ； Cell alignment ； Electrical stimulation ； NGF release

參考文獻

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