靜電紡紗谷胺酸苯酯-谷胺酸無規共聚物用於PC-12軸突生長研究

Abstract

隨著可攜帶式電子產品的普及人們對其依賴日益加深，然而這無非對我們的眼睛造成極大負擔，最近眼科門診指出，年輕族群由於長時間近距離使用3C產品導致黃斑部病變、青光眼年輕化，由於視神經細胞一旦受損萎縮無法自行修復，因此我們期望利用組織工程，透過體外培養視神經細胞再移植到受損區完成修復。在組織工程上，支架必須模仿細胞外基質的特性具有生物相容性、細胞可辨識的官能基、生物降解性以及互相連通的孔洞。靜電紡絲為一種常用於製備奈米纖維支架的技術，其奈米纖維支架的結構與細胞外基質極為相似，具備極高的表面積供細胞貼附和相互連通的孔洞代謝廢物。而谷胺酸苯酯-谷胺酸無規共聚物兼具細胞可辨識的谷胺酸官能基，因此我們認為在培養環境下藉由支架的化學刺激對神經軸突的生長有良好的助益。 在本篇論文中，我們利用谷胺酸苯酯 (L-glutamic acid γ-benzyl ester)和三光氣(triphosgene)反應得到谷胺酸苯酯羧酸酐 (γ-benzyl glutamate-N-carboxy anhydride)。之後將谷胺酸苯酯羧酸酐溶解在無水苯中，加入甲醇鈉強鹼進行開環聚合，得到纖維狀聚谷胺酸苯酯。最後控制酸解時間得到不同比例的谷胺酸苯酯-谷胺酸無規共聚物。我們透過核磁共振光譜分析酸解程度和時間的關係並且利用凝膠滲透層析分析分子量。另外我們利用四氫呋喃 (THF)和二甲基乙醯胺 (DMAc)的混合溶劑製備電紡絲纖維支架，我們發現高分子纖維支架導入神經傳遞因子谷胺酸不僅能提高生物相容性，PC-12在其上分化出的軸突長度和數目也有所提高，其中以PBGA30的效果最為顯著。但視神經軸突傳遞訊息的方向性相當重要，因此我們藉由更換滾筒式收集器可以得到單一方向性排列之纖維支架，並發現單一方向性纖維可以誘導軸突順著纖維生長，我們認為單一方向性排列的谷胺酸苯酯-谷胺酸無規共聚物可做為視神經修復的高潛力性生醫材料。

As portable electronic devices popularize, people seems to be addicted to them for getting new information or watching videos. However, using eye for long time that causes optic nerve damage. Clinical research reports that macular degeneration and glaucoma have been found on people in their thirties more often than before which may be using electronic devices without proper distance and adequate eye relaxation. As long as optic nerve is damaged, it will not regenerate by itself. We would like to culture optic nerves in vitro on scaffold by tissue engineering, then transfer the scaffold to damaged part of nerves for regeneration. In tissue engineering, the scaffold must be porous, cell-recognized motif, biodegradable, biocompatible which is mimic native tissue. Electrospin is a promising technique to fabricate nanofibrous scaffold which is mimic the structure of extracellular matrix (ECM) and provides high surface area with interconnected pores. Poly(benzyl glutamate)-r-poly(glutamic acid) (PBGA) have glutamic acid, which is a excitatory neurotransmitter that plays the principal role in neural activation. We expect it can benefit neuron differentiation by glutamic acid motif during cell culture. In this study, we have synthesized monomer: γ-benzyl glutamate-N-carboxy anhydride from L-glutamic acid γ-benzyl ester and triphosgene. Then we used sodium methoxide as an initiator to polymerize the monomer into poly(benzyl-glutamate) in anhydrous benzene. Finally, we controlled the ratio of glutamic acid on the main chain by hydrolysis time. After getting product, we use NMR and GPC to analyze the ratio of glutamic acid in copolymer and molecular weight of PBGA. The PBG and PBGA were further fabricated into fibrous scaffolds by electrospinning using tetrahydrofuran (THF) and dimethylacetamide (DMAc) cosolvent. We investigated the effect of glutamic acid ratio on PC-12 cells proliferation and neuron differentiation. We found that the biocompatibility and neuron differentiation can be improved by glutamic acid. In our study, The copolymer contained 30% molar ratio of glutamic acid (PBGA30) exhibited best biocompatibility and the longest nerve outgrowth. However, neuron transmit direction plays important role on nerve tissue functionality. We fabricated scaffold with aligned fibers by employing a rotating drum collector to guide the growth of nerve. We found that neurite is growth along the fiber. In summary, the aligned electrospun PBGA scaffold has potential for optic nerve regeneration.