A novel chitosan nerve conduit with micro and nano hybrid patterns

神經導管已廣泛應用於修復較長範圍損壞之神經束，然而其修復率仍遠小於預期。故為促進神經細胞於導管之增生，神經導管製作已趨向仿生化之探討，藉由不同之材料應用與表面結構之變化，以模擬神經束之原生環境。神經細胞原生環境之表面結構為奈微米尺度，可影響神經細胞之生長方向，故需製備高度圖案化之表面結構，引導神經細胞規則的排列生長，使訊息可有效的向下傳遞。故本研究由半導體製程中黃光微影技術著手，搭配奈米製程與電鑄製程，提出圖案化奈微米結構之幾丁聚醣神經導管製作流程，除兼具可快速製作、低成本、可重複使用且易於量產之特性外，且可使神經細胞有效的生長於導管內部進而發展為神經束。主要製程為以表面具奈米結構之陽極氧化鋁模作為模仁，並以黃光微影方式產生圖案化之微米結構，再以奈米電鑄方式製備具圖案化奈微米結構之鎳模具，接著以天然高分子幾丁聚醣進行翻模，製備出具有奈微米結構之神經導管。本研究選用具生物相容性與生物可降解特性之幾丁聚醣天然高分子材料做為神經導管之材料，而後將大鼠神經母細胞(B35)植覆培養於幾丁聚醣神經導管支架上，觀察不同表面結構之幾丁聚醣對神經細胞生長之影響，並研究奈微米結構是否能促進細胞之增生與引導其生長方向。實驗結果顯示，本研究之方法可成功製備具圖案化奈微米結構、奈米結構、與微米結構等三種薄膜支架；實際細胞培養顯示神經細胞於奈米結構薄膜支架可有較佳之貼附與生長，而圖案化的微米結構也確實具有引導神經細胞生長方向的作用，奈微米結構薄膜(線寬30 mm、間距30 mm)支架則有最佳促進神經細胞生長與引導神經細胞之作用。Nerve conduits have been widely used for repairing damaged nerve bundles. However, the repair rate is still far below expectation currently. To enhance the proliferation of nerve cells on nerve conduits, nerve conduits that can mimic the natural environment of human body is a feasible solution. Since the primitive living environment, that is in the scale of nano/micro meter, can influence the growth of nerve cell, it is desirable to fabricate a scaffold mimicking the primitive living environment such that the growth of nerve cells can be well directed. Therefore, a novel chitosan nerve conduit with micro and nano hybrid patterns is proposed in this study. The microelectromechanical system (MEMS) and nickel electroforming techniques were used for the fabrication of the chitosan nerve conduits. The hemispheric array of the barrier layer of an anodic aluminum oxide (AAO) film was used as the substrate. The MEMS process was then used to fabricate micro-structure pattern on the surface of the barrier layer. Following, a nickel replica mold was produced through electroforming using the patterned AAO barrier layer as the template. Scaffolds of chitosan nerve conduit were formed by casting using the synthesized nickel replica mold. Nerve cells were then cultured on the scaffolds. The WST-1 test was used to illustrate the cells proliferation rate. The cell adhesion and morphology were observed through the Hoechst (staining nucleus) and phalloidin (staining cytoskeleton) labeling. It is observed that a micro-structure can only guide the nerve cells to grow along a certain direction, while the proposed micro and nano hybrid structure can successfully guide the growth direction and enhance the proliferation of nerve cells.誌謝 i 摘要 ii Abstract iv 目錄 vi 圖目錄 ix 第1章 緒論 1 1.1. 前言 1 1.2. 文獻回顧與背景 1 1.2.1. 組織工程 1 1.2.2. 神經系統 2 1.2.3. 奈微米結構對細胞的影響 9 1.2.4. 奈微米結構製程 12 1.3. 研究動機與目的 15 第2章 研究方法與材料 17 2.1. 材料與藥品 17 2.1.1. 藥品 17 2.1.2. 儀器 18 2.2. 實驗製程 18 2.2.1. 陽極氧化鋁膜製備 19 2.2.2. 黃光微影 22 2.2.3. 濺鍍金薄膜電極 23 2.2.4. 高溫退火 24 2.2.5 試片封裝 24 2.2.6. 電鑄鎳 25 2.2.7. 幾丁聚醣具奈微米結構薄膜之製備 26 2.2.8. 奈微米複合結構支架之細胞培養 27 2.2.9. 量測與觀察 28 2.3. 統計分析 30 第3章 實驗結果與討論 31 3.1. 奈微米複合結構陽極氧化鋁膜製備結果 31 3.2. 奈微米複合結構鎳模具製備結果 34 3.3. 奈微米複合結構之幾丁聚醣支架製備結果 36 3.3.1. 奈微米複合結構之幾丁聚醣支架之表面觀測 36 3.3.2. 奈微米複合結構幾丁聚醣支架之水滴接觸角 38 3.3.3. 奈微米複合結構幾丁聚醣支架之表面元素分析 39 3.4. 奈微米複合結構幾丁聚醣薄膜之細胞培養 42 3.4.1. 細胞型態 43 3.4.2. 細胞增生 48 第4章 結論與未來展望 55 4.1. 結論 55 4.2. 未來展望 55 參考文獻 5