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標題: | 以幾丁聚醣為材料之功能性神經導管的製備與應用 Fabrication and Application of Functional Chitosan Nerve Conduits |
作者: | Yi-Cheng Huang 黃意真 |
指導教授: | 黃義侑(Yi-You Huang) |
關鍵字: | 神經導管,幾丁聚醣,聚乳酸-甘醇酸,冷凍乾燥及金屬絲加熱方式,鎳鉻絲,許旺細胞,氧氣電漿,脊髓損傷, Nerve Conduits (NCs),Chitosan,PLGA,Lyophilizing and Wire-Heating Process,Ni-Cr Wires,Schwann Cells(SCs),Laminin(LN),Oxygen Plasma,Spinal Cord Injury(SCI),Laminin coated Nerve Conduits(LN-NCs),Microcontact Printing(μCP), |
出版年 : | 2006 |
學位: | 博士 |
摘要: | 我們利用冷凍乾燥及金屬絲加熱方式,在可降解性高分子材料(幾丁聚醣(Chitosan)、聚乳酸-甘醇酸(PLGA))中製備具方向性的通道(Channel),做為神經導管。此法以鎳鉻絲(Ni-Cr Wires)為軸心,其製備過程安全、有效且再現性高。相較於聚乳酸-甘醇酸,幾丁聚醣的結構具多孔洞及高滲透的特性,提供較大的表面積以支撐並引導神經軸突的生長。調控鎳鉻絲之尺寸及量即能控制材料中通道之數目、直徑及式樣,更重要的是,此技術亦適用於其他高分子溶劑系統,應用相當廣泛。
為刺激神經生長,許旺細胞(Schwann cells)在短時間內快速且大量的的增殖是必需的,為達此目的,材料必需具有利於細胞貼附的特質,Laminin正是神經系統常用來促進胞貼附的蛋白質,存在於細胞外間質中。此研究的目的是探討以化學方式及電漿處理,將Laminin塗覆在聚乳酸-甘醇酸及幾丁聚醣膜的表面後,材料表面性質的改變及細胞生長的情況。相較於傳統化學處理,電漿是較佳的製備方式,已塗覆Laminin之幾丁聚醣膜亦可明顯地增加許旺細胞貼附的傾向。 為修復脊損傷,我們結合前述兩項技術,以冷凍乾燥及金屬絲加熱的方式製備多孔性的幾丁聚醣神經導管,並利用氧氣電漿處理,將Laminin塗覆在導管的內層表面。電漿處理的時間不能太長,否則材料之微結構將會被破壞。實驗結果顯示,將塗覆Laminin的神經導管植入老鼠的脊髓損傷處後,可觀察到行為改善的趨勢。組織學及免疫化學分析發現,神經導管可引導受損之軸突生長,穿過受傷部位,達到修復的目的,且不誘發發炎及細胞凋零。藉神經軸突標誌(GAP-43)之Western Blot Analysis亦說明塗覆Laminin之神經導管的修復效果,優於未塗覆的導管,是較佳的選擇。 為使神經的修復最佳化,生物系統之微環境之考量亦是一重要因素。我們利用Microcontact Printing (μCP)技術控制材料上微米級或奈米級的結構變化,促進細胞貼附及生長。實驗顯示,在幾丁聚醣膜的表面製備具特定樣式之Laminin,的確影響許旺細胞的排列及型態。μCP技術有效地將利於細胞貼附的分子(ex: Laminin),以特定的樣式塗覆在材料上,可直接、方便地應用於組織再生的研究。 For fabricating functional nerve conduits, we have developed a method using longitudinally oriented channels within biodegradable polymers, chitosan and ploy (d,l-lactide-co-glycolide) (PLGA), created by a combined lyophilizing and wire-heating process. This high permeability and the characteristic porous structure of chitosan scaffold provide increased area to support and guide extending axons subsequent to nerve injury. Utilizing Ni-Cr wires as mandrels to create channels in scaffold increased safety, effectiveness, and reproducibility. Regulating the size and quantity of the Ni-Cr wired allow us to control the number, diameter and pattern of the channels. Furthermore, the techniques can be easily tailored to other solvent and polymer systems. Fast proliferation of large numbers of schwann cells (SCs) in a short time appears to be promising for stimulating nerve regeneration. Good attachment is a prerequisite for the SCs to survive. Laminin, the extracellular matrix protein, is a permissive protein for SCs adhesion used in neural regrowth. The aim of this study was to investigate the surface effects of laminin coated PLGA and Chitosan membranes after chemical method and plasma treatment. Our results indicated that oxygen plasma is indeed a better method to incorporate laminin onto the surface of membrane. Laminin coated Chitosan membrane significantly increases SCs attachment and affinity for directing nerve regeneration. To better direct repair following spinal cord injury (SCI), we designed a porous chitosan nerve conduit (NC) created by lyophilizing and wire-heating process, and then incorporated with laminin (LN) on the inner surface by oxygen plasma treatment. To preserve the microstructure of the scaffold, prolonging plasma treatment time was unsuitable. Implantation of the laminin coated nerve conduit (LN-NC) into an adult rat hemisection model of SCI indicated the tendency of behavior improvement. Histology and immunocytochemical analysis suggested that the NCs could lead the damaged axons crossing through the lesion area, but not trigger inflammation or apoptosis. Together with significantly enhanced local GAP-43 expression evidenced by western blot analysis, we found functional recovery mediated by LN-NCs compared better than NCs alone. For optimizing nerve regeneration through nerve conduits, microenvironment mimicking biological situation is a vital issue. We use microcontact printing (μCP) technique to pattern the bio-molecule, laminin, on the chitosan film surface. Patterned laminin affect the alignment and morphology of Schwann cells. The microcontact printing provides a suitable micro- or nano-scale biological and topological control for cell attachment and outgrowth. This technique is straightforward and convenient for cell-recognition molecules to be micropattern-bound onto the scaffold for tissue regeneration. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33077 |
全文授權: | 有償授權 |
顯示於系所單位: | 醫學工程學研究所 |
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