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研究生:林建成
研究生(外文):Chien Chen Lin
論文名稱:經去細胞與脫鈣之魚鱗作為人工眼角膜之研究
論文名稱(外文):A New Artificial Cornea Derived from Acellularized & Decalcified Fish Scale
指導教授:林峰輝林峰輝引用關係
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:醫學工程學研究所
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:66
中文關鍵詞:人工眼角膜魚鱗膠原蛋白組織工程生醫材料
外文關鍵詞:Artificial corneabiocorneacorneal regenerationtissue engineeringscaffoldcollagen
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角膜移植術是施行於因混濁而導致視力障礙者,將病患角膜組織切除,而置換他人之正常眼角膜以改善視力。隨著醫學的進步與眼科顯微手術的發展,角膜移植已是一種為常見的手術;根據統計,台灣以往每年角膜移植個案約有一千七百例,但因進口的眼角膜移植數量變少了,如今只剩下一千多例可手術,未來可能都要靠國人自行捐贈的眼角膜,而國人因為傳統信仰因素願意捐贈眼角膜的人數非常有限。
因此,本研究之主要目的在於希望開發出一種人工眼角膜能夠彌補角膜捐贈的不足,本實驗選擇魚鱗做為材料,經過去細胞與脫鈣處理,利用SEM觀察其特殊的微渠道與向心圓的顯微結構並不會受到上述處裡的影響,並且能夠具備極佳的親水性與透明性;為了進一步觀察這種材料的細胞親和性(cytocompatibility),將兔子角膜細胞培養在材料上,先以SEM觀察細胞在材料上的生長情形,再以共軛焦顯微鏡確認細胞在材料上的增生情形,最後另外也以細胞株進行MTT定量上述結果。
在本研究中發現隨手可得的魚鱗特殊結構,除了有表面特殊結構外,其相互交錯的膠原蛋白天然結構更提供了極佳的機械強度,而經過處裡過後的魚鱗仍然可以保有相當的透明度,這也初步證實了該天然結構的確適合應用在人工眼角膜的開發。


The purpose of this study is to develop a novel scaffold, derived from fish scales, as an alternative functional material with sufficient mechanical strength for corneal regenerative applications. Fish scales, which are usually considered as marine wastes, were acellularized, decalcified and fabricated into collagen scaffolds. The microstructure of the acellularized scaffold was imaged by scanning electron microscopy (SEM). The acellularization and decalcification treatments did not affect the naturally 3-dimentional, highly centrally-oriented micropatterned structure of the material. To assess the cytocompatibility of the scaffold with corneal cells, rabbit corneal cells were cultured on the scaffold and examined under SEM and confocal microscopy at different time periods. Rapid cell proliferation and migration on the scaffold were observed under SEM and confocal microscopy. The highly centrally-oriented micropatterned structure of the scaffold was beneficial for efficient nutrient and oxygen supply to the cells cultured in the three-dimensional matrices, and therefore it is useful for high-density cell seeding and spreading. Collectively, we demonstrate the superior cellular conductivity of the newly developed material. We provide evidences for the feasibility of the scaffold as a template for corneal cells growth and migration, and thus the fish scale-derived scaffold can be developed as a promising material for tissue-engineering of cornea.

Contents

中文摘要 ii
Abstract iii
Contents iv
Abbreviations list xi
Chapter 1 Introduction
1-1 Cornea ------------------------------------- 1
1-1-1 Anatomy of Cornea------------------------ 1
1-1-2 Cornea Disease --------------------- 10
1-1-3 Surgery Treatment of Cornea ------------- 12
1-2 Cornea Transplant----------------------------- 18
1-2-1 Synthetic Cornea Implant----------------- 18
1-2-2 Bio-Engineered Cornea Implant------------ 30
1-3 Purpose of the Study------------------------- 32
Chapter 2 Basic Science and Theory 34
2-1 Tissue Engineering-------------------------- 34
2-2 Characteristic of Fish Scales--------------- 37
Chapter 3 Materials & Methods 39
3-1 Acellular scaffold preparation ------------- 39
3-2 Decalcification of the scaffolds------------ 40
3-3 Harvesting corneal cells ------------------- 40
3-4 Scanning electron microscopy ---------------- 41
3-5 Confocal microscopy ------------------------- 42
3-6 Hydrophilicity test ------------------------- 43
3-7 Oxygen permeability test--------------------- 44
3-8 DAPI staining and direct cell nuclei counting -44
Chapter 4 Results 46
4-1 Characterization of the scaffolds----------- 46
4-2 Cell morphology analysis by SEM ------------ 48
4-3 Fluorescence microscopy -------------------- 51
4-4 Hydrophilicity & oxygen permeability ------- 53
4-5 Corneal cell proliferation on the scaffold-----53
Chapter 5 Discussions 55
Chapter 6 Conclusions 60
References 61


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