以生物聚合物水膠載體建立角膜內皮細胞層片組織工程之多功能傳輸系統

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研究生：	盧佩琳 Pei-Lin Lu
論文名稱：	以生物聚合物水膠載體建立角膜內皮細胞層片組織工程之多功能傳輸系統 Biopolymer-Based Hydrogel Carriers as Multifunctional Delivery System in Corneal Endothelial Cell Sheet Engineering
指導教授：	薛敬和 Ging-Ho Hsiue
口試委員:
學位類別：	博士 Doctor
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2007
畢業學年度：	95
語文別：	英文
論文頁數：	185
中文關鍵詞：	生醫材料、動物明膠、透明質酸、交聯、多功能水膠載體傳輸系統、人類眼角膜內皮層、移植、細胞層片組織工程、再生醫學
外文關鍵詞：	Biopolymers, Gelatin, Hyaluronic acid, Cross-linking, Multifunctional carrier system, Human corneal endothelial cell sheet, Transplantation, Ocular regenerative medicine
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由於人類眼角膜內皮細胞在體內具有不會分化生長的特性，正常角膜內皮組織的細胞密度自出生後即不斷減少。當密度低於每平方毫米1000個細胞時，角膜內皮組織的排水生理功能將無法發揮，進而導致角膜水腫混濁及視覺喪失。目前臨床上，全層角膜移植術為治療角膜內皮組織病變的主要方式。然而，捐贈角膜之來源不足與全層角膜移植的術後相關併發症仍是此法應用時必須克服之瓶頸。因此，進行組織工程之人類眼角膜內皮細胞層片移植，以達到受損角膜內皮組織的單層置換乃一種極具潛力的替代方案。本研究之目的即藉由設計功能性生醫材料，以克服細胞層片移植之瓶頸。
由於細胞層片組織相當柔軟易碎，研究首先設計一多功能水膠載體傳輸系統，以進行生醫工程人類眼角膜內皮細胞層片之眼內移植應用。實驗採用不同等電點(5.0或9.0)及分子量(3至100 kDa)的動物明膠為原料，製備水膠載體並接受γ射線照射消毒。藉由機械性質、含水率、分解率與細胞相容性等測試進行各種水膠載體之功能性評估。結果顯示具有等電點5.0及分子量100 kDa的動物明膠最適合作為細胞層片移植治療之水膠載體與發展穩定的眼內傳輸系統。
由於動物明膠來源具有引發牛海綿狀腦病之風險，本研究亦同時進行替代生醫材料的開發。藉由1-乙基-3-(3-二甲氨基丙基)碳二亞胺交聯處理程序，透明質酸水膠將可獲得適當修飾改質。進一步以形態學、機械性質、熱性質、含水率、分解率與細胞相容性等測試評估此材料是否具有作為細胞層片傳輸載體之可行性。本實驗對照組為戊二醛交聯之透明質酸。
在體內試驗方面，本研究以兔子為動物實驗模式，進行生醫工程人類眼角膜內皮細胞層片之眼內移植治療可行性分析。經由臨床觀測與病理組織切片檢視，人類眼角膜內皮細胞層片在體內能夠順利貼覆結合於宿主病變組織上，並有效發揮其細胞特有生理功能。與僅製造眼角膜內皮傷口的對照組相較下，動物經植入細胞層片後，其受損角膜之病態水腫及其澄清度均可獲得大幅改善。此外，兔子眼角膜厚度幾乎恢復至原始狀態，也意謂著植入的細胞層片組織在體內確實能夠展現功能。這些實驗結果指出具有良好結構與功能之生醫工程細胞層片相當適合應用於眼角膜內皮組織修復。
基於上述研究發現，利用感溫性培養界面與多功能水膠載體，以製備及移植生醫工程人類眼角膜內皮細胞層片，能夠建立一套角膜內皮細胞治療之新策略。此外，功能性生醫材料在人類眼角膜內皮細胞層片組織工程及其再生醫學之開發極具潛力。最後，本研究希望此細胞層片新療法未來能有效改善角膜內皮相關病變，並應用於眼組織再生重建與臨床工程。

Human corneal endothelium in vivo demonstrates an age-related decrease in cell density and cannot be compensated due to its limited regenerative capacity. When the cell density is less than a critical level of 1000 cells/mm2, the endothelial monolayer no longer functions, causing corneal edema and loss of visual acuity. Penetrating keratoplasty (PK) is currently the common way to treat corneas that are opacified due to endothelial dysfunction. However, insufficient supplies of donor corneas and several complications associated with PK remain a worldwide problem. Therefore, transplantation of tissue-engineered human corneal endothelial cell (HCEC) sheets to replace damaged corneal endothelium alone is a promising alternative to PK. This work is devoted to overcome the limitations of cell sheet transplantation.
Because of the soft and fragile nature of bioengineered HCEC sheets, we have designed and developed a multifunctional hydrogel carrier system for intraocular delivery of these sheet grafts. The functionality of gamma-sterilized cell carriers made from raw gelatins with a different isoelectric point (IEP = 5.0 and 9.0) and a molecular weight (MW) range from 3 to 100 kDa, was investigated by the determination of mechanical properties, water content, dissolution degree, and cytocompatibility. The results of our study indicate that the gamma-sterilized hydrogel discs consisting of raw gelatins (IEP = 5.0, MW = 100 kDa) are promising candidates as cell sheet carriers for effective corneal endothelial cell transplantation and therapy.
Development of alternative biomaterials to bovine-based gelatin vehicles can potentially eliminate the risk of bovine spongiform encephalopathy. To investigate whether it was appropriate for use as cell sheet delivery vehicles, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) cross-linked hyaluronic acid (HA) hydrogels were studied by determinations of morphological characteristic, mechanical and thermal property, water content, in vitro degradability, and cytocompatibility. Glutaraldehyde (GTA) cross-linked HA samples were used for comparison. It is concluded that EDC can be successfully applied for HA cross-linking to fabricate structurally stable, mechanically reinforced, readily deformable, transparent, and cytocompatible HA hydrogel discs with the potential to be applied as delivery vehicles for corneal endothelial cell therapy.
On the other hand, intraocular implantation in the anterior chamber has received much attention for the determination of the interactions between the immune privileged tissues and biomaterial implants. A novel methodology based on the anterior chamber of rabbit eyes model was developed to evaluate the in vivo biocompatibility of biomaterials in an immune privileged site. The 7-mm-diameter membrane implants made from either a biological tissue material (amniotic membrane, AM group) or a biomedical polymeric material (gelatin, GM group) were inserted in rabbit anterior chamber for 36 months and characterized by biomicroscopic examinations, intraocular pressure measurements, and corneal thickness measurements. The noninvasive ophthalmic parameters were scored to provide a quantitative grading system. Our data suggest that the anterior chamber of rabbit eyes model is an efficient method for noninvasively determining the immune privileged tissue/biomaterial interactions.
In the present study, we have demonstrated that the multifunctional carrier system is beneficial for transportation and surgical handling of bioengineered human corneal endothelium. Cell sheet transplantation with biopolymer-based hydrogels can potentially offer a new therapeutic strategy for corneal endothelial cell loss. In addition, a novel methodology based on the anterior chamber of rabbit eyes model has great potential for evaluating the ocular biocompatibility and safety of biomaterial carriers. We hope this work will lead to insights into cell sheet-based therapy for ocular regenerative medicine and will open an exciting new door to the future.

ENGLISH ABSTRACT...........................................I
CHINESE ABSTRACT..........................................IV
DEDICATION.............................................. VII
ACKNOWLEDGMENTS.........................................VIII
TABLE OF CONTENTS..........................................X
LIST OF FIGURES......................................... XVI
LIST OF TABLES......................................... XXII
CHAPTER 1 BACKGROUND AND LITERATURE REVIEW.................1
1 Motivation and Objectives..............................2
2 Corneal Endothelium....................................7
2.1 Cornea...............................................7
2.2 Corneal Endothelium..................................9
2.2.1 Structure and Function.............................9
2.2.2 Proliferative Capacity............................10
3 Corneal Endothelial Cell Therapy......................15
3.1 Penetrating Keratoplasty............................15
3.2 Cell-Based Therapy..................................17
3.2.1 Injection of Isolated Cell Suspensions............17
3.2.2 Transplantation of Tissue-Engineered Constructs...18
4 Cell Sheet Engineering................................28
4.1 Tissue Engineering..................................28
4.2 Fabrication of Tissue-Engineered Cell Sheets........31
4.3 Rapid Cell Detachment and Cell Sheet Transfer.......32
4.4 Biomedical Applications.............................34
5 Biopolymers...........................................41
5.1 Biopolymer-based Hydrogel Carrier System............44
5.2 Gelatin.............................................47
5.3 Hyaluronic Acid.....................................50
6 Immune Privilege......................................53
6.1 Principles of Transplantation Immunology............54
6.2 Impact of Immune Privilege on Transplantation.......55
6.3 The Eye as a Site of Immune Privilege...............57
6.4 The Eye as a Source of Immune Privileged Tissues....59
6.5 Biocompatibility Studies on the Immune Privileged Sites.....................................................60
7 Dissertation Layouts..................................62
CHAPTER 2 EFFECT OF CHARGE AND MOLECULAR WEIGHT ON THE FUNCTIONALITY OF GELATIN CARRIERS FOR CORNEAL ENDOTHELIAL CELL THERAPY..............................................64
1 Introduction..........................................65
2 Experimental..........................................69
2.1 Materials...........................................69
2.2 Preparation of Gelatin Hydrogel Discs...............71
2.3 Tensile Tests.......................................71
2.4 Determination of Water Content and Dissolution Degree....................................................72
2.5 Cell Culture........................................72
2.6 Cell Proliferation Assay............................73
2.7 Cell Viability Assay................................74
2.8 Cytokine Expression.................................75
2.9 Gelatin Carriers for Cell Sheet Delivery............76
2.10 Statistical Analysis...............................78
3 Results and Discussion................................79
3.1 Tensile Tests.......................................79
3.2 Determination of Water Content and Dissolution Degree....................................................80
3.3 Cell Proliferation Assay............................83
3.4 Cell Viability Assay................................88
3.5 Cytokine Expression.................................91
3.6 Gelatin Carriers for Cell Sheet Delivery............91
4 Conclusions...........................................96
CHAPTER 3 CARBODIIMIDE CROSS-LINKED HYALURONIC ACID HYDROGELS AS CELL SHEET DELIVERY VEHICLES: CHARACTERIZATION AND INTERACTION WITH CORNEAL ENDOTHELIAL CELLS............99
1 Introduction.........................................100
2 Experimental.........................................104
2.1 Materials..........................................104
2.2 Preparation of HA Hydrogel Discs...................104
2.3 Cross-linking of HA Discs by EDC or GTA............105
2.4 Scanning Electron Microscopy.......................105
2.5 Tensile Tests......................................105
2.6 Differential Scanning Calorimetry..................106
2.7 Water Content Measurements.........................106
2.8 In Vitro Degradation Tests.........................107
2.9 Corneal Endothelial Cell Culture...................107
2.10 Cell Proliferation Assays.........................108
2.11 Statistical Analysis..............................109
3 Results and Discussion...............................110
3.1 Scanning Electron Microscopy.......................110
3.2 Tensile Tests......................................112
3.3 Differential Scanning Calorimetry..................115
3.4 Water Content Measurements.........................115
3.5 In Vitro Degradation Tests.........................118
3.6 Cell Proliferation Assays..........................121
4 Conclusions..........................................125
CHAPTER 4 A METHODOLOGY BASED ON THE “ANTERIOR CHAMBER OF RABBIT EYES” MODEL FOR NONINVASIVELY DETERMINING THE BIOCOMPATIBILITY OF BIOMATERIALS IN AN IMMUNE PRIVILEGED SITE.....................................................126
1 Introduction.........................................127
2 Experimental.........................................130
2.1 Materials..........................................130
2.2 Preparation of Implants............................131
2.3 Animals............................................132
2.4 Surgery............................................132
2.5 Postoperative Evaluations..........................133
2.6 Ocular Grading System..............................134
2.7 Statistical Analysis...............................134
3 Results and Discussion...............................136
3.1 Biomicroscopic Examinations........................136
3.2 Intraocular Pressure Measurements..................140
3.3 Corneal Thickness Measurements.....................142
3.4 Ocular Grading System..............................144
4 Conclusions..........................................147
CHAPTER 5 SUMMARY AND FUTURE DIRECTIONS..................148
REFERENCES...............................................153
BIBLIOGRAPHY.............................................186

                                

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