以功能性生醫材料開發人類眼角膜內皮細胞層片之組織工程及其再生醫學之應用

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研究生：	賴瑞陽 Lai, Jui-Yang
論文名稱：	以功能性生醫材料開發人類眼角膜內皮細胞層片之組織工程及其再生醫學之應用 Functional Biomaterials for Human Corneal Endothelial Cell Sheet Engineering and Regenerative Medicine
指導教授：	薛敬和 Hsiue, Ging-Ho
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：	2006
畢業學年度：	94
語文別：	英文
論文頁數：	275
中文關鍵詞：	生醫材料、細胞層片組織工程、再生醫學、感溫性聚異丙基丙烯醯胺接枝培養表面、多功能性動物明膠傳輸載體、人類眼角膜內皮層、電漿化學、眼科學、移植
外文關鍵詞：	Biomaterials, Cell sheet engineering, Regenerative medicine, Thermo-responsive poly(N-isopropylacrylamide)-grafted culture surface, Multi-functional gelatin carrier, Human corneal endothelium, Plasma chemistry, Ophthalmology, Transplantation
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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。在目前臨床上，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，以體外大量培養之人類眼角膜內皮細胞進行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。本研究之目的即是藉由製備與移植生醫工程的人類眼角膜內皮細胞層片，以開發一新穎性治療法進行眼組織重建。
研究首先根據電漿化學反應設計一套兩階段的材料表面改質方法，以製備感溫性聚異丙基丙烯醯胺高分子接枝表面，並應用於人類眼角膜內皮細胞之培養。進一步藉由外界溫度改變所產生的熱刺激達到有效控制細胞於培養界面上之貼覆與分離。經由能量分散式X射線光譜學、霍氏全反射紅外線光譜學、原子力顯微鏡學及靜態接觸角量測等表面鑑定分析，聚異丙基丙烯醯胺高分子於材料表面之最佳接枝量為每平方公分1.6微克。此外，導入丙烯酸高分子鏈段作為材料接枝的間距分子將可有效加速細胞於培養表面的分離，並防止細胞因過度低溫處理而衍生之不良效應。
接續探討以感溫性培養基材所製備之生醫工程人類眼角膜內皮細胞層片是否具有作為天然組織替代物之可行性。將來自眼庫捐贈組織的成體人類眼角膜內皮細胞在37°C下培養於感溫性高分子接枝表面。三週後，長滿之細胞可經由降溫至20°C而脫離培養表面，並獲得一層片狀組織。此生醫工程細胞層片的體外性質以存活率試驗、掃瞄式電子顯微鏡學、免疫組織化學及組織學進行評估。結果指出在與眼庫角膜內皮組織比較下，人類眼角膜內皮細胞層片於形態、結構、活性與功能等方面均有相似特徵，適合作為天然組織替代物。
由於細胞層片組織相當柔軟易碎，本研究亦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。
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 in vitro cultured human corneal endothelial cells (HCECs) to replace damaged corneal endothelium alone is a promising alternative to PK. In this study, we developed a novel therapy technique to fabricate and transplant cultured HCEC sheets for corneal endothelial reconstruction.
On the basis of plasma chemistry, we have designed a two-step method to prepare a thermo-responsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted culture surface for controlling HCEC adhesion and detachment via a thermal stimulus. The results of surface characterization including energy-dispersive X-ray spectroscopy (EDX), attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), atomic force microscopy (AFM), and static contact angle measurements show that an optimal grafting amount of PNIPAAm is 1.6 μg/cm2. We have also demonstrated that the introduction of AAc segment as short spacers onto the culture support can accelerate the cell detachment, which is beneficial to protect these harvested cells from functional damage.
We further investigated whether the bioengineered HCEC sheets harvested from thermo-responsive culture supports could be used as biological tissue equivalents. Untransformed adult HCECs derived from eye bank corneas were cultivated on PNIPAAm-grafted surfaces for 3 weeks at 37°C. Confluent cell cultures were detached as a laminated sheet by lowering culture temperature to 20°C. In vitro characteristics of HCEC sheets were evaluated by viability, scanning electron microscopy, immunohistochemistry, and histology. Similar to the native corneal endothelium from eye bank donors, the fabricated HCEC monolayers having normal morphology, structure and viability are suitable to be used as tissue replacements for transplantation.
Because of the soft and fragile nature of bioengineered HCEC sheets, we have designed and developed a multi-functional 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.
In the in vivo tests, we evaluated the feasibility of HCEC transplantation by harvesting the cell sheets from the thermo-responsive culture supports and delivering with multi-functional gelatin hydrogel discs in a rabbit model. We have shown that the transplanted HCEC sheets could be integrated into rabbit corneas denuded of endothelium. Additionally, when endothelium alone was removed, the rabbit corneas became cloudy and remained opaque throughout the course of the experiment. Once receiving tissue-engineered HCEC sheets, the corneas have returned to a nearly normal thickness. These results imply the biological function of transplanted cell sheets. Our findings indicated that a well-organized and functional HCEC sheet is feasible to be used as tissue equivalents for replacing compromised endothelium.
In the present study, we have demonstrated that the bioengineered human corneal endothelium fabricated from thermo-responsive culture supports and delivered by multi-functional hydrogel carriers can potentially offer a new therapeutic strategy for corneal endothelial cell loss. In addition, functional biomaterials have great potential for development of HCEC sheet engineering and regenerative medicine in ophthalmology. We hope this work will lead to insights into cell sheet-based therapy for corneal endothelial dysfunction and will open an exciting new door to the future.

TABLE OF CONTENTS

ENGLISH ABSTRACT………………………………………………………I
CHINESE ABSTRACT………………………………………………………IV
DEDICATION……………………………………………………………VII
ACKNOWLEDGMENTS……………………………………………………VIII
TABLE OF CONTENTS………………………………………………………X
LIST OF FIGURES……………………………………………………XVIII
LIST OF TABLES……………………………………………………XXVIII
CHAPTER 1 BACKGROUND AND LITERATURE REVIEW……………………1
1.1 Motivation and Objectives..............................2
1.2 Corneal Endothelium....................................5
1.2.1 Cornea...............................................5
1.2.2 Endothelium: Structure and Function..................7
1.2.3 Lack of Proliferation in the Endothelial Monolayer In Vivo......................................................11
1.2.3.1 The Decrease in Endothelial Cell Density: Age and Trauma....................................................11
1.2.3.2 Mechanisms for Inhibition of Cell Proliferation In Vivo......................................................13
1.2.3.3 Endothelial Monolayer Repair: Cell Enlargement and Migration.................................................15
1.2.3.4 Endothelial Dysfunction from Excessive Cell Loss..17
1.3 Corneal Endothelial Cell Therapy......................18
1.3.1 Penetrating Keratoplasty............................18
1.3.2 Keratoprosthesis....................................20
1.3.2.1 Before 1900.......................................22
1.3.2.2 1950s.............................................22
1.3.2.3 1960s.............................................23
1.3.2.4 1970s and 1980s...................................25
1.3.2.5 After 1990........................................25
1.3.3 Cell-Based Therapy..................................30
1.3.3.1 Injection of Isolated Cell Suspensions............30
1.3.3.2 Transplantation of Tissue-Engineered Constructs...31
1.4 Cell Sheet Engineering................................41
1.4.1 Tissue Engineering..................................41
1.4.2 Cell-Culture Substrate Interaction..................44
1.4.3 Fabrication of Tissue-Engineered Cell Sheets........46
1.4.4 Rapid Cell Detachment and Cell Sheet Transfer.......48
1.4.5 Biomedical Applications.............................50
1.5 Biomaterials..........................................57
1.5.1 Poly(N-isopropylacrylamide).........................57
1.5.2 Thermo-Responsive Culture Support...................60
1.5.3 Gelatin.............................................63
1.5.4 Biofunctional Hydrogel Carrier System...............65
1.6 Dissertation Layouts..................................68
CHAPTER 2 THERMO-RESPONSIVE POLY(N-ISOPROPYLACRYLAMIDE)(PNIPAAM)-GRAFTED CULTURE SURFACES FOR CONTROLLING CELL ADHESION AND DETACHMENT……………………………………………70
2.1 Introduction..........................................71
2.2 Experimental..........................................77
2.2.1 Materials...........................................77
2.2.2 Preparation of Thermo-Responsive Culture Substrates.78
2.2.2.1 Glow Discharge....................................78
2.2.2.2 PAAc-g-PE Surfaces: Thermal Graft Polymerization..79
2.2.2.3 PNIPAAm-g-PAAc-g-PE Surfaces: Photografting Polymerization............................................80
2.2.3 Surface Characterization............................81
2.2.3.1 Peroxide Determination............................81
2.2.3.2 Attenuated Totoal Reflection-Fourier Transform Infrared Spectroscopy.....................................81
2.2.3.3 Energy-Dispersive X-Ray Spectroscopy..............81
2.2.3.4 Atomic Force Microscopy...........................82
2.2.3.5 Static Contact Angle Measurements.................82
2.2.4 Cell Resource.......................................82
2.2.5 Cell Adhesion/Detachment Assay......................83
2.3 Results and Discussion................................84
2.3.1 Peroxide Determination..............................84
2.3.2 PAAc-g-PE Surfaces: Thermal Graft Polymerization....85
2.3.3 PNIPAAm-g-PAAc-g-PE Surfaces: Photografting Polymerization............................................89
2.3.3.1 Attenuated Totoal Reflection-Fourier Transform Infrared Spectroscopy.....................................89
2.3.3.2 Energy-Dispersive X-Ray Spectroscopy..............89
2.3.3.3 Atomic Force Microscopy...........................91
2.3.3.4 Static Contact Angle Measurements.................93
2.3.4 Cell Adhesion/Detachment Assay......................94
2.4 Conclusions...........................................99
CHAPTER 3 BIOENGINEERED HUMAN CORNEAL ENDOTHELIAL CELL (HCEC) SHEETS AS FUNCTIONAL TISSUE EQUIVALENTS……………101
3.1 Introduction.........................................102
3.2 Experimental.........................................107
3.2.1 Materials..........................................107
3.2.2 Cell Preparation...................................108
3.2.3 Harvest of HCEC Monolayers from Thermo-Responsive Culture Supports.........................................109
3.2.4 Viability Bioassay.................................110
3.2.5 Scanning Electron Microscopy.......................111
3.2.6 Immunohistochemistry...............................111
3.2.7 Histology..........................................112
3.3 Results and Discussion...............................113
3.3.1 Cell Preparation...................................113
3.3.2 Harvest of HCEC Monolayers from Thermo-Responsive Culture Supports.........................................117
3.3.3 Viability Bioassay.................................121
3.3.4 Scanning Electron Microscopy.......................124
3.3.5 Immunohistochemistry...............................126
3.3.6 Histology..........................................128
3.4 Conclusions..........................................130
CHAPTER 4    MULTI-FUNCTIONAL GELATIN HYDROGEL CARRIERS FOR INTRAOCULAR DELIVERY OF HUMAN CORNEAL ENDOTHELIAL CELL (HCEC) SHEET GRAFTS…………………………………………………132
4.1 Introduction.........................................133
4.2 Experimental.........................................137
4.2.1 Materials..........................................137
4.2.2 Preparation of Gelatin Hydrogel Discs..............139
4.2.3 Tensile Tests......................................139
4.2.4 Determination of Water Content and Dissolution Degree...................................................140
4.2.5 Cell Culture.......................................141
4.2.6 Cell Proliferation Assay...........................141
4.2.7 Cell Viability Assay...............................142
4.2.8 Cytokine Expression................................143
4.2.9 Gelatin Carriers for Cell Sheet Delivery...........144
4.2.10 Statistical Analysis..............................146
4.3 Results and Discussion...............................147
4.3.1 Tensile Tests......................................147
4.3.2 Determination of Water Content and Dissolution Degree...................................................148
4.3.3 Cell Proliferation Assay...........................151
4.3.4 Cell Viability Assay...............................156
4.3.5 Cytokine Expression................................159
4.3.6 Gelatin Carriers for Cell Sheet Delivery...........159
4.4 Conclusions..........................................164
CHAPTER 5    HUMAN CORNEAL ENDOTHELIAL CELL (HCEC) SHEET TRANSPLANTATION FOR OCULAR RECONSTRUCTION……………………167
5.1 Introduction.........................................168
5.2 Experimental.........................................171
5.2.1 Materials..........................................171
5.2.2 HCEC Cultivation...................................172
5.2.3 PKH26 Labeling of Cultivated HCECs.................173
5.2.4 Cultivation and Harvest of HCEC Sheets from Thermo-Responsive Culture Surfaces..............................173
5.2.5 Electron Microscopy................................174
5.2.6 Western Blot Analyses of Na+/K+-ATPase and ZO-1....175
5.2.7 Preparation and Characterization of Gelatin Discs..175
5.2.8 Establishment of Corneal Endothelium Trauma Model of Rabbits..................................................176
5.2.9 Transplantation of HCEC Monolayers Using Gelatin Discs as Carriers........................................177
5.2.10 Histological Examination and Localization of HCECs....................................................178
5.3 Results and Discussion...............................179
5.3.1 Cultivation of PKH26 Labeled HCECs on PNIPAAm-Grafted Surfaces.................................................179
5.3.2 Detachment of HCEC Monolayers from PNIPAAm-Grafted Surfaces.................................................180
5.3.3 Electron Microscopy................................180
5.3.4 Western Blot Analyses of Na+/K+-ATPase and ZO-1....183
5.3.5 Delivery of HCEC Monolayers by Gelatin Discs.......185
5.3.6 Clinical Observations of the Surgical Corneas......188
5.3.7 Central Corneal Thickness Measurements.............188
5.3.8 Histological Examination and Localization of HCECs.189
5.4 Conclusions..........................................198
CHAPTER 6    SUMMARY AND FUTURE DIRECTIONS………………………200
REFERENCES……………………………………………………………205
BIBLIOGRAPHY…………………………………………………………231

LIST OF FIGURES

Figure 1.1 A novel strategy for corneal endothelial reconstruction with bioengineered cell sheets by utilizing functional biomedical materials............................4
Figure 1.2 Diagrammatic horizontal cross-section of a vertebrate eye.............................................6
Figure 1.3 Schematic transverse section of the cornea......7
Figure 1.4 Schematic illustration of the fine structure of human corneal.endothelium..................................9
Figure 1.5 Corneal endothelial barrier and pump function..11
Figure 1.6 Age-related decrease in corneal endothelial cell density in 56 pre- and postnatal samples, ranging in age from 16 weeks of gestation to 98 years old................12
Figure 1.7 The average cell density of human corneal endothelium at different stages of lifespan...............13
Figure 1.8 Mechanisms for inhibition of corneal endothelial cell proliferation in vivo................................14
Figure 1.9 Schematic illustration of altered corneal endothelial cell shape (A) and different corneal endothelial cell density (B)..............................16
Figure 1.10 Schematic illustration of penetrating keratoplasty..............................................19
Figure 1.11 Schematic illustration of the experimental keratoprosthesis design...................................21
Figure 1.12 Schematic illustration of implantation of keratoprosthesis..........................................21
Figure 1.13 Schematic illustration of Cardona “through-and-through” keratoprosthesis................................24
Figure 1.14 Schematic illustration of Girard keratoprosthesis..........................................24
Figure 1.15 Schematic illustration of a homobifunctional SR membrane..................................................27
Figure 1.16 Schematic illustration for biofunction of a heterobifunctional membrane...............................28
Figure 1.17 Cross-sectional view of preparation of a heterobifunctional membrane...............................29
Figure 1.18 Injection of 5 × 105 cells into the anterior chamber of an eye which has had the corneal endothelium chemically destroyed with benzalkonium chloride...........31
Figure 1.19 A button seeded with a suspension of corneal endothelial cells from tissue culture can be used as the donor source for standard PK..............................32
Figure 1.20 Flow diagram illustrating the procedures used to produce donor corneas with either full human endothelial cell enhancement or replacement...........................34
Figure 1.21 A thin carrier membrane, serves as substrate for growth of tissue cultured corneal endothelial cells in the case of autologous transplantation....................35
Figure 1.22 Schematic representation of corneal button glued to underlying membrane by a continuous peripheral ring of cyanoacrylate adhesive............................37
Figure 1.23 Diagram of surgical technique used to implant the cell carrier device...................................39
Figure 1.24 In one approach to open-system implants, 3-dimensional highly porous scaffolds composed of synthetic polymers serve as cell transplant devices.................43
Figure 1.25 Mechanism of the cell attachment to and detachment from material surfaces.........................44
Figure 1.26 Cell harvest mechanism by using temperature-responsive culture surfaces...............................46
Figure 1.27 Cell sheet release from temperature-responsive culture surfaces..........................................47
Figure 1.28 An illustration of cell sheet detachment by different types of water supply to (a) the PNIPAAm-grafted TCPS surface and (b) the PNIPAAm-grafted porous membrane..................................................49
Figure 1.29 Schematic illustration of cell sheet transfer.50
Figure 1.30 Three contexts in cell sheet engineering......51
Figure 1.31 Schematic illustration of 3-dimensional manipulation of viable cultured cardiomyocyte sheets (CMS).....................................................52
Figure 1.32 Corneal epithelial cell sheet transplantation.54
Figure 1.33 Tissue-engineered epithelial cell sheets fabricated from oral mucosal epithelium...................56
Figure 1.34 Chemical structure of a representative thermo-sensitive polymer, PNIPAAm................................58
Figure 1.35 The concept of release mechanism from thermo-sensitive PNIPAAm-g-PHEMA gel particles...................60
Figure 1.36 A plasma treatment apparatus was constructed for processing of materials and forming a film on the surface of a substrate....................................62
Figure 1.37 Preparative process for acidic (negatively charged) and basic (positively charged) gelatins from collagen..................................................64
Figure 1.38 Release of protein drug from biodegradable polymer carrier on the basis of polyion complexation......65
Figure 1.39 Schematic illustration of retinal transplantation with sandwich and trephination............66
Figure 1.40 Histological examination of sandwich-like sheet grafts in the rabbit subretinal space 2 weeks postoperatively, frozen-sectioned, and H&E stained........67
Figure 1.41 Dissertation layouts..........................69
Figure 2.1 Schematic illustration of a two-step method based on plasma-induced graft polymerization for preparation of thermo-responsive PNIPAAm-grafted culture surfaces..................................................76
Figure 2.2 Schematic diagram of the plasma treatment apparatus.................................................79
Figure 2.3 Formation of peroxide groups (mol/cm2) on the PE substrate surfaces after activation by Ar plasma with different treatment times (s) and discharge powers (W)....85
Figure 2.4 Dependence of the grafted amount of PAAc on the concentration of monomer aqueous solution at Ar plasma treatment (120 W, 90 s)...................................86
Figure 2.5 Effect of the concentration of peroxides on the grafted amount of PAAc on the PE substrates...............87
Figure 2.6 Effect of the concentration of Mohr's salt in the redox system on the grafted amount of PAAc on the PE substrates................................................88
Figure 2.7 ATR-FTIR spectra of (a) untreated PE specimens, and (b) PNIPAAm-grafted PE surfaces (1.6 μg/cm2) with the characteristic structures of isopropyl group, amide I, and amide II..................................................90
Figure 2.8 AFM tapping mode images from surfaces of (a) untreated PE specimens, and (b) PNIPAAm-grafted PE samples (1.6 μg/cm2).............................................92
Figure 2.9 Representative photographs of PNIPAAm-grafted PE substrates with high grafting yield (1846 μg/cm2) at different temperatures....................................96
Figure 2.10 HCECs cultured on the PNIPAAm-grafted PE surfaces with different grafting yields...................97
Figure 2.11 Cell detachment assay by the low-temperature treatment for (A) untreated PE, and (B) PNIPAAm-grafted PE culture substrates (1.6 μg/cm2)..........................98
Figure 3.1 Representative topographic image of PNIPAAm-grafted surface is observed by tapping-mode atomic force microscopy...............................................105
Figure 3.2 Fabrication of bioengineered human corneal endothelium from thermo-responsive culture supports.................................................106
Figure 3.3 Morphological features of cultured, untransformed human corneal endothelial cells, corneal epithelial cells, and stromal fibroblasts................114
Figure 3.4 RT-PCR detection of mRNA for G3PDH (452 bp), keratin 12 (409 bp), and collagen VIII α-1 (496 bp) in fresh human corneal epithelium, fresh human corneal endothelium, cultured human corneal epithelium, and cultured human corneal endothelium.......................115
Figure 3.5 Transmission electron micrographs of human corneal endothelial cells 1 (A) and 2 weeks (B) post-transplantation..........................................116
Figure 3.6 Phase-contrast micrographs of HCEC cultures on the thermo-responsive supports after incubation at 37°C for different time intervals.................................118
Figure 3.7 Gross observations of HCEC monolayer detachment from thermo-responsive culture supports after incubation at 20°C for (A) 10 min, (B) 25 min, and (C) 45 min..........120
Figure 3.8 Phase-contrast micrograph of HCEC monolayer separation from thermo-responsive culture support at 20°C........................................................121
Figure 3.9 Viability of detached HCEC monolayers was assessed by staining with a LIVE/DEAD Viability/Cytotoxicity Kit in which the live cells fluoresce green and the dead cells fluoresce red.........123
Figure 3.10 SEM micrographs of human eye bank corneas (control samples) and HCEC sheets........................125
Figure 3.11 Fluorescence micrographs of immunolocalization of ZO-1 in HCECs within detached cell sheets compared to control samples..........................................127
Figure 3.12 Fluorescence micrographs of immunolocalization of Na+/K+-ATPase in HCECs within detached cell sheets compared to control samples..............................128
Figure 3.13 Histological examination of control samples (A) and HCEC sheets (B) with DAPI (blue fluorescence) labeling show a monolayer of endothelial cells (arrows) is located on corneal stroma (asterisk) and cell carrier membrane (double asterisk), respectively..........................129
Figure 4.1 After cell separation from thermo-responsive PNIPAAm-grafted culture surfaces via thermal stimulus, a gelatin hydrogel carrier exhibiting multi-functions was used for the transfer of HCEC sheets.....................136
Figure 4.2 Time course of water content of various gelatin hydrogel discs after incubation in BSS at 34°C...........149
Figure 4.3 Typical gross photographs of gelatin discs (MW = 100 kDa) are shown (A) before testing, and after incubation in BSS at 34°C for distinct time periods (B) 5, (C) 60, (D) 360, and (E) 1440 min....................................150
Figure 4.4 Time course of dissolution degree of various gelatin hydrogel discs after incubation in BSS at 34°C...153
Figure 4.5 Phase-contrast micrographs of HCEC cultures in (A) controls (without materials) after a 2-day exposure to different types of gelatin discs (B) G-5-3, (C) G-5-8, (D) G-5-100, and (E) G-9-100.................................154
Figure 4.6 Cell proliferation assay of HCEC cultures incubated for 2 days at 37°C with various gelatin hydrogel discs....................................................155
Figure 4.7 Live/Dead assay of the thermally detached HCEC sheets in (A) controls (without materials) after direct contact with different types of gelatin discs (B) G-5-3, (C) G-5-8, (D) G-5-100, and (E) G-9-100 for 2 days at 37°C........................................................157
Figure 4.8 Mean percentage of live cells in the HCEC sheets incubated in direct contact with various gelatin hydrogel discs as determined by the Live/Dead assay...............158
Figure 4.9 The level of IL-6 released from HCEC cultures after incubation for 2 days at 37°C with various gelatin hydrogel discs...........................................162
Figure 4.10 Gelatin carriers for intraocular delivery of thermally detached HCEC sheets...........................163
Figure 5.1 The morphology of cultivated HCECs on PNIPAAm-grafted surfaces.........................................181
Figure 5.2 Gross observations of HCEC sheet detachment...182
Figure 5.3 Morphology of the harvested HCEC monolayers was observed by SEM and TEM..................................184
Figure 5.4 Western blot analyses of ZO-1 and Na+/K+-ATPase α1 subunit expression in HCEC sheets....................185
Figure 5.5 Surgical procedures of HCEC monolayer delivery using gelatin carriers...................................187
Figure 5.6 Representative slit-lamp biomicroscopic images of rabbit corneas denuded of endothelium (wound groups)..190
Figure 5.7 Representative slit-lamp biomicroscopic images of rabbit corneas receiving bioengineered HCEC sheets (HCEC sheet groups)............................................191
Figure 5.8 Central corneal thickness measurements during the follow-up period of 6 months.........................192
Figure 5.9 Histological examination of rabbit corneas after implantation of HCEC sheets for different time intervals.193
Figure 5.10 Gross examination of excised rabbit corneas 1 week after surgery.......................................194
Figure 5.11 Immunohistochemistry of the excised corneas after transplantation for 2 weeks........................195

LIST OF TABLES

Table 1.1 A brief history of artificial corneas...........30
Table 1.2 Corneal endothelial cell transplantation using donor corneal buttons.....................................33
Table 1.3 Corneal endothelial cell transplantation using carrier substrates........................................36
Table 1.4 A brief history of corneal endothelial cell therapy...................................................40
Table 2.1 Elemental composition of control and PNIPAAm-grafted PE surfaces analyzed by EDX.......................91
Table 2.2 Water contact angles of control and PNIPAAm-grafted PE surfaces measured by sessile drop method.......93
Table 4.1 Physicochemical characteristics of raw gelatins.................................................138
Table 4.2 Tensile properties of the gelatin hydrogel carriers.................................................147

                                

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