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研究生:王鵬元
研究生(外文):Peng-Yuan Wang
論文名稱:細胞在奈米/次微米溝槽表面的行為研究
論文名稱(外文):Modulation of Cellular Behaviors by Nano/Submicron Groove/Ridge Surfaces
指導教授:蔡偉博
指導教授(外文):Wei-Bor Tsai
口試委員:林睿哲謝學真陳志平李澤民鍾次文王孟菊游佳欣
口試日期:2011-07-08
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:化學工程學研究所
學門:工程學門
學類:化學工程學類
論文出版年:2011
畢業學年度:99
語文別:英文
論文頁數:166
中文關鍵詞:微/奈米溝槽地形平行排列骨骼肌肉心肌間葉幹細胞硬骨/脂肪/肌肉分化
外文關鍵詞:grooved topographyanisotropicalignmentstem cellsdifferentiation
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利用細胞周圍微環境的物理因素,例如材料表面地形,調控細胞的生長和分化行為對於生醫裝置、人體植入物、和組織工程的應用具有重要的影響。物理刺激,例如細胞貼附在具有溝槽的表面,已經證實對於細胞表現具有不同生物程度的影響。本論文的目地為瞭解不同尺寸的微/奈米溝槽表面地形,對於骨骼肌肉細胞、心肌細胞、與間葉幹細胞生長與分化的影響。結果顯示,微/奈米溝槽表面對於三種細胞的排列有顯著的影響,其主要受到溝槽深度的支配,而並非寬度。骨骼肌肉細胞在溝槽表面產生順延後,肌小管融合指數比在平坦表面高,推斷是由於平行排列的骨骼肌肉細胞增加了頭尾相接的機會,進而促進融合行為。而將溝槽表面接枝胜肽後,不但產生平行的肌小管,也提升了整體肌小管融合指數。心肌細胞在溝槽表面產生順延後,細胞的收縮速率較平坦表面高,推斷是由於平行排列的心肌細胞收縮的方向較一致,力量不會互相抵銷導致。而將溝槽表面轉印在較柔軟的材料上,可以延長心肌細胞的收縮功能,證明地形與材料硬度均很重要。間葉幹細胞在微/奈米溝槽表面產生順延後,生長速率和硬骨分化的程度並沒有顯著的改變。但是,對於早期肌肉分化與脂肪分化,在溝槽表面均要比在平坦表面要高。化學的因子與物理的溝槽地形均會對間業幹細胞的分化產生影響,但前者的效應可能更大。本研究證實,微/奈米溝槽表面改變了細胞貼附行為、細胞型態、細胞分化、與細胞的功能。本研究的結果,希望能對於生醫材料的設計、細胞生理學、與組織工程的發展有所幫助。

Directing mammalian cell behavior using biophysical cues such as topography in the extracellular microenvironment is crucial for the successes in the biomedical devices, implants, and tissue engineering. It has been suggested that groove/ridge topography modulate cellular behaviors in different biological level. The goal of this dissertation is to understand the effect of nano/submicron grooved surface on the behaviors of skeletal myoblasts, cardiomyocytes, and rat mesenchymal stem cells (rMSCs). Nano/submicron grooved surfaces affect focal adhesion, cell alignment, and function of cell in a remarkable manner, dominating by the depth of groove. Myogenic index of aligned skeletal myoblasts is enhanced on the grooved surfaces compared to the flat control, due to increase in the end-to-end fusion. Peptide conjugation further enhances the myogenic index and meantime morphologically parallel myotubes. The contractile function of aligned cardiomyocytes is upregulated on the grooved surface compared to the flat control, because the anisotropic morphology facilitates synchronous contraction of cardiomyocytes. The rigidity of substrate also affects the contraction of cardiomyocytes. Grooved surfaces have minor effect on cell proliferation and osteogenesis of rMSCs compared to the flat control, while these surfaces enhance the early myogenesis and adipogenesis of rMSCs. Chemical factor and topographic cue both affect the rMSCs differentiation, while the former plays a more momentous role. Taken together, nano/submicron grooved surface modulated focal adhesion and cell morphology anisotropically, depending on the feature size, which in turn changes the differentiation and functionality of cells in a cell-type dependent manner.

ACKNOWLEDGMENTS I
摘要 II
ABSTRACT III
TABLE OF CONTENTS V
LIST OF FIGURES IX
LIST OF TABLES XI
CHAPTER 1 INTRODUCTION 1
1.1. EXTRACELLULAR MATRIX (ECM) 1
1.2. CELL-ECM INTERACTION 2
1.3. TOPOGRAPHIC FEATURES OF ECM 4
1.4. IN VIVO ANISOTROPIC ECM TOPOGRAPHIES 6
1.5. IN VITRO MIMIC NANO/SUBMICRON ANISOTROPIC TOPOGRAPHIES 7
1.6. CELLULAR RESPONSES TO NANO/SUBMICRON ANISOTROPIC TOPOGRAPHY 9
1.7. RESEARCH MOTIVATION 16
1.8. RESEARCH FRAMEWORK 17
1.9. FIGURES 19
CHAPTER 2 MATERIALS AND METHODS 23
2.1 MATERIALS 23
2.2. FABRICATION AND CHARACTERIZATION OF GROOVED POLYSTYRENE 23
2.3. FLUORESCENT STAINING 24
2.4. STATISTIC ANALYSIS 24
CHAPTER 3 MODULATION OF ALIGNMENT AND DIFFERENTIATION OF SKELETAL MYOBLASTS BY NANO/SUBMICRON RIDGE/GROOVE SURFACE STRUCTURE 27
3.1. ABSTRACT 27
3.2. INTRODUCTION 28
3.3. MATERIALS AND METHODS 30
3.3.1. Materials 30
3.3.2. Fabrication and characterization of grooved polystyrene 30
3.3.3. Cell culture and characterization of cell morphology 31
3.3.4. Differentiation of myoblasts into myotubes 32
3.3.5. Fluorescent staining of nuclei, myosin heavy chain and F-actin of myotubes 32
3.4. RESULTS 33
3.4.1. Surface characterization by water contact angle measurement 33
3.4.2. Morphology of C2C12 cells prior to differentiation 34
3.4.3. Proliferation and differentiation of myoblasts 35
3.5. DISCUSSION 36
3.6. CONCLUSIONS 40
3.7. FIGURES AND TABLES 41
CHAPTER 4 MODULATION OF NANOGROOVED TOPOGRAPHY AND LIGAND PRESENTATION ON C2C12 MYOBLAST DIFFERENTIATION 53
4.1. ABSTRACT 53
4.2. INTRODUCTION 54
4.3. MATERIALS AND METHODS 56
4.3.1. Materials 56
4.3.2. Fabrication and Characterization of Grooved Surface 57
4.3.3. Surface characterization 57
4.3.4. C2C12 Myoblasts Culture 58
4.3.5. Scanning Electronic Microscopy 58
4.3.6. Immunofluorescent Staining for Focal Adhesion and Myosin Heavy Chain 58
4.3.7. Myogenic index 59
4.4. RESULTS 59
4.4.1. Preparation and Characterization of nanogrooved Substrates 59
4.4.2. C2C12 myoblasts attachment 60
4.4.3. C2C12 myoblasts fusion and myogenic index 61
4.5. DISCUSSION 63
4.6. CONCLUSIONS 66
4.7. FIGURES AND TABLES 67
CHAPTER 5 MODULATION OF ALIGNMENT, ELONGATION AND CONTRACTION OF CARDIOMYOCYTES THROUGH COMBINATION OF NANOTOPOGRAPHY AND RIGIDITY OF SUBSTRATES 77
5.1. ABSTRACT 77
5.2. INTRODUCTION 78
5.3. MATERIALS AND METHODS 80
5.3.1. Materials 80
5.3.2. Fabrication and Characterization of Nanogrooved Substrates 81
5.3.3. Isolation and Culture of Cardiomyocytes 82
5.3.4. Analysis of Cell Morphology 82
5.3.5. Immunofluorescence Staining 83
5.3.6. Contraction of cardiomyocytes 84
5.4. RESULTS 84
5.4.1. Preparation and Characterization of nanogrooved Substrates 84
5.4.2. Cardiomyocyte Culture at Low Seeding Density 84
5.4.3. Cardiomyocyte Culture at High Seeding Density 85
5.5. DISCUSSION 87
5.6. CONCLUSIONS 93
5.7. FIGURES 94
CHAPTER 6 MODULATION OF ALIGNMENT AND DIFFERENTIATION OF RAT MESENCHYMAL STEM CELL BY NANO/SUBMICRON GROOVED SURFACES 103
6.1. ABSTRACT 103
6.2. INTRODUCTION 104
6.3. MATERIALS AND METHODS 107
6.3.1. Materials 107
6.3.2. Preparation of the Nano/Submicron Grooved Surfaces 107
6.3.3. Rat Mesenchymal Stem Cell 107
6.3.4. rMSCs Culture and Proliferation on the Nano/Submicron Grooved Surfaces 108
6.3.5. Cell Morphology and Focal Adhesion Staining 109
6.3.6. Differentiation of rMSCs on the Nano/Submicron Grooved Surfaces 109
6.3.7. ALP Activity and ALP Deposition Staining for Osteogenesis 110
6.3.8. Alizarin Red S Staining and Calcium Quantification for Osteogenesis 111
6.3.9. Oil Red-O Staining and Triacylglycerols Quantification for Adipogenesis 111
6.3.10. Desmin and Myosin Heavy Chain Staining for Myogenesis 111
6.4. RESULTS 112
6.4.1. Nano/Submicron Grooved surfaces fabrication 112
6.4.2. Focal adhesion and Cell Morphology on the Grooved Surfaces 113
6.4.3. Cell Proliferation on the Grooved Surfaces 113
6.4.4. Osteogenesis of rMSCs on the Grooved Surfaces 114
6.4.5. Adipogenesis of rMSCs on the Grooved Surfaces 115
6.4.6. Myogenesis of rMSCs on the Grooved Surfaces 115
6.5. DISCUSSION 116
6.6. CONCLUSIONS 123
6.7. FIGURES 125
CHAPTER 7 GENERAL CONCLUSIONS 133
CHAPTER 8 FUTURE WORKS 141
CHAPTER 9 REFERENCES 143
APPENDIX 161
AUTOBIOGRAPHY 162
PUBLICATION LISTS 163

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