Hydrogel Scaffolds to Modulate Smooth Muscle Cell Phenotype
Total Page：16
File Type：pdf, Size：1020Kb
ENGINEERING POLY(ETHYLENE GLYCOL) HYDROGEL SCAFFOLDS TO MODULATE SMOOTH MUSCLE CELL PHENOTYPE by JEFFREY ALAN BEAMISH Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisors: Dr. Roger E. Marchant and Dr. Kandice Kottke-Marchant Department of Biomedical Engineering CASE WESTERN RESERVE UNIVERSITY August, 2009 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ______________________________________________________ candidate for the ________________________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. To my loving wife, Susan, and my parents, who have provided unwavering support for all my endeavors Table of Contents Table of Contents ...............................................................................................................i List of Tables ....................................................................................................................iii List of Figures................................................................................................................... iv Acknowledgements .......................................................................................................... vi List of Abbreviations ....................................................................................................... ix Abstract........................................................................................................................... xiv CHAPTER 1: Cardiovascular Disease, Bypass Grafting, and Intimal Hyperplasia. 1 1.1. Summary of Cardiovascular Disease in the United States....................................... 1 1.2. Atherosclerosis......................................................................................................... 1 1.3. Treatment Approaches for Cardiovascular Disease................................................. 2 1.4. Anastomotic Intimal Hyperplasia............................................................................. 5 1.5. References .............................................................................................................. 15 CHAPTER 2: Regulation of SMC Phenotype: Implications for Vascular Tissue Engineering...................................................................................................................... 20 2.1. Introduction ............................................................................................................ 20 2.2. The Continuum of SMC Phenotypes ..................................................................... 21 2.3. Markers of Contractile SMC Phenotype ................................................................ 24 2.4. Mediators of SMC Phenotype................................................................................ 30 2.5. Molecular Regulation of SMC Gene Expression................................................... 48 2.6. Engineered Biomaterial Approaches to Regulate SMC Phenotype....................... 54 2.7. Conclusions ............................................................................................................ 58 2.8. References .............................................................................................................. 61 CHAPTER 3: Vascular Tissue Engineering................................................................. 83 3.1. Introduction ............................................................................................................ 83 3.2. Normal Blood Vessel Histology ............................................................................ 83 3.3. Design Criteria ....................................................................................................... 84 3.4. Tissue Engineered Modification of Existing Graft Materials ................................ 84 3.5. Fully Tissue Engineered Blood Vessels (TEBVs) ................................................. 87 3.6. Discussion .............................................................................................................. 99 3.7. PEG-based Hydrogels for Tissue Engineering..................................................... 103 3.8. Specific Aims ....................................................................................................... 108 3.9. References ............................................................................................................ 111 CHAPTER 4: The Effects of Monoacrylated Poly(Ethylene Glycol) on the Properties of Poly(Ethylene Glycol) Diacrylate Hydrogels Used for Tissue Engineering.................................................................................................................... 119 4.1. Introduction .......................................................................................................... 119 4.2. Materials and Methods......................................................................................... 123 4.3. Results .................................................................................................................. 129 4.4. Discussion ............................................................................................................ 136 4.5. Conclusions .......................................................................................................... 142 i 4.6. Acknowledgements .............................................................................................. 142 4.7. References ............................................................................................................ 143 CHAPTER 5: The Influence of RGD-Bearing Hydrogels on the Re-expression of Contractile Vascular Smooth Muscle Cell Phenotype............................................... 146 5.1. Introduction .......................................................................................................... 146 5.2. Materials and Methods......................................................................................... 148 5.3. Results .................................................................................................................. 156 5.4. Discussion ............................................................................................................ 167 5.5. Conclusions .......................................................................................................... 172 5.6. Acknowledgements .............................................................................................. 172 5.7. References ............................................................................................................ 174 CHAPTER 6: The Effects of Heparin Releasing Hydrogels on Vascular Smooth Muscle Cell Phenotype ................................................................................................. 178 6.1. Introduction .......................................................................................................... 178 6.2. Materials and Methods......................................................................................... 180 6.3. Results .................................................................................................................. 189 6.4. Discussion ............................................................................................................ 201 6.5. Conclusions .......................................................................................................... 207 6.6. Acknowledgments................................................................................................ 207 6.7. References ............................................................................................................ 208 CHAPTER 7: Conclusions and Future Directions .................................................... 213 7.1. Summary and Conclusions of Completed Work.................................................. 213 7.2. Engineering of Improved Scaffold Systems......................................................... 215 7.3. Modulation of Cultured SMCs Toward a Contractile Phenotype........................ 226 7.4. References ............................................................................................................ 233 Bibliography .................................................................................................................. 238 ii List of Tables Table 3.1-Design criteria for tissue engineered blood vessels 86 Table 4.1-Chemical structures of PEGDA and PEGMA 124 Table 5.1-Primers used for real-time RT-PCR 153 Table 6.1-Summary of heparin release from PEGDA hydrogels 193 Table 6.2-Network properties of PEGDA hydrogels 196 Table 6.3-Heparin concentration in medium during transwell insert study 199 iii List of Figures Figure 1.1-Typical distribution of intimal hyperplasia 7 Figure 2.1-Summary of characteristics of SMC phenotypes 23 Figure 2.2-Mechanisms of SMC phenotype modulation 32 Figure 2.3-Chemical composition and structure of heparin 34 Figure 2.4-Molecular regulation of smooth muscle α-actin transcription 53 Figure 3.1-Structure