GUIDING CHONDROGENESIS THROUGH CONTROLLED GROWTH FACTOR PRESENTATION WITH POLYMER MICROSPHERES IN HIGH DENSITY STEM CELL SYSTEMS by LORAN DENISE SOLORIO Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Eben Alsberg Department of Biomedical Engineering CASE WESTERN RESERVE UNIVERSITY May 2012 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Loran Denise Solorio candidate for the Ph.D degree*. (signed) Eben Alsberg (chair of the committee) Jean Welter Melissa Knothe Tate Edward Greenfield Arnold Caplan (date) Jan. 10, 2012 *We also certify that written approval has been obtained for any proprietary material contained therein. To my husband, Luis, my sister, Eran, and my parents Table of Contents Table of Contents.......................................................................................................... 1 List of Tables................................................................................................................. 4 List of Figures............................................................................................................... 5 Acknowledgements....................................................................................................... 7 List of Abbreviations.................................................................................................... 8 Abstract.......................................................................................................................... 10 Chapter 1: Introduction................................................................................................ 12 1.1 Problem Statement.................................................................................................... 12 1.2 Hypothesis................................................................................................................. 13 1.3 Specific Aims and Strategy....................................................................................... 14 1.4 Significance............................................................................................................... 16 1.5 References................................................................................................................. 17 Chapter 2: Key Components of Cartilage Tissue Engineering Approaches.......... 20 2.1 Introduction............................................................................................................... 20 2.2 Cell Sources.............................................................................................................. 22 2.3 Chondrogenic Growth Factors.................................................................................. 25 2.4 Polymeric Biomaterials............................................................................................. 28 2.5 Mechanical Stimulation............................................................................................ 30 2.6 Conclusions............................................................................................................... 32 2.7 References................................................................................................................. 32 Chapter 3: High Density Cell Systems Incorporating Polymer Microspheres as Microenvironmental Regulators in Engineered Cartilage Tissues........................... 50 3.1 Introduction............................................................................................................... 50 3.2 Microspheres as Matrix Spacers................................................................................ 51 3.3 Microspheres as Cell Carriers.................................................................................... 55 3.4 Microspheres as Bioactive Factor Delivery Vehicles................................................ 57 1 3.5 Surface-Active Microspheres.................................................................................... 61 3.6 Conclusions............................................................................................................... 64 3.7 References................................................................................................................. 66 Chapter 4: Chondrogenic Differentiation of Human Mesenchymal Stem Cell Aggregates via Controlled Release of TGF-β1 from Incorporated Poly(Lactide-co- Glycolide) Microspheres................................................................................................ 74 4.1 Introduction.............................................................................................................. 74 4.2 Materials and Methods............................................................................................. 77 4.3 Results and Discussion............................................................................................. 80 4.4 Conclusions............................................................................................................... 85 4.5 Acknowledgements................................................................................................... 85 4.6 References................................................................................................................. 86 Chapter 5: Temporal Regulation of Chondrogenic Differentiation via Controlled Delivery of TGF- β1 from Gelatin Microspheres in Mesenchymal Stem Cell Aggregates..................................................................................................................... 88 5.1 Preface...................................................................................................................... 88 5.2 Introduction.............................................................................................................. 93 5.3 Materials and Methods............................................................................................. 95 5.4 Results....................................................................................................................... 99 5.5 Discussion.................................................................................................................. 104 5.6 Conclusions............................................................................................................... 110 5.7 Acknowledgements................................................................................................... 111 5.8 References................................................................................................................. 111 Chapter 6: Engineered Cartilage via Self-Assembled hMSC Sheets with Incorporated Biodegradable Gelatin Microspheres Releasing Transforming Growth Factor-β1......................................................................................................................... 116 6.1 Preface....................................................................................................................... 116 6.2 Introduction............................................................................................................... 116 2 6.3 Materials and Methods............................................................................................. 120 6.4 Results....................................................................................................................... 126 6.5 Discussion.................................................................................................................. 134 6.6 Conclusions............................................................................................................... 142 6.7 Acknowledgements.................................................................................................... 142 6.8 References.................................................................................................................. 143 Chapter 7: Conclusions and Future Directions......................................................... 149 7.1 Conclusions and Implications for Cartilage Tissue Engineering.............................. 149 7.2 Future Directions....................................................................................................... 152 7.3 References................................................................................................................. 153 Appendix I: Self-Assembling Human Adipose-Derived Stem Cell Sheets Incorporated with Transforming Growth Factor-β1-Releasing Biodegradable Polymer Microspheres for Cartilage Tissue Engineering.......................................... 154 I.1 Preface........................................................................................................................ 154 I.2 Introduction............................................................................................................... 155 I.3 Materials and Methods.............................................................................................. 157 I.4 Results........................................................................................................................ 164 I.5 Discussion.................................................................................................................. 169 I.6 Conclusions................................................................................................................ 174 I.7 Acknowledgements...................................................................................................