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A Multiscale Approach to Stem Cell-Based

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A Multiscale Approach to Stem Cell-Based A MULTISCALE APPROACH TO STEM CELL-BASED CHONDROGENESIS FOR CARTILAGE REPAIR by CHIH-LING CHOU Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Harihara Baskaran Department of Chemical Engineering CASE WESTERN RESERVE UNIVERSITY May, 2013 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Chih-Ling Chou candidate for the Doctor of Philosophy degree *. (signed) Dr. Harihara Baskaran . (chair of the committee) Dr. Jean F. Welter . Dr. Chung-Chiun Liu . Dr. Heidi B. Martin . Date of Defense March 14, 2013. *We also certify that written approval has been obtained for any proprietary material contained therein. TABLE OF CONTENTS Table of contents .................................................................................................................. I List of tables ...................................................................................................................... VI List of figures ................................................................................................................... VII Acknowledgements ........................................................................................................... XI Abstract .......................................................................................................................... XIII Chapter 1. Introduction ....................................................................................................... 1 1.1 Purpose of the Study ............................................................................................ 1 1.2 Significance and Motivation of the study .......................................................... 2 1.2.1 Articular Cartilage Injury ................................................................................. 2 1.2.2 Limitations of Current Treatment Options ....................................................... 2 1.2.3 Challenges for Tissue-Engineered (TE) Cartilage ............................................ 3 1.3 Overall Research Goals ........................................................................................ 6 1.4 Thesis Outline ...................................................................................................... 8 1.5 Summary .............................................................................................................. 9 1.6 References ............................................................................................................ 9 Chapter 2. Background ..................................................................................................... 12 2.1 Articular Cartilage ................................................................................................... 12 2.1.1 Knee joint and Cartilage ................................................................................... 12 I 2.1.2 Ultrastructure and Constituents of Articular cartilage ...................................... 13 2.1.3 Articular Cartilage Injury and Repair ............................................................... 17 2.1.4 Current Treatment for Articular Cartilage Injury ............................................. 20 2.2 Cartilage Tissue engineering ................................................................................... 25 2.2.1 Cell Sourcing for Cartilage Tissue engineering ............................................... 27 2.2.2 Biomaterials for Cartilage Tissue Engineering ................................................ 29 2.2.3 Challenges in Cartilage Tissue Engineering ..................................................... 31 2.3 Cartilage Structure and Guidance ........................................................................... 34 2.3.1 Contact Guidance ............................................................................................. 34 2.3.2 Biomechanics of Articular Cartilage ................................................................ 36 2.3.3 Structure and Guidance .................................................................................... 38 2.4 References ............................................................................................................... 39 Chapter 3. Control of human mesenchymal stem selective adhesion on collagen- glycosaminoglycan and Poly(dimethyl siloxane) surfaces .............................................. 51 3.1 Introduction ............................................................................................................. 51 3.2 Materials and Methods ............................................................................................ 54 3.2.1 Materials ........................................................................................................... 54 3.2.2 CG Solution and EDC Solution Formation ...................................................... 55 3.2.3 Design of Microchannels .................................................................................. 55 3.2.4 Fabrication of Silicon Substrate Template with Microchannels ...................... 56 II 3.2.5 Fabrication of Collagen and PDMS Microchannels ........................................ 59 3.2.6 Selective Cell Attachment in Collagen Microchannels .................................... 63 3.2.7 Selective Cell Attachment in PDMS Microchannels ....................................... 65 3.2.8 Cell Culture....................................................................................................... 66 3.2.9 MSC Viability, and Adhesion Assessment....................................................... 68 3.3 Results .................................................................................................................. 68 3.3.1 CG Membrane Patterning ................................................................................. 68 3.3.2 MSC Viability and Adhesion Assessment....................................................... 70 3.2.3 Selective Cell Attachment ................................................................................ 71 3.4 Discussion ............................................................................................................ 74 3.5 Conclusion ............................................................................................................ 78 3.6 References ............................................................................................................ 78 Chapter 4. Investigate the effect of microscale guidance on mesenchymal stem cell-based chondrogenesis .................................................................................................................. 81 4.1 Introduction ............................................................................................................. 81 4.2 Materials and Methods ............................................................................................ 89 4.2.1 Materials ........................................................................................................... 89 4.2.2 Fabrication of Collagen-hMSC construct and PDMS-hMSC construct ........... 90 4.2.3 Image Acquisition............................................................................................. 91 4.2.4 Histology and Immunohistochemistry.............................................................. 91 III 4.2.5 Cell alignment analysis ..................................................................................... 92 4.2.6 Mechanical Property Testing ............................................................................ 94 4.2.7 Biochemical Measurements .............................................................................. 96 4.3 Result ...................................................................................................................... 98 4.3.1 Cellular Growth and Organization in Microchannels ....................................... 98 4.3.2 Microscale Guidance on Cell alignment .......................................................... 99 4.3.3 ECM Production ............................................................................................. 105 4.3.4 Mechanical Properties .................................................................................... 108 4.4 Discussion ............................................................................................................ 111 4.5 Conclusion ............................................................................................................ 115 4.6 References ............................................................................................................. 116 Chapter 5. Design of large-scale 3-dimensional cartilage constructs with microchannels for preclinical studies ...................................................................................................... 123 5.1 Introduction ........................................................................................................... 123 5.2 Materials & Methods ............................................................................................. 127 5.2.4 Autonomous Rolled-up CG-hMSC Construct (ARCGs) Formation .............. 130 5.2.5 Induced Rolled-up CG-hMSC construct (IRCGs) Formation ........................ 131 5.2.6 Compression Testing ...................................................................................... 132 5.2.7 Histological and Immunohistochemistry Analysis ........................................
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