University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Summer 2010 Enhancing Mesenchymal Stem Cell Chondrogenesis for Cartilage Tissue Engineering Alice H. Huang University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Molecular, Cellular, and Tissue Engineering Commons Recommended Citation Huang, Alice H., "Enhancing Mesenchymal Stem Cell Chondrogenesis for Cartilage Tissue Engineering" (2010). Publicly Accessible Penn Dissertations. 183. https://repository.upenn.edu/edissertations/183 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/183 For more information, please contact [email protected]. Enhancing Mesenchymal Stem Cell Chondrogenesis for Cartilage Tissue Engineering Abstract Articular cartilage provides a bearing surface for transmitting forces across joints. The poor ability of cartilage to self-repair has motivated efforts to engineer replacement tissues, and mesenchymal stem cells (MSCs), which can undergo chondrogenesis, have emerged as a promising cell source. To date however, the functional properties of MSC-based constructs remain lower than those of the native tissue and of chondrocyte-based constructs cultured identically. Therefore, the overall objective of this thesis is to better understand the transcriptional and functional limitations underlying chondrogenic differentiation and enhance MSC chondrogenesis. Toward this end, established tissue engineering strategies from the chondrocyte literature were applied. Specifically, the effects of cell seeding density, media formulation and mechanical stimulation were examined with respect to functional growth. Transient application of TGF-β3 improved the compressive properties of MSC-laden constructs, but only when constructs were formed at a higher seeding density. Long-term dynamic compression initiated 3 days after MSC encapsulation impaired functional properties; in contrast, dynamic compression initiated after 3 weeks of chondrogenic pre-culture improved mechanical function. While these strategies enhanced functional chondrogenesis, the compressive properties achieved were ~50% of native tissue levels and did not reach chondrocyte levels. To understand the basis of this difference, microarray analysis was carried out to compare these two cells types and a set of molecular factors were identified as mis-expressed during MSC chondrogenesis. Although work up to this point focused on optimizing compressive properties, the tensile properties of articular cartilage are also critical to its functional role. In this work, we characterized the tensile properties of MSC-based constructs and demonstrated functional parity with chondrocyte-based constructs. To further enhance these properties, a novel sliding contact bioreactor was developed to better replicate physiologic joint loading conditions. Long-term application of loading to MSC-laden constructs improved not only tensile properties, but instilled biochemical inhomogeneity, reminiscent of native articular cartilage. Overall, the work outlined in this thesis represents a significant advancement in engineering cartilage replacements as well as in understanding MSC chondrogenesis. Using a multi-faceted approach, we explored potential routes toward overcoming limitations in chondrogenesis and demonstrated that MSCs are responsive to their chemical and mechanical environment. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Bioengineering First Advisor Robert L. Mauck Keywords mesenchymal stem cells, tissue engineering, bioreactors, chondrogenesis, mechanical stimulation Subject Categories Molecular, Cellular, and Tissue Engineering This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/183 ENHANCING MESENCHYMAL STEM CELL CHONDROGENESIS FOR CARTILAGE TISSUE ENGINEERING Alice H. Huang A Dissertation in Bioengineering Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy, 2010 Supervisor of Dissertation _________________________________ Robert L. Mauck Assistant Professor of Orthopaedic Surgery and Bioengineering University of Pennsylvania Graduate Group Chair _________________________________ Susan S. Margulies Professor of Bioengineering and Neurosurgery University of Pennsylvania Dissertation Committee Jason A. Burdick, Associate Professor of Bioengineering, University of Pennsylvania Maurizio Pacifici, Professor of Orthopaedic Surgery, Thomas Jefferson University Dawn M. Elliott, Associate Professor of Orthopaedic Surgery, University of Pennsylvania Kurt Hankenson, Assistant Professor of Cell Biology, University of Pennsylvania ENHANCING MESENCHYMAL STEM CELL CHONDROGENESIS FOR CARTILAGE TISSUE ENGINEERING COPYRIGHT 2010 Alice H. Huang ACKNOWLEDGEMENTS I would first like to thank my advisor, Dr. Robert Mauck. Given Rob’s penchant for farm animal idioms, it seems appropriate to co-opt one of his favorites – you can’t make a silk purse from a sow’s ear. If there is anyone who has proven this idiom wrong, it is Rob, as whatever progress I have made from sow’s ear toward silk purse is largely due to his tireless support and guidance. During my time here, his dedication to his students and his enthusiasm for science was a constant source of energy and created an environment that was both fun and intellectually rewarding; I will always be grateful for this experience. I would also like to thank the other members of my thesis committee, Drs. Dawn Elliott, Jason Burdick, Maurizio Pacifici and Kurt Hankenson for their thoughtful suggestions and advice. Not only have they improved my work significantly, but they have also provided valuable career guidance as I transition to the next stage of my studies. This work would also not have been possible without the assistance of several talented undergraduate and graduate students. In particular, I would like to acknowledge Ashley Stein, an undergraduate student who was involved in nearly every aspect of my work, as well as the other members (past and present) of the cartilage tissue engineering group, Isaac Erickson, Megan Farrell, Minwook Kim, Jenny Yuan and Meira Yeger-McKeever, for helpful discussions and moral support. In addition, I’d like to thank several current and former members of the McKay Orthopaedic Research Laboratory for facilitating my studies; in no particular order, these include Nelly Andarawis, Ashwin Nathan, Albert Gee, Lachlan Smith, Barbara Gibson, Eileen Shore, and Lou Soslowsky. And finally, I iii am fortunate to have conducted my entire graduate career alongside my two close friends and officemates, Brendon Baker and Nandan Nerurkar. Always available for discussion of data or to lend a hand in experiments, they have made me a better scientist, writer and student. They have also helped me maintain my sense of humor and mental equilibrium, mainly through TTA. Last but not least, I would like to thank my mom, Betty, and my sister, Jessica for their constant love and support. Even from a distance of 3000 miles, their presence has been an important part of my life. I also thank my aunt in Taiwan, Pi-Chin, for giving me many opportunities that would not have been otherwise possible and my brother in law, Ryan. Finally I acknowledge Tony the cat for being an ideal roommate and source of jolliness. iv ABSTRACT ENHANCING MESENCHYMAL STEM CELL CHONDROGENESIS FOR CARTILAGE TISSUE ENGINEERING Alice H. Huang Robert L. Mauck Articular cartilage provides a bearing surface for transmitting forces across joints. The poor ability of cartilage to self-repair has motivated efforts to engineer replacement tissues, and mesenchymal stem cells (MSCs), which can undergo chondrogenesis, have emerged as a promising cell source. To date however, the functional properties of MSC- based constructs remain lower than those of the native tissue and of chondrocyte-based constructs cultured identically. Therefore, the overall objective of this thesis is to better understand the transcriptional and functional limitations underlying chondrogenic differentiation and enhance MSC chondrogenesis. Toward this end, established tissue engineering strategies from the chondrocyte literature were applied. Specifically, the effects of cell seeding density, media formulation and mechanical stimulation were examined with respect to functional growth. Transient application of TGF-β3 improved the compressive properties of MSC-laden constructs, but only when constructs were formed at a higher seeding density. Long-term dynamic compression initiated 3 days after MSC encapsulation impaired functional properties; in contrast, dynamic compression initiated after 3 weeks of chondrogenic pre-culture improved mechanical function. While these strategies enhanced functional chondrogenesis, the compressive properties achieved were ~50% of native tissue levels v and did not reach chondrocyte levels. To understand the basis of this difference, microarray analysis was carried out to compare these two cells types and a set of molecular factors were identified as mis-expressed during MSC chondrogenesis. Although work up to this point focused on optimizing compressive properties, the tensile properties of articular cartilage are also critical to its functional role. In this work, we characterized the tensile properties of MSC-based constructs and demonstrated functional parity with chondrocyte-based constructs.