Viscoelastic Hydrazone Covalent Adaptable Networks to Study Chondrocyte Mechanobiology for Cartilage Tissue Engineering by Benjamin McGlenn Richardson B.A., Rensselaer Polytechnic Institute, 2015 M.S., University of Colorado Boulder, 2017 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirement for a degree of Doctor of Philosophy Department of Chemical and Biological Engineering 2020 Thesis Committee Adviser: Dr. Kristi S. Anseth Dr. Stephanie J. Bryant Dr. Virginia L. Ferguson Dr. Jeffry W. Stansbury Dr. Franck J. Vernerey i Abstract Richardson, Benjamin M. (Ph.D., Chemical Engineering) Viscoelastic Hydrazone Covalent Adaptable Networks to Study Chondrocyte Mechanobiology for Cartilage Tissue Engineering Thesis directed by Professor Kristi S. Anseth Symptomatic knee osteoarthritis is estimated to affect nearly 1 in 5 Americans over the age of 45. Osteoarthritis patients often experience pain caused by damage to articular cartilage in load-bearing joints. Matrix-assisted autologous chondrocyte transplantation (MACT) has emerged as a promising tissue engineering strategy to enhance the ability of chondrocytes to repair cartilage defects. This strategy often employs water-swollen polymer networks, known as hydrogels, as delivery vehicles to support chondrocytes and permit extracellular matrix (ECM) deposition. Hydrogels used for cartilage tissue engineering can be covalently crosslinked to withstand compressive forces experienced in articulating joints. However, traditional covalent crosslinks exhibit elastic responses to mechanical deformation and can limit ECM deposition to pericellular space. One potential strategy to improve regenerative outcomes of MACT is to incorporate viscoelastic properties, making hydrogels more similar to the viscoelastic ECM chondrocytes experience in vivo. However, few covalent hydrogels used for cartilage tissue engineering exhibit viscoelastic properties. Moreover, the effects of viscoelasticity (e.g., stress relaxation, creep compliance) on cartilage tissue engineering remain largely understudied. Covalent adaptable networks (CANs) represent a rapidly growing class of polymers with reversible covalent crosslinks which potentially offer both robust mechanical support and viscoelastic network reorganization for cartilage tissue engineering. In this thesis, we aim to add to this growing body of research by examining the effects of mechanobiological cues on chondrocytes encapsulated in hydrazone CANs. First, we sought to engineer hydrazone CANs with user-defined control over the viscoelastic properties by ii leveraging differences in the equilibrium kinetics of alkyl-hydrazone and benzyl-hydrazone crosslinks. Next, viscoelastic stress relaxation timescales of these networks are investigated to modulate ECM deposition by encapsulated chondrocytes. Then viscoelastic creep compliance is examined to temporally direct chondrocyte morphology during mechanical deformation. Finally, mechanobiological interactions between viscoelasticity and dynamic compression on chondrocytes in hydrazone CANs are studied using dynamic compression bioreactors to simulate biomechanical forces experienced within articulating joints. Overall, this work lends insight about how viscoelastic material properties influence chondrocyte behavior in hydrazone CANs with the hope of informing the design of polymer matrices for cartilage tissue engineering to treat osteoarthritis in load-bearing joints. iii Acknowledgments First and foremost, I would like to thank my thesis adviser Dr. Kristi Anseth. I remember meeting with you during my first semester at the University of Colorado and being blown away. I said to myself after that meeting, this woman is amazing how can I work with and learn from her. Your mentorship over the last five years, and the experience of being a member of your world-class research group has without a doubt been one of the richest learning experiences of my life. I am a better scientist and a better person through your dedicated efforts. I also want to thank my thesis committee Dr. Stephanie Bryant, Dr. Virginia Ferguson, Dr. Jeff Stansbury, and Dr. Franck Vernerey and my collaborator Mark Randolph for the guidance you have provided during my time at the University of Colorado. I would also like to thank the other members of the Anseth Research Group. Specifically, I would like to thank Dr. Kemal Arda Gűnay, Dr. Laura Macdougall, and Dr. Tobin Brown for mentoring me and helping Kristi shape my scientific identity. I would also like to thank my by graduate and undergraduate student collaborators, Cierra Walker, Mollie Maples, Daniel Wilcox, and Jack Hoye. I would also like to give a special thanks to my desk mate Ben Carberry for late night talks in the lab. In addition to my professional life, I also need to thank my friends. I have had the privilege to know many special people during my life and the people I have met in Colorado are no exception. I want to name Mike Hjortness, Katie Manduca, Jacob Fenster, Alex Delluva, Adrianne Blevins, Archish Muralidharan, David Bull, Alexandra Morris, Lyle Bliss, Ben Coscia, Jenna Wagenblatt, the rest of my classmates and the LaCoix Bois. You have made my time here immensely more enjoyable and I am excited for many more adventures with you in the future. I also need to give special thanks to Bryce Manubay and James Gallant for being there for me at my best and at my worst. And to Grace Ramsey: thank you for supporting me. Whether you were making sure I ate food while writing my thesis or just lending emotional support, your presence has been indispensable these last few weeks. Finally, I also want to thank my friends from back in Maine, my friends from New York, and all of the people I have met along the way that have made my life rich and fulfilling but are too numerous to list here. iv And finally, to my family. To my siblings Josh, Julia, and Karina: thank you for being there for me. I am so unbelievably proud to be related to each of you and each of you give me valuable perspective on life, the world, and everything. To my mother and father: You have made me the person I am today more so than anyone else and I am eternally grateful. I couldn’t wish for parents who care more about me and I will never forget the sacrifices you made for me. I am extremely grateful for the love and support that has enabled me to pursue this degree. Thank you. v Table of Contents Chapter 1 - Introduction ............................................................................................................................ 1 1. Cartilage tissue engineering to treat osteoarthritis ........................................................................ 1 1.1. Articular cartilage structure and function ................................................................................. 1 1.1.1. Chondrocytes .................................................................................................................... 3 1.1.2. Extracellular matrix (ECM) .............................................................................................. 4 1.1.3. Mechanical properties of articular cartilage ...................................................................... 5 1.2. Prevalence, symptoms and etiology of osteoarthritis ................................................................ 6 1.3. Limitations of current osteoarthritis treatment strategies .......................................................... 7 1.3.1. Joint arthroplasty ............................................................................................................... 8 1.3.2. Surgical intervention procedures ...................................................................................... 8 1.3.3. Cell and matrix based treatments .................................................................................... 10 1.4. Polymer scaffold design considerations for MACT ................................................................ 13 1.5. Dissertation approach .............................................................................................................. 21 1.6. References ............................................................................................................................... 21 Chapter 2 - Background ........................................................................................................................... 34 2. Dynamic covalent hydrogels as biomaterials to mimic the viscoelasticity of soft tissues .......... 34 2.1. Abstract ................................................................................................................................... 34 2.2. Introduction ............................................................................................................................. 34 2.3. Mechanical signatures of viscoelasticity ................................................................................. 37 2.4. Viscoelasticity of native tissues .............................................................................................. 41 2.5. Viscoelastic networks with reversible crosslinks and their relaxation mechanisms ............... 45 2.6. Viscoelastic hydrogels with dynamic chemical bonds ............................................................ 50 2.6.1. Diels-Alder reactions ...................................................................................................... 53 2.6.2. Imines .............................................................................................................................