RICE UNIVERSITY Novel Exogenous Agents for Improving Articular Cartilage Tissue Engineering by Donald Joseph Responte A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE Doctor of Philosophy APPROVED, THESIS COMMITTEE: Kyriacos thanas u, PH. ., Thesis Director Distinguis ed Professor Department of Biomedical Engineering University of California, Davis Antonios Mikos, Ph.D., Committee Chair Louis Calder Professor Department of Bioengineering Rice University Junl;G.~~ Assistant Professor Department of Mechanical Engineering Rice University HOUSTON, TEXAS OCTOBER 2011 ii ABSTRACT Novel Exogenous Agents for Improving Articular Cartilage Tissue Engineering by Donald Joseph Responte This thesis demonstrated the effects of exogenous stimuli on engineered articular cartilage constructs and elucidated mechanisms underlying the responses to these agents. In particular, a series of studies detailed the effects of chondroitinase-ABC (C-ABC), hyaluronic acid (HA), and TGF-~1 on the biochemical and biomechanical properties of self-assembled articular cartilage. Work with C-ABC showed that this catabolic agent can be employed to improve the tensile properties of constructs. When constructs were cultured for 6 weeks, treating with C-ABC at 2 weeks enhanced the tensile stiffness. Furthermore, treating at 2 and 4 weeks synergistically increased tensile properties and allowed compressive stiffness to recover to control levels. Another study showed that combining C-ABC and TGF-~1 synergistically enhanced the biochemical and biomechanical properties of neotissue. Microarray analysis demonstrated that TGF-~1 increased MAPK signaling in self-assembled neocartilage whereas C­ ABC had minimal effects on gene expression. SEM analysis showed that C-ABC increased collagen fibril diameter and fibril density, indicating that C-ABC potentially acts via a biophysical mechanism. Constructs treated with C-ABC and TGF-~1 also showed stability and maturation in vivo, exhibiting a tensile stiffness of 3.15±0.47 MPa compared to a pre-implantation stiffness of 1.95±0.62 MPa. To iii assess the response to HA application, studies were conducted to optimize HA administration and examine its effects in conjunction with TGF-~1. Applying HA increased the compressive stiffness 1-fold and increased GAG content by 35%, with these improvements depending on HA molecular weight, application commencement time, and concentration. Microarray and PCR analyses showed that HA also influenced genetic signaling, up-regulating multiple genes associated with the TGF-~1 pathway. In addition to genetic effects, the enhanced GAG retention due to HA treatment could increase the fixed charge density of the matrix and thereby increase resistance to compressive loading. Additive effects were observed when HA was applied in conjunction with TGF-~ 1, with the combined treatment increasing compressive stiffness and GAG content by 150% and 65%, respectively. In general, results demonstrated mechanisms underlying C-ABC, HA, and TGF-~1 treatments and showed how these agents can be applied to improve cartilage regeneration efforts. iv Acknowledgments This dissertation depended on the contributions of many individuals both within and outside of our laboratory. A wide spectrum of people, from undergraduates to seasoned faculty members, made this thesis project possible. I would like to acknowledge all of these individuals for their personal and academic support over the past four years. First I would like to thank Dr. Athanasiou for providing guidance and support throughout the entire process over the years. I would also like to acknowledge my other committee members, Dr. Tony Mikos and Dr. Jun Lou, for their assistance during graduate school. Additionally, Dr. Jerry Hu has provided a wealth of scientific knowledge. The guidance of these individuals has added considerable perspective to work described in this thesis. My primary mentor upon joining the lab was Dr. Roman Natoli; I credit him with teaching me the fundamentals of laboratory work and, more importantly, introducing me to the conduct of good science. The early morning starts with our esteemed undergraduate Ben Lu also taught me the sacrifices needed to get data! The projects and ideas explored during those early years eventually became a large portion of my current work; in many ways, my experience in the Roman Legion formed the foundation of this thesis. Several current colleagues in the lab also contributed greatly to my thesis work. Dena Wiltz and Sriram Eleswarapu helped me navigate the Athanasiou lab shortly after I started graduate school and have provided continual support v throughout the process. Dr. Eleftherios Makris, Michael Higashioka, and Gordon Peng have worked with me on various HPLC projects as we established this new assay. Dr. Boaz Arzi has assisted extensively with in vivo work that provided some exciting additions to our conventional assays. Jeni Lee, in addition to gracing our office with stellar baked goods, did an outstanding job working on substantial review papers and contributing to our SHG collaboration soon after starting in the lab. All of these individuals and the other members of the Athanasiou lab have provided invaluable assistance. Last but not least, I would like to thank family and friends who have helped me over the years. This support has been crucial during the ups and downs of graduate school, particularly in light of the lab move to California, and it has been vital to my successes over the past several years. Your academic and personal encouragement has played a pivotal role in my graduate school career. vi Table of Contents List of Tables ......................................................................................................ix List of Figures .....................................................................................................x Introduction and background ........•.............................................•.....................1 Articular cartilage: basic biology ........................................................................ 1 Articular cartilage disease and repair ................................................................. 3 Exogenous stimuli for cartilage tissue engineering ............................................ 5 Cell sources for cartilage engineering .............................................................. 10 Tissue engineering approaches ....................................................................... 11 Thesis aims ..................................................................................................... 16 Chapter 1: Effects of multiple chondroitinase ABC applications on tissue engineered articular cartilage ....•....................•........•....•...•..............•••..........•..18 Abstract ........................................................................................................... 18 Introduction ...................................................................................................... 19 Methods ...........................................................................................................20 Results .............................................................................................................27 Discussion .......................................................................................................32 Chapter 2: Mechanisms underlying the synergistic enhancement of self­ assembled neocartilage treated with chondroitinase-ABC and TGF-P1 ...••. 42 Abstract ...........................................................................................................42 Introduction ......................................................................................................43 Results .............................................................................................................45 Discussion .......................................................................................................52 Methods ...........................................................................................................56 Chapter 3: Identification of potential biophysical and molecular signaling mechanisms underlying hyaluronic shows that acid enhancement of cartilage formation ............................................................................................68 Abstract ...........................................................................................................68 Results .............................................................................................................71 Discussion .......................................................................................................77 Materials and methods ....................................................................................81 Chapter 4: Additive effects of exogenous hyaluronic acid and TGF-P1 on tissue engineered articular cartilage .......•...............••..•••...••...••...••....•..........•.. 92 Abstract ...........................................................................................................92 Introduction ......................................................................................................93 Materials and methods .................................................................................... 95 Results .............................................................................................................99 vii Discussion ....................................................................................................