Bone Tissue Engineering using Colloidal Gels and Native Extracellular Matrix Biomaterials By S. Connor Dennis B. S., Chemical and Petroleum Engineering, The University of Kansas, 2010 Submitted to the Bioengineering Program and the Graduate Faculty of the University of Kansas in partial fulfillment of the degree requirements for the degree of Doctor of Philosophy Committee Members: _________________________________ Dr. Cory J. Berkland, Committee Chair _________________________________ Dr. Michael Detamore _________________________________ Dr. Sarah Kieweg _________________________________ Dr. Teruna Siahaan _________________________________ Dr. Stevin Gehrke January 12th, 2015______________ Date defended ii The Dissertation Committee for Stephen Connor Dennis certifies that this is the approved version of the following dissertation: Bone Tissue Engineering using Colloidal Gels and Native Extracellular Matrix Biomaterials Committee Chair _________________________________ Dr. Cory J. Berkland, Committee Chair February 3rd, 2015________________ Date approved iii ABSTRACT Self-assembling and shear-responsive biomaterials possess favorable rheological and viscoelastic properties to be injectable and facilitate minimally invasive surgery. The current thesis work describes the evaluation of malleable colloidal gel scaffolding technology for the regeneration of bone tissue in non-load bearing critical-sized defects. This represents the first attempt to form colloids exclusively from biomaterials found in the microenvironment of healing bone fractures including hyaluronic acid (HA), hydroxyapatite (HAP), bone and cartilage extracellular matrix (ECM). Work in the current thesis evaluated preliminary in vitro and in vivo efficacy of several injectable colloids. Specifically, HA-HAP colloids exhibiting desirable rheological, viscoelastic, and swelling properties for surgical placement and defect site retention were identified from an array of formulations. Subsequent formulation refinement incorporated micronized decellularized cartilage (DCC) and demineralized bone matrix (DBM) particles into HA-HAP colloids, which did not result in any significant decreases in measured fluid properties. Coupling ECM microparticles within a colloidal fluid carrier was hypothesized to enhance the regenerative capacity of HA-HAP colloidal formulations. In vitro studies demonstrated evidence of temporal chondrogenic, hypertrophic, and osteogenic gene expression in response to changes in colloidal composition. More specifically, the inclusion of DCC led to hypertrophic chondrogenesis while both HAP and demineralized bone matrix DBM colloidal formulations appeared to direct cell lineage down an osteogenic pathway in a temporal manner similar to expression profiles observed in native bone fracture healing. In vivo studies demonstrated the feasibility and efficacy of colloidal scaffolds in critical-sized rat calvarial defects. Although no significant differences in regenerated bone were observed in defects treated with colloidal formulations compared to negative control, the presence of endochondral (EC) derived ossification foci were only observed in HA-HAP and iv HA-HAP-ECM treated defects. Thus, definite advantages of using colloidal gel technology were observed in these preliminary studies, but future iterations of the implant formulation design may yield enhanced bone tissue regeneration. Ultimately, the idea of colloidal-based tissue implants has been taken from concept to practice, produced promising results for the treatment of non-load bearing bone defects, and has given rise to numerous areas of tangential research to refine the technology. v ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my advisor, Dr. Cory Berkland, for his continued support over the course of my graduate studies. His advice and encouragement in my professional and personal life have been instrumental in the shaping of my scientific career. I will forever be in your debt for the opportunities and experiences you have given me, thank you. I would like to also acknowledge the other members of my dissertation committee, Dr. Michael Detamore, Dr. Sarah Kieweg, Dr. Stevin Gehrke, and Dr. Teruna Siahaan for their support with regard to the development of this thesis along with their continued guidance of my scientific research in the laboratory and in the classroom. I would also like to thank the faculty and staff of the Bioengineering Graduate Program for giving me the opportunity to pursue my passion for biomaterials, tissue engineering, and regenerative medicine. This research would not have been possible without the generous funding from the National Institutes of Health (NIH RO1 DE022471) and the NIGMS Pre-doctoral Training Grant Program (NIH/NIGMS T32-GM08359), and the University Of Kansas School Of Engineering. Special thanks are needed for all of my undergraduate research assistants who made my graduate experience more enlightening and rewarding: Jon Whitlow, Addison Schile, Austin Childress, Kathryn Scherich, Michael Holtz, Martin Wind, and Jake Hopkins. Their contributions in the laboratory were essential to the completion of this thesis. I would like to thank current and former graduate and post-doctorate collaborators for their scientific insight, persistent work ethic, and extraordinary kindness that have made my graduate studies rich in knowledge and friendship. I would especially like to thank Emily Beck, Amanda Sutherland, and Dr. Brad Sullivan for their assistance with in vitro characterization along with Dr. Ye Feng for his surgical work and management of in vivo studies and Dr. Prem Thapa and Heather Shinogle in the KU Microscopy vi Lab for their assistance in SEM and TEM imaging. In addition, I would like to thank Chris Kuehl, Sharadvi Thati, Vineet Gupta, Amanda Renth, BanuPriya Sridharan, Cate Wisdom, Dr. Nathan Dormer, Dr. Amir Fakhari, Dr. Joshua Sestak, and Dr. Nashwa El-Gendy. I would also like to thank my industry mentors, Dr. Mark Menning and Sean Ritchie, as well as former KU alumni, Dr. Diana Davey and Dr. Eric Gorman, at Gilead Sciences Inc. for broadening my scientific experience. There are two final groups of people that I need to acknowledge. The first are the inspirational mentors and teachers that have shaped my academic story. The University of Kansas in Lawrence has been my home throughout my undergraduate and graduate career, and special thanks are needed for the numerous educators that have opened my eyes and mind to the world. There truly is no place like home. Lastly, I want to thank my family. I want to thank my parents, Jan and Steve, along with my step-parents, Chris and Melanie, for their unconditional love and support which has molded me into the person I am today and allowed me to pursue my academic dreams in science and engineering. Finally, I want to thank my wife, Elise. She is my muse, my confidant, and the mother of my beautiful child, Sloane. She challenges me to become the person I want to be in the world. Let this be another chapter, another memory in the book of our lives as we look forward to making new ones together. Adventure is out there! vii TABLE OF CONTENTS ACCEPTANCE PAGE ................................................................................................................. ii ABSTRACT .................................................................................................................................. iii ACKNOWLEDGEMENTS ..........................................................................................................v TABLE OF CONTENTS ........................................................................................................... vii CHAPTER 1: Introduction to Thesis ...........................................................................................1 CHAPTER 2: Mapping Glycosaminoglycan-Hydroxyapatite Colloidal Gels as Potential Tissue Defect Fillers .......................................................................................................................5 Abstract .......................................................................................................................................6 Introduction .................................................................................................................................7 Experimental .............................................................................................................................10 Materials ...........................................................................................................................10 Characterization of HAP nanoparticles ............................................................................10 Preparation of colloidal gels .............................................................................................10 Swelling Characterization ..................................................................................................11 Rheological Characterization ............................................................................................11 Rheological Modeling .......................................................................................................12 Statistical Analysis .............................................................................................................12 Results .......................................................................................................................................13 Characterization of HAP nanoparticles