Enhancement of peripheral nerve regeneration with controlled release of glial cell line-derived neurotrophic factor (GDNF) by Kasra Tajdaran B.Sc., University of Toronto, 2013 A thesis submitted in conformity with the requirements for the degree of master of applied science Institute for Biomaterials and Biomedical Engineering University of Toronto © Copyright by Kasra Tajdaran, 2015 Enhancement of peripheral nerve regeneration with controlled release of glial cell line-derived neurotrophic factor (GDNF) Kasra Tajdaran Master of Applied Science (MASc) Institute of Biomaterials and Biomedical Engineering University of Toronto 2015 Abstract Nerve injuries cause severe disability. The present investigational drug delivery strategies for enhancing peripheral nerve regeneration after nerve transection are not yet clinically translatable due to lack of efficiency or biocompatibility. We developed a local delivery system using drug-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres (MS) embedded in a fibrin gel. This drug delivery system (DDS) could be applied at the nerve injury site to deliver exogenous glial cell line-derived neurotrophic factor (GDNF) to the regenerating axons. We used our developed DDS to enhance nerve regeneration in clinically applicable models of severe nerve injuries, including cases with delayed nerve repair and with large nerve defects. ii Declaration of co-authorship The original scientific content of the thesis is comprised of two articles that are submitted to peer-reviewed internationally recognized journals. In both cases these contributions were primarily the work of Kasra Tajdaran. The contributions of the co-authors are declared in the following sections in conformity with the requirements for the degree of Master’s of Applied Science. iii Acknowledgement I would like to express my sincere gratitude to my supervisors, Dr. Gregory Borschel and Dr. Tessa Gordon for giving me an exceptional opportunity during my graduate studies. Thank you for your support and mentorship during the last two years. Thank you for teaching me how to better practice scientific approach and critical thinking. Your advice and constructive criticism has taught me how to become a scientist and remain dedicated to my research. I would like to acknowledge my committee members, Dr. Mike Salter and Dr. Molly Shoichet for contributing to this project. Dr. Salter, thank you for your insightful comments and guidance during our meetings. Dr. Shoichet, since my first undergraduate biochemistry course, you have always inspired me and made me passionate about my research. In addition, thank you for allowing to me attend your weekly team meetings anytime I was facing challenges in my project. I would also like to thank my best friends for the past two years and lab members at the Borschel lab. Cecilia Alvarez-Veronesi, Joseph Catapano, Mike Willand, Mike Hendry Steve Kemp, Cameron Chiang and Jennifer Zhang, thank you for your warm welcome and kindness from the first day we met. Cecilia, without your help and teaching me how to do everything just perfectly, I would not have been able to complete my project. I wish you the best of luck in your medical school quest. Joseph, Mike Willand, and Mike Hendry, thank you for always making me excited to come to the lab. I am very happy that I had a chance to know you guys and have friends like you who I can always look up to. Jennifer, your kindness and tremendous help during the past two years has helped me to overcome some of the toughest and busiest periods of my project. You have always helped me out with anything that I’ve asked for and I cannot thank you enough for it. iv Cameron Chiang and David Scholl, thank you for helping me with cryo-cutting throughout my project and thanks for always bringing a positive energy to the lab. I would like to thank members of the Shoichet lab for letting me use their equipment and lab space and providing me with guidance through out my project. Thank you, Mike Cook for your helpful suggestions on designing the in vitro cell viability assay. Thank you, Anup Tuladhar for helping me with working with the mass spectrometry and lyophilizer devices. Thank you Jackie Obermeyer and Ying Fang Cheng, for helping me with the neurite extension assays. Finally, I want to thank my family. My parents have always taught me what it means to be hardworking. You have always encouraged me to find my passion in life. Thank you for always providing me with the best advice and support. v Table of contents Abstract ............................................................................................................................................... ii Declaration of co-authorship ........................................................................................................ iii Acknowledgement ............................................................................................................................ iv List of figures .................................................................................................................................. viii List of tables..................................................................................................................................... xii 1 Introduction ................................................................................................................................. 1 1.1 Overview ................................................................................................................................................ 1 1.2 Anatomy of peripheral nervous system...................................................................................... 4 1.3 Injuries to peripheral nervous system ..................................................................................... 10 1.4 Tissue response to peripheral nerve injuries ....................................................................... 12 1.4.1 Peripheral nerve regeneration after chronic axotomy ......................................................... 14 1.4.2 Peripheral nerve regeneration after chronic denervation .................................................. 16 1.5 Current approaches for treating nerve injuries ................................................................... 17 1.5.1 Acellular allografts ................................................................................................................................... 18 1.6 Neurotrophic factors support ...................................................................................................... 23 1.7 Local neurotrophic factor delivery to the injured peripheral nerve ............................ 28 1.8 Hydrogels in drug delivery applications ................................................................................. 30 1.9 Use of polymeric microspheres as a drug vehicle ................................................................ 31 1.9.1 Poly(esters) ............................................................................................................................................. 32 1.10 Methods of PLGA microsphere synthesis for protein delivery ................................. 34 1.11 Summary and research goal .................................................................................................. 35 1.11.1 Project objective and hypothesis ................................................................................................. 35 1.11.2 Specific aims ......................................................................................................................................... 35 1.11.3 Scope of Thesis .................................................................................................................................... 36 2 An engineered biocompatible drug delivery system enhances nerve regeneration after delayed repair ................................................................................................................. 37 2.1 Abstract ............................................................................................................................................... 38 2.2 Introduction ....................................................................................................................................... 39 2.3 Materials and Methods ................................................................................................................... 41 2.3.1 GDNF encapsulation in PLGA microsphere ............................................................................... 41 2.3.2 GDNF microsphere characterization ............................................................................................ 42 2.3.3 GDNF DDS composite construction and in vitro release ...................................................... 42 2.3.4 Cell seeding and culture ..................................................................................................................... 43 2.3.5 Cell viability ............................................................................................................................................ 44 2.3.6 Experimental animals ......................................................................................................................... 44 2.3.7 Experimental design ............................................................................................................................ 44 vi 2.3.8 Operative procedure ..........................................................................................................................