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Directed Adult Neural Stem/Progenitor Cell Fate in Microsphere-Loaded Chitosan Channels

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Directed Adult Neural Stem/Progenitor Cell Fate in Microsphere-Loaded Chitosan Channels Directed Adult Neural Stem/Progenitor Cell Fate in Microsphere-Loaded Chitosan Channels by Howard Kim A thesis submitted in conformity with the requirements for the degree of Doctorate of Philosophy Institute of Medical Science University of Toronto © Copyright by Howard Kim 2011 Directed Adult Neural Stem/Progenitor Cell Fate in Microsphere- Loaded Channels Howard Kim Doctor of Philosophy Institute of Medical Science University of Toronto 2011 Abstract Spinal cord injury (SCI) is a devastating condition characterized by the loss of neuronal pathways responsible for coordinating motor and sensory information between the brain and the rest of the body. The mammalian spinal cord is limited in its ability to repair itself, so treatments devised to replace damaged tissue and promote regeneration are essential towards developing a cure. This work describes the development of a guidance channel strategy for spinal cord transection. Chitosan guidance channels were designed as a delivery vehicle for neural stem/progenitor cell (NSPC) transplants and drug-eluting poly(lactic-co-glycolic acid) (PLGA) microspheres. PLGA microspheres were embedded into chitosan channels by a spin-coating method. These microsphere-loaded channels demonstrated the ability for controlled short-term bioactive release of the small molecule drug dibutyryl cyclic-AMP (dbcAMP) and long-term bioactive release of the protein alkaline phosphatase. NSPCs were shown to be responsive to dbcAMP delivery, which results in greatly enhanced differentiation into neurons. The effect of directed neuronal differentiation was investigated after spinal cord transection in rat, resulting in a dramatic increase in NSPC transplant survival. Guidance channels containing NSPCs treated with dbcAMP resulted in robust tissue bridge formation after SCI, demonstrating extensive axonal regeneration and promoting functional recovery. ii Acknowledgments My motivation for entering the field of spinal cord research begins first and foremost from my younger brother Jonathan, who suffered a spinal cord injury during high school. His perseverance and independence has been a great inspiration for me throughout my degree, and has served as a constant reminder of the importance of translational research. I would also like to deeply thank my supervisors Dr. Molly Shoichet and Dr. Charles Tator, who both have been instrumental in my growth as a scientist, and without whose guidance this work would not be possible. Thank you also to lab members, past and present, of the Shoichet and Tator labs, who are too numerous to name individually. I would also like to express gratitude to my committee member Dr. Cindi Morshead, who provided valuable insights and direction for this work. Finally, I would also like to acknowledge my friends and family, particularly my parents Sue and Vince Kim, who all have been so supportive of me during my graduate career. Many thanks, Howard Kim iii Table of Contents Acknowledgments .......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ ix List of Appendices ........................................................................................................................ xv 1 Background and Introduction ..................................................................................................... 1 1.1 Rationale ............................................................................................................................. 1 1.2 Hypothesis and Objectives .................................................................................................. 2 1.3 Anatomy of the Spinal Cord ............................................................................................... 3 1.4 Pathology of Spinal Cord Injury ......................................................................................... 5 1.5 Animal Models of Spinal Cord Injury ................................................................................ 7 1.6 Treatment of Spinal Cord Injury ......................................................................................... 8 1.7 Cell-Based Therapies for SCI ............................................................................................. 9 1.7.1 Overview ................................................................................................................. 9 1.7.2 Neural Stem Cells ................................................................................................. 10 1.7.3 Directed Differentiation of NSPCs ....................................................................... 12 1.8 Biomaterials for SCI ......................................................................................................... 13 1.8.1 Overview ............................................................................................................... 13 1.8.2 Biomaterial Use in SCI ......................................................................................... 14 1.8.3 Chitosan ................................................................................................................ 17 1.8.4 Fibrin ..................................................................................................................... 19 1.8.5 Poly(lactic-co-glycolic acid) ................................................................................. 20 1.9 Drug Therapies for SCI ..................................................................................................... 21 1.9.1 Overview ............................................................................................................... 21 iv 1.9.2 Dibutyryl Cyclic-AMP .......................................................................................... 23 1.9.3 Drug Delivery Systems for the Spinal Cord ......................................................... 25 1.10 The Entubulation Strategy ................................................................................................ 26 2 Evaluation of Chitosan in the Spinal Cord ............................................................................... 29 2.1 Abstract ............................................................................................................................. 29 2.2 Introduction ....................................................................................................................... 29 2.3 Materials and Methods ...................................................................................................... 31 2.3.1 Material Processing ............................................................................................... 31 2.3.2 In vivo Implantation .............................................................................................. 32 2.3.3 Tissue Preparation ................................................................................................. 33 2.3.4 Explant Analysis ................................................................................................... 33 2.3.5 Staining of Paraffin-Embedded Tissue ................................................................. 34 2.3.6 Statistics ................................................................................................................ 34 2.4 Results ............................................................................................................................... 34 2.4.1 Intrathecal Implantation ........................................................................................ 34 2.4.2 Intramedullary Implantation ................................................................................. 38 2.5 Discussion ......................................................................................................................... 41 3 Incorporating a Drug Delivery System into Chitosan Guidance Channels.............................. 45 3.1 Abstract ............................................................................................................................. 45 3.2 Introduction ....................................................................................................................... 45 3.3 Materials and Methods ...................................................................................................... 47 3.3.1 Materials ............................................................................................................... 47 3.3.2 Microsphere Preparation ....................................................................................... 48 3.3.3 Chitosan Guidance Channel Preparation .............................................................. 48 3.3.4 Microsphere-loaded Channels .............................................................................. 49 3.3.5 In Vitro Protein Release ........................................................................................ 50 v 3.4 Results and Discussion ..................................................................................................... 50 3.5 Conclusion .......................................................................................................................
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