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University of Florida Thesis Or Dissertation Formatting ENGINEERING AN IN SITU OXYGEN GENERATING MATERIAL FOR EXTRAHEPATIC ISLET TRANSPLANTATION By MARIA MARGARITA CORONEL SALAS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2016 © 2016 Maria M. Coronel To my mother, for her love and support ACKNOWLEDGMENTS Time flies. I first joined the Stabler lab during my junior year as an undergrad. I had no idea of how to hold a pipette but was very excited to be given a chance to immerse myself in the beautiful, and most times frustrating, world of research. Fast forward 8 years and here I am still fascinated with the work I’ve chosen. First and foremost, I owe a deep sense of gratitude to my mentor Dr. Cherie Stabler. I would not have been able to complete this work without her support both personally, and professionally. Her dedication, professionalism, scientific approach, and above all her profound interests for her students are some of the values I have learned from her, and will take with me in my new scientific endeavors. It has been a great honor to work under your supervision. I would like to express my deepest thanks and sincere appreciation to Eileen Pedraza, Jessica Weaver and Irayme Labrada. You three have provided me with invaluable guidance, support, and encouragement through different stages of my career. For all this and for more, thank you. I would also like to thanks all members of the Stabler lab past and present. The time I spent working along you has definitely made me a better scientist. I would also like to express my deepest thanks and sincere appreciation to my committee members, Dr. Benjamin Keselowsky, Clayton Matthews, and Peter McFetridge for taking time out of their busy schedules to be part of my dissertation committee as well as their constructive criticism and direction. To Enrico, my rock in this last year, thank you for your patience and unconditional love. I would not have made it without you. Last but not least, I can never be grateful enough to my mother who supported my dream as a teenager to leave her 4 side to achieve my professional dreams. I would not be here today without her constant encouragements and support. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 10 LIST OF ABBREVIATIONS ........................................................................................... 16 ABSTRACT ................................................................................................................... 18 CHAPTER 1 RATIONALE ........................................................................................................... 20 Introductory Remarks.............................................................................................. 20 Specific Aims .......................................................................................................... 21 Contents of Dissertation .......................................................................................... 23 2 ENGINEERING AN EXTRAHEPATIC TRANSPLANT SITE ................................... 25 Background ............................................................................................................. 25 Selecting the Optimal Engraftment Site .................................................................. 26 3D Scaffolding ........................................................................................................ 28 Oxygenation and Vascularization ........................................................................... 31 Conclusion .............................................................................................................. 34 3 ENGINEERING AN IN SITU OXYGEN GENERATOR ........................................... 37 Background ............................................................................................................. 37 Methods .................................................................................................................. 39 Materials ........................................................................................................... 39 Fabrication of Oxygen Generator ..................................................................... 39 Leaching Studies .............................................................................................. 40 Macroscopic Analysis ....................................................................................... 40 Microscopic Analysis ........................................................................................ 40 Surface Characterization .................................................................................. 41 Hydrogen Peroxide Measurements .................................................................. 42 Calcium Concentration ..................................................................................... 43 Non-invasive Oxygen Measurements ............................................................... 43 Mathematical Model ......................................................................................... 44 Computational Modeling ................................................................................... 46 Islet Isolation and Culture ................................................................................. 47 In Vitro Cellular Assessments ........................................................................... 48 Statistics ........................................................................................................... 49 Results .................................................................................................................... 49 6 Conclusion .............................................................................................................. 63 4 MITIGATING HYPOXIC STRESS ON PANCREATIC ISLETS VIA IN SITU OXYGEN GENERATION ........................................................................................ 91 Background ............................................................................................................. 91 Research Design and Methods ............................................................................... 92 Materials ........................................................................................................... 92 Islet Isolation and Culture ................................................................................. 92 Fabrication of Oxygen Generating Material, OxySite ....................................... 93 Oxygen Studies with Pancreatic Islets ............................................................. 93 In Vitro Cellular Assessments ........................................................................... 94 Islet Transplant and Graft Assessment ............................................................ 97 Histological Assessment................................................................................... 98 Statistical Analysis ............................................................................................ 99 Results .................................................................................................................... 99 Conclusion ............................................................................................................ 109 5 MACROPORUS SCAFFOLDS WITH ENHANCED OXYGENATION FOR EXTRAHEPATIC ISLET TRANSPLANTATION .................................................... 127 Background ........................................................................................................... 127 Methods ................................................................................................................ 129 Materials ......................................................................................................... 129 Composite Scaffold-OxySite Fabrication ........................................................ 129 Non-invasive Oxygen Measurements ............................................................. 130 Scanning Electron Microscopy ....................................................................... 131 Islet Isolation and Culture ............................................................................... 131 Islet Transplant and Graft Assessment .......................................................... 131 Histological Assessment................................................................................. 133 Statistical Analysis .......................................................................................... 134 Results .................................................................................................................. 134 Conclusions .......................................................................................................... 139 6 IN SITU OXYGENATION OF IMMUNOISOLATING DEVICES ............................ 149 Background ........................................................................................................... 149 Methods ................................................................................................................ 151 Materials ........................................................................................................
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