Scaffolds with Oriented Microchannels for Angioneural Regenerative Engineering A Thesis Submitted to the Faculty of Drexel University by Aybike Saglam in partial fulfillment of the requirements for the degree of Master of Science in Biomedical Engineering December 2010 To my father… iii iv ACKNOWLEDGMENTS It is very difficult to express how grateful I am to the people who contributed to this project. First of all, I want to thank to my committee members: Dr. Peter I. Lelkes, Dr. Philip Lazarovici, Dr. Anat Perets Katsir and Dr. Fred Allen. This thesis would have been impossible without all of you. Dr. Lelkes was a great advisor and mentor who was a big influence in my academic life. Dr. Perets was an amazing advisor and friend with constant optimism and encouragement. Dr. Lazarovici was an inspiration for good academic character and integrity and I hope to be as good an academian some day. Dr. Allen was a particularly positive source of encouragement in the beginning of my degree. Special thanks to Dr. Onaral, Dean of Biomedical Engineering, Science, and Health Systems, for serving as such a positive and tenacious role model for me throughout my degree. Thanks to many individuals and groups who have helped me considerably along the way: Dr. Itzhak Fisher,Dr. Gianluca Gallo, and to their laboratory members for their help on primary neuron extraction and culture, particularly Dr. Katherine Kollins; Dr. J. Yasha Kresh and Anant for letting me use the rheometer even for extended hours; Dr. Yvonne Mueller for her help on Fluorescence Activated Cell Sorting technique; and Dr. Qingwei Zhang and Gozde Senel for their help on Scanning Electron Microscopy. Thanks, too, to my friends and labmates: Mie was a great friend, advisor, mentor, roommate, classmate, and labmate; Greg, Diane and Leko who were never too busy v to help me; Adam who was personally involved in some of my experiments and designed the controlled freeze-drying machine with Ho-Lung Li according to Dr. Peret’s directives; my “sisters,” Gozde, Seda and particularly Asli made me feel at home; our program manager, Susan; our program coordinator, Tantri; Dr. Har-el was a source of advice and support; labmates Mike, Collin, Patrick, Gaurav, Sean, Jingjia, Tracy, Jessica, and Anna; and finally, Paul for fixing the lyophilization machine. Thanks to many institutions and programs on campus for their logistical support: the Biomed office, specifically Aylin, Natalia, Danielle and Lisa; and the ISSS office, especially to Adrienne. Finally, but not least, to my family and friends to whom I am grateful for their support. vi TABLE OF CONTENTS ACKNOWLEDGMENTS ............................................................................................................................................. iv TABLE OF CONTENTS ............................................................................................................................................. vi List of Figures ........................................................................................................................................................... vii ABSTRACT ................................................................................................................................................................ viii INTRODUCTION ......................................................................................................................................................... 1 .1 Overall goal & hypothesis ............................................................................................................................... 2 .2 Aims of the thesis ............................................................................................................................................... 3 .3 Definitions ............................................................................................................................................................ 4 .4 Rationale .............................................................................................................................................................. 4 BACKGROUND ............................................................................................................................................................ 8 .1 Past & current tissue engineering strategies for SCI ............................................................................. 8 .2 Spinal cord organization ............................................................................................................................... 10 .3 Spinal cord injury ............................................................................................................................................ 11 .4 Overview on Scaffolds for SCI ...................................................................................................................... 12 .5 Hydrogels for engineered scaffolds ........................................................................................................... 16 .6 Freeze­drying techniques ............................................................................................................................. 16 .7 The effect of pore diameter on axon extension and ECM secretion ................................................ 19 .8 Angiogenesis & vasculogenesis: .................................................................................................................. 20 .9 Neuritogenesis .................................................................................................................................................. 22 .10 Neurovascular crosstalk ................................................................................................................................ 24 .10.1 Interactions between nervous & vascular systems: ................................................................................. 24 .11 Cross­talk between neurons and ECs ..................................................................................................... 25 .11.1 Angiogenic factors’ effects on the nervous system: .................................................................................. 27 .11.2 Neurogenic factors’ effects on the vascular system .................................................................................. 28 MATERIALS AND METHODS ................................................................................................................................ 30 .1 Preparation of genipin crosslinked gelatin polymer (GCG) .............................................................. 30 .2 Scaffold production by regular and controlled freeze­drying .......................................................... 30 .3 Measurements of mechanical properties of the scaffold .................................................................... 33 .4 In­vitro degradation of the GCG­CFDSs ..................................................................................................... 33 .5 Cells ...................................................................................................................................................................... 34 .6 Cell proliferation ............................................................................................................................................. 37 .7 Fluorescence activated cell sorting (FACS) ............................................................................................. 37 .8 Endothelial cell labeling ................................................................................................................................ 38 .9 Populating the scaffold with cells and treatment with angioneurins ............................................ 39 .10 Confocal Microscopy ....................................................................................................................................... 39 .11 Scanning Electron Microscopy .................................................................................................................... 40 .12 Statistics .............................................................................................................................................................. 40 RESULTS & DISCUSSION ........................................................................................................................................ 41 .1 The morphology of the scaffolds ................................................................................................................ 41 .2 The mechanical properties of the scaffolds ............................................................................................ 43 .3 The degradability of the GCG­CFDS in the serum .................................................................................. 44 .4 Biocompatibility of the GCG­CFDS for homogeneous cell populations .......................................... 45 .5 Biocompatibility of the GCG­CFDS for heterogeneous cell populations ........................................ 49 SUMMARY .................................................................................................................................................................. 53 CONCLUSION& FUTURE WORK........................................................................................................................... 56 LIST of REFERENCES .............................................................................................................................................