Neural Biomimetic Materials for Investigating Cell Behaviors in 3D DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Shreyas S. Rao, B.E., M.S. Graduate Program in Chemical and Biomolecular Engineering The Ohio State University 2012 Dissertation Committee: Dr. Jessica O. Winter, Advisor Dr. Stuart L. Cooper Dr. John J. Lannutti Dr. Atom Sarkar Copyright by Shreyas S. Rao 2012 Abstract Central nervous system (CNS) insult because of neurodegenerative disorders or cancer can be devastating. To restore lost neuronal function, prosthetic devices are commonly employed; however achieving robust tissue-electrode interfacing has been difficult because of mismatch in device-tissue properties. Likewise, CNS glial cancers are challenging to treat because of our limited understanding of cancer cell behaviors. To address these challenges, biomimetic materials that can serve as coatings to improve the tissue-electrode interface or as three dimensional (3D) physiologically relevant in vitro models to examine cancer behaviors are needed. This dissertation, therefore, has developed and evaluated 3D biomimetic materials composed of hydrogels, electrospun nanofibers, or a combination of the two. To enhance the tissue-prostheses interface, synthetic poly(ethylene) glycol based- biomimetic hydrogel coatings endowed with adhesion molecules (polylysine) were developed. Polylysine modified hydrogels were shown to promote better cell adhesion over unmodified controls. Coatings were stable for at least four weeks in vitro suggesting that these biocompatible hydrogels hold potential to enhance the stability of chronic neural interfaces. In addition to coatings, biomimetic hydrogels were also evaluated as in vitro tumor cell culture models to understand glioblastoma multiforme (GBM) glial cancer cell behaviors ii in 3D that closely mimic in vivo behaviors as opposed to traditionally studied two dimensional behaviors. Using experimental and computational techniques, we demonstrated that edge effects or mechanical cues resulting from a rigid support-soft hydrogel interface significantly influence GBM morphology, spreading, elongation, migration, and actin organization in a 3D Matrigel biomaterial. These results have import for hydrogel-based, 3D cell culture and suggest that such inherent mechanical gradients should be considered while evaluating 3D cell behaviors. Next, we developed composite hydrogels composed of collagen and hyaluronic acid (HA), one of the major components of the brain tumor microenvironment. To mimic increased HA levels observed in GBM tumors, composite hydrogels with increasing HA content were synthesized and characterized. Patient derived GBM cell morphology, spreading, and migration were all strongly dependent on HA density in 3D with higher compositions promoting little or no migration. These findings suggest that the interplay of these hydrogel components guide cell behavior in 3D. To mimic white matter topography, a major GBM migration highway, we developed an electrospun nanofiber platform and performed a comprehensive investigation of the influence of mechanics and chemistry on GBM behaviors utilizing core-shell electrospinning. Modulating nanofiber mechanics using different polymers (gelatin, polyethersulfone, polydimethylsiloxane) in the ‘core’ with a common poly (ε- caprolactone) (PCL) ‘shell’ revealed GBM elongation, migration, focal adhesion kinase and myosin light chain 2 expression sensitivity to nanofiber mechanics. Similarly, modulating nanofiber chemistry using HA, collagen, and Matrigel as a ‘shell’ on PCL iii ‘core’ nanofibers revealed GBM sensitivity to HA, specifically, in which a negative effect on migration was observed. To incorporate multiple cues and more importantly, mimic multiple key features of the in vivo microenvironment within a single 3D system, an integrated nanofiber-hydrogel model was also developed. Preliminary results indicate that GBMs align with nanofibers within 3D hydrogels allowing us to further investigate highly directed migration processes in 3D. Taken together, we have identified several factors influencing tumor cell behaviors in 3D by developing novel in vitro models as well as developed biomimetic coatings that can potentially enhance neural prosthesis biocompatibility. Thus, these findings should have far reaching implications in neural engineering and oncology with the potential for clinical translation. iv Dedication To my late grandparents, my parents, and Almighty God. v Acknowledgments I would like to acknowledge all my mentors, colleagues and friends who provided me with great advice and support throughout my graduate life. Firstly, I would like thank Prof. Jessica O. Winter for her excellent mentorship as my thesis advisor. In the last five years, I have learnt a great deal from her. She has always encouraged us to think out of the box, think big and positive and come up with solutions that will potentially have a great impact on the scientific community. I cannot be more thankful and I must admit that it has been an honor to work with you. I would also like to thank my other mentors, Dr. Atom Sarkar and Prof. John J. Lannutti. Dr. Sarkar has been a great clinical mentor answering all my clinical questions and providing crucial clinical inputs to my projects. I also appreciate help and invaluable suggestions from Prof. Lannutti on the work with electrospun fibers who was virtually a “second” advisor for small period during Prof. Winter’s absence during later half of my graduate life. Also, I would like thank Prof. Mariano Viapiano, for taking out time to answer some of my neurobiology related questions and also helping with molecular biology techniques described in this work. I look forward to interacting with all of you in the future. I would sincerely like to thank all my qualifier and candidacy committee members, Prof. Stuart L. Cooper, Prof. John J. Lannutti, Prof. Jeffery J. Chalmers, Dr. vi Atom Sarkar and Prof. Jessica O. Winter for providing critical inputs on my research. I would also like to thank Prof. David Wood for all his help and career advice. I would also like to thank all members of the Winter lab who have helped me in my research endeavors. First and foremost, I would like to thank Dr. Ning Han and Dhananjay Thakur who have been great colleagues providing research advice and help when I first started as a graduate student in the lab in 2008. Thanks Dhananjay for those stimulating research discussions in our office. I would like to thank my awesome undergraduate researchers Kunal Parikh, John Larison, Alex Hissong, and Caroline Dahlem for providing experimental help as well as for taking on new research directions. I would also like to thank Dr. Shuang Deng, Dr. Gang Ruan, Dr. Jianquan Xu, Joe Grodecki, Aaron Short, Kalpesh Mahajan, Jenny Dorcena, Craig Buckley and Qirui Fan for making my stay in the lab enjoyable. My thanks also goes to Sarah Bentil and Dr. Rebecca Dupaix for providing their FEM simulation expertise to my projects and helping me understand the mechanics of hydrogels using finite element models. I would like to thank the Lannutti lab members Dr. Jed Johnson, Carol Lee, Tyler Nelson, Ruipeng Xue and Jason Drexler (Nanofiber Solutions) for providing me with electrospun fibers for my research. I would also like to thank the Sarkar lab members, Jessica DeJesus, Lisa Denning and Kristin Dahl for all their help in confocal microscopy and related experiments. I would also like to thank Dr. Sara Cole (Campus Microscopy and Imaging Facility (CMIF), OSU) for her help and assistance with confocal reflectance microscopy and Henk Colijn (Campus Electron vii Optics Facility (CEOF), OSU) for help and assistance with transmission electron microscopy. I would like to thank all my Columbus friends for making me feel at home during my graduate study. More specifically, I would like to thank my roommates Kartik and Ashutosh, colleagues Preshit and Shweta for spending and sharing 5 years of graduate life both as colleagues and friends. Thanks to Dr. Vikas Khanna and Dr. Somnath Sinha for making my initial years in Columbus enjoyable. I would also like to acknowledge my gym buddies Ganita, Varun, and Anshuman. Gymming at the Jesse Owens Recreation Center could not have been more fun! Also, my special thanks to Dr. Khanna, Kartik, Archana, and Harshit for being a positive sounding board and providing advice in times of need. Thanks Guys! I would also like to acknowledge financial assistance from the National Science Foundation (NSF), Women in Philanthropy at the Ohio State University, fellowship support via a Pelotonia Graduate Fellowship and a Joseph H. Koffolt Graduate Fellowship. I am particularly indebted to the Pelotonia Fellowship program for making these last two years of graduate study enriching via symposiums enabling efficient exchange of highly inter-disciplinary research ideas in the cancer field. Last but not the least; I would like thank my parents, Mr. Subhaschandra Rao and Mrs. Subhashini Rao and my brother, Sandesh Rao for their unconditional love and support. My heartfelt thanks and gratitude also goes to my late grandparents Mr. Srinivas Rao and Mrs. Indira Rao for inculcating in me a scientific curiosity since my early days. viii Vita September, 1985 ............................................Born in Mumbai, India June, 2001 ......................................................Little Angels High School, Mumbai, India June, 2003