Investigation of Magnesium-based Interventions for Central and Peripheral Nervous Tissue Regeneration A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY (Ph.D.) In the Department of Biomedical Engineering Of the College of Engineering and Applied Science February 2013 by John James Vennemeyer B.S., University of Cincinnati, Cincinnati, OH 2008 Dissertation Committee Sarah K. Pixley, Ph.D., Committee Chair, Associate Professor, Department of Cancer Biology, College of Medicine, University of Cincinnati. Joseph F. Clark, Ph.D., Professor, Department of Neurology, College of Medicine, University of Cincinnati. Daria A. Narmoneva, Ph.D., Associate Professor, Biomedical Engineering, School of Energy, Environmental, Biological & Medical Engineering, University of Cincinnati. Mark J. Schulz, Ph.D. Professor, College of Engineering and Applied Science, School of Dynamic Systems; Mechanical Engineering, University Of Cincinnati 1 Abstract Damage to tissues in the central nervous system (CNS) and peripheral nervous system (PNS) is of serious concern since they possess a limited capacity to regenerate. A new generation of interventions centered on the essential element magnesium (Mg) offers a promising proposition to improve treatment options for nervous tissue trauma. The long-term goal of our research is to understand how ionic and metallic Mg can be used to therapeutically interact with CNS and PNS tissues. Initial experiments focused on studying the interaction between Mg ion (Mg2+) solutions and CNS-derived neural stem/progenitor cells (NSCs) in vitro. Latter studies focused on using Mg metal as a novel biomaterial in peripheral nerve (PN) tissue engineering. The use of Mg2+ solutions to treat traumatic brain injury (TBI) and stroke is well documented and shows tremendous promise in animal models; however, there are still obstacles to translating these findings into effective treatments for humans. When administered systemically, Mg2+ can attenuate a host of complex, harmful biochemical cascades that occur in CNS tissue in the hours and days after injury, though the exact mechanisms are still unclear. With this in mind, we sought to develop a culture system that would be responsive to changes in Mg2+ in an effort to better understand the interactions between Mg2+ and CNS tissue (in the form of NSCs). The central hypothesis of this work was that neural cells (NSCs) would show altered biological responses, including beneficial effects, to moderately elevated Mg2+ concentrations in vitro. Severe injuries to the PNS are also resistant to current treatment methods. There is a great clinical need for an engineered alternative to autografts, the current standard of treatment for management of nerve defects greater than 3 cm. It has been hypothesized that one of the principal reasons that nerve conduits are ineffective at bridging defects greater than 3 cm is that there is inadequate mechanical support for tissue regeneration in the lumen of the conduit. We proposed that metallic Mg, which has been shown to i be biocompatible and biodegradable in orthopedic and vascular applications, could serve as a physical support, a scaffold, within a nerve conduit. The central hypothesis of this work was that metallic Mg, placed in the lumen of a nerve conduit, would improve nerve regeneration by serving as a scaffold that would safely resorb into the body over time, after mechanical support was no longer required by regenerating tissue. The results from the research in this dissertation identify a culture system for studying Mg2+ supplementation using primary CNS-derived stem cells and present a novel use for metallic Mg as a biodegradable scaffold in PN tissue engineering. New methods for monitoring neural cell health in vitro are described, as are methods for monitoring the response of nerve tissue in the presence of Mg metal and the resorption of Mg metal surrounded by nerve tissue in situ. The findings of this research will contribute toward the development of more sophisticated culture systems for studying the interaction between neural cells and Mg2+ and also toward the further development of biodegradable metals as tissue engineering scaffolds for the improvement of PN tissue repair. ii iii Acknowledgements I would like to acknowledge the support given to me by parents, siblings, family, friends, mentors and peers over the last 5 years. The work presented in this document is the result of a tremendous, collaborative effort from a very strong team. I would especially like to acknowledge Tracy Hopkins, for taking the technical lead on these projects and educating me in many cell culture ‘tricks of the trade’; the undergraduate and graduate students who spent time in the Pixley lab contributing to these projects: Erin Armao, Andrea Bobadillo, Ami Cohen, John Galvin, Dr. Xuefei Guo, Ben Hogan, Dr. Julia Kuhlmann, John Mast and Chris Valentic; our surgeons: Dr. Matt Hershcovitch, Dr. Kevin Little and Dr. David Hom, our in vivo projects would not have been possible without your steady hands and generous support; and Kati LaSance, your skills with the microCT are amazing; thank you for providing us with so much good data! Thank you to our external collaborators, especially Danielle Minteer, Da-Tren Chou, Dr. Lauren Kokai and Dr. Kacey Marra from the University of Pittsburgh - your insight, instruction and materials support were invaluable to the success of our in vivo work. To the other students and faculty of the NSF ERC for Revolutionizing Metallic Biomaterials at the University of Cincinnati, the University of Pittsburgh, North Carolina A&T State University and Hannover Medical School, this journey would not have been half as special without your support and companionship. One Team, One Dream! I extend a special thanks to the esteemed faculty who generously agreed to serve on my committee: to Dr. Daria Narmoneva for her excellent mentorship in my undergraduate education and input on quantifying biological phenomena, to Dr. Joe Clark for providing an essential clinical perspective and Dr. Mark Schulz for taking time to share his outlook on the business of engineering and for his steadfast support of Student Leadership and Professional Development. It is with great respect and gratitude that I also thank my mentor, Dr. Sarah Pixley, for welcoming me into her lab and guiding me through this process over the iv last 4 years. Your unwavering drive for knowledge and passion for science are remarkable and this work would not have been possible without your relentless support, patience and superb instruction. Finally, to our sponsors, thank you: This research was supported by the lab of Dr. Sarah Pixley and the National Science Foundation Engineering Research Center for Revolutionizing Metallic Biomaterials (EEC-0812348 and NCAT 260116C supplement). v Table of Contents Abstract……………………………………….…………………………………….……………………...i Acknowledgements…………………………………….…………………………………….……...….…iv Table of Contents…………………………………….…………………………………….………………….........vi List of Figures..................………………….…………………………………….……………………....vii List of Tables....................………………….…………………………………….……………………..xvii 1.0 Introduction and Specific Aims…………………………………….……………………......…....1 2.0 Background……………………….…………………………………….……………………….10 3.0 Effects of Mg2+ supplementation of neural stem/progenitor cells in vitro…………………........59 4.0 Digital methods for evaluating cellular response to magnesium filaments………………….......86 in peripheral nerve tissue in vivo 5.0 Magnesium metal as a scaffolding material for peripheral nerve regeneration..........................126 6.0 Discussion and Conclusions….…………………………………….…………….....................165 References….…………………………………….…………….............................................................170 vi List of Figures Figure 1 Functional organization of the nervous system. Receptors in peripheral nerves send sensory information to the central nervous system via the afferent division of the peripheral nervous system. After processing in the central nervous system, motor commands travel out to distal target through the efferent division of the peripheral nervous system 1. .................................................................... 11 Figure 2 Neuron structure. The neuron is a complex, electrically excitable cell that is the principle functional building block of nervous tissues. Neurons communicate with other neurons and all other organ systems in the body to regulate the processes of life. Image from 2. ....................................... 13 Figure 3 Anatomy of the spinal cord. The spinal cord resides in, and is protected by, the vertebral column (A – Superior transverse view). Nerves project from the left and right sides to innervate the body (B). The ventral root carries motor commands from the CNS to the periphery whereas the dorsal root carries sensory information from the periphery to the spinal cord. Modified from 3. These views are in an upside-down position because that is the way that information comes in from an MRI machine scan. So ‘dorsal’ (toward the back) is actually down in this picture and ‘ventral’ (the belly side) is up 256. ...................................................................................................................................... 16 Figure 4 Nerve architecture. Nerve fibers (axons) are supported and encased in myelin by Schwann cells within the endoneurium. The