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Bipolymer-Microglia Cell Compositions for Neural Tissue Repair

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Title:
Bipolymer-Microglia Cell Compositions for Neural Tissue Repair
Creator:
STOPEK, JOSHUA BENJAMIN ( Author, Primary )
Copyright Date:
2008

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Subjects / Keywords:
Alginates ( jstor )
Biomaterials ( jstor )
Dosage ( jstor )
Lesions ( jstor )
Magnetic resonance imaging ( jstor )
Microglia ( jstor )
Physical trauma ( jstor )
Rats ( jstor )
Signals ( jstor )
Spinal cord ( jstor )

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University of Florida
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University of Florida
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Copyright Joshua Benjamin Stopek. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
12/1/2003
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434595875 ( OCLC )

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BIOPOLYMER-MICROGLIA CELL COMPOSITIONS FOR NEURAL TISSUE REPAIR By JOSHUA BENJAMIN STOPEK 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 2003

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Copyright 2003 by Joshua Benjamin Stopek

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This work is dedicated to my father, Alan Stopek, for his love, support, and guidance. This journey would not have been possible without him.

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ACKNOWLEDGMENTS I would first like to acknowledge my parents, Alan and Goldie Stopek, and Dorrit Stopek, who offered much love and wisdom during my entire academic career. I would not have obtained this degree without their support. I would like to thank my advisor and committee chair, Dr. Eugene P. Goldberg, for his knowledge, support, enthusiasm, and patience. Working under his guidance has been a wonderful learning experience. I would like to thank my committee, Dr. Wolfgang “Jake” Streit; Dr. Anthony Brennan; Dr. Christopher Batich and Dr. Amelia Dempere for their advice and insight. I would especially like to thank my co-mentor, Jake Streit, for his friendship, support, and dedication, which without this experience would not have been possible. I would like to extend a special thanks to Dr. Parker Mickle for all of his help, hard work, and friendship, as well as for feeding and fueling me on a regular basis. I would like to thank several good friends and colleagues who played a vital role during my graduate experience. I would like to especially thank Brian Cuevas, my partner in crime, for his never-ending support, encouragement, sense of humor, and uncanny ability to say the right thing at the right time, usually in hidden code (English). He has been there through thick and thin, and I would not be where I am today without his friendship. I would like to thank Amin Elachchabi, our protg, for his friendship, loyalty (to BMW), and dedication. But, most importantly, I would like to thank him for his philosophical diatribes and our countless arguments over the significance of concentration. I would like to acknowledge Paul Martin for his expertise, curiosity, friendship, and generosity, especially during mischief missions. iv

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I would also like to acknowledge Dr. Bob Hadba and Dr. Chris Widenhouse for being excellent mentors and friends, and for introducing me to the world of biomaterials, drug delivery, and expensive beer. Special appreciation is extended to my friends and colleagues for their assistance and encouragement and include: Tim Shepard, Amanda York, Patrick Leamy, Jason Coleman, Lori White, Tanya McGraw, Toby Ferguson, Julia and Mark Winkler, Cheska Cuevas, Xeve Silver, Raquel Torres, David Peace, Clay Bohn, Brian Hatcher, Lynn Peck, James Marotta, Drew Amery, Kaustabh Rau, Mike Grumski, Barry Flannary, Chris Mariani, Amanda Kuhns, Jessica Conde, Brad Willenberg, Jamie Rhodes, Mike Ollinger, Nabil Bassim, Dan Urbaniak, Brett Almond, Mike Langford, Rey Rivera, Ronny Reddy, Amy Guinn, Daphne Stoker, Jessica Mata, Megan Prommersberger, Jeremy Snyder, Shema Freeman, Adam Feinberg, Amy Gibson, Mark and Matt Swick, Steve Nemeth, and Jeff Kuno. I would also like to thank the Christopher Reeve Paralysis Foundation for giving me the opportunity to conduct and contribute this research. v

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS....................................................................................................iv LIST OF TABLES...............................................................................................................ix LIST OF FIGURES..............................................................................................................x ABSTRACT......................................................................................................................xiv 1 INTRODUCTION.............................................................................................................1 2 BACKGROUND...............................................................................................................4 Human Clinical Trials....................................................................................................9 Experimental Repair and Regeneration Concepts....................................................14 Neuroprotective and Neurotrophic Microglia and Macrophages...............................14 Rubrospinal Tract.......................................................................................................18 Previous Use of Biomaterials for SCI Repair.............................................................19 Alginates..............................................................................................................21 Polymeric Phospholipids.....................................................................................23 Radiation Polymerization of Hydrophilic Biopolymers........................................24 Chitosan...............................................................................................................25 Deoxyribonucleic acid (DNA)..............................................................................25 3 ALGINATE-BASED COMPOSITIONS FOR THE REPAIR OF INJURED NEURAL TISSUE.......................................................................................................................28 Specific Aims..............................................................................................................28 Aim 1....................................................................................................................29 Aim 2....................................................................................................................29 Materials and Methods...............................................................................................30 Polysaccharide Solutions....................................................................................30 Porous Alginate Foam Synthesis........................................................................30 Porous Alginate Thin Film Synthesis..................................................................31 Semi-solid Alginate Gel Preparations.................................................................32 MPC Monomer....................................................................................................32 MPC Presoak......................................................................................................33 -Radiation Polymerization..................................................................................33 Post-Irradiation Cleaning.....................................................................................33 Polymer Characterization...........................................................................................34 Inductively Coupled Plasma Emission (ICP)......................................................34 Electron Microscopy............................................................................................35 vi

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Multi-Angle Light Scattering (MALS)...................................................................35 Fourier-Transform Infrared Spectroscopy (FTIR)...............................................35 X-Ray Photoelectron Spectroscopy (XPS).........................................................36 In Vitro Evaluations.....................................................................................................36 Cell Culture Reagents.........................................................................................36 Microglial Cell Cultures........................................................................................37 In Vitro Cell Seeding............................................................................................38 In Vivo Evaluations.....................................................................................................39 Surgical Implantation...........................................................................................39 Non-invasive Magnetic Resonance Imaging (MRI)............................................39 Behavioral Analysis.............................................................................................40 Retrograde Tract Tracing....................................................................................41 Immunohistochemical Staining...........................................................................41 Results and Discussion..............................................................................................42 Porous Alginate Compositions............................................................................42 Pilot -Polymerizations and MPC Modifications..................................................48 MALS Study of -Polymerized MPC....................................................................50 MPC Modification of Porous Alginate.................................................................51 In Vitro Evaluations..............................................................................................63 In Vivo Evaluations..............................................................................................64 Noninvasive Magnetic Resonance Imaging (MRI).............................................64 Behavioral Analysis.............................................................................................75 Retrograde Tract Tracing....................................................................................77 Anatomical and Histological Analyses................................................................78 Summary.....................................................................................................................95 4 CHITOSAN-BASED COMPOSITIONS FOR THE REPAIR OF INJURED NEURAL TISSUE.......................................................................................................................96 Introduction.................................................................................................................96 Specific Aims..............................................................................................................96 Aim 1....................................................................................................................97 Aim 2....................................................................................................................97 Materials and Methods...............................................................................................97 Chitosan Solutions...............................................................................................97 Genipin Solutions................................................................................................98 Microporous Foam Synthesis..............................................................................98 In Vivo Evaluations.....................................................................................................99 Surgical Implantation...........................................................................................99 Non-invasive Magnetic Resonance Imaging (MRI)..........................................100 Behavioral Analysis...........................................................................................100 Retrograde Tract Tracing and Tissue Processing............................................100 Results and Discussion............................................................................................101 Non-invasive Magnetic Resonance Imaging (MRI)..........................................101 Behavioral Analysis...........................................................................................103 Retrograde Tract Tracing..................................................................................103 Anatomical and Histological Analyses..............................................................104 Summary...................................................................................................................113 vii

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5 DNA-BASED COMPOSITIONS FOR REPAIRING INJURED NEURAL TISSUE......114 Introduction...............................................................................................................114 Specific Aims............................................................................................................116 Aim 1..................................................................................................................117 Aim 2..................................................................................................................117 Materials and Methods.............................................................................................117 DNA Isolation.....................................................................................................117 Preparation of Nano/Microstructured Porous DNA...........................................118 Characterization........................................................................................................119 Electron Microscopy..........................................................................................119 GPC and Multi-angle Light Scattering (MALS).................................................119 In Vivo Evaluation.....................................................................................................119 Surgical Implantation.........................................................................................119 Noninvasive Magnetic Resonance Imaging (MRI)...........................................120 Spontaneous Vertical Exploration.....................................................................120 Retrograde Tract Tracing..................................................................................120 Immunohistochemical Analysis.........................................................................121 Results and Discussion............................................................................................122 Porous DNA Preparation...................................................................................122 Noninvasive Magnetic Resonance Imaging (MRI)...........................................123 Behavioral Analysis...........................................................................................124 Retrograde Tract Tracing..................................................................................124 Immunohistochemical Staining.........................................................................125 Summary...................................................................................................................139 6 CONCLUSIONS...........................................................................................................140 7 FUTURE DIRECTIONS...............................................................................................143 LIST OF REFERENCES..................................................................................................145 BIOGRAPHICAL SKETCH..............................................................................................158 viii

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LIST OF TABLES Table page 2-1 Human SCI Clinical Trials......................................................................................13 2-2 Growth factors, cytokines and chemokines produced by microglia.......................16 3-1 -Radiation polymerization conditions of MPC.......................................................34 3-2 Molecular vibrations associated with the polymeric phospholipid MPC.................37 3-3 XPS binding energies of interest...........................................................................38 3-4 Radiation dose response on MPC Mw and Rg......................................................49 3-5 Effects of dose (Mrad) and dose rate (rad/min) on MPC molecular weight (Mw) and radius of gyration (Rg)...........................................................................................55 ix

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LIST OF FIGURES Figure page 2-1 T2 diffusion weighted MR image of SCI............................................................8 2-2 Nissyl stained contused spinal cord..................................................................9 2-3 Molecular structure of methylprednisolone (MP)..............................................10 2-4 Adult rat red nucleus labeled with the retrograde tract tracer Fluorogold............19 2-5 Molecular structure of alginate.......................................................................23 2-6 Molecular structure of 2-methacryloyloxyethyl phosphorylcholine......................24 2-7 Molecular structure of the cationic polysaccharide chitosan..............................25 2-8 Molecular structure of DNA and ability to base pair..........................................27 3-2 Multi-angle lightscattering chromatogram of alginate........................................44 3-3 Optical micrographs of porous freeze-dried and tubular gel alginates................44 3-4 SEM micrographs of porous alginate foam compositions..................................45 3-5 SEM micrographs detailing pore and surface morphology................................46 3-6 SEM micrographs of semi-solid tubular alginate gel compositions.....................47 3-8 MALS chromatograms for MPC polymerized under varied dose........................50 3-9 FTIR spectra of MPC modified alginate..........................................................50 3-10 MALS chromatogram illustrating the effects of dose rate on molar mass............54 3-11 MALS chromatogram illustrating the effects of total dose on molar mass...........54 3-12 Effects of dose and dose rate on molar mass..................................................56 3-13 Effects of dose and dose rate on radius of gyration..........................................57 3-14 FT-IR spectrum of MPC modified alginate.......................................................58 x

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3-15 FT-IR spectrum of MPC modified alginate.......................................................59 3-16 EDS spectrum for a representative MPC modified alginate...............................60 3-17 MALS 3-D chromatograms............................................................................61 3-18 Pilot AFM images of MPC gamma modified alginate........................................62 3-19 PC-12 cell differentiation...............................................................................63 3-20 T2 diffusion weighted MRI serial sections of a normal control...........................67 3-21 T2 diffusion weighted MR image of an untreated SCI.......................................68 3-22 T2 diffusion weighted MRI 4 months following injury........................................69 3-23 T2 diffusion weighted MRI serial sections of porous alginate.............................70 3-24 T2 diffusion weighted MRI 4 months following injury........................................71 3-25 T2 MRI of MPC modified alginate implant 24 hrs after implantation...................72 3-26 T2 diffusion weighted serial MRI....................................................................73 3-27 T2 diffusion weighted MRI.............................................................................74 3-28 Spontaneous vertical exploration at 4 months post-injury.................................77 3-29 Spontaneous vertical exploration 4 wks post-injury..........................................80 3-30 Spontaneous vertical exploration 8 wks post-injury..........................................80 3-31 Spontaneous vertical exploration 12 wks post-injury........................................81 3-32 Spontaneous vertical exploration 16 wks post-injury........................................81 3-33 Serial sections of the magnocellular red nucleus.............................................82 3-34 Serial sections of the contralateral magnocellular red nucleus..........................83 3-35 3-D reconstruction of the magnocellular red nucleus........................................84 3-36 Gross anatomical analyses 4 months post-implantation...................................85 3-37 Cresyl violet staining of the lesion site for a representative untreated control subject........................................................................................................86 3-38 Lesion site morphology 4 wks post-implantation..............................................87 3-39 Lesion and implant interface for porous alginate treated animals at 4 wks..........88 xi

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3-41 Lesion site morphology for MPC modified porous alginate................................90 3-42 Lesion site morphology for semi-solid alginate-microglial cell gel implants.........91 3-43 Details of lateral lesion site morphology for semi-solid alginate-microglial cell gel implants.......................................................................................................92 3-44 Ependymal cell migration..............................................................................93 3-45 HA-alginate blend gel compositions 4 wk following implantation........................94 4-1 Molecular structure of chitosan......................................................................98 4-2 Molecular structure of genipin........................................................................98 4-3 T2 diffusion weighted MR image of an untreated SCI subject.........................105 4-4 T2 diffusion weighted MRI serial sections of an untreated animal in the sagittal, coronal and horizontal planes 4 months following injury.................................106 4-5 T2 weighted MRI 4wks following implantation of a porous chitosan.................107 4-6 T2 weighted MRI 4wks following implantation of a porous chitosan.................108 4-7 T2 weighted MRI 1 yr post-implantation........................................................109 4-8 T2 weighted MR image of genipin-crosslinked chitosan implant......................110 4-9 Spontaneous vertical exploration 4 wk post-implantation................................111 4-10 FG retrograde tract tracing 4wk post-injury....................................................111 4-11 Cresyl violet stained spinal cord section at the implant/tissue interface............112 5-1 Molecular structure of DNA..........................................................................115 5-2 MALS chromatograms of Herring teste DNA following high-speed mixing........127 5-3 SEM micrographs of a porous DNA composition...........................................128 5-4 Optical images of solution cast DNA thin films...............................................129 5-5 SEM micrographs of solution cast DNA thin films..........................................130 5-6 T2 weighted MR images.............................................................................131 5-7 T2 weighted MR images 3 days post-implantation.........................................132 5-8 Fluorogold labeled neurons in the magnocellular red nucleus.........................133 xii

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5-9 Cresyl violet staining of a DNA treated lesion................................................134 5-10 Dense cellular infiltrate and NF-M immunolabeled nerve fibers in the lesion site epicenter...................................................................................................135 5-11 Neurofilament M immunoreactive axons and gray matter cell bodies 4 months post-injury..................................................................................................136 5-12 Immunopositve macrophages and microglia..................................................137 5-13 Maisson trichrome staining for collagen and connective tissue........................138 xiii

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Abstract of Dissertation Presented to the Graduate School of the University of Florida { TC "ABSTRACT" }in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy BIOPOLYMER-MICROGLIA CELL COMPOSITIONS FOR NEURAL TISSUE REPAIR By Joshua Benjamin Stopek August 2003 Chair: Eugene P. Goldberg Major Department: Materials Science and Engineering The design of clinically effective treatments following traumatic spinal cord injury (SCI) remains a challenge. The excitement of recent advances in the understanding of CNS development and pathologies has been tempered by a lack of neurological recovery following current attempts to repair SCI. Our primary objective was therefore to conduct a tissue engineering study using composite microglial cell-biopolymer scaffold compositions for the repair of injured neural tissue following SCI. The basic concept for this research was to use neural cell-biopolymer systems which might control the complex repair processes after CNS trauma. Novel nano-mesoporous alginate/phospholipid and DNA-based scaffold implants were synthesized and implanted into the injured adult rat spinal cord using a partial cervical SCI model. New methods for the phospholipid surface modification of alginate were developed. This work also marks the first reported use of DNA as a scaffold biomaterial for treatment following SCI. Dorsolateral funiculotomies at C4/C5 were made in order to transect the right rubrospinal tract. Some implants were seeded with primary rat microglia. Alginate-based implants showed significant signal voiding xiv

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(hypointense) under MRI, which histologically correlated to a dense cellular infiltrate with minimal associated inflammation. Untreated control lesions showed hyperintense signal at the lesion, indicative of cerebral spinal fluid and cyst formation and a characteristic pathological response following SCI. Implant treated subjects showed minimal cystic cavitation and promoted tissue regeneration. Alginate-microglial cell implant subjects retained increased neurological function and use of the affected right limb (10 to 15 %) versus untreated controls (p<0.05). Although, this motor function recovery was encouraging, treated subjects still exhibited significant functional deficits during reaching, grasping, and walking. They were found to be statistically different than normal healthy animals (p<0.05). High field MRI and histology corroborated tissue regeneration. Retrograde labeling with Fluorogold was demonstrated in the injured red nucleus for alginate and DNA-based implant-treated subjects indicating some ability for axonal transport to the brain. Untreated controls did not show this behavior. Results of studies to date with our composite implants suggest that optimization of compositions, implant design, and surgical procedures may lead to a clinically important medical technology for spinal cord injury. xv

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CHAPTER 1 INTRODUCTION There is currently no effective treatment following traumatic spinal cord injury (SCI). There are approximately 10,000 new SCI patients annually in the United States, with 250,000 Americans currently suffering from some sort of SCI. Methylprednisolone, a synthetic corticosteroid and the standard of care, is the only clinically available treatment given immediately after injury. However, its safety and efficacy have been recently questioned. (Rabchevsky et al, 2002). The pathological events of traumatic SCI are also poorly understood, making the design of clinically effective treatments challenging. Where once it was thought that damage to the central nervous system (CNS) was irreversible and irrepairable, it is now understood that the CNS’s apparent lack of regenerative ability can be manipulated (Savio and Schwab, 1990; Richardson et al., 1980). Experimental repair strategies to promote spontaneous regeneration and neuronal survival have emphasized the transplantation of neural tissue and cells, as well as the delivery of neurotrophic proteins or genes, antibodies (IN -1) against Nogo or the Nogo receptor (Ng-R), chrondroitinase ABC, and anti-inflammatory or excitatory agents (Schwab, 2002). However, it is improbable that any single intervention, (i.e., the delivery of a single protein or gene) will completely reverse the consequences of traumatic SCI. It is also unlikely that any of these strategies would be candidates for systemic administration. Multi-faceted treatments providing localized biomechanical and biochemical support need to be designed. In view of this, the strategy of this research was to use viable CNS cells, microglia, to control the synthesis and regulation of the highly complex neurorepair process. 1

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2 Our primary objective was to conduct a tissue engineering study using composite cell-biopolymer scaffold compositions for preventing post-traumatic cystic cavitation and repair of injured neural tissue after SCI. Novel microporous alginate/phospholipid and DNA-based scaffold implants were synthesized and acutely implanted into the injured adult rat spinal cord using a partial SCI model. Some implants were seeded with primary rat microglia. Emphasis was placed on noninvasive evaluation of implants by high field magnetic resonance imaging (MRI) and functional evaluations. Retrograde tract tracing, and immunohistochemical staining were also used. Our study combined perspectives from biomaterials science and neurobiology in an effort to provide both biomechanical and biochemical support, in a favorable surface terrain and structure (biopolymer implant), as well as in the form of various pro-regenerative molecules produced by microglia. Important broader implications of results stemming from this project include the application of implant compositions for the treatment of optic nerve disease and trauma, as well as macular degeneration and retinal disease. The specific aims were as follows: Aim 1 Synthesis and characterization of novel porous polysaccharide, phospholipid, and polynucleotide biopolymer-based implant compositions. Porous alginate, chitosan and DNA biopolymer compositions were prepared using freeze dry/lyophilization and film casting techniques. Some porous alginate compositions were surface/bulk modified with the polymeric phospholipid, 2-methacryloyloxyethyl phosphorylcholine (MPC) using gamma radiation initiation polymerization. Fourier Transform Infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), elemental analysis for C, O, P and N atomic content, atomic force

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3 microscopy (AFM), and multi-angle light scattering (MALS) for molecular weight determinations were used. The effects of gamma radiation, ethylene oxide and autoclave sterilization on implant stability were also investigated. Aim 2 In vivo evaluations of biopolymer-microglia scaffold compositions in an adult rat partial SCI model. We tested the hypothesis that intraspinal porous polysaccharide, phospholipid, and DNA biopolymer implants can facilitate endogenous tissue regeneration, leading to the prevention of post-traumatic cystic cavitation and functional recovery of injured axons. Dorsolateral funiculotomies at level C4/C5 were performed in the adult rat cervical spinal cord in order to transect the right rubrospinal tract. Implant compositions, some containing primary rat microglia, were placed immediately after injury. Animals were allowed to recovery for 1 week to 1 year post-implantation. Analyses included high field magnetic resonance imaging (MRI), behavioral analysis by spontaneous vertical exploration, retrograde tract tracing, and immunohistochemical staining.

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CHAPTER 2 BACKGROUND There is currently no cure or effective therapies for traumatic spinal cord injury (SCI). There are 10,000 new spinal cord injury (SCI) patients annually in the United States. Approximately 250,000 Americans currently suffer from some sort of SCI, with an annual health care cost in excess of $10-12 billion. Methylprednisolone (MP), a synthetic corticosteroid used to attenuate the acute inflammatory response, is the only clinically available treatment given immediately following SCI. However, its safety and efficacy are now debated (Rabchevsky et al, 2002). The pathological events of traumatic SCI are also poorly understood, making the design of clinically effective treatments challenging. The intricate electrical and biochemical circuitry responsible for rhythmic coordinated movements, such as locomotion, are intrinsic within the spinal cord. Descending and segmental afferent inputs modulate locomotion, and after insult this is severely altered. Recent reports in neurobiology suggest that the mature mammalian central nervous system (CNS) remodels continually throughout aging, and retains its intrinsic capacity to respond to signals that promote survival and regeneration after injury or disease. However, this capacity appears to be inhibited by a nonpermissive extracellular post-lesion microenvironment including glial scarring, lack of sustained neurotrophic support, a suppressed post-injury inflammatory response, and progressive secondary injury mechanisms. In addition, several putative growth inhibitory molecules have been identified, including myelin inhibitory proteins such as Nogo-A, MAG and the family of chrondroitin sulfate proteoglycans. The restrictive nature of these inhibitory 4

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5 molecules has been reported by several in vitro and in vivo developmental studies (Karim et al., 2001). In addition to myelin debris, glial scarring presents a dense mechanical and biochemical barrier at the lesion site interface, making it increasingly difficult for regenerating axons to traverse the injury in route to their targets. Reactive astrocytes, activated microglia, oligodendrocytes and fibroblasts compose the bulk of the scar, which has been reported to contain a variety of potentially axonal regrowth inhibitory factors including semaphorins, ephrins and tenascin. Until recently, little was understood concerning the mechanisms and consequences of acute and chronic inflammatory responses after insult to the CNS. While the inflammatory responses observed in CNS tissue shares some similarity to the responses seen in peripheral tissues, there are marked differences in intensity (Perry et al., 1995). An equivalent external stimulus has been reported to result in a much weaker response in the CNS (Andersson et al., 1992). In particular, the accumulation of mononuclear phagocytes in response to CNS injury is delayed and limited in comparison to injury in the PNS. This limited CNS mononuclear phagocyte response may in turn lead to inefficient removal of inhibitory myelin and blood component debris; and suboptimal release of macrophage/microglia-derived factors that would promote modulation of Schwann cells, astrocytes and oligodendrocytes so as to support spontaneous axonal regeneration. Microglia, brain macrophages of monocytic origin, constitute the resident CNS immune system and have drawn increased attention in the last decade in their ability for scavenging dead cells, antigen-presentation, and immunomodulation. They have been reported to promote spontaneous, endogenous regeneration by enhancing the non-permissive nature of the CNS through the production of various neurotrophic factors, chemokines, cytokines, extracellular matrix (ECM) molecules, and other factors.

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6 However, activated microglia have also been reported to exhibit neurotoxic behavior since they retain the capacity to produce potentially cytotoxic molecules in pathological conditions including pro-inflammatory cytokines, reactive oxygen intermediates, nitric oxide, glutamate and eicosanoids. The reasons for the limited and attenuated CNS immune response and the role of multi-potential microglial post-injury remain unclear. After axotomy of most peripheral nervous system (PNS) neurons, there is an upregulation of specific regeneration-associated genes (RAGs) and molecular repair programs leading to regeneration and functional recovery. In stark contrast, the axotomy of CNS neurons (those primarily affected by SCI) leads to little, transient, or no upregulation of comparable RAGs and cellular repair programs (Hiebert et al, 2000; Houle et al., 1998; Kobayashi et al., 1997; Fernandes et al., 1999; Tetzlaff et al., 1988, 1991, 1992, 1994, 1996). Axotomized adult CNS neurons either atrophy or die, and nerve impulse conduction across the lesion site ceases (Bregman, 1998; Reier et al., 1983; Caroni and Schwab, 1988a, b; Rudge and Silver; 1990; Crowe et al., 1997). The poor regenerative properties of CNS axons also seem to worsen with distance from the terminal cell body (Lazarov-Spiegler et al., 1998). Most importantly, the pathology of SCI is not limited to the initial insult. Secondary mechanisms of injury including free-radical formation, excitotoxicity, anoxia and ischemia may occur over days and weeks following injury (Beattie et al., 2000). The further dynamic loss of spared tissue and inadequate spinal wound healing frequently result in post-traumatic syringomyelia (SM) and post-traumatic spinal cord tethering (SCT). Both states have been suggested to be a pathophysiological cause for the formation of fluid-filled cystic cavities, which may further jeopardize surviving tissue and cause various sorts of chronic pain (Figures 2-1 and 2-2). Post-traumatic cysts occur in approximately 50% of SCI patients, with a 10% incidence of post-traumatic SM occurring in all SCI patients.

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7 As mentioned previously, no current treatment affords significant functional or morphological recovery after traumatic SCI. Clinical treatments are aimed mostly at minimizing the acute inflammatory response (standard of care high-dose MP) after the initial mechanical insult and attenuating the later consequences of chronic pain and spasticity. These treatments generally are not geared at repairing and regenerating neural tissue, but rather at maintaining and stabilizing via various drug regimens and neurosurgical procedures. Central deafferent or neurogenic pain is common after SCI. These pains are intrinsically generated by the injured spinal cord itself. Pain may be experienced in areas where normal sensation has ceased following injury. This pain may be either proximal or distal to the spinal injury. These pains are classically described as burning, stinging, stabbing, sharp, shooting, squeezing and/or tight. Additionally, pain may occur immediately following injury or years following. Pain may be initially treated with antidepressants or antiseizure medications; and if unsuccessful, neurosurgical intervention may be required. Additionally, the presence of spinal “tethering” or scarring may cause abnormal and enhanced activity of pain-associated fibers within the spinal cord. The surgical release of scar tissue (untethering) and/or shunting of cystic cavities have been reported to afford some pain relief. A variety of surgical procedures using radio frequency (RF) heat induced lesions, including computer-assisted dorsal root entry zone microcoagulation and percutaneous thermal rhizotomies, have been used to target pain fibers in people suffering pain in dermatomes and unilateral pain, and spasticity. However, results have been less than satisfactory in patients with sacral or diffuse pain.

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8 Figure 2-1 T2 diffusion weighted MR images of a SCI in the adult rat with significant cavitation noted by a lesion-localized hyperintense signal likely from CSF accumulation (FOV = 6 x 4 cm, TR = 3 s and TE = 60 ms) in the coronal (A) and sagittal (B) planes.

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9 Figure 2-2 Nissyl stained contused spinal cord displaying the classical case of cystic cavitation and gray matter necrosis (coronal plane). Invasive surgical procedures for the scission of specific sensory nerve rootlets or placement of intrathecal drug pumps have been used for the treatment of spasticity. It is important to note that these procedures may also be associated with a variety of additional neurological complications. Human Clinical Trials As previously mentioned, there are approximately 10,000 new SCI patients annually in the United States alone. Of these, men suffer roughly 80% of SCI, and 60% of injuries (para and quadraplegia) occur between the ages of 16 and 30 yr, with 80% of all injuries occurring between the ages of 16 and 45 yr. Motor vehicular accidents (47%) and falls (21%) are reported to be the leading causes of trauma. Sports (16%) and violence (14%) related injuries represent 30% of SCI. Several SCI clinical trials have been completed (Table 2-1) with some therapies progressing to the clinic, including the standard of care, a 30 mg/kg bolus of MP (Figure 2-3), followed by 5.4 mg/kg/hr over 23 hr when initiated within 3 hr of injury. MP

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10 treatment continues for 48 hr if initiated within 3 to 8 hr of trauma. The primary mechanism of action of MP is the inhibition of hydrolysis and lipid peroxidation at the lesion site as reported by the National Acute Spinal Cord Injury Study (Bracken et al. 1990, 1992; Bracken and Holford, 1993; Hall and Braughler, 1987; Young, 1990). Similar studies have been performed using tirilazad mesylate (TM), also reported to be a potent inhibitor of lipid peroxidation. However, decreased neurological recovery has been reported compared to a 48 hr regimen of MP (Bracken et al., 1997). Monosialotetrahexosylganglioside (GM-1) is currently in open-label phase III clinical trials for treatment of chronic SCI. Gangliosides represent complex acidic glycolipids present in CNS cells as a major constituent of the outer leaflet of the cell membrane bilayer. Acute delivery of GM-1 has been reported to improve neurological recovery in patients suffering stroke or subarachnoid hemorrhage, and more recently SCI (Goldstein et al., 1995; Geisler, 1998). O OH H CH3 CO H CH3 H OH CH2OH CH3 Figure 2-3 Molecular structure of methylprednisolone (MP). Whole body hypothermia and local cord cooling have been reported to offer promising results following phase l trials. Hypothermia is thought to be neuroprotective for specific cell populations with increased secondary sensitivity to neuronal death or prolonged inflammation, which may decrease immediate neuronal mortality (Hansebout et al., 1984; Hayes et al., 1993). Local acute (within 8 hr) cooling of the cord in 10

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11 patients significantly enhanced the rate of sensory and motor recovery, accompanied with a decrease in mortality in comparison to conventional methods of treatment (Hansebout et al., 1984). These results corroborated previous spinal irrigation studies with 5 o C saline in 8 patients (Bricolo et al., 1976). The local infusion of the voltage-sensitive K channel blocker 4-aminopyridine (4-AP) in 6 to 30 mg/kg doses has been examined for blocking exposed K channels in an effort to partially restore functional conduction of surviving demyelinated axons. Phase I clinical trials of 4-AP reported increased sensory and motor control caudal to the injury, reduction in spasticity and chronic pain, and restored voluntary bowel control in some patients (Hayes et al., 1994; Hansebout et al., 1993). Phase ll trials are currently underway with 200 incomplete SCI patients (Accorda Therapeutics, Inc.). However, the determination of 4-AP efficacy using the American Spinal Injury Association (ASIA) sensory and motor scoring systems has been difficult. Pilot safety and feasibility studies exploring the implantation of human fetal tissue transplants in patients suffering post-traumatic SM have shown some promise in treating cavitation following SCI. Similar results were reported following preclinical animal trials (Diener and Bregman, 1998; Bregman et al., 1997; Anderson et al., 1995; Reier et al., 1994). Embryonic en bloc transplants may also provide a rich source of factors that have been reported to mediate growth, astrogliosis and host nerve fiber conduction (Zompa et al., 1997). However, regenerating host fibers in adult animal studies have been found to terminate within the fetal transplants themselves, rather than projecting into the host cord. This may be attributed partly to the growth inhibition of the host microenvironment (Bregman et al., 1989; Reier et al., 1983). Eight human patients have been treated with 6 to 9 week postconception human fetal tissues; and data for the first 2 patients have been reported through 18 months after surgery (Wirth et al, 2001). Patient outcome measures included magnetic resonance

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12 imaging (MRI), standardized measures of neurological impairment and functional disability, pain assessment, and neurophysiological testing. The first 2 patients were reported to be neurologically stable. Detailed MRIs showed evidence of the successful incorporation of graft tissues without donor tissue overgrowth. Voluntary locomotor function and reduction of chronic central pain were not reported. Thus, fetal tissue transplants, while possibly inhibiting progressive neural tissue loss due to chronic injury, do not facilitate significant functional restoration of white matter long tracts (Falci et al., 1997b; Wirth et al., 1995). The transplantation of myelin basic protein (MBP) activated peripheral macrophages is currently in phase I clinical trials in Israel (Proneuron Biotechnology, USA; Weizmann Institute of Science, Rehovot, Israel). It is thought that the implantation of patient-derived macrophages may provide local neurotrophic support through the secretion of neurotrophins, ECM, cytokines, and chemokines; and in the ability to mediate reactive gliosis and the phagocytosis of dying cells and debris. Promising preliminary results in a limited number of patients have been reported; and the FDA has recently accepted macrophage-based therapy as an Investigational New Drug. The development of chronic pain syndromes occurs in most patients and represents a frequently neglected aspect of SCI (Christensen and Hulsebosch, 1997). The use of baclofen and the anticonvulsant gabapentin are currently used as analgesics for the treatment of central pain (Ashburn and Staats, 1999; Attal et al., 1998). Antidepressants including tricylic amitripyline have proven to be clinically effective in the treating dysesthetic pain, which may be related to the inhibition of norepinephrine and serotonin reuptake.

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13 Table 2-1 Human SCI Clinical Trials. Treatment Action Methylprednisolone (MP) Anti-inflammatory Tirilazad mesylate (TM) Anti-inflammatory 4-aminopyridine (4-AP) K channel blocker Monosialotetrahexosylganglioside (GM-1) Ganglioside Gacyclidine Excitotoxicity Baclofen Spasticity/ chronic pain Gabopentin Spasticity/ chronic pain Amitriptyline Spasticity/ chronic pain Nortriptyline Spasticity/ chronic pain Clonidine Spasticity/ chronic pain Hypothermia/local SC cooling Anti-inflammatory/SecondaryInjury Fetal tissue transplantation Cystic cavitation Cell transplantation Neurotrophic factors/phagocytosis

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14 Experimental Repair and Regeneration Concepts Once it was thought that damage to the central nervous system was irreversible and irrepairable. Now it is understood that the CNS’s apparent lack of regenerative ability can be manipulated (Savio and Schwab, 1990; Richardson et al., 1980). Experimental repair strategies to promote spontaneous regeneration and survival have emphasized the transplantation of neural tissue and cells, as well as the delivery of neurotrophic proteins or genes, antibodies (IN -1) against Nogo or the Nogo receptor (Ng-R), chrondroitinase ABC, and anti-inflammatory agents (Schwab, 2002). The implantation of various biomaterials and medical and bioelectrical devices has also been reported. However, it is improbable that any single intervention, i.e. the delivery of a single protein or gene, will completely reverse the consequences of traumatic SCI. It is also unlikely that any of these strategies would be candidates for systemic administration. Thus, the design of multi-faceted combinatorial treatments that provide localized biomechanical and biochemical support is necessary. Delivery of regeneration associated genes and proteins including NGF, BDNF, NT-3, NT-4/5 and CNTF (Romero et al., 2001; Kwon and Tetzlaff, 2001), anti-apoptotic agents such as bcl-2 (Takahashi et al., 1999), anti-inflammatory agents such as IL-10 (Bethea et al., 1999), acute delivery of subunit specific excitatory amino acid receptor antagonists (Rosenberg et al., 1999; Wrathall et al., 1997) and glial and stem cell therapies (Liu et al., 1999; McDonald et al., 1999; Zompa et al., 1997) are being investigated. However, these interventions have not led to significant functional or morphological recovery. Neuroprotective and Neurotrophic Microglia and Macrophages Microglia are uniformly distributed in the normal CNS, where they are identified as “resting”, or ramified, microglia. In times of injury or disease, microglia expediently transform from the resting state into the activated microglia (Streit and Kreutzberg, 1988;

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15 Streit et al., 1988). Microglial activation is evident by cellular hypertrophy and retraction of cytoplasmic processes, transforming to a more ameboid morphology. This is accompanied by an increase in cell surface antigens including complement receptors and MHC antigens (Streit et al., 1989; Graeber at al., 1988). Conflicting ideas exist pertaining to both neurotoxic and neuroprotective roles of activated microglia in association with injury in the CNS. Their presence alone in large numbers at injury sites has led to the notion that these mononuclear phagocytes are intrinsically destructive. It is thought that they exacerbate lesions through the secretion of cytotoxins and noxious agents such as nitric oxide, glutamate and reactive oxygen intermediates (Chao et al., 1992; Colton and Gilbert, 1987; Piani et al., 1991). However, there is a considerable body of data that supports a neurotrophic and neuroprotective role for microglia in the post-traumatic microenvironment. This may be attributed to the expression of microglia-derived cytokines, neurotrophic factors, extracellular matrix (ECM) molecules and recruitment of cells from the periphery (Table 2-2) (Lazar et al., 1999). Microglia have been reported to secrete growth factors such as NGF (Mallat et al., 1989), bFGF (Shimojo et al., 1991), BDNF (Bachelor et al., 2002), NT-3 and NT-4/5 (Elkabes et al., 1996). They have also been reported to secrete ECM molecules including laminin and thrombospondin, both known to be potent promoters of neuron regeneration (Rabchevsky and Streit, 1997, 1998: Mller et al., 1996; Chamak et al., 1994; Masuda-Nakagawa et al., 1993; O’Shea et al., 1991; Neugebauer et al., 1991). Nagata et al. (1993) reported microglia-conditioned medium promoted the survival and development of cultured neurons. In addition, microglia derived IL-1 and TGFhas been reported to induce NGF production in astrocytes and Schwann cells (Yoshida and Gage, 1992; Lindholm et al., 1990; Lindholm et al., 1987), demonstrating a pro-regenerative role for these cytokines. It has also been shown that the astroglial

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16 Table 2-2 Growth factors, cytokines and chemokines produced by microglia. GFs Cytokines Chemokines ECM NGF IL-1/IL-1 MCP-1 Laminin BDNF IL-3 MIP-1 Thrombospondin NT-3 IL-6 MIP-1 KSPGs NT-4/5 IL-8 MIP-2 PDGF IL-10 RANTES EGF IL-12 bFGF IL-15 M-CSF TGFTNFtPA

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17 response may be modified by activated microglia via the secretion of IL-1 and IL-6 (Giulian et al., 1986; Woodroofe et al., 1991). These studies led to a number of in vivo transplantation experiments involving microglia/macrophages. When placed into the acutely injured spinal cord, purified microglia were shown to increase neuritic growth compared to astrocytic grafts (Rabchevsky and Streit, 1997). In addition, spontaneous nerve fiber growth into controls not containing grafted microglia almost always occurred in regions near the transplant that showed intense infiltration of host microglia/macrophages. The presence of OX-42 positive microglia within transplants was not coincident with the presence of GFAPpositive astrocytes. GFAP immunoreactivity was usually restricted to surviving tissue surrounding the implantation site. Microglia transplanted into the injured spinal cord in conjunction with fetal neural transplants were reported to increase regeneration of dorsal root ganglion (DRG) sensory fibers (Prewitt et al., 1997). Regeneration of descending tracts accompanied by improved locomotor function was observed when macrophages alone were implanted into the transected spinal cord (Rapalino et al., 1998). Other authors have observed similar beneficial effects of macrophage transplants following spinal cord injury and have attributed these primarily to the enhanced removal of myelin debris, production of extracellular matrix molecules, and the promotion of neovascularization by the grafted cells (Franzen et al., 1998). In other studies, injections of activated peripheral macrophages were used to overcome the inherent non-permissive nature of the optic nerve for regeneration (Lazarov-Spiegler et al., 1996, 1998). The results from these transplantation studies are perhaps not surprising since a critical role of macrophages in peripheral nerve regeneration has been known for some time (for review see Griffin et al., 1993). One of the primary pro-regenerative functions attributed to peripheral macrophages in the PNS is the recruitment and activation of

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18 Schwann cells (Griffin et al., 1993), which was observed following intraspinal grafting of purified microglia into the injured adult rat spinal cord (Rabchevsky and Streit, 1997). In all, studies with grafts consisting of microglia/macrophages strongly support the use of these cells for enhancing the generally poor regenerative potential of CNS axons. Rubrospinal Tract The rubrospinal tract (RST) is analogous in many ways to the human corticospinal tract, and arises primarily from a well-defined pool of neurons in the red nucleus (RN). In addition to locomotor responsibilities, the RST has been reported to be associated with exerting control of fine limb movements including reaching and grasping. RST axons exert an excitatory effect on flexor motoneurons, and in all species, the majority of the RST originates from the magnocellular region of the RN (Figure 2-4). Axons exit the ventromedial aspect of the RN, traverse through the ventral tegmental decussation, and descend in the dorsolateral funiculus of the spinal cord. The tract is reported to be approximately 90-95% contralateral, with a small, ipsilateral component reported in the rat and other species (Shieh et al., 1983; Holstege, 1987). Evidence of a somatotopic arrangement has also been reported (Murray and Gurule, 1979). The RST has been characterized in neonatal, developing and adult rats, and more recently following SCI (Shieh et al, 1983; Kobayashi et al., 1997). Rubrospinal neurons have been reported to undergo significant atrophy within 2 weeks following cervical axotomy. This has been correlated with a decreased expression of regeneration-associated genes (RAGs) and trk receptors including GAP-43 and Talpha1-tubulin. TrkB family receptor mRNA expression has been reported to decline within 7 days post-axotomy. Tetzlaff has reported low level expression of full-length trkC mRNA, undetectable trkA mRNA, and localized p75 expression in a subpopulation of axotomized neurons (Kobayashi et al., 1997). Differences in RN neuron RAG expression have also been noted to vary with proximity of the axotomy to the terminal

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19 cell body. In vivo evaluations included in this work were conducted using a partial transection model in the adult rat, which included axotomy of the rubrospinal tract at C4/C5. Figure 2-4 The adult rat red nucleus (A) labeled with the retrograde tract tracer Fluorogold shown in detail (B). Previous Use of Biomaterials for SCI Repair A variety of conceptual approaches utilizing biomaterials, most notably biopolymers, have been applied to attempt to provide a permissive microenvironment for the recovery and regrowth of injured axons. Permanent and degradable materials with varied architectures have been designed, some with intent to deliver growth factors, gene therapy agents, cells or other therapeutic agents (Teng et al., 2002; Plant et al., 1995; Bellamkonda et al., 1995; Woerly et al., 2001). Other interventions have focused on preparing tailored surface chemistries and features, which may facilitate preferential cell adhesion and spreading, as well as directed growth (Patel et al., 1998; Biran et al., 2001). The use of biopolymer devices presents a possible advantage in being capable of not only providing physical scaffolding and a favorable surface terrain for regenerating tissue, but also in the ability to locally deliver a host of the therapeutic agents previously mentioned including tissue, cells, gene therapy and neurotrophic agents, and drugs. Surfaces modified with the amino acid sequence RGD were reported to direct primary nerve cell adhesion and neurite outgrowth in culture (Saneinejad and Shoichet,

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20 2000). Similar results have been observed for PC-12 nerve cells cultured on RGD (Patel et al., 1998 and Ranieri et al., 1995). Aligned micro-contact printing of micrometer-scale features and electrically conducting polymers are also under consideration (James et al., 2000; Kotwal et al., 2001). Electrical stimulation and bioelectricity has been shown to enhance spontaneous regeneration in many areas of wound healing. Electrical stimulation may alter the local fields of ECM molecules, which has been reported to increase the expression of neurites. A large number of studies have reported that ECM-derived proteins are potent promoters of neurite outgrowth (Archibald et al., 1991; Webb et al., 2001). Novel methods of depositing surfactant-immobilized fibronectin have been shown to enhance bioactivity and sensory neurite outgrowth in vitro (Webb et al., 2001b; Biran et al., 2001). Collagen devices/matrices carrying large payloads of Schwann cells or growth factors have been grafted into the lesioned adult spinal cord and a substantial amount of nerve fiber in-growth, both myelinated and unmyelinated, has been noted immunohistochemically (Paino et al., 1994; Joosten et al., 1995; Liu et al., 1998). Locally applied NT-3, released from a collagen type I matrix, has been shown to stimulate and direct the regrowth of corticospinal tract fibers (CST), but no CST fibers grew into areas caudal to the collagen implant (Houweling et al., 1998). A variety of neurotrophins, including brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), NT-3 and NT4/5 have been delivered in like fashions, typically in in vitro cell culture experiments. Extracellular matrix or ECM-like polysaccharides have been of interest in tissue repair and wound healing, including the use of hyaluronic acid (HA), the alginates and agarose. Polysaccharide hydrogels have been reported to stimulate and guide neuronal process extension in three dimensions in vitro and in vivo (Yu et al., 1999; Bellamkonda et al., 1995). Bellamkonda et al. (1995) considered the importance of gel concentration

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21 and pore size, and reported that primary neural cells fail to extend neurites above threshold agarose gel concentrations. In addition, Yu has also reported that neurite extension on similar biopolymer surfaces in vitro can be significantly increased with the covalent coupling of laminin to the scaffold surface. These results are not surprising since a critical role for ECM, most specifically laminin, has been recognized in the regeneration of both PNS and CNS tissues for quite some time. Neurogel (Organogel, Canada Lte), a synthetic hydrogel of (PHPMA), has been extensively investigated. The polymer has been reported to bridge tissue defects and interface well with host tissues. These investigations have also included the immobilization of neuronal and glial cells within the PHPMA, as well as the incorporation of the RGD sequence (Woerly et al., 1996; Woerly et al., 1996b, Woerly et al., 1998; Woerly et al., 2001; Woerly et al., 2001b). Cook et al. (1997) has suggested similar behavior in vitro. Alginates A co-polymer of -D-mannuronic acid (M) and -L-guluronic acid (G), alginate is functionalized with carboxylic acid and hydroxyl groups, making chemical coupling or modification possible (Figure 2-5). The molecular weight (MW) averages and distributions, as well as the M and G ratios, dictate the polymers properties, including gelation characteristics, viscosity and mechanical attributes. It is thought that the gel-forming capabilities and gel pore size correlate with the poly-G content of the polymer. Bivalent cations (calcium, barium and strontium) bridge the negatively charged guluronic acid residues on the alginate backbone. It is thought the M residues only play a subordinate role in the gel architecture (Grant et al., 1973). Increasing G content is reported to produce a gel with increased pore size (Martinsen et al., 1989). Originally found useful in dentistry, alginate has found broad application in cell encapsulation, drug delivery, immunoisolation and tissue engineering, and has been well characterized by

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22 NMR, FTIR and multi-angle light scattering (Seifert and Phillips, 1997; Sartori et al., 1996; Johnson et al., 1997; Shapiro and Cohen, 1996). The use of alginate for neural tissue repair has been reported (Wu et al., 2001; Suzuki et al., 2002; Suzuki et al., 2000; Suzuki et al., 1999; Kataoka et al., 2001). Freeze dried alginate sponges, covalently crosslinked, have been demonstrated to enhance spontaneous nerve regeneration in both the PNS and CNS. Suzuki et al. (1999) implanted freeze-dried alginate foams covered by a poly (glycolic acid) mesh and evaluated its efficacy on peripheral nerve regeneration. Functional reinnervation of motor and sensory nerves occurred 13 weeks post-implantation, characterized by recovery of compound muscle action potential. Similar results were shown by Kataoka et al. (2001) who transversely resected the spinal cord at T7-T8 to produce a 2mm gap. When filled with an alginate foam, the recovery of evoked electromyogram and sensory-evoked potentials 6 weeks after surgery indicated that elongation of axons could establish electrophysiologically functional projections through the gap. Histology revealed that both myelinated and unmyelinated axons had elongated across the lesion. Anterograde tract tracing studies revealed both ascending and descending fibers localized within the alginate. However, these studies were conducted in postnatal day 8 rats, which do not accurately represent the chronic condition in the adult. Suzuki et al. (1999 and 2001) excised the complete spinal cord at T9-T10 and filled the gap by implanting a non-degradable alginate sponge. Horseradish peroxidase tract tracing at 21 weeks after injury illuminated numerous ascending and descending fibers traversing the alginate-filled gap, which extended haphazardly over long distances from the distal stump. These findings, including those by Novikov et al. (2002), show promise in the ability of alginate to maintain a pro-regeneration response in the lesion environment.

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23 4O1 14O 1O4 O H 41O OH H H H OH H O H O O O O H OH H H OH H O H OH H H H O OH H O O OH H H H H O H OH O O M M G G Figure 2-5 Molecular structure of alginate composed of (M) mannuronic and (G) guluronic acid residues. Polymeric Phospholipids From a biomaterials perspective, the use of phospholipid biopolymers has been primarily focused on reducing the thrombogenicity of blood contact devices. Arterial recanalization for stented balloon angioplasty has demonstrated significant restoration of function to diseased coronary and peripheral arteries using catheter based techniques. However, primary failure of stented angioplasty is attributed to neointimal cell growth (restenosis) and an inherently thrombogenic luminal stent surface. Many attempts have been made to resolve this problem and several researchers have reported favorable properties for phospholipid coated-devices. It is important to note that the implantation of an intraspinal device will be in contact with blood due to spinal bleeding emphasizing the ancillary importance of blood compatible materials. In collaborations with our laboratory, Ishihara et al. (1990, 1991, 1998, 1998b, 2000), from the Tokyo Medical and Dental Institute, have synthesized phospholipid polymers including 2-methacryloyloxyethyl phosphorylcholine (MPC) (Figure 2-6). Traditional polymers used for cardiovascular applications, when coated with polyMPC, demonstrated increased endogenous blood phospholipid adhesion and decreased platelet activation (Iwasaki et al., 1999; Iwasaki et al., 1999b: Zhang et al., 1998). Dacron vascular grafts (2mm), coated with polyMPC demonstrated in vivo patency after

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24 5 days in a rabbit model. A significant reduction in platelet adhesion was also observed (Furuzono et al., 2000). These surfaces are thought to improve hemocompatiblity since increased endogenous phospholipid adsorption has been correlated with reduced thrombus formation and chronic inflammation. In our lab, the radical and gamma-radiation induced polymerization and copolymerizations of MPC have been studied, especially for nano/meso surface modifications, which applicable to favorably altering the porous surfaces of alginate and DNA compositions. POO-OCH2CH2+NCH3CH3CH3 H2CCCH3COOCH2CH2O Figure 2-6 Molecular structure of 2-methacryloyloxyethyl phosphorylcholine (MPC) monomer. Radiation Polymerization of Hydrophilic Biopolymers Polymerization by ionizing radiation has the capacity to initiate radical polymerization at ambient temperature in the absence of chemical radical initiators, of which residuals may be cytotoxic and carcinogenic in vivo. The initiation step of radiation polymerization has been reported to be temperature independent, and the net activation energies may be considerably smaller than in chemically initiation processes (Chapiro, 1962 and 1979). Initiation mechanisms via the radiolysis of water and production of hydroxyl radicals, hydrogen atoms and hydrated electron intermediates in the polymerization of aqueous acrylates, methacrylate derivatives and acrylamide type monomers have been reported (Biro et al., 1996; Wojnarovits, et al., 1999; Takacs et al., 1999; Takacs et al., 1999). Additionally, gamma radiation initiation polymerization results in homogenous polymer, free of any impurities, and control may be exerted over the molar mass and

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25 molecular architecture by varying the total dose (Mrad) and dose rate (rad/min). Control may also be placed over polymer solubility and radiation-induced crosslinking by tailoring the respective radiation conditions. Polymerization and surface modification using gamma grafting and polymerization techniques has been previously reported by our laboratory. It may uniquely afford the surface modification of complex and porous geometries. However, little is currently known concerning the synthesis and surface modification of polysaccharide hydrogel materials by radiation-induced polymerization. Chitosan Chitosan is a natural biodegradable copolymer of glucosamine and N-acetylglucosamine derived from the exoskeleton of marine crustaceans. In its highly deacetylated form it is soluble in dilute acids, where it carries a strong positive charge due to the protonation of the amino functionality. Its charge density and solubility are pH dependent and exert control over chitosan’s ability to form hydrogels under neutralizing conditions. The molecular structure of chitosan is shown in Figure 2-7. O O NH2 OH OH O NH2 OH OH O O NH2 OH OH O Figure 2-7 Molecular structure of chitosan. Originally discovered to accelerate wound-healing mechanisms, chitosan has been broadly investigated for drug and gene delivery, as well tissue engineering. Limited data exists for application in CNS repair following injury or disease. Deoxyribonucleic acid (DNA) Nucleotides, the monomers of nucleic acids, are composed of a pentose sugar, phosphate group, and nitrogenous base, either a purine (adenine or guanine) or a pyrimidine (cytosine or thymine). In the DNA polymer (Figure 2-8), nucleotides are

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26 joined by phosphodiester linkages between the phosphate of one nucleotide and the sugar of the next to synthesize a regular backbone. Conversely, base sequence is unique for each gene. In addition, uninterrupted DNA of a single human chromosome may constitute 2 x 10 8 base pairs. This DNA, as well as the DNA of the other 45 human chromosomes, is neatly compacted into a multilevel packing system contained within the cell nucleus. Since the publication of Watson and Crick’s letter to Nature in 1953 elucidating its double helix structure, DNA has been widely studied and the human genome mapped. However, little is known concerning DNA’s capabilities as a structural biomaterial scaffold for the repair of injured or diseased tissue.

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27 NCHN NCN N O O CH2 OH H O NCHN NCN H N H H O O CH2 O P H O O NCHN NCN N O O O CH2 O P H O O NCHN NCN H N H H O O PO O O O PO O O PO O O CH2 O P H O H H H H H H O PO O O PO O O NN O H O CH2 O P O O O H N H H H NN O H O CH2 O P O H C O H H H H NN O H H N O H O CH2 O P O H H NN O H OH CH2 O P O O O H C O H H H H O O O Figure 2-8 Molecular structure of DNA and ability to base pair.

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CHAPTER 3 ALGINATE-BASED COMPOSITIONS FOR THE REPAIR OF INJURED NEURAL TISSUE Specific Aims The primary objective of this research was to conduct a tissue engineering study utilizing composite cell-biopolymer scaffold compositions for the prevention of post-traumatic cystic cavitation and repair of injured neural tissue following SCI. Unique porous alginate scaffold compositions were synthesized using freeze dry/lyophilization techniques. Some compositions were modified with polymeric phospholipids by gamma radiation initiation polymerization under various radiation conditions. Characterization included scanning electron microscopy, energy dispersive spectroscopy, elemental analysis by inductively coupled plasma emission, Fourier transform infrared spectroscopy, X-ray photoelectric spectroscopy, and multi-angle light scattering. Implants, some seeded with primary rat microglia, were acutely placed into the injured adult rat spinal cord using a partial SCI model. Emphasis was placed on noninvasive evaluation of implants by high field magnetic resonance imaging (MRI) and neurological function. Retrograde tract tracing and immunohistochemical staining were also used. This research combined perspectives from biomaterials science and neurobiology in effort to provide both biomechanical and biochemical support, in the forms of a favorable surface terrain and structure (biopolymer implant), as well as from the various pro-regenerative molecules produced by microglia. Important broader implications of results stemming from this project include the application of implant compositions for the 28

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29 treatment of optic nerve disease and trauma, as well as macular degeneration and retinal disease. The specific aims were as follows: Aim 1 Synthesis and characterization of novel porous polysaccharide and phospholipid biopolymer-based implant compositions. Porous alginate biopolymer implant compositions were prepared using freeze dry/lyophilization and film casting techniques. Some implant compositions were surface/bulk modified with the polymeric phospholipid, 2-methacryloyloxyethyl phosphorylcholine (MPC) using gamma radiation initiation polymerization. Fourier Transform Infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), elemental analysis for C, O, P and N atomic content, atomic force microscopy (AFM), and multi-angle light scattering (MALS) for molecular weight determinations were used. The effects of gamma radiation, ethylene oxide and autoclave sterilization on implant stability were also investigated. Aim 2 In vivo evaluations of biopolymer-microglia scaffold compositions in an adult rat partial SCI model. The hypothesis that intraspinal porous biopolymer implants, some containing primary rat microglial cells, can facilitate endogenous tissue regeneration, leading to the prevention of post-traumatic cystic cavitation and functional recovery of injured axons was tested. Dorsolateral funiculotomies at level C4/C5 were performed in the adult rat cervical spinal cord in order to transect the right rubrospinal tract. Implant compositions were placed immediately following injury. Animals were allowed to recovery for 1 week to 1 year post-implantation. Analyses included high field magnetic resonance imaging (MRI), behavioral analysis by spontaneous vertical exploration, retrograde tract tracing, and immunohistochemical staining.

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30 Materials and Methods Polysaccharide Solutions Kelco HV alginate (Mw = 5 x 10 5 g/mol) (Kelco, NJ) was used in the preparation of all microporous foams and semi-solid gels. Alginate (10 – 20 mg/ml) was dissolved in ultrapure water (resistivity > 17.4 M) using Lightnin and Caframo high-speed mechanical mixers and in-house built 3-blade propellers. Some solutions were prepared in Tris buffered saline (TBS), Dubellco’s minimal essential medium (DMEM) (Gibco) or microglia conditioned media for some pilot studies. Solutions were vigorously stirred at 700 – 1500 rpm for a minimum of 12 hours. Solutions were filtered into clean 250 ml screw cap glass bottles or sterile 50 ml polypropylene centrifuge tubes using a stainless steel air-pressure apparatus (Gelman Sciences) and 70 m Spectra filters. Solution concentration (mg/ml) was verified using a Mettler LJ16 Moisture Analyzer. Solutions were autoclaved (Steris) on a programmed liquid cycle (240 o C, 25 minutes) and stored at 4 o C until further use. Solution viscometry was conducted using dynamic Brookfield dynamic viscometers. Alginate molecular weight was determined using a Waters gel permeation chromatography (GPC) system and a Wyatt Dawn EOS multi-angle light scattering (MALS) detector. Hyaluronic acid (HA) (Mw = 1 x 10 6 g/mol) (Genzyme Corporation), 7HF carboxymethyl cellulose (CMC) (Mw = 7.5 x 10 5 g/mol) and alginate blend solutions were prepared using similar methods. Porous Alginate Foam Synthesis Microporous alginate compositions were prepared using lyophilization techniques. Although a variety of processing conditions were examined, a typical preparation is given here. One ml of viscous alginate solution was gently pressure injected into each well of a 4 well Nunc cell culture plate. The plates were sealed in parafilm and allowed to settle at room temperature in a Class III biological cabinet, upon which they were either frozen (-20 o C) overnight, flash frozen in liquid N 2 , or some combination of both.

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31 Fast freezing with liquid N 2 has been reported to produce meso-microporous foams with a smaller mean pore size and more uniform distribution. In contrast, slow freezing has been shown to result in an increased pore size and distribution, by permitting prolonged ice crystal nucleation and growth (Shapiro and Cohen, 1997). Plates were packed unidirectional into Labconco 900 ml flash freeze flasks, insulated with gel packs and ice, and lyophilized (-50 o C, 10 m Hg) for a minimum of 24-36 hrs using an established protocol in order to produce a nano-microporous, channeled-foam disc (Stopek et al, 2002). Plates were resealed with parafilm and samples were maintained dessicated. Foams were ionically crosslinked with 0.01M CaCl 2 or Ca phosphorylcholine in nanopure H 2 O (resistivity > 17.4 M) by immersion in 50 ml centrifuge tubes at RT. Foams were tumbled overnight on a clinical rotator and repeatedly washed with nanopure H 2 O for a minimum of 3 days to remove excess salt. Samples were stored fully hydrated and suspended at 4 o C. Porous alginate foams subject to MPC modification were prepared using slow freezing methods (-20 o C overnight) and crosslinked with 0.01M CaCl 2 . Porous Alginate Thin Film Synthesis Thin (< 20 m) films were cast by solvent evaporation under a Class III laminar flow biological cabinet for a minimum of 24 – 36 hrs. Known volumes (500 – 1000 l) of sterile alginate were injected into 30 mm Falcon polystyrene culture plates. The plates were briefly rocked and allowed to settle to a uniform coating. Films were crosslinked with 0.01M CaCl 2 or Ca phosphorylcholine by immersion, gently peeled from the dish with microforceps, and repeatedly washed with ultrapure water for a minimum of 2 days. Films were autoclaved (240 o C, 25 min) on a programmed liquid cycle fully hydrated and suspended in ultrapure water, and stored at 4 o C.

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32 Semi-solid Alginate Gel Preparations Alginate, alginate/HA, and alginate/CMC gel compositions were prepared using methods to produce tubular structures. Twenty ml syringes were loaded with sterile 20 mg/ml polysaccharide solutions and fitted with needles of 15 – 27 gauge based on the desired extruded tube diameter. The needle tip was submerged approximately 1 cm into 25 ml of sterile 0.01M CaCl 2 or Ca phosphorylcholine solution contained within 50 ml centrifuge tubes. Solutions were gently pressure injected (1ml/s) and instantly formed an insoluble semi-solid gel tube. Tube diameter was controlled by injection rate and needle gauge. They were allowed to tumble for 1hr at RT. Tubes were repeatedly washed with nanopure H 2 O to remove excess salt. These compositions were not subject to MPC modification. Hollow gel tubes were prepared by using a new spin casting method. Alginate solution was pressure injected at a controlled rate through a blunt 19-gauge hypodermic needle into the vortex of 400 ml beaker stirring 0.01 M calcium phosphorylcholine or chloride at 1200 rpm. Gel tubes were spun around the mandrel of 3-blade propellers, collected, and washed with nanopure water. MPC Monomer Two-methacryloyloxyethyl phosphorylcholine (MPC) monomer was generously donated by Dr. K. Ishihara, Department of Materials Engineering, University of Tokyo (Tokyo, Japan), and stored dry at o C. All gravimetric determinations were made at ambient temperature under an argon atmosphere in a dry box (Labconco) to prevent water adsorption. All MPC monomer solutions (50 – 100 mg/ml) were prepared in ultrapure water (resistivity > 17.4 M). Following solvation, solutions were micropipetted into clean 15 ml borosilicate test tubes and capped. Solutions were degassed (5 – 10 min) and backfilled with argon to remove dissolved O 2 prior to -polymerization.

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33 MPC Presoak Monomer presoak methods for surface and bulk modifications used were similar to those reported by Yahiaoui (1990). Individual porous alginate substrates were soaked at RT for 24hrs in 4 ml of 100 mg/ml MPC aqueous monomer solutions in clean 16 x 125 mm borosilicate test tubes to facilitate a rich monomer environment within the bulk alginate and surface. Samples were periodically agitated to remove air bubbles, degassed (10 – 15 min) and backfilled with argon to remove dissolved O 2 prior to -polymerization. -Radiation Polymerization Polymerization was conducted using a Wisconsin Type 60 Co source reactor (Figure 3-1). Samples were orientated around a target consisting of concentric circles corresponding to known dose rates (Table 3-1). Dosimetry was conducted prior to all experiments in order to calculate target radii for respective dose rates (295 – 1180 rad/min). The total dose varied between 0.05 – 0.15 Mrad. The 60 Co source was lowered to 1” above the reactor floor and imprints were made to verify target centering. Samples (minimum n=3 per condition) were evenly distributed around targets to account for dose rate inhomogeneity. Post-Irradiation Cleaning Following irradiation, alginate substrates were gently removed from the grafting solution and repeatedly rinsed with ultrapure water to remove free polyMPC (PMPC) homopolymer. Samples were transferred to 50 ml polypropylene centrifuge tubes and washed in water for a minimum of 1wk at RT. Some samples were dried in vacuo for 12 hrs at 40 o C prior to characterization. PMPC solutions were transferred to sterile 15 ml polypropylene centrifuge tubes and their intrinsic viscosity, molecular weight and radius of gyration were determined using GPC and MALS.

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34 Polymer Characterization Inductively Coupled Plasma Emission (ICP) Elemental analysis by ICP was utilized to determine N and P atomic content following gamma radiation modifications. Samples were dried to a constant weight at 40 o C and burned under optimal combustion conditions. Roberstson Microlit Laboratories (Madison, NJ) conducted all ICP analyses. Figure 3-1. 60 Co source stored in a Wisconsin Type reactor used in -polymerization studies. Table 3-1. -Radiation polymerization conditions of MPC. Dose Rate (rad/min) Total Dose (Mrad) 295 0.05 525 0.10 1180 0.15

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35 Electron Microscopy Scanning electron microscopy (SEM) was employed to qualitatively examine pore size and morphology of all synthesized microporous foams. Dry samples were gently adhered to aluminum SEM stubs with double-sided sticky tape and carbon paint. The samples were coated with a gold/palladium alloy using the Technix Hummer V sputter coater. The samples were analyzed using the JEOL SEM-6400 scanning electron microscope (JEOL, Ltd., Peabody, MA), operated at an accelerated voltage of 5 KeV, a condenser lens setting of 8 to 10, and a working distance of 15 mm. Energy Dispersive Spectroscopy (EDS) was used to determine the presence of phosphorus following MPC gamma modification. EDS samples were analyzed uncoated and under full-scale resolution. Paul Martin assisted with all SEM. Multi-Angle Light Scattering (MALS) Gel permeation chromatography (GPC) was conducted in conjunction with MALS to determine the weight-average molecular weight (MW), radius of gyration (Rg), intrinsic viscosity and molecular architecture. An 18-angle Wyatt Dawn EOS MALS detector was used for experiments. A Waters 600E system controller equipped with a Waters 966 Photo Diode Array, a Waters 410 Differential Refractometer and a Waters 717 autosampler was used. Injection volumes and flow rates were optimized for each analysis. Dilute polymer solutions (1 – 10 mg/ml) were prepared and filtered with 0.2 m cellulose acetate filters. PBS (pH 7.4, 300 mOsm) was used as the solvent. Phenomenex Shodex OH pak KB-803, SB-805 and SB-G columns were used. These experiments were conducted with the assistance of Brian Cuevas. Fourier-Transform Infrared Spectroscopy (FTIR) Absorption FTIR spectra were collected using a Nicolet Magna 60SC FT-IR spectrometer using Omnic ESP software for analysis (Nicolet Instrument Company). All

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36 spectra were collected in the range 4000 cm -1 to 400 cm -1 with a spectral resolution of 4 cm -1 , averaging 200 scans per sample. Functional group vibrations of interest are listed below in Table 3-1. Each spectrum was corrected for CO 2 and H 2 O absorption since data collection was carried out in laboratory atmosphere. Unmodified alginate substrates were run as baselines and used for all corrections. X-Ray Photoelectron Spectroscopy (XPS) XPS was performed using a Kratos XSAM 800mcd spectrometer. This system uses Mg K x-ray source (h =1253.6), a 127 mm hemispherical analyzer and a multi-channel detector. The x-ray source was operated at a power level of 108 W. Analysis was conducted pass energies of 12 25 eV for spectral collection. A photoelectron takeoff angle of 90 o was used. Peak identification and quantitation was accomplished using the peak integration package of the DS800 software. A Savitsky-Golay 25 point filter was used to smooth data. XPS was used primarily for the qualitative determination of the presence of molecular N and P (Table 3-2). Characterization using XPS was conducted with the assistance of Paul Martin. In Vitro Evaluations Cell Culture Reagents Dissociation solution used in the preparation for microglial cultures included the following: 0.137 M NaCl, 5.4 mM KCl, 0.2 M NaH 2 PO 4 , 0.2 M KH 2 PO 4 , 5 mM glucose, 58 M sucrose, 0.25 g/ml Fungizone (Gibco), and 1 x 10 6 U penicillin-G/streptomycin. Solutions were filter-sterilized with 0.22 m cellulose acetate filters into autoclaved 500 ml screw cap bottles and stored at o C. Dubelco’s minimal essential medium containing L-glutamine (DMEM) (Gibco) supplemented with 10% (v/v) sterile fetal bovine serum (FBS) and 1 x 10 6 U penicillin-G/streptomycin was used for all cell cultures. Media was filter sterilized and stored at 4 o C. This preparation is referred to as complete media (CM). The polyamino acid PLL (Gibco) was used to pre-coat culture flasks prior

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37 to microglia seeding. Flasks were treated with 0.01 g/L PLL in double distilled H 2 O for a minimum of 1 hour at 37 o C prior to plating. The PLL solution was aspirated and flasks washed with Solution D. Immediately prior to plating, flasks were aspirated and filled with CM. Microglial Cell Cultures Primary microglial cell cultures were prepared from perinatal rat brains. Isolated whole brains were dissected and stripped of their meninges while immersed in Solution D (Streit and Rabchevsky, 1997). Clean fragments were mechanically minced with a #10 blade scapel, transferred to a sterile 50 ml centrifugel tube, and incubated under bi-directional rotation in 0.05% (v/v) trypsin in Solution D for 20 minutes at 37 o C. An equal volume of CM was added to the suspension to quench the trypsin reaction. The tissue suspension was triturated again, passed through a 130 m Nitex filter and Table 3-2. Molecular vibrations associated with the polymeric phospholipid MPC. Functional Groups Vibration Wavenumber Range (cm -1 ) CH 3 Asymmetric Stretch 2972 – 2952 CH 3 Symmetric Stretch 2882 – 2862 CH 2 Asymmetric Stretch 2936 – 2916 CH 2 Symmetric Stretch 2865 – 2845 CH 3 Asymmetric Bend 1470 – 1450 CH 3 Asym. Bend (Umbrella) 1385 – 1365 CH 2 Scissors 1465 – 1445 Alkanes CH 2 Rock 730 – 710 C-O (1 o ) 1075 – 1000 O-H Stretch 3300 – 3400 Alcohols O-H Bend 1300 – 1400, 600 – 700 C=O Stretch 1750 – 1735 C-(C=O)-O Stretch 1210 – 1160 Esters O-C-C Stretch 1100 – 1030 C=O Stretch 1730 – 1700 C-O Stretch 1320 – 1210 O-H Stretch 3500 – 2500 O-H In-plane Bend 1440 – 1395 Carboxylic Acid O-H Out-of-plane Bend 960 – 900 -P-O-CH21070 Phosphorus Bonds P=O 1240 N-Methyl -N + -(CH 3 ) 3 Stretch 970

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38 Table 3-3. XPS binding energies of interest. Atomic Species Functional Group Binding Energy (eV) C-N+-C 286.0 COOH 289.3 C-O-*C=O 289.0 * C-O-C=O 286.6 Carbon (C1s) C-C 285.0 *O-(CO)-C 532.2 O-(C*O)-C 533.6 C-OH 532.9 C-O-P 533.4 Oxygen (O1s) O=C-N 531.3 C-N+-(CH 3 ) 3 402.1 Nitrogen (N1s) N-C=O 400.0 Phosphorus (P2p) C-O-P 133.0 centrifuged (400 x g, 10 minutes). The cell pellet was resuspended in 10 ml of CM, filtered using 40 m Nitex filters and plated on PLL coated 75 cm 2 culture flasks. The plating density was approximately 2 brains per 75 cm 2 flask. Cultures were incubated (37 o C, 8% CO 2 ) for 3 days, after which the culture media was exchanged with fresh CM. The cultures were incubated for an additional 7 days prior to harvesting. Microglia were collected every third day following the first harvest. Microglia were harvested by mechanical detachment. Flasks were placed on an orbital shaker in an incubator (37 o C, 200 rpm) for 1 hour. Following vigorous shaking, the media was collected and centrifuged (400 x g, 10 minutes), yielding a high concentration of microglia (typically 25 – 35 x 10 6 cells/harvest). Cells were suspended in a known volume of CM, stained with vital dye and counted with a hemacytometer, and adjusted to the desired final concentration. In Vitro Cell Seeding Implant compositions were seeded with second harvest primary rat microglia as previously described (Stopek et al., 2002, 2003). Microglia, harvested by mechanical detachment, were washed and resuspended with FBS-free DMEM to a final

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39 concentration of 2 x 10 6 cells/ml. Cell viability was determined by vital staining with trypan blue. Sterile implant compositions were trimmed to ~ 1 mm 3 under an operating microscope, incubated in 150 l of microglial suspension contained in sterile microfuge tubes, and placed on a bi-directional rotator for approximately 30 minutes (8 % CO 2 , 37 o C). Immediately prior to surgery, microfuge tubes were placed on ice. In Vivo Evaluations Surgical Implantation Adult male Wistar rats (175 g) were anesthetized with gaseous isoflurane (2-3 %, 0.8-1.0 L/min flow O 2 ) and operated in the prone position on a heating pad. The skin was sterilized with alcohol/betadine paint and a 2 cm skin incision was made with a #10 surgical blade centered over the C5 spinal level. The paraspinal musculature was subperiosteally elevated on the right side to expose the C4-C5 interspace from the midline to the facet joint. A partial laminectomy of C4 was performed with a small ronger. The dura was carefully opened with a #11 surgical blade and a 1 mm deep incision was made in the exposed spinal cord with the #11 knife from the midline to the midlateral cord. The cut was completed with several sweeps of a needle dissector within the same 1 mm depth, carefully avoiding the dorsal rootlets entering the cord. Sterile implants were sized under an operating microscope to a 1 mm diameter, shaped to conform to the lesion and carefully inserted into the cordotomy. The muscles and fascia were closed with suture and the skin edges approximated with wound clips. Animals received 5 cc sterile saline intraperitoneally and penicillin and torbutrol intramuscularly. Dr. Parker Mickle performed all surgeries. Non-invasive Magnetic Resonance Imaging (MRI) MRI was conducted at the Advanced Magnetic Resonance Imaging and Spectroscopy Center (AMRIS) on a 4.7T Bruker system with a 116 mm gradient insert. Briefly, animals were anesthetized with Isoflurane gas (2-3 %) and oxygen (0.8 1 %

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40 L/min) and given 1.5 cc of sterile saline prior to imaging to prevent dehydration. Animals were positioned on a Plexiglass cradle on an in-house built quadrature birdcage RF coil optimized for the cervical spinal cord. Tri-planar localizers were acquired using a RARE phase-encoded spin echo sequence in 1 minute with FOV = 7 x 7 cm, 1.5 mm thick slices and a 256 x 128 matrix with TR = 3 sec and TE (effective) = 55 ms. Sagittal images were acquired using TR = 3 sec and TE = 60 ms FOV = 6 x 4 cm with five 1-mm-thick slices (256 x 128 matrix). A T1 weighted 3-D gradient echo sequence was acquired (27 min) with a matrix size (256 x 128 x 64) and FOV 5 x 3.5 x 3.5 cm with TR = 200 ms and TE = 8 ms. All animals were imaged within 1 hour. All data was processed using the Paravision software package. Timothy Shepard, Xeve Silver, and Raquel Torres assisted in MRI investigations. Behavioral Analysis Behavioral analysis was conducted using a test reported to be sensitive to forelimb use asymmetries during spontaneous vertical exploration following injury to the rubrospinal tract (Schallert et al., 2000; Liu et al., 1999). Subjects were placed in a clear Plexiglass cylinder. The session was videotaped for blind scoring of forelimb use. Forelimb use during vertical exploration was scored as one of the following: independent-left, independent-right, or simultaneous (both). A forelimb that came into full weight-support contact with a wall during a rear was scored as independent-left or independent-right. Delayed placement of the second limb onto the wall was counted as an additional movement and scored as simultaneous. Both limbs had to be removed from the wall before another movement was scored. For example, if the rat moved along the wall laterally alternating both limbs, each complete two-limb movement (removes right while supporting on left, removes left while supporting right) was scored as simultaneous. Ratios of independent movements/total movements were calculated and statistically analyzed using ANOVA and multiple comparisons techniques (Tukey

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41 test). Animals were observed over varied periods of time dependent upon their respective survival period. Retrograde Tract Tracing Retrograde tract tracing utilizing 30mg/ml Fluorogold (FG) in PBS (pH 7.4, 300 mOsm) was performed via injection into the spinal cord with a 10 l Hamilton microsyringe at the T1 spinal level. The interval between T1 and T2 was exposed through a small incision using a #11 surgical blade. The spinal cord was exposed and the junction between the dorsal lateral spinal columns was injected bilaterally with 1 l of FG at a depth of 1 mm. A stereotactic injection apparatus was used to drive injections for long-term survival animals (> 7 months) in excess of 400 g body mass. Immunohistochemical Staining Serial vibratome and cryostat sections were prepared of the red nucleus and spinal cord, respectively. Floating vibratome 50 and frozen 20 m sections were immunostained using monoclonal antibodies (Abs) against neurofilament M (NF-M), glial fibrillary acidic protein (GFAP), OX-42, ED-1, S-100, -internexin, -2 laminin, serotonin and polyclonal Abs against calcitonin gene related peptide (CGRP). Endogenous peroxidases were quenched in 1% (v/v) H 2 O 2 in methanol for 15 min and blocked in 10% (v/v) normal goat serum (NGS) in PBS for 1 hr. Sections were washed briefly with PBS and PBS with 5mg/ml Triton-X for 15 min at room temperature (RT) for enhanced Ab permeation. Sections were blocked against secondary Abs with 10% (v/v) NGS in PBS containing 3 mg/ml Triton-X for 1 hr at RT. Samples were incubated with 1 o Ab in PBS containing 3 mg/ml Triton-X and 3% (v/v) NGS for 3 hr at RT. Samples were washed 3X in PBS (first 2 containing 3 mg/ml Triton-X) in 5 min intervals. Biotinylated 2 o Abs (1:500) were applied in PBS containing 3 mg/ml Triton-X and 3% (v/v) NGS for 1 hr at RT. Samples were washed 3X again with PBS (first 2 containing 3 mg/ml Triton-X) in 5 min intervals. Horseradish peroxidase-Avidin D (1:500)

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42 in PBS was applied for 30 min at RT. Samples were washed 3X with PBS. Sections were developed with 0.5 mg/ml diaminobenzidine (DAB) activated with 1 l/ml of 3% (v/v) aqueous H 2 O 2 for a maximum of 10 min. Sections were washed 3X with PBS, some counterstained with 30 mg/ml cresyl violet in acetate buffer (pH 4.5), dehydrated through graded ethanols and xylene, and slides covered in glass using Permount solvated in xylene. Maisson Trichrome and H&E staining were also conducted. Results and Discussion Porous Alginate Compositions The synthesis of porous biopolymers by salt leaching, freeze-drying or lyophilization has been reported, including the effects of freezing conditions and heat transfer rates on pore size and morphology (Shapiro and Cohen, 1997; Park et al, 2001). In these particular studies, porous alginate compositions were prepared using freeze-dry/lyophilization techniques, with water acting as the porogen upon freezing. Porous alginate gel compositions were also prepared, and some were subjected to freeze-drying and qualitatively assessed using optical microscopy and SEM. EDS was used to qualitatively determine phosphorus (P) atomic content prior to MPC modifications. The weight average molecular weight (Mw) and radius of gyration (Rg) of Kelco HV alginate in PBS was determined by GPC and MALS prior to fabrication. Mw and Rg were found to be 5 x 10 5 g/mol and 26 nm, respectively (Figure 3-2). Optical micrographs depicting nano-mesoporous alginate foams and semi-solid gels are shown in Figure 3-3. Porous freeze-dried alginate compositions were found to have a heterogeneous pore distribution and pore size and exhibited a honeycomb-like morphology when imaged in cross-section (Figure 3-4). Pore size varied (10 – 80 m) as determined by

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43 SEM. In contrast, exterior surfaces displayed a ridged or channeled morphology. Higher magnification images reveal small, finger-like projections and pores orientated perpendicular to the channel direction. Additionally, a skin effect was noticeable by sight and optical microscopy. Pore surfaces were found to be relatively smooth and resembled surface morphologies of analogous thin alginate film compositions as shown in Figure 3-5. Similar results were found for alginate blend compositions. It is noted that SEM images of dry preparations may not accurately represent their hydrated, in vivo characteristics. Semi-solid alginate and alginate-blend gel compositions were prepared by multiple techniques. New spin casting methods were developed for the preparation of hollow gel tubes (Figure 3-6). It was also found that gel compositions could be prepared in similar fashions using cell culture media (protein containing) or cell conditioned medium as the solvent for either alginate or crosslinking solutions, without detrimental effects to gel formation. Figure 3-7 depicts a representative EDS spectrum of unmodified calcium alginate.

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44 51.0x1061.0x1071.0x1012.014.016.018.020.0Molar Mass vs. Volume ALGIN Molar Mass (g /mol ) Elution Volume ( ml ) Figure 3-2 Multi-angle (18) lightscattering chromatogram of alginate determined to have Mw = 5 x 10 5 g/mol and Rg = 26 nm. Figure 3-3 Optical micrographs of (A) porous freeze-dried and (B) tubular gel alginate compositions. Freeze-dried compositions may be prepared in varied size and geometry and seeded with biologically active cells or factors. Gel tubes have the potential to be loaded with a variety of therapeutic agents including cells, neurotrophins, viral vectors or drugs.

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45 Figure 3-4 SEM micrographs of porous alginate foam compositions revealing honeycomb-like pore morphology in cross section (A) and ridge or channel-like surface skin features (B and C).

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46 Figure 3-5 SEM micrographs detailing pore (A) and surface morphology (B).

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47 Figure 3-6 SEM micrographs of semi-solid tubular alginate gel compositions (A – F). Low magnification depicts a relatively smooth outer surface (A). Higher magnification reveals calcium crystals derived from CaCl 2 crosslinking (D – E). Panel F represents an inverted tube.

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48 Figure 3-7 Representative EDS spectra of porous alginate compositions with no detection of phosphorus as also seen by inductively coupled plasma emission. Pilot -Polymerizations and MPC Modifications Following preparation of porous and thin film alginate substrates, samples were modified using radiation initiation polymerization methods and characterized. MALS analysis of MPC solution polymers determined a significant dose response on molecular weight (Mw, weight average) and radius of gyration (Rg). Increased total dose (0.05 – 0.15 Mrad at 1180 rad/min) was found to decrease molecular weight and radius of gyration (8.2 – 3.3 x 10 5 g/mol). MALS chromatograms are shown in Figure 3-8. These results conflicted with our previous findings for the radiation polymerization of the vinyl monomer N-2-vinyl pyrrolidone (NVP). Increased dose (0.05 – 0.15 Mrad) in the gamma

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49 polymerization of NVP synthesized PVP homopolymers with statistically larger Mws and Rgs (p<0.001). Dose response data is presented in Table 3-4 and provoked a detailed study of the effects of dose (0.05 – 0.15 Mrad) and dose rate (295 – 1180 rad/min) on MPC Mw and Rg. Table 3-4 Dose responses of MPC Mw and Rg following gamma radiation initiation solution polymerization. Dose Dose Rate Mw Rg (Mrad) (rad/min) (g/mol) (nm) 0.05 1180 8.2 x 10 5 30 0.075 1180 4.6 x 10 5 24 0.15 1180 3.3 x 10 5 19 The surface modification of polysaccharide substrates with hydrophilic monomers such as MPC using gamma radiation polymerization and grafting techniques has not been reported. Pilot studies in the gamma surface modification of porous alginate reported promising results under varied radiation and presoak conditions. FTIR analyses of modified samples revealed characteristic MPC absorptions including P=O and P-O-CH 2 (Figure 3-9). Weak P2p peaks (133 eV) were detected by XPS and contact angle measurements determined significant changes in hydrophilicity for some modified samples. In parallel experiments, alginate was also surface modified with PVP, HEMA, MAA, and co-polymers with MPC. Together, these results demonstrated proof of concept and feasibility for the surface modification of polysaccharide compositions with hydrophilic, water-soluble, vinyl monomers using gamma radiation initiation polymerization.

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50 0.061.0x1062.0x1063.0x1064.0x1065.0x1012.014.016.018.020.0Custom Plot 0.05 0.075 0.15 Molar Mass (g/mol) Elution Volume (ml) Figure 3-8 MALS chromatograms (molar mass versus elution volume) for MPC polymerized under varied dose (Mrad). Wavenumbers (cm -1 ) Figure 3-9 FTIR spectra of MPC modified alginate-revealing absorptions at 1240 cm -1 characteristic to MPC polymers and uncharacteristic of alginate. MALS Study of -Polymerized MPC MPC was synthesized by gamma initiation polymerization using a 60 Co source stored in a Wisconsin Type reactor. A 3 x 3 factorial was designed to study the effects of dose (Mrad) and dose rate (rad/min) on weight-average molecular weight (Mw) and radius of gyration (Rg) as determined by gel permeation chromatography and multi-angle lightscattering (MALS).

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51 Samples (n=3) were orientated around a target of concentric circles associated with specified dose rates (295 – 1180 rad/min) and were exposed to a known total dose (0.05 – 0.15 Mrad) as previously detailed. Following polymerization, 200 l aliquots of MPC polymerization solution was added to 1800 l of PBS (pH 7.4, 300 mOsm), 0.2 m filtered, and analyzed by GPC and MALS (18 angle). MALS chromatograms displaying the effects of varied dose rate and total dose are shown in Figures 3-10 and 3-11, respectively. Statistical analysis using 2-way ANOVA suggested there was a significant effect of both dose and dose rate on MPC Mw and Rg (p<0.001). Pair-wise comparisons (Tukey test) suggested there were significant differences between levels of total dose (0.05, 0.10 and 0.15 Mrad) and dose rate (295, 525 and 1180 rad/min) on Mw and Rg (p<0.05). Increasing the total dose was found to lower both molecular weight and radius of gyration. For example, at 295 rad/min, doses of 0.05, 0.10, and 0.15 Mrad resulted in MPC polymers with molecular weights of 1.9 x 10 6 , 1.3 x 10 6 and 1.1 x 10 6 g/mol, respectively. Likewise, at a total dose of 0.05 Mrad, dose rates of 295, 525, and 1180 rad/min resulted in polymers with molecular weights of 1.9 x 10 6 , 1.5 x 10 6 and 1.1 x 10 6 g/mol, respectively. Similar results were observed for Rg as seen in Figure 3-13. Both increased total dose and dose rate resulted in high molar mass MPC polymers with statistically smaller Rgs. The data for these experiments are reported in Table 3-5 and Figures 3-12 and 3-13. MPC Modification of Porous Alginate Following MALS analysis of the gamma radiation initiation polymerization of MPC, porous alginate substrates were modified using gamma-grafting methods under various radiation polymerization conditions. Based on pilot studies and in order to conserve MPC monomer, a single dose rate (1180 rad/min) was selected for these experiments. The total dose was varied as described previously (0.05 – 0.15 Mrad).

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52 Porous alginate substrates were subjected to a pre-soak 100 mg/ml MPC monomer incubation solution for 24 hrs at RT prior to polymerization. Substrates were irradiated, thoroughly washed, dried and characterized by FT-IR, elemental analysis by inductively coupled plasma emission, EDS, GPC and MALS, and XPS for MPC content. Pilot studies using optical profilometry and AFM were also conducted. FT-IR evaluations of calcium alginate have been previously reported, however, the modification of alginate with MPC has not as of yet. IR spectra illustrated obvious qualitative differences between all MPC modified alginate and unmodified controls. The MPC modified spectra revealed absorptions at frequencies characteristic of MPC and not alginate, including P=O, P-O-CH 2 , and –N + -(CH 3 ) 3 at 1240 cm -1 , 1075 cm -1 , and 970 cm -1 , respectively (Figures 3-14 and 3-15). Peak intensity was qualitatively observed to increase with increasing dose (0.05 – 0.15 Mrad). Similar results have been previously reported in the surface modification of Dacron and PMMA with MPC (Grumski, 2000). Elemental analysis by inductively coupled plasma emission and EDS were used as expedient techniques to determine N and P elemental content, both characteristic to MPC and not alginate, following gamma modifications. ICP data correlated well with the IR results and revealed the presence of elemental P in all samples under experimental MPC modification conditions. As anticipated, no P was detected in the unmodified porous alginate controls, which was later corroborated by further characterization techniques. EDS was conducted on uncoated samples and most spectra were collected in full-scale. X-ray analysis of unmodified porous calcium alginate failed to detect P in any samples as observed by ICP evaluations. Intense C and O peaks were observed, with moderate counts for Ca. In contrast, EDS detected C, O, Ca and P in all MPC modified alginate substrates (Figure 3-16). There was no clear distinction of a dose rate effect on P content, and the magnitude of the P peak (counts) varied with beam location on the

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53 sample. This may be attributed to a non-homogeneous distribution of MPC phospholipid graft or interpenetrating/co-interpenetrating network (IPN) formation. The magnitude of detectable X-ray signal due may have varied due to changes in sample surface morphology and terrain (pore peak versus valley). XPS was employed for surface analysis and detection of N and P. Very small P2p peaks at 133 eV were observed for some MPC modified samples, although signal was on average only twice the magnitude of the background noise. The detection of N was unsuccessful. ICP and EDS proved to be more reliable methods for the detecting the incorporation of MPC following modification. Further XPS experiments were abandoned. GPC and MALS analyses of PBS eroded MPC surface modified substrates typically resulted in noisy scattering peaks with large polydispersities and likely represented a mix of MPC, MPC-alginate graft, and alginate polymers. Microfiltration of gel particles prior to analysis introduced extraneous variables into the molecular weight determinations. Although, some samples displayed relatively clean, multi-peak chromatograms in the range of 3 x 10 5 to 1 x 10 6 g/mol (Figure 3-17). In addition, it is noted that GPC analysis was conducted at the upper molar mass exclusion limits of the columns for some experiments. Pilot atomic force microscopy experiments (AFM) (n=3) were conducted under contact mode and dry conditions. Thin porous alginate films modified with MPC (0.05 Mrad, 1180 rad/min) exhibited significant differences in surface topography and roughness compared to unmodified controls. MPC surface features were considerably smoother and larger in multiple directions (1 m versus 200 – 800 nm Y direction). Unmodified alginate samples were composed of small, rough, pillar-like projections. Images are shown in Figure 3-18. These images do not accurately depict the surface properties in a fluid environment and the biological consequences of such surface features are not clear.

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54 51.0x1061.0x1071.0x1012.013.014.015.016.017.0Molar Mass vs. Volume 295 525 1180 Molar Mass (g/mol) Elution Volume (ml) Figure 3-10 MALS chromatogram illustrating the effects of dose rate (rad/min) on molar mass (g/mol). 51.0x1061.0x1071.0x1012.013.014.015.016.017.0Molar Mass vs. Volume 0.05 0.10 0.15 Molar Mass (g/mol) Elution Volume (ml) Figure 3-11 MALS chromatogram illustrating the effects of total dose (Mrad) on molar mass (g/mol).

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55 Table 3-5 Effects of dose (Mrad) and dose rate (rad/min) on PMPC molecular weight (Mw) and radius of gyration (Rg). Statistical analysis using 2-way ANOVAs suggested there was a significant effect of both dose and dose rate on MPC Mw and Rg (p<0.001). Pair-wise comparisons (Tukey test) suggested there were significant differences between levels of total dose (0.05, 0.10 and 0.15 Mrad) and dose rate (295, 525 and 1180 rad/min) on Mw and Rg (p<0.05). Dose Dose Rate Mw SD Rg SD (Mrad) (rad/min) (g/mol) (g/mol) (nm) (nm) 0.05 295 1,900,000 12,000 57 6 0.05 525 1,500,000 42,000 53 0.6 0.05 1180 1,100,000 53,000 43 1.1 0.10 295 1,300,000 14,000 49 0.2 0.10 525 1,200,000 32,000 45 1.0 0.10 1180 890,000 25,000 37 0.1 0.15 295 1,100,000 14,000 42 0.1 0.15 525 970,000 10,000 40 0.2 0.15 1180 780,000 16,000 33 0.1

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56 0.00E+002.00E+054.00E+056.00E+058.00E+051.00E+061.20E+061.40E+061.60E+061.80E+062.00E+062955251180Dose Rate (rad/min)Molar Mass (g/mol) 0.05 Mrad 0.10 Mrad 0.15 Mrad Figure 3-12 Effects of dose (0.05 – 0.15 Mrad) and dose rate (295 – 1180 rad/min) on molar mass (g/mol). 2-way ANOVA suggested there was a statistically significant effect of dose and dose rate on Mw and Rg (p<0.001). Increasing both the total dose and dose rate resulted in statistically lower molar mass MPC polymers with smaller Rgs (P<0.05).

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57 01020304050600.050.10.15Total Dose (Mrad)Radius of Gyration (nm ) 295 rad/min 525 rad/min 1180 rad/min 01020304050602955251180Dose Rate (rad/min)Radius of Gyration (nm) 0.05 Mrad 0.1 Mrad 0.15 Mrad Figure 3-13 Effects of dose (0.05 – 0.15 Mrad) and dose rate (295 – 1180 rad/min) on radius of gyration Rg (nm). 2-way ANOVA suggested there was a statistically significant effect of dose and dose rate on Mw and Rg (p<0.001). Increasing both the total dose and dose rate resulted in statistically smaller Rgs (P<0.05).

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58 Figure 3-14 FT-IR spectrum of MPC modified alginate observed at 970 cm -1 for the N + -(CH 3 ) 3 absorption characteristic to MPC and not alginate. Increasing dose (0.05 (pink), 0.10 (green) and 0.15 Mrad (blue)) coincided qualitatively with increased absorbance versus unmodified alginate (red).

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59 Figure 3-15 FT-IR spectrum of MPC modified alginate observed at 1240 cm -1 for the P=O absorption characteristic to MPC and not alginate. Increasing dose (0.05 (pink), 0.10 (green) and 0.15 Mrad (blue)) coincided qualitatively with increased absorbance versus unmodified alginate (red).

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60 Figure 3-16 EDS spectrum for a representative MPC modified alginate sample with detection of phosphorus indicative of the phosphorylcholine moiety of MPC (0.05 Mrad, 1180 rad/min). X-ray analysis failed to detect phosphorus in unmodified alginate control samples.

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61 Figure 3-17 MALS 3-D chromatograms for representative MPC solution polymers (left) and PBS eroded MPC surface modified alginate polymers (right). Multiple scattering peaks were observed in some MPC graft samples.

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62 Figure 3-18 Pilot dry AFM images illustrating differences in surface feature size, morphology, and roughness in thin unmodified (A) and MPC gamma modified alginate films (B).

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63 In Vitro Evaluations In vitro studies involving the inclusion of MPC polymers in cell culture media, as well as the seeding of microglia, PC-12 cells, and mixed brain cultures on various biopolymer substrates, indicated that MPC surfaces exhibit favorable growth properties. MPC surfaces appeared to stimulate microglial proliferation and differentiation, as well as guided growth. Inclusion of gamma polymerized MPC solution polymers in culture with primary microglia or mixed brain cultures typically rendered microglia with a “resting” state morphology. Favorable astrocyte growth was also noted, and pilot studies with microglia-neuron co-cultures exhibited acceptable biocompatibility and minimal toxicity. Subsequent seeding of PC-12 cells onto microglia cultures resulted in excellent survival and differentiation, without the addition of exogenous NGF (Figure 3-19). These data have also shown that lipopolysaccharide mediated induction of IL-1 synthesis by microglia may be attenuated by culture with MPC. These results have been detailed previously (Stopek et al., 2002). Future studies using MTT-based proliferation and live/dead assays may provide a quantitative method of modeling the effects of MPC inclusion on cell viability and proliferation, including dose response effects. B A Figure 3-19 PC-12 cell differentiation on an MPC biopolymer surface cultured with primary rat microglia (A) and an unmodified PDMS surface cultured with primary microglia (B).

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64 In Vivo Evaluations In our preliminary studies, dorsolateral funiculotomies were performed in the cervical spinal cord of the adult rat at level C4-C5 in order to transect the right rubrospinal tract (RST). The rubrospinal system consists of a well-defined pool of supraspinal neurons in the red nucleus of the midbrain, which project their axons bilaterally (90% contralateral, 10% ipsilateral) to the cervical and thoracic spinal cord. This system has been utilized for regeneration studies by many laboratories and regeneration-associated genes have been characterized (Tetzlaff et al., 1992). Following transection of rubrospinal axons in the cervical spinal cord, neuronal cell bodies in the red nucleus normally atrophy and fail to regenerate their severed axons. Surgically placed implants were found to elicit a dense cellular infiltration composed of mixed glial and peripheral blood borne cells, with evidence of axonal growth. Serial high field MRI (4.7 T) found all implant compositions to exhibit significant signal void behavior (dark images) under T2 weighted MRI. Conversely, untreated control subjects exhibited a lesion localized hyperintense signal (bright images) indicative of free water and CSF. Improved use of the affected limb during functional evaluations was observed for those subjects treated with biopolymer implants cultured with primary rat microglia. Enhanced retrograde tracing of Fluorogold was observed in treated subjects at 7 and 14 weeks post-implantation indicating some biochemical transport to the brain. Noninvasive Magnetic Resonance Imaging (MRI) With the greater resolution provided by non-invasive imaging techniques such as MRI, it is possible to follow the progress of material transplanted following traumatic SCI. MRI was used in these studies to provide a more clinically relevant method of monitoring injury progression and determining outcomes following experimental treatment. In addition, these studies attempted to correlate MR data with motor function and post

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65 mortem histology including the assessment of axonal retrograde transport. The translation of post-operative MRI data into clinical outcomes is necessary if human SCI patients are to be treated with experimental biomaterial implants. MRI was conducted at the AMRIS facility on Bruker 4.7T and 17T systems. Pilot studies at 17T were initially conducted on ex vivo spinal cord specimens implanted with porous alginate implants under acute recovery periods (6 – 7 wks). Noninvasive in vivo imaging was conducted on a 200 MHz 4.7T system with a 116 mm gradient insert and a quadrature birdcage RF coil optimized for the cervical cord. Images were acquired in the coronal, sagittal and horizontal planes. Briefly, ex vivo pilot studies of acute animals using a variety of pulse sequences failed to show the presence of cystic cavitation in subjects treated with porous alginate implant compositions. Some cords displayed significant signal voiding (hypointense signal) on T2 weighted MR, which masked particular segments of the implantation site and increased the difficulty in discerning lesion and implant interfaces. All T2 diffusion and proton density weighted MR data was lost in these regions. Iron and manganese rich blood components or entrapped air may have been responsible for these artifacts. The cause still remains unclear and other investigators have reported similar results. Initial in vivo experiments were conducted to determine optimum TE and TR times for imaging of the cervical adult rat spinal cord. Normal adult rats (200-250 g) were used in these studies and were evaluated for neurological function prior to imaging. Figure 3-20 shows an MR image of a normal animal in the sagittal, coronal and horizontal planes. Tri-planar localizers were acquired using a RARE phase-encoded spin echo sequence in 1 min with a 7 x 7 cm field of view (FOV), 1.5 cm thick slices and a 256 X 128 matrix with TR = 3 sec and TE (effective) = 55 ms. T2 weighted images were collected in the sagittal, coronal and horizontal planes under multiple pulse sequences.

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66 Some experimental animals were imaged 1 day to 1 yr post-injury/implantation and most results were similar to those observed in preliminary ex vivo experiments. The implantation/lesion site was apparent in all subjects by T2 and T1 weighted MRI. Untreated SCI animals were observed to suffer various degrees of atrophy and cystic cavitation, as evidenced by a lesion localized hyperintense signal (white) on T2, which was most likely accumulation of CSF (Figure 3-21). This was found to occur within 1 month post-injury. In addition, significant morphological deviations and loss of gray matter organization caudal to the injury level was noted over several millimeters. This was later corroborated by gross anatomical and histological analyses. Conversely, animals treated with porous alginate implants exhibited significant signal voiding (hypointense) and loss of T2 data at the implantation site when imaged beyond 3 – 4 wks post-implantation (Figures 3-22 – 3-27). No evidence of cavitation or significant atrophy was found at any time point for implant treatment group subjects by MRI, or later by post-mortem analyses. Implant treated subjects maintained a more normal dorsal horn morphology caudal to the lesion site compared to untreated controls, which partially account for their improved neurological function (Figures 3-24 through 3-27). This was observed in both acute and long-term survival animals. Further studies using stronger main magnetic fields (B o ) or implantable coils may allow for finer spatial resolution and a greater signal to noise ratio, while limiting volume averaging effects. This may permit a plethora of dynamic assessments including implant integration, cellular infiltration, tissue maturation or rejection, and scaffold bioerosion. These MR studies presented proof of concept and further evidence that MRI has potential in being an effective, noninvasive diagnostic following implantation of biopolymer-neural cell compositions.

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67 Figure 3-20 T2 diffusion weighted MRI serial sections of a normal control animal in the sagittal, coronal and horizontal planes using a 200 MHz Bruker 4.7T spectrometer with a 116mm gradient insert and an RF quadrature birdcage coil optimized for the cervical spinal cord. The distinction between white and gray matter and the dorsal and ventral horns are apparent.

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68 Figure 3-21 T2 diffusion weighted MR image of an untreated SCI animal with significant cavitation noted by the lesion-localized hyperintense signal (FOV = 6 x 4 cm, TR = 3 s and TE = 60 ms) in the coronal (A) and sagittal (B) planes.

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69 Figure 3-22 T2 diffusion weighted MRI serial sections of an untreated animal in the sagittal, coronal and horizontal planes 4 months following injury. The normal morphology of the dorsal horn is lost for several mms caudal to the injury and displays significant atrophy (easiest seen in coronal plane).

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70 Figure 3-23 T2 diffusion weighted MRI serial sections of a porous alginate treated animal in the sagittal, coronal and horizontal planes 4 months following injury. The normal morphology of the dorsal horn is partially maintained immediately caudal to the injury and displays some atrophy (easiest seen in coronal plane).

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71 Figure 3-24 T2 diffusion weighted MRI serial sections of an animal treated with a porous alginate implant seeded with primary rat microglia in the sagittal, coronal and horizontal planes 4 months following injury. The normal morphology of the dorsal horn is well maintained immediately caudal to the injury and does not display atrophy (easiest seen in coronal plane).

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72 Figure 3-25 MRI serial sections of an animal acutely treated with a MPC modified alginate implant (0.10 Mrad, 1180 rad/min) in the sagittal, coronal and horizontal planes using a 200 MHz Bruker 4.7T spectrometer with a 116mm gradient insert and an RF quadrature birdcage coil optimized for the cervical spinal cord. T2 diffusion weighted images were acquired within 24 hrs of implantation (TE = 48.6 – 56.7 ms).

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73 Figure 3-26 T2 diffusion weighted MRI serial sections of an animal treated with a porous alginate implant modified with MPC in the sagittal, coronal and horizontal planes 4 wks following injury. The normal morphology of the dorsal horn is well maintained immediately caudal to the injury and does not display atrophy or CSF accumulation (easiest seen in coronal plane).

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74 Figure 3-27 T2 diffusion weighted MRI serial sections of an animal treated with a porous alginate implant modified with MPC in the sagittal, coronal and horizontal planes 1 yr following injury. The normal morphology of the dorsal horn is well maintained immediately rostral and caudal to the injury and does not display any atrophy or CSF accumulation (easiest seen in coronal plane).

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75 Behavioral Analysis Biopolymer implant (Stopek et al., 2002, 2003) and microglial graft compositions (Rabchevsky et al, 1997) have shown promise in the repair of the injured spinal cord. However, little is known concerning their capacity to promote functional neurological recovery. These studies evaluated the long-term efficacy of alginate/microglial compositions implanted in the adult rat spinal cord using a partial transection injury model. Rats were examined for forelimb use during spontaneous vertical exploration using a test reported to be highly sensitive to chronic limb use asymmetries (Schallert et al., 2000; Liu et al., 1999). Repeated tested has not been reported to influence asymmetry scoring. MRI and FG tract tracing were used to complement these studies. Male adult Wistar rats (n=40) were divided into 4 experimental groups (n=10) including control (normal), lesion only (LO) (no treatment), porous alginate implant only (AO) and porous alginate implants seeded with primary rat microglia (AM). Briefly, dorsolateral funiculotomies were performed on the right side in order to transect the rubrospinal tract at C4/C5. Implants were placed immediately following SCI and animals were allowed to recover for 4 to 7 months. Animals were observed weekly for forelimb use asymmetries during spontaneous vertical exploration. Animals were placed in a clear Plexiglas cylinder (20 x 30 cm) for 5 minutes. The cylinder encouraged the use of the forelimbs during vertical exploration and the following movements were scored: (1) independent use of the left or right forelimb for contacting the cylinder wall during a full rear, to initiate a weight-shifting move, or to reestablish center of gravity while laterally moving along the cylinder wall; and (2) the simultaneous or near-simultaneous use of both forelimbs during a full rear or any movement along the cylinder wall. Individual movements were scored as percentages of (1) ipsilateral, (2) contralateral and (3) simultaneous use relative to the total number of observed movements.

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76 Healthy adult Wistar rats were used as a baseline study for determining normal behavior. Rats were observed to independently initiate (single forelimb) approximately 50% of the time, evenly distributed between “independent left” and “independent right”. The remaining movements (50%) were attributed to the simultaneous use of both forelimbs, termed “simultaneous both” (p<0.05). Dorsolateral funiculotomy at C4/C5 produced asymmetry in forelimb use in all experimental groups. Walking, reaching, grasping, and grooming related movements were also affected. Statistical analysis by two-way ANOVA followed by Tukey multiple comparisons suggested treatment had a statistically significant effect on vertical exploration (p<0.001). Following a 4 wk recovery, LO (no treatment) animals were infrequently observed to use the forelimb ipsilateral to the lesion, including simultaneous both movements, which represented approximately 5 – 10% of their movements. LO forelimb use was restricted to the contralateral left forelimb (90 – 95% independent left) during exploration (Figure 3-28). Pair-wise multiple comparison tests suggested LO rats were statistically different than normal and implant-treated rats (p<0.05). Animals in both porous alginate groups, with and without primary microglia, were found to use the injured ipsilateral forelimb more frequently (20 – 25% simultaneous both) than LO animals (5 % simultaneous both) at 4 wks. Pair-wise comparisons suggested AO and AM rats were statistically different than normal and LO animals (p<0.05). Although AO and AM animals exhibited increased simultaneous use of both forelimbs versus LO during exploration, the overwhelming majority of movements involving the ipsilateral limb were initiated with the contralateral. Independent initiation of contact using the contralateral limb remained the predominate movement for all groups versus normal rats at 4 wks (p<0.05).

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77 Figure 3-28 Porous alginate implant treatment group (A) versus lesion only (no treatment) (B) during spontaneous vertical exploration at 4 months post-injury. Over the course of the study (4 – 28 wk) AO and AM animals were infrequently found to use the affected ipsilateral limb independently (Figures 3-29 through 3-32), with greater symmetry found in AM versus LO and AO (p<0.05). Some AM animals were found to initiate independent right by 4 wks and maintained to 16 wks. Statistical analysis indicated there was no significant interaction between time and treatment (p<0.05). Similar statistics were found through 28 wks post-injury. Future studies incorporating physical rehabilitation and environmental enrichment may prove to further enhance functional recovery. Retrograde Tract Tracing Tract tracing using Fluorogold (FG) was conducted to assess retrograde axonal transport to the red nucleus (RN) in the midbrain following injury and treatment. Untreated control animals exhibited minimal FG labeling in the injured contralateral magnocellular region of the RN at 6 and 16 wk. These neurons are most likely attributed to those fibers (5 – 10 %) representing ipsilateral (uncrossed) projections of the left uninjured rubrospinal tract and not those from the injured side.

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78 In contrast, rubrospinal neurons in the injured magnocellular red nucleus in porous alginate-treated animals were found to be FG positive as early as 4 wk. This is shown in Figures 3-33 through 3-35 and implied that some ability for retrograde transport to the brain existed for injured rubrospinal neurons in implant treated subjects. Many of these cell bodies retained a normal neuronal morphology, and their presence alone at 4 months was promising in that these implant compositions did not induce or promote further retrograde cell death following chronic injury. These results corroborate well with MRI, functional, and post-mortem anatomical and histological analyses and indicated our implant compositions afford tissue regeneration. Further experiments including tracing axonal projections from the brain to the injured spinal cord may provide insight to the regenerative nature of these implant compositions. Anatomical and Histological Analyses Alginate and alginate-microglial cell implants were apparent by gross anatomical inspection following spinal cord dissection under all recovery periods as shown in Figure 3-36. Implant treated subjects displayed no evidence of cavitation or significant atrophy at the lesion site. In stark contrast, untreated control lesions exhibited significant atrophy and cystic cavitation as previously seen by MRI, as well as poor motor function (Figure 3-36). Thin cryosections found untreated control lesions to display moderate tissue loss at the lesion site as shown in Figure 3-37. Porous implant compositions induced a dense glial and peripheral cellular infiltrate with minimal inflammation (Figures 3-38 and 3-39). It was thought that neural cells including astrocytes, endogenous microglia, Schwann cells, and blood borne macrophages constituted the majority of the cellular infiltrate. NF-M immunoreactive nerve fibers (Figure 3-40) and moderate glial fibrillary acidic protein (GFAP) reactive astrocytes were found. While some mononuclear cells were infrequently observed at the implantation level, the density of these cells was no

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79 greater than that of distant uninjured tissue. Motor neurons in the immediate vicinity, rostral and caudal to the implantation site, were apparent in most cases. Some porous implants were found to partially fragment or develop voids during histological procedures using PBS, a known chelator of calcium alginate (Figure 3-41). This was termed the “Swiss cheese effect.” Further experiments are required to determine if this is processing artifact or implant fragmentation in vivo. MRI failed to detect these voids during T2 and T1 weighted imaging. Implants, especially semi-solid alginate-microglia gels, were found to interface well with the host tissue and there was no evidence of a profuse cellular accumulation or barrier along exterior implant surfaces (Figures 3-42 through 3-44). Gel erosion was clearly diffusion controlled. The lateral edges of the neural cell containing gels were largely eroded by 4 weeks (Figure 3-43), while the bulk of the gel core remained (Figure 3-42). A cellular infiltration gradient and an ependymal response were also observed as shown in Figure 3-44. Alginate-hyaluronic acid gel compositions exhibited significant fragmentation problems, although NF-M immunoreactivity was found juxtaposed to implant edges (Figure 3-45). Second-generation injectable gels may offer the most promising strategy by requiring less invasive surgical procedures and having the ability to conform to complex geometries. These are currently under investigation in our laboratories.

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80 Spontaneous Vertical Exploration (4wks)00.20.40.60.811.2ControlLOAOAMTreatment% Use Independent Left Independent Right Simultaneous Both Figure 3-29 Spontaneous vertical exploration 4 wks post-injury expressed as a function of percent total forelimb use. Lesion only (LO), porous alginate (AO), and porous alginate seeded with primary rat microglia (AM) treatment groups were found to be statistically different than control (normal) group. Pair-wise comparisons suggested AO and AM were statistically different than LO (p<0.05). Spontaneous Vertical Exploration (8wks)00.20.40.60.811.2ControlLOAOAMTreatment% Use Independent Left Independent Right Simultaneous Both Figure 3-30 Spontaneous vertical exploration 8 wks post-injury expressed as a function of percent total forelimb use. Lesion only (LO), porous alginate (AO), and porous alginate seeded with primary rat microglia (AM) treatment groups were found to be statistically different than control (normal) group. Pair-wise comparisons suggested AO and AM were statistically different than LO (p<0.05).

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81 Spontaneous Vertical Exploration (12wks)00.20.40.60.811.2ControlLOAOAMTreatment% Use Independent Left Independent Right Simultaneous Both Figure 3-31 Spontaneous vertical exploration 12 wks post-injury expressed as a function of percent total forelimb use. Lesion only (LO), porous alginate (AO), and porous alginate seeded with primary rat microglia (AM) treatment groups were found to be statistically different than control (normal) group. Pair-wise comparisons suggested AO and AM were statistically different than LO (p<0.05). Spontaneous Vertical Exploration (16wks)00.20.40.60.811.2ControlLOAOAMTreatment% Use Independent Left Independent Right Simultaneous Both Figure 3-32 Spontaneous vertical exploration 16 wks post-injury expressed as a function of percent total forelimb use. Lesion only (LO), porous alginate (AO), and porous alginate seeded with primary rat microglia (AM) treatment groups were found to be statistically different than control (normal) group. Pair-wise comparisons suggested AO and AM were statistically different than LO (p<0.05).

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82 Figure 3-33 Serial sections of the first 150 microns of the magnocellular red nucleus in untreated control (lesion only), alginate implant treated (implant), and alginate cultured with primary microglia (implant w/microglia) following injection with the retrograde tract tracer Fluorogold. Note increased FG labeling in implant treatment groups.

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83 Figure 3-34 Serial sections (A – C, caudal to rostral) of the first 150 microns of the contralateral magnocellular red nucleus for alginate treated subjects 4 months. Untreated control subjects did not exhibit this behavior.

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84 Figure 3-35 3-D reconstruction of the first 150 microns of the magnocellular red nucleus in an alginate treated subject 4-months post-implantation.

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85 Figure 3-36 Gross anatomical analyses of representative untreated control (A) and implant treated subjects (B) at 4 months post-implantation.

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86 Figure 3-37 Cresyl violet staining of the lesion site for a representative untreated control subject (A – B). Fields C – D are details of A and B. Note extensive tissue loss in fields A and B, which correlated with poor functional results and hyperintense T2 weighted MR images indicative of cavitation. This behavior is not found in implant treated subjects in which implants induced a robust cellular infiltrate with minimal or no resulting cavitation.

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87 Figure 3-38 Lesion site morphology via cresyl violet staining for representative porous alginate treated subjects 4 wks post-implantation (A). Panel B is a detail. Implants induced a dense cellular infiltration at all time points.

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88 Figure 3-39 Lesion and implant interface as shown by cresyl violet staining for porous alginate treated animals 4 wks post-implantation (A and B). Implants induced a dense cellular infiltration and interfaced well with host tissue.

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89 Figure 3-40 Neurofilament M (NF-M) immunopositive nerve fibers within the center of the alginate implant.

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90 Figure 3-41 Lesion site morphology for MPC modified porous alginate via cresyl violet staining (A-D). NF-M immunopositive fibers were found localized with the implant.

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91 Figure 3-42 Lesion site morphology (implant core) for semi-solid alginate-microglial cell gel implants (A – F).

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92 Figure 3-43 Details of lateral lesion site morphology for semi-solid alginate-microglial cell gel implants (A – C).

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93 Figure 3-44 Ependymal cell migration towards the interface of semi-solid alginate-microglial cell gel implant (A). Panel B presents a detail. The central canal has been reported to be one of the origins of CNS derived stem cells.

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94 Figure 3-45 HA-alginate blend gel compositions (residual stained purple) 4 wk following implantation (A – D). Fragmentation artifacts are apparent in fields A and B. Neurofilament M (NF-M) immunoreactivity surrounded and apposed implant external surfaces. These implants did not induce as robust a cellular infiltrate as porous counterparts.

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95 Summary Novel nano-mesoporous alginate scaffold compositions were prepared and surface modified with the polymeric phospholipid MPC using radiation polymerization methods. FTIR revealed absorptions characteristic of MPC and not alginate, including P=O, P-O-CH 2 , and –N + (CH 3 ) 3 . Peak intensity increased with increasing total dose (0.05 – 0.15 Mrad). Elemental analysis by ICP and energy dispersive spectroscopy determined the presence of phosphorus and nitrogen, contrary to unmodified control substrates. Weak P2p peaks at 133 eV were observed by XPS for some MPC gamma modified substrates, while detection of N1s was unsuccessful. MALS determinations suggested that increased dose and dose rate attenuates MPC molecular weight and radius of gyration (p<0.001). In addition, solid-state NMR may further elucidate surface properties following MPC modifications. Experiments are ongoing. Morphologically, porous alginate compositions were found to have a heterogeneous pore ultrastructure and distribution, and exhibited a honeycomb-like structure in cross-section. Exterior surfaces displayed a ridged morphology, which contained finger-like projections orientated perpendicular to the main pore/channel direction. Contact mode AFM under dry conditions suggested MPC surface modified compositions exhibited smoother, larger, nanosurface structural features. The neurobiological consequences of these differences, especially during chronic neurodegenerative disease or injury, require further study.

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CHAPTER 4 CHITOSAN-BASED COMPOSITIONS FOR THE REPAIR OF INJURED NEURAL TISSUE Introduction Biopolymer compositions have shown promise in the repair of the injured spinal cord. Chitosan, a biodegradable copolymer of glucosamine and N-acetylglucosamine derived from the exoskeleton of marine crustaceans, has been reported to possess good biocompatibility and minimal toxicity (Figure 4-1). In its highly deacetylated form it is soluble in dilute acids, where it carries a strong positive charge due to the protonation of the amino functionality. Its charge density and solubility are pH dependent and exert control over chitosan’s ability to form hydrogels under neutralizing conditions. Degraded enzymatically by lysozyme, chitosan by-products are reported to be noncarcinogenic, nonimmunogenic, and nontoxic. Originally reported in the early 1970’s to accelerate wound-healing mechanisms, chitosan has been used in a variety of in vitro drug delivery and cell culture studies. In vitro studies have reported favorable outcomes for chitosan and chitosan-derived biomaterials, with primary application in skin, bone, and connective tissue. Neural investigations appear to have been limited to in vitro cell culture and in vivo peripheral nerve experiments. Little is known concerning the in vivo biocompatibility of chitosan in the repair of the injured spinal cord and brain. Specific Aims The main objective of the proposed exploratory work was to determine whether chitosan-based implants may be used to prevent post-traumatic cavitation and achieve functional improvements with regard to axonal regeneration as determined by MRI, 96

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97 behavioral analysis by spontaneous vertical exploration, retrograde tract tracing, and histology. Important broader implications of results stemming from this project include the potential application of implant concepts for treatment of brain disease and trauma. The specific aims were as follows: Aim 1 Synthesis and characterization of novel microporous bioerodable chitosan implant compositions. Porous implant compositions were prepared using viscous chitosan and freeze dry/lyophilization processes. All chitosan implants were covalently crosslinked with a novel biological compound, genipin (derived from the hydrolysis of geniposide), which has been reported to display neurotogenic effects on PC-12h cells in vitro. The effect of autoclave sterilization on implant stability was investigated. Aim 2 Pilot in vivo evaluations of porous genipin-crosslinked chitosan compositions implanted into the injured adult rat spinal cord. The hypothesis that intraspinal implants consisting of porous genipin-crosslinked chitosan may facilitate the functional regeneration of rubrospinal axons was tested. Dorsolateral funiculotomies were performed at C4/C5 in the adult rat cervical spinal cord in order to transect the rubrospinal tract. Chitosan compositions were placed acutely following injury. Survival times ranged from 1 week to 1 year post-implantation. Analyses included high field magnetic resonance imaging (MRI), retrograde tract tracing, and behavioral analysis. Materials and Methods Chitosan Solutions Chitosan (Polysciences) (10 – 20 mg/ml) was dissolved in dilute 3 % (v/v) acetic acid in ultrapure water (resistivity > 17.4 M) using Lightnin and Caframo high-speed mechanical mixers and in-house built 3-blade propellers. Solutions were vigorously stirred at 700 – 1500 rpm for a minimum of 12 hours. Solutions were filtered into clean

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98 O O NH2 OH OH O NH2 OH OH O O NH2 OH OH O Figure 4-1 Molecular structure of chitosan. 250 ml screw cap glass bottles or sterile 50 ml polypropylene centrifuge tubes using a stainless steel air-pressure apparatus (Gelman Sciences) and 70 m Spectra filters. Solution concentration (mg/ml) was verified using a Mettler LJ16 Moisture Analyzer. Solutions were autoclaved on a liquid cycle (240 o C, 25 min) and stored at 4 o C. Viscometry was conducted on a Brookfield dynamic viscometer. Genipin Solutions Genipin (Figure 4-2) solutions were prepared by dissolving solid genipin in dry acetone to a final concentration of 60 mg/ml. Figure 4-2 Molecular structure of genipin. Microporous Foam Synthesis Microporous chitosan compositions were prepared using freeze dry/lyophilization techniques. One ml of viscous chitosan solution was gently pressure injected into each well of a 4 well Nunc cell culture plate. The plates were sealed with parafilm and allowed to settle at room temperature in a Class III biological cabinet, upon which they were either frozen (-20 o C) overnight. Plates were packed unidirectional into Labconco

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99 900ml flash freeze flasks, insulated with gel packs and ice, and lyophilized (-50 o C, 10 m Hg) for a minimum of 24-36 hrs using an established protocol in order to produce a nano-microporous, channeled-foam disc (Stopek et al, 2001). Plates were resealed with parafilm. Samples were maintained dry, in the dark, and refrigerated. Foams were covalently crosslinked using genipin. Porous chitosan samples were covalently crosslinked by immersion in 60 mg/ml genipin in dry acetone in 50 ml centrifuge tubes. Swollen samples were tumbled overnight until genipin’s brilliant blue chromaphore was observed. Samples were repeatedly washed with nanopure H 2 O for a minimum of 3 days to remove excess genipin. Implants were autoclave stable and sterilized (240 o C, 25 min) on a programmed liquid cycle and stored (4 o C, dark) fully hydrated and suspended in supplemented minimal essential medium containing phenol red for pH indication. In Vivo Evaluations Surgical Implantation Adult male Wistar rats (175 g) were anesthetized with gaseous isoflurane (2-3 %, 0.8-1.0 L/min flow O 2 ) and operated in the prone position on a heating pad. The skin was sterilized with alcohol/betadine paint and a 2 cm skin incision was made with a #10 surgical blade centered over the C5 spinal level. The paraspinal musculature was subperiosteally elevated on the right side to expose the C4-C5 interspace from the midline to the facet joint. A partial laminectomy of C4 was performed with a small ronger. The dura was carefully opened with a #11 surgical blade and a 1 mm deep incision was made in the exposed spinal cord with the #11 knife from the midline to the midlateral cord. The cut was completed with several sweeps of a needle dissector within the same 1 mm depth, carefully avoiding the dorsal rootlets entering the cord. Sterile implants were sized under an operating microscope to a 1 mm diameter, shaped to conform to the lesion and carefully inserted into the cordotomy. The muscles and fascia

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100 were closed with suture and the skin edges approximated with wound clips. Animals received 5 cc sterile saline IP and penicillin and torbutrol IM. Non-invasive Magnetic Resonance Imaging (MRI) MRI was conducted on a 4.7T Bruker system with a 116mm gradient insert. Briefly, animals were anesthetized with Isoflurane gas (2-3 %) and oxygen (0.8-1 % L/min) and given 1.5 cc of sterile saline prior to imaging to prevent dehydration. Animals were positioned on a Plexiglass cradle on an in-house built quadrature birdcage RF coil optimized for the cervical spinal cord. Tri-planar localizers were acquired using a RARE phase-encoded spin echo sequence in 1 minute with FOV = 7 x 7 cm, 1.5 mm thick slices and a 256 x 128 matrix with TR = 3 sec and TE (effective) = 55 ms. T2 weighted images were acquired using TR = 3 sec and TE = 60 ms FOV = 6 x 4 cm with five 1mm thick slices (256 x 128 matrix). A T1 weighted 3-D gradient echo sequence was acquired (27 minutes) with a matrix size (256 x 128 x 64) and FOV 5 x 3.5 x 3.5 cm with TR = 200 ms and TE = 8ms. All animals were imaged within 1 hour. All data was processed using the Paravision software package. Behavioral Analysis Behavioral analysis was conducted using a test reported to be sensitive to forelimb use asymmetries during spontaneous vertical exploration following injury to the rubrospinal tract (Schallert et al., 2000; Liu et al., 1999). Retrograde Tract Tracing and Tissue Processing Retrograde tract tracing utilizing 3 % (v/v) Fluorogold (FG) in PBS was performed via injection into the spinal cord with a 10 l Hamilton microsyringe at the T1 spinal level. The interval between T1 and T2 was exposed through a small incision using a #11 surgical blade. The spinal cord was exposed and the junction between the dorsal lateral spinal columns was injected bilaterally with 1 l of FG at a depth of 1 mm. A stereotactic

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101 injection apparatus was used to drive injections for long-term survival animals (> 7 months) in excess of 400 g body mass. Animals were euthanized by intracardial perfusion under sodium pentobarbital (150mg/kg) with PBS and 4% (w/v) paraformaldehyde (PFA). The brain was dissected and post-fixed for 24 hours in 4% (w/v) PFA. Vibratome serial sections (50 m thick) of the entire RN were collected, mounted on gelatin-coated slides, and coverslipped with Permount in dry xylene. FG labeled magnocellular red nucleus neurons (contralateral and ipsilateral) were imaged under DAPI fluorescence using the SPOT camera and Image Pro Plus analysis software. Results and Discussion Non-invasive Magnetic Resonance Imaging (MRI) Post-operative evaluation with MRI is recognized as a vital component of any future human clinical protocol involving the implantation of any tissue or biomaterials following traumatic SCI. Correlating complex MRI data with clinical and functional outcomes is necessary if human SCI patients are to be treated with experimental biomaterial implants. In addition, serial confirmation of tissue viability or necrosis within biopolymer implants over time would benefit functional evaluations in living subjects. MRI was used in these pilot studies to provide a more clinically relevant method of monitoring injury progression and determining outcomes following experimental treatment. Briefly, pilot studies of acute animals at 4.7T using a variety of pulse sequences failed to detect the presence of cystic cavitation in subjects treated with porous chitosan implant compositions. Most cords displayed significant signal voiding (hypointense signal) which masked segments of the implantation site and lesion and implant interfaces. All T2 diffusion (long TE and TR) weighted MR data was lost in these regions (images were black). Blood components or entrapped air may have been responsible

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102 for these artifacts, although the cause still remains unclear and other investigators have reported similar results. Wirth has reported differences in T1 and T2 weighted images in cats treated with fetal tissue grafts compared to untreated control cats suffering from cavitation (hyperintense signal, white images). Further in vivo experiments were conducted to determine optimum TE and TR times for imaging of the cervical adult rat spinal cord. Normal adult rats (200-250 g) were used in these studies and were evaluated for neurological function prior to imaging. Tri-planar localizers were acquired using a RARE phase-encoded spin echo sequence in 1 min with a 7 x 7 cm field of view (FOV), 1.5 cm thick slices and a 256 X 128 matrix with TR = 3 sec and TE (effective) = 55 ms. T2 weighted images were collected in the sagittal, coronal and horizontal planes under multiple pulse sequences. Untreated SCI animals were observed to suffer various degrees of atrophy and cystic cavitation, as evidenced by a lesion localized hyperintense signal (white) on T2, indicative of CSF (Figure 4-3). This was found to occur within 1 month post-injury. In addition, significant morphological deviations and loss of gray matter organization caudal to the injury level was noted over several millimeters as shown in Figure 4-4. This was later corroborated by post-mortem histological analyses. Conversely, chitosan-treated animals again exhibited significant signal voiding (hypointense) resulting in dark T2 images of the lesion/implantation site (Figure 4-5). This was observed for all time points (4wks – 1yr). No indication of cavitation (hyperintense CSF signal) or atrophy was found at any time point for implant treatment group animals by serial MRI, or later by histological methods. Most importantly, a more normal dorsal horn gray matter morphology caudal to the lesion site was observed for chitosan-treated animals versus untreated control animals, which may vindicate enhanced functional performance when compared to injured controls (Figures 4 through 4-8).

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103 Behavioral Analysis Functional evaluations by spontaneous vertical exploration were conducted in a limited number of animals (n = 3) and without statistical analysis. As previously detailed, axotomy at C4/C5 produced significant functional deficits in tasks including reaching, grasping, forelimb placement, walking, and normal posture. Untreated control animals were restricted to using the uninjured, contralateral (left) limb during vertical exploration throughout the course of these studies (~ 90 % at 4 wks). Conversely, chitosan-treated animals displayed some use of their affected limb by 4 wks (~ 55 % simultaneous both) as shown in Figure 4-9. These results corroborate well with the MRI results and suggest that chitosan compositions may exert a rescuing effect of rubrospinal axons following cervical SCI by preserving tissue and minimizing secondary damage and cavitation. Retrograde Tract Tracing Retrograde tract tracing experiments using 30 mg/ml aqueous Fluorogold (FG) were conducted by bilateral injection at T1. Untreated control animals exhibited minimal FG labeling in the injured contralateral magnocellular region of the red nucleus (RN) at 4 months. Control contralateral RN neurons labeled with FG are most likely derived from those fibers (5 – 10 %) representing ipsilateral (uncrossed) projections of the left uninjured rubrospinal tract. These results corroborate well with the MR and behavioral data. Some rubrospinal neurons in the injured contralateral magnocellular red nucleus in chitosan-treated animals were found to be FG positive at 4 months post-implantation. This is shown in Figure 4-10 and implied that some capacity for retrograde transport of existed for injured rubrospinal neurons. Many of the FG labeled RN neuron cell bodies retained normal morphological features, and their presence alone at 4 months was promising in that chitosan-based compositions did not induce or promote further retrograde cell death following axotomy.

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104 Anatomical and Histological Analyses Genipin crosslinked chitosan implants were apparent by gross anatomical inspection and no evidence of atrophy or cyst formation was found. Cryosections found implants to induce a robust glial and peripheral cellular infiltrate. A higher concentration of neutrophils was noted compared to alginate systems at 4 weeks. Implants interfaced well, and as previously determined by MRI, no signs of cavitation were found. Figure 4-11 shows lesion site morphology and implants were found to fluoresce at wavelengths similar to fluoroscein, making residual implant apparent.

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105 Figure 4-3 T2 diffusion weighted MR images of an untreated SCI animal with significant cavitation 4 months post-injury noted by the lesion-localized hyperintense signal (FOV = 6 x 4 cm, TR = 3 s and TE = 60 ms) in the coronal (A) and sagittal planes (B).

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106 Figure 4-4 T2 diffusion weighted MRI serial sections of an untreated animal in the sagittal, coronal and horizontal planes 4 months following injury. The normal morphology of the dorsal horn is lost for several mms caudal to the injury and displays significant atrophy (easiest seen in coronal plane).

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107 Figure 4-5 T2 weighted MRI 4wks following implantation of a porous chitosan implant compositions in the sagittal plane. The lesion/implantation site is marked by signal void (hypointense signal, black image). TE = 56.7 ms (top row) or 68.9 ms.

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108 Figure 4-6 T2 weighted MRI 4wks following implantation of a porous chitosan implant compositions in the sagittal and coronal planes. The lesion/implantation site is marked by signal void (hypointense signal, black images). Morphology of the cord caudal to the lesion does not display significant atrophy.

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109 Figure 4-7 T2 weighted MRI 1 year post-implantation in the sagittal, coronal and horizontal planes. The lesion/implantation site does not display signal void (hypointense signal) as seen in acute imaging. Morphology of the cord caudal to the lesion was well maintained.

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110 Figure 4-8 T2 weighted MR image of an acutely placed genipin-crosslinked chitosan implant in the sagittal plane (A) and respective cresyl violet stained sections revealing a dense cellular infiltrate of mixed glial and peripheral cells (B). Field (C) is a detail of (B).

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111 Spontaneous Vertical Exploration (4wks)00.20.40.60.811.2CLOCHTreatment% Use Independent Left Independent Right Simultaneous Both Figure 4-9 Spontaneous vertical exploration 4 wks post-implantation for control (normal) (C), lesion only (LO), and chitosan treated (CH) subjects. Figure 4-10 FG retrograde tract tracing of an untreated control (A) and porous chitosan treated animal (B – D). Panel (B) displays the first 50 m of the magnocellular RN and (C and D) higher magnifications at 100 m depth. No labeling was detected in untreated controls (A) at 4 wks post-injury.

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112 Figure 4-11 Cresyl violet stained spinal cord section at the implant/tissue interface showing a dense cellular infiltrate into the implant (A). Same field under FTIC fluorescence (B). Fields (C) and (D) represent details of above.

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113 Summary Chitosan has been identified as a promising biopolymer for application in tissue engineering and drug delivery. Microsphere compositions crosslinked with the gardenia derived compound, genipin, have been reported. In addition, free genipin has been shown to possess neurotogenic properties under particular biochemical conditions. However, to our current knowledge, the use of similar compositions as porous scaffolds or semi-solid gels for tissue repair has not been previously reported. Novel porous genipin-crosslinked chitosan implant compositions were prepared using new lyophilization techniques. Chitosan compositions exhibited a brilliant blue chromaphore following reaction with genipin, which afforded enhanced visual localization in the injured spinal cord during placement and enucleation. Following acute implantation, porous chitosan compositions elicited a permissive glial and peripheral cellular infiltrate, although a neutrophil population was still present at 4 wks. Genipin-crosslinked chitosan implants exhibited significant signal void (hypointense signal) at the lesion level as determined by serial T2 weighted MRI. No evidence of cavitation or hyperintense signal indicative of free water or CSF was found in any treated subjects. In addition, increased preservation of the normal anatomical and morphological structure of both gray and adjacent white matter caudal to the lesion/implantation site was observed in chitosan treated animals. Serial X-ray by energy dispersive spectroscopy and E beam of normal and lesion level tissue may aid in determining if T2 signal void characteristic to porous chitosan is related to blood components or byproducts including iron and manganese. Increased retrograde transport of Fluorogold was found in chitosan treated subjects versus untreated controls. Together, these exciting results warrant further investigations for the use of genipin-crosslinked chitosan compositions for neural tissue repair.

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CHAPTER 5 DNA-BASED COMPOSITIONS FOR REPAIRING INJURED NEURAL TISSUE Introduction DNA is perhaps the most important biopolymer and genetic material of living organisms. It offers a unique double-stranded structure composed of nitrogenous bases, pentose sugars, and phosphate groups, which make chemical modification, complexing and crosslinking feasible. Recent DNA research has focused primarily on genome mapping, gene therapy by transfection or transduction, or altering gene expression ex vivo or in vivo. However, little is currently understood concerning the application of DNA as a structural biomaterial or scaffold for tissue repair. Considering its molecular structure (Figure 5-1) and functionality, hydrophilicity, intrinsically high molecular weight, biodegradability, and availability, it may be an ideal matrix for incorporation of bioactive neural pro-regenerative molecules or cells. In addition, DNA implants may be relatively non-immunogenic when compared to antigen-presenting protein counterparts. Very high molecular weight DNA may be readily purified from essentially all living tissues, including a plethora of inexpensive, high-yield sources such as vegetables (onions), poultry (chicken blood), plants, bovine by-products and salmon milts or shellfish gonads. Enormous amounts of DNA-enriched materials are discarded everyday as waste. DNA from common vegetable and animal sources may be a potentially economic and valuable biomaterial for scaffold/implant compositions and drug delivery vehicles. It is important to note that many of the natural biopolymers previously studied for SCI repair are derived from a wide-range of biological sources including FDA approved 114

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115 NCHN NCN N O O CH2 OH H O NCHN NCN H N H H O O CH2 O P H O O NCHN NCN N O O O CH2 O P H O O NCHN NCN H N H H O O PO O O O PO O O PO O O CH2 O P H O H H H H H H O PO O O PO O O NN O H O CH2 O P O O O H N H H H NN O H O CH2 O P O H C O H H H H NN O H H N O H O CH2 O P O H H NN O H OH CH2 O P O O O H C O H H H H O O O Figure 5-1 DNA and its ability to hydrogen bond.

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116 alginates (brown seaweed, bacteria), chitosan (crustacean shell), collagen and gelatin (bovine), hyaluronic acid (bacteria), agarose (agar gums) and commercially available Matrigel (ECM from mouse sarcoma). The extraction and purification of many of these biopolymers has often been more complex than for polynucleotides. DNA has not been investigated to date as a structural scaffold material for the repair of tissues including the spinal cord and brain. DNA is most recognized for its wealth of genetic information. Association with biomaterials science has been focused predominately on delivering gene therapy agents or encapsulated compounds such as dyes or drugs by intercalation or groove binding. Complexes of DNA and highly cationic polysaccharides such as chitosan have been reported. Polyamine, polycationic lipids and neutral polymers capable of condensing DNA into small particles have also been reported. However, the DNA molecular structure may afford a major untapped opportunity for devices to provide localized biomechanical biochemical support. This may include the delivery of neurotrophic factors, antibodies, drugs or other therapeutic agents not ideally suited for systemic administration. In this research, DNA-based structures were found to be bioerodable, most likely by enzymatic mechanisms, and appear to be cleared through non-inflammatory means. Most importantly, DNA’s intrinsically high MW requires very small amounts of DNA in the synthesis of multifunctional scaffolds, which could possibly utilize the patients’ own DNA from simple blood or tissue collections. Inexpensive, non-human sources may prove to be the most invaluable sources for DNA-based devices. Specific Aims The main objective of the proposed exploratory work was to determine whether DNA-based implants may be used to achieve functional improvements with regard to axonal regeneration as determined by MRI, behavioral analysis by spontaneous vertical exploration and histology. Important broader implications of results stemming from this

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117 project include the potential application of implant concepts for treatment of brain disease and trauma. The specific aims were as follows: Aim 1 Synthesis and characterization of novel nano/mesoporous bioerodable DNA-based implant compositions. Vegetable and animal DNA were isolated. Porous DNA samples were prepared using viscous DNA solution film casting and freeze/dry lyophilization processes. Scanning electron microscopy (SEM), X-ray by energy dispersive spectroscopy (EDS) and multi-angle light scattering (MALS) for DNA molecular weight determinations were used. The effects of ethylene oxide and autoclave sterilization on implant stability were investigated. Aim 2 Pilot In vivo evaluations of porous DNA compositions implanted into the injured adult rat spinal cord. The hypothesis that intraspinal implants consisting of microporous DNA implants can facilitate the formation of a dense cellular infiltrate into the lesion cavity with minimal chronic inflammation or scarring, leading to the functional regeneration of rubrospinal axons was tested. Dorsolateral funiculotomies at level C4/C5 were performed in the adult rat cervical spinal cord in order to transect the rubrospinal tract. DNA compositions were placed immediately following injury. Survival times ranged from 1 week to 16 weeks post-implantation. Analyses included high field magnetic resonance imaging (MRI), detailed immunohistochemical staining, retrograde tract tracing, and behavioral analysis. Materials and Methods DNA Isolation DNA was extracted from vegetables including Spanish onion, broccoli and plum tomato by separation. DNA derived from calf thymus and herring testes was also used for in vitro and in vivo experiments (Sigma Chemical Company). Vegetables were

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118 chopped into small pieces and incubated in a homogenization medium (HM) containing (in mg/ml) 0.3 EDTA, 8.8 NaCl, 4.4 Na citrate and 60 SDS. Tissue fragments were stirred in 400ml HM for approximately 15 minutes at 60 o C. Mixtures were cooled on ice for 10 minutes and transferred to a chilled blender (4 o C). Mixtures were blended for 1 minute and foam settled. Mixtures were microfiltered through 70 m Spectra filters into sterile 50 ml polypropylene centrifuge tubes and centrifuged (4g) for 5 minutes. The supernatant was transferred to a clean beaker and ice-cold 95% (v/v) isopropanol was slowly added to form an upper alcohol layer. DNA was collected following precipitation from the alcohol layer and lyophilized (-50 o C, 10m Hg). Preparation of Nano/microstructured Porous DNA Although a variety of processing conditions were examined, a typical preparation is given here. 5 – 20 mg/ml solutions were prepared in nanopure water using low torque, high-speed mechanical mixing (1200 – 1500 rpm). Solutions were transferred to sterile 50 ml centrifuge tubes, tumbled overnight at ambient T, and stored at 4 o C until further use. Solution concentration (mg/ml) was verified by moisture analysis. 500 l of DNA was pressure injected into each well of multi-well Nunc cell culture plates, sealed with parafilm and frozen overnight at o C. Plates were loaded into 900 ml Labconco flash freeze flasks insulated with frozen gel packs and lyophilized (-50 o C, 10m Hg) overnight. Thin DNA films (< 20 m) were prepared by solvent evaporation under a laminar flow biological cabinet (25 o C) and at elevated temperatures (37 – 80 o C). Samples were ionically crosslinked with 0.0025 – 0.025 M CrK(SO 4 ) 2 , FeCl 3 , or GdCl 3 . Samples were incubated in the salt solution for 12 – 24 hours. However, crosslinking was found to be essentially instantaneous (immediate water insolubility). Samples were repeatedly washed with ultrapure water to remove excess salt and dried in vacuo (40 o C). Samples were sterilized by autoclaving (fully hydrated and suspended in nanopure water) or ethylene oxide.

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119 Characterization Electron Microscopy Samples were coated with a gold/palladium alloy using the Technix Hummer V sputter coater. The samples were analyzed using the JEOL SEM-6400 scanning electron microscope (JEOL Ltd., Peabody, MA), operated at an accelerated voltage of 5 KeV, a condenser lens setting of 8 to 10, and a working distance of 15 mm. EDS was conducted on uncoated samples. GPC and Multi-angle Light Scattering (MALS) GPC with MALS was used to determine M w , radius of gyration (R g ) and molecular architecture using an 18-angle Wyatt Dawn EOS MALS detector with a Waters 600E system controller equipped with a Waters 966 Photo Diode Array, a Waters 410 Differential Refractometer, and a Waters 717 autosampler. PBS (pH 7.4, 300 mOsm) was used as the solvent. Phenomenex Shodex OH pak KB-803, SB-805 and SB-G columns were used. In Vivo Evaluation Surgical Implantation Adult male Wistar rats (175 g) were operated in the prone position under isoflurane (2-3 %, 0.8-1.0 L/min flow O 2 ) on a heating pad. The skin was sterilized with alcohol/betadine paint and a 2 cm skin incision was made with a #10 surgical blade centered over the C5 spinal level. The paraspinal musculature was subperiosteally elevated on the right side to expose the C4-C5 interspace from the midline to the facet joint. A partial laminectomy of C4 was performed with a small ronger. The dura was carefully opened with a #11 surgical blade and a 1 mm deep incision was made in the exposed spinal cord with the #11 knife from the midline to the midlateral cord. The cut was completed with several sweeps of a needle dissector within the same 1 mm depth, carefully avoiding the dorsal rootlets entering the cord. Sterile implants were sized

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120 under an operating microscope to a 1 mm diameter, shaped to conform to the lesion and carefully inserted into the cordotomy. The muscles and fascia were closed with suture and the skin edges approximated with wound clips. Animals were given 5 cc saline IP and procaine/penicillin and Torbutrol IM. Noninvasive Magnetic Resonance Imaging (MRI) MRI was conducted on a 4.7T 200 MHz Bruker system with a 116mm gradient insert and quadrature RF birdcage coil optimized for the cervical spinal cord. Tri-planar localizers were acquired using a RARE phase-encoded spin echo and T2 weighted images were acquired in the sagittal, coronal, and horizontal planes. A T1 weighted 3-D gradient echo sequence was also acquired. Spontaneous Vertical Exploration Behavioral analysis was conducted using a test reported to be sensitive to forelimb use asymmetries during spontaneous vertical exploration following injury to the rubrospinal tract (Schallert et al., 2000; Liu et al., 1999). Retrograde Tract Tracing Retrograde tract tracing with 30mg/ml Fluorogold (FG) in PBS (pH 7.4, 300 mOsm) as performed via injection into the spinal cord with a 10 l Hamilton microsyringe at the T1 spinal level. The interval between T1 and T2 was exposed through a small incision using a #11 surgical blade. The spinal cord was exposed and the junction between the dorsal lateral spinal columns was injected bilaterally with 1l of aqueous FG at a depth of 1 mm. A stereotactic injection apparatus was used to drive injections for long-term survival animals (> 4 months) in excess of 400g body mass. Animals were recovered 3 days post-injection prior to euthanasia. Animals were euthanized by intracardial perfusion under sodium pentobarbital (150mg/kg) with PBS and 4% (w/v) paraformaldehyde (PFA). The brain was dissected and post-fixed for 24 hours in 4% (w/v) PFA. Vibratome serial sections (50 m thick) of

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121 the entire RN were collected, mounted on gelatin-coated slides, and coverslipped with Permount in dry xylene. FG labeled magnocellular red nucleus neurons (contralateral and ipsilateral) were imaged under DAPI fluorescence using the SPOT camera and Image Pro Plus analysis software. Immunohistochemical Analysis Serial vibratome and cryostat sections were prepared of the red nucleus and spinal cord, respectively. Floating vibratome 50 and frozen 20 m sections were immunostained using monoclonal antibodies (Abs) against neurofilament M (NF-M), glial fibrillary acidic protein (GFAP), OX-42, ED-1, S-100, and -internexin. Endogenous peroxidases were quenched in 1% (v/v) H 2 O 2 in methanol for 15 min and blocked in 10% (v/v) normal goat serum (NGS) in PBS for 1 hr. Sections were washed briefly with PBS and PBS with 5mg/ml Triton-X for 15 min at room temperature (RT) for enhanced Ab permeation. Sections were blocked against secondary Abs with 10% (v/v) NGS in PBS containing 3mg/ml Triton-X for 1 hr at RT. Samples were incubated with 1 o Ab in PBS containing 3mg/ml Triton-X and 3% (v/v) NGS for 3 hr at RT. Samples were washed 3X in PBS (first 2 containing 3mg/ml Triton-X) in 5 min intervals. Biotinylated 2 o Abs (1:500) were applied in PBS containing 3mg/ml Triton-X and 3% (v/v) NGS for 1 hr at RT. Samples were washed 3X again with PBS (first 2 containing 3mg/ml Triton-X) in 5 min intervals. Horseradish peroxidase-Avidin D (1:500) in PBS was applied for 30 min at RT. Samples were washed 3X with PBS. Sections were developed with 0.5mg/ml diaminobenzidine (DAB) activated with 1l/ml of 3% (v/v) aqueous H 2 O 2 for a maximum of 10 min. Sections were washed 3X with PBS, some counterstained with 30mg/ml cresyl violet in acetate buffer (pH 4.5), dehydrated through graded ethanols and xylene,

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122 and slides covered in glass using Permount solvated in xylene. Maisson Trichrome and H&E staining were also conducted. Results and Discussion Porous DNA Preparation Porous DNA samples were prepared using lyophilization and solution casting techniques using various sources of DNA including Herring teste, calf thymus, Spanish onion, broccoli and tomato. Prior to sample fabrication, GPC using MALS was conducted on aqueous DNA solutions used in foam and film preparations. MALS chromatograms are shown in Figure 5-2. Herring teste DNA (initially 26 x 10 6 g/mol) was found to degrade to a much lower molecular weight following high shear mixing at 1200 – 1500 rpm for 4 hr (Mw = 2.7 x 10 6 g/mol, Rg = 100 nm). The PDI was observed to be approximately 2. These data indicate that DNA Mw may be tailored by controlled high shear mixing conditions. SEM micrographs of porous DNA scaffolds are shown in Figure 5-3. Microporous foams shared similar features with previously described polysaccharide substrates; however, distinct differences in surface morphologies were observed. Foam pore ultrastructure and distribution was observed to be heterogeneous in nature. DNA surfaces were semi-organized with a grain-like topography, possibly attributed to intermixed regions of crystalline and amorphous DNA. Further studies with solution cast thin films pronounced this behavior. These compositions were found to support a dense cellular infiltrate of mixed glia and peripheral macrophages in later in vivo implantations. Thin films were solution cast at temperatures ranging from 25 o C – 80 o C. Dendritic growth was observed at lower casting temperatures (Figure 5-4). As the temperature was raised, crystal size and morphology was observed to be more regular (Figure 5-5). Future studies with X-ray may afford insight into their inherent crystal structure.

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123 Noninvasive Magnetic Resonance Imaging (MRI) In order to non-invasively evaluate porous DNA implants, T2 and T1 weighted spin echo images were acquired at times ranging 1 day to 4 months post-implantation. Images were collected in the sagittal, coronal and horizontal planes under multiple pulse sequences. The injury site was clearly visible in all subjects. Untreated controls exhibited hyperintense signal behavior at the lesion level, in which the normal anatomical morphology of gray and surrounding white matter were lost to a homogenous signal zone, characteristic of CSF signal (hyperintense T2). Subsequent histology determined this to be due to significant tissue loss and cavitation, which corroborates well with the bright images since T2 images are intrinsically sensitive to free water. In addition, animals displaying hyperintense MR images retained little or no use of the affected right forelimb for the duration of the study. As previously noted following polysaccharide implantation studies, DNA implants exhibited significant signal void (hypointense signal) at the lesion level (Figures 5-6 and 5-7). The cause of the T2 hypointense signal is unclear, but is interestingly observed regardless of biopolymer implant composition. Serial X-ray analysis by energy dispersive spectroscopy may be used to determine metal content and distribution throughout normal and injured tissue, i.e., Fe and Mn, of which may directly contribute to this phenomena. Serial MRI confirmed these observations through 4 months post-implantation. Post-mortem histology revealed a dense cellular infiltrate and evidence of neurofilament immunopositive axons. Conversely, acute imaging, i.e., within 24 – 36 hours of DNA implantation, results in implant MR images with more moderate signal. These experiments confirm the importance of the translation of serial MRI into useful clinical data and outcome measures for biopolymer implants.

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124 Behavioral Analysis Pilot functional evaluations by spontaneous vertical exploration were conducted in a limited number of animals (n = 3) and without statistical analysis. As previously detailed, axotomy at C4/C5 produced significant functional deficits in tasks including reaching, grasping, forelimb placement, walking, and normal posture. Untreated control animals were restricted to using the uninjured, contralateral (left) limb during vertical exploration throughout the course of these studies (~ 90 % at 4 wks). Conversely, DNA-treated animals displayed some use of their affected limb by 4 wks (~ 55 % simultaneous both). In addition, untreated control subjects frequently suffered self-mutilation complications. DNA-treated subjects were not observed to exhibit this behavior. These results coupled with MRI and post-mortem anatomical and histological analyses suggest that DNA compositions may exert a rescuing effect of rubrospinal axons following cervical SCI through tissue regeneration and the minimization of secondary injury mechanisms. Retrograde Tract Tracing Retrograde tract tracing experiments using 30 mg/ml aqueous Fluorogold (FG) were conducted by bilateral injection at T1. Untreated control animals exhibited minimal FG labeling in the injured contralateral magnocellular region of the red nucleus (RN) at 4 months. Labeled neurons in the control contralateral RN were most likely attributed to those fibers (5 – 10 %) representing ipsilateral (uncrossed) projections of the left uninjured rubrospinal tract. Neurons in the injured magnocellular RN in porous DNA-treated animals were found to be FG positive at 4 months post-implantation. This is shown in Figure 5-8 and implied that some ability for biochemical transport to the midbrain existed. Many FG labeled RN neuron cell bodies retained a normal morphology, and their presence alone

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125 at 4 months was promising in that foreign DNA compositions did not induce or promote retrograde cell death following axotomy. Immunohistochemical Staining Porous DNA compositions induced a dense cellular infiltration following implantation and cervical SCI. Within 1 wk post-implantation the lesion epicenter was filled with large, globular macrophages filled with myelin debris. By 4 wks, implants had completely eroded or degraded, and the lesion site resembled newly formed amorphous tissue with evidence of microvascularity and angiogenesis (Figure 5-9). These implants exhibited intense signal void under T2 weighted MRI. Similar results were found at 4 months. Neurofilament M (NF-M) immunopositive axons were found throughout the implantation site, with robust staining focal to the interfaces between injured and spared tissue (Figure 5-10). Motor neurons in the immediate vicinity, rostral and caudal to the implantation site, were apparent in most cases as shown in Figure 5-11. -Internexin positive fibers were observed within the space of the primary injury. Injured contralateral RN neurons were found to robustly express NF-M, and appeared slightly swollen and enlarged compared to those in the control ipsilateral nucleus. The reason for this is unclear. Unreactive OX-42 immunopositive microglia were found in close proximity. While some mononuclear cells were infrequently observed at the implantation level, the density of these cells was no greater than that of distant uninjured tissue. A moderate astrocyte response was observed in the spinal cord and GFAP immunoreactivity was found within and surrounding the lesion site, as were ED-1 and OX-42 positive macrophages and microglia, respectively (Figure 5-12). Staining with Maisson Trichrome failed to identify connective tissue or collagen as a constituent of the infiltrate (Figure 5-13).

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126 The infiltration of glial and blood-borne peripheral cells, which likely included Schwann cells, may have provided a neurotrophic environment that is permissive to axonal growth and tissue wound healing. All of which have been reported to play pro-regenerative roles following SCI. Further immunohistochemical studies are required including staining for immature glial or progenitor cells.

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127 51.0x1061.0x1071.0x1081.0x1011.512.012.513.013. 5 Molar Mass vs. Volume DNA 51.0x1061.0x1071.0x1081.0x1091.0x104080120160200Custom Plot DNA Radius of Gyration (nm) Molar Mass (g/mol) Elution Volume (ml) Molar Mass (g/mol) Figure 5-2 MALS chromatograms of Herring teste DNA following high-speed mixing (Mw = 2.7 x 10 6 g/mol, Rg = 100 nm).

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128 Figure 5-3 SEM micrographs of a porous DNA composition in cross-section (A) and of the pore surface (B).

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129 B A Figure 5-4 Optical images of solution cast DNA thin films revealing the dendritic morphology at RT in 2-dimensions (A) and in a 3-dimensional surface plot (B). Scale bars = 100 m.

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130 Figure 5-5 SEM micrographs of solution cast DNA thin films (80 o C) with A – F increasing in magnification.

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131 Figure 5-6 T2 weighted MR images of an untreated control (A) and a porous DNA treated animal (B) 4 months post-implantation. The untreated control displays a significant hyperintense signal focal to the lesion. Porous DNA MRI displays significant hypointense (signal void) at the lesion/implantation site (TE = 56.7 ms).

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132 Figure 5-7 T2 weighted MR images (A – C) 3 days post-DNA implantation displaying moderate signal with small hypointense regions (signal void). Panel C shows regions of possible CSF accumulation distal and caudal to the lesion.

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133 Figure 5-8 Fluorogold labeled neurons in the magnocellular red nucleus in untreated controls (A) and DNA treated (B) following retrograde tract tracing at 4 months.

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134 Figure 5-9 Cresyl violet staining of a DNA treated lesion showing myelin filled macrophages (A) and evidence of angiogenesis and microvascularity (B).

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135 Figure 5-10 Dense cellular infiltrate (A) and NF-M immunolabeled nerve fibers in the lesion site epicenter (B).

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136 Figure 5-11 NF-M immunoreactive axons (A) and gray matter cell bodies (B) caudal to the implantation site 4 months post-injury.

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137 Figure 5-12 ED-1 (A) and OX-42 (B) immunopositve macrophages and microglia.

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138 Figure 5-13 H and E (fields A and B) and Maisson Trichrome (C and D). Tissue stained negative for collagen and connective tissue following Maisson Trichrome.

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139 Summary Recent DNA research has focused primarily on gene mapping, expression, and targeted therapy. However, DNA has not been investigated as a structural biomaterial or scaffold for neural tissue repair. This work marks the first reported use of implantable DNA compositions for treatment following spinal cord injury. Following implantation, porous DNA induced a glial and peripheral cellular infiltration, with evidence of axonal regrowth. Serial MR images failed to detect cavitation at the lesion level and treated animals exhibited limited function of the affected limb. Serial X-ray by energy dispersive spectroscopy of normal and lesion level tissue may aid in determining if T2 signal void characteristic to porous DNA is related to blood components including iron and manganese. These intriguing results warrant further investigation for the use of DNA as a structural biomaterial. In addition, DNA may have many other biomaterial applications, i.e., for drug delivery vehicles, nano-mesosphere preparations, and tissue protective solutions for the prevention of post-operative adhesions.

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CHAPTER 6 CONCLUSIONS The excitement of recent advances in the understanding of CNS development and pathologies has been tempered by a lack of neurological recovery following current attempts to repair SCI. Recent repair strategies have emphasized fetal tissue and cell transplantation, as well as local protein, gene, antibody, and enzyme delivery. However, it is clear that no single intervention, i.e., the expression of a single gene or delivery of a neurotrophic protein alone will reverse the consequences of traumatic SCI. The strategy of this research was to combine perspectives from biomaterials science and neurobiology in effort to provide physical/structural support and a favorable surface terrain (biopolymer implant), and neurotrophic support in the form of various growth factors, cytokines, extracellular matrix molecules and other factors produced by viable microglia. The basic concept was to use neural cell-biopolymer compositions to regulate the complex and dynamic repair processes following CNS injury. Novel nano-mesoporous alginate/phospholipid, chitosan, and DNA-based scaffold implants were synthesized. New methods for phospholipid surface modification of polysaccharide implant compositions by radiation initiation polymerization were developed. This work also marks the first reported use of DNA as a scaffold biomaterial for treatment following SCI. Results of studies to date with our polysaccharide, phospholipid, and polynucleotide composite implants suggest that optimization of compositions, implant design, and surgical procedures may lead to a clinically important medical technology for spinal cord repair. Noninvasive MRI found all implant compositions to prevent or greatly minimize cystic cavitation and atrophy. Untreated control subjects were found to suffer 140

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141 significant cavitation, which correlated with poor neurological function. Implants, regardless of specific biopolymer composition, exhibited significant signal voiding behavior (dark images) on T2 and T1 weighted MRI versus hyperintense untreated lesions (bright images) indicative of free water (CSF) and characteristic to cystic cavitation. Implant treated subjects were found to have increased neurological function versus untreated controls during spontaneous vertical exploration at recovery times as early as 4 weeks. Microglia treated subjects had some ability to independently use their affected limbs. Results from DNA implant behavioral studies are also promising in that vegetable or animal DNAs may promote regeneration and prevent secondary injury mechanisms (cavitation). Although this motor function recovery was encouraging, treated subjects still exhibited significant functional deficits during reaching, grasping, and walking. Their behavior was found to be statistically different than normal healthy animals. However, increased retrograde transport was found for implant treated subjects, indicating some axonal transport to the midbrain. Post-mortem anatomical and histological analyses correlated dark T2 weighted signal void MR images, improved function, and retrograde axonal transport with a dense glial and peripheral cellular infiltration. Future locomotor evaluations using open-field testing or complete transections may prove to be the most valuable method of assessing recovery of neurological function. Together, these data indicate the strategy of using neural cell-biopolymer compositions is valid and warrants further investigation. Ideal compositions have not been identified as of yet, and may include the incorporation of other cell types or therapeutic agents to existing or new implants. In addition there are important broader implications of results stemming from this research including the application of similar

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142 implant compositions for the treatment of optic nerve disease and trauma, as well as macular degeneration and retinal disease.

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CHAPTER 7 FUTURE DIRECTIONS During the course of this research a plethora of interests arose, some of which may directly benefit the foregoing research. Possible future endeavors are described in the following in no particular order. 1. Synthesis of protein based scaffold implants using neurofilament cytoskeleton proteins including NF-L, NF-M, NF-L, and -INT. The use of proteins in the fabrication of scaffolds including collagen and ECM fibronectin and laminin has been reported. However, the use of the most abundantly expressed proteins in the CNS, i.e., cytoskeletal filament proteins, has not to date. NF proteins are largely homologous between species (> 80 %) and high yields (grams) of protein can be easily extracted from a variety of sources including bovine and porcine brain. The synthesis of NF based scaffold compositions is intriguing and represents an opportunity to prepare implants from materials endogenous to the CNS. 2. Synthesis of imaging enhancing implants for increased MRI resolution. The use of gadolinium containing or crosslinked implants may afford an enhanced signal to noise ratio for improved evaluation of implant integration and erosion by noninvasive MRI. 3. Application of implant compositions for delivery of gene therapy in treatment of inherited disease. Recent pilot studies have shown that our implant compositions can be in situ loaded with high payloads of retrovirus and delivered to a focal area. Our implant compositions may afford controlled, localized release of virus to early stage tissue in which systemic transduction is not desired. 143

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144 4. Application of implant compositions for treatment of ophthalmic nervous disease and disorder including macular degeneration and optic nerve disease. 5. X-ray/E Beam analyses of iron and manganese content at lesion sites containing cellular infiltrates exhibiting T2 weighted signal void behavior during MRI. Characteristic implant signal void behavior may be elucidated by detailed X-ray analysis of the lesion/implantation site and new experiments may determine if our implants themselves have a strong affinity for Fe or Mn. 7. Use of bioelectricity and nanocurrents in conjunction with biopolymer implant compositions. Small direct current electric potentials (DCEFs) are important in development, injury, and regeneration of tissues including the brain and spinal cord. Recently, experiments have shown these currents may promote regeneration of damaged nerve fibers in the spinal cords of rats and dogs, with some improvement in motor function in these models. The combined use of bioelectrical stimulators and biopolymer scaffolds may afford greater return of neurological function than either alone.

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BIOGRAPHICAL SKETCH Joshua Benjamin Stopek was born on February 16, 1976 in Hollywood, Florida. He attended the School of the Arts in Palm Beach County in 1990 and graduated in 1994 with honors in the Advanced Placement program. During these years he discovered science and his passion for painting. He continued his academic career at the University of Florida in 1994 and was introduced to the field of Materials Science and Engineering. He earned his master’s degree in material science and engineering, with a specialization in biomaterials, in 2001. He continued his academic journey at the university and earned his PhD in biomaterials science and engineering in 2003. He has consistently and intentionally combined his creative and intuitive attributes with his analytical and intellectual pursuits. 158