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Hyaluronic Acid-Olfactory Ensheathing Cell Compositions for Spinal Cord Injury Nerve Regeneration


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HYALURONIC ACID-OLFACOTRY ENSH EATHING CELL COMPOSITIONS FOR SPINAL CORD INJURY NERVE REGENERATION By JENNIFER DE TOLEDO MALLEK A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Jennifer de Toledo Mallek

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This document is dedicated to my cat, Angel.

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iv ACKNOWLEDGMENTS First, I would like to tha nk my parents, Howard and Mara Mallek, for showing to me the true value of education and supporting me throughout my academic career. I would like to thank my advisor and committee chair, Dr. Eugene P. Goldberg, for his knowledge, support, help, and advice. I am grateful to Dr. Paul J. Reier, for providing all the necessary laboratory tools and knowledge in neuroscience to complete my work. I would also like to thank Dr. Kenneth Wage ner, for sparking my interest in polymers through excellent instruction and advice. I w ould also like to thank Dr. Johnny Bengston, for his emphasis on individuality and pers onality development, for offering great literature and advice, and for his lively attitude towards life and those around him. I would like to thank Paul Martin, for va luable advice and excellent instruction on the electron microscope. I woul d like to thank Jennifer Wri ghton, for taking care of all the backstage issues. I would also like to thank Dr. Ishihara for generously donating the phophorylcoline used for my project, and Dr. Jaccoberger, for allowing us to use his immortalized olfactory ensheathi ng cell line. I would especially like to thank Lisa Tanzer for preparing and shipping cells, and for providi ng protocols and advi ce to culture cells. I would also like to thank Troy Sendler, my partner, for encouraging me, listing to me, having a great sense of humor, helping me in my path, and cooking some great food for me on days I had to work late. He has been with me through thick and thin and I would not be who I am and where I am today without his love and advice. And, finally, I would like to thank my dog, Buffy, for being womans best friend.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT....................................................................................................................... ix CHAPTER 1 INTRODUCTION........................................................................................................1 1.1 Background.............................................................................................................1 1.2 Specific Aims..........................................................................................................7 1.2.1 Aim 1: Synthesis and Charact erization of Novel Porous 2Methacryloyloxyethyl Phosphorylcho line (MPC) Modified Hyaluronic Acid (HA) Based Compositions..................................................................7 1.2.2 Aim 2: In Vitro and In Vivo Evaluations of HA Scaffolds as Olfactory Ensheathing Cell (OEC) Carriers in the Treatment of Spinal Cord Injury(SCI)...................................................................................................8 2 HYALURONIC ACID SCAFFOLDS FOR TREATMENT OF SPINAL CORD INJURY........................................................................................................................9 2.1 Hyaluronic Acid (HA) Solutions............................................................................9 2.2 Uniaxial Porous Scaffold Synthesis........................................................................9 2.3 Thin Film Synthesis..............................................................................................11 2.4 2-Methacryloyloxyethyl Phosphorylch oline (MPC) Surface Modifications........12 2.4.1 Monomer Solutions....................................................................................12 2.4.2 Gamma-Radiation Polymerization.............................................................12 2.4.3 Post Irradiation Cleaning............................................................................13 2.5 Scaffold Characterization.....................................................................................13 2.5.1 Scanning Electron Microscopy (SEM).......................................................13 2.5.2 Optical Microscopy....................................................................................14 2.5.3 Equilibrium Water Content........................................................................15 2.5.4 Fourier Transform-Infrared Spectroscopy (FT-IR)....................................15 2.5.5 Atomic Force Microscopy (AFM)..............................................................16 2.6 Results and Discussion.........................................................................................16

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vi 2.6.1 Scaffolds Directionality & Porosity...........................................................16 2.6.2 2-Methacryloyloxyethyl Phos phorylcholine (MPC) Modified Compositions.............................................................................................20 3 IN VITRO AND IN VIVO EVALUA TIONS OF HYALURONIC ACID SCAFFOLDS AS OEC CARRIERS FOR TREATMENT INJURED NEURONAL SPINAL CORD TISSUE.....................................................................28 3.1 Specific Aim.........................................................................................................28 3.2 Thin Film Synthesis..............................................................................................29 3.3 In Vitro Evaluations..............................................................................................29 3.3.1 Cell Cultures...............................................................................................29 3.3.2 Cell Culture Reagents.................................................................................30 3.4 Scaffold Cell Seeding...........................................................................................30 3.5 In Vivo Trial..........................................................................................................31 3.5.1 Surgical Procedure......................................................................................31 3.5.2 Surgical Implantation.................................................................................32 3.6 Postmortem Evaluations.......................................................................................32 3.6.1 Perfusions and Tissue Storage....................................................................32 3.6.2 Plastic Embedding of Tissue......................................................................33 3.5 Results and Discussion.........................................................................................34 3.5.1 In Vitro Thin Film Evaluations...................................................................34 3.5.2 Histological Analysis..................................................................................34 4 CONCLUSIONS........................................................................................................36 5 FUTURE DIRECTIONS............................................................................................38 LIST OF REFERENCES...................................................................................................40 BIOGRAPHICAL SKETCH.............................................................................................45

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vii LIST OF TABLES Table page 2-1 Pore area and degree of circ ularity of hydrated and porous hyaluronic acid scaffolds..........................................................................................18 2-2 Atomic force microscopy (AFM) values obtained for average height of surface of modified hyaluronic acid (HA) films.......................................23

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viii LIST OF FIGURES Figure page 1-1 Structure of hyaluronic acid.(HA)..............................................................................4 1-2 Structure of 2-methacryloyloxye thyl phosphorylcholine monomer..........................6 2.1 Freezing apparatus....................................................................................................11 2-2 Microscopy images of HA scaffolds........................................................................18 2.3 Electronic dispersive spectra (EDS ) of unmodified crosslinked HA.......................20 2-4 Percent equilibrium water content............................................................................22 2-5 Atomic force microscopy images.............................................................................23 2-6 Plot of average roughness versus total radiation dose..............................................25 2-7 Fourier transform-infrared sp ectra of percent transmittance....................................25 2-8 Scanning electron microscope (S EM) images of scaffold pieces............................26 2-9 SEM images at high magnification of scaffold pieces.............................................27 3-1 Optical image at lesion site of hya luronic acid scaffold implant at 2.5x.................35

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ix Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science HYALURONIC ACID-OLFACTORY ENSH EATHING CELL COMPOSITIONS FOR SPINAL CORD INJURY NERVE REGENERATION By Jennifer de Toledo Mallek August 2006 Chair: Eugene P. Goldberg Major Department: Chemistry There are approximately a quarter of a million people suffering from spinal cord injury (SCI) today in the United States a nd there are over 10,000 new cases reported each year however a clinically eff ective treatment for traumatic SCI is yet to be developed. Although recent advances in the neurological research has yielde d positive findings on the regenerative capacity of axons within th e central nervous system (CNS), functional regeneration of the injured spinal cord rema ins a current challenge, due to its chemical environment, which is highly non-permissive to growth and regeneration. Our primary objective of this study was to conduct a tissue-engineering study using composite olfactory ensheathing cell-hyal uronic acid (OEC-HA) scaffold compositions for the spinal cord. Anisotropic HA scaffolds were prepared by a freeze-drying method for the first time. Selected scaffolds ionically crossl inked with trivalent chromium were surface modified with 2-methacryloyloxyethyl phosphor ylcholine (MPC), in order to enhance

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x biocompatible properties and increase cell at tachment and viability on the substrate material prior to implantation. In order to verify the reproducibility of the implant material and to maximize its biocompatibility, chemical and physical prop erties of the scaffold implants were characterized by scanning electron microscopy (SEM), electron dispersive spectrometry (EDS), atomic force microscopy (AFM), four ier transforminfrar ed spectrometry (FTIR), gas permeation chromatography (GPC) and multi-angle light scattering (MALS). Plastic embedding of tissue in Epon was conducte d to visualize the im plant in vivo and to assess stability of the polymer stability a nd verify the absence of an inflammatory response and other immune events. Novel biodegradable micro-uniaxialmesoporous hyalurnonan/phospholipid scaffold implants were synthesized and implan ted into the rat spinal cord following a left cervical hemisection at C2/C3 in order to assess proof of prin ciple for this method. OECs regenerative-promoting capacity has be en highly investigated and have been found to facilitate axonal elongation at grafthost interfaces than Schwann cells; making them good candidates in SCI applications. Op timal OEC cell attachment and viability was found in vitro on the HA films which were subject to a total gamma irradiation dose of 0.15Mrad. Implant treated subjects showed no immunological res ponse to the implant and showed minimal cystic cavitation and promoted tissue regeneration. Although implants seeded with cells has been done, th e analysis is not c overed in this study.

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1 CHAPTER 1 INTRODUCTION 1.1 Background Today in the United States there are approximately 250,000 persons suffering from spinal cord injury (SCI) and approximately 11,000 new cases are reported each year (1). Clinically the only neurologically targeted tr eatment even consider ed is the systemic delivery of methylprednisolone. However, the drugs safety and efficacy has been recently questioned (2) and results from clinical trials have been openly criticized (3). Traumatic SCI is most commonly the result of a single displacement or penetration of the spinal cord which is referred to as primary injury. The effects of primary injury are sign ificantly enhanced by the destructive secondary injury events which can extend for days after the initial traumatic event (4). Secondary injury involves a complex a nd destructive cascade of pathophysiological processes involving a variety of vascular changes including edema, eschemia and hypoxia, cellular responses such as glia l cell activation and scar formation, and biochemical events including nitric oxid e release and free radical formation (5). Immediate scar tissue formation as a normal wo und healing response is a major factor in limiting SCI repair. Strategies to overcome this problem are needed. In this regard, acute animal models with immediate treatment may not reflect the real clinical setting for more effective SCI repair. (6). Biodegradable polymer implant materials have the potential to ameliorate a variety of the problems associated with SCI. Impl ants prevent cystic cavitation, and can be

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2 seeded with a variety of drugs to be slowly released as the material degrades which can either promote growth of injured axons such as NGF and BGNF, or inhibit unwanted cellular responses such as g lial scar formation. These scaffolds can also act as cell carriers so that the desired cells have a substr ate to adhere to. The micro-architecture of the implant can be engineered for optim al axonal growth and organization through various methods such as the freeze-dry proces s (7), particulate-le aching techniques (8), heat compression and extrusion (9) while pr oviding a physical support for delivery of various cell types including st em cells (15, 16), microglial ce lls (17), Schawnn cells (SC) (18), and potentially others cells such as olfactory ensheathing cel ls (OEC) which have shown the ability to restore axonal conduction after injections into the dorsal column lesions in rats (19, 20) and ar e being currently used in clinical trials overseas (21). Another possibility, which has not yet been investigated, is the seeding of multiple cell types. For example, implanted grafts show that SC are more efficient than OEC in promoting axonal regeneration/sparing and re myelination of spared axons; however, OECs seemed to facilitate axonal elongation at graft-host interfaces better than SCs. These findings suggest that a combination of SC-OEC grafting might be more beneficial in the contused spinal cord than one involving OECs or SCs alone. OECs, however, have the unique ability to meld with astrocytes, lacking in SCs and peripheral nerve grafts, making them ideal candidates for SCI. (22) The implant material may also serve as a conduit for sustained-release delivery of therap eutic agents such as vascular endothelial growth factor (VEGF) (10), nerve growth f actor (NGF) (11), ciliar y-neurotrophic factor (CNTF) (12) and a variety of other neur otrophic factors ( 23, 24, 25). The delivery through biomaterials may provide an attractive alternative to the intrathecal pump, as

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3 delivery is reproducible and immobilized concen tration gradients can be created (13, 14). With these factors taken into consideration, the use of a modi fied biomaterial implant for treatment of SCI can significantly aid the patients recovery by attacking the multiple issues involving SCI with a single implant. A material that is a good ca ndidate as a scaffold for SCI should be a biocompatible polymer which is biodegradable, so that he althy neural tissue can gradually replace the implant as it degrades, and provides both chemical and physical guidance for regenerating axons and related cells. Implan t compositions of natural polymers such as alginate, collagen, hyaluronic acid and DNA as well as synthetic polymers such as poly( -hydroxyacids), polyethylene glycol and 2hydroxyethyl methacrylate have been previously synthesized and ch aracterized and are shown to have a potential uses as biomaterials (17, 26). Natural materials such as collagen and hyaluronic acid (HA) are at an advantage since they convey chemical signa ls within the structure that can enhance tissue formation (27). The optimal time for scaffold biodegradation is dependent on the rate of regeneration of the spinal cord tissu e. Ideally, the scaffold should degrade at the same rate that the neural tissue reconstitutes itself however degradation rate is hard to control and is difficult to assess in situ (7). HA is a naturally occurring polymer which was first found in the vitreous humor of the eye in cattle (30) and la ter was discovered to be wi dely distributed in the body, notably in synovial fluid, extr acellular matrix and loose c onnective tissues such as the umbilical cord, dermis and rooster combs. (31, 32, 33) HA is also known to interact with cellular surface receptors including well known CD44, RHAMM and ICAM-1 which are involved in processes such as morphogenesi s, wound repair, inflammation and metastasis

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4 (28). HA is found naturally throughout the spin al cord and is synthesized predominantly by astrocytes, which contain the RHAMM a nd CD4 receptors, and are localized around white matter fibers in the interstitial space between myelin and astrocyte processes. In spinal gray matter, HA surrounds neuron cell bodies. (36, 37) The role of HA in the nervous system has not been extensively ex amined; however, recent studies demonstrate that high-molecular weight form of HA can i nhibit astrocyte prolif eration in vitro and promotes astrocyte quiescence in vivo (38). Uncrosslinked HA injected into a fibrin matrix nerve guide tube has been shown to enhance peripheral nerve regeneration in vivo due to the influence on the motility of growing cells by way of its cell surface interactions with extracellular matrix fibroblasts and by its ability to distort the fibrin matrix into a more porous microenviroment, wh ich thus facilitates infiltra tion of cells responsible for tissue regrowth. (34, 35) Figure 1-1. Structure of hyaluronic acid com posed of repeating un its of d-glucuronic acid and N-acetyl d-glycosamine (44). HA is a linear, unbranched high molecu lar weight, up to 7 million Daltons, polysaccharide of repeating disaccharide units composed of N-acetyld-glucosamine and d-glucuronic acid linked by a 1-4 glycosidic bond as shown in Figure 1-1. The disaccharides units are linked together by 1-3 bonds (28, 29). The polymer backbone contains carboxyl, acetoamino and alcohol func tional groups which are free for chemical

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5 crosslinking or surface modifications of a ny kind. It has been found through preliminary experiments that HA can be crosslinked with polyvalent chromium (III) under specified conditions to form a water insoluble scaffol d. It is likely that chromium (III) crosslinkes HA via electrostatic interactions between the cation and the negatively charged carboxylic acid groups, leaving the alcohol groups available for further chemical modifications (43). It is hypothesized that the probability of achieving f unctional recovery, by having growing axons reconnect to the appropriate (o riginal) neurons, is increased by retaining the native organization of regenerating axons across the lesion site. This is achieved by, in addition to providing biochemical and/or cellular support, providi ng physical guidance for the linear growth of axons across a site of injury. The production of linear guidance pores, utilizing freeze-drying techniques, extendi ng through the entire length of a scaffold for use in SCI has been previously reporte d with the natural pol ymer agarose; (11) however, this engineering method has not yet been applied to other natural polymers, including hyaluronic acid. A freeze-drying technique is of preferred use for pore formation and manipulation since no additional, potentially toxic, chemicals are required and the direction of growth and size of ice crystals may be controlled by manipulating the temperature gradient applied to polymer so lution (7). HA has never been previously reported in the form of an insoluble scaffo ld with an intentionally defined tertiary structure. 2-methacryloyloxyethyl phosphorylcholine (M PC), and its copolymers have been proved to interact mildly with proteins a nd cells on their surfaces and have show biocompatibility and hemocompatibility in vivo (17, 48 49 ). Polymers used for

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6 cardiovascular applications demonstrated increased endogenous blood phospholipid adhesion and decreased platelet activation when coated with polyMPC (40, 41, 42). In addition, MPC copolymers have been shown to be a promising cytocompatible material for tissue engineering, because the MPC polymer prevents the cells on biomaterials from inducing an inflammatory response or ot her foreign body response (50, 51, 52) Also, cells cultured on an MPC-Al ginate copolymer previously in our lab, among other MPC copolymers, showed high cell viability in vitro ( 17, 53, 54). Since reduced thrombus formation and chronic inflammation has b een correlated with endogenous phospholipid adsorption it is hypothesized that the surface modification of hyaluronic acid scaffolds will improve hemocompatibility of the sca ffolding material, as well as improving the cellular proliferation and adhe sion onto its surface. The stru cture of the MPC monomer is shown in figure 1-2. Results from previous st udies demonstrated proof of principle for the surface modification of pol ysaccharide compositions with hydrophilic, water soluble, vinyl monomers using gamma radiati on initiation polymerization (17). P O O-O CH2CH2 +N CH3CH3CH3 H2CC CH3CO O CH2CH2O Figure 1-2. Structure of 2-methacr yloyloxyethyl phosphorylcholine monomer. Ionizing radiation may be used to ini tiate radical polymer ization of MPC at ambient temperature in the absence of ot her chemical radical initiators. Residual chemical from chemical initiations may poten tially exhibit cytotoxic and carcinogenic responses in vivo Polymerization of aqueous acryla tes, methacrylate and acrylamide monomers have been previously report ed to be polymeri zed through initiation

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7 mechanisms via the radiolysis of water. (45, 46, 47). In addition to the lack of residual chemicals, the molar mass of the polymer, or extent of surface modifications to be irradiated onto a substrate, may be controll ed by varying the total dose and dose rate of gamma irradiation a solution is exposed t o. Although surface modifications using gamma grafting have been previously reported in our laboratory (17), lit tle is currently known concerning surface modification of hydrogel materials by gamma-induced polymerization. With these factors taken into consideration, gamma radiation initiation polymerization is the ideal method for HA surface modifications of previously crosslinked insoluble scaffolds. 1.2 Specific Aims 1.2.1 Aim 1: Synthesis and Charact erization of Novel Porous 2Methacryloyloxyethyl Phosphorylcholine (MPC ) Modified Hyaluronic Acid (HA) Based Compositions Porous hyaluronic biopolymer implant com positions were prepared using freezedry/lyophilization and film casting techni ques. Implant compositions were surface modified with the polymeric phospholipi d, MPC using gamma radiation initiation polymerization. Fourier Transform Infrared spectroscopy (FTIR), a nd energy dispersive spectroscopy (EDS) were used in order to ve rify the presence of the MPC substrate on the modified samples by functional group and elemental analysis, respectively. Scanning electron microscopy (SEM) was used to visua lize porous structure of the material and analyze surface features of dehydrated films. Gas permeation chromatography (GPC) and multi-angle light scattering (MALS) were us ed for molecular weight determinations. Atomic force microscopy (AFM) was performed on hydrated films in order to determine, quantitatively, the surface feat ures introduced by varying dos es of MPC. For comparison with both hydrated data and SEM data, AFM was performed on dehydrated film as well.

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8 The effects of gamma radiation and autoclave sterilization on implant stability were also investigated. 1.2.2 Aim 2: In Vitro and In Vivo Evaluations of HA Scaffolds as Olfactory Ensheathing Cell (OEC) Carriers in the Treatment of Spinal Cord Injury(SCI) In order to determine if OECs are able to adhere to and grow on HA scaffolds, In Vitro studies were conducte d on HA films of the same composition as the scaffolds. Films were soaked in a solution of MPC and gamma irradiated with doses varying from 0.5 to 1.5Mrads. Cells were then placed on unm odified films and film s modified with a gradation of MPC. Adhesion studies and vita l staining with trypan blue were conducted to determine adhesion and viability of cells onto specific substrates. As an advance toward future tests of therapeutic efficacy, we first wanted to determine the stability of this polymer in vivo and whether it w ould elicit an adverse inflammatory response. The second issue e xplored was how stable the cell-polymer construct would be in vivo. To address these i ssues, left sided hemisections were made at the cervical C2/C3 level in the adult ra t cord. Implant compositions were placed immediately following injury. Animals were allowed to recover for 1 week postimplantation. Analysis was conducted by plastic embedding in order to determine scaffold regenerative effects.

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9 CHAPTER 2 HYALURONIC ACID SCAFFOLDS FOR TREATMENT OF SPINAL CORD INJURY 2.1 Hyaluronic Acid (HA) Solutions Low molecular weight hyaluronic ac id (reported MW=560 KDa) donated by Genzyme was used in the prep aration of all microporous fo ams and films. 40mg/mL of HA was dissolved in ultrapure water w ith a resistivity of approximately 17.4M using Lightnin and Caframo high-speed mechanical mixers and in-house built 3-blade propellers at a speed of 400 revolutions per minute overnig ht. 10mg/mL solutions were prepared with less aggressive magnetic sti rrer at the maximum speed setting. The 1% solutions were used for film casting and the more viscous 4% solutions were used for foam synthesis. Solutions were filtered into clean 100 ml screw cap glass bottles using a stainless steel air-pressure apparatus ma nufactured by Gelman Sciences and 70 m Spectra filters. Solution concentration (mg/ml) was verified using a Mettler LJ16 Moisture Analyzer. Solutions were stored at 4oC until further use. Hyaluronic acid molecular weight was verified by using a Waters gel permeation chromatography (GPC) system and a Wyatt Dawn EOS multi-angle light scattering (MALS) detector. 2.2 Uniaxial Porous Scaffold Synthesis Approximately 4mL of the 4% HA solu tion was injected into flat-bottom borosilicate tubes measuring 8mm in diameter and 4.5cm tall. Solutions were allowed to sit at room temperature covered with a piece of Kimwipe for approximately 45 minutes in order to allow for gravitati onal removal of air bubbles trapped within the viscous solution. If air bubbles still remained, tubes were placed inside 50mL centrifuge tubes

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10 and centrifuged at approximately 1500rpm or der to remove residual air bubbles. The physical setup used for the formation of lin ear pores by freeze drying was similar to the one reported by Stokols in 2004 (11). A piece of Styrofoam (10.85cm x 12.7cm x 4.7cm) which serves as a 15mm polypropylene centrifuge test tube holder (x25) from Fishers centrifuge packaging was used as the insulating test tube holder in the experiment. Four holes measuring 8mm in diameter (flush with borosilicate tube) were cut through the Styrofoam into respective centrifuge tube slots as shown in Figure 2-1. Each HA filled tube was then firmly pressed into its respective hole so that the bottom of the tube was flush with the bottom of the Styrofoam piece (both surfaces were in cont act with the bench). A piece of dry ice measuring approximately (15cm x 13cm x 3cm) which contains a flat surface that is parallel to the ground, was placed inside a small vacuum oven hooked up to a vacuum pump. The Styrofoam setup containing the HA solutions was then placed on top of the block of dry ice which is s itting inside the vacuum-oven. The entire setup may be visualized in Figure 2-1. Immediately after inserting the sample the vacuum was turned on and adjusted to 500mmHg in order to remove gaseous carbon di oxide that could potentially freeze the solution laterally. Solutions were left to fr eeze in for two hours and linear ice crystals were clearly visible when removed. Scaffold s were then lyophilized overnight, gently removed from their glass tubes and cut into sections measuring 2mm in using a #10 stainless steel surgical blade. The bottom 1cm and top 2mm of the scaffold was discarded. Individual foam pieces were then crosslinked for 3 days on rotary tumbler in solution of 70% ethanol and 30% 0.0025M CrK(SO4)2.

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11 a. b. Figure 2.1. Freezing apparatus. A) A flat block of dry ice wa s placed in the Styrofoam container inside the vacuum oven. The two Styrofoam sample holders were then placed on top of the dr y ice. B) Borosilicate flat bottom test tube inserted into a hole within the St yrofoam holder (right) next to a vacant spot (left) 2.3 Thin Film Synthesis The dilute 1% HA solution was used for casting of all films. Two milliliters of the HA solution was injected into individual wells of a 12 well cell culture flask (Corning Inc.) and flask was covered with a piece of Kimwipe in order to prevent contamination. Flask was gently tapped in order to ensure an even distribution of the solution throughout the bottom of each well. Flask was then left overnight under biological hood in order to allow for the films to dry. Once dry, 2mL of 0.0025M CrK(SO4)2 was injected to each well and allowed again to rest in the hood c overed with a piece of Kimwipe for 3 days. Dried films were then peeled off the bottom of the dish using microforceps and washed with ultrapure water in 50mL centrifuge tube s. Films were then transferred to a new 12 well cell culture flask and stored at 4oC until further use or into a single detached wells from what was originally a 12 well cell culture flask for MPC modifications.

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12 2.4 2-Methacryloyloxyethyl Phosphorylcho line (MPC) Surface Modifications 2.4.1 Monomer Solutions MPC monomer was generously donated by Dr. K. Ishihara, Department of Materials Engineering, University of Tokyo (Tokyo, Japan), and st ored dry at oC. 10% MPC solutions (100 mg/mL) were prep ared using nanopure wa ter (resistivity > 17.4M ) and used for all modifications. Each sc affolding piece subject to modifications was washed for only one day inside a 50mL centrifuge tubing using nanopure water. The scaffold was then immersed in 4mL of the MPC solution inside a 15mL borosilicate test tube, capped, and left on the bench at room temperature for 48 hours. Films were also washed for one day and placed into single de tached wells of a 12 well flask. Two mL of the MPC solution was pipetted into each well an d covered with Parafilm. Films were also left to soak for 48 hours. Tubes or wells were gently agitated periodically in order to remove air bubbles. Prior to the gamma-polymeri zation, solutions inside the test tube and wells were degassed for approximately 5 minutes and 3 minutes respectively in order to remove dissolved O2. 2.4.2 Gamma-Radiation Polymerization Polymerizations were conduc ted using a Wiscon Type 60Co source reactor. Source was lowered to 3cm above the reactor floor and dosimetry was conducted prior to initial experiment to determine target radii. Prelim inary studies found no si gnificant effect on dose rate variability therefore a constant dose rate of 1000rad/min was used for all modifications. Samples were evenly distribut ed around sources cente r and rotated on its axis 180o every 0.05Mrad and 1/3 the circumference of the test tube slots in order to account for dose rate inhomogeneity. The th ree total doses of exposure were 0.05, 0.10 and 0.15Mrad.

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13 2.4.3 Post Irradiation Cleaning Once modifications were complete 1mL aliquots of each polyMPC solution was transferred to 2mL vials and stored at 4oC for further experimentation in order determination the molecular weight and radi us of gyration of the polymer through GPC and MALS. The scaffolds and/or films were transferred to 50mL centrifuge tubes and washed for one week at room temperature using ultrapure water. Once radiation was complete scaffolds and/or films were stored in the refrigerator until further use. 2.5 Scaffold Characterization 2.5.1 Scanning Electron Microscopy (SEM) Scanning electron microscopy (SEM) was empl oyed in order to analyze the three dimensional microstructure of the MPC modified and unmodified samples and to qualitatively analyze the materials pores. L ongitudinal sections of the HA scaffold were imaged in order to analyze pore distribution, area, and degree of ci rcularity. Transverse sections were imaged in order to verify the c ontinuity and directionali ty of the pores. The major (dmj) and minor (dmn) diameter of 30 pores were measured per scaffold piece utilizing ImajeJ software. Three longitudina l pieces of MPC modified and unmodified were analyzed along with two transverse cro ss-sections running the entire length of the center of the scaffold prior to slicing into 2mm sections (~4cm) The area of each pore was calculated using the gene ral equation for calculating the area of an ellipse: mn mjd d A General pore structure was found to have an ellipsoid nature therefore the degree of circularity (C) of each pore measured was also calculated by the following expression: mj mnd d C

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14 Upon completion of the washes, samples we re placed in the lyophilizer overnight in order to ensure sample dryness. Sample s were then adhered onto a piece of double sided carbon tape on an aluminum stub and sputter coated with gold-palladium using the Technix Hummer V sputter coater. All im aging was conducted using the JOEL SEM6400 scanning electron microscope (JOEL, Ltd., Peabody, MA) at an accelerating voltage of 5KeV, working distance of 15 mm and condenser le ns setting of 8. Electron Dispersive Spectroscopy (EDS) was us ed in order to verify the presence of phosphorous, not found on HA, on the surface of a MPC modified scaffold. EDS was conducted on uncoated sample and all parameters are the same as above except for the accelerating voltage was increased to 10KeV in order to visualize the K and K lines for Cr. 2.5.2 Optical Microscopy Optical microscopy was employed in orde r to analyze pore distribution and to measure pore diameters for scaffold pieces hyd rated in PBS. A Zeiss Axioplan2 imaging optical microscope was utilized for all im aging and pictures were captured by a Zeiss AxioCamHR camera, version 5.05.10, utilizing the AxioVission 3.1 software. The AxioVission software, in conjunction with Imag eJ software, was also used to measure the major and minor diameters of 10 pores per sca ffold piece. Three longitudinal pieces of MPC modified and unmodified scaffolds were hydrated in PBS for at 48hrs in order to ensure they were fully hydrated prior to pore diameter measurement. The same equations used for calculating area of an ellipse and degree of circularity in SEM were used for hydrated pore analysis.

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15 2.5.3 Equilibrium Water Content The equilibrium water content of sc affolds (n=5) was measured for both unmodified and MPC modified with a total radiation dose of 0.10Mrad. Scaffold pieces were dried overnight in an oven at 45oC to ensure sample dryness and weighed immediately afterwards. Each piece was then pla ced in a 50mL centrifuge tube filled with phosphate buffered saline (PBS) at a pH of 7.4. Water content was measured at the same time each day for five consecutive days, beginning 24 hours after initial immersion, and on the eighth day. Each scaffold was remove d from PBS solution, gently blotted onto a piece of Kimwipe until no more water was visi bly being absorbed by the Kimwipe, and weighed. Pieces were then immediately return ed to its original PBS solution in the centrifuge tube. The percent equilibrium water content is equivalent to the percent mass increase due to water absorption and was cal culated using the expression bellow. The wet and dry masses are represented by Mw and Md respectively. 100 % d d wM M M EWC 2.5.4 Fourier Transform:Infra red Spectroscopy (FT-IR) FT-IR was utilized in order to qualita tively verify the presence of MPC on modified thin films subject to gamma radiation doses of 0.05, 0.10 and 0.15Mrad. Absorption frequencies characteristic of MPC, which are not present in HA, include P=O, P-O-CH2, and N+-(CH3)3 at 1240cm-1, 1075cm-1 and 970cm-1 respectively (17). FT-IR spectra was collected using a Nicolet Magna 60SC FT-IR spectrometer using Omnic ESP software. Spectra were scanned 200 times per sample with a spectra l resolution of 4cm-1 in the range of 2000cm-1 to 800cm-1. After thin films were placed inside the sealed sample holder and prior to al l spectra collection, 5 minutes was allowed to pass in order

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16 for the majority of CO2 and H2O to be removed from the chamber with AIR PUMP THINGIE. Prior to film analysis, a background spectra was obtained in order to correct for the absorption of the remaining CO2 and H2O still present in the chamber. 2.5.5 Atomic Force Microscopy (AFM) AFM experiments were conducted on a Veeco Dimension 3100 Atomic Force Microscope under liquid cell cont act mode with a s ilicon nitride tip in order to analyze the difference in surface topography between hydrated modified and unmodified thin films. This method of analysis will allow fo r relative quantitative assessment of MPC on the films surface by correlating gradual changes in surface topography with the increasing dose rate of gamma irradiation experienced by each sample. All scans were conducted with a scan area of 10m2, a scan rate of 4.069 Hz, and a z range of 30.0nm. Three spots were chosen at random and an alyzed under doses analyzed (0.05-0.15Mrad). Andrew Gerger assisted r unning all AFM experiments. AFM was also conducted on dried films under contact mode in order to analyze the difference in surface topography of hydrated a nd dry films as well as a direct comparison with SEM data obtained. Films were placed on microscope slides, covered with a piece of Kimwhipe and allowed to dry overni ght in the hood prior to AFM. 2.6 Results and Discussion 2.6.1 Scaffolds Directionality & Porosity For a biomaterial implant to be suitable fo r tissue repair, not only must the material exhibit biocompatibility but it must also cont ain a porous structure and a relatively large surface to volume area, in order to suppor t cellular and sustain tissue ingrowth. The biocompatibility of the HA has been previous ly demonstrated in numerous publications, as well as the freeze drying processes used in this experiment to innavertantly control

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17 direction and size of pores by controlling the rate and temperature water freezes within the matrix (7). Intact scaffolds initially measured appr oximately 4cm tall and were cut into 2mm slices prior to any crosslinking. In the part icular experimental task of verifying the directionality and continuity of pores through SEM, intact sc affold were carefully cut in half longitudinally prior to any imaging. Av erage pores of lyophili zed unmodified and modified (0.15Mrad) samples we re found to have area of 0.100.049mm2 and 0.160.078mm2 respectively when imaged under SEM. Through light microscopy imaging of scaffolds hydrated in PBS the area of unmodified and modified samples were found to be 0.0700.058mm2 and 0.100.058mm2 respectively. Summary of pore circularity and area can be found summarized in Table 1 bellow. Pore area was found to slightly decrease as the materi al was hydrated. This can be at tributed to the swelling of the HA walls as the material swells. Erro r obtained through light microscopy, however, was significantly greater than error obta ined through SEM. These results could be partially attributed to the fact that there s eems to be a larger discrepancy between large and small pores when the material is hydrate d. In addition, two differe nt focus setting per section had to be used in order to image the entire section properly due to the 3D nature of the scaffold, which inadvertently enters some errors when measuring pore diameter. Figure 2-2 A and B are examples of two differe nt focus settings used when imaging an MPC modified scaffold. This dual focusing is needed in order to properly visualize and measure pore diameters.

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18 Table 2-1. Pore area and de gree of circularity of hydrat ed and porous HA scaffolds. Plain HA Scaffold MPC modified (0.15Mrad) Dehydrated Hydrated Dehydrated Hydrated Porea Area (mm2) 0.100.049 0.0700.058 0.160.078 0.100.058 Deg. Circ 0.300.12 0.360.19 0.300.13 0.340.18 Transverse sections, imaged solely thr ough SEM, confirmed dire ctionality of the porous material to be consistent 0.8-1cm above the bottom of an intact scaffold. Some interconnection is seen through the pores however the directionality is clearly conserved. Gamma irradiation shows no difference in s caffold directionality when modified transverse sections were imaged through SEM. Figure 2-2 C exhibits the directionality observed of an SEM image of a transverse sect ion of an unmodified substrate. Transverse sections were too thick in order to be visu alized hydrated under a light microscope. A. B. Figure 2-2. Microscopy images of HA scaffolds. A)Light microscopy of MPC modified (0.15Mrad) scaffold at 40x zoom at a cer tain focal length. B) Same image as A however using a different focal length. C).SEM image of a transverse crosssection of a HA scaffold

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19 C. Figure 2-2. Continued The majority of pores in the tertiary stru cture assumed an ellipsoid shape. It is hypothesized that perfectly circular pores w ould not have a significant effect, if any effect, on the neurogenerative properties of the material relative to the importance of polymer composition and an observed directiona lity throughout the material. Degree of circularity values ranges from 0 to 1 where 0 represents a straight line and 1 represents a perfect circle. The average circularity of the unmodified and modified sample through SEM was determined to be 0.300.12 and 0.300.13 respectively. When pores were fully hydrated, they assumed a sligh tly more circular shape with a degree of circularity of 0.360.19 and 0.340.18 respectively. The relatively larg e standard deviation is due to the fact that there was a somewhat significan t range of pore shape however, the majority of pores were clearly ellipsoid in shape. Similarity in pore structure between MPC modified and unmodified subs trates can be seen in the comparative SEM images in Figure 2-8.

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20 2.6.2 2-Methacryloyloxyethyl Phosphorylcho line (MPC) Modified Compositions A main objective of this experiment was to determine the physical properties that differentiates between the different doses of gamma irradiated MPC scaffolds and unmodified samples. In order to determin e if MPC modifications on a HA is feasible electron dispersive spectroscopy (EDS) was pr imarily employed in order to verify the presence of MPC. The elemental compositi on of a HA scaffold consists of carbon, oxygen, hydrogen and the crosslinking agent chromium. MPC elemental composition differs from HA only in that it cont ains phosphorous; which has a distinct K value. An accelerating voltage of 10KeV was used in order to visualiz e the chromium K line. Uncoated samples were used in order to e liminate interference of the gold-palladium coating. EDS spectra seen in Figure 2-3 clea rly demonstrates the MPC modified sample (total dose = 0.10Mrad) contai ning a phosphorous peak which was not initially present in an unmodified sample. Figure 2.3. EDS spectra of unmodified crosslinked HA (top) and MPC modified (0.10Mrad) scaffold slices (bottom).

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21 Figure 2-3. Continued HA is a very hydrophilic polymer that is known to absorb and retain water efficiently. It has been re ported that 1g of uncrosslinked HA has the capacity of absorbing up to 6kg of water. Since the HA scaffolds are to be placed in an aqueous environment of the spinal cord, the equilibrium water content of the material is crucial in predicting the swelling propertie s of the scaffold in vivo. The equilibrium water content was obtained by immersion of scaffold pieces in PBS at pH=7.4. Since PBS at this pH has a similar salt and hydroxide composition to fluids in the body, it is hypothesized to yield more accurate results relative to nanopure water. Two conditions were used for this study, an MPC modified scaffold with a total irradiation dose of 0.10Mrad and an unmodi fied scaffold. Upon immersion of dried scaffolds slices in PBS, 48 hours was necessary in order for the pieces to become fully hydrated. Figure 2-4 shows the equilibrium wate r content (EWC) values obtained through eight consecutive days of immersion of 5 di fferent scaffolds in the same PBS solution. The average EWC for the unmodified and modified scaffolds was 635% and 576% respectively. This value for EWC wa s computed by taking the average water

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22 content for all days after the second date to ensure scaffold was completely hydrated in the calculations. Equilibrium Water Content MPC Mod No MPC 500 550 600 650 700 750 800 123458 Day% EWC Figure 2-4. Percent equilibrium water conten t for MPC modified (t otal dose = 0.10Mrad) or unmodified scaffolds for five consecutive days and on the eighth day. Although dehydrated SEM images gives us info rmation on pore size a nd distribution, it is not a valid representation of the surface feat ures which will be hydrat ed when in direct contact with animal tissue in vivo. The surface features of hydrated modified and unmodified samples were examined through at omic force microscopy (AFM) in order to access the effect of MPC on surface roughness of various HA films. It was found that surface roughness of hydrated samples gradually increased as the MPC content of the scaffold increased. Figure 2-5 shows 3D AF M images of a typical film surface with different MPC contents. Table 2-1 shows th e numerical values corresponding to the average of all the conditions that were r un for the hydrated and dehydrated samples. Dehydrated samples followed a roughness topogra phy change similar to the one observed for hydrated sample of the modified substrat e, however the unmodified sample had an average roughness significantly larger than any condition of the modified samples. This data indicates that there is a clear change in the rearrang ement of MPC along the surface

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23 when the surface is hydrated. Therefore, one must be skeptical of any conclusions drawn from data obtained solely of dehydrated samples, as is often the case in biomaterial publications. Table 2-2. Atomic force microscopy (AFM) values obtained for average height of surface of modified hyaluronic acid (HA) films. Hydrated Avg Height (nm) Dehydrated Avg Height (nm) No MPC 2.6190.545 No MPC 5.3840.596 0.05Mrad 5.0180.733 0.05Mrad 1.4620.176 0.10Mrad 7.1060.145 0.10Mrad 1.8060.188 0.15Mrad 8.1371.849 0.15Mrad 1.8190.143 Figure 2-5. AFM images taken under liquid cell contact mode of a. unmodified and b. 0.15Mrad MPC modified sample.

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24 Figure.2-5. Continued A quantitative assessment of MPC a dhered to the surface through gamma irradiation can be deduced by inspection of liquid cell AFM and thin film fourier transform infrared spectroscopy (FT-IR) data Absolute quantitativ e measurements are not possible through these methods due to th e lack of known standa rds however relative quantitative results yielded useful informati on. If average roughness is correlated directly with MPC content of the material, the plot shown in Figure 2-6 bellow shows an initial constant increase of MPC adhered to the surf ace of the material. At the highest radiation dose, the materials surface is beginning to get saturated with MPC and the rate of MPC adhesion begins to decrease sl ightly. FT-IR data provides us not only with the qualitative confirmation of the presence of MPC but it also gives us some insight as to the amount of MPC adhered to the films surface. The FT-IR shows peaks for P=O, P-O-CH2, and N+(CH3)3 at 1240cm-1, 1075cm-1 and 970cm-1 respectively for all MPC modified samples.

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25 These peaks are not present in unmodified f ilms of HA. Figure 2-7 shows superimposed spectra lines for all four e xperimental conditions ran in FT-IR from a range of 2000 to 800cm-1. Similar to the AFM data, the increase of MPC is observed as the transmittance peaks get stronger however the strongest abso rbance line (green) corr esponded to a total dose of 0.10Mrad. This could be due to varyi ng thickness between films, or microscopic scratches on the surface that may deflect the IR beam. Average Roughness0 2 4 6 8 10 00.050.10.15 Total Radiation Dose (Mrad)Avg Roughness (nm) Figure 2-6. Plot of average roughne ss versus total radiation dose Figure 2-7. Fourier transform-infrared spect ra of percent transmittance through a range of 2000-800cm-1 for unmodified samples, 0.05Mrad, 0.10Mrad and 0.15Mrad total doses corresponding to the brown, re d, green, and blue lin es respectively.

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26 Scanning electron microscopy (SEM) images al so yielded interesting visual cues of the MPC adhered to the surface. By visu al inspection the MPC modified surfaces exhibited a less uniform and rough topography that more closely resembles materials which favor cellular adhesion. The polymerized MPC on the surface of a 0.15Mrad irradiated scaffold can be clearly seen when compared with an unm odified scaffold piece, shown in Figure 2-8. These same features are not visible when observed under SEM. Images taken at higher magnification, shown in Figure 2-9, also allowed for visual inspection of the MPC adhered to the surface of the material. One can deduce from the visual inspection of the surface topography of the MPC modified substrates that the material is suitable for cellular adhesion. An important note to keep in mind is the fact that SEM images are taken of dehydrated samp les that do not clearly exhibit the tertiary conformation of the material when hydrate d. Ideally, environmental SEM images should to be taken in order to properly access the configuration of the MPC on the surface of the material. Figure 2-8. SEM images of scaffold pieces unm odified (left) and modified with a total dose of 0.15Mrad (right) samples exhibiti ng clearly the MPC adhered onto its surface. \

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27 Figure 2-9. SEM images at high magnification of scaffold pieces of unmodified (left) and modified with a total dose of 0.15M rad (right) exhibiting MPC adhesion.

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28 CHAPTER 3 IN VITRO AND IN VI VO EVALUATIONS OF HYALURONIC ACID SCAFFOLDS AS OEC CARRIERS FOR TREATMENT INJURED NEURONAL SPINAL CORD TISSUE 3.1 Specific Aim The purpose of this chapter is to explore the casting of hyaluronic acid (HA) films and the 2-methacryloyloxyethyl phosphorylchol ine (MPC) modification of such films in what is considered to be a st erile environment. These films we re then used to determine if olfactory ensheathing cells (OEC) will adhere to and grow on HA films. It is hypothesized that since the film is of the same composition as the scaffold, the cells would demonstrate similar adhesion properties on scaffolds prior to implantation. Films were soaked in a solution of MPC and gamma irradiated with doses varying from 0.5 to 1.5Mrads. Cells were allowed to grow for four da ys prior to analysis of vital staining with trypan blue and cell adhesion experimentation. In order to first determine the stabilit y of the polymer in vivo and if adverse inflammatory responses are present, implants were sectioned in a sterile environment and implanted into a rat spinal cord. The stability of the cell-biopolymer construct, as well as the differing regeneration effect s, in vivo is also of interest To address these issues, left sided hemisections were made at the cervical C2/C3 level in the adult rat cord. Implant compositions were placed immediately followi ng acute injury. Animals were allowed to recover for 1 post-implantation. Tissue anal ysis was conducted by plastic embedding in Epon.

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29 3.2 Thin Film Synthesis The same 1% HA solution described in chapter 2, which was used for thin film characterization, was utilized for in vitro st udies. Individual wells were mechanically detached from a 12 well cell culture flask a nd washed with 95% et hanol prior to any work. Two milliliters of the HA solution was then injected into each well and each was gently tapped in order to ensure an even di stribution of the soluti on. The wells were then placed in a large Petri dish and covered w ith a piece of Kimwipe in order to prevent contamination. The dish was placed in a bi ological hood and films were allowed to dry overnight. Once dry, 2mL of 0.0025M CrK(SO4)2 was injected to each well and allowed again to rest in the hood covered with a pi ece of Kimwipe for 3 days. Dried films were then washed inside the wells repeatedly for two days. Wells that were not going to be furthe r modified were washed for two more consecutive days and placed in 4oC for storage. Three milliliters of 10% MPC solution were injected into the wells designated fo r MPC modification. The wells were covered with Parafilm, backfilled with Argon, and then covered with two more pieces of Kimwhipes to ensure no gas exchange was o ccurring prior to modifi cations. Twenty four to forty eight hours was allowed to pass prio r to gamma irradiation of each well for a total dose of 0.5, 1.0 and 1.5Mrad. MPC solutions were then discarded and HA films were again washed inside wells with ultra pure water continuously for 3 days. Films were stored at 4oC fully hydrated in water until st erilization with EtO gas. 3.3 In Vitro Evaluations 3.3.1 Cell Cultures The immortalized OEC cell line utilized was created by Dr. Jacobberger (54) and donated by Dr. Kathy Jones. Lisa Tanzer, a cu rrent member of Dr. Jones lab, generously

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30 prepped cells for shipment, provided tissue culture protocols, and was available for generalized questions in handling thes e cells. Cells arrived frozen at -70oC and remained in -70oC until further use. One vial was thawed a nd utilized for all in vitro and in vivo experiments. Cell viability and attachme nt of each experimental condition was determined through vital st aining with trypan blue. 3.3.2 Cell Culture Reagents All materials were obtained from Sigma-Aldrich. All filterware was obtained through Fisher Scientific. Gr owing media was utilized to multiply OECs upon thawing and splitting. The growth media consisted of 500mL of Dulbeccos Modified Eagles Medium (DMEM) containing high glucose sodium pyruvate, 5mL of a penicillinstreptomycin mixture, 5mL of glutamine-gent amicin, and 50mL of heat inactivated Fetal Bovine Serum (FBS). Solution was filtered through a 0.22m filter into a previously autoclaved screw cap 500mL glass bottle. FB S was added afterwards in order to avoid filtration of essential proteins. Experimental media was used for all in vitro and in vivo experiments and was prepared exactly as described above howe ver FBS addition was omitted. Cell detachment was accomplished by utilizing a 0.25% tryspin-EDTA solution to wash cells of T75 cell culture flasks. Grow ing media was used to inhibit the trypsin in the processing of splitting and collecting cells for experiments. Freezing media was prepared by adding 1mL of 10% cell culture grade DMSO a nd 3mL of FBS to 7mL of DMEM. 3.4 Scaffold Cell Seeding Prepared scaffolds were stored in the refrigerator at 4oC. Prior to any seeding or implantation 2m scaffold pieces were suspe nded in a 50mL centrifuge tube containing ultrapure water (R =17.4Mohm) and autoclave d. Sealed tubes were then placed in the

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31 refrigerator until further use. Inside a biol ogical hood sterile implants were trimmed to approximately 1mm3 pieces and placed insi de a well of a 96 well plate. OECs were trypsanized and washed with FBS-free DMEM three times prior to use. Cells were diluted to a final concentration of 2*106cells/mL and viability was determined by vital staining with trypan blue. 150L of the OE C suspension was pipetted onto sterile implants within the well and placed on a bi -directional rotator for approximately 1 hour (8% CO2 & 37oC) prior to implantation. Tissue culture flask was removed from incubator and taken to surgery immediately prior to the implants need. 3.5 In Vivo Trial 3.5.1 Surgical Procedure An adult female Sprague Dawley rat weighing approximately 225g was used as the subject of this study. Careful attention was made to ensure that proper handling and sterile techniques were utili zed at all times prior, duri ng, and post surgery. 0.15mL of Xylazine was administered subcutaneously in order for the rats to become sedated. The rats weight was then obtained once sedated (approximately 5-15 minutes), in order to determine the correct Ketamine dose to be administered intraparitoneally. Animal clippers were used to shave from the top of the head to T2 and ophthalmic ointment was applied to each eye. Rat was placed supine on a wooden block under the microscope and rats level of sedation was checked before any incisions we re performed. A #15 scalpel blade was used to perform a skin incision at the base of the skull running 5cm caudally along the midline. Surgical scissors were used to cut away the fi rst three layers of muscle from C1 to C5 and forceps were used to separate the final layer, exposing the vertebral process over C2 and the surrounding tissue. Curved Dumont was us ed to hold the dura which was cut with a

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32 #11 blade. A 1-mm-long left-sided hemisection was made in the cranial segment of C2, and the section was aspirated with a fine-ti pped glass pipette. The dura was sutured with Ethicon 10-0 and muscle was then sutured using a Vicryl 4-0 and a cutting needle. Finally skin staples were used to close th e wound. Rat was moved to heated water pad on post-op table and 0.2mL Yobine, 0.4mL Bupinex and 6mL of warmed Lactated Ringers were administered subcutaneously. Rat ear was wiped with alcohol soaked gauze and appropriate ear holes were punched out. Surgery was performed by Kevin Siegel. 3.5.2 Surgical Implantation Sterile implants were sized under an opera ting microscope to a ~1mm diameter and carefully inserted into the cardotomy. The data analyzed was from the trail animal with an unmodified HA implant containing no cells a nd the animal was allowed to survive for a week. Implants to be placed with cells were sized and soaked in FBS free DMEM for two days prior to trimming and cell seeding. Implants were placed in spinal cord by Dr. Paul J Reier and Todd White. Once proof of principle was achieved four MPC modified HA scaffolds were implanted through the sa me surgery, however, as mentioned above, that data was not anal yzed in this study. 3.6 Postmortem Evaluations 3.6.1 Perfusions and Tissue Storage All perfusions were performed under the fume hood utilizing a Millipore mechanical perfusion pump. Rat was anesthe tized with 0.3-0.5mL of Beuthanasia D and her weight was logged. If rat had no response to a pinch reflex pe rfusion was begun. If rat continued to respond after a pproximately 10minutes, about 0.1mL of Beuthanasia-D was administered. Each rat was perfused with 250mL of saline followed by 500mL of fixative solution made with 4% paraformaldehyde and 3.5% glutalaldehyde in 0.1M phosphate

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33 buffer. Following perfusion the entire spin al cord was removed the following day and stored at 4oC in a fresh batch of the same fixativ e solution as used in the perfusion process. 3.6.2 Plastic Embedding of Tissue Lesion area of the cord, running approxima tely 5mm in length, was removed under Zeiss dissecting microscope along with 2mm se ctions caudal and raustral to the lesion site. Each section was then subject to plastic embedding in hard Epon. Tissue sections were first washed with saline solution and we re stored in 2% Osmium tetrachloride at 4oC for 2 -4 hours depending on section size. Ti ssues were then washed with 0.8% NaCl and 8% glucose solutions, with distilled wa ter, and then dehydrat ed in progressively increasing concentrations of ethanol. Sections were then washed with propylene oxide and left overnight in a mixture of 50% propylene oxide and 50% Epon. The following day tissue was placed in pure Epon, baked at 60oCfor 15 minutes and allowed to cool to room temperature. Tissue sections were th en each placed in a plastic embedding mold containing Epon ensuring that the raustral to caudal directiona lity of the spinal cord ran perpendicular to the benchs surface. Tissue was then baked overnight at 60oC and ready for further processing the following day, once cooled to room temperature. All sections were trimmed down and slic es cut using a LKB Nova Ultratome. 24 slices (2 slides) measuring 2m thick were consecutivel y sectioned every 100m through the entire lesion site. Sections were visualized from 1.25-40x their original size using a Zeiss Axioplan microscope. Images were obtained with the KS400 Image Analysis System (Zeiss, KONTRON) utilizing a Ha mamatsu Color Chilled 3CCD camera.

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34 3.5 Results and Discussion 3.5.1 In Vitro Thin Film Evaluations Preliminary in Vitro studies involving the seedin g of OEC surface modified HA substrates with varying degrees of MP C via gamma irradiation were conducted. Inspection through an inverted light micros cope yielded results which indicate MPC surfaces exhibit favorable growth and adhesion properties. Superior growth and adhesion properties were found on control samples of OEC growing on Petri dish however this result is to be expected since cells most co mmonly grow preferenti ally in tissue culture plates. When looking at the MPC modified sa mples relative to each other, however, a gradually increasing number of OECs a dhered to HA surface was found as the MPC content of the films were increased. Thes e results support the hypothesis stating MPC modified surfaces would be preferential to OEC attachment and growth relative to unmodified substrates. 3.5.2 Histological Analysis Hyaluronic acid implants were apparent by gross anatomical inspection following spinal cord dissection. Analysis of the sl ices through light micr oscopy indicated the implant did not provoke an inflammatory res ponse and has proven to be biocompatible in vivo. An example of a slice image at 2.5x is seen in Figure 3.1. It was also apparent that the scaffold had degraded with some time, indicating the entire material would degrade with time. No infiltration of tissue was seen and a rather large air bubble was trapped within the lesion area during the embedding process, which introduced some error. Proof of feasibility is seen and a different study now in place is analyzi ng the effects of OEC alone, OEC seeded onto a MPC modified impl ant and modified implant alone implanted into a rat spinal cord.

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35 Figure 3-1. Optical image at lesion site of hyaluronic acid scaffold implant at 2.5x

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36 CHAPTER 4 CONCLUSIONS The lack of neurological r ecovery in spinal cord in jury (SCI) following current attempts has enticed research which yielde d advances in the u nderstanding of the developing CNS and associated pathologies. Cu rrent repair strategies have emphasized fetal tissue and specific cell transplantation, as well as a variety of sp ecific biochemicals. The more knowledge that is gained by SCI research, the more apparent is the multifaceted nature of the problem. This being so, a combination of interventions would have to be utilized to reverse th e variety of issues encountered. Biomaterials offer the possibility to deal with more than one of the associated problems in SCI simultaneously. The surface ca n provide physical/structural support, a favorable surface terrain, an architecture which offers directionality to ingrowing neurons and also act as support for cells which offer a variety of neuroregenerative advantages. The basic concept was to use neural cell-b iopolymer compositions to help control the dynamic and complex repair process following SCI. Novel anisotropic, water insoluble hyaluron ic acid (HA) scaffolds were engineered. The architecture of the linear pores of the HA scaffold is also unique. Although polysaccharides have been surface modified with MPC, this is the first time specifically HA has been gamma irradiated 2-methacryl oyloxyethyl phosphorylcholine (MPC) onto its surface. Results of studies to date with our polysaccharide, phospholipid, and polynucleotide composite implants suggest th at optimization of compositions, implant

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37 design, and surgical procedures may lead to a clinically important medical technology for spinal cord repair. In vitro studies show ed that olfactory en sheathing cells (OEC) preferentially adhered and grew on the polysty rene cell culture well superiorly to any HA film condition. Within the HA films, however, the MPC modified films showed superior adhesion and growth relative to the unmodifi ed films. The film modified with 0.15Mrad had the most healthy cells growing on its su rface. The In vivo trial showed proof of principle and the feasibi lity of such strategy. The accumulated data indicate the strate gy of using neural OEC-HA compositions can address a number of issues involved in SCI and warrants further investigation. Ideal compositions have not been id entified as of yet, and may include th e incorporation of other multiple cell types and other therapeutic agents to existing or novel implants. Also, there are important broader imp lications of results stemming fr om this research including the application of similar implant compositi ons for the treatment of peripheral nerve damage and optic nerve disease and trauma, as well as macular degeneration and retinal disease.

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38 CHAPTER 5 FUTURE DIRECTIONS While working on this research a series of questions arose that could be experimentally answered thr ough future research. Bellow are some examples of potential experimentation that would yield in in teresting and applicable information. 1. Environmental Scanning Electron Mi croscopy (ESEM) experiments on hydrated scaffolds The tertiary structure of the scaffold implants investigated through scanning electron microscopy (SEM) was conducted on dehydrated samples. If one wants to examine the hydrated microstructure of the 3D scaffold, which resembles more closely the hydrated implant that would be placed in an injured spinal cord, then the structure must be examined hydrated. The only way to achieve such goal would be through ESEM. There would also be the possibility of visu alizing the olfactory ensheathing cells (OEC) directly onto the scaffolds, instead of cast films which have a different geometry. 2. In Vitro and In Vivo evaluations of hyal uronic acid (HA) implants crosslinked with Gadolinium. Through my research I conducted trial studies with Gd3+ crosslinked scaffolds and films and determined it is feasible although re latively more short lived than chromium cations. Recent literature ha s reported gadolinium to be a more biocompatible ion than chromium. It would be interesting to analy ze the effects of cell se eding onto films and implanted scaffolds of HA that have been crosslinked with Gd3+ relative to Cr3+ to see if there are advantages associated with such.

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39 3. Seeding multiple cell types (Schwann ce lls + OEC) onto a single implant. OEC cells have been shown to facilitate axonal elongation at gr aft-host interfaces more effectively than Schwann cells however Schwann cells promoted axonal regeneration/sparing and remyelination better than OECs. Never has there been a study which evaluated more than one cell type seeded onto a biomaterial substrate simultaneously and implanted into the spinal cord It is expected that the seeding of these two cell types together would result in increas ed regeneration, remyelination and axonal elongation more than only one cell type. 4. Hyaluronic acid scaffolds covalentl y crosslinked after being ionically crosslinked in order to increase biod egradation time of implant. By following the methods described in chapter 2 it is possible to make anisotropic insoluble HA scaffolds when crosslinked with Cr3+. The uncrosslinked HA scaffold dissolves in solution prior to crosslinking using the same technique with covalent crosslinking such as divinyl sulfone (DVS) or a carbodiamide. Covalently crosslinking HA scaffolds would most likely results in sc affolds with a longer in vivo degradation time. A possible investigation would be th e crosslinking with DVS and a carbodiamide, among other known covalent crosslinkers of HA, after crosslinking with Cr3+. These covalent crosslinkers use HA s hydroxyl groups which are ava ilable for reacting since the carboxylic acid groups is what is in volved in the ionic crosslinking.

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40 LIST OF REFERENCES 1. Bracken MB, Shepard MJ, Holford TR, Leo-Summers L, Aldrich EF, Fazl M. Methylprednisolone or tirilazad mesylate administration after acute spinal cord injury. 1-year follow up. Results of the th ird national acute spinal cord injury randomized controlled trial. J Neurosurg 1998;89(5):699 2. Rabchevsky AG, Fugaccia I, Sullivan PG, Blades DA, Scheff SW. Efficacy of methylprednisolone therapy for the inju red rat spinal cord. J. Neurosci Res 2002;68:7 3. Ramer LM, Ramer MS, Steeves JD. Setting the stage for functional repair of spinal cord injuries: a cas t of thousands. Spinal Cord 2005;43:131 4. Hausmann ON. Post-traumatic inflammation following spinal cord injury. Spinal Cord 2003;41:369 5. Bunge MB. Bridging areas of injury in the spinal cod. Neur oscientist 2001;7:325 339 6. Madihally SV, Matthew HW, Porous chito san scaffolds for tissue engineering. Biomaterials 1999;20:1133 7. Hadlock T, Sundback C, Koka R, Hunter D, Cheney M, Vacanti J. A novel biodegradable polymer c onduit delivers neurotrophins and promotes nerve regeneration. Laryngos cope 1999;109(9):1412 8. Madihaly SV, Matthew HW. Porous chito san scaffolds for tissue engineering. Biomaterials 1993;24(3): 481 9. Murphy WL, Peters MC, Kohn DH, Mooney DJ. Sustained release of vascular endothelial growth factor fr om mineralized poly(lactideco-glycolide) scaffolds for tissue engineering. Biom aterials 2000;21:2521 10. Stokols S, Tuszynski MH. The fabrication a nd characterization of linearly oriented nerve guidance scaffolds for spinal co rd injury. Biomat erials 2004;25(27):5839 11. Burdick JA, Ward M, Liang E, Young MJ, Langer R. Stimulation of neurite outgrowth by neurotrophins delivered fr om degradable hydrogels. Biomaterials 2006;27(3):452

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41 12. Jimenez-Hamann MC, Tsai EC, Tator CH, S hoichet MS. Novel intrathecal delivery system for treatment of spinal cord injury. Exp Neurol 2003;182:300 13. Kapur TA, Shoichet MS. Immobilized con centration gradients of nerve growth factor guide neurite outgrowt h. J Biomed Mater Res 2004;68A:235. 14. Newman KD, McBurney MW. Poly (d,l lact ic-co-glycolic acid) microspheres as biodegradable microcarriers for pluri potent stem cells Biomaterials 2004;25:5763 71 15. Teng YD, Lavik EB, Qu X, Park KI, Oure dnik J. Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded with neural stem cells. Proc Na tl Acad Sci 2002;99(5):3024 16. Stopek, JB (2003). Biopolymer-microglia cell compositions for neural tissue repair. Unpublished doctorate thesis, University of Florida, Gainesville, Florida, USA. 2003 17. Hurtado A, Moon LDF, Maquet V. Poly (d,l-lactic acid) macroporous guidance scaffolds seeded with Schwann cells geneti cally modified to secrete a bi-functional neurotrophin implanted in the completely transected adult rat thoracic spinal cord Biomaterials 2006;27(3):430 18. Imaizumi T, Lankford KL, Kocsis JD. Tr ansplantation of olfactory ensheathing cells or Schwann cells rest ores rapid and secure conduc tion across the transected spinal cord. Brain Res 2000;854:70 19. Imaizumi T, Lankford KL, Burton WV, F odor WL, Kocsis JD. Xenotransplantation of transgenic pig olfactory ensheathing cells promotes axonal regeneration in rat spinal cord. Nat Biotechnol 2000;18:949 20. OEC Transplantation. [Internet] Laurance J. (China); [updated 2004 April 11; cited 2006 July 15] Available from: http://www.healingtherapies.info/OEC-Huang.htm 21. Reier RJ. Cellular Transplantation Strategies for Spinal Cord Injury and Translational Neurobiol ogy NeuroRx 2004;1:424 22. Goraltchouk A, Scanga V, Morshead CM, Shoichet MS. Incorporation of proteineluting microspheres into biodegradable nerve guidance channels for controlled release. J Control Release 2006;110(2):400 23. Jain A, Kim YT, McKeon RJ, Bellamkonda RV. In situ gelling hydrogels for conformal repair of spinal cord defects, and local delivery of BDNF after spinal cord injury. Biomat erials 2006;27(3):497 24. Whittlesey KJ, Shea LD. Delivery systems fo r small molecule drugs, proteins, and DNA: the neuroscience/biomaterial interface. Exp Neurol 2004;190(1)

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42 25. Novikova LN, Novikov LN, Kellerth JO. Biopolymer and biodegradable smart implants for tissue regeneration after sp inal cord injury. Curr Opin Neurol. 2003;16(6):711 26. Segura T, Anderson BC, Chung PH, Webber RE, Shull KR, Shea LD. Crosslinked hyaluronic acid hydrogels: a strategy to functionalize and pattern. Biomaterials 2005;26:359 27. Entwistle J, Hall CL, Turley EA. HA receptors: Regulators of signaling to cytoskeleton. J Cell Biochem 1998;61(4):569 28. Rapport MM, Weismann B, Linker A, Me yer K. Isolation of crystalline disaccharide, hyalobiuronic acid, from hyaluronic acid. Nature 1951;168(4284):996 29. Meyer K, Palmer JW. The polysaccharide of the vitreous humor. J. Biol. Chem. 1934;107(3):629 30. McDonald JA. Extracellular matrix assembly. Annu. Rev. Cell Biol. 1988;4:183 207 31. Laurent TC, Fraser JRE. Hyal uronan. FASEB J. 1992;6(7):2397 32. Hay ED. Cell Biology of the Extracellular Matrix; Plenum Press: New York, 1991. 33. Wang KK, Nemeth IR, Seckel BR, Chakalis-Haley DP, Swann DA, Kuo JW, Bryan DJ, Cetrulo CL Jr. Hy aluronic acid enhances peri pheral nerve regeneration in vivo. Microsurgery. 1998;18(4):270 34. Laurent TC. The biology of hyalur onan: Introduction. Ciba Found Symp 1989;143:1 35. Bignami A, Asher R. Some observations on the localization of hyaluronic acid in adult, newborn and embryonal rat br ain. Int J Dev Neurosci 1992;10:45 36. Eggli PS, Lucocq J, Ott P, Graber W, va n der Zypen E. Ultras tructural localization of hyaluronan in myelin sheaths of the rat central and rat and human peripheral nervous systems using hyaluronan-bindi ng protein-gold and link protein-gold. Neuroscience 1992;48:737 37. Struve J, Maher PC, Li Y, Kinnery S, Fehlings MG, Kuntz CIV, Sherman LS. Disruption of hyaluonan-based extracellula r matrix in spinal cord promotes astrocyte proliferation. Glia 2005;52(1):16 38. Lapcik L Jr, Lapcik L. Hyaluronan: Pr eparation, Structure, Properties and Applications. Chemical Reviews 1998;98(8):2663

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43 39. Iwasaki Y, Ijuin M, Mikami A, Nakabayash i N, Ishihara K. Behavior of blood cells in contact with water soluble phospholip id polymer. J. Biomed. Mater. Res 1999;46:360. 40. Iwasaki Y, Sawada S, Nakabayashi N, Khang G, Lee H B, Ishihara K. The effect of the chemical structure of the phospholipid polymer on fibronectin adsorption and fibroblast adhesion on the gradient phospholipid surface. Biomaterials 1999;20:2185 41. Zhang SF, Rolfe P, Wright G, Lian W, M illing AJ, Tanaka S, Ishihara K. Physical and biological properties of compound membranes incorp orating a copolymer with a phosphorycholine head group. Biomaterials. 1998;19:691 42. Huang Ionically crosslinked carboxyl-c ontaining polysacchar ides for adhesion prevention. U.S Patent #5,532,221. July 2nd, 1996. 43. Associates of Cape Cod Inc. [Internet]. Bulk reagents: sodium hyaluronate. [cited 2006 July 17]. Available from http://www.acciusa.com/bio/sodium_hyaluronate.html 44. Takacs E, Emmi SS, Wojnarovits L. Radiat. Phys. Chem. 1999;55:621 45. Biro A, Takacs E, Wojnarovits L. Macromol. Rapid Commun. 1996;17:353 46. Takacs E, Emmi SS, Wojnarovits L. Nucl. Inst. And Meth. In Phys. Res 1999;151:346 47. Ishihara K, Aragaki R, Ueda T, Watanabe A, Nakabayashi N. Reduced thrombogenicity of polymers having phos pholipid polar group. J Biomed Mater Res 1990;24:1069. 48. Ishihara K, Oshida H, Ueda T, E ndo Y, Watanabe A, Nakabayashi N. Hemocompatibility of human whole bl ood on polymers with a phospholipid polar group and its mechanism, J Biomed Mater Res 1992;26(12):1543 49. Iwasaki S, Sawada K, Ishihara G, Khang L, Lee H.B. Reduction of surface-induced inflammatory reaction on PLGA/MPC pol ymer blend, Biomaterials 2002;23:3897 3903. 50. Sawada S, Sakaki S, Iwasaki Y, Nakabaya shi N, Ishihara K. Suppression of the inflammatory response from adherent cells on phospholipid polymers. J Biomed Mater Res 2003;64:411. 51. Moro T, Takatori Y, Ishihara K, Konno T, Takigawa Y, Matsushita T. Surface grafting of artificial joints with biocompatible polymer for preventing periprosthetic osteolysis Nat Mater 2004;3:829.

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44 52. Watanabe J, Ishihara K. Change in cell adhesion property on cytocompatible interface using phospholipid polymer grafted with poly(d,l-lactic acid) segment for tissue engineering. Sci Technol Adv Mater 2003;4:539. 53. Watanabe J, Ishihara K. Cell engieering biointerface focusing on cytocompatibility using phospholipid polymer with an is omeric oligo (lactic acid) segment, Biomacromolecules 2005;6:1797 54. Goodman MN, Silver J, Jacobberger JW. 1993. Establishment and neurite outgrowth properties of neonatal and adult ra t olfactory bulb glia l cell lines. Brain Res 619:199

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45 BIOGRAPHICAL SKETCH Jennifer de Toledo Mallek was born on Ma rch 13, 1982 in Sao Paulo, Brazil. She moved to the Boca Raton in 1998 and complete d her last two years of high school at Boca Raton Community High. During these year s, she discovered a passion for sports, science and understanding the human body. She obtained her Bachlor of Science in chemistry within three years at the University of Florida and was admitted to UFs graduate program in 2003. During these years she found a passion for medicine and a desire to teach. She earned dual masters degree in 2006: a Master of Sc ience, with a thesis focusing on biomaterial implants for application in spinal cord injury; and a Mast er of Science in teach ing, with an emphasis of overcoming misconceptions in in troductory chemistry classes.


Permanent Link: http://ufdc.ufl.edu/UFE0015880/00001

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Title: Hyaluronic Acid-Olfactory Ensheathing Cell Compositions for Spinal Cord Injury Nerve Regeneration
Physical Description: Mixed Material
Copyright Date: 2008

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Holding Location: University of Florida
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Permanent Link: http://ufdc.ufl.edu/UFE0015880/00001

Material Information

Title: Hyaluronic Acid-Olfactory Ensheathing Cell Compositions for Spinal Cord Injury Nerve Regeneration
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0015880:00001


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HYALURONIC ACID-OLFACOTRY ENSHEATHING CELL COMPOSITIONS FOR
SPINAL CORD INJURY NERVE REGENERATION
















By

JENNIFER DE TOLEDO MALLEK


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

Jennifer de Toledo Mallek

































This document is dedicated to my cat, Angel.















ACKNOWLEDGMENTS

First, I would like to thank my parents, Howard and Mara Mallek, for showing to

me the true value of education and supporting me throughout my academic career. I

would like to thank my advisor and committee chair, Dr. Eugene P. Goldberg, for his

knowledge, support, help, and advice. I am grateful to Dr. Paul J. Reier, for providing all

the necessary laboratory tools and knowledge in neuroscience to complete my work. I

would also like to thank Dr. Kenneth Wagener, for sparking my interest in polymers

through excellent instruction and advice. I would also like to thank Dr. Johnny Bengston,

for his emphasis on individuality and personality development, for offering great

literature and advice, and for his lively attitude towards life and those around him.

I would like to thank Paul Martin, for valuable advice and excellent instruction on

the electron microscope. I would like to thank Jennifer Wrighton, for taking care of all

the "backstage" issues. I would also like to thank Dr. Ishihara for generously donating the

phophorylcoline used for my project, and Dr. Jaccoberger, for allowing us to use his

immortalized olfactory ensheathing cell line. I would especially like to thank Lisa Tanzer

for preparing and shipping cells, and for providing protocols and advice to culture cells.

I would also like to thank Troy Sendler, my partner, for encouraging me, listing to

me, having a great sense of humor, helping me in my path, and cooking some great food

for me on days I had to work late. He has been with me through thick and thin and I

would not be who I am and where I am today without his love and advice. And, finally, I

would like to thank my dog, Buffy, for being woman's best friend.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TA BLE S .................. .......... ............ .... ............. .......... vii

LIST OF FIGU RE S ............. .............. .............. .................... .. ......... .. viii

ABSTRACT ........ .............. ............. ...... ...................... ix

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

1.1 B background .................... ................................ ........ .. ..... .......... 1
1.2 Specific A im s.................. .................................. .... ........ ............... 7
1.2.1 Aim 1: Synthesis and Characterization of Novel Porous 2-
Methacryloyloxyethyl Phosphorylcholine (MPC) Modified Hyaluronic
A cid (H A ) B ased C om positions ........................................ .......................7
1.2.2 Aim 2: In Vitro and In Vivo Evaluations of HA Scaffolds as Olfactory
Ensheathing Cell (OEC) Carriers in the Treatment of Spinal Cord
Injury(SC I) ................................................................ .. ......... 8

2 HYALURONIC ACID SCAFFOLDS FOR TREATMENT OF SPINAL CORD
IN JU R Y ...................................... .................................... ................ .. 9

2.1 Hyaluronic Acid (HA) Solutions ................. ...................................9
2.2 U niaxial Porous Scaffold Synthesis.................................... ........................ 9
2 .3 T h in F ilm Sy nth esis ........... .... .............. ................ .. .... ........... ...... ............1 1
2.4 2-Methacryloyloxyethyl Phosphorylcholine (MPC) Surface Modifications........12
2.4.1 M onom er Solutions ............................................... .......... ............... 12
2.4.2 Gamma-Radiation Polymerization .....................................................12
2.4.3 P ost Irradiation C leaning ......... ................. ................... ..................... 13
2.5 Scaffold Characterization ................................................................ 13
2.5.1 Scanning Electron Microscopy (SEM).............. ........ ...............13
2.5.2 O ptical M icroscopy ............................................................................. 14
2.5.3 Equilibrium W ater Content ......................................... ..........15
2.5.4 Fourier Transform-Infrared Spectroscopy (FT-IR).................................15
2.5.5 Atomic Force Microscopy (AFM).......................................................16
2.6 R results and D discussion ........................................................... ............... 16









2.6.1 Scaffolds D irectionality & Porosity ................................... ............... .16
2.6.2 2-Methacryloyloxyethyl Phosphorylcholine (MPC) Modified
C o m p o sitio n s ............................................... ................ 2 0

3 IN VITRO AND IN VIVO EVALUATIONS OF HYALURONIC ACID
SCAFFOLDS AS OEC CARRIERS FOR TREATMENT INJURED
NEURONAL SPINAL CORD TISSUE.......................................... .....................28

3 .1 S p ecific A im ..................................................... ................ 2 8
3.2 T hin F ilm Synthesis.......... ............................................................... ..... ..... .. 29
3.3 In Vitro Evaluations ....... .......................... ........ ...... ...... .. ........ .... 29
3.3.1 Cell Cultures ............. .. .... ..... ..........29
3.3.2 Cell Culture Reagents ......... ............................................. ............... 30
3 .4 S caffold C ell S eeding ........................................ ............................................30
3.5 In Vivo Trial ...........3...................... ..........31
3.5.1 Surgical Procedure.......... .................................... ...... .. .... .............
3.5.2 Surgical Im plantation ........................................ ........................... 32
3.6 P ostm ortem E valuations ............................................................ .....................32
3.6.1 Perfusions and Tissue Storage......... ............... ...............32
3.6.2 Plastic Em bedding of Tissue ............................ ................................... 33
3.5 R results and D discussion ................................................. ............. ............... 34
3.5.1 In Vitro Thin Film Evaluations........................ .......................... 34
3.5.2 H istological A nalysis............................................ ........... ............... 34

4 CON CLU SION S ..................................... .......... .......... ........... 36

5 FU TU R E D IR E C T IO N S ........................................ .............................................38

L IST O F R EFE R E N C E S ............................................................................. ............. 40

B IO G R A PH IC A L SK E TCH ..................................................................... ..................45
















LIST OF TABLES

Table p

2-1 Pore area and degree of circularity of hydrated and porous
hyaluronic acid scaffolds................................................. ............................. 18

2-2 Atomic force microscopy (AFM) values obtained for average
height of surface of modified hyaluronic acid (HA) films................................23
















LIST OF FIGURES


Figure page

1-1 Structure of hyaluronic acid.(H A ).................................... .......................... .......... 4

1-2 Structure of 2-methacryloyloxyethyl phosphorylcholine monomer. .......................

2.1 Freezing apparatus ..... ...... .................................. ........ .... .. ............ 11

2-2 M icroscopy im ages of HA scaffolds ................................................ ............... 18

2.3 Electronic dispersive spectra (EDS) of unmodified crosslinked HA.....................20

2-4 Percent equilibrium water content...................................................................... 22

2-5 Atom ic force m icroscopy im ages.................................... .......................... ......... 23

2-6 Plot of average roughness versus total radiation dose........................................25

2-7 Fourier transform-infrared spectra of percent transmittance...............................25

2-8 Scanning electron microscope (SEM) images of scaffold pieces ..........................26

2-9 SEM images at high magnification of scaffold pieces............... ................27

3-1 Optical image at lesion site of hyaluronic acid scaffold implant at 2.5x .................35















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

HYALURONIC ACID-OLFACTORY ENSHEATHING CELL COMPOSITIONS FOR
SPINAL CORD INJURY NERVE REGENERATION
By

Jennifer de Toledo Mallek

August 2006

Chair: Eugene P. Goldberg
Major Department: Chemistry

There are approximately a quarter of a million people suffering from spinal cord

injury (SCI) today in the United States and there are over 10,000 new cases reported each

year however a clinically effective treatment for traumatic SCI is yet to be developed.

Although recent advances in the neurological research has yielded positive findings on

the regenerative capacity of axons within the central nervous system (CNS), functional

regeneration of the injured spinal cord remains a current challenge, due to its chemical

environment, which is highly non-permissive to growth and regeneration. Our primary

objective of this study was to conduct a tissue-engineering study using composite

olfactory ensheathing cell-hyaluronic acid (OEC-HA) scaffold compositions for the

spinal cord. Anisotropic HA scaffolds were prepared by a freeze-drying method for the

first time. Selected scaffolds ionically crosslinked with trivalent chromium were surface

modified with 2-methacryloyloxyethyl phosphorylcholine (MPC), in order to enhance









biocompatible properties and increase cell attachment and viability on the substrate

material prior to implantation.

In order to verify the reproducibility of the implant material and to maximize its

biocompatibility, chemical and physical properties of the scaffold implants were

characterized by scanning electron microscopy (SEM), electron dispersive spectrometry

(EDS), atomic force microscopy (AFM), fourier transform- infrared spectrometry (FT-

IR), gas permeation chromatography (GPC) and multi-angle light scattering (MALS).

Plastic embedding of tissue in Epon was conducted to visualize the implant in vivo and to

assess stability of the polymer stability and verify the absence of an inflammatory

response and other immune events.

Novel biodegradable micro-uniaxial-mesoporous hyalumonan/phospholipid

scaffold implants were synthesized and implanted into the rat spinal cord following a left

cervical hemisection at C2/C3 in order to assess proof of principle for this method.

OECs' regenerative-promoting capacity has been highly investigated and have been

found to facilitate axonal elongation at graft-host interfaces than Schwann cells; making

them good candidates in SCI applications. Optimal OEC cell attachment and viability

was found in vitro on the HA films which were subject to a total gamma irradiation dose

of 0.15Mrad. Implant treated subjects showed no immunological response to the implant

and showed minimal cystic cavitation and promoted tissue regeneration. Although

implants seeded with cells has been done, the analysis is not covered in this study.














CHAPTER 1
INTRODUCTION

1.1 Background

Today in the United States there are approximately 250,000 persons suffering from

spinal cord injury (SCI) and approximately 11,000 new cases are reported each year (1).

Clinically the only neurologically targeted treatment even considered is the systemic

delivery of methylprednisolone. However, the drug's safety and efficacy has been

recently questioned (2) and results from clinical trials have been openly criticized (3).

Traumatic SCI is most commonly the result of a single displacement or penetration of the

spinal cord which is referred to as primary injury.

The effects of primary injury are significantly enhanced by the destructive

secondary injury events which can extend for days after the initial traumatic event (4).

Secondary injury involves a complex and destructive cascade of pathophysiological

processes involving a variety of vascular changes including edema, eschemia and

hypoxia, cellular responses such as glial cell activation and scar formation, and

biochemical events including nitric oxide release and free radical formation (5).

Immediate scar tissue formation as a normal wound healing response is a major factor in

limiting SCI repair. Strategies to overcome this problem are needed. In this regard, acute

animal models with immediate treatment may not reflect the real clinical setting for more

effective SCI repair. (6).

Biodegradable polymer implant materials have the potential to ameliorate a variety

of the problems associated with SCI. Implants prevent cystic cavitation, and can be









seeded with a variety of drugs to be slowly released as the material degrades which can

either promote growth of injured axons such as NGF and BGNF, or inhibit unwanted

cellular responses such as glial scar formation. These scaffolds can also act as cell

carriers so that the desired cells have a substrate to adhere to. The micro-architecture of

the implant can be engineered for optimal axonal growth and organization through

various methods such as the freeze-dry process (7), particulate-leaching techniques (8),

heat compression and extrusion (9) while providing a physical support for delivery of

various cell types including stem cells (15, 16), microglial cells (17), Schawnn cells (SC)

(18), and potentially others cells such as olfactory ensheathing cells (OEC) which have

shown the ability to restore axonal conduction after injections into the dorsal column

lesions in rats (19, 20) and are being currently used in clinical trials overseas (21).

Another possibility, which has not yet been investigated, is the seeding of multiple

cell types. For example, implanted grafts show that SC are more efficient than OEC in

promoting axonal regeneration/sparing and remyelination of spared axons; however,

OECs seemed to facilitate axonal elongation at graft-host interfaces better than SCs.

These findings suggest that a combination of SC-OEC grafting might be more beneficial

in the contused spinal cord than one involving OECs or SCs alone. OECs, however, have

the unique ability to meld with astrocytes, lacking in SCs and peripheral nerve grafts,

making them ideal candidates for SCI. (22) The implant material may also serve as a

conduit for sustained-release delivery of therapeutic agents such as vascular endothelial

growth factor (VEGF) (10), nerve growth factor (NGF) (11), ciliary-neurotrophic factor

(CNTF) (12) and a variety of other neurotrophic factors (23, 24, 25). The delivery

through biomaterials may provide an attractive alternative to the intrathecal pump, as









delivery is reproducible and immobilized concentration gradients can be created (13, 14).

With these factors taken into consideration, the use of a modified biomaterial implant for

treatment of SCI can significantly aid the patient's recovery by attacking the multiple

issues involving SCI with a single implant.

A material that is a good candidate as a scaffold for SCI should be a biocompatible

polymer which is biodegradable, so that healthy neural tissue can gradually replace the

implant as it degrades, and provides both chemical and physical guidance for

regenerating axons and related cells. Implant compositions of natural polymers such as

alginate, collagen, hyaluronic acid and DNA as well as synthetic polymers such as poly-

(a-hydroxyacids), polyethylene glycol and 2- hydroxyethyl methacrylate have been

previously synthesized and characterized and are shown to have a potential uses as

biomaterials (17, 26). Natural materials such as collagen and hyaluronic acid (HA) are at

an advantage since they convey chemical signals within the structure that can enhance

tissue formation (27). The optimal time for scaffold biodegradation is dependent on the

rate of regeneration of the spinal cord tissue. Ideally, the scaffold should degrade at the

same rate that the neural tissue reconstitutes itself however degradation rate is hard to

control and is difficult to assess in situ (7).

HA is a naturally occurring polymer which was first found in the vitreous humor of

the eye in cattle (30) and later was discovered to be widely distributed in the body,

notably in synovial fluid, extracellular matrix and loose connective tissues such as the

umbilical cord, dermis and rooster combs. (31, 32, 33) HA is also known to interact with

cellular surface receptors including well known CD44, RHAMM and ICAM-1 which are

involved in processes such as morphogenesis, wound repair, inflammation and metastasis










(28). HA is found naturally throughout the spinal cord and is synthesized predominantly

by astrocytes, which contain the RHAMM and CD4 receptors, and are localized around

white matter fibers in the interstitial space between myelin and astrocyte processes. In

spinal gray matter, HA surrounds neuron cell bodies. (36, 37) The role of HA in the

nervous system has not been extensively examined; however, recent studies demonstrate

that high-molecular weight form of HA can inhibit astrocyte proliferation in vitro and

promotes astrocyte quiescence in vivo (38). Uncrosslinked HA injected into a fibrin

matrix nerve guide tube has been shown to enhance peripheral nerve regeneration in vivo

due to the influence on the motility of growing cells by way of its cell surface interactions

with extracellular matrix fibroblasts and by its ability to distort the fibrin matrix into a

more porous microenviroment, which thus facilitates infiltration of cells responsible for

tissue regrowth. (34, 35)



COONa CH20H

O O
OH OH O

OH
NH Ac
n

Figure 1-1. Structure of hyaluronic acid composed of repeating units of d-glucuronic
acid and N-acetyl d-glycosamine (44).

HA is a linear, unbranched high molecular weight, up to 7 million Daltons,

polysaccharide of repeating disaccharide units composed of N-acetyld-glucosamine and

d-glucuronic acid linked by a P 1-4 glycosidic bond as shown in Figure 1-1. The

disaccharides units are linked together by 0 1-3 bonds (28, 29). The polymer backbone

contains carboxyl, acetoamino and alcohol functional groups which are free for chemical









crosslinking or surface modifications of any kind. It has been found through preliminary

experiments that HA can be crosslinked with polyvalent chromium (III) under specified

conditions to form a water insoluble scaffold. It is likely that chromium (III) crosslinkes

HA via electrostatic interactions between the cation and the negatively charged

carboxylic acid groups, leaving the alcohol groups available for further chemical

modifications (43).

It is hypothesized that the probability of achieving functional recovery, by having

growing axons reconnect to the appropriate (original) neurons, is increased by retaining

the native organization of regenerating axons across the lesion site. This is achieved by,

in addition to providing biochemical and/or cellular support, providing physical guidance

for the linear growth of axons across a site of injury. The production of linear guidance

pores, utilizing freeze-drying techniques, extending through the entire length of a scaffold

for use in SCI has been previously reported with the natural polymer agarose; (11)

however, this engineering method has not yet been applied to other natural polymers,

including hyaluronic acid. A freeze-drying technique is of preferred use for pore

formation and manipulation since no additional, potentially toxic, chemicals are required

and the direction of growth and size of ice crystals may be controlled by manipulating the

temperature gradient applied to polymer solution (7). HA has never been previously

reported in the form of an insoluble scaffold with an intentionally defined tertiary

structure.

2-methacryloyloxyethyl phosphorylcholine (MPC), and its copolymers have been

proved to interact mildly with proteins and cells on their surfaces and have show

biocompatibility and hemocompatibility in vivo (17, 48, 49). Polymers used for









cardiovascular applications demonstrated increased endogenous blood phospholipid

adhesion and decreased platelet activation when coated with polyMPC (40, 41, 42). In

addition, MPC copolymers have been shown to be a promising cytocompatible material

for tissue engineering, because the MPC polymer prevents the cells on biomaterials from

inducing an inflammatory response or other foreign body response (50, 51, 52) Also,

cells cultured on an MPC-Alginate copolymer previously in our lab, among other MPC

copolymers, showed high cell viability in vitro ( 17, 53, 54). Since reduced thrombus

formation and chronic inflammation has been correlated with endogenous phospholipid

adsorption it is hypothesized that the surface modification of hyaluronic acid scaffolds

will improve hemocompatibility of the scaffolding material, as well as improving the

cellular proliferation and adhesion onto its surface. The structure of the MPC monomer is

shown in figure 1-2. Results from previous studies demonstrated proof of principle for

the surface modification of polysaccharide compositions with hydrophilic, water soluble,

vinyl monomers using gamma radiation initiation polymerization (17).

,CH3
H2C=C
C=0 0 C ,H3
11 _. C --CNfcH3
CH-CH2. O 2 CHCH3


Figure 1-2. Structure of 2-methacryloyloxyethyl phosphorylcholine monomer.

Ionizing radiation may be used to initiate radical polymerization of MPC at

ambient temperature in the absence of other chemical radical initiators. Residual

chemical from chemical initiations may potentially exhibit cytotoxic and carcinogenic

responses in vivo. Polymerization of aqueous acrylates, methacrylate and acrylamide

monomers have been previously reported to be polymerized through initiation









mechanisms via the radiolysis of water. (45, 46, 47). In addition to the lack of residual

chemicals, the molar mass of the polymer, or extent of surface modifications to be

irradiated onto a substrate, may be controlled by varying the total dose and dose rate of

gamma irradiation a solution is exposed to. Although surface modifications using gamma

grafting have been previously reported in our laboratory (17), little is currently known

concerning surface modification of hydrogel materials by gamma-induced

polymerization. With these factors taken into consideration, gamma radiation initiation

polymerization is the ideal method for HA surface modifications of previously

crosslinked insoluble scaffolds.

1.2 Specific Aims

1.2.1 Aim 1: Synthesis and Characterization of Novel Porous 2-
Methacryloyloxyethyl Phosphorylcholine (MPC) Modified Hyaluronic Acid (HA)
Based Compositions

Porous hyaluronic biopolymer implant compositions were prepared using freeze-

dry/lyophilization and film casting techniques. Implant compositions were surface

modified with the polymeric phospholipid, MPC using gamma radiation initiation

polymerization. Fourier Transform Infrared spectroscopy (FTIR), and energy dispersive

spectroscopy (EDS) were used in order to verify the presence of the MPC substrate on

the modified samples by functional group and elemental analysis, respectively. Scanning

electron microscopy (SEM) was used to visualize porous structure of the material and

analyze surface features of dehydrated films. Gas permeation chromatography (GPC) and

multi-angle light scattering (MALS) were used for molecular weight determinations.

Atomic force microscopy (AFM) was performed on hydrated films in order to determine,

quantitatively, the surface features introduced by varying doses of MPC. For comparison

with both hydrated data and SEM data, AFM was performed on dehydrated film as well.









The effects of gamma radiation and autoclave sterilization on implant stability were also

investigated.

1.2.2 Aim 2: In Vitro and In Vivo Evaluations of HA Scaffolds as Olfactory
Ensheathing Cell (OEC) Carriers in the Treatment of Spinal Cord Injury(SCI)

In order to determine if OECs are able to adhere to and grow on HA scaffolds, In

Vitro studies were conducted on HA films of the same composition as the scaffolds.

Films were soaked in a solution of MPC and gamma irradiated with doses varying from

0.5 to 1.5Mrads. Cells were then placed on unmodified films and films modified with a

gradation of MPC. Adhesion studies and vital staining with trypan blue were conducted

to determine adhesion and viability of cells onto specific substrates.

As an advance toward future tests of therapeutic efficacy, we first wanted to

determine the stability of this polymer in vivo and whether it would elicit an adverse

inflammatory response. The second issue explored was how stable the cell-polymer

construct would be in vivo. To address these issues, left sided hemisections were made at

the cervical C2/C3 level in the adult rat cord. Implant compositions were placed

immediately following injury. Animals were allowed to recover for 1 week post-

implantation. Analysis was conducted by plastic embedding in order to determine

scaffold regenerative effects.














CHAPTER 2
HYALURONIC ACID SCAFFOLDS FOR TREATMENT OF SPINAL CORD INJURY

2.1 Hyaluronic Acid (HA) Solutions

Low molecular weight hyaluronic acid (reported MW=560 KDa) donated by

Genzyme was used in the preparation of all microporous foams and films. 40mg/mL of

HA was dissolved in ultrapure water with a resistivity of approximately 17.4MQ using

LightninTM and Caframo high-speed mechanical mixers and in-house built 3-blade

propellers at a speed of 400 revolutions per minute overnight. 10mg/mL solutions were

prepared with less aggressive magnetic stirrer at the maximum speed setting. The 1%

solutions were used for film casting and the more viscous 4% solutions were used for

foam synthesis. Solutions were filtered into clean 100 ml screw cap glass bottles using a

stainless steel air-pressure apparatus manufactured by Gelman Sciences and 70[lm

SpectraTM filters. Solution concentration (mg/ml) was verified using a Mettler LJ16

Moisture Analyzer. Solutions were stored at 40C until further use. Hyaluronic acid

molecular weight was verified by using a Waters gel permeation chromatography (GPC)

system and a Wyatt Dawn EOS multi-angle light scattering (MALS) detector.

2.2 Uniaxial Porous Scaffold Synthesis

Approximately 4mL of the 4% HA solution was injected into flat-bottom

borosilicate tubes measuring 8mm in diameter and 4.5cm tall. Solutions were allowed to

sit at room temperature covered with a piece of Kimwipe for approximately 45 minutes in

order to allow for gravitational removal of air bubbles trapped within the viscous

solution. If air bubbles still remained, tubes were placed inside 50mL centrifuge tubes









and centrifuged at approximately 1500rpm order to remove residual air bubbles. The

physical setup used for the formation of linear pores by freeze drying was similar to the

one reported by Stokols in 2004 (11).

A piece of Styrofoam (10.85cm x 12.7cm x 4.7cm) which serves as a 15mm poly-

propylene centrifuge test tube holder (x25) from Fisher's centrifuge packaging was used

as the insulating test tube holder in the experiment. Four holes measuring 8mm in

diameter (flush with borosilicate tube) were cut through the Styrofoam into respective

centrifuge tube slots as shown in Figure 2-1. Each HA filled tube was then firmly pressed

into its respective hole so that the bottom of the tube was flush with the bottom of the

Styrofoam piece (both surfaces were in contact with the bench). A piece of dry ice

measuring approximately (15cm x 13cm x 3cm), which contains a flat surface that is

parallel to the ground, was placed inside a small vacuum oven hooked up to a vacuum

pump. The Styrofoam setup containing the HA solutions was then placed on top of the

block of dry ice which is sitting inside the vacuum-oven. The entire setup may be

visualized in Figure 2-1.

Immediately after inserting the sample the vacuum was turned on and adjusted to

500mmHg in order to remove gaseous carbon dioxide that could potentially freeze the

solution laterally. Solutions were left to freeze in for two hours and linear ice crystals

were clearly visible when removed. Scaffolds were then lyophilized overnight, gently

removed from their glass tubes and cut into sections measuring 2mm in using a #10

stainless steel surgical blade. The bottom 1cm and top 2mm of the scaffold was

discarded. Individual foam pieces were then crosslinked for 3 days on rotary tumbler in

solution of 70% ethanol and 30% 0.0025M CrK(S04)2.






















a. b.

Figure 2.1. Freezing apparatus. A) A flat block of dry ice was placed in the Styrofoam
container inside the vacuum oven. The two Styrofoam sample holders were
then placed on top of the dry ice. B) Borosilicate flat bottom test tube inserted
into a hole within the Styrofoam holder (right) next to a vacant spot (left)

2.3 Thin Film Synthesis

The dilute 1% HA solution was used for casting of all films. Two milliliters of the

HA solution was injected into individual wells of a 12 well cell culture flask (Corning

Inc.) and flask was covered with a piece of Kimwipe in order to prevent contamination.

Flask was gently tapped in order to ensure an even distribution of the solution throughout

the bottom of each well. Flask was then left overnight under biological hood in order to

allow for the films to dry. Once dry, 2mL of 0.0025M CrK(SO4)2 was injected to each

well and allowed again to rest in the hood covered with a piece of Kimwipe for 3 days.

Dried films were then peeled off the bottom of the dish using microforceps and washed

with ultrapure water in 50mL centrifuge tubes. Films were then transferred to a new 12

well cell culture flask and stored at 40C until further use or into a single detached wells

from what was originally a 12 well cell culture flask for MPC modifications.









2.4 2-Methacryloyloxyethyl Phosphorylcholine (MPC) Surface Modifications

2.4.1 Monomer Solutions

MPC monomer was generously donated by Dr. K. Ishihara, Department of

Materials Engineering, University of Tokyo (Tokyo, Japan), and stored dry at -20oC.

10% MPC solutions (100 mg/mL) were prepared using nanopure water (resistivity >

17.4MQ) and used for all modifications. Each scaffolding piece subject to modifications

was washed for only one day inside a 50mL centrifuge tubing using nanopure water. The

scaffold was then immersed in 4mL of the MPC solution inside a 15mL borosilicate test

tube, capped, and left on the bench at room temperature for 48 hours. Films were also

washed for one day and placed into single detached wells of a 12 well flask. Two mL of

the MPC solution was pipetted into each well and covered with Parafilm. Films were also

left to soak for 48 hours. Tubes or wells were gently agitated periodically in order to

remove air bubbles. Prior to the gamma-polymerization, solutions inside the test tube and

wells were degassed for approximately 5 minutes and 3 minutes respectively in order to

remove dissolved 02.

2.4.2 Gamma-Radiation Polymerization

Polymerizations were conducted using a Wiscon Type 60Co source reactor. Source

was lowered to 3cm above the reactor floor and dosimetry was conducted prior to initial

experiment to determine target radii. Preliminary studies found no significant effect on

dose rate variability therefore a constant dose rate of 1000rad/min was used for all

modifications. Samples were evenly distributed around source's center and rotated on its

axis 1800 every 0.05Mrad and 1/3 the circumference of the test tube slots in order to

account for dose rate inhomogeneity. The three total doses of exposure were 0.05, 0.10

and 0.15Mrad.









2.4.3 Post Irradiation Cleaning

Once modifications were complete ImL aliquots of each polyMPC solution was

transferred to 2mL vials and stored at 4C for further experimentation in order

determination the molecular weight and radius of gyration of the polymer through GPC

and MALS. The scaffolds and/or films were transferred to 50mL centrifuge tubes and

washed for one week at room temperature using ultrapure water. Once radiation was

complete scaffolds and/or films were stored in the refrigerator until further use.

2.5 Scaffold Characterization

2.5.1 Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) was employed in order to analyze the three

dimensional microstructure of the MPC modified and unmodified samples and to

qualitatively analyze the material's pores. Longitudinal sections of the HA scaffold were

imaged in order to analyze pore distribution, area, and degree of circularity. Transverse

sections were imaged in order to verify the continuity and directionality of the pores. The

major (dmj) and minor (dn) diameter of 30 pores were measured per scaffold piece

utilizing ImajeJ software. Three longitudinal pieces of MPC modified and unmodified

were analyzed along with two transverse cross-sections running the entire length of the

center of the scaffold prior to slicing into 2mm sections (-4cm). The area of each pore

was calculated using the general equation for calculating the area of an ellipse:

A = r dm d,,

General pore structure was found to have an ellipsoid nature therefore the degree of

circularity (C) of each pore measured was also calculated by the following expression:


c dmn
d
M in









Upon completion of the washes, samples were placed in the lyophilizer overnight

in order to ensure sample dryness. Samples were then adhered onto a piece of double

sided carbon tape on an aluminum stub and sputter coated with gold-palladium using the

Technix Hummer V sputter coater. All imaging was conducted using the JOEL SEM-

6400 scanning electron microscope (JOEL, Ltd., Peabody, MA) at an accelerating

voltage of 5KeV, working distance of 15 mm and condenser lens setting of 8.

Electron Dispersive Spectroscopy (EDS) was used in order to verify the presence

of phosphorous, not found on HA, on the surface of a MPC modified scaffold. EDS was

conducted on uncoated sample and all parameters are the same as above except for the

accelerating voltage was increased to 10KeV in order to visualize the K, and Kp lines for

Cr.

2.5.2 Optical Microscopy

Optical microscopy was employed in order to analyze pore distribution and to

measure pore diameters for scaffold pieces hydrated in PBS. A Zeiss Axioplan2 imaging

optical microscope was utilized for all imaging and pictures were captured by a Zeiss

AxioCamHR camera, version 5.05.10, utilizing the AxioVission 3.1 software. The

AxioVission software, in conjunction with ImageJ software, was also used to measure the

major and minor diameters of 10 pores per scaffold piece. Three longitudinal pieces of

MPC modified and unmodified scaffolds were hydrated in PBS for at 48hrs in order to

ensure they were fully hydrated prior to pore diameter measurement. The same equations

used for calculating area of an ellipse and degree of circularity in SEM were used for

hydrated pore analysis.









2.5.3 Equilibrium Water Content

The equilibrium water content of scaffolds (n=5) was measured for both

unmodified and MPC modified with a total radiation dose of 0.10Mrad. Scaffold pieces

were dried overnight in an oven at 45C to ensure sample dryness and weighed

immediately afterwards. Each piece was then placed in a 50mL centrifuge tube filled with

phosphate buffered saline (PBS) at a pH of 7.4. Water content was measured at the same

time each day for five consecutive days, beginning 24 hours after initial immersion, and

on the eighth day. Each scaffold was removed from PBS solution, gently blotted onto a

piece of Kimwipe until no more water was visibly being absorbed by the Kimwipe, and

weighed. Pieces were then immediately returned to it's original PBS solution in the

centrifuge tube. The percent equilibrium water content is equivalent to the percent mass

increase due to water absorption and was calculated using the expression bellow. The wet

and dry masses are represented by Mw and Md respectively.


%EWC M -Md 100
Md

2.5.4 Fourier Transform:Infrared Spectroscopy (FT-IR)

FT-IR was utilized in order to qualitatively verify the presence of MPC on

modified thin films subject to gamma radiation doses of 0.05, 0.10 and 0.15Mrad.

Absorption frequencies characteristic of MPC, which are not present in HA, include P=0,

P-O-CH2, and -N+-(CH3)3 at 1240cm-1, 1075cm-1 and 970cm-1 respectively (17). FT-IR

spectra was collected using a Nicolet Magna 60SC FT-IR spectrometer using Omnic ESP

software. Spectra were scanned 200 times per sample with a spectral resolution of 4cm-1

in the range of 2000cm-1 to 800cm-1. After thin films were placed inside the sealed

sample holder and prior to all spectra collection, 5 minutes was allowed to pass in order









for the majority of CO2 and H20 to be removed from the chamber with AIR PUMP

THINGIE. Prior to film analysis, a background spectra was obtained in order to correct

for the absorption of the remaining CO2 and H20 still present in the chamber.

2.5.5 Atomic Force Microscopy (AFM)

AFM experiments were conducted on a Veeco Dimension 3100 Atomic Force

Microscope under liquid cell contact mode with a silicon nitride tip in order to analyze

the difference in surface topography between hydrated modified and unmodified thin

films. This method of analysis will allow for relative quantitative assessment of MPC on

the film's surface by correlating gradual changes in surface topography with the

increasing dose rate of gamma irradiation experienced by each sample. All scans were

conducted with a scan area of 10lm2, a scan rate of 4.069 Hz, and a z range of 30.0nm.

Three spots were chosen at random and analyzed under doses analyzed (0.05-0.15Mrad).

Andrew Gerger assisted running all AFM experiments.

AFM was also conducted on dried films under contact mode in order to analyze the

difference in surface topography of hydrated and dry films as well as a direct comparison

with SEM data obtained. Films were placed on microscope slides, covered with a piece of

Kimwhipe and allowed to dry overnight in the hood prior to AFM.

2.6 Results and Discussion

2.6.1 Scaffolds Directionality & Porosity

For a biomaterial implant to be suitable for tissue repair, not only must the material

exhibit biocompatibility but it must also contain a porous structure and a relatively large

surface to volume area, in order to support cellular and sustain tissue ingrowth. The

biocompatibility of the HA has been previously demonstrated in numerous publications,

as well as the freeze drying processes used in this experiment to innavertantly control









direction and size of pores by controlling the rate and temperature water freezes within

the matrix (7).

Intact scaffolds initially measured approximately 4cm tall and were cut into 2mm

slices prior to any crosslinking. In the particular experimental task of verifying the

directionality and continuity of pores through SEM, intact scaffold were carefully cut in

half longitudinally prior to any imaging. Average pores of lyophilized unmodified and

modified (0.15Mrad) samples were found to have area of 0.100.049mm2 and

0.160.078mm2 respectively when imaged under SEM. Through light microscopy

imaging of scaffolds hydrated in PBS the area of unmodified and modified samples were

found to be 0.0700.058mm2 and 0.100.058mm2 respectively. Summary of pore

circularity and area can be found summarized in Table 1 bellow. Pore area was found to

slightly decrease as the material was hydrated. This can be attributed to the swelling of

the HA walls as the material swells. Error obtained through light microscopy, however,

was significantly greater than error obtained through SEM. These results could be

partially attributed to the fact that there seems to be a larger discrepancy between large

and small pores when the material is hydrated. In addition, two different focus setting per

section had to be used in order to image the entire section properly due to the 3D nature

of the scaffold, which inadvertently enters some errors when measuring pore diameter.

Figure 2-2 A and B are examples of two different focus settings used when imaging an

MPC modified scaffold. This dual focusing is needed in order to properly visualize and

measure pore diameters.









Table 2-1. Pore area and degree of circularity of hydrated and porous HA scaffolds.
Plain HA Scaffold MPC modified (0.15Mrad)
Dehydrated Hydrated Dehydrated Hydrated
Porea Area (mm2) 0.100.049 0.070+0.058 0.160.078 0.100.058
Deg. Circ 0.30+0.12 0.36+0.19 0.30+0.13 0.340.18

Transverse sections, imaged solely through SEM, confirmed directionality of the

porous material to be consistent 0.8-1cm above the bottom of an intact scaffold. Some

interconnection is seen through the pores however the directionality is clearly conserved.

Gamma irradiation shows no difference in scaffold directionality when modified

transverse sections were imaged through SEM. Figure 2-2 C exhibits the directionality

observed of an SEM image of a transverse section of an unmodified substrate. Transverse

sections were too thick in order to be visualized hydrated under a light microscope.
















A. B.

Figure 2-2. Microscopy images of HA scaffolds. A)Light microscopy of MPC modified
(0.15Mrad) scaffold at 40x zoom at a certain focal length. B) Same image as
A however using a different focal length. C).SEM image of a transverse cross-
section of a HA scaffold



























Figure 2-2. Continued

The majority of pores in the tertiary structure assumed an ellipsoid shape. It is

hypothesized that perfectly circular pores would not have a significant effect, if any

effect, on the neurogenerative properties of the material relative to the importance of

polymer composition and an observed directionality throughout the material. Degree of

circularity values ranges from 0 to 1 where 0 represents a straight line and 1 represents a

perfect circle. The average circularity of the unmodified and modified sample through

SEM was determined to be 0.300.12 and 0.300.13 respectively. When pores were fully

hydrated, they assumed a slightly more circular shape with a degree of circularity of

0.360.19 and 0.340.18 respectively. The relatively large standard deviation is due to

the fact that there was a somewhat significant range of pore shape however, the majority

of pores were clearly ellipsoid in shape. Similarity in pore structure between MPC

modified and unmodified substrates can be seen in the comparative SEM images in

Figure 2-8.










2.6.2 2-Methacryloyloxyethyl Phosphorylcholine (MPC) Modified Compositions

A main objective of this experiment was to determine the physical properties that

differentiates between the different doses of gamma irradiated MPC scaffolds and

unmodified samples. In order to determine if MPC modifications on a HA is feasible

electron dispersive spectroscopy (EDS) was primarily employed in order to verify the

presence of MPC. The elemental composition of a HA scaffold consists of carbon,

oxygen, hydrogen and the crosslinking agent chromium. MPC elemental composition

differs from HA only in that it contains phosphorous; which has a distinct K, value. An

accelerating voltage of 10KeV was used in order to visualize the chromium K, line.

Uncoated samples were used in order to eliminate interference of the gold-palladium

coating. EDS spectra seen in Figure 2-3 clearly demonstrates the MPC modified sample

(total dose = 0.10Mrad) containing a phosphorous peak which was not initially present in

an unmodified sample.

Courts






0 0




C1
2 4 5
E nr ,Vp (ln-U

Figure 2.3. EDS spectra of unmodified crosslinked HA (top) and MPC modified
(0.1OMrad) scaffold slices (bottom).









Country










Cr

0 2 4 B

Figure 2-3. Continued

HA is a very hydrophilic polymer that is known to absorb and retain water

efficiently. It has been reported that Ig of uncrosslinked HA has the capacity of

absorbing up to 6kg of water. Since the HA scaffolds are to be placed in an aqueous

environment of the spinal cord, the equilibrium water content of the material is crucial in

predicting the swelling properties of the scaffold in vivo. The equilibrium water content

was obtained by immersion of scaffold pieces in PBS at pH=7.4. Since PBS at this pH

has a similar salt and hydroxide composition to fluids in the body, it is hypothesized to

yield more accurate results relative to nanopure water.

Two conditions were used for this study, an MPC modified scaffold with a total

irradiation dose of 0.10Mrad and an unmodified scaffold. Upon immersion of dried

scaffolds slices in PBS, 48 hours was necessary in order for the pieces to become fully

hydrated. Figure 2-4 shows the equilibrium water content (EWC) values obtained through

eight consecutive days of immersion of 5 different scaffolds in the same PBS solution.

The average EWC for the unmodified and modified scaffolds was 63555% and

57643% respectively. This value for EWC was computed by taking the average water









content for all days after the second date to ensure scaffold was completely hydrated in

the calculations.


Equilibrium Water Content
800
750
700
~650
S600
550
500
1 2 3 4 5 8
E MPC Mod Day No MPC
Figure 2-4. Percent equilibrium water content for MPC modified (total dose = 0. 10Mrad)
or unmodified scaffolds for five consecutive days and on the eighth day.

Although dehydrated SEM images gives us information on pore size and distribution, it is

not a valid representation of the surface features which will be hydrated when in direct

contact with animal tissue in vivo. The surface features of hydrated modified and

unmodified samples were examined through atomic force microscopy (AFM) in order to

access the effect of MPC on surface roughness of various HA films. It was found that

surface roughness of hydrated samples gradually increased as the MPC content of the

scaffold increased. Figure 2-5 shows 3D AFM images of a typical film surface with

different MPC contents. Table 2-1 shows the numerical values corresponding to the

average of all the conditions that were run for the hydrated and dehydrated samples.

Dehydrated samples followed a roughness topography change similar to the one observed

for hydrated sample of the modified substrate, however the unmodified sample had an

average roughness significantly larger than any condition of the modified samples. This

data indicates that there is a clear change in the rearrangement of MPC along the surface











when the surface is hydrated. Therefore, one must be skeptical of any conclusions drawn

from data obtained solely of dehydrated samples, as is often the case in biomaterial

publications.

Table 2-2. Atomic force microscopy (AFM) values obtained for average height of
surface of modified hyaluronic acid (HA) films.
Hydrated Avg Height (nm) Dehydrated Avg Height (nm)
No MPC 2.6190.545 No MPC 5.3840.596
0.05Mrad 5.0180.733 0.05Mrad 1.4620.176
0.1OMrad 7.1060.145 0.1OMrad 1.8060.188
0.15Mrad 8.137+1.849 0.15Mrad 1.819+0.143


Digital InstruMents NanoScope
Scan size 10.00 UM
Scan rate 4.069 Hz
NuMber of samples 512
uIage Data Height
Data scale 30.00 nM


X 2.000 PM/div
2 30.000 nM/div


view angle
light angle










0 0


noMpc.002


Figure 2-5. AFM images taken under liquid cell contact mode of a. unmodified and b.
0.15Mrad MPC modified sample.










Digital InstruMents NanoScope
Scan size 10.00 ON
Scan rate 4.069 Hz
Number of saMples 512
Image Data Height
Data scale 80.00 nM







view angle
light angle






4\
"6

8 X 2.000 PM/div 0
2 80.000 nm/div

Mpc015b.005


Figure.2-5. Continued

A quantitative assessment of MPC adhered to the surface through gamma

irradiation can be deduced by inspection of liquid cell AFM and thin film fourier

transform infrared spectroscopy (FT-IR) data. Absolute quantitative measurements are

not possible through these methods due to the lack of known standards however relative

quantitative results yielded useful information. If average roughness is correlated directly

with MPC content of the material, the plot shown in Figure 2-6 bellow shows an initial

constant increase of MPC adhered to the surface of the material. At the highest radiation

dose, the material's surface is beginning to get saturated with MPC and the rate of MPC

adhesion begins to decrease slightly. FT-IR data provides us not only with the qualitative

confirmation of the presence of MPC but it also gives us some insight as to the amount of

MPC adhered to the films surface. The FT-IR shows peaks for P=O, P-O-CH2, and -N+-

(CH3)3 at 1240cm-1, 1075cm-1 and 970cm-1 respectively for all MPC modified samples.







These peaks are not present in unmodified films of HA. Figure 2-7 shows superimposed
spectra lines for all four experimental conditions ran in FT-IR from a range of 2000 to
800cm-1. Similar to the AFM data, the increase of MPC is observed as the transmittance
peaks get stronger however the strongest absorbance line (green) corresponded to a total
dose of 0.10Mrad. This could be due to varying thickness between films, or microscopic
scratches on the surface that may deflect the IR beam.


Average Roughness


Figure 2-6. Plot of average roughness versus total radiation dose


vt\
"1 I

,"


i ,'~


_I


1000 15'00 I uD'
Figure 2-7. Fourier transform-infrared spectra of percent transmittance through a range
of 2000-800cm'1 for unmodified samples, 0.05Mrad, 0.10Mrad and 0.15Mrad
total doses corresponding to the brown, red, green, and blue lines respectively.


I
-9 .4
I









Scanning electron microscopy (SEM) images also yielded interesting visual cues of

the MPC adhered to the surface. By visual inspection the MPC modified surfaces

exhibited a less uniform and rough topography that more closely resembles materials

which favor cellular adhesion. The polymerized MPC on the surface of a 0.15Mrad

irradiated scaffold can be clearly seen when compared with an unmodified scaffold piece,

shown in Figure 2-8. These same features are not visible when observed under SEM.

Images taken at higher magnification, shown in Figure 2-9, also allowed for visual

inspection of the MPC adhered to the surface of the material. One can deduce from the

visual inspection of the surface topography of the MPC modified substrates that the

material is suitable for cellular adhesion. An important note to keep in mind is the fact

that SEM images are taken of dehydrated samples that do not clearly exhibit the tertiary

conformation of the material when hydrated. Ideally, environmental SEM images should

to be taken in order to properly access the configuration of the MPC on the surface of the

material.














Figure 2-8. SEM images of scaffold pieces unmodified (left) and modified with a total
dose of 0.15Mrad (right) samples exhibiting clearly the MPC adhered onto its
surface. \






27














Figure 2-9. SEM images at high magnification of scaffold pieces of unmodified (left)
and modified with a total dose of 0.15Mrad (right) exhibiting MPC adhesion.














CHAPTER 3
IN VITRO AND IN VIVO EVALUATIONS OF HYALURONIC ACID SCAFFOLDS
AS OEC CARRIERS FOR TREATMENT INJURED NEURONAL SPINAL CORD
TISSUE

3.1 Specific Aim

The purpose of this chapter is to explore the casting of hyaluronic acid (HA) films

and the 2-methacryloyloxyethyl phosphorylcholine (MPC) modification of such films in

what is considered to be a sterile environment. These films were then used to determine if

olfactory ensheathing cells (OEC) will adhere to and grow on HA films. It is

hypothesized that since the film is of the same composition as the scaffold, the cells

would demonstrate similar adhesion properties on scaffolds prior to implantation. Films

were soaked in a solution of MPC and gamma irradiated with doses varying from 0.5 to

1.5Mrads. Cells were allowed to grow for four days prior to analysis of vital staining with

trypan blue and cell adhesion experimentation.

In order to first determine the stability of the polymer in vivo and if adverse

inflammatory responses are present, implants were sectioned in a sterile environment and

implanted into a rat spinal cord. The stability of the cell-biopolymer construct, as well as

the differing regeneration effects, in vivo is also of interest. To address these issues, left

sided hemisections were made at the cervical C2/C3 level in the adult rat cord. Implant

compositions were placed immediately following acute injury. Animals were allowed to

recover for 1 post-implantation. Tissue analysis was conducted by plastic embedding in

Epon.









3.2 Thin Film Synthesis

The same 1% HA solution described in chapter 2, which was used for thin film

characterization, was utilized for in vitro studies. Individual wells were mechanically

detached from a 12 well cell culture flask and washed with 95% ethanol prior to any

work. Two milliliters of the HA solution was then injected into each well and each was

gently tapped in order to ensure an even distribution of the solution. The wells were then

placed in a large Petri dish and covered with a piece of Kimwipe in order to prevent

contamination. The dish was placed in a biological hood and films were allowed to dry

overnight. Once dry, 2mL of 0.0025M CrK(SO4)2 was injected to each well and allowed

again to rest in the hood covered with a piece of Kimwipe for 3 days. Dried films were

then washed inside the wells repeatedly for two days.

Wells that were not going to be further modified were washed for two more

consecutive days and placed in 4C for storage. Three milliliters of 10% MPC solution

were injected into the wells designated for MPC modification. The wells were covered

with Parafilm, backfilled with Argon, and then covered with two more pieces of

Kimwhipes to ensure no gas exchange was occurring prior to modifications. Twenty four

to forty eight hours was allowed to pass prior to gamma irradiation of each well for a

total dose of 0.5, 1.0 and 1.5Mrad. MPC solutions were then discarded and HA films

were again washed inside wells with ultrapure water continuously for 3 days. Films were

stored at 4C fully hydrated in water until sterilization with EtO gas.

3.3 In Vitro Evaluations

3.3.1 Cell Cultures

The immortalized OEC cell line utilized was created by Dr. Jacobberger (54) and

donated by Dr. Kathy Jones. Lisa Tanzer, a current member of Dr. Jones' lab, generously









prepped cells for shipment, provided tissue culture protocols, and was available for

generalized questions in handling these cells. Cells arrived frozen at -700C and remained

in -70C until further use. One vial was thawed and utilized for all in vitro and in vivo

experiments. Cell viability and attachment of each experimental condition was

determined through vital staining with trypan blue.

3.3.2 Cell Culture Reagents

All materials were obtained from Sigma-Aldrich. All filterware was obtained

through Fisher Scientific. Growing media was utilized to multiply OECs upon thawing

and splitting. The growth media consisted of 500mL of Dulbecco's Modified Eagle's

Medium (DMEM) containing high glucose sodium pyruvate, 5mL of a penicillin-

streptomycin mixture, 5mL of glutamine-gentamicin, and 50mL of heat inactivated Fetal

Bovine Serum (FBS). Solution was filtered through a 0.221im filter into a previously

autoclaved screw cap 500mL glass bottle. FBS was added afterwards in order to avoid

filtration of essential proteins. Experimental media was used for all in vitro and in vivo

experiments and was prepared exactly as described above however FBS addition was

omitted. Cell detachment was accomplished by utilizing a 0.25% tryspin-EDTA solution

to wash cells of T75 cell culture flasks. Growing media was used to inhibit the trypsin in

the processing of splitting and collecting cells for experiments. Freezing media was

prepared by adding ImL of 10% cell culture grade DMSO and 3mL of FBS to 7mL of

DMEM.

3.4 Scaffold Cell Seeding

Prepared scaffolds were stored in the refrigerator at 40C. Prior to any seeding or

implantation 2[im scaffold pieces were suspended in a 50mL centrifuge tube containing

ultrapure water (R =17.4Mohm) and autoclaved. Sealed tubes were then placed in the









refrigerator until further use. Inside a biological hood sterile implants were trimmed to

approximately lmm3 pieces and placed inside a well of a 96 well plate. OECs were

trypsanized and washed with FBS-free DMEM three times prior to use. Cells were

diluted to a final concentration of 2*106cells/mL and viability was determined by vital

staining with trypan blue. 150tL of the OEC suspension was pipetted onto sterile

implants within the well and placed on a bi-directional rotator for approximately 1 hour

(8% CO2 & 37C) prior to implantation. Tissue culture flask was removed from incubator

and taken to surgery immediately prior to the implant's need.

3.5 In Vivo Trial

3.5.1 Surgical Procedure

An adult female Sprague Dawley rat weighing approximately 225g was used as the

subject of this study. Careful attention was made to ensure that proper handling and

sterile techniques were utilized at all times prior, during, and post surgery. 0.15mL of

Xylazine was administered subcutaneously in order for the rats to become sedated. The

rat's weight was then obtained once sedated (approximately 5-15 minutes), in order to

determine the correct Ketamine dose to be administered intraparitoneally. Animal

clippers were used to shave from the top of the head to T2 and ophthalmic ointment was

applied to each eye.

Rat was placed supine on a wooden block under the microscope and rat's level of

sedation was checked before any incisions were performed. A #15 scalpel blade was used

to perform a skin incision at the base of the skull running 5cm caudally along the midline.

Surgical scissors were used to cut away the first three layers of muscle from Cl to C5 and

forceps were used to separate the final layer, exposing the vertebral process over C2 and

the surrounding tissue. Curved Dumont was used to hold the dura which was cut with a









#11 blade. A 1-mm-long left-sided hemisection was made in the cranial segment of C2,

and the section was aspirated with a fine-tipped glass pipette. The dura was sutured with

Ethicon 10-0 and muscle was then sutured using a Vicryl 4-0 and a cutting needle.

Finally skin staples were used to close the wound. Rat was moved to heated water pad on

post-op table and 0.2mL Yobine, 0.4mL Bupinex and 6mL of warmed Lactated Ringers

were administered subcutaneously. Rat ear was wiped with alcohol soaked gauze and

appropriate ear holes were punched out. Surgery was performed by Kevin Siegel.

3.5.2 Surgical Implantation

Sterile implants were sized under an operating microscope to a -Imm diameter and

carefully inserted into the cardotomy. The data analyzed was from the trail animal with

an unmodified HA implant containing no cells and the animal was allowed to survive for

a week. Implants to be placed with cells were sized and soaked in FBS free DMEM for

two days prior to trimming and cell seeding. Implants were placed in spinal cord by Dr.

Paul J Reier and Todd White. Once proof of principle was achieved four MPC modified

HA scaffolds were implanted through the same surgery, however, as mentioned above,

that data was not analyzed in this study.

3.6 Postmortem Evaluations

3.6.1 Perfusions and Tissue Storage

All perfusions were performed under the fume hood utilizing a Millipore

mechanical perfusion pump. Rat was anesthetized with 0.3-0.5mL ofBeuthanasia D and

her weight was logged. If rat had no response to a pinch reflex perfusion was begun. If rat

continued to respond after approximately minutes, about 0. lmL of Beuthanasia-D was

administered. Each rat was perfused with 250mL of saline followed by 500mL of fixative

solution made with 4% paraformaldehyde and 3.5% glutalaldehyde in 0.1M phosphate









buffer. Following perfusion the entire spinal cord was removed the following day and

stored at 40C in a fresh batch of the same fixative solution as used in the perfusion

process.

3.6.2 Plastic Embedding of Tissue

Lesion area of the cord, running approximately 5mm in length, was removed under

Zeiss dissecting microscope along with 2mm sections caudal and raustral to the lesion

site. Each section was then subject to plastic embedding in hard Epon. Tissue sections

were first washed with saline solution and were stored in 2% Osmium tetrachloride at

4C for 2 -4 hours depending on section size. Tissues were then washed with 0.8% NaCl

and 8% glucose solutions, with distilled water, and then dehydrated in progressively

increasing concentrations of ethanol. Sections were then washed with propylene oxide

and left overnight in a mixture of 50% propylene oxide and 50% Epon. The following

day tissue was placed in pure Epon, baked at 60Cfor 15 minutes and allowed to cool to

room temperature. Tissue sections were then each placed in a plastic embedding mold

containing Epon ensuring that the raustral to caudal directionality of the spinal cord ran

perpendicular to the bench's surface. Tissue was then baked overnight at 600C and ready

for further processing the following day, once cooled to room temperature.

All sections were trimmed down and slices cut using a LKB Nova Ultratome. 24

slices (2 slides) measuring 2[tm thick were consecutively sectioned every 100pm through

the entire lesion site. Sections were visualized from 1.25-40x their original size using a

Zeiss Axioplan microscope. Images were obtained with the KS400 Image Analysis

System (Zeiss, KONTRON) utilizing a Hamamatsu Color Chilled 3CCD camera.









3.5 Results and Discussion

3.5.1 In Vitro Thin Film Evaluations

Preliminary in Vitro studies involving the seeding of OEC surface modified HA

substrates with varying degrees of MPC via gamma irradiation were conducted.

Inspection through an inverted light microscope yielded results which indicate MPC

surfaces exhibit favorable growth and adhesion properties. Superior growth and adhesion

properties were found on control samples of OEC growing on Petri dish however this

result is to be expected since cells most commonly grow preferentially in tissue culture

plates. When looking at the MPC modified samples relative to each other, however, a

gradually increasing number of OECs adhered to HA surface was found as the MPC

content of the films were increased. These results support the hypothesis stating MPC

modified surfaces would be preferential to OEC attachment and growth relative to

unmodified substrates.

3.5.2 Histological Analysis

Hyaluronic acid implants were apparent by gross anatomical inspection following

spinal cord dissection. Analysis of the slices through light microscopy indicated the

implant did not provoke an inflammatory response and has proven to be biocompatible in

vivo. An example of a slice image at 2.5x is seen in Figure 3.1. It was also apparent that

the scaffold had degraded with some time, indicating the entire material would degrade

with time. No infiltration of tissue was seen and a rather large air bubble was trapped

within the lesion area during the embedding process, which introduced some error. Proof

of feasibility is seen and a different study now in place is analyzing the effects of OEC

alone, OEC seeded onto a MPC modified implant and modified implant alone implanted

into a rat spinal cord.



























Figure 3-1. Optical image at lesion site of hyaluronic acid scaffold implant at 2.5x














CHAPTER 4
CONCLUSIONS

The lack of neurological recovery in spinal cord injury (SCI) following current

attempts has enticed research which yielded advances in the understanding of the

developing CNS and associated pathologies. Current repair strategies have emphasized

fetal tissue and specific cell transplantation, as well as a variety of specific biochemicals.

The more knowledge that is gained by SCI research, the more apparent is the

multifaceted nature of the problem. This being so, a combination of interventions would

have to be utilized to reverse the variety of issues encountered. .

Biomaterials offer the possibility to deal with more than one of the associated

problems in SCI simultaneously. The surface can provide physical/structural support, a

favorable surface terrain, an architecture which offers directionality to ingrowing neurons

and also act as support for cells which offer a variety of neuroregenerative advantages.

The basic concept was to use neural cell-biopolymer compositions to help control the

dynamic and complex repair process following SCI.

Novel anisotropic, water insoluble hyaluronic acid (HA) scaffolds were engineered.

The architecture of the linear pores of the HA scaffold is also unique. Although

polysaccharides have been surface modified with MPC, this is the first time specifically

HA has been gamma irradiated 2-methacryloyloxyethyl phosphorylcholine (MPC) onto

its surface.

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. In vitro studies showed that olfactory ensheathing cells (OEC)

preferentially adhered and grew on the polystyrene cell culture well superiorly to any HA

film condition. Within the HA films, however, the MPC modified films showed superior

adhesion and growth relative to the unmodified films. The film modified with 0.15Mrad

had the most healthy cells growing on its surface. The In vivo trial showed proof of

principle and the feasibility of such strategy.

The accumulated data indicate the strategy of using neural OEC-HA compositions

can address a number of issues involved in SCI and warrants further investigation. Ideal

compositions have not been identified as of yet, and may include the incorporation of

other multiple cell types and other therapeutic agents to existing or novel implants. Also,

there are important broader implications of results stemming from this research including

the application of similar implant compositions for the treatment of peripheral nerve

damage and optic nerve disease and trauma, as well as macular degeneration and retinal

disease.














CHAPTER 5
FUTURE DIRECTIONS

While working on this research a series of questions arose that could be

experimentally answered through future research. Bellow are some examples of potential

experimentation that would yield in interesting and applicable information.

1. Environmental Scanning Electron Microscopy (ESEM) experiments on
hydrated scaffolds.

The tertiary structure of the scaffold implants investigated through scanning

electron microscopy (SEM) was conducted on dehydrated samples. If one wants to

examine the hydrated microstructure of the 3D scaffold, which resembles more closely

the hydrated implant that would be placed in an injured spinal cord, then the structure

must be examined hydrated. The only way to achieve such goal would be through ESEM.

There would also be the possibility of visualizing the olfactory ensheathing cells (OEC)

directly onto the scaffolds, instead of cast films which have a different geometry.

2. In Vitro and In Vivo evaluations of hyaluronic acid (HA) implants crosslinked
with Gadolinium.

Through my research I conducted trial studies with Gd3+ crosslinked scaffolds and

films and determined it is feasible although relatively more short lived than chromium

cations. Recent literature has reported gadolinium to be a more biocompatible ion than

chromium. It would be interesting to analyze the effects of cell seeding onto films and

implanted scaffolds of HA that have been crosslinked with Gd3+ relative to Cr3+ to see if

there are advantages associated with such.









3. Seeding multiple cell types (Schwann cells + OEC) onto a single implant.

OEC cells have been shown to facilitate axonal elongation at graft-host interfaces

more effectively than Schwann cells however Schwann cells promoted axonal

regeneration/sparing and remyelination better than OECs. Never has there been a study

which evaluated more than one cell type seeded onto a biomaterial substrate

simultaneously and implanted into the spinal cord. It is expected that the seeding of these

two cell types together would result in increased regeneration, remyelination and axonal

elongation more than only one cell type.

4. Hyaluronic acid scaffolds covalently crosslinked after being ionically
crosslinked in order to increase biodegradation time of implant.

By following the methods described in chapter 2 it is possible to make anisotropic

insoluble HA scaffolds when crosslinked with Cr3+. The uncrosslinked HA scaffold

dissolves in solution prior to crosslinking using the same technique with covalent

crosslinking such as divinyl sulfone (DVS) or a carbodiamide. Covalently crosslinking

HA scaffolds would most likely results in scaffolds with a longer in vivo degradation

time. A possible investigation would be the crosslinking with DVS and a carbodiamide,

among other known covalent crosslinkers of HA, after crosslinking with Cr3+. These

covalent crosslinkers use HA's hydroxyl groups which are available for reacting since the

carboxylic acid groups is what is involved in the ionic crosslinking.















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BIOGRAPHICAL SKETCH

Jennifer de Toledo Mallek was born on March 13, 1982 in Sao Paulo, Brazil. She

moved to the Boca Raton in 1998 and completed her last two years of high school at

Boca Raton Community High. During these years, she discovered a passion for sports,

science and understanding the human body.

She obtained her Bachlor of Science in chemistry within three years at the

University of Florida and was admitted to UF's graduate program in 2003. During these

years she found a passion for medicine and a desire to teach. She earned dual master's

degree in 2006: a Master of Science, with a thesis focusing on biomaterial implants for

application in spinal cord injury; and a Master of Science in teaching, with an emphasis

of overcoming misconceptions in introductory chemistry classes.