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Injectable Biopolymer Gel Compositions for Neural Tissue Repair

Permanent Link: http://ufdc.ufl.edu/UFE0024088/00001

Material Information

Title: Injectable Biopolymer Gel Compositions for Neural Tissue Repair
Physical Description: 1 online resource (123 p.)
Language: english
Creator: Barnes, Samesha
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: acid, alginate, biomaterial, carboxymethylcellulose, central, cord, engineering, gel, hyaluronic, injectable, injury, nervous, spinal, system, tissue
Materials Science and Engineering -- Dissertations, Academic -- UF
Genre: Materials Science and Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Injuries to the brain, spinal cord or other central nervous system (CNS) tissues trigger a cascade of biochemical events that result in an environment that is unfavorable for axonal regeneration and re-establishment of functional connections. Advances in understanding of the cellular and molecular mechanisms underlying spinal cord injury (SCI) over the past twenty years have resulted in the development of a number of therapeutic approaches to treating this critical problem. Biomaterial constructs represent an important and perhaps essential component of spinal cord repair strategies; however the functional and restorative potential of these approaches has not yet been realized. This research focused on the development, synthesis and properties of biopolymer gel compositions for neural tissue repair. The primary goal was to prepare injectable gels which could function to bridge the lesion, prevent development or progression of a cystic cavity and provide a favorable terrain for axonal regeneration by delivering cells or other growth-promoting factors to the injured spinal cord. Homogeneous alginate (ALG), alginate-carboxymethylcellulose (ALG-CMC) and alginate-hyaluronic acid (ALG-HA) gels suitable for soft tissue engineering applications were synthesized via ionic crosslinking. Gradual gelation was achieved by slow liberation of calcium ions from calcium carbonate by reaction with D-glucono-delta-lactone (GDL). In situ-forming ALG, ALG-CMC and ALG-HA gels have not previously been studied as biopolymer matrices for SCI repair. All compositions were injectable through a 22-gauge needle prior to crosslinking. Gelation timing was evaluated as a function of biopolymer composition, calcium content, and temperature, and ranged from one to three hours for the conditions studied. Swelling and stability of gels were evaluated in vitro, and oscillatory tests were used to examine rheological properties. The potential for ALG, ALG-CMC and ALG-HA gels as transplantation matrices was investigated by incorporating Schwann cells in gel compositions in vitro. A pilot animal study was conducted to demonstrate proof of concept in vivo using a clinically relevant SCI model in adult rats. Study animals received midline cervical contusion injuries at C3/C4 using an Infinite Horizon impactor and were treated with an ALG-CMC gel one week later. Histology revealed that the compositions integrated well with host spinal cord tissue and did not initiate a significant inflammatory response. Treated animals also showed minimal evidence of cystic cavitation. Results suggest that injectable alginate-based compositions have significant potential for minimally-invasive treatment of SCI and should undergo further investigation and optimization for neural tissue repair.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Samesha Barnes.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Goldberg, Eugene P.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024088:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024088/00001

Material Information

Title: Injectable Biopolymer Gel Compositions for Neural Tissue Repair
Physical Description: 1 online resource (123 p.)
Language: english
Creator: Barnes, Samesha
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: acid, alginate, biomaterial, carboxymethylcellulose, central, cord, engineering, gel, hyaluronic, injectable, injury, nervous, spinal, system, tissue
Materials Science and Engineering -- Dissertations, Academic -- UF
Genre: Materials Science and Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Injuries to the brain, spinal cord or other central nervous system (CNS) tissues trigger a cascade of biochemical events that result in an environment that is unfavorable for axonal regeneration and re-establishment of functional connections. Advances in understanding of the cellular and molecular mechanisms underlying spinal cord injury (SCI) over the past twenty years have resulted in the development of a number of therapeutic approaches to treating this critical problem. Biomaterial constructs represent an important and perhaps essential component of spinal cord repair strategies; however the functional and restorative potential of these approaches has not yet been realized. This research focused on the development, synthesis and properties of biopolymer gel compositions for neural tissue repair. The primary goal was to prepare injectable gels which could function to bridge the lesion, prevent development or progression of a cystic cavity and provide a favorable terrain for axonal regeneration by delivering cells or other growth-promoting factors to the injured spinal cord. Homogeneous alginate (ALG), alginate-carboxymethylcellulose (ALG-CMC) and alginate-hyaluronic acid (ALG-HA) gels suitable for soft tissue engineering applications were synthesized via ionic crosslinking. Gradual gelation was achieved by slow liberation of calcium ions from calcium carbonate by reaction with D-glucono-delta-lactone (GDL). In situ-forming ALG, ALG-CMC and ALG-HA gels have not previously been studied as biopolymer matrices for SCI repair. All compositions were injectable through a 22-gauge needle prior to crosslinking. Gelation timing was evaluated as a function of biopolymer composition, calcium content, and temperature, and ranged from one to three hours for the conditions studied. Swelling and stability of gels were evaluated in vitro, and oscillatory tests were used to examine rheological properties. The potential for ALG, ALG-CMC and ALG-HA gels as transplantation matrices was investigated by incorporating Schwann cells in gel compositions in vitro. A pilot animal study was conducted to demonstrate proof of concept in vivo using a clinically relevant SCI model in adult rats. Study animals received midline cervical contusion injuries at C3/C4 using an Infinite Horizon impactor and were treated with an ALG-CMC gel one week later. Histology revealed that the compositions integrated well with host spinal cord tissue and did not initiate a significant inflammatory response. Treated animals also showed minimal evidence of cystic cavitation. Results suggest that injectable alginate-based compositions have significant potential for minimally-invasive treatment of SCI and should undergo further investigation and optimization for neural tissue repair.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Samesha Barnes.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Goldberg, Eugene P.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024088:00001


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INJECTABLE BIOPOLYMER GEL COMPOSITIONS FOR NEURAL TISSUE REPAIR By SAMESHA ROSNNE BARNES A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009 1

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2009 Samesha Rosnne Barnes 2

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This work is dedicated to my mother, Mrs. Rosemary Dorsey Barnes, who gave me wings and allowed me to fly. 3

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ACKNOWLEDGMENTS First and foremost I would like to acknowledge my lord and savior, Jesus Christ, who has brought me to this expected end. Apart from hi m I can do nothing. I especially thank my mother, Mrs. Rosemary D. Barnes, father, Mr. Samuel A. Barnes, brothers Reginald and Vincent Barnes, sister Tiara Barnes, and godsiste r Sarah Stubblefield for all of their love and support throughout my academic career. I would like to thank my advisor and chair, Dr. Eugene Goldberg, for his continued support and patience durin g this process. I appreciate him for believing in me to the end and pushing me across the finish line. I also thank the members of my supervisory committee, Dr. Chris Batich, Dr. Anthony Brennan, Dr. Henry Hess, and Dr. Paul Reier for helping me to reach this goal. It was truly an honor to be connected to such giants in the field. I would also like to acknowledge the late Dr. Abbas Zaman, Dr. Brij Moudgil and Dr. Charles Beatty for their contributions to my academic development at the University of Florida. I acknowledge Gil Sanchez and Dr. Scott Brown from the Particle Engineering Research Center for assistance with rheological character ization. From the McKnight Brain Institute, I acknowledge Dr. Michael Lane, Barbara OSteen, a nd Alex Jones from the Reier Research group and Drs. Irina Madorsky and Lucia Notterpek for their expertise a nd generosity in conducting the animal and Schwann cell studies. I also acknowledge Dr. Taili Thul a, Paul Martin, Scott Cooper and Chelsea Magin from the Depart ment of Materials Science and Engineering for their research assistance. I especially thank my former undergraduate mentees, David Cepeda, Julianne Huegel, David Walker and Bequita Gaines, for all of their contributions which were vital to the completion of this work. I also thank all th e members of the Goldberg, Brennan and Batich research groups, past and present, that have been an essential part of my graduate experience. I would like to give special acknowledgement to Ayana Johnson, Tara Washington, Dr. Shema Freeman, Jennifer Wrighton, Dr. Anne Donnelly, Dr. Todd White, Dr. Danyell Wilson, 4

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Dr. Charlee Bennett, Dr. Erika Kn ight Styles, Dr. Anika Odukale Edwards, Dr. John Azeke, and Dr. Jompo Moloye-Olabisi, Dr. Margaret Kayo, and Dr. Daniel Urbaniak for being my cheerleaders and support system throughout my gra duate career. Special appreciation is also given to my extended family, friends and coll eagues for their support and encouragement along the way: Bill and Cynthia Jones, Shirley Benning, Carolyn Hayes, Delores Merriweather, the late Robert Howard, Geneva Stal lings, Lily Hamilton, Linda McClellan, Penny McCloud, Monique McCane, Kecia and Cedric Rouse, Tambra Rain ey, Pastor Eric Thomas, Kimberly Hayes, Kimberly Coward, Katara Starkey, Dani sha Duncan-Phillips, Gwendolyn Saffo, Wendy Fletcher-Shannon, Dr. Sally Williams, Dr. Barbara Henry, Betty Floyd, Christina Scott, Kyana Stewart, Ashon and Takisha Nesbitt, Antoinet te Black, Kevin Holloway, Leah Woodward, Kennesha Adolphin, Ian Fletcher, Anntwanique Ed wards, Dr. Carla Phillips, Dr. Iris EnriquezSchumaker, Dr. Amanda Ely, Dr. Leslie Wils on, Dr. Lizandra Williams, Dr. Jeremiah Abiade, Dr. Robert Crosby and the members of BGSO. I would also like to thank the Department of Materials Scie nce and Engineering Academic Service Coordinators, Martha McDonald, Dori s Harlow and Jennifer Horton, the Southeast Alliance for Graduate Education and the Profe ssoriate (SEAGEP), and the Office of Graduate Minority Programs (OGMP) for their s upport and commitment to my success. 5

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4TABLE OF CONTENTS ............................................................................................................. ....6LIST OF TABLES ...........................................................................................................................9LIST OF FIGURES .......................................................................................................................10LIST OF ABBREVIATIONS ........................................................................................................1 2ABSTRACT ...................................................................................................................... .............14 CHAPTER 1 INTRODUCTION ................................................................................................................ ..16Significance of Spinal Cord In jury in the United States .........................................................16Clinical Treatment of Acute SCI ............................................................................................17Specific Aims ..........................................................................................................................17Aim 1: Synthesis and Characterization of Injectable Biopolymer Gel Compositions via Gradual Ionic Crossl inking of Alginate .................................................................18Aim 2: In Vitro and In Vivo Evaluations of Injectable Biopolymer Gel Compositions as a Matrix for Neural Tissue Repair ....................................................182 BACKGROUND ....................................................................................................................19Approaches to SCI Repair ...................................................................................................... 20Cellular Transplantation Strategies .........................................................................................21Schwann Cells .................................................................................................................21Olfactory Ensheathing Cells ............................................................................................22Macrophages/Microglia ...................................................................................................23Stem and Progenitor Cells ...............................................................................................24Tissue Engineering in the Injured CNS ..................................................................................25Natural Biopolymers .......................................................................................................26Agarose .....................................................................................................................26Alginate ....................................................................................................................27Chitosan ....................................................................................................................28Collagen ...................................................................................................................29Hyaluronic Acid .......................................................................................................30Synthetic Polymers ..........................................................................................................32PEG ..........................................................................................................................3 2PHEMA ....................................................................................................................33PHPMA (Neurogel ) .............................................................................................35 6

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PLA/PLGA ...............................................................................................................35Injectable Gels for Neural Tissue Repair ................................................................................363 SYNTHESIS AND CHARACTERIZATION OF INJECTABLE CARBOXYMETHYLCELLULOSE-ALGINATE AND HYALURONIC ACIDALGINATE GEL COMPOSITIONS .....................................................................................43Introduction .................................................................................................................. ...........43Materials and Methods ...........................................................................................................46Polysaccharide Solution and Blend Preparation ..............................................................46Crosslinked Gel Preparation ............................................................................................47Gelation Time ................................................................................................................. .47Statistical Analysis .......................................................................................................... 48Rheological Characterization ..........................................................................................48Electron Microscopy .......................................................................................................49Results and Discussion ........................................................................................................ ...49Injectable ALG-CMC and ALG-HA Gel Compositions .................................................49Gelation Time ................................................................................................................. .50Rheology ...................................................................................................................... ....52Morphology .....................................................................................................................55Summary ....................................................................................................................... ..........554 IN VITRO EVALUATION OF INJECTABLE GEL COMPOSITIONS AS A TRANSPLANTATION MATRIX FOR NEURAL TISSUE REPAIR .................................70Introduction .................................................................................................................. ...........70Materials and Methods ...........................................................................................................70In Vitro Swelling and Dissolution ...................................................................................70Primary Schwann Cell Culture ........................................................................................71Cell Viability ...................................................................................................................72Cell Entrapment Feasibility Study ...................................................................................73Results and Discussion ........................................................................................................ ...74Swelling and Dissolution .................................................................................................74Cell Viability ...................................................................................................................74Cell Entrapment Feasibility Study ...................................................................................75Summary ....................................................................................................................... ..........765 IN VIVO EVALUATION OF AN INJECT ABLE ALGINATE-BASED GEL COMPOSITION IN A CERVICAL SPINAL CORD CONTUSION INJURY MODEL .....82Introduction .................................................................................................................. ...........82Materials and Methods ...........................................................................................................83Biopolymer Gel Preparation ............................................................................................83Contusion Injuries ...........................................................................................................84Biopolymer Gel Injection ................................................................................................85Tissue Resection and Histology ......................................................................................85Histology .........................................................................................................................86 7

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Microscopy .................................................................................................................... ..86Results and Discussion ........................................................................................................ ...87Contusion Injuries ...........................................................................................................87Biopolymer Gel Injection ................................................................................................87Histology .........................................................................................................................88Summary ....................................................................................................................... ..........896 CONCLUSIONS ................................................................................................................. ...997 FUTURE WORK ................................................................................................................. .100LIST OF REFERENCES .............................................................................................................101BIOGRAPHICAL SKETCH .......................................................................................................122 8

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LIST OF TABLES Table page 2-1 Summary of key natural biopolymer s investigated for CNS repair ...................................412-2 Summary of key synthetic biopolym ers investigated for CNS repair ...............................413-1 Concentration of polysaccharides in biopolymer solutions ...............................................583-2 Concentration of polysacchar ides in biopolymer gels .......................................................583-3 Viscosity and gelation times of ALG-CMC compositions at 25 C ..................................633-4 Gel point (G-G crossover) and gela tion time (inverted tube) for biopolymer compositions at 37 C. .......................................................................................................6 94-1 Artificial cerebrospinal fluid formulation ..........................................................................78 9

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LIST OF FIGURES Figure page 1-1 Structure of me thylprednisolone. .......................................................................................182-1 Cellular response to injury in the nervous system .............................................................382-2 Chemical structure of agarose. ...........................................................................................392-3 Chemical structure of alginate. ..........................................................................................392-4 Chemical structure of chitosan. ..........................................................................................392-5 Chemical structure of hyaluronic acid. ..............................................................................392-6 Chemical structure of pol yethylene glycol (PEG). ............................................................402-7 Chemical structure of poly(2hydroxyethylmethacrylate) (PHEMA). ..............................402-8 Chemical structure of poly(N-[2hydroxypropyl methyacrylamide]) (PHPMA). .............402-9 Chemical structures of poly(glycolic acid) (PGA) and poly(la ctic acid) (PLA). ..............402-10 Methods of transplantation/implan tation in the injured spinal cord. .................................423-1 Structure of alginate showing guluronic acid (G) and mannuronic acid (M) residues. Consecutive G residues form a V-shaped cavity. ..............................................................573-2 Egg-box model of ionic crossl inking of alginate. Divalent cations (circles) sit in Vshaped cavities formed by G blocks of adjacent chains. ...................................................573-3 Chemical structure of glucono-lactone (GDL). ..............................................................573-4 Schematic of alginate cross linking procedure with GDL and CaCO3. ..............................593-5 Chemical structure of ca rboxymethylcellulose (CMC). ....................................................603-6 Photograph of ALG cro sslinking progression by the in ternal gelation method.. ..............613-7 Gelation time at 37 C as a function of biopolymer composition......................................623-8 Effect of temperature on gelation time of ALG-CMC compositions at high crosslink density (8 mM Ca2+). .........................................................................................................633-9 Modulus (G, G) of ALG gel as a function of time at 37 C. ..........................................643-10 Modulus (G, G) of ALG-CMC gel as a function of time at 37 C .................................65 10

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3-11 Modulus (G, G) of ALG-HA gel as a function of time at 37 C. ...................................663-12 Storage modulus (G) of biopolymer co mpositions as a function of time at low crosslink density (6 mM Ca2+) at 37 C. ............................................................................673-13 Storage modulus (G) of biopolymer co mpositions as a function of time at high crosslink density (8 mM Ca2+) at 37 C. ............................................................................683-14 SEM micrographs of snap-froze n, freeze-dried ALG gel compositions ...........................694-1 Swelling of biopolymer compositions in ar tificial cerebrospinal fluid at 37 C. ..............794-2 Relative viability of Schwa nn cells after 24 and 48 hours.. ...............................................804-3 Inverted microscope image of Schwann cells encapsulated within an ALG gel. ..............815-1 Infinite Horizon spinal cord impactor ................................................................................915-2 Cresyl violet stain of ALGCMC1 gel showing pink coloration. ......................................925-3 Tissue near the lesion epicenter stained w ith cresyl violet one week post-treatment with ALG-CMC1 gel (cryopr otected but not fixed). .........................................................935-4 Tissue rostral to lesion epi center stained with cresyl vi olet one week post-treatment with ALG-CMC1 gel (cryopr otected but not fixed). .........................................................945-5 Tissue near lesion epicenter stained with cresyl violet one week post-treatment with ALG-CMC1 gel (not cry oprotected or fixed). ...................................................................955-6 Tissue near lesion epicenter stained via cr esyl violet one week post-treatment with ALG-CMC1 gel (cryoprot ected and fixed).. ......................................................................965-7 Cresyl violet staining of contusion lesion tissue from an untreated subject one week post-injury in a prior study.. ...............................................................................................9 75-8 Cresyl violet staining of longitudinal section from an untreated subject one week post-contusion injury in a prior study.. ..............................................................................98 11

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LIST OF ABBREVIATIONS ALG alginate ANOVA analysis of variance BDNF brain derived neurotrophic factor BSA bovine serum albumin CMC carboxymethylcellulose CNS central nervous system CSPG chondroitin sulfate proteoglycan DRG dorsal root ganglion ECM extracellular matrix ESC embryonic stem cell G guluronic acid residue in alginate GDL glucono-delta-lactone HA hyaluronic acid IKVAV Ile-Lys-Val-Ala-Val peptide sequence M mannuronic acid residue in alginate MP methylprednisolone NGF nerve growth factor NSC neural stem cell NT-3 neurotrophin-3 OEC olfactory ensheathing cell PEG polyethylene glycol PHEMA poly(2-hydroxyethylmethacrylate) PHPMA poly(N-[2-hydroxypropyl] methacrylamide) PGA poly(glycolic acid) 12

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PLA poly(lactic acid) PLGA poly(lactic acid-co-glycolic acid) PNS peripheral nervous system RGD Arg-Gly-Asp peptide sequence SCI spinal cord injury SEM scanning electron microscopy 13

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INJECTABLE BIOPOLYMER GEL COMPOSITIONS FOR NEURAL TISSUE REPAIR By Samesha Rosnne Barnes December 2009 Chair: Eugene Goldberg Major: Materials Scie nce and Engineering Injuries to the brain, spinal cord or other central nervous system (CNS) tissues trigger a cascade of biochemical events that result in an environment that is unfavorable for axonal regeneration and re-establishment of functional connections. Adva nces in understanding of the cellular and molecular mechanisms underlying spinal cord injury (SCI) over the past twenty years have resulted in the development of a num ber of therapeutic approaches to treating this critical problem. Biomaterial constructs repres ent an important and perhaps essential component of spinal cord repair strategies; however th e functional and restora tive potential of these approaches has not yet been realized. This research focused on the development, s ynthesis and properties of biopolymer gel compositions for neural tissue repair. The primary goal was to prepare injectable gels which could function to bridge the lesion, prevent deve lopment or progression of a cystic cavity and provide a favorable terrain for axonal regeneration by delivering cells or other growth-promoting factors to the injured spinal cord. Ho mogeneous alginate (ALG), alginatecarboxymethylcellulose (ALG-CMC) and alginate-hyaluronic acid (A LG-HA) gels suitable for soft tissue engineering applica tions were synthesized via ioni c crosslinking. Gradual gelation was achieved by slow liberation of calcium i ons from calcium carbonate by reaction with D14

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15 glucono-delta-lactone (GDL). In situ-formi ng ALG, ALG-CMC and AL G-HA gels have not previously been studied as biopol ymer matrices for SCI repair. All compositions were injectable through a 22gauge needle prior to crosslinking. Gelation timing was evaluated as a function of bi opolymer composition, calcium content, and temperature, and ranged from one to three hours for the conditions studied. Swelling and stability of gels were evaluated in vitro and oscillatory tests were used to examine rheological properties. The potential for ALG, ALGCMC and ALG-HA gels as transplantation matrices was investigated by incorporating Sc hwann cells in gel compositions in vitro A pilot animal study was conducted to demonstrate proof of concept in vivo using a clinically relevant SCI model in adult rats. Study animals received midline cervical contusion injuries at C3/C4 using an Infinite Horizon im pactor and were treated with an ALG-CMC gel one week later. Histology revealed that the co mpositions integrated well with host spinal cord tissue and did not initiate a significant inflam matory response. Treated animals also showed minimal evidence of cystic cavitation. Results suggest that inject able alginate-based compositions have significant potential for mi nimally-invasive treatment of SCI and should undergo further investigation and optim ization for neural tissue repair.

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CHAPTER 1 INTRODUCTION Significance of Spinal Cord In jury in the United States Damage to the spinal cord is a life-alteri ng experience for those who suffer from these injuries. An insult to the spinal cord disrupts communication with the brain below the level of injury and frequently leaves SCI victims with limited use of limbs, chronic pain, and other complications; the higher the injury along the spinal cord, the more catastrophic the consequences. Over 250,000 people in the United States live with SCI, and more than half of these patients were young adults between the ages of 16 and 30 at the time of injury (NSCISC, 2007). Most SCIs are caused by automobile accidents, falls, a nd gun shots, and approximately 12,000 new injuries occur each year resulting in lifelong medical expenses that often exceed $1 million per patient. It was once believed that the CNS had no inherent ability to repair itself. However, decades of research has proven otherwise. It has been discovered that bot h regenerative and degenerative processes are triggered following CNS trauma, but the end result is an environment that is usually inhibitory to neural tissue repair. The permanent nature of SCI is largely due to the inability of axons to regenerate in the presence of physical and chemical barriers that develop around the wounded area; and the progres sive nature of these injuries results in damage that is exacerbated over time. There has been no cure or effective treatment for SCI, but a number of factors have been identified to encourage axonal growth and re-e stablish functional connections. Hence, a growing body of research aimed at CN S tissue repair and re generation. Accordingly, research proposed here was dedicated to ach ieving CNS repair by development of biopolymercell compositions with neur oregenerative properties. 16

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Clinical Treatment of Acute SCI The current clinical standard for acute SCI is high-dose treatment w ith methylprednisolone (MP) (Figure 1-1), a synthetic gl ucocorticosteroid. MP is administ ered within 8 hours of injury as a bolus of 30 mg/kg followed by 23hour infusion of 5.4 mg/kg/hour based on recommendations from the National Acute Spinal Cord Injury Studies (NASCIS I, II, III) (Bracken et al., 1984; 1990; 1997; Bracken, 2002). The primary neuroprotective benefit of MP is believed to be reduction of inflammation and inhibition of oxygen free radical-induced lipid peroxidation (Hall, 1992; Kwon et al., 2004). MP was found to improve mo tor function recovery when delivered within 8 hours of injury and fu rther improvement was reported when therapy is extended for 48 hours, especially wh en the initial bolus cannot be de livered within the first 3 to 8 hours after injury (Bracken, 2002). Others argue that MP offers only modest improvements in neurological function and when the risks of side-effects associated with high-dose steroid therapy are considered, the use of MP therapy as the standard of care for acute SCI cannot be justified (Gerndt et al., 1997; Short et al., 2000; Rabchevsky et al., 2002 ; Hugenholtz, 2003; Qian et al., 2005; Miller, 2008). Sustai ned delivery of MP from poly(l actic-co-glycolic acid) (PLGA) nanoparticles has recently been investigated a nd found to result in decreased lesion volume and enhanced behavioral recovery compared to standard MP treatment (Kim et al., 2009). Specific Aims The objective of this research was to develop biopolymer gel compositions for neural tissue repair using SCI as a m odel. Our primary goal was to s ynthesize injectable gels which would provide a favorable terrain for axonal regeneration by bridging the lesion site, preventing development (acute SCI) or pr ogression (chronic SCI) of a cystic cavity, and functioning as a carrier matrix for transplanted cells or growth -promoting substances. Th is project was divided into two specific aims: 17

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Aim 1: Synthesis and Characterization of Injectable Biopolymer Gel Compositions via Gradual Ionic Crosslinking of Alginate Injectable alginate-carboxymethylcellulose (ALG-CMC) and alginate-hyaluronic acid (ALG-HA) gels were prepared via ionic cr osslinking. Homogeneous gels of varying compositions were prepared and gradually crosslinked by the slow release of Ca2+ ions from CaCO3 by reaction with D-glucono-lactone (GDL). Gelation time was assessed at room and physiological temperatures using the inverted tu be method. Modulus of gels was determined using a Paar-Physica UDS 200 rheometer with a cone-and-plate measuring cell, and morphology was examined by scanning electron microscopy (SEM). In vitro swelling and dissolution were evaluated for select compositions in artificial cerebrospinal fluid (aCSF) at 37 C. Aim 2: In Vitro and In Vivo Evaluations of Injectable Biopolymer Gel Compositions as a Matrix for Neural Tissue Repair Suitability of gel compositions for cellular tr ansplantation was evaluated by incorporating Schwann cells in vitro. Cell viability was examined using an alamarBlue assay. Cervical contusion lesions were introduced in the spinal cord of adult rats at C3/C4 using an Infinite Horizon spinal cord impactor, and biopolymer gel was injected one week post-injury and allowed to gel in situ. Rats were allowed to recover for one week before histological evaluation was conducted. 18 Figure 1-1. Structure of methylprednisolone.

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CHAPTER 2 BACKGROUND Physical trauma to the spinal cord caus es immediate mechanical damage, vascular disruption and cell death, typically as a result of compression or tearing of tissue by the vertebral column. An intricate sequence of cellular and molecular events is subsequently triggered that includes both regenerative and degenerative processe s. As early as 15 minutes after injury axons begin to swell causing surrounding my elin to peel away and ruptur e (Profyris et al., 2004). As a result, axonal contents are rel eased into the extrace llular space along with myelin fragments. Crushing or severing of a xons physically divides the axon into two parts leading to the eventual degeneration of the distal segment in a proce ss called Wallerian degene ration (Reier and Lane, 2008). Within hours, activated macrophages and microg lia invade the area and clear out myelin and axonal debris (Fawcett and Asher, 1999; Darian-Smith, 2009) but also secrete proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-2 (IL-6) that can compound tissue damage (Kwon et al., 2004). Breakdown of myelin debris by phagocytic cells elevates levels of myelin-derived proteins Nogo, myelinassociated glycoprotein (MAG) and oligodendrocyte myelin gl ycoprotein (OMgp) which have been shown to suppress plasticity and inhibi t axonal growth and regeneration in the CNS (Grandpre and Strittmatter, 2001). Immediately after injury astroc ytes undergo reactive gliosis and migrate to the damaged area. Within 3 to 5 days of injury, these reactive astrocytes begin to wall-off the injury and upregul ate chondroitin sulfate proteogl ycans (CSPGs) which are potent inhibitors of axonal regeneration (McGraw et al., 2001). The resulting glial scar is prominent in areas where the blood-brain barr ier (BBB) has been most significantly breached accompanied by 19

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a larger population of activated macrophages (Busch and Silver, 2007). The result is an inhibitory environment that is not conducive for repair. Approaches to SCI Repair The peripheral and central ner vous systems differ in their re sponse to injury which impacts the extent of axonal regeneration and functiona l recovery following trauma (Figure 2-1). Damage to the adult mammalian CNS was consid ered to be permanen t until the early 1980s when its regenerative potential was demonstrat ed by elongation of transected CNS axons into peripheral nerve grafts (Richardson et al., 1980; David and Aguayo, 1981; Benfey and Aguayo, 1982). These landmark studies unambiguously sh owed that some CNS axons could undergo significant regrowth following injury if presented with a favorable environment. Since that time, advancement in the understandi ng of SCI mechanisms has led to considerable progress in developing treatments to promote axonal regeneration and functi onal recovery. The progressive nature of CNS injury makes it imperative that an effective strategy be developed that will minimize or halt further development of secondary damage and provide a favorable environment for regrowth of spared axons through the lesion. A number of strategies been investigated that have b een be categorized to offer neuroprotection, promote axonal growth, bridge the lesion, restore axon al conduction or to promote plasticity (Ramer et al., 2005), and ma ny recent studies have focused on combining more than one approach (Ikegami et al., 2005; Ji et al., 2005; Lpez-Va les et al., 2006; Bunge, 2008). Neuroprotective pharmaceutical agents such as methylprednisolone, GM-1 ganglioside, and minocycline have been studied to prevent loss of undamaged neur al cells to se condary injury processes but the efficacy of some of these ag ents has been debated (Hall and Springer, 2004; Baptiste and Fehlings, 2006). Axonal rege neration has been encouraged through the neutralization of grow th inhibitors, includi ng antibody blocking of Nogo-66 receptor function 20

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(Grandpre and Strittmatter, 2001; Li et al., 2005 ; Tian et al., 2005; Yu et al., 2008) and enzymatic degradation of CSPGs in the glial scar by chondroitinase ABC (Yick et al., 2003; Chau et al., 2004; Garca-Alas et al., 2008; Tester and Howl and, 2008) Also nerve growth factor (NGF), neurotrophin-3 (NT-3), brainderived neurotrophic f actor (BDNF) and other neurotrophic substances have b een extensively used in partners hip with other treatments to support axonal elongation. Due to the complexity of SCI, an effective treatment will ultimately consist of complementary approaches and will like ly incorporate transpla nted cells (Reier, 2004; Willerth and Sakiyama-Elbert, 2008). Major cell-b ased therapies are reviewed in the following section. Cellular Transplantation Strategies Schwann Cells Unlike the CNS, axons in the peripheral nervous system (PNS) undergo significant elongation and regeneration following injury. Th e robust regenerative capacity of the PNS has largely been linked to Schwann cells, myelin -producing glia surrounding peripheral nerves, making them a prime candidate for transplantati on in the injured spinal cord. When peripheral nerves are severed, macrophages invade the area and remove myelin and axonal debris resulting from Wallerian degeneration. Schwa nn cells in the distal segment proliferate and line up in tubes (bands of Bunger) along the basal lamina, up-re gulating neurotrophins an d ECM molecules that promote axonal regeneration. Growth cones in th e proximal segment are directed through the Schwann cell tubes towards the distal segment: ultimately th e ends are reconnected and remeyelinated and function is restored (Hall, 2001; Dezawa, 2002). In severe injuries where there is no residual distal segment, Schwann cel ls can be generated by precursors in proximal nerves, and these can stimulate a limited degree of regeneration and functional recovery (Purves et al., 2008). 21

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Schwann cell transplantation in the spinal cord has been extensively investigated over the past twenty years and has been shown to promot e repair and functional recovery (Martin et al., 1996; Guest et al., 1997; Xu et al., 1997; Oude ga et al., 2001; Dezawa, 2002; Oudega et al., 2005; Rasouli et al., 2006; Agudo et al., 2008; Lavdas et al., 2008). Recently, intraspinal injection was found to be a more effective route for deliverin g autologous activated Schwann cells to the contused rat spinal cord, resulting in enhanced axonal regeneration, myelination and hindlimb locomotor recovery compared to intrav enous or intrathecal deli very (Ban et al., 2009). In recent clinical trials, four adult patients wi th chronic mid-thoracic SCI received rehabilitation and treatment with autologous Schwann cells fr om the sural nerve (Sab eri et al., 2008). Study patients experienced transient paresthesia and musc le spasms in lower limbs, but did not show signs of infection or neurologi cal decline up to one year following cell transplantation. These results suggest that autologous Schwann cell transplantation is safe; however no sensory or motor function improvement was demonstrated. Olfactory Ensheathing Cells The mammalian olfactory neuroepithelium is unique in its ability to continuously renew itself for a lifetime, with neurogenesis occurri ng into adulthood (Ramn-Cueto and Avila, 1998), so when the olfactory system is injured, neur ogenesis is enhanced and new axonal connections are established (Franssen et al., 2007). Olfactor y ensheathing cells are g lial cells that enwrap axons of olfactory neurons and guide them from the neuroepithelium in the nasal cavity to the olfactory bulb in the brain. The ability of OECs to permit axona l growth across the PNS to CNS interface into adulthood is a key factor in the regenerative capacity of the olfactory system (Franssen et al., 2007), and these cells may work in concert with other olfactory cells such as meningeal cells, olfactory nerve fibroblasts and olfactory mucosa cells (Barnett and Chang, 2004). OECs are similar to Schwann cells in many re spects in that they exhibit: secretion of 22

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neurotrophic factors, expression of ECM and cell adhesion molecules, stimulation of axonal regeneration and remyelination (Kocsis et al ., 2009), and both are av ailable for autologous transplantation. However, OECs have been show n to intermingle better with astrocytes and induce lower expression of inhib itory CSPGs than Schwann cells in vitro and in vivo (Lakatos et al., 2000; Lakatos et al., 2003). There has been considerable research involving acute and chronic transplantation of OECs in a variety of spinal cord injury models during the past 15 years (Ramn-Cueto and Nieto-Sampedro, 1994; Li et al., 1997, 1998; Bartolomei and Greer, 2000; Ramn-Cueto et al., 2000; Garca-Alas et al ., 2004; Ramer et al., 2004; Lpez-Vales et al., 2006; Franssen et al., 2007; Guest et al., 2008) as well as in the optic nerve (Li et al., 2003; Li et al., 2008). Clinical trials involving transplantation of OECs or ol factory tissue have also been conducted in Australia (Feron et al., 2005; Mackay-Sim et al., 2008), China (Zheng et al., 2007) and Portugal (Lima et al., 2006). Macrophages/Microglia Microglia are brain macrophage found throughout the adult CNS that play a key role in its immune function. There has been ongoing deba te over whether these cells behave in a neuroprotective or neurotoxic manner in the in jured CNS. Following CNS trauma, microglia become activated and secrete cytotoxic pro-infl ammatory cytokines and free radicals (Stopek, 2003; Kitamura et al., 2009); however a pro-re generative role is supported by activated microglial expression of neurotrophins, grow th factors and ECM molecules as well as phagocytosis of cellular debris (Dougherty et al., 2000; Stopek et al., 2002; Kitamura et al., 2009). It has been proposed that microglia may ex ist in more than one type of activated state depending on the trigger which determines the effect these cells will have in response to trauma (Streit, 2002). The neuroprotective train of thought has lead to c onsideration of microglia for SCI repair in a few studies. 23

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Prewitt et. al. co-transplanted microglia-coate d nitrocellulose membranes along with fetal spinal cord tissue into adult rat lesions and found that increasing the concentration of activated macrophage/microglia at the injury site enhanced growth of sensory a xons (1997). These results were presumed to be due to expression of cy tokines and interaction with surrounding cells. Rabchevsky and Streit observed enhanced regeneration and substantial axonal extension into microglia-gelfoam grafts implante d into the injured adult rat sp inal cord (1997; Streit, 2001). The authors concluded that cultur ed microglia in the grafts creat ed a favorable environment for regeneration determined from neurite growth, va scularization, laminin expression and infiltration of growth-promoting host cells, e.g. Schwann cells. Microglial grafts were later revisited in studies by Stopek who implanted microglia-seeded alginate scaffolds into partial transection lesions in rats (2003). Seeded scaffolds were found to increase neurological function during spontaneous vertical exploration compared to un treated controls and to integrate well with host tissue. Transplantation of microglia for SCI re pair has not been reported in recent years. Stem and Progenitor Cells Because of their ability to differentiate into specialized cells, stem and progenitor cells are of great interest for replacement of lost, injured or diseased cells and tissues. Embryonic stem cells (ESCs) are pluripotent and self-renewing and can be directed to differentiate into neurons and glia. This makes ESCs particularly attract ive for SCI repair. Transplanted ESC-derived neurons have been shown survive and differentiate in the spinal cord and to promote functional recovery following injury (McDonald et al ., 1999; Deshpande et al., 2006). Potential development of teratomas and risk of immunorejection in addition ethical issues have somewhat hampered ESC research for use in the spinal cord (Tewarie et al., 2009). However the first human clinical trials involving ESCs were re cently approved by the United States FDA (Alper, 2009). 24

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Neural stem cells (NSCs) are multipotent, self-renewing cells that generate neurons, astrocytes and glia that have b een isolated from the developing and adult brain, spinal cord and optic nerve (Coutts and Keirstead, 2008). Adult NSCs have gained popu larity as an alternative to embryonic cells as they have potential for autologous or donor-mat ched treatment and circumvent ethical concerns (Louro and P earse, 2008). Also, a numbe r of studies have investigated transplantat ion of neural progenitor cells as we ll as adult stems cells derived from bone marrow (e.g. hematopoietic stem cells and bone marrow stromal cells) and dermis as reviewed by Bareyre (Bareyre, 2008 ). Clinical trials involving stem and progenitor cells have also been conducted. Tissue Engineering in the Injured CNS The limited availability of autologous neural tissue and immunologi cal risks related to allografts have led to investigation of tissue engineering approaches to CNS repair employing natural or synthetic polymeric scaffolds (Novikova et al., 2003). Development of biopolymer matrices is especially important as interest in cellular replacement therapies for SCI discussed above continues to grow (Reier, 2004; Ronsyn et al., 2008; Willerth and Sakiyama-Elbert, 2008). Therefore methods will be required for more effective delivery of donor tissue or bioactive substances into human spinal cord lesions whic h are typically larger and more complex than those created in the laboratory setting (Friedman et al., 2002; Ka kulas, 2004; Guo et al., 2007). The overall goal of neural tissue engineering is to provide an environment where scaffolds, cells, and bioactive molecules interact s ynergistically to promote tissue repair (Samadikuchaksaraei, 2007). This is appro ached by bridging the lesion with biomaterial constructs that will create a more permissive terrain for axonal regeneration either alone or in combination with growth-promoting cell types, ne urotrophic molecules, or substances that neutralize inhibitory fact ors. There has been extensive explor ation of biomaterial constructs for 25

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CNS repair over the past decade as reviewed in the literature (Harvey, 20 00; Geller and Fawcett, 2002; Nomura et al., 2006; Samadikuchaksaraei, 2007; Nisb et et al., 2008; Zhong and Bellamkonda, 2008). A variety of natural and synthetic polymers in the form of sponges or gels have been utilized, and effects of scaffold prope rties, e.g. scaffold architecture (Wong et al., 2008), have been explored. Linear ly-oriented axonal grow th and encapsulation of stem cells into biocompatible polymers have been issues of great interest (Teng et al., 2002; Silva et al., 2004; Stokols and Tuszynski, 2004; Nisbet et al., 2009). However, failure to achieve significant axonal growth beyond inhibitory host-implant glial inte rfaces is a challenge that has not been fully resolved (Geller and Fawcett, 2002). The following sections review some of the key polymers that have been investigated for CNS repair. Natural Biopolymers Agarose Agarose (Figure 2-2) is a linear polysaccharide derived from red algae that forms thermoreversible gels in the temperature range of 1740 C. Agarose gels have been shown to support outgrowth of dorsal root ganglion (DRG) neurites in vitro, and neurite extension was found to be inversely related to gel concentration and stiffness (Bellamkonda et al., 1995; Dillon et al., 1998; Balgude et al., 2001). Laminin-modi fied agarose hydrogel scaffolds enhanced DRG and PC12 neurite outgrowth in vitro (Yu et al., 1999). Additionally, loading the modified agarose scaffolds with NGF-loaded lipid microtubules stimulated directional DRG neurite extension. Freeze-dried agarose scaffolds w ith linearly oriented channels filled with collagen gel supported axonal ingrowth when implanted in the injure d spinal cord (Stokols and Tuszynski, 2006). Regeneration was enhanced for scaffolds contai ning BDNF. In situ-forming agarose gels loaded with BDNF-loaded microtubules allowed axona l ingrowth and reduced the inflammatory response when injected in hemis ection spinal cord lesions in rats (Jain et al., 2006). The low 26

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gelling temperature of agarose (SeaPrep) nece ssitated the use of a c ooling system to induce thermal gelation of in vivo after injection into the cord whic h is a drawback of this approach. This limitation was overcome by combining agarose with another thermosensitive polymer with opposite gelling behavior, methylcellulose, produ cing a blend that gels near physiological temperature (Martin et al., 2008). These blends are undergoing furt her investigation for neural tissue repair. Alginate Alginate (Figure 2-3) is a lin ear, anionic polysaccharide from brown algae that has been used for controlled drug delivery, cell encap sulation, wound dressings and other biomedical applications because of its many favorable properties which include being nontoxic, biocompatible, and able to form gels under mild conditions via ionic crosslinking with divalent ions (dAyala et al., 2008). A number of studies have examined the use of alginate for repair in both the PNS and CNS. Alginate hydrogels ha ve been reported to support DRG neurite outgrowth in vitro with enhanced neuroregenerative properties when Schwann cells were incorporated (Mosahebi et al ., 2001). Similarly, YIGSR peptid e-modified alginate gels stimulated a significant increase in attachment of NB2a neuroblastoma cells as well as enhanced neuritie differentiation and outgrowth compared to unmodified alginate gels (Dhoot et al., 2004). In the cochlea, ionically crossl inked alginate beads were shown to be an effective matrix for delivery of NT-3 and afforded protection of auditory neurons afte r round window and intracochlear implantation in deafened guinea pigs (N oushi et al., 2005). Neural progenitor cells have also been cultured and expanded in vitro in enzymatically degradable alginate hydrogels containing poly(lactide-co-glycolide) (PLGA) micr ospheres loaded with alginate lyase (Ashton et al., 2007). The degrad ation rate of alginate was tunable by adjusting the concentration and release rate of the enzyme. 27

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Suzuki and colleagues synthesized freeze-drie d, covalently crosslinked alginate sponges via reaction of ethylenediami ne and water soluble carbodiimide and demonstrated their usefulness for wound healing and peripheral nerve regeneration. In later studies freeze dried alginate sponges were implanted into the resected spinal cord and reportedly reduced glial scar formation and enhanced outgrowth of axons (Suzuki et al., 1999; Kataoka et al., 2001; Suzuki et al., 2002; Kataoka et al., 2004). Additionally, th ese alginate sponges were shown to be an effective matrix for transplantation of hippocam pus-derived neurospheres which were injected into alginate sponge-filled spin al cord lesions (Wu et al., 2001). Extensive migration and differentiation of neurospheres we re observed as well as good inte gration with host spinal cord tissue. Self-assembling alginate anisot ropic capillary hydrogels have been shown to promote directional axonal regeneration in vitro and after implantation into cervical spinal cord transection lesions (Prang et al., 200 6). Enhanced regeneration was observed in vitro when the anisotropic alginate scaffolds were seeded with adult neural progeni tor cells. Choi et al. prepared injectable alginate gels by di ssolving covalently crosslinked al ginate sponges in physiological saline (2006). When injected in to acute spinal cord lesions in rats a reduction in CSPG expression was observed compar ed to untreated controls. Chitosan Chitosan (Figure 2-4) is a cationic polysacchar ide derived from chitin. It is found in the exoskeleton of crustaceans and is the second most abundant natural polymer. Chitosan-based membranes have been reported to have good nerve cell affinity in vitro (Haipeng et al., 2000), which is enhanced by combining chitosan with gelatin (Cheng et al., 2003) or poly-L-lysine (Mingyu et al., 2004). Genipin-cros slinked chitosan scaffolds prev ented cystic cavitation and promoted some functional recovery following acut e SCI but produced a grea ter concentration of 28

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inflammatory neutrophils compared to alginate scaffolds (Stopek, 2003). Minimal axonal regeneration was observed after de layed implantation of chitosan cha nnels in spinal cord lesions. However extensive regeneration was obtained when the chitosan channels were filled with peripheral nerve grafts, but w ithout evidence of functional re covery (Nomura et al., 2008). Similarly, axonal regeneration and partial locomo tor functional recovery were observed after implantation of collagen-filled chitosan tubes after SCI, but not for chitosan tubes alone (Li et al., 2009). In situ-forming chitosan gels which fo rm gels at physiological pH and temperature have been synthesized using -glycerophosphate salt (Chenite et al., 2000; Chenite et al., 2001). In vitro studies indicated that these thermally-gelling chitosans s upported limited growth of fetal mouse cortical cells which was enhanced by at tachment of poly-D-lys ine (Crompton et al., 2007). However after implantation in the brain these thermosensitive chitosan gels stimulated significant activation of macrophages which may lim it their use for CNS repair (Crompton et al., 2006). Collagen Collagen is the most abundant protein in the human body and the primary component of the extracellular matrix and connective tissues. Collagen supports neurona l cell attachment and growth (Tomaselli et al., 1987) and is used as a substrate for neuronal cell culture (Letourneau, 2001). Collagen has also been used for peripheral nerve repair (Archibald et al., 1995; Navarro et al., 2001; Stang et al., 2005) and is the basis of comm ercial NeuroGenTM nerve guides (Integra, 2005). In the injured spinal cord, corticospinal axons did not gr ow into collagen gels; however substantial ingrowth of axons was stimulated wh en the gels were combined with gray matter extracts from the cervical spinal cord (Joos ten et al., 1995). Collage n grafts containing the neurotrophins BDNF and NT-3 increased locomoto r function and stimulated axonal ingrowth in 29

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the transected spinal cord (Houweling et al., 1998a; 1998b). Injured spinal axons regrew through implanted collagen tubes and reconnected with ta rgets in the ventral ro ot (Liu et al., 2001). Collagen filament scaffolds were reported to bri dge spinal cord lesions and promote functional recovery in rats (Yoshii et al., 2003; 2004) and rabbits (Yosh ii et al., 2009) when implanted parallel to the spinal cord axis. Collagen gels have also been used as a matrix for intrathecal delivery of growth factors to the spinal cord (Jimen ez Hamann et al., 2003; 2005). Hyaluronic Acid Hyaluronic Acid (HA) is a non-sulfated glyc osaminoglycan (GAG) that is ubiquitous in the body and found in abundance in the extracellular matrix (ECM) of most animal tissues. It is a linear anionic polysaccharide consisting of D-glucuronic acid and N-acetylglucosamine disaccharide repeat units (Figure 2-5). HA functions as a lubricant for all joints and interacts with the cell surface receptors CD44, RHAMM and ICAM-1 in the regulation of biological processes such as morphogenesis, wound healing and in flammation (Toole, 2001). Also, HA degradation products have been shown to stimulate angiogenesis (West et al., 1985) HA has been employed extensively in biomedical applications becau se it is biodegradable, biocompatible, and nonimmunogenic in addition to its unique pseudoplas tic rheological properties. Major uses of HA include viscosupplementation, ophthalmic visc osurgery, wound healing and drug delivery (Balazs and Denlinger, 1989) a nd for the prevention of post-surg ical adhesions (Burns et al., 1995). Poor mechanical properties and rapid enzymatic degradation in vivo necessitate crosslinking or combination of HA with othe r polymers for tissue engi neering applications (Segura et al., 2005). In the CNS, HA is produced mostly by astrocyt es and surrounds myelinated axons in white matter and neuronal cell bodies in grey matter of the spinal cord (A sher and Bignami, 1991; Bignami and Asher, 1992; Bignami et al., 1992). The functions of HA in the CNS are not fully 30

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understood, however it has been associated w ith neuronal and glial cell migration, axonal guidance and synaptic modulation (Sherman et al., 2002). In the normal rat spinal cord, high molecular weight HA has been demonstrated to ma intain astrocytes in a state of quiescence but is degraded after SCI which stimulates astr ocyte proliferation (Struve et al., 2005). These findings suggest that degradation of HA plays a signi ficant role in the formation of the glial scar following injury. HA has been investigated as a matrix for cellu lar transplantation to the CNS. For example, attachment of OECs on HA thin films has been investigated in vitro (Mallek, 2006). Modification with the phospho lipid 2-methacryloyloxyethyl phos phorylcholine (MPC) resulted in increased OEC attachment compared to unm odified films. In a recent study, HA-Polylysine hydrogels were shown to support survival and diffe rentiation of neural st em cells into neurons and astrocytes in culture (Ren et al., 2009). DRGs cultured in 3-D crosslinked thiolated HA hydrogels exhibited enhanced su rvival and neurite extension in vitro when compared to DRGs cultured in fibrin matrices. However there was no difference in histol ogical or functional outcome between rats implante d with the thiolated HA hydrogels following transection SCI and untreated control animals. HA has also been invesigated as a carrier for a Nogo-66 receptor (NgR) antibody to promote axona l regeneration (Tian et al., 2005). HA hydrogels coupled with PDL and the NgR antagonist were shown to suppor t neural cell attachment and to induce DRG neurite outgrowth in vitro (Hou et al., 2006). HA hydrogel scaffolds conjugated with bioactiv e molecules such as laminin (Hou et al., 2005), poly-D-lysine (PDL) (Tian et al., 2005), RGD (Cui et al ., 2006) and IKVAV (Wei et al., 2007) have been implanted in the injured br ain. The modified HA scaffolds were found to support cellular infiltration a nd angiogenesis, to reduce glia l scarring and to improve DRG 31

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neurite extension verses HA alone. HA has also been combined with methylcellulose (MC), a thermoreversible polymer that gels around 37 C, to create a rapidly gelling matrix for localized, intrathecal drug delivery to the injured spinal cord (Gupta et al., 2006; Baumann et al., 2009; Kang et al., 2009). Synthetic Polymers PEG Polyethylene glycol (PEG) (Fi gure 2-6) is a hydrophilic polymer with surfactant properties that is biocompatible and used in a nu mber of products for human use. Several in vitro studies have been conducted to determ ine the suitability of PEG ge ls for delivery of cells and neurotrophins to the injured CNS. Photopolymer izable gels have been explored for their potential for minimally invasive delivery as they can be injected in the site of injury and cured in vivo by exposure to light. Photopolym erized PEG gels have been st udied for controlled release of CNTF and were reported to enha nce neurite outgrowth from retinal explants compared to PEG gels without CNTF (Burdick et al., 2006). Microspheres embedde d within the photopolymerized PEG gels were also able to simultaneously deliver multiple neurotrophins with distinct release profiles. Photopolymerized PEG (Mahoney and An seth, 2006) and poly-L-lysine-PEG (Hynes et al., 2007) gels have also b een synthesized and supported in vitro survival of ne ural progenitor cells. In vivo PEG solutions containing NT-3 were inj ected into the rat spinal cord following hemisection injury and subsequently polymeri zed by exposure to light (Piantino et al., 2006). The gels promoted axonal regeneration and improvement in open field BBB scoring and horizontal ladder walk tests. In jectable poly(N-isopropylacrylamide) (PNIPAAm)-PEG gels have also been synthesized for sustained release of BDNF and NT-3 in the injured CNS. These gels were reported to preserve neurotrophin bioactivity and support survival and attachment of BMSCs in vitro (Comolli et al., 2008). 32

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Because of its fusogenic properties, PEG solu tions have been investigated for topical application to the acutely inured spinal cord where they have been demonstrated to promote rapid functional recovery and restoration of axonal conduction through the lesion (Shi and Borgens, 1999; Borgens and Shi, 2000; Shi and Borgens, 2000; Borgens et al., 2002). Similar results have been obtained for subcutaneous (Borgens and Bohnert, 2001) and intravenous delivery of PEG (Laverty et al., 2004; Baptiste et al., 2009). The neuroprot ective benefits of PEG are not fully understood but are beli eved to be due to its ability to rapidly restore integrity of damaged cells/membranes and to suppress second ary injury mechanisms such as the production of reactive oxygen species (Borge ns et al., 2002; Luo et al., 2002; Liu-Snyder et al., 2007). PEG must be administered early afte r injury while prior to glial scarring (Nisbet et al., 2008), and may be useful in combination with other approaches to SCI repair rather than as an independent treatment (Baptiste et al., 2009). PHEMA Poly(2-hydroxyethylmethacrylate) (PHEMA) (Figure 2-7) is a soft, hydrophilic, nonbiodegradable polymer and the most highly used hydrogel. PHEMA properties are highly tunable by varying the water cont ent, degree of crosslinking and method of preparation (Peppas, 2009). Biomedical uses of PHEMA include soft contact lenses, drug delivery, wound healing, articulating surfaces for joint pros theses and artificial interverte bral discs (Meakin et al., 2003). PHEMA sponges with 85% water cont ent have been engineered by Bakshi et al. to match the mechanical properties (compressive modulus) of the spinal cord (2004). When implanted into the hemisected cord the hydrogel scaffolds elicited a modest inflammatory response, had minimal scarring around the implant and supported consid erable angiogenesis. Also, axonal regeneration was improved by soaking the PHEMA sponge in BDNF. 33

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HEMA has been copolymerized with methyl methacrylate (MMA) to improve mechanical strength and application to spinal cord repair (Dalton et al., 2002). Unfilled guidance tubes synthesized PHEMA-PMMA implanted between the stumps of the transected rat spinal cord were found to promote regeneration of axons from brainstem motor nuclei with minimal inflammatory response and minimal scarring at th e tube-spinal cord interface (Tsai et al., 2004). The PHEMA-PMMA channels coll apsed over time due to inadequate mechanical strength and were subsequently reinforced with coils which increased the channel strength by seven times (Nomura et al., 2006). However, the coil-rein forced channels offered no regenerative improvement due to complications with syringomye lia and caudal migration of the rostral stump. Tsai et al. demonstrated that filling the PHEMA-PMMA channels with a matrix material, e.g. collagen, fibrin, Matrigel, impacted the numbe r of regenerating axons, the original of regenerating axons and also impr oved functional recovery (2006). In recent studies, macroporous PHEMA scaffo lds with positive charges were prepared by copolymerization with [2-(methacryloyloxy)ethyl] trimethylammonium chloride and implanted into the transected spinal cord (Hej l et al., 2008). The positively-charged hydrogels adhered well to spinal cord tissue and permitted inf iltration of blood vessels, neurofilaments and Schwann cells. Delayed implantati on (1 week) was shown to result in a statistically significant decrease in cystic cavitation compared to implantation immediately following injury. PHEMA scaffolds of varying surface charges have also been compared in the same injury model (Hej l et al., 2009). It was concluded that axonal regene ration within PHEMA hydr ogels is promoted by charged functional groups, whether positive or negative, compared to uncharged scaffolds. However, regeneration was most significant in positively charged scaffolds. 34

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PHPMA (Neurogel ) Poly(N-[2-hydroxypropyl methyacrylamide]) (PHPMA) (Figure 2-8) hydrogels with interconnected pores and viscoelastic properties si milar to nervous tissue have been synthesized by Woerly et al. These hydrogels have been shown to support cellula r ingrowth and axonal regeneration after acute implant ation in the transected rat (W oerly et al., 1998; 1999) and cat (Woerly et al., 2001) spinal cord as well as in brain lesions (Woerly et al., 1999). PHPMA hydrogels also promoted tissue reconstructi on and functional recove ry following delayed implantation in compression lesions three months after injury (Woerly et al., 2001) and are reported to suppress glial scar formation (Woe rly et al., 2004). RGD peptide-functionalized PHPMA hydrogels have also been synthesized under the trademark (NeuroGel) and implanted into the injured spinal cord (Woerly et al., 2001). Schwann cells, astr ocytes and embryonic neural cells entrapped within PHPMA had limited vi ability which was believed to be due to the polymerization conditions, e.g. monomer toxicity and heat production (Woerly et al., 1996a; 1996b), indicating that these hydrog els are not suitable for in situ cell immobilization. However, fibroblasts modified to express BDNF and ciliary neurotrophic factor (CNTF) have been infused into RGD-PHPMA hydrogels and reported to enha nce axonal regeneration in the injured optic nerve (Loh et al., 2001). PLA/PLGA Poly(lactic acid) (PLA), poly( glycolic acid), PGA, and their copolymers (PLGA), are the most used synthetic, biodegra dable. These polymers (Figure 2-9) are members of the poly( hydroxyacid) family and have been used in sutu res, bone fixation device s, microspheres for drug delivery, and scaffolds for tissue engineeri ng (Athanasiou et al., 1996). The mechanical properties and degradation ra te (rate of hydrolysis) of PLGA copolymers can be easily 35

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manipulated by adjusting molecula r weight and monomer ratios, as well as the stereochemistry of PLA which exists in D and L isomeric forms (PDLA, PLLA). The potential of poly( -hydroxyacids) for spinal cord re pair was first demonstrated by Gautier et al. who found that a PLGA copolymer and its degradation products did not adversely affect Schwann cell morphology and proliferation in vitro (1998). When implante d in the spinal cord PLGA cylinders had an inflammatory response similar to controls and showed evidence of axonal regeneration. Two types of PLA tubes cont aining Schwann cell cables were found to have inadequate mechanical propertie s and permeability for spinal cord repair (Oudega et al., 2001). Later, macroporous PLA foam scaffolds were s ynthesized which have been investigated for delivery of BDNF (Patist et al., 2004) and gene tically-modified Schwann cells (Hurtado et al., 2006) to the injured cord Porous PLGA scaffold s have be studied for Schwann cell delivery in recent studies (Chen et al., 2009). Also, PLGA nanopart icles have been shown to be effective for instraspinal delivery of the neurotrophic fact or GDNF with enhanced neuronal survival and hindlimb locomotor recovery (Wang et al., 2008 ). The key natural and synthetic polymers investigated for CNS repair are summarized in Table 2-2 and Table 2-3, respectively. Injectable Gels for Neural Tissue Repair Gels derived from natural and synthetic polymer s have been investigated as scaffolding for neural tissue repair as reviewed above because of their high water content, porous structure and soft, tissue-like mechanical properties (Woe rly et al., 1999). For vinyl monomers such as HEMA, crosslinking is achieved with polyfunctiona l acrylic monomers such as the bisacrylates by free radical polymerization which results in covalently crosslinked molecular structures which are insoluble but read ily swollen by water. Hydrogel prope rties (i.e. strength and water absorption) may be varied by adjusting the degree of crosslinking; i.e. th e crosslink density. Gel 36

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implants are frequently used in the form of spong es or soft, semi-solid jelly-like solids that may be cut to size for surgical implantation into the injured CNS tissues and organs such as the spinal cord. Because they are often synthesized with radical initiators and cytotoxic covalent crosslinking agents they must be extensively purified to be suit able for implantation. In addition, a number of studies have examined the use of gel implants in conjunction with cells, enzymes, or growth factors. However, incorporation of th ese agents during hydrogel synthesis subjects them to the free radical reactio n chemistry which may be damaging or degrading. It has been suggested that an ideal hydrogel scaffold for spinal cord regeneration should have the ability to be shaped in situ in a ddition to being biocompatible, biodegradable or bioresorbable, and permissive for cell migr ation and axonal outgrowth (Zhong and Bellamkonda, 2008). An in situ-forming gel has several advantag es. The gel can be injected in a minimallyinvasive procedure compared to sponge-like sc affolds which require lesioning the injured cord for implantation. Unlike foam or pre-formed gel sca ffolds, an injectable gel can completely fill the irregular geometry of a contused lesion cavity (Figure 2-10) facilitatin g integration with host tissue. Also, multiple cells and substances can be easily dispersed within the material prior to gelation for simultaneous deliv ery in a single injection. 37

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Injury to peripheral nerve A Macrophages rapidly remove myelin debris Expression of growth-related g enes Figure 2-1. Cellular response to injury in the nervous system. A) Secretion of neurotrophins and other growth-promoting factors by Schwann cells in the PNS is essential to peripheral nerve regeneration. B) Myelin debris and inhibitory factors secreted by reactive astrocytes contribute to failure of CNS re generation. [Reprinted with permission from Sinauer Associates, Inc. Purves, D., Augustin e, G. J., Fitzpatrick, D., Hall, W. C., LaMantia, A.-S., McNamara, J. O., and Wh ite, L. E., 2008. Neuroscience. (Page 641, Figure 25.5; Page 648, Figure 25.10) Sinauer Associates, Inc., Sunderland, MA.] B Injury to CNS axon Prolonged clearing of myelin debris Inhibitory factors disrupt axon extension 38

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Figure 2-2. Chemical structure of agarose. Figure 2-3. Chemical structure of alginate. Figure 2-4. Chemical structure of chitosan. Figure 2-5. Chemical stru cture of hyaluronic acid. 39

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Figure 2-6. Chemical structure of polyethylene glycol (PEG). Figure 2-7. Chemical structure of poly(2-hydroxyethylmethac rylate) (PHEMA). Figure 2-8. Chemical structure of poly( N-[2-hydroxypropyl methyacrylamide]) (PHPMA). PLA PGA Figure 2-9. Chemical structures of poly(gly colic acid) (PGA) and pol y(lactic acid) (PLA). 40

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Table 2-1. Summary of ke y natural biopolymers invest igated for CNS repair Polymer Source Advantages Disadvantages Agarose Red algae Thermoreversible gelation Poor cell adhesion, cooling required to induce gelation Alginate Brown algae Mild (ionic) crosslinking Poor cell adhesion Chitosan Crustaceans Cell adhesive (cationic) Significant inflammatory response Collagen ECM Supports cell attachment Immunogenicity concerns Hyaluronic acid ECM Bioactiv e Poor cell adhesion, Poor mechanical properties Table 2-2. Summary of ke y synthetic biopolymers investigated for CNS repair Polymer Degradable Use in CNS repair PEG Yes Injectable (photopolymerized) gels, topical application PHEMA No Sponge-like scaffolds, tubular channels PHPMA No Gel scaffolds PLA/PLGA Yes Sponge-like scaffolds, tubular channels, nanoparticles 41

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42 Figure 2-10. Methods of transpla ntation/implantation in the injured spinal cord. Scaffolds, preformed gels or whole pieces of tissue are inserted into A) resection or B) contusion lesions after making an incision in the spinal cord. C) Injectable gels or cellular suspensions may be injected into chronic c ontusions lesions direct ly through the dura. [Reprinted with permission from Elsevier: Gi ovanini, M. A., Reier, P. J., Eskin, T. A., Wirth, E., and Anderson, D. K., 1997. Char acteristics of human fetal spinal cord grafts in the adult rat spin al cord: influences of lesi on and grafting conditions. Exp. Neurol. 148, 523-543 (Page 524, Figure 1).]

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CHAPTER 3 SYNTHESIS AND CHARACTERIZATION OF INJECTABLE CARBOXYMETHYLCELLULOSE-ALGINAT E AND HYALURONIC ACID-ALGINATE GEL COMPOSITIONS Introduction Alginates are composed of -D-mannuronic acid (M) and -L-guluronic acid (G) residues (Figure 3-1) that are distributed in blocks of G, blocks of M and alternating GM sequences. The proportion and distribution of M, G and GM vary with the s ource from which alginate was obtained. These polymers are abundant in nature non-toxic, biocompatible, bioresorbable and inexpensive, making them practical for a variety of food, pharmaceutical, cosmetic and biomedical applications. One of the key properties of alginate is the ability to form gels via ionic crosslinking with multivalent cations. Ca2+ ions for example convenientl y fit in the cavity formed by blocks of G residues in adjacent polymer chains and form an egg-box structure (Grant et al., 1973) (Figure 3-2). Because the content and a rrangement of G residues are responsible for gelling behavior of alginates, alginate source is a knob for varyi ng properties (porosity, stiffness, swelling, and stability) of the final gel in add ition to the selection a nd concentration of the crosslinking cation. For example, high G alginates form rigid, brittle gels compared to alginates with higher M content which form softer, defo rmable gels (ISP, 2000; Simpson et al., 2004). Because of its mild ionic crosslinking conditions alginate is considered an ideal matrix for encapsulating cells (Smidsrd and Skjk-Brk, 1990). For example, alginate gels have been extensively investigated for imm obilization of a number of cell types, including chondrocytes for cartilage tissue engineering (B onaventure et al., 1994; Marijnisse n et al., 2000; Stevens et al., 2004; Xu et al., 2007) and pancrea tic islet cells for treatment of type I diabetes (Soon-Shiong, 1999; Trivedi et al., 2001; Sambanis, 2003; Figliu zzi et al., 2006; Qi et al., 2008). Cells are typically entrapped by dripping alginate with dispersed cells into a solution of CaCl2 or CaSO4 43

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(Smidsrd and Skjk-Brk, 1990; Tnnesen an d Karlsen, 2002; dAy ala et al., 2008). Crosslinking takes place from the outside in as th e alginate solution contacts calcium ions which continue to diffuse into the gel bead over time. The resulting alginate beads are non-uniform as a result of depletion of alginate at the center of the gel (Skjk-Brk et al., 1989; Moe et al., 1995). Similarly, alginate gels have been prepared by di ffusion of calcium ions into alginate solutions via the dialysis method, which also produces inhomogeneous gels (Skjk-Brk et al., 1989; Smidsrd and Skjk-Brk, 1990), which is not de sirable for tissue engineering applications. Draget et al. described an internal gelation method for producing homogeneous alginate gels by employing a sparingly soluble calcium salt, CaCO3, and glucono-lactone (GDL) (Draget et al., 1989; Draget et al ., 1990). GDL (Figure 3-3) is a non-toxic substance used in the food industry for curing, pickling, leavening, and pH control (FDA, 2002). GDL gradually hydrolyses to -gluconic acid (Pocker and Green, 1973), and Ca2+ ions are slowly liberated from CaCO3 in an acid-base reaction with the formation of CO2 gas and water (Equation 3-1). Subsequently, carboxyl groups (in G blocks) of two adjacent alginate chains are complexed by the Ca2+ ions (Equation 3-2) and a 3-D network is formed over time. Gels of neutral pH are obtained when equivalent amounts of CaCO3 and GDL are used, i.e. when the molar ratio of GDL to CaCO3 is equal to two (Draget et al., 198 9). Crosslinking of alginate by CaCO3-GDL is illustrated schematically in Figure 3-4. OH2 2 H 3 2 H 3CO HCO Ca CaCO ( 3-1) (complex) Ca(COO) COO2Ca2 2 ( 3-2) Gradual crosslinking allows ce lls to be uniformly disperse d within the gel, making the internal gelation method an attractive approach to developing injectable homogeneous alginate 44

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gels suitable for spinal cord repair and other tissue engineerin g applications. These in situ forming hydrogels are of interest as a novel matrix for spinal cord repair due to several key factors. First, these gels are comprised of natural polymers that are biocompatible, biodegradable/bioreso rbable and can be modified via av ailable functional groups to impart bioactivity. Second, an injectable matrix can be de livered directly into th e cord through the dura minimizing the invasiveness of the procedure. Third, the gels can serve as a bridge for regenerating axons by filling and conforming to th e irregular geometry of the cystic cavity in a contusion lesion. Finally, therap eutic cells and pro-regenerative substances can be easily incorporated simultaneously prior to gelation for a combined approach that targets multiple factors to create an environment favorable fo r repair and re-establi shment of functional connections. CMC (Figure 3-5) is an anioni c, water-soluble derivative of cellulose whose properties are easy varied with the degree of carboxymethyl su bstitution (Hercules, 1999). CMC is used as a thickening agent in a number of food, cosmetic and pharmaceutical products and is also used for barriers for prevention of post-surgical adhesions (Peck et al., 1995; Guo et al., 1998; Zeng et al., 2007). CMC is considered to be a low cost al ternative to HA (Ogushi et al., 2007); however it has not been significantly examined for CNS repa ir. This chapter focuses on the synthesis and characterization of ionically crosslinked AL G, ALG-CMC and ALG-HA gels. The primary goal was to synthesize injectable gel compositions vi a internal gelation of ALG combined with CMC or HA to modulate properties and to evaluate the suitability of these compositions as injectable cell-biopolymer matrices for neural tissue repair. This work is the first reported investigation of in situ-forming ALG, ALG-CMC and ALG-HA com positions for spinal cord repair. Aqueous ALG, CMC and HA solutions were prepared, mixed in varying ratios by volume and crosslinked 45

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with Ca2+ ions released using the CaCO3-GDL system. The effect of biopolymer composition, Ca2+ concentration and temperatur e on gelation time and mechanic al properties was studied. Morphology of freeze-dried gels was also examined. Materials and Methods Polysaccharide Solution and Blend Preparation Viscous (~1000 3000 cps) polys accharide solutions were prepared with alginic acid sodium salt (high viscosity from Macrocystis pyrifera MP Biomedical) and sodium carboxymethylcellulose (7HF PH, Hercules) usin g a procedure adapted from Mentak (Mentak, 1993). Ultrapure wa ter (resistivity > 17.4 M ), 600 mL, was vigorously stirred in a 1000 mL Pyrex beaker at 1800 rpm us ing Caframo high speed mechanical mixers (Models BDC 6015 and BDC 1850) and 3-blade propellers. Powdered ALG (2.5% w/v) or CMC (1.5% w/v) was gently sifted into the vortex and stirred at 1800 rpm for the first 15 minutes. The propeller speed was reduced to 1000 rpm, and the solutions were covered with parafilm and allowed to mix for 12 hours. The moisture content of each powder was determined prior to solution preparation using a Mettler LJ16 moisture anal yzer to determine the correct we ight of each polymer needed to give the desired concentration. ALG and CMC solutions were filtered into 250-mL Pyrex bottles with screw caps using a stainless steel air pressure filtra tion funnel (Gelman Sciences) and 10 m Spectra Mesh nylon filters. The filtered solutions were allowed to remain at room temperature for 24 hours and were sterilized in a Tuttnauer 2540 EA autoclave on a programmed liquid cycle (20 minutes at 240F). After sterilization ALG and CMC solutions we re combined to give 75% ALG-25% CMC and 50% ALG-%-50% CMC compositions by volume. A ppropriate volumes of each solution were 46

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injected into in 100 mL Pyrex bottles using 20 mL syringes and magnetically stirred for 1 hour to mix. Hyaluronic acid solutions (0.5 % w/v) were prepared by adding HA powder (molecular weight 1.5 MDa, Genzyme) to ultrapure wa ter and stirring magneti cally for 12 hours. HA solutions were sterile-filtered through a 0.22 m filter, and combined with ALG solutions to obtain 75% ALG-25% HA and 50% ALG-%-50% HA by volume. Final polysaccharide blend concentrations are shown in Table 3-1. Crosslinked Gel Preparation Crosslinked gels were prepared in 15 mL Nalgene containers All mixture concentrations were based on a total volume of 10 mL. CaCO3 was added to 1 mL of ultrapure water and sterilized by autoclaving. Sterile ALG, ALG-CMC or ALG-HA solutions, 8 mL, were added to the CaCO3 suspension and magnetically stirred for 1 minute to mix. A freshly made solution of GDL (Sigma), 1 mL, was injected thr ough a 1 cc syringe fitted with a 0.22 m filter to initiate gelation, and the mixture was stirred for an additional 20 seconds. Low (6 mM) and high (8 mM) concentrations of Ca2+ were evaluated, and the concentrati on of GDL was adjusted for a CaCO3 to GDL molar ratio of 1:1 to ma intain neutral pH. The concentr ations of ALG, CMC and HA in the final gels are given in Table 3-2. Gelation Time The rate of gel formation was determined by observing flow behavior of the gels as a function of time. A timer was started immediatel y after crosslinking was initiated by addition of GDL solution to polymer solution-CaCO3 mixture. One mL of biopolymer blend solution was injected into 16 x 150 mm test tube s and allowed to gel. The tubes were tilted every 5 minutes to observe the change in flow of the polymer so lutions. The time when the gel became too viscous 47

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to flow was considered to be the gelation time Sample flow was compared against a visual standard to minimize error since the same level of gelation was not achieved for all samples due to differences in composition. For gelation at phys iological temperature, polymer solutions were equilibrated at 37 C in a cons tant temperature bath for 2 hours prior to crosslinking. Samples were quickly returned to the bath after in itiating gelation to maintain temperature. Statistical Analysis Average gelation times and standard error of th e mean were calculated in Microsoft Excel. Statistical analysis was done using SigmaStat 3.1 software with assistance from Scott Cooper. A two-way ANOVA was conducted to determine the statistical influence of composition and Ca2+ concentration on gelation time at 37 C. Tukeys te st was used to determine which blends had a gelation rate different from that of pure ALG. Rheological Characterization The viscosity of each biopolymer solution a nd blend was measured with a Brookfield RVTDV-IICP cone-and-plate viscometer. Measurements were taken at room temperature as a function of different shear rates. Rheological evaluation of injectable gel compositions was carried out using a Paar-Physica UDS 200 rheometer with a cone-and-plate measuring system (MK226: diameter = 50 mm, angle = 1). An amplitude sweep ( = 0.01 to 100%, f = 1 Hz) was conducted at 37 C to determine the region of near linear viscoelasticity (LVE) using the most viscous gel composition (ALG, high crosslink density). The ALG gel was injected onto the plate immediately after initiating cro sslinking and was allowed to gel for 3 hours before initiating the test. Subsequently, time tests were performed on all biopolymer compositions at constant temperature (37 C) using a frequency of 1 Hz and = 1% (below the LVE limit) to measure storage modulus (G) and loss modus (G) as a function of time. Each sample was freshly 48

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prepared and injected onto the plate usi ng a 10-mL syringe immedi ately after initiating crosslinking. To allow time to load the sample and lower the cone, each test was started exactly 5 minutes after GDL was mixed into the sample. G and G and were recorded every minute for a total of 2 hours. Electron Microscopy The morphology of freeze dried gels was exam ined by SEM with assistance from Paul Martin. One mL of biopolymer was injected into the wells of a 24-well cell culture plate immediately after initiating crossl inking and allowed to gel for 24 hours at 37 C. The gels were then snap-frozen with liquid nitrogen and freeze -dried. Lyophilized gels were sectioned and mounted onto SEM stubs with c onductive carbon adhesive tabs. A dusting of a gold/palladium alloy was applied to the samples (< 2 minutes) using a Technix Hummer V sputter coater. Images were aquired using the JEOL SEM6400 scanning electron microscope with an accelerating voltage of 2 KeV, condenser lens setting of 8 to 10, and a 15 mm working distance. Results and Discussion Injectable ALG-CMC and ALG-HA Gel Compositions In situ-forming gels based on ALG containi ng CMC or HA were prepared via internal gelation for the first time in th is work. In this method, CaCO3, a poorly soluble salt, was combined with GDL which slowly hydrolyzes to D-gluconic acid in a queous solution. Slow release of calcium ions is triggered by the gradua l decrease in pH associat ed with the hydrolysis of GDL. Neutral gels were obtai ned by maintaining a GDL to CaCO3 molar ratio equal to 2. The gradual release of Ca2+ ions resulted in gradual cross linking of ALG and produced uniform, transparent gels that visibly increased in s tiffness over time (Figure 3-6). All compositions studied were injectable through a 22-gauge needle prior to crosslinking. The incorporation of 49

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CMC or HA within the solution did not hinder the ability of ALG to gel in the presence of calcium ions. Gelation rate was characterized by the time to solidify using the inverted tube method and was used a screening criteria to determine the appropriate range of compositions and crosslink densities (i.e. calcium ion concen trations) to consider for furthe r study. In the screening studies ALG-CMC blends (25/75, 50/50, 75/25, 100/0 ALG/CMC by volume) were prepared from 2% ALG and 1.5% CMC (w/v) solutions. Three Ca2+ concentrations were initially examined: low (2 mM), medium (4 mM) and high (6 mM). Conditions under which observed gelation time exceeded 3 hours were excluded from further study. The ALG-CMC3 composition (25/75 ALG/CMC) did not produce gels when observed 24 hours after the addition GDL to initiate crosslinking but instead resulted in a non-uniform viscous fluid with gelatinous regions formed. It is believed that the concentration of AL G in the ALG-CMC3 composition (0.5% w/v) was below the critical gelling concentration and ther efore a continuous gel did not form. This is consistent with observations by Straatmann et al. who prepared ALG gels (0.2 to 2 wt%) in a similar manner and reported gel formation when the ALG concentration was above 0.5 wt%. (2003). The results of our prelimin ary studies suggested that calci um ion concentration should be at least 6 mM and that ALG concentration shou ld be above a minimum concentration of 0.5 % w/v in the final composition to obtain gels that solidified within 3 hours for the specific polymers used in this research. Gelation Time Gelation time was determined via the inverted tube method as a measure of gelation rate. This is important clinically as it is preferable that the gelling solution stiffen in a reasonable timeframe to maintain uniform distribution of in corporated cells, etc. di spersed within the gel once implanted. At the same time, gradual gelati on affords injectability for an extended amount 50

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of time after crosslinking has been initiated. Ge lation time was generally observed to increase as the amount of ALG in the blend decreased for th e compositions studied (F igure 3-7, 3-8). Also, gelation rate increased as the concentration of CaCO3 increased. Two-way ANOVA indicated that the influence of bi opolymer composition and Ca2+ concentration on gelation time at 37 C were both statistically significan t (p<0.001). Tukeys comparison test indicated that gelation rate of all blend compositions was significantly differe nt (p<0.05) from ALG w ith the exception of ALG-HA1. This was true for both low (6 mM) and high (8 mM) Ca2+ concentrations. Kuo and Ma prepared injectable ALG gels using the internal gelation method and demonstrated that the gelation ra te, homogeneity and mechanical pr operties can be controlled by combining CaCO3 with a more soluble calcium salt, specifically CaSO4, and manipulating the ratio of the two salts (2001). Gelation rate increased as the proportion of CaSO4 increased but the resulting gels were weaker and less homogeneous than those crosslinked with CaCO3 alone. Osteoblasts were uniformly incorporated within the ALG gels in vitro indicating the potential for the ALG-CaCO3-GDL system to support cells. Kuo and Ma also observed more rapid gelation as the concentration of ALG decreased which was attributed to a decrease in viscosity. In our studies with ALG, ALG-CMC and ALGHA gels, gelation rate decreased as ALG concentration decreased due to the decreasing proportion of AL G available for crosslinking relative to CMC or HA in solution. There did not a ppear to be a correlatio n between viscosity of the parent biopolymer solutions and gelation rate (Table 3-3). Gelation rates for ALG and ALG-CMC were evaluated at both 25 C and 37 C and were observed to increase with increasing temperature for all compositions (Figure 3-8). This is also consistent with previously reported data (K uo and Ma, 2001). The increased rate is due to acceleration of GDL hydrolysis with increasing temperature (Pocker and Green, 1973) which 51

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results in a corresponding increased rate of Ca2+ release within the solutions. The compositional effect on gelation rate was more pronounced at lo wer temperature. Gelation rate is important clinically as it is preferable that the gelling so lution stiffen in a reasonable timeframe to prevent leakage and ensure uniform distri bution of incorporated cells, etc. dispersed within the gel. At the same time, gradual gelation affords inje ctability for an extended amount of time. Rheology Oscillatory testing in the linear viscoelas tic region was conducted at 37 C using a rheometer with a cone-and-plate attachment. The da ta revealed a gradual increase in storage (G) and loss moduli (G) of all compositions as a function of time (Figures 3-9 through 3-13). The storage modulus G represents elastic behavior and is a measure of stiffness whereas the loss modulus G represents the visc ous behavior of the sample. As more alginate chains are complexed by calcium ions slowly liberated from CaCO3, the resulting polymer network becomes more rigid. A more rapid increase in G was observed for compositions crosslinked with 8 mM CaCO3 than with 6 mM CaCO3 which is in agreement with the in crease in gelation time observed with increasing calcium as discussed earlier. Ho wever, no relationship was observed between rheological behavior and gela tion time measured by the invert ed tube method. Instead the modulus G remained fairly constant for all com positions in the region near the recorded gelation time. It was not expected that these data would correspond as the samples were subjected to different types and magnitudes of deformation in these tests. The modulus at test completion (120 minutes) was generally an order of magnit ude greater for corresponding compositions at higher vs. lower calcium concentra tion. ALG gels were significantly stiffer for the initial 60 to 80 minutes of the oscillatory te sts; however there was little difference between ALG and ALG52

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CMC after 120 minutes. ALG-HA gels had the lowest modulus of all three compositions at both crosslink densities. In oscillatory tests the intersec tion of G and G, G-G crossover (green circles in Figures 3-9 through 3-11), is considered to be a measur e of gel point (Tung and Dynes, 1982; Metzger, 2006). This point indicates the temperature or time (depending on the sample and test) at which there is a phase transition from viscous liquid behavior to elastic solid behavior at a critical extent of crosslinking reaction (Winter and Ch ambon, 1986). Beyond this point G is greater than G and the polymer behaves more like a viscous solid/gel than a fluid. The gel points for ALG, ALG-CMC and ALG-HA compositions ranged from 10 to 30 minutes (Table 3-4). The recorded values include five minutes added back to the ob served G-G crossover times to compensate for the time delay during test startup. The gel poi nts measured by rheometry were significantly lower than the gelation times observed by the inverted tube method. This is expected as the inverted tube method is not a measure of the actual onset of gelation but is rather an indication of the time at which the gel strength is suffi cient enough that the gel no longer flows upon inversion. Strong gels have been reported to show permanent rupture and failure under large deformations whereas weak gels are able to flow without fracture and rec over their solid, gel-like behavior (Ross-Murphy, 1995). This is consistent with observations from the inverted tube data, and it is believed that our compositions formed weak gels at the G-G crossover which were still deformable or pourable for an extended period of time beyond the gel point. Additional testing including replicates for each condition should be performed to get a more accurate representation of the rheological behavior of the ALG, ALG-CMC a nd ALG-HA compositions. An adequate estimate of the stiffness of the spinal cord is needed to effectively design biomaterials for neural tissue engineering to avoid a mismatch in properties that can exert 53

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physical stress on the cord. Also, it has been demonstrated in vitro that mechanical stiffness is inversely related to the rate of neurite extens ion within a gel (Balgude et al., 2001). Therefore implanted gels should be sufficiently complia nt to permit axonal re gneration. Mechanical properties are difficult to measure accurately due to the delicate nature of the tissue and significant stiffening of the cord even short time s after death (Dalton et al., 2002). A number of mechanical tests have been employed to investigat e the mechanical properties of the spinal cord under in vitro or in vivo conditions with great variability in the results. Despite the fact that tensile loading is not the primary mechanism of inju ry to the spinal cord, the majority of studies to estimate spinal cord modulus have been tensile tests with fewe r studies devoted to compression testing or other techniques (Cheng et al., 2008). The elastic modulus of the spinal cord includ ing the pia mater has been estimated to be between ~200 and 600 kPa depending on the host sp ecies, method of measurement and elapsed time before measurement after death of the host (Dalton et al., 2002). Th e modulus of the feline spinal cord has been appr oximated at 230 kPa using an in vivo tensile test (Chang et al., 1988). In later studies by Ozawa et al., elastic moduli of both white and gray matter of rabbit spinal cord were estimated to be ~ 3 kPa using an in situ pipette aspiration tec hnique developed for the assessment of soft biological tissue, and the pi a was estimated to have a modulus of ~2000 kPa (2001; 2004). The compressive modulus of the spinal cord has been estimated at 5 kPa using an in vivo technique (Hung et al., 1982). These results demonstrate the diversity in published data on the properties of the cord. To date, oscillatory testing as performed in the present research has been reported for brain tissue but not for the spin al cord, and therefore we are unable to directly compare our results to published data (Cheng et al., 2008). It is recomm ended that compressive 54

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modulus of the compositions studied here be measured under simulated physiological conditions in future studies to optimize the gel properties for use in the spinal cord. Morphology Pore structure is an important design consider ation for materials used in tissue engineering applications. The implanted scaffold or gel must have high fractional porosity and sufficiently large pores for optimal cell attachment and growth and to allow infiltration of host constituents to promote ingrowth of regenerating tissue (Woe rly et al., 1999). Scanning electron micrographs were taken of samples that were allowed to gel overnight in multi-well cell culture plates that were subsequently snap-frozen in liquid nitrogen and lyophilized. To get an accurate representation of the structure of a gel the morphology should be examined in a hydrated state under representative physiological conditions; however standard SEM was utilized to qualitatively examine pore size and mor phology of the ALG, ALG-CMC and ALG-HA compositions. SEM of a representative ALG sample is shown in Figure 3-14. The freeze-dried compositions were observed to have a porous st ructure with fairly homogeneous pore sizes uniformly distributed throughout. The pore structur e appeared to vary with gel composition but could not be confirmed due to distortion of some of the samples during sectioning. Further evaluation using a more appropriate technique for hydrated gels is recommended to elucidate the morphology and obtain quantifiable results. Summary Novel in situ-forming ALG-CMC and ALG-HA ge ls were prepared for the first time via gradual ionic crosslinking of ALG. Calcium ions were slowly released using the CaCO3-GDL internal gelation technique. Ge lation time was measur ed using the inverted tube method and ranged from ~1 to 2 hours at 37 C for all compos itions. The presence of CMC or HA in solution did not prevent crosslinking but did decrease gelation rate compared to pure ALG gels. 55

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Increasing the calcium ion concentr ation resulted in an increase d rate of gelation. Mechanical properties of the gels were evaluated using a c one-and-plate rheometer. The storage modulus G of the biopolymer gels gradually increased with time and wa s on the order of 104 and 105 Pa for low and high crosslink densities, respectively, after two hours. The compositions were concluded to form weak gels at the gel point which ranged from 10 to 30 minutes as determined by the crossover of G and G. SEMs of snap-frozen, fr eeze-dried gels revealed a fairly uniform pore structure but further study is re quired using an appropriate met hod to examine morphology in the hydrated state. 56

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Figure 3-1. Structure of algina te showing guluronic acid (G) and mannuronic acid (M) residues. Consecutive G residues form a V-shaped cavity. Figure 3-2. Egg-box model of ionic crosslinking of alginate. Divalent cations (circles) sit in Vshaped cavities formed by G blocks of adjacent chains. Figure 3-3. Chemical structure of glucono-lactone (GDL). 57

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Table 3-1. Concentration of polys accharides in biopolymer solutions Blend Designation [ALG] wt% [CMC] wt% [HA] wt% ALG 2.5% ALG-CMC1 1.9% 0.4 ALG-CMC2 1.3% 0.8 ALG-HA1 1.9% 0.1 ALG-HA2 1.3% 0.3 Table 3-2. Concentration of polysaccharides in biopolymer gels Gel Designation [ALG] wt% [CMC] wt% [HA] wt% ALG 2.0% ALG-CMC1 1.5% 0.3 ALG-CMC2 1.0% 0.6 ALG-HA1 1.5% 0.1 ALG-HA2 1.0% 0.2 58

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Figure 3-4. Schematic of alginate cr osslinking procedure with GDL and CaCO3. 59

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Figure 3-5. Chemical structure of carboxymethylce llulose (CMC). 60

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A B C Figure 3-6. Photograph of AL G crosslinking progression by the internal gelation method. A) Initially the ALG solution is a viscous liquid. B) and C) The gel stiffens with time as calcium ions are released and complex with -COOgroups in G residues. 61

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99 125 169 112 140 0 30 60 90 120 150 180 ALG ACM1 ACM2 AHA1 AHA2Gelation Time (min) 6 mM Ca2+A Figure 3-7. Gelation time at 37 C as a f unction of biopolymer composition. A) low Ca2+ (6 mM) B) high Ca2+ (8 mM). Compositions with gelati on time significantly different (p<0.05) from ALG are i ndicated by an asterisk. 64 83 105 73 87 0 30 60 90 120 150 180 ALG ACM1 ACM2 AHA1 AHA2Gelation Time (min) 8 mM Ca2+B 62

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Figure 3-8. Effect of temperature on gelation time of ALG-CMC compositions at high crosslink density (8 mM Ca2+). 85 134 173 64 83 105 0 30 60 90 120 150 180 210 ALG ACM1 ACM2Gelation Time (min) 25 C 37 C Table 3-3. Viscosity and gelation times of ALG-CMC compositions at 25 C Solution Viscosity at 25 C (cps) Gelation Time at 25 C (min) ALG 2860 85 ALG-CMC1 987 134 ALG-CMC2 1263 173 63

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1.0E 01 1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05 1.0E+06 0102030405060708090100110120G', G" (Pa)Time (min) G' (Pa) G" (Pa) 1.0E 01 1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05 1.0E+06 0102030405060708090100110120G', G" (Pa)Time (min)A G' (Pa) G" (Pa) B Figure 3-9. Modulus (G, G) of ALG gel as a function of time at 37 C. A) Low Ca2+ (6 mM ). B) High Ca2+ (8 mM). 64

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Figure 3-10. Modulus (G, G) of ALG-CMC ge l as a function of time at 37 C. A) Low Ca2+ (6 mM ) B) High Ca2+ (8 mM) 1.0E 01 1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05 1.0E+06 0102030405060708090100110120G', G" (Pa)Time (min) G' (Pa) G" (Pa) 1.0E 01 1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05 1.0E+06 0102030405060708090100110120G', G" (Pa)Time (min)G' (Pa) G" (Pa) 65

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1.0E 01 1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05 0102030405060708090100110120G', G" (Pa)Time (min) G' (Pa) G" (Pa)Figure 3-11. Modulus (G, G) of ALG-HA ge l as a function of time at 37 C. A) Low Ca2+ (6 mM ). B) High Ca2+ (8 mM). 1.0E 01 1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05 0102030405060708090100110120G', G" (Pa)Time (min)G' (Pa) G" (Pa) 66

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Figure 3-12. Storage modulus (G) of biopolymer compositions as a function of time at low crosslink density (6 mM Ca2+) at 37 C. 1.0E 01 1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05 0102030405060708090100110120G' (Pa)Time (min)ALG ALG CMC1 ALG HA1 67

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Figure 3-13. Storage modulus (G) of biopolymer compositions as a function of time at high crosslink density (8 mM Ca2+) at 37 C. 1.0E 01 1.0E+00 1.0E+01 1.0E+02 1.0E+03 1.0E+04 1.0E+05 1.0E+06 0102030405060708090100110120G' (Pa)Time (min) ALG ALG CMC1 ALG HA1 68

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Table 3-4. Gel point (G-G crossover) and gelation time (i nverted tube) for biopolymer compositions at 37 C. Solution Ca2+ concentration (mM) Gel point (min) Gelation time (min) ALG 6 11 99 ALG-CMC1 6 30 125 ALG-HA1 6 19 112 ALG 8 10 64 ALG-CMC1 8 20 83 ALG-HA1 8 13 73 69 Figure 3-14. SEM micrographs of snap-frozen freeze-dried ALG gel compositions. A) low (6 mM) Ca2+. B) high (8 mM) Ca2+.

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CHAPTER 4 IN VITRO EVALUATION OF INJECTABLE GEL COMPOSITIONS AS A TRANSPLANTATION MATRIX FOR NEURAL TISSUE REPAIR Introduction When designing materials for biomedical appl ications, evaluating behavior and properties under physiological conditions is critical to ensure th at the implant is non-toxic, will not elicit an adverse response in the body, and is adequate or e ffective for the intended application. It is not always feasible to conduct in vivo experiments, especially for sc reening or feasibility studies, therefore in vitro testing is an alternative for evaluating materials. It is important to match the experimental conditions to the actual in vivo environment as closely as possible to ensure reliability of the results. This chapter focuses on in vitro evaluation of injectable alginate-based gel compositions as matrices for neural tissue repair The primary objective was to ch aracterize the properties of the gels under simulated physiological conditions to determine their suitability for use as an injectable matrix for repairing the injured spinal cord. Swelling and dissolution of gels were evaluated in artificial cerebrospinal fluid (a CSF) at 37 C. The viability of Schwann cells dispersed in the gels was evaluate d using an alamarBlue assay. Materials and Methods In Vitro Swelling and Dissolution Swelling and dissolution of gels were evaluated in vitro in aCSF, ph 7.4 (Table 4-1) to mimic the physiological environment of the sp inal cord. The weights of 144 empty microcentrifuge tubes (1.5 mL) were measured a nd 0.5 mL of freshly prepared biopolymer composition was injected into each centrifuge tube (n = 3) immediatel y after crosslinking was initiated. The compositions were incubated overn ight at 37 C to ensu re that gelation was complete, and the centrifuge tube weights with ge l were determined. Initial wet gel weight, Wwet 70

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(0), was determined by subtracting the weight of the tubes from the weight of tubes plus gel. A set of tubes were then frozen and lyophilized an d used to determine the dry control gel weight, Wdry(0). To the remaining tubes 0.5 mL of aCSF wa s added which was pre-equilibrated to 37 C, and the tubes were placed in a biological cabin et. The fluid was change d daily, and the swollen gel weights, Wwet(t), were determined by subtraction af ter 1, 4, 7, 14 and 28 days of incubation. The corresponding dry gel weights, Wdry(t) were determined at the same timepoints on lyophilized gels. The degree of sw elling and dissolution were dete rmined as a function of time according to (4-1) and (4-2). % Swelling = W wet (t) W wet (0) x 100 (4-1) Wwet (0) % Dissolved = W dry (0) W dry (t) x 100 (4-2) Wdry (0) Primary Schwann Cell Culture Schwann cells were isolated from neonatal ra ts. P4 rat pups were sacrificed according to IACUC approved methods, and the sciatic nerves were dissected and dissociated according to established protocols (Notterpek et al., 1999). Nerves were dissected, stripped of connective tissue and epineurium, minced and digested at 37 C in a humidified atmosphere of 5% CO2 The digestion medium consisted of Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Grand Island, NY), 15% fetal calf serum (FCS) (Hyclone, Logan, UT), penicillin streptomycin (Gibco) and an enzyme cocktail of 0.03% coll agenase type III (Worthington, Lakewood, NJ), 0.1% hyaluronidase (Sigma), 1.25 units/mL dispase (Worthington). Cell suspensions were washed once and resuspended in culture medium (DMEM containing 10% FCS). Cells were then plated on poly-L-lysine (Sigma) coated plastic Petri-dish es in growth medium [DMEM with 10% FCS + 50 L/5mL bovine pituitary extract (glial growth factor) (Biomedical Technologies, Inc., 71

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Stroughton, MA) + 1 L/5mL forskolin (Calbioche m, La Jolla, CA)]. The medium was changed every other day. Cells were allowed to attach overnight and proliferat e for 7 or 8 days. Schwann cells were harvested by trypsinizati on. Culture medium was aspirated and plates were rinsed twice with DMEM. Trypsin/ethylen ediaminetetraacetic acid (EDTA) was added to detach cells, and plates were incubated at 37 C for 2 minutes. Proteolysis was stopped by adding 8 mL DMEM/10% fetal calf serum (FCS). Cell suspensions were transferred to 15 mL conical tubes and centrifuged for 5 minutes at 1200 rpm. Medium was aspirated, and the cell pellet was re-suspended in a known volume of Schwann cell growth medium. Cells were counted with a hemocytometer and adjusted to the desired final concentration. Cell Viability Sterile ALG, ALC-CMC and ALG-HA blends were prepared and combined with CaCO3 and GDL to initiate gelation. Immediately after adding GDL, 400 L of each biopolymer mixture was injected into the wells of a 24-well culture plate and allowed to gel overnight in a biological hood. Primary Schwann cells (pass 6) were harvested and re-suspended in growth medium at a concentration of 1 x 106cells in 25 mL. 400 L of cell suspension was added on top of gels and onto poly-L-lysine-coated plates (controls) at a density of 2 x 104 cells/well (n = 6 per condition). Viability of Schwann cells seeded on select biopolymer gels was determined using an alamarBlue assay (AbD Serotec, Oxford, UK) which incorporates a non toxic oxidation-reduction (REDOX) indicator that changes color in response to reduction by metabolically active cells. The assay protocol was adapted from Novikova et al (2006). 0Twenty four hour s after cell seeding, medium from control wells was replaced with 300 L of fresh growth medium containing 5% alamarBlue. Assay medium was added on top of gel compositions as the old medium was 72

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absorbed by the gels and could not be removed. Th e plates were returned to the incubator for 4 hours, and 100 L of the assay-containing medium was removed and transferred to a 96-well culture plate. Absorbance was measured at 570 nm and 600 nm using a Molecular Devices SpectraMax M5 muliplate reader, and viability was determined as percentage reduction of alamarBlue according to the suppliers protocol Viability was assayed at 24 and 48 hours after seeding. Cell Entrapment Feasibility Study An experiment was conducted to determine th e effects of culture medium on the gelling behavior of ALG gel. Four mL of sterile ALG solution and 4 mL of DMEM were added to CaCO3 (1 mL) in a 15 mL Nalgene container and ma gnetically stirred for 1 minute. One mL of a fresh GDL solution was added to initiate cr osslinking, and gelling behavior was visually observed by inverting tubes periodically. To assess the feasibility of entrapping cells within gel compositions, an ALG composition was combined with a Schwann cell suspension a nd crosslinked under aseptic conditions. Four mL of sterile ALG solution and 4 mL of Schwan n cells suspended in growth media were added to CaCO3 (1 mL) and magnetically s tirred for 1 minute. One mL of a fresh GDL solution was injected through a syringe fitted with a 0.22 m filter to initiate crosslinking, and the Schwann cell-biopolymer mixture was stir red for 20 seconds. Four hundred L of cell-gel mixture was injected into the wells of a 24 well culture plate and allowed to gel for 30 minutes in a biological cabinet at 37 C. Fresh media was then added, and Schwann cells were examined the next day using an inverted microscope. 73

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Results and Discussion Swelling and Dissolution Swelling and dissolution were ex amined at 37 C for biopolymer gels that were covered with aCSF 24 hours after crosslinking was initiated. The fluid was changed at the same time daily, and samples were weighe d after 1, 4, 7, 14 and 28 days. Swelling was apparent for many compositions as the gels expanded noticeably be yond their initial volume during the course of the experiment. The degree of swelling graduall y increased over time (Figure 4-1) and was greatest for gels crosslinked with low (6 mM) CaCO3. This behavior is consistent with what is expected for crosslinked polymers as the chains have greater flexibility when the crosslink density is low. As the degree of crosslinking increases, the gel becomes more rigid and the polymer network is less able to expand. This was also demonstrated by the rheological data presented in Chapter 3. After 28 days, swelling was greatest for ALG gels and decreased with decreasing ALG content. ALG-HA gels showed the lowest degree of swelling, and the ALGHA2 gel showed a minimal degree of swelling. HA is known to have poor mechanical properties and may have contributed to this behavior. The mass of the gels was found to be stable during the course of the study and did not undergo significant dissolution. For application in SCI repair, a minor degree of swelling is desi rable as significant expansion of an implanted gel or scaffold can a pply pressure to the cord that could potentially cause damage. Therefore the swelling behavior of the gel is an impor tant factor to be considered in optimizing composition. Cell Viability Schwann cell viability was assayed using al amarBlue (AB) at 24 and 48 hours after seeding onto biopolymer compositions. The AB assay is based on conversion of a nonfluorescent indicator dye, resazuri n (blue), which is converted to a fluorescent dye, resorufin 74

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(red), when reduced by metabolically active cells The assay is non-toxic and therefore can be used to continuously monitor cell viability over time (Ahmed et al., 1994). Schwann cell viability was determined as % reduction of AB according to: % reduction of alamarBlue = (O2 A1) (O1 A2) x 100 (4.1) (R1 N2) (R2 N1) where O1 = molar extinction coefficient of oxidized AB at 570 nm (80586), O2 = molar extinction coefficient of oxidized AB at 600 nm (117216), A1 = absorbance of test wells at 570 nm, A2 = absorbance of test wells at 600 nm, R1 = molar extinction coeffici ent of reduced AB at 570 nm (155677), R2 = molar extinction coefficient of reduced AB at 600 nm (14652), N1 = absorbance of negative control well at 570 nm, and N2 = absorbance of negative control well at 600 nm. The coefficients O1, O2, R1 and R2 we re obtained from the a ssay product literature. The absorbances measured for the blank wells used as references in determining cell viability indicate that these well may have been contaminated with cells during the plating procedure. As a result, the calcu lated reduction of AB did not range from 0 to 100% as expected, and therefore Schwann cell viability could not be quantitatively determined. However, it could be determined from the data (Figure 4-2) that Schwann cells survived on all compositions and increased in number after 48 hour s in culture. Viability appear ed to be the same for all biopolymer compositions but definite conclusions cannot be drawn without repeating the experiment. The assay procedure should be further improved to take into account absorption of medium into the gels which can also skew the results. Cell Entrapment Feasibility Study The effect of cell culture medium on the gelling behavior of the ALG-CaCO3-GDL system was investigated by mixing an ALG solution with DMEM prior to crossl inking. Crosslinking of ALG was observed to progress in a similar mann er to previous experiments without culture 75

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medium. The ALG composition did not flow after approximately one hour as reported for ALG gels prepared in water (Chapter 3). The DM EM formulation used (Gibco 11995) contains 1.8 mM CaCl2, which is an additional source of calcium ions for crosslinking ALG. This Ca2+ concentration should be taken into account when formulating and optimizing the composition in future experiments. ALG gels with dispersed Schwann cells had solidified when examin ed after 24 hours of incubation. A number of live Schw ann cells were apparent by microscopy as well as a smaller number of dead cells which were clus tered together (Fig ure 4-3). Previous in vitro studies have reported on the survival of Schwann cells incorpor ated within alginate gels prepared in DMEM containing 0.1 M CaCl2 (Mosahebi et al., 2001; Novikova et al., 2006). Our results are consistent with these findings and indicate that ALG-based solutions can be mixed with cells suspended in culture medium prior to adding CaCO3 and GDL to initiate crosslinking and that these compositions have the potential to support Schwann cells or other cells for transplantation in the injured CNS. Viability, proliferation and mor phology of encapsulated cells should be examined in depth in future studies. Summary Swelling and stability of ALG, ALG-CMC, and ALG-HA gels were evaluated in vitro as a function of composition and calcium ion concentr ation. Compositions were allowed to gel in microcentrifuge tubes, covered with aCSF, and in cubated at 37 C for 28 days. The swollen and freeze-dried weights of each gel were determined in triplicate at 1, 4, 7, 14 and 28 days of incubation with aCSF changed daily. The gels were found to be stable du ring the course of the study. Maximum swelling was observed for ALG ge ls while the degree of swelling increased with increasing ALG content and decreasing cross link density. Viability of Schwann cells seeded on top of biopolymer gels was examined usi ng an AlamarBlue assay. The potential for 76

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injectable ALG-CaCO3-GDL gels as transplantation matri ces was demonstrated by survival of encapsulated Schwann cells in vitro ; however viability could not be quantified. Future studies should investigate the viability, proliferation a nd morphology of Schwann ce lls or other cells of interest encapsulated within the gels. 77

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Table 4-1. Artificial cerebr ospinal fluid formulation Component Concentration Unit NaCl 148.0 mM KCl 3.0 mM CaCl2 2H20 1.4 mM MgCl2 0.8 mM Na2HPO4 1.5 mM NaH2PO4 0.2 mM BSA 0.1 mg/mL 78

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0% 20% 40% 60% 80% 100% 120% 140% 160% 180% 0 7 14 21 28SwellingTime (days) ALG ALG CMC1 ALG HA1 0% 20% 40% 60% 80% 100% 120% 140% 160% 180% 0 7 14 21 28SwellingTime (Days) ALG ALG CMC1 ALG CMC2 ALG HA1 ALG HA2A B Figure 4-1. Swelling of biopolymer compositions in artificial cerebrospinal fluid at 37 C. A) low (6 mM) Ca2+. B) high (8mM) Ca2+. 79

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0 50 100 150 200 250ALG loALG CMC1 loALG HA1 locontrolRelative Viability of Schwann Cells 24 hours 48 hours Figure 4-2. Relative viability of Schwann ce lls after 24 and 48 hours. A) low (6 mM) Ca2+. B) high (8mM) Ca2+. 0 50 100 150 200 250ALG hiALG CMC1 hiALG HA1 hicontrolRelative Viability of Schwann Cells 24 hours 48 hours 80

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Figure 4-3. Inverted microscope image of Schw ann cells encapsulated within an ALG gel. A number of live cells are visible as well as a clusters of dead cells (black). 81

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CHAPTER 5 IN VIVO EVALUATION OF AN INJECTABLE ALGINATE-BASED GEL COMPOSITION IN A CERVICAL SPINAL CORD CONTUSION INJURY MODEL Introduction There are approximately 12,000 new cases of SCI in the United States each year, primarily affecting young adults between the ages of 16 a nd 30 (NSCISC, 2008). The majority of SCIs are caused by contusion or compression of the cord due to impingement by the vertebral column and most frequently occur at the cervical level. Re spiratory impairment and associated complications are some of the most severe consequences of SCI, especially for upperto mid-cervical spinal cord trauma, and are the leadi ng causes of SCI mortality and morbidity (Zimmer et al., 2007). This makes evaluation of potential treatments for SCI in cervical injury models with application to respiratory function recove ry of utmost importance. A number of in vivo studies evaluating biopol ymer substrates for SCI repair have utilized transection type models in which an incision is ma de to partially or completely sever the spinal cord or to remove an entire se ction. A polymeric scaffold typical ly in the form of a sponge or pre-formed gel is subsequently implanted in to the lesion. The nature of hemisection and transection injuries make them id eal models for assessing transpla ntation of pre-formed gels and scaffolds that can be placed into the lesion. Furthermore, given the precision of these lesions, they serve as valuable proof-of-principles mode ls for studies aimed at promoting and evaluating axonal regeneration (Steward et al., 2003; Talac et al., 2004). However, this type of injury rarely occurs in human SCI. Compression or contusion in juries more closely resemble those injuries seen in humans, but few studies have investigat ed the use of biomatrices in such injuries. (Woerly et al., 2001; Borgens et al., 2002; Tysseling-Mat tiace et al., 2008). The objective of this study wa s to evaluate the potentia l of injectable ALG-based compositions as a matrix for neural tissue repair in vivo using a clinically relevant SCI model 82

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with important practical implications. Proof-o f-concept was demonstrated by injecting ALGCMC1 biopolymer gel into the contused adult rat cervical spinal cord. Animals received contusion injuries using an Infi nite Horizon (IH) Impactor at C3/C4 and were treated with gel one week later. Animals were allowed to reco ver for one week post-implantation, and tissue was examined after cresyl violent staining. Materials and Methods Biopolymer Gel Preparation ALG-CMC1 composition was prepared as prev iously described. Brie fly, ALG (2.5% w/v) and CMC (1.5% w/v) solutions were prepared in ultrapure water by high speed mechanical mixing at 1000 rpm for 12 hours. The moisture content of each pow der was determined beforehand to determine the correct weight of each polymer needed to give the desired concentration. ALG and CMC soluti ons were filtered into 250-mL Pyrex bottles using an air pressure filtration funnel and 10 m nylon filters. The filtered solutions were allowed to remain at room temperature for 24 hours and were sterilized in an autoclave on a programmed liquid cycle (20 minutes at 240F). Under sterile conditions in a biological hood, ALG (30 mL) and CMC (10 mL) solutions were combined by injecting into a 100 mL Pyre x bottle using 20 mL syringes and magnetically stirred for 1 hour to mix. A suspension of CaCO3 (8 mg) in 1 mL of ultrapure water was prepared in a 15 mL Nalgene container and sterilized by au toclaving. All materials were stored at room temperature and kept sealed to maintain sterility. On the day of animal surgery ALG-CMC1 solution and CaCO3 suspension were equilibrated to 37 C for 2 hours in a water bath. All procedures were carried out in a biological hood. The polysaccharide solution, 8 mL, was added to the CaCO3 suspension using a 10 mL syringe and magnetically stirred fo r 1 minute. A fresh solution of GDL (28 mg in 1 mL ultrapure 83

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water) was prepared and added by syringe filtration (0.22 m filter) to initiate gelation, and the mixture was stirred for an additional 20 seconds. The sterile ALG-CMC1 biopolymer composition was then immediately transporte d to the surgery suite for implantation. Contusion Injuries All procedures involving animals were conducted following NIH guidelines and with approval from the Institutional Anim al Care and Use Committee at th e University of Florida. All animal surgeries and histology were conducted w ith assistance from Barbara OSteen, Alex Jones and Drs. Paul Reier and Michael Lane (College of Medicine, Department of Neuroscience). Adult Female Sprague-Dawley rats (230 to 250g; n = 3) were obtained from Harlan-Scientific and housed in the Animal Care Facility at the McKnight Brain Institute, University of Florida. Animals were anesthetiz ed for all surgical pro cedures by injection of xylazine (3 mg subcutaneous; Phoenix Pharmaceu tical, Inc., St. Joseph, MO) and ketamine (90 mg/kg intraperitoneal; Fort Dodge An imal Health, Fort Dodge, IA). Following anesthesia, the surgical site was shaved and sterilized. All animals were then laid in the prone position on a heat-pad (maintai ned at 36 C). A dorsal incision was made from approximately the first to fifth cervical segm ent (C1-C5) and muscle overlying the vertebral column retracted. A laminectomy was then performed to remove the entire C3 vertebral segment and most of C4. A contusion was subsequently made at C3/C4 using the Infinite Horizon Pneumatic Impactor (Precision Systems & In strumentation, Lexington, KY) (Figure 5-1). Animals were secured in sterile clamps to posi tion the exposed cord beneath the impactor probe and the probe was raised approximately 5mm above the intact dura. Spinal cord contusions were then made in air at a preset force of 150 kilodyn es with zero dwell time The resulting impact force ranged from 151 to 159 kdynes. Following c ontusion, the muscle layers overlying the 84

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exposed cord were sutured with 4-0 Vicryl (Ethicon, Inc., Somerville, NJ), and the skin was closed with wound clips. Each ra t was given subcutaneous injectio ns of Lactated Ringers (5 ml) to prevent dehydration, yohimbine (0.4 mg; Lloyd La boratories, Shenandoah, IA) to reverse the action of xylazine, and buprenorphine (0.012 mg; Reckitt Benckiser Pharmaceutical, Inc., Richmond, VA) to reduce any post-operative pain. Rats were evaluated daily for overall postoperative health and well-being. Biopolymer Gel Injection One week following, animals were again anesth etized and the injury site re-exposed. The site of injury and cavitation was identified by bruising at the laminectomy site. A sterile, freshly prepared ALG-CMC1 gelling mixture was drawn in to a 1 mL Tuberculin syringe and backfilled into a Hamilton syringe with a 30-gauge needle. The filled syringe was mounted above the animal in a micromanipulator, and approximately 40-50 L of the biopolymer gelling composition was injected through the dura into the injury site of each animal. The needle was retracted when reflux was observed. The muscles were then re-sutured and the skin closed with would clips. Animals were left to recover for 1 week at which time they received a lethal dose of anesthetic. Tissue Resection and Histology Two weeks post injury (one week post-gel injection) the rats were euthanized with 0.4 ml of Beuthanasia-D Special euthanasia solution (78 mg pentobarbital sodium and 10 mg phenytoin sodium; Schering-Plough Animal Health Corp., Union, NJ). Spinal cord tissue taken from each rat was processed in a different manner to i nvestigate the best met hod for obtaining a quality section without loss of the gel. The methods were as follows: Animal 1) Tissue was cryoprotected through a seri es of sucrose steps without fixation. The animal was intracardially perfused with 15% sucr ose, dissected and stored in 15% sucrose for 24 85

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hours, then transferred into 30% sucrose. Once the tissue began to sink in the sucrose solution, it was considered ready to be frozen for sectioni ng. The tissue was frozen by immersion in cooled isopentane in a beaker on dry-ice. Animal 2) Tissue was dissected immediately following euthanasia and snap-frozen by immersion in cooled isopentane, without fixation or cryoprotection. Animal 3) Tissue was processed by perfusion with paraformaldehyde (4% w/v in 0.1 M PBS). The fixed tissue was subsequently dissected and stored in the same solution until ready for cryoprotections. The tissue was then cryoprotected by immersion in a series of sucrose solutions (as described above) and frozen in isopentane. Histology Frozen tissue was mounted in O.C.T. (Optimal Cutting Temperature, TissueTek) and sectioned (20 m thick, either transverse or longitudinal as indicated) at -20 C in a cryostat. Sections were immediately slide mounted and air dried. All sections were subsequently stained with cresyl violet. Following brie f immersion in distilled water, th e sections were incubated in cresyl violet for 2 minutes. Sections were then rinsed in distilled water, dehydrated in a series of alcohols, followed by xylene (2-5 minutes in each). Upon completi on all slides were coverslipped with permanent mounting medium (Richard-Allen Scientific). To assess the appearance of the gel stained with Cresyl Violet, some gel material was applied directly to clean microscope slides and processed fo r staining as described above. Microscopy All tissue was examined by brightfield microscopy using a Zeiss AxioPhot microscope. All images were taken with a Zeiss AxioCam digita l camera connected to a PC. Images were color corrected for white balance only. No other color correction profile s were used at any stage. Sections from each animal were qualitatively revi ewed for the presence of scaffold material. 86

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Results and Discussion Contusion Injuries In this study, injuries were in troduced in the cervical spinal cord of adult rats at C3/C4 using an Infinite Horizon (IH) impactor. The IH impactor is a compute r-controlled device that applies a set force to the exposed spinal cord of an immobilized subject, resulting in damage to both white and grey matter (Scheff et al., 2003; Scheff and Roberts, 2009). This device has the demonstrated ability to produce consistent cont usion injuries in small rodents and can impose graded degrees of damage by adjusting user-defined force settings. The type of injuries produced by the IH impactor and similar devices, rapid application of force to produce a contusion, are reported to accurately mimic injuries seen in humans (Stokes and Jakeman, 2002). The cervical contusion injury model used in this study has been well characterized in the laboratory of Dr. P. J. Reier (Department of Neuroscience at the Un iversity of Florida) a nd has proven useful for quantitative evaluation of resp iratory dysfunction following cerv ical SCI (El-Bohy et al., 1998; Lane et al., 2008). Because most incidents of spinal cord trauma are contusion/compression injuries, and over half of these in juries occur at cervical levels the model used in this work closely matches the majority of human cases of SCI and has significant clinical relevance and translational potential. Biopolymer Gel Injection This study represents the first reported study of injectable, in situ-forming ALG-based biopolymer compositions in the injured spinal co rd. Three rats received injections of ALGCMC1 gel crosslinked with 8 mM Ca2+ one week post-injury. Immediately after incorporating a fresh GDL solution, the ALG-CMC1 gel was brought into the surgical suite, and the first rat was treated within 15 minutes. Alt hough the biopolymer solutions exhi bit pseudoplastic behavior, the viscosity was too high to allow as piration directly into a Hamilton syringe fitted with a 33-gauge 87

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needle. Therefore the solution was loaded into a 1-mm syringe attached to a 20-gauge needle. The gel was then backfilled into the Hamilton sy ringe and injected through the dura until reflux was observed. The remaining animals were treat ed approximately 10 minutes apart using the same ALG-CMC1 sample. Pseudoplastic behavior and gradual increase in viscosity (see chapter 4) facilitated injection for at least 30 minutes after crosslinking was initiated, which allowed sufficient time to load the syringe before treating each animal. This flexibility in working time may prove critical in surgi cal situations where unexpected delays could arise. The gels used in this study are also advant ageous for treating actua l human cases of SCI because they are able to completely fill the ir regularly shaped volume of contusion lesions and can be delivered in a minimally invasive procedure. Scaffolds and semi-solid gels require sizing before implantation and cannot conform to the sh ape of the lesion which can affect integration with host tissue (Stabenfeldt et al., 2006). Mini mizing mismatch between the scaffold and host tissue may also be important for reducing the infl ammatory response (Hanson et al., 1996; Jain et al., 2006; Anderson, 2008). Histology In contrast to the deep violet appearance of cel ls within the spinal cord stained with cresyl violet, ALG-CMC1 gel was labeled a distinctive pink color (Figure 5-2). Tissue from Animal 1 was cryoprotected by a series of sucrose steps without fixation. This histological technique preserved and allowed clear visu alization of the gel which was differentiated from host tissue due to color of stain, confirming that the gel wa s successfully delivered to the injured spinal cord. ALG-CMC1 gel appeared to intermingle w ith host tissue and completely fill the contusion cavity (Figure 5-3). No rigid boundary between impla nt and host spinal cord tissue was apparent and evidence of gel was visible in sections rostra l to the lesion epicenter (Figure 5-4). This is likely due to prolonged liquid-li ke behavior of the ALG-CMC1 composition which would have 88

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allowed flow throughout the lesion before extensiv e crosslinking im mobilized the gel. Also, the gel did not appear to elicit a significant inflammatory response as there was no noticeable accumulation of cells al ong gel-tissue interfaces. Tissue from Animal 2 underwent the least am ount of histological processing whereby the tissue was snap-frozen immediately after the an imal was euthanized. ALG-CMC1 gel was not visible in this animal (Figure 5-5). Anim al 3 tissue was cryoprotected and fixed with paraformaldehyde. There was no gross evidence of gel in the lesion, however pink fragments were visible at higher magnificati ons indicating that gel was presen t in the lesion and most likely dissolved during the fixati on process (Figure 5-6). Previous studies in the laborat ory of Dr. Paul Reier demons trated the development of a significant cystic cavity one week post-injury in rats subjected to cervical contusion injury model used in this research (Figure 5-6, Figure 5-7). In this work, cy stic cavitation appeared to be minimal in animals treated with ALC-CMC1 gel. This suggests that the gel may have suppressed cavity progression; however this cannot be de termined conclusively without further study. Summary Contusion injuries represent the most common cases of human SCI, and the majority of these injuries occur at cervical levels. Howeve r, most neural tissue e ngineering studies have focused on thoracic level injuries and have utilized tr ansection lesions whic h are convenient for implantation of sponges or semi-solid gel scaffolds. This work is the first reported use of injectable ALG-based gel compositions for CNS repair. Feasibility of this approach was demonstrated in a clinically-relevant SCI model by successful injection of an ALG-CMC composition into cervical spinal cord contusion injuries in adult rats one week post-injury. Injectability allowed the gels to be delivered into the injured cord through the dura, minimizing the invasiveness of the procedure. The biopolymer composition integrated well with host tissue 89

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and did not appear to stimulate a significant inflammatory res ponse. Also, treatment with the ALG-CMC gel appeared to suppres s cystic cavitation. The results of this study indicate that insitu forming ALG-based compositions have sign ificant potential for CNS tissue repair and should undergo further investigation. 90

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Figure 5-1. Infinite Horizon spinal cord imp actor. [Reprinted with permission from Springer Science + Business Media: Scheff, S., and Roberts, K. N., 2009. Infinite horizon spinal cord contusion model. In: Chen, J ., Xu, X.-M., Xu, Z. C., and Zhang, J. H., (Eds.), Animal models of acute neurologi cal injuries (Page 424, Figure 1). Humana Press, New York.] 91

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Figure 5-2. Cresyl violet stain of ALG-CMC1 gel showing pink coloration. 92

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Figure 5-3. Tissue near the lesion epicenter stained with cresyl violet one week post-treatment with ALG-CMC1 gel (A). Panels (B) and (C) are details. Tissue was cryoprotected, but not fixed. Gel implants integrated well with host tissue. 93

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A B Figure 5-4. Tissue rostral to lesi on epicenter stained with cresyl violet one week post-treatment with ALG-CMC1 gel (A). Panel (B) is a deta il showing a fragment of the gel (arrow). Tissue was cryoprotected, but not fixed. 94

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Figure 5-5. Tissue near lesion epi center stained with cresyl violet one week post-treatment with ALG-CMC1 gel. Tissue was not cryoprotected or fixed. 95

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Figure 5-6. Tissue near lesion epi center stained via cresyl violet one week post-treatment with ALG-CMC1 gel. Tissue was fixed with paraformaldye and subsequently cryoprotected. Evidence of ge l (pink) is not apparent (A) except under higher magnification (B) and (C). 96

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Figure 5-7. Cresyl violet staining of contusion lesion tissue from an untreated subject one week post-injury (from a prior st udy). Substantial tissue loss is apparent at the lesion epicenter (B) and in rostral (A) and caudal (C) directions. 97

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98 Figure 5-8. Cresyl violet staining of longitudina l section from an untre ated subject one week post-contusion injury in a prior study. Signi ficant tissue loss is apparent close to the injured surface (A) which declines moving ventrally thr ough the cord (B) and (C).

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CHAPTER 6 CONCLUSIONS A variety of biomaterials constr ucts have been investigated for neural tissue repair mostly in the form of sponge-like scaffolds or pre-formed gels which must be cut to size and require incision of the spinal cord for implantation. The purpose of this research was to develop and characterize biopolymer gel compositions for mini mally-invasive delivery to injured CNS tissue. The primary goal was to prepare injectable ALG-ba sed gels that can be combined with viable cells, bioactive molecules or drugs and bridge the lesion, prevent or reduce cystic cavitation and provide a favorable terrain fo r axonal regeneration. The follo wing conclusions were drawn: In situ-forming ALG, ALG-CMC and ALG-HA ge ls suitable for soft tissue engineering were synthesized via gradual ioni c crosslinking of ALG with CaCO3 and GDL. The compositions studied were injectable through a 22-gauge needle prior to crosslinking. Gelation was studied using the inverted tube method as a function of composition, calcium ion concentration, and temperature. Gelation time was extended as the amount of alginate in the gel decreased (p<0.05). Increasing the concentration of Ca2+ from 6 mM to 8 mM resulted in a significant decrease in gelation time (p<0.05). Gelation rate at 37 C was faster th an at room temperature (25 C). Swelling behavior in artifici al cerebrospinal fluid varied by composition and increased with increasing ALG content and decreasing concentration of CaCO3. Potential for cellular transplantation was shown by survival of encapsulated Schwann cells in vitro Proof of concept was demonstrated by injec tion of ALG-CMC1 gel into the contused cervical spinal cord of adult rats one week post-injury. The compositions integrated well with host tissue and do not stimulate a significant inflammatory response. In situ-forming gels based on ALG-CaCO3-GDL are promising candidates for neural tissue repair and should unde rgo further investigation. 99

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CHAPTER 7 FUTURE WORK The focus of this work was to explore the suitability and feasibility of utilizing injectable, in situ-forming ALG, ALC-CMC an d ALG-HA gels for repair of in jured nervous system tissue. The promising results of the current studies s uggest that these biopolymer compositions should undergo further investigation. The following are possible future resear ch directions for consideration: Extended in vivo studies of gel compositions. Pilot studies have shown that ALG-based gel compositions can be injected into the inju red spinal cord and integrate well with host tissue. More extensive studies implanting gels with and without Schwann cells should be conducted. Immunohistochemical analysis and eval uation of functional outcomes at several time points are necessary for fully eval uating the suitability of these gels. Attachment of bioactive peptides. Covalent attachment of bioactive peptides, e.g. laminin, fibronectin, or peptide sequences e.g. IKVAV, to biopolymer compositions should be explored to enhance cellular adhesion within the gel environment. Incorporation of porous CaCO3 microparticles. Porous CaCO3 microparticles embedded within alginate solutions have recently been shown to be an effective carrier for sustained release of ibuprofen in vitro (Wang et al., 2008). These porous microparticles could potentially be utilized as a reservoir for controlled delivery of neurotrophins, chondroitinase ABC to degrade inhibitory C SPGs, or drugs (e.g. methylprednisolone) while simultaneously serving as a source of calcium ions for in situ gelation of alginate. 100

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LIST OF REFERENCES Agudo, M., Woodhoo, A., Webber, D., Mirsky, R ., Jessen, K. R., and Mcmahon, S. B., 2008. Schwann cell precursors transplanted into the injured spinal cord multiply, integrate and are permissive for axon growth. Glia 56 1263-1270. Ahmed, S. A., R. M. Gogal, J., and Walsh, J. E., 1994. A new rapid and simple non-radioactive assay to monitor and determine the prolifer ation of lymphocytes: an alternative to [3H]thymidine incorporation as say. J. Immunol. Methods 170 211-224. Alper, J., 2009. Geron gets green light for hum an trial of ES cell-derived product. Nat. Biotechnol. 27 213-214. Anderson, J. M., 2008. Biocompatibility and biorespons e to biomaterials. In: Atala, A., Lanza, R., Nerem, R., and Thomson, J. A., (Eds.) Principles of regenerative medicine. Academic Press, New York, pp. 704-723. Archibald, S. J., Shefner, J., Krarup, C., and Madison, R. D., 1995. Monkey median nerve repaired by nerve graft or collagen nerve guide tube. J. Neurosci. 15 4109-4123. Asher, R., and Bignami, A., 1991. Localization of hyaluronate in primary glial cell cultures derived from newborn ra t brain. Exp. Cell Res. 195 401-411. Ashton, R. S., Banerjee, A., Punyani, S., Schaffer, D. V., and Kane, R. S., 2007. Scaffolds based on degradable alginate hydrogels and poly(l actide-co-glycolide) microspheres for stem cell culture. Biomaterials 27 5518-5525. Athanasiou, K. A., Niederauer, G. G., and Agrawal, C. M., 1996. Sterilization, toxicity, biocompatibility and clinical applications of polylactic acid/ polyglycolic acid copolymers. Biomaterials 17 93-102. Bakshi, A., Fisher, O., Dagci, T., Himes, B. T ., Fischer, I., and Lowman, A., 2004. Mechanically engineered hydrogel scaffolds for axonal growth and angiogenesis afte r transplantation in spinal cord injury. J. Neurosurg. Spine 1 322-329. Balazs, E. A., and Denlinger, J. L., 1989. Clin ical uses of hyaluronan. In: Evered, D., and Whelan, J., (Eds.), The biology of hyaluronan. Wiley, New York, pp. 265-280. Balgude, A. P., Yu, X., Szymanski, A., and Bellamkonda, R. V., 2001. Ag arose gel stiffness determines rate of DRG neurite extens ion in 3D cultures. Biomaterials 22 1077-1084. Ban, D.-X., Kong, X.-H., Feng, S.-Q., Ning, G.-Z., Chen, J.-T., and Guo, S.-F., 2009. Intraspinal cord graft of autologous activ ated Schwann cells efficiently promotes axonal regeneration and functional recovery after rat's spinal cord injury. Brain Res. 1256 149-161. Baptiste, D. C., Austin, J. W., Zhao, W., Na hirny, A., Sugita, S., and Fehlings, M. G., 2009. Systemic polyethylene glycol promotes neurol ogical recovery and ti ssue sparing in rats after cervical spinal cord inju ry. J. Neuropathol. Exp. Neurol. 68 661-676. 101

PAGE 102

Baptiste, D. C., and Fehlings, M. G., 2006. Pharm acological approaches to repair the injured spinal cord. J. Neurotrauma 23 318-334. Bareyre, F. M., 2008. Neuronal repair and replacement in spinal co rd injury. J. Neurol. Sci. 265 63-72. Barnett, S. C., and Chang, L., 2004. Olfactory ensheathing cells and CNS repair: going solo or in need of a friend? Trends Neurosci. 27 54-60. Bartolomei, J. C., and Greer, C. A., 2000. Olfactor y ensheathing cells: bridging the gap in spinal cord injury. Neurosurgery 47 1057. Baumann, M. D., Kang, C. E., Stanwick, J. C., Wang, Y., Kim, H., Lapitsky, Y., and Shoichet, M. S., 2009. An injectable drug delivery platfo rm for sustained combination therapy. J. Controlled Release 138 205-213. Bellamkonda, R., Ranieri, J. P., Bouche, N., and Aebischer, P., 1995. Hydrogel-based threedimensional matrix for neural cells. J. Biomed. Mater. Res. 29 663-671. Benfey, M., and Aguayo, A. J., 1982. Extensive elongation of axons from rat brain into peripheral nerve grafts. Nature 296 150-152. Bignami, A., and Asher, R., 1992. Some observations on the locali zation of hyaluronic acid in adult, newborn and embryonal rat brain. Int. J. Dev. Neurosci. 10 45-57. Bignami, A., Asher, R., and Perides, G., 1992. Th e extracellular matrix of rat spinal cord: a comparative study on the localization of hyaluronic aci d, glial hyaluronate-binding protein, and chondroitin sulfate proteoglycan. Exp. Neurol. 117 90-93. Bonaventure, J., Kadhom, N., Cohen-Solal, L., Ng, K. H., Bourguignon, J., Lasselin, C., and Freisinger, P., 1994. Reexpression of cartilage -specific genes by de differentiated human articular chondrocytes cultured in alginate beads. Exp. Cell Res. 212 97-104. Borgens, R. B., and Bohnert, D., 2001. Rapid recovery from spinal cord injury after subcutaneously administered polyeth ylene glycol. J. Neurosci. Res. 66 1179-1186. Borgens, R. B., and Shi, R., 2000. Immediate recove ry from spinal cord in jury through molecular repair of nerve membranes with polyethylene glycol. FASEB J. 14 27-35. Borgens, R. B., Shi, R., and Bohnert, D., 2002. Be havioral recovery from spinal cord injury following delayed application of pol yethylene glycol. J. Exp. Biol. 205 1-10. Bracken, M., 2002. Steroids for acute spinal cord injury, Cochrane Database of Systematic Reviews CD001046. John Wiley & Sons, Ltd. Bracken, M. B., Collins, W. F., Freeman, D. F., Shepard, M. J., Wagner, F. W., Silten, R. M., Hellenbrand, K. G., Ransohoff, J., Hunt, W. E ., Perot, P. L., Jr., and et, a., 1984. Efficacy of methylprednisolone in acute spinal cord injury. JAMA 251, 45-52. 102

PAGE 103

Bracken, M. B., Shepard, M. J., Collins, W. F., Holford, T. R., Young, W., Baskin, D. S., Eisenberg, H. M., Flamm, E., Leo-Summers, L., Mar oon, J., and et al., 1990. A randomized, controlled trial of methylprednisolo ne or naloxone in the treatment of acute spinal-cord injury. Results of the Second Na tional Acute Spinal Cord Injury Study. N. Engl. J. Med. 322 1405-1411. Bracken, M. B., Shepard, M. J., Holford, T. R., Leo-Summers, L., Aldrich, E. F., Fazl, M., Fehlings, M., Herr, D. L., Hitchon, P. W., Mars hall, L. F., Nockels, R. P., Pascale, V., Perot, P. L., Jr., Piepmeier, J., Sonntag, V. K., Wagner, F., Wilberger, J. E., Winn, H. R., and Young, W., 1997. Administration of met hylprednisolone for 24 or 48 hours or tirilazad mesylate for 48 hours in the treatment of acute spinal cord injury. Results of the Third National Acute Spinal Cord Injury Randomized Controlled Trial. National Acute Spinal Cord Injury Study. JAMA 277 1597-1604. Bunge, M. B., 2008. Novel combination strategies to repair the injured mammalian spinal cord. J. Spinal Cord Med. 31, 262-269. Burdick, J. A., Ward, M., Liang, E., Young, M. J ., and Langer, R., 2006. Stimulation of neurite outgrowth by neurotrophins delivered fr om degradable hydrogels. Biomaterials 27 452459. Burns, J. W., Skinner, K., Colt, J., Sheidlin, A., Bronson, R., Yaacobi, Y., and Goldberg, E. P., 1995. Prevention of tissue injury and postsurgi cal adhesions by precoating tissues with hyaluronic acid solutions. J. Surg. Res. 59 644-652. Busch, S. A., and Silver, J., 2007. The role of extracellular matrix in CNS regeneration. Curr. Opin. Neurobiol. 17, 120-127. Chang, G.-L., Hung, T.-K., and Feng, W. W., 1988. An in-vivo measurement and analysis of viscoelastic properties of the spinal cord of cats. J. Biomech. Eng. 110 115-122. Chau, C. H., Shum, D. K. Y., Li, H., Pei, J., Lui, Y. Y., Wirthlin, L., Chan, Y. S., and Xu, X. M., 2004. Chondroitinase ABC enhances axonal re growth through Schwann cell-seeded guidance channels after spinal cord injury. FASEB J. 18 194-196. Chen, B. K., Knight, A. M., de Ruiter, G. C. W., Spinner, R. J., Yaszemski, M. J., Currier, B. L., and Windebank, A. J., 2009. Axon regeneration thr ough scaffold into distal spinal cord after transection. J. Neurotrauma In Press. Cheng, M., Deng, J., Yang, F., Gong, Y., Zh ao, N., and Zhang, X., 2003. Study on physical properties and nerve cell affinity of compos ite films from chitosan and gelatin solutions. Biomaterials 24 2871-2880. Cheng, S., Clarke, E. C., and Bilston, L. E., 2008. Rheological properties of the tissues of the central nervous system: a review. Med. Eng. Phys. 30, 1318-1337. 103

PAGE 104

Chenite, A., Buschmann, M., Wang, D., Chaput, C., and Kandani, N., 2001. Rheological characterisation of thermogelling chitosa n/glycerol-phosphate solutions. Carbohydr. Polym. 46, 39-47. Chenite, A., Chaput, C., Wang, D., Combes, C., Buschmann, M. D., Hoemann, C. D., Leroux, J. C., Atkinson, B. L., Binette, F., and Selmani, A., 2000. Novel injectab le neutral solutions of chitosan form biodegradable gels in situ. Biomaterials 21 2155-2161. Choi, G.-H., Youn, Y.-H., Kim, D.-Y., Son, H.-S., Kim, H.-T., and Kim, J.-K., 2006. Alginate gel decreases chondroitin sulfate proteoglyc an immunoreactivity following spinal cord injury: preliminary study. Tissue Eng. Reg. Med. 3 472-477. Comolli, N., Neuhuber, B., Fischer, I., and Lowm an, A., 2008. In vitro analysis of PNIPAAmPEG, a novel, injectable scaffold for spinal cord repair. Acta Biomater. 5 1046-1055. Coutts, M., and Keirstead, H. S., 2008. Stem cells for the treatment of sp inal cord injury. Exp. Neurol. 209, 368-377. Crompton, K. E., Goud, J. D., Bellamkonda, R. V., Gengenbach, T. R., Finkelstein, D. I., Horne, M. K., and Forsythe, J. S., 2007. Polylysi ne-functionalised ther moresponsive chitosan hydrogel for neural tissue e ngineering. Biomaterials 28 441-449. Crompton, K. E., Tomas, D., Finkelstein, D. I., Marr, M., Forsythe, J. S., and Horne, M. K., 2006. Inflammatory response on injection of ch itosan/GP to the brain. J. Mater. Sci. Mater. Med. 17 633-639. Cui, F. Z., Tian, W. M., Hou, S. P., Xu, Q. Y., and Lee, I.-S., 2006. Hyaluronic acid hydrogel immobilized with RGD peptides for brain tissue engineering. Journal of Materials Science Materials in Medicine 17 1393. dAyala, G. G., Malinconico, M., and Laurienzo, P., 2008. Marine derived polysaccharides for biomedical applications: chemical modification approaches. Molecules 13, 2069-2106. Dalton, P. D., Flynn, L., and Shoichet, M. S., 2002. Manufacture of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) hydrogel tubes for use as nerve guidance channels. Biomaterials 23 3843-3851. Dalton, P. D., Flynn, L., and Shoichet, M. S., 2002. Manufacture of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) hydrogel tubes for use as nerve guidance channels. Biomaterials 23 3843-3851. Darian-Smith, C., 2009. Synaptic plasticity, neuroge nesis, and functional r ecovery after spinal cord injury. Neuroscientist 15 149-165. David, S., and Aguayo, A. J., 1981. Axonal elongati on into peripheral nervous system "bridges" after central nervous system in jury in adult rats. Science 214, 931-933. 104

PAGE 105

Deshpande, D. M., Kim, Y. S., Martinez, T., Carmen, J., Dike, S., Shats, I., Rubin, L. L., Drummond, J., Krishnan, C., Hoke, A., Maragaki s, N., Shefner, J., Rothstein, J. D., and Kerr, D. A., 2006. Recovery from paralysis in adult rats using embryonic stem cells. Ann. Neurol. 60, 32-55. Dezawa, M., 2002. Central and peripheral nerve regeneration by transplantation of Schwann cells and transdifferentiated bone marro wstromal cells. Anat. Sci. Int. 77 12-25. Dhoot, N. O., Tobias, C. A., Fischer, I., and Wheatley, M. A., 2004. Peptide-modified alginate surfaces as a growth permissive substrate for neurite outgrowth. J. Biomed. Mater. Res. A 71A 191-200. Dillon, G. P., Xiaojun, Y., Sridharan, A., Rani eri, J. P., and Bellamkonda, R. V., 1998. The influence of physical structure and charge on neurite extens ion in a 3D hydrogel scaffold. J. Biomater. Sci. Polym. Ed. 9 1049-1069. Dougherty, K. D., Dreyfus, C. F., and Black, I. B., 2000. Brain-derived neur otrophic factor in astrocytes, oligodendrocytes, and microgl ia/macrophages after spinal cord injury. Neurobiol. Dis. 7, 574-585. Draget, K. I., stgaard, K., and Smidsrd, O., 1989. Alginate-based solid media for plant-tissue culture. Appl. Micr obiol. Biotechnol. 31 79-83. Draget, K. I., stgaard, K., and Smidsrd, O., 1990. Homogeneous alginate gels: A technical approach. Carbohydr. Polym. 14, 159-178. El-Bohy, A. A., Schrimsher, G. W., Reier, P. J., and Goshgarian, H. G., 1998. Quantitative assessment of respiratory function following cont usion injury of the cervical spinal cord. Exp. Neurol. 150 143-152. Fawcett, J. W., and Asher, R. A., 1999. The glial scar and central nervous system repair. Brain Res. Bull. 49 377-391. FDA, 2002. 21CFR184.1318 Title 21 food and drugs, pp. 502. Feron, F., Perry, C., Cochrane, J., Licina, P ., Nowitzke, A., Urquhart, S., Geraghty, T., and Mackay-Sim, A., 2005. Autologous olfactory ensheathing cell transp lantation in human spinal cord injury. Brain 128 2951-2960. Figliuzzi, M., Plati, T., Cornolti, R., Adobati, F., Fagiani, A., Rossi, L., Remuzzi, G., and Remuzzi, A., 2006. Biocompatibility and function of microencapsulated pancreatic islets. Acta Biomater. 2, 221-227. Franssen, E. H. P., de Bree, F. M., and Verh aagen, J., 2007. Olfactory ensheathing glia: their contribution to primary olfactory nervous sy stem regeneration and their regenerative potential following transplantation into th e injured spinal cord. Brain Res. Rev. 56 236258. 105

PAGE 106

Friedman, J. A., Windebank, A. J., Moore, M. J., Spinner, R. J., Currier, B. L., and Yaszemski, M. J., 2002. Biodegradable polymer grafts for su rgical repair of the injured spinal cord. Neurosurgery 51, 742-752. Garca-Alas, G., Lin, R., Akrimi, S. F., Stor y, D., Bradbury, E. J., and Fawcett, J. W., 2008. Therapeutic time window for the application of chondroitinase ABC after spinal cord injury. Exp. Neurol. 210 331-338. Garca-Alas, G., Lpez-Vales, R., Fors, J., Navarro, X., and Verd, E., 2004. Acute transplantation of olfactory en sheathing cells or Schwann cells promotes recovery after spinal cord injury in the rat. J. Neurosci. Res. 75 632-641. Gautier, S. E., Oudega, M., Fragoso, M., Chapon, P., Plant, G. W., Bunge, M. B., and Parel, J.M., 1998. Poly(alpha-hydroxyacids) for applicati on in the spinal cord: resorbability and biocompatibility with adult rat Schwann cells and spinal cord. J. Biomed. Mater. Res. 42 642-654. Geller, H. M., and Fawcett, J. W., 2002. Building a bridge: engineering spinal cord repair. Exp. Neurol. 174 125-136. Gerndt, S. J., Rodriguez, J. L., Pawlik, J. W., Taheri, P. A., Wa hl, W. L., Micheals, A. J., and Papadopoulos, S. M., 1997. Consequences of high -dose steroid therapy for acute spinal cord injury. J. Trauma 42 279-284. Giovanini, M. A., Reier, P. J., Eskin, T. A., Wirth, E., and Anderson, D. K., 1997. Characteristics of human fetal spinal cord gr afts in the adult rat spinal cord: influences of lesion and grafting conditions. Exp. Neurol. 148 523-543. Grandpre, T., and Strittmatter, S. M., 2001. Nogo: a molecular determinant of axonal growth and regeneration. Neuroscientist 7 377-386. Grant, G. T., Morris, E. R., Rees, D. A., Smith, P. J. C., and Thom, D., 1973. Biological interactions between polysacch arides and divalent cations: the egg-box model. FEBS Lett. 32, 195-198. Guest, J. D., Herrera, L., Margitich, I., Oliv eria, M., Marcillo, A., and Casas, C. E., 2008. Xenografts of expanded primate olfactory ensh eathing glia support transient behavioral recovery that is independent of serotonergic or corticospinal axona l regeneration in nude rats following spinal cord transection. Exp. Neurol. 212 261-274. Guest, J. D., Rao, A., Olson, L., Bunge, M. B ., and Bunge, R. P., 1997. The ability of human Schwann cell grafts to promote regeneration in the transected nude rat spinal cord. Exp. Neurol. 148, 502-522. Guo, J.-H., Skinner, G. W., Harcum, W. W., and Barnum, P. E., 1998. Pharmaceutical applications of naturally occurring water-so luble polymers. Pharm. Sci. Tech. Today 1 254-261 106

PAGE 107

Guo, J., Su, H., Zeng, Y., Liang, Y.-X., Wong, W. M., Ellis-Behnke, R. G., So, K.-F., and Wu, W., 2007. Reknitting the injured spinal cord by self-assembling peptide nanofiber scaffold. Nanomed. Nanotechnol. Biol. Med. 3, 311-321. Gupta, D., Tator, C. H., and Shoichet, M. S., 2006. Fast-gelling injectable blend of hyaluronan and methylcellulose for intrathecal, locali zed delivery to the injured spinal cord. Biomaterials 27 2370-2379. Haipeng, G., Yinghui, Z., Jianchun, L., Yandao, G ., Nanming, Z., and Xiufang, Z., 2000. Studies on nerve cell affinity of chitosan-derived materials. J. Biomed. Mater. Res. A 52 285295. Hall, E. D., 1992. The neuroprotective pharmacol ogy of methylprednisolone. J. Neurosurg. 76 13-22. Hall, E. D., and Springer, J. E., 2004. Neuroprotecti on and acute spinal cord injury: a reappraisal. NeuroRX 1 80-100. Hall, S., 2001. Nerve repair: a neurobiologist's view. J. Hand Surg. B. 26 129-136. Hanson, S., Lalor, P. A., Niemi, S. M., Northup, S. J., Ratner, B. D., Spector, M., Vale, B. H., and Willson, J. E., 1996. Testing biomaterials. In: Ratner, B. D., Hoffman, A. S., Schoen, F. J., and Lemons, J. E., (Eds.), Biomateria ls science: an introduction to materials in medicine. Academic Press, San Diego, pp. 215-242. Harvey, A. R., 2000. Use of cell/polymer hybrid stru ctures as conduits for regenerative growth in the central nervous system. In: Saunders, N., and Dziegielewska, K. M., (Eds.), Degeneration and regeneration in the ner vous system Harwood Academic Publishers, Amsterdam, pp. 191-203. Hej l, A., Lesn, P., P dn, M., ed, J., Zme nk, J., Jendelov, P., Michlek, J., and Sykov, E., 2009. Macroporous hydrogels based on 2-hydr oxyethyl methacrylate. part 6: 3D hydrogels with positive and negative surface charges and polyelectrolyte complexes in spinal cord injury repair J. Mater. Sci. Mater. Med. 20 1571-1577. Hej l, A., Urdzikova, L., ed, J., Lesn, P., P dn, M., Michlek, J., Burian, M., Hajek, M., Zme nk, J., Jendelov, P., and Sykov, E., 2008. Acute and delayed implantation of positively charged 2-hydroxyethyl methacrylate sca ffolds in spinal cord injury in the rat. J. Neurosurg. Spine 8 67-73. Hercules, 1999. Aqualon(R) sodium carboxymethylcel lulose: physical and chemical properties. Wilmington, DE. Hou, S., Tian, W., Xu, Q., Cui, F., Zhang, J., Lu, Q., and Zhao, C., 2006. The enhancement of cell adherence and inducement of neurite outgrowth of dorsal root ganglia co-cultured with hyaluronic acid hydrogels modified with Nogo-66 receptor antagonist in vitro. Neuroscience 137, 519-529. 107

PAGE 108

Hou, S., Xu, Q., Tian, W., Cui, F., Cai, Q., Ma, J., and Lee, I.-S., 2005. The repair of brain lesion by implantation of hyaluronic acid hydrogels modified with laminin. J. Neurosci. Methods 148, 60-70. Houweling, D. A., Lankhorst, A. J., Gispen, W. H., Br, P. R., and Joosten, E. A. J., 1998. Collagen containing neurotrophin -3 (NT-3) attracts regrowin g injured corticospinal axons in the adult rat spinal cord and promotes partial functi onal recovery. Exp. Neurol. 153 49-59. Houweling, D. A., van Asseldonk, J. T. H., Lankhorst, A. J., Hamers, F. P. T., Martin, D., Br, P. R., and Joosten, E. A. J., 1998. Local applic ation of collagen cont aining brain-derived neurotrophic factor decreases th e loss of function after spinal cord injury in the adult rat. Neurosci. Lett. 251, 193-196. Hugenholtz, H., 2003. Methylprednisolone for acute sp inal cord inju ry: not a standard of care. CMAJ 168, 1145-1146. Hung, T. K., Lin, H. S., Bunegin, L., and Albi n, M. S., 1982. Mechanical and neurological response of cat spinal cord under static loading. Surg. Neurol. 17 213-217. Hurtado, A., Moon, L. D. F., Maquet, V., Blits, B., Jerome, R., and Oudega, M., 2006. Poly (d,llactic acid) macroporous guidance scaffolds seeded with Schwann cells genetically modified to secrete a bi-functional neurotroph in implanted in the completely transected adult rat thoracic spinal cord. Biomaterials 27 430-442. Hynes, S. R., McGregor, L. M., Rauch, M. F., and Lavik, E. B., 2007. Photopolymerized poly(ethylene glycol)/poly(L-lys ine) hydrogels for the delivery of neural progenitor cells. J. Biomater. Sci.-Polym. Ed. 18 1017-1030. Ikegami, T., Nakamura, M., Yamane, J., Katoh, H., Okada, S., Iwanami, A., Watanabe, K., Ishii, K., Kato, F., Fujita, H., Toyomi, T., Okano, H. J., Toyama, Y., and Okano, H., 2005. Chondroitinase ABC combined with neural stem /progenitor cell transplantation enhances graft cell migration and outgrow th of growth-associated prot ein-43-positive fibers after rat spinal cord injur y. Eur. J. Neurosci. 22 3036-3046. Integra, 2005. NeuraGen nerve guide: advanced so lutions for peripheral nerve repair. Integra LifeSciences Corp., Plainsboro, NJ, pp. 1-4. ISP, 2000. Alginates: products for scientific water control. Internationa l Specialty Products, San Diego, CA. Jain, A., Kim, Y.-T., McKeon, R. J., and Bellam konda, R. V., 2006. In situ gelling hydrogels for conformal repair of spinal cord defects, a nd local delivery of BDNF after spinal cord injury. Biomaterials 27 497-504. Ji, B., Li, M., Budel, S., Pepinsky, R. B., Walus, L., Engber, T. M., Strittmatter, S. M., and Relton, J. K., 2005. Effect of combined treatm ent with methylprednisolone and soluble Nogo-66 receptor after rat spinal co rd injury. Eur. J. Neurosci. 22 587-594. 108

PAGE 109

Jimenez Hamann, M. C., Tator, C. H., and Shoichet M. S., 2005. Injectable intrathecal delivery system for localized administra tion of EGF and FGF-2 to the injured rat spinal cord. Exp. Neurol. 194, 106-119. Jimenez Hamann, M. C., Tsai, E. C., Tator, C. H., and Shoichet, M. S., 2003. Novel intrathecal delivery system for treatment of sp inal cord injury. Exp. Neurol. 182 300-309. Joosten, E. A. J., Br, P. R., and Gispen, W. H., 1995. Directional regrowth of lesioned corticospinal tract axons in adult rat spinal cord. Neuroscience 69 619-626. Kakulas, B. A., 2004. Neuropathology: the foundati on for new treatments in spinal cord injury. Spinal Cord 42 549-563. Kang, C. E., Poon, P. C., Tator, C. H., and Shoi chet, M. S., 2009. A new paradigm for local and sustained release of therapeutic molecules to the injured spin al cord for neuroprotection and tissue repair. Tissue Eng. Part A 15 595-604. Kataoka, K., Suzuki, Y., Kitada, M., Hashimoto, T ., Chou, H., Bai, H., Ohta, M., Wu, S., Suzuki, K., and Ide, C., 2004. Alginate enhances elongat ion of early regenerating axons in spinal cord of young rats. Tissue Eng. 10, 493-504. Kataoka, K., Suzuki, Y., Kitada, M., Ohnishi, K ., Suzuki, K., Tanihara, M., Ide, C., Endo, K., and Nishimura, Y., 2001. Alginate, a bioresorbable material de rived from brown seaweed, enhances elongation of amputated axons of spinal cord in infant rats. J. Biomed. Mater. Res. 54, 373-384. Kim, Y.-t., Caldwell, J.-M., and Bellamkonda, R. V., 2009. Nanoparticle-mediated local delivery of methylprednisolone after spin al cord injury. Biomaterials 30 2582-2590. Kitamura, Y., Yanagisawa, D., Takata, K., and Taniguchi, T., 2009. Neuropr otective function in brain microglia. Curr. Anaesthesia & Crit. Care 20 142-147. Kocsis, J. D., Lankford, K. L., Sasaki, M., and Radtke, C., 2009. Unique in vivo properties of olfactory ensheathing cells that may contribute to neural repair a nd protection following spinal cord injury. Neurosci. Lett. 456 137-142. Kuo, C. K., and Ma, P. X., 2001. Ionically crossli nked alginate hydrogels as scaffolds for tissue engineering: part 1. structure, gelation rate and mechanical properties. Biomaterials 22 511-521. Kwon, B. K., Tetzlaff, W., Grau er, J. N., Beiner, J., and Vaccaro, A. R., 2004. Pathophysiology and pharmacologic treatment of acute spin al cord injury. The Spine Journal 4 451-464. Lakatos, A., Barnett, S. C., and Franklin, R. J. M., 2003. Olfactory ensh eathing cells induce less host astrocyte response and chondroitin sulpha te proteoglycan expression than schwann cells following transplantation into a dult cns white matter. Exp. Neurol. 184 237-246. 109

PAGE 110

Lakatos, A., Franklin, R. J. M., and Barne tt, S. C., 2000. Olfactor y ensheathing cells and Schwann cells differ in their in vitro interactions with astrocytes. Glia 32 214-225. Lane, M. A., Fuller, D. D., White, T. E., and Re ier, P. J., 2008. Respiratory neuroplasticity and cervical spinal cord injury: translatio nal perspectives. Trends Neurosci. 31 538-547. Lavdas, A. A., Papastefanaki, F., Thomaidou, D., and Matsas, R., 2008. Schwann cell transplantation for CNS re pair. Curr. Med. Chem. 15 151-160. Laverty, P. H., Leskovar, A., Br eur, G. J., Coates, J. R., Bergman, R. L., Widmer, W. R., Toombs, J. P., Shapiro, S., and Borgens, R. B., 2004. A preliminary study of intravenous surfactants in paraplegic dogs: polymer therapy in canine clinical SCI. J. Neurotrauma 21, 1767. Letourneau, P. C., 2001. Preparation of substrata for in vitro culture of neurons. In: Fedoroff, S., and Richardson, A., (Eds.), Protocols for neur al cell culture. Humana Press, Totowa, NJ, pp. 245-254. Li, S., Kim, J.-E., Budel, S., Hampton, T. G., a nd Strittmatter, S. M., 2005. Transgenic inhibition of Nogo-66 receptor function allows axonal sprouting and improved locomotion after spinal injury. Mol. Cell. Neurosci. 29 26-39. Li, X., Yang, Z., Zhang, A., Wang, T., and Chen, W ., 2009. Repair of thoracic spinal cord injury by chitosan tube implantation in adult rats. Biomaterials 30 1121-1132. Li, Y., Field, P. M., and Raisman, G., 1997. Repair of adult rat corticospi nal tract by transplants of olfactory ensheathing cells. Science 277 2000-2002. Li, Y., Field, P. M., and Raisman, G., 1998. Re generation of adult rat corticospinal axons induced by transplanted olfactory ensheathing cells. J. Neurosci. 18 10514-10524. Li, Y., Li, D., Khaw, P. T., a nd Raisman, G., 2008. Transplanted olfactory ensheathing cells incorporated into the optic nerve head ensheathe retinal ganglion cell axons: Possible relevance to glaucoma. Neurosci. Lett. 440 251-254. Li, Y., Sauve, Y., Li, D., Lund, R. D., a nd Raisman, G., 2003. Transplanted olfactory ensheathing cells promote regeneration of cut adult rat optic nerve axons. J. Neurosci. 23 7783-7788. Lima, C., Pratas-Vital, J., Escada, P., Hasse-F erreira, A., Capucho, C., and Peduzzi, J. D., 2006. Olfactory mucosa autografts in human spinal cord injury: a pilot c linical study. J. Spinal Cord Med. 29, 191. Liu-Snyder, P., Logan, M. P., Shi, R., Smith, D. T., and Borgens, R. B., 2007. Neuroprotection from secondary injury by polyethylene glycol requires its internaliz ation. J. Exp. Biol. 210, 1455-1462. 110

PAGE 111

Liu, S., Said, G., and Tadie, M., 2001. Regrowth of the rostral spinal axons into the caudal ventral roots through a collagen tube implante d into hemisected adult rat spinal cord. Neurosurgery 49, 143-151. Loh, N. K., Woerly, S., Bunt, S. M., Wilton, S. D., and Harvey, A. R., 2001. The regrowth of axons within tissue defects in the CNS is pr omoted by implanted hydrogel matrices that contain BDNF and CNTF producin g fibroblasts. Exp. Neurol. 170, 72-84. Lpez-Vales, R., Fors, J., Navarro, X., and Ve rd, E., 2006. Olfactory ensheathing glia graft in combination with FK506 administration prom ote repair after spinal cord injury. Neurobiol. Dis. 24 443-454. Lpez-Vales, R., Fors, J., Verd, E., and Na varro, X., 2006. Acute and delayed transplantation of olfactory ensheathing cells promote partial recovery after complete transection of the spinal cord. Neurobiol. Dis. 21 57-68. Louro, J., and Pearse, D. D., 2008. Stem and progen itor cell therapies: rece nt progress for spinal cord injury repair. Neurol. Res. 30, 5-16. Luo, J., Borgens, R., and Shi, R., 2002. Polyethylene glycol immediat ely repairs neuronal membranes and inhibits free radical production after acute spinal cord injury. J. Neurochem. 83, 471-480. Mackay-Sim, A., Fron, F., Cochrane, J., Bassingt hwaighte, L., Bayliss, C., Davies, W., Fronek, P., Gray, C., Kerr, G., Licina, P., Nowitzke, A., Perry, C., Silburn, P. A. S., Urquhart, S., and Geraghty, T., 2008. Autologous olfactory en sheathing cell transplantation in human paraplegia: a 3-year cl inical trial. Brain 131, 2376. Mahoney, M. J., and Anseth, K. S., 2006. Three-di mensional growth and function of neural tissue in degradable polyethylene glycol hydrogels. Biomaterials 27 2265. Mallek, J. D., 2006. Hyaluronic acid-olfactory en sheathing cell compositions for spinal cord injury nerve regeneration, (Thesis), Univ ersity of Florida, Gainesville, FL. Marijnissen, W. J. C. M., van Osch, G. J. V. M., Aigner, J., Verwoerd-Verhoef, H. L., and Verhaar, J. A. N., 2000. Tissue-engineered ca rtilage using serially passaged articular chondrocytes. Chondrocytes in alginate, comb ined in vivo with a synthetic (E210) or biologic biodegradable carrier (DBM). Biomaterials 21 571-580. Martin, B. C., Minner, E. J., Wiseman, S. L., Kl ank, R. L., and Gilbert, R. J., 2008. Agarose and methylcellulose hydrogel blends for nerve rege neration applications. Journal of Neural Engineering 5 221-231. Martin, D., Robe, P., Franzen, R., Delre, P., Schoenen, J., Stevenaert, A., and Moonen, G., 1996. Effects of Schwann cell tran splantation in a contusion model of rat spinal cord injury. J. Neurosci. Res. 45 588-597. 111

PAGE 112

McDonald, J. W., Liu, X.-Z., Qu, Y., Liu, S., Mi ckey, S. K., Turetsky, D., Gottlieb, D. I., and Choi, D. W., 1999. Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord. Nat. Med. 5 1410-1412. McGraw, J., Hiebert, G. W., and Steeves, J. D ., 2001. Modulating astroglios is after neurotrauma. J. Neurosci. Res. 63 109-115. Meakin, J. R., Hukins, D. W. L., Aspden, R. M., and Imrie, C. T., 2003. Rheological properties of poly(2-hydroxyethyl methacr ylate) (pHEMA) as a func tion of water content and deformation frequency Journal of Materi als Science Materials in Medicine 14 783-787. Mentak, K., 1993. Tissue-protecti ve hydrophilic polymer solutions and surface modifications, (Dissertation), University of Florida, Gainesville, FL. Metzger, T. G., 2006. The rheology handbook: for us ers of rotational and oscillatory rheometers. Vincentz Network GmbH & Co. KG, Hannover, Germany. Miller, S. M., 2008. Methylpredisolone in acute sp inal cord injury: a ta rnished standard. J. Neurosurg. Anesthesiol. 20 140-142. Mingyu, C., Kai, G., Jiamou, L., Yandao, G., Nanming, Z., and Xiufang, Z., 2004. Surface modification and characterization of chito san film blended with poly-L-lysine. J. Biomater. Appl. 19 59-75. Moe, S. T., Draget, K. I., Skjak-Braek, G., and Smidsrod, O., 1995. Alginates. In: Stephen, A. M., (Ed.), Food polysaccharides and their a pplications. Marcel Dekker, New York, pp. 245-286. Mosahebi, A., Simon, M., Wiber g, M., and Terenghi, G., 2001. A novel use of alginate hydrogel as Schwann cell matrix. Tissue Eng. 7 525-534. Navarro, X., Verd, E., Rodrguez, F. J., and Ce ballos, D., 2001. Artificial nerve graft for the repair of peripheral nerve injuries. Neurol. Sci. 22 S7-S13. Nisbet, D. R., Crompton, K. E., Horne, M. K., Finkelstein, D. I., and Forsythe, J. S., 2008. Neural tissue engineering of the CNS usi ng hydrogels. J. Biomed. Mater. Res. B 87B 251-263. Nisbet, D. R., Moses, D., Gengenbach, T. R., Fors ythe, J. S., Finkelstein, D. I., and Horne, M. K., 2009. Enhancing neurite outgrowth from primary neurones and neural stem cells using thermoresponsive hydrogel scaffolds for the repair of spinal cord injury. J Biomed Mater Res A 89 24-35. Nomura, H., Baladie, B., Katayama, Y., Morshea d, C. M., Shoichet, M. S., and Tator, C. H., 2008. Delayed implantation of intramedullary chitosan channels containing nerve grafts promotes extensive axonal regeneration af ter spinal cord inju ry. Neurosurgery 63 127143. 112

PAGE 113

Nomura, H., Katayama, Y., Shoichet, M. S., a nd Tator, C. H., 2006. Complete spinal cord transection treated by implantation of a reinforced synthetic hydroge l channel results in syringomelia and caudal migration of the rostral stump. Neurosurgery 59 183-192. Nomura, H., Tator, C. H., and Shoichet, M. S., 2006. Bioengineered strate gies for spinal cord repair. J. Neurotrauma 23 496-507. Notterpek, L., Snipes, G. J., and Shooter, E. M ., 1999. Temporal expression pattern of peripheral myelin protein 22 during in vivo a nd in vitro myelination. Glia 25, 358-369. Noushi, F., Richardson, R. T., Hardman, J., Clark, G., and O'Leary, S., 2005. Delivery of neurotrophin-3 to the co chlea using alginate bead s. Otology & Neurotology 26 528-533. Novikova, L. N., Mosahebi, A., Wiberg, M., Te renghi, G., Kellerth, J.-O., and Novikov, L. N., 2006. Alginate hydrogel and matrigel as potential cell carriers for neurotransplantation. J. Biomed. Mater. Res. A 77A 242-252. Novikova, L. N., Novikov, L. N., and Kellert h, J.-O., 2003. Biopolymers and biodegradable smart implants for tissue regeneration after spinal cord injury. Curr. Opin. Neurol. 16 711-715. NSCISC, 2007. The 2007 annual statis tical report for the spinal co rd injury model systems. University of Alabama at Birmingham, Birmingham, AL. NSCISC, 2008. Spinal cord injury facts and figur es at a glance. University of Alabama at Birmingham, Birmingham, AL, ( http://images.main.uab.edu/spin alcord/pdffiles/Facts08.pdf) accessed August 2008. Ogushi, Y., Sakai, S., and Kawakami, K ., 2007. Synthesis of enzymatically-gellable carboxymethylcellulose for biomedical ap plications. J. Biosci. Bioeng. 104 30-33. Oudega, M., Gautier, S. E., Chapon, P., Fragoso, M., Bates, M. L., Parel, J.-M., and Bartlett Bunge, M., 2001. Axonal regeneration into Sc hwann cell grafts within resorbable poly([alpha]-hydroxyacid) guidance channels in the adult rat spinal cord. Biomaterials 22, 1125-1136. Oudega, M., Moon, L. D. F., and de Almeida Leme R. J., 2005. Schwann cells for spinal cord repair. Braz. J. Med. Biol. Res. 38, 825-835. Ozawa, H., Matsumoto, T., Ohashi, T., Sat o, M., and Kokubun, S., 2001. Comparison of spinal cord gray matter and white matter softness: measurement by pipette aspiration method. J. Neurosurg. Spine 2 95, 221. Ozawa, H., Matsumoto, T., Ohashi, T., Sat o, M., and Kokubun, S., 2004. Mechanical properties and function of the spinal pia mater. J. Neurosurg. Spine 1 1 122. 113

PAGE 114

Patist, C. M., Mulder, M. B., Gautier, S. E., Maquet, V., Jrme, R., and Oudega, M., 2004. Freeze-dried poly(D,L-lactic acid) macropor ous guidance scaffolds impregnated with brain-derived neurotrophic f actor in the transected adult rat thoracic spinal cord. Biomaterials 25 1569-1582. Peck, L. S., Quigg, J. M., Fossum, G. T., a nd Goldberg, E. P., 1995. Evaluation of CMC and HA solutions for adhesiolysis. J. Invest. Surg. 8 337-348. Peppas, N. A., 2009. Hydrogels. In: Ratner, B. D., Dyro, J., Grimnes, S. J., and Schoen, F. J., (Eds.), Biomedical engineering desk reference. Academic Press, pp. 188-195. Piantino, J., Burdick, J. A., Gol dberg, D., Langer, R., and Benowitz, L. I., 2006. An injectable, biodegradable hydrogel for tr ophic factor delivery enhances axonal rewiring and improves performance after spinal cord injury. Exp. Neurol. 201 359-367. Pocker, Y., and Green, E., 1973. Hy drolysis of D-glucono-delta-lac tone I. general acid-base catalysis, solvent deuterium isotope effects, and transition state characterization. J. Am. Chem. Soc. 95 113-119. Prang, P., Mller, R., Eljaouhari, A., Heckmann, K ., Kunz, W., Weber, T., Faber, C., Vroemen, M., Bogdahn, U., and Weidner, N., 2006. The prom otion of oriented axonal regrowth in the injured spinal cord by alginate-based anisotropic capillary hydrogels. Biomaterials 27 3560-3569. Prewitt, C. M. F., Niesman, I. R., Kane, C. J. M., and Houl, J. D., 1997. Activated macrophage/microglial cells can promote the regeneration of sensory axons into the injured spinal cord. Exp. Neurol. 148 433-443. Profyris, C., Cheema, S. S., Zang, D., Azari, M. F., Boyle, K., and Petratos, S., 2004. Degenerative and regenerative mechanisms gove rning spinal cord injury. Neurobiol. Dis. 15, 415-436. Purves, D., Augustine, G. J., Fitzpatrick, D., Hall, W. C., LaMantia, A.-S., McNamara, J. O., and White, L. E., 2008. Neuroscience. Sinauer Associates, Inc., Sunderland, MA. Qi, M., Strand, B. L., Morch, Y., Lacik, I., Wa ng, Y., Salehi, P., Barbaro, B., Gangemi, A., Kuechle, J., Romagnoli, T., Hansen, M. A., R odriguez, L. A., Benedetti, E., Hunkeler, D., Skjak-Brajk, G., and Oberholzer, J., 2008. Encapsulation of human islets in novel inhomogeneous alginate-Ca2+/Ba2+ Microbead s: in vitro and in vivo function. Artif. Cells. Blood Substit. Immobil. Biotechnol. 36 403-420. Qian, T., Guo, X., Levi, A. D., Vanni, S., Sh ebert, R. T., and Sipski, M. L., 2005. High-dose methylprednisolone may cause myopathy in acute spinal cord injury patients. Spinal Cord 43, 199-203. Rabchevsky, A. G., Fugaccia, I., Sullivan, P. G., Blades, D. A., and Scheff, S. W., 2002. Efficacy of methylprednisolone therapy for the inju red rat spinal cord. J. Neurosci. Res. 68 7-18. 114

PAGE 115

Rabchevsky, A. G., and Streit, W. J., 1997. Grafting of cultured microglial cells into the lesioned spinal cord of adult rats enhances neurite outgrowth. J. Neurosci. Res. 47 34-48. Ramer, L., Ramer, M., and Steeves, J., 2005. Setting the stage for functional repair of spinal cord injuries: a cast of thousands. Spinal Cord 43 134-161. Ramer, L. M., Au, E., Richter, M. W., Liu, J., Tetzlaff, W., and Roskams, A. J., 2004. Peripheral olfactory ensheathing cells reduce scar and cavity formation and promote regeneration after spinal cord inju ry. J. Comp. Neurol. 473 1-15. Ramn-Cueto, A., and Avila, J., 1998. Olfactory ensheathing glia: properties and function. Brain Res. Bull. 46 175-187. Ramn-Cueto, A., Cordero, M. I., Santos-Benito, F. F., and Avila, J., 2000. Functional recovery of paraplegic rats and motor axon regene ration in their spinal cords by olfactory ensheathing glia. Neuron 25 425-435. Ramn-Cueto, A., and Nieto-Sampedro, M., 1994 Regeneration into the spinal cord of transected dorsal root axons is promoted by ensheathing glia tran splants. Exp. Neurol. 127, 232-244 Rasouli, A., Bhatia, N., Suryadevara, S., Cah ill, K., and Gupta, R., 2006. Transplantation of Preconditioned Schwann Cells in Peripheral Ne rve Grafts After Cont usion in the Adult Spinal Cord. Improvement of Recovery in a Rat Model. J. Bone Joint Surg. Am. 88 2400-2410. Reier, P. J., 2004. Cellular transplantation strategi es for spinal cord injury and translational neurobiology. NeuroRX 1 424-451. Reier, P. J., 2004. Cellular transplantation strategi es for spinal cord injury and translational neurobiology. NeuroRx 1, 424-451. Reier, P. J., and Lane, M. A., 2008. Degeneratio n, regeneration, and plasticity in the nervous system In: Conn, P. M., (Ed.), Neuroscience in medicine. Humana Press, Totowa, NJ, pp. 691-727. Ren, Y.-J., Zhou, Z.-Y., Cui, F.-Z., Ying, W., Zhao, J.-P., and Xu, Q.-Y., 2009. Hyaluronic acid/polylysine hydrogel as a transfer system for transplantation of neural stem cells. J. Bioact. Compatible Polym. 24 56-62. Richardson, P. M., McGuinness, U. M., and Aguayo, A. J., 1980. Axons from CNS neurons regenerate into PNS grafts. Nature 284 264-265. Ronsyn, M. W., Berneman, Z. N., Van Tendeloo, V. F. I., Jorens, P. G., and Ponsaerts, P., 2008. Can cell therapy heal a spinal cord injury? Spinal Cord 46 532-539. Ross-Murphy, S. B., 1995. Structure-property relationships in food biopolymer gels and solutions. J. Rheol. 39, 1451-1463. 115

PAGE 116

Saberi, H., Moshayedi, P., Aghayan, H.-R., Arjm and, B., Hosseini, S.-K., Emami-Razavi, S.-H., Rahimi-Movaghar, V., Raza, M., and Firouzi, M., 2008. Treatment of chronic thoracic spinal cord injury patients with autologous Schwann cell tr ansplantation: an interim report on safety considerations and po ssible outcomes. Neurosci. Lett. 443 46-50. Samadikuchaksaraei, A., 2007. An overview of tissue engineering approaches for management of spinal cord injuries. Journal of NeuroEngineering and Rehabilitation 4 15. Sambanis, A., 2003. Encapsulated islets in diab etes treatment. Diabetes Technol. Ther. 5 665668. Scheff, S., and Roberts, K. N., 2009. Infinite hori zon spinal cord contusion model. In: Chen, J., Xu, X.-M., Xu, Z. C., and Zhang, J. H., (Eds .), Animal models of acute neurological injuries. Humana Press, New York. Scheff, S. W., Rabchevsky, A. G., Fugaccia, I., Main, J. A., and James E. Lumpp, J., 2003. Experimental modeling of spinal cord injury : characterization of a force-defined injury device. J. Neurotrauma 20 179-193. Segura, T., Anderson, B. C., Chung, P. H., Webber R. E., Shull, K. R., and Shea, L. D., 2005. Crosslinked hyaluronic acid hydrogels: a strategy to functionalize and pattern. Biomaterials 26 359-371. Sherman, L. S., Struve, J. N., Rangwala, R., Wallingford, N. M., Tuohy, T. M. F., and Kuntz, C., 2002. Hyaluronate-based extracellular matrix : keeping glia in their place. Glia 38 93102. Shi, R., and Borgens, R. B., 1999. Acute repair of crushed guinea pig spinal cord by polyethylene glycol. J. Neurophysiol. 81 2406-2414. Shi, R., and Borgens, R. B., 2000. Anatomical repair of nerve membranes in crushed mammalian spinal cord with polyethylen e glycol. J. Neurocytol. 29 633. Short, D. J., Masry, W. S. E., and Jones, P. W., 2000. High dose methylprednisolone in the management of acute spinal cord injury a sy stematic review from a clinical perspective. Spinal Cord 38 273-286. Silva, G. A., Czeisler, C., Niece, K. L., Beni ash, E., Harrington, D. A., Kessler, J. A., and Stupp, S. I., 2004. Selective differentiation of neural progenitor cells by high-epitope density nanofibers. Science 303 1352-1355. Simpson, N. E., Stabler, C. L., Simpson, C. P ., Sambanis, A., and Constantinidis, I., 2004. The role of the CaCl2-guluronic acid interact ion on alginate encapsulated [beta]TC3 cells. Biomaterials 25 2603-2610. Skjk-Brk, G., Grasdalen, H., and Smidsr d, O., 1989. Inhomogeneous polysaccharide ionic gels. Carbohydr. Polym. 10 31-54. 116

PAGE 117

Smidsrd, O., and Skjk-Brk, G., 1990. Alginate as immobilization matrix for cells. Trends Biotechnol. 8 71-78. Soon-Shiong, P., 1999. Treatment of type I diabet es using encapsulated islets. Adv. Drug Del. Rev. 35, 259-270. Stabenfeldt, S. E., Garca, A. J., and La Placa, M. C., 2006. Thermoreversible lamininfunctionalized hydrogel for neural tissue e ngineering. J. Biomed. Mater. Res. A 77A 718-725. Stang, F., Fansa, H., Wolf, G., Reppin, M., and Keilhoff, G., 2005. Structural parameters of collagen nerve grafts influence peripher al nerve regeneration. Biomaterials 26 30833091. Stevens, M. M., Qanadilo, H. F., Langer, R ., and Prasad Shastri, V., 2004. A rapid-curing alginate gel system: utilit y in periosteum-derived cartilage tissue engineering. Biomaterials 25 887-894. Steward, O., Zheng, B., and Tessier-Lavigne, M., 2003. False resurrec tions: distinguishing regenerated from spared axons in the injured central nervous syst em. J. Comp. Neurol. 459, 1-8. Stokes, B. T., and Jakeman, L. B., 2002. Experiment al modelling of human spinal cord injury: a model that crosses the species barrier and mimics the spectrum of human cytopathology. Spinal Cord 40 101-109. Stokols, S., and Tuszynski, M. H., 2004. The fabrica tion and characterization of linearly oriented nerve guidance scaffolds for spinal cord injury. Biomaterials 25 5839-5846. Stokols, S., and Tuszynski, M. H., 2006. Freeze-dried agarose scaffolds with uniaxial channels stimulate and guide linear axonal growth following spinal cord injury. Biomaterials 27 443-451. Stopek, J. B., 2003. Biopolymer-microglia cell compositions for neural tissue repair, (Dissertation), University of Florida, Gainesville, FL. Stopek, J. B., Streit, W. J., and Goldberg, E. P ., 2002. Opportunities for axon repair in the CNS: use of microglia and biopolymer compositions. In: Streit, W. J., (Ed.), Microglia in the regenerating and degenerating central ne rvous system. Springer, New York, pp. 227-244. Straatmann, A., and Borchard, W., 2003. Phase se paration in calcium alginate gels. Eur. Biophys. J. 32 412-417. Streit, W. J., 2001. Microglia and macrophages in the developing CNS. Neurotoxicology 22 619-624. 117

PAGE 118

Streit, W. J., 2002. Physiology & pathophysiology of microglial cell function. In: Streit, W. J., (Ed.), Microglia in the regene rating and degenerating central nervous system. Springer, New York, pp. 1-14. Struve, J., Maher, P. C., Li, Y. Q., Kinney, S., Fehlings, M. G., Kuntz, C., and Sherman, L. S., 2005. Disruption of the hyaluronan-based extrace llular matrix in spinal cord promotes astrocyte proliferation. Glia 52 16-24. Suzuki, K., Suzuki, Y., Ohnishi, K., Endo, K., Tanihara, M., and Nishimura, Y., 1999. Regeneration of transected spinal cord in young adult rats using freez e-dried alginate gel. Neuroreport 10 2891-2894. Suzuki, Y., Kitaura, M., Wu, S., Kataoka, K., Su zuki, K., Endo, K., Nishimura, Y., and Ide, C., 2002. Electrophysiological and horseradish peroxidase-tracing studies of nerve regeneration through alginate-filled gap in adult rat spinal cor d. Neurosci. Lett. 318 121124. Talac, R., Friedman, J. A., Moore, M. J., Lu, L., Jabbari, E., Windebank, A. J., Currier, B. L., and Yaszemski, M. J., 2004. Animal models of spinal cord injury for evaluation of tissue engineering treatment stra tegies. Biomaterials 25 1505-1510. Teng, Y. D., Lavik, E. B., Qu, X., Park, K. I., Ourednik, J., Zurakowski, D., Langer, R., and Snyder, E. Y., 2002. Functional recovery followi ng traumatic spinal co rd injury mediated by a unique polymer scaffold seeded with neur al stem cells. Proceedings of the National Academy of Sciences of the United States of America 99 3024-3029. Tester, N. J., and Howland, D. R., 2008. Chondroitinase ABC improves basic and skilled locomotion in spinal cord injured cats. Exp. Neurol. 209 483-496. Tewarie, R. S. N., Hurtado, A., Bartels, R. H., Grotenhuis, A., and Oudega, M., 2009. Stem cell based therapies for spinal cord injury. J. Spinal Cord Med. 32 105-114. Tian, W. M., Hou, S. P., Ma, J., Zhang, C. L., Xu, Q. Y., Lee, I. S., Li, H. D., Spector, M., and Cui, F. Z., 2005. Hyaluronic acidpoly-D-ly sine-based three-dimensional hydrogel for traumatic brain injury. Tissue Eng. 11 513-525. Tian, W. M., Zhang, C. L., Hou, S. P., Yu, X., Cui, F. Z., Xu, Q. Y., Sheng, S. L., Cui, H., and Li, H. D., 2005. Hyaluronic acid hydrogel as Nogo-66 receptor an tibody delivery system for the repairing of injured rat brain: in vitro. J. Controlled Release 102 13-22. Tomaselli, K. J., Damsky, C. H., and Reichardt, L. E., 1987. Interactions of a neuronal cell line (PC12) with laminin, collagen IV, and fibr onectin: identification of integrin-related glycoproteins involved in attachment and process outgrowth. J. Cell Biol. 105 23472358. Tnnesen, H. H., and Karlsen, J., 2002. Alginate in drug delivery sy stems. Drug Dev. Ind. Pharm. 28, 621-630. 118

PAGE 119

Toole, B. P., 2001. Hyaluronan in morphogenesis. Semin. Cell Dev. Biol. 12 79-87. Trivedi, N., Keegan, M., Steil, G. M., Hollis ter-Lock, J., Hasenkamp, W. M., Colton, C. K., Bonner-Weir, S., and Weir, G. C., 2001. Islets in alginate macrobeads reverse diabetes despite minimal acute insulin secr etory responses. Transplantation 71 203-211. Tsai, E. C., Dalton, P. D., Shoichet, M. S., a nd Tator, C. H., 2004. Synthetic hydrogel guidance channels facilitate regeneration of adult rat brainstem motor axons after complete spinal cord transection. J. Neurotrauma 21 789. Tsai, E. C., Dalton, P. D., Shoichet, M. S., a nd Tator, C. H., 2006. Matrix inclusion within synthetic hydrogel guidance channels impr oves specific supraspinal and local axonal regeneration after complete spinal cord transection. Biomaterials 27 519-533. Tung, C.-Y. M., and Dynes, P. J., 1982. Relatio nship between viscoela stic properties and gelation in thermosetting systems. J. Appl. Polym. Sci. 27 569-574. Tysseling-Mattiace, V. M., Sahni, V., Niece, K. L., Birch, D., Czeisler, C., Fehlings, M. G., Stupp, S. I., and Kessler, J. A., 2008. Self-a ssembling nanofibers in hibit glial scar formation and promote axon elongation after spinal cord injury. J. Neurosci. 28 38143823. Wang, C., Liu, H., Gao, Q., Liu, X., and Tong, Z., 2008. Alginate-calcium carbonate porous microparticle hybrid hydrogels with versatil e drug loading capabilities and variable mechanical strengths. Carbohydr. Polym. 71 476-480. Wang, Y.-C., Wu, Y.-T., Huang, H.-Y., Lin, H.-I ., Lo, L.-W., Tzeng, S.-F., and Yang, C.-S., 2008. Sustained intraspinal delivery of neurotro phic factor encapsula ted in biodegradable nanoparticles following contusive spin al cord injury. Biomaterials 29 4546-4553. Wei, Y. T., Tian, W. M., yu, X., Cui, F. Z., Hou, S. P., Xu, Q. Y., and Lee, I.-S., 2007. Hyaluronic acid hydrogels w ith IKVAV peptides for tissue repair and axonal regeneration in an injured rat brain. Biomed. Mater. 2 S142-S146. West, D. C., Hampson, I. N., Arnold, F., and Kumar, S., 1985. Angiogenesis induced by degradation products of hya luronic acid. Science 228 1324-1326. Willerth, S. M., and Sakiyama-Elbert, S. E., 2008. Cell therapy for spinal cord regeneration. Adv. Drug Del. Rev. 60, 263-276. Winter, H. H., and Chambon, F., 1986. Analysis of linear viscoelastic ity of a crosslinking polymer at the gel point. J. Rheol. 30 367-382. Woerly, S., Doan, V. D., Evans-Martin, F., Paramore, C. G., and Peduzzi, J. D., 2001. Spinal cord reconstruction using NeuroGel (TM) impl ants and functional r ecovery after chronic injury. J. Neurosci. Res. 66 1187-1197. 119

PAGE 120

Woerly, S., Doan, v. D., Sosa, N., de Vellis, J ., and Espinosa, A., 2001. Reconstruction of the transected cat spinal cord following Ne uroGel(TM) implantation: axonal tracing, immunohistochemical and ultrastructural studies. Int. J. Dev. Neurosci. 19 63-83. Woerly, S., Doan, V. D., Sosa, N., Vellis, J. d., and Espinosa-Jeffrey, A., 2004. Prevention of gliotic scar formation by NeuroGel (TM) allows partial endogenous re pair of transected cat spinal cord. J. Neurosci. Res. 75 262-272. Woerly, S., Petrov, P., Sykova, E., Roitbak, T., Simonova, Z., and Harvey, A. R., 1999. Neural tissue formation within porous hydrogels impl anted in brain and spinal cord lesions: ultrastructural, immunohistochemical and diffusion studies. Tissue Eng. 5 467-488. Woerly, S., Pinet, E., de Robertis, L., Bousmina M., Laroche, G., Roitback, T., Vargov, L., and Sykov, E., 1998. Heterogeneous PHPMA hydroge ls for tissue repair and axonal regeneration in the inju red spinal cord J. Biomater. Sci., Polym. Ed. 9 681-711. Woerly, S., Pinet, E., de Rober tis, L., Van Diep, D., and Bousmina M., 2001. Spinal cord repair with PHPMA hydrogel containing RGD peptides (NeuroGel(TM)). Biomaterials 22 1095-1111. Woerly, S., Plant, G. W., and Harvey, A. R., 1996. Cultured rat neuronal and glial cells entrapped within hydrogel polymer matri ces: a potential tool for neural tissue replacement. Neurosci. Lett. 205 197-201. Woerly, S., Plant, G. W., and Harvey, A. R., 19 96. Neural tissue engineering: from polymer to biohybrid organs. Biomaterials 17 301-310. Wong, D. Y., Leveque, J. C., Brumblay, H., Krebsb ach, P. H., Hollister, S. J., and Lamarca, F., 2008. Macro-architectures in spinal cord s caffold implants influence regeneration. Journal of neurotrauma 25 1027-1037. Wu, S., Suzuki, Y., Kitada, M., Kitaura, M., Kata oka, K., Takahashi, J., Ide, C., and Nishimura, Y., 2001. Migration, integration, and differentiation of hippo campus-derived neurosphere cells after transplantation into injured rat spinal cord. Neurosci. Lett. 312 173-176. Xu, X., Urban, J. P. G., Browning, J. A., Tirlapur U., Wilkins, R. J., Wu M. H., Cui, Z., and Cui, Z., 2007. Influences of buffer system s on chondrocyte growth during long-term culture in alginate. Oste oarthritis Cartilage 15 396-402. Xu, X. M., Chen, A., Guenard, V., Kleitma n, N., and Bunge, M. B., 1997. Bridging Schwann cell transplants promote axonal regeneration fr om both the rostral and caudal stumps of transected adult rat spinal cord J. Neurocytol. 26, 1-16. Yick, L.-W., Cheung, P.-T., So, K.-F., and Wu, W., 2003. Axonal regeneration of Clarke's neurons beyond the spinal cord injury scar after treatment with chondroitinase ABC. Exp. Neurol. 182, 160-168. 120

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121 Yoshii, S., Ito, S., Shima, M., Taniguchi, A ., and Akagi, M., 2009. Functional restoration of rabbit spinal cord using collagen-filame nt scaffold. J.Tissue Eng. Regen. Med. 3 19-25. Yoshii, S., Oka, M., Shima, M., Akagi, M., and Taniguchi, A., 2003. Br idging a spinal cord defect using collagen filament. Spine 28 2346-2351. Yoshii, S., Oka, M., Shima, M., Taniguchi, A., Taki, Y., and Akagi, M., 2004. Restoration of function after spinal cord tran section using a collagen bridge J. Biomed. Mater. Res. A 70A 569-575. Yu, P., Huang, L., Zou, J., Yu, Z., Wang, Y., Wang, X., Xu, L., Liu, X., Xu, X.-M., and Lu, P.H., 2008. Immunization with recombinant Nogo-66 receptor (NgR) promotes axonal regeneration and recovery of function after spinal cord inju ry in rats. Neurobiol. Dis. 32 535-542. Yu, X., Dillon, G. P., and Bellamkonda, R. V., 1999. A laminin and nerve growth factor-laden three-dimensional scaffold for enhanced neurite extension. Tissue Eng. 5 291-304. Zeng, Q., Yu, Z., You, J., and Zhang, Q., 2007. Effi cacy and safety of Seprafilm for preventing postoperative abdominal adhesion: systematic review and meta-analysis. World J. Surg. 31, 2125-2131. Zheng, Z., Liu, C., Zhang, L., Gao, R., Wei, S., Zhang, K., and Zhang, L., 2007. Olfactory ensheathing cell transplantation in 106 patients with old spinal cord injury: differences in ages, sexes, disease courses, injured types and sites. Neural Regen.Res. 2 380-384. Zhong, Y., and Bellamkonda, R. V., 2008. Biomaterials for the central nervous system. J. R. Soc. Interface 5 957. Zimmer, M. B., Nantwi, K., and Goshgarian, H. G., 2007. Effect of spinal cord injury on the respiratory system: basic research and current clinical treatment options. J. Spinal Cord Med. 30, 319-330.

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BIOGRAPHICAL SKETCH Samesha R. Barnes was born in Fort Gordon, Georgia. She was reared in the Richmond County school system in Augusta, Georgia where she attended the award-winning John S. Davidson Fine Arts Magnet High School. Samesha excelled in academics and in the arts and was especially gifted in violin, flute, French and Japanese. Samesha had the opportunity to travel to Takarazuka, Japan as a representa tive of the Foreign Langauge Department on a school exchange visit for which she was selected on the basis of her academic achievement in Japanese Studies during her senior. In addition to her affection for the arts, Samesh a had a love for science, math and engineering which was enhanced by her part icipation in the Research and Engineering Apprenticeship Program (REAP). She graduated with honors and m oved to Atlanta, Georgia to follow her dream of becoming a chemical engineer. Samesha enrolled in the Atlanta University Centers Dual-Degree Engineering Program where she pursued degrees in chemistry and chemical engineering from Clark Atlanta University (CAU) and the Georgia Institute of Technology. Sh e was a member of the CAU Honors Program and a High Performance Polymers and Ceramics (HiPPAC) fellow which afforded the opportunity to conduct scholarly research throughout her undergraduate matriculation. In addition, Samesha participated in undergraduate research progra ms in the summer, including the Summer Undergraduate Research Pr ogram (SURP) at Virginia Tech. After graduating in 1996, Samesha began her professional car eer as a Product Development Engineer in Hewlett-Packards In kjet Media Division in San Diego, California where she previously worked as an intern. She served on the recruiting team and also filed an invention disclosure for a product idea. After two years in industry Samesha decided to pursue an advanced degree in materials science and engineering. 122

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123 Samesha continued her education at the Univ ersity of Florida and earned a master of science in 2003. During her tenure at the uni versity she received an Alumni Association Fellowship, an NSF Alliance for Graduate Educa tion and the Professoriate (AGEP) Fellowship and was a part of the inaugural class of Bill & Melinda Gates Foundation Millennium Scholars. She completed two internships with Kimberly-C lark in Neenah, WI and Roswell, GA and was also active in leadership role s in organizations on campus and in the Gainesville community. Samesha was competitively selected to serve as a teaching assistant for the College of Engineerings pilot Chem Teach program to prom ote freshman retention in engineering, which stirred her passion for teaching and mentoring. She earned a PhD in materials science and engineering in 2009 with a concentration in biom aterials and plans to pursue a career in academia.