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Modular Tissue Scaffolding Tools: A New Family of Self-Assembled Biomaterials Derived from Copper-Capillary Alginate Gels

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MODULAR TISSUE SCAFFOLDING TOOLS: A NEW FAMILY OF SELF-A SSEMBLED BIOMATERIALS DERIVED FROM COPPER-CAPILLARY ALGINATE GELS By BRADLEY JAY WILLENBERG 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 2005

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Copyright 2005 by Bradley Jay Willenberg

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To the old man who drowned so near the shore.

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iv ACKNOWLEDGMENTS I would like to sincerely thank Dr. Chri stopher Batich, my supervising committee chairman. His hands-off approach and wealth of scientific and engineering knowledge propelled me and this project faster and fa rther than initially e nvisioned. I thank Dr. Anthony Brennan, Dr. Robert DeHoff and Dr. T homas Mareci for their time and efforts as my teachers and committee members. I al so specially thank Dr. Naohiro Terada and Dr. Takashi Hamazaki for their tremendous e fforts, input and profound support of this work. Much praise and thanks go to Marina Scot ti for giving me roots; split a piece of wood and she is there. Lift a stone and you will find her. I thank my family for their consistent support, guidance, criticism and st rength. I especially thank, Mom, Jimbo, Dan and Ryan, Dr. Amelia Dempere and Speci alist Wayne Acree (“The Lab Dude”). I also thank the entire Major Analytical Inst rumentation Center (MAIC) staff for their scientific and social insights. I further tha nk all those students, f aculty and staff who took the time to know and talk with me. Special thanks go to Charlie Murphey (Pr ecision Tool & Engineering, Gainesville, FL) for custom machining all my tools and re actors, and to Dr. Charles (Chuck) Seegert for shaping my early scientific and tissue-engineering thinking.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES.........................................................................................................viii LIST OF OBJECTS.............................................................................................................x ABSTRACT....................................................................................................................... xi CHAPTER 1 INTRODUCTION........................................................................................................1 2 BACKGROUND AND SIGNIFICANCE....................................................................5 3 MATERIALS AND METHODS...............................................................................15 Scaffold Synthesis......................................................................................................15 Raw Copper-Capillary Alginate Gel (RCCAG)..................................................15 Classic descending growth technique..........................................................16 Time-lapse videoscopy.................................................................................17 Barium Stabilized Copper-Capi llary Alginate Gel (BCCAG)............................17 Exchange-reactor design and setup..............................................................17 Barium hydroxide processing.......................................................................18 Oligochitosan-Barium (OBCCAG) a nd Oligochitosan (OCCAG) Stabilized RCCAG............................................................................................................19 Scaffold Characterization....................................................................................20 Optical microscopy......................................................................................20 Scanning electron microscopy.....................................................................21 Percent Water Conten t Determination.................................................................22 Biological Assesment.................................................................................................22 In Vitro Study: Swiss Albino Embr yonic Mouse Fibroblasts Expressing Green Fluorescent Protein (GFP-3T3).............................................................24 In Vitro Study: Mouse Embryonic Stem Cells Expressing Green Fluorescent Protein (GFP-mES)..........................................................................................24 Evaluation of mES cell growth, su rvival and morphology vs. time.............24 Comparison of ES maintenance (LIF+) and ES differentiation (LIF-) media conditions.....................................................................................25

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vi 4 RESULTS AND DISCUSSION.................................................................................26 Scaffold Synthesis and Processing.............................................................................26 RCCAG...............................................................................................................26 Growth videos..............................................................................................27 Growth kinetics............................................................................................27 Storage concerns..........................................................................................28 BCCAG...............................................................................................................29 Colorimetric changes during ba rium hydroxide treatment..........................29 Exchange-reactor advantages and difficulties..............................................30 OBCCAG and OCCAG.......................................................................................31 Consequences of Media Wash.............................................................................32 Scaffold Characterization...........................................................................................32 Optical Microscopy.............................................................................................33 RCCAG........................................................................................................33 Evidence of precipita tes within BCCAG.....................................................34 Scanning Electron Microscopy/Energy Di spersive Spectroscopy (SEM/EDS) and X-ray Mapping..........................................................................................35 Consequences of freeze-drying....................................................................35 RCCAG Data................................................................................................35 BCCAG Data................................................................................................37 OCCAG Data...............................................................................................39 Summary of morphologic and compositional analysis................................39 Equilibrium Water Weig ht Percent Analysis......................................................40 Biologic Assessment...................................................................................................41 Mouse Embryonic Fibroblasts (GFP-3T3)..........................................................41 Mouse Embryonic Stem Cells (GFP-mES).........................................................42 Evaluation of cell Growth, survival and morphology vs. time....................43 Confocal microscopy and video data...........................................................43 Comparison of ES maintenance (Lif+) and ES differentiation (Lif-) media conditions................................................................................................44 5 CONCLUSIONS........................................................................................................62 Introduction.................................................................................................................62 Scaffold Synthesis: Th e Agony and the Ecstasy........................................................62 Characterization: CCAG Scaffolds as Subtle Composites.........................................63 Biological Assessment: Living with Success and Failure..........................................64 6 FUTURE WORK........................................................................................................66 Introduction.................................................................................................................66 Synthesis: Expanding the CCAG Scaffold Family.....................................................66 Scaffold Characterization: Quantitative Bulk and Surface Compositional Analysis.66 Biologic Assessment: Standardized Methods and Controls for Stem Cell Studies....67 APPENDIX PUBLICATIONS PRESENTATIONS AND PATENTS.........................69

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vii LIST OF REFERENCES...................................................................................................70 BIOGRAPHICAL SKETCH.............................................................................................76

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viii LIST OF FIGURES Figure page 2-1 Peripheral nerve hier archical structure.....................................................................13 2-2 Molecular structures of algi nate and oligochitosan polymers..................................14 3-1 Raw-CCAG (RCCAG) classical desce nding technique synthesis scheme..............17 3-2 Teflon™ exchange-reactor.......................................................................................18 4-1 Plot of RCCAG growth as a function of time..........................................................46 4-2 First derivative plot of RCCAG growth data...........................................................47 4-3 Low magnification optical micrographs of representative RCCAG sample discs...47 4-4 Increased magnification optical microgra phs of RCCAG at sections at different parent gel levels........................................................................................................48 4-5 Graph of RCCAG average capillary di ameter vs. parent gel thickness. ................48 4-6 Graph of calculated RCCAG metr ics vs. parent gel thickness.................................49 4-7 Optical micrograph of BCCAG showing brown precipitate....................................49 4-8 Optical micrograph of BCCAG s howing shimmering precipitate. ........................50 4-9 Summary of RCCAG SEM/ED S and X-ray mapping data......................................51 4-10 Summary of BCCAG SEM/ED S and X-ray mapping data......................................52 4-11 Higher magnification BCCAG mo rphologic and compositional study...................53 4-12 Large false color compositional map of a BCCAG.................................................54 4-13 Summary of OCCAG SEM/ED S and X-ray mapping data.....................................55 4-14 A 4000X secondary electron image hi ghlighting the “hairy” OCCAG surface character...................................................................................................................56 4-15 Equilibrium water weight percents of different CCAG derivatives........................56

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ix 4-16 Confocal microscope image of live GFP-3T3 cells seeded within an OCCAG scaffold at day 2 in culture.......................................................................................57 4-17 Phase contrast and complementary fluor escence microscope image series of GFPmES cultured in OCCAG over nine days.................................................................58 4-18 Confocal microscope image of live GFP-mES cells seeded within an OCCAG scaffold at day 7 in culture.......................................................................................59 4-19 Hoechst stained nuclei of mES cells in an OCCAG capillary at day 4 in culture....60 4-20 Growth of GFP-mES cells in OCCAG s caffolds cultured in maintenance (M) or differentiation (D) media or a combination (M/D) over 4 days...............................61

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x LIST OF OBJECTS Object page 4-1 Time-Lapse Video of RCCAG Growth...................................................................46 4-2 Confocal microscope video of live GFP-3T3 cells seeded within an OCCAG scaffold at day 2 in culture.......................................................................................57 4-3 Confocal microscope video of live GFP-mES cells seeded within an OCCAG scaffold at day 7 in culture.......................................................................................59

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xi 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 MODULAR TISSUE SCAFFOLDING TOOLS: A NEW FAMILY OF SELF-A SSEMBLED BIOMATERIALS DERIVED FROM COPPER-CAPILLARY ALGINATE GELS By Bradley Jay Willenberg August 2005 Chair: Christopher Batich Major Department: Biomedical Engineering Tissue engineering aims to regenerate or replace lost/damaged cells, tissues and organs. Biomaterial scaffolds are ofte n fundamental components of many tissue engineering strategies. Devel opment of advanced biomaterial scaffolds is crucial to the continued progress and ultimate success of the field. Motivated to aid peripheral nerve regeneration/engineering, our study offers an innovative way to produce advanced biomaterial scaffolds derived from coppe r-capillary alginate gel (CCAG). These novel materials possess regular, con tinuous microtubular ar chitectures that relatively few fabrication techniques can ach ieve. These hydrogel materials have been morphologically and compositionally characte rized using scanning electron microscopy and energy dispersive spectroscopy (SEM/EDS ). We conducted X-ray mapping studies yielding the spatial distribution of elements within the different scaffolds. Fluorescence

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xii and confocal microscopy studies detail th e unique growth and survival of mouse embryonic stem cells (mES) and fibroblasts (3T3) in and on CCAG sc affolds in vitro.

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1 CHAPTER 1 INTRODUCTION Clinical motivation. Peripheral nerve injuries are extremely prevalent. Injury is often the result of trauma (e.g., lacerations gunshot wounds, motor vehicle accidents), acute compression, stretching and tension or dise ase (e.g., cancer, leprosy). Each year, an estimated 50,000 peripheral nerve repair proce dures are performed in the United States alone [1]. Much of what has been learned about peripheral nerve re pair has grown out of the treatment of warfare injuries [2]. Unfo rtunately, despite many advances and creative repair strategies, functional outcomes of ne rve repairs are still far from optimal, and motor nerves tend to be more refractory than sensory to full recovery [1]. Ideally, surgeons attempt a neurorrhaphy (direct suture of the nerve ends without tension) for all laceration or avulsive injuries [3, 4]. When transected or resected nerve ends cannot be coapted without tension, a ga p defect results requi ring nerve grafting to restore neural continuity [5]. Autograft (autologous nerve) is the “gold standard” graft material, and is preferentially obtained from harvest of the sural nerve, antebrachial cutaneous radial nerve or superficial sens ory radial (SSR) nerve [5]. Fundamental determinates of functional re generation for autograft are the endoneurium and remaining Schwann cells, since the epiand perineural elements are trimmed from harvested nerve before engraftment. Although reported to facilitate neuroregen eration over substantial distances (2-15 cm) [6], autograft has some disadvantages, including lack of donor supply, donor-site morbidity, need for a secondary surgical site and insuffici ent functional outcomes [1, 6-

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2 7]. Harvesting donor nerve is also time-consuming and often the fascicles do not match the target nerve in both number and diameter Central or segmental necrosis can also occur in large diameter grafts [8]. Tissue engineering could be a promising approach to functional neurorepair. Many tissue engineering strategies have already been used to facilitate neuroregeneration (chapter 2). Pinpointing the first tissue engineering experiments is difficult; however, most credit Langer and Vacanti [9] with cr ystallizing the central dogma and fundamental strategies of the field. They define tissue e ngineering as “. . an interdisciplinary field that applies the principals of engineering a nd the life sciences towa rd the development of biological substitutes that restore, ma intain or improve tissue function.” A primary thrust of tissue engineering is to develop three-dimensional biomaterials for use as scaffoldstemplates to format growing/regenerating cells and tissues. Scaffolds are becoming integral component s of tissue reparative, restorative and regenerative strategies [9], and development of advanced biomaterial scaffolds is crucial to the continued progress and success of the tissue engineering field. The ability to impose structural order on growing/regenerati ng cells and tissues via scaffold architecture and geometry is a key feature of advanced scaffolds. According to a review by Ma [10], scaffo lds are usually highly porous with large surface areas. Biodegradability is also generally required, with degradation rates designed to match the rate of neotissue forma tion. Further, the scaffold material(s) and possible degradation products should be non-to xic (i.e., biocompatible), especially to target cells and tissues. Finally, scaffo lds should maintain adequate mechanical properties and enhance cell adhe sion, growth, migration and differentiated function. The

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3 underlying idea of the above design guidelines is to produce biomaterials that bring together large numbers of cells in comfortable close quarters, and provide an environment that facilitates growth/regen eration/remodeling into functional target tissue(s). Scaffolds are essentially modular biomaterial tools. The word modular is intended to convey flexibility, customizability and dynamic range. To illustrate this concept, consider a computer software progr am. At its most basic level, the program comes with some set of featur es that perform needed functio ns. If more than the basic features are required to address specific needs, then often times one can enable or install additional program modules (for a small fee, of course) adding the n eeded functionality. This concept is well articulated in current microsphere technology, ye t is still nascent in current scaffolding designs. Modular homes are another example of the concept discussed above. The home analogy is particularly instru ctive from a biological perspect ive; no longer simply tools, scaffolds are homes for regenerating cells and tissues. One wants to encourage cells/tissues to take up orderly, productive re sidence and integrate into a much larger community. Using this logic, combinations of differ ent biomaterial modules (e.g., architecture, modulus, surface chemistry) are used to create a family of related scaffolds. These tailormade tools can then be implemented in tissue engineering. The scienc e is to know (at the molecular level) the effects of specific combinations of scaffold modules on cells/tissues. Only with this knowledge can we engineer scaffolds with tremendous flexibility and broad applicability.

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4 Project-specific achievements. Our study introduces a ne w family of biomaterials derived from copper-capillary alginate gels. These hydrogels have regular, continuous microtubular architectures similar to those of the endoneurium. To date, relatively few fabrication techniques produce such biomateria ls [7, 10-13]. Although we did not test the neuroregenerative potential of these materials, their treme ndous scaffolding potential was demonstrated through in vitro experiment s using mouse embryonic stem cells (mES).

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5 CHAPTER 2 BACKGROUND AND SIGNIFICANCE Classification of peripheral nerve injury. In 1943, Sir Herbert Seddon introduced a peripheral nerve injury classi fication system comp rising 3 categories: neurapraxia, axonotmesis and neurotmesis [1]. In 1951, Sundeland expanded the Seddon system to five categories by further subdividi ng axonotmesis [3, 4]. A first-degree injury (neurapraxia) involves a temporary conduc tion block with local demyelination. Complete recovery occurs and may take up to 12 weeks. A second-degree injury (axonotmesis) involves more-severe trau ma or compression causing Wallerian degeneration. The endoneurial tubes remain int act and therefore recovery is expected to be complete, but could take months. A th ird-degree injury also involves Wallerian degeneration, however the endoneurial tubes ar e not intact. Therefore, axons may not reinnervate their origin al motor/sensory target s and recovery is incomplete. A fourthdegree injury is a partial transection of the ne rve, ultimately resulting in a large scar area at the site of injury. This scar preclu des axons from advancing distally, and requires surgery for any chance at meaningful func tional recovery. A fi fth-degree injury (neurotmesis) is a complete transection of the nerve and requires surgery to restore neural continuity. MacKinnon added a sixth degree that combines th e other degrees to describe a mixed nerve injury [3]. Age and locati on are also key factors governing functional recovery, with poorer results expected for increasing age and more-proximal injuries. Anatomy and biology of adult periph eral nerve in the healthy state. Figure 2-1 illustrates peripheral nerve hierarchy. Nerves are composed of motor, sensory and

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6 sympathetic components [1]. Nerves may be designated as primarily motor or sensory; however, no nerve is purely one or the othe r [1]. Myelinated and unmyelinated axons comprise the nerve fibers. Motor fibers ar e primarily myelinated and are outnumbered 4 to 1 by unmyelinated sensory fibers [1]. Myelin ated fibers range in size from 1 to 20 m in diameter, while umyelinated fibers ar e typically below 1 m diameter [14, 15]. The edoneurium is composed mainly of l ongitudinally aligned collagen fibers 30 to 60 nm in diameter [14, 16-17]. Tiny capilla ries (<10 m), fibroblast, mast cells and macrophages are also found in the endoneuriu m. The innermost endoneurial layer is often observed to be in close contact with Schwann cell basal laminae. Compared to the epinerium and endoneur ium the perineurium is unique [14, 1617]. Cells composing the perineurium exhib it both myoid and epith elioid features and express basal lamina on both surfaces. The cells are interlocked in successive sheets via tight junctions. Blood vessels also infiltrate this layer, wi th the perineurim functioning as a selectively permeable barrier. The outermost perineurial layers are composed of dense concentric layers of mostly l ongitudinally arranged collagen fibrils ~50 nm diameter with a few fibroblasts and macr ophages among the strands. The epineurium is a dense collagenou s layer surrounding all peripheral nerve trunks [14, 16-17]. Fibers in this layer are di sposed mainly longitudinally with diameters between 70 and 85 nm. Elastin fibers are also present, with diameters ranging from 250 to 500 nm. Fibroblast and mast cells ar e scattered throughout this layer. Peripheral nerve in the injured state. Axotomy (axon severance) occurs after any 2nd degree injury and beyond. The cel l body then undergoes chromatolysis (swelling) and increased prot ein and RNA metabolism [1, 14, 16]. Later, axonal sprouts

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7 grow from the proximal stump, and the distal stump undergoes Wallerian degeneration (a process in which the distally remaining seve red axon swells and br eaks apart). During Wallerian degeneration, Schwann cells in the distal stump concomita ntly dedifferentiate, reduce myelin protein synthesis, fragment remaining myelin sheaths into ovoids, phagocytize myelin debris along with macropha ges, and proliferate to form tubular structures termed bands of Bngner that gui de regenerating axon sp routs. Regenerating axons typically grow at a rate of 1 to 4 mm per day, and the events of degeneration and regeneration overlap. Schwann cells and macrophages also play a role in degeneration/regeneration at the molecular level through cytokine and growth factor production [18]. Immediately after a crushing injury, Schwann cells s how increased levels of IL-1 IL-6, LIF (leukemia inhibitory factor) and IL10 mRNA transcripts. IL-1 possibly induces nerve growth factor (NGF) synthesis while IL-6 appears to affect sensory fi ber regeneration. LIF appears to affect the conducti on velocity of regenerating fi bers reportedly increasing the size and number of myelinated fibers. Schwann cells also produce basal lamina components laminin and collagen type IV wh ich are required for neuroregeneration. Furthermore, Schwann cells secrete a cock tail of neurotrophic factors like NGF, neurotrophin-3, brain-derived growth factor (BDGF), neur egulin, fibroblast growth factors (FGF) 1 and 2, insulin-like grow th factors (IGF) 1 and 2, and ciliary neurotrotrophic factor (CNTF) that play active roles in neuroregeneration [19]. Transcript levels for the IL-18, IFNand TNF(pro-inflammatory cytokines) describe a more persistent upregulation p eaking ca. 1 to 2 weeks post injury [18]. Infiltrating macrophages appear to be the ce llular source of IL-18, but the source of

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8 IFNis less clear. Schwann cells, fibrobl asts, endothelial cells and macrophages all express TNFfollowing injury. Strong evidence supports the contention that TNFplays a significant role in macrophage recr uitment [18]. Transcripts for the antiinflammatory cytokine transfor ming growth factor-beta-1 (TGF1), the p40 subunit of IL-12 also peek 14 days following injury. Murine macrophages stimulated with myelin in vitro were shown to release IL-12 and TNF, suggesting that IL-12 expression is potentially a consequence of myelin phagocyt osis and part of macrophage autoregulation [18]. Previously studied biomaterial nerve conduits. Entubulation is the most common alternative to autograft repair [20]. In entubulation, severed nerve ends are inserted into a hollow or filled-lumen biomater ial tube employed to protect, facilitate and guide neuroregeneration. Gaps of centim eters have been regenerated successfully depending upon the specific materials used [ 6, 9, 19, 21-28]. Ideally, conduits [6] should be: Easily available Resorbable Readily vascularized Non-immunogenic Permeable to oxygen and other nutrients Able to block infiltrating scar tissue Able to function as depots fo r biologically active compounds Clinically investigated biomaterial conduits. According to a clinical review by Meek and Coert [6], vein, denatured muscle combination vein filled with muscle, silicone, Gore-Tex™, and polyglyc olic acid (PGA) tubes have been used clinically (in humans) for nerve reconstruction with success. Vein grafts were found suitable for gap lengths of 4.5 cm depending upon the nerve under repair. Muscle grafts appeared

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9 suitable for reconstruction of >6 cm gaps in leprosy patients and were judged superior to conventional nerve grafting for repairing 1.5 to 2.8 cm gaps resulting from laceration injuries. Combination vein filled with musc le conduits have been used successfully to reconstruct 6 cm gaps. The ready supply of vein and muscle makes them attractive graft material choices, and combination vein-muscle gr afts have shown superi or results to vein alone in similar defects. A llografts in combination with systemic immunosuppressive therapy have also been used successfully in the clinic to reconstr uct massive (>10 to 20 cm) peripheral nerve defects [29]. Hollow Gore-Tex™ conduits are indicated in reconstructions up to 4 cm and cause less tissue irritation than silicone tubes. S ilicone tubes were only shown successful for 4 mm gaps, and 29% of the tubes had to be remove d because of (compressive) irritation. In clinical studies using PGA tubes, the maximu m defect that could be reconstructed was 3 cm and the conduits performed significantly be tter than autograft. The PGA conduits permitted reconstruction of larger gaps perhaps because they were porous, permeable to oxygen and less likely to collapse. Also, b ecause these tubes were bioabsorbed, there was no need to re-operate for compression/irritation. Hence, clinical studies show that conduits (natural or synthetic) are at least comparable to autograft for repairing short defects ( ~3 cm). However, the ideal conduit milieu has not been established for repairing larger nerve gaps [20]. The studies also indicate that bioresorbable s ynthetic conduits are preferable to biodurable ones; filling the conduit lumen with a permissive tissue (e.g., mu scle) appears to yield significantly better regenerative outcomes. Although allografts ha ve been used successfully to reconstruct

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10 large nerve defects, the need for systemic immunosuppressive therapy is a serious drawback. Experimentally investigated biomaterials. Many experimental studies in animal models (primarily rat) aimed to improve c onduit design and performance. Essential facts gained from that literature are as follow s: Permeable conduits and conduits possessing smooth inner walls significantly outperformed impermeable conduits or conduits with rough inner walls [28, 30]. More importa ntly and perhaps not surprisingly, culturing/seeding autologous Schwann cells in conduits before implantation positively impacted regeneration, improving recovery [ 19, 26, 27]. Combining Schwann cells with a basement membrane gel such as Matrigel in the conduit lumen also positively affects nerve regeneration [9, 24]. The assertion that nerve conduits need to function as a scaffold more for Schwann cells than axons is gaining stre ngth in the experime nt literature. A few researchers have fashioned scaffolds that induce cultured/seeded Schwann cells to form structures reminiscent of Bngner bands [7, 12, 26], although results of these studies are preliminary. The multi-lumen PLGA-Schwann cell seeded conduits constructed to implement this strategy are of particular inte rest [7, 12]. Hadlock et al. [7, 12] have produced conduits incorporat ing both fundamental determinants of functional regeneration present in autograf t. Thin stainless steel wires in the polymer injection mold were used to approximate the continuous, t ubular microstructure of the endoneurium. Autologous Schwann cells were then flow-seed ed into these lamini n-coated conduits and the cellularized implant was placed in a 7 mm rat sciatic nerve defect. After 6 weeks, these conduits had statistically similar amount s of neural tissue per cross-sectional open

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11 area compared to autograft. However, the mean myelinated fiber diameter of 3.73 0.51 m was significantly higher than the 2.3 0.24 m mean diameter found in autograft controls (p < 0.05). Although these multilumen conduits are innovative and show promising initial results, studies using these conduits are far from comprehensive, and the requisite production methods could ultimately limit their widespread use. Alginate (Figure 2-2A) is a linear polys accharide discovered by E.C.C. Stanford in 1880 obtained from alkali digestion of various brown sea algae [ 31, 32]. The polymer chain is composed of -1,4 linked D-mannuronic acid (M) and -1,4 linked L-guluronic acid (G) monosaccharides found in three distinct blocks: polyM, polyMG and PolyG blocks [33]. Compositional variation is a re flection of source and processing. The pKa’s of the C5 epimers are 3.38 and 3.65 for M and G respectively, with the pKa of an entire alginate molecule somewhere inbetween [31, 32]. Alginate forms colloidal gels (high-wate r-content gels, hydrogels) with divalent cations. In the alginate ion affinity series Pb2+>Cu2+>Ba2+>Ca2+>Zn2+>Ni2+>Co2+>Mn2+, Ca2+ is perhaps the most used and characterized to form gels [34]. Studies indicate that Ca-alginate gels form vi a cooperative binding of Ca2+ ions by polyG blocks on adjacent polymer chains, the so-called “egg-box” mode l [32, 33]. G-rich alginates tend to form thermally stable, strong, yet britt le Ca-gels that are likely to undergo syneresis. M-rich alginates tend to form less thermally stable, weaker but more elastic gels. Alginate is commercially used as a bi nding, stabilizing and/or thickening additive in many foods and cosmetics [ 32]. Clinically, alginate is used in dental-impression materials and hemostatic wound dressings [35, 36]. Alginate:poly-L-lysine polyelectrolyte complex (PEC) encapsulated panc reatic islet cells were also evaluated in

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12 a human clinical trial for treatment of type I diabetes [37, 38]. Alginate:chitosan PEC beads and films have been made experime ntally for cellular immunoprotective capsules and drug release devices [39, 40]. Ionically (Ca2+) and covalently (e.g., ethylene diamine) crosslinked freeze-dried foams and gels have been developed and implemented as tissue scaffolds [39-43]. Copper algina te gel beads have been used for enzyme immobilization with success [44]. Barium and oligochitosan (Figure 2-2B) crosslinked alginate microspheres have al so been previously synthesi zed and investigated [45-47]. Copper-capillary alginate gel(s) (CCAG) have been previously described and studied in the scientif ic literature [31, 48-51] These self-assembled gels are essentially formed by allowing solutions of Cu2+ to diffuse uniformly into viscous solutions of alginate. During this diffusion process, T humbs and Kohler [48] state that fluid instabilities arise from the friction forces involved in the contraction of alginate polymer chains to the newly forming gel front. Convec ting tori (similar to those observed in the Raleigh-Benard model of heat convection) result from these hydrodyna mic instabilities. In a sense, these tori tunnel parallel capilla ries through the forming gel in the direction of diffusion. A continuous, tubular microstructure is mapped onto the forming gel because of the convective-like proce ss the system undergoes to diss ipate energy. Gel capillary diameter can be adjusted by ma nipulating (singly or in combin ation) the initial alginate concentration, initial Cu2+ concentration or system pH [31, 48-49]. Surprisingly, no previous reports descri be CCAG-derived hydrogels synthesized and implemented as tissue scaffolds; this beau tiful material and all its tissue engineering potential uninvestigated. This could be because raw CCAG (RCCAG) dissolves in several hours under cell culture conditions. However, many studies have already

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13 described chemical crosslinking of RCCAG [49], and ceramics derived from CCAGs have been produced and suggested as potential implants [52, 53]. Figure 2-1. Peripheral nerve hierarchical structure.

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14 A B O OHHO O O CO2H OHHO O n CO2H x y M GO NH2CH2OH O OH NHCOCH3CH2OH O OH O n x y Figure 2-2. Molecular structures of alginate and oligochitosan polymers. A) Alginate, B) Oligochitosan

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15 CHAPTER 3 MATERIALS AND METHODS Scaffold Synthesis All alginate used was Keltone LV obtaine d from ISP Alginates, Inc. (formally known as Keltone, MW range: 12,000-80,000 g/mol). Coppe r sulfate pentahydrate ACS grade was obtained from Acros Organics NJ. Barium hydroxide monohydrate was obtained from Aldrich Chemical Company, In c., Milwaukee, WI. Oligochitosan was a kind gift from Dr. Dong-Won L ee who originally obtained it from E-ZE Co., Ltd., Korea; manufacturer-reported average molecular we ight and moisture content were 1150 g/mol and 8%, respectively. Dr. Lee reported a 70% degree of deacetylation measured by 1HNMR [54]. Raw Copper-Capillary Alginate Gel (RCCAG) Preparation of 2% w/v alginate solution. 4 g of Keltone LV sodium alginate was dispersed in 170 mL of distilled water in a 500-mL Erlenmeyer flask. The suspension was stirred with a stir plate at medium-hi gh speed until a clear, homogenous solution was obtained. Distilled water was then added to the solution until the final solution volume was 200 mL, yielding a 2% w/v solution of sodium alginate (manufacturer-reported viscosity, 100-300 centipoise (cP)). The algina te solution was stirre d for 2 h and allowed to stand for an additional 2 h to minimize so lution bubbles. Solutions were either used immediately or stored for no more than a week at 4C. Petri dish preparation. A thin coat of alginate n eeds to be baked onto the petri dish to prevent gel separation from the vesse l wall during growth [31, 49-51]. Five thin

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16 coats of freshly prepared 2% w/v alginate solution were smeared onto the entire inner surface and rim of a Pyrex™ petri dish (9 cm di ameter 2 cm height or 9 cm diameter 3.25 mm height). A few minutes for air-dryi ng were allowed between coats. Once the 5 coats were applied, the coated petri dish wa s baked in an oven heated at 120C for 10 minutes. The dish was then removed, allowed to cool and the procedure was repeated 3 additional times. Classic descending growth technique The method below resembles the methods described previously [31, 49-51]. An alginate-coated petri dish was carefully fi lled to the brim, almost overflowing, with freshly prepared 2% w/v sodium alginate solution (Figure 3-1). A large Kimwipe™ soaked with freshly prepared 0.5M copper su lfate solution was pulled taut like a drum (using a needlepoint hoop) and brought down di rectly on top of the alginate-filled petri dish. The entire surface of the alginate solution and rim of th e petri dish were assured to be in good contact with the soaked Kimwipe ™. Over the course of 5-7 minutes at approximately 10-15 second intervals, 1-2 mL of 0.5M copper sulfate solution was dripped onto the soaked Kimwipe™ now cove ring the alginate-filled petri dish. The soaked Kimwipe™ was then slowly and gently peeled off the alginate filled petri dish. A solid membrane, contiguous with the rim of the petri dish (~ 1 mm thick) completely covered the top of the alginate -filled petri dish. This memb rane (the primary membrane) was a little rough, approximately the color of the 0.5M copper sulfate solution and contained no visible voids. Taking extreme car e not to jar the gell ing solution, the filled petri dish was transferred to a large covered tank. The ta nk’s geometry allowed for a 1.5 to 2 cm submersion of an alginate filled petri dish in 700 mL of the 0.5M copper sulfate

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17 solution. The tank was slowly filled with 700 mL of 0.5M copper sulfate, covered, placed on a leveled table w/anti-fatigue padding and left undisturbed for 36 hours. 9 cm 2 cm Thin Layer of Baked on Alginate Pyrex ™ Glass Petri Dish 2% w/v Alginate Solution Large Kimwipe ™ Soaked in 0.5M CuSO4Solution 0.5M CuSO4Solution Polystyrene Fish Tank 9 cm Thin Layer of Baked on Alginate Pyrex ™ Glass Petri Dish 2% w/v Alginate Solution Large Kimwipe ™ Soaked in 0.5M CuSO4Solution 0.5M CuSO4Solution Polystyrene Fish Tank Figure 3-1. Raw-CCAG (RCCAG) classical descending technique synthesis scheme. Time-lapse videoscopy Time-lapse videos documenting RCCAG gel growth were made using a Panasonic model PV-L658 Palmcorder, ATI Rage Fury Pro video capture card, and a Windows 98 PC running C3 Systems WinTLV dig ital time-lapse videography software. Barium Stabilized Copper-Capillary Alginate Gel (BCCAG) We chose barium for the ion-exchange process because it forms an extremely stable complex with alginate u nder physiological conditions [45, 46]. Also, previous experience showed that treatment with ba rium hydroxide did not grossly alter RCCAG morphology. However, special precautions were needed because soluble barium is toxic, and barium hydroxide reacts with carbon dioxide present in air. Exchange-reactor design and setup A Teflon™ reactor (~ 250 mL void volume) was used for the barium hydroxide treatment of RCCAG (Figure 3-2). This mi nimized the potential for personal contact

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18 with the toxic barium hydroxide solution and provided a rela tively air-free processing atmosphere. The reactor was then coupled with reservoirs, a sm all peristaltic pump, silicone tubing as plumbing and an ultra-hi gh purity (UHP) nitrogen gas bottle for the complete setup. A B Figure 3-2. Teflon™ exchange-reactor. A) T op-down view. B) Side view. A Buna-N rubber gasket was inserted into the th in grove, and a glass plate was clamped down on top of the reactor to form a sealed system. Barbed polypropylene screw-in connectors and Buna-N rubber ga skets were also inserted into the inlet-outlet ports. Barium hydroxide processing An RCCAG parent gel was cut into thin st rips (~ 3 mm) parallel to the capillary long axis with a stainless-steel kitchen knife Three long strips were sealed in the exchange-reactor and washed by flushing a to tal of 2.3 L of deioni zed (DI) water through

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19 the reactor over 72 hours. The sealed reac tor was then purged w ith UHP nitrogen and filled (250 mL) with freshly prepared 0.5M Ba(OH)2 solution. The filled reactor was then placed on an orbital shaker (btbBack to Basics, Bellco Biotechnology, Vineland, NJ) for 24 h at 75 RPM. The reactor was ag ain purged with UHP nitrogen and refilled with 200 mL of 0.5M Ba(OH)2 and shaken for an addition 24 h at 75 RPM on an orbital shaker. The sealed reactor was again purged with UHP nitrogen, filled with DI water and shaken for 24 h at 75 RPM; this DI water so ak was repeated one additional time. A total of 3 L of DI water were then flushed th rough the reactor over 72 hours. The exchangereactor was finally unsealed, and BCCAG sample s were extracted and stored in DI water at 4oC for further processing or experimentation. Oligochitosan-Barium (OBCCAG) and Oli gochitosan (OCCAG) Stabilized RCCAG Chitosan, (a polysaccharide polymer composed of -1,4' linked glucosamine and N-acetylglucosamine residues) was chosen for PEC stabilization because much work has been done producing and characterizing algi nate-chitosan multilayer microspheres [40, 55]. Chitosan also appears to have excel lent biocompatibility [56], and alginate microspheres crosslinked with oligochitosan have also been previously reported [47]. Preparation of 2% w/v oligochitosan solution. 2g of oligochitosan were dispersed in 80 mL of DI wate r in a 250-mL Erlenmeyer flask. The suspension was then stirred vigorously until a clear, yellow-brown solution was obtained. DI water was then added to the solution until the final soluti on volume was 100 mL yielding a 2% w/v solution of oligochitosan. Solutions were ei ther used immediately or stored for no more than a week at 4oC. Preparation of OCCAG/ OBCCAG. RCCAG samples (3-5) cut into rectangles (~ 7 mm 5 mm 3 cm) were placed into 50 mL centrifuge tubes. Freshly prepared

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20 oligochitosan solution (45 mL, 2% w/v) was th en added to each and the tubes were then placed on an orbital shaker for 17-19 hours. Next, the oligochitosan solution was poured off and the samples were rinsed three times with small volumes (5-10 ml) of DI water. DI water was then added (45 ml/tube) and th e tubes were placed on an orbital shaker overnight. The DI water was fully exchange d at least once over the next 8-12 hours. Samples were then stored in a small volume of DI water at 4oC. The procedure to produce OBCCAG was identical to the above except BCCAG was used as the starting material instead of RCCAG. OCCAG/ OBCCAG washing in cell culture medium. Samples were placed singly in the wells of 6-well cell-culture plat es. Three milliliters of cell culture (either fibroblast or ES differentiation, see below) media containing serum were then added and the plates were placed in a 37oC incubator overnight. The media was completely exchanged and the plates were returned to th e incubator overnight. After this point, the scaffolds were used for cell-biology experiments. Scaffold Characterization All optical microscopy was conducted on samp les just submerged in distilled water (as this provided the clearest, most consistent images). To freeze-dry materials for SEM analysis, samples were placed individually in 50 mL polypropylene centrifuge tubes with 3 mL of DI water. Samples were then flas h frozen by placing the tubes in liquid nitrogen for 5 minutes. The flash frozen samples were then freeze-dried (-40oC, 10-15 m Hg) on a Labconco lyophilizer (Kansas C ity, MO) for at least 48 hours. Optical microscopy Using a 1 cm inner-diameter stainle ss-steel cork bore (Precision Tool & Engineering, Gainesville, FL), a plug the entire height of the parent RCCAG gel was

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21 quickly punched out, starting from the bottom parent gel face. The sample plug was gently pushed out of the bore, and the thin layer of the primary membrane was cut off with a stainless steel-kitchen knife and aluminum miter box. The core was then progressively sectioned into discs approximate ly 3 mm thick. Upper, middle and lower samples were placed separately in Pyrex™ glass bowls (5 per bowl) and submerged in 100 mL of DI water. Each bowl was covered and stirred on an orbital shaker at 100 rpm for 72 hours. The water in each bowl was completely changed every 12 hours. After washing, three discs from upper, middle and lowe r core sections were observed with an Olympus SZ stereomicroscope (Tokyo, Japan) equipped with a Mi niVID digital camera (LW Scientifc, Lawrenceville, GA). Optical mi crographs were recorded and stored on a Windows 98 PC using an ATI Rage Fury Pr o video capture card running ATI Multimedia Center software version 6.2. An image of a 25 mm reticle (0.010 mm gradations, Klarmann Rulings, Inc., Manchest er, NH) was also captured to scale the sample images. Determination and comparison of average capillary diameter as a function of parent gel thickness. Thirty (30) capillaries from each micrograph were measured using NIH ImageJ freeware version 1.28u. That da ta was inputted into Microsoft Excel 97 spreadsheets and average capillary sizes and standard deviations were calculated using internal Excel functions. ANOVA analysis was performed with Mi nitab Release 14.12. Differences were judged significant for p 0.05. Scanning electron microscopy Freeze-dried samples of RCCAG, BCCAG a nd OCCAG produced previously were mounted separately onto aluminum SEM stubs with double-sided carbon tabs (SPI Supplies, West Chester, PA). The mount ed specimens were then carbon coated (Ion Equipment Corp., Santa Clara, CA) and stor ed until analyzed in a desiccator. All

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22 samples were analyzed using a JEOL JSM-6400 SEM (JEOL USA, Peabody, MA) equipped with an Oxford energy dispersive spectroscopy (EDS) system and a LINK ISIS software package version 3.35 (Oxford Instru ments USA, Concord MA). All samples were analyzed at 20 KeV accelerating volta ge to maintain consistency with the standardless digital library. This accelerati ng voltage is more than sufficient to observe all X-ray peaks of interest with EDS. Image processing was performed utilizing features available in the LINK ISIS software package. Percent Water Content Determination Five small, previously washed samp les from RCCAG, BC CAG and OCCAG each were equilibrated in a minimum of distilled water in 50 ml conical centrifuge tubes for one week. After equilibration, the sample s were removed, blotted to dryness on a Kimwipe™, placed in a pre-weighed 15 ml conical centrifuge tube, weighed and recorded. The samples were then re-submerge d in a minimum of distilled water and flash frozen in liquid nitrogen and lyophilized for 48 hours. After l yophilization, the tubes w/sample were re-weighed and recorded. The difference between the initial and final weights was attributed solely to the loss of water during dryin g. ANOVA analysis was performed with Minitab Re lease 14.12. Differences were judged significant for p 0.05. Biological Assesment All regular cell maintenance such as media changing, cell splitting, etc. was performed solely by Dr. Takashi Hamazaki of the Terada group, Department of Pathology, University of Florida. All Cell seeding, maintenance and documentation of scaffold culture experiments were done jointly with Dr. Hamazaki. Fluorescence microscopy was performed with an IX70 Olympus/C Squared equipped with a MagnaFire digital camera system and soft ware package (Optronics). Confocal

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23 microscopy was performed by Marda Jorgense n, Departement of PathologyStem Cell and Regenerative Medicine Program, Univer sity of Florida us ing a Leica TCS SP2 AOBS Spectral confocal microscope equippe d with laser point scanning (405-633 nm) and proprietary software (Leica Micr osystems Inc., Buffalo, New York). Maintenace of mouse Swiss Albino em bryonic fibroblasts expressing greenfluorescing protein (GFP-3T3) cells. GFP-3T3 cells were main tained in tissue culture dishes (6 cm, 2 X 105 cells) in Dulbecco’s Modified Eagle Media (DMEM, GIBCO BRL, Grand Island, NY) containing 10% fetal bo vine serum (FBS, Atlanta biologicals, Norcross, GA), 2 mM L-glutamine, 100 units /ml penicillin, 100 g/ml streptomycin, 25 mM HEPES (GIBCO BRL). Media (termed fibroblast media) was changed every two days and the cells were split upon reaching ~ 2 X 106, 80% confluence. Maintenace of mouse embryonic stem cells expressing green-fluorescing protein (GFP-mES). GFP-mES cells were maintained in an undifferent iated state on gelatin-coated dishes (6 cm, 4 X104 cells) in Knock-out DM EM (GIBCO BRL, Grand Island, NY) containing 10% knockout serum replacement (KSR, GIBCO BRL), 1% FBS (Atlanta biologicals, Norcross, GA), 2 mM L-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, 25 mM HEPES (GIBCO BRL), 300 M monothioglycerol (Sigma, St. Louis, MO), and 1000 unit/ml recombinant mouse Leukemia inhibitory factor (LIF, ESGRO) (Chemicon, Temecula, CA). Me dia (termed ES maintenace media) was changed every two days and the cells were split upon reaching ~ 2 X 106, 80% confluence.

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24 In Vitro Study: Swiss Albino Embryonic Mouse Fibroblasts Expressing Green Fluorescent Protein (GFP-3T3) It was not known if CCAG-de rived scaffold would be re latively non-toxic to cells in in-vitro cell culture. Hence, two circul ar OCCAG scaffolds (~ 8 X 3 mm) were placed singly into a six-well tissue culture plate (N alge Nunc International, Rochester, NY). GFP-3T3 cells were first dissociated by us ing 0.25% trypsin/EDTA (GIBCO BRL) and then re-suspended in the GFP-3T3 culture media (see above). To seed the cells, a total of 200 l of the cell suspension (1 X 106 cells/ml) was applied to one end of the capillaries while applying vacuum to the other capillar y ends. Cell-scaffold combos were then cultured for one week in fibroblast media. The combos were observed daily with the fluorescence microscope and the media wa s changed every two days. Confocal microscopy was performed on select samples at day 2 in culture. In Vitro Study: Mouse Embryonic Stem Cell s Expressing Green Fluorescent Protein (GFP-mES) Undifferentiated ES cells were dissoci ated using 0.25% trypsin/EDTA (GIBCO BRL). ES cells were suspended in Iscove 's Modified Dulbecco's Medium (IMDM), supplemented with 20% fetal bovine serum (A tlanta biologicals), 2 mM L-glutamine, 100 units/ml penicillin, 100 g/ml strept omycin (GIBCO BRL), and 300 M monothioglycerol (Sigma). This media was te rmed “ES differentiation media”. To seed ES cells into OCCAG scaffolds, a to tal 200 l of the cell suspension (1x106 cells/ml) was applied from one end of the capillaries whil e suctioning the fluid fr om the other end of the capillaries. Evaluation of mES cell growth, su rvival and morphology vs. time No previous data was available to inform one’s intuition about mES cell behavior when seeded and cultured in CCAG-derived sc affolds. Therefore, 4 OCCAG scaffolds

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25 cut into rectangular blocks were seeded, placed singly into a six-well culture plate (Nalge Nunc International) a nd cultured in ES maintenance medi a for nine days. Cell-scaffold combos were observed daily and the media was changed every two days. Fluorescence micrographs were recorded at days 0, 6 a nd 9 and confocal microscopy was performed on select samples at day 7 in culture. Comparison of ES maintenance (LIF+) and ES differentiation (LIF-) media conditions It was not known if mES cells seeded in OCCAG scaffolds and cultured under cell differentiation conditions behaved differently in terms of cell survival and proliferation than cells seeded and cultured under cell main tenance (undifferentiated) conditions. To that end, we conducted a four day study us ing ES maintenance or ES differentiation media. The four day end point was chosen because previous experience and reported studies [57] indicated that mES cells are in the early phases of cell fate determination when cultured in ES maintenance me dia, i.e. media lacking LIF. Six OCCAG scaffolds cut into rectangular blocks (~ 10 X 5 X 3 mm) were seeded, placed singly into a six-well culture plate (N alge Nunc International) and cultured over four days under one of three different media conditions: mES maintenance medium (LIF+) only mES maintenance (LIF+) / mES differentiation (LIF-), switched at day 2 in culture. mES differentiation (LIF-) only

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26 CHAPTER 4 RESULTS AND DISCUSSION Scaffold Synthesis and Processing Scaffold synthesis was successful overall, albeit an underestimated challenge. Early on it was discovered that RCCAG dissolv ed over the course of several hours (<24 h) in standard cell culture media likely due the chelation and/or ion-exchange of Cu2+. An attempt to covalently crosslink RCCAG with ethylene diamine utilizing carbodiimide chemistry was made, however, the attempt fa iled due again to the rapid chelation of Cu2+ by ethylene diamine. Ethylene diamine solu tion dissolved the RCCAG within a matter of minutes. Other synthetic techniques to cros slink the raw material were avoided in deference to the end-use as a biomaterial. These complications led to the successful attempts to stabilize RCCAG via ion-exchange with Ba2+ ions and/or formation of a polyelectrolyte complex with oligochitosan (below). Although the synthesis procedures described in the Materials and Methods section of this text are far from optimized, they were adequate to produce gram scale quanti ties of all the new scaffolding materials. RCCAG Production of this material was the most straightforward because it had already been investigated. As previous ly reported, coating the petri di sh with a thin film of baked alginate proved necessary. However, the te mporary use of a Kimwipe™ to aid in the formation of a regular primary membrane was a new addition to the general RCCAG synthesis method. Significant amounts of waste copper sulfate solution were also produced and the raw material had to be wa shed extensively to rid it of excess copper

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27 sulfate. The amount of waste produced makes the material less attr active for large scale production. RCCAG was a homogenous transl ucent sky blue due the Cu2+ ions crosslinking it and was also the most durable of the material s produced in this work. It did not tear easily and regained its original shape after co mpression. Cutting the material parallel to the long capillary axis was much more difficu lt than cutting perpendicular to the long axis. This anisotropy is presumably due the fact that the alginate chai ns are preferentially oriented perpendicular to the long capillary axis [31, 48-49]. Growth videos Successful video monitoring of the entir e RCCAG synthesis process was achieved. The use of time-lapse videoscopy (TLV, vide o 4-1) was a powerful technique yielding not only kinetic data, but also clearly illustra ted the fact that RCCAG “grows” via a selfassembly process. The most fundamental reaction occurring is the binding of Cu2+ ions by alginate molecules in solu tion; all other processes (cha in contraction, formation of convective tori) resulting in the material’s st ructure and anisotropic properties stem from this action. Growth kinetics Kinetic data obtained from RCCAG growth TLVs are of particular engineering interest. Figures 4-1 and 4-2 summarize our key kinetic findings. In a previous study, Schuberth [31] puts forward the idea that ge l growth follows the so-called square law (equation 4-1and 4-2). It was confirmed in that study that RCCAG growth behavior is approximated by the square law at least within the first hour of gel growth. 1) (4 Dt 2 y

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28 Where D: Diffusion Coefficient (cm2/s) and t: Diffusion Time (s) y: Diffusion Path Length/Gel Thickness (cm) Hence, 2) (4 t y Our kinetic data do confirm that equati on 4-2 reasonably approximates the very beginning of gel growth assuming D 2 0.02 cm/min. However at longer growth times past this initial phase the square law mode l significantly under-predict s the gel thickness. A power fitted model of the observed data sugge sts a value of 0.6 rather than 0.5 for the value of the exponent. The 1st derivative plots also support this contention. The gel growth rate is also apparently decreasi ngly settling within a range of 0.001 0.002 cm/min. It is unclear how this reported value of the RCCAG growth rate specifically compares with other studies [31, 58], but Sc huberth does report that growth rates are ca. 25% slow in gels lacking capillaries and a sserts that an overlay of convection in the capillaries could account for this difference. Storage concerns During the early phases of this project, it was decided to store the RCCAG in some formation buffer (0.5M CuSO4) at 4oC in a commercial polymer container called FoodKeepers™ by Anchor Hocking. At the time it was tacitly assumed that the container was “resistant” and would not contaminate the RCCAG with soluble degradation products. Later re search into the exchange re actor material design however indicated that CuSO4 solution could be caustic to a wide range of polymers over long

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29 exposure of times. Unfortunately, details of the Foodkeepers™ polymer composition were difficult to find since the brand has been discontinued for many years, but the dishes are very rigid and heat resistant, perhaps sim ilar to a phenolic or melamine type resin. Hence, at the current time it is impossible to rule out the possibility that the storage container contaminated the RCCAG with bi ologically active de gradation products; though it can be stated with confidence that this possibility is remote at best given the extensive washing regime undertaken during production. BCCAG This material was the most challengin g to produce. BCCAG was also the most interesting from a materials perspective and proved to be quite stable in cell culture media. However, BCCAG had the poorest ha ndling qualities tending to crumble or fracture readily. This limita tion, coupled with its demandi ng synthesis, limited the study of BCCAG as a scaffold to a few rough ce ll culture experiment s (data not shown). Colorimetric changes during barium hydroxide treatment When RCCAG samples were initial submerged into 0.05M Ba(OH)2 solution they floated due to a difference in density. Howe ver, within minutes the samples sank and this sinking was accompanied by a color change of samp le edges from sky blue to royal blue. This color change uniformly proceeded into the core of the samples over the course of many hours. Sample cores then began to blacken sometime between 12-24 h of treatment, and this blackening was further enhanced during the washing of the newly formed BCCAG in DI water. The above colorimetric changes can be explained by the formation of different copper compounds within RCCAG samples duri ng barium hydroxide processing. The initial color change from tran slucent sky blue to royal bl ue corresponded to the reaction

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30 of Cu2+ ions with hydroxide ions to form copper hydroxide (Reaction 4.1) which is often described as a pale blue gelatinous water insoluble precipitate. Cu2+(aq) + 2OH-(aq) Cu(OH)2(s) 4.1 Although the term “pale” seems inconsistent with the above descri ption, concentration and matrix effects presumably influence the ap parent copper hydroxide color intensity. The progressive blackening of the core was due to the progressive formation of copper II oxide (Reaction 4.2). Cu(OH)2(s) + Heat CuO(s) + H2O(l) 4.2 Copper II oxide is often descri bed as a black or golden brown insoluble precipitate formed by heating copper hydroxide. The h eat released by the formation of copper hydroxide in the RCCAG possibly drove its ow n decomposition to copper II oxide within the gel (Figure 4-7). Exchange-reactor advantages and difficulties The exchange reactor was a tremend ous advantage during barium hydroxide possessing. Barium hydroxide is caustic and toxic and r eadily absorbs carbon dioxide from the air. The excha nge reactor provided a means of exposing large amounts of RCCAG to barium hydroxide solution under an atmosphere of UHP nitrogen, and a means of flushing the solution directly to waste. The newly produced BCCAG could then be extensively washed with water under nitrogen as well. What was at the onset a tedious and precarious task of filling and draini ng flasks of toxic solutions was reduced to filling appropriate reservoirs; the plumbing of the exchange-reactor minimized the human interaction with toxic effluents. This system was far from perfect though.

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31 The placement of the inlet and outlet ports co mplicated reactor filling and draining. Also, the silicone tubing serving as pl umbing had a tendency to split during long pumping cycles resulting in a significant reactor leak. Finally, it was difficult to maintain a low, consistent flow rate with the perist altic pump used. Despite these short comings, BCCAG was reproducibly produced in suffici ent quantity for further study. OBCCAG and OCCAG Only OCCAG was successfully produced utilizing the synthesis protocols described in this work. Fortunately, this ma terial was well suited for use in biological experiments (below) due to its optical clar ity and ease/reproducibility of production. OCCAG was inhomogeneously colored in crosssection, composed of a yellowed outer surface with a blue-green core. This inhomoge neity is likely the result of differential crosslinking of the exterior and core by the 2% w/v oligochitosan solution. Apparently, a more densely crosslinked skin of alginate :oligochitosan PEC formed around samples of OCCAG. Overexposure or overreaction is a concer n and possibility for any chemical crosslinking procedure of polymeric ma terials and CCAGs are no exception. The oligochitosan is a multifunctional crosslinker forming ionic rather than covalent bonds. The electrostatic bonding between the RCCA G or BCCAG and oligochitosan happens essentially instantaneously. Slightly to moderately overexposed rectangular samples began to round at the corners and distort as the overcrosslinked PEC skin contracted on the low modulus gel core. This skin was also darkly stained brown which negatively impacted its optical qualities. It was also found in an early set of experiments that RCCAG stiffened, turned dark brown and profoundly shrunk when severely overreacted

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32 in an excess of 2% w/v oligoc hitosan solution (reaction times 24 hours). Syneresis of the gel likely accompanied these profound changes. A reaction time range of 17 to 19 hours was th erefore used in this study to stabilize both RCCAG and BCCAG with 2% w/v oligoch itosan solution. This exposure time resulted in no significant change in the mate rials’ original size and morphology, and the materials were only slightly yellowed in color after the reaction. OBCCAG synthesis however failed during the cell culture media wash. Consequences of Media Wash Media washing is technically the final step in scaffold processing because material changes occur during the process. All of the copper present in the scaffolds as free ions or otherwise appears to be removed with succe ssive washes in media. This effectively dissolved the water insolubl e copper hydroxide and oxide pa rticles present in BCCAG. Free copper ions are also apparently leached or chelated serving to decolorize the scaffold. The result is a translucent scaffold colored the same as the phenol red spiked media itself. This result was great because it facilitated the use of advance microscopic techniques to observe the cells in situ, alive and dynamic. The OCCAG results were similar the BCCAG results, but OBCCAG collapsed and stiffened when washed in media, reminiscent of the earlier overcro sslinked RCCAG and consequently was never used in cell culture experiments. Scaffold Characterization A broader characterization regime was initially envisioned for the materials developed in this project. In fact, XPS, XRD and TEM measurements were all attempted but yielded poor and/or inconc lusive results. Technical difficulties encountered in sample preparation were essentially to bl ame for the poor results. As the project

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33 progressed, timing and the high demand for scaffolds for the biological assessment stunted further pursuit of XPS, XRD and TEM measurements. Also, at the time it was not clear what measures were most germane and how best to design experiments aimed at acquiring them. Therefore, the characterization data pr esented below is not comprehensive; however, the optical microscopy (OM), scan ning electron microscopy (SEM) and the small swelling studies reported here provide a solid foundation for future work. The OM studies essentially report on RCCAG mor phology; the SEM studies describe the morphology and composition (via energy dispersive spectr oscopy, EDS) of RCCAG, BCCAG and OCCAG before cell culture media processing. The swelling study was an attempt to elucidate possible differences in the materials’ equilibrium water contents resulting from the different cros slinking/ stabilization methods. Optical Microscopy The Olympus SZ equipped with the Mini VID camera provided an effective and comparatively inexpensive digital capture microscope system. The images captured with this setup were more than adequate for obtai ning quantitative measurem ents. This digital stereomicroscope also gave a fair idea of surface topography. RCCAG Figure 4-3 and 4-4 show representative mi crographs of RCCAG samples cut from a parent gel. The average capillary diameter of three representative areas at each gel level is given in figure 4-5. This graph also show s that the average capillary diameters for all samples in the upper gel level group differed significantly from each other (p < 0.05); only one sample was judged to have a significa ntly different average capillary diameter in the middle and lower gel level groups.

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34 The fact that at least one of the averag e capillary diameters within each group in figure 4-5 differed from the others supports th e idea that either the capillary diameters within a given gel levels are not uniform or that there was significant systematic error involved in sample sectioning. The latter is lik ely the case for this set of experiments. In fact, the sectioning method described in the Ma terials and Methods ch apter turned out to be somewhat crude, yielding both imprecise and inaccurate results. Section thickness was therefore variable, sometimes differing by a millimeter or more. It is also unclear if the above data is comparable to pr eviously conducted studies [31, 49]. Figure 4-6 presents calculated RCCAG me trics germane to tissue engineering derived from the average capillary data. Because all of the uppe r gel level samples differed significantly from each other, only th e smallest average capillary diameter was used to calculate the subsequent metrics. Middle and lower gel level metrics were calculated from pooled capillary diameter data of samples that were judged statistically similar by ANOVA (p 0.05). The error bars given for the average capillary diameters are the standard deviations. Despite the limitations discussed above, cap illary diameter is undeniably a function of gel thickness. Hence, it is tempting to c onclude that the apparent differences in the calculated metrics are significant and are also subsequently a function of gel thickness; the story is less clear for the percent free spa ce. The following trends are clear from the graph: capillary density with gel thickness and the average surface area in a standard disc with thickness. Evidence of precipitates within BCCAG Figure 4-7 and 4-8 are repr esentative micrographs s howing brown and shimmering particles within BCCAG samples. The prec ipitate shown in figure 4-7 appears golden-

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35 brown (presumably due to lighting) and is the same formation responsible for the progressive blackening describe d in the scaffold synthesis section above. This datum further supports the claim that copper II oxide has precipitat ed with the capillaries of BCCAG due to processing in barium hydroxide solution. The shimmering particles are believed to be insoluble barium sulfate and/or carbonate crys tals formed within the walls of BCCAG during synthesis. Scanning Electron Microscopy/Energy Di spersive Spectroscopy (SEM/EDS) and Xray Mapping The SEM/EDS and X-ray mapping studies yiel ded a wealth of data concerning the sample morphology, bulk composition and elemen tal distribution within samples Despite the analysis not being optimized for im aging, high quality secondary (SEI) and backscatter (BSE) images were still obtained Relatively short collection times (ca. 20 min.) coupled with image processing functi ons like smoothing and contrast enhancement provided informative X-ray maps with an economy of time. Consequences of freeze-drying Freeze-drying had several effects on the st udied materials. RCCAG densified and the once circular capillaries turned pentagonal or hexagonal. This is especially visible in the BSE image in figure 4-9B. BCCAG became very fragile and powdered if handle too much, but maintained circular capillaries (Figure 4-10A). OCCAG did not become fragile but maintained circular capillaries (Figure 4-12A). All ma terials however tended to flake into sheets perpendicu lar to the capillary long axis. RCCAG Data Figure 4-9 is a summary of represen tative RCCAG SEM/EDS and X-ray mapping data. The secondary image (4-9A) shows irregularly shaped capillaries, but the

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36 backscatter image (4-9B) shows that these shapes are likely artifacts of sample preparation. The backscatter signal is more sensitive to mass-thickness than is the secondary signal, and it appears that the irre gularly shaped capillarie s are really the result of thin sheets or flaps of RCCAG that have fall en over the capillaries at some point in the sample preparation. Hence, the densif ied hexagonal and pentagonal walls or RCCAG beneath the thin sheets is highlighted in th e BSE image. This morphology has been seen previously in earlier experime nts with RCCAG (d ata not shown). EDS analysis shows that RCCAG is esse ntially composed of carbon, oxygen and copper (Figure 4-9C). The carbon and oxygen ar e the sole components of the alginate polymer and the copper is the crosslinker res ponsible for the capillary structure. Low amounts of silicon and sulfur al so appear in the representa tive spectrum and presumably came from the alginate powder used to make the 2% w/v sodium alginate solution and residual copper sulfate used in gel synthesis respectively (Ch. 3, Materials and Methods). Caution should be exercised when attempting to assess the amounts of elements from any EDS spectrum. Peaks of elements that ar e actually present in high concentrations (particularly low Z elements) can appear smalle r than peaks of elements present in small concentrations. The correction matrix is co mplicated and the variables used are usually poorly known for low atomic number elements leadi ng to semi-quantitative data at best. The X-ray map group (figure 4-9D) shows low levels of silicon within the RCCAG walls; sulfur is also present to a lesser degr ee in the walls, but appears more concentrated in discrete particles. Copper appears unifo rmly distributed over the whole map area and it is not possible to discern the capillary structure in the copper map. This is due to the large interaction volume of th e X-ray signal (especially in a polymeric material) and the

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37 relatively energetic nature of the copper K X-ray. The X-ray signal comes form “deep” within the material (perhaps > 1 m) and the Cu K is not significantly absorbed by anything else in the sample resultin g in a homogenous looking copper map. BCCAG Data BCCAG was the most complex material produc ed in this study. Figure 4-10 is a representative summary of BCCAG SEM/ED S and X-ray mapping data. Figure 4-11 is a higher magnification study highlighting copperrich nano-particulate formations within BCCAG. The SEI BCCAG image (figure 4-10A) shows that the capillaries have remained circular and the material does not appear to have densified like RCCAG. Excellent BSE images were obtained due to the material’s barium content (Figure 410B). The representative EDS spectrum (4-10C) indicates that BCCAG is mainly composed of carbon, oxygen, copper and barium. Strontium also appears in the spectrum, overlapping in the same energy rang e as silicon (Figure 4-9C). Silicon could be present but masked by strontium that apparently came from the barium hydroxide solution. The small aluminum peak is po ssibly due to scatter from the SEM mount. Figure 4-10D, the BCCAG X-ray map group, is markedly different from the RCCAG map group (figure 4-9D). Barium now appears homogenously distributed throughout BCCAG, supplanti ng copper; strontium also app ears uniformly distributed. The majority of the copper signal is locali zed to the “bumpy” particles lining inner capillary diameters. Figure 4-11A and C are higher magnification SEI images of the copper-particles; figure 4-11B is the compleme ntary BSE of 4-11A. 4-11E is an X-ray map group of the same area as 4-11A taken to more clearly illustrate the distribution of elements within BCCAG. Sulfur rich areas also appear in this map group which are

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38 believed to correspond to the shimmering part icles noted in BCCAG optical microscopy above. A complex series of physicochemical events occur during barium hydroxide processing of RCCAG. Upon submersion in the barium hydroxide solution, copper hydroxide begins to form in the outer surfaces and edges of the material. As the barium and hydroxide ions diffuse into th e RCCAG matrix and capillaries, Cu2+ ions react with the OHions forming insoluble copper hydroxide at all material-solution interfaces. As the reaction proceeds, Cu2+ ions at the interface are depl eted, stimulating migration of Cu2+ ions from within the material down th eir concentration gradient. Copper ions migrating to the material-solution interface re act with the essentia lly infinite sink of solution hydroxide ions forming more inso luble copper hydroxide concentrated at the interface. Heat produced from the formation reaction is not dissipate d efficiently within the RCCAG sample, and thus drives the dehydration of the newly formed copper hydroxide to copper oxide over time. The above ideas are not intended to apply to all copper within BCCAG. Examination of the BCCAG X-ray map groups clearly shows copper within the BCCAG matrix. However, a comparison of the RCCAG and BCCAG X-ray map groups also shows a change in the distribution of copper a nd it is this change th at the above theory attempts to explain. Concomitantly, barium ions exchange w ith copper ions and/or form new ionic crosslinks within the gel, stabilizing its stru cture. This exchange presumably influences the migration of Cu2+ ions to material-solution interfaces. Residual SO4 2and dissolved CO3 2ions also react with diffusing Ba2+ ions forming insoluble salt crystals within the

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39 RCCAG matrix (Figures 4-11D and 4-12). These crystals result in the “shimmering” optical micrographs discussed above. OCCAG Data Figure 4-13 is a summary of the repr esentative OCCAG SEM/EDS and X-ray mapping. 4-13A shows that OCCAG has also retained circular capillaries, but the material’s surface appears “hairy”. This surf ace character can be mo re easily seen in the higher magnification SE image shown in figure 414. The striated la yer structure of the RCCAG has also been preserved. The OCCAG EDS spectrum (4-13C) appears similar to the RCCAG EDS spectrum shown earlier with th e addition of small amounts of chlorine. Closer inspection of 4-13C shows a much hi gher carbon and oxygen intensity as well as a reduced copper intensity in contrast to figure 4-9C, probably due to oligochitosan processing. The oligochitosan is a polymeric cros slinker composed mainly of carbon and oxygen. Oligochitosan ionically crossli nks RCCAG (from the surface inwards) via a positively charged amine functionality. Hence, oligochitosan processing results in a carbonaceous film on the surface of OCCAG which would contribute to the higher carbon and oxygen intensities, as well as damp ing the measured copper intensity. The amine residues could also leach Cu2+ ions from the RCCAG, fu rther contributing to the drop in measured copper signal. Summary of morphologic and compositional analysis RCCAG has a smooth surface with pentagona l and hexagonal shaped capillaries and a densified structure due to freeze-drying. The material is composed of mainly carbon, oxygen and copper with low amounts of silicon and sulfur. All elements, with

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40 the exception of sulfur, appear uniformly dispersed in RCCAG; sulfur appears concentrated in particles. BCCAG has circular capillaries and the stru cture does not appear to have changed due to dehydration. This material is co mposed mainly of carbon, oxygen, barium and copper with low amounts of strontium and sulfur. Barium and strontium appear homogenously distributed throughout the ma terial. Copper is concentrated in nanoparticles located on the inner capillary surfaces gi ving a bumpy appearance and sulfur appears as particles in and on BCCAG. OCCAG has circular capillaries and the st ructure also does not appear to have changed due to drying. Sim ilarly to RCCAG, OCCAG is composed mainly of carbon, oxygen and copper, however the relatively intens ities of these elements are different. Carbon and oxygen appear greater while copper appears lower presumably due to the carbonaceous surface coating resulting from oligochitosan crosslinking. The copper content of OCCAG may have al so been depleted via comple xation with the oligochitosan in solution. Equilibrium Water Weight Percent Analysis Figure 4-15 shows the results of the sma ll swelling study comparing the different CCAG crosslinking methods. Although the graph suggests that RCCAG contains the highest equilibrium water weight percen t followed by OCCAG and then BCCAG, no significant differences between the groups were indicated by ANOVA analysis (p 0.05). The study should be repeated with larg er sample sizes and a revised experimental procedure. The procedure implemented for wet sample weight measurement in this experiment was the largest source of systematic error.

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41 Biologic Assessment Initially, this work was motivated to aid peripheral nerve regeneration by developing synthetic biomateria l mimics of the endoneurium. The requisite materials were developed, but neuroregenerative testing was frustrated mainly due to the lack of a motivated, expert collaborator. This was a bl essing in disguise howev er, as expert stem cell biologist collaborators (The Terada Group, Department of Pathology, University of Florida) did enthusiastically particip ate in in vitro scaffold testing. The main point of the biological experime nts discussed below was to assess 1) if cells could be seeded in and on the newly developed scaffolds and 2) if these seeded cells could survive and proliferate ov er the course of several days Initially it was hoped that BCCAG, OBCCAG and OCCAG could all be test ed, however the only reportable data came only from OCCAG experiments. First we tried to seed and gr ow green fluorescent mouse embryonic fibroblast (GFP-3T3) cells in OCCAG scaffolds, but obtained a limited data set. Later we switched to a green fluorescent mouse embryonic stem cell line and were able to conduct multiple experiments. The results presented below show that cells, mouse embryonic stem cells (mES) mainly, can be seeded into OCCAG scaffolds, and that these cells survive and proliferate over the course of many days. Furtherm ore, mES cells form ordered cylindrical structures when seeded and grown in OCC AG scaffolds. Hence, the claim that CCAGderived scaffolds can impose structural or der on growing cells vi a architecture and geometry is well supported. Mouse Embryonic Fibroblasts (GFP-3T3) GFP-3T3 cells were initially chosen becau se they are robust, highly proliferative cells that were in good supply. Also, fibrobl ast migratory behavior has been reported on

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42 previously [59] and it was hoped that this behavior would be observed for direct comparison. Unfortunately, the GFP-3T3s avai lable appeared to have the same diameter as the capillaries (~ 25 m) and were not easily seeded into the OCCAG scaffolds. This significantly limited the possible experimental work. Figure 4-16 and video 4-2 are a representative micrograph and confocal mi croscopy video from an early GFP-3T3 study respectively. The relative clarity and translucence of the OCCAG scaffold provided for reasonably good fluorescent and confocal images. The confocal video shows the morphology of GFP-3T3 cells up to ca. 100 m d eep (perpendicular to the capillary long axis) in the scaffold after tw o days in culture. Cells with in capillaries usually appear deformed, taking on a pill-like shape. Multi cellular aggregates appear clumped on the outer surface of the OCCAG sample. This possibly indicates that OCCAG is not very adhesive to cells as they prefer clump toge ther rather than attach and spread on the materials surface. Cells confined within th e capillaries typically did not survive more than 2-3 days and did not appear to proliferat e. Large vacuous regions observed within the GFP-3T3s were also taken as a sign of poor cell health. Given the above results, it was decided to switch to a differ ent GFP expressing cell line. Mouse Embryonic Stem Cells (GFP-mES) Mouse embryonic stem cells are ~12 m in di ameter, ca. half that of the GFP-3T3s used above. It was therefore hoped that sin ce the cells would no longer be squeezed into capillaries, they would survive and prolif erate better. GFP-mES were also in good supply and the collaborating researchers were well published mES experts. This hope was realized and the results are shown in Figures 4-17 – 4-20 and video 4-3.

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43 Evaluation of cell Growth, survival and morphology vs. time Figure 4-18 shows phase contrast and co mplementary fluorescence micrographs documenting the survival, pro liferation and morphology of GFP-mES cells cultured in OCCAG scaffolds over nine days. Since the GFP-mES cells constitutively expressed GFP, expression past 36 hours was taken as an indicator of cell vi ability. The cells usually seeded as small groups lined up in the capill aries (Figure 4-17A, B). At day 6, the cells had proliferated heartily and formed cylindrical structures within a few OCCAG capillaries. The cells had proliferat ed so well in some cases that they had escaped from the ends of capillaries and clumped into spherical structures (Figure 417D). These “Papillon” cell structures were judged to resemble embryoid bodies, a formation seen regularly in ES cell culture. Day 9 shows an extension of the behavior observed at day 6 with more capillaries fille d. A group of cellular bulges seen in the central portion of the 4-17F toward the top po ssibly shows the expans ion of cells out of their initial capillary. Although care was taken to use OCCAG w ith an average capillary diameter between 20-30 m (mid parent gel level) for this experiment, inspection of the micrographs indicates that the gels used possessed capillaries with diameters > 35m (lower parent gel level). The mES cylindrical formations also appeared to have expanded the capillary diameter to ~ 40-50 m. Fortunate ly, this larger than expected capillary diameter did not critically affect the pres ent experiment, but be tter tracking of this variable will be required in the future. Confocal microscopy and video data Figure 4-18 and video 4-3 are a representa tive confocal micrograph and confocal microscopy video taken at day 7 of the sample s in the nine day experiment above. The

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44 confocal video shows the morphology of mES ce lls up to ca. 200 m deep (perpendicular to the capillary long axis) in the scaffold. Cylindrical and Papilli on mES cell structures are again observed. A set of ongoing experiments conducted by Dr. Takashi Hamazaki of the Terada group has provi ded insight on the specific pl acement of individual mES cells within a single capi llary. Figure 4-19 is a fl uorescence micrograph showing Hoechst stained nuclei of mES cells in an O CCAG capillary at day 4 in culture. The cells take up a staggered formation, almost a ppearing to spiral within the capillary. Comparison of ES maintenance (Lif+) and ES differentiation (Lif-) media conditions Figure 4-20 shows the results of a small four day experiment exploring the effect of three different media condition on mES cell gr owth in OCCAG scaffolds. A four day time period was chosen because mES cells are in the early phases of cell fate determination when cultured in media lacki ng leukemia inhibiting factor (LIF) [57], a cytokine essential for preventing mES cell differentiation and maintaining them in a pleuri-potent state. Cell-seeded scaffolds were cultured in ES differentiation media (LIF-) for the first culture condi tion shown in figure 4-20A, E and G. Scaffolds in the intermediate second conditi on were cultured first in ES maintenance media (LIF+) and then switched at day 2 to ES differentiation media (figure 4-20B, E, H); ES maintenance media was used exclusively in the fi nal condition (Figure 4-20C, F, I). Despite the best efforts and many refineme nts of the seeding process, examination of figures 4-20A, B and C still shows an a pparent difference in the number of cells loaded per gel. The culture using differen tiation media alone appears to have the most cells seeded in the capillaries of the gel followed by the maintenance media alone condition; the combination condition however wa s a very close third. With these seeding caveats in mind, day 2 shows that greate r mES cell proliferation occurred in

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45 differentiation media than either of th e other conditions. The combination and maintenance media conditions appear similar in cell proliferation. However, by day 4, the differentiation and combination media cond itions were judged similar in terms of cell proliferation as well as the patchy GFP expression by the mES cell cylindrical formations. Low cell proliferation was still evident at day 4 for the maintenance media condition, but the cylindrical formations had more intense, homogenous GFP expression. The mES cell cylindrical formations in the ma intenance also appeared to have expanded the initial scaffold capillary diameter. One can observe an impact on the cellula r growth within the scaffolds due to different culture media despite the seeding differences between the conditions. Aside from containing LIF or not, th e next biggest difference between the media types used was that differentiation media was 20% FCS wh ile the maintenance media only contained only 10% KSR. It is expected that a highe r concentration of se rum will support faster, more robust cell growth in flat culture and appears to be the result for this experiment as well. It is difficult to speculate on the effect of LIF on mES cell proliferation given the significant difference in serum concentrati ons. It does appear clear though that maintaining mES cells in a pluri-potent stat e makes for healthier cells as indicated by GFP expression. The results with the mES cells differ markedly from that of the 3T3s. A fundamental difference between the cell lines is that 3T3s are cont act dependent and mES cells can grow in a suspension/aggregation type culture. This difference might help explain why mES cells grow comparativel y well on the OCCAG scaffolds despite the material’s apparent lack of adhesivity. Furthermore, the difference between the number

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46 of seeded cells in figure 4-20C and the numbe r of cylindrical cell formation seen in 4-20I indicates that seeding in the OCCAG functions as some sort of a selective pressure. Only the mES cells best suited for this environment survive and proliferate. Video 4-1. Time-Lapse Vi deo of RCCAG Growth. 0. 5M CuSO4 and 2% w/v NaAlginate solutions were used in creation of the above gel. Note the enhanced contrast of the growing gel boundary with the alginate sol due to a subtle change in lighting appearing a third of the way through the video. (8,067 KB, RCCAG18HrTLV.AVI). RCCAG Growth as a Function of Time y = 0.0207x0.601R2 = 0.9989 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 020040060080010001200Growth Time (min)Gel Thickness (cm) Observed Gel Thickness Square Law Model Power Fitted Model y=0.02x0.5 Figure 4-1. Plot of RCCAG growth as a function of time. *Error bars = 0.05cm, the error associated with determining a single gel thickness.

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47 RCCAG Growth Rate as a Function of Time0 0.002 0.004 0.006 0.008 0.01 0.012 020040060080010001200 Time (Min)Growth Rate (cm/min) 1st Derivative of Growth Data 1st Derivate of Square Law Model 1st Derivative of Power Fitted Model y'=0.01x-0.5y'=0.012x-0.4 Figure 4-2. First derivative plot of RCCAG growth data. A B Figure 4-3. Low magnification optical micr ographs of representative RCCAG sample discs. A) Sample plane perpendicular to long capillary axis, B) Sample plane containing capillary long axis.

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48 A B C Figure 4-4. Increased magnifi cation optical micrographs of RCCAG at sections at different parent gel levels. A) Upper gel sec tion. B) Middle gel se ction. C) Lower gel section. Average Capillary Diameter of Raw-CCAG vs. Parent Gel Thicknesses 19.5 21.2 23.1 26.7 27.2 30.0 31.2 32.8 34.20 5 10 15 20 25 30 35 40Parent Gel Level Average Capillary Diameter ( m) Upper Middle Lower* * * Figure 4-5. Graph of RCCAG average capillary diameter vs. parent gel thickness. A “*” indicates significant differen ces within gel level groups, p 0.05.

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49 Calculated RCCAG Metrics vs. Parent Gel Thickness12.0 5.0 3.2 29 28 33*19.5*26.9*33.4 36 58 26 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 Upper Gel Level Middle Gel Level Lower Gel LevelAvg. Capillary Diameter ( m)Capillary Density (#Caps/10,000 m2)% Free S p aceAvg. SA in Std. Disc (cm2) Figure 4-6. Graph of calculate d RCCAG metrics vs. parent gel thickness. A “*” indicates significant differences within gel level groups, p 0.05. SA = surface area, Std. Disc = 1 cm in diameter X 1 mm thick. Figure 4-7. Optical microgra ph of BCCAG showing brown pr ecipitate. S cale bar = 100 m.

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50 Figure 4-8. Optical microgra ph of BCCAG showing shimmering precipitate. Scale bar = 100 m.

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51 A B C D Figure 4-9. Summary of RCC AG SEM/EDS and X-ray mappi ng data. A) Secondary electron image, B) Backscatter elec tron image, C) Representative EDS spectrum, D) X-ray map group. Scale bar = 50 m.

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52 SrLa1 ,2A B C D Figure 4-10. Summary of BC CAG SEM/EDS and X-ray mapp ing data. A) Secondary electron image, B) Backscatter elec tron image, C) Representative EDS spectrum, D) X-ray map group. Scale bars = 50 m.

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53 A B C D E Figure 4-11. Higher magnification BCCAG morphologic and compositional study. A) 4000X SEI image, B) Complementary BSE image to A (*note the charge wave distortion in the central region of this micrograph), C) 15000X SEI image of copper-rich nanoparticle form ations D) Small combination false color image of X-ray map group, E) X -ray map group. Scale bar for C = 2 m; all others = 5 m. *Image C courtesy of Wayne A. Acree.

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54 Figure 4-12. Large false color compositional map of a BCCAG. Green represents the barium crosslinked alginate matrix. Vo ids are black, and the blue within the voids is the material behind the curr ent imaging plane. The red-pink spots often with yellow borders are sulfur rich crystalline particles. The yellow “eyelash” formations seen mainly at the upper edge of the voids are copper rich nanoparticle aggregates. The yellow -orange streak present at the bottom of the image is a charging artifact, while the blue highlights in the upper portion of the image are a result of local specimen topography and tilt. Neither feature is indicative of sample composition.

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55 A B C D A Figure 4-13. Summary of OC CAG SEM/EDS and X-ray mapp ing data. A) Secondary electron image, B) Backscatter elec tron image, C) Representative EDS spectrum, D) X-ray map group. Scale bars = 50 m.

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56 Figure 4-14. A 4000X secondary electron image highlighting the “hairy” OCCAG surface character. Equilibrium Water Weight Percents of Crosslinked CCAG 94.0% 90.3% 88.3% 75.0% 80.0% 85.0% 90.0% 95.0% 100.0% CCAG Type Water Weight Precent RCCAG OCCAG BCCAG Figure 4-15. Equilibrium water weight percents of differe nt CCAG derivatives.

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57 Figure 4-16. Confocal microscope image of live GFP-3T3 cells seeded within an OCCAG scaffold at day 2 in culture. The capillary long axis is oriented topbottom. Scale bar = 200 m Video 4-2. Confocal microsc ope video of live GFP-3T3 cells seeded within an OCCAG scaffold at day 2 in culture. Refe rence figure 4-16 for scale (31,363 KB, GFP-3T3Confo.AVI).

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58 A B CD EF Figure 4-17. Phase contrast and complementar y fluorescence microscope image series of GFP-mES cultured in OCCAG over nine days. A) Day 0 phase contrast image, B) Complementary day 0 GFP-filtered image, C) Day 6 phase contrast image, D) Complementary da y 6 GFP-filtered image, E) Day 9 phase contrast image, F) Complementary day 9 GFP-filtered image. Scale bar = 100 m for all images.

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59 Figure 4-18. Confocal microscope image of live GFP-mES cells seeded within an OCCAG scaffold at day 7 in culture. Scale bar = 200 m Video 4-3. Confocal microsc ope video of live GFP-mES cells seeded within an OCCAG scaffold at day 7 in culture. Refe rence figure 4-18 for scale. (127,532 KB, GFP-mESConfo.AVI).

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60 Figure 4-19. Hoechst stained nuclei of mES cells in an OCCAG capillary at day 4 in culture. Scale bar = 75 m. *Image courtesy of Dr. Takashi Hamazaki

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61 A BC DEF GH I Figure 4-20. Growth of GFP-mES cells in OCCAG scaffolds cultured in maintenance (M) or differentiation (D) media or a combination (M/D) over 4 days. A) Day 0D, B) Day 0M/D C) Day 0M D) Day 2D, E) Day 2M/D, F) Day 2M, G) Day 4D, H) Day 4M/D, I) Day4M. Scale bar = 100 m for all images.

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62 CHAPTER 5 CONCLUSIONS Introduction This project developed and biologically assessed new biomaterial tissue scaffolds derived from copper-capillary alginate gels. These scaffolds are related members of a new family of biomaterials best thought of as modular tissue engine ering tools. Though many of the studies presented are in the early stages and much experimental work remains, the project was overall successful and the potential for significant future developments is high. Scaffold Synthesis: The Agony and the Ecstasy Time lapse videoscopy provided an excel lent means of tracking RCCAG growth. These videos clearly demonstr ate the dynamic of raw gel formation. RCCAG growth did however result in the production of significan t quantities (liters) of copper sulfate hazardous waste. Treatment with barium hydroxide or oligochitosan was sufficient to crosslink RCCAG for experiment al biology, however scaffold stabilization proved an underestimated challenge. The flow-reactor did improve the BCCAG production process but fell short of performing as initially envi sioned. OCCAG in comparison was easier to produce and yielded much smaller quantities of waste. OBCCAG was tricky to make with overexposure to the oligoc hitosan solution ultimately resulting in scaffold collapse. Hence, the synthesis protocols described in this work produce the new biomaterials, but are not optimized.

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63 Characterization: CCAG Scaffolds as Subtle Composites Material colorimetric changes were happy accidents of the synthesis process. The change from light blue to royal blue while processing th e RCCAG in barium hydroxide provided a crude means of monitoring the proc ess. This change was likely the result of copper hydroxide formation within the gel. RCCAG also turned a yellowish-brown after soaking in oligochitosan solution for mu ltiple hours, probabl y the result of the oligochitosan:alginate complexa tion rather than formation of a copper salt. All scaffolds clarified and become almost “invisible” when washed in cell culture media due to the loss of copper. Crystalline and golden brown-black gela tinous precipitates also formed during barium hydroxide treatment. The gelatinous precipitate was proba bly copper II oxide and the X-ray mapping data at least support that the particles were copper rich. Following this logic, copper ions apparently migrated or diffused toward materialsolution interfaces, perhaps driven by the formation of copper hydroxide and copper II oxide. Copper II oxide ultimately formed po ssibly due to the relatively poor thermal conductivity at the core of the CCAG-derived material. The thermal energy released during copper hydroxide formation may have thus been “trapped” and enough for the formation of copper II oxide. X-ray mapping and backscatter images s upport the idea that th e crystalline or “shimmering” precipitates are likely bari um compounds perhaps barium sulfate or carbonate. BCCAG was also more brittle than the other materials resulting in poor handling properties. Barium cr osslinked alginate spheres have been previously shown to have a higher modulus than copper cross linked counterparts [3 4]. The barium crosslinked material tending to crumble or sh atter hydrated or freeze-dried. This behavior

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64 could also be due in part to the precipitate s formed within the gel, degradation of the alginate through base cata lyzed hydrolysis or a combination of both. X-ray maps also tell us the RCCAG a nd BCCAG are uniformly crosslinked by their respective metal ions (at least at the s cale of the interaction volume). Interestingly silicon also appears uniformly distributed within the gels, presumably in the form of silicon dioxide. The raw alginate material is likely the source of this contamination. The silica could also be thought of as a filler, perhaps affecting mechanical properties. Cast in this light, CCAG scaffolds are subtle co mposites and this perspective could provide interesting avenues for future material exploitation. Biological Assessment: Living with Success and Failure This study clearly demonstrates that m ouse embryonic stems cells can survive and proliferate in OCCAG scaffolds for many days, and these cells form cylindrical structures in the scaffolds. This is the first report of any (mammalian) cell type cultured in CCAG materials. The formation of the mES cel l cylindrical structures proves that CCAGderived scaffolds can guide in vitro cell growth If the growth behavior of the mES cells describes generally how cells grow in CCAG scaffolds, then the average capillary size will likely need to be increased (perhaps to 100 m) for Schwann cell/ peripheral nerve regeneration studies. Furthermore, the us e of GFP expressing cells coupled with the CCAG structure and relative op tical clarity of the scaffolds made possible confocal microscopy studies. The use of confocal mi croscopy has become increasingly popular in experimental cell biology and will undoubtedly continue to provide valuable insights in future CCAG scaffold experiments. Studies utilizing GFP-3T3 cells were not as successful however, probably due to the size of these cells compared to that of th e average scaffold capillary diameter. Also,

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65 the 3T3 study clearly shows that the cells prefer to adhere to each other rather than the scaffold indicating a need to adjust/improve material surface chemistry. Successful experiments with fibroblasts in CCAG scaffolds will likely be critical for further evaluation of these new biomaterials as much is known about fibrobl ast cell behavior in other settings.

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66 CHAPTER 6 FUTURE WORK Introduction The neuroregenerative potential of the new CCAG scaffolds remains to be investigated. These investigations will like ly be secondary priorities however, as the intriguing results using mES cells have excited all involved. Hence, stem cell-scaffold interaction studies will be the dominant future research direction. Unfortunately, so much curiosity has been generate d by the results of this work that defining a clear future research path has been difficult. Synthesis: Expanding the CCAG Scaffold Family New additions to the CCAG scaffold family must be made in order to fully evaluate the tissue engineering potential of these new biomaterials. Synthesizing scaffolds with a different average capillary di ameters and oligochitosan crosslink times is the next logical step since adjusting these s caffold modules is straightforward. These materials will be critical to future stem ce ll–CCAG scaffold interaction studies. Further work could utilize derivitized oligochitosan cross linkers, protein additives and diffusible cues to make a host of CCAG-derived scaffold s. Production processes will also need to be optimized to reduce intrinsic scaffold variability. Scaffold Characterization: Quantitative Bulk and Surface Compositional Analysis The qualitative characterization work pe rformed here provided a wealth of interesting data that will se rve as a springboard for quantit ative studies. Although EDS is a powerful qualitative elemental characterization technique, it is a cumbersome means of

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67 obtaining quantitative information. A more suitable means of obtaini ng quantitative bulk elemental analysis of CCAG-derived biomater ials is laser ablativ e inductively coupled plasma mass spectrometry (ICP-MS). This an alysis would be part icularly focused on copper and its potential release from the scaffold since copper has been reported to have in vivo angiogenic potential [60, 61]. X-ra y photoelectron spectro scopy (XPS) and/or Fourier transform infrared spectroscopy (FTIR) studies s hould also be conducted to obtain quantitative surface composition data. Tallying nitrogen concentrations at the surfaces of CCAG scaffolds provides a means of tracking oligochitosan and/or protein treatments. Biologic Assessment: Standardized Methods and Controls for Stem Cell Studies A difficulty recognized early in the course of mES cell experiments was the lack of established protocols for seeding precise num bers of cells. The development of the vacuum seeding method was a large improvement, but it was still exc eedingly difficult to determine the number of cells actually seeded Mouse ES cell behavior is, to some degree, a function of cell culture density (unpub lished observations); it will therefore be important to solve this problem in order to draw firm conclusions about scaffold influence on cell behavior(s). Defining proper comparative controls is a nother area that requires attention for further mES cell experiments. For example, is it fair to compare mES cell behavior in flat culture to that of cells grown in CCAG scaffolds? O ngoing studies underway in the Terada laboratory at the University of Flor ida, Department of Pathology comparing the average cell fates present in the two conditions are mixed. There is simply too much variability to draw a confident conclusi on. The mES cell behavior is, after all mysterious.

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68 Given all of the above, some long range goals will be to demonstrate that stem cell fate can be modulated via CCAG scaffold architecture, modulus and surface chemistry and to quantify the interaction(s). A few r ecent stem cell-scaffold studies have claimed as much [62-64], putting this goal within achievable range. CCAG-derived scaffolds provide a unique and elegant means of controll ing the cell-cell interactions felt to be crucial to stem cell differentiation. The regular, adjustable capillary microstructure of these novel scaffolds provides a robust model system not currently available for studying cell-cell and cell-scaffold interactions. Tailori ng the system to propagate adult stem cells in vitro, especially hematopoietic stem cells would be directly be neficial to patients requiring bone marrow transplantation. Fina lly, in vivo studies using CCAG scaffolds alone and loaded with various cells types need to be conducte d pursuant to any attempt at organoid synthesis.

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69 APPENDIX : PUBLICATIONS, PRESENTATIONS AND PATENTS 1. Tollon, M.H., Hamazaki, T., Willenberg, B. J., Batich, C., Terada, N., Fabrication of Coated Polycaprolactone Scaffolds and Their Effects on Murine Embryonic Stem Cells. Materials Research Societ y Spring Conference Proceedings, 2005. K9.14. Willenberg, B.J., A New Family of Tissu e Engineering Scaffolds Derived From Copper-Capillary Alginate Gels: Synthe sis and Characterization. FLAVS-FSM Annual Joint Symposium, 2005. Invited presentation.3. Batich, C., Willenberg, B.J., Hamazaki, T., Terada, N., Novel tissue engineered scaffolds derived from copper capillary algi nate gels. US patent application, 2005. Serial No. 11/074,285.

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73 36. Rives, J.M., Pannier M., Castede J.C., Ma rtinot V., Le Touze A., Romana M.C., Guitard J., Bohbot S., Ebel C., Calcium al ginate versus paraffin gauze in the treatment of scalp graft d onor sites. Wounds-a Compendi um of Clinical Research and Practice, 1997. 9(6): p. 199-205. 37. Soon-Shiong, P., Treatment of type I diab etes using encapsulated islets. Advanced Drug Delivery Reviews, 1999. 35(2-3): p. 259-270. 38. Sandford, P.A. and P. Spoonshiong, Al ginate Encapsulation Update on 1st Human Clinical-Trial with Encapsulated Hu man Islets in a Type-I-Diabetic Patient with Sustained Islet Function 16 Months Post Encapsulated Islet Transplant. Abstracts of Papers of the American Chemical Society, 1995. 209: p. 44-CELL. 39. Yan, X.L., E. Khor, and L.Y. Lim, PEC films prepared from chitosan-alginate coacervates. Chemical & Pharmaceutical Bulletin, 2000. 48(7): p. 941-946. 40. Gaserod, O., A. Sannes, and G. Skjak-Br aek, Microcapsules of alginate-chitosan. II. A study of capsule stability and pe rmeability. Biomaterials, 1999. 20(8): p. 773783. 41. Kuo, C.K. and P.X. Ma, Ionically crossl inked alginate hydrogels as scaffolds for tissue engineering: Part 1. Structure, gelation rate and mechanical properties. Biomaterials, 2001. 22(6): p. 511-521. 42. Suzuki, K., Suzuki Y., Ohnishi K., Endo K., Tanihara M., Nishimura Y., Regeneration of transected spinal cord in young adult rats using freeze-dried alginate gel. Neuroreport, 1999. 10(14): p. 2891-2894. 43. Suzuki, Y., Kataoka K., Kitada M., Wu S ., Hashimoto T., Ohnishi K., Suzuki K., Ide C., Endo K., Nishimura Y., Tanihara M., Spinal cord regeneration through alginate, a polysaccharide from seaweed. European Journal of Neuroscience, 2000. 12: p. 287-287. 44. Palmieri, G., GiardinA.P., Desiderio B., Ma rzullo L., Giamberini M., Sannia G., A New Enzyme Immobilization Procedure Us ing Copper Alginate Gel Application to a Fungal Phenol Oxidase. Enzyme a nd Microbial Technology, 1994. 16(2): p. 151-158. 45. Widere, H. and S. Danielsen, Evaluation of the use of Sr2+ in alginate immobilization of cells. Naturwisse nschaften, 2001. 88: p. 224-228. 46. Duvivier-Kali, V.F., Omer A., Parent R.J., O'Neil J.J., Weir G.C., Complete Protection of Islets Against Allorecti on and Autoimmunity by a Simple BariumAlginate Membrane. Diab etes, 2001. 50: p. 1698-1705. 47. Bartkowiak, A. and D. Hunkeler, New Mi crocapsules Based on Oligoelectrolyte Complexation. Annals of the New York Academy of Sciences, 1999. 875: p. 36-45.

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75 61. Giavaresi, G., Torricelli P., Fornasari P.M., Giardino R., Barbucci R., Leone G., Blood vessel formation after soft-tissue implantation of hyaluronan based hydrogel supplemented with copper ions. Biomaterials, 2005. 26: p. 3001–3008. 62. Dang, S.M., Gerecht-Nir S., Chen J., Itskovi tz-Eldor J., Zandstra P.W., Controlled, Scalable Embryonic Stem Cell Differentiati on Culture. Stem Cells, 2004. 22: p. 275-282. 63. Gerecht-Nir, S., Cohen S., Ziskind A., Itskovitz-Eldor J., Three-Dimensional Porous Alginate Scaffolds Provide a Conducive Environment for Generation of Well-Vascularized Embryoid Bodies From Human Embryonic Stem Cells. Biotechnology and Bioengineer ing, 2004. 88(3): p. 313-320. 64. Levenberg, S., Huang N.F., Lavik E., Roge rs A.B., Itskovitz-Eldor J., Langer R., Differentiation of human embryonic stem cells on three-dimensional polymer scaffolds. Proceedings of the National Academy of Sciences of the USA, 2003. 100(22): p. 12741–12746.

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76 BIOGRAPHICAL SKETCH Bradley Jay Willenberg spent the first 13 years of his life growing up privileged in Bloomfield Hills, Michigan. Here he spent much time in a garage-based laboratory contemplating fire, disassembling toys, repair ing bikes, experimenting with electrical motors, staring at the sun with a telescope and exploring the un iverse in a drop of standing ground water. Brad had significant difficulty reading and writing and therefore also spent considerable time deeply immers ed. When he was 9 years old, he was fortunately injected into a well-funded public middle school that was able to redress his apparent lack of educational progress. Brad spent his high school years attending th ree different schools in three different states. After graduating from First Colonial High in Virginia Beach, Brad was granted admission and attended college at the Universi ty of Florida. He graduated Phi Beta Kappa with highest honors, receiving a bachel or’s degree in interdisciplinary studies focused on biochemistry and molecular biology. Brad then worked for a little over a year at a small biotech start-up in Alachua, Florida. He returned to the University of Florida to pursue a doctorate in biomedical e ngineering. Over the course of his doctoral work, Brad developed a promising family of biomaterial scaffolds that he hopes to continue studying and developi ng. He has also worked as a materials analyst at the Major Analytical Instrumentation Center (MAIC) since June 2003.

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77 All of his life, Brad has shared a very deep connection with music. He is an accomplished bass player and a budding singer a nd guitar player. Brad currently lives with his family in Gainesville, FL.


Permanent Link: http://ufdc.ufl.edu/UFE0010981/00001

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Title: Modular Tissue Scaffolding Tools: A New Family of Self-Assembled Biomaterials Derived from Copper-Capillary Alginate Gels
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Title: Modular Tissue Scaffolding Tools: A New Family of Self-Assembled Biomaterials Derived from Copper-Capillary Alginate Gels
Physical Description: Mixed Material
Copyright Date: 2008

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Holding Location: University of Florida
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System ID: UFE0010981:00001

Full Text












MODULAR TISSUE SCAFFOLDING TOOLS:
A NEW FAMILY OF SELF-ASSEMBLED BIOMATERIALS
DERIVED FROM COPPER-CAPILLARY ALGINATE GELS
















By

BRADLEY JAY WILLENBERG


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Bradley Jay Willenberg

































To the old man who drowned so near the shore.















ACKNOWLEDGMENTS

I would like to sincerely thank Dr. Christopher Batich, my supervising committee

chairman. His hands-off approach and wealth of scientific and engineering knowledge

propelled me and this project faster and farther than initially envisioned. I thank Dr.

Anthony Brennan, Dr. Robert DeHoff and Dr. Thomas Mareci for their time and efforts

as my teachers and committee members. I also specially thank Dr. Naohiro Terada and

Dr. Takashi Hamazaki for their tremendous efforts, input and profound support of this

work.

Much praise and thanks go to Marina Scotti for giving me roots; split a piece of

wood and she is there. Lift a stone and you will find her. I thank my family for their

consistent support, guidance, criticism and strength. I especially thank, Mom, Jimbo,

Dan and Ryan, Dr. Amelia Dempere and Specialist Wayne Acree ("The Lab Dude"). I

also thank the entire Major Analytical Instrumentation Center (MAIC) staff for their

scientific and social insights. I further thank all those students, faculty and staff who took

the time to know and talk with me.

Special thanks go to Charlie Murphey (Precision Tool & Engineering, Gainesville,

FL) for custom machining all my tools and reactors, and to Dr. Charles (Chuck) Seegert

for shaping my early scientific and tissue-engineering thinking.
















TABLE OF CONTENTS

page

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

L IST O F F IG U R E S ................ ................................................................... viii

LIST OF OBJECTS ........................................................................ ....

ABSTRACT ........ .............. ............. ...... ...................... xi

CHAPTER

1 IN T R O D U C T IO N ............................................................................. .............. ...

2 BACKGROUND AND SIGNIFICANCE.................................................................5

3 M ATERIALS AND M ETHOD S ........................................ ......................... 15

Scaffold Synthesis ...............................................................15
Raw Copper-Capillary Alginate Gel (RCCAG).................. ......... ...........15
Classic descending growth technique .................................. ............... 16
Tim e-lapse videoscopy ........................................................ ............... 17
Barium Stabilized Copper-Capillary Alginate Gel (BCCAG) ............................17
Exchange-reactor design and setup ....................... ....... ..................... 17
Barium hydroxide processing..................................................18
Oligochitosan-Barium (OBCCAG) and Oligochitosan (OCCAG) Stabilized
R C C A G .................................................... ................ 19
Scaffold Characterization ............................................................................. 20
O ptical m icroscopy ...................................... ...... ................ ..............20
Scanning electron m icroscopy ........................................ ............... 21
Percent Water Content Determination................................ ............ 22
B biological A ssesm ent .............. .. .... .............................. ............. ................. 22
In Vitro Study: Swiss Albino Embryonic Mouse Fibroblasts Expressing
Green Fluorescent Protein (GFP-3T3)................... ........................24
In Vitro Study: Mouse Embryonic Stem Cells Expressing Green Fluorescent
Protein (G FP-m E S) ........................................... ................ ........ .. .......... 24
Evaluation of mES cell growth, survival and morphology vs. time.............24
Comparison of ES maintenance (LIF) and ES differentiation (LIF-)
m edia conditions ................................................. .... .... .. ...... .... 25









4 RESULTS AND DISCU SSION ........................................... .......................... 26

Scaffold Synthesis and Processing ........................................ ......... ............... 26
R C C A G ............................................................................................................... 2 6
G row th videos ......................... .. .................... ......... ........... 27
G row th kinetics ........................ .. ...................... .. ...... .... ..... ...... 27
Storage concerns .................................... .................. ......... 28
BCCA G ............................... .................. .. ...............29
Colorimetric changes during barium hydroxide treatment ........................29
Exchange-reactor advantages and difficulties............................................30
O B C CA G and O C C A G ................................................ ............ ............... 31
Consequences of M edia W ash................................................................. ...... 32
Scaffold C haracterization ........................................ ............................................32
O ptical M icroscopy .............................. ........................ .. ........ .... ............33
RCCAG .................................... .................. ..................33
Evidence of precipitates within BCCAG .....................................................34
Scanning Electron Microscopy/Energy Dispersive Spectroscopy (SEM/EDS)
and X -ray M apping .............................................. .... .. .. ......... .... 35
Consequences of freeze-drying ............ ...............................................35
R CCA G D ata ........................................................... .. ...............35
BCCA G D ata ............. ............... ................................................ 37
OCCAG D ata .............. ....... ........... ........ ................ .. .. .......... 39
Summary of morphologic and compositional analysis ..............................39
Equilibrium Water Weight Percent Analysis ............................................... 40
Biologic A ssessm ent.......... ................... ..................... ..... .... 41
M house Embryonic Fibroblasts (GFP-3T3)............................... ............... 41
Mouse Embryonic Stem Cells (GFP-mES)......................................................42
Evaluation of cell Growth, survival and morphology vs. time ..................43
Confocal m icroscopy and video data ................................................... .... 43
Comparison of ES maintenance (Lif) and ES differentiation (Lif) media
conditions ................................................................... .......... 44

5 CON CLU SION S ............................ ........ ... ......... ........ ..... ...... 62

Introduction .................................................................. .... ................... 62
Scaffold Synthesis: The Agony and the Ecstasy ................................................62
Characterization: CCAG Scaffolds as Subtle Composites.............. ...................63
Biological Assessment: Living with Success and Failure .......................................64

6 FUTURE W ORK ......................... ........... .. ........... ... ...... 66

Introduction ......................................... ........................................... 66
Synthesis: Expanding the CCAG Scaffold Family.................................................66
Scaffold Characterization: Quantitative Bulk and Surface Compositional Analysis .66
Biologic Assessment: Standardized Methods and Controls for Stem Cell Studies....67

APPENDIX PUBLICATIONS, PRESENTATIONS AND PATENTS.......................69









L IST O F R E F E R E N C E S ...................................... .................................... ....................70

B IO G R A PH IC A L SK E TCH ...................................................................... ..................76
















LIST OF FIGURES


Figure pge

2-1 Peripheral nerve hierarchical structure....................... .......... ......... .... ........... 13

2-2 Molecular structures of alginate and oligochitosan polymers ..............................14

3-1 Raw-CCAG (RCCAG) classical descending technique synthesis scheme ............17

3-2 TeflonTM exchange-reactor ........... ... ....... ................................. ............... 18

4-1 Plot of RCCAG growth as a function of time. ................................. ... ..................46

4-2 First derivative plot of RCCAG growth data. .................................. ... ..................47

4-3 Low magnification optical micrographs of representative RCCAG sample discs...47

4-4 Increased magnification optical micrographs of RCCAG at sections at different
p aren t g el lev els................................................. ................ 4 8

4-5 Graph of RCCAG average capillary diameter vs. parent gel thickness. ................48

4-6 Graph of calculated RCCAG metrics vs. parent gel thickness..............................49

4-7 Optical micrograph of BCCAG showing brown precipitate ..................................49

4-8 Optical micrograph of BCCAG showing shimmering precipitate. ......................50

4-9 Summary of RCCAG SEM/EDS and X-ray mapping data............................... 51

4-10 Summary of BCCAG SEM/EDS and X-ray mapping data...................................52

4-11 Higher magnification BCCAG morphologic and compositional study .................53

4-12 Large false color compositional map of a BCCAG. ..............................................54

4-13 Summary of OCCAG SEM/EDS and X-ray mapping data. ...................................55

4-14 A 4000X secondary electron image highlighting the "hairy" OCCAG surface
character. ............................................................................56

4-15 Equilibrium water weight percent of different CCAG derivatives......................56









4-16 Confocal microscope image of live GFP-3T3 cells seeded within an OCCAG
scaffold at day 2 in culture. ..... ........................... ....................................... 57

4-17 Phase contrast and complementary fluorescence microscope image series of GFP-
mES cultured in OCCAG over nine days........................................................... 58

4-18 Confocal microscope image of live GFP-mES cells seeded within an OCCAG
scaffold at day 7 in culture. ..... ........................... ....................................... 59

4-19 Hoechst stained nuclei of mES cells in an OCCAG capillary at day 4 in culture....60

4-20 Growth of GFP-mES cells in OCCAG scaffolds cultured in maintenance (M) or
differentiation (D) media or a combination (M/D) over 4 days............................. 61
















LIST OF OBJECTS


Object page

4-1 Time-Lapse Video of RCCAG Growth. ..............................................................46

4-2 Confocal microscope video of live GFP-3T3 cells seeded within an OCCAG
scaffold at day 2 in culture. ..... ........................... ....................................... 57

4-3 Confocal microscope video of live GFP-mES cells seeded within an OCCAG
scaffold at day 7 in culture. ..... ........................... ....................................... 59















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

MODULAR TISSUE SCAFFOLDING TOOLS:
A NEW FAMILY OF SELF-ASSEMBLED BIOMATERIALS
DERIVED FROM COPPER-CAPILLARY ALGINATE GELS

By

Bradley Jay Willenberg

August 2005

Chair: Christopher Batich
Major Department: Biomedical Engineering

Tissue engineering aims to regenerate or replace lost/damaged cells, tissues and

organs. Biomaterial scaffolds are often fundamental components of many tissue

engineering strategies. Development of advanced biomaterial scaffolds is crucial to the

continued progress and ultimate success of the field. Motivated to aid peripheral nerve

regeneration/engineering, our study offers an innovative way to produce advanced

biomaterial scaffolds derived from copper-capillary alginate gel (CCAG).

These novel materials possess regular, continuous microtubular architectures that

relatively few fabrication techniques can achieve. These hydrogel materials have been

morphologically and compositionally characterized using scanning electron microscopy

and energy dispersive spectroscopy (SEM/EDS). We conducted X-ray mapping studies

yielding the spatial distribution of elements within the different scaffolds. Fluorescence









and confocal microscopy studies detail the unique growth and survival of mouse

embryonic stem cells (mES) and fibroblasts (3T3) in and on CCAG scaffolds in vitro.














CHAPTER 1
INTRODUCTION

Clinical motivation. Peripheral nerve injuries are extremely prevalent. Injury is

often the result of trauma (e.g., lacerations, gunshot wounds, motor vehicle accidents),

acute compression, stretching and tension or disease (e.g., cancer, leprosy). Each year, an

estimated 50,000 peripheral nerve repair procedures are performed in the United States

alone [1]. Much of what has been learned about peripheral nerve repair has grown out of

the treatment of warfare injuries [2]. Unfortunately, despite many advances and creative

repair strategies, functional outcomes of nerve repairs are still far from optimal, and

motor nerves tend to be more refractory than sensory to full recovery [1].

Ideally, surgeons attempt a neurorrhaphy (direct suture of the nerve ends without

tension) for all laceration or avulsive injuries [3, 4]. When transected or rejected nerve

ends cannot be coapted without tension, a gap defect results requiring nerve grafting to

restore neural continuity [5]. Autograft autologouss nerve) is the "gold standard" graft

material, and is preferentially obtained from harvest of the sural nerve, antebrachial

cutaneous radial nerve or superficial sensory radial (SSR) nerve [5]. Fundamental

determinates of functional regeneration for autograft are the endoneurium and remaining

Schwann cells, since the epi- and perineural elements are trimmed from harvested nerve

before engraftment.

Although reported to facilitate neuroregeneration over substantial distances (2-15

cm) [6], autograft has some disadvantages, including lack of donor supply, donor-site

morbidity, need for a secondary surgical site and insufficient functional outcomes [1, 6-









7]. Harvesting donor nerve is also time-consuming and often the fascicles do not match

the target nerve in both number and diameter. Central or segmental necrosis can also

occur in large diameter grafts [8].

Tissue engineering could be a promising approach to functional neurorepair.

Many tissue engineering strategies have already been used to facilitate neuroregeneration

(chapter 2). Pinpointing the first tissue engineering experiments is difficult; however,

most credit Langer and Vacanti [9] with crystallizing the central dogma and fundamental

strategies of the field. They define tissue engineering as ". an interdisciplinary field

that applies the principals of engineering and the life sciences toward the development of

biological substitutes that restore, maintain or improve tissue function."

A primary thrust of tissue engineering is to develop three-dimensional biomaterials

for use as scaffolds- templates to format growing/regenerating cells and tissues.

Scaffolds are becoming integral components of tissue reparative, restorative and

regenerative strategies [9], and development of advanced biomaterial scaffolds is crucial

to the continued progress and success of the tissue engineering field. The ability to

impose structural order on growing/regenerating cells and tissues via scaffold architecture

and geometry is a key feature of advanced scaffolds.

According to a review by Ma [10], scaffolds are usually highly porous with large

surface areas. Biodegradability is also generally required, with degradation rates

designed to match the rate of neotissue formation. Further, the scaffold materials) and

possible degradation products should be non-toxic (i.e., biocompatible), especially to

target cells and tissues. Finally, scaffolds should maintain adequate mechanical

properties and enhance cell adhesion, growth, migration and differentiated function. The









underlying idea of the above design guidelines is to produce biomaterials that bring

together large numbers of cells in comfortable close quarters, and provide an

environment that facilitates growth/regeneration/remodeling into functional target

tissue(s).

Scaffolds are essentially modular biomaterial tools. The word modular is

intended to convey flexibility, customizability and dynamic range. To illustrate this

concept, consider a computer software program. At its most basic level, the program

comes with some set of features that perform needed functions. If more than the basic

features are required to address specific needs, then often times one can enable or install

additional program modules (for a small fee, of course) adding the needed functionality.

This concept is well articulated in current microsphere technology, yet is still nascent in

current scaffolding designs.

Modular homes are another example of the concept discussed above. The home

analogy is particularly instructive from a biological perspective; no longer simply tools,

scaffolds are homes for regenerating cells and tissues. One wants to encourage

cells/tissues to take up orderly, productive residence and integrate into a much larger

community.

Using this logic, combinations of different biomaterial modules (e.g., architecture,

modulus, surface chemistry) are used to create a family of related scaffolds. These tailor-

made tools can then be implemented in tissue engineering. The science is to know (at the

molecular level) the effects of specific combinations of scaffold modules on cells/tissues.

Only with this knowledge can we engineer scaffolds with tremendous flexibility and

broad applicability.






4


Project-specific achievements. Our study introduces a new family of biomaterials

derived from copper-capillary alginate gels. These hydrogels have regular, continuous

microtubular architectures similar to those of the endoneurium. To date, relatively few

fabrication techniques produce such biomaterials [7, 10-13]. Although we did not test the

neuroregenerative potential of these materials, their tremendous scaffolding potential was

demonstrated through in vitro experiments using mouse embryonic stem cells (mES).














CHAPTER 2
BACKGROUND AND SIGNIFICANCE

Classification of peripheral nerve injury. In 1943, Sir Herbert Seddon

introduced a peripheral nerve injury classification system comprising 3 categories:

neurapraxia, axonotmesis and neurotmesis [1]. In 1951, Sundeland expanded the Seddon

system to five categories by further subdividing axonotmesis [3, 4]. A first-degree injury

(neurapraxia) involves a temporary conduction block with local demyelination.

Complete recovery occurs and may take up to 12 weeks. A second-degree injury

(axonotmesis) involves more-severe trauma or compression causing Wallerian

degeneration. The endoneurial tubes remain intact and therefore recovery is expected to

be complete, but could take months. A third-degree injury also involves Wallerian

degeneration, however the endoneurial tubes are not intact. Therefore, axons may not

reinnervate their original motor/sensory targets and recovery is incomplete. A fourth-

degree injury is a partial transaction of the nerve, ultimately resulting in a large scar area

at the site of injury. This scar precludes axons from advancing distally, and requires

surgery for any chance at meaningful functional recovery. A fifth-degree injury

(neurotmesis) is a complete transaction of the nerve and requires surgery to restore neural

continuity. MacKinnon added a sixth degree that combines the other degrees to describe

a mixed nerve injury [3]. Age and location are also key factors governing functional

recovery, with poorer results expected for increasing age and more-proximal injuries.

Anatomy and biology of adult peripheral nerve in the healthy state. Figure 2-1

illustrates peripheral nerve hierarchy. Nerves are composed of motor, sensory and









sympathetic components [1]. Nerves may be designated as primarily motor or sensory;

however, no nerve is purely one or the other [1]. Myelinated and unmyelinated axons

comprise the nerve fibers. Motor fibers are primarily myelinated and are outnumbered 4

to 1 by unmyelinated sensory fibers [1]. Myelinated fibers range in size from 1 to 20 [am

in diameter, while umyelinated fibers are typically below 1 [m diameter [14, 15].

The edoneurium is composed mainly of longitudinally aligned collagen fibers 30 to

60 nm in diameter [14, 16-17]. Tiny capillaries (<10 km), fibroblast, mast cells and

macrophages are also found in the endoneurium. The innermost endoneurial layer is

often observed to be in close contact with Schwann cell basal laminae.

Compared to the epinerium and endoneurium the perineurium is unique [14, 16-

17]. Cells composing the perineurium exhibit both myoid and epithelioid features and

express basal lamina on both surfaces. The cells are interlocked in successive sheets via

tight junctions. Blood vessels also infiltrate this layer, with the perineurim functioning as

a selectively permeable barrier. The outermost perineurial layers are composed of dense

concentric layers of mostly longitudinally arranged collagen fibrils -50 nm diameter with

a few fibroblasts and macrophages among the strands.

The epineurium is a dense collagenous layer surrounding all peripheral nerve

trunks [14, 16-17]. Fibers in this layer are disposed mainly longitudinally with diameters

between 70 and 85 nm. Elastin fibers are also present, with diameters ranging from 250

to 500 nm. Fibroblast and mast cells are scattered throughout this layer.

Peripheral nerve in the injured state. Axotomy (axon severance) occurs after

any 2nd degree injury and beyond. The cell body then undergoes chromatolysis

(swelling) and increased protein and RNA metabolism [1, 14, 16]. Later, axonal sprouts









grow from the proximal stump, and the distal stump undergoes Wallerian degeneration (a

process in which the distally remaining severed axon swells and breaks apart). During

Wallerian degeneration, Schwann cells in the distal stump concomitantly dedifferentiate,

reduce myelin protein synthesis, fragment remaining myelin sheaths into ovoids,

phagocytize myelin debris along with macrophages, and proliferate to form tubular

structures termed bands of Bingner that guide regenerating axon sprouts. Regenerating

axons typically grow at a rate of 1 to 4 mm per day, and the events of degeneration and

regeneration overlap.

Schwann cells and macrophages also play a role in degeneration/regeneration at the

molecular level through cytokine and growth factor production [18]. Immediately after a

crushing injury, Schwann cells show increased levels of IL-10, IL-6, LIF (leukemia

inhibitory factor) and IL-10 mRNA transcripts. IL-10 possibly induces nerve growth

factor (NGF) synthesis while IL-6 appears to affect sensory fiber regeneration. LIF

appears to affect the conduction velocity of regenerating fibers reportedly increasing the

size and number of myelinated fibers. Schwann cells also produce basal lamina

components laminin and collagen type IV which are required for neuroregeneration.

Furthermore, Schwann cells secrete a cocktail of neurotrophic factors like NGF,

neurotrophin-3, brain-derived growth factor (BDGF), neuregulin, fibroblast growth

factors (FGF) 1 and 2, insulin-like growth factors (IGF) 1 and 2, and ciliary

neurotrotrophic factor (CNTF) that play active roles in neuroregeneration [19].

Transcript levels for the IL-18, IFN-y and TNF-a (pro-inflammatory cytokines)

describe a more persistent upregulation peaking ca. 1 to 2 weeks post injury [18].

Infiltrating macrophages appear to be the cellular source of IL-18, but the source of









IFN-y is less clear. Schwann cells, fibroblasts, endothelial cells and macrophages all

express TNF-a following injury. Strong evidence supports the contention that TNF-a

plays a significant role in macrophage recruitment [18]. Transcripts for the anti-

inflammatory cytokine transforming growth factor-beta-1 (TGF-P1), the p40 subunit of

IL-12 also peek 14 days following injury. Murine macrophages stimulated with myelin

in vitro were shown to release IL-12 and TNF-a, suggesting that IL-12 expression is

potentially a consequence of myelin phagocytosis and part of macrophage autoregulation

[18].

Previously studied biomaterial nerve conduits. Entubulation is the most

common alternative to autograft repair [20]. In entubulation, severed nerve ends are

inserted into a hollow or filled-lumen biomaterial tube employed to protect, facilitate and

guide neuroregeneration. Gaps of centimeters have been regenerated successfully

depending upon the specific materials used [6, 9, 19, 21-28]. Ideally, conduits [6] should

be:

* Easily available
* Resorbable
* Readily vascularized
* Non-immunogenic
* Permeable to oxygen and other nutrients
* Able to block infiltrating scar tissue
* Able to function as depots for biologically active compounds

Clinically investigated biomaterial conduits. According to a clinical review by

Meek and Coert [6], vein, denatured muscle, combination vein filled with muscle,

silicone, Gore-TexTM, and polyglycolic acid (PGA) tubes have been used clinically (in

humans) for nerve reconstruction with success. Vein grafts were found suitable for gap

lengths of <4.5 cm depending upon the nerve under repair. Muscle grafts appeared









suitable for reconstruction of >6 cm gaps in leprosy patients and were judged superior to

conventional nerve grafting for repairing 1.5 to 2.8 cm gaps resulting from laceration

injuries. Combination vein filled with muscle conduits have been used successfully to

reconstruct 6 cm gaps. The ready supply of vein and muscle makes them attractive graft

material choices, and combination vein-muscle grafts have shown superior results to vein

alone in similar defects. Allografts in combination with systemic immunosuppressive

therapy have also been used successfully in the clinic to reconstruct massive (>10 to 20

cm) peripheral nerve defects [29].

Hollow Gore-TexTM conduits are indicated in reconstructions up to 4 cm and cause

less tissue irritation than silicone tubes. Silicone tubes were only shown successful for 4

mm gaps, and 29% of the tubes had to be removed because of compressivee) irritation. In

clinical studies using PGA tubes, the maximum defect that could be reconstructed was 3

cm and the conduits performed significantly better than autograft. The PGA conduits

permitted reconstruction of larger gaps perhaps because they were porous, permeable to

oxygen and less likely to collapse. Also, because these tubes were bioabsorbed, there

was no need to re-operate for compression/irritation.

Hence, clinical studies show that conduits (natural or synthetic) are at least

comparable to autograft for repairing short defects (< -3 cm). However, the ideal conduit

milieu has not been established for repairing larger nerve gaps [20]. The studies also

indicate that bioresorbable synthetic conduits are preferable to biodurable ones; filling the

conduit lumen with a permissive tissue (e.g., muscle) appears to yield significantly better

regenerative outcomes. Although allografts have been used successfully to reconstruct









large nerve defects, the need for systemic immunosuppressive therapy is a serious

drawback.

Experimentally investigated biomaterials. Many experimental studies in animal

models (primarily rat) aimed to improve conduit design and performance. Essential facts

gained from that literature are as follows: Permeable conduits and conduits possessing

smooth inner walls significantly outperformed impermeable conduits or conduits with

rough inner walls [28, 30]. More importantly and perhaps not surprisingly,

culturing/seeding autologous Schwann cells in conduits before implantation positively

impacted regeneration, improving recovery [19, 26, 27]. Combining Schwann cells with

a basement membrane gel such as Matrigel in the conduit lumen also positively affects

nerve regeneration [9, 24].

The assertion that nerve conduits need to function as a scaffold more for Schwann

cells than axons is gaining strength in the experiment literature. A few researchers have

fashioned scaffolds that induce cultured/seeded Schwann cells to form structures

reminiscent of Bungner bands [7, 12, 26], although results of these studies are

preliminary. The multi-lumen PLGA-Schwann cell seeded conduits constructed to

implement this strategy are of particular interest [7, 12]. Hadlock et al. [7, 12] have

produced conduits incorporating both fundamental determinants of functional

regeneration present in autograft. Thin stainless steel wires in the polymer injection mold

were used to approximate the continuous, tubular microstructure of the endoneurium.

Autologous Schwann cells were then flow-seeded into these laminin-coated conduits and

the cellularized implant was placed in a 7 mm rat sciatic nerve defect. After 6 weeks,

these conduits had statistically similar amounts of neural tissue per cross-sectional open









area compared to autograft. However, the mean myelinated fiber diameter of 3.73 0.51

[tm was significantly higher than the 2.3 0.24 [tm mean diameter found in autograft

controls (p < 0.05). Although these multi-lumen conduits are innovative and show

promising initial results, studies using these conduits are far from comprehensive, and the

requisite production methods could ultimately limit their widespread use.

Alginate (Figure 2-2A) is a linear polysaccharide discovered by E.C.C. Stanford in

1880 obtained from alkali digestion of various brown sea algae [31, 32]. The polymer

chain is composed of P-1,4 linked D-mannuronic acid (M) and a-1,4 linked L-guluronic

acid (G) monosaccharides found in three distinct blocks: polyM, polyMG and PolyG

blocks [33]. Compositional variation is a reflection of source and processing. The pKa's

of the C5 epimers are 3.38 and 3.65 for M and G respectively, with the pKa of an entire

alginate molecule somewhere inbetween [31, 32].

Alginate forms colloidal gels (high-water-content gels, hydrogels) with divalent

cations. In the alginate ion affinity series Pb2+>CU2+>Ba2>Ca2+>Zn2+>Ni2+>C2+ >Mn2

Ca2+ is perhaps the most used and characterized to form gels [34]. Studies indicate that

Ca-alginate gels form via cooperative binding of Ca2+ ions by polyG blocks on adjacent

polymer chains, the so-called "egg-box" model [32, 33]. G-rich alginates tend to form

thermally stable, strong, yet brittle Ca-gels that are likely to undergo syneresis. M-rich

alginates tend to form less thermally stable, weaker but more elastic gels.

Alginate is commercially used as a binding, stabilizing and/or thickening additive

in many foods and cosmetics [32]. Clinically, alginate is used in dental-impression

materials and hemostatic wound dressings [35, 36]. Alginate:poly-L-lysine

polyelectrolyte complex (PEC) encapsulated pancreatic islet cells were also evaluated in









a human clinical trial for treatment of type I diabetes [37, 38]. Alginate:chitosan PEC

beads and films have been made experimentally for cellular immunoprotective capsules

and drug release devices [39, 40]. Ionically (Ca2+) and covalently (e.g., ethylene

diamine) crosslinked freeze-dried foams and gels have been developed and implemented

as tissue scaffolds [39-43]. Copper alginate gel beads have been used for enzyme

immobilization with success [44]. Barium and oligochitosan (Figure 2-2B) crosslinked

alginate microspheres have also been previously synthesized and investigated [45-47].

Copper-capillary alginate gel(s) (CCAG) have been previously described and

studied in the scientific literature [31, 48-51]. These self-assembled gels are essentially

formed by allowing solutions of Cu2+ to diffuse uniformly into viscous solutions of

alginate. During this diffusion process, Thumbs and Kohler [48] state that fluid

instabilities arise from the friction forces involved in the contraction of alginate polymer

chains to the newly forming gel front. Convecting tori (similar to those observed in the

Raleigh-Benard model of heat convection) result from these hydrodynamic instabilities.

In a sense, these tori tunnel parallel capillaries through the forming gel in the direction of

diffusion. A continuous, tubular microstructure is mapped onto the forming gel because

of the convective-like process the system undergoes to dissipate energy. Gel capillary

diameter can be adjusted by manipulating (singly or in combination) the initial alginate

concentration, initial Cu2+ concentration or system pH [31, 48-49].

Surprisingly, no previous reports describe CCAG-derived hydrogels synthesized

and implemented as tissue scaffolds; this beautiful material and all its tissue engineering

potential uninvestigated. This could be because raw CCAG (RCCAG) dissolves in

several hours under cell culture conditions. However, many studies have already







13


described chemical crosslinking of RCCAG [49], and ceramics derived from CCAGs

have been produced and suggested as potential implants [52, 53].


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Figure 2-1. Peripheral nerve hierarchical structure.
___ 2






Fiue2] eihrlnrv irrhclsrcue




























Figure 2-2. Molecular structures of alginate and oligochitosan polymers. A) Alginate, B)
Oligochitosan














CHAPTER 3
MATERIALS AND METHODS

Scaffold Synthesis

All alginate used was Keltone LV obtained from ISP Alginates, Inc. (formally

known as Keltone, Mw range: 12,000-80,000 g/mol). Copper sulfate pentahydrate ACS

grade was obtained from Acros Organics, NJ. Barium hydroxide monohydrate was

obtained from Aldrich Chemical Company, Inc., Milwaukee, WI. Oligochitosan was a

kind gift from Dr. Dong-Won Lee who originally obtained it from E-ZE Co., Ltd., Korea;

manufacturer-reported average molecular weight and moisture content were 1150 g/mol

and 8%, respectively. Dr. Lee reported a 70% degree of deacetylation measured by

1HNMR [54].

Raw Copper-Capillary Alginate Gel (RCCAG)

Preparation of 2% w/v alginate solution. 4 g of Keltone LV sodium alginate was

dispersed in 170 mL of distilled water in a 500-mL Erlenmeyer flask. The suspension

was stirred with a stir plate at medium-high speed until a clear, homogenous solution was

obtained. Distilled water was then added to the solution until the final solution volume

was 200 mL, yielding a 2% w/v solution of sodium alginate (manufacturer-reported

viscosity, 100-300 centipoise (cP)). The alginate solution was stirred for 2 h and allowed

to stand for an additional 2 h to minimize solution bubbles. Solutions were either used

immediately or stored for no more than a week at 40C.

Petri dish preparation. A thin coat of alginate needs to be baked onto the petri

dish to prevent gel separation from the vessel wall during growth [31, 49-51]. Five thin









coats of freshly prepared 2% w/v alginate solution were smeared onto the entire inner

surface and rim of a PyrexTM petri dish (9 cm diameter x 2 cm height or 9 cm diameter x

3.25 mm height). A few minutes for air-drying were allowed between coats. Once the 5

coats were applied, the coated petri dish was baked in an oven heated at 1200C for 10

minutes. The dish was then removed, allowed to cool and the procedure was repeated 3

additional times.

Classic descending growth technique

The method below resembles the methods described previously [31, 49-51]. An

alginate-coated petri dish was carefully filled to the brim, almost overflowing, with

freshly prepared 2% w/v sodium alginate solution (Figure 3-1). A large KimwipeTM

soaked with freshly prepared 0.5M copper sulfate solution was pulled taut like a drum

(using a needlepoint hoop) and brought down directly on top of the alginate-filled petri

dish. The entire surface of the alginate solution and rim of the petri dish were assured to

be in good contact with the soaked KimwipeTM. Over the course of 5-7 minutes at

approximately 10-15 second intervals, 1-2 mL of 0.5M copper sulfate solution was

dripped onto the soaked KimwipeTM now covering the alginate-filled petri dish. The

soaked KimwipeTM was then slowly and gently peeled off the alginate filled petri dish. A

solid membrane, contiguous with the rim of the petri dish (- 1 mm thick) completely

covered the top of the alginate-filled petri dish. This membrane (the primary membrane)

was a little rough, approximately the color of the 0.5M copper sulfate solution and

contained no visible voids. Taking extreme care not to jar the gelling solution, the filled

petri dish was transferred to a large covered tank. The tank's geometry allowed for a 1.5

to 2 cm submersion of an alginate filled petri dish in 700 mL of the 0.5M copper sulfate









solution. The tank was slowly filled with 700 mL of 0.5M copper sulfate, covered,

placed on a leveled table w/anti-fatigue padding and left undisturbed for 36 hours.


























PC running C3 Systems WinTLV digital time-lapse videography software.

Barium Stabilized Copper-Capillary Alginate Gel (BCCAG)

We chose barium for the ion-exchange process because it forms an extremely stable

complex with alginate under physiological conditions [45, 46]. Also, previous

experience showed that treatment with barium hydroxide did not grossly alter RCCAG

morphology. However, special precautions were needed because soluble barium is toxic,

and barium hydroxide reacts with carbon dioxide present in air.

Exchange-reactor design and setup

A TeflonTM reactor (- 250 mL void volume) was used for the barium hydroxide

treatment of RCCAG (Figure 3-2). This minimized the potential for personal contact










with the toxic barium hydroxide solution and provided a relatively air-free processing

atmosphere. The reactor was then coupled with reservoirs, a small peristaltic pump,

silicone tubing as plumbing and an ultra-high purity (UHP) nitrogen gas bottle for the

complete setup.































MAT'L IL LON :., -s.
inlet-outlet ports.
1 "3 V -.. -------- K1./34 -------------



1 ^ 469 :


^ <33a> BRILL THRU
MAT'LL ILFLN pDpTNp B
Figure 3-2. TeflonTM exchange-reactor. A) Top-down view. B) Side view. A Buna-N
rubber gasket was inserted into the thin grove, and a glass plate was clamped
down on top of the reactor to form a sealed system. Barbed polypropylene
screw-in connectors and Buna-N rubber gaskets were also inserted into the
inlet-outlet ports.

Barium hydroxide processing

An RCCAG parent gel was cut into thin strips (- 3 mm) parallel to the capillary

long axis with a stainless-steel kitchen knife. Three long strips were sealed in the

exchange-reactor and washed by flushing a total of 2.3 L of deionized (DI) water through









the reactor over 72 hours. The sealed reactor was then purged with UHP nitrogen and

filled (250 mL) with freshly prepared 0.5M Ba(OH)2 solution. The filled reactor was

then placed on an orbital shaker (btb- Back to Basics, Bellco Biotechnology, Vineland,

NJ) for 24 h at 75 RPM. The reactor was again purged with UHP nitrogen and refilled

with 200 mL of 0.5M Ba(OH)2 and shaken for an addition 24 h at 75 RPM on an orbital

shaker. The sealed reactor was again purged with UHP nitrogen, filled with DI water and

shaken for 24 h at 75 RPM; this DI water soak was repeated one additional time. A total

of 3 L of DI water were then flushed through the reactor over 72 hours. The exchange-

reactor was finally unsealed, and BCCAG samples were extracted and stored in DI water

at 4C for further processing or experimentation.

Oligochitosan-Barium (OBCCAG) and Oligochitosan (OCCAG) Stabilized RCCAG

Chitosan, (a polysaccharide polymer composed of 0-1,4' linked glucosamine and

N-acetylglucosamine residues) was chosen for PEC stabilization because much work has

been done producing and characterizing alginate-chitosan multilayer microspheres [40,

55]. Chitosan also appears to have excellent biocompatibility [56], and alginate

microspheres crosslinked with oligochitosan have also been previously reported [47].

Preparation of 2% w/v oligochitosan solution. 2g of oligochitosan were

dispersed in 80 mL of DI water in a 250-mL Erlenmeyer flask. The suspension was then

stirred vigorously until a clear, yellow-brown solution was obtained. DI water was then

added to the solution until the final solution volume was 100 mL yielding a 2% w/v

solution of oligochitosan. Solutions were either used immediately or stored for no more

than a week at 40C.

Preparation of OCCAG/ OBCCAG. RCCAG samples (3-5) cut into rectangles

(~ 7 mm x 5 mm x 3 cm) were placed into 50 mL centrifuge tubes. Freshly prepared









oligochitosan solution (45 mL, 2% w/v) was then added to each and the tubes were then

placed on an orbital shaker for 17-19 hours. Next, the oligochitosan solution was poured

off and the samples were rinsed three times with small volumes (5-10 ml) of DI water.

DI water was then added (45 ml/tube) and the tubes were placed on an orbital shaker

overnight. The DI water was fully exchanged at least once over the next 8-12 hours.

Samples were then stored in a small volume of DI water at 40C. The procedure to

produce OBCCAG was identical to the above except BCCAG was used as the starting

material instead of RCCAG.

OCCAG/ OBCCAG washing in cell culture medium. Samples were placed

singly in the wells of 6-well cell-culture plates. Three milliliters of cell culture (either

fibroblast or ES differentiation, see below) media containing serum were then added and

the plates were placed in a 37C incubator overnight. The media was completely

exchanged and the plates were returned to the incubator overnight. After this point, the

scaffolds were used for cell-biology experiments.

Scaffold Characterization

All optical microscopy was conducted on samples just submerged in distilled water

(as this provided the clearest, most consistent images). To freeze-dry materials for SEM

analysis, samples were placed individually in 50 mL polypropylene centrifuge tubes with

3 mL of DI water. Samples were then flash frozen by placing the tubes in liquid nitrogen

for 5 minutes. The flash frozen samples were then freeze-dried (-40C, 10-15 [im Hg) on

a Labconco lyophilizer (Kansas City, MO) for at least 48 hours.

Optical microscopy

Using a 1 cm inner-diameter stainless-steel cork bore (Precision Tool &

Engineering, Gainesville, FL), a plug the entire height of the parent RCCAG gel was









quickly punched out, starting from the bottom parent gel face. The sample plug was

gently pushed out of the bore, and the thin layer of the primary membrane was cut off

with a stainless steel-kitchen knife and aluminum miter box. The core was then

progressively sectioned into discs approximately 3 mm thick. Upper, middle and lower

samples were placed separately in PyrexTM glass bowls (5 per bowl) and submerged in

100 mL of DI water. Each bowl was covered and stirred on an orbital shaker at 100 rpm

for 72 hours. The water in each bowl was completely changed every 12 hours. After

washing, three discs from upper, middle and lower core sections were observed with an

Olympus SZ stereomicroscope (Tokyo, Japan) equipped with a MiniVID digital camera

(LW Scientifc, Lawrenceville, GA). Optical micrographs were recorded and stored on a

Windows 98 PC using an ATI Rage Fury Pro video capture card running ATI Multimedia

Center software version 6.2. An image of a 25 mm reticle (0.010 mm gradations,

Klarmann Rulings, Inc., Manchester, NH) was also captured to scale the sample images.

Determination and comparison of average capillary diameter as a function of

parent gel thickness. Thirty (30) capillaries from each micrograph were measured using

NIH ImageJ freeware version 1.28u. That data was inputted into Microsoft Excel 97

spreadsheets and average capillary sizes and standard deviations were calculated using

internal Excel functions. ANOVA analysis was performed with Minitab Release 14.12.

Differences were judged significant for p < 0.05.

Scanning electron microscopy

Freeze-dried samples of RCCAG, BCCAG and OCCAG produced previously were

mounted separately onto aluminum SEM stubs with double-sided carbon tabs (SPI

Supplies, West Chester, PA). The mounted specimens were then carbon coated (Ion

Equipment Corp., Santa Clara, CA) and stored until analyzed in a desiccator. All









samples were analyzed using a JEOL JSM-6400 SEM (JEOL USA, Peabody, MA)

equipped with an Oxford energy dispersive spectroscopy (EDS) system and a LINK ISIS

software package version 3.35 (Oxford Instruments USA, Concord MA). All samples

were analyzed at 20 KeV accelerating voltage to maintain consistency with the

standardless digital library. This accelerating voltage is more than sufficient to observe

all X-ray peaks of interest with EDS. Image processing was performed utilizing features

available in the LINK ISIS software package.

Percent Water Content Determination

Five small, previously washed samples from RCCAG, BCCAG and OCCAG each

were equilibrated in a minimum of distilled water in 50 ml conical centrifuge tubes for

one week. After equilibration, the samples were removed, blotted to dryness on a

KimwipeTM, placed in a pre-weighed 15 ml conical centrifuge tube, weighed and

recorded. The samples were then re-submerged in a minimum of distilled water and flash

frozen in liquid nitrogen and lyophilized for 48 hours. After lyophilization, the tubes

w/sample were re-weighed and recorded. The difference between the initial and final

weights was attributed solely to the loss of water during drying. ANOVA analysis was

performed with Minitab Release 14.12. Differences were judged significant for p < 0.05.

Biological Assesment

All regular cell maintenance such as media changing, cell splitting, etc. was

performed solely by Dr. Takashi Hamazaki of the Terada group, Department of

Pathology, University of Florida. All Cell seeding, maintenance and documentation of

scaffold culture experiments were done jointly with Dr. Hamazaki. Fluorescence

microscopy was performed with an IX-70 Olympus/C Squared equipped with a

MagnaFire digital camera system and software package (Optronics). Confocal









microscopy was performed by Marda Jorgensen, Departement of Pathology- Stem Cell

and Regenerative Medicine Program, University of Florida using a Leica TCS SP2

AOBS Spectral confocal microscope equipped with laser point scanning (405-633 nm)

and proprietary software (Leica Microsystems Inc., Buffalo, New York).

Maintenance of mouse Swiss Albino embryonic fibroblasts expressing green-

fluorescing protein (GFP-3T3) cells. GFP-3T3 cells were maintained in tissue culture

dishes (6 cm, 2 X 105 cells) in Dulbecco's Modified Eagle Media (DMEM, GIBCO

BRL, Grand Island, NY) containing 10% fetal bovine serum (FBS, Atlanta biologicals,

Norcross, GA), 2 mM L-glutamine, 100 units/ml penicillin, 100 [tg/ml streptomycin, 25

mM HEPES (GIBCO BRL). Media (termed fibroblast media) was changed every two

days and the cells were split upon reaching 2 X 106, 80% confluence.

Maintenance of mouse embryonic stem cells expressing green-fluorescing

protein (GFP-mES). GFP-mES cells were maintained in an undifferentiated state on

gelatin-coated dishes (6 cm, 4 X104 cells) in Knock-out DMEM (GIBCO BRL, Grand

Island, NY) containing 10% knockout serum replacement (KSR, GIBCO BRL), 1% FBS

(Atlanta biologicals, Norcross, GA), 2 mM L-glutamine, 100 units/ml penicillin, 100

[tg/ml streptomycin, 25 mM HEPES (GIBCO BRL), 300 [aM monothioglycerol (Sigma,

St. Louis, MO), and 1000 unit/ml recombinant mouse Leukemia inhibitory factor (LIF,

ESGRO) (Chemicon, Temecula, CA). Media (termed ES maintenance media) was

changed every two days and the cells were split upon reaching 2 X 106, 80%

confluence.









In Vitro Study: Swiss Albino Embryonic Mouse Fibroblasts Expressing Green
Fluorescent Protein (GFP-3T3)

It was not known if CCAG-derived scaffold would be relatively non-toxic to cells

in in-vitro cell culture. Hence, two circular OCCAG scaffolds (- 8 X 3 mm) were placed

singly into a six-well tissue culture plate (Nalge Nunc International, Rochester, NY).

GFP-3T3 cells were first dissociated by using 0.25% trypsin/EDTA (GIBCO BRL) and

then re-suspended in the GFP-3T3 culture media (see above). To seed the cells, a total of

200 al of the cell suspension (1 X 106 cells/ml) was applied to one end of the capillaries

while applying vacuum to the other capillary ends. Cell-scaffold combos were then

cultured for one week in fibroblast media. The combos were observed daily with the

fluorescence microscope and the media was changed every two days. Confocal

microscopy was performed on select samples at day 2 in culture.

In Vitro Study: Mouse Embryonic Stem Cells Expressing Green Fluorescent Protein
(GFP-mES)

Undifferentiated ES cells were dissociated using 0.25% trypsin/EDTA (GIBCO

BRL). ES cells were suspended in Iscove's Modified Dulbecco's Medium (IMDM),

supplemented with 20% fetal bovine serum (Atlanta biologicals), 2 mM L-glutamine, 100

units/ml penicillin, 100 [ag/ml streptomycin (GIBCO BRL), and 300 [aM

monothioglycerol (Sigma). This media was termed "ES differentiation media". To seed

ES cells into OCCAG scaffolds, a total 200 [l of the cell suspension (1x106 cells/ml) was

applied from one end of the capillaries while suctioning the fluid from the other end of

the capillaries.

Evaluation of mES cell growth, survival and morphology vs. time

No previous data was available to inform one's intuition about mES cell behavior

when seeded and cultured in CCAG-derived scaffolds. Therefore, 4 OCCAG scaffolds









cut into rectangular blocks were seeded, placed singly into a six-well culture plate (Nalge

Nunc International) and cultured in ES maintenance media for nine days. Cell-scaffold

combos were observed daily and the media was changed every two days. Fluorescence

micrographs were recorded at days 0, 6 and 9 and confocal microscopy was performed on

select samples at day 7 in culture.

Comparison of ES maintenance (LIF+) and ES differentiation (LIF) media
conditions

It was not known if mES cells seeded in OCCAG scaffolds and cultured under cell

differentiation conditions behaved differently in terms of cell survival and proliferation

than cells seeded and cultured under cell maintenance (undifferentiated) conditions. To

that end, we conducted a four day study using ES maintenance or ES differentiation

media. The four day end point was chosen because previous experience and reported

studies [57] indicated that mES cells are in the early phases of cell fate determination

when cultured in ES maintenance media, i.e. media lacking LIF.

Six OCCAG scaffolds cut into rectangular blocks (- 10 X 5 X 3 mm) were seeded,

placed singly into a six-well culture plate (Nalge Nunc International) and cultured over

four days under one of three different media conditions:

* mES maintenance medium (LIF ) only
* mES maintenance (LIF) / mES differentiation (LIF-), switched at day 2 in culture.
* mES differentiation (LIF-) only














CHAPTER 4
RESULTS AND DISCUSSION

Scaffold Synthesis and Processing

Scaffold synthesis was successful overall, albeit an underestimated challenge.

Early on it was discovered that RCCAG dissolved over the course of several hours (<24

h) in standard cell culture media likely due the chelation and/or ion-exchange of Cu2+

An attempt to covalently crosslink RCCAG with ethylene diamine utilizing carbodiimide

chemistry was made, however, the attempt failed due again to the rapid chelation of Cu2+

by ethylene diamine. Ethylene diamine solution dissolved the RCCAG within a matter of

minutes. Other synthetic techniques to crosslink the raw material were avoided in

deference to the end-use as a biomaterial. These complications led to the successful

attempts to stabilize RCCAG via ion-exchange with Ba2+ ions and/or formation of a

polyelectrolyte complex with oligochitosan (below). Although the synthesis procedures

described in the Materials and Methods section of this text are far from optimized, they

were adequate to produce gram scale quantities of all the new scaffolding materials.

RCCAG

Production of this material was the most straightforward because it had already

been investigated. As previously reported, coating the petri dish with a thin film of baked

alginate proved necessary. However, the temporary use of a KimwipeTM to aid in the

formation of a regular primary membrane was a new addition to the general RCCAG

synthesis method. Significant amounts of waste copper sulfate solution were also

produced and the raw material had to be washed extensively to rid it of excess copper









sulfate. The amount of waste produced makes the material less attractive for large scale

production.

RCCAG was a homogenous translucent sky blue due the Cu2+ ions crosslinking it

and was also the most durable of the materials produced in this work. It did not tear

easily and regained its original shape after compression. Cutting the material parallel to

the long capillary axis was much more difficult than cutting perpendicular to the long

axis. This anisotropy is presumably due the fact that the alginate chains are preferentially

oriented perpendicular to the long capillary axis [31, 48-49].

Growth videos

Successful video monitoring of the entire RCCAG synthesis process was achieved.

The use of time-lapse videoscopy (TLV, video 4-1) was a powerful technique yielding

not only kinetic data, but also clearly illustrated the fact that RCCAG "grows" via a self-

assembly process. The most fundamental reaction occurring is the binding of Cu2+ ions

by alginate molecules in solution; all other processes (chain contraction, formation of

convective tori) resulting in the material's structure and anisotropic properties stem from

this action.

Growth kinetics

Kinetic data obtained from RCCAG growth TLVs are of particular engineering

interest. Figures 4-1 and 4-2 summarize our key kinetic findings. In a previous study,

Schuberth [31] puts forward the idea that gel growth follows the so-called square law

(equation 4-land 4-2). It was confirmed in that study that RCCAG growth behavior is

approximated by the square law at least within the first hour of gel growth.


y = 2Dt (4-1)
n









Where

D: Diffusion Coefficient (cm2/s) and

t: Diffusion Time (s)

y: Diffusion Path Length/Gel Thickness (cm)

Hence,

yoc (4-2)

Our kinetic data do confirm that equation 4-2 reasonably approximates the very


beginning of gel growth assuming 2,- D 0.02 cm/min. However at longer growth times


past this initial phase the square law model significantly under-predicts the gel thickness.

A power fitted model of the observed data suggests a value of 0.6 rather than 0.5 for the

value of the exponent. The 1st derivative plots also support this contention. The gel

growth rate is also apparently decreasingly settling within a range of 0.001 0.002

cm/min. It is unclear how this reported value of the RCCAG growth rate specifically

compares with other studies [31, 58], but Schuberth does report that growth rates are ca.

25% slow in gels lacking capillaries and asserts that an overlay of convection in the

capillaries could account for this difference.

Storage concerns

During the early phases of this project, it was decided to store the RCCAG in some

formation buffer (0.5M CuSO4) at 40C in a commercial polymer container called

FoodKeepersTM by Anchor Hocking. At the time it was tacitly assumed that the

container was "resistant" and would not contaminate the RCCAG with soluble

degradation products. Later research into the exchange reactor material design however

indicated that CuSO4 solution could be caustic to a wide range of polymers over long









exposure of times. Unfortunately, details of the FoodkeepersTM polymer composition

were difficult to find since the brand has been discontinued for many years, but the dishes

are very rigid and heat resistant, perhaps similar to a phenolic or melamine type resin.

Hence, at the current time it is impossible to rule out the possibility that the storage

container contaminated the RCCAG with biologically active degradation products;

though it can be stated with confidence that this possibility is remote at best given the

extensive washing regime undertaken during production.

BCCAG

This material was the most challenging to produce. BCCAG was also the most

interesting from a materials perspective and proved to be quite stable in cell culture

media. However, BCCAG had the poorest handling qualities tending to crumble or

fracture readily. This limitation, coupled with its demanding synthesis, limited the study

of BCCAG as a scaffold to a few rough cell culture experiments (data not shown).

Colorimetric changes during barium hydroxide treatment

When RCCAG samples were initial submerged into 0.05M Ba(OH)2 solution they

floated due to a difference in density. However, within minutes the samples sank and this

sinking was accompanied by a color change of sample edges from sky blue to royal blue.

This color change uniformly proceeded into the core of the samples over the course of

many hours. Sample cores then began to blacken sometime between 12-24 h of

treatment, and this blackening was further enhanced during the washing of the newly

formed BCCAG in DI water.

The above colorimetric changes can be explained by the formation of different

copper compounds within RCCAG samples during barium hydroxide processing. The

initial color change from translucent sky blue to royal blue corresponded to the reaction









of Cu2+ ions with hydroxide ions to form copper hydroxide (Reaction 4.1) which is often

described as a pale blue gelatinous water insoluble precipitate.

Cu2+(aq) + 20H-(aq) -> Cu(OH)2(s) 4.1

Although the term "pale" seems inconsistent with the above description, concentration

and matrix effects presumably influence the apparent copper hydroxide color intensity.

The progressive blackening of the core was due to the progressive formation of

copper II oxide (Reaction 4.2).

Cu(OH)2(s) + Heat -> CuO(s) + H20(1) 4.2

Copper II oxide is often described as a black or golden brown insoluble precipitate

formed by heating copper hydroxide. The heat released by the formation of copper

hydroxide in the RCCAG possibly drove its own decomposition to copper II oxide within

the gel (Figure 4-7).

Exchange-reactor advantages and difficulties

The exchange reactor was a tremendous advantage during barium hydroxide

possessing. Barium hydroxide is caustic and toxic and readily absorbs carbon dioxide

from the air. The exchange reactor provided a means of exposing large amounts of

RCCAG to barium hydroxide solution under an atmosphere of UHP nitrogen, and a

means of flushing the solution directly to waste. The newly produced BCCAG could

then be extensively washed with water under nitrogen as well. What was at the onset a

tedious and precarious task of filling and draining flasks of toxic solutions was reduced to

filling appropriate reservoirs; the plumbing of the exchange-reactor minimized the human

interaction with toxic effluents. This system was far from perfect though.









The placement of the inlet and outlet ports complicated reactor filling and draining.

Also, the silicone tubing serving as plumbing had a tendency to split during long

pumping cycles resulting in a significant reactor leak. Finally, it was difficult to maintain

a low, consistent flow rate with the peristaltic pump used. Despite these short comings,

BCCAG was reproducibly produced in sufficient quantity for further study.

OBCCAG and OCCAG

Only OCCAG was successfully produced utilizing the synthesis protocols

described in this work. Fortunately, this material was well suited for use in biological

experiments (below) due to its optical clarity and ease/reproducibility of production.

OCCAG was inhomogeneously colored in cross-section, composed of a yellowed outer

surface with a blue-green core. This inhomogeneity is likely the result of differential

crosslinking of the exterior and core by the 2% w/v oligochitosan solution. Apparently, a

more densely crosslinked skin of alginate:oligochitosan PEC formed around samples of

OCCAG.

Overexposure or overreaction is a concern and possibility for any chemical

crosslinking procedure of polymeric materials and CCAGs are no exception. The

oligochitosan is a multifunctional crosslinker forming ionic rather than covalent bonds.

The electrostatic bonding between the RCCAG or BCCAG and oligochitosan happens

essentially instantaneously. Slightly to moderately overexposed rectangular samples

began to round at the corners and distort as the overcrosslinked PEC skin contracted on

the low modulus gel core. This skin was also darkly stained brown which negatively

impacted its optical qualities. It was also found in an early set of experiments that

RCCAG stiffened, turned dark brown and profoundly shrunk when severely overreacted









in an excess of 2% w/v oligochitosan solution (reaction times > 24 hours). Syneresis of

the gel likely accompanied these profound changes.

A reaction time range of 17 to 19 hours was therefore used in this study to stabilize

both RCCAG and BCCAG with 2% w/v oligochitosan solution. This exposure time

resulted in no significant change in the materials' original size and morphology, and the

materials were only slightly yellowed in color after the reaction. OBCCAG synthesis

however failed during the cell culture media wash.

Consequences of Media Wash

Media washing is technically the final step in scaffold processing because material

changes occur during the process. All of the copper present in the scaffolds as free ions

or otherwise appears to be removed with successive washes in media. This effectively

dissolved the water insoluble copper hydroxide and oxide particles present in BCCAG.

Free copper ions are also apparently leached or chelated serving to decolorize the

scaffold. The result is a translucent scaffold colored the same as the phenol red spiked

media itself. This result was great because it facilitated the use of advance microscopic

techniques to observe the cells in situ, alive and dynamic. The OCCAG results were

similar the BCCAG results, but OBCCAG collapsed and stiffened when washed in

media, reminiscent of the earlier overcrosslinked RCCAG and consequently was never

used in cell culture experiments.

Scaffold Characterization

A broader characterization regime was initially envisioned for the materials

developed in this project. In fact, XPS, XRD and TEM measurements were all attempted

but yielded poor and/or inconclusive results. Technical difficulties encountered in

sample preparation were essentially to blame for the poor results. As the project









progressed, timing and the high demand for scaffolds for the biological assessment

stunted further pursuit of XPS, XRD and TEM measurements. Also, at the time it was

not clear what measures were most germane and how best to design experiments aimed at

acquiring them.

Therefore, the characterization data presented below is not comprehensive;

however, the optical microscopy (OM), scanning electron microscopy (SEM) and the

small swelling studies reported here provide a solid foundation for future work. The OM

studies essentially report on RCCAG morphology; the SEM studies describe the

morphology and composition (via energy dispersive spectroscopy, EDS) of RCCAG,

BCCAG and OCCAG before cell culture media processing. The swelling study was an

attempt to elucidate possible differences in the materials' equilibrium water contents

resulting from the different crosslinking/ stabilization methods.

Optical Microscopy

The Olympus SZ equipped with the MiniVID camera provided an effective and

comparatively inexpensive digital capture microscope system. The images captured with

this setup were more than adequate for obtaining quantitative measurements. This digital

stereomicroscope also gave a fair idea of surface topography.

RCCAG

Figure 4-3 and 4-4 show representative micrographs of RCCAG samples cut from a

parent gel. The average capillary diameter of three representative areas at each gel level

is given in figure 4-5. This graph also shows that the average capillary diameters for all

samples in the upper gel level group differed significantly from each other (p < 0.05);

only one sample was judged to have a significantly different average capillary diameter

in the middle and lower gel level groups.









The fact that at least one of the average capillary diameters within each group in

figure 4-5 differed from the others supports the idea that either the capillary diameters

within a given gel levels are not uniform or that there was significant systematic error

involved in sample sectioning. The latter is likely the case for this set of experiments. In

fact, the sectioning method described in the Materials and Methods chapter turned out to

be somewhat crude, yielding both imprecise and inaccurate results. Section thickness

was therefore variable, sometimes differing by a millimeter or more. It is also unclear if

the above data is comparable to previously conducted studies [31, 49].

Figure 4-6 presents calculated RCCAG metrics germane to tissue engineering

derived from the average capillary data. Because all of the upper gel level samples

differed significantly from each other, only the smallest average capillary diameter was

used to calculate the subsequent metrics. Middle and lower gel level metrics were

calculated from pooled capillary diameter data of samples that were judged statistically

similar by ANOVA (p > 0.05). The error bars given for the average capillary diameters

are the standard deviations.

Despite the limitations discussed above, capillary diameter is undeniably a function

of gel thickness. Hence, it is tempting to conclude that the apparent differences in the

calculated metrics are significant and are also subsequently a function of gel thickness;

the story is less clear for the percent free space. The following trends are clear from the

graph: capillary density [ with 1 gel thickness and the average surface area in a standard

disc [ with 1 thickness.

Evidence of precipitates within BCCAG

Figure 4-7 and 4-8 are representative micrographs showing brown and shimmering

particles within BCCAG samples. The precipitate shown in figure 4-7 appears golden-









brown (presumably due to lighting) and is the same formation responsible for the

progressive blackening described in the scaffold synthesis section above. This datum

further supports the claim that copper II oxide has precipitated with the capillaries of

BCCAG due to processing in barium hydroxide solution. The shimmering particles are

believed to be insoluble barium sulfate and/or carbonate crystals formed within the walls

of BCCAG during synthesis.

Scanning Electron Microscopy/Energy Dispersive Spectroscopy (SEM/EDS) and X-
ray Mapping

The SEM/EDS and X-ray mapping studies yielded a wealth of data concerning the

sample morphology, bulk composition and elemental distribution within samples Despite

the analysis not being optimized for imaging, high quality secondary (SEI) and

backscatter (BSE) images were still obtained. Relatively short collection times (ca. 20

min.) coupled with image processing functions like smoothing and contrast enhancement

provided informative X-ray maps with an economy of time.

Consequences of freeze-drying

Freeze-drying had several effects on the studied materials. RCCAG densified and

the once circular capillaries turned pentagonal or hexagonal. This is especially visible in

the BSE image in figure 4-9B. BCCAG became very fragile and powdered if handle too

much, but maintained circular capillaries (Figure 4-10A). OCCAG did not become

fragile but maintained circular capillaries (Figure 4-12A). All materials however tended

to flake into sheets perpendicular to the capillary long axis.

RCCAG Data

Figure 4-9 is a summary of representative RCCAG SEM/EDS and X-ray mapping

data. The secondary image (4-9A) shows irregularly shaped capillaries, but the









backscatter image (4-9B) shows that these shapes are likely artifacts of sample

preparation. The backscatter signal is more sensitive to mass-thickness than is the

secondary signal, and it appears that the irregularly shaped capillaries are really the result

of thin sheets or flaps of RCCAG that have fallen over the capillaries at some point in the

sample preparation. Hence, the densified hexagonal and pentagonal walls or RCCAG

beneath the thin sheets is highlighted in the BSE image. This morphology has been seen

previously in earlier experiments with RCCAG (data not shown).

EDS analysis shows that RCCAG is essentially composed of carbon, oxygen and

copper (Figure 4-9C). The carbon and oxygen are the sole components of the alginate

polymer and the copper is the crosslinker responsible for the capillary structure. Low

amounts of silicon and sulfur also appear in the representative spectrum and presumably

came from the alginate powder used to make the 2% w/v sodium alginate solution and

residual copper sulfate used in gel synthesis respectively (Ch. 3, Materials and Methods).

Caution should be exercised when attempting to assess the amounts of elements from any

EDS spectrum. Peaks of elements that are actually present in high concentrations

(particularly low Z elements) can appear smaller than peaks of elements present in small

concentrations. The correction matrix is complicated and the variables used are usually

poorly known for low atomic number elements leading to semi-quantitative data at best.

The X-ray map group (figure 4-9D) shows low levels of silicon within the RCCAG

walls; sulfur is also present to a lesser degree in the walls, but appears more concentrated

in discrete particles. Copper appears uniformly distributed over the whole map area and

it is not possible to discern the capillary structure in the copper map. This is due to the

large interaction volume of the X-ray signal (especially in a polymeric material) and the









relatively energetic nature of the copper Ka X-ray. The X-ray signal comes form "deep"

within the material (perhaps > 1 [tm) and the Cu Ka is not significantly absorbed by

anything else in the sample resulting in a homogenous looking copper map.

BCCAG Data

BCCAG was the most complex material produced in this study. Figure 4-10 is a

representative summary of BCCAG SEM/EDS and X-ray mapping data. Figure 4-11 is a

higher magnification study highlighting copper-rich nano-particulate formations within

BCCAG. The SEI BCCAG image (figure 4-10A) shows that the capillaries have

remained circular and the material does not appear to have densified like RCCAG.

Excellent BSE images were obtained due to the material's barium content (Figure 4-

10B).

The representative EDS spectrum (4-10C) indicates that BCCAG is mainly

composed of carbon, oxygen, copper and barium. Strontium also appears in the

spectrum, overlapping in the same energy range as silicon (Figure 4-9C). Silicon could

be present but masked by strontium that apparently came from the barium hydroxide

solution. The small aluminum peak is possibly due to scatter from the SEM mount.

Figure 4-10D, the BCCAG X-ray map group, is markedly different from the

RCCAG map group (figure 4-9D). Barium now appears homogenously distributed

throughout BCCAG, supplanting copper; strontium also appears uniformly distributed.

The majority of the copper signal is localized to the "bumpy" particles lining inner

capillary diameters. Figure 4-11A and C are higher magnification SEI images of the

copper-particles; figure 4-11B is the complementary BSE of4-11A. 4-1 1E is an X-ray

map group of the same area as 4-11A taken to more clearly illustrate the distribution of

elements within BCCAG. Sulfur rich areas also appear in this map group which are









believed to correspond to the shimmering particles noted in BCCAG optical microscopy

above.

A complex series of physicochemical events occur during barium hydroxide

processing of RCCAG. Upon submersion in the barium hydroxide solution, copper

hydroxide begins to form in the outer surfaces and edges of the material. As the barium

and hydroxide ions diffuse into the RCCAG matrix and capillaries, Cu2+ ions react with

the OH- ions forming insoluble copper hydroxide at all material-solution interfaces. As

the reaction proceeds, Cu2+ ions at the interface are depleted, stimulating migration of

Cu2+ ions from within the material down their concentration gradient. Copper ions

migrating to the material-solution interface react with the essentially infinite sink of

solution hydroxide ions forming more insoluble copper hydroxide concentrated at the

interface. Heat produced from the formation reaction is not dissipated efficiently within

the RCCAG sample, and thus drives the dehydration of the newly formed copper

hydroxide to copper oxide over time.

The above ideas are not intended to apply to all copper within BCCAG.

Examination of the BCCAG X-ray map groups clearly shows copper within the BCCAG

matrix. However, a comparison of the RCCAG and BCCAG X-ray map groups also

shows a change in the distribution of copper and it is this change that the above theory

attempts to explain.

Concomitantly, barium ions exchange with copper ions and/or form new ionic

crosslinks within the gel, stabilizing its structure. This exchange presumably influences

the migration of Cu2+ ions to material-solution interfaces. Residual SO42- and dissolved

CO32- ions also react with diffusing Ba2+ ions forming insoluble salt crystals within the









RCCAG matrix (Figures 4-11D and 4-12). These crystals result in the "shimmering"

optical micrographs discussed above.

OCCAG Data

Figure 4-13 is a summary of the representative OCCAG SEM/EDS and X-ray

mapping. 4-13A shows that OCCAG has also retained circular capillaries, but the

material's surface appears "hairy". This surface character can be more easily seen in the

higher magnification SE image shown in figure 4-14. The striated layer structure of the

RCCAG has also been preserved. The OCCAG EDS spectrum (4-13C) appears similar to

the RCCAG EDS spectrum shown earlier with the addition of small amounts of chlorine.

Closer inspection of 4-13C shows a much higher carbon and oxygen intensity as well as a

reduced copper intensity in contrast to figure 4-9C, probably due to oligochitosan

processing.

The oligochitosan is a polymeric crosslinker composed mainly of carbon and

oxygen. Oligochitosan ionically crosslinks RCCAG (from the surface inwards) via a

positively charged amine functionality. Hence, oligochitosan processing results in a

carbonaceous film on the surface of OCCAG which would contribute to the higher

carbon and oxygen intensities, as well as damping the measured copper intensity. The

amine residues could also leach Cu2+ ions from the RCCAG, further contributing to the

drop in measured copper signal.

Summary of morphologic and compositional analysis

RCCAG has a smooth surface with pentagonal and hexagonal shaped capillaries

and a densified structure due to freeze-drying. The material is composed of mainly

carbon, oxygen and copper with low amounts of silicon and sulfur. All elements, with









the exception of sulfur, appear uniformly dispersed in RCCAG; sulfur appears

concentrated in particles.

BCCAG has circular capillaries and the structure does not appear to have changed

due to dehydration. This material is composed mainly of carbon, oxygen, barium and

copper with low amounts of strontium and sulfur. Barium and strontium appear

homogenously distributed throughout the material. Copper is concentrated in

nanoparticles located on the inner capillary surfaces giving a bumpy appearance and

sulfur appears as particles in and on BCCAG.

OCCAG has circular capillaries and the structure also does not appear to have

changed due to drying. Similarly to RCCAG, OCCAG is composed mainly of carbon,

oxygen and copper, however the relatively intensities of these elements are different.

Carbon and oxygen appear greater while copper appears lower presumably due to the

carbonaceous surface coating resulting from oligochitosan crosslinking. The copper

content of OCCAG may have also been depleted via complexation with the oligochitosan

in solution.

Equilibrium Water Weight Percent Analysis

Figure 4-15 shows the results of the small swelling study comparing the different

CCAG crosslinking methods. Although the graph suggests that RCCAG contains the

highest equilibrium water weight percent followed by OCCAG and then BCCAG, no

significant differences between the groups were indicated by ANOVA analysis (p >

0.05). The study should be repeated with larger sample sizes and a revised experimental

procedure. The procedure implemented for wet sample weight measurement in this

experiment was the largest source of systematic error.









Biologic Assessment

Initially, this work was motivated to aid peripheral nerve regeneration by

developing synthetic biomaterial mimics of the endoneurium. The requisite materials

were developed, but neuroregenerative testing was frustrated mainly due to the lack of a

motivated, expert collaborator. This was a blessing in disguise however, as expert stem

cell biologist collaborators (The Terada Group, Department of Pathology, University of

Florida) did enthusiastically participate in in vitro scaffold testing.

The main point of the biological experiments discussed below was to assess 1) if

cells could be seeded in and on the newly developed scaffolds and 2) if these seeded cells

could survive and proliferate over the course of several days. Initially it was hoped that

BCCAG, OBCCAG and OCCAG could all be tested, however the only reportable data

came only from OCCAG experiments. First we tried to seed and grow green fluorescent

mouse embryonic fibroblast (GFP-3T3) cells in OCCAG scaffolds, but obtained a limited

data set. Later we switched to a green fluorescent mouse embryonic stem cell line and

were able to conduct multiple experiments.

The results presented below show that cells, mouse embryonic stem cells (mES)

mainly, can be seeded into OCCAG scaffolds, and that these cells survive and proliferate

over the course of many days. Furthermore, mES cells form ordered cylindrical

structures when seeded and grown in OCCAG scaffolds. Hence, the claim that CCAG-

derived scaffolds can impose structural order on growing cells via architecture and

geometry is well supported.

Mouse Embryonic Fibroblasts (GFP-3T3)

GFP-3T3 cells were initially chosen because they are robust, highly proliferative

cells that were in good supply. Also, fibroblast migratory behavior has been reported on









previously [59] and it was hoped that this behavior would be observed for direct

comparison. Unfortunately, the GFP-3T3s available appeared to have the same diameter

as the capillaries (- 25 pm) and were not easily seeded into the OCCAG scaffolds. This

significantly limited the possible experimental work. Figure 4-16 and video 4-2 are a

representative micrograph and confocal microscopy video from an early GFP-3T3 study

respectively.

The relative clarity and translucence of the OCCAG scaffold provided for

reasonably good fluorescent and confocal images. The confocal video shows the

morphology of GFP-3T3 cells up to ca. 100 pm deep (perpendicular to the capillary long

axis) in the scaffold after two days in culture. Cells within capillaries usually appear

deformed, taking on a pill-like shape. Multicellular aggregates appear clumped on the

outer surface of the OCCAG sample. This possibly indicates that OCCAG is not very

adhesive to cells as they prefer clump together rather than attach and spread on the

materials surface. Cells confined within the capillaries typically did not survive more

than 2-3 days and did not appear to proliferate. Large vacuous regions observed within

the GFP-3T3s were also taken as a sign of poor cell health. Given the above results, it

was decided to switch to a different GFP expressing cell line.

Mouse Embryonic Stem Cells (GFP-mES)

Mouse embryonic stem cells are -12 [tm in diameter, ca. half that of the GFP-3T3s

used above. It was therefore hoped that since the cells would no longer be squeezed into

capillaries, they would survive and proliferate better. GFP-mES were also in good

supply and the collaborating researchers were well published mES experts. This hope

was realized and the results are shown in Figures 4-17 4-20 and video 4-3.









Evaluation of cell Growth, survival and morphology vs. time

Figure 4-18 shows phase contrast and complementary fluorescence micrographs

documenting the survival, proliferation and morphology of GFP-mES cells cultured in

OCCAG scaffolds over nine days. Since the GFP-mES cells constitutively expressed

GFP, expression past 36 hours was taken as an indicator of cell viability. The cells

usually seeded as small groups lined up in the capillaries (Figure 4-17A, B).

At day 6, the cells had proliferated heartily and formed cylindrical structures within

a few OCCAG capillaries. The cells had proliferated so well in some cases that they had

escaped from the ends of capillaries and clumped into spherical structures (Figure 4-

17D). These "Papillon" cell structures were judged to resemble embryoid bodies, a

formation seen regularly in ES cell culture. Day 9 shows an extension of the behavior

observed at day 6 with more capillaries filled. A group of cellular bulges seen in the

central portion of the 4-17F toward the top possibly shows the expansion of cells out of

their initial capillary.

Although care was taken to use OCCAG with an average capillary diameter

between 20-30 tm (mid parent gel level) for this experiment, inspection of the

micrographs indicates that the gels used possessed capillaries with diameters > 35tm

(lower parent gel level). The mES cylindrical formations also appeared to have expanded

the capillary diameter to 40-50 am. Fortunately, this larger than expected capillary

diameter did not critically affect the present experiment, but better tracking of this

variable will be required in the future.

Confocal microscopy and video data

Figure 4-18 and video 4-3 are a representative confocal micrograph and confocal

microscopy video taken at day 7 of the samples in the nine day experiment above. The









confocal video shows the morphology of mES cells up to ca. 200 [m deep (perpendicular

to the capillary long axis) in the scaffold. Cylindrical and Papillion mES cell structures

are again observed. A set of ongoing experiments conducted by Dr. Takashi Hamazaki

of the Terada group has provided insight on the specific placement of individual mES

cells within a single capillary. Figure 4-19 is a fluorescence micrograph showing

Hoechst stained nuclei of mES cells in an OCCAG capillary at day 4 in culture. The cells

take up a staggered formation, almost appearing to spiral within the capillary.

Comparison of ES maintenance (Lif) and ES differentiation (Lif) media conditions

Figure 4-20 shows the results of a small four day experiment exploring the effect of

three different media condition on mES cell growth in OCCAG scaffolds. A four day

time period was chosen because mES cells are in the early phases of cell fate

determination when cultured in media lacking leukemia inhibiting factor (LIF) [57], a

cytokine essential for preventing mES cell differentiation and maintaining them in a

pleuri-potent state. Cell-seeded scaffolds were cultured in ES differentiation media

(LIF-) for the first culture condition shown in figure 4-20A, E and G. Scaffolds in the

intermediate second condition were cultured first in ES maintenance media (LIF) and

then switched at day 2 to ES differentiation media (figure 4-20B, E, H); ES maintenance

media was used exclusively in the final condition (Figure 4-20C, F, I).

Despite the best efforts and many refinements of the seeding process, examination

of figures 4-20A, B and C still shows an apparent difference in the number of cells

loaded per gel. The culture using differentiation media alone appears to have the most

cells seeded in the capillaries of the gel followed by the maintenance media alone

condition; the combination condition however was a very close third. With these seeding

caveats in mind, day 2 shows that greater mES cell proliferation occurred in









differentiation media than either of the other conditions. The combination and

maintenance media conditions appear similar in cell proliferation. However, by day 4,

the differentiation and combination media conditions were judged similar in terms of cell

proliferation as well as the patchy GFP expression by the mES cell cylindrical

formations. Low cell proliferation was still evident at day 4 for the maintenance media

condition, but the cylindrical formations had more intense, homogenous GFP expression.

The mES cell cylindrical formations in the maintenance also appeared to have expanded

the initial scaffold capillary diameter.

One can observe an impact on the cellular growth within the scaffolds due to

different culture media despite the seeding differences between the conditions. Aside

from containing LIF or not, the next biggest difference between the media types used was

that differentiation media was 20% FCS while the maintenance media only contained

only 10% KSR. It is expected that a higher concentration of serum will support faster,

more robust cell growth in flat culture and appears to be the result for this experiment as

well. It is difficult to speculate on the effect of LIF on mES cell proliferation given the

significant difference in serum concentrations. It does appear clear though that

maintaining mES cells in a pluri-potent state makes for healthier cells as indicated by

GFP expression.

The results with the mES cells differ markedly from that of the 3T3s. A

fundamental difference between the cell lines is that 3T3s are contact dependent and mES

cells can grow in a suspension/aggregation type culture. This difference might help

explain why mES cells grow comparatively well on the OCCAG scaffolds despite the

material's apparent lack of adhesivity. Furthermore, the difference between the number











of seeded cells in figure 4-20C and the number of cylindrical cell formation seen in 4-201

indicates that seeding in the OCCAG functions as some sort of a selective pressure. Only

the mES cells best suited for this environment survive and proliferate.


P'-:-:a*; 1 Hr TL

Video 4-1. Time-Lapse Video of RCCAG Growth. 0.5M CuSO4 and 2% w/v Na-
Alginate solutions were used in creation of the above gel. Note the enhanced
contrast of the growing gel boundary with the alginate sol due to a subtle
change in lighting appearing a third of the way through the video. (8,067 KB,
RCCAG1 8HrTLV.AVI).


RCCAG Growth as a Function of Time


1.60

1.40

1.20 y=0.0207X 601 _
-1.00R2= 0.9989 T
. 1.00 __

I 0.80

0.60

0.40 -_____-

0.20

0.00
0 200 400 600
Growth Time (min)


o Observed Gel Thickness
- Square Law Model
-- Power Fitted Model


800 1000 1200


Figure 4-1. Plot of RCCAG growth as a function of time.
associated with determining a single gel thickness.


Error bars


0.05cm, the error








47




RCCAG Growth Rate as a Function of Time


0012


001


.E 0008
E

0 006


S0004


0002


o 1st Derivative of Growth Data
- 1st Derivate of Square Law Model
1st Derivative of Power Fitted Model


0 200 400 600 800 1000 1200
Time (Min)


Figure 4-2. First derivative plot of RCCAG growth data.


Figure 4-3. Low magnification optical micrographs of representative RCCAG sample
discs. A) Sample plane perpendicular to long capillary axis, B) Sample plane
containing capillary long axis.



































Figure 4-4. Increased magnification optical micrographs of RCCAG at sections at
different parent gel levels. A) Upper gel section. B) Middle gel section.
C) Lower gel section.


Average Capillary Diameter of Raw-CCAlG vs

-J0 i


Figure 4-5. Graph of RCCAG average capillary diameter vs. parent gel thickness. A "*"
indicates significant differences within gel level groups, p < 0.05.












Metrics vs. Parent Gel Thickness


40.0
r -4
30.0

20.0

10.0

0.0 -


Figure 4-6.


1 2' 0




1-apinari,. Den;it.


I .


reac


Graph of calculated RCCAG metrics vs. parent gel thickness. A "*" indicates
significant differences within gel level groups, p < 0.05. SA = surface area,
Std. Disc = 1 cm in diameter X 1 mm thick.


Figure 4-7. Optical micrograph of BCCAG showing brown precipitate. Scale bar







50






I. ,
















Figure 4-8. Optical micrograph of BCCAG showing shimmering precipitate. Scale bar
100 [m.
100 ym.





































Energy (eV)


D

cis


Figure 4-9. Summary ofRCCAG SEM/EDS and X-ray mapping data. A) Secondary
electron image, B) Backscatter electron image, C) Representative EDS
spectrum, D) X-ray map group. Scale bar = 50 am.






























0 B
C
0-



0-
Ba
CU
0-
- C B B

Cu Al I B BaC
0-1L. :_


2 4


6 8 10
Energy (keV)


Figure 4-10. Summary ofBCCAG SEM/EDS and X-ray mapping data. A) Secondary
electron image, B) Backscatter electron image, C) Representative EDS
spectrum, D) X-ray map group. Scale bars = 50 km.


Coun


150



100



50


D


cis


i







53















2000- CuKa SKa












IAE255 SKa'2










aal 7 CuKa5










Figure 4-11. Higher magnification BCCAG morphologic and compositional study. A)
4000X SEI image, B) Complementary BSE image to A (*note the charge
wave distortion in the central region of this micrograph), C) 15000X SEI
image of copper-rich nanoparticle formations D) Small combination false
color image of X-ray map group, E) X-ray map group. Scale bar for C = 2
[tm; all others = 5 [tm. *Image C courtesy of Wayne A. Acree.
































Figure 4-12. Large false color compositional map of a BCCAG. Green represents the
barium crosslinked alginate matrix. Voids are black, and the blue within the
voids is the material behind the current imaging plane. The red-pink spots
often with yellow borders are sulfur rich crystalline particles. The yellow
"eyelash" formations seen mainly at the upper edge of the voids are copper
rich nanoparticle aggregates. The yellow-orange streak present at the bottom
of the image is a charging artifact, while the blue highlights in the upper
portion of the image are a result of local specimen topography and tilt.
Neither feature is indicative of sample composition.
































Counts

3000-




2000-




1000-


Cu

CI Cu
Cu

0 2 4 6 8 10
Energy (keV)


MAGE, 255


Figure 4-13. Summary of OCCAG SEM/EDS and X-ray mapping data. A) Secondary

electron image, B) Backscatter electron image, C) Representative EDS

spectrum, D) X-ray map group. Scale bars = 50 km.
































Figure 4-14. A 4000X secondary electron image highlighting the "hairy" OCCAG
surface character.


Figure 4-15. Equilibrium water weight percent of different CCAG derivatives.






























Figure 4-16. Confocal microscope image of live GFP-3T3 cells seeded within an
OCCAG scaffold at day 2 in culture. The capillary long axis is oriented top-
bottom. Scale bar = 200 tm


I GFP-3T3Confo
Video 4-2. Confocal microscope video of live GFP-3T3 cells seeded within an OCCAG
scaffold at day 2 in culture. Reference figure 4-16 for scale (31,363 KB,
GFP-3T3Confo.AVI).


















































Figure 4-17. Phase contrast and complementary fluorescence microscope image series of
GFP-mES cultured in OCCAG over nine days. A) Day 0 phase contrast
image, B) Complementary day 0 GFP-filtered image, C) Day 6 phase
contrast image, D) Complementary day 6 GFP-filtered image, E) Day 9
phase contrast image, F) Complementary day 9 GFP-filtered image. Scale
bar = 100 tm for all images.































Figure 4-18. Confocal microscope image of live GFP-mES cells seeded within an
OCCAG scaffold at day 7 in culture. Scale bar = 200 [tm


Video 4-3. Confocal microscope video of live GFP-mES cells seeded within an OCCAG
scaffold at day 7 in culture. Reference figure 4-18 for scale. (127,532 KB,
GFP-mESConfo.AVI).







































figure 4-1i. Hoecnst stainea nuclei or mts cells in an UCCAuT capillary at aay 4 in
culture. Scale bar = 75 pm. *Image courtesy ofDr. Takashi Hamazaki




































Figure 4-20. Growth of GFP-mES cells in OCCAG scatfolds cultured in maintenance
(M) or differentiation (D) media or a combination (M/D) over 4 days. A)
Day OD, B) Day OM/D, C) Day OM D) Day 2D, E) Day 2M/D, F) Day 2M,
G) Day 4D, H) Day 4M/D, I) Day4M. Scale bar = 100 tm for all images.














CHAPTER 5
CONCLUSIONS

Introduction

This project developed and biologically assessed new biomaterial tissue scaffolds

derived from copper-capillary alginate gels. These scaffolds are related members of a

new family of biomaterials best thought of as modular tissue engineering tools. Though

many of the studies presented are in the early stages and much experimental work

remains, the project was overall successful and the potential for significant future

developments is high.

Scaffold Synthesis: The Agony and the Ecstasy

Time lapse videoscopy provided an excellent means of tracking RCCAG growth.

These videos clearly demonstrate the dynamic of raw gel formation. RCCAG growth did

however result in the production of significant quantities (liters) of copper sulfate

hazardous waste. Treatment with barium hydroxide or oligochitosan was sufficient to

crosslink RCCAG for experimental biology, however scaffold stabilization proved an

underestimated challenge. The flow-reactor did improve the BCCAG production process

but fell short of performing as initially envisioned. OCCAG in comparison was easier to

produce and yielded much smaller quantities of waste. OBCCAG was tricky to make

with overexposure to the oligochitosan solution ultimately resulting in scaffold collapse.

Hence, the synthesis protocols described in this work produce the new biomaterials, but

are not optimized.









Characterization: CCAG Scaffolds as Subtle Composites

Material colorimetric changes were happy accidents of the synthesis process. The

change from light blue to royal blue while processing the RCCAG in barium hydroxide

provided a crude means of monitoring the process. This change was likely the result of

copper hydroxide formation within the gel. RCCAG also turned a yellowish-brown after

soaking in oligochitosan solution for multiple hours, probably the result of the

oligochitosan:alginate complexation rather than formation of a copper salt. All scaffolds

clarified and become almost "invisible" when washed in cell culture media due to the loss

of copper.

Crystalline and golden brown-black gelatinous precipitates also formed during

barium hydroxide treatment. The gelatinous precipitate was probably copper II oxide

and the X-ray mapping data at least support that the particles were copper rich.

Following this logic, copper ions apparently migrated or diffused toward material-

solution interfaces, perhaps driven by the formation of copper hydroxide and copper II

oxide. Copper II oxide ultimately formed possibly due to the relatively poor thermal

conductivity at the core of the CCAG-derived material. The thermal energy released

during copper hydroxide formation may have thus been "trapped" and enough for the

formation of copper II oxide.

X-ray mapping and backscatter images support the idea that the crystalline or

"shimmering" precipitates are likely barium compounds perhaps barium sulfate or

carbonate. BCCAG was also more brittle than the other materials resulting in poor

handling properties. Barium crosslinked alginate spheres have been previously shown to

have a higher modulus than copper crosslinked counterparts [34]. The barium

crosslinked material tending to crumble or shatter hydrated or freeze-dried. This behavior









could also be due in part to the precipitates formed within the gel, degradation of the

alginate through base catalyzed hydrolysis or a combination of both.

X-ray maps also tell us the RCCAG and BCCAG are uniformly crosslinked by

their respective metal ions (at least at the scale of the interaction volume). Interestingly

silicon also appears uniformly distributed within the gels, presumably in the form of

silicon dioxide. The raw alginate material is likely the source of this contamination. The

silica could also be thought of as a filler, perhaps affecting mechanical properties. Cast

in this light, CCAG scaffolds are subtle composites and this perspective could provide

interesting avenues for future material exploitation.

Biological Assessment: Living with Success and Failure

This study clearly demonstrates that mouse embryonic stems cells can survive and

proliferate in OCCAG scaffolds for many days, and these cells form cylindrical structures

in the scaffolds. This is the first report of any (mammalian) cell type cultured in CCAG

materials. The formation of the mES cell cylindrical structures proves that CCAG-

derived scaffolds can guide in vitro cell growth. If the growth behavior of the mES cells

describes generally how cells grow in CCAG scaffolds, then the average capillary size

will likely need to be increased (perhaps to 100 tm) for Schwann cell/ peripheral nerve

regeneration studies. Furthermore, the use of GFP expressing cells coupled with the

CCAG structure and relative optical clarity of the scaffolds made possible confocal

microscopy studies. The use of confocal microscopy has become increasingly popular in

experimental cell biology and will undoubtedly continue to provide valuable insights in

future CCAG scaffold experiments.

Studies utilizing GFP-3T3 cells were not as successful however, probably due to

the size of these cells compared to that of the average scaffold capillary diameter. Also,






65


the 3T3 study clearly shows that the cells prefer to adhere to each other rather than the

scaffold indicating a need to adjust/improve material surface chemistry. Successful

experiments with fibroblasts in CCAG scaffolds will likely be critical for further

evaluation of these new biomaterials as much is known about fibroblast cell behavior in

other settings.














CHAPTER 6
FUTURE WORK

Introduction

The neuroregenerative potential of the new CCAG scaffolds remains to be

investigated. These investigations will likely be secondary priorities however, as the

intriguing results using mES cells have excited all involved. Hence, stem cell-scaffold

interaction studies will be the dominant future research direction. Unfortunately, so

much curiosity has been generated by the results of this work that defining a clear future

research path has been difficult.

Synthesis: Expanding the CCAG Scaffold Family

New additions to the CCAG scaffold family must be made in order to fully

evaluate the tissue engineering potential of these new biomaterials. Synthesizing

scaffolds with a different average capillary diameters and oligochitosan crosslink times is

the next logical step since adjusting these scaffold modules is straightforward. These

materials will be critical to future stem cell-CCAG scaffold interaction studies. Further

work could utilize derivitized oligochitosan crosslinkers, protein additives and diffusible

cues to make a host of CCAG-derived scaffolds. Production processes will also need to

be optimized to reduce intrinsic scaffold variability.

Scaffold Characterization: Quantitative Bulk and Surface Compositional Analysis

The qualitative characterization work performed here provided a wealth of

interesting data that will serve as a springboard for quantitative studies. Although EDS is

a powerful qualitative elemental characterization technique, it is a cumbersome means of









obtaining quantitative information. A more suitable means of obtaining quantitative bulk

elemental analysis of CCAG-derived biomaterials is laser ablative inductively coupled

plasma mass spectrometry (ICP-MS). This analysis would be particularly focused on

copper and its potential release from the scaffold since copper has been reported to have

in vivo angiogenic potential [60, 61]. X-ray photoelectron spectroscopy (XPS) and/or

Fourier transform infrared spectroscopy (FTIR) studies should also be conducted to

obtain quantitative surface composition data. Tallying nitrogen concentrations at the

surfaces of CCAG scaffolds provides a means of tracking oligochitosan and/or protein

treatments.

Biologic Assessment: Standardized Methods and Controls for Stem Cell Studies

A difficulty recognized early in the course of mES cell experiments was the lack of

established protocols for seeding precise numbers of cells. The development of the

vacuum seeding method was a large improvement, but it was still exceedingly difficult to

determine the number of cells actually seeded. Mouse ES cell behavior is, to some

degree, a function of cell culture density (unpublished observations); it will therefore be

important to solve this problem in order to draw firm conclusions about scaffold

influence on cell behavior(s).

Defining proper comparative controls is another area that requires attention for

further mES cell experiments. For example, is it fair to compare mES cell behavior in

flat culture to that of cells grown in CCAG scaffolds? Ongoing studies underway in the

Terada laboratory at the University of Florida, Department of Pathology comparing the

average cell fates present in the two conditions are mixed. There is simply too much

variability to draw a confident conclusion. The mES cell behavior is, after all

mysterious.









Given all of the above, some long range goals will be to demonstrate that stem cell

fate can be modulated via CCAG scaffold architecture, modulus and surface chemistry

and to quantify the interactionss. A few recent stem cell-scaffold studies have claimed

as much [62-64], putting this goal within achievable range. CCAG-derived scaffolds

provide a unique and elegant means of controlling the cell-cell interactions felt to be

crucial to stem cell differentiation. The regular, adjustable capillary microstructure of

these novel scaffolds provides a robust model system not currently available for studying

cell-cell and cell-scaffold interactions. Tailoring the system to propagate adult stem cells

in vitro, especially hematopoietic stem cells, would be directly beneficial to patients

requiring bone marrow transplantation. Finally, in vivo studies using CCAG scaffolds

alone and loaded with various cells types need to be conducted pursuant to any attempt at

organoid synthesis.














APPENDIX:
PUBLICATIONS, PRESENTATIONS AND PATENTS

1. Tollon, M.H., Hamazaki, T., Willenberg, B.J., Batich, C., Terada, N., Fabrication
of Coated Polycaprolactone Scaffolds and Their Effects on Murine Embryonic
Stem Cells. Materials Research Society Spring Conference Proceedings, 2005.
K9.14.

2. Willenberg, B.J., A New Family of Tissue Engineering Scaffolds Derived From
Copper-Capillary Alginate Gels: Synthesis and Characterization. FLAVS-FSM
Annual Joint Symposium, 2005. Invited presentation.

3. Batich, C., Willenberg, B.J., Hamazaki, T., Terada, N., Novel tissue engineered
scaffolds derived from copper capillary alginate gels. US patent application, 2005.
Serial No. 11/074,285.
















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BIOGRAPHICAL SKETCH

Bradley Jay Willenberg spent the first 13 years of his life growing up privileged in

Bloomfield Hills, Michigan. Here he spent much time in a garage-based laboratory

contemplating fire, disassembling toys, repairing bikes, experimenting with electrical

motors, staring at the sun with a telescope, and exploring the universe in a drop of

standing ground water. Brad had significant difficulty reading and writing and therefore

also spent considerable time deeply immersed. When he was 9 years old, he was

fortunately injected into a well-funded public middle school that was able to redress his

apparent lack of educational progress.

Brad spent his high school years attending three different schools in three different

states. After graduating from First Colonial High in Virginia Beach, Brad was granted

admission and attended college at the University of Florida. He graduated Phi Beta

Kappa with highest honors, receiving a bachelor's degree in interdisciplinary studies

focused on biochemistry and molecular biology. Brad then worked for a little over a

year at a small biotech start-up in Alachua, Florida. He returned to the University of

Florida to pursue a doctorate in biomedical engineering. Over the course of his doctoral

work, Brad developed a promising family of biomaterial scaffolds that he hopes to

continue studying and developing. He has also worked as a materials analyst at the

Major Analytical Instrumentation Center (MAIC) since June 2003.






77


All of his life, Brad has shared a very deep connection with music. He is an

accomplished bass player and a budding singer and guitar player. Brad currently lives

with his family in Gainesville, FL.