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Tissue repair and regeneration is a complex physiological process that requires multiple biological and physico-chemical cues acting together. Electrical regimes are particularly effective in controlling the cellular response of electrically sensitive tissues such as nerve, highlighting the need to develop new electroconductive/active microenvironments. This thesis describes the development of electroconductive/active micro/nano- fibrous scaffolds based on Bombyx mori silk fibroin (SF) for neural tissue engineering applications. Composites based on the incorporation of graphene oxide (GO) at controlled loadings (1 to 10% wt.) followed by in situ post-reduction into reduced GO (rGO) were first explored, but the electrical conductivity achieved was limited. On the other hand, functionalisation with poly(3,4-ethylenedioxythiophene)- polystyrene sulfonate (PEDOT-PSS) led to much higher conductance, readily tuned in terms of the coating concentration or by treating it with dimethyl sulfoxide (DMSO). Decoration of silk with intrinsically conductive recombinant reflectin, reported among the highest proton conductors in nature and involved in cephalopod brain neurogenesis, was also investigated. Experimental work was conducted to characterise the physico-chemical properties of the developed scaffolds. In vitro studies with neuroma NG108-15 cells showed that cellular viability was maintained in all scaffold groups, while metabolic activity and proliferation were greatly promoted from the early stages of the cell culture. Furthermore, these families of electroconductive/active scaffolds supported cell differentiation with neurite sprouting. Neurite outgrowth was observed after 5 days of culture, with neurite extensions up to 150-250 um. The data reported here suggests that these electroconductive/active microenvironments may be beneficial and could potentially outperform unmodified silk scaffolds. Overall, the study conducted here provides useful information about the combined use of silk and various electroconductive/active moieties that could be useful for the regeneration and repair process of peripheral nerves, and hint at the potential of using these electroconductive/active scaffolds in combination with exogenous electrical regimes to allow direct delivery of electrical signals and trigger the controlled release of therapeutics to the site of interest.

The failure of mammalian CNS neurons to regenerate their axons derives from a combination of intrinsic deficits and extrinsic obstacles. Following injury, chondroitin sulfate proteoglycans (CSPGs) accumulate within the glial scar that forms at the lesion site in response to the insult. CSPGs inhibit axonal growth and regeneration, an action mediated by their sulfated glycosaminoglycan (GAG) chains, especially those with 4-sulfated (4S) sugars. Arylsulfatase B (ARSB) selectively cleaves 4S groups from the non-reducing ends of GAG chains without disrupting other, potentially growth-permissive motifs. In this thesis, "Modifying Chondroitin Sulfation Enhances Retinal Ganglion Cell Axon Regeneration," I, Craig Pearson, seek to determine the time course and spatial distribution of CSPG accumulation in the glial scar following acute injury, and then to demonstrate that ARSB is effective in reducing the inhibitory actions of CSPGs. I examine the effects of ARSB in an in vitro model of the glial scar and in vivo, using optic nerve crush (ONC) in adult mice. ARSB is clinically approved for replacement therapy in patients with mucopolysaccharidosis VI and therefore represents an attractive candidate for translation to the human CNS. My findings illustrate the importance of CSPGs as a barrier to axon extension following injury, and show compelling evidence that selective modification of the sulfation pattern on GAG chains results in significant enhancement of RGC axonal regeneration. Finally, I combine ARSB treatment with a host of intrinsic pro-regenerative stimuli and show robust, long-distance regeneration of RGC axons through the optic chiasm and into the optic tract. Taken together, the results of this thesis argue for the therapeutic potential of modifying the extracellular matrix to promote regeneration of axons in the CNS.

Unlike the peripheral nervous system (PNS), adult neurons in the central nervous system (CNS) have limited regenerative capacity after injury. One interesting phenomenon observed nearly four decades ago was that lesion of a peripheral nerve can significantly enhance the regenerative capacity of the central axons of the corresponding dorsal root ganglion (DRG) neurons, termed a ‘conditioning lesion’, but the underlying mechanism is still not fully understood. Since ATP is released after nerve injury and extracellular ATP has a broad range of biological activities, we postulated that ATP might be the injury signalling molecule that triggers the regenerative machinery in the injured neurons. If that were the case, injection of ATP into a peripheral nerve should be able to mimic the effect of a conditioning lesion. To test this theory, we injected ATP into a peripheral (sciatic) nerve after a dorsal column transection and found that ATP injection did promote the regeneration of injured axons into the lesion cavity. We also found that ATP injection activated transcription factor STAT3 and increased the expression of growth associated protein 43 (GAP43) in the corresponding DRG neurons. ATP injection increased the concentrations of ciliary neurotrophic factor and interleukin-6 in sciatic nerve and DRG. These results indicate that intraneural injection of ATP can mimic conditioning lesion to a certain degree. Most interestingly, we found that a second injection of ATP one week after the first one markedly boosted the effects of the first injection as many more axons grew into or across the lesion compared with double saline injection or ATP plus saline injection. Double ATP injection is also more effective in sustaining the expression of phospho- STAT3 and GAP43. Immunohistochemical analysis showed ATP injection caused little Wallerian degeneration at the injection site. Behavioural tests showed no long-term adverse effects to the injected sciatic nerve. In order to explore the underlying mechanism of ATP induced elevation of the regeneration state of DRG neurons and look for more potent purinoceptor agonists to stimulate axonal regeneration, we first tried to identify the expression of purinoceptor subtypes in sciatic nerves using quantitative PCR and immunohistochemistry. We found that mRNAs for all the four P1 and fourteen P2 purinoceptor subtypes were expressed in the sciatic nerve, DRG or dissociated Schwann cells at various levels. Immunohistochemical analysis showed that purinoceptor subtypes are expressed by different types of cells. Due to the expression of nearly all purinoceptor subtypes in the sciatic nerve, it will be a big challenge to identify the receptor subtype(s) responsible for ATP induced axonal regeneration. We have set up a compartmented co-culture system to test various agonists/antagonists of purinoceptors. Taken together, we have shown that intraneural ATP injection can mimic conditioning lesion in promoting sensory axonal regeneration. Identification of the receptor subtype(s) and other molecules involved in the enhanced regeneration capacity of injured neurons may lead to the development of therapeutic agents to effectively promote the axonal regeneration of both peripheral and central neurons.

The use of biomaterials for tissue repair applications presents immense potential for regenerative medicine. Soft tissue such as skin, muscle, and nerves share a similar overarching molecular paradigm for initiating the process of repair after injury. Namely, the interaction between individual cells and the surrounding microenvironment is a clear predictor of regenerative success. Phenotypic changes in cells such as stem cells, glial cells, or other progenitor cells are, largely, determined by changes in the deposited extracellular matrix. However, as cells often play some role (whether direct or indirect) in secreting, assembling, and modifying the connective matrix, this interaction is dynamic and reflects the litany of considerations for human induced changes to the microenvironment. While there is great variance in the repair success of individual damage, the microenvironment of cells is typically a useful target for improving regenerative outcomes. This work models the interdisciplinary reach of biomaterial design by exploring the use of numerous engineered substrates to direct the behavior of cell line models for soft tissue applications. A piezoelectric, electrospun polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE) was specifically identified as a model substrate for nerve repair. With extensive materials, chemical, and electrical characterizations, it was emphatically determined that PVDF-TrFE fibrous scaffolds can be used to precisely influence the microenvironment of 3T3 Fibroblasts and RT4-D6P2T Schwann Cells to promote a prolonged regenerative phenotype. Additional modifications in scaffold fabrication produced numerous methods for functionalization with bioactive, decellularized extracellular matrix (dECM) proteins that are known to contribute significantly to repair. Current clinical solutions for nerve damage consist of nerve grafts, nerve conduits, and neurorrhaphies. While surgical repair is capable of retaining functionality in many patients, there are concerns related to donor site morbidity, incomplete axonal resection, and limited availability. Thus, after confirming the efficacy of various biomaterials for in Vitro models, a clinically feasible nerve conduit design was constructed entirely of electrospun, PVDF-TrFE scaffolds that maintained the relevant characteristics necessary for tissue repair. Further in Vivo testing will provide proof-of-concept for scaffold implementation, with the objective to use electrospun conduits for repair of severely transected nerves. This foundation introduces an alternative strategy to current nerve repair strategies with a cost-effective and relatively simplistic design structure. Further work will continue to improve on implementation parameters to ultimately produce a novel biomaterial for tissue repair, drug delivery, and therapeutic targeting.

Severe peripheral nerve injuries are currently limited in their available treatment approaches. Standard clinical therapies include the use of nerve grafts or nerve conduits to guide axons across the injury gap, however many of these approaches show variable outcomes in functional recovery, possibly due to a lack of cues directing the nerve regeneration. Polyvinylidene flouride triflouroethylene (PVDF-TrFE) is a piezoelectric polymer that generates electric charge in response to mechanical deformation, and can provide a suitable regenerative microenvironment by imparting electrical stimulation to direct peripheral nervous system cells and function. In this thesis, PVDF-TrFE nanofibrous scaffolds are fabricated through electrospinning and are extensivelycharacterized for their material and electrical properties. Further, the PVDF-TrFE scaffolds are functionalized with decellularized extracellular matrix (dECM), presenting a promising outlook for a piezoelectric biomaterial for tissue engineering.

Surgical repair of ablation-type peripheral nerve injuries by using peripheral nerve allografts (PNAs) has been hindered for decades due to slow and ineffective axon regeneration from proximal nerve ends as well as immunological rejection of PNAs. We have developed a polyethylene glycol (PEG)-fusion repair protocol for sciatic PNAs in rats that results in maintenance of myelinated axons that do not degenerate, neuromuscular junction innervation, and significantly improved behavioral recovery, as compared to current nerve repair methods. These phenotypes are maintained for weeks postoperatively without using tissue-matching, decellularization, or immunosuppressive drugs. That is, PEG-fused PNAs are functionally tolerated by the host immune system. This dissertation presents work performed to characterize and investigate mechanisms underlying immunological responses to PNAs treated with PEG-fusion with the aim of understanding how and why PEG-fused allografts are not rejected by the host immune system in rat sciatic nerve injury models. Chapter 1 provides a review of the clinical significance of peripheral nerve injury, including: Biological processes of axonal degeneration and regeneration, current methods of nerve repair, immunological rejection of PNAs, concepts underlying PEG-fusion repair and current experimental results of PEG-fusion in rat PNAs, as well as the hypotheses and aims explored. Chapter 2 characterizes innate and adaptive immune responses to PEG-fused PNAs using primarily immunohistochemistry, electron microscopy, and quantitative reverse transcription PCR (RT-qPCR). Our results suggest that PEG-fused PNAs achieve immunotolerance via attenuated innate and adaptive immune responses. Chapter 3 examines via RNA sequencing the coding transcriptome of PEG-fused PNAs to determine which biological processes, protein families, pathways, and protein-protein interaction networks differentiate PEG-fused PNAs from negative control PNAs not treated with PEG with the goal of identifying potential mechanisms underlying immunotolerance. This work provides a critical molecular foundation for future studies investigating PEG-fusion-mediated immunosuppression in PNAs. Chapter 4 investigates whether treatment of PNAs with PEG alone without axonal fusion induces similar immunosuppressive effects. Our results suggest that PEG treatment alone does not prevent Wallerian degeneration or attenuation of innate and adaptive immune responses. Chapter 5 provides a summary of the dissertation work and describes future directions for research.

The ideal wound closure device should restore the original integrity of the tissue, offer easy application and seamless, fluid-tight seal. Commonly used wound closure devices including sutures, staples and tissue adhesives do not offer effective sealing of the wound and possess a range of associated disadvantages including dehiscence, infection, toxicity and iatrogenic related trauma. Laser tissue welding has been proposed as an alternative and is becoming increasingly popular. This technique uses laser irradiation to initiate chemical reactions in a target material, thereby activating chemical components within the material to form an immediate seal. However, laser tissue welding is reliant upon temperatures exceeding the collagen denaturation point and is associated with some tissue damage. SurgiLux is a chitosan-based thin film surgical adhesive that relies upon laser irradiation to increase the strength of chitosan binding to tissue without thermal damage to tissue and avoiding many of the disadvantages of current wound closure devices. Previous in vitro and in vivo studies of SurgiLux has demonstrated the potential of SurgiLux in sutureless repair of peripheral nerves. Recent approaches to regenerative medicine and tissue engineering involve the use of decellularised extracellular matrix as biological scaffolds to augment the formation of new functional tissue and facilitate successful tissue reconstruction. The aim of the work reported here was to combine SurgiLux with an extracellular matrix scaffold derived from porcine urinary bladder matrix to potentially improve the capacity of the SurgiLux technology to enhance wound healing by promoting functional tissue regeneration, for potential applications in peripheral nerve repair. The bio-scaffold was incorporated into the SurgiLux film in a variety of ways; bio-scaffold embedded into SurgiLux had a greater tensile strength (32.4 ± 5.2 MPa), crystallinity (12.1 ± 1.3 %) and hydrophilicity (75.0 ± 2.0)° than the chitosan adhesive alone (8.5 ± 3.1 MPa, 10.7 ± 1.2% crystallinity, Ө = 98.1 ± 2.03°). Tissue adhesion strengths using these hybrid biomaterials were maintained at ~15 kPa compared to 3 kPa for fibrin glue. Furthermore, histological analysis demonstrated that laser irradiation of the UBM-SurgiLux adhesive caused no thermal damage to tissue. In vitro biocompatibility of the composite films was assessed by examining their influence on the proliferation and health of olfactory ensheathing cells and human monocyte-derived macrophages. Incorporation of the bio-scaffold into the SurgiLux films increased the attachment and proliferation of olfactory ensheathing cells and decreased the cytotoxicity of the films. Similarly, while chitosan films induced a cell population to undergo early apoptotic activation, the composite films apparently increased biocompatibility, preventing the cells from undergoing necrosis. Similarly, while SurgiLux showed a significantly reduced macrophage response compared to chitosan film, introduction of the bio-scaffold into the SurgiLux reduced their response further. A quantitative real time PCR approach was undertaken to identify polarised macrophage phenotype, M1 (pro-inflammatory) and M2 (anti-inflammatory) through the detection of specific cytokines expressed by the macrophages. Reduced cell spread (6.2e3 ± 7.0e2 μm2) and lack of foreign body giant cell formation lead to significantly reduced expression of M1 markers, IL-23p19, IL-12p40 and IL-12p35; thereby suggesting the presence of an alternative anti-inflammatory, tissue remodelling pathway. A protein expression profile of the UBM scaffold was generated to identify novel proteins within the UBM via an advanced mass spectrometry methodology. A total of 129 proteins were identified with the majority of these revealing a role in maintaining cell structure (19%) and adhesion (13%), while the smallest groups (1 %) had remodelling and stimulatory roles. A number of growth promoting proteins including galectins 1 and 7, obscurin, fibulin and have been identified that may have enhanced cellular proliferation on the UBM-SurgiLux composite scaffolds compared to chitosan films alone. UBM also contains proteins that have neurotrophic, anti-angiogenic, tumour suppressor activity and proteins known to promote tissue remodelling and morphogenesis. Therefore, coalescence of the bio-scaffold within SurgiLux matrix resulted in a surgical adhesive with enhanced biocompatibility and reduced cytotoxicity compared to chitosan films. The results suggest that the unique combination of extracellular matrix bio-scaffold with SurgiLux technology has the potential to promote functional tissue regeneration leading to enhanced sutureless nerve repair.

Abstract: This study explores the effects of surgical reconstruction and nerve repair on sensorimotor function and quality of life (QOL) for patients with base of tongue (BOT) cancer compared to healthy, age-matched adults. Sensations were tested on the anterior two-thirds of the oral tongue for two-point discrimination, light touch, taste, temperature, form and texture on 30 patients with BOT reconstruction with radial forearm free-flap and on 30 controls. Results indicated sensation for the unaffected tongue side and affected side with lingual nerve intact was comparable to controls, with poorer sensory outcomes for nerve repair. However, lingual nerves repaired with reanastomosis provided superior results to cable-grafting and severed nerves. Patients had decreased motor function only when the hypoglossal and lingual nerves were affected. Patients' QOL responses on the UW-QOL and EORTC QLQ-H&N35 revealed involvement of lingual and hypoglossal nerves resulted in poorer QOL outcomes. QOL interviews revealed additional problematic issues in this population not identified by standardized questionnaires.

Regenerative biology seeks to understand the molecular mechanisms that differentiate several animals with the ability to regenerate tissues. An approach to evaluate these mechanisms is via a comparative analysis of regeneration-competent versus regeneration-deficient tissues. This thesis analyses the regenerative capacity in the axolotl blastema during the dedifferentiation phase. Currently, there is an equivocal understanding of the molecular factors expressed during the regenerative process and further analysis is warranted. Comprehensive profiles of the proteins expressed during this process are presented. Nerve derived factors such as neuregulin-1 and neurotrophin-3 were identified in the axolotl blastema during the time of dedifferentiation. Additionally, this study investigated if there are any correlation between the nerve determinants, such as neurotrophins, from the regenerating blastema and neurogenesis that occurs throughout the lifespan of mammals in the olfactory system. This was achieved by comparing the expression proteomes of the regeneration-competent axolotl and olfactory ensheathing cells. Collectively, the study proposes that there is an influence by neurotrophins on neurogenesis in the olfactory system that are comparable to the dynamic nerve signaling networks that occur in the axolotls regenerating limb. It provides a window into the complex signaling pathways during the two processes that can be clinically translated to further develop better therapeutic strategies that involve peripheral nervous tissue of the spinal cord. The current clinical outcomes usually result in limited functional restoration. One of the most promising techniques is to induce a regenerative capacity in the affected tissues using a tissue engineering strategy. The combination of factors identified may serve as candidates to augment in vivo peripheral nerve microenvironments and to achieve a regenerative capacity in human tissues. These neurotrophins should be considered for regenerative medicine applications to induce regenerative competence in human peripheral nerve tissues.