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The hypothesis underlying the work to be presented here is:’…that a large animal (sheep) may successfully be used as a model for peripheral nerve injury, repair and regeneration and that laboratory studies using this model may furnish information useful in predicting clinical outcome’. There are nine chapters. In the first chapter the history of nerve regeneration is briefly discussed. The second chapter is a critique of the use of the sheep as a model for nerve injury and repair and a comparison of this large animal model with small animal models such as the laboratory rat. The conclusion is that the ovine model is a nearer representation of the human case than small animal models. In the third chapter the various clinical and laboratory tests which may be used to evaluate the outcome of nerve injury and repair are discussed. Chapter 4 is a consideration of the case of a mixed (median) nerve and in Chapter 5 a purely motor (facial) nerve is investigated using the tests outlined in Chapter 3. In Chapter 6 there is a discourse upon the use of a technique for estimating the distribution of conduction velocities (CVDist) in the human. In Chapter 7 the special case of obstetrical brachial plexus injury is discussed in the light of the clinical and laboratory tests which may be used to assess it and a similar treatment is given in Chapter 8 to nerve injuries complicated by cavitation, fibrosis, haematoma, long-bone fracture and arterial injury. In the final chapter (9), all of the above are discussed and the value of both the ovine model and the tests described in Chapter 3 are evaluated. It is concluded that the ovine model is an excellent substrate for the evaluation of the pathophysiology of nerve injuries, surgical methods for their repair and of the tests which may be used to assess the outcome of these procedures.

Abstract: Introduction: The timing of nerve transfer or graft after peripheral nerve injury is critical, and accepted to be approximately 3 to 6 months. However, in practice, patients often present in a delayed manner for surgical intervention. This study describes the timing of surgery after peripheral nerve injury for adult patients in Alberta, and explores factors influencing time to surgical intervention and clinical outcomes. Design and Methods: A retrospective analysis of adult patients undergoing peripheral nerve transfer or grafting in Alberta from 2005 to 2017 was completed. One hundred and sixty-six patients who underwent distal nerve transfers or grafts for either upper or lower limb peripheral nerve injuries were included in the analysis of time to surgery. One hundred and twenty-nine patients with a minimum of one year follow up, after peripheral nerve surgery, were included in the analysis of factors affecting clinical outcomes. Additionally, qualitative semi-structured interviews were conducted with patients who underwent surgery through the Northern Peripheral Nerve Clinic in order to explore patient perceived barriers and experiences accessing care for severe peripheral nerve injuries. Intervention(s) and Outcome Measures: A Cox Proportional Hazard Regression was completed to determine correlation of patient, injury and systemic factors with time to surgical intervention. Additionally, a multivariable logistic regression analysis was completed to examine the association of time to surgery, patient, injury and operative characteristics on MRC strength outcomes. Thematic analysis was utilized to examine the qualitative data collected from the semi-structured interviews. Results: The mean (SD) time to surgery was 221 (118.1) days. A referral made by a surgeon approximately doubled the hazard of earlier surgery as compared to a general practitioner (p=0.006). An increase in one comorbidity resulted in the adjusted hazard of earlier surgery decreasing by 16% (p=0.014). Patients identified three main themes of concern: delays in diagnosis, issues with resource accessibility, and lack of support as barriers to accessing the Peripheral Nerve Clinic and subsequent surgery. Numerous factors are associated with post-operative strength outcomes including: time to operative intervention, operative procedure, and injury. For every week increase from injury to time of surgery, the adjusted odds of the patient achieving a MRC strength grade ≥ 3 decreases by 3% (p=0.02). If a patient received a nerve transfer instead of a nerve graft the adjusted odds of the patient achieving a MRC strength grade ≥ 3 was 388% (p=0.003). The adjusted odds of achieving a MRC ≥ 3 decreased 65% if the injury sustained had a pre-ganglionic injury component (p=0.05). Conclusions: The timing of operative intervention after peripheral nerve injury is critical, and delays in surgical intervention are best explained by both patient and systemic factors. These areas of deficiency in the peripheral nerve injury service pathway require further exploration and improvement in order to optimize patient care.

Peripheral nerve injury results in debilitating physical impairments, including loss of motor, sensory, and autonomic functions, which are often permanent. The greatest advancement to clinical repair of traumatic peripheral nerve injury came in the 19th century with the implementation of microsurgical suturing to re-approximate severed nerve ends. However, even upon surgical repair, the distal nerve segment undergoes degeneration and therefore requires the slow regeneration of proximal axons (at a rate of 1-2mm per day) for restoration of functions. This results in slow and often unsatisfactory recovery. To overcome these obstacles, we have developed a method of nerve repair utilizing the fusogen polyethylene glycol (PEG) to fuse severed axons and rapidly restore lost functions. The work within this dissertation characterizes the mechanisms underlying PEG-fusion restoration of function for injuries to peripheral nerves. Chapter 1 provides background on the events that occur after nerve injury, current clinical strategies aimed at improving nerve function after injury, and the development of the PEG-fusion procedure from previously published work. Chapter 2 examines the functional and morphological changes that occur after PEG-fusion of transected peripheral nerves, extending our mechanistic understanding of PEG-fusion mediated repair. Here, we found PEG-fusion restores axonal continuity across the lesion site, Wallerian degeneration is prevented for many distal axons, and muscle innervation is maintained. Chapter 3 examines alterations in motoneuron morphology and circuity after PEG-fusion repair of peripheral nerves. PEG-fusion repair results in no changes to motoneuron soma size or number but does alter dendritic arborizations. Surprisingly, PEG-fusion results in the mismatch of proximal axons and distal muscle and sensory targets yet results in significant functional recovery. Chapter 4 highlights studies to translate our findings to the clinical setting. Here we have optimized the PEG-fusion procedure using a series of FDA approved solutions and precise surgical resection of axons after initial severance, leading to increased functional recovery and easy adaptation of methods for clinical trials. Chapter 5 provides a summary of the dissertation and future directions of the research, including the adaptation of PEG-fusion technology for vascularized composite allotransplantation and spinal cord injury.

After peripheral nerve injuries patients lose and do not recover the stretch reflex which leads to altered locomotor function. The focus of this thesis is to investigate the structural integrity of the central connection between Ia afferents and alpha motoneurons that mediate the stretch reflex. The overall hypothesis is that the density and distribution of Ia synapses on motoneurons is altered after peripheral nerve injuries. Analysis of Ia afferent-motoneuron contacts, revealed by vesicular glutamate transporter 1 (VGLUT1) immunoreactivity, on the soma and dendritic arbor of motoneurons after peripheral nerve injuries revealed major reorganizations in the distribution and density of Ia synapses. Synaptic stripping of Ia afferent synapses occurred on the soma and proximal dendrites and appeared to be permanent even after reinnervation; in contrast, VGLUT1 synapses on distal dendrites were unchanged. In conclusion, after peripheral nerve injuries motoneurons are contacted by fewer Ia synapses and they are more distally located. This overall reorganization likely weakens the input and may contribute to stretch reflex anomalies.

Peripheral nerve injuries have traditionally been treated with a variety of different surgical procedures, but the use of allografts for these injuries remain to be a largely unexplored concept. As a result, there has yet to be a successful protocol created for imaging cells on the surface of nerve allografts. In this thesis, I developed a z-stack projection imaging protocol for nerve allografts and demonstrated the ability to take clear and focused images of cell retention and proliferation on the surface of the allograft. I also optimized the preparation of the nerve graft to improve cell and connective progenitor cell (CTP) retention rates.

The current gold standard treatment for large peripheral nerve injuries is an autograft but this method comes with several downsides such as donor site morbidity and limited donor tissue availability. Autografts are also not always successful with patients left with long-term impacts on quality of life such as paralysis and chronic pain. A tissue engineered alternative to the autograft could greatly improve outcomes, avoid the detrimental effects of an autograft, and hopefully lead to improve quality of life and socioeconomic outcomes. The components of the tissue engineered construct developed in this project include Schwann cells or Schwann cell precursors. Repair Schwann cells are a key cell type in supporting axon regeneration and using cells expressing genes involved in the repair phenotype is likely to be beneficial. As an autograft contains nerve extracellular matrix and aligned columns of Schwann cells and endothelial cells, the Schwann cells and precursors were also combined with collagen hydrogel and formed into an aligned cylindrical construct. Schwann cells and Schwann cell precursors were successfully differentiated from two clinically relevant hiPSC lines and characterised using immunocytochemistry and RT-qPCR. This protocol was optimised to generate sufficient cell numbers for tissue engineering applications. Conditional immortalisation methods were also assessed, and those cells derived from hiPSCs containing a conditional immortalising transgene characterised. The proliferation rates of the differentiated cells in permissive and non-permissive conditions were tracked over time to determine the tumorigenic properties of the cells to be implanted. These differentiated cells were successfully incorporated into an aligned engineered nerve construct by gel aspiration-ejection. Co-culture experiments with primary rat dorsal root ganglia showed these stable constructs can support and guide neurons along the 12mm long cylindrical gel. This project provides a method for producing human Schwann cells from clinically relevant iPSC sources and demonstrates the first use in GAE.

Peripheral nerve injuries result in a disconnection in the nervous system communication and a consequent loss in neurological function. Currently, there is very limited treatment for these conditions. Neuroprosthetics and cell transplantation are promising approaches to restore lost neurological function: the former aims to bypass the site of injury, connecting directly one part of the nervous system to another (or a prosthetic limb); while the latter aims to repair the injury site. To date, both strategies have shown limited efficacy and lifetime due to several challenges. However, a combinational approach of implantable electronics and stem cell-derived cells for functional neurological restoration could address these issues. The integration between implantable electronics and existing tissue is of paramount importance, this biohybrid strategy with the incorporation of cells may allow for a 'controllable' synaptic integration between implanted cells and existing circuitry. Attributes to such a biohybrid implant are: an ability to host and interact with stem-cell derived cells; promotion of organised functional cellular integration with living tissue; and restoration of lost function. Here I report the new design of a biohybrid peripheral nerve multielectrode neural interface device. My devices are fabricated using photolithography and chemical etching techniques to establish a functional electronic device. This multielectrode neural device contains 32 electrodes. Each electrode consisting of planar gold, coated with a conducting polymer (PEDOT:PSS) to decrease the electrode impedance and improve the signal to noise ratio. The layers of Parylene C give a desirable stiffness compatible with that of native nerve. The device hosts iPSC derived myocytes on the electrically active surface, allowing their efficient electrical recording. I have shown survival of human iPSC derived muscle in a rat for up to seven days post implantation in a rat model. I have developed a strong bonded hydrogel that allows for biofabrication on top of flexible electronics. Finally, I showed long term recordings from my biohybrid neural interfaces in a chronic rat peripheral nerve injury model for 30 days duration. These findings strongly suggest that biohybrid peripheral neural interfaces could be an efficient way to record the peripheral nervous system in a rat peripheral nerve injury model.

Peripheral nerve injury is a severe condition that affects the quality of life of a patient as sensory and motor function are impaired. Current treatments include end-to-end suturing and end-to-side suturing of the injured nerve to restore function. The gold standard treatment for larger peripheral nerve injuries is the use of autografts, however, these are limited by donor site morbidity and size mismatches. Alternatively, allografts and xenografts have also been tried, but they might promote immunological responses that lead to rejection. To overcome these disadvantages, a hollow tube called nerve guide conduit (NGC) has been designed and fabricated, with natural and synthetic polymers, to aid peripheral nerve repair by connecting the proximal and distal stumps. Nonetheless, nerve guide conduits are limited to repair nerve gaps shorter than 20 mm. Research has focused on innovating the design of NGCs to improve their performance in a variety of ways. For example, topographical cues, such as channels and intraluminal fibres, have been included inside the lumen of the NGC. Even though promising results have been obtained, for both in vitro and in vivo evaluation, the regeneration potential of these NGCs is still not enough, as it has been recognized that peripheral regeneration is a complex process in which different cells and molecules play a significant role to achieve complete functional regeneration. For this reason, chemical cues have been added to NGCs. Chemical cues added to NGCs include mainly growth factors, peptides, and extracellular matrix components, such as collagen and laminin. These molecules have been incorporated into NGCs through different approaches, such as coatings on the inner wall, matrices filling the lumen, microspheres, adding the molecules within the wall of the NGC, and immobilisation, either on the wall or onto intraluminal fibres. Growth factors, such as nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), and ciliary neurotrophic factor (CNTF), play an important role during peripheral nerve repair, therefore, these have been included in NGC to enhance neurite outgrowth and Schwann cell migration. Nevertheless, techniques to add these growth factors usually involve the use of solvents, which decrease the bioactivity of the growth factors. Thus, a method to introduce growth factors to NGCs that extends their half-life and preserves their bioactivity would be a promising approach to enhance neurite outgrowth and Schwann cell migration during nerve repair. The use of heparin has been reported to extend the half-life of growth factors as well as maintaining their bioactivity. Usually, heparin is bound covalently on different polymer surfaces, however, this technique could impair the bioactivity of heparin as covalent binding techniques also use solvents. Hence, binding heparin through electrostatic interactions to polymeric surfaces is an encouraging alternative. Heparin is a negatively charged glycosaminoglycan, thus, binding it to a positively charged surface would be the path to follow. Plasma deposition is a widely used technique to modify the surface of a material, making it hydrophilic or hydrophobic, depending on the application. Furthermore, plasma can be used to modify the charge on polymeric surfaces, either to encourage cell adhesion and proliferation of cells, or to bind other charged functional groups to the surface. Therefore, by using plasma deposition technique on a surface, it is possible to bind heparin, and then to bind growth factors, which would create a bioactive surface capable of acting as a local delivery system of growth factors. Specifically, growth factors such as NGF and BDNF can be used to aid neurite outgrowth and Schwann cell migration during nerve repair. This bioactive surface would deliver growth factors in a sustained manner, avoiding undesirable high burst release. Furthermore, taking advantage of the flexibility of the plasma deposition technique, this bioactive surface could be fabricated on the surface of any polymer of choice, regardless of the shape, which would make this bioactive surface an attractive delivery system to adapt to any polymeric scaffold. The work in this thesis consisted of, firstly, fabricating the bioactive surface on a commercially available amine (NH2+) coated plates (positively charged), to which heparin was bound (NH2+ + Heparin). Then, NGF, BDNF, or a combination of NGF plus BDNF, at different concentrations, were immobilised on heparin by electrostatic interactions (NH2+ + Heparin + Immobilised NGF, NH2+ + Heparin + Immobilised BDNF, and NH2+ + Heparin + Immobilised NGF plus BDNF). Contact angle and XPS analysis were used to characterise the bioactive surface and to confirm the presence of heparin. ELISA was performed to characterise the release of NGF and BDNF from the bioactive surface. The bioactive surface was then evaluated using NG108-15 neuronal cells and PC12 adh neuronal cells, finding that immobilised NGF and BDNF encouraged significant neurite outgrowth. Later, this bioactive surface was tested using dorsal root ganglia (DRG) and primary Schwann cells to evaluate neurite outgrowth and Schwann cell migration. The result of these experiments revealed that immobilised NGF at 1 ng/mL encouraged the growth of the longest neurites, in comparison to other test and control groups. Positive results were achieved so far, hence, it was important to implement this bioactive surface to an approach that, in the future, could be implemented in an NGC. Thus, this bioactive surface was added to polycaprolactone (PCL) electrospun fibres. To achieve this, plasma deposition was used to add positively charged amine (NH2+) functional groups on the surface of PCL electrospun scaffolds. Then, heparin was bound to the NH2+, and finally, NGF, BDNF, and a combination of NGF plus BDNF, at different concentrations, were immobilised onto the PCL + NH2+ + Heparin surface. XPS analysis was performed to confirm the successful addition of NH2+ and heparin. Moreover, ELISA showed that there was a sustained delivery of NGF and BDNF from the bioactive surface for up to21 days. Additionally, XPS analysis revealed that heparin was still present on the bioactive surface after 3 months of incubation at 4°C and 21°C. Furthermore, DRG were seeded on these PCL + NH2+ + Heparin + Immobilised NGF/BDNF/NGF plus BDNF scaffolds and neurite outgrowth and Schwann cell migration were evaluated. The results of this experiment revealed that PCL + NH2+ + Heparin + Immobilised NGF 1 ng/mL encouraged the longest neurite outgrowth as well as the furthest Schwann cell migration in comparison to other test groups, control groups, and importantly, to other results found in the literature. Even though PCL + NH2+ + Heparin + Immobilised BDNF did not encourage the longest neurite outgrowth, this surface stimulated the growth of longer neurites in comparison to when the bioactive surface was fabricated on flat, commercially available NH2+ coated plates, revealing that topological and chemical cues further improve the growth of neurites and migration of Schwann cells. Nevertheless, PCL + NH2+ + Heparin + Immobilised NGF plus BDNF did not stimulate an accumulative effect regarding neurite outgrowth and Schwann cell migration. In conclusion, the bioactive surface NH2+ + Heparin + Immobilised NGF/BDNF/NGF plus BDNF was successfully fabricated on PCL electrospun fibres. Then, it was found that NGF and BDNF were delivered in a sustained manner for 21 days. In addition, it was found that by immobilising a relatively low concentration of NGF (1 ng/mL), neurite length of 3 mm was achieved in vitro. Therefore, the use of this bioactive surface on PCL electrospun fibres makes this approach directly applicable and scalable for improving the function of NGCs.

Surgical treatment of major peripheral nerve injuries always result in poor functional outcomes for patients. Although bioengineered nerve conduits offer promise in bridging nerve gaps, current lab research has not translated into routine surgical practice. Schwann cells implanted into nerve conduits improve regenerative outcomes in experimental conduits. As a result of their neurotrophic capabilities, but morbid harvest procedures and specialised maintenance renders them unsuitable for cell therapy. Stimulated, differentiated adipose stem cells (dhASCs) exhibit a Schwann-like phenotype, capable of regenerative outcomes comparable to native Schwann cells. However, there are several important drawbacks including prolonged, costly chemical stimulation and regression towards the undifferentiated phenotype following withdrawal. Furthermore, the hypoxic, inflammatory damaged peripheral nerve environment causes cell detachment from conduits, subsequently leading to cell death. Graphene Oxide (GO) is a 2-dimensional nanomaterial with potential in tissue engineering. Its large surface area with oxygen groups provides a platform for further chemical functionalisation and biochemical stimulation. Moreover, GO can enhance stem cell attachment in vitro and stimulate differentiation towards desired phenotypes. As such, GO has potential for enhancing dhASC function and survival in damaged nerve environments. We hypothesise that GO substrates can prolong dhASC neuro-regenerative stimulation and improve the survival rate in hostile environments, this work was primarily aimed at producing consistent GO platforms, investigating the effects upon dhASC maintenance and survival, and assessing their ability for further functionalisation. Using spin-coating and thermal reduction, consistent GO substrates were engineered for use in in vitro experiments with dhASCs. The platforms were shown to trigger upregulation in neurotrophic protein secretion for nerve growth factor and glial-derived growth factor. Moreover, GO is protective against cell detachment, prolonging attachment and metabolic activity in simulated injured microenvironment. Furthermore, GO can be functionalised with fibronectin using micro-contact spotting which, which was stable under in vitro conditions and maintained functionality, promoting cell attachment. The results demonstrate the potential for using and developing GO-based substrates as enhanced interfaces in novel peripheral nerve conduits. This provides a strong basis for future studies to elucidate dhASC attachment mechanism and effects upon viability observed in damaged nerve environments for their use in peripheral nerve regeneration.

Breakthroughs in nanotechnology and nanomedicine are driving researchers to create novel, precision-medicine based approaches. These advancements can be specifically tuned for various applications, such as treating peripheral nerve injury. Appropriate functioning of the nervous system is tremendously important to overall quality of life. The nerves within our body branch from the spinal column and can extend up to one meter away. Peripheral nerve injuries are a common occurrence and can readily heal in most cases. These injuries come in many flavors and can be targeted at various levels. Therapeutics can be developed to target the soma that can be in the spinal cord (motor neurons) or in the dorsal root ganglia (sensory neurons), intervene locally at the direct site of injury, or target the downstream aspects at the nerve terminals. The work described later will focus on aspects of treating the local injury through a sciatic nerve crush model, maintaining the downstream health of skeletal muscle, and developing strategies to provide immune protection to cellularized nerve allografts. The work discussed within utilizes the tissue nano-technology platform developed by Dr. Gallego-Perez and team to deliver plasmid DNA cargos to intact nerves, skeletal muscle, and nerves for transplantation. We hypothesized that 1) the local induction of endothelial cells in a crush model can lead to more rapid functional recovery through an earlier restoration of vascularization, 2) the delivery of neurogenic factors to skeletal muscle can locally induce neurons to re-innervate the motor endplates and extend the muscles window to remain functional once complete regeneration can occur, and 3) TNT delivery of Fas ligand can mitigate immune cell invasion to grafted nerve tissue to avoid apoptosis of the needed cell populations and to prevent host rejection. Goal one uses a plasmid cocktail containing Etv2, Fli1, and Foxc2 (EFF) that has previously been validated to reprogram fibroblasts into endothelial cells within one week. TNT has previously been demonstrated to work in mouse skin. The applications discussed within this work focused on the translation of this approach to nerve tissue. As such, in addition to evaluating the impact of endothelial cell reprogramming, the optimization of transfection parameters is a large focus in answering our first hypothesis. In looking at downstream impact of nerve denervation, we sought to develop strategies aimed at maintaining muscle health as axons regenerate across the great distance they cover. In humans, the terminal location of nerves can be up to one meter or three feet away from the cell body. Tissues and organs existing beyond ~18 inches away from the nerve body, are highly susceptible to poor reinnervation outcomes and long-term detriments. A contributing cause for these deficits is the slow growth rate of the axons (1-2mm/day or 1 inch/month). Literature has reported that human skeletal muscle can remain receptive to reinnervation for 18-24 months. Current intervention strategies are focused on addressing the injury along the nerve bundle and developing methods for more complete regeneration. A shortcoming of these approaches that we sought to address is extending the window that skeletal muscle is responsive to reinnervation once the process is completed. Thus, the primary goal discussed in Chapter 3 centers around an approach to locally induce neurons, through cell reprogramming, capable of synapsing on the motor endplates as a strategy to preserve the muscle in a healthy state. From the two hypotheses and goals of Chapters 2 & 3, there is a demonstrated need for new local and downstream interventions. Another plausible intervention strategy for injuries disrupting the integrity of the nerve, is the inclusion of nerve grafts. The gold standard, when available, is to source nerve segments from another region of the body. The use of nerves from the patient (autograft), is a great strategy as the cells, vasculature, and other components remain intact to support regeneration. When the gap defect is too larger (>5 cm), nerves can be taken from other sources (i.e., cadavers). The use of cellularized grafts from other than the patient includes the requirement for immunosuppressant drug usage from weeks up to two-years based on time for regeneration and function to be restored. Developing a strategy to eliminate the need for long-term drug use while allowing the use of cell-laden nerve segments from other donors can greatly improve outcomes and lower overall costs of transplants. The work to be discussed covers a variety of approaches for peripheral nerve injury. While focused on this one field of medicine, TNT has great potential beyond treating nerves. Additionally, when demonstrating efficacy in these various tissues, we can begin thinking of delivering other cargos and enhancing repair in other conditions. Specifically, the ability to locally deliver cargos to mitigate the immune response and limit host rejection has great implications for the transplant world. Enjoy!

Peripheral nerve injuries (PNI) result from traumatic injury, surgery or repetitive compression, and are reported in 3-5% of all trauma patients. The impact ranges from severe (major loss of sensory/motor function and/or intractable neuropathic pain) to mild (some sensory and/or motor deficits) and in both cases, is devastating for the patient. PNI affect ∼1M people in Europe and the US p.a. of whom 660,000 have surgery. PNI has high healthcare, unemployment, rehabilitation, societal costs and affects mostly young people. The current surgical practice for nerve gaps >3 cm is to bridge the site of injury with a graft taken from the patient. However, this involves additional time, cost and damage to a healthy nerve, limited supply, and unsatisfactory functional recovery (50% of the cases). For these reasons, research has focused on developing artificial nerve conduits to replace grafts, but to-date those available for clinical use do not match and/or exceed the functional performance of the autograft. This project develops a rational basis for promoting neurite growth through tissue-engineered conduits for peripheral nerve repair, by exploiting the response of cells to spatial variations in mechanical properties of conduits to inform their design. This is achieved through an interdisciplinary approach, that combines in vitro experimentation with mathematical modelling. First of all, the mechanical and structural properties of RAFTStabilised collagen gels (RsC) are explored, physiologically coherent RsC stiffness gradients are fabricated and characterised as well as the neuronal response to them. Finally, a predictive framework to inform the design of nerve conduits is parameterised and tested using experimental results and literature. The use of this multidisciplinary approach can help tissue engineers in the development of novel tissue repair solutions, as well as informing mathematical models of neurite behaviour which can contribute to the design process.

Peripheral nerve injuries affect a large proportion of the population and are often associated with prolonged disability. Current surgical interventions, including autografts and nerve guidance conduits (NGCs), are associated with a number of limitations. Decellularised nerve grafts offer considerable potential for peripheral nerve repair, by providing a non-immunogenic extracellular matrix (ECM) scaffold with biological, biochemical and biomechanical characteristics similar to native nerve. The aim of this study was to develop a decellularised peripheral nerve allograft with a native ECM histioarchitecture and biochemical composition, able to support viable cell populations. A previously established protocol for the decellularisation of porcine peripheral nerves, utilising a combination of hypotonic buffers, low concentration sodium dodecyl sulphate (SDS) and nuclease enzymes, was further developed to maintain the efficacy of decellularisation whilst minimising structural changes to the ECM. The improved protocol was shown to achieve sufficient cell and DNA removal, and decellularised porcine peripheral nerves were shown to be biocompatible and retain a native ECM histioarchitecture and composition. The protocol was then translated for the decellularisation of human femoral nerves. Following further development, the protocol was shown to achieve sufficient cell and DNA removal, and decellularised human femoral nerves were also found to be biocompatible and retain a native ECM histioarchitecture and composition. Human femoral nerves were then cultured in vitro with primary rat dorsal root ganglion (DRG) explants to assess long term biocompatibility and neurite outgrowth. The results presented suggest that decellularised human femoral nerve is a suitable substrate to support cellular infiltration and axon regeneration. Decellularised human femoral nerve was shown to support the long-term cell viability, migration, and neurite outgrowth of DRG explants, for up to 28 days in vitro. In this study, a novel decellularised human femoral nerve graft was developed with clinical potential for use in peripheral nerve repair. Although the results presented in this study are promising, further studies are required prior to clinical translation. A sterilisation process must be developed and evaluated for the decellularised nerve graft, and the biomechanical properties of decellularised and sterilised human femoral nerves must be investigated. The in vivo biocompatibility and efficacy of a decellularised and sterilised human femoral nerve graft for peripheral nerve repair must also be evaluated.

Peripheral nerve injuries represent one of the leading causes of disability globally; due to limitations of current therapies, there is a clear clinical need for a novel peripheral nerve graft . Decellularised porcine peripheral nerves may represent a suitable material; however, they would require terminal sterilisation prior to use. The main aim of this study was to identify a sterilisation method which minimally impacted upon decellularised nerve extracellular matrix (ECM) structure, biochemical composition, biomechanical properties and biocompatibility. Peracetic acid (PAA) solution 0.1 % (v/v) caused disruption to the structure of the endoneurium and reductions in the intensity of antibody labelling for basement membrane components, particularly collagen IV (with increased severity after a 12 month storage period). Supercritical carbon dioxide (SCCO2) treatment (with an additive solution containing 13.5 - 18.5 % [v/v] PAA and 4.5 - 6 % [v/v] H2O2) under standard conditions induced similar effects to the histioarchitecture and significant alterations to tensile mechanical properties; processing of the tissue whilst submerged in phosphate-buffered saline appeared to protect against such effects. Treatment with gamma radiation (25 - 28 kGy) and E Beam (33 - 37 kGy) mediated retention of the decellularised nerve ECM structure, and changes to the localisation and intensity of labelling for basement membrane components were minimal compared with those observed following treatment with PAA solution or SCCO2 under standard conditions. However, gamma radiation caused the stiffness of the nerves to increase. Contact culture experiments did not yield evidence of cytotoxicity following any of the sterilisation methods; furthermore, the sterilised nerves did not induce any significant changes in the secretion of key cytokines by a murine macrophage cell line. These data indicate that E Beam could be an optimal sterilisation method for use with the decellularised nerves, notwithstanding preliminary results indicating that SCCO2 processing under submerged conditions may enable superior basement membrane preservation.

Peripheral nerve injuries, caused mainly by traumas, affect over 200,000 people yearly in the US. There is a great need to find an alternative to autografts, the current clinical standard treatment, for the injuries, especially the ones resulting in long gaps (> 3 cm). In this research, we investigated on applying Magnesium (Mg) metal filaments as physical guidance to direct nerve regenerations in long-gap defects. Our overall hypothesis is that with the combination of porous conduits, Mg filaments, with additional factors like CNTF or Mg2+ are a promising method in treating long-gap peripheral nerve injuries. On the basis of our previous results, we first proposed that a novel polycaprolactone (PCL) mesh electrospun with Mg metal particles will be a promising candidate as a conduit material. Multiple mechanical and biological features of the mesh were characterized, as well as in vivo tissue response after subcutaneous implantation. The data revealed the presence of Mg metal was beneficial in immune-modulation and tissue repair. One mesh type, PM10, has the desirable properties and was then made into conduits. We hypothesized the conduits made from PM10 would support the repair and later tested it in the sciatic injured rats with stranded Mg Resoloy® wire inserted inside the conduits. The behaviors of the animals were monitored for 14 weeks before collecting the nerve tissues for histological assessments. Though histology shown that the regenerated tissue was healthy, no equivalent functional recovery has been observed compared to isografts. MicroCT scanning revealed that the conduits swelled and blocked the inner space for tissue growth. Besides altering to a more suitable conduit material (polysulfone), we proposed adding a growth factor, with or without the presence of Mg2+ salt solution, will improve the repair outcomes. The behavioral data indicated that empty polysulfone conduits with/without CNTF gave the best repairs. Only behavioral results were included due to the time limited and we expect the future histology will reveals more detail about the effects of Mg. In general, this study shed lights on nerve regenerating effects of Mg and tissue response to Mg when using it in peripheral nerve repair.

Peripheral nerve injuries affect 2.8% of all trauma patients which amounts to 8.5 million 'restricted activity days' every year. Unlike the central nervous system, nerves of the peripheral nervous system can regenerate after injury, at a rate of around 1 mm/day. Current treatments involve direct end to end suturing or using an autograft, which is the current 'gold standard' treatment to bridge large gaps (over 20mm). However, there is limited donor nerve available and limited nerve function is restored. Current nerve guide conduits (NGCs) are fabricated from both natural and synthetic materials. However, the efficiency of current nerve guide conduits is limited and conduits only treat defects up to 20mm. Research focuses primarily on improving hollow nerve guide conduits in several different ways. These include: the addition of intraluminal guidance scaffolds, coatings to modify surface chemistry or changing the conduit design by incorporating channels and pores. The aim of this work was to investigate different blends of synthetic polymers for their potential use as an intraluminal nerve guidance scaffold. It is hypothesised that the addition of electrospun fibres to hollow nerve guide conduits will increase nerve regeneration length by providing aligned guidance for Schwann cell attachment, migration and nerve regeneration. Blends of synthetic polymers were characterised to assess surface properties, mechanical properties, biocompatibility and cytotoxicity. Electrospinning was performed to determine if polymers, and their blends, could be fabricated into aligned fibres of varying diameters. Polymers which had a higher Young's modulus, Polyhydroxybutyrate-co-Valerate (PHBV), Polycaprolactone (PCL), Polyhydroxybutyrate (P(3HB) and Polylactic Acid (PLLA) could all be fabricated into aligned fibres of varying diameters. Polymers with more elastomeric properties Polyhydroxyoctanoate (P(3HO) and Polyhydroxyoctanoate-cohydroxydecanoate (P(3HO-co-3HD) could not be electrospun into aligned fibres. Overall, the most efficient blend ratio of the brittle polymers with the elastomeric polymers was 50:50. This ratio fabricated aligned fibres and maintained a lower Young's modulus (closer to that of native nerve). The effect of fibre diameter on neuronal and Schwann cell adherence, viability and differentiation was investigated by culturing cells on electrospun Polyhydroxybutyrate-coValerate (PHBV) fibres. In vitro analysis concluded that 5 and 8μm diameter fibres supported neuronal cell viability and differentiation most effectively. The effect of different material composition on neuronal and Schwann cell adherence, viability and differentiation was investigated by culturing cells onto 5 and 8 μm fibres of PHBV, PCL, P(3HB): P(3HO) (50:50) and P(3HO-co-3HD):PLLA (50:50). PCL was investigated as a control material. PHBV, P(3HB):P(3HO) (50:50) and P(3HO-co-3HD):PLLA (50:50) were investigated further due to success with regards to electrospinning, excellent cell viability and mechanical properties compared to other investigated blends of polymers. It was hypothesised that using polyhydroxyalkanoates, and novel blends of these polymers, would improve neuronal and Schwann cell attachment and support cell proliferation and differentiation more effectively than known FDA approved biopolymers, due to their chemical composition. The novelty in this work is the use of PHA blends and the ability to electrospin them. NG108-15 neuronal cell neurite outgrowth was significantly higher when cells were cultured on the 8μm PHBV fibres compared to the other materials and fibre diameters. Schwann cell viability was significantly decreased when cultured on the 5 and 8μm P(3HOco-3HD):PLLA (50:50) fibres, compared to the other polymer blends. Polymer fibres were further investigated using a novel 3D ex vivo fibre testing model. 5mm Polyethylene glycol hollow tubes were manufactured using microstereolithography. Polymer fibres, of 5 and 8μm diameters, were threaded into tubes, and an explanted DRG body placed on the proximal end. The use of DRGs in neuronal cell culture mimics a nerve injury, as neurite extensions grow from the DRG body after removing the nerve roots. It was hypothesised that the addition of electrospun fibres to hollow nerve guide conduits would increase nerve regeneration length. Both Schwann cell migration lengths and neurite outgrowth lengths were greater on the 5μm fibres, compared to their 8μm counterparts. Schwann cell migration length was statistically greater on the 5μm PHBV fibres and P(3HO):P(3HB) (50:50) fibres. PHBV 5μm fibres supported greater neurite lengths, compared to the 8μm PCL and 8μm PHBV fibres. This study highlights the difference between using a neuronal cell line, compared to using a primary neuron (DRG) model, and using a 3D in vitro culture model compared to 2D in vitro culture. This work also reveals the potential of 5μm P(3HO):P(3HB) (50:50) and PHBV fibres, as intraluminal fibrous scaffolds to improve nerve repair and would support the use of this blend for in vivo investigations. Additionally, surface modification is used to improve current nerve guide conduits, by improving biocompatibility of the surfaces of bulk biodegradable polymers with the addition of reactive chemical groups. Compared to plasma polymerisation, aminosilanisation is an alternative method to modify surfaces with amine groups as it is cost effective, scalable and cheaper. Two different chain lengths, long and short, of aminosilanes were investigated, to determine their potential use in peripheral nerve repair. It was hypothesised that the addition of aminosilanes plain glass would improve neuronal and Schwann cell adherence and neuronal cell differentiation. The novelty of this work is that aminosilanes have not been investigated for use in peripheral nerve repair and that they could be a useful addition to biopolymer NGCs. Long chain aminosilanes supported significantly longer neurite lengths from NG108-15 neuronal cells, compared to the short chain and plain glass control. Both chain lengths supported NG108-15 neuronal and Schwann cell viability. A co-culture of primary neurons and Schwann cells was established by dissociating rat DRGs. Long chain aminosilanes supported significantly longer neurite lengths from primary neurons, compared to the short chain aminosilane, plain glass and tissue culture plastic controls. This work highlights the potential use of long chain aminosilanes for use in peripheral nerve repair, to coat conduits or intraluminal guidance scaffolds. Besides electrospinning, pressurized gyration has recently been reported to manufacture fibrous scaffolds for tissue engineering applications. The set up established at The Department of Mechanical Engineering, University College London, was used to manufacture P(3HB) and P(3HB):P(3HO-co-3HD) (80:20) fibres. Gyrated fibres were investigated with electrospun P(3HB) fibres, to determine which manufacturing method was the most efficient in manufacturing aligned fibres, and in vitro and ex vivo analysis was performed. SEM analysis confirmed that the P(3HB) electrospun fibres were more aligned compared to both gyrated fibre types. Both gyrated and electrospun fibres supported NG108-15 neuronal cell and Primary Schwann cell proliferation and viability. However, greater neurite lengths were observed on the electrospun P(3HB) fibres compared to the gyrated PHA fibres, when culturing DRGs. Schwann cell migration length was also significantly larger on the electrospun fibres compared to the gyrated fibres. This work highlights the potential of gyration for fabricating fibrous scaffolds. However, future work will need to be performed to fabricate aligned fibre scaffolds which compare with electrospun fibres. Overall, this thesis demonstrates that Polyhydroxyalkanoates, and blends of PHAs, can be fabricated into aligned fibre scaffolds by electrospinning. It also demonstrates that the addition of PHAs to polymer blends supports neuronal and Schwann cell attachment, proliferation and differentiation more effectively than the biopolymer alone. Although pressurized gyration is a new technique to fabricate fibre scaffolds, this thesis has shown that it does not fabricate fibres with the required alignment for nerve repair and therefore, more optimisation of the technique is required. Surface modification, using silanisation, has also been shown to be an effective technique to modify a glass surface with amine reactive groups, and therefore, can be applied to polymer surface modification.

Abstract: Peripheral nerve injuries (PNI) and peripheral nerve diseases/neuropathy (PND) are common clinical conditions characterized by varying degrees of sensory, autonomic and motor dysfunctions. Despite significant advancement in microsurgical techniques to repair the injured nerves, functional recovery from PNI and PND is often suboptimal. Disabilities ranging from muscle weakness, skin numbness to chronic neuropathic pain often accompany PNI and PND patients, severely affecting their quality of life. A series of molecules and pathways are recognized to participate in axon regeneration, but one unexplored area is how those newly regrowing sensory axons reconnect and reinnervate one of their chief targets: the skin. Building on preliminary work from my former graduate student colleague, Dr. Rebecca Long, my project focuses on protocadherin-gamma (Pcdh-γ), a molecule that has been proposed by researchers to be a strong candidate to serve as cell identity marker in the mammalian nervous system. Pcdh-γ has been shown to play a role in regulating interneuron survival and synaptogenesis during developmental stages in the central nervous system (CNS). In adult mice, conditional knockout of Pcdh-γ in retinal starburst amacrine cells (SACs) and Purkinje neurons is associated with neurite self-avoidance defects and fasciculation of dendrites. In pyramidal neurons, however, conditional knockout of Pcdh-γ led to dendritic simplification and thinning of cortical layers. Current literature has yet to study Pcdh-γ in the peripheral nervous system (PNS), especially in the context of axon regeneration, given its role of directing dendrite morphology in the CNS. In this project, by using both in vitro and in vivo experiments, we explored the roles played by Pcdh-γ in sensory axons. Our in vitro experiment showed that knockdown of Pcdh-γ in uninjured DRG neurons is associated with greater neurite extension and more branchpoints per neuron. Our in vivo experiment revealed to us that footpads of mice that received Pcdh-γ II knockdown through siRNA administration displayed greater epidermal reinnervation: higher intraepidermal nerve fiber density (IENFD). These exciting findings shed light on the potential roles of Pcdh-γ in axon regeneration/growth and skin epidermal reinnervation. In the final experiment, we studied Rac1 as a potential downstream signaling molecule of Pcdh-γ knockdown. We proposed that Rac1 inhibition might attenuate those axon morphological changes we observed from the knockdown of Pcdh-γ. The greater total neurite extension and branchpoints per neuron that resulted from Pcdh-γ knockdown were indeed nullified with Rac1 inhibition. Ultimately, by understanding the molecular mechanisms of Pcdh-γ in skin epidermal reinnervation, a new molecular approach to treat PNI and PND could be discovered.

Peripheral nerve injuries (PNIs) are prevalent in healthcare worldwide and are commonly caused by traumatic accidents. Traumatic PNIs lead to loss of function that is often never fully regained. While treatments exist to alleviate this issue, such as neuropathy or end-to-end nerve suturing and nerve grafting, drawbacks arise in the size of the injury gap, limited functional recovery, low success rates, damage to nerve graft sites, and the availability of donor locations. Thus, these drawbacks have led to the development of alternative materials and treatments, such as nerve guidance conduits. This work addresses this need by using a piezoelectric polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE) polymer that produces an electric charge when mechanically deformed without the need for an external source. The PVDF-TrFE polymer was electrospun to produce a nanofiber scaffold with aligned fibers designed to direct cell migration. The scaffolds were rolled to produce nerve guidance conduits to aid in peripheral nerve repair by acting as a bridge between the proximal and distal ends of the damaged nerve, to align and guide cell migration to the nerve ends and provide electrical stimulation to aid in nerve regeneration, allowing nutrient and waste to diffuse through the conduit wall. The PVDF-TrFE conduits were further characterized for their biocompatibility, material, mechanical, electrical, and physical properties, establishing the efficacy of the constructs. Additional in vitro and in vivo work will provide proof-of-concept and necessary results for conduit viability to repair transected PNIs. This work discusses an alternative treatment for PNIs, and future work will improve on these material properties to develop a piezoelectric nanofiber nerve guidance conduit for peripheral nerve repair.

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.

Poor functional outcomes are frequent after peripheral nerve injuries despite the regenerative support of Schwann cells. Whilst motoneurons and to a lesser extent, sensory neurons survive the injuries, outgrowth of axons across the injury site is slow and the neuronal regenerative capacity is progressively reduced when neurons remain without targets and chronically denervated Schwann cells fail to support axon growth. Strategies including brief low frequency electrical stimulation that accelerates axon outgrowth and, in turn, target reinnervation and functional recovery, have excellent potential for translation to human patients. Other strategies including the insertion of cross-bridges between a donor nerve and a recipient denervated nerve stump, are effective in promoting functional outcomes after complete injuries. During muscle reinnervation the properties of the motoneurons and muscle fibers that they supply are rematched that provide some control of muscle force even when regenerating axons are misdirected to foreign targets. Axon sprouting from intact nerves is effective, although limited, in reinnervating denervated muscle fibers after incomplete injuries and in poliomyelitis. Studies in mouse models of amyotrophic lateral sclerosis however, indicate that sprouting is very limited with rapid and preferential loss of the largest and fastest contracting motor units during the asymptomatic phase of the disease.

Autologous nerve grafts are the current gold standard for the repair of peripheral nerve injuries. However, there is a need to develop an alternative to this technique, as donor-site morbidities such as neuroma formation and permanent loss of function are a few of the limitations concerned with this technique. Artificial nerve conduits have therefore emerged as an alternative for the repair of short peripheral nerve defects of less than 30 mm, however they do not surpass autologous nerve grafts clinically. To develop a nerve conduit that supports regeneration over long nerve gaps and in large diameter nerves, researchers have focused on functionalizing of the conduits by studying the components that enhance nerve regeneration such as micro/nano-topography, growth factor delivery systems, supportive cells and extracellular matrix (ECM) proteins as well as understanding the complex biological reactions that take place during peripheral nerve regeneration. This thesis presents strategies to improve peripheral nerve interfaces to better the regenerative potential by using dorsal root ganglions (DRGs) isolated from neonatal rats as an in vitro model of nerve regeneration. The work started off by investigating the usefulness of a frog foam protein Ranaspumin-2 (Rsn2) to coat biomaterials for compatibility, this lead to the discovery of temporary cell adhesion on polydimethylsiloxane (PDMS), which was investigated as a suitable tool to derive cell-sheets for nerve repair. The influence of Rsn2 anchored to specific adhesion peptide sequences, such as isoleucine-lysine-valine-alanine-valine (IKVAV), a sequence derived from laminin proven to promote cell adhesion and neurite outgrowth, was tested as a useful means to influence nerve regeneration. This approach improves the axonal outgrowth and maintains outgrowth long term. Based on the hypothesis that combinational modulation of substrate topography, stiffness and neurotrophic support, affects axonal outgrowth in whole DRGs, dissociated DRGs were used to assess if these factors similarly act at the single cell level. Rho associated protein kinase (ROCK) and myosin II inhibitors, which affect cytoskeletal contractility, were used to influence growth cone traction forces and have shown that these factors work in combination by interfering with growth cone dynamic creating a different response in axonal outgrowth at the single cell level.

Peripheral nerve injuries can lead to a major loss of function and affect quality of life. Current autologous treatments and commercially available products such as nerve guide conduits all have limitations. The aim of this study was to develop biocompatible, non-immunogenic nerve grafts using low concentration SDS to decellularise porcine peripheral nerves. Initially the porcine nerve anatomy was defined. Acellular nerves were then used as the basis for an in vitro study of perfused flow within the tissue for the introduction of Schwann cells - as the delivery of these cells is reported to stimulate axon regeneration. Furthermore, an in vivo study using a rat sciatic nerve injury model was conducted to evaluate the regenerative capacity of acellular porcine grafts. Histology confirmed an absence of cells and retention of the native nerve histioarchitecture. DNA levels were reduced by >95 % throughout the decellularised tissue. Immunohistochemistry showed retention of important extracellular matrix proteins such as collagen, laminin and fibronectin. In vitro biocompatibility studies indicated the acellular nerves were not cytotoxic to human dermal fibroblasts and primary rat Schwann cells. Uniaxial tensile testing showed a significant increase in ultimate tensile strength (UTS) and strain between native and acellular nerve tissues. In addition, mechanical testing of the nerves revealed porcine peroneal nerves to have a higher UTS value in comparison to the porcine tibial nerves. Analysis of the nerves using transmission electron microscopy concluded that the mechanical properties of nerves could not be determined exclusively by their ultrastructure or collagen fibril diameter. Porcine acellular nerve grafts were used as an in vitro and in vivo model for the introduction of primary rat Schwann cells. The in vitro nerve model confirmed that reseeded Schwann cells under perfusion maintained their phenotype and had a lower rate of cell death when compared to static conditions. A rat sciatic nerve in vivo gap injury model demonstrated that porcine grafts were able to promote axonal regeneration; however, they were not as effective as the autologous graft. In summary, acellular peripheral nerve tissue was found to have excellent potential for development of a tissue engineered graft to aid peripheral nerve regeneration.

Peripheral nerve injuries (PNI) affect 20 million people, with over 200,00 nerve repairs performed annually in the United States alone. Nerve conduits (NCs) are a commonly employed treatment for injured nerve segments under 3 cm, but unsatisfactory outcomes have been reported for larger gaps. This is in part due to the lack of bioactive agents such as stem cells or growth factors incorporated into FDA-approved NCs. While they have been experimentally shown to promote peripheral nerve regeneration (PNR), their inherent variability, harvesting complexity, and sometimes inadvertently high biological activity pose great regulatory hurdles. Therefore, plant extracts have become an attractive alternative area of bioactive agents proven to improve PNR while overcoming these issues, one of which is curcumin. However, studies largely use unoptimized curcumin which presents an issue as it is hydrophobic and degrades quickly in aqueous solutions. In this study, a nano-formulation comprised of tannic acid and polyvinylpyrrolidone (TA-PVP) was developed to encapsulate curcumin and optimize its delivery to cells. Results show that the developed Cur-TA-PVP nanoparticles (NPs) had an average size of 220 nm, a surface charge of -35mV, and a polydispersity index of 0.3. Further, curcumin was shown to be localized within the core of the NP. This significantly increased the colloidal stability, decreased the degradation rate, and promoted the slow release of curcumin in aqueous solutions. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the Cur-TA-PVP NPs was also shown to be superior as compared to free curcumin. Finally, fluorescence microscopy revealed a significantly higher concentration of curcumin in SH-SY5Y, S16 rat Schwann cells, and J774 murine macrophages when delivered via NPs. These results collectively provide evidence for the improved physicochemical properties of the nano-curcumin, which led to improved delivery to cells. Therefore, this formulation has the necessary attributes for incorporation into NCs to promote functional recovery following PNI.

Peripheral nerve injuries in adults can trigger neuropathic pain which coincides with alterations in dorsal horn neuronal activity and glial cells residing in the dorsal horn, and infiltrating T-lymphocytes. These cells synthesise and release pro-inflammatory mediators that can directly and indirectly sensitize dorsal horn neurones and contribute to neuropathic pain. Neuropathic pain is rare in infants and studies presented in this thesis show that a spared nerve injury (SNI) at postnatal day (P) 10 does not result in pain-like behaviour but triggers an anti-inflammatory immune response in the dorsal horn, not observed in nerve injured adults. However, if infant SNI treated animals are intrathecally administered with the pro-inflammatory mediator tumor necrosis factor (TNF) or lipopolysaccharide-activated microglia they develop pain-like behaviour while blockade of anti-inflammatory activity after infant SNI 'unmasks' neuropathic pain-like behaviour. Thus, nerve injury induced pain-like hypersensitivity in infants is actively suppressed by dominant anti-inflammatory neuro-immune activity. The anti-inflammatory response can also be evoked by direct C-fibre nerve stimulation in the infant, but not in adult rodents. However, mechanical hypersensitivity does eventually develop following early life nerve injury in the rat at adolescence (Vega-Avelaira et al., 2012). Longitudinal studies presented in this thesis indicate that hypersensitivity emerges in response to not only mechanical stimulation but also following innocuous and noxious cold stimulation of the hind paw, and contralateral weight bearing. The emergence of behavioural hypersensitivity at adolescence coincides with an increase in spontaneous and evoked- activity of wide dynamic dorsal horn neurons, that is absent in sham controls. In addition, the immune response in the dorsal horn switches from an anti-inflammatory response to pro-inflammatory, characterised by an increase in the expression of TNF and Brain-derived neurotrophic factor. This explains why neuropathic pain is rare in infants, but complex regional pain syndromes can emerge, for no observable reason at adolescence.

The peripheral nervous system has a limited ability to repair after injuries (1 mm/day). Current therapies for peripheral nerve injuries include suturing, autografting and nerve guide conduits (NGC). NGC's can be implanted into defects up to 20 mm in length. Possible improvements to the existing designs include material alterations as well as various coatings, fillers and cellular components. Plasma polymerisation is a chemical deposition technique, which can coat any surface with nano to micro-metre layers of desired chemistry. The aim of the thesis was to investigate the effect of the plasma polymers acrylic acid (AAc) and maleic anhydride on neuronal and Schwann cell metabolic rate and morphology in vitro. Evaluation of AAc and maleic anhydride surfaces was assessed using contact angle, X-ray Photoelectric Spectroscopy (XPS) and Time of Flight Secondary Mass Spectrometry (ToF-SIMS). NG108-15 neuronal cell staining protocols were optimised, then out of three maleic anhydride duty cycles (MA1, 2 and 3), MA1 supported neuronal cells with longer neurites and a higher metabolic rate. Glass and laminin/poly-L-lysine were used throughout as basal and positive controls respectively. Investigations then compared AAc with MA1 coatings under serum and serum free conditions. Whilst there were similar results under serum conditions, NG108-15 neuronal cells on MA1 under serum free conditions had significantly longer neurites compared to glass, also longer than laminin and acrylic acid. Primary rat Schwann cells were then studied, due to their integral role in peripheral nerve regeneration, on AAc and MA1. It was observed that Schwann cells had a longer, thinner phenotype and a higher total cell count on poly-L-lysine and glass compared to the plasma polymers. Primary rat dorsal root ganglion (DRG) culture was then optimised to look into the effect of both neuronal and glial types, initially using explants. DRG on AAc showed similar neurite outgrowth characteristics to those on glass, whilst DRG on MA 1 had comparatively fewer and shorter neurites. Dissociated DRG culture on MA1 and AAc surfaces were then observed to have longer neurite lengths and a higher percentage of neurons with neurites compared to glass. In conclusion, NG108-15 neuronal cells had longer and more numerous neurites on the plasma polymers compared to glass, which was corroborated using results from dissociated DRG. As the length and number of neurite outgrowths per neuron has been shown to be indicative of neuronal differentiation[100], plasma polymer coatings may support neuronal differentiation. Conversely, primary rat Schwann cells showed a longer and thinner phenotype on glass compared to the plasma polymers. DRG explants that contain both neuronal and glial cell types extended further on glass compared to the plasma polymers. Using a number of cell types was therefore a very relevant method of evaluating AAc and MA1 for neuronal applications. This work demonstrates the suitability of MA1 and AAc as coatings for nerve guide conduits, and would support further investigations into the effect of plasma polymers on neuronal/glial progenitor cells regarding differentiation and in vivo work.

Peripheral nerve injuries occur with high incidence and often result in profound and permanent impact on the life of patients and on healthcare expenditure. Schwann cells (SC) play a promoting role in peripheral nerve regeneration providing physical and neurotrophic support that aids axon re-growth. However, these beneficial properties are not exploitable in nerve tissue engineering due to the difficulties in SC harvesting and expansion in culture. Adult stem cells derived from bone marrow (BM-MSC) and from adipose tissue (ASC) can be differentiated in SC-like cells and be used as SC substitutes in bioengineered nerve conduits for the improvement of peripheral nerve regeneration. Pharmacological intervention approaches for the treatment of nerve injury are still not clinically available. Nevertheless, γ-Aminobutyric acid (GABA) receptors have been recently suggested as a putative target for such purpose. GABA is the main inhibitory neurotransmitter of the adult brain and interacts with two different receptor types. However, both GABA-A and GABA-B receptor types are functionally expressed also in SC, where they are involved in the regulation of SC physiology and in the development of the peripheral nervous system (PNS).The aim of this thesis was to characterise the GABAergic system of BM-MSC and ASC differentiated into a SC-like phenotype and to evaluate changes in the expression levels following differentiation. Moreover, the effect of specific GABA receptor ligands on cell proliferation and neurotrophic potential of differentiated stem cells were assessed. Using reverse transcriptase polymerase chain reaction, western blot analysis and immunohistochemistry we demonstrated that adult stem cells express several subunits of both GABA-A and GABA-B receptor systems such as GABA-B1a, GABA-B1b and GABA-B2, as well as GABA-A α2 and GABA-A β3. Expression levels and cellular localisation were comparable with adult and neonatal SC cultures used as positive controls, and protein expression levels for some of the subunits changed following glial differentiation. Interestingly, stimulation of GABA receptors with specific agonists influenced stem cell proliferation in two opposite ways. Baclofen, a GABA-B receptor agonist decreased proliferation of SC and differentiated ASC (dASC), but not of SC-like BM-MSC (dBM-MSC). By contrast, muscimol, a GABA-A receptor agonist, increased proliferation in SC and in both dASC and dBM-MSC. This suggests that GABAergic signalling could be a potential player in the mechanisms regulating stem cell differentiation and proliferation as reported in SC. Finally, baclofen treatments on SC and dASC modulated the expression levels and the release of the neurotrophins BDNF and NGF, which are key actors in the processes involved with peripheral nerve regeneration. Although further studies will be needed to clarify the role of GABA receptors in the PNS, the presence of functional GABA receptors on SC-like adult stem cells could represent an exploitable pharmacological target to modulate stem cell physiology and improve their neurotrophic potential for peripheral nerve regeneration.

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.

Pain is a complex experience involving both sensory and emotional aspects. Neuropathic pain syndromes, which remain a major area of unmet clinical need, are often associated with behavioural co-morbidities such as anxiety and depression. Recent studies have demonstrated changes in a number of affect-related outcome measures in rodent models of neuropathic pain. The expansion of such outcome measures offers one possible route for the improvement of studies aimed at creating novel therapies for neuropathic pain. Bias reduction through randomisation requires mixed housing of animals with peripheral nerve injuries with non-injured conspecifics. Such mixed housing was shown to alter behaviour in the open field. Therefore, in the following studies treatments were allocated to cage groups as a whole. Anxiety-like behaviour was demonstrated in L5 spinal nerve transected (L5 SNT) rats in a novel dark/light preference test and the utility of this test in detecting efficacious analgesics was investigated. The possible presence of neuropathy-induced depression-like behaviour following L5 SNT was then investigated using the forced swim test. However, animals showed no increase in depression-like behaviour, as measured by time spent immobile in the forced swim test, at either two or three weeks post-neuropathy. Assessment of burrowing behaviour, a novel behavioural outcome measure thought to reflect over all well being in rodents, was then carried out in rats following L5 SNT and partial sciatic nerve ligation. A reduction in burrowing behaviour was demonstrated in both models of neuropathic pain compared to naive animals. The use of novel outcome measures, such as those detailed above, both as outcome measures in drug development and in studies into pathophysiology will improve the quality of studies aimed at creating novel therapies for neuropathic pain.

Volumetric muscle loss (VML) is the traumatic or surgical loss of skeletal muscle comprising 20-30% or more of the muscle volume. By definition, VML exceeds the muscle’s capacity for self-repair and results in persistent functional deficits. Significantly, no treatment options exist that can fully restore native structure and function. To address the limitations of current treatments, our laboratory has developed tissue-engineered skeletal muscle units (SMUs) as a novel treatment for VML repair. SMUs have shown promising regenerative potential in a rat VML model; however, limitations of rodent models necessitated transitioning our technology to a large animal (sheep) model. Despite substantial heterogeneity of muscle progenitor cell populations obtained from craniofacial, trunk, and limb muscle, engineered skeletal muscle tissues are almost exclusively fabricated from cells derived from hindlimb muscle, making the effects of cell source on engineered muscle tissue unknown. Thus, we conducted a comparison of the development (myogenesis), structure (histology), and function (biomechanics) of SMUs fabricated from muscle cells isolated from both craniofacial and hindlimb muscle sources. Specifically, we showed that the semimembranosus muscle was best suited for the fabrication of sheep-derived SMUs. We also sought to develop a method to scale our SMUs to clinically relevant sizes. We developed a modular fabrication method that combines multiple smaller SMUs into a larger implantable graft. Consequently, we successfully fabricated of one of the largest engineered skeletal muscle tissues to date while avoiding the formation of a necrotic core. To treat peripheral nerve injuries that accompany VML, we also developed engineered neural conduits (ENCs) to bridge gaps between native nerve and the injury site. We used scaled-up SMUs and ENCs to treat a 30% VML in the ovine peroneus tertius muscle. After a 3-month recovery, we performed in situ biomechanical testing and histological analyses on explanted muscles. Results showed that SMU-treated groups restored muscle mass and force production to a level that was statistically indistinguishable from the uninjured contralateral muscle. Lastly, we evaluated the efficacy of SMUs in repairing craniofacial VML. Despite reported differences in the regenerative capacity of craniofacial muscle compared to limb muscle, prior to the work described herein, there were no models of craniofacial VML in either large or small animal models. Thus, we introduced the first model of craniofacial VML and evaluated the ability of SMUs to treat a 30% VML in the zygomaticus major muscle. Despite using the same injury and repair model in both implantation studies, results showed differences in pathophysiology between craniofacial and hindlimb VML. The fibrotic response was greater in the facial muscle model, and there was tissue tethering and intramuscular fat deposition that was not observed in the hindlimb study. The craniofacial model was also confounded by concomitant denervation and ischemia injuries which were too severe for our SMUs to repair. Overall, this work significantly contributed to the field of skeletal muscle tissue engineering by evaluating the effects of muscle source on the structure and function of SMUs, creating a modular fabrication method for tissue scale-up, and introducing a new large animal model and a craniofacial model of VML. The success of this technology demonstrates its potential for treating clinical VML in the future.

Neurological disorders are the primary cause of disability and the second leading cause of deaths worldwide, with an ever-increasing burden as populations grow and age. The peripheral nervous system (PNS) possesses great regenerative capability, but even so, no effective therapy exists for severe peripheral nerve injuries (PNIs) and underlying mechanisms are incompletely understood. Conversely, treatment for central nervous system (CNS) disease is crippled by little innate regenerative ability of the brain and inaccessibility of patient neural tissue. Direct reprogramming or transdifferentiation of adult somatic cells into neuronal fate (“induced neurons”) has great potential for overcoming these barriers, but still suffers from low and variable efficiencies of conversion. My thesis harnessed principles of cell engineering, neuroengineering, and regenerative medicine to address these issues. In the PNS, we demonstrated a novel proof-of-concept that engineered synthetic neuromuscular tissue (SyNMT) can be used to guide development of and screening for stem cell therapies to treat denervation injuries. We compared neural crest stem cells (NCSCs) to bone marrow-derived mesenchymal stem cells (MSCs). Three-dimensional (3D) multicellular spheroids of NCSCs enhanced in vitro regenerative functions and dramatically increased in vivo longevity, with significant functional improvement in rat trials that recapitulated SyNMT findings of NCSC but not MSC improvements in neuromuscular junction innervation. In the CNS, we demonstrated that 3D spheroids promoted the direct reprogramming of human fibroblasts into neurons, increasing conversion efficiency by over 67 times. Moreover, reprogramming displayed distinct spatial patterns dependent on adhesive polarity that could be rescued by dual BMP & TGF-β pathway inhibition. Overall, this work demonstrated the significant impact of engineering biophysical cues to improve regeneration and survival of neuromuscular cell therapies as well as to improve neural reprogramming, enhancing translational potential for biomedical applications.

Peripheral nerve injuries are damaging nerve conditions that can result in pain and motor and sensory deficits. A number of biological processes, including structural, transcriptional, translational and growth-related pathways within nerve cells are affected by injury. In this study, we examine a peripheral nerve injury to compare protein expression level changes among control uninjured nerves and two different injury repair strategies at an early regenerative time point; such changes may be useful in predicting the eventual success of a particular repair strategy. UPLC-ESI mass spectrometry and MaxQuant LFQ and PEAKS v8.5 bioinformatic algorithms were performed on samples from the proximal stump of peripheral nerves injured and acutely repaired by autograft or a new nerve lengthening strategy, as well as uninjured contralateral control nerves. The proteins overexpressed in the lengthened sample by two-fold or greater were analyzed in DAVID, and the output data was manually clustered into 15 pathways relevant to nerve injury and regenerative processes. We present evidence of upregulated structural, immune response, bioenergetic, and protein synthesis pathways in the lengthened condition to support the stretch-activated growth hypothesis for nerves. We also highlight candidate proteins for future diagnostic or manipulation studies to characterize the nerve regeneration environment. These findings are important in advancing our understanding of the peripheral nerve regeneration process post-injury.

The repair of peripheral nerve injuries is a major reconstructive problem, particularly when direct suturing of a transected nerve is not feasible option. This arises because mechanisms of nerve repair are not fully understood. The purpose of the first group of experiments was to compare methods of nerve repair, including use of novel, biodegradable glass tubes. The possible use of these tubes as a means of confining humoral or cellular substances at the site of repair was also assessed. In a further study, experiments were designed to establish first, the optimal timing of nerve repair (immediate or delayed); second, whether there was a difference in the level of recovery between neonates and adults after nerve repair; third, to assess the freeze-thawed muscle graft (FTMG) method as a surgical technique and to determine whether the FTMG is at least as good as a conventional nerve graft. This investigation was carried out in an animal model of obstetrical brachial plexus palsy (OBPP). Assessment of experimental outcome in both studies was by measurement of the structural and cytochemical changes which occurred in the target muscle after peripheral nerve injury and repair. Morphometric, histochemical and immunocytochemical measurements showed alterations in muscle fibre size and architecture, as well as connective tissue content and the proportions and the distributions of the different fibre types. These changes indicated that after repair with controlled-release glass tubes there was reinnervation of the target muscle, although the results were superior after repair by FTMG. As to whether the potential of nerve to regenerate after repair decreases with age, or whether immediate or delayed repair is best in the treatment of OBPP, the experiments have contributed to solving but have not resolved the dilemma associated with these issues.

Chimera: (Biology) An organism consisting of at least two genetically different kinds of tissue as a result of mutation, grafting, etc. Collins English Dictionary This thesis has set out to investigate the regenerative potential of peripheral nerve allografting in a rodent model. In particular, the strategy of reconstructing a nerve defect using a novel chimeric graft of mixed antigenicity (Sandwich graft) was explored as an alternative to autografting or immunosuppression. Peripheral nerve injury produces significant individual and community morbidity. Despite technical and surgical advances, even the results from urgent repair of single modality nerves are sub-optimal. This situation is compounded when nerve structure is lost and a bridging conduit is required to reconstruct the defect. While many organic and inorganic materials have been investigated, autografting remains the mainstay of current clinical practice. However, suitable autografts are in limited supply and associated with donor site morbidity. Like autografts, peripheral nerve allografts (PNAG) possess all the features required of an ideal nerve conduit, especially Schwann cells. Studies with acellular conduits have shown the addition of Schwann cells is advantageous to regeneration. However, immunological rejection of antigenic Schwann cells within nerve allografts renders the graft acellular, inhibiting regeneration. Immunosuppression preserves allogenic Schwann cells and results in regeneration equivalent to autografts. But immunosuppressants are associated with serious side effects such that their use in peripheral nerve injuries is deemed unethical. Temporary immunosuppression has been proposed as a solution to this problem until the non-antigenic host axon regeneration is established. However this has produced conflicting results although an axonopathic process following drug withdrawal is acknowledged. The aims of this project were firstly to assess the regenerative potential of non- immunosuppressed PNAGs. The hypothesis being that rejection would produce acellular grafts as donor Schwann cells were destroyed. Host Schwann cells are known to repopulate acellular conduits from adjacent nerve ends. By using a chimeric Sandwich graft, the second aim of the project was to assess whether augmenting host Schwann cell graft repopulation could improve axonal regeneration and reduce immunosuppressant requirements. A clinically reproducible chimeric construct of a nerve allograft with an intervening segment of autologous nerve (a Sandwich graft) was used to bridge a standard traumatic nerve gap within the experimental groups. Comparisons were made between simple and sandwich grafts composed of autologous, isogeneic and allogenic nerves split into immunosuppressed and non-immunosuppressed limbs, incompatible rat strains were employed using Cyclosporin (CyA) as the sole immunosuppressant. Qualitative and quantitative analyses of immunohistochemical and conventional histological specimens, along with measurement of Gastrocnemius muscle mass, were used to assess the outcome of nerve regeneration. Analyses were undertaken within the early and late regeneration periods of 21 days and 32 weeks respectively. Immunohistochemistry and conventional histological techniques were employed, along with measurements of muscle mass recovery to indicate target organ reinnervation. Cyclosporin (CyA) was the sole immunosuppressants used. Non-immunosuppressed PNAGs were shown to support less early axonal regeneration than their immunosuppressed counterparts and non-allogenic controls. This was associated with a reduction in Schwann cells and increased inflammation indicating rejection. However, the pattern of regeneration followed that of the other groups and improved with time. Sandwich allografting was associated with improved axonal regeneration, increased Schwann cells and less inflammation suggesting reduced antigenicity of the sandwich construct. The increased neurorrhaphies compared to simple grafts did not appear deleterious. Long-term myelinated nerve counts and target organ reinnervation produced similar results from all experimental groups regardless of immunosuppression. This work validates the use of a chimeric sandwich graft within this model. The addition of a depot of host Schwann cells within an allograft without immunosuppression produces similar regeneration to non-allogenic grafts and reduces the early delay period associated with rejection. Further analysis of the formulations of CyA used indicated that although oily and parenteral CyA did adequately immunosuppress the allografts, the results from parenteral CyA were marginally better. Also, CyA use did contribute to model morbidity and mortality within these experiments.

Peripheral nerve injuries commonly result in a surgically irreducible gap and represent a serious problem in surgery. Clinically, autologous nerve grafts are the most effective means of promoting axonal regeneration across the space, but normally produce donor site morbidity. Other variations of experimental models have been used with the aim of directing rapid nerve regeneration, including entubation, artery and vein grafts and Millipore. However, each of these have significant limitations which affect complete regrowth. Fibronectin (Fn), a large extracellular matrix cell adhesion glycoprotein has been made into three-dimensional mats with a predominant fibre direction and shown to successfully enhance peripheral nerve regeneration in a rat model. The aim of this study was to identify the main features of Fn-implants which are important for rapid nerve repair. This involved testing implanted versions of Fn-mat from rat and monkey models, assessment of cell-matrix interaction in vitro and modification of Fn-materials by chemical and growth factor addition. The results from this study show that new materials can be made from fibronectin which have a potential use in repair of long peripheral nerve lesions. These fibronectin-based materials may be stabilised with micromolar concentrations of copper and zinc which also support strong growth of Schwann cells within these materials in culture. Fibronectin mats may also be soaked with neurotrophins to enhance nerve cell survival and enhance nerve regeneration. The speed of Schwann cell migration and alignment (upto 50 μm away from the original fibre) was increased. Migration speed was further enhanced when the fibres were treated with micromolar concentrations of copper or made, with a substantial content of fibrinogen (optimum 50:50). Taken together, all these results suggest a first design for an ideal conduit material for peripheral nerve repair. This would be (a) dimensions (fibrous), (b) fibre orientation (c) 50:50 fibronectin: fibrinogen, (d) treated with micromolar concentrations of copper, (e) seeded with Schwann cells in culture, and (f) soaked with a 'cocktail' of neurotrophic growth factors including nerve growth factor and neurotrophin-3.

Scope and Method of Study: Neuropathic pain is a common complication of peripheral nerve injuries. In pain studies, it has been revealed that glutamate neurotransmission has a critical role in the noxious stimulus sensation. The glutamate cycle have several well established markers: vesicular glutamate transporters 1 & 2 and glutaminase. However, systemic studies for vesicular glutamate transporters 1 & 2 and glutaminase changes after peripheral nerve injuries have not been done. Thus, in this thesis project we studied the changes in vesicular glutamate transporters 1 & 2 and glutaminase systemically through immunohistochemistry at six different time points that represent the acute (day 1 & 2), subacute (day 7 & 14), and chronic (day 28 & 70) phases in peripheral nerve regeneration. Our peripheral nerve injury models, nerve crush and nerve transection, imitate two types of peripheral nerve injury in human, axonotmesis and neurotemsis, respectively.Findings and Conclusions: The study of subacute phase of peripheral nerve regeneration in sciatic nerve crush model showed an elevation of dorsal root ganglia neurons VGLUT 2 immunoreactivity only at day 7 post injury. On the other hand, the study of VGLUT 1 & 2 and glutaminase dorsal root ganglia neurons immunoreactivity in the acute, subacute, and chronic phase of regeneration showed a pattern of elevation and reduction in the glutamate metabolism cycle. A minor increase in the three markers immunoreactivity at day 1 followed by marked decrease at day 2. In subacute phase VGLUT 1 & 2 and glutaminase immunoreactivity levels return to control levels at day 7, however, at day 14 they showed an increase above control levels. Finally at the chronic phase of regeneration, the three markers show a decrease in immunoreactivity levels initially (day 28) then return to control levels at day 70 post injury. In conclusion, our immunohistochemistry studies for vesicular glutamate transporters 1 & 2 and glutaminase gave insight into part of the story of neuronal response to injury. By understanding the molecular and physiological changes after peripheral nerve injuries, potential pharmacological or other treatments could be designed to improve the functional outcome of those injuries.

Peripheral nerve injuries are a significant clinical challenge due to the limited capacity of nerve tissue to regenerate.[1] Recently, research has focused its attention on the polymer nanofiber substrates since they mimic ECM topography and can be functionalized with bioactive molecules to overcome the limitations of both naturally occurring proteins and synthetic polymers used in nerve tissue regeneration applications. In the present work, we have developed poly(ԑ-caprolactone) (PCL) nanofiber substrates, modified with peptides to control the cellular activity. High molecular mass PCLs with a 4-dibenzocyclooctynol initiator was synthesized via ring-opening polymerization using magnesium 2,6-di-tert-butyl-4-methylphenoxide catalyst. The efficient chemical methods of nanofiber surface functionalization, such as strain-promoted azide-alkyne cycloaddition (SPAAC), developed by the Becker Lab[2], and thiol-ene reaction were utilized to introduce biomolecules.The dissertation is separated into four parts and demonstrates the combination of topographical cues and surface-tethered bioactive factors in effort to understand the optimal substrates for nerve regeneration or repair. We utilized the two most promising clinical approaches from our point of view for nerve tissue regeneration. The first approach is the differentiation of stem cells in vitro, described in the first two parts. Substrates that facilitate the differentiation and support long term culture of mature neural cells would advance the field of regenerative medicine. First, we demonstrated that YIGSR-functionalized aligned nanofibers can be potentially used for stem cells neural differentiation in vitro. The commitment and maturity of mESCs on these substrates were characterized using gene and protein expression. Then we compared influence of nanofiber topography and bioactive factors on neural differentiation of mESCs. These results highlight the influence of RGD binding versus topography in differentiation through high levels of glial fibrillary acidic protein expression.The second approach is improving Schwann cell migration and is described in the last two parts. Improved Schwann cell response on functionalized nanofibers in vitro was demonstrated in our lab.3 In the present study, we examined RGD-functionalized fibers in vivo, on the rat sciatic nerve model and demonstrated enhancement of Schwann cell infiltration and functional outcomes. To further improve Schwann cell migration, we created nanofiber substrates with gradients of bioactive factors along the aligned fiber direction.All the parts describe efforts to increase control over cellular activity (stem cell neural differentiation or Schwann cell migration) through combination of nanofiber topography and chemically tethered bioactive peptides. Each of these studies help to get an insight into optimal tissue engineering constructs for nerve regeneration.

As we age it becomes more difficult for our bodies to repair and recover from illness or injury; specifically, injuries to the peripheral nervous system. In addition, cellular and systemic dysfunction can lead to disorders that cause incomplete regeneration including movement and sensory disorders like neuralgia and vision loss1. Consequently, the extent of neurological recovery decreases, with the best recovery being for patients from 20-25 years of age, with a progressive non-linear decline as we age2. Annually, the majority of the 18 million peripheral nerve injuries occur in patients over the age of 45, and less than half of those ever regain full functional recovery1. Even when controlling for the type and severity of the nerve injury, the strongest determinant of recovery is the age of the subject3.

Peripheral nerve injuries, particularly transection injuries, are difficult to repair and treatment strategy is decided on a case-by-case basis. When there is a large gap between the proximal and distal stumps of the nerve, the use of a graft to bridge the distance between the stumps is the current clinical gold standard of treatment. However, grafts are often either expensive (allografts) or necessitate loss of function (autografts). Axons must also grow from the proximal stump, through the graft, to the distal stump, and so outcomes are often poor. Correspondingly, substantial clinical and pre-clinical evidence demonstrates that graft-free end-to-end repair of nerve endings would be preferable when possible. In this dissertation, I examine lengthening of injured peripheral nerves followed by end-to-end repair as a new method to bridge large nerve gaps, using a novel device to directionally stretch the proximal nerve stump. Though tension is often considered to be damaging to the nerve, studies have shown that axons can in fact grow more quickly when under modest tension. We first showed that for moderate gaps, an end-to-end nerve repair under tension had equivalent functional and growth outcomes when compared to tension-free repairs. Following an acute large-gap injury, nerves that were lengthened had equal or better outcomes when compared to a graft. Following a chronic large-gap injury, nerves that were lengthened had equivalent outcomes when compared to a graft, but, consistent with prior literature, outcomes as a whole were inferior to those following acute injury. Finally, we used proteomic profiling to probe biological pathways upregulated during tension-based regeneration. Cumulatively, we found that nerve lengthening followed by end-to-end repair is a new and potentially efficacious approach for nerve regeneration. Future work will examine the possibility of lengthening the nerve over very large gaps as well as new strategies for regeneration following chronic injury.

Electrical stimulation (ES) has previously demonstrated promising effects on peripheral nerve repair through enhanced neurite growth in vitro and shortened recovery time in vivo. In this study, we aimed to evaluate the effect of intraoperative short term ES on a clinically relevant isograft-repair model of a rodent peripheral nerve. In our model, an isograft was used to repair a 13 mm sciatic nerve gap-defect in adult male rats. Intraoperative ES was applied for 10 min at 24 V/m-DC to the experimental group and no stimulation was applied to the control group. We evaluated biweekly functional recovery over 12 weeks for motor function, using the sciatic functional index and external postural thrust. Sensory function was evaluated using a thermal stimulus. Motor nerves are more heavily myelinated and regenerate more quickly, while sensory nerves are less myelinated and have a slower recovery time. Structural repair outcomes were evaluated through histological examination of the sciatic nerves and gastrocnemius muscles at 6 and 12-week time points. The ES group had a significantly better motor recovery than the control group in weeks 4 and 6 after surgery. In addition, the ES group had 7% fewer paw contractures than the control group. Paw contractures form when the flexor muscles are innervated more quickly than extensor muscles, leaving the paw in a chronically curled over position that is representative of a clinical challenge in human nerve recovery. Furthermore, sensory functional testing and histology evaluation confirmed that ES was safe to use, as the ES- treated group had comparable recovery outcomes to the no-ES control group. Our previous study showed that 10 minutes of ES was effective in promoting functional recovery through a collagen scaffold bridging a 10-mm nerve defect. Here, we extend our findings by showing that ES can speed up motor recovery in an isograft-repair model, while slowing contracture formation. Demonstrating the benefits of applying short term intraoperative ES in a clinically relevant nerve injury model, creates an early point of translation to improve the current standard of care for peripheral nerve injuries. Future work is necessary to evaluate the sensory functional recovery response to ES and evaluate the mechanisms of ES that may lead to a reduction in contracture formation.

Peripheral nerve injuries are relatively common with broad-ranging aetiologies that often produce debilitating functional consequences. In its most severe form, nerve trauma involves complete transection of the nerve causing denervation of the target tissue with corresponding functional deficit. While the body possesses an ability to regenerate the severed axons through the mechanism of axon budding, such process is generally incomplete and fails to fully restore sensory and/or motor functions. The outcomes are, among other things, influenced by the surgical repair technique used. For example, standard surgical approach involves suturing the approximated nerve ends, which does not always ensure good alignment and leads to retention of permanent non-absorbable suture material, which often leads to intraneural scarring. Here I have tested a novel sutureless nerve repair technique using a biodegradable collagen membrane bonded with a photochemically activated dye. This process avoids the tissue tension/compression and foreign material retention commonly associated with non-absorbable sutures. In a transected rat sciatic nerve model, this technique has demonstrated superior histological and functional recovery when compared to a standard suturing approach. In future it may form a viable and, potentially, better alternative for surgical treatment of nerve injuries in clinical practice. Additional research will be required to further quantify functional sensory and motor recovery process, as well as histological changes and outcomes in regard to inflammation and regeneration.

Peripheral nerve injuries (PNI) cause alternations in central synapses leading to loss of function. The C-bouton synapses onto a-motoneurons in the ventral horn, and has a role in regulating motor output. Following tibial nerve ligation, the somatic C-bouton coverage is depleted (Alvarez et al., 2011), however, it is unknown what happens following crush type injuries. PNI causes neuroglia activation and proliferation that contribute to synaptic alterations, a response that has not been well-characterized in the ventral horn, where motoneurons are located. Therefore, I hypothesize that glia activation following peripheral nerve injury correlates to the degree of depletion of synaptic coverage of C-boutons. To test, I performed Immunohistochemical analysis of rat spinal cord to characterize C-bouton coverage, and activation of glia following two types of PNI. Our results indicate less C-bouton depletion following tibial nerve crush than ligation injuries. In addition, we found less glia activation following crush than ligation injuries.

Nerve conduits, allografts, patches, and autografts are commonly used in clinical settings to treat peripheral nerve injuries (PNI) via sutured microsurgery. Injuries caused by trauma such as crush and laceration require regenerative and protective measures. Studies present different outcome profiles reflecting high failure rates and complications associated with autografts and conduits. Proteins naturally expressed by nerve epineurium induce axonal regeneration toward the degenerating stump while providing flexibility and biocompatibility. Harvesting and manufacturing human donor epineural sheath can be cost effective as exhibited by existing partnership agreements and profit margins within the marketplace. This thesis focuses on the development and commercialization of two Cleveland Clinic peripheral nerve repair (PNR) products for surgical use. The applications of both products will address associated high health care costs in the U.S. and efficacy across mid to large nerve gaps. Gap coverage in each the civilian and military market is pending FDA approval.

Background: Nerve injuries can occur due to trauma, tumor removal, and accidental surgical resection. Standard care for a peripheral nerve transection involves excising a portion of a non-essential sensory nerve (i.e., an autograft), such as the sural nerve. This donor nerve is then transplanted into the defect. Although autografts are considered the clinical standard of peripheral nerve repair, they also pose significant disadvantages for patients. Nerve autografts contribute to loss of sensory function and the production of neuromas at the donor site. Surgeons are limited in the number and diameter of nerves that can be harvested. This can complicate trauma situations in which multiple peripheral nerve injuries are sustained. When an autograft is not available, nerve guides are often used to reconstruct defects less than 3 em. To date, there is no commercially available guide that is Food and Drug Administration (FDA)-approved to repair long gap peripheral nerve defects (i.e. gaps > 3 em). Our research has aimed to counteract this deficit in care by investigating nerve conduit therapies that are able to promote repair over long gaps. The combination of synthetic poly(caprolactone) with a native nerve guide structure and a neurotrophic factor delivery system should promote peripheral nerve repair over long gaps. Methods: Poly(caprolactone) nerve guides were fabricated with an internal drug delivery system in the form of double-walled microspheres. Double-walled microspheres were encapsulated with glial cell line-derived neurotrophic factor (GDNF), a prominent growth factor indicated in nerve repair. The poly(caprolactone) was embedded with double-walled microspheres and wrapped around a decellularized nerve allograft to form a novel nerve guide with synthetic and native components. A prototype of this novel guide was developed and scanning electron microscopy was used to further assess nerve guide structure. Results: Scanning electron microscopy revealed smooth and round microspheres with a double-walled morphology. Scanning electron microscopy also revealed the presence of intact endoneurial tubules within the acellular nerve allograft. Conclusion: A novel synthetic-native nerve guide with an internal neurotrophic factordelivery system was created to support repair of large peripheral nerve defects (i.e. defects greater than 3 centimeters).

Peripheral nerve injuries are frequently encountered in trauma, sports accidents, military activities, and degenerative muscle diseases. Like most neurological conditions, patients exhibit pain, sensory and motor deficits, and functional disability. Despite all the advances in biomedical science and technology, achieving full function and organ reinnervation after these injuries remains a major challenge. High costs of healthcare, loss of employment, and social disruption have provided the impetus for active research focusing on improved strategies for repair and regeneration. Stem cell therapy holds tremendous potential for the treatment of pathologic conditions and has consequently emerged as a new area of focus in regenerative medicine. Stem cells isolated from skeletal muscle have been shown to be both pluripotent and of significant therapeutic value; however, their ability to undergo neurogenic differentiation has yet to be investigated. Here, we report that progenitor cells isolated from skeletal muscles of both mouse and human, using our established preplate technique, adopt neuronal and glial phenotypes under controlled culture conditions. Transplantation of these cells into a critical-size sciatic nerve defect allowed full nerve restoration with induction of axonal regeneration through myelin-producing Schwann-like cells. Functional recovery resulted in improved gait of cell-transplanted mice. Multi-lineage progenitor cells have been recently identified in blood vessel walls, notably in skeletal muscle, and venous grafts have been used effectively to bridge nerve defects experimentally and clinically, through unknown cellular mechanisms. In a sex-mismatch model, we identified donor-derived Y chromosomes co-localized with host Schwann cells' nuclei, indicating nerve repair through vein grafting are mediated by vascular cells. A sustained decrease in nerve regeneration by decellularized or irradiated venous grafts also highlights the contribution of blood vessel-derived cells to nerve repair. Together, these findings not only identify the cellular basis for the efficacy of therapeutic vein wrapping, but also reinforce the emerging view of muscle cell-mediated therapy for peripheral neuropathies.