No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

Keratins are a family of structural proteins that can be extracted from a variety of sources including wool, nails, skin, hooves, and hair. Keratin can be processed into different constructs such as coatings, scaffolds, and hydrogels, and has shown favorable results when placed in in vitro and in vivo settings for different tissue regeneration applications. Over three decades, keratin extraction technology has been continuously modified, and these differences in extraction processes have distinct effects on the characteristics of the end product. In this work, we examine the effect of keratin aggregation during a widely-used purification step, dialysis ultra-filtration, on material characteristics of the final keratin product when fabricated into a hydrogel. Two distinct dialysis procedures were applied during the extraction of oxidized keratin (keratose): one promoting protein aggregation and the other mitigating it. Analyses of material properties such as mechanical and enzymatic stability were conducted in addition to observing the differences in solution behavior between products. Data revealed that protein aggregation during the extraction process has a profound effect on keratose hydrogel material properties. After determination of the effect of protein aggregation during extraction on keratose hydrogels, investigation of how a blended material comprised of said keratose and type I collagen was undertaken. It was hypothesized that a blend would result in mixing at the molecular level, resulting in improved properties compared to either pure material alone. A protocol was created to make stable keratose/type I collagen blends and material characterization techniques were applied to determine the inherent properties of samples with differing ratios. Crosslinking density, mechanical properties, enzymatic degradation properties, water uptake capacity, structural architecture, and thermal properties were all assessed. In addition, the ability of this material to maintain cell viability was conducted. Results showed that the addition of type I collagen has a significant effect on the properties of hydrogel blends with keratose compared to the pure keratose system. This was mostly evident with hydrogel mechanical stability and material architecture. Finally, the ability to use this hybrid material as a luminal filler for a nerve conduit during peripheral nerve regeneration was explored in an in vitro setting. The ability of this blend to promote Schwann cell viability was assessed in addition to determining the ability of these cells to attach and migrate through the material matrix. These experiments demonstrate proof-of-concept for the application of using keratose/type I collagen matrices as a luminal filler in peripheral nerve guidance conduits. / Doctor of Philosophy / Keratins are a family of structural proteins that can be extracted from wool, skin, nails, and hair, and that have been investigated in the field of tissue regeneration. Humans make several types of keratins, so it has a natural acceptance by the body and its inflammatory and immune systems. However, keratins can be hard to make and process into useful products. Many methods for producing keratin biomaterials have been developed over the past 30 years, but most of them are not ideal. This work sought to explore a production method that addresses a particular problem, that of protein aggregation during purification. In so doing, methods can be optimized to create more useful keratin biomaterials. Experiments comparing preparation methods that maximize and minimize protein aggregation were compared. Data showed that minimizing aggregation leads to better biomaterial characteristics, thus demonstrating the potential impact of targeting this processing step. However, even after optimization of purification, keratins still have limitations. Most notably their mechanical strength is not as great as some other materials. A typical approach to address this in other systems has been by blending. In the present work, we explored a blend made from keratin and type 1 collagen. A method was developed to effectively blend keratin and collagen and create stable mixtures that yielded protein-to-protein coordination. Such interactions typically yield beneficial material characteristics such as increased strength. Data showed that intimate mixing of the two proteins was achieved, and resulting characteristics were improved compared to either pure material. Finally, studies were conducted to assess the potential for keratin/collagen blends to be used to regenerate injured nerves. A common method is to enclose the ends of a cut nerve into a tube and let the nerve re-grow through the tube to its target muscle. An important characteristic is an ability for cells to populate the interior of the tube and help the nerve fibers grow. In the present study, we investigated the behavior of a particularly important cell, the Schwann cell, to attach, move and grow through a keratin/collagen biomaterial. Data showed good cell behavior, suggesting that the material could be used in a medical product for nerve repair.

Yes / Current nerve tissue engineering applications are adopting xenogeneic nerve tissue as potential nerve grafts to help aid nerve regeneration. However, there is little literature that describes the exact location, anatomy and physiology of these nerves to highlight their potential as a donor graft. The aim of this study was to identify and characterise the structural and extracellular matrix (ECM) components of porcine peripheral nerves in the hind leg. Methods included the dissection of porcine nerves, localisation, characterisation and quantification of the ECM components and identification of nerve cells. Results showed a noticeable variance between porcine and rat nerve (a commonly studied species) in terms of fascicle number. The study also revealed that when porcine peripheral nerves branch, a decrease in fascicle number and size was evident. Porcine ECM and nerve fascicles were found to be predominately comprised of collagen together with glycosaminoglycans, laminin and fibronectin. Immunolabelling for nerve growth factor receptor p75 also revealed the localisation of Schwann cells around and inside the fascicles. In conclusion, it is shown that porcine peripheral nerves possess a microstructure similar to that found in rat, and is not dissimilar to human. This finding could extend to the suggestion that due to the similarities in anatomy to human nerve, porcine nerves may have utility as a nerve graft providing guidance and support to regenerating axons.

Following peripheral nerve transection and microsurgical repair (PNIr) most patients retain significant sensorimotor impairments, a proportion of which also develop chronic neuropathic pain. Individual psychological factors may contribute to the development, intensity and duration of chronic pain. Furthermore, a large body of evidence has indentified beneficial and maladaptive cortical plasticity following disease or injury. The general aim of this thesis was to determine the extent of sensory and motor recovery, functional and structural brain changes, and the impact of chronic neuropathic pain on sensorimotor outcomes following upper limb PNIr. Towards this main aim a sensorimotor psychophysical assessment (that included psychological assessments), nerve conduction testing, and an MRI session that examined brain function and structure was performed in patients with peripipheral nerve injury induced neuropathic pain (PNI-P) and those with no neuropathic pain (PNI-NP). Nerve conduction testing demonstrated that all patients had incomplete peripheral nerve regeneration, and that PNI-P patients had worse sensory nerve regeneration. Psychophysical assessment confirmed that all PNIr patients had significant sensorimotor deficits. Additionally, deficits on tests of vibration detection, sensorimotor integration, and fine dexterity were significantly greater in PNI-P patients. Psychological measures clearly distinguished PNI-P from PNI-NP and healthy controls (HC). Vibrotactile stimulation of the deafferented territory in PNI-NP patients results in reduced BOLD activation within the primary and secondary somatosensory cortices. Interestingly, the regions of reduced BOLD corresponded with gray matter thinning which was negatively correlated with behavioural measures of sensory recovery. Structural abnormalities were also identified in the right insula. PNI-P patients had thinning within the right middle insula and a corresponding decrease in white matter pathways projecting into/out of that region. PNI-P patients also had white matter abnormalities in pathways feeding into/out of the contralesional primary somatosensory cortex and thalamus. In conclusion, PNIr is clearly associated with sensorimotor impairments and brain plasticity. Furthermore, neuropathic pain is associated with worse peripheral nerve regeneration, sensorimotor deficits, different psychological profiles, and structural alterations in brain regions involved in pain perception and somatosensation. These results provide insight into peripheral regeneration, the development of chronic pain, brain plasticity and structure-function-behavioural relationships following nerve injury and have important therapeutic implications.

Following peripheral nerve transection and microsurgical repair (PNIr) most patients retain significant sensorimotor impairments, a proportion of which also develop chronic neuropathic pain. Individual psychological factors may contribute to the development, intensity and duration of chronic pain. Furthermore, a large body of evidence has indentified beneficial and maladaptive cortical plasticity following disease or injury. The general aim of this thesis was to determine the extent of sensory and motor recovery, functional and structural brain changes, and the impact of chronic neuropathic pain on sensorimotor outcomes following upper limb PNIr. Towards this main aim a sensorimotor psychophysical assessment (that included psychological assessments), nerve conduction testing, and an MRI session that examined brain function and structure was performed in patients with peripipheral nerve injury induced neuropathic pain (PNI-P) and those with no neuropathic pain (PNI-NP). Nerve conduction testing demonstrated that all patients had incomplete peripheral nerve regeneration, and that PNI-P patients had worse sensory nerve regeneration. Psychophysical assessment confirmed that all PNIr patients had significant sensorimotor deficits. Additionally, deficits on tests of vibration detection, sensorimotor integration, and fine dexterity were significantly greater in PNI-P patients. Psychological measures clearly distinguished PNI-P from PNI-NP and healthy controls (HC). Vibrotactile stimulation of the deafferented territory in PNI-NP patients results in reduced BOLD activation within the primary and secondary somatosensory cortices. Interestingly, the regions of reduced BOLD corresponded with gray matter thinning which was negatively correlated with behavioural measures of sensory recovery. Structural abnormalities were also identified in the right insula. PNI-P patients had thinning within the right middle insula and a corresponding decrease in white matter pathways projecting into/out of that region. PNI-P patients also had white matter abnormalities in pathways feeding into/out of the contralesional primary somatosensory cortex and thalamus. In conclusion, PNIr is clearly associated with sensorimotor impairments and brain plasticity. Furthermore, neuropathic pain is associated with worse peripheral nerve regeneration, sensorimotor deficits, different psychological profiles, and structural alterations in brain regions involved in pain perception and somatosensation. These results provide insight into peripheral regeneration, the development of chronic pain, brain plasticity and structure-function-behavioural relationships following nerve injury and have important therapeutic implications.