Peripheral nerve injuries are often associated with significant loss of nervous tissue leading to poor restoration of function following repair of injured nerves. Although the injury gap could be bridged by autologous nerve graft, the limited access to donor material and additional morbidity such as loss of sensation and scarring have prompted a search for biosynthetic nerve transplants. The present thesis investigates the effects of a synthetic matrix BD™ PuraMatrix™ peptide (BD)hydrogel, alginate/fibronectin gel and fibrin glue combined with cultured rat Schwann cells or human bone marrow derived mesenchymal stem cells (MSC) on neuronal regeneration and muscle recovery after peripheral nerve injury in adult rats. In a sciatic nerve injury model, after 3 weeks postoperatively, the regenerating axons grew significantly longer distances within the conduits filled with BD hydrogel if compared with the alginate/fibronectin gel. The addition of rat Schwann cells to the BD hydrogel drastically increased regeneration distance with axons crossing the injury gap and entering into the distal nerve stump. However, at 16 weeks the number of regenerating spinal motoneurons was decreased to 49% and 31% in the BD hydrogel and alginate/fibronectin groups respectively. The recovery of the gastrocnemius muscle was also inferior in both experimental groups if compared with the nerve graft. The addition of the cultured Schwann cells did not further improve the regeneration of motoneurons and muscle recovery. The growth-promoting effects of the tubular conduits prepared from fibrin glue were also studied following repair of short and long peripheral nerve gaps. Retrograde neuronal labeling demonstrated that fibrin glue conduit promoted regeneration of 60% of injured sensory neurons and 52% of motoneurons when compared with the autologous nerve graft. The total number of myelinated axons in the distal nerve stump in the fibrin conduit group reached 86% of the nerve graft control and the weight of gastrocnemius and soleus muscles recovered to 82% and 89%, respectively. When a fibrin conduit was used to bridge a 20 mm sciatic nerve gap, the weight of gastrocnemius muscle reached only 43% of the nerve graft control. The morphology of the muscle showed a more atrophic appearance and the mean area and diameter of fast type fibres were significantly worse than those of the corresponding 10 mm gap group. In contrast, both gap sizes treated with nerve graft showed similar fiber size. The combination of fibrin conduit with human MSC and daily injections of cyclosporine A enhanced the distance of regeneration by four fold and the area occupied by regenerating axons by three fold at 3 weeks after nerve injury and repair. In addition, the treatment also significantly reduced the ED1 macrophage reaction. At 12 weeks after nerve injury the treatment with cyclosporine A alone or cyclosporine A combined with hMSC induced recovery of the muscle weight and the size of fast type fibres to the control levels of the nerve graft group. In summary, these results show that a BD hydrogel supplemented with rat Schwann cells can support the initial phase of neuronal regeneration across the conduit. The data also demonstrate an advantage of tubular fibrin conduits combined with human MSC to promote axonal regeneration and muscle recovery after peripheral nerve injury.

The goal of nerve tissue engineering is to promote and guide axon growth across a site of nerve injury without misdirection. Bioengineered tissue scaffolds have been shown to be promising for the regeneration of damaged peripheral nerves. Schwann cells play a pivotal role following nerve injury by forming aligned “bands of Büngner” that promote and guide axon regeneration into the distal nerve segment. The incorporation of living Schwann cells into various hydrogels has therefore been urged during the fabrication of tissue engineered nerve scaffolds. The aim of this research is to characterize biomaterials suitable for 3D bioplotting of nerve repair scaffolds. Here a novel technique of scaffold fabrication has been optimized to print alginate-based three-dimensional tissue scaffolds containing hyaluronic acid and living Schwann cells. Alginate/hyaluronic acid scaffolds were successfully fabricated with good printability and cell viability. Addition of the polycation polyethyleneimine (PEI) during the fabrication process stabilized the structure of alginate through the formation of a polyelectrolyte complex and had a significant influence on the degree of swelling, degradation rate, mechanical property, and release kinetics of incorporated protein within the scaffolds. A preliminary in vivo study showed the feasibility of implanting 3D printed alginate/hyaluronic acid scaffolds as nerve conduits in Sprague-Dawley (SD) rats with resected sciatic nerves. However alginate/hyaluronic acid scaffolds were found to be unsuitable for axonal regeneration. Further in vitro culture of Schwann cells was performed in collagen type-I, fibrin, fibrin/hyaluronic acid, and their combination with alginate. It was found that Schwann cells had more favorable cell morphology in fibrin/hyaluronic acid or collagen without alginate. Schwann cell proliferation and alignment were better in fibrin/hyaluronic acid. Therefore fibrin/hyaluronic acid is more ideal than most other hydrogel formulations for use in the bioprinting of nerve repair tissue engineering scaffolds, which incorporate cellular elements. As Schwann cells also align along the long axis of the printed fibrin/hyaluronic acid strands, 3D bioprinting of multiple layers of crosslinked fibrin strands can be used to fabricate a nerve conduit mimicking the bands of Büngner.

Acetylcholine is a neurotransmitter that regulates neurite branching, induces neurite outgrowth, and synapse formation. Because of its various roles in neuronal activities, acetylcholine-based materials may also be useful in nerve repair. We present a series of biodegradable polymers with varying concentrations of acetylcholine-like motifs. We hypothesize that neurite sprouting and extension can be enhanced by using materials to present biochemical and physical cues. Acetylcholine-like motifs were incorporated by the polycondensation of diglycidyl sebacate, aminoethyl acetate, and leucine ethyl ester, which permitted control over acetylcholine motif concentration. Interactions between the polymers and neurons were characterized using rat dorsal root ganglia explants (DRG). We screened the potential application of these materials in nerve tissue engineering using the following criteria: 1) neurite sprouting, 2) neurite length, and 3) distribution of the neurite lengths. The ability of DRG to sprout neurites was influenced by the concentration of acetylcholine motifs of the polymer. Addition of acetylcholine receptor antagonists to DRG cultured on the polymers significantly decreased neurite sprouting, suggesting acetylcholine receptors mediate sprouting on the polymers. Future studies may examine how neurons on acetylcholine-based polymers exhibit changes in downstream signaling events and cell excitability that are associated with receptor activation. In preparation for testing the acetylcholine-based polymers in vivo, porous scaffolds with longitudinally oriented channels were fabricated using fiber templating and salt leaching. Micro computed tomography, scanning electron microscopy, and cryo-sectioning revealed the presence of longitudinally oriented channels. Channel volume and average pore size of the scaffolds were controlled by the number of fibers and salt fusion time. Future studies may involve testing the effect of acetylcholine-motifs by coating polymers onto such scaffolds or assessing the effect of the scaffold's dimensional properties on nerve regeneration.

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.

Nerve injury is a serious clinical problem that has a direct impact on the quality of life. Nerve tissue engineering (NTE) is one of the most promising methods in human health care to restore the function of damaged neural tissues. The current state of the art involves the construction of a tissue engineered, nano or micropatterned 3-D nerve tube that has fibers or channels in the inside. The scope of this study is to construct a 3-D, biodegradable nerve tube which consists of an aligned, electrospun mat seeded with stem cells that is wrapped in a porous micropatterned film which contains support cells. In two separate approaches human mesenchymal stem cells (MSCs) and mouse neural stem cells (NSCs) were used. In the design with the MSCs, the micropatterned exterior part of the nerve tube contained undifferentiated MSCs as support cells and this was wrapped around the fibers seeded with MSCs which were induced to neural differentiation. In the other case, NSCs differentiated into astrocytes were used as support cells seeded on the micropatterned film and the mat was loaded with undifferentiated NSCs. Differentiation into neural cells and astrocytes were shown with immunocytochemistry and RT-PCR. The neuron-like MSCs and NSCs were shown to express neural marker &amp / #946 / -Tubulin III whereas astrocytes expressed glial fibrillary acidic protein (GFAP), an astrocyte marker. RT-PCR showed that early neural markers, nestin and Nurr 1, were expressed at passage 4 by undifferentiated MSCs and by MSCs induced to neural differentiation, while these markers were not expressed in undifferentiated MSCs at passages 2 and 3. The cells aligned along the axis of the micropattern of the film and along the axis of the fiber on the fibrous mat. This behavior was also maintained after construct formation. MTS and confocal microscopy revealed that the cells were viable and homogeneously distributed over the two parts of the scaffold. This indicates that the construct has a potential to be tested in vivo for nerve tissue engineering purposes.

There are different methods used in the surgical treatment of peripheral nerve injury. In this respect, end-to-end surgical reconnection of the damaged nerve ends or autologous nerve grafts are applied as soon as possible after the injury. When autologous tissue transplant is considered, there are some medical devices available generally for relatively short nerve defects. As a solution for this problem, different tissue engineered nerve conduits have been developed. In the current study, a pHEMA hydrogel membranes were designed to mimic the tubular conduits and they were loaded with 1-6% (w/w) multiwalled carbon nanotubes (mwCNTs) to obtain electrical conductivity. The most important reason for the use of CNTs in peripheral nerve injury is their electrical conductivity. Within the context of the study, the degree of swelling, contact angles, electrical conductivity and mechanical properties of the membranes were analyzed. As the amount of mwCNTs were increased, the contact angles, indicating higher hydrophobicity and the electrical conductivity increased. The tensile test of the mwCNT-pHEMA composite membranes showed that the membranes have viscoelastic structure similar to the structure of the soft tissues. The structure of the mwCNT containing pHEMA composite membranes were analyzed with different microscopical techniques such as SEM, CSLM and microCT. MwCNTs on the hydrogels were morphologically similar to the original. SEM micrographs also showed that the mwCNTs were grouped in clumps on hydrogel surfaces. No mwCNT leaching was observed because the mwCNTs were embedded in the hydrogel, therefore, no cytotoxic effect was observed. The pHEMA hydrogels were porous which is suitable for transportation of materials, electrolytes and gas needed for cell nutrition and growth. In the in vitro studies, SHSY5Y neuroblastoma cells were seeded on the membranes to determine the sustainability and effects of the membranes on the cell growth. Electrical potential of 1 and 2 V were used to stimulate the cells. Microscopical examination with SEM and CSLM, and MTT viability assay were used. The SHSY5Y neuroblastoma cells were attached and proliferated on both the composite and the hydrogel membranes. The cells on pHEMA membranes without mwCNTs, however, were not able to survive after application of electrical potential. As a conclusion, use of composite membranes in the treatment of peripheral nerve injury as a nerve conduit is appropriate. Electrical stimulation, however, did not induce the cells to align in contrast to the expected results, indicating potential and current application regime needs to be optimized to obtain the desired results.

Spinal cord injury results in complete failure of the central neurons to regenerate and is associated with cyst formation and enlargement of the trauma zone. In contrast to the spinal cord, axons in the injured peripheral nerve have the capacity to undergo some spontaneous regeneration. However, significant post-traumatic loss of nervous tissue causing long nerve gap is one of the main reasons for the poor restoration of function following microsurgical repair of injured nerves. The present thesis investigates the effects of biodegradable conduits prepared from fibrin glue and poly-beta-hydroxybutyrate (PHB) in combination with cultured Schwann cells, mesenchymal stem cells and extracellular matrix molecules on regeneration after spinal cord and peripheral nerve injury in adult rats. At 4-8 weeks after transplantation into the injured spinal cord, the PHB conduit was well integrated into the cavity but regenerating axons were found mainly outside the PHB. When suspension of BrdU-labeled Schwann cells was added to the PHB, regenerating axons filled the conduit and became associated with the implanted cells. Modification of the PHB surface with extracellular matrix molecules significantly increased Schwann cell attachment and proliferation but did not alter axonal regeneration. To improve the labeling technique of the transplanted cells, the efficacy of fluorescent cell tracers Fast Blue, PKH26, Vibrant DiO and Cell Tracker™ Green CMFDA was evaluated. All tested dyes produced very efficient initial labeling of olfactory ensheathing glial cells in culture. The number of Fast Blue-labeled cells remained largely unchanged during the first 4 weeks whereas the number of cells labeled with other tracers was significantly reduced after 2 weeks. After transplantation into the spinal cord, Fast Blue-labeled glial cells survived for 8 weeks but demonstrated very limited migration from the injection sites. Additional immunostaining with glial and neuronal markers demonstrated transfer of the dye from the transplanted cells to the host tissue. In a sciatic nerve injury model, the extent of axonal regeneration through a 10mm gap bridged with tubular PHB conduit was compared with a fibrin glue conduit. At 2 weeks after injury, the fibrin conduit supported similar axonal regeneration and migration of the host Schwann cells compared with the PHB conduit augmented with a diluted fibrin matrix and GFP-labeled Schwann cells or mesenchymal stem cells. The long-term regenerative response was evaluated using retrograde neuronal labeling. The fibrin glue conduit promoted regeneration of 60% of sensory neurons and 52% of motoneurons when compared with the autologous nerve graft. The total number of myelinated axons in the distal nerve stump in the fibrin conduit group reached 86% of the nerve graft control and the weight of gastrocnemius and soleus muscles recovered to 82% and 89%, respectively. When a fibrin conduit was used to bridge a 20mm sciatic nerve gap, the weight of gastrocnemius muscle reached only 43% of the nerve graft control. The morphology of the muscle showed more chaotic appearance and the mean area and diameter of fast type fibers were significantly worse than those of the corresponding 10mm gap group. In contrast, both gap sizes treated with nerve graft showed similar fiber size. In summary, these results show that a PHB conduit promotes attachment, proliferation and survival of adult Schwann cells and supports marked axonal growth after transplantation into the injured spinal cord. The data suggest an advantage of the fibrin conduit for the important initial phase of peripheral nerve regeneration and demonstrate potential of the conduit to promote long-term neuronal regeneration and muscle recovery.

Yes / The peripheral nervous system has a limited innate capacity for self-repair following injury, and surgical intervention is often required. For injuries greater than a few millimeters autografting is standard practice although it is associated with donor site morbidity and is limited in its availability. Because of this, nerve guidance conduits (NGCs) can be viewed as an advantageous alternative, but currently have limited efficacy for short and large injury gaps in comparison to autograft. Current commercially available NGC designs rely on existing regulatory approved materials and traditional production methods, limiting improvement of their design. The aim of this study was to establish a novel method for NGC manufacture using a custom built laser-based microstereolithography (muSL) setup that incorporated a 405 nm laser source to produce 3D constructs with approximately 50 mum resolution from a photocurable poly(ethylene glycol) resin. These were evaluated by SEM, in vitro neuronal, Schwann and dorsal root ganglion culture and in vivo using a thy-1-YFP-H mouse common fibular nerve injury model. NGCs with dimensions of 1 mm internal diameter x 5 mm length with a wall thickness of 250 mum were fabricated and capable of supporting re-innervation across a 3 mm injury gap after 21 days, with results close to that of an autograft control. The study provides a technology platform for the rapid microfabrication of biocompatible materials, a novel method for in vivo evaluation, and a benchmark for future development in more advanced NGC designs, biodegradable and larger device sizes, and longer-term implantation studies.