Electrospinning Nanofibers for Neural Tissue Engineering and Injuries: From Benchtop to Bedside IntroductionNeural tissue engineering (NTE) seeks to address the limited treatment options for those who have suffered from peripheral nerve injuries. Peripheral nerves have a limited capacity for regeneration following physical damage. To restore function to the damaged or transected nerve, a continuous pathway must be re-established to regenerate the axons across the nerve gap. Current clinical strategies include autologous grafts, which involve the harvesting and implantation of a patient-derived donor nerve. However, this procedure requires multiple surgeries, leave the donor site nonfunctional, and limited availability of donor nerves. Allografts are nerves harvested from other humans or animals but are limited by transplant rejection. To overcome the limitations of current clinical options, an area of focus in the development of nerve regeneration technologies is electrospinning biocompatible polymeric nanofibers into nerve guidance conduits (NGCs).  The main objective of this article is to brief the potential of electrospun nanofibers for neural tissue engineering and the utility of NGCs to repair peripheral nerve injuries.Process and Mechanism of Electrospinning NanofibersElectrospinning nanofibers is a relatively simple and versatile procedure that has been applied successfully to a variety of tissue engineering studies. The essential components of a typical electrospinning setup are a high voltage power supply, a spinneret (typically a hypodermic needle), syringe pump, and electrically conductive collector surface.  Figure 1 displays a schematic view of the essential components of a typical electrospinning apparatus. Electrospinning nanofibers has tunable properties and are determined by the setting of certain variables. The syringe pump is the main control unit of the electrospinning process and establishes the flow rate of the polymer solution. The high voltage power supply is then attached to the spinneret tip, which applies charge to the polymer solution droplet. The polymer droplet then elongates to form the Taylor cone, where the finely charged jet is formed at the tip. Taylor cone formation indicates that electrospinning has initiated, and the electrostatic repulsive force exceeds the surface tension of the droplet [Lohani et al., 2014]. The whipping jet motion of the pumped polymer solution cause nanofibers to accumulate on the surface of the grounded collector.  The mechanism of the instability jet region is thought to form from the electrostatic interactions between the electric field and the charges on the surface of the jet [Xie J et al., 2010]. The polymer and solvent of choice must have the optimum viscoelastic properties to maintain its morphology during this whipping process. This continuous acceleration and stretching of the polymer solution produce nanofibers, which are generally as thin as tens of nanometers in diameter [Xie J et al., 2010].  The geometry, kinetics, and electrostatic interactions between the charged polymer solution and grounded conductive collector determine the orientation of the produced nanofibers. Generally, static fiber collectors produce randomly-oriented nanofibers, while aligned nanofibers are typically produced via rapidly rotating collector devices, or by manipulation of the electric field. Figure1: Typical setup of electrospinning device with a flat collector. The collector geometry, kinetics, and electrostatic interactions determine the orientation of electrospun nanofibers. [Lohani et al. 2014].Neural Cell behavior on electrospun nanofibers: From Differentiation to ProliferationElectrospun nanofibers have been used extensively in tissue engineering studies because the topography of the nanofibers mimics the natural extracellular matrix (ECM), while aligned nanofibers can direct neurite growth and improve axon regeneration [Han et al., 2011]. The use of stem cells on nanofibrous structures has been studied extensively and research continues to grow in this area. In one study, the team of researchers were able to drive the differentiation of human mesenchymal stem cells (hMSCs) into cells of neuronal lineage [Prabhakaran et al., 2009]. Figure 2a below shows the differentiated hMSC into a neuronal cell type on collagen coated PLCL nanofibers. Compared to the undifferentiated hMSC seen in figure 2b, the differentiated hMSC displays multipolar elongations characteristics of neuronal cells. The differentiation of hMSC to neuronal cell type was driven by a variety of inducing and growth factors such as ?-mercaptoethanol, epidermal growth factor, nerve growth factor and brain derived growth factor [Prabhakaran et al., 2009]. The one limitation of this study was the fact that the cells were only cultured on randomly oriented nanofibers.  Figure 2: (A) Differentiated hMSC into neuronal cell type on PLCL/Collagen scaffold. (B) Undifferentiated hMSC on PLCL/Collagen scaffold.  [Prabhakaran et al., 2009] In another study using human Schwann cells (hSCs), it was demonstrated that aligned polycaprolactone (PCL) nanofibers provided improved hSCs contact guidance and elongation compared to randomly oriented nanofibers [Wang X et al., 2013]. Figure 3 shows how the hSCs cultured onto the random nanofibers appeared to grow and elongate in a haphazard fashion, while hSCs cultured on aligned nanofibers elongated along the nanofibers. By manipulating the orientation of the nanofibers from random to aligned, better contact guidance cues can be provided for the cell so that the nuclei and cytoskeletal elements can elongate along the orientation of the fibers [Chew et al, 2008].                       Figure 3: (A, B) Schematic view of hSC behavior on randomly oriented and aligned nanofibers. (C, D) SEM images of random and aligned nanofibers. (E, F) Fluorescent images of hSCs on random and aligned nanofibers. [Wang X et al., 2013].Fabrication of Nerve Guidance Conduits: From Benchtop to BedsideFor nanofibrous structures to fully translate into the clinical setting, research continues towards the development of biocompatible and biodegradable nerve guidance conduits (NGCs). The ideal NGC should also provide the mechanical and chemical cues to promote cell adhesion, proliferation, migration and axonal extension [Zhu et al., 2015].  Figure 4 shows the most important properties to consider when designing an NGC.Figure 4: Ideal properties of a tissue engineered NGC [Zhu et al, 2015].When a peripheral nerve is damaged, several physiological events occur at the injury site. In the cell body, signals from damaged axons upregulate regeneration-associated genes (RAGs) [Lopez-Cebral et al, 2017]. The proximal axon stump then degenerates to the node of Ranvier, while the distal axon stump undergoes a process called Wallerian degeneration, where debris from the damaged axon is cleared by macrophages and proliferating Schwann cells [Faroni et al., 2015]. Figure 5 shows a schematic view of the Wallerian degeneration process and it is imperative that the NGC designed can strengthen the nerves natural regeneration process. Figure 5: Schematic view of the Wallerian degeneration process [Faroni et al., 2015].In one study, an NGC with aligned nanofibers was developed for an in vivo nerve regeneration study using rats with an injured sciatic nerve [Huang et al. 2015]. In order to fabricate the NGC via electrospinning, two small diameter rotating mandrels were used as the collection device. In figure 6, it shows how the NGC performed following implantation of the NGC. The NGC was based off the polymer cellulose acetate butyrate (CAB) and was 15 mm long. Figures 6a-6c shows how the NGC was sutured to each nerve end and bridged the nerve gap. Figure 6d shows the same NGC starting to become covered with native tissue, while figure 6e shows the regenerated nerve 6 weeks after implantation. The dotted ovals show the regenerated nerve. Figure 6f shows the statistical date for the retention rate of nerve diameter in the smooth and grooved CABs. The main difference between the smooth and grooved CABs is the molecular weight of the model polymer [Huang et al., 2015]. It is apparent that the longitudinally grooved NGC increased the retention rate of the regenerated nerve diameter. Figure 6: (A) Surgical procedure to where the right femur of the experimental rats was shaved. (B) image of the transected sciatic nerve before implantation of the NGC. (C) Image of implanted NGC, bridging the nerve gap. (D) Image of the implanted NGC. Native tissue begins to regenerate. (E) Nerve fully regenerated after 6 weeks. (F) 6-week and 12-week statistical analysis for the retention rate of nerve diameter.  [Huang et al, 2015].In addition, functional recovery tests were performed to further analyze the nerve regeneration capabilities of the smooth and grooved CAB NGCs.  Walking track analysis, action potential measurements, and analysis of the gastrocnemius muscle are seen in figure 7. NCV provides an objective measurement of the conduction of action potentials in peripheral nerves and DCAAMP is used to quantify the number of regenerated motor nerve fibers. In these tests, it shows that the grooved CAB NGCs were significantly greater than the smooth CAB NGCs at 6 weeks. However, after 12 weeks, the difference between each group decreased. Also, the cross-sectional images of the gastrocnemius muscle show that the grooved CAB had greater growth inside the grooved CAB compared to the smooth CAB. Further quantification determined that there was a significant difference between these groups. SFI measures quantify the walking track data, with the value ~0 as an indicator for normal nerve function, while –100 indicated total nerve dysfunction [Huang et al, 2015].  SFI decreased significantly following implantation in both CAB groups within 12 weeks.Figure 7: (A) Nerve and muscle compound action potential measurement, cross-section H&E stain of gastrocnemius muscle, and footprint of rats 12 weeks after implantation. (B) NCV from regenerated nerve. (C) DCAMP from regenerated nerve. (D) Quantification of wet weight ration of the gastrocnemius muscle. (E) Average SFI calculated from the walking track analysis at different time intervals. [Huang et al, 2015].Conclusions and Future ProspectsThe versatility of the electrospinning process provides several ways to produce more robust scaffolds for neural tissue engineering. Parameters such Parameters such as input voltage of the power supply, the flow rate of the syringe pump, the type of polymer being electrospun and collector geometry play important roles to determine what kind of nanofiber construct is produced. By combining the electrospun fibers with other proteins, growth factors, and neural stem cells, the outgrowth of neurites can be more controlled and understood. Other properties such as the polymer composition and morphology of the nanofibers greatly determine whether neural cell proliferation and differentiation can occur. Overall, NGCs fabricated via electrospinning have great potential in producing more functional outcomes following peripheral nerve injuries. Further in vivo studies in the future will help translate this technology into human peripheral nerve injury cases.ReferencesChew SY, Mi R, Hoke A, Leong KW: The effect of the alignment of electrospun fibrous scaffolds on Schwann cell maturation. 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