Traumatic peripheral nerve injuries affect 50,000 patients annually with USD 7 billion in healthcare costs. Current treatments like the gold standard autograft and commercially available nerve guide conduits (NGC) are insufficient to repair long gap peripheral nerve injuries. Autografts have a severe size mismatch limitation resulting in incomplete functional recovery further confounded by harvest site co-morbidities. NGCs can aid recovery but lack key microenvironment cues that promote nerve regeneration over long gap injuries. We hypothesized that providing topographical, mechanical, and electrical guidance cues into a nanofibrous composite biopolymer would result in improve neuron growth metrics in an in vitro model. We embedded hydrophilic carbon nanotubes (CNT) within hyaluronic acid (HA) nanofibers by electrospinning. The aims of this study were (1) to define the topographical, nanomechanical, and electrochemical material properties of HA-CNT nanofibers and (2) to determine the electrical stimulus parameters required to elicit increased neurite outgrowth on our nanofibrous scaffold.

HA-CNT toppography was visualized as smooth nanofibers, nanoscale in diamter, with CNTs contained within the nanofiber core, as characterized by scanning electron microscopy and tunneling electron microscopy. Mechanical properties were evaluated under physiological conditions by hydrating nanofibers to equilibrium, then testing samples fully immersed in electrolyte buffer. A reduced elastic modulus was obtained by fitting quantitative nanomechanical mapping data to the Sneddon model using atomic force microscopy imaging in fluid. Local modulus was measured to The electrochemical characterization performed was electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV). EIS resulted in a decreased resistance to current flow by a factor of 1.7 at 20 Hz and 1.2 at 1kHz. CV revealed a 2.1-fold increase in specific capacitance (mF/cm²) of HA-CNT relative to HA nanofibers.

Chick dorsal root ganglia neurons grown on HA or HA-CNT substrates for 24h were either unstimulated or stimulated at 20Hz for 30min or 60min using a bi-phasic 150, 200, or 250mV/mm square wave. Significant effects of fiber type and stimulus amplitude and time were observed when measuring neuron viability and neuron outgrowth after 72 h. Neuron outgrowth was significantly longer on HA-CNT substrates electrically stimulated for 60min at all stimulus amplitudes versus all other groups (p < 0.01 3-Way ANOVA, α). This study demonstrates the potential of combining electrical stimulation with material based repair strategies for neural regeneration. Further, the results contribute to defining the electrical stimulus parameters necessary for regeneration in the peripheral nerve environment. Incorporating well-dispersed hydrophilic CNTs in HA nanofibers significantly enhances neural regeneration following electrical stimulation. Future work encompasses characterizing glial responses to electrical stimulation including electrophysiological calcium imaging assays to elucidate the governing molecular mechanisms for both neuronal and glial behavior.