Peripheral nerve injuries annually affect hundreds of thousands of people globally. Current treatments like the gold standard autograft and commercially available nerve guide conduits (NGC) are insufficient to repair long gap peripheral nerve injuries. NGCs can aid recovery but lack key microenvironment cues that promote nerve regeneration. We hypothesized that providing topographical, mechanical, and electrical guidance cues through a nanofibrous composite biopolymer would result in improved neuron growth metrics using 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.
Mechanical properties were evaluated under physiological conditions using nanofiber samples hydrated to equilibrium. Local elastic modulus was measured by fitting atomic force microscopy quantitative nanomechanical mapping data to the Sneddon model. The mean and standard error for Local Young's modulus was 74.93±12.6 kPa for HA nanofibers and 174.85±31.9 kPa for HA-CNT nanofibers. The electrochemical characterization performed was electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Conductivity and charge storage capacity of HA-CNT nanofibers were significantly increased. 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 charge balanced 150, 200, or 250mV/mm square wave. Neuron outgrowth after 72h was significantly longer on HA-CNT substrates electrically stimulated for 60min at all stimulus amplitudes versus all other groups (p < 0.01). Significant effects of fiber type, time, and stimulus amplitude were also observed when measuring neuron viability. 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 in vitro. 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.
|Advisor:||Sundararaghavan, Harini G.|
|Commitee:||Cheng, Mark Ming-Cheng, Matthew, Howard W.T., Ren, Wei-Ping|
|School:||Wayne State University|
|School Location:||United States -- Michigan|
|Source:||DAI-B 80/02(E), Dissertation Abstracts International|
|Subjects:||Biomedical engineering, Biomechanics, Materials science|
|Keywords:||Biomaterial, Carbon nanotubes, Conductive nanofibers, Hyaluronic acid, Neurons, Tissue engineering|
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