Objective: The objective of this work was to test the synergistic effects of substrate stiffness, electro-conductivity, composition and electrical stimulation on the morphology, alignment and directional neurite outgrowth of neuron-like PC12 cells. The use of exogenous electrical stimulation has emerged as a promising new intervention to promote neural regeneration following injury. For critical gap size nerve injuries, a permissive biomaterial coupled to electrical stimulation may be needed to provide guidance and support for neurite outgrowth. Thus, the combinatorial effects of biomaterial composition and properties and exogenous electrical stimulation need interrogation to develop successful therapeutic interventions. Carefully designed in vitro models are ideally suited to perform such multidimensional detailed studies.
Approach: We assembled a simple electrical stimulation device to deliver uniform electrical current with minimum voltage field variation through a hydrogel. We used polyacrylamide (PA), polyethylene glycol (PEG), and multi-walled carbon nanotubes (MWCNT)-PEG nanocomposite hydrogels of varying stiffness, resistivity and MWCNT concentration. Cells were seeded on the substrates for 24 h, stimulated for 1 h at 30 V m-1 DC, and then cultured for additional 24 h. Non-stimulated cells were used as controls. To induce neurite outgrowth, cells were primed with nerve growth factor (100 ng ml-1).
Main results: For all substrates tested, electrical stimulation induced neurite alignment at 60-90° angle to the applied current. It also increased total neurite outgrowth by 18%-49% and mean neurite length by 20%-46% (increase dependent on the underlying substrate) compared to non-stimulated cells. The nanocomposite composed of 20% w/v PEG and 0.1% w/v MWCNTs resulted in the highest total neurite outgrowth and mean neurite length, which were further significantly enhanced by electrical stimulation by 2-fold and 1.8-fold, respectively.
Significance: Our results indicate that nanocomposites, where carbon nanotubes have been added to hydrogel substrates, in combination with electrical stimulation provided improved conditions for neural growth and regeneration.