(585ad) Effect of Electrical Stimulation on Nerve Cells As a Function of Hydrogel Stiffness and Electrical Conductivity with a Custom Designed Device

Electrical stimulation of nerve cells has shown promising results related to cell migration, alignment and neurite outgrowth, which can be used to promote and guide axon regeneration across an injury. Polymer scaffolds are also used to direct and support nerve regeneration. The goal of this study was to design a device for electrically stimulating nerve cells seeded on a polymeric substrate and test the combined effect of substrate properties and electrical stimulation on cell alignment as a means to achieve directional cell growth.

Here, we designed a custom device for electrically stimulating neural cells seeded on a hydrogel. Polyacrylamide (PA) hydrogels were prepared by first mixing bis-acrylamide and acrylamide in various ratios for gels of varying stiffness. Then, 0.1% w/v Irgacure 2959 was added and the solution was degassed for 60 min. Hydrogels were formed by exposure to 365 nm UV light for 10 min. 4-arm polyethylene glycol acrylate (4-arm PEG-Ac)-carbon nanotube (CNT) gels (7.5% and 20% w/v in PEG and 0.05% and 0.1% w/v in CNTs) were prepared. PEG-dithiol was used as a crosslinker (thiol and acrylate were kept at 1:1 molar ratio). Bovine serum albumin (0.25% w/v) was used as a surfactant to aid CNT dispersion. PC12 cells were seeded on top of the gels at a density of 5×10⁴ cells/cm² for 24 h, stimulated with a direct current at 0.07 mA and voltage of 6 V/cm for 2 h and imaged at 24 h post-stimulation. MATLAB was used to simulate electrical current density, direction, and distribution through the hydrogels.

We first determined the effect of electrode geometry and positioning on electrical current density and distribution through the hydrogel. We determined that rectangular shape electrodes spaced 26 mm apart could uniformly distribute current density within the hydrogel. For the electrodes, we used biocompatible and cost-effective Ni-Cr alloys. Neural-like PC12 cells were seeded on polyacrylamide hydrogels of varying stiffness (2-70 kPa) as well as resistivity (0.08 to 1 Ωm) and stimulated for 2 h. Cells exhibited alignment along the applied current direction at 24 h post-stimulation, where the extent of alignment was dependent on hydrogel stiffness and resistivity. The number of PC12 cells aligned to the direction of applied current at 60 ± 10 was most frequent on soft hydrogels ranging from 2-10 kPa with higher conductivity. We also studied the alignment of PC12 cells on PEG-CNT nanocomposite gels of various stiffness and resistivity. Cells showed more uniform alignment on soft substrates with higher CNT concentration (i.e. higher conductivity).

In this study, we designed a custom device to electrically stimulate cells seeded on a hydrogel substrate. We optimized electrode geometry and spacing to achieve a uniform current density and distribution through the substrate. We applied the device towards the stimulation of neural-like PC12 cells on hydrogels of varying stiffness and electro-conductivity. We showed that cells aligned along the direction of the applied current at 24 h post-stimulation and that cell alignment was inversely proportional to substrate stiffness and proportional to substrate conductivity.