Influence of Biphasic Stimulation on Olfactory Ensheathing Cells for Neuroprosthetic Devices

Abstract

The recent success of olfactory ensheathing cell (OEC) assisted regeneration of injured spinal cord has seen a rising interest in the use of these cells in tissue-engineered systems. Previously shown to support neural cell growth through glial scar tissue, OECs have the potential to assist neural network formation in living electrode systems to produce superior neuroprosthetic electrode surfaces. The following study sought to understand the influence of biphasic electrical stimulation (ES), inherent to bionic devices, on cell survival and function, with respect to conventional metallic and developmental conductive hydrogel (CH) coated electrodes. The CH utilized in this study was a biosynthetic hydrogel consisting of methacrylated poly(vinyl-alcohol) (PVA), heparin and gelatin through which poly(3,4-ethylenedioxythiophene) (PEDOT) was electropolymerised. OECs cultured on Pt and CH surfaces were subjected to biphasic ES. Image-based cytometry yielded little significant difference between the viability and cell cycle of OECs cultured on the stimulated and passive samples. The significantly lower voltages measured across the CH electrodes (147 ± 3 mV) compared to the Pt (317 ± 5 mV), had shown to influence a higher percentage of viable cells on CH (91-93%) compared to Pt (78-81%). To determine the functionality of these cells following electrical stimulation, OECs co-cultured with PC12 cells were found to support neural cell differentiation (an indirect measure of neurotrophic factor production) following ES.

Keywords: PEDOT; electrical stimulation of nervous system; living electrodes; neural interfaces; olfactory ensheathing cells.

Figure 1

The individual and assembled components of the electrical stimulation rigs with Pt and CH coated electrode substrates.

Figure 2

A schematic of a cathodic first biphasic current and transient voltage waveforms.

Figure 3

The total voltage (V_t) and the residual voltage (E_m) at the of the cathodic phase for both the CH and Pt electrode systems. Error bars represent one standard deviation. (t-test, ^****significant difference, p ≤ 0.0001, n = 3).

Figure 4

The estimated OEC density on each sample as determined by the cell concentration measured with the Tali. Error bars represent the standard error of the mean. (t-test, ^*significant difference, p < 0.05, N = 3).

Figure 5

Relative viable, necrotic, and apoptotic cell populations. Error bars represent standard deviation. (Tukey, Two-way ANOVA multiple comparisons test, ^*p < 0.05, n = 3).

Figure 6

The relative distribution of OECs cultured on Pt and CH electrodes found to be in the cell cycle phases G0/G1, S, and G2/M. Cell health controls in serum deprived media and full serum media with the addition of aphidicolin (S phase control) and nocodazole (G2/M phase control). Error bars represent the standard error of the mean.

Figure 7

Quantification of PC12 cell attachment when cultures with and without OECs on passive and electrically stimulated Pt and CH electrodes. Statistical analysis was in the form of Tukey one-way ANOVA, ^*p < 0.05. Error bars represent the standard error of the mean.

Figure 8

Quantification of neurite outgrowth density for PC12 cells cultures with and without OECs on passive and electrically stimulated Pt and CH electrodes. Statistical analysis was in the form of Tukey one-way ANOVA. ^*Shows significant differences for p < 0.05. Error bars represent the standard error of the mean.

Figure 9

Magnified immunofluorescence images of PC12 cultures on passive and electrically stimulated Pt and CH substrates. Nuclei were stained with Hoechst 33,342 (blue) and PC12 cell bodies and neurites stained with anti-βIII-tubulin (red). Scale bars = 150 μm.

Figure 10

Magnified immunofluorescence images of PC12 and OEC co-cultures on passive and electrically stimulated Pt and CH substrates (200 × magnification). Nuclei of both cell types were stained with Hoechst 33,342 (blue) and PC12 only cell bodies and neurites stained with anti-βIII-tubulin (red). Scale bars = 150 μm.