Experimental model for functional evaluation of the effects of electrical stimulation in human dermal fibroblasts

Abstract

Skin injuries originate small electric currents due to a potential difference between epithelial layers, generating electric fields (EFs) that trigger wound healing by directing cell migration towards the wound. Exogenous EFs can accelerate wound closure via increased fibroblast migration rates, gene expression, and growth factor secretion. However, optimal parameters for electrical stimulation (ES) have not been established; and, although it is known that healing outcomes vary depending on the waveform employed, alternating current (AC) remains unexplored. An experimental model for simultaneous ES of cell cultures has been developed, facilitating the systematic evaluation of ES parameters. Primary human dermal fibroblasts were subjected to AC intensities of 10, 25, 50, 100, 150, 200, and 300 mV mm−1 at 1 Hz for 12 hours; the EF was monitored and regulated using MATLAB. A scratch assay was used to quantify cell migration, with microscope images taken pre-and post-ES. Cellular markers for cytotoxicity, metabolic activity, and apoptosis were measured using colorimetric or fluorometric assays whilst cell cycle was analysed by f luorescence activated cell sorting. Microscope images processed using ImageJ revealed slightly increased migration speed in stimulated fibroblasts (p < .1); however, higher EF intensities also resulted in considerable cell loss. While lactate dehydrogenase levels confirmed high cytotoxicity (p < .005), caspase 3/7 activation was not significant (p > .5), discarding apoptosis as the main mechanism of cell death. Cell cycle remained unaltered (p > .1), and interestingly, cell metabolism was upregulated after ES according to a resazurin-based assay. Nonetheless, the impact of these findings requires further research. This is the first systematic exploration of AC effects in human dermal fibroblasts. Different waveforms are to be studied in order to profile optimal ES parameters to enhance wound healing. The outcome of this research will expand the current knowledge on the effect of different electrical waveforms on skin cells and may lead to the development of more effective novel therapies for wound healing.

Date of Award	31 Dec 2018
Original language	English
Awarding Institution	The University of Manchester
Supervisor	M.T. Alonso-Rasgado (Supervisor) & Ardeshir Bayat (Supervisor)