Accelerating Skin Wound Healing in vitro - Establishment of an External Electrical Field System for Stimulating Keratinocytes

Abstract

There is developing proof that skin wound healing can be affected by external electrical fields (EFs). This study pointed toward researching the impact of EFs (200 mV/mm) on the cell migration, proliferation, and secretion of healing-associated factors of HaCaT cells (a human skin-derived cell line), as these are fundamental stages of wound healing. We established an experimental ES system that can distribute EFs with a strength of 200V/mm to HaCaT cells, meanwhile keeping a steady cell culture climate. Compared to the works of pioneers, the newly designed system had a more straightforward structure. Moreover, it could transmit identical EF signals to up to four parallel wells, which significantly increased the reliability of the system and the reproducibility of the experiments. The newly designed system was applied to experiments. The scratch wound closure assay was performed to evaluate HaCaT cell migration changes after exposure to EFs. Cell proliferation was assessed by calcein AM and Hoechst staining, resazurin assay, SRB staining, and DNA quantification. Expression of growth factors with the potential to assist in healing was assessed by cytokine array assay, and Western blot was performed to examine the possible signaling pathway molecules involved in responding to EF signals (Lu et al., 2021). The result showed that, after 72 h of monitoring, the pH value and temperature change in the newly designed ES system was acceptable, which means a stable cell culture environment could be kept between two medium changes (Lu et al., 2021). When contrasted with the non-ES groups, the migration rate of the cells fundamentally expanded (up to 46.51%, p < 0.05) after 36 hours of continuous stimulation. For cell proliferation, with the application of EFs, the uptrends of the indicators above are more pronounced during the 7-day experimental period. The mitochondrial activity (up to 26.7%, p < 0.05), total protein content (up to 21.4%, p < 0.05) and total DNA content (up to 17.2%, p < 0.05) on day 7 in the ES groups increased significantly when compared with the non-ES groups (Lu et al., 2021). The results of the cytokine array demonstrated that several growth factors were upregulated after exposure to EFs, with significant increases in GM-CSF (up to 328.07%, p < 0.05) and PDGF-AB (up to 167.31%, p < 0.05), which are emerging as crucial players in the wound healing process and tissue reconstruction. To further investigate the potential molecular mechanism of the response of HaCaT cells to ES, phosphorylation levels of p-ERK1/2, p-P38, p-90RSK, and p-27HSP were investigated via Western blot after 30-, 60-, 90-, and 120-min exposures to EFs, and the results showed the Western blot signals for p-ERK1/2 and p-P38 increased significantly. To confirm the role of ERK 1/2 in migration, we treated the HaCaT cells with signaling inhibitors of ERK1/2i (U0126) and P38i (SB203580), and the migration assay showed that ERK1/2i could significantly inhibit EF-induced migration. In contrast, inhibition of P38 did not show a noticeable influence on the migration of HaCaT cells, which suggested that EFs improved the migration of HaCaT cells by phosphorylating ERK1/2 signaling (Lu et al., 2021). In this study, our study demonstrates that 200 mV/mm EFs have a solid potential to accelerate wound healing by improving the migration and proliferation of keratinocytes, as well as optimizing the healing-related microenvironment, which provides an experimental basis for clinical workers to understand and apply EFs for wound healing acceleration (Lu et al., 2021). Although research on mechanisms still has a long path to explore, we do not doubt that, soon, we will witness exciting developments in ‘electrotherapy’ in the field of wound healing or even tissue engineering.