Improvement of human keratinocyte migration by a redox active bioelectric dressing

Abstract

Exogenous application of an electric field can direct cell migration and improve wound healing; however clinical application of the therapy remains elusive due to lack of a suitable device and hence, limitations in understanding the molecular mechanisms. Here we report on a novel FDA approved redox-active Ag/Zn bioelectric dressing (BED) which generates electric fields. To develop a mechanistic understanding of how the BED may potentially influence wound re-epithelialization, we direct emphasis on understanding the influence of BED on human keratinocyte cell migration. Mapping of the electrical field generated by BED led to the observation that BED increases keratinocyte migration by three mechanisms: (i) generating hydrogen peroxide, known to be a potent driver of redox signaling, (ii) phosphorylation of redox-sensitive IGF1R directly implicated in cell migration, and (iii) reduction of protein thiols and increase in integrinαv expression, both of which are known to be drivers of cell migration. BED also increased keratinocyte mitochondrial membrane potential consistent with its ability to fuel an energy demanding migration process. Electric fields generated by a Ag/Zn BED can cross-talk with keratinocytes via redox-dependent processes improving keratinocyte migration, a critical event in wound re-epithelialization.

Conflict of interest statement

Competing Interests: The authors would like to declare that this work was partly supported by research funding from the manufacturer Procellera, Vomaris Innovations, by means of an unrestricted gift to The Ohio State University where the donor had no control over scientific experiments or reporting. Additional financial competing interest includes ownership of shares and paid consultancy. There is no board membership, employment or patent applications involved. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Characterization of the Ag/Zn BED.

(A, B) Schematic diagram of the design, application and electric fields generated by the bioelectric dressing, (C) EDS spectrum of a silver dot on a BED (D) EDS spectrum of a zinc dot on a BED. (E) Photographs of silver and (F) zinc dots of the BED mounted using carbon tape in an aluminum holder for energy dispersive spectroscopic (EDS) X-ray analysis. (G) Scanning electron microscope image of a silver dot on a BED (H) Scanning electron microscope image of a zinc dot on a BED.

Figure 2. Experimental determination of electric fields generated by the bioelectric dressing.

(A) Voltage measurements from an experiment with a Zn (99.994% pure) and silver (99.998% pure) foil immersed in 100 mL of de-ionized water. (B) Schematic showing the computational grid and boundary conditions used to calculate the magnitude and extent of the electric fields under the bioelectric dressing presumed to be in contact with H2O. (C, D) Contour plots of calculated values of the electric potential for 0.2 V and 1 V potential difference, respectively. (E, F) Electric fields from the solution of the 2-D Laplace equation for the particular case of 0.2 V and 1 V potential difference, respectively between the silver and the zinc dots. (G,H) Expanded view of the electric field vectors between adjacent pairs of electrodes for potential difference of 0.2 V and 1 V respectively.

Figure 3. Increased rate of migration of human keratinocytes under an Ag/Zn bioelectric dressing (BED).

(A) HaCaT cells were treated with Ag/Zn BED or polyester printer printed with only silver or only zinc for 24 h followed by scratch assay and migration of cells was observed at 6 h and 9 h following scratch. (B) % closure at 6 h, (C) % closure at 9 h. BED significantly increased rate of migration while placebo or silver alone or zinc alone did not change cell migration. (n = 4).

Figure 4. Increased generation of H2O2 under the effect of a Ag/Zn bioelectric dressing.

(A) H2O2 production on immersing placebo/BED in PBS for 1 hr.H2O2 production increases with increase in the no. of Ag/Zn couples and attenuated on inactivating BED by boiling for 15 mins (n = 3). (B) HaCaT cells loaded by 5 µM PF6 (H2O2 indicator) showed increase in green fluorescence indicating generation of intracellular H2O2 (n = 3). (C–D) Ad-Catalase (Ad-Catal) attenuates Ag/Zn BED induced faster cell migration (n = 4). (E–F) N-acetyl cysteine attenuates Ag/Zn BED induced faster cell migration (n = 4).

Figure 5. Ag/Zn bioelectric dressing energizes mitochondria in keratinocytes.

(A) After 24 h of treatment with Ag/Zn BED, HaCaT cells were stained with JC-1 dye and analyzed using a flow cytometer (n = 3). (B) After 24 h of treatment with Ag/Zn BED, HaCaT cells were stained with TMRM and PMPI and fluorescence was observed using a Zeiss microscope. Increased relative mitochondrial membrane potential was observed under the Ag/Zn BED indicated by higher red fluorescence (n = 3).[TMRM = Tetramethylrhodamine methyl ester; PMPI = plasma membrane potential indicator] (C) Increased glucose uptake in Ag/Zn BED treated HaCaT cells (n = 3).

Figure 6. Increase in phosphorylation of IGF1 receptor under Ag/Zn bioelectric dressing.

(A) Receptor tyrosine kinase phosphorylation array was performed on lysate from HaCaT cells treated with Ag/Zn BED for 24 h. Black boxes denote housekeeping controls, blue box denotes IGF1R. (B–C) Template for receptor tyrosine kinase phosphorylation assay.

Figure 7. BED induces IGF1 receptor phosphorylation which helps in migration.

(A) Validation of increased phosphorylation of IGF1R using ELISA. (B–C) IGF1R inhibitor attenuates Ag/Zn BED induced faster keratinocyte migration (n = 3).

Figure 8. Ag/Zn BED increases integrinαV expression and protein thiol in keratinocytes but does not increase glutathione.

(A–C) Flow cytometry analysis demonstrates increase in fluorescence in HaCaT cells loaded with monobromobimane (MBB). (D) No significant change in fluorescence was observed in HaCaT cells loaded with monochlorobimane (MCB) (n = 4). (E) Integrinαv expression at the scratch edge under placebo or Ag/Zn BED 6 h post scratch (n = 3).

Figure 9. Summary figure.