Galvanic microparticles increase migration of human dermal fibroblasts in a wound-healing model via reactive oxygen species pathway

Highlights

• A 3-layer skin model (fibroblasts, hydrogel, galvanic particles/hydrogel) was studied.

• Wound closure speed, reactive oxygen species (ROS), and gene expression were explored.

• Data suggest ROS-mediated enhancement of fibroblast migration via BMP/SMAD signaling.

• Results imply that topical electric currents via microparticles may enhance wound healing.

Abstract

Electrical signals have been implied in many biological mechanisms, including wound healing, which has been associated with transient electrical currents not present in intact skin. One method to generate electrical signals similar to those naturally occurring in wounds is by supplementation of galvanic particles dispersed in a cream or gel. We constructed a three-layered model of skin consisting of human dermal fibroblasts in hydrogel (mimic of dermis), a hydrogel barrier layer (mimic of epidermis) and galvanic microparticles in hydrogel (mimic of a cream containing galvanic particles applied to skin). Using this model, we investigated the effects of the properties and amounts of Cu/Zn galvanic particles on adult human dermal fibroblasts in terms of the speed of wound closing and gene expression. The collected data suggest that the effects on wound closing are due to the ROS-mediated enhancement of fibroblast migration, which is in turn mediated by the BMP/SMAD signaling pathway. These results imply that topical low-grade electric currents via microparticles could enhance wound healing.

Introduction

Electrical signals regulate the behavior of excitable cells including those found in skin, heart, skeletal muscle, neural and vascular tissue. These signals are essential to a range of biological processes, including vision, hearing, heartbeat, and digestion [1]. Furthermore, transient bioelectrical signals have been implicated in cell proliferation, migration and differentiation during embryogenesis, wound healing, and tissue regeneration [2].

Electric fields were detected within endogenous wounds more than 150 years ago [3]. During wound healing, outward electrical currents of 10–100 µA/cm² create a voltage drop of ~600 mV/cm within the first 125 µm of the extracellular space [4]. These fields are due to a short-circuiting of the transepithelial potential gradient, which arises due to separation of ionic charges across the intact epithelium. When the skin is cut, a large steady electric field arises immediately and persists for hours at the wound edge, as currents pour out from underneath the wounded epithelium [5].

Many types of cells [6], [7], including some skin cell types (e.g., fibroblasts [8], [9], [10] and keratinocytes [11], [12] but not melanocytes [13]), exhibit directional migration (i.e., galvanotaxis) when exposed to direct currents of physiologically relevant magnitudes. These effects have implicated that wound-generated electric fields provide migrational cues that contribute to wound healing (see [14] for a review), and motivated the application of exogenous electric fields to enhance the healing of chronic wounds, in clinical practice for many decades [15], [16].

Some of the new treatment modalities currently under research include topical application of galvanic particles dispersed in cream or gel. In one application, zinc micro-particles were deposited with copper specks and used to deliver low-level electrical stimulation to intact skin and primary keratinocytes [17]. Topical application on intact skin was shown to generate an electric potential of 1.5 V/cm, and application to primary keratinocytes in culture enhanced the production of reactive oxygen species (ROS), and inhibited secretion of inflammatory cytokines. These results are quite interesting when considering that after the initial phase of wound healing, inflammation is reduced, and cellular interactions are dominated by the interplay of keratinocytes and fibroblasts [18]. In fact, during skin wound healing, keratinocytes, which make up the predominant cell type in the epidermis, stimulate fibroblasts to synthesize growth factors, which in turn stimulates keratinocyte proliferation in a double paracrine manner [19].

Starting from this evidence, we were interested in exploring the mechanism of action of galvanic microparticles on human dermal fibroblasts. We sought the putative mediator between the microparticles and the cells, which could trigger responses promoting/hindering wound healing. For example, it is known that reactive oxygen species (ROS) are potent effectors in directing cell fate. It is likely that the galvanic microparticles could generate microcurrents capable of inducing or mimicking ROS production. We thus hypothesized that these microcurrents will enhance the migration of dermal fibroblasts, and thereby accelerate wound healing, via increased production of ROS. To test this hypothesis, we treated adult dermal fibroblasts with low concentrations of zinc–copper galvanic microparticles, in a model system designed to mimic the skin dermis, epidermis, and the topical cream. We observed a ROS-mediated increase in fibroblast migration, and determined that this increase was induced by a BMP/Smad signaling pathway.