Cold plasma on full-thickness cutaneous wound accelerates healing through promoting inflammation, re-epithelialization and wound contraction

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Abstract

We investigated cold plasma effects on acute wounds of mice. The mice were classified into experimental and control groups. In the former, wounds were treated using cold plasma once daily for 1 min, and then covered with hydrocolloid dressing; wounds in the control were left to heal under hydrocolloid dressing. Daily evaluation was conducted for 15 days. General and specific staining was applied to evaluate re-epithelialization, neutrophil, macrophage, myofibroblast and transforming growth factor beta. It was found that cold plasma accelerated wound healing by 1 day. Plasma may promote the late phase of inflammation, accelerate re-epithelialization and increase wound contraction.

Introduction

Cutaneous wound healing is a complex physiological process consisting of orchestrated events communicated by collaborative factors [1]. The utilization of various exogenous agents from natural products like Indonesian honey [2] and oleic or linoleic acid [3] to physical tools like light [4] and laser [5] has been shown to enhance the overlapping healing phases, including inflammation, proliferation and remodeling [1]. Among these, wound therapy based on cold plasma, that is, non-equilibrium plasma (with an electron temperature much higher than the gas temperature), with a low temperature of ionized gas [6], has opened the possibility of a paradigm shift in biomedical therapy [7]; it has drawn substantial attention from both plasma and wound care scientists since its feasibility to work through living tissue [8], [9] and its potency for resolving problems in contemporary wound care were demonstrated [10]. As the fourth state of matter [6], plasma has the ability to produce controllable reactive species, like nitric oxide (NO) and hydroxyl radicals (OH), upon contacting the open air [11], as well as OH radicals and hydrogen peroxide (H2O2) upon contacting an aqueous solution [12]. Although the clinical efficacy of carefully controlled treatment with cold plasma for killing bacteria colonizing chronic wounds [13], [14] and improving wound healing [15] has been demonstrated, there have been few studies about the effects of cold plasma and its mechanism of action on acute wounds in mouse models.

Re-epithelialization and wound contraction are two key events in the healing of full-thickness wounds. The former is central to wound closure, which is closely connected to granulation tissue formation in a spatiotemporal manner [16], and the latter may account for up to a 40% decrease in wound size, correlated with the expression of myofibroblasts [17]. It is well established that these processes are influenced by the presence of growth factors like epidermal growth factor (EGF), keratinocyte growth factor (KGF) and transforming growth factor (TGF) [15], which are likely mediated by reactive oxygen species (ROS) and NO [18], [19], [20].

Although the mechanism of the interaction between cold plasma and cells or living tissue is still unclear [21], several studies have reported the effects of cold plasma on key wound-related cells or sub-cells, included promoting the proliferation of fibroblasts [22] and endothelial cells [23], as well as the growth of epithelial cells [24], inhibiting the migration of fibroblasts [25] and their surface expression [26], and activating integrin of fibroblasts and epithelial cells [27]. Interestingly, some of these effects are likely to be similar to the activities of natural ROS and/or NO during wound healing, particularly cold plasma׳s effects on the proliferation of both fibroblasts and endothelial cells [19]. Therefore, the aim of this study was to assess the effects of cold plasma on acute cutaneous wound healing in an in vivo scenario with a focus on re-epithelialization and wound contraction.

Section snippets

Cold plasma jet characterization and mouse wound positioning

The cold atmospheric pressure plasma jet system that we used here is similar to the device developed by Teschke et al. [28]. Two metal ring electrodes were used around the quartz tube for the cold atmospheric pressure plasma jet system provided by the Division of Electrical Engineering and Computer Science, Kanazawa University, Kanazawa, Japan. It had a quartz tube with a 1.6 mm inner diameter. A low-frequency (~20 kHz) AC high voltage, with a peak-to-peak voltage of 25 kV, was applied to the two

Macroscopic evaluation

Immediately after cold plasma irradiation, the wound surfaces in the experimental group seemed drier than those in the control group. Wounds were observed daily (Fig. 2). There were no particular differences regarding the appearance of the wound surfaces between the experimental and control groups during this observation. Apparent differences just in the wound size were found for several days of this observation period.

Wounds in both the experimental and the control groups experienced slight

Discussion

This research design separated between treated and untreated mice because it was considered that cold plasma produced not only reactive species like nitric oxide (NO) and hydrogen peroxide (H2O2) that in appropriate dosage may have efficacy for wound healing [20], but also that temperature change may have the same effect [30]. While the former may work in a locally specific manner [25], the latter may operate at a physiologically systematic level under a hypothalamic regime [31]. In this

Conflict of interest

The authors have no conflicts of interest to report in regard to this manuscript.

Acknowledgments

Nasruddin would like to acknowledge the help of the Directorate General of Higher Education (DIKTI), Indonesia, which supported him financially during his Ph.D. study through the Joint Scholarship Program DIKTI, Indonesia-Kanazawa University, Japan.

Part of this work was supported by Grants-in-Aid for Scientific Research, Japan (nos. 22592363 and 25293430) and a Grant from The Mitani Foundation for Research and Development.

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