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. 2013 Nov 12;8(11):e79325.
doi: 10.1371/journal.pone.0079325. eCollection 2013.

Cold atmospheric plasma (CAP) changes gene expression of key molecules of the wound healing machinery and improves wound healing in vitro and in vivo

Stephanie Arndt  1 , Petra Unger, Eva Wacker, Tetsuji Shimizu, Julia Heinlin, Yang-Fang Li, Hubertus M Thomas, Gregor E Morfill, Julia L Zimmermann, Anja-Katrin Bosserhoff, Sigrid Karrer
Affiliations

Cold atmospheric plasma (CAP) changes gene expression of key molecules of the wound healing machinery and improves wound healing in vitro and in vivo

Stephanie Arndt et al. PLoS One. .

Abstract

Cold atmospheric plasma (CAP) has the potential to interact with tissue or cells leading to fast, painless and efficient disinfection and furthermore has positive effects on wound healing and tissue regeneration. For clinical implementation it is necessary to examine how CAP improves wound healing and which molecular changes occur after the CAP treatment. In the present study we used the second generation MicroPlaSter ß® in analogy to the current clinical standard (2 min treatment time) in order to determine molecular changes induced by CAP using in vitro cell culture studies with human fibroblasts and an in vivo mouse skin wound healing model. Our in vitro analysis revealed that the CAP treatment induces the expression of important key genes crucial for the wound healing response like IL-6, IL-8, MCP-1, TGF-ß1, TGF-ß2, and promotes the production of collagen type I and alpha-SMA. Scratch wound healing assays showed improved cell migration, whereas cell proliferation analyzed by XTT method, and the apoptotic machinery analyzed by protein array technology, was not altered by CAP in dermal fibroblasts. An in vivo wound healing model confirmed that the CAP treatment affects above mentioned genes involved in wound healing, tissue injury and repair. Additionally, we observed that the CAP treatment improves wound healing in mice, no relevant side effects were detected. We suggest that improved wound healing might be due to the activation of a specified panel of cytokines and growth factors by CAP. In summary, our in vitro human and in vivo animal data suggest that the 2 min treatment with the MicroPlaSter ß® is an effective technique for activating wound healing relevant molecules in dermal fibroblasts leading to improved wound healing, whereas the mechanisms which contribute to these observed effects have to be further investigated.

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Conflict of interest statement

Competing Interests: Shimizu and Morfill are holders of the patent for the MicroPlasTer ß®. The MicroPlasTer ß® was developed by the Max-Planck-Institute for Extraterrestrial Physics (MPE) and ADTEC Plasma Technology Co. Ltd., Hiroshima and the MPE provided the MicroPlaSter ß®. There are no further patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Figure 1
Figure 1. Expression of Cytokines and Growth Factors after CAP treatment.
(a I–III) mRNA expression analysis of IL-6, IL-8 and MCP-1 performed 6 h, 24 h, 48 h and 72 h after CAP treatment for 2 min by LightCycler® 1.2 technology. (b I–III) Protein expression analysis of IL-6, IL-8 and MCP-1 24 h, 48 h and 72 h after CAP treatment for 2 min by FlowCytomix™ Technology. (c I–II and d I–II) Expression profile of TGF-ß1 and TGF-ß2 on mRNA level analyzed by LightCycler® 1.2 technology and on protein level analyzed by ELISA technique is shown in accordance to the above mentioned time points after CAP treatment for 2 min. *p<0.05; **p<0.01; ***p<0.001; ns: not significant.
Figure 2
Figure 2. Fibroblast migration, proliferation and ECM production after CAP treatment.
(a I) A wound healing assay shows that CAP treatment for 30 sec promotes fibroblast migration. Representative images are shown immediately after culture insert was removed (0 h) and 12 h and 22 h later. (a II) The migration rate was calculated 12 h and 22 h after culture insert was removed and is displayed as the percentage relative to untreated control (ctr. set 100%). The results are measurements of the “wound area” from at least four separate visual fields from three separate experiments. (b) Cell proliferation was determined using XTT proliferation assay 24 h, 48 h and 72 h after CAP exposure for 2 min. (c I–II) mRNA expression analysis of collagen type I and alpha-SMA was determined 6 h, 24 h, 48 h and 72 h after CAP treatment for 2 min by LightCycler® 1.2 technology. *p<0.05; **p<0.01; ***p<0.001; ns: not significant.
Figure 3
Figure 3. Wound healing after CAP therapy in a mouse wound healing model.
(a I) Schematic overview of the wound generation in 129/Sv/Ev mice. (a II) CAP treatment of wounds with the MicroPlaSter ß® device (2 min daily); control mice were placebo-treated. (b I) Representative photographs of placebo or CAP treated wounds at the indicated days after wounding. (b II) Quantification of the wound area at the indicating times after wounding. Results represent the mean+/− s.e.m; n = 4 for each time point. *p<0.05; **p<0.01. Representative examples of (c) CD68 and (d) Ly6G immunohistological staining of skin wounds 5 days after wounding with 5 × placebo or 5 × CAP therapy. The amount of neutrophils was determined by counting the positive stained cells per 4 different fields of view in magnification as indicated (e) mRNA expression of MCP-1, IL-6, IL-8, TGF-ß1 and TGF-ß2 in wound tissue on day 5 after wounding after 5 × placebo or 5 × CAP therapy. *p<0.05; **p<0.01; ns: not significant; nd: not detectable. Representative examples of H&E (f I–II), Sirius Red/Fast Green (f III–IV) and alpha-SMA (f V–VI) staining of skin wounds 15 days after wounding with 10 × placebo or with 10 × CAP therapy. Magnification as indicated. (g) Dermal mRNA expression of collagen type I and alpha-SMA 15 days after wounding with 10 × CAP therapy compared to placebo control. *p<0.05; **p<0.01.
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Grants and funding

The MicroPlasTer ß® was developed by the Max-Planck-Institute for Extraterrestrial Physics (MPE) and ADTEC Plasma Technology Co. Ltd., Hiroshima and the MPE provided the MicroPlaSter ß®. ADTEC Plasma Technology Co. Ltd. did not participate in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The Max-Planck-Institute for Extraterrestrial Physics contributed to the study design, data collection and analysis, decision to publish, and preparation of the manuscript, but had no financial interest concerning the study.