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.

Over the past few years, the application of cold atmospheric plasma (CAP) in medicine has developed into an innovative field of research of rapidly growing importance. One promising new medical application of CAP is cancer treatment. Different studies revealed that CAP may potentially affect the cell cycle and cause cell apoptosis or necrosis in tumor cells dependent on the CAP device and doses. In this study, we used a novel hand-held and battery-operated CAP device utilizing the surface micro discharge (SMD) technology for plasma production in air and consequently analysed dose-dependent CAP treatment effects on melanoma cells. After 2 min of CAP treatment, we observed irreversible cell inactivation. Phospho-H2AX immunofluorescence staining and Flow cytometric analysis demonstrated that 2 min of CAP treatment induces DNA damage, promotes induction of Sub-G1 phase and strongly increases apoptosis. Further, protein array technology revealed induction of pro-apoptotic events like p53 and Rad17 phosphorylation of Cytochrome c release and activation of Caspase-3. Interestingly, using lower CAP doses with 1 min of treatment, almost no apoptosis was observed but long-term inhibition of proliferation. H3K9 immunofluorescence, SA-ß-Gal staining and p21 expression revealed that especially these low CAP doses induce senescence in melanoma cells. In summary, we observed differences in induction of apoptosis or senescence of tumor cells in respond to different CAP doses using a new CAP device. The mechanism of senescence with regard to plasma therapy was so far not described previously and is of great importance for therapeutic application of CAP.

Cold atmospheric plasma (CAP) has been gaining increasing interest as a new approach for the treatment of skin diseases or wounds. Although this approach has demonstrated promising antibacterial activity, its exact mechanism of action remains unclear. This study explored in vitro and in vivo whether CAP influences gene expression and molecular mechanisms in keratinocytes. Our results revealed that a 2 min CAP treatment using the MicroPlaSter ß in analogy to the performed clinical studies for wound treatment induces expression of IL-8, TGF-ß1, and TGF-ß2. In vitro and in vivo assays indicated that keratinocyte proliferation, migration, and apoptotic mechanisms were not affected by the CAP treatment under the applied conditions. Further, we observed that antimicrobial peptides of the ß-defensin family are upregulated after CAP treatment. In summary, our results suggest that a 2 min application of CAP induces gene expression of key regulators important for inflammation and wound healing without causing proliferation, migration or cell death in keratinocytes. The induction of ß-defensins in keratinocytes describes an absolutely new plasma strategy. Activation of antimicrobial peptides supports the well-known antibacterial effect of CAP treatment, whereas the mechanism of ß-defensin activation by CAP is not investigated so far.

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