Background. The worldwide increasing number of patients suffering from nonhealing wounds requires the development of new safe strategies for wound repair. Recent studies suggest the possibility of nonthermal (cold) plasma application for the acceleration of wound closure. Methods. An in vitro wound healing model with upper airway S9 epithelial cells was established to determine the macroscopically optimal dosage of tissue-tolerable plasma (TTP) for wound regeneration, while a 2D-difference gel electrophoresis (2D-DIGE) approach was used to quantify the proteomic changes in a hypothesis-free manner and to evaluate the balance of beneficial and adverse effects due to TTP application. Results. Plasma doses from 30 s up to 360 s were tested in relation to wound closure after 24 h, 48 h, 72 h, 96 h, and 120 h, in which lower doses (30, 60, and 120 s) resulted in dose-dependent improved wound healing rate compared to untreated cells. Thereby, the 120 s dose caused significantly the best wound healing properties after 96 and 120 h. The proteome analysis combined with IPA revealed that a lot of affected stress adaptation responses are linked to oxidative stress response emphasizing oxidative stress as a possible key event in the regeneration process of epithelial cells as well as in the adaptation to plasma exposure. Further cellular and molecular functions like proliferation and apoptosis were significantly up- or downregulated by all TTP treatments but mostly by the 120 s dose. Conclusions. For the first time, we were able to show plasma effects on cellular adaptation of upper airway epithelial S9 cells improving wound healing. This is of particular interest for plasma application, for example, in the surgery field of otorhinolaryngology or internal medicine.

The promising potential of cold atmospheric plasma (CAP) treatment as a new therapeutic option in the field of medicine, particularly in Otorhinolaryngology and Respiratory medicine, demands primarily the assessment of potential risks and the prevention of any direct and future cell damages. Consequently, the application of a special intensity of CAP that is well tolerated by cells and tissues is of particular interest. Although improvement of wound healing by CAP treatment has been described, the underlying mechanisms and the molecular influences on human tissues are so far only partially characterized. In this study, human S9 bronchial epithelial cells were treated with cold plasma of atmospheric pressure plasma jet that was previously proven to accelerate the wound healing in a clinically relevant extent. We studied the detailed cellular adaptation reactions for a specified plasma intensity by time-resolved comparative proteome analyses of plasma treated vs. nontreated cells to elucidate the mechanisms of the observed improved wound healing and to define potential biomarkers and networks for the evaluation of plasma effects on human epithelial cells. K-means cluster analysis and time-related analysis of fold-change factors indicated concordantly clear differences between the short-term (up to 1 h) and long-term (24-72 h) adaptation reactions. Thus, the induction of Nrf2-mediated oxidative and endoplasmic reticulum stress response, PPAR-alpha/RXR activation as well as production of peroxisomes, and prevention of apoptosis already during the first hour after CAP treatment are important cell strategies to overcome oxidative stress and to protect and maintain cell integrity and especially microtubule dynamics. After resolving of stress, when stress adaptation was accomplished, the cells seem to start again with proliferation and cellular assembly and organization. The observed strategies and identification of marker proteins might explain the accelerated wound healing induced by CAP, and these indicators might be subsequently used for risk assessment and quality management of application of nonthermal plasma sources in clinical settings.