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Der Einsatz von atmosphärischem Niedertemperaturplasma zur Behandlung chronischer Wunden stellt eine erfolgversprechende, in Entwicklung befindliche Therapieoption dar. Dabei wird von der Hypothese ausgegangen, dass chronische Wunden durch die Stimulation mittels Plasma aktiviert werden können, wodurch die über die Stufe der resorptiven Inflammation die physiologische Heilung wieder in Gang gesetzt werden kann. Daher wurde zur Identifizierung des Reiz- und Inflammationspotential des Plasmas und zur Ableitung geeigneter Behandlungsparameter Plasma im HET-CAM geprüft. Zunächst wurden mit einem Atmosphärendruck Plasma-Jet unter Verwendung von Argongas und einer Dielectric-Barrier Discharge (DBD)-Plasmaquelle die Reizwirkungen an der CAM durch mäanderförmige Plasmabehandlung und punktuelle Anwendung getestet. Dabei wurden neben mäanderförmigen und punktuellen Applikationsmustern unterschiedliche Anwendungsfrequenzen und damit unterschiedliche Plasmadosen untersucht. Die Temperatur des Plasmas nahm v. a. bei längeren Behandlungszeiten Einfluss und führte zu einer Wirkungsverstärkung bis hin zu thermischen Schädigungen. Der Gasfluss erwies sich als limitierend für das geprüfte Modell. Der gepulste Modus war bei mäanderförmiger Anwendung am verträglichsten. Die Überprüfung auf Reversibilität der Effekte ergab, dass geringe Reizungen vollständig reversibel waren, währen schwere Reizungen zu nicht reversiblen Koagulationen führten. Aufgrund der unbefriedigenden Ergebnisse mit sehr schneller Bildung von Thrombosen, geringem Arbeitsabstand und fehlender Potentialfreiheit wurde die DBD-Elektrode von der weiteren Untersuchung ausgenommen und der kINPen09 als Weiterentwicklung des Plasma-Jets mit geringerer Plasmatemperatur auf inflammatorische Wirkung bei punktueller Anwendung mit verschiedenen Expositionszeiten im kontinuierlichen Modus und im gepulsten Modus mit Argon als Trägergas, sowie im kontinuierlichen Modus unter Zusatz von 0,1 % Sauerstoff, getestet. In dieser Testreihe war ebenfalls das gepulste Plasma am verträglichsten, das mit Sauerstoff angereicherte Plasma führte zu den schwersten Inflammationen. Alle Reaktionen wurden durch die kombinierte Anwendung mit Hydrocortison deutlich abgeschwächt. Mit der Inflammationsinduktion konnten Gewebeprozesse in Form von Kontraktion, Koagulation und inflammationsassoziierter Angiogenese auf der CAM erzeugt werden, die für Aktivierung der sekundären Wundheilung relevant sein dürften. Ableitend aus den Untersuchungen kann für die Anwendung an chronischen Wunden eine Behandlungszeit von maximal 5 s pro qcm Wundfläche für den kontinuierlichen Modus empfohlen werden. Dabei sollte wegen der besseren Verträglichkeit der mäanderförmigen Behandlung gegenüber der punktuellen Anwendung der Vorzug gegeben werden. Unter Berücksichtigung der vorliegenden Untersuchung zur Reiz- und Inflammationswirkung von Plasma im HET-CAM und weiteren Prüfungen zur Wirksamkeit und Verträglichkeit in vivo und in vitro erweist sich die Plasmaapplikation mit dem kINPen09 als vielversprechende Option zur Behandlung chronischer Wunden.
Non-healing wounds pose a major burden to patients and health care systems alike. These wounds are chronically stuck in the inflammatory phase of the healing process without transitioning to the proliferative phase. They are also characterized by the excessive presence of leukocytes which are assumed to provoke the persistent inflammation observed in pathological wound healing. Recent studies suggested a beneficial role of cold physical plasma in the treatment of chronic wounds. Hence, it was the central question, whether exposure to cold physical plasma would affect the viability and/or function of human leukocytes. Cold plasma displays various properties of which the generation of reactive molecules, such as reactive oxygen and nitrogen species (ROS/RNS), where found to be central in mediating redox changes in leukocytes. Oxidative stress was present especially in lymphocytes that readily underwent apoptosis after exposure to plasma. This was largely a direct consequence of plasma-generated hydrogen peroxide but not superoxide or RNS. Amount of apoptosis was comparable among several lymphocyte subpopulations, with the wound healing-relevant γδ T cells being least affected. Lymphocyte apoptosis was accompanied by mitochondrial membrane depolarization, caspase 3 activation, DNA fragmentation, and phosphatidylserine exposure. These results are in line with previous characterizations of the intrinsic apoptotic pathway in redox biology, and suggest that plasma-induced apoptosis was not mediated by alternative molecular mechanisms. An important immune response mechanism, the proliferation of lymphocytes, was not interrupted in plasma-treated but non-apoptotic cells. In wounds, a central role of leukocytes is to orchestrate the healing response via the release of small communication molecules called cytokines. Non-healing wounds are associated with elevated amounts of pro-inflammatory IL-1β, IL-6, and TNFα, and plasma-treatment of leukocytes strongly decreased their concentrations. At the same time, the expression of anti inflammatory cytokines (IL-10, TGFβ) was markedly increased. The pro inflammatory chemokine IL-8 was the only molecule to be significantly increased in supernatants of plasma-treated cells. IL-8 is the major chemo-attractant for neutrophil granulocytes. Neutrophils are frequently associated with non-healing wounds. These professional phagocytes are the first to migrate to the site of injury where they inactivate invading pathogens by various mechanisms. Importantly, highly relevant effector functions remained mostly unaffected by plasma treatment: the phagocytosis of bacteria, the oxidative burst, and the intracellular killing of microbes. Of note, plasma induced a strong induction of neutrophil extracellular traps (NETs). Decorated with antimicrobial proteins, NETs are web-like chromatin extrusions that entrap pathogens. These results have several implications for wound healing. Plasma-treated neutrophils were still capable of eradicating bacteria, which are frequently associated with non-healing wounds. In addition, plasma-induced NETs could aid in wound healing by providing an antibacterial scaffold to safeguard against further dissemination of microorganisms. Chronic wounds display a state of sustained inflammation and plasma induced apoptosis but not necrosis in lymphocytes. This was an important finding as necrosis, the involuntary cell death, is associated with the release of intracellular content, enhancing inflammation. By contrast, apoptosis dampens it as dead cells are cleared by macrophages inducing anti inflammatory responses. Further, the cytokine signature of plasma-treated leukocytes was largely non inflammatory, which could further decrease inflammation in wounds. Altogether, this work provided first insight with regard to effects and mechanisms of cold physical plasma treatment of wound-relevant leukocytes. Generally, these cells were affected by a plasma mediated modulation of their redox state. Future studies should include the possibility of redox modulation into their experimental approach to further elucidate the role of ROS/RNS in inflammation and possibly to improve existing wound healing therapies.
There is a growing interest in the application of non-thermal atmospheric pressure plasma for the treatment of wounds. Due to the generation of various ROS and RNS, UV radiation and electric fields plasma is a very promising tool which can stimulate skin and immune cells. However, not much is known about the mammalian cell responses after plasma treatments on a molecular level. The present work focusses on the impact of plasma on cell signaling in the human keratinocyte cell line HaCaT by using the methods DNA microarray, qPCR, ELISA and flow cytometry. Here, cell signaling mediators such as cytokines and growth factors which could promote wound healing by enhancing angiogenesis, reepithelization, migration and proliferation were of major interest. Additionally, the crosstalk between keratinocytes and monocytes was studied using a co-culture. For the first time extensive investigations on the impact of plasma on cell signaling in human keratinocytes were conducted. The most prominent cytokines and growth factors which were regulated by plasma at gene and protein level were VEGF-A, GM-CSF, HB-EGF, IL-8, and IL-6. The latter was not activated due to the JAK/STAT-pathway but probably by a combined activation of MAPK- and PI3K/Akt-pathways. By the use of conditioned medium it was found out that ROS and RNS generated directly after plasma treatment induced larger effects on cell signaling in keratinocytes than the subsequently secreted growth factors and cytokines. Furthermore, monocytes and keratinocytes hardly altered their secretion profiles in co-culture. From these results it is deduced that the plasma generated reactive species are the main actors during cell signaling. In order to differentiate the impact of ROS and RNS on the cellular response the ambience of the plasma effluent was controlled, varying the ambient gas composition from pure nitrogen to pure oxygen. Thereby a first step towards the attribution of the cellular response to specific plasma generated reactive species was achieved. While IL-6 expression correlated with ROS generated by the plasma source, the cell signaling mediators VEGF-A, GM-CSF and HB-EGF were significantly changed by RONS. Above all hydrogen peroxide was found to play a dominant role for observed cell responses. In summary, plasma activates wound healing related cell signaling mediators as cytokines and growth factors in keratinocytes. It was also shown that the generated reactive species mainly induced cell signaling. For the first time cell responses can be correlated to ROS and RONS in plasma treated cells. These results underline the potential of non-thermal atmospheric pressure plasma sources for their applications in wound treatment.