Einleitung
Die Heilung einer Wunde unterscheidet sich in der Narbenbildung, dem Einfluss von Risikofaktoren, dem funktionellen und Ă€sthetischen Ergebnis und in der Wundheilungsdauer. Eine Vielzahl wissenschaftlicher Studien beschĂ€ftigt sich mit Möglichkeiten, die Dauer der Wundheilung zu verkĂŒrzen. Dabei fĂ€llt auf, dass bisher keine klinischen Daten zur Wundheilungsdauer existieren, insbesondere keine, die die Dauer der Re-Epithelialisierung ohne spezifische Wundbehandlung wiedergeben. Ziel dieser Studie ist daher die Ermittlung der Re-Epithelialisierungszeit einer klinischen Wunde in Tagen und wie diese durch individuelle Faktoren beeinflusst wird. Die vorliegende Arbeit umfasst Ergebnisse einer klinischen Studie von Spalthautentnahmestellen hinsichtlich der durchschnittlichen Dauer des epithelialen Wundverschlusses in AbhĂ€ngigkeit verschiedener individueller Einflussfaktoren.
Material und Methoden
Die verwendeten Daten entstammen zweier multizentrischer Phase-III-Studien zur Untersuchung der klinischen Wirksamkeit und Sicherheit eines topischen BetulinGels bezĂŒglich der Heilung von Spalthautentnahmestellen (EudraCT no. 2012-003390-26, EudraCT no. 2012-000777-23). Die Spalthautentnahmestellen wurden halbiert und nach Randomisierung eine HĂ€lfte mit Betulin Gel behandelt und die andere HĂ€lfte als Kontrollseite mit einem Standardwundverband verschlossen. Gegenstand dieser Studie waren ausschlieĂlich die Kontrollseiten. Die Beurteilung der Wundheilung erfolgte mittels Auswertung von Fotos durch drei unabhĂ€ngige und verblindete Wundexperten via eines Online-Tools. Das Studienprotokoll schloss 198 Patienten ein. Die Beobachtung endete nach Erreichen einer Re-Epithelialisierung von ĂŒber 95% der WundflĂ€che oder spĂ€testens 28 Tagen. Die statistische Auswertung erfolgte mittels Kaplan-Meier-Ăberlebenszeitanalysen mit einem Konfidenzintervall von 95%. Dabei wurde die kumulative Ereignisrate fĂŒr verschiedene Einflussfaktoren aufgetragen und mittels Logrank Test statistisch analysiert.
Ergebnisse
Die Re-Epithelialisierung der Wunden dauerte mindestens sieben Tage. Die schnellsten 25% der Patienten (âfast healersâ) zeigten einen epithelialen Wundverschluss innerhalb von elf Tagen, wĂ€hrend die Re-Epithelialisierung im Median 14 Tage andauerte und die langsamsten 25% (âslow healersâ) der Studienpopulation 18 Tage benötigten. Patienten unter 40 Jahren zeigten einen signifikant schnelleren epithelialen Wundverschluss von zwölf Tagen wĂ€hrend die Re-Epithelialisierung bei Patienten ĂŒber 60 Jahren 18 Tage dauerte (p=0). GebĂ€rfĂ€higkeit reduziert die Dauer signifikant um vier Tage (p=0,002), wĂ€hrend Krebserkrankungen und begleitende Glucocorticoid-Therapien die oberflĂ€chliche Wundheilungsdauer um zwölf beziehungsweise zehn Tage verlĂ€ngern (p=0).
Zusammenfassung
Eine oberflĂ€chliche, artifiziell durch eine Spalthautentnahme entstandene Wunde zeigt ohne gezielte Wundbehandlung einen Schluss der Epitheldecke nach frĂŒhestens sieben Tagen, in der Gruppe der âfast healersâ nach elf Tagen, im Median nach 14 Tagen und benötigt 18 Tage in der Gruppe der âslow healersâ. Die Wundheilung wird um vier bis zwölf Tage verlĂ€ngert durch ein Patientenalter ĂŒber 60 Jahren, eine maligne Grunderkrankung und eine Begleitmedikation mit Glucocorticoiden. Die Wundheilung wird durch ein Patientenalter unter 40 Jahren und die GebĂ€rfĂ€higkeit von Frauen um zwei bis vier Tage beschleunigt. Diese ergab die Analyse von 198 Patientendaten aus 32 europĂ€ischen KrankenhĂ€usern basierend auf einer verblindeten Beurteilung standardisierter fotografischer Wunddokumentation.
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 γΎ 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-1ÎČ, 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, TGFÎČ) 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.
