Thomas von Woedtke

Atmospheric-pressure plasma sources: Prospective tools for plasma medicine

Plasma-based treatment of chronic wounds or skin diseases as well as tissue engineering or tumor treatment is an extremely promising field. First practical studies are promising, and plasma medicine as an independent medical field is emerging worldwide. While during the last years the basics of sterilizing effects of plasmas were well studied, concepts of tailor-made plasma sources which meet the technical requirements of medical instrumentation are still less developed. Indeed, studies on the verification of selective antiseptic effects of plasmas are required, but the development of advanced plasma sources for biomedical applications and a profound knowledge of their physics, chemistry, and parameters must be contributed by physical research. Considering atmospheric-pressure plasma sources, the determination of discharge development and plasma parameters is a great challenge, due to the high complexity and limited diagnostic approaches. This contribution gives an overview on plasma sources for therapeutic applications in plasma medicine. Selected specific plasma sources that are used for the investigation of various biological effects are presented and discussed. Furthermore, the needs, prospects, and approaches for its characterization from the fundamental plasma physical point of view will be discussed.

Non-Thermal Atmospheric-Pressure Plasma Possible Application in Wound Healing

Non-thermal atmospheric-pressure plasma, also named cold plasma, is defined as a partly ionized gas. Therefore, it cannot be equated with plasma from blood; it is not biological in nature. Non-thermal atmospheric-pressure plasma is a new innovative approach in medicine not only for the treatment of wounds, but with a wide-range of other applications, as e.g. topical treatment of other skin diseases with microbial involvement or treatment of cancer diseases. This review emphasizes plasma effects on wound healing. Non-thermal atmospheric-pressure plasma can support wound healing by its antiseptic effects, by stimulation of proliferation and migration of wound relating skin cells, by activation or inhibition of integrin receptors on the cell surface or by its pro-angiogenic effect. We summarize the effects of plasma on eukaryotic cells, especially on keratinocytes in terms of viability, proliferation, DNA, adhesion molecules and angiogenesis together with the role of reactive oxygen species and other components of plasma. The outcome of first clinical trials regarding wound healing is pointed out.

Atmospheric pressure plasma: a high-performance tool for the efficient removal of biofilms.

Introduction The medical use of non-thermal physical plasmas is intensively investigated for sterilization and surface modification of biomedical materials. A further promising application is the removal or etching of organic substances, e.g., biofilms, from surfaces, because remnants of biofilms after conventional cleaning procedures are capable to entertain inflammatory processes in the adjacent tissues. In general, contamination of surfaces by micro-organisms is a major source of problems in health care. Especially biofilms are the most common type of microbial growth in the human body and therefore, the complete removal of pathogens is mandatory for the prevention of inflammatory infiltrate. Physical plasmas offer a huge potential to inactivate micro-organisms and to remove organic materials through plasma-generated highly reactive agents. Method In this study a Candida albicans biofilm, formed on polystyrene (PS) wafers, as a prototypic biofilm was used to verify the etching capability of the atmospheric pressure plasma jet operating with two different process gases (argon and argon/oxygen mixture). The capability of plasma-assisted biofilm removal was assessed by microscopic imaging. Results The Candida albicans biofilm, with a thickness of 10 to 20 µm, was removed within 300 s plasma treatment when oxygen was added to the argon gas discharge, whereas argon plasma alone was practically not sufficient in biofilm removal. The impact of plasma etching on biofilms is localized due to the limited presence of reactive plasma species validated by optical emission spectroscopy.

Identification of the biologically active liquid chemistry induced by a nonthermal atmospheric pressure plasma jet

The mechanism of interaction of cold nonequilibrium plasma jets with mammalian cells in physiologic liquid is reported. The major biological active species produced by an argon RF plasma jet responsible for cell viability reduction are analyzed by experimental results obtained through physical, biological, and chemical diagnostics. This is complemented with chemical kinetics modeling of the plasma source to assess the dominant reactive gas phase species. Different plasma chemistries are obtained by changing the feed gas composition of the cold argon based RF plasma jet from argon, humidified argon (0.27%), to argon/oxygen (1%) and argon/air (1%) at constant power. A minimal consensus physiologic liquid was used, providing isotonic and isohydric conditions and nutrients but is devoid of scavengers or serum constituents. While argon and humidified argon plasma led to the creation of hydrogen peroxide dominated action on the mammalian cells, argon-oxygen and argon-air plasma created a very different biological action and was characterized by trace amounts of hydrogen peroxide only. In particular, for the argon-oxygen (1%), the authors observed a strong negative effect on mammalian cell proliferation and metabolism. This effect was distance dependent and showed a half life time of 30 min in a scavenger free physiologic buffer. Neither catalase and mannitol nor superoxide dismutase could rescue the cell proliferation rate. The strong distance dependency of the effect as well as the low water solubility rules out a major role for ozone and singlet oxygen but suggests a dominant role of atomic oxygen. Experimental results suggest that O reacts with chloride, yielding Cl2(-) or ClO(-). These chlorine species have a limited lifetime under physiologic conditions and therefore show a strong time dependent biological activity. The outcomes are compared with an argon MHz plasma jet (kinpen) to assess the differences between these (at least seemingly) similar plasma sources.

Cold plasma is well‐tolerated and does not disturb skin barrier or reduce skin moisture

Background: Cold plasma, a new treatment principle in dermatology based on ionic discharge delivering reactive molecular species and UV‐light, exhibits strong antimicrobial efficacy in vitro and in vivo. Before implementing plasma as new medical treatment tool, its safety must be proven, as well as assessing skin tolerance and patient acceptance.

Experimental Recovery of CO2-Laser Skin Lesions by Plasma Stimulation

In a series of 5 experimental case reports with identical settings in terms of methods and materials, nonthermal atmospheric pressure plasma stimulation of laser skin lesion recovery looks promising.

Non-thermal plasma activates human keratinocytes by stimulation of antioxidant and phase II pathways.

Background: Non-thermal plasma provides an interesting therapeutic opportunity to control redox-based processes, e.g. wound healing. Results: The transcription factor NRF2 and downstream signaling molecules were found to act as key controllers orchestrating the cellular response. Conclusions: Plasma triggers hormesis-like processes in keratinocytes. Significance: These findings facilitate the understanding of plasma-tissue interaction and its deduced clinical application. Non-thermal atmospheric pressure plasma provides a novel therapeutic opportunity to control redox-based processes, e.g. wound healing, cancer, and inflammatory diseases. By spatial and time-resolved delivery of reactive oxygen and nitrogen species, it allows stimulation or inhibition of cellular processes in biological systems. Our data show that both gene and protein expression is highly affected by non-thermal plasma. Nuclear factor erythroid-related factor 2 (NRF2) and phase II enzyme pathway components were found to act as key controllers orchestrating the cellular response in keratinocytes. Additionally, glutathione metabolism, which is a marker for NRF2-related signaling events, was affected. Among the most robustly increased genes and proteins, heme oxygenase 1, NADPH-quinone oxidoreductase 1, and growth factors were found. The roles of NRF2 targets, investigated by siRNA silencing, revealed that NRF2 acts as an important switch for sensing oxidative stress events. Moreover, the influence of non-thermal plasma on the NRF2 pathway prepares cells against exogenic noxae and increases their resilience against oxidative species. Via paracrine mechanisms, distant cells benefit from cell-cell communication. The finding that non-thermal plasma triggers hormesis-like processes in keratinocytes facilitates the understanding of plasma-tissue interaction and its clinical application.

Atmospheric pressure plasma jet treatment evokes transient oxidative stress in HaCaT keratinocytes and influences cell physiology

Modern non‐thermal atmospheric pressure plasma sources enable controllable interaction with biological systems. Their future applications – e.g. wound management – are based on their unique mixture of reactive components sparking both stimulatory as well as inhibitory processes. To gain detailed understanding of plasma–cell interaction and with respect to risk awareness, key mechanisms need to be identified. This study focuses on the impact of an argon non‐thermal atmospheric pressure plasma jet (kINPen 09) on human HaCaT keratinocytes. With increasing duration, cell viability decreased. In accordance, cells accumulated in G2/M phase within the following 24 h. DNA single‐strand breaks were detected immediately after treatment and receded in the aftermath, returning to control levels after 24 h. No directly plasma‐related DNA double‐strand breaks were detected over the same time. Concurrently, DNA synthesis decreased. Coincident with treatment time, an increase in intracellular 2′,7′‐dichlorodihydrofluorescein diacetate (H2DCFDA) conversion increased reactive oxygen species (ROS) levels. The radical scavenging activity of culture medium crucially influenced these effects. Thus, ROS changed DNA integrity, and the effectiveness of cellular defence mechanisms characterises the interaction of non‐thermal plasma and eukaryotic cells. Effects were time‐dependent, indicating an active response of the eukaryotic cells. Hence, a stimulation of eukaryotic cells using short‐term non‐thermal plasma treatment seems possible, eg in the context of chronic wound care. Long‐term plasma treatments stopped in cell proliferation and apoptosis, which might be relevant in controlling neoplastic conditions.

Surface molecules on HaCaT keratinocytes after interaction with non-thermal atmospheric pressure plasma.

Non‐thermal atmospheric‐pressure plasmas have been developed that will be used in future for several purposes, e.g. medicine. Living tissues and cells are at the focus of plasma treatment, e.g. to improve wound healing, or induce apoptosis and growth arrest in tumour cells. Detailed investigations of plasma‐cell interactions are needed. Cell surface adhesion molecules as integrins, cadherins or the EGFR (epidermal growth factor receptor) are of importance in wound healing and also for development of cancer metastasis. This study has focused on measurement of cell surface molecules on human HaCaT keratinocytes (human adult low calcium temperature keratinocytes) promoting adhesion, migration and proliferation as one important feature of plasma‐cell interactions. HaCaT keratinocytes were treated with plasma by a surface dielectric barrier discharge in air. Cell surface molecules and induction of intracellular ROS (reactive oxygen species) were analysed by flow cytometry 24 h after plasma treatment. Besides a reduction of cell viability a significant down‐regulation of E‐cadherin and the EGFR expression occurred. The influence on α2‐ and β1‐integrins was less pronounced, and expression of ICAM‐1 (intercellular adhesion molecule 1) was unaffected. The extent of effects depended on the exposure time of cells to the plasma and the treatment regimen. Intracellular level of ROS detected by the fluorescent dye H2DCFDA (2′,7′‐dichlorodihydrofluorescein diacetate) increased by plasma treatment, but it was neither dependent on the treatment time nor related to the different treatment regimens. Two‐dimensional cultures of HaCaT keratinocytes appear to be a suitable method of investigating plasma‐cell interactions.

Non-thermal atmospheric-pressure plasma can influence cell adhesion molecules on HaCaT-keratinocytes

Abstract:  Non‐thermal atmospheric‐pressure plasmas provide new hope for improvement in chronic wound management because of their potency in killing microorganisms. However, the effectiveness of the procedure has to be verified and negative effects on healthy tissues have to be excluded. In wound healing adhesion molecules play a crucial role for cell migration and proliferation. We investigated whether an atmospheric‐pressure plasma jet (kINPen09) influences the expression of adhesion molecules responsible for cell‐cell and cell‐matrix interactions after treatment of HaCaT‐keratinocytes for 10 and 30 s. Twenty‐four hours after plasma treatment expression of α2‐ and β1‐integrin, E‐cadherin and the epidermal growth factor receptor (EGFR) was determined by flow cytometry. Plasma‐treated HaCaT‐cells were characterized by normal α2‐integrin and increased β1‐integrin expression. E‐cadherin and EGFR expression was reduced after the 30‐s treatment. We did not observe any effects following the 10‐s plasma treatment. In conclusion, short‐term plasma treatment can be applied without effects for cell‐cell and cell‐matrix adhesion.