René Bussiahn

Tissue Tolerable Plasma (TTP) induces apoptosis in pancreatic cancer cells in vitro and in vivo

BackgroundThe rate of microscopic incomplete resections of gastrointestinal cancers including pancreatic cancer has not changed considerably over the past years. Future intra-operative applications of tissue tolerable plasmas (TTP) could help to address this problem. Plasma is generated by feeding energy, like electrical discharges, to gases. The development of non-thermal atmospheric plasmas displaying spectra of temperature within or just above physiological ranges allows biological or medical applications of plasmas.MethodsWe have investigated the effects of tissue tolerable plasmas (TTP) on the human pancreatic cancer cell line Colo-357 and PaTu8988T and the murine cell line 6606PDA in vitro (Annexin-V-FITC/DAPI-Assay and propidium iodide DNA staining assay) as well as in the in vivo tumour chorio-allantoic membrane (TUM-CAM) assay using Colo-357.ResultsTTP of 20 seconds (s) induced a mild elevation of an experimental surface temperature of 23.7 degree Celsius up to 26.63+/−0.40 degree Celsius. In vitro TTP significantly (p=0.0003) decreased cell viability showing the strongest effects after 20s TTP. Also, TTP effects increased over time levelling off after 72 hours (30.1+/−4.4% of dead cells (untreated control) versus 78.0+/−9.6% (20s TTP)). However, analyzing these cells for apoptosis 10s TTP revealed the largest proportion of apoptotic cells (34.8+/−7.2%, p=0.0009 versus 12.3+/−6.6%, 20s TTP) suggesting non-apoptotic cell death in the majority of cells after 20s TTP. Using solid Colo-357 tumours in the TUM-CAM model TUNEL-staining showed TTP-induced apoptosis up to a depth of tissue penetration (DETiP) of 48.8+/−12.3μm (20s TTP, p<0.0001). This was mirrored by a significant (p<0.0001) reduction of Ki-67+ proliferating cells (80.9+/−13.2% versus 37.7+/−14.6%, p<0.0001) in the top cell layers as well as typical changes on HE specimens. The bottom cell layers were not affected by TTP.ConclusionsOur data suggest possible future intra-operative applications of TTP to reduce microscopic residual disease in pancreatic cancer resections. Further promising applications include other malignancies (central liver/lung tumours) as well as synergistic effects combining TTP with chemotherapies. Yet, adaptations of plasma sources as well as of the composition of effective components of TTP are required to optimize their synergistic apoptotic actions.

The hairline plasma: An intermittent negative dc-corona discharge at atmospheric pressure for plasma medical applications

A cold atmospheric pressure plasma source, called hairline plasma, for biological and medical applications has been developed. Using the physical effect of the negative dc corona discharge, a nanosecond pulsed microplasma has been created. The device produces a very thin (d∼30 μm) plasma filament with a length of up to 1.5 cm. Due to this geometrical parameters this plasma is particularly suitable for the treatment of microscopic cavities. The low plasma temperature allows to treat the human skin without any heating or painful irritation.

Antimicrobial efficacy of two surface barrier discharges with air plasma against in vitro biofilms.

The treatment of infected wounds is one possible therapeutic aspect of plasma medicine. Chronic wounds are often associated with microbial biofilms which limit the efficacy of antiseptics. The present study investigates two different surface barrier discharges with air plasma to compare their efficacy against microbial biofilms with chlorhexidine digluconate solution (CHX) as representative of an important antibiofilm antiseptic. Pseudomonas aeruginosa SG81 and Staphylococcus epidermidis RP62A were cultivated on polycarbonate discs. The biofilms were treated for 30, 60, 150, 300 or 600 s with plasma or for 600 s with 0.1% CHX, respectively. After treatment, biofilms were dispensed by ultrasound and the antimicrobial effects were determined as difference in the number of the colony forming units by microbial culture. A high antimicrobial efficacy on biofilms of both plasma sources in comparison to CHX treatment was shown. The efficacy differs between the used strains and plasma sources. For illustration, the biofilms were examined under a scanning electron microscope before and after treatment. Additionally, cytotoxicity was determined by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay with L929 mouse fibroblast cell line. The cell toxicity of the used plasma limits its applicability on human tissue to maximally 150 s. The emitted UV irradiance was measured to estimate whether UV could limit the application on human tissue at the given parameters. It was found that the UV emission is negligibly low. In conclusion, the results support the assumption that air plasma could be an option for therapy of chronic wounds.

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.

Phase-resolved measurement of electric charge deposited by an atmospheric pressure plasma jet on a dielectric surface

The surface charge distribution deposited by the effluent of a dielectric barrier discharge driven atmospheric pressure plasma jet on a dielectric surface has been studied. For the first time, the deposition of charge was observed phase resolved. It takes place in either one or two events in each half cycle of the driving voltage. The charge transfer could also be detected in the electrode current of the jet. The periodic change of surface charge polarity has been found to correspond well with the appearance of ionized channels left behind by guided streamers (bullets) that have been identified in similar experimental situations. The distribution of negative surface charge turned out to be significantly broader than for positive charge. With increasing distance of the jet nozzle from the target surface, the charge transfer decreases until finally the effluent loses contact and the charge transfer stops.

Impact of plasma jet vacuum ultraviolet radiation on reactive oxygen species generation in bio-relevant liquids

Plasma medicine utilizes the combined interaction of plasma produced reactive components. These are reactive atoms, molecules, ions, metastable species, and radiation. Here, ultraviolet (UV, 100–400 nm) and, in particular, vacuum ultraviolet (VUV, 10–200 nm) radiation generated by an atmospheric pressure argon plasma jet were investigated regarding plasma emission, absorption in a humidified atmosphere and in solutions relevant for plasma medicine. The energy absorption was obtained for simple solutions like distilled water (dH2O) or ultrapure water and sodium chloride (NaCl) solution as well as for more complex ones, for example, Rosewell Park Memorial Institute (RPMI 1640) cell culture media. As moderate stable reactive oxygen species, hydrogen peroxide (H2O2) was studied. Highly reactive oxygen radicals, namely, superoxide anion (O2•−) and hydroxyl radicals (•OH), were investigated by the use of electron paramagnetic resonance spectroscopy. All species amounts were detected for three different treatmen...

On the Use of Atmospheric Pressure Plasma for the Bio-Decontamination of Polymers and Its Impact on Their Chemical and Morphological Surface Properties

Low temperature atmospheric pressure plasma processes can be applied to inactivate micro-organisms on products and devices made from synthetic and natural polymers. This study shows that even a short-time exposure to Ar or Ar/O2 plasma of an atmospheric pressure plasma jet leads to an inactivation of Bacillus atrophaeus spores with a maximum reduction of 4 orders of magnitude. However, changes in the surface properties of the plasma exposed material have to be considered, too. Therefore, polyethylene and polystyrene are used as exemplary substrate materials to investigate the effect of plasma treatment in more detail. The influence of process parameters, such as type of operating gas or jet-nozzle to substrate distance, is examined. The results show that short-time plasma treatment with Ar and Ar/O2 affects the surface wettability due to the introduction of polar groups as proofed by X-ray photoelectron spectroscopy. Furthermore, atomic force microscopy images reveal changes in the surface topography. Thus, nanostructures of different heights are observed on the polymeric surface depending on the treatment time and type of process gas.

Influence of the capillary on the ignition of the transient spark discharge

A self-pulsing negative dc discharge in argon generated in a needle-to-plane geometry at open atmosphere is investigated. Additionally, the needle electrode can be surrounded by a quartz capillary. It is shown that the relative position of the capillary end to the needle tip strongly influences the discharge inception and its spatio-temporal dynamics. Without the capillary for the selected working parameters a streamer corona is ignited, but when the capillary surrounds the needle, the transient spark (TS) discharge is ignited after a pre-streamer (PS) occurs. The time between PS and TS discharge depends on the relative capillary end position. The existence of the PS is confirmed by electro-optical characterization. Furthermore, spectrally and spatio-temporally resolved cross-correlation spectroscopy is applied to show the most active region of pre-phase emission activity as indicators for high local electric field strength. The results indicate that with a capillary in place, the necessary energy input of the pre-phase into the system is mainly reduced by additional electrical fields at the capillary edge. Even such a small change as a shift of dielectric surface close to the plasma largely changes the energy balance in the system.

Persistent effectivity of gas plasma-treated, long time-stored liquid on epithelial cell adhesion capacity and membrane morphology.

Research in plasma medicine includes a major interest in understanding gas plasma-cell interactions. The immediate application of gas plasma in vitro inhibits cell attachment, vitality and cell-cell contacts via the liquid. Interestingly, in our novel experiments described here we found that the liquid-mediated plasma effect is long-lasting after storage up to seven days; i. e. the liquid preserves the characteristics once induced by the argon plasma. Therefore, the complete Dulbeccos Modified Eagle cell culture medium was argon plasma-treated (atmospheric pressure, kINPen09) for 60 s, stored for several days (1, 4 and 7 d) at 37°C and added to a confluent mouse hepatocyte epithelial cell (mHepR1) monolayer. Impaired tight junction architecture as well as shortened microvilli on the cell membrane could be observed, which was accompanied by the loss of cell adhesion capacity. Online-monitoring of vital cells revealed a reduced cell respiration. Our first time-dependent analysis of plasma-treated medium revealed that temperature, hydrogen peroxide production, pH and oxygen content can be excluded as initiators of cell physiological and morphological changes. The here observed persisting biological effects in plasma-treated liquids could open new medical applications in dentistry and orthopaedics.

Time-resolved ion density determination by electrical current measurements in an atmospheric-pressure argon plasma

High-resolution current signal measurements of a self-pulsing transient spark discharge revealed oscillations in the pulse decay phase. These oscillations appear with decreasing frequency between 250 MHz and 50 MHz for a specific gas flow range. The frequencies are well within the range of the plasma ion frequency. Consequently, ion acoustic waves are discussed as a possible reason. Considering molecular argon ions as the dominant ion, densities are found to decrease from to . Evaluation of the plasma ion frequency creates a new possibility to determine ion densities.