Investigating the Mutagenicity of a Cold Argon-Plasma Jet in an HET-MN Model.

OBJECTIVE: So-called cold physical plasmas for biomedical applications generate reactive oxygen and nitrogen species and the latter can trigger DNA damage at high concentrations. Therefore, the mutagenic risks of a certified atmospheric pressure argon plasma jet (kINPen MED) and its predecessor model (kINPen 09) were assessed. METHODS: Inner egg membranes of fertilized chicken eggs received a single treatment with either the kINPen 09 (1.5, 2.0, or 2.5 min) or the kINPen MED (3, 4, 5, or 10 min). After three days of incubation, blood smears (panoptic May-Grünwald-Giemsa stain) were performed, and 1000 erythrocytes per egg were evaluated for the presence of polychromatic and normochromic nuclear staining as well as nuclear aberrations and binucleated cells (hen's egg test for micronuclei induction, HET-MN). At the same time, the embryo mortality was documented. For each experiment, positive controls (cyclophosphamide and methotrexate) and negative controls (NaCl-solution, argon gas) were included. Additionally, the antioxidant potential of the blood plasma was assessed by ascorbic acid oxidation assay after treatment. RESULTS: For both plasma sources, there was no evidence of genotoxicity, although at the longest plasma exposure time of 10 min the mortality of the embryos exceeded 40%. The antioxidant potential in the egg's blood plasma was not significantly reduced immediately (p = 0.32) or 1 h (p = 0.19) post exposure to cold plasma. CONCLUSION: The longest plasma treatment time with the kINPen MED was 5-10 fold above the recommended limit for treatment of chronic wounds in clinics. We did not find mutagenic effects for any plasma treatment time using the either kINPen 09 or kINPen MED. The data provided with the current study seem to confirm the lack of a genotoxic potential suggesting that a veterinary or clinical application of these argon plasma jets does not pose mutagenic risks.

Efficacy of Tissue Tolerable Plasma (TTP) against Ixodes ricinus.

The efficacy of Tissue Tolerable Plasma (TTP) against ticks was tested, as data from the literature has demonstrated its efficacy against other acari. The study was carried out by using the KINPen09 (Argon as carrier gas) on Ixodes ricinus (n=24). Treatment times of 1 and 3 minutes led to a reversible inactivation of the ticks. After 5 min of treatment, they died. Thanks to the acaricidal effect of TPP, a new treatment strategy using the KINPen09 for tick-infested pets is now available.

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.

Tissue tolerable plasma (TTP) induces apoptosis in pancreatic cancer cells in vitro and in vivo.

BACKGROUND: The 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. METHODS: We 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. RESULTS: TTP 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. CONCLUSIONS: Our 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.

Pilot-study on the influence of carrier gas and plasma application (open resp. delimited) modifications on physical plasma and its antimicrobial effect against Pseudomonas aeruginosa and Staphylococcus aureus.

INTRODUCTION: Physical plasma is a promising new technology regarding its antimicrobial effects. This is especially accounting for treatment of bacterial infection of chronic wounds. Plasma can be generated with different carrier gases causing various biological effects. Screening of different carrier gases and plasma generation setups is therefore needed to find suitable compositions for highly effective antimicrobial plasma treatments and other applications. METHOD: The plasma source used was a radio-frequency plasma jet which generates tissue tolerable plasma (TTP). The study compared the antimicrobial efficacy of air, argon, or helium plasma alone or admixed with 0.1%, 0.5%, and 1% oxygen against Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus). Treatment took place in an environmentally open and delimited system. Therefore, bacteria were plated on agar and treated with plasma in a punctiform manner. The resulting inhibition zones were measured and the reduction factors were calculated by colony counting, respectively. RESULTS: For S. aureus and P. aeruginosa, inhibition zones and overall reduction of colony forming units (CFU) on the agar plate were observed while an accumulative reduction of CFU dominated for S. aureus. The highest antimicrobial effect was shown in form of an inhibition zone for argon plasma with 0.1% oxygen admixture for both species. S. aureus was more sensitive for helium plasma with >0.1% oxygen admixture compared to P. aeruginosa which in turn was more sensitive for argon plasma with and without oxygen. The efficacy of air plasma was very low in comparison to the other gases. The treatment in a closed system predominantly enhanced the antimicrobial effect. The effect intensity varied for each treatment time and gas mixtures. DISCUSSION: As expected, the antimicrobial effect mostly increased when increasing oxygen admixture to the carrier gases. The variation in bacterial growth and inhibition after exposure to different plasma gas compositions could be due to a varying generation of reactive oxygen species or radiation. CONCLUSION: The applied plasma in a "closed system" accumulates bactericidal plasma species and might increase antimicrobial efficacy in clinical settings as in wound management involving multi-drug resistant bacteria.

The modified HET-CAM as a model for the assessment of the inflammatory response to tissue tolerable plasma.

Plasma medicine is a novel, highly interdisciplinary field of research. Although the knowledge is rare concerning plasma based biomedical mechanisms, correct dosages and treatment times, animal experiments have been carried out. To follow the 3Rs (reduction, refinement, replacement), it is necessary to define methods for the screening of plasma parameters. In order to determine a reliable test and validate the use of tissue tolerable plasma (TTP) for the treatment of chronic wounds, we have selected the modified hen's egg test on the chorioallantoic membrane (mod. HET-CAM) as a model to benchmark the inflammation potential of plasma. Inflammations of different intensities provoked by using an HF-plasma jet corresponded to the time of plasma-tissue interaction. Additionally, the plasma mode and the gas composition were changed to assess their influence on the efficacy of treatment. Pulsed plasma led to the mildest inflammation, while the addition of 0.1% oxygen to the argon carrier gas led to the most distinct reaction. It was found that the influence of the exposure time was greater than that of the mode and the gas composition. All inflammations were alleviated, when hydrocortisone (HC) was added immediately after plasma treatment. The results of the study demonstrate that the modified HET-CAM test is suitable for screening plasma sources and for the determination of the optimum parameters for treatment of chronic wounds. To transfer the chronic wound into an acute healing wound without harmful inflammation, the maximum time for plasma-tissue interaction should not exceed 5s with the tested plasma source. Furthermore, it is possible to induce defined plasma-inflammations on the chorioallantoic membrane and to determine the anti-inflammatory potential of test substances.

Prevention of post-operative infections after surgical treatment of bite wounds.

After reviewing the literature about the microbial spectrum, the risk factors of post-operative infections, and the results of surgical interventions, the following recommendation can be made for the management of bite wounds:FRESH, OPEN WOUNDS: surgical debridement, if appropriate, then an antiseptic lavage with a fluid consisting of povidone iodine and ethanol (e.g., Betaseptic(®)), no antibiotics, primary wound closurenearly closed fresh wounds (e.g., cat bites): surgical debridement, if appropriate, dressing with an antiseptic-soaked compress for ~60 minutes with repeated soaking (e.g., Betaseptic(®)), no antibioticsolder wounds after ~4 hours: surgical debridement, if appropriate, dressing with an antiseptic-soaked compress or bandage for ~60 minutes with repeated soaking (e.g., Betaseptic(®)), at the same time intravenous or dose-adapted oral antibiotics (Amoxicillin and/or clavulanic acid)older wounds after ~24 hours: surgical debridement, then antiseptic lavage (Betaseptic(®)), in case of clinically apparent infection or inflammation surgical revision with opening of wound and treatment with antibiotics according to resistogram (empirical start with Amoxicillin and/or clavulanic acid).For each kind of bite wound, the patient's tetanus immunization status as well as the risk of exposure to rabies have to be assessed. Similarly, the possibility of other infections, such as lues (Syphilis), hepatitis B (HBV), hepatitis C (HBC), hepatitis D (HDV) and HIV, in the rare case of a human bite wound, has to be taken into account.

Screening test for quality control of surface barrier discharged plasma sources with the microorganism-agar test (MAT).

In the recent past, the antimicrobial applicability of tissue tolerable plasma (TTP) has been examined, and is increasing in importance. Many different plasma sources can be used medically. In order to obtain reliable test results by using plasma sources, it is essential not only to assess physical parameters but also to screen a use-oriented application of plasma sources. It is necessary to both determine the biological efficacy and establish uniform coverage of the treated surfaces. This article shows a simple screening method for this. We used newly inoculated agar plates with bacterial test organisms and treated the agar plates with two surface barrier discharged (SDB) plasmas. The carrier gas was argon and air.