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- ItemDifferential influence of components resulting from atmospheric-pressure plasma on integrin expression of human HaCaT keratinocytes(New York, NY : Hindawi, 2013) Haertel, B.; Straßenburg, S.; Oehmigen, K.; Wende, K.; Von Woedtke, T.; Lindequist, U.Adequate chronic wound healing is a major problem in medicine. A new solution might be non-thermal atmospheric-pressure plasma effectively inactivating microorganisms and influencing cells in wound healing. Plasma components as, for example, radicals can affect cells differently. HaCaT keratinocytes were treated with Dielectric Barrier Discharge plasma (DBD/air, DBD/argon), ozone or hydrogen peroxide to find the components responsible for changes in integrin expression, intracellular ROS formation or apoptosis induction. Dependent on plasma treatment time reduction of recovered cells was observed with no increase of apoptotic cells, but breakdown of mitochondrial membrane potential. DBD/air plasma increased integrins and intracellular ROS. DBD/argon caused minor changes. About 100 ppm ozone did not influence integrins. Hydrogen peroxide caused similar effects compared to DBD/air plasma. In conclusion, effects depended on working gas and exposure time to plasma. Short treatment cycles did neither change integrins nor induce apoptosis or ROS. Longer treatments changed integrins as important for influencing wound healing. Plasma effects on integrins are rather attributed to induction of other ROS than to generation of ozone. Changes of integrins by plasma may provide new solutions of improving wound healing, however, conditions are needed which allow initiating the relevant influence on integrins without being cytotoxic to cells.
- ItemTissue Tolerable Plasma (TTP) induces apoptosis in pancreatic cancer cells in vitro and in vivo(London : BioMed Central, 2012) Partecke, L.I.; Evert, K.; Haugk, J.; Doering, F.; Normann, L.; Diedrich, S.; Weiss, F.-U.; Evert, M.; Huebner, N.O.; Guenther, C.; Heidecke, C.D.; Kramer, A.; Bussiahn, R.; Weltmann, K.-D.; Pati, O.; Bender, C.; von Bernstorff, W.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.
- ItemPeriodic Exposure of Keratinocytes to Cold Physical Plasma: An In Vitro Model for Redox-Related Diseases of the Skin(London: Hindawi, 2016) Schmidt, Anke; von Woedtke, Thomas; Bekeschus, SanderOxidative stress illustrates an imbalance between radical formation and removal. Frequent redox stress is critically involved in many human pathologies including cancer, psoriasis, and chronic wounds. However, reactive species pursue a dual role being involved in signaling on the one hand and oxidative damage on the other. Using a HaCaT keratinocyte cell culture model, we investigated redox regulation and inflammation to periodic, low-dose oxidative stress after two, six, eight, ten, and twelve weeks. Chronic redox stress was generated by recurrent incubation with cold physical plasma-treated cell culture medium. Using transcriptome microarray technology, we identified both acute ROS-stress responses as well as numerous adaptions after several weeks of redox challenge. We determined a differential expression (2-fold, FDR < 0.01, p < 0.05) of 260 genes that function in inflammation and redox homeostasis, such as cytokines (e.g., IL-6, IL-8, and IL-10), growth factors (e.g., CSF2, FGF, and IGF-2), and antioxidant enzymes (e.g., HMOX, NQO1, GPX, and PRDX). Apoptotic signaling was affected rather modestly, especially in p53 downstream targets (e.g., BCL2, BBC3, and GADD45). Strikingly, the cell-protective heat shock protein HSP27 was strongly upregulated (p < 0.001). These results suggested cellular adaptions to frequent redox stress and may help to better understand the inflammatory responses in redox-related diseases.
- ItemRemoving biofilms from microstructured titanium Ex Vivo: A novel approach using atmospheric plasma technology(San Francisco, CA : Public Library of Science, 2011) Rupf, S.; Idlibi, A.N.; Marrawi, F.A.; Hannig, M.; Schubert, A.; von Mueller, L.; Spitzer, W.; Holtmann, H.; Lehmann, A.; Rueppell, A.; Schindler, A.The removal of biofilms from microstructured titanium used for dental implants is a still unresolved challenge. This experimental study investigated disinfection and removal of in situ formed biofilms from microstructured titanium using cold atmospheric plasma in combination with air/water spray. Titanium discs (roughness (Ra): 1.96 μm) were exposed to human oral cavities for 24 and 72 hours (n = 149 each) to produce biofilms. Biofilm thickness was determined using confocal laser scanning microscopy (n = 5 each). Plasma treatment of biofilms was carried out ex vivo using a microwave-driven pulsed plasma source working at temperatures from 39 to 43°C. Following plasma treatment, one group was air/water spray treated before re-treatment by second plasma pulses. Vital microorganisms on the titanium surfaces were identified by contact culture (Rodac agar plates). Biofilm presence and bacterial viability were quantified by fluorescence microscopy. Morphology of titanium surfaces and attached biofilms was visualized by scanning electron microscopy (SEM). Total protein amounts of biofilms were colorimetrically quantified. Untreated and air/water treated biofilms served as controls. Cold plasma treatment of native biofilms with a mean thickness of 19 μm (24 h) to 91 μm (72 h) covering the microstructure of the titanium surface caused inactivation of biofilm bacteria and significant reduction of protein amounts. Total removal of biofilms, however, required additional application of air/water spray, and a second series of plasma treatment. Importantly, the microstructure of the titanium discs was not altered by plasma treatment. The combination of atmospheric plasma and non-abrasive air/water spray is applicable for complete elimination of oral biofilms from microstructured titanium used for dental implants and may enable new routes for the therapy of periimplant disease.