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- ItemConductive Gas Plasma Treatment Augments Tumor Toxicity of Ringer’s Lactate Solutions in a Model of Peritoneal Carcinomatosis(Basel : MDPI, 2022) Miebach, Lea; Freund, Eric; Cecchini, Alessandra Lourenço; Bekeschus, SanderReactive species generated by medical gas plasma technology can be enriched in liquids for use in oncology targeting disseminated malignancies, such as metastatic colorectal cancer. Notwithstanding, reactive species quantities depend on the treatment mode, and we recently showed gas plasma exposure in conductive modes to be superior for cancer tissue treatment. However, evidence is lacking that such a conductive mode also equips gas plasma-treated liquids to confer augmented intraperitoneal anticancer activity. To this end, employing atmospheric pressure argon plasma jet kINPen-treated Ringer’s lactate (oxRilac) in a CT26-model of colorectal peritoneal carcinomatosis, we tested repeated intraabdominal injection of such remotely or conductively oxidized liquid for antitumor control and immunomodulation. Enhanced reactive species formation in conductive mode correlated with reduced tumor burden in vivo, emphasizing the advantage of conduction over the free mode for plasma-conditioned liquids. Interestingly, the infiltration of lymphocytes into the tumors was equally enhanced by both treatments. However, significantly lower levels of interleukin (IL)4 and IL13 and increased levels of IL2 argue for a shift in intratumoral T-helper cell subpopulations correlating with disease control. In conclusion, our data argue for using conductively over remotely prepared plasma-treated liquids for anticancer treatment.
- ItemBiological Risk Assessment of Three Dental Composite Materials following Gas Plasma Exposure(Basel : MDPI, 2022) Bekeschus, Sander; Miebach, Lea; Pommerening, Jonas; Clemen, Ramona; Witzke, KatharinaGas plasma is an approved technology that generates a plethora of reactive oxygen species, which are actively applied for chronic wound healing. Its particular antimicrobial action has spurred interest in other medical fields, such as periodontitis in dentistry. Recent work has indicated the possibility of performing gas plasma-mediated biofilm removal on teeth. Teeth frequently contain restoration materials for filling cavities, e.g., resin-based composites. However, it is unknown if such materials are altered upon gas plasma exposure. To this end, we generated a new in-house workflow for three commonly used resin-based composites following gas plasma treatment and incubated the material with human HaCaT keratinocytes in vitro. Cytotoxicity was investigated by metabolic activity analysis, flow cytometry, and quantitative high-content fluorescence imaging. The inflammatory consequences were assessed using quantitative analysis of 13 different chemokines and cytokines in the culture supernatants. Hydrogen peroxide served as the control condition. A modest but significant cytotoxic effect was observed in the metabolic activity and viability after plasma treatment for all three composites. This was only partially treatment time-dependent and the composites alone affected the cells to some extent, as evident by differential secretion profiles of VEGF, for example. Gas plasma composite modification markedly elevated the secretion of IL6, IL8, IL18, and CCL2, with the latter showing the highest correlation with treatment time (Pearson’s r > 0.95). Cell culture media incubated with gas plasma-treated composite chips and added to cells thereafter could not replicate the effects, pointing to the potential that surface modifications elicited the findings. In conclusion, our data suggest that gas plasma treatment modifies composite material surfaces to a certain extent, leading to measurable but overall modest biological effects.
- ItemCombined toxicity of gas plasma treatment and nanoparticles exposure in melanoma cells in vitro(Basel : MDPI, 2021) Bekeschus, SanderDespite continuous advances in therapy, cancer remains a deadly disease. Over the past years, gas plasma technology emerged as a novel tool to target tumors, especially skin. Another promising anticancer approach are nanoparticles. Since combination therapies are becoming increas-ingly relevant in oncology, both gas plasma treatment and nanoparticle exposure were combined. A series of nanoparticles were investigated in parallel, namely, silica, silver, iron oxide, cerium oxide, titanium oxide, and iron-doped titanium oxide. For gas plasma treatment, the atmospheric pressure argon plasma jet kINPen was utilized. Using three melanoma cell lines, the two murine non-metastatic B16F0 and metastatic B16F10 cells and the human metastatic B-Raf mutant cell line SK-MEL-28, the combined cytotoxicity of both approaches was identified. The combined cytotoxicity of gas plasma treatment and nanoparticle exposure was consistent across all three cell lines for silica, silver, iron oxide, and cerium oxide. In contrast, for titanium oxide and iron-doped titanium oxide, significantly combined cytotoxicity was only observed in B16F10 cells.
- ItemPatient-derived human basal and cutaneous squamous cell carcinoma tissues display apoptosis and immunomodulation following gas plasma exposure with a certified argon jet(Basel : Molecular Diversity Preservation International, 2021) Saadati, Fariba; Moritz, Juliane; Berner, Julia; Freund, Eric; Miebach, Lea; Helfrich, Iris; Stoffels, Ingo; Emmert, Steffen; Bekeschus, SanderReactive oxygen species (ROS) have been subject of increasing interest in the pathophysiology and therapy of cancers in recent years. In skin cancer, ROS are involved in UV-induced tumorigenesis and its targeted treatment via, e.g., photodynamic therapy. Another recent technology for topical ROS generation is cold physical plasma, a partially ionized gas expelling dozens of reactive species onto its treatment target. Gas plasma technology is accredited for its wound-healing abilities in Europe, and current clinical evidence suggests that it may have beneficial effects against actinic keratosis. Since the concept of hormesis dictates that low ROS levels perform signaling functions, while high ROS levels cause damage, we investigated herein the antitumor activity of gas plasma in non-melanoma skin cancer. In vitro, gas plasma exposure diminished the metabolic activity, preferentially in squamous cell carcinoma cell (SCC) lines compared to non-malignant HaCaT cells. In patient-derived basal cell carcinoma (BCC) and SCC samples treated with gas plasma ex vivo, increased apoptosis was found in both cancer types. Moreover, the immunomodulatory actions of gas plasma treatment were found affecting, e.g., the expression of CD86 and the number of regulatory T-cells. The supernatants of these ex vivo cultured tumors were quantitatively screened for cytokines, chemokines, and growth factors, identifying CCL5 and GM-CSF, molecules associated with skin cancer metastasis, to be markedly decreased. These findings suggest gas plasma treatment to be an interesting future technology for non-melanoma skin cancer topical therapy.
- ItemOral SARS-CoV-2 reduction by local treatment: A plasma technology application?(Weinheim : Wiley-VCH, 2022) von Woedtke, Thomas; Gabriel, Gülsah; Schaible, Ulrich E.; Bekeschus, SanderThe SARS-CoV-2 pandemic reemphasized the importance of and need for efficient hygiene and disinfection measures. The coronavirus' efficient spread capitalizes on its airborne transmission routes via virus aerosol release from human oral and nasopharyngeal cavities. Besides the upper respiratory tract, efficient viral replication has been described in the epithelium of these two body cavities. To this end, the idea emerged to employ plasma technology to locally reduce mucosal viral loads as an additional measure to reduce patient infectivity. We here outline conceptual ideas of such treatment concepts within what is known in the antiviral actions of plasma treatment so far.
- ItemEndothelial Differentiation of CCM1 Knockout iPSCs Triggers the Establishment of a Specific Gene Expression Signature(Basel : Molecular Diversity Preservation International, 2023) Pilz, Robin A.; Skowronek, Dariush; Mellinger, Lara; Bekeschus, Sander; Felbor, Ute; Rath, MatthiasCerebral cavernous malformation (CCM) is a neurovascular disease that can lead to seizures and stroke-like symptoms. The familial form is caused by a heterozygous germline mutation in either the CCM1, CCM2, or CCM3 gene. While the importance of a second-hit mechanism in CCM development is well established, it is still unclear whether it immediately triggers CCM development or whether additional external factors are required. We here used RNA sequencing to study differential gene expression in CCM1 knockout induced pluripotent stem cells (CCM1−/− iPSCs), early mesoderm progenitor cells (eMPCs), and endothelial-like cells (ECs). Notably, CRISPR/Cas9-mediated inactivation of CCM1 led to hardly any gene expression differences in iPSCs and eMPCs. However, after differentiation into ECs, we found the significant deregulation of signaling pathways well known to be involved in CCM pathogenesis. These data suggest that a microenvironment of proangiogenic cytokines and growth factors can trigger the establishment of a characteristic gene expression signature upon CCM1 inactivation. Consequently, CCM1−/− precursor cells may exist that remain silent until entering the endothelial lineage. Collectively, not only downstream consequences of CCM1 ablation but also supporting factors must be addressed in CCM therapy development.
- ItemIn-Vitro Biofilm Removal Efficacy Using Water Jet in Combination with Cold Plasma Technology on Dental Titanium Implants(Basel : Molecular Diversity Preservation International, 2023) Matthes, Rutger; Jablonowski, Lukasz; Miebach, Lea; Pitchika, Vinay; Holtfreter, Birte; Eberhard, Christian; Seifert, Leo; Gerling, Torsten; Schlüter, Rabea; Kocher, Thomas; Bekeschus, SanderPeri-implantitis-associated inflammation can lead to bone loss and implant failure. Current decontamination measures are ineffective due to the implants’ complex geometry and rough surfaces providing niches for microbial biofilms. A modified water jet system (WaterJet) was combined with cold plasma technology (CAP) to achieve superior antimicrobial efficacy compared to cotton gauze treatment. Seven-day-old multi-species-contaminated titanium discs and implants were investigated as model systems. The efficacy of decontamination on implants was determined by rolling the implants over agar and determining colony-forming units supported by scanning electron microscopy image quantification of implant surface features. The inflammatory consequences of mono and combination treatments were investigated with peripheral blood mononuclear cell surface marker expression and chemokine and cytokine release profiles on titanium discs. In addition, titanium discs were assayed using fluorescence microscopy. Cotton gauze was inferior to WaterJet treatment according to all types of analysis. In combination with the antimicrobial effect of CAP, decontamination was improved accordingly. Mono and CAP-combined treatment on titanium surfaces alone did not unleash inflammation. Simultaneously, chemokine and cytokine release was dramatically reduced in samples that had benefited from additional antimicrobial effects through CAP. The combined treatment with WaterJet and CAP potently removed biofilm and disinfected rough titanium implant surfaces. At the same time, non-favorable rendering of the surface structure or its pro-inflammatory potential through CAP was not observed.
- ItemSynergistic In Vitro Anticancer Toxicity of Pulsed Electric Fields and Glutathione(Basel : Molecular Diversity Preservation International (MDPI), 2022) Wolff, Christina M.; Bekeschus, SanderDespite continuous advancement in skin cancer therapy, the disease is still fatal in many patients, demonstrating the need to improve existing therapies, such as electrochemotherapy (ECT). ECT can be applied in the palliative or curative setting and is based on the application of pulsed electric fields (PEF), which by themselves exerts none to low cancer toxicity but become potently toxic when combined with low-dosed chemotherapeutics such as bleomycin and cisplatin. Albeit their favorable side-effect profiles, not all patients respond to standard ECT, and some responders experience tumor recurrence. To identify potential adjuvant or alternative agents to standard electrochemotherapy, we explored the possibility of combining PEF with a physiological compound, glutathione (GSH), to amplify anticancer toxicity. GSH is an endogenous antioxidant and is available as a dietary supplement. Surprisingly, neither GSH nor PEF mono treatment but GSH + PEF combination treatment exerted strong cytotoxic effects and declined metabolic activity in four skin cancer cell lines in vitro. The potential applicability to other tumor cells was verified by corroborating results in two leukemia cell lines. Strikingly, GSH + PEF treatment did not immediately increase intracellular GSH levels, while levels 24 h following treatment were enhanced. Similar tendencies were made for intracellular reactive oxygen species (ROS) levels, while extracellular ROS increased following combination treatment. ROS levels and the degree of cytotoxicity could be partially reversed by pre-incubating cells with the NADPH-oxidase (NOX) inhibitor diphenyleneiodonium (DPI) and the H2O2-degrading enzyme catalase. Collectively, our findings suggest a promising new “endogenous” drug to be combined with PEF for future anticancer research approaches.
- ItemPeriodic Exposure of Plasma-Activated Medium Alters Fibroblast Cellular Homoeostasis(Basel : Molecular Diversity Preservation International (MDPI), 2022) Bhartiya, Pradeep; Kaushik, Neha; Nguyen, Linh N.; Bekeschus, Sander; Masur, Kai; Weltmann, Klaus-Dieter; Kaushik, Nagendra Kumar; Choi, Eun HaExcess amounts of redox stress and failure to regulate homeostatic levels of reactive species are associated with several skin pathophysiologic conditions. Nonmalignant cells are assumed to cope better with higher reactive oxygen and nitrogen species (RONS) levels. However, the effect of periodic stress on this balance has not been investigated in fibroblasts in the field of plasma medicine. In this study, we aimed to investigate intrinsic changes with respect to cellular proliferation, cell cycle, and ability to neutralize the redox stress inside fibroblast cells following periodic redox stress in vitro. Soft jet plasma with air as feeding gas was used to generate plasma-activated medium (PAM) for inducing redox stress conditions. We assessed cellular viability, energetics, and cell cycle machinery under oxidative stress conditions at weeks 3, 6, 9, and 12. Fibroblasts retained their usual physiological properties until 6 weeks. Fibroblasts failed to overcome the redox stress induced by periodic PAM exposure after 6 weeks, indicating its threshold potential. Periodic stress above the threshold level led to alterations in fibroblast cellular processes. These include consistent increases in apoptosis, while RONS accumulation and cell cycle arrest were observed at the final stages. Currently, the use of NTP in clinical settings is limited due to a lack of knowledge about fibroblasts’ behavior in wound healing, scar formation, and other fibrotic disorders. Understanding fibroblasts’ physiology could help to utilize nonthermal plasma in redox-related skin diseases. Furthermore, these results provide new information about the threshold capacity of fibroblasts and an insight into the adaptation mechanism against periodic oxidative stress conditions in fibroblasts.
- ItemBiocompatible Gas Plasma Treatment Affects Secretion Profiles but Not Osteogenic Differentiation in Patient-Derived Mesenchymal Stromal Cells(Basel : Molecular Diversity Preservation International (MDPI), 2022) Fischer, Maximilian; Schoon, Janosch; Freund, Eric; Miebach, Lea; Weltmann, Klaus-Dieter; Bekeschus, Sander; Wassilew, Georgi I.Cold physical plasma (CPP), a partially ionized gas that simultaneously generates reactive oxygen and nitrogen species, is suggested to provide advantages in regenerative medicine. Intraoperative CPP therapy targeting pathologies related to diminished bone quality could be promising in orthopedic surgery. Assessment of a clinically approved plasma jet regarding cellular effects on primary bone marrow mesenchymal stromal cells (hBM-MSCs) from relevant arthroplasty patient cohorts is needed to establish CPP-based therapeutic approaches for bone regeneration. Thus, the aim of this study was to derive biocompatible doses of CPP and subsequent evaluation of human primary hBM-MSCs’ osteogenic and immunomodulatory potential. Metabolic activity and cell proliferation were affected in a treatment-time-dependent manner. Morphometric high content imaging analyses revealed a decline in mitochondria and nuclei content and increased cytoskeletal compactness following CPP exposure. Employing a nontoxic exposure regime, investigation on osteogenic differentiation did not enhance osteogenic capacity of hBM-MSCs. Multiplex analysis of major hBM-MSC cytokines, chemokines and growth factors revealed an anti-inflammatory, promatrix-assembling and osteoclast-regulating secretion profile following CPP treatment and osteogenic stimulus. This study can be noted as the first in vitro study addressing the influence of CPP on hBM-MSCs from individual donors of an arthroplasty clientele.