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In musculoskeletal surgery, the treatment of large bone defects is challenging and can require the use of bone graft substitutes to restore mechanical stability and promote host-mediated regeneration. The use of bone allografts is well-established in many bone regenerative procedures, but is associated with low rates of ingrowth due to pre-therapeutic graft processing. Cold physical plasma (CPP), a partially ionized gas that simultaneously generates reactive oxygen (O2) and nitrogen (N2) species, is suggested to be advantageous in biomedical implant processing. CPP is a promising tool in allograft processing for improving surface characteristics of bone allografts towards enhanced cellularization and osteoconduction. However, a preclinical assessment regarding the feasibility of pre-therapeutic processing of allogeneic bone grafts with CPP has not yet been performed. Thus, this pilot study aimed to analyze the bone morphology of CPP processed allografts using synchrotron radiation-based microcomputed tomography (SR-µCT) and to analyze the effects of CPP processing on human bone cell viability and function. The analyzes, including co-registration of pre- and post-treatment SR-µCT scans, revealed that the main bone morphological properties (total volume, mineralized volume, surface area, and porosity) remained unaffected by CPP treatment if compared to allografts not treated with CPP. Varying effects on cellular metabolic activity and alkaline phosphatase activity were found in response to different gas mixtures and treatment durations employed for CPP application. It was found that 3 min CPP treatment using a He + 0.1% N2 gas mixture led to the most favourable outcome regarding a significant increase in bone cell viability and alkaline phosphatase activity. This study highlights the promising potential of pre-therapeuthic bone allograft processing by CPP prior to intraoperative application and emphasizes the need for gas source and treatment time optimization for specific applications.
Cold physical plasma (CPP) technology is of high promise for various medical applications.
The interplay of specific components of physical plasma with living cells, tissues and organs on a structural and functional level is of paramount interest with the aim to induce therapeutic effects in a controlled and replicable fashion.
In contrast to other medical disciplines such as dermatology and oromaxillofacial surgery, research reports on CPP application in orthopaedics are scarce.
The present implementation of CPP in orthopaedics involves surface modifications of orthopaedic materials and biomaterials to optimize osseointegration. In addition, the influence of CPP on musculoskeletal cells and tissues is a focus of research, including possible adverse reactions and side effects. Its bactericidal aspects make CPP an attractive supplement to current treatment regimens in case of microbial inflammations such as periprosthetic joint infections. Attributed anticancerogenic and pro-apoptotic effects underline the clinical relevance of CPP as an additive in treating malignant bone lesions.
The present review outlines ongoing research in orthopaedics involving CPP; it distinguishes considerations for safe application and the need for more evidence-based research to facilitate robust clinical implementation.
: Human osteosarcoma (OS) is the most common primary malignant bone tumor occurring
most commonly in adolescents and young adults. Major improvements in disease-free survival have
been achieved by implementing a combination therapy consisting of radical surgical resection of the
tumor and systemic multi-agent chemotherapy. However, long-term survival remains poor, so novel
targeted therapies to improve outcomes for patients with osteosarcoma remains an area of active
research. This includes immunotherapy, photodynamic therapy, or treatment with nanoparticles.
Cold atmospheric plasma (CAP), a highly reactive (partially) ionized physical state, has been shown
to inherit a significant anticancer capacity, leading to a new field in medicine called “plasma oncology.”
The current article summarizes the potential of CAP in the treatment of human OS and reviews the
underlying molecular mode of action.
Multiple evidence in animal models and in humans suggest a beneficial role of cold physical
plasma in wound treatment. Yet, risk assessment studies are important to further foster therapeutic
advancement and acceptance of cold plasma in clinics. Accordingly, we investigated the long-term
side effects of repetitive plasma treatment over 14 consecutive days in a rodent full-thickness ear
wound model. Subsequently, animals were housed for 350 days and sacrificed thereafter. In blood,
systemic changes of the pro-inflammatory cytokines interleukin 1β and tumor necrosis factor α
were absent. Similarly, tumor marker levels of α-fetoprotein and calcitonin remained unchanged.
Using quantitative PCR, the expression levels of several cytokines and tumor markers in liver,
lung, and skin were found to be similar in the control and treatment group as well. Likewise,
histological and immunohistochemical analysis failed to detect abnormal morphological changes
and the presence of tumor markers such as carcinoembryonic antigen, α-fetoprotein, or the neighbor
of Punc 11. Absence of neoplastic lesions was confirmed by non-invasive imaging methods such as
anatomical magnetic resonance imaging and positron emission tomography-computed tomography.
Our results suggest that the beneficial effects of cold plasma in wound healing come without apparent
side effects including tumor formation or chronic inflammation.
Despite continuous advances in therapy, malignant melanoma is still among the deadliest
types of cancer. At the same time, owing to its high plasticity and immunogenicity, melanoma is
regarded as a model tumor entity when testing new treatment approaches. Cold physical plasma is a
novel anticancer tool that utilizes a plethora of reactive oxygen species (ROS) being deposited on the
target cells and tissues. To test whether plasma treatment would enhance the toxicity of an established
antitumor therapy, ionizing radiation, we combined both physical treatment modalities targeting
B16F10 murine melanoma cell in vitro. Repeated rather than single radiotherapy, in combination
with gas plasma-introduced ROS, induced apoptosis and cell cycle arrest in an additive fashion. In
tendency, gas plasma treatment sensitized the cells to subsequent radiotherapy rather than the other
way around. This was concomitant with increased levels of TNFα, IL6, and GM-CSF in supernatants.
Murine JAWS dendritic cells cultured in these supernatants showed an increased expression of cell
surface activation markers, such as MHCII and CD83. For PD-L1 and PD-L2, increased expression
was observed. Our results are the first to suggest an additive therapeutic effect of gas plasma and
radiotherapy, and translational tumor models are needed to develop this concept further.
Medical gas plasmas are of emerging interest in pre-clinical oncological research. Similar to an array of first-line chemotherapeutics and physics-based therapies already approved for clinical application, plasmas target the tumor redox state by generating a variety of highly reactive species eligible for local tumor treatments. Considering internal tumors with limited accessibility, medical gas plasmas help to enrich liquids with stable, low-dose oxidants ideal for intratumoral injection and lavage. Pre-clinical investigation of such liquids in numerous tumor entities and models in vitro and in vivo provided evidence of their clinical relevance, broadening the range of patients that could benefit from medical gas plasma therapy in the future. Likewise, the application of such liquids might be promising for recurrent BRAF(V600E) papillary thyroid carcinomas, resistant to adjuvant administration of radioiodine. From a redox biology point of view, studying redox-based approaches in thyroid carcinomas is particularly interesting, as they evolve in a highly oxidative environment requiring the capability to cope with large amounts of ROS/RNS. Knowledge on their behavior under different redox conditions is scarce. The present study aimed to clarify resistance, proliferative activity, and the oxidative stress response of human papillary thyroid cancer cells K1 after exposure to plasma-oxidized DMEM (oxDMEM). Cellular responses were also evaluated when treated with different dosages of hydrogen peroxide and the RNS donor sodium nitroprusside (SNP). Our findings outline plasma-oxidized liquids as a promising approach targeting BRAF(V600E) papillary thyroid carcinomas and extend current knowledge on the susceptibility of cells to undergo ROS/RNS-induced cell death.
Reactive 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.
Gas 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.
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.
Cold medical gas plasmas are under pre-clinical investigation concerning their hemostatic activity and could be applied for intra-operative bleeding control in the future. The technological leap innovation was their generation at body temperature, thereby causing no thermal harm to the tissue and ensuring tissue integrity. This directly contrasts with current techniques such as electrocautery, which induces hemostasis by carbonizing the tissue using a heated electrode. However, the necrotized tissue is prone to fall, raising the risk of post-operative complications such as secondary bleedings or infection. In recent years, various studies have reported on the ability of medical gas plasmas to induce blood coagulation, including several suggestions concerning their mode of action. As non-invasive and gentle hemostatic agents, medical gas plasmas could be particularly eligible for vulnerable tissues, e.g., colorectal surgery and neurosurgery. Further, their usage could be beneficial regarding the prevention of post-operative bleedings due to the absence or sloughing of eschar. However, no clinical trials or individual healing attempts for medical gas plasmas have been reported to pave the way for clinical approvement until now, despite promising results in experimental animal models. In this light, the present mini-review aims to emphasize the potential of medical gas plasmas to serve as a hemostatic agent in clinical procedures. Providing a detailed overview of the current state of knowledge, feasible application fields are discussed, and possible obstacles are addressed.
AbstractCold physical plasma is a partially ionized gas that generates various components identified as potential anticancer compounds. Due to its topical application, cold plasmas are suitable, especially in dermatological applications. We, therefore, tested the cold plasma effects in skin cancer cells in vitro. An atmospheric pressure argon plasma jet was used as the plasma source. The plasma exposure alone reduced the metabolic activity and induced lethal effects in a treatment time-dependent fashion in both cell lines investigated. This was accompanied by executioner caspases 3 and 7, cleavage indicative of apoptosis and reduced cell migration and proliferation. Recent research also indicated roles of novel indirubin derivatives with potent anticancer effects. Three candidates were tested, and reduced metabolic activity and viability in a dose-dependent manner were found. Strikingly, one compound exerted notable synergistic toxicity when combined with plasma in skin cancer cells, which may be promising for future in vivo experiments.
The loss of skin integrity is inevitable in life. Wound healing is a necessary sequence of events to reconstitute the body’s integrity against potentially harmful environmental agents and restore homeostasis. Attempts to improve cutaneous wound healing are therefore as old as humanity itself. Furthermore, nowadays, targeting defective wound healing is of utmost importance in an aging society with underlying diseases such as diabetes and vascular insufficiencies being on the rise. Because chronic wounds’ etiology and specific traits differ, there is widespread polypragmasia in targeting non-healing conditions. Reactive oxygen and nitrogen species (ROS/RNS) are an overarching theme accompanying wound healing and its biological stages. ROS are signaling agents generated by phagocytes to inactivate pathogens. Although ROS/RNS’s central role in the biology of wound healing has long been appreciated, it was only until the recent decade that these agents were explicitly used to target defective wound healing using gas plasma technology. Gas plasma is a physical state of matter and is a partially ionized gas operated at body temperature which generates a plethora of ROS/RNS simultaneously in a spatiotemporally controlled manner. Animal models of wound healing have been vital in driving the development of these wound healing-promoting technologies, and this review summarizes the current knowledge and identifies open ends derived from in vivo wound models under gas plasma therapy. While gas plasma-assisted wound healing in humans has become well established in Europe, veterinary medicine is an emerging field with great potential to improve the lives of suffering animals.
The requirements for new technologies to serve as anticancer agents go far beyond their toxicity potential. Novel applications also need to be safe on a molecular and patient level. In a broader sense, this also relates to cancer metastasis and inflammation. In a previous study, the toxicity of an atmospheric pressure argon plasma jet in four human pancreatic cancer cell lines was confirmed and plasma treatment did not promote metastasis in vitro and in ovo. Here, these results are extended by additional types of analysis and new models to validate and define on a molecular level the changes related to metastatic processes in pancreatic cancer cells following plasma treatment in vitro and in ovo. In solid tumors that were grown on the chorion-allantois membrane of fertilized chicken eggs (TUM-CAM), plasma treatment induced modest to profound apoptosis in the tissues. This, however, was not associated with a change in the expression levels of adhesion molecules, as shown using immunofluorescence of ultrathin tissue sections. Culturing of the cells detached from these solid tumors for 6d revealed a similar or smaller total growth area and expression of ZEB1, a transcription factor associated with cancer metastasis, in the plasma-treated pancreatic cancer tissues. Analysis of in vitro and in ovo supernatants of 13 different cytokines and chemokines revealed cell line-specific effects of the plasma treatment but a noticeable increase of, e.g., growth-promoting interleukin 10 was not observed. Moreover, markers of epithelial-to-mesenchymal transition (EMT), a metastasis-promoting cellular program, were investigated. Plasma-treated pancreatic cancer cells did not present an EMT-profile. Finally, a realistic 3D tumor spheroid co-culture model with pancreatic stellate cells was employed, and the invasive properties in a gel-like cellular matrix were investigated. Tumor outgrowth and spread was similar or decreased in the plasma conditions. Altogether, these results provide valuable insights into the effect of plasma treatment on metastasis-related properties of cancer cells and did not suggest EMT-promoting effects of this novel cancer therapy.
Molecular Mechanisms of the Efficacy of Cold Atmospheric Pressure Plasma (CAP) in Cancer Treatment
(2020)
Non-healing wounds pose a major burden to patients and health care systems alike. These wounds are chronically stuck in the inflammatory phase of the healing process without transitioning to the proliferative phase. They are also characterized by the excessive presence of leukocytes which are assumed to provoke the persistent inflammation observed in pathological wound healing. Recent studies suggested a beneficial role of cold physical plasma in the treatment of chronic wounds. Hence, it was the central question, whether exposure to cold physical plasma would affect the viability and/or function of human leukocytes. Cold plasma displays various properties of which the generation of reactive molecules, such as reactive oxygen and nitrogen species (ROS/RNS), where found to be central in mediating redox changes in leukocytes. Oxidative stress was present especially in lymphocytes that readily underwent apoptosis after exposure to plasma. This was largely a direct consequence of plasma-generated hydrogen peroxide but not superoxide or RNS. Amount of apoptosis was comparable among several lymphocyte subpopulations, with the wound healing-relevant γδ T cells being least affected. Lymphocyte apoptosis was accompanied by mitochondrial membrane depolarization, caspase 3 activation, DNA fragmentation, and phosphatidylserine exposure. These results are in line with previous characterizations of the intrinsic apoptotic pathway in redox biology, and suggest that plasma-induced apoptosis was not mediated by alternative molecular mechanisms. An important immune response mechanism, the proliferation of lymphocytes, was not interrupted in plasma-treated but non-apoptotic cells. In wounds, a central role of leukocytes is to orchestrate the healing response via the release of small communication molecules called cytokines. Non-healing wounds are associated with elevated amounts of pro-inflammatory IL-1β, IL-6, and TNFα, and plasma-treatment of leukocytes strongly decreased their concentrations. At the same time, the expression of anti inflammatory cytokines (IL-10, TGFβ) was markedly increased. The pro inflammatory chemokine IL-8 was the only molecule to be significantly increased in supernatants of plasma-treated cells. IL-8 is the major chemo-attractant for neutrophil granulocytes. Neutrophils are frequently associated with non-healing wounds. These professional phagocytes are the first to migrate to the site of injury where they inactivate invading pathogens by various mechanisms. Importantly, highly relevant effector functions remained mostly unaffected by plasma treatment: the phagocytosis of bacteria, the oxidative burst, and the intracellular killing of microbes. Of note, plasma induced a strong induction of neutrophil extracellular traps (NETs). Decorated with antimicrobial proteins, NETs are web-like chromatin extrusions that entrap pathogens. These results have several implications for wound healing. Plasma-treated neutrophils were still capable of eradicating bacteria, which are frequently associated with non-healing wounds. In addition, plasma-induced NETs could aid in wound healing by providing an antibacterial scaffold to safeguard against further dissemination of microorganisms. Chronic wounds display a state of sustained inflammation and plasma induced apoptosis but not necrosis in lymphocytes. This was an important finding as necrosis, the involuntary cell death, is associated with the release of intracellular content, enhancing inflammation. By contrast, apoptosis dampens it as dead cells are cleared by macrophages inducing anti inflammatory responses. Further, the cytokine signature of plasma-treated leukocytes was largely non inflammatory, which could further decrease inflammation in wounds. Altogether, this work provided first insight with regard to effects and mechanisms of cold physical plasma treatment of wound-relevant leukocytes. Generally, these cells were affected by a plasma mediated modulation of their redox state. Future studies should include the possibility of redox modulation into their experimental approach to further elucidate the role of ROS/RNS in inflammation and possibly to improve existing wound healing therapies.