Oxidized Melanoma Antigens Promote Activation and Proliferation of Cytotoxic T-Cell Subpopulations.

Increasing evidence suggests the role of reactive oxygen and nitrogen species (RONS) in regulating antitumor immune effects and immunosuppression. RONS modify biomolecules and induce oxidative post-translational modifications (oxPTM) on proteins that can alarm phagocytes. However, it is unclear if and how protein oxidation by technical means could be a strategy to foster antitumor immunity and therapy. To this end, cold gas plasma technology producing various RONS simultaneously to oxidize the two melanoma-associated antigens MART and PMEL is utilized. Cold plasma-oxidized MART (oxMART) and PMEL (oxPMEL) are heavily decorated with oxPTMs as determined by mass spectrometry. Immunization with oxidized MART or PMEL vaccines prior to challenge with viable melanoma cells correlated with significant changes in cytokine secretion and altered T-cell differentiation of tumor-infiltrated leukocytes (TILs). oxMART promoted the activity of cytotoxic central memory T-cells, while oxPMEL led to increased proliferation of cytotoxic effector T-cells. Similar T-cell results are observed after incubating splenocytes of tumor-bearing mice with B16F10 melanoma cells. This study, for the first time, provides evidence of the importance of oxidative modifications of two melanoma-associated antigens in eliciting anticancer immunity.

Multi-Cohort Transcriptomic Profiling of Medical Gas Plasma-Treated Cancers Reveals the Role of Immunogenic Cell Death.

The therapeutic potential of cold physical gas plasma operated at atmospheric pressure in oncology has been thoroughly demonstrated in numerous preclinical studies. The cytotoxic effect on malignant cells has been attributed mainly to biologically active plasma-generated compounds, namely, reactive oxygen and nitrogen species. The intracellular accumulation of reactive oxygen and nitrogen species interferes strongly with the antioxidant defense system of malignant cells, activating multiple signaling cascades and inevitably leading to oxidative stress-induced cell death. This study aims to determine whether plasma-induced cancer cell death operates through a universal molecular mechanism that is independent of the cancer cell type. Using whole transcriptome data, we sought to investigate the activation mechanism of plasma-treated samples in patient-derived prostate cell cultures, melanoma, breast, lymphoma, and lung cancer cells. The results from the standardized single-cohort gene expression analysis and parallel multi-cohort meta-analysis strongly indicate that plasma treatment globally induces cancer cell death through immune-mediated mechanisms, such as interleukin signaling, Toll-like receptor cascades, and MyD88 activation leading to pro-inflammatory cytokine release and tumor antigen presentation.

Cold Physical Plasma Reduces Motility of Various Bone Sarcoma Cells While Remodeling the Cytoskeleton.

BACKGROUND/AIM: Cold physical plasma (CPP) has emerged as an effective therapy in oncology by inducing cytotoxic effects in various cancer cells, including chondrosarcoma (CS), Ewing's sarcoma (ES), and osteosarcoma (OS). The current study investigated the impact of CPP on cell motility in CS (CAL-78), ES (A673), and OS (U2-OS) cell lines, focusing on the actin cytoskeleton. MATERIALS AND METHODS: The CASY Cell Counter and Analyzer was used to study cell proliferation and determine the optimal concentrations of fetal calf serum to maintain viability without stimulation of cell proliferation. CellTiter-BlueCell viability assay was used to determine the effects of CPP on the viability of bone sarcoma cells. The Radius assay was used to determine cell migration. Staining for Deoxyribonuclease I, G-actin, and F-actin was used to assay for the effects on the cytoskeleton. RESULTS: Reductions in cell viability and motility were observed across all cell lines following CPP treatment. CPP induced changes in the actin cytoskeleton, leading to decreased cell motility. CONCLUSION: CPP effectively reduces the motility of bone sarcoma cells by altering the actin cytoskeleton. These findings underscore CPP's potential as a therapeutic tool for bone sarcomas and highlight the need for further research in this area.

Reactive Species Risk Assessment Using Optimized HET-CAM Safety Evaluation of Feed Gas-Modified Gas Plasma Technology and Anticancer Drugs.

Clinical therapies, including dermatology and oncology, require safe application. In vitro experiments allow only limited conclusions about in vivo effects, while animal studies in, e.g., rodents have ethical constraints at a large scale. Chicken embryos lack pain reception until day 15 postfertilization, making the in ovo model a suitable alternative to in vivo safety assessment. In addition, the hen's egg test on chorioallantoic membrane assay allows irritation potential analysis for topical treatments, but standardized analysis has been limited so far. Medical gas plasma is a topical, routine, approved dermatology treatment. Recent work suggests the potential of this technology in oncology. Its main mode of action is the release of various reactive species simultaneously. Intriguingly, varying plasma feed gas compositions generates customized reactive species profiles previously shown to be optimized for specific applications, such as skin cancer treatment. To support clinical implications, we developed a novel chicken embryo CAM scoring and study scheme and employed the model to analyze 16 different plasma feed gas settings generated by the atmospheric pressure plasmajet kINPen, along with common anticancer drugs (e.g., cisplatin) and physiological mediators (e.g., VEGF). Extensive gas- and liquid-phase plasma reactive species profiling was done and was found to have a surprisingly low correlation with irritation potential parameters. Despite markedly different reactive species patterns, feed gas-modulated kINPen plasma was equally tolerated compared to standard argon plasma. CAM irritation with gas plasmas but not anticancer agents was reversed 48 h after treatment, underlining the only temporary tissue effects of medical gas plasma. Our results indicate a safe therapeutic application of reactive species.

Nanoplastic Size and Surface Chemistry Dictate Decoration by Human Saliva Proteins.

Environmental pollution with plastic polymers has become a global problem, leaving no continent and habitat unaffected. Plastic waste is broken down into smaller parts by environmental factors, which generate micro- and nanoplastic particles (MNPPs), ultimately ending up in the human food chain. Before entering the human body, MNPPs make their first contact with saliva in the human mouth. However, it is unknown what proteins attach to plastic particles and whether such protein corona formation is affected by the particle's biophysical properties. To this end, we employed polystyrene MNPPs of two different sizes and three different charges and incubated them individually with saliva donated by healthy human volunteers. Particle zeta potential and size analyses were performed using dynamic light scattering complemented by nanoliquid chromatography high-resolution mass spectrometry (nLC/HRMS) to qualitatively and quantitatively reveal the protein soft and hard corona for each particle type. Notably, protein profiles and relative quantities were dictated by plastic particle size and charge, which in turn affected their hydrodynamic size, polydispersity, and zeta potential. Strikingly, we provide evidence of the latter to be dynamic processes depending on exposure times. Smaller particles seemed to be more reactive with the surrounding proteins, and cultures of the particles with five different cell lines (HeLa, HEK293, A549, HepG2, and HaCaT) indicated protein corona effects on cellular metabolic activity and genotoxicity. In summary, our data suggest nanoplastic size and surface chemistry dictate the decoration by human saliva proteins, with important implications for MNPP uptake in humans.

Exploring the Influence of Cold Plasma on Epidermal Melanogenesis In Situ and In Vitro.

Epidermal melanin synthesis determines an individual's skin color. In humans, melanin is formed by melanocytes within the epidermis. The process of melanin synthesis strongly depends on a range of cellular factors, including the fine-tuned interplay with reactive oxygen species (ROS). In this context, a role of cold atmospheric plasma (CAP) on melanin synthesis was proposed due to its tunable ROS generation. Herein, the argon-driven plasma jet kINPen® MED was employed, and its impact on melanin synthesis was evaluated by comparison with known stimulants such as the phosphodiesterase inhibitor IBMX and UV radiation. Different available model systems were employed, and the melanin content of both cultured human melanocytes (in vitro) and full-thickness human skin biopsies (in situ) were analyzed. A histochemical method detected melanin in skin tissue. Cellular melanin was measured by NIR autofluorescence using flow cytometry, and a highly sensitive HPLC-MS method was applied, which enabled the differentiation of eu- and pheomelanin by their degradation products. The melanin content in full-thickness human skin biopsies increased after repeated CAP exposure, while there were only minor effects in cultured melanocytes compared to UV radiation and IBMX treatment. Based on these findings, CAP does not appear to be a useful option for treating skin pigmentation disorders. On the other hand, the risk of hyperpigmentation as an adverse effect of CAP application for wound healing or other dermatological diseases seems to be neglectable.

Sonicated polyethylene terephthalate nano- and micro-plastic-induced inflammation, oxidative stress, and autophagy in vitro.

The environmental presence of nano- and micro-plastic particles (NMPs) is suspected to have a negative impact on human health. Environmental NMPs are difficult to sample and use in life science research, while commercially available plastic particles are too morphologically uniform. Additionally, this NMPs exposure exhibited biological effects, including cell internalization, oxidative stress, inflammation, cellular adaptation, and genotoxicity. Therefore, developing new methods for producing heterogenous NMPs as observed in the environment is important as reference materials for research. Thus, we aimed to generate and characterize NMPs suspensions using a modified ultrasonic protocol and to investigate their biological effects after exposure to different human cell lines. To this end, we produced polyethylene terephthalate (PET) NMPs suspensions and characterized the particles by dynamic light scattering and scanning electron microscopy. Ultrasound treatment induced polymer degradation into smaller and heterogeneous PET NMPs shape fragments with similar surface chemistry before and after treatment. A polydisperse suspension of PET NMPs with 781 nm in average size and negative surface charge was generated. Then, the PET NMPs were cultured with two human cell lines, A549 (lung) and HaCaT (skin), addressing inhalation and topical exposure routes. Both cell lines interacted with and have taken up PET NMPs as quantified via cellular granularity assay. A549 but not HaCaT cell metabolism, viability, and cell death were affected by PET NMPs. In HaCaT keratinocytes, large PET NMPs provoked genotoxic effects. In both cell lines, PET NMPs exposure affected oxidative stress, cytokine release, and cell morphology, independently of concentration, which we could relate mechanistically to Nrf2 and autophagy activation. Collectively, we present a new PET NMP generation model suitable for studying the environmental and biological consequences of exposure to this polymer.

Cold Atmospheric Plasma Is a Promising Alternative Treatment Option in Case of Split Skin Graft Failure.

Cold atmospheric plasma (CAP) has shown promising potential in promoting wound healing. This case report presents the successful application of CAP in a 42-year-old female patient with extensive wound healing disorders and superinfections following the excision of an abscess in the left thoracic region. After several failed split skin graft attempts, the implementation of CAP led to significant improvements in wound healing. This report highlights the wound healing-promoting effects of CAP and discusses its potential mechanisms of action.

Lactic acid in macrophage polarization: A factor in carcinogenesis and a promising target for cancer therapy.

Cancer remains a formidable challenge, continually revealing its intricate nature and demanding novel treatment approaches. Within this intricate landscape, the tumor microenvironment and its dynamic components have gained prominence, particularly macrophages that can adopt diverse polarization states, exerting a profound influence on cancer progression. Recent revelations have spotlighted lactic acid as a pivotal player in this complex interplay. This review systematically explores lactic acid's multifaceted role in macrophage polarization, focusing on its implications in carcinogenesis. We commence by cultivating a comprehensive understanding of the tumor microenvironment and the pivotal roles played by macrophages. The dynamic landscape of macrophage polarization, typified by M1 and M2 phenotypes, is dissected to reveal its substantial impact on tumor progression. Lactic acid, a metabolic byproduct, emerges as a key protagonist, and we meticulously unravel the mechanisms underpinning its generation within cancer cells, shedding light on its intimate association with glycolysis and its transformative effects on the tumor microenvironment. Furthermore, we decipher the intricate molecular framework that underlies lactic acid's pivotal role in facilitating macrophage polarization. Our review underscores lactic acid's dual role in carcinogenesis, orchestrating tumor growth and immune modulation within the tumor microenvironment, thereby profoundly influencing the balance between pro-tumor and anti-tumor immune responses. This duality highlights the therapeutic potential of selectively manipulating lactic acid metabolism for cancer treatment. Exploring strategies to inhibit lactic acid production by tumor cells, novel approaches to impede lactic acid transport in the tumor microenvironment, and the burgeoning field of immunotherapeutic cancer therapies utilizing lactic acid-induced macrophage polarization form the core of our investigation.

Effective combination of cold physical plasma and chemotherapy against Ewing sarcoma cells in vitro.

Ewing's sarcoma (ES) is the second most common bone tumor in children and adolescents and is highly malignant. Although the new chemotherapy has significantly improved the survival rate for ES from about 10 to 75%, the survival rate for metastatic tumors remains around 30%. This treatment is often associated with various side effects that contribute to the suffering of the patients. Cold physical plasma (CPP), whether used alone or in combination with current chemotherapy, is considered a promising adjunctive tool in cancer treatment. This study aims to investigate the synergistic effects of CPP in combination with cytostatic chemotherapeutic agents that are not part of current ES therapy. Two different ES cell lines, RD-ES and A673, were treated with the determined IC20 concentrations of the chemotherapeutic agents cisplatin and methotrexate (MTX) in combination with CPP. The effects on population doubling, cell viability, and apoptotic processes within these cell lines were assessed. This combination therapy has led to a reduction of population doubling and cell viability, as well as an increase in apoptotic activity in cells compared to CPP monotherapy. The results of this study provide evidence that combining CPP with non-common chemotherapy drugs such as MTX and CIS in the treatment of ES enhances the anticancer effects of these drugs. These findings open up new possibilities for the effective use of these drugs against ES.

Convolutional neuronal network for identifying single-cell-platelet-platelet-aggregates in human whole blood using imaging flow cytometry.

Imaging flow cytometry is an attractive method to investigate individual cells by optical properties. However, imaging flow cytometry applications with clinical relevance are scarce so far. Platelet aggregation naturally occurs during blood coagulation to form a clot. However, aberrant platelet aggregation is associated with cardiovascular disease under steady-state conditions in the blood. Several types of so-called antiplatelet drugs are frequently described to reduce the risk of stroke or cardiovascular diseases. However, an efficient monitoring method is missing to identify the presence and frequency of platelet-platelet aggregates in whole blood on a single cell level. In this work, we employed imaging flow cytometry to identify fluorescently labeled platelets in whole blood with a conditional gating strategy. Images were post-processed and aligned. A convolutional neural network was designed to identify platelet-platelet aggregates of two, three, and more than three platelets, and results were validated against various data set properties. In addition, the neural network excluded erythrocyte-platelet aggregates from the results. Based on the results, a parameter for detecting platelet-platelet aggregates, the weighted platelet aggregation, was developed. If employed on a broad scale with proband and patient samples, our method could aid in building a future diagnostic marker for cardiovascular disease and monitoring parameters to optimize drug prescriptions in such patient groups.

Package delivered: folate receptor-mediated transporters in cancer therapy and diagnosis.

Neoplasias pose a significant threat to aging society, underscoring the urgent need to overcome the limitations of traditional chemotherapy through pioneering strategies. Targeted drug delivery is an evolving frontier in cancer therapy, aiming to enhance treatment efficacy while mitigating undesirable side effects. One promising avenue utilizes cell membrane receptors like the folate receptor to guide drug transporters precisely to malignant cells. Based on the cellular folate receptor as a cancer cell hallmark, targeted nanocarriers and small molecule-drug conjugates have been developed that comprise different (bio) chemistries and/or mechanical properties with individual advantages and challenges. Such modern folic acid-conjugated stimuli-responsive drug transporters provide systemic drug delivery and controlled release, enabling reduced dosages, circumvention of drug resistance, and diminished adverse effects. Since the drug transporters' structure-based de novo design is increasingly relevant for precision cancer remediation and diagnosis, this review seeks to collect and debate the recent approaches to deliver therapeutics or diagnostics based on folic acid conjugated Trojan Horses and to facilitate the understanding of the relevant chemistry and biochemical pathways. Focusing exemplarily on brain and breast cancer, recent advances spanning 2017 to 2023 in conjugated nanocarriers and small molecule drug conjugates were considered, evaluating the chemical and biological aspects in order to improve accessibility to the field and to bridge chemical and biomedical points of view ultimately guiding future research in FR-targeted cancer therapy and diagnosis.

Oxali-palladium nanoparticle synthesis, characterization, protein binding, and apoptosis induction in colorectal cancer cells.

This paper focuses on the synthesis of nano-oxali-palladium coated with turmeric extract (PdNPs) using a green chemistry technique based on the reduction in the Pd (II) complex by phytochemicals inherent in turmeric extract. PdNPs were examined and characterized using Field Emission Scanning Electron Microscopy (FESEM), Dynamic Light Scattering (DLS), Fourier Transform Infrared (FTIR), and Atomic Force Microscopy (AFM). Using different spectroscopic and molecular dynamics simulations, a protein-binding analysis of the produced nanoparticle was conducted by observing its interaction with human serum albumin (HSA). Lastly, the cytotoxic effects and apoptotic processes of PdNPs were studied against the HCT116 human colorectal cell line using the MTT assay and flow cytometry tests. According to the findings, PdNPs with spherical and homogenous morphology and a size smaller than 100 nm were generated. In addition, they can induce apoptosis in colorectal cancer cells in a dose-dependent manner with a lower Cc50 (78 µL) than cisplatin and free oxali-palladium against HCT116 cells. The thermodynamic characteristics of protein binding of nanoparticles with HSA demonstrated that PdNPs had a great capacity for quenching and interacting with HSA through hydrophobic forces. In addition, molecular dynamics simulations revealed that free oxali-palladium and PdNP attach to the same area of HSA via non-covalent interactions. It is conceivable to indicate that the synthesized PdNPs are a potential candidate for the construction of novel, nature-based anticancer treatments with fewer side effects and a high level of eco-friendliness.

Gas Plasma Exposure Alters Microcirculation and Inflammation during Wound Healing in a Diabetic Mouse Model.

Diabetes can disrupt physiological wound healing, caused by decreased levels or impaired activity of angiogenic factors. This can contribute to chronic inflammation, poor formation of new blood vessels, and delayed re-epithelialization. The present study describes the preclinical application of medical gas plasma to treat a dermal, full-thickness ear wound in streptozotocin (STZ)-induced diabetic mice. Gas plasma-mediated effects occurred in both sexes but with gender-specific differences. Hyperspectral imaging demonstrated gas plasma therapy changing microcirculatory parameters, particularly oxygen saturation levels during wound healing, presumably due to the gas plasma's tissue delivery of reactive species and other bioactive components. In addition, gas plasma treatment significantly affected cell adhesion by regulating focal adhesion kinase and vinculin, which is important in maintaining skin barrier function by regulating syndecan expression and increasing re-epithelialization. An anticipated stimulation of blood vessel formation was detected via transcriptional and translational increase of angiogenic factors in gas plasma-exposed wound tissue. Moreover, gas plasma treatment significantly affected inflammation by modulating systemic growth factors and cytokine levels. The presented findings may help explain the mode of action of successful clinical plasma therapy of wounds of diabetic patients.

ß-Caryophyllene Inhibits Endothelial Tube Formation by Modulating the Secretome of Hypoxic Lung Cancer Cells-Possible Role of VEGF Downregulation.

ß-Caryophyllene (BCP), a bicyclic sesquiterpene that is a component of the essential oils of various spice and food plants, has been described as a selective CB2 cannabinoid receptor agonist. In the present study, the effect of BCP on angiogenesis was investigated. It was found that conditioned media (CM) from BCP-treated hypoxic A549 lung cancer cells exhibited a concentration-dependent inhibitory effect on human umbilical vein endothelial cell (HUVEC) tube formation induced by CM from vehicle-treated hypoxic A549 cells. There was an associated concentration-dependent decrease in the proangiogenic factor vascular endothelial growth factor (VEGF) in the CM, with both BCP inhibitory effects (tube formation, VEGF secretion) being CB2 receptor-dependent. A reduction of the transcription factor hypoxia-inducible factor 1α (HIF-1α) was furthermore detected. The antiangiogenic and VEGF-lowering properties of BCP were confirmed when CM from another lung cancer cell line, H358, were tested. When directly exposed to HUVECs, BCP showed no significant effect on tube formation, but at 10 µM, impaired VEGF receptor 2 (VEGFR2) phosphorylation triggered by recombinant VEGF in a CB2 receptor-independent manner. In summary, BCP has a dual antiangiogenic effect on HUVECs, manifested in the inhibition of tube formation through modulation of the tumor cell secretome and additionally in the inhibition of VEGF-induced VEGFR2 activation. Because the CB2 agonist has no psychoactive properties, BCP should continue to be evaluated preclinically for further antitumor effects.

Synergistic effect of cold gas plasma and experimental drug exposure exhibits skin cancer toxicity in vitro and in vivo.

INTRODUCTION: Skin cancer is often fatal, which motivates new therapy avenues. Recent advances in cancer treatment are indicative of the importance of combination treatments in oncology. Previous studies have identified small molecule-based therapies and redox-based technologies, including photodynamic therapy or medical gas plasma, as promising candidates to target skin cancer. OBJECTIVE: We aimed to identify effective combinations of experimental small molecules with cold gas plasma for therapy in dermato-oncology. METHODS: Promising drug candidates were identified after screening an in-house 155-compound library using 3D skin cancer spheroids and high content imaging. Combination effects of selected drugs and cold gas plasma were investigated with respect to oxidative stress, invasion, and viability. Drugs that had combined well with cold gas plasma were further investigated in vascularized tumor organoids in ovo and a xenograft mouse melanoma model in vivo. RESULTS: The two chromone derivatives Sm837 and IS112 enhanced cold gas plasma-induced oxidative stress, including histone 2A.X phosphorylation, and further reduced proliferation and skin cancer cell viability. Combination treatments of tumor organoids grown in ovo confirmed the principal anti-cancer effect of the selected drugs. While one of the two compounds exerted severe toxicity in vivo, the other (Sm837) resulted in a significant synergistic anti-tumor toxicity at good tolerability. Principal component analysis of protein phosphorylation profiles confirmed profound combination treatment effects in contrast to the monotherapies. CONCLUSION: We identified a novel compound that, combined with topical cold gas plasma-induced oxidative stress, represents a novel and promising treatment approach to target skin cancer.

Gas plasmas technology: from biomolecule redox research to medical therapy.

Physical plasma is one consequence of gas ionization, i.e. its dissociation of electrons and ions. If operated in ambient air containing oxygen and nitrogen, its high reactivity produces various reactive oxygen and nitrogen species (RONS) simultaneously. Technology leap innovations in the early 2010s facilitated the generation of gas plasmas aimed at clinics and operated at body temperature, enabling their potential use in medicine. In parallel, their high potency as antimicrobial agents was systematically discovered. In combination with first successful clinical trials, this led in 2013 to the clinical approval of first medical gas plasma devices in Europe for promoting the healing of chronic and infected wounds and ulcers in dermatology. While since then, thousands of patients have benefited from medical gas plasma therapy, only the appreciation of the critical role of gas plasma-derived RONS led to unraveling first fragments of the mechanistic basics of gas plasma-mediated biomedical effects. However, drawing the complete picture of effectors and effects is still challenging. This is because gas plasma-produced RONS not only show a great variety of dozens of types but also each of them having distinct spatio-temporal concentration profiles due to their specific half-lives and reactivity with other types of RONS as well as different types of (bio) molecules they can react with. However, this makes gas plasmas fascinating and highly versatile tools for biomolecular redox research, especially considering that the technical capacity of increasing and decreasing individual RONS types holds excellent potential for tailoring gas plasmas toward specific applications and disease therapies.

Multi-Oxidant Environment as a Suicidal Inhibitor of Myeloperoxidase.

Tissue inflammation drives the infiltration of innate immune cells that generate reactive species to kill bacteria and recruit adaptive immune cells. Neutrophil activation fosters the release of myeloperoxidase (MPO) enzyme, a heme-containing protein generating hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride ions. MPO-dependent oxidant formation initiates bioactive oxidation and chlorination products and induces oxidative post-translational modifications (oxPTMs) on proteins and lipid oxidation. Besides HOCl and H2O2, further reactive species such as singlet oxygen and nitric oxide are generated in inflammation, leading to modified proteins, potentially resulting in their altered bioactivity. So far, knowledge about multiple free radical-induced modifications of MPO and its effects on HOCl generation is lacking. To mimic this multi-oxidant microenvironment, human MPO was exposed to several reactive species produced simultaneously via argon plasma operated at body temperature. Several molecular gas admixes were used to modify the reactive species type profiles generated. MPO was investigated by studying its oxPTMs, changes in protein structure, and enzymatic activity. MPO activity was significantly reduced after treatment with all five tested plasma gas conditions. Dynamic light scattering and CD-spectroscopy revealed altered MPO protein morphology indicative of oligomerization. Using mass spectrometry, various oxPTMs, such as +1O, +2O, and +3O, were determined on methionine and cysteine (Cys), and -1H-1N+1O was detected in asparagine (Asp). The modification types identified differed between argon-oxygen and argon-nitrogen plasmas. However, all plasma gas conditions led to the deamidation of Asp and oxidation of Cys residues, suggesting an inactivation of MPO due to oxPTM-mediated conformational changes.

Bone marrow from periacetabular osteotomies as a novel source for human mesenchymal stromal cells.

BACKGROUND: Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are used in regenerative medicine and related research involving immunomodulatory, anti-inflammatory, anti-fibrotic and regenerative functions. Isolation of BM-MSCs from samples obtained during total hip arthroplasty (THA) is routinely possible. Advanced age and comorbidities of the majority of patients undergoing THA limit their applicability. Our study aimed to evaluate the potential of bone marrow obtained during periacetabular osteotomy (PAO) as a novel source of BM-MSCs from young donors by analyzing cell yield and cell characteristics. METHODS: Bone samples were obtained from the anterior Os ilium or superior Os pubis during PAO and from the femoral cavity during primary THA. Isolation of bone marrow-derived mononuclear cells (BM-MNCs) was performed by density gradient centrifugation. The samples from PAO and THA patients were compared in terms of BM-MSC yield, colony formation and the proportion of BM-MSCs within the BM-MNC population using flow cytometry analysis. The cells were characterized based on the expression of BM-MSC-specific surface markers. The functionality of the cells was compared by quantifying post-thaw viability, metabolic activity, proliferation capacity, senescence-associated beta galactosidase (SA-ß-gal) expression, trilineage differentiation potential and major secretome proteins. RESULTS: Isolation of BM-MNCs was possible in a reliable and reproducible manner when using bone from PAO containing more than 0.24 g bone marrow. PAO patients were younger than patients of the THA group. Bone obtained during PAO contained less bone marrow and led to a lower BM-MSC number after the first cell culture passage compared to BM-MSCs obtained during THA. BM-MSCs from PAO samples are characterized by a higher proliferation capacity. This results in a higher yield in cell culture passage two, when normalized to the sample weight. BM-MSCs from PAO patients showed increased secretion of TGF-ß1, TIMP2, and VEGF upon osteogenic differentiation. BM-MSCs from PAO and THA patients revealed similar results regarding the onset of SA-ß-gal expression and trilineage differentiation capacity. CONCLUSIONS: We suggest that bone obtained during PAO is a promising novel source for BM-MSCs from young donors. Limited absolute cell yield due to low sample weight must be considered in early cell culture passages and might be critical for the range of clinical applications possible for BM-MSCs from this source. The higher proliferation capacity and increased growth factor secretion of BM-MSCs from young donors may be beneficial for future regenerative cell therapies, in vitro models, and tissue engineering.