Plasma-generated Reactive Species Research Articles

On a heavy path - determining cold plasma-derived short-lived species chemistry using isotopic labelling.

Cold atmospheric plasmas (CAPs) are promising medical tools and are currently applied in dermatology and epithelial cancers. While understanding of the biomedical effects is already substantial, knowledge on the contribution of individual ROS and RNS and the mode of activation of biochemical pathways is insufficient. Especially the formation and transport of short-lived reactive species in liquids remain elusive, a situation shared with other approaches involving redox processes such as photodynamic therapy. Here, the contribution of plasma-generated reactive oxygen species (ROS) in plasma liquid chemistry was determined by labeling these via admixing heavy oxygen 18O2 to the feed gas or by using heavy water H218O as a solvent for the bait molecule. The inclusion of heavy or light oxygen atoms by the labeled ROS into the different cysteine products was determined by mass spectrometry. While products like cysteine sulfonic acid incorporated nearly exclusively gas phase-derived oxygen species (atomic oxygen and/or singlet oxygen), a significant contribution of liquid phase-derived species (OH radicals) was observed for cysteine-S-sulfonate. The role, origin, and reaction mechanisms of short-lived species, namely hydroxyl radicals, singlet oxygen, and atomic oxygen, are discussed. Interactions of these species both with the target cysteine molecule as well as the interphase and the liquid bulk are taken into consideration to shed light onto several reaction pathways resulting in observed isotopic oxygen incorporation. These studies give valuable insight into underlying plasma–liquid interaction processes and are a first step to understand these interaction processes between the gas and liquid phase on a molecular level.

The effect of pH on the aqueous reactive species in sodium phosphate buffers induced by surface air discharge

The solution pH is an important parameter for the applications of atmospheric plasmas such as sewage disposal, food processing and biomedicine, mainly because the pH affects both the liquid-phase chemistry and the oxidation potential of the plasma-generated aqueous reactive species. In order to unravel the correlation between the aqueous reactive species and the solution pH, a numerical model was developed in this paper to study the sodium phosphate buffers treated by the surface plasma in atmospheric air, and the initial concentrations of phosphates in the buffers were altered to make the initial pH change from 3.0 to 11.9. After treated by the surface air plasma for 1 min, it was found that the concentrations of aqueous O3, HNO2, HNO3 and NO3 decreased with an increase in the initial pH, while those of , O−, , , , ONOO− and O2NOO− anions increased. Additionally, the concentrations of aqueous H2O2, HO3, and NO2 changed little with the initial pH. The oxidation potentials of O3, OH, H2O2 and HO2 decreased with the increasing initial pH, but those of ONOOH and ONOO− peaked at pH ~ 8. Some phosphorous compounds such as were also produced, which had an undeniable effect on the liquid-phase chemistry of the reactive species at pH ⩾ 6.0.

Degradation effect and mechanism of gas-liquid phase dielectric barrier discharge on norfloxacin combined with H2O2 or Fe2+

Norfloxacin (NOR) is a typical kind of Fluoroquinolone antibiotics which is extensively used around the world. Residues of NOR have been found continuously increasing in the water systems thus posing detrimental effects to the aquatic ecosystem and human health. In this study, the degradation effect and mechanism on NOR in aqueous solution are investigated by gas-liquid phase dielectric barrier discharge (DBD) plasma combined with different catalysts (H2O2 or Fe2+). The concentrations of three representative plasma-generated long-lived reactive species (H2O2, O3 and NO3-) were measured in dH2O and found enhancing with the lengthening discharge period. Variations on the treatment parameters were carried out in discharge power, initial solution pH and the concentration of catalysts. A discharge power at 60 W provided optimal balance on the degradation effect and energy efficiency would be beneficial, while there is not much impact from the initial solution pH on NOR degradation. Both H2O2 and Fe2+ at low concentration (0.5–10 mg/L of Fe2+, 0.1–1.0 mmol/L of H2O2) were observed contributing to the improvement on the NOR degradation rate, while inhibition would occur at high concentration (10–20 mg/L of Fe2+, 1.0–2.0 mmol/L of H2O2). The DBD/H2O2 (0.5 mmol/L)/Fe2+ (10 mg/L) system was found the optimal condition to achieve a high NOR degradation rate of more than 98% after plasma treatment for only 0.5 min at discharge power of 60 W. Moreover, 72.3 ± 1.7% of the total organic carbon (TOC) in NOR solution have been removed in the DBD/Fe2+ (10 mg/L) system at 15 min which signifies a high mineralization efficiency. The cleavage of piperazine ring and quinolone ring, defluorination and hydroxylation are considered the major pathways for NOR degradation. The biological toxicity of the plasma/Fe2+-induced degradation intermediates has been detected and it fluctuated with the treatment time and finally declined. Therefore, the DBD/catalyst system could be an effective and promising method on pharmaceutical wastewater treatment in the future.

Plasma for cancer treatment: How can RONS penetrate through the cell membrane? Answers from computer modeling

Plasma is gaining increasing interest for cancer treatment, but the underlying mechanisms are not yet fully understood. Using computer simulations at the molecular level, we try to gain better insight in how plasma-generated reactive oxygen and nitrogen species (RONS) can penetrate through the cell membrane. Specifically, we compare the permeability of various (hydrophilic and hydrophobic) RONS across both oxidized and non-oxidized cell membranes. We also study pore formation, and how it is hampered by higher concentrations of cholesterol in the cell membrane, and we illustrate the much higher permeability of H2O2 through aquaporin channels. Both mechanisms may explain the selective cytotoxic effect of plasma towards cancer cells. Finally, we also discuss the synergistic effect of plasma-induced oxidation and electric fields towards pore formation.

Uptake and diffusion of plasma-generated reactive nitrogen species through keratinized membrane

We propose a mathematical model for uptake and diffusion of air plasma-generated reactive nitrogen species (RNS) in a model keratinized membrane, such as a thin slice of bovine hoof or human nail material. An experimental system consisting of a surface microdischarge (SMD) in air was designed for the purpose of developing and validating a reaction-diffusion model to describe this system. Key variables such as membrane effective diffusivity, surface reaction rate coefficients, and other parameters are determined through comparison between the model predictions and experimental measurements. The model results yield spatial and temporal concentration profiles of RNS inside the keratinized membrane, leading to an improved understanding of transport and reaction of plasma generated RNS through the membrane. This work offers insights into possible mechanisms underlying plasma treatment of toenail fungus.

Plasma-generated reactive oxygen and nitrogen species can lead to closure, locking and constriction of the Dionaea muscipula Ellis trap.

Reactive oxygen and nitrogen species (RONS) can influence plant signalling, physiology and development. We have previously observed that an argon plasma jet in atmospheric air can activate plant movements and morphing structures in the Venus flytrap and Mimosa pudica similar to stimulation of their mechanosensors in vivo. In this paper, we found that the Venus flytrap can be activated by plasma jets without direct contact of plasma with the lobe, midrib or cilia. The observed effects are attributed to RONS, which are generated by argon and helium plasma jets in atmospheric air. We also found that application of H2O2 or HNO3 aqueous solutions to the midrib induces propagation of action potentials and trap closing similar to plasma effects. Control experiments showed that UV light or neutral gas flow did not induce morphing or closing of the trap. The trap closing by plasma is thus likely to be associated with the production of hydrogen peroxide by the cold plasma jet in air. Understanding plasma control of plant morphing could help design adaptive structures and bioinspired intelligent materials.

The impact of radicals in cold atmospheric plasma on the structural modification of gap junction: a reactive molecular dynamics study

ABSTRACTCold atmospheric plasmas are currently gaining increasing attention for cancer therapy. However, very limited studies regarding the interaction mechanisms between plasma species and tissues are available. We report the interaction of plasma produced species ( and ) with gap junction by employing reactive molecular dynamics simulations. Our results indicate and radicals can chemically react with N-terminal of gap junction resulting in its structural damage. There are two breaking mechanisms being identified: C-N peptide bonds and C-C bonds can be damaged by and radicals, respectively. Our findings could be particularly important for understanding the plasma-generated reactive species triggering bystander effect based on gap junction intercellular communication.

Analysis of Short-Lived Reactive Species in Plasma-Air-Water Systems: The Dos and the Do Nots.

This Feature addresses the analysis of the reactive species generated by nonthermal atmospheric pressure plasmas, which are widely employed in industrial and biomedical research, as well as first clinical applications. We summarize the progress in detection of plasma-generated short-lived reactive oxygen and nitrogen species in aqueous solutions, discuss the potential and limitations of various analytical methods in plasma-liquid systems, and provide an outlook on the possible future research goals in development of short-lived reactive species analysis methods for a general nonspecialist audience.

Effects of the Red Pigment in Cordyceps pruinosa on the Biological, Structural and Optical Properties of Fungal Spores and Scavenging of Plasma-Generated Reactive Species

Effects of the Red Pigment in<i> Cordyceps pruinosa</i> on the Biological, Structural and Optical Properties of Fungal Spores and Scavenging of Plasma-Generated Reactive Species

Steam reforming of methane in a temperature-controlled dielectric barrier discharge reactor: the role of electron-induced chemistry versus thermochemistry

While classic reforming processes rely on heat and chemical equilibrium, plasma-based reforming processes possess the ability to induce non-equilibrium and reactive chemistry at low temperatures using high energy electrons. To better understand the distinctive roles of both electron-induced chemistry and thermochemistry during plasma-assisted fuel reforming, we previously developed a temperature-controlled dielectric barrier discharge (DBD) reactor, which controlled the gas temperature and the electron temperature independently. Here, we investigate plasma-assisted steam reforming of methane using the temperature-controlled DBD reactor and electron-kinetics calculations. We investigated the individual effects of the determining factors for electron-induced chemistry (i.e. reduced electric field intensity and discharge power) and for thermochemistry (i.e. background gas temperature) by varying the discharge power, gas temperature, and pressure inside the reactor. As a result, we found that both the electron-induced chemistry and thermochemistry governed the reactant conversions. Thermochemistry positively affected the methane conversion in particular, but negatively affected the water conversion as the gas temperature increased. The electron-induced chemistry weakly affected the product distribution, while the background temperature (thermochemistry) strongly influenced the product selectivity and composition by altering the chemical pathways involving the plasma-generated reactive species at the given temperature.

Adaptation of Operational Parameters of Cold Atmospheric Plasma for in Vitro Treatment of Cancer Cells.

Cold atmospheric plasma (CAP), an ionized gas operated at near-ambient temperatures, has been introduced as a new therapeutic opportunity for treating cancers. The effectiveness of the therapy has been linked to CAP-generated reactive oxygen and nitrogen species such as hydrogen peroxide and nitrite. In this study, we monitor in real-time cancer cell response to CAP over the course of 48 h. The results demonstrate a correlation between cell viability, exposure time (30, 60, 90, and 180 s), and discharge voltage (3.16 and 3.71 kV), while stressing the likely therapeutic role of plasma-generated reactive species. A 30-60 s increase in CAP exposure time and/or a discharge voltage adjustment from 3.16 to 3.71 kV is consistently accompanied by a significant reduction in cell viability. Comparably, levels of hydrogen peroxide and nitrite vary as a function of voltage with elevated levels detected at the highest tested voltage condition of 3.71 kV. CAP ultimately initiates a reduction in cell viability and triggers apoptosis via damage to the mitochondrial membrane, while also deregulating protein synthesis. The findings presented in this study are discussed in the context of facilitating the development of an adaptive CAP platform which could improve treatment outcomes.

Where Do Ros Go? Oxidation Cascades In Melanoma Exposed To Cold Physical Plasma

In plasma medicine, physical atmospheric cold plasma is used for application in therapeutic appliances such as wound healing or decontamination of infected skin.[1] Over the last decade, plasmas became a valuable research tool for cancer therapy.[2] Many groups have not only reported an efficient killing of cancer cells with plasma but also a selectivity of the killing of malignant over non-malignant cells.[3] Yet, following the trajectories of plasma-derived reactive oxygen and nitrogen species has only begun to be explored. First modeling studies suggest an important role of lipid peroxidation and phosphatidylserine content in the cell membrane.[4] Using fluorescent reporter dyes, many groups have reported cytosolic oxidation following plasma treatment. Some groups claim that DNA double-stranded breaks are a direct product of reactive species deposition by plasmas.[5] Using human melanoma cells, the purpose of this work was to study in detail how different cell compartments are affected reactive species derived the atmospheric pressure argon plasma jet kINPen. Using confocal laser scanning microscopy, several versions of fluorescent reporter dyes were used to assess oxidation qualitatively and quantitatively. We hope our study to contribute to the understanding how plasma-generated reactive species penetrate cells and affect their behavior and viability, especially for future plasma cancer treatment.

Influence of plasma-generated reactive species on the plasmid DNA structure and plasmid-mediated transformation of Escherichia coli cells

The influence of plasma-generated reactive species on the conformation of plasmid DNA (pDNA) and the transformation efficiency of Escherichia coli cells were studied. An atmospheric-pressure plasma jet (APPJ) was used to generate reactive oxygen and nitrogen species (RONS) in an aqueous solution. When E. coli cells were transformed, the transformation efficiency of E. coli with the APPJ-treated plasmid was lower than with the APPJ-untreated plasmid. Transformation efficiency was reduced due to structural modification and degradation of the pDNA by the APPJ. Plasma treatment caused structural modification of the plasmid from the supercoiled form to the linear form, and also decreased the amount of plasmid by degrading the deoxyribonucleic acid (DNA) structure accompanied by disruption of nucleobases and DNA strand breakage. The formation of linear plasmid from supercoiled plasmid by the APPJ treatment was verified through electrophoretic analysis of the NdeI restriction enzyme-cut supercoiled plasmid. The structural modification and/or decrease in the amount of pDNA are attributed to the RONS from the plasma itself and to those derived from the interaction of plasma radicals with the aqueous solution. The effect of plasma treatment on the transformation efficiency of E. coli cells was more pronounced with the linear plasmid than with the supercoiled plasmid, indicating that the linear plasmid is more vulnerable to RONS. Overall, these results revealed that plasma-generated RONS can modify the structural and optical properties of bacterial pDNA, thus affecting its biological function.

Special issue: Plasma Conversion

With growing concern of energy and environmental issues, the combination of plasma and heterogeneous catalysts receives special attention in greenhouse gas conversion, nitrogen fixation and hydrocarbon chemistry. Plasma gas conversion driven by renewable electricity is particularly important for the electrification of the chemical industry, which is currently dominated by water electrolysis, producing CO2-free renewable hydrogen. Meanwhile, electrochemical conversion of CO2, CH4 and other gaseous materials has yet to be successful because the material and energy conversion efficiency as well as productivity of the electrochemical processes greatly depend on the electrolyte material in terms of the solubility of gaseous materials and mass transport capability, which needs to be dramatically improved. As for fuel processing, the majority of reactions are categorized as uphill (endothermic) reactions where energy input is indispensable due to the conservation of energy. Ideally, such energy should be supplied by low temperature heat while plasma-generated reactive species promote initiation reactions. One promising approach is the use of vibrationally excited molecules which enhance the probability of chemisorption onto the catalyst surfaces. The role of vibrational species has been well investigated through molecular beam studies. However, the way to create such species has not been studied yet. Non-thermal plasma creates an oversaturated amount of vibrational species even at low temperature, showing great promises towards low temperature plasma catalysis of stable molecules. Furthermore, vibrational species uniquely create reaction pathways in homogeneous reactions via stepwise energy transfer gradually populating higher vibrational levels which eventually leads to the dissociation of stable molecules such as CO2. The special issue entitled “Plasma Conversion” consists of three review articles and 11 original research papers, focusing on the state-of-the-art plasma technology that is used for enhancing homogeneous/heterogeneous plasma catalytic conversion of greenhouse gases, nitrogen fixation and hydrocarbon chemistry. Review articles include the new catalyst synthesis approach using nonthermal plasma processes and the potential application of such unique catalysts is introduced (Rahimpour et al.) [1]. CH4 and CO2 reforming is one of the hot topics in plasma catalysis and research has been conducted mostly by experimental approaches. Bogaerts et al. discuss CH4 reforming and CO2 splitting and various mixtures from a modeling perspective, paying special attention to plasma chemistry and the role of plasma-generated vibrationally excited molecules [2]. Reactions involving vibrationally excited species need to be explored further and a numerical approach is truly beneficial providing insightful information towards the reaction mechanisms. Liquefaction of CH4 is to produce higher hydrocarbons via CH4 polymerization at ambient conditions (not for cryogenic methane condensation) [3]. Carbon containing liquid fuels such as gasoline and diesels produced via renewable electricity improve transportability and storage of renewable energy. Two research papers are related to heterogeneous plasma catalysis of CH4 and CO2 where nonthermal plasma is combined with Ni-supported reforming catalysts [4,5]. Research was conducted experimentally and plasma induced synergistic effects are highlighted. Van Rooij and Bongers discuss the homogeneous CO2 conversion with warm discharge, or low pressure microwave plasma [6,7]. Special emphasis on the vibrationally excited CO2 chemistry and CO2 dissociation by the latter mechanism is discussed. A novel heterogeneous plasma catalysis approach of CO2, where nonthermal plasma is combined with an oxygen permeating solid electrolyte membrane, is presented by Mori [8]. O2 is extracted directly from the plasma reaction field, and thus the CO2 conversion and energy efficiency were greatly improved. Moreover, CO2 microwave plasma diagnostics by optical emission spectroscopy [9], diagnostics plasma-catalyst interfacial phenomena [10], and atomistic simulations of plasma catalytic interactions [11] are included in the collection of this special issue. Three other papers focus on ammonia synthesis with atmospheric pressure plasma combined with heterogeneous catalysts [12–14]. Finally we would like to thank all contributors to this special issue and the editorial staff of Plasma Processes and Polymers for their great support. We hope that this issue contributes to exploring the new frontiers of plasma chemistry, and also to promoting the interdisciplinary interaction among the plasma science, chemical catalysis and surface science community, as well as other science specialists.

Investigation and control of the - to -transition in a novel sub-atmospheric pressure dielectric barrier discharge

A novel flow-driven dielectric barrier discharge concept is presented, which uses a Venturi pump to transfer plasma-generated reactive oxygen and nitrogen species from a sub-atmospheric pressure () discharge region to ambient pressure and can be operated with air. By adjusting the working pressure of the device, the plasma chemistry can be tuned continuously from an ozone ()-dominated mode to a nitrogen oxides ()-only mode. The plasma source is characterized focusing on the mechanisms effecting this mode change. The composition of the device’s output gas was determined using Fourier-transform infrared spectroscopy. The results are correlated to measurements of discharge chamber pressure and temperature as well as of input power. It is found that the mode-change temperature can be controlled by the discharge chamber pressure. The source concept is capable of generating an -dominated plasma chemistry at gas temperatures distinctly below . Through mixing of the processed gas stream with a second flow of pressurized air required for the operation of the Venturi pump, the resulting product gas stream remains close to room temperature. A reduced zero-dimensional reaction kinetics model with only seven reactions is capable of describing the observed pressure- and temperature-dependence of the to mode-change.

Nanosecond-Pulsed DBD Plasma-Generated Reactive Oxygen Species Trigger Immunogenic Cell Death in A549 Lung Carcinoma Cells through Intracellular Oxidative Stress.

A novel application for non-thermal plasma is the induction of immunogenic cancer cell death for cancer immunotherapy. Cells undergoing immunogenic death emit danger signals which facilitate anti-tumor immune responses. Although pathways leading to immunogenic cell death are not fully understood; oxidative stress is considered to be part of the underlying mechanism. Here; we studied the interaction between dielectric barrier discharge plasma and cancer cells for oxidative stress-mediated immunogenic cell death. We assessed changes to the intracellular oxidative environment after plasma treatment and correlated it to emission of two danger signals: surface-exposed calreticulin and secreted adenosine triphosphate. Plasma-generated reactive oxygen and charged species were recognized as the major effectors of immunogenic cell death. Chemical attenuators of intracellular reactive oxygen species successfully abrogated oxidative stress following plasma treatment and modulated the emission of surface-exposed calreticulin. Secreted danger signals from cells undergoing immunogenic death enhanced the anti-tumor activity of macrophages. This study demonstrated that plasma triggers immunogenic cell death through oxidative stress pathways and highlights its potential development for cancer immunotherapy.

Plasma cell treatment device Plasma-on-Chip: Monitoring plasma-generated reactive species in microwells

We have developed a plasma cell treatment device called Plasma-on-Chip that enables the real-time monitoring of a single cell culture during plasma treatment. The device consists of three parts: 1) microwells for cell culture, 2) a microplasma device for generating reactive oxygen and nitrogen species (RONS) for use in cell treatment, and 3) through-holes (microchannels) that connect each microwell with the microplasma region for RONS delivery. Here, we analysed the delivery of the RONS to the liquid culture medium stored in the microwells. We developed a simple experimental set-up using a microdevice and applied in situ ultraviolet absorption spectroscopy with high sensitivity for detecting RONS in liquid. The plasma-generated RONS were delivered into the liquid culture medium via the through-holes fabricated into the microdevice. The RONS concentrations were on the order of 10–100 μM depending on the size of the through-holes. In contrast, we found that the amount of dissolved oxygen was almost constant. To investigate the process of RONS generation, we numerically analysed the gas flow in the through-holes. We suggest that the circulating gas flow in the through-holes promotes the interaction between the plasma (ionised gas) and the liquid, resulting in enhanced RONS concentrations.

Plasma inactivation of microorganisms on sprout seeds in a dielectric barrier discharge

Fresh produce is frequently contaminated by microorganisms, which may lead to spoilage or even pose a threat to human health. In particular sprouts are considered to be among the most risky foods sold at retail since they are grown in an environment practically ideal for growth of bacteria and usually consumed raw. Because heat treatment has a detrimental effect on the germination abilities of sprout seeds, alternative treatment technologies need to be developed for microbial inactivation purposes. In this study, non-thermal plasma decontamination of sprout seeds is evaluated as a promising option to enhance food safety while maintaining the seed germination capabilities. In detail, investigations focus on understanding the efficiency of non-thermal plasma inactivation of microorganisms as influenced by the type of microbial contamination, substrate surface properties and moisture content, as well as variations in the power input to the plasma device. To evaluate the impact of these parameters, we studied the reduction of native microbiota or artificially applied E. coli on alfalfa, onion, radish and cress seeds exposed to non-thermal plasma in an atmospheric pressure pulsed dielectric barrier discharge streamed with argon. Plasma treatment resulted in a maximum reduction of 3.4 logarithmic units for E. coli on cress seeds. A major challenge in plasma decontamination of granular food products turned out to be the complex surface topology, where the rough surface with cracks and crevices can shield microorganisms from plasma-generated reactive species, thus reducing the treatment efficiency. However, improvement of the inactivation efficiency was possible by optimizing substrate characteristics such as the moisture level and by tuning the power supply settings (voltage, frequency) to increase the production of reactive species. While the germination ability of alfalfa seeds was considerably decreased by harsh plasma treatment, enhanced germination was observed under mild conditions. In conclusion, the results from this study indicate that cold plasma treatment represents a promising technology for inactivation of bacteria on seeds used for sprout production while preserving their germination properties.

Effects of Atmospheric-Pressure N2, He, Air, and O2 Microplasmas on Mung Bean Seed Germination and Seedling Growth

Atmospheric-pressure N2, He, air, and O2 microplasma arrays have been used to investigate the effects of plasma treatment on seed germination and seedling growth of mung bean in aqueous solution. Seed germination and growth of mung bean were found to strongly depend on the feed gases used to generate plasma and plasma treatment time. Compared to the treatment with atmospheric-pressure O2, N2 and He microplasma arrays, treatment with air microplasma arrays was shown to be more efficient in improving both the seed germination rate and seedling growth, the effect attributed to solution acidification and interactions with plasma-generated reactive oxygen and nitrogen species. Acidic environment caused by air discharge in water may promote leathering of seed chaps, thus enhancing the germination rate of mung bean, and stimulating the growth of hypocotyl and radicle. The interactions between plasma-generated reactive species, such as hydrogen peroxide (H2O2) and nitrogen compounds, and seeds led to a significant acceleration of seed germination and an increase in seedling length of mung bean. Electrolyte leakage rate of mung bean seeds soaked in solution activated using air microplasma was the lowest, while the catalase activity of thus-treated mung bean seeds was the highest compared to other types of microplasma.

Chemical modification of extracellular matrix by cold atmospheric plasma-generated reactive species affects chondrogenesis and bone formation.

The goal of this study was to investigate whether cold plasma generated by dielectric barrier discharge (DBD) modifies extracellular matrices (ECM) to influence chondrogenesis and endochondral ossification. Replacement of cartilage by bone during endochondral ossification is essential in fetal skeletal development, bone growth and fracture healing. Regulation of this process by the ECM occurs through matrix remodelling, involving a variety of cell attachment molecules and growth factors, which influence cell morphology and protein expression. The commercially available ECM, Matrigel, was treated with microsecond or nanosecond pulsed (μsp or nsp, respectively) DBD frequencies conditions at the equivalent frequencies (1kHz) or power (~1W). Recombinant human bone morphogenetic protein-2 was added and the mixture subcutaneously injected into mice to simulate ectopic endochondral ossification. Two weeks later, the masses were extracted and analysed by microcomputed tomography. A significant increase in bone formation was observed in Matrigel treated with μsp DBD compared with control, while a significant decrease in bone formation was observed for both nsp treatments. Histological and immunohistochemical analysis showed Matrigel treated with μsp plasma increased the number of invading cells, the amount of vascular endothelial growth factor and chondrogenesis while the opposite was true for Matrigel treated with nsp plasma. In support of the in vivo Matrigel study, 10T1/2 cells cultured in vitro on μsp DBD-treated type I collagen showed increased expression of adhesion proteins and activation of survival pathways, which decreased with nsp plasma treatments. These results indicate DBD modification of ECM can influence cellular behaviours to accelerate or inhibit chondrogenesis and endochondral ossification. Copyright © 2016 John Wiley & Sons, Ltd.