Non-thermal plasma has gained significant popularity in the field of biology due to its ability to generate reactive oxygen and nitrogen species, which can alter gene expression within cells. Plasma treatment has been used to address various medical issues, such as cancer treatment, blood clotting, and bacterial sterilization. In our study, we investigated the effect of non-thermal plasma treatment on monocyte-derived macrophages (U937) in vitro. To create an in vivo-like environment, the cells were embedded in a collagen matrix and treated with a di-electric barrier non-thermal plasma system using a combination of air and helium flow. Our results showed that plasma in air might modulate macrophages through changes in gene expression. Further experiments revealed the presence of reactive species responsible for these changes. By evaluating cell viability and gene expression, we determined that 120 s of plasma treatment in air is optimal for Fibroblast cells within collagen. Our study suggests that non-thermal plasma in air can modulate monocyte-derived macrophages embedded in collagen.

The formation of bacterial biofilm on implanted devices or damaged tissues leads to biomaterial-associated infections often resulting in life-threatening diseases and implant failure. It is a challenging process to eradicate biofilms as they are resistant to antimicrobial treatments. Conventional techniques, such as high heat and chemicals exposure, may not be suitable for biofilm removal in nosocomial settings. These techniques create surface degradation on the treated materials and lead to environmental pollution due to the use of toxic chemicals. A novel technique known as non-thermal plasma has a great potential to decontaminate or sterilize those nosocomial biofilms. This article aims to provide readers with an extensive review of non-thermal plasma and biofilms to facilitate further investigations. A brief introduction summarizes the problem caused by biofilms in hospital settings with current techniques used for biofilm inactivation followed by the literature review strategy. The remainder of the review discusses plasma and its generation, the role played by plasma reactive species, various factors affecting the antimicrobial efficacy of non-thermal plasma and summarizes many studies published in the field.

Non-thermal plasma has been a promising new cancer treatment modality in plasma oncology field. It generates extracellular and intracellular reactive species which are key factors for the treatment of cancer cells. In this study, we investigated the differential effect of non-thermal plasma on both A549 lung adenocarcinoma and MRC-5 lung fibroblast cells. Extracellular generation of reactive species in both A549 lung cancer and MRC-5 normal lung fibroblast cells were similar, whereas intracellular penetration of reactive species generated by plasma in A549 cancer cells were almost fourfold higher than the normal cells. Interestingly, A549 cancer cells treated for shorter (15 and 30 s) and longer durations (60 and 120 s) get arrested in S-phase (19%) and G2/M phase (28%) respectively. In a healthy MRC-5 cells, few cells arrested in S and G2/M phase in relative to A549 cells for all treatment time. Finally, we evaluated the expression of apoptosis-related genes, H2AX, BAX, P53, Caspase-8, and ATM on normal and cancer cells. There was a higher expression of BAX gene for 120 s plasma treated A549 cell samples at day 1 relative to MRC-5 cells. These findings demonstrate that non-thermal plasma generated reactive species creates intracellular stress, that arrests cell cycle and induces apoptosis in cancer cells. This study suggests that non-thermal plasma could be a potential therapy for lung cancer treatment.

Biofilm formation on implanted medical devices is the reason for most of the nosocomial infections in clinical settings. Biofilms are more resistant to antimicrobials than their planktonic cells mainly because of the presence of the matrix of extracellular polymeric substances (EPSs), which acts as a physical barrier that limits the transport of antimicrobials inside the biofilm. A combinatorial antimicrobial approach of a non-thermal plasma and chlorhexidine (CHX) digluconate can be used to sterilize those surfaces contaminated with biofilm. However, the reason behind achieving this combinatorial decontamination is not known. Thus, in this study, we developed a mathematical model to explain the reason behind sterilization with the combinatorial treatment approach. It was found that the application of plasma prior to treatment with CHX is disrupting the biofilm and making it very porous. This is allowing CHX to penetrate deeper inside the porous biofilm, which is then effective at sterilizing the biofilm.

Bacterial biofilm formation on medical implants is a major cause of illness in patients and is, therefore, increasing healthcare costs due to extended hospital stays and the failure/disposal of contaminated implants. Only limited progress has been made to prevent or eradicate this growing problem. Effective approaches include inhibiting the initiation of biofilm growth by killing planktonic bacteria and breaking down existing biofilms. We propose to employ atmospheric pressure nonthermal regular dielectric-barrier discharge (DBD) and jet plasma to kill bacteria that have been grown planktonically or in biofilms. In this paper, Pseudomonas aeruginosa was grown either planktonically or on titanium coupons in a bioreactor under dynamic conditions to form mature biofilms. The planktonic bacteria and biofilms were exposed to regular DBD and jet plasma and bacterial survival was evaluated after treatment for various times and at different distances. Within 5 min of plasma treatment, we observed complete decontamination of planktonically grown bacteria, and within 15 min, we observed more than a 3 log reduction (99.9%) of bacteria grown as biofilms. The efficacy of plasma treatment was also visualized using scanning electron microscopy, where disruption of biofilm was found with an increasing treatment time. This paper also determined that jet plasma is more effective in treating biofilms than regular DBD plasma.

Recurrence of intervertebral disc (IVD) herniation is the most important factor leading to chronic low back pain and subsequent disability after discectomy. Efficacious annulus fibrosus (AF) repair strategy that delivers cells and biologics to IVD injury site is needed to limit the progression of disc degeneration and promote disc self-regeneration capacities after discectomy procedures. In this study, a biphasic mechanically-conditioned scaffold encapsulated with human adipose-derived stem cells (ASCs) is studied as a potential treatment strategy for AF defects. Equiaxial strains and frequencies were applied to ASCs-encapsulated scaffolds to identify the optimal loading modality to induce AF differentiation. Equiaxial loading resulted in 2–4 folds increase in secretion of extracellular matrix proteins and the reorganization of the matrix fibers and elongations of the cells along the load direction. Further, the equiaxial load induced region-specific differentiation of ASCs within the inner and outer regions of the biphasic scaffolds. Gene expression of AF markers was upregulated with 5–30 folds within the equiaxially loaded biphasic scaffolds compared to unstrained samples. The results suggest that there is a specific value of equiaxial strain favorable to differentiate ASCs towards AF lineage and that ASCs-embedded biphasic scaffold can potentially be utilized to repair the AF defects.

Recent breakthroughs in plasma medicine have identified a potential application for the non-thermal plasma in cancer therapy. Most studies on the effects of non-thermal plasma on cancer cells have used traditional two-dimensional (2D) monolayer cell culture. However, very few studies are conducted employing non-thermal plasma in animal models. Two dimensional models do not fully mimic the three-dimensional (3D) tumor microenvironment and animal models are expensive and time-consuming. Therefore, we used 3D collagen matrices that closely resemble the native geometry of cancer tissues and provide more physiologically relevant results than 2D models, while providing a more cost effective and efficient precursor to animal studies. We previously demonstrated a role for non-thermal plasma application in promoting apoptotic cell death and reducing the viability of A549 lung adenocarcinoma epithelial cells cultured upon 2D matrices. In this study, we wished to determine the efficacy of non-thermal plasma application in driving apoptotic cell death of A549 lung cancer cells encapsulated within a 3D collagen matrix. The percentage of apoptosis increased as treatment time increased and was time dependent. In addition, the anti-viability effect of plasma was demonstrated. Twenty-four hours post-plasma treatment, 38% and 99% of cell death occurred with shortest (15 s) and longest treatment time (120 s) respectively at the plasmatreated region. We found that plasma has a greater effect on the viability of A549 lung cancer cells on the superficial surface of 3D matrices and has diminishing effects as it penetrates the 3D matrix. We also identified the nitrogen and oxygen species generated by plasma and characterized their penetration in vertical and lateral directions within the 3D matrix from the center of the plasma-treated region. Therefore, the utility of non-thermal dielectric barrier discharge plasma in driving apoptosis and reducing the viability of lung cancer cells in 3D collagen matrix indicates a therapeutic potential that warrants further research.

Traditional cancer treatments like radiotherapy and chemotherapy have drawbacks and are not selective for killing only cancer cells. Nonthermal atmospheric pressure plasmas with dielectric barrier discharge (DBD) can be applied to living cells and tissues and have emerged as novel tools for localized cancer therapy. The purpose of this study was to investigate the different effects caused by miniature DBD (mDBD) plasma to A549 lung cancer cells. In this study, A549 lung cancer cells cultured in 12 well plates were treated with mDBD plasma for specified treatment times to assess the changes in the size of the area of cell detachment, the viability of attached or detached cells, and cell migration. Furthermore, we investigated an innovative mDBD plasma-based therapy for localized treatment of lung cancer cells through apoptotic induction. Our results indicate that plasma treatment for 120 sec causes apoptotic cell death in 35.8% of cells, while mDBD plasma treatment for 60 sec, 30 sec, or 15 sec causes apoptotic cell death in 20.5%, 14.1%, and 6.3% of the cell population, respectively. Additionally, we observed reduced A549 cell migration in response to mDBD plasma treatment. Thus, mDBD plasma system can be a viable platform for localized lung cancer therapy.

Nosocomial infections caused by opportunistic bacteria pose major healthcare problem worldwide. Out of the many microorganisms responsible for such infections, Pseudomonas aeruginosa is a ubiquitous bacterium that accounts for 10-20% of hospital-acquired infections. These infections have mortality rates ranging from 18 to 60% and the cost of treatment ranges from $20,000 to $ 80,000 per infection. The formation of biofilms on medical devices and implants is responsible for the majority of those infections. Only limited progress has been made to prevent this issue in a safe and cost-effective manner. To address this, we propose employing jet plasma to break down and inactivate biofilms in vitro. Moreover, to improve the antimicrobial effect on the biofilm, a treatment method using a combination of jet plasma and a biocide known as chlorhexidine (CHX) digluconate was investigated. We found that complete sterilization of P. aeruginosa biofilms can be achieved after combinatorial treatment using plasma and CHX. A decrease in biofilm viability was also observed using confocal laser scanning electron microscopy (CLSM). This treatment method sterilized biofilm-contaminated surfaces in a short treatment time, indicating it to be a potential tool for the removal of biofilms present on medical devices and implants.

Biofilm formation on medical devices and implants is a major cause of patient illness leading it to be one of the key nosocomial problems worldwide. The infection caused by antibiotic resistant bacteria pseudomonas aeruginosa accounts for majority of hospital acquired infections. These infections have been one of the serious problems in the hospital in terms of morbidity, mortality and health care costs1. The infections lead to a mortality rate of 18 to 60%, and the cost of treatment is considerably large ranging from $20,000 to $80,000 per infection1. Only limited progress has been made to prevent or eradicate this progressive issue in a safe and cost-effective mode. To address this, we propose to employ atmospheric pressure jet plasma to break down and inactivate bacterial biofilm in vitro. Moreover, to improve the antimicrobial effect on the biofilm, a treatment method using the combination of jet plasma and a biocide known as Chlorhexidine (CHX) digluconate was investigated.

In this study we aim to address treatment of Staphylococcus aureus, one of the most common causes of infection after injury or surgery. Besides the infecting wounds, Staphylococcus aureus may even spread to the bone and antibiotic resistance in Staphylococcus aureus is a serious problem. The application of atmospheric pressure non-thermal dielectric barrier discharge is an effective method in decontamination of the living tissue. In this project we apply non-thermal plasma on contaminated liquid and surfaces to evaluate sterilization efficacy with log reduction in bacterial concentration as a function of different parameters such as plasma treatment time and power density. We test the effects of non-thermal plasma treatment in decontamination of Staphylococcus aureus, methicillinresistant Staphylococcus aureus and Escherichia coli (as benchmark species) in different media and on different surfaces. Mechanisms of plasma decontaminations were studied using various assays and characterization methods. All three bacteria species were studied in their early logarithmic growth phase and late lag phase. In conclusion, we found that atmospheric pressure non-thermal plasma can effectively sterilize methicillin-resistant Staphylococcus aureus.

Surface properties of biopolymers are crucial for providing topographical and chemical cues to affect cellular behaviors, such as attachment, spreading, viability, proliferation, and differentiation. As an effective surface modification technique, plasma treatment is often applied to enhance surface wettability, adhesion, and biocompatibility of polymers. In this study, an atmospheric-pressure microplasma jet based on dielectric barrier discharge was installed on an automated arm which allows movement in the x-y-z directions at various trajectory presets. Polycaprolactone (PCL) samples were functionalized with helium-oxygen plasma generated by this system and characterized via water contact angle, x-ray photoelectron spectroscopy, and scanning electron microscopy. Mouse osteoblast cells (7F2) were cultured on both treated and native PCL samples and examined by MarkerGene (TM) Live: Dead/Cytotoxicity and alamarBlue (R) assaying techniques. The surface and biological characterization results indicate that microplasma treatment improved surface hydrophilicity, as well as cell viability and proliferation. The localized microplasma treatment can lead to the application of bioactive scaffolds with selective surface functionalization.

In this study, we investigated whether nitric oxide (NO) generated using a non-thermal plasma system can mediate osteoblastic differentiation of osteoprogenitor cells without creating toxicity. Our objective was to create an NO delivery mechanism using NO-dielectric barrier discharge (DBD) plasma that can generate and transport NO with controlled concentration to the area of interest to regulate osteoprogenitor cell activity. We built a non-thermal atmospheric pressure DBD plasma nozzle system based on our previously published design and similar designs in the literature. The electrical and spectral analyses demonstrated that N2 dissociated into NO under typical DBD voltage–current characteristics. We treated osteoprogenitor cells (MC3T3-E1) using NO-plasma treatment system. Our results demonstrated that we could control NO concentration within cell culture media and could introduce NO into the intracellular space using NO-plasma treatment with various treatment times. We confirmed that NO-plasma treatment maintained cell viability and did not create any toxicity even with prolonged treatment durations. Finally, we demonstrated that NO-plasma treatment induced early osteogenic differentiation in the absence of pro-osteogenic growth factors/proteins. These findings suggest that through the NO-plasma treatment system we are able to generate and transport tissue-specific amounts of NO to an area of interest to mediate osteoprogenitor cell activity without subsequent toxicity. This opens up the possibility to develop DBD plasma-assisted tissue-specific NO delivery strategies for therapeutic intervention in the prevention and treatment of bone diseases.

Increasing emergence of antibiotic resistance in bacteria during the past few decades has become a major public health concern and has reduced the efficacy of conventional therapies. The objective of the current study is to examine the bactericidal efficacy of atmospheric pressure nonthermal dielectric barrier discharge (DBD) plasma on methicillin-resistant Staphylococcus aureus (MRSA) as a common musculoskeletal pathogen and Escherichia coli (E. coli) as another cause of bone, joint, and soft tissue infections in planktonic phase and in colonies. In planktonic phase, results demonstrated complete decontamination of E. coli and MRSA cells on agar plates with a bacterial surface density of 1.27 × 105 colony-forming units (CFU)/cm2 following 30 seconds and 60 seconds of plasma treatment, respectively. Reduction rates of 7 log10 steps in the number of viable CFUs in E. coli colonies was observed following 5 minutes of plasma treatment. The colony growth was halted, and no more growth in colony size was observed during a 24-hour monitoring period. For MRSA colonies, results demonstrated 2 log10 to 7 log10 steps reduction in the number of viable CFUs in each colony after 5 minutes of plasma treatment. Results indicated that plasma-treated colonies will have prolonged lag time during generation of the growth curves. Finally, diffusion of nitric oxide into the agarose gel was confirmed as a reactive agent that was effective in the decontamination process.

In the field of tissue engineering, regenerative medicine, and life sciences, the topological biochemical cues regulate cell attachment and alignment within the construct. In a native biological system, these cues are inherent. However, most of the biological materials utilized in the fabrication of tissue construct do not possess the appropriate cues required to develop an architecture to support the cell attachment and growth of a functional tissue Therefore the ability to manipulate structural and biochemical cues plays an important role in biofabrication process, and it is a key element to evaluate a engineered cellular model. Plasma surface functionalization and biologics printing have been investigated and validated as two effective techniques to guide cell functions by creating microenvironments. The objective of this work is to develop a novel dual functional platform for freeform microplasma surface patterning and biologics printing process as well as to study the underlying process science and the process induced cellular functions. The microplasma jet system was assembled by two parts. The upper part is a plastic NPT connector surrounding an extending high voltage copper electrode. The lower part is a dielectric Pasteur pipette connected with a capillary micro-scale nozzle tip. The lower part is interchangeable and the diameter of the tip ranges from 50 μm to 1 mm. With up to 20 kV output capability, a high-voltage power supply was connected to the copper electrode through the NPT connector which also served as gas inlet. A high-voltage probe linked to an oscilloscope is used to monitor the real time voltage. The whole microplasma jet system was set up on automation platform, which allows X-Y-Z motion control and switch control. This integrated system operates at atmospheric pressured environment. All tissue constructs could be fabricated at room temperature without the use of a mask. Clear polystyrene microplates were used as plasma treatment substrates. After O2-He mixed microplasma treatment, 7F2 mouse osteoblastic cells were cultured in the microplates for cell biology studies. We demonstrated the capability of our dual functional platform by applying microplasma in the polystyrene wells and control group (without any treatment) in other wells of the same microplate substrate. The results show that the microplasma treatment changed the surface properties and improved cell attachment. This dual functional freeform system allows for surface patterning and printing of cells, proteins, growth factors, etc. to fabricate three-dimensional tissue constructs.

A maskless microplasma surface patterning system for biologics printing has been developed. The novel system integration includes a low-temperature atmospheric plasma jet and a multi-nozzle freeform biomolecule printing apparatus. Microplasma surface patterning was applied on polystyrene and characterized biologically.

An atmospheric pressure non-thermal microplasma jet (Ø 50 μm) was developed for localized functionalization of various substrates, including polymers, to allow maskless freeform cell printing. The applied microplasma jet power ranged from 0.1 to 0.2 W without causing any damage to the polyethylene substrate. The surface characterization results demonstrate that the microplasma treatment locally changes the surface roughness and the concentration of oxygen-containing functional groups on the polyethylene surface. The biological characterization confirms that the osteoblast cells attach and survive on the plasma activated line while untreated surfaces show almost no attachment and viability.

Summary form only given. It has been demonstrated recently that direct treatment of relatively smooth surfaces by non-thermal dielectric barrier discharge (DBD) in air is highly effective in killing bacteria and fungi. The key aspect of the direct treatment was shown to be contact with electrical charges. These results hold significant promise for medical applications of direct DBD such as sterilization of wound surfaces. However, a typical DBD in air can be highly non-uniform, particularly on topographically non-uniform surfaces such as most of the living tissues. As a result, it is not clear that pathogens can be destroyed as effectively on the recessed areas between the ridges of real tissue by the conventional DBD in air. In this study authors have investigated effectiveness of a DBD excited by nanosecond rise and fall time voltage pulses in killing bacteria covering topographically non-uniform surfaces. Sterilization experiments were conducted on the E. coli covered agar surface acting as one of the DBD electrodes. The nanosecond-pulsed DBD (ns-DBD) was tested on non-uniform surfaces and produced uniform plasma independent of the surface topography. Sterilization effectiveness of ns-DBD also has been compared with that of a conventional DBD, i.e. microsecond-pulsed DBD (ms- DBD). Experiments reveal that the ns-DBD sterilizes a larger surface area than the ms-DBD does for the same duration and power. Moreover, experiments on non-uniform surfaces showed that the ns-DBD can penetrate into the recessed areas and sterilizes completely whereas the ms-DBD fails to do so. In summary, ns-DBD with short rise time and high overvoltage is insensitive to the morphological non-uniformities of the surface. Thus DBDs with nanosecond rise times are potentially more convenient for in vivo and hospital sterilization. Although several investigators did report uniform DBD systems, to the authors' knowledge the uniform discharge reported here is the only one to have been demonstra- ed at atmospheric pressure in open moist air over topographically non-uniform surfaces.

For some period of time the use of plasma in medicine has been limited to thermal discharges for cauterization and dissection. The effects of thermal plasma on tissue are entirely related to local heating. Non-thermal plasma, on the other hand, can have many different modes of interaction with tissue. It has been recently demonstrated that direct treatment of smooth surfaces by non-thermal dielectric barrier discharge (DBD) in air is highly effective in killing pathogens. Moreover, DBD can create different sub-lethal and selective effects. These results hold significant promise for medical applications such as sterilization of wound surfaces. However, a typical DBD in air can be highly non-uniform, particularly on topographically non-uniform surfaces such as in most living tissues. This creates significant limitations for use of DBDs in wound care and other biomedical applications. In this thesis, a novel non-thermal plasma system, namely nanosecondpulsed DBD, has been developed and investigated to address this important limitation. Nanosecond-pulsed DBD is shown to be uniform in air at atmosphericpressure and much more effective in killing bacteria than conventional DBDs, particularly on topographically non-uniform surfaces. Thus, this new plasma system is potentially convenient for in vivo and hospital sterilization cases.%%%%Ph.D., Mechanical Engineering – Drexel University, 2009