Wearable Plasma Pads for Biomedical Applications

applied sciences

Article Wearable Plasma Pads for Biomedical Applications

Junggil Kim 1,†, Kyong-Hoon Choi 2,†, Yunjung Kim 1, Bong Joo Park 1,2,* and Guangsup Cho 1,* 1 Department of Electrical and Biological Physics, Kwangwoon University, 20 Kwangwoon-Ro, Nowon Wallgye, Seoul 01897, Korea; [email protected] (J.K.); [email protected] (Y.K.) 2 Institute of Biomaterials, Kwangwoon University, 20 Kwangwoon-Ro, Nowon Wallgye, Seoul 01897, Korea; [email protected] * Correspondence: [email protected] (B.J.P.); [email protected] (G.C.); Tel.: +82-2940-5233 (B.J.P. & G.C.) † These authors contributed equally to this work.

Received: 20 November 2017; Accepted: 14 December 2017; Published: 17 December 2017 Abstract: A plasma pad that can be attached to human skin was developed for aesthetic and dermatological treatment. A polyimide film was used for the dielectric layer of the flexible pad, and high-voltage and ground electrodes were placed on the film surface. Medical gauze covered the ground electrodes and was placed facing the skin to act as a spacer; thus, the plasma floated between the gauze and ground electrodes. In vitro and in vivo biocompatibility tests of the pad showed no cytotoxicity to normal cells and no irritation of mouse skin. Antibacterial activity was shown against Staphylococcus aureus and clinical isolates of methicillin-resistant S. aureus. Furthermore, skin wound healing with increased hair growth resulting from increased exogenous nitric oxide and capillary tube formation induced by the plasma pad was also confirmed in vivo. The present study suggests that this flexible and wearable plasma pad can be used for biomedical applications such as treatment of wounds and bacterial infections.

Keywords: biomedicine; dielectric barrier discharge; plasma devices; plasma pads; plasma treatment

1. Introduction Non-thermal atmospheric-pressure plasmas (NAPPs) have been studied for potential applications in dermatology and wound healing [1–6]. Plasma components such as ultraviolet (UV) rays, electric current (EC), reactive oxygen species (ROS), and reactive nitrogen species (RNS) are involved in skin treatment. In the most recent review papers [1,7–10], plasma sources for cell and tissue research have been introduced. At least three different NAPP sources have been developed for biomedical applications [11–14]. Direct plasma sources—primarily dielectric barrier discharge (DBD) technology—use the human body as a counter electrode [6,15]. Indirect plasma sources generate plasma between two electrodes within a delivery device and the active components created by the plasma are transported to the target area [16–21]. These indirect delivery devices can vary from thin plasma needles to plasma jets and large torches, with different arrangements of electrodes. A recent approach uses the so-called hybrid plasma source, which combines the benefits of both of the above techniques and enables large and flexible plasma designs. Thus far, the plasma sources used for medical purposes have been based on both direct and indirect approaches. Future trends seem to be shifting toward hybrid plasmas, as these enable large and flexible designs. In the early stage of ﬂoating electrode DBD (FE-DBD) plasma generation, a high voltage (10–30 kV) with a low frequency (200 Hz–1 kHz) is applied to an electrode protected by a 1-mm-thick quartz plate as a dielectric barrier [2,19]. Technical (operating voltage) parameters for DBD sources developed at the Leibniz Institute for Plasma Science and Technology (INP Greifswald) are 10 kV at 20 kHz for surface DBD in ambient air [7]. High-voltage (10–30 kV) non-thermal plasma discharge generation in ambient air to treat living animals and humans requires high-level safety precautions. Safety and guaranteed non-injurious regimens are essential in plasma medicine applications.

Appl. Sci. 2017, 7, 1308; doi:10.3390/app7121308 www.mdpi.com/journal/applsci Appl. Sci. 2017, 7, 1308 2 of 13

Our laboratory developed two types of FE-DBD devices [18]. One uses a 1-mm-thick quartz plate and the other uses a copper wire coated with 1-mm-thick foam rubber for insulation against high voltage. The applied voltage is reduced to the range of 2–3 kV at a higher frequency of 40–60 kHz using a direct current to alternative current (DC-AC) inverter. Further development of the FE-DBD involves a reduced operating voltage. The other parameter involved in voltage reduction is dielectric barrier thickness. A thin film of polyimide can reduce thickness by replacing the solid dielectric plate, which is fragile. In this study of hybrid-DBD plasma, the operating voltage was reduced to 1 kV at a frequency of 50 kHz using a flexible 125-µm-thick polyimide film. The lower voltage enables development of a miniaturized wearable power supply with a DC-AC inverter. For the last two decades, the focus of biomedical plasma research has been on plasma jets and DBD devices. Plasma jets have been developed in pencil structures, while DBD devices comprising a finite surface area have been produced in plane structures [3,16–19]. Both devices are utilized for direct skin treatments by physicians and other healthcare professionals; however, these devices require prolonged treatment. For example, in the case of atopic eczema, itching decreases after a 4 h plasma treatment, or after applications of several minutes per day for 30 days [3]. Long-term plasma treatment is required for wound healing and treatment of skin diseases. It is also important to note that these devices can only cover a limited area in a single treatment. However, because they are not limited by treatment time and the size of the treatment area, DBDs integrated in an adhesive device for application on human extremities have been proposed [20–22]. In this study, a plasma pad was introduced as another hybrid-DBD plasma treatment method, whereby the restrictions on treatment time and size of the treated area are eliminated. A flexible, wearable plasma pad of various sizes can be applied to human skin; therefore, it was expected that the proposed pad could be used as an effective treatment option at various sites, thereby reducing both time and spatial restrictions. Here, a plasma pad system, as well as the power system and spectroscopic plasma emissions, is described. The biocompatibility of the plasma pad and the effect of plasma treatment on wound healing and antibacterial activity were tested.

2. Experimental Methods

2.1. Fabrication and Characterization of a Plasma Pad System

2.1.1. Fabrication of the Plasma Pad For the dielectric layer of the plasma pad, a commercial polyimide film (Kapton® film, DuPont, Wilmington, DE, USA) was selected, with a thickness ranging from several tens to hundreds of µm. Kapton film has good electrical and physical characteristics, including a thermal resistance up to +400 ◦C, high chemical resistance, and a high breakdown voltage exceeding 6 kV for a film thickness of 120 µm, with a field strength of 5 × 105 V/cm [23]. For a hybrid-DBD plasma pad, the high-voltage electrode and the ground electrode were fabricated on the upper and lower surfaces of the 120-µm-thick polyimide film of the plasma pad. The pad is electrically safe because the high-voltage film surface is covered with silicone rubber for insulation, while the cold plasma that is generated on the ground electrode surface is in contact with the skin and medical gauze covers the film surface of the ground electrode. To attach the pad to the skin, adhesive paste is applied around the periphery of the gauze, except in the area covering the ground electrode.

2.1.2. Characteristics of Applied Power A sinusoidal voltage in the range of 1–2 kV at a frequency of 50 kHz was applied to the high-voltage electrode through a DC-AC inverter. The operating voltage was modulated with the duty ratio of an on-and-off time control as deﬁned by

β = (τ/T) × 100 (%) (1) Appl. Sci. 2017, 7, 1308 3 of 13 with an on-time of τ and a modulation period of T = 4 s. For a duty ratio β = 100%, respective voltage and current values measured at the terminal of the inverter were approximately 1.1 kV rms and 5.5 mA rms. The input power to the inverter was Po = 6 W for β = 100% and P = βPo for a variable β.

2.1.3. Measurement Method for Temperature and Ozone Concentration The temperature of the plasma pad was measured with a thermocouple attached to the gauze on the surface of the plasma pad, and high voltage was applied to measure the temperature change. The temperature was measured for 1 h under both conditions of β = 50% and β = 100%. The ozone concentration in the plasma pad was measured using ozone detectors (Detector Tube No. 18 L, GASTEC, Ayase, Japan). The measuring distance between the plasma pad and the ozone detector was 10 cm or 20 cm, and the duty ratio of the applied power was 25%, 50%, 75%, or 100%.

2.1.4. Optical Emission Spectroscopy of the Plasma Pad The emitted surface light from the plasma pad was collected by a spectroscopic diagnostic system, composed of a collimating lens pair, optical ﬁber (QP400-2-SR), and spectrophotometer (HR4000CG-UV-NIR, Ocean Optics, Largo, FL, USA) with a linear charge-coupled device (CCD) detector array. Emission spectra were measured over a wide wavelength range of 200–1100 nm. The spectral focal length was 101 mm, and the best spectral resolution was 0.75 nm (Full Width Half Max). This system was calibrated with deuterium and mercury lamps. The measured data were integrated over 1 s or more. The recorded data were observed using Origin 8.5 software.

2.2. In Vitro and In Vito Biocompatibilty Testing of the Plasma Pad For in vitro biocompatibility tests, cytotoxicity was assessed using green fluorescent protein (GFP)-transfected keratinocytes (HaCaT cells) and fibroblasts (L-929 cells), as previously described [24–26]. Briefly, the GFP-transfected HaCaT and L-929 cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotic solution. Each cell line was seeded on cover glasses (ϕ 12 mm) in 24-well plates (Coster Corp., 5 ◦ Bethesda, MD, USA) at 1.5 × 10 cells/mL and incubated at 37 C in 5% CO2 for 24 h. After incubation, the cover glasses were transferred into a non-coated 35 mm dish with 2 mL of DMEM media and the plasma pad was placed about 1 to 2 mm distant from the media in the dish. Each cell line on the cover glass was exposed to plasma by treating the media surface for 0 min, 1 min, 3 min, 5 min, ◦ and 10 min and the cover glass was transferred into a 24-well plate and incubated at 37 C in 5% CO2 for another 24 h. Cell viability was calculated using the absorbance value of each well at 450 nm after incubation with a Cell Counting Kit-8 (CCK-8) solution (Dojindo Laboratories, Kumamoto, Japan). To confirm morphological changes, each cell was examined with an imaging reader (Cytation 3; BioTek Instruments, Inc., Winooski, VT, USA). The data shown for biocompatibility in this study were obtained from two independent experiments (sample number n = 4) and were expressed as mean ± standard deviation (SD) for quantitative data. Statistical comparisons were performed with a Student’s t-test, and P < 0.05 was considered statistically significant. To evaluate in vivo biocompatibility, a skin irritation test was conducted on hairless mice using a method described in the ISO 10993-10 [27]. Briefly, 10 healthy hairless mice were used for in vivo experiments and were divided into 2 groups. Group 1 served as the control, and Group 2 was treated with plasma for 10 min. After plasma treatment, signs of edema and/or erythema in each mouse were checked at 24 h and 48 h, and any responses in the plasma-treated group were also evaluated according to the method in the ISO 10993-10. Histological analysis was also performed after staining with hematoxylin and eosin (H&E).

2.3. Detection of Nitric Oxide Generated by the Plasma Pad In order to detect exogenous nitric oxide (NO), an absorption-based Griess reagent method was − used to determine total amounts of NOx generated by the plasma pad. Griess reaction solutions and Appl. Sci. 2017, 7, 1308 4 of 13 processes were prepared as in a previous report [28]. Brieﬂy, 2 samples (1 mL each) of Dulbecco’s phosphate buffered solution (DPBS) and DMEM with 10% FBS were exposed to plasma for different treatment times (0 min, 1 min, 3 min, 5 min, and 10 min). Then, 100 µL of each plasma-treated sample was placed into the 96-well microplate and 100 µL of the two Griess reagents was added to each sample solution. The mixed solutions were left for at least 30 min to terminate this reaction. After mixing, the absorbance was measured at 540 nm using a multimode microplate reader (CYTATION 3, BioTek Instruments, Inc., Winooski, VT, USA). Calibration was performed using a sodium nitrate solution for each measurement.

2.4. Analysis of Capillary Tube Formation with Plasma Treatment Human dermal microvascular endothelial cells (HDMVECs, Lonza, Walkersville, MD, USA) were purchased from Lonza Japan Ltd. and cultured in endothelial cell-basal medium-2 (EBM-2) supplemented with 2% FBS and endothelial cell growth supplements (Lonza, Walkersville, MD, USA), ◦ and a 1% antimycotic solution at 37 C and 5% CO2. The capillary tube formation study was performed with the HDMVECs between Passages 5 and 6. For cell differentiation in HDMVECs, each well of a 24-well plate was coated with 180 µL of Matrigel (growth factor reduced type; BD Biosciences, Rockville, MD, USA) and incubated at 37 ◦C for 30 min to harden the Matrigel in each well. Pre-cultured HDMVECs were plated at 0.3 × 105 cells/mL in non-coated 35 mm dishes with 2 mL of EBM-2 media and the plasma pad was placed as described above. Then, the HDMVECs were also exposed to plasma by treating the media surface for 0 min, 1 min, 3 min, 5 min, and 10 min. The plasma-treated HDMVECs were transferred into the Matrigel-coated 24-well plate and incubated at 37 ◦C to form capillary tubes. After incubation for 8 h, each well was examined with an automated live cell imager (LIONHEART FX; BioTek Instruments, Inc., Winooski, VT, USA) to conﬁrm the formation of new capillary tubes from HDMVECs by plasma treatment.

2.5. In Vivo Wound Healing Experiment To confirm the in vivo wound healing effect of the plasma pad, an animal experiment was conducted using a full-thickness excisional wound model as previously described [26,29–31]. Briefly, 16 healthy male mice (C57BL/6J, Charles River Corp. Inc., Barcelona, Spain) were divided into 2 groups and the mice in each group were bred in individual cages. To induce full-thickness excisional wounds, the skin was disinfected with povidone-iodine and 70% (v/v) ethanol and a sterile biopsy punch (8 mm in diameter) was used to create a full-thickness excisional wound. Then, each wound site was carefully splinted using a silicone-splinting ring (12 mm inner diameter and 20 mm outer diameter) and the plasma pad was attached on the silicone ring. After plasma treatment for 10 min, the wounds in each mouse were dressed with a transparent wound dressing (Tegaderm film, 3M Corp.) and the dressing was changed every other day. The negative control mice were treated with PBS only. The wound sites were observed using a stereomicroscope (SZX16, Olympus, Tokyo, Japan) every other day and histological analysis of each skin sample was performed after 21 days with H&E staining to evaluate the wound healing effect of the plasma treatment.

2.6. Evaluation of Antibacterial Activity Using Plasma Treatment To confirm the antibacterial activity of the plasma pads, Staphylococcus aureus (S. aureus ATCC 14458) and methicillin-resistant S. aureus (MRSA), isolated from patients at the Yonsei Medical Center in Seoul [26], were used. S. aureus was grown on plate count agar (PCA, Becton, Dickinson and Company, Sparks, MD, USA) at 35 ◦C for 24 h and MRSA was grown on Brain Heart Infusion agar (BHIA, Becton, Dickinson and Company, Sparks, MD, USA) at 35 ◦C for 24 h. Pre-cultured bacterial cells were suspended at 1 × 106–1 × 107 colony forming units (CFU)/mL in nutrient broth for (S. aureus) and BHI broth for MRSA. The bacterial cells were inoculated into 24-well plates after 10-fold dilution to yield 1 × 105–1 × 106 CFU/mL and were incubated at 35 ◦C after plasma treatment for 0 min, 1 min, 3 min, 5 min, and 10 min. After incubation for 24 h, the bacterial cells were serially diluted by 10-fold Appl. Sci. 2017, 7, 1308 5 of 13 incrementsAppl. (1 Sci.× 201710,1 7,to 1305 1 × 107) and inoculated into PCA or BHIA to confirm cell viability.5 of 13 Bacterial cell viabilityand was BHI broth determined for MRSA. by The counting bacterial cells the CFUwere inoculated of each bacterial into 24‐well type, plates and after the 10‐fold antibacterial dilution activity was definedto yield as a 1 >3-log × 105–1 × decrease 106 CFU/mL in and CFU/mL. were incubated at 35 °C after plasma treatment for 0 min, 1 min, 3 min, 5 min, and 10 min. After incubation for 24 h, the bacterial cells were serially diluted by 10‐fold 3. Resultsincrements and Discussion (1 × 101 to 1 × 107) and inoculated into PCA or BHIA to confirm cell viability. Bacterial cell viability was determined by counting the CFU of each bacterial type, and the antibacterial activity 3.1. Descriptionwas defined of the as Plasma a >3‐log Pad decrease in CFU/mL.

The plasma3. Results pad and introducedDiscussion in this paper was produced in the form of a wearable device with a flexible thin-film plasma sheet and an electric power unit. Figure1 shows a schematic of the overall 3.1. Description of the Plasma Pad concept of the plasma pad system, including the wearable power-supply unit stored in a band-type pocket. ElectricalThe plasma power pad is introduced supplied in to this the paper pad was through produced a cable in the form with of a a connector, wearable device and with the a connector flexible thin‐film plasma sheet and an electric power unit. Figure 1 shows a schematic of the overall is detachableconcept for of pad the plasma disposal. pad Aftersystem, the including system the has wearable been power charged‐supply with unit the stored secondary in a band‐type battery of the power-supplypocket. unit, Electrical it can power be usedis supplied in a to wireless the pad through form without a cable with external a connector, power; and therefore,the connector the patient can moveis freely detachable while for wearing pad disposal. the After plasma the system pad system.has been charged Although with plasmathe secondary is generated battery of the in the voids of the gauzepower space‐supply between unit, it can the be skin used and in a wireless the film form surface, without the external plasma power; pad therefore, has good the patient electrical safety. can move freely while wearing the plasma pad system. Although plasma is generated in the voids of In addition,the gauze it is protectedspace between from the skin low and temperature the film surface, thermal the plasma damage pad has as good the electrical film surface safety. In constantly ◦ maintainsaddition, a temperature it is protected below from 40 C.low temperature thermal damage as the film surface constantly maintains a temperature below 40 °C.

Figure 1. FigureSchematic 1. Schematic illustration illustration of theof the wearable wearable plasma plasma pad pad kit and kit andphotographs photographs of the thin of‐film the thin-film plasma padplasma discharge. pad discharge. A schematic of the plasma film layers and the discharge of the flexible plasma pad are shown in A schematicFigure 2a. ofA sinusoidal the plasma voltage film of 50 layers kHz and and 1.1 the kV rms discharge is applied of to the high flexible‐voltage plasma electrode. pad The are shown cold plasma is readily generated even on a curved flexible surface, as shown in Figure 2a, and no in Figure2a. A sinusoidal voltage of 50 kHz and 1.1 kV rms is applied to the high-voltage electrode. electrical problems are evident when the flexible pad is attached to the skin. The cold plasmaTo confirm is readily the formation generated of the even ROS on and a RNS curved generated flexible in the surface, discharge, as the shown plasma in pad Figure surface2 a, and no electrical problemslight was analyzed are evident by optical when emission the flexiblespectroscopy pad in is the attached wavelength to therange skin. of 200–900 nm (Figure To confirm2b). The identification the formation of these of emission the ROS lines andwas performed RNS generated according to in previous the discharge, reports [32,33]. the plasma The strongest emission peaks were the nitrogen lines at 316 nm, 337 nm, and 358 nm. It should be pad surface light was analyzed by optical emission spectroscopy in the wavelength range of noted that emission lines from OH (309 nm) and N (391 nm) appeared, but their intensities were 200–900 nmvery (Figure weak. 2Theseb) . The emission identification spectra indicate of these that emission the processed lines chemical was performed and physical according reaction to previous ∙ reports [32produced,33]. The various strongest ROS such emission as NO, OH, peaks and were O via the the nitrogenplasma pad. lines at 316 nm, 337 nm, and 358 nm. Figure 2c shows the variation of temperature inside the plasma pad+ versus the operating time It should be noted that emission lines from OH (309 nm) and N2 (391 nm) appeared, but their under the same operating conditions of β = 100% and β = 50. The temperature was measured using a intensities were very weak. These emission spectra indicate that the processed chemical and physical thermocouple inside the pad attached to the skin. Temperature· saturating occurred at 50 °C for β = reaction produced100% and at various 38.6 °C for ROS β = 50% such after as 10 NO, min. OH, During and a long O2 ‐viarun thetest of plasma several pad.hours for stability and Figuresafety2c shows at an operating the variation voltage of of 1.1 temperature kV, failures, such inside as thermal the plasma deformation pad of versus the film, the were operating not time under theobserved, same operating but slight conditionserosion‐staining of βdue= to 100% plasma and onβ the= ground 50. The electrode temperature was noted. was Concerns measured using about safety and failure due to aerial ozone or thermal heat can be readily reduced through use of a thermocouple inside the pad attached to the skin. Temperature saturating occurred at 50 ◦C for the power control for voltage modulation. β = 100% and at 38.6 ◦C for β = 50% after 10 min. During a long-run test of several hours for stability and safety at an operating voltage of 1.1 kV, failures, such as thermal deformation of the film, were not observed, but slight erosion-staining due to plasma on the ground electrode was noted. Concerns about safety and failure due to aerial ozone or thermal heat can be readily reduced through use of the power control for voltage modulation. Appl. Sci. 2017, 7, 1308 6 of 13 Appl. Sci. 2017, 7, 1305 6 of 13

Appl. Sci. 2017, 7, 1305 6 of 13

Figure 2.Figure(a) Schematic 2. (a) Schematic illustration illustration of of the the plasma plasma pad and and photographs photographs of the of thin the‐film thin-ﬁlm plasma plasma pad pad discharge. (b) Optical emission spectroscopy of plasma pad discharge. (c) Variation of temperature discharge; (b) Optical emission spectroscopy of plasma pad discharge; (c) Variation of temperature measured inside the pad attached to the skin with β = 100% and β = 50% during 1 h operation. (d) β β measuredOzone inside concentration the pad according attached to to the the duty skin ratio with at the distances= 100% of and d = 10 cm= and 50% d during= 20 cm over 1 h the operation; (d) Ozoneplasma concentration surface. according to the duty ratio at the distances of d = 10 cm and d = 20 cm over the plasma surface. An important issue could be the adjustability of aerial ozone to maintain an allowable concentration below 0.08 ppm and an ozone odor detection threshold below 0.03 ppm [34,35]. Figure An important issue could be the adjustability of aerial ozone to maintain an allowable 2d showsFigure the assessment 2. (a) Schematic of illustrationozone concentration of the plasma pad in andaerial photographs space over of the the thin plasma‐film plasma pad pad with voltage concentrationmodulation belowdischarge. for 0.08 power (b) ppm Optical control and emission an during ozone spectroscopy plasma odor of detectionplasmageneration. pad discharge. threshold Figure ( c)2d Variation below shows of 0.03 ozonetemperature ppm concentration [ 34,35]. Figure 2d shows theversus assessment dutymeasured ratio atinside of distances ozone the pad attachedof concentration d = 10 to cmthe andskin withd in = β 20 aerial= cm.100% When and space β β = 50%= over100%, during thethe 1 h ozone plasmaoperation. concentration (d pad) with is voltage slightly >0.10Ozone ppm. concentration When β according<50%, theto the concentration duty ratio at the is distances <0.05 ppm, of d = nearly10 cm and below d = 20 odorcm over detection the level. modulation forplasma power surface. control during plasma generation. Figure2d shows ozone concentration versus duty ratioFigure at 2d distances shows the of possibility d = 10 cmof ozone and control d = 20 in cm.the plasma When pad.β =Even 100%, if the the concentration ozone concentration over the is plasma surface was 0.10 ppm at a distance d = 10 cm with β = 100%, the measured level was <0.05 slightly >0.10 ppm.An important When β issue< 50%, could the be concentrationthe adjustability isof <0.05aerial ppm,ozone to nearly maintain below an allowable odor detection level. ppm concentrationwith the pad below attached 0.08 ppmto the and skin. an ozone odor detection threshold below 0.03 ppm [34,35]. Figure Figure2d showsIn2d thisshows the study, the possibility assessment the use of of ofmedical ozone ozone concentration gauze control is a distinctive in in theaerial plasma space feature over pad. of the the plasma Even plasma pad if pad. the with While concentration voltage the pad over the plasmais attachedmodulation surface to skin, was for plasma power 0.10 ppm controlis generated at during a distance in plasma the gauze generation. d = between 10 cm Figure the with pad 2dβ andshows= the 100%, ozone skin. the Theconcentration measured medical gauze level was <0.05 ppmplays withversus the role the duty padof ratio a spacer, attached at distances securing to of the d =a 10void skin. cm spaceand d =for 20 thecm. Whengeneration β = 100%, of plasma. the ozone Another concentration advantage is is that medicalslightly >0.10 gauze ppm. can When be used β <50%, for the various concentration skin care is <0.05 purposes ppm, nearly by applying below odor medication detection level.as a gel or In this study,Figure 2d the shows use the of possibility medical of gauze ozone control is a distinctive in the plasma feature pad. Even of if the concentration plasma pad. over While the the pad is ointment, which will not prevent plasma ignition in the gauze. As shown in Figure 3, plasma attached to skin,plasma plasma surface was is generated0.10 ppm at a in distance the gauze d = 10 cm between with β = the100%, pad the andmeasured the level skin. was The <0.05 medical gauze generation between the pad and skin is visually verified by attaching the plasma pad to a glass plate plays the roleppm of with a spacer, the pad securing attached to a the void skin. space for the generation of plasma. Another advantage is that rather thanIn thisto human study, the skin. use Figure of medical 3a shows gauze isthe a distinctive layers comprising feature of thethe plasma pad. Because pad. While the the discharge pad is medicalnot gauze visibleis attached can when be to used skin,the pad plasma for is various attached is generated skin to skin, in care the the gauze purposes pad between is placed by the applyingonpad the and glass the medicationskin. plate The to medical visually as gauze a verify gel or the ointment, which willdischarge notplays prevent theunderneath role of plasma a spacer, the glass ignition securing plate. ina Figurevoid the space gauze. 3b showsfor the As generationa shown photograph in of Figureplasma. taken 3Anotherfrom, plasma over advantage the generation glass is plate. between the padThe and photographthat skin medical is visually showsgauze can that verified be the used plasma by for attachingvarious was generated skin the care plasma purposes in the gauze pad by applying tospace, a glass evenmedication plate with the ratheras apad gel attachedor than to human ointment, which will not prevent plasma ignition in the gauze. As shown in Figure 3, plasma skin. Figureto the3a glass shows plate. the The layers ring comprisingpattern of plasma the pad. reflects Because the pattern the discharge of the ground is not electrode visible placed when on the pad is the polyimidegeneration filmbetween as shown the pad in and Figure skin is2a. visually verified by attaching the plasma pad to a glass plate attached to skin,rather the than pad to human is placed skin. Figure on the 3a glass shows plate the layers to visually comprising verify the pad. the Because discharge the discharge underneath is the glass plate. Figurenot3b visible shows when a photograph the pad is attached taken to skin, from the pad over is placed the glass on the plate. glass plate The to photograph visually verify the shows that the discharge underneath the glass plate. Figure 3b shows a photograph taken from over the glass plate. plasma wasThe generated photograph in shows the gauze that the space, plasma even was generated with the in padthe gauze attached space, even to the with glass the pad plate. attached The ring pattern of plasma reflectsto the glass the patternplate. The of ring the pattern ground of plasma electrode reflects placed the pattern on theof the polyimide ground electrode film as placed shown on in Figure2a. the polyimide film as shown in Figure 2a.

Figure 3. (a) Schematic of the plasma pad attached to a glass plate and (b) a photograph of plasma pad discharge under a glass plate.

Figure 3. (a) Schematic of the plasma pad attached to a glass plate and (b) a photograph of plasma Figure 3. (a)pad Schematic discharge under of the a glass plasma plate. pad attached to a glass plate and (b) a photograph of plasma pad discharge under a glass plate. Appl. Sci. 2017, 7, 1308 7 of 13

Appl. Sci. 2017, 7, 1305 7 of 13 3.2. Biocompatibilty of the Plasma Pad 3.2. Biocompatibilty of the Plasma Pad ForFor biomedical biomedical applicationsapplications ofof the plasma pad, pad, tests tests recommended recommended by by the the International International OrganizationOrganization for for Standardization-10993 Standardization‐10993 (ISO (ISO-10993)‐10993) are are essential essential for assessing for assessing safety. safety. Therefore, Therefore, we weperformed performed inin vitro vitro andand inin vivo vivo biocompatibilitybiocompatibility experiments experiments on onthe the plasma plasma pad pad using using two two kinds kinds of of skinskin cells cells and and hairless hairless mice. mice. AsAs shown shown in in Figure Figures4a,b, 4a,b, there there was was no cytotoxicityno cytotoxicity in GFP-transfectedin GFP‐transfected HaCaT HaCaT cells cells or or mouse mouse skin ﬁbroblastsskin fibroblasts (L-929 cells),(L‐929 and cells), morphological and morphological changes were changes also not were observed also withnot observed plasma treatment, with althoughplasma celltreatment, viability although was slightly cell viability decreased was comparedslightly decreased to that ofcompared controls to due that to of plasma controls treatment. due to plasmaNone treatment. of the plasma treatments triggered skin irritation at any site, nor did plasma treatment induceNone intracutaneous of the plasma damage, treatments as shown triggered in Figure skin irritation4c. Additionally, at any site, no nor symptoms did plasma related treatment to skin tissueinduce toxicity intracutaneous or abnormal damage, behaviors, as shown such in as Figure convulsions 4c. Additionally, or prostration, no symptoms were observed, related to even skin after treatmenttissue toxicity for 10 or min. abnormal The results behaviors, demonstrate such as convulsions that treatment or prostration, with a plasma were padobserved, is minimally even after toxic totreatment skin cells, for HaCaT 10 min. and The L-929 results cells, demonstrate and mouse that skin, treatment and the with plasma a plasma pad pad is biocompatible is minimally toxic for use to in skinskin treatment. cells, HaCaT and L‐929 cells, and mouse skin, and the plasma pad is biocompatible for use in skin treatment.

Figure 4. In vitro and in vivo biocompatibility of the plasma pad. (a) Quantitative analysis of in vitro Figure 4. In vitro and in vivo biocompatibility of the plasma pad. (a) Quantitative analysis of cytotoxicity of the plasma pad on GFP‐transfected human keratinocytes (HaCaT cells) and mouse in vitro cytotoxicity of the plasma pad on GFP-transfected human keratinocytes (HaCaT cells) and fibroblasts (L‐929 cells). (n = 4, * P < 0.05 compared to control). (b) Images of plasma‐treated GFP‐ mouse fibroblasts (L-929 cells). (n = 4, * P < 0.05 compared to control); (b) Images of plasma-treated transfected HaCaT cells and L‐929 cells. The images are magnified from those inserted in each picture GFP-transfectedtaken with a 4× optical HaCaT lens. cells Scale and bars L-929 are cells. 100 μ Them. (c images) Schematic are magnifiedillustration fromfor evaluating those inserted the in vivo in each pictureskin irritation taken with test a using 4× optical the plasma lens. Scale pad. bars(d) Photographs are 100 µm; and (c) Schematic histological illustration images of formouse evaluating skin at the inday vivo 2 skinpost‐ irritationplasma treatment test using for the10 min. plasma Scale pad; bars ( drepresent) Photographs 200 μm. and histological images of mouse skin at day 2 post-plasma treatment for 10 min. Scale bars represent 200 µm. 3.3. Exogenous Nitric Oxide and Capillary Tube Formation in Hdmvecs Induced by the Plasma Pad 3.3. Exogenous Nitric Oxide and Capillary Tube Formation in Hdmvecs Induced by the Plasma Pad Plasma is known to catalyze and regulate biochemical activities and cellular responses such as proliferation,Plasma is knowndifferentiation, to catalyze and andcell death regulate or apoptosis biochemical [36]. activities The responses and cellular may be due responses to the ROS such as proliferation,and RNS generated differentiation, by non and‐thermal cell death plasma or apoptosis[37]. Among [36 the]. The various responses reactive may species, be due NO to the can ROS andmodulate RNS generated angiogenesis, by non-thermal resulting plasmain the [promotion37]. Among of the wound various healing. reactive Therefore, species, NO we can analyzed modulate angiogenesis,exogenous NO resulting generated in the from promotion plasma ofby wound using Griess healing. reagents. Therefore, As shown we analyzed in Figure exogenous 5a, plasma NO − NO 2 generatedtreatment from significantly plasma by increased using Griess exogenous reagents. As in shown both inDPBS Figure and5a, cell plasma culture treatment media in significantly a time‐ dependent manner, and the amount of generated NO reached a maximum 60 mM with 10 min of increased exogenous NO− in both DPBS and cell culture media in a time-dependent manner, and the plasma treatment. This 2indirectly indicates that the NO generated from plasma plays a key role in amount of generated NO− reached a maximum 60 mM with 10 min of plasma treatment. This indirectly vascularization and wound2 healing. indicates that the NO generated from plasma plays a key role in vascularization and wound healing. Generally, vascularization or angiogenesis plays a key role in both wound healing and tissue recovery.Generally, Non vascularization‐thermal plasma recently or angiogenesis emerged playsas a useful a key method role in in both biomedical wound applications healing and due tissue recovery.to the ROS Non-thermal and RNS produced. plasma recently With exogenous emerged NO as adata, useful angiogenesis method in activity biomedical was also applications confirmed due to the ROS and RNS produced. With exogenous NO data, angiogenesis activity was also confirmed Appl. Sci. 2017, 7, 1308 8 of 13

Appl. Sci. 2017, 7, 1305 8 of 13 with plasma treatment. Although angiogenesis is a very complex process, capillary tube formation by endothelialwith plasma cells treatment. is a key step Although [38,39 ].angiogenesis is a very complex process, capillary tube formation byTo endothelial evaluate cells the potentialis a key step for [38,39]. angiogenesis with plasma treatment, a capillary tube formation assay wasTo evaluate performed the using potential HDMVECs for angiogenesis on Matrigel with for plasma 8 h. Astreatment, shown ina capillary Figure5b, tube the formation HDMVECs treatedassay with was plasmaperformed formed using well-deﬁned HDMVECs on capillary Matrigel tubes for 8 withh. As enhanced shown in alignmentFigure 5b, the and HDMVECs organization in atreated time-dependent with plasma manner; formed well moreover,‐defined thecapillary total tubes length with of theenhanced capillary alignment tubes alsoand organization increased with in a time‐dependent manner; moreover, the total length of the capillary tubes also increased with plasma treatment, while the negative control HDMVECs formed randomly short and disconnected plasma treatment, while the negative control HDMVECs formed randomly short and disconnected capillary tubes. This result demonstrates that plasma treatment of HDMVECs may have potential for capillary tubes. This result demonstrates that plasma treatment of HDMVECs may have potential for enhancement of vascularization in the ﬁeld of tissue engineering. enhancement of vascularization in the field of tissue engineering

− FigureFigure 5. 5.Exogenous Exogenous nitric nitric oxide oxide ((NONO2) )and and inin vitro vitro capillarycapillary tube tube formation formation with with plasma plasma treatment. treatment. − (a)( Quantitativea) Quantitative analysis analysis of of NO NO2 generated generated by by a plasmaa plasma pad pad (n (n= = 4, 4, * *P P< < 0.005, ** P < 0.0005 0.0005 compared compared to control);to control). (b) Images (b) Images of capillary of capillary tube formation tube formation by HDMVECs by HDMVECs with plasma with treatment plasma treatment and quantitative and analysisquantitative of capillary analysis tube of length capillary (n = 5,tube * P length< 0.005, (n ** =P 5,< * 0.0005 P < 0.005, compared ** P < to 0.0005 control). compared Scale bars to control). are 100 µ m. Scale bars are 100 μm. 3.4. Wound Healing Effect of the Plasma Pad 3.4. Wound Healing Effect of the Plasma Pad To evaluate the in vivo wound healing effect of the plasma pad, treatment was applied at each site To evaluate the in vivo wound healing effect of the plasma pad, treatment was applied at each after full-thickness excisional wounds were made on mouse back skin; wound closure in each group site after full‐thickness excisional wounds were made on mouse back skin; wound closure in each was observed using a stereomicroscope and the wound area was observed for 21 days after treatment. group was observed using a stereomicroscope and the wound area was observed for 21 days after treatment.Plasma treatment significantly enhanced the rate of wound closure at Days 6 and 9 compared to that in negativePlasma treatment controls significantly (Figure6a). Theenhanced rate of the wound rate of closurewound closure in the plasma-treated at Days 6 and 9 groupcompared reached to aboutthat 85.8%in negative and 94.8%,controls respectively (Figure 6a). The (* P rate < 0.05) of wound compared closure to in that the plasma in the‐ controltreated group group reached (72% and 84%).about This 85.8% might and be 94.8%, due respectively to rapid re-epithelialization (* P < 0.05) compared resulting to that inin the control promotion group of (72% cell proliferationand 84%). and/orThis might migration, be due thereby to rapid facilitating re‐epithelialization wound resulting healing. in Additionally, the promotion we of assume cell proliferation that the increasedand/or exogenousmigration, NO thereby in the facilitating wound area wound and healing. around Additionally, tissues promoted we assume angiogenesis that the increased and enhanced exogenous cellular growthNO in for the wound wound healing. area and This around is supported tissues promoted by several angiogenesis previous and studies enhanced reporting cellular that growth NO resulted for in angiogenesiswound healing. triggering This is cellularsupported growth by several and differentiation previous studies in a woundreporting model that [ 38NO–41 resulted]. in angiogenesisAs shown intriggering Figure6 b,cellular the wounded growth and skin differentiation fully recovered in a without wound model any contraction [38–41]. or crusting in plasma-treatedAs shown mice, in Figure whereas 6b, the some wounded crusting skin was fully found recovered in the negative without controlany contraction group at or Day crusting 21. in Toplasma confirm‐treated wound mice, whereas healing some efficacy, crusting recovery was found was in the evaluated negative by control using group the at rate Day of21. skin re-epithelialization,To confirm wound remodeling, healing and efficacy, repair inrecovery each slide was sample evaluated after by staining using the with rate H&E. of Theskin ratere‐ of epithelialization, remodeling, and repair in each slide sample after staining with H&E. The rate of wound repair in the plasma-treated group was 1.3-fold higher than that in the negative control group, wound repair in the plasma‐treated group was 1.3‐fold higher than that in the negative control group, calculated as 12.5% in negative controls and 16.7% in the plasma-treated group. This indicates that calculated as 12.5% in negative controls and 16.7% in the plasma‐treated group. This indicates that plasma treatment induced accelerated wound repair at Day 21 compared to the controls. Histological plasma treatment induced accelerated wound repair at Day 21 compared to the controls. Histological analysis also exhibited the same result as shown in Figure6c. The histological images of skin treated analysis also exhibited the same result as shown in Figure 6c. The histological images of skin treated withwith plasma plasma showed showed re-epithelialization re‐epithelialization with thick epidermis epidermis and and increased increased dermal dermal fibroblasts, fibroblasts, resultingresulting in in dense dense dermal dermal fiber, fiber, remodeling remodeling withwith some skin skin adnexa adnexa including including hair hair follicles follicles and and dense dense dermaldermal fibroblast, fibroblast, and and complete complete wound wound repairrepair with skin adnexa adnexa and and increased increased hair hair follicles. follicles. Hair Hair folliclesfollicles in in anagen anagen and and catagen catagen stages stages werewere significantlysignificantly increased increased compared compared with with the the control control group, group, asas shown shown in in Figure Figure6d. 6d. On On the the other other hand, hand, compared compared with the plasma plasma-treated‐treated group, group, the the negative negative Appl. Sci. 2017, 7, 1308 9 of 13

Appl. Sci. 2017, 7, 1305 9 of 13 control group showed re-epithelialization of thin epidermis, skin remodeling with fewer dermal ﬁbroblasts,control group less deposition showed re of‐epithelialization collagen, and wound of thin repairepidermis, with skin fewer remodeling total hair follicleswith fewer and dermal increased telogen-stagefibroblasts, less follicles. deposition of collagen, and wound repair with fewer total hair follicles and increased telogenThe overall‐stage follicles. rate of wound healing in the plasma-treated group was enhanced in comparison with thatThe in overall negative rate of controls. wound healing These in results the plasma indicate‐treated that group remodeling was enhanced of skin in tissue comparison through re-epithelializationwith that in negative is faster controls. in the These plasma-treated results indicate group that than remodeling in the negative of skin control tissue group, through resulting re‐ in anepithelialization increased rate is of faster wound in the repair. plasma‐treated group than in the negative control group, resulting in an increased rate of wound repair.

FigureFigure 6. In6. vivoIn vivowound wound healing healing effects effects of plasma of plasma pads pads in the in full-thickness the full‐thickness excisional excisional wound wound splinting model.splinting (a) Quantiﬁcation model. (a) Quantification of the wound of the closure wound rate closure following rate following wounding. wounding. (n = 8, * P(n< = 0.058, * P compared < 0.05 to control);compared ( bto) control). Representative (b) Representative stereomicroscopic stereomicroscopic images of images full-thickness of full‐thickness wound wound sites andsites wound and wound contraction at Days 0 and 21. Scale bars are 2 mm. (c) Histological images of hematoxylin and contraction at Days 0 and 21. Scale bars are 2 mm; (c) Histological images of hematoxylin and eosin eosin (H&E) staining of negative control and plasma‐treated skin. Scale bars are 500 μm. (d) (H&E) staining of negative control and plasma-treated skin. Scale bars are 500 µm; (d) Quantitative Quantitative hair follicle assessment in repaired wound sites. Total and hair follicles at each hair follicle assessment in repaired wound sites. Total and hair follicles at each stage (anagen, catagen, stage (anagen, catagen, and telogen) were determined after H&E staining (n = 8, * P < 0.05 compared and telogen) were determined after H&E staining (n = 8, * P < 0.05 compared to control). to control).

3.5.3.5. Antibacterial Antibacterial Activity Activity of of the the Plasma Plasma Pad Pad TheThe plasma plasma pad pad antibacterial antibacterial propertiesproperties were evaluated using using quantitative quantitative and and qualitative qualitative methodsmethods that that involved involved counting counting the the numbernumber of CFUs and and performing performing inhibition inhibition zone zone assays assays after after plasmaplasma treatment, treatment, as as shown shown in in Figure Figure7. 7. The The antibacterial antibacterial activity activity using using aa quantitative method method shows shows a similara similar trend trend to the to one the shown one shown for the for treatment the treatment time-dependent time‐dependent antibacterial antibacterial effect in effect the qualitative in the analysisqualitative and theanalysis plasma and pads the plasma showed pads signiﬁcant showed significant killing efﬁciency killing efficiency toward S. toward aureus S.as aureus well as well clinical MRSAas clinical isolates. MRSA The isolates. strong antibacterialThe strong antibacterial effects of the effects plasma of the pad plasma were pad also were observed also observed in qualitative in images.qualitative These images. results These indicate results that indicate plasma that pads plasma can also pads be can used also for be skin used disinfection. for skin disinfection. Appl. Sci. 2017, 7, 1308 10 of 13 Appl. Sci. 2017, 7, 1305 10 of 13

FigureFigure 7. 7.Antibacterial Antibacterial activity of of the the plasma plasma pad. pad. Viable Viable bacterial bacterial cells were cells counted were counted after 24 afterh post 24‐ h post-plasmaplasma treatment. treatment. Differences Differences of P of < P0.05< 0.05 were were considered considered statistically statistically significant signiﬁcant (n = 3, (n * =P 3,< 0.05,*P < ** 0.05, ** PP< < 0.0050.005 compared to to the the control). control).

Overall, these results suggest that the biocompatible plasma pad, which causes no cytotoxicity orOverall, skin irritation, these results has a wound suggest healing that the effect biocompatible and antibacterial plasma activity pad, which against causes S. aureus no cytotoxicity and drug‐ or skinresistant irritation, bacteria, has a woundsuch as MRSA, healing indicating effect and great antibacterial potential activity as a method against of S.skin aureus treatment.and drug-resistant bacteria, such as MRSA, indicating great potential as a method of skin treatment. 4. Conclusions 4. Conclusions A wearable hybrid‐DBD plasma pad with a wearable power supply was developed. Unlike earlierA wearable plasma hybrid-DBD jets or DBD plasmadevices, pad this with wearable a wearable plasma power pad offers supply a wasvariety developed. of applications Unlike and earlier plasmaconvenience jets or DBD in terms devices, of treatment this wearable area and plasma time. pad offers a variety of applications and convenience in termsThe of treatmentflexible thin area‐film and plasma time. pad was fabricated with a polyimide film as a dielectric layer in a DBD,The where flexible two thin-film electrodes plasma were padformulated was fabricated on each surface. with a polyimide Protection filmfrom as electrical a dielectric hazard layer was in a DBD,ensured where because two electrodes the high‐ werevoltage formulated electrode was on each covered surface. with Protectionan insulation from layer, electrical and the hazard plasma was ensuredwas generated because the on high-voltage a ground electrode electrode that was was covered covered with with an insulationmedical gauze layer, facing and the the plasma skin. A was generateddistinctive on feature a ground of the electrode proposed that plasma was coveredpad was withthe containment medical gauze of the facing plasma the in skin.the gauze A distinctive space, featureeven of when the proposedthe pad was plasma attached pad to was human the containment skin. During of operation the plasma at approximately in the gauze space, 1 kV, evenstability when theand pad safety was attachedwere ensured, to human as electrical skin. During and thermal operation damage at approximatelyto the skin and aerial 1 kV, stabilityozone or andthermal safety wereheat ensured, could be as readily electrical reduced and thermal through damagepower modulation. to the skin and aerial ozone or thermal heat could be From in vitro and in vivo biocompatibility tests, the potential of the plasma pad for biomedical readily reduced through power modulation. applications was confirmed, with no cytotoxicity for normal cells and no irritation of mouse skin. From in vitro and in vivo biocompatibility tests, the potential of the plasma pad for biomedical Skin wound healing with hair growth was also observed after in vivo plasma treatment. Moreover, applications was confirmed, with no cytotoxicity for normal cells and no irritation of mouse skin. the activity of the plasma pad against two bacteria present in normal or damaged skin, including SkinMRSA, wound was healing demonstrated. with hair growth was also observed after in vivo plasma treatment. Moreover, the activityFuture of research the plasma will padbe conducted against two to confirm bacteria the present actual intreatment normal effect or damaged of plasma skin, in the including form MRSA,of a patch, was demonstrated. bandage, socks, and cap. Future research will be conducted to confirm the actual treatment effect of plasma in the form of a Acknowledgments: This work was supported in part by the Kwangwoon University under a Research Grant in patch, bandage, socks, and cap. 2017 and by a grant provided from the National Research Foundation of Korea (NRF‐2017R1A2B4012590 and NRF‐2015M3A9E2066855). Acknowledgments: This work was supported in part by the Kwangwoon University under a Research Grant in 2017Author and by Contributions: a grant provided Guangsup from theCho National conceived Research and designed Foundation the experiments; of Korea (NRF-2017R1A2B4012590 Junggil Kim, Kyong‐Hoon and NRF-2015M3A9E2066855).Choi, and Bong Joo Park performed the experiments and wrote the manuscript; Yunjung Kim analyzed the data.

AuthorConflicts Contributions: of Interest: GuangsupThe authors Cho declare conceived no conflict and of designed interest. the experiments; Junggil Kim, Kyong-Hoon Choi and Bong Joo Park performed the experiments and wrote the manuscript; Yunjung Kim analyzed the data. ConﬂictsReferences of Interest: The authors declare no conﬂict of interest. Appl. Sci. 2017, 7, 1308 11 of 13

References

1. Heinlin, J.; Morfill, G.; Landthaler, M.; Stolz, W.; Isbary, G.; Zimmermann, J.L.; Shimizu, T.; Karrer, S. Plasma medicine: Possible applications in dermatology. J. Dtsch. Dermatol. Ges. 2010, 8, 968–976. [CrossRef] [PubMed] 2. Isbary, G.; Köritzer, J.; Mitra, A.; Li, Y.F.; Shimizu, T.; Schroeder, J.; Schlegel, J.; Morfill, G.E.; Solz, W.; Zimmermann, J.L. Ex vivo human skin experiments for the evaluation of safety of new cold atmospheric plasma devices. Clin. Plasma Med. 2013, 1, 36–44. [CrossRef] 3. Emmert, S.; Brehmer, F.; Hänßle, H.; Helmke, A.; Mertens, N.; Ahmed, R.; Simon, D.; Wandke, D.; Friedrichs, W.M.; Däschlein, G.; et al. Atmospheric pressure plasma in dermatology: Ulcus treatment and much more. Clin. Plasma Med. 2013, 1, 24–29. [CrossRef] 4. Kalghatgi, S.U.; Fridman, G.; Fridman, A.; Friedman, G.; Clyne, A.M. Non-Thermal Dielectric Barrier Discharge Plasma Treatment of Endothelial Cells. In Proceedings of the 30th Annual International Conference of the IEEE Engineering in Medicine and Biology Society, Vancouver, BC, Canada, 20–24 August 2008. 5. Helmke, A.; Hoffmeister, D.; Mertens, N.; Emmert, S.; Schuette, J.; Vioel, W. The acidification of lipid film surfaces by non-thermal DBD at atmospheric pressure in air. New J. Phys. 2009, 11, 115025–115034. [CrossRef] 6. Fridman, G.; Shereshevsky, A.; Jost, M.M.; Brooks, A.D.; Fridman, A.; Gutsol, A.; Vasilets, V.; Friedman, G. Floating Electrode Dielectric Barrier Discharge Plasma in Air Promoting Apoptotic Behavior in Melanoma Skin Cancer Cell Lines. Plasma Chem. Plasma Process. 2007, 27, 163–176. [CrossRef] 7. Haertel, B.; von Woedtke, T.; Weltmann, K.-D.; Lindequist, U. Non-Thermal Atmospheric-Pressure Plasma Possible Application in Wound Healing. Biomol. Ther. 2014, 22, 477–490. [CrossRef][PubMed] 8. Isbary, G.; Zimmermann, J.L.; Shimizu, T.; Li, Y.-F.; Morfill, G.E.; Thomas, H.M.; Steffes, B.; Heinlin, J.; Karrer, S.; Stolz, W. Non-thermal plasma—More than five years of clinical experience. Clin. Plasma Med. 2013, 1, 19–23. [CrossRef] 9. Heinlin, J.; Isbary, G.; Stolz, W.; Morfill, G.; Landthaler, M.; Shimizu, T.; Steffes, B.; Nosenko, T.; Zimmermann, J.L.; Karrer, S. Plasma applications in medicine with a special focus on dermatology. J. Eur. Acad. Dermatol. Venereol. 2010, 25, 1–11. [CrossRef][PubMed] 10. Tiede, R.; Hirschberg, J.; Daeschlein, G.; von Woedtke, T.; Vioel, W.; Emmert, S. Plasma Applications: A Dermatological View. Contrib. Plasma Phys. 2014, 54, 118–130. [CrossRef] 11. Weltmann, K.-D.; Brandenburg, R.; von Woedtke, T.; Ehlbeck, J.; Foest, R.; Stieber, M.; Kindel, E. Antimicrobial treatment of heat sensitive products by miniaturized atmospheric pressure plasma jets (APPJs). J. Phys. D Appl. Phys. 2008, 41, 194008. [CrossRef] 12. Hahnel, M.; von Woedtke, T.; Weltmann, K.-D. Influence of the Air Humidity on the Reduction of Bacillus Spores in a Defined Environment at Atmospheric Pressure Using a Dielectric Barrier Surface Discharge. Plasma Process. Polym. 2010, 7, 244–249. [CrossRef] 13. Ehlbeck, J.; Schnabel, U.; Polak, M.; Winter, J.; von Woedtke, T.; Brandenburg, R.; von dem Hagen, T.; Weltmann, K.-D. Low temperature atmospheric pressure plasma sources for microbial decontamination. J. Phys. D Appl. Phys. 2011, 44, 013002. [CrossRef] 14. Wu, Z.; Chen, M.; Li, P.; Zhu, Q.; Wang, J. Dielectric barrier discharge non-thermal micro-plasma for the excitation and emission spectrometric detection of ammonia. Analyst 2011, 136, 2552–2557. [CrossRef] [PubMed] 15. Fridman, G.; Fridman, G.; Gutsol, A.; Shekhter, A.; Vasilets, V.; Fridman, A. Applied Plasma Medicine. Plasma Process. Polym. 2008, 5, 503–533. [CrossRef] 16. Shashurin, A.; Keidar, M.; Bronnikov, S.; Jurjus, R.A.; Stepp, M.A. Living tissue under treatment of cold plasma atmospheric jet. Appl. Phys. Lett. 2008, 98, 181501. [CrossRef] 17. Weltmann, K.-D.; Kindel, E.; Brandenburg, R.; Meyer, C.; Bussiahn, R.; Wilke, C.; von Woedtke, T. Atmospheric Pressure Plasma Jet for Medical Therapy: Plasma Parameters and Risk Estimation. Contrib. Plasma Phys. 2009, 49, 631–640. [CrossRef] 18. Kim, Y.; Jin, S.; Han, G.; Kwon, G.; Choi, J.; Choi, E.; Uhm, H.; Cho, G. Plasma Apparatuses for Biomedical Applications. IEEE Trans. Plasma Sci. 2015, 43, 944–950. [CrossRef] 19. Fridman, G.; Peddinghaus, M.; Ayan, H.; Fridman, A.; Balasubramanian, M.; Gutsol, A.; Brooks, A.; Friedman, G. Blood Coagulation and Living Tissue Sterilization by Floating-Electrode Dielectric Barrier Discharge in Air. Plasma Chem. Plasma Process. 2006, 26, 425–442. [CrossRef] Appl. Sci. 2017, 7, 1308 12 of 13

20. Weltmann, K.-D.; Fricke, K.; Stieber, M.; Brandenburg, R.; von Woedtke, T.; Schnabel, U. New Nonthermal Atmospheric-Pressure Plasma Sources for Decontamination of Human Extremities. IEEE Trans. Plasma Sci. 2012, 40, 2963–2969. [CrossRef] 21. Von Woedtke, T.; Reuter, S.; Masur, K.; Weltmann, K.-D. Plasmas for medicine. Phys. Rep. 2013, 530, 291–320. [CrossRef] 22. Weltmann, K.-D.; Polak, M.; Masur, K.; von Woedtke, T.; Winter, J.; Reuter, S. Plasma Processes and Plasma Sources in Medicine. Contrib. Plasma Phys. 2012, 52, 644–654. [CrossRef] 23. Diaham, S.; Zelmat, S.; Locatelli, M.L.; Dinculescu, S.; Decup, M.; Lebey, T. Dielectric Breakdown of Polyimide Films—Area, Thickness and Temperature Dependence. IEEE Trans. Dielectr. Electr. Insul. 2010, 17, 18–27. [CrossRef] 24. International Standard ISO 10993-5. Part 5: Tests for In Vitro Cytotoxicity; International Organization for Standardization: Geneva, Switzerland, 2009. 25. Park, B.J.; Choi, K.H.; Nam, K.C.; Min, J.; Lee, K.D.; Uhm, H.S.; Choi, E.H.; Kim, H.J.; Jung, J.S. Photodynamic

Anticancer Activity of CoFe2O4 Nanoparticles Conjugated with Hematoporphyrin. J. Nanosci. Nanotechnol. 2015, 15, 7900–7906. [CrossRef][PubMed]

26. Lee, Y.; Choi, K.H.; Park, K.Y.; Lee, J.M.; Park, B.J.; Park, K.D. In situ forming and H2O2-releasing hydrogels for treatment of drug-resistant bacterial infections. ACS Appl. Mater. Interfaces 2017, 9, 16890–16899. [CrossRef][PubMed] 27. International Standard ISO 10993-10. Part 10: Tests for Irritation and Skin Sensitization; International Organization for Standardization: Geneva, Switzerland, 2010. 28. Giustarini, D.; Rossi, R.; Milzani, A.; Dalle-Donne, I. Nitrite and Nitrate Measurement by Griess Reagent in Human Plasma: Evaluation of Interferences and Standardization. Methods Enzymol. 2008, 440, 361–380. [CrossRef][PubMed] 29. Wang, X.; Ge, J.; Tredget, E.E.; Wu, Y. The mouse excisional wound splinting model including applications for stem cell transplantation. Nat. Protoc. 2013, 8, 302–309. [CrossRef][PubMed] 30. Asai, J.; Takenaka, H.; Kusano, K.F.; Ii, M.; Luedemann, C.; Curry, C.; Eaton, E.; Iwakura, A.; Tsutsumi, Y.; Hamada, H.; et al. Topical sonic hedgehog gene therapy accelerates wound healing in diabetes by enhancing endothelial progenitor cell-mediated microvascular remodeling. Circulation 2006, 113, 2413–2424. [CrossRef] [PubMed] 31. Luo, J.D.; Wang, Y.Y.; Fu, W.L.; Wu, J.; Chen, A.F. Gene therapy of endothelial nitric oxide synthase and manganese superoxide dismutase restores delayed wound healing in type 1 diabetic mice. Circulation 2004, 110, 2484–2493. [CrossRef][PubMed] 32. Kim, S.J.; Joh, H.M.; Chung, T.H. Production of intracellular reactive oxygen species and change of cell viability induced by atmospheric pressure plasma in normal and cancer cells. Appl. Phys. Lett. 2013, 103, 153705. [CrossRef] 33. Cheng, X.; Murphy, W.; Recek, N.; Yan, D.; Cvelbar, U.; Vesel, A.; Mozetiˇc,M.; Canady, J.; Keidar, M.; Sherman, J.H. Synergistic effect of gold nanoparticles and cold plasma on glioblastoma cancer therapy. J. Phys. D Appl. Phys. 2014, 47, 335402. [CrossRef] 34. Wilska, S. Ozone. Its Physiological Effects and Analytical Determination in Laboratory Air. Acta Chem. Scand. 1951, 5, 1359–1367. [CrossRef] 35. Diggle, W.M.; Cage, J.C. The toxicity of ozone in the presence of oxides of nitrogen. Br. J. Ind. Med. 1955, 12, 60–64. [CrossRef][PubMed] 36. Graves, D.B. Low temperature plasma biomedicine: A tutorial review. Phys. Plasmas 2014, 21, 080901. [CrossRef] 37. Niemi, K.; O’Connell, D.; de Oliveira, N.; Joyeux, D.; Nahon, L.; Booth, J.P.; Gans, T. Absolute atomic oxygen and nitrogen densities in radio-frequency driven atmospheric pressure cold plasmas: Synchrotron vacuum ultra-violet high-resolution Fouriertransform absorption measurements. Appl. Phys. Lett. 2013, 103, 034102. [CrossRef] 38. Lucia, M.; Sandra, D.; Marina, Z. Role of nitric oxide in the modulation of angiogenesis. Curr. Pharm. Des. 2003, 9, 521–530. [CrossRef] 39. Luo, J.-D.; Chen, A.F. Nitric oxide: A newly discovered function on wound healing. Acta Pharmacol. Sin. 2005, 26, 259–264. [CrossRef][PubMed] Appl. Sci. 2017, 7, 1308 13 of 13

40. Zhu, H.; Wei, X.; Bian, K.; Murad, F. Effects of nitric oxide on skin burn wound healing. J. Burn Care Res. 2008, 29, 804–814. [CrossRef][PubMed] 41. Stefen, F.; Heiko, K.; Christian, W.; Josef, P. Nitric oxide drives skin repair: Novel functions of an established mediator. Kidney Int. 2002, 61, 882–888. [CrossRef]

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).