wound healing with nonthermal microplasma jets generated in arrays of hourglass microcavity devices

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Wound healing with nonthermal microplasma jets generated in arrays of hourglass

microcavity devices

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2014 J. Phys. D: Appl. Phys. 47 435402

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1. Introduction

Low temperature plasma ejected into ambient air as a jet appears to have considerable promise as a medical thera-peutic tool [1, 2]. Early work in this area suggested that such

non-equilibrium plasma jets are effective in deactivating spe-cific cells (and viral and bacterial pathogens, in particular) and in promoting the healing of wounds [1–3]. Although the fun-damental mechanisms responsible for such beneficial effects remain speculative [2], one critical function of the atmospheric

Journal of Physics D: Applied Physics

Wound healing with nonthermal microplasma jets generated in arrays of hourglass microcavity devices

Chan Hum Park1,2, Joong Seob Lee1,2, Ji Heui Kim1,2, Dong-Kyu Kim1,2, Ok Joo Lee1, Hyung Woo Ju1, Bo Mi Moon1, Jin Hoon Cho3, Min Hwan Kim3, Peter Peng Sun3, Sung-Jin Park3 and J Gary Eden3

1 Nano-Bio Regenerative Medical Institute, Hallym University, Chuncheon, Korea2 Department of Otorhinolaryngology-Head and Neck Surgery, Chuncheon Sacred Heart Hospital, Hallym University College of Medicine, Chuncheon, Korea3 Laboratory for Optical Physics and Engineering, Department of Electrical and Computer Engineering, University of Illinois, Urbana, IL 61801, USA

E-mail: hlpch@paran.com

Received 19 January 2014, revised 25 August 2014Accepted for publication 9 September 2014Published 9 October 2014

AbstractClinical studies are reported in which artificial wounds in rat epidermal and dermal tissue have been treated by arrays of sub-500 µm diameter, low temperature plasma microjets. Fabricated in Al/nanoporous alumina (Al2O3) by wet chemical and microablation processes, each plasma jet device has a double parabolic (hourglass) structure, and arrays as large as 6  ×  6 devices with 500 µm diameter apertures have been tested to date. Treatment of 1 cm2 acute epidermal wounds for 20–40 s daily with an array of microplasma jets generated in He feedstock gas promoted wound recovery significantly, as evidenced by tissue histology and measured wound area. Seven days after wound formation, the wound area of the untreated control was 40  ±  2% of its initial value, whereas that for an identical wound treated twice daily for 20 s was 9  ±  2% of its original surface area. No histological distinctions were observed between wounds treated twice each day for 10 or 20 s – only the full recovery time differed. Spectra produced in the visible and ultraviolet by He jets in room air are dominated by atomic oxygen (3p 5P → 3s 5S) at 777 nm and violet fluorescence (391.4 nm) from N2

+, a species produced when the He (2s 3S1) metastable is deactivated by Penning ionization of N2. Although the combined cross-sectional area of the jets in the array is only 7% of the wound area, the microplasma treatment results in spatially uniform, and accelerated, wound healing. Both effects are attributed to the increased surface area of the jet array (relative to a single jet having an equivalent diameter) and the concomitant enhancement in the generation of molecular radicals, and metastable atoms and molecules (such as Σ +AN ( )2

3u ).

Keywords: microplasma, wound healing, plasma device

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pressure plasma jet is presumably the generation of nitrogen and/or oxygen-bearing radicals, including NO and OH [2], at the interface between the plasma jet and the surrounding gas-eous atmosphere, Consequently, the radical production rate is constrained by the surface area of the plasma jet. Molecular precursor (N2, O2) number densities are depleted at the inte-rior of the jet, and the reactive species beneficial to plasma therapeutics are apparently not primarily delivered to tissue along the axis of the plasma column.

Virtually all previous studies of the medical applications of low temperature plasma jets have involved a single cylindrical plasma column having a diameter of several millimetres [2, 4]. In recent experiments [5], researchers at Old Dominion University and the University of Illinois investigated the lat-eral extent of the biomedical impact of atmospheric pressure microjets. With an array of 355 µm diameter jets, the spatial variation of the deactivation of E. coli K12 cells by the plasma array was examined, and a synergistic effect between neigh-bouring jets was observed. Specifically, the spatial map of E. coli deactivation demonstrated that the biochemical effect of the microplasmas could not be represented as the sum of the jets acting independently. Thorough inactivation of the bacteria lying in regions between adjacent jets was interpreted in terms of associative reactions (such as → ++ −B2N ( ) N e2 4 ) having rates that scale quadratically with the reactant number density.

Insofar as germicidal and wound healing applications are concerned, therefore, dense arrays of low temperature micro-plasma jets are preferable to a single jet of larger diameter because of: (1) the scaling of the plasma column surface area-to-volume ratio as the cross-sectional dimensions of the jet are reduced, and (2) the gas phase chemical synergies that are available. Over the past 17 years, microcavity plasmas [6] have been shown to embody characteristics quite unlike their macroscopic counterparts, particularly with respect to their operating pressure range, abnormal glow behaviour, and the electron densities obtainable. Fabrication processes developed at the University of Illinois have resulted in precise control of the device and array structure, as well as the realization of arrays of jets as large as 11  ×  11 (121 jets) with a packing density above 150 cm-2. No barrier to scaling the cross- sectional area of arrays appears to exist at present, and values of the plasma jet pitch (centre-to-centre spacing) and diameter as small as 1.0 mm and 355  ±  10 µm, respectively, have been achieved to date [5, 7].

We report here the first clinical evaluation of arrays of microplasma jets for accelerating the healing of any wound, and acute epidermal wounds, specifically. Artificial wounds produced in the skin tissue of Sprague-Dawley rats have been treated for 20–40 s per day with a 6  ×  6 array of microplasma jets generated in He feedstock gas. Despite the intentionally short treatment times adopted for the protocol described here, the wound area was reproducibly reduced to 9  ±  2% of its original value with only two, 20 s treatments of the wound each day for one week. Details concerning the experiments, the histological assessment of the array’s impact, and the significance of these results, as well as their implications for other therapeutic applications of microplasma jet arrays, will be discussed in the sections to follow.

2. Experimental methods and analysis

2.1. Arrays of microplasma jet devices fabricated in Al/Al2O3 layered structures

Arrays of microplasma jet devices as large as 6   ×  6 have been fabricated in nanoporous alumina (Al2O3). Comprising hexagonal pores with controllable dimensions, nanoporous alumina was grown by electrochemical processing of Al foil in oxalic acid. Several years of developing this process have yielded precise control of the alumina quality, and its thickness, through the composition and temperature of the oxalic acid solution, processing time, and the electrical cur-rent. Following the growth of the oxide to a thickness of typically ~70 µm, microcavities having the form of truncated paraboloids [8, 9] are produced within the oxide by a single photolithographic step and a micropowder ablation process sequence similar to that described recently for glass substrates

Figure 1. Structural details for the microplasma jet arrays employed in this study: (a) diagram in cross-section of a single jet device, comprising two truncated paraboloidal cavities in an hourglass configuration. Notice that the orifice diameter is 300 µm and the exit aperture is 500 µm in diameter; (b) SEM recorded in plan view of a 5  ×  4 segment of a 6  ×  6 array of microcavity plasma devices having the double parabolic structure; (c) optical micrograph of a 6  ×  6 array of microplasma jets, viewed at an estimated angle of 40◦ with respect to the surface normal. The jets are produced in He at a flow rate of 6 slm and are propagating to the left in room air.

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[10]. As illustrated in figure 1, bonding of two paraboloidal microcavities back to back results in an hourglass structure that has recently also been developed for microthruster appli-cations [11]. For the experiments reported here, an orifice 300  ±  10 µm in diameter is defined by the smaller aperture of each truncated paraboloid. The larger aperture is 500  ±  10 µm in diameter, and Al electrodes are buried in the wall of each paraboloid by an electrochemical process. Surrounding the paraboloid, each electrode is 20 µm in thickness and its inner face is flared and recessed from the cavity wall by 30 µm of nanoporous alumina. In each row of the array, the device pitch (centre-to-centre spacing) is 700 µm. Therefore, the separation between individual devices is 200 µm as measured at the out-ermost apertures (perimeters) of the paraboloids. It is clear from figure 1(a) that, although the fabrication process begins with Al foil 127 µm in thickness, virtually all of the aluminum is eventually converted into Al2O3. Figure 1(b) is a scanning electron micrograph (SEM) in plan view of a 5  ×  4 portion of a 6  ×  6 array of the microplasma jet devices.

A short section  of glass tubing, flared at one end, pro-vided the transition from the gas flow system to the micro-plasma jet array. Research grade He served as the feedstock gas throughout these experiments and the flow rate was maintained in the range of 1.5–6 standard litres per minute (slm). Further details regarding the device fabrication process sequence can be found in [12]. The finished arrays are light-weight and flexible, thus permitting (if desired) the substrate to be shaped so as to conform the array to the contour of a surface to be treated. Throughout these experiments, the array surface was flat.

2.2. Animal study protocol

In order to assess the potential of microplasma array treat-ment for wound healing, 12 adult male Sprague-Dawley rats, each weighing between 250 and 300 g, served as the animal model for the experiments. After anesthetizing the animal with zoletil (virbac, 300 mg kg−1, i.p.) and confirming full anesthetization, the back of the rat was shaved and sterilized with povidone solution. Three square wounds, 1 cm2 in area, were then made with a dermatome by adopting the procedure of Suzuki et al [12]. The epidermis and a portion of the dermis were removed in this procedure, and the wound closest to the head of the animal was designated as the control. No treat-ment was applied to the control but the other two wounds were subjected to 10 s and 20 s exposures of the microplasma jet array, and are denoted as P10 and P20, respectively, in figure 2. The twice daily treatments were separated by 8 h, and the distance between the array and the surface of the tissue was fixed at 3 mm for all treatments. With the He flow rate of 1.5 slm per microcavity, the length of the visible plume associated with every jet in the array was ~2 mm. Most of the experimental data presented here were obtained with a He flow rate of 0.35 slm/microcavity or less than 14 slm overall. It should also be noted that only a gauze dressing was applied to the wounds during these tests. Blood crusts were not removed from the wounds because doing so induces a secondary injury to the wound.

The extent of the unhealed tissue surface was determined 1, 3 and 7 d after wound formation by imaging each square (con-trol, P10, and P20) with a charge-coupled device (CCD) camera and analysing the images with INNERVIEW 2.0 software. On each of the test days (days 1, 3 and 7), three rats were termi-nated and the harvested skin specimens were fixed in a 10% buffered formalin solution. After embedding each specimen in a paraffin block and preparing a thin tissue section, these sam-ples were stained with hematoxylin and eosin (H and E) and assessed histologically with an optical microscope. This experi-mental animal study protocol was approved by the institutional review board of Hallym University, Chuncheon, Korea.

2.3. Spectroscopic and electrical measurements

Spectra of the emission produced by the microplasma jets expanding into room air were recorded in the 250–800 nm region with a collimating lens and a 0.75 m spectrom-eter equipped with an 1800 lines mm−1 blazed holographic grating. For a slit width of 30 µm, the estimated resolution of the spectrometer is 1.2  ×  10−2 nm to first order, and because the fluorescence was observed end-on to the jets, the spectra to be presented in the next section should be regarded as being spatially averaged over the entire array.

During the microplasma treatment tests, the voltage and cur-rent waveforms for the 6  ×  6 array were monitored while the microplasma device arrays were driven with a 20 kHz sinusoidal voltage having an RMS magnitude of 190–374 V. Also, the temporal history of the microplasma jet fluorescence in room air was recorded with a photodiode prior to the animal model experiments. Figure 3 presents waveforms that are representa-tive of those observed throughout the experiments. Owing to the capacitive nature of the microcavity plasma device design, the conduction component of the total current (red trace, figure 3) is masked by the dominant displacement current. Fort this reason, fluorescence waveforms provide an unambiguous measure of the discharge current, and the blue curve of figure 3 shows the current pulse produced during the positive half-cycle of the driving voltage waveform to have a width (FWHM) of ~1 µs. During the negative half-cycle, however, the current pulse dis-plays considerably less structure and broadens (relative to the

Figure 2. Photograph of an animal model immediately after the formation of the control wound (left square). The locations of P10 (centre) and P20 (right) are indicated as black squares.

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positive half-cycle waveform) to ~8.5 µs FWHM. The duty cycle for this dielectric barrier device structure is, therefore, almost 20%, and the power consumed per microcavity is esti-mated to be approximately 40 mW.

3. Data and discussion

3.1. Wound recovery measurement and spectral data

The data acquired in these experiments demonstrate con-sistently that microplasma array treatment of animal tissue accelerates wound healing. Figure 4(a) is a photograph of the control (C), P10 and P20 wounds (in one animal) after 7 d of microplasma treatment. This image and all optical inspections of the artificial wounds during the one week trial indicate that the control heals more slowly than the plasma-treated tissue. Three days after wound formation, for example, considerable tissue damage was still observed in the control. Figure 4(b) summarizes the results of measurements of the wound surface area at days 1, 3 and 7. At the end of a set of experiments—7 d after the wounds were produced—the area of artificial wounds P10 and P20 had decreased to 27  ±  3% and 9  ±  2%, respec-tively, of their initial values. Since the square of the P10 nor-malized wound area at 7 d (27%) is 7.3%, a value consistent with the P20 value of 9   ±  2%, one concludes that the rate of shrinkage of the tissue wound area is linear in the total microplasma treatment time. It should also be noted that the introduction of the microplasma array results in the wounds healing in a more spatially uniform manner, despite the fact that the combined cross-sectional area of the jets (~7 mm2) is less than 3% of the array cross-section and 7% of the wound area. Furthermore, the array was fixed in position during the treatments and was not rastered (or scanned in any way) over the wound.

Histologic evaluations of the tissue samples confirmed the conclusion that the wounds treated with the microplasma array recovered more quickly than the control wounds. Images of

tissue samples prepared after 3 and 7 d of treatment are pre-sented in the left and right columns, respectively, of figure 5. During the treatment period, the control was fully covered by blood clots, which are absent from the P10 and P20 sam-ples at day 7. Furthermore, no damage to the subepithelium regions of P10 and P20 is detectable at the microscopic level. In fact, no histologic differences were observed between the P10 and P20 tissue samples. The only distinction between the two is the wound recovery time. These conclusions are also consistent with the observation that none of the animals exhibited signs of skin anomalies such as erythema, necrosis, or swelling.

In an effort to monitor the reactive species environment produced by the jet array in the vicinity of the tissue wound, optical emission spectra were recorded with the array oper-ating at an RMS voltage of 310 V, 0.35 slm of He flowing through each microcavity (<13 slm for the entire array), and an overall detection system resolution of less than 0.5 Å. Figure 6 is a panoramic view of the fluorescence spectrum, observed end-on to the array of jets, in the 250–800 nm region. Although laser-induced fluorescence measurements will be necessary ultimately to identify the non-radiating species (such as the metastable A Σ+3

u state of N2, and NO in its ground state) and

Figure 3. Voltage (V), current (I) and spontaneous emission (photodiode) waveforms representative of those observed throughout the experiments. The current waveform (red trace) includes the displacement current which partially obscures the contribution of the conduction current.

Figure 4. Observations of wound healing after exposure of rat tissue to microplasma jet arrays: (a) photograph of the control (C), P10 and P20 wounds after 7 d of microplasma treatment (10 or 20 s of exposure, twice daily); (b) data illustrating the acceleration of wound healing by exposure of animal model tissue to He microplasma expanding in room air. The error bars represent ±1σ for the measurements made at days 1, 3 and 7.

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determine their relative abundances, the spontaneous emis-sion spectrum of figure 6 shows the ultraviolet to near-infrared region to be dominated by atomic emission from oxygen at λ~777 nm (3p 5P → 3s 5S transition) and violet/blue fluores-cence from the nitrogen dimer ion ( Σ → Σ+ +B X2

u2

g transition at 391.4 and 427.8 nm). The latter is produced by Penning ionization of N2 by the He (2s 3S) metastable species, and N2

+ generation is followed by recombination of the ion (or N4

+, formed by the associative, two-body collision mentioned ear-lier) and the population of long-lived triplet states of the mol-ecule. In contrast to intense fluorescence observed from N2

+, only weak emission is produced by the OH (A 2Σ+ → X 2Π) transition at 308 nm and the nitrogen second positive system (C → B) at 337.1, 357.7 and 380.5 nm.

3.2. Discussion of results, relation to prior work

Several recent studies have examined the influence of low temperature, atmospheric pressure plasmas on wound healing [4, 13–17]. Experiments evaluating donor site recovery in skin grafts found that re-epithelialization was improved signifi-cantly in the plasma-treated wound area. Similar results were reported for the treatment of chronic wounds. In particular, the healing of venous ulcers was promoted by the applica-tion of Ar plasma to the affected tissue [15]. Another prom-ising aspect of the treatment of tissue with low temperature plasma is that wound healing does not come at the expense of tissue damage by the plasma. On the contrary, the histo-logical studies described in section 3.1 found no differences between the P10 and P20 wounds except for wound recovery

time, which varies inversely with the total time the tissue is exposed to plasma. No signs of sub-epithelial damage were evident. Such results are consistent with the report of the treatment of human cadaver tissue with plasma generated by a macroscopic dielectric barrier discharge (DBD) [3]. Exposure of the tissue for time periods up to 5 min did not result in any observable macroscopic or microscopic alterations. Studies of ex vivo human skin exposed to atmospheric plasma for up to 2 min yielded the same result.

Although previous studies, as well as the present data, point to the ability of low-temperature plasma to accelerate wound healing, the mechanisms and species responsible have not been identified unambiguously. One factor in expe-diting tissue healing appears to be the bactericidal proper-ties of low-temperature plasma [2, 5]. Infections are known to impair wound healing [18, 19] and, therefore, reducing the bacterial load in wounds is expected to be conducive to enhancing the recovery rate. Other studies have interpreted the beneficial impact of plasma treatment on the wound healing process in terms of enhanced cytokine levels. Upon exposing fibroblasts to low-temperature plasma, an accel-erated expression of collagen type I, alpha-SMA, TGF-β1 and TGF-β2 was observed [20]. Further clues regarding the wound healing mechanisms promoted by microplasmas are provided by recent experiments [21] in which bovine serum albumin (BSA) samples were exposed to microplasma arrays. Probing the treated surfaces with x-ray photoelectron spectrometry (XPS) found that plasma treatment is effective in the removal of protein. Desmet et al concluded that, in addition to protein ablation, the He microplasmas induced

Figure 5. Histologic images of tissue samples acquired 3 and 7 d (left and right columns, respectively) following wound formation. On the seventh day, blood clots still cover the surface of the control.

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surface oxidation and cross-linking in the samples. Each of these is presumably a factor in the accelerated wound healing process described earlier.

With regard to the atomic and molecular species driving these effects, previous experiments with atmospheric pres-sure plasma jets have shown that atomic oxygen is generated preferentially when the plasma interacts with air [7], and it is well known that oxygen atoms play an important role in both biomedical applications and physiological responses [22]. Combined with the strength of the relative atomic oxygen emission evident in figure 6, these considerations sug-gest that plasma jet production of oxygen atoms is another factor contributing to the improvement of the efficacy of the treatment described here. From a broader perspective, other reactive oxygen and/or nitrogen-bearing radicals produced by the plasma jets have been identified as likely candidates for promoting the healing of tissue. The NO molecule, for example, is known to stimulate regenerative processes and the specific contributions of the radical to the wound healing process potentially include vasodilation and stabilization of microcirculation, enhancement of bacterial phagocytosis and nerve conductance, vascular growth by secretion of cytokines, the proliferation of fibroblast and keratinocytes, and increased collagen production [2].

Of particular significance in the present studies, however, is the small cross-sectional area of the jets, relative to the wound area. The wound area and histological results of figures  4 and 5, respectively, confirm the conclusions of [5] that the species beneficial to plasma jet therapies are not delivered to the tissue primarily along the jet axis but rather diffuse radi-ally outward from the plasma and ultimately to the biolog-ical sample. This scenario is analogous to that of the indirect dielectric barrier discharge (DBD) configuration reviewed by Graves [2] but has the advantage that the jets, and thus

the source of the radicals and excited species, can extend to the tissue surface (if desired). A final comment regarding the species responsible for the acceleration of wound healing is that the therapeutic effect is generally attributed to radicals and polyatomics, all of which are in their ground electronic states. Often overlooked is the potential role of long-lived (metastable) states of diatomics, such as the Σ+A3

u state of N2 that lies at 6.2 eV and has a radiative lifetime of 1.4–2.7 s [23]. Having estimated (time-averaged) number densities of ~1014 cm−3, such energetic long-lived species are capable not only of reaching the tissue surface but also of inactivating bacteria and promoting the formation of species such as NO. Known as a signalling molecule, NO is one of the few mem-bers of the reactive oxygen–nitrogen species (RONS) family to be effective in several therapeutic applications, including the treatment of pulmonary hypertension [2].

4. Conclusions

Clinical studies have been reported in which acute skin wounds in Sprague-Dawley rats were treated with low-temperature plasma generated by an array of microcavity jet devices. Arrays comprising 36 microcavities (in a 6  ×  6 configuration), having an hourglass cross-sectional geometry, produce diffuse plasma jets in air with a diameter of ~500 µm and exhibiting a visible plasma plume ~2 mm in length when the He feed-stock gas flow rate is 1.5 slm. Exposing acute tissue wounds to the plasma for time periods of up to 40 s per day clearly accelerates healing of the wounds. After 7 d of two, 20 s expo-sures per day to the microplasma array, rat tissue wounds are reduced in area to 9  ±  2% of that for the original wound. The corresponding value for the control wound was found to be 24  ±  5%. Histological images confirmed significant plasma-induced regeneration of the epidermis and underlying dermis, but no signs of tissue damage by the plasma were detected.

One limitation of the present study is that the locations of the animal wounds were not randomized. Rather, a cephalic wound served as the control in all experiments. Furthermore, no standardized measurement for plasma-assisted wound healing exists, and our assessment of wound healing efficacy is linked primarily to the measured surface area of the wound. Nevertheless, the data demonstrate consistently that exposure of the wound to the plasma array promotes tissue healing. Although the total cross-sectional area of the jets is only 7% of the wound area, and the array was not rastered over the wound during treatment, the introduction of the microplasma arrays promotes spatially uniform healing of the wound. This result is attributed to the increased surface area of the jet array, rela-tive to a single jet, and the concomitant enhancement in the production of molecular radicals and long-lived, electronically excited species.

Acknowledgements

This work was supported by Hallym University Research Fund and the National Research Foundation of Korea (NRF) funded

Figure 6. Spontaneous emission spectrum produced by a 6  ×  6 array of microplasma jets and recorded over the 250–800 nm spectral region. The measured resolution for the detection system is less than 0.5 Å and the He flow rate though each hourglass microcavity was 0.35 slm.

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by the Ministry of Science, ICT & Future Planning (no NRF-2013R1A1A2074849). The Illinois portion of this work was supported by the National Science Foundation and the US Department of Energy under grant no. DE-SC0008333.

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