Evaluation of Skin Tissue Effects of Two-Pass Helium Plasma Treatment in Comparison to Nitrogen Plasma Using a Swine Model

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Introduction
The pursuit of smooth, youthful skin with even texture and tone is common in aesthetic practice. Numerous modalities through time have been used to achieve this end including chemical peeling, dermabrasion, lasers, radiofrequency and plasma sources. Each procedure has a unique interaction with the skin to achieve the desired result. Within the skin, the dermis contains well-organized collagen fibers that are responsible for the firmness and smoothness of the skin. As people age, collagen fibers become reduced in numbers and less organized, resulting in lax and wrinkled skin.
In most skin resurfacing procedures, an energy source is used to selectively damage the skin, prompting a healing response that stimulates the growth of new, well-organized collagen fibers in the dermis resulting in smoother and firmer skin.
Physical plasma is an ionized gas, commonly referred to as the fourth state of matter to distinguish it from solids, liquids, and gases.
Plasma is generated by applying a continuous supply of energy to a neutral gas until an excited state is achieved. The energy source needed to create plasma may be thermal, chemical or electrical in nature [1]. Based on its thermal properties, the role of plasma in medicine has expanded to include a wide range of applications including cancer [2], wound healing [3], infectious disease [4], and dentistry [5]. Plasma is also being used with increasing frequency in dermatology [6].
A nitrogen-based plasma device (NeoGen PSR System; Energist Medical Group formerly Rhytec, Swansea, United Kingdom) has FDA clearance for the treatment of dermatologic conditions including wrinkles and rhytides [7]. The system converts nitrogen gas into plasma energy within the tip of the handpiece which results in the expulsion of controlled pulses of energy to the tissue. The energy is not chromophore dependent and does not vaporize tissue [8,9]. The upper layers of the skin are desiccated during treatment and remain intact to serve as a biological dressing during the initial stages of healing [8,9]. The technology has demonstrated efficacy for the treatment of mild-to-moderate periorbital wrinkles and photodamaged facial skin with reported complications typical for resurfacing procedures such as postinflammatory hyperpigmentation, focal areas of delayed healing, and rare instances of hypertrophic scarring [8][9][10][11][12][13]. to the nitrogen plasma system in a porcine model [15]. The results of this pre-clinical study were then translated into a prospective, 55-patient FDA investigational device exemption (IDE) clinical study in which subjects treated with a single pass from the helium device achieved significant improvements in facial appearance with rapid recovery and relatively few unanticipated adverse events [16]. Successful clinical use has also been reported outside of a controlled clinical study [17,18].
The objective of this pre-clinical study was to evaluate the tissue effects of a second treatment pass of the helium plasma device using a naïve swine model. The tissue effects were compared to those of the established predicate nitrogen plasma device to support an assessment of the safety profile of two treatment passes prior to inclusion in additional clinical studies.

Methods
This study was performed in compliance with the US Public Treatment delivery for the helium plasma system occurred as linear/non-overlapping strokes with both single pass and twopass protocols using a treatment speed of approximately 1 cm/ sec. Although the primary focus of this study was the evaluation of the tissue effects caused by the two-pass protocol, the single pass treatment was included to allow for comparison to the data previously reported by Holcomb and Schucker [15]. Device settings for the helium plasma system consisted of 20% Power (energy density 8.6 J/cm 2 ) and 40% power (energy density 17.8 J/cm 2 ) with 4 lpm of helium flow. For the treatment protocols requiring two treatment passes, a saline soaked gauze was used to wipe the desiccated tissue between the first and second pass and the surface was dried of any moisture. Treatment using the nitrogen plasma system was performed using the highest energy (4.0J, 2.5 Hz pulse rate and energy density 14.1 J/cm 2 ) double pass (PSR3) treatment protocol to establish the maximum treatment depth in clinical use for the device.
The spacer available for the system was used to ensure consistent distance of the treatment nozzle from the tissue, and proper spacing of the energy pulses was maintained to prevent overtreatment due to overlapping of treatment spots. Table 1 summarizes the treatment settings used for both devices. Six sites were treated for each of the five device settings tested, and six untreated sites were used as controls to provide a baseline for epidermal and dermal thicknesses for comparison when performing the histomorphometric evaluation.
Following treatment, skin tissue samples were harvested.

Results
The maximum of the three measurements taken for each treatment location are reported in Table 1 Increasing the number of passes of helium plasma treatment from one to two led to an increase in the maximum depth of thermal effect. Although there was a slight trend of higher maximum depth for both the 20% and 40% two-pass helium treatments when compared to the nitrogen plasma, the results were not statistically different.  have an acceptable safety profile. Recently, helium plasma has been studied in both pre-clinical and prospective clinical studies using a low energy, single pass treatment protocol [15,16]. The results of these studies indicated that the helium plasma device has the potential for effective, safe treatment of facial rhytidosis. Treated subjects achieved significant improvement in facial appearance, relatively few adverse events, and had high overall satisfaction with the procedure and aesthetic results [16].

Conclusion
Helium-based plasma systems are relatively new in the aesthetic toolbox. Surface application of helium plasma energy may be a promising way to achieve dermal resurfacing. The results of this study indicated that there are not significant differences in the depth of thermal effect associated with a two-pass helium plasma treatment when compared to the depth of thermal effect associated with nitrogen-based plasma skin resurfacing systems. These results combined with other data provide reasonable assurance that a two-pass helium plasma treatment protocol can be used safely for certain skin types, facial regions, and/or clinical conditions. Further basic science and clinical research is warranted. A formal clinical study evaluating the safety and efficacy of the helium plasma device for dermal resurfacing is ongoing.