Microplasma radio frequency technology using stationary tips on pig skin: A histological study

To investigate the histological properties of microplasma radiofrequency (MPRF) using a stationary tip in different treatment strategies on porcine skin.


| INTRODUC TI ON
Microplasma radiofrequency (MPRF), as a fractional ablative energybase device, has a definite effect in improving superficial atrophic acne scars and pigmentation. 1MPRF is equipped with two types of treatment heads: roller tip and stationary tips.The stationary tip is divided into with and without socket tips (Figure.1).The comparison between the two is detailly shown in Table 1.Among which, the area of the stationary tip with non-socket tip (STNS) is smaller, and the area of the stationary tip with socket tip (STS) is larger.The roller tip is commonly used to treat mild atrophic and mild hypertrophic scars with large area coverage, while the stationary tip is often used as a strengthening treatment for mild hypertrophic scars or atrophic scars.
The safety and efficacy of MPRF have been confirmed to some extent, but the safety threshold of intensive therapy has not been explored, let alone the systematic, basic experimental studies on the histological response of the skin associated with MPRF, which have rarely been reported.Understanding the effect of MPRF on the degree of tissue thermol damage under different parameters is the basis of clinical application for dermatologists and laser physicians.In this study, porcine skin is used as a model by obtaining histopathological images of the interaction between tissues and two kinds of stationary tips of MPRF, analyzing the range of thermal damage and thermal effect in detail, evaluating and comparing the wound healing process, and investigating the difference as well as the influence of different treatment strategies (power, duration, pass) on tissues in the short and long term, which can provide solid evidence for safe and effective clinical treatment.

| Animals and equipment
Two Bama miniature pigs aged 6 months, female, weighing 17-18 kg.MPRF was performed using the Accent XL (Alma Lasers, Caesarea, Israel).The Ethics Committee of the Plastic Surgery Hospital of the Chinese Academy of Medical Sciences approved the study.All experiments were performed in accordance with the approved guidelines.

| Study design
The back was divided into 8 treatment areas.Each treatment area was set as 3 × 12 cm in size, and the distance between each treatment area was 2 cm.The specific treatment parameters of each treatment area are shown in Table 2. Methylene blue was used to pierce the edge for later observation of the treatment area.
Two types of stationary tips were investigated in this experiment: the stationary tip with a non-socket tip (STNS) and the stationary tip with a socket tip (STS).The STNS has a total diameter of 7 mm, 74 needles, and a needle spacing of 0.7 mm.It was used for the treatment of groups 1-5.The STNS has a total diameter of 12 mm, 112 needles, and a needle distance of 1.0 mm for the treatment of 6-8 groups.In the treatment, the STNS is placed 1 mm away from the skin to emit energy, while for the STS, it is gently touched to the skin during the treatment.

Device
The Accent XL (Alma Lasers, Caesarea, Israel) was shaved with a razor, gently cleaned with soap, and disinfected with iodophor and alcohol.The general appearance of the skin was observed immediately, 1 week, 1 month, 3 months, and 6 months after treatment.
Appropriate skin tissue was collected from each treatment area.The incision was closed in two layers using 3-0 sutures.After each operation, 1.6 million units of penicillin sodium were injected intramuscularly for 3 days.The pigs were euthanized at the end of the experiment.

| Histological observation
After the samples were fixed in 4% paraformaldehyde for 24-48 h, the

| General injury observation
The skin appearance of the total eight groups immediately, 1 week, 1 month, 3 months, and 6 months after treatment is shown in Figure 2.
After treatment, the skin in all groups was red and swollen with spot scab.In the STNS groups, the energy setting of group 1 was twice as high compared to group 2, and it can be concluded that the increase in energy led to some exacerbation of the trauma.However, there was no significant difference in the immediate response and the recovery of the scab after 1 week.After tripling the energy (group 5), the tissue trauma only showed more scabs, but the scab shape and wound healing time did not increase significantly.In groups 1, 2, and 5, the scab had essentially fallen off and healed 1 month after treatment, but a small amount of erythema was still present.Healing was complete in 3 to 6 months, with no residual color remaining.Comparison between groups 2 and 3 (increasing the number of treatment passes) and group 4 (increasing the pulse width) showed that the skin wounds caused by these two groups were significantly more severe than those caused by simply increasing the energy.The skin wounds in group 2 showed only thin scabs after 1 week and were essentially healed after 1 month, while thick black scabs formed in groups 3 and 4. The scab had fused over a large area and practically fell off after 3 months.After 6 months, there was a scarring change in the skin texture.
In the STS groups, compared to group 6, there was no significant difference in the immediate erythema response with the increase of pulse width (group 7) or the energy (group 8).The scab basically fell off after 1 month.However, one of the pigs in group 7 was left with a white scar after 6 months.

| Histological staining observation
Comparison of skin HE staining between groups at different time points after treatment was shown in Figure 3 laser, 3 electron beam irradiation, 4 and photodynamic therapy 5 ) for the enhancement of hypertrophic scars.Some believe that plasma forms a non-evaporative microablation on the skin surface while maintaining the epidermis as a natural dressing to promote skin healing and repair, and that exfoliation eventually lasts several days. 6wever, in this experiment, it was observed that the tissue was damaged after treatment with HE staining as a tissue defect, which was similar to the damage of an ablative CO 2 laser.
Currently, MPRF technology has been widely used and has proven effective in improving acne scars, pigmentation, periocular wrinkles and photoaging. 1,7MPRF also has the effect of skin rejuvenation and is a good substitute for fractional ablative CO 2 laser.
First, plasma is a non-color-based energy source, so it is of high clinical value for Asians with high Fitzpatrick skin typing.In addition, compared with CO 2 laser, MPRF is safer, the duration of erythematosus and downtime after treatment is shorter, and the incidence of pigmentary abnormalities is lower. 8,9From the point of view of histological evidence, the ablation column produced by MPRF is wider but shallower.And the area of thermal damage is smaller but wider, which promotes wound healing and tissue re-epithelialization and reduces the inflammatory response and the incidence of postinflammatory pigmentation. 10e MPRF was usually equipped with three treatment heads: the roller tip, the stationary tip with and without socket tip.The roller tip is a rolling type of treatment, while the stationary tip is a stamping type.We have done research on the roller tip previously, 11 so this experiment is mainly based on the direction of the stationary tip.At the same time, we chose pigs as subjects mainly because the skin structure of pigs is very similar to that of humans, and the skin response and changes after treatment can also be compared with human skin.In addition, the skin area of the pig's back is large, so the changes in skin appearance can be clearly observed, and the need for multiple treatment groups and pathological biopsies is met.
In this study, by observing the skin wound response, healing process, collagen increase, and heat shock protein expression at different time points after treatment, the injury characteristics and healing process of the two treated heads can be described as follows.

| Skin trauma after MPRF
In the STNS group, a small area of vaporization damage formed due to flake discharge, with a large defect depth and slow repair rate.The scab also fell off slowly, and the collagen hyperplasia reaction and HSP expression were stronger than in the STS group.
After double-pass treatment in group 3 and prolonged treatment in group 4, the skin reaction was excessively enhanced, healing was delayed, and even the skin texture had changed.This brings us to an important point; because of the severe tissue trauma, the stationary tip is only suitable for local treatment.It should not be used for large-scale treatment to avoid excessive skin trauma, delayed healing, and even permanent scarring.In particular, doublepass treatment significantly aggravated the skin trauma, resulting in a change in skin texture, so repeated treatment at the same site should be avoided.
When treating with STS, the tip is close to the skin.There is basically no plasma phenomenon visible to the naked eye.We observed significant ring erythema and delayed healing with pulse-width increase alone (group 7), which has more severe trauma than energy increase alone (group 8).The histological observation revealed that there was a small spot gasification injury in addition to the thermal effect in HE staining (immediate reaction of group 8 in Figure 2).We think that the slight curvature of the treatment tip resulted in incomplete contact between the outer ring of the treatment head and the skin, creating a small gap where weak plasma could still be generated, albeit undetectable by visual inspection.The circular scab on the skin tissue immediately after treatment also confirmed this point.

| Changes of collagen and expression of heat shock protein after MPRF
The dynamic changes in heat shock proteins may represent cellular metabolism and collagen regulation to some extent.As a precollagen companion, it can promote fibroblast formation when upregulated while reducing oxidative damage and inflammation and playing an important role in long-term wound healing.HSP72/74 is expressed at low levels in the physiological state. 12It was weakly positive in the superficial dermis immediately after the therapy.In the early phase, it was restricted to the adjacent thermal area and gradually distributed to the superficial middle layer of the dermis in the later phase.
Masson staining showed a parallel increase in collagen, indicating that the collagen changes and remodeling caused by the treatment were extensive.
In this study, we compared the histological differences between treatments with different power, duration, and pass.Increasing treatment duration and repeated treatment were significantly worse for trauma than increasing therapeutic power.Therefore, to increase the intensity of the treatment, the power should be increased in clinical application instead of increasing the duration of the treatment.
From the perspective of tissue healing, the appropriate power of STNS is 40-80 W, and the duration is about 0.4 ms.The thermal effect of STS on tissue is obvious, so low energy and short duration are recommended.The suitable power is less than 40 W, and the duration is less than 0.2 ms.And both of the two tips should avoid repeated passes.
This study also has some limitations.The total power is not fully controlled in the design of parameters.Since this study is mainly a preliminary investigation based on clinical needs, appropriate adjustments are made within the range of clinical parameters.

| CON CLUS ION
Microplasma radiofrequency can cause epidermal reconstruction and an increase in dermal collagen and remodeling.It is suitable for the treatment of scars and for skin rejuvenation.To ensure the effectiveness and safety of the treatment, different treatment tips and appropriate treatment parameters should be used, depending on the clinical conditions.If it is necessary to increase the treatment intensity, it is better to increase the power rather than increase the treatment duration or repeat the treatment to avoid excessive trauma to the skin.
Pigs were generally anesthetized by intramuscular injection of 0.5 mg/kg tiletamine and zolazepam (Zoletil 50, VIRBAC, FR) with atropine (0.05 mg/kg) before each treatment or sampling.Then the back F I G U R E 1 The stationary tip of MPRF.(A) The stationary tip with non-socket tip (STNS).(B) The stationary tip with socket tip (STS).(C) Treatment handle.TA B L E 1 Comparison of the stationary tip with non-socket tip (STNS) and the stationary tip with socket tip (STS).
tissues were embedded in paraffin and sliced.Hematoxylin and eosin (HE), as well as Masson's three-color staining, were used to examine the changes in skin histopathology and the distribution and arrangement of collagen fibers.the Immunohistochemical staining of heat shock protein 47 (HSP47, Jackson, US) and heat shock protein 72 (HSP72, Jackson, US) was performed and visualized by incubating sections for 3-5 minutes in DAB chromogen.Sections were measured and photographed under an optical microscope (DM3000, LEICA, Germany) at 50× magnification to evaluate the thermal damage and wound repair of the skin.

2423 FENG
Skin appearance in each group at each time after MPRF.The red arrow indicates the remaining white scar in group 7 at 6 months.|et al.

F I G U R E 3
, and Masson staining was shown in Figure 4A.Immediately after treatment, a scaly ablative injury was observed at the dermis-epidermis junction in all treatment groups.In the damaged basement membrane zone of the epidermis, there were many wedge-shaped injury sites with irregular depth.The depth of the tissue defect and the depth of thermal injury were measured immediately after treatment.Under different parameter settings, the depth of tissue defect in STNS groups was 70-200 μm and the depth of thermal injury was 150-1400 μm.The depth of tissue defect in the STS treatment group was 100-200 μm, and the depth of thermal injury was 200-800 μm.One week after treatment, epidermal re-epithelialization was complete in groups 1 and 2 of the STNS group and in all STS groups.One month after treatment, the number of fibroblasts had increased near the superficial layer of the dermis.At 3 months after treatment, the number of dermal fibroblasts in groups 1 and 2 of the STNS group and in all STS groups had essentially returned to normal, with a regular arrangement of collagen fibers.But groups 3, 4, and 5 of the STNS group basically recovered only 6 months after treatment.Positive signals of the immunohistochemical staining were brown or brownish yellow in color.Almost no expression of HSP47 or HSP 72 was found in normal tissue, but expression appeared in the dermis of each group immediately after the session, albeit sparsely.Brown-yellow particles increased on day 7 and were concentrated mainly in the superficial dermis near the area of thermal injury.Expression peaked at month 1 and showed a diffuse distribution.Expression began to decrease after 3 months, but 6 months after treatment, the HSPs expression was still higher than baseline in all groups, especially in the STNS groups, as shown in Figure 4B.Using the same treatment head, the response was enhanced with an increase in treatment intensity and duration.Comparison of skin in Hematoxylin and eosin staining in each group at different time after MPRF.Scale bar = 200 μm.F I G U R E 4 Masson staining and HSP47/72 immunohistochemical staining among groups at different time periods after MPRF.(A) Masson staining for group 5 and 7. Scale bar = 200 μm.(B) Immunohistochemical staining of HSP47 for group 4 and 7 (left), and the immunohistochemical staining of HSP72 for group 2 and 8 (right).Scale bar = 300 μm.The principle of MPRF is to combine RF energy with plasma technology and excite nitrogen in the air to generate a high-energy plasma using micro-unipolar RF.Due to the high conductivity of the plasma current, microsparks can be triggered to create multiple and controllable RF plasma microdischarges between the treatment head and the skin tissue.The effect of the plasma on the skin is mainly microablation and thermal effect.2Due to the ultra-high temperature effect of the plasma, the heat output to the surrounding tissue is low while the tissue is vaporized and ablated to avoid excessive damage to the surrounding tissue.Microablation can create a hot channel in the dermis.Due to the dual benefits of RF and plasma, it can further enhance the therapeutic thermal effect, promote collagen regeneration and restructuring, and skin regeneration and repair.The MPRF is currently employed in clinical combination therapy (including CO 2