Randomized Trial of Cold Plasma and Vitamin C Synergy: Effects on Skin Hydration and Wrinkle Reduction

Randomized Trial of Cold Plasma and Vitamin C Synergy: Effects on Skin Hydration and Wrinkle Reduction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Randomized Trial of Cold Plasma and Vitamin C Synergy: Effects on Skin Hydration and Wrinkle Reduction Najmeh Eskandari, Farzane Ostovarpour, Mohammad Ali Nilfrosh Zadeh, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5237210/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract As the skin ages, it naturally becomes drier and loses elasticity, resulting in wrinkles. Over time, the production of collagen and elastin in the skin decreases. Several methods exist for reducing skin wrinkles, including cold plasma technology. Plasma is an advanced treatment employed for skin rejuvenation and aesthetic enhancement. This technique can enhance skin appearance by boosting collagen and elastin production, thereby diminishing wrinkles. This study utilizes a FEDBD device for hand skin rejuvenation. The plasma treatment was administered over 8 sessions spanning 8 weeks. Skin analyses and laboratory tests such as elasticity, oxygenation, perfusion, tissue water index, and hemoglobin were performed before the plasma treatment, immediately after the treatment, at the fourth and eighth weeks of treatment, and six weeks post-final session. The findings of this study suggest that plasma can assist in cleansing the skin surface through the production of CO and OH. Additionally, an increase in the maintenance of skin hydration and enhanced oxygenation and perfusion was reported. The data reveal that, while vitamin C alone does not significantly improve skin elasticity, combining it with plasma treatments, particularly at 7.4 W, leads to a notable enhancement in skin elasticity over time that proves the synergy effect of cold plasma. The combination therapy is the most effective in improving skin elasticity compared to other treatments. Physical sciences/Physics/Plasma physics Health sciences/Medical research/Pre clinical studies Cold atmospheric pressure plasma FEDBD plasma ambient cold atmospheric plasma skin rejuvenation collagen production Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 1. Introduction Plasma, often referred to as the fourth state of matter [ 1 – 5 ], finds versatile applications across various scientific domains owing to its unique properties. Plasma applications extend beyond materials science, medicine, and astrophysics into environmental remediation, energy production, and agriculture. In environmental science, plasma-based technologies offer efficient air and water purification solutions by degrading pollutants and disinfecting contaminants. Diverse applications underscore the interdisciplinary nature of plasma science and its potential to address pressing challenges across multiple fields [ 6 – 13 ]. In the field of medicine, plasma plays a crucial role in sterilization processes [ 14 ], wound healing therapies [ 15 ], and plasma-based cancer treatments [ 16 ], leveraging its antimicrobial and tissue-regenerative properties. The integumentary system, the most conspicuous and susceptible organ in the human body, experiences anatomical and functional alterations as a consequence of the aging process. Maintaining the integrity of the skin initiates a comprehension of its anatomical, physiological, and functional aspects, along with the recognition of conditions and caregiving factors that might influence its structure and performance [ 17 – 19 ]. Scientific investigations suggest that implementing appropriate skin cleansing and protective strategies and nutritional and environmental interventions can reduce certain aging-related effects and yield positive psychological and health implications [ 20 , 21 ]. The skin, constituting the body's largest organ, spans an average area of around 2 square meters in adults [ 22 ]. The composition of the skin might exhibit quantitative differences contingent upon variables such as location, age, health status, and gender, among other factors [ 23 ]. Moreover, the skin [ 24 , 25 ] is vital in preventing water and electrolyte loss. From an immunological standpoint, the skin functions as a dynamic sensory and excretory organ, playing a role in regulating body temperature. Daily, the skin is subjected to many factors, such as prolonged exposure to excessive sunlight, rigorous cleanliness practices, overly dry environmental conditions, and environmental impacts during leisure and work activities. Consequently, prioritizing skin care is crucial for maintaining optimal health. The aging phenomenon is identified by the build-up of macro-molecular damage, disruptions in tissue regeneration, and the progressive decline in physiological coherence. The aging process [ 26 , 27 ] arises due to a synergistic interplay of internal factors, including hormone levels, genotypes, endocrine gland metabolism, and external factors, such as ultraviolet radiation, nutritional status, chemical pollutants, etc. Reactive oxygen and nitrogen species (RONS), exemplified by hydrogen peroxide, ozone, and nitric oxide, manifest diverse biological effects, motivating researchers to explore the application of Cold Atmospheric Plasma (CAP) in various biomedical contexts. Cold plasma has shown promising results in cancer treatment by selectively inducing apoptosis in cancer cells, decreasing tumor volume, and inhibiting metastasis [ 28 – 30 ]. Nevertheless, FEDBD exhibited significantly superior efficacy compared to all the other apparatuses. FEDBD plasma plays a role in enhancing blood coagulation, indicating that plasma can catalyze intricate biochemical processes involved in blood coagulation [ 31 ]. To revitalize the skin on the hands and face and address clinical manifestations throughout different stages of the pathogenesis of vulgaris acne, characterized by the simultaneous involvement of multiple follicles in the affected area, employing atmospheric cold plasma emerges as a suitable approach [ 32 ]. Charipour et al. employed the FEDBD plasma device for skin rejuvenation in animal models. Their study's primary accomplishment was the preservation of skin elasticity [ 33 ]. The current study evaluated the effectiveness of a floating electrode dielectric barrier discharge (FEDBD) device in rejuvenating the skin on human hands. Active species were identified using optical spectroscopy, and the thermal impact of the plasma was assessed by measuring skin temperature with FLIR. The Elastometer instrument was used to measure the elastic properties of the skin. The gas analyzer was employed to quantify the levels of various gases present in the environment. Besides, this project employed a hyperspectral imaging system to analyze changes in oxygen levels, perfusion, hemoglobin content, and tissue water. 2. Results 2.1 Plasma characterization One of the most effective techniques for identifying active plasma species and examining their properties is spectroscopy. Before initiating the research, the FEDBD plasma was analyzed at power levels of 3.3 and 7.4 W. The observed peaks (Figure 2) in this analysis fell within the 280-420 nanometer range, with the wavelengths and origins of the primary emission peaks in the spectra specified for each. According to the OES analysis, most species present and produced in the FEDBD plasma are active nitrogen species, likely due to the 78% nitrogen content in the air. Other detected species include OH and CO at 295 and 297 nanometers, respectively, N₂ at 313, N 2 and N 337.1, 380.46, and 405 nanometers, N 2 at 315.01 nanometers, and excited N₂⁺ species at 258.08 nanometers, formed through increased collisions with high-energy electrons [38]. The presence of these active species is crucial in dermatological and regenerative medicine due to their impact on skin cell function. Specifically, they enhance the activity of fibroblast cells, which play a key role in maintaining skin structure and integrity. Fibroblasts are responsible for producing collagen, a primary structural protein that provides strength and elasticity to the skin. When these cells are more active, collagen production increases, improving skin firmness and resilience. Additionally, the regeneration of the epidermis, the outermost layer of the skin, is accelerated, promoting faster healing and rejuvenation. This process is particularly important in wound healing, anti-aging treatments, and therapies to improve skin health and appearance. The enhanced activity of fibroblasts and the resulting increase in collagen synthesis and epidermal regeneration underscore the therapeutic potential of these active species in clinical applications [33]. The relative intensity of the species was compared at power levels of 3.3 and 7.4 W. As the power increases, the particles' energy and collisions also increase, leading to greater ionization. Consequently, the emission intensity of the species increases. As previously stated, a thermal camera (FLIR) was employed to monitor the skin temperature during plasma processing to prevent skin damage. Skin temperature readings were taken before and after plasma treatment and 3 and 5 minutes during treatment (Figure 3(a) and (b)). This protocol was executed for both 3.3 W and 7.4 W power levels. As illustrated in Figure 3(c), at a power level of 3.3 W prior to plasma treatment, the temperature was recorded at 31.5°C. For 3 minutes of plasma exposure, the temperature rose by 2.4°C, peaking at 36.6°C after 5 minutes of plasma treatment. Following the treatment's conclusion, a downward temperature trend was observed, with it settling at 32.9°C. This pattern was also evident at a power level of 7.4 W, albeit with slightly higher temperature variations. Initially, before treatment, the temperature was 31.3°C. During 3 minutes of exposure to 7.4 W plasma, the temperature increased by 5.6°C, reaching 36.9°C. Subsequently, after 5 minutes of treatment, the temperature further increased to 38.8°C, eventually decreasing to 32.8°C post-treatment. Exposure of the skin to a temperature of 45 degrees Celsius results in the denaturation of proteins, leading to loss of function and structural alterations, ultimately causing skin burns. Conversely, an elevation in temperature up to around 39 degrees Celsius induces the proliferation of keratinocytes, which can be advantageous in tissue repair and regeneration processes. As observed at power levels of 3.3 and 7.4 W, these temperatures are conducive to stimulating and increasing fibroblast cells. The skin's surface contains particles such as various pollutants, oils, and dust. To quantify these particles pre- and post-plasma processing, the HT-9600 dust meter device was employed. This apparatus features a silent internal pump designed for ambient air suction. The device functions by conducting a 60-second suction initially, followed by displaying the particle count on its monitor in three ranges: 0.3, 2.5, and 10 micrometers, demonstrating the atmospheric particle levels. In both power settings, the particle count in the specified ranges was initially reported as 0 before processing. However, after plasma processing at 3.3 W, the particle count increased to 3,820,000 in the 2.5-micrometer range and 116,000 in the 10-micrometer range. Furthermore, at 7.4 W, the particle counts were recorded at 4,347,000 in the 2.5-micrometer range and 733,000 in the 10-micrometer range. Figure 4(a) illustrates the performance outcomes of the dust meter device before and after plasma processing at power levels of 3.3 and 7.4 W, highlighting superior cleaning efficiency at 7.4 W. The PTM600 series portable composite gas analyzer detects diverse gas concentrations in the surrounding environment. It can identify a mixture of one to six gases and includes a sampling pump. The tube connected to the gas analyzer was positioned on the skin during plasma processing for six minutes. The carbon monoxide production at the end of the process was measured and recorded at power levels of 3.3 and 7.4 W. The carbon monoxide generated by the plasma reacts with particles on the skin, effectively removing oils and various contaminants. CO concentration, measured in ppm, is higher at 7.4 W compared to 3.3 W. This suggests that the higher power level leads to more efficient cleaning of oils from the skin surface. The CO levels generated during the six-minute FEDBD plasma processing were 3.2 ppm at 3.3 W and 6.9 ppm at 7.4 W, which is entirely safe. Low concentrations of CO gas can have beneficial effects on health. In addition to cleansing the skin surface, it can increase blood circulation and oxygen delivery to the skin, stimulating fibroblast proliferation and promoting collagen synthesis. The amount of ozone generated by the FEDBD plasma device at power levels of 3.3 and 7.4 W was measured using an ozone detector. This detector, equipped with a suction pump, calculated the ozone gas concentration. The tube connected to the detector was placed on the skin surface, and different ozone concentrations were recorded by moving the plasma device probe. As the probe neared the tube connected to the detector, the amount of ozone produced by the device increased, while moving the probe away from the tube resulted in a lower ozone level. Ozone production was zero at three centimeters from the target surface. Thus, the gas's effect is confined to the skin surface, posing no respiratory risks and ensuring safety. Ozone gas [39] enhances metabolism, accelerates healing, slow aging, and improves blood circulation and the immune system. Moreover, it has antioxidant effects, reduces inflammation, and stimulates microcirculation. Thanks to its immunological effects, this boosts the tissue's oxygen delivery capacity and enhances regenerative abilities. Therefore, stimulating and proliferating fibroblasts, the cells responsible for collagen, glycosaminoglycans, and proteoglycans production, helps strengthen the cellular matrix's main components responsible for skin integrity [40]. The average ozone concentrations measured at power levels of 3.3 and 7.4 W were reported to be 31.67 and 33.5 ppm, respectively. To examine the results of water absorption, a droplet of water measuring two microliters was applied to the skin, and measurements were taken. This process was repeated after plasma processing for both power levels of 3.3, and 7.4 W. ROS and RNS produced by plasma induce changes in the superficial layer of the skin. Nitrides and nitrates resulting from plasma lead to skin acidification, causing the removal of lipids and existing carbons, thus resulting in skin cleansing. Additionally, bonds with new chemical compounds primarily based on oxygen increase the polar components of the branched surface layer. Besides, UV radiation within plasma prompts the creation of hydroxides and epoxides, the hydrogenation of double bonds, and the fragmentation of carbon chains. As a result, the physical and chemical interactions between plasma components and the branched surface layer result in heightened wettability [41]. A larger contact angle signifies hydrophobicity, while a smaller contact angle indicates greater hydrophilicity and chemical affinity of the surface. As depicted in Figure 4(b), at a power of 7.4 W, the skin surface demonstrates higher hydrophilicity at a power of 3.3 W. In the realm of plasma physics, the electrical characteristics of plasma are significantly influenced by the temporal progression of voltage and current. Typically visualized graphically, this correlation is represented by a curve delineating alterations in voltage and current with respect to time. Comprehending these fluctuations is essential for scrutinizing the dynamic attributes of plasma. The voltage-current relationship offers critical insights into plasma behavior, facilitating researchers in advancing plasma-based technologies and applications across diverse domains. The FEDBD plasma is a type of glow discharge. We will have high current and voltage peaks when the plasma is ignited, so they will not change significantly. However, they will change over time due to voltage fluctuations. The wavefront within the FEDBD plasma apparatus manifests as sinusoidal, representing the periodic oscillations of the electrical field across the discharge gap, thereby leading to periodic alterations in plasma properties. Figures 5(a) and 5(b) illustrate voltage and current temporal fluctuations at power levels of 3.3 and 7.4 W, respectively. 2.2 Skin Analysis Skin elasticity [42] denotes the capability of the skin to stretch and revert to its original form, primarily attributed to the presence of the protein elastin in the dermal layer. Elastin fibers [43] create a network that imparts elastic properties to the skin, enabling it to return to its normal structure after stretching or contracting. To evaluate the Elastometer under various conditions, the variable was measured at five different time points: before treatment, immediately post-treatment, at four and eight weeks during the treatment period, and at the follow-up session, employing the repeated measures method . In the 3.3 W plasma group, skin elasticity increased by 3.13% immediately after treatment compared to pre-treatment levels, while no changes were observed in the control group. Furthermore, in the 3.3 W plasma group, skin elasticity increased by approximately 5% four weeks post-treatment compared to immediately after treatment. In the 3.3 W plasma group, skin elasticity increased by 3% after eight weeks of treatment. Furthermore, skin elasticity decreased by 0.08% in this group six weeks after the final treatment compared to the eighth session. It can be concluded that a significant difference in skin elasticity exists between the control group and the 3.3 W plasma treatment group. In the control group, skin elasticity remained relatively stable or occasionally decreased throughout the treatment sessions. Conversely, in the 3.3 W plasma treatment group, skin elasticity gradually increased and sustained over time as the treatment progressed (Figure 6(a)). In the 7.4 W plasma group, skin elasticity decreased by 5% immediately post-treatment compared to pre-treatment levels, while no changes were observed in the control group. Furthermore, in the 7.4 W plasma group, skin elasticity increased by about 16% four weeks after treatment compared to immediately post-treatment, whereas the control group showed a 2% decrease. The 7.4 W plasma group continued to show an upward trend in skin elasticity after eight weeks of treatment. At the follow-up session (six weeks after the last treatment), skin elasticity decreased by 4% compared to the eighth session, while the control group experienced a 0.7% decrease. In the control group, skin elasticity remained relatively stable before and after treatment but started to decline four weeks post-treatment. Conversely, skin elasticity initially decreased post-treatment in the 7.4 W plasma treatment group but increased over time, with improvements noted at both four and eight weeks post-treatment (Figure 6(b)). In the vitamin C group, skin elasticity increased by 0.3% immediately after treatment compared to pre-treatment levels, whereas the control group showed no change. After four weeks of treatment, skin elasticity in the vitamin C group decreased by approximately 0.8% relative to the immediate post-treatment measurement, while the control group experienced a 2.4% increase. Additionally, skin elasticity increased by 1.3% over the eight-week treatment period. Furthermore, six weeks after the final treatment, skin elasticity decreased compared to both the eighth session and the pre-treatment levels in both the control and vitamin C groups. The data indicate no significant difference in skin elasticity between the control and vitamin C treatment groups. Skin elasticity levels in both groups are nearly identical. Thus, vitamin C treatment alone does not significantly impact skin elasticity statistically (Figure 6(c)). In the plasma treatment group, applying 7.4 W of power along with vitamin C resulted in an immediate 4% reduction in skin elasticity relative to baseline measurements, while no significant change was detected in the control group. After four weeks, skin elasticity in the treatment group increased by 19% compared to the immediate post-treatment measurement, whereas the control group experienced a 4.7% decline. The findings demonstrated that the increase in skin elasticity continued in the plasma plus vitamin C group, with a 9% improvement compared to the fourth session after eight weeks of treatment. In contrast, the control group only showed a 2.3% improvement. Six weeks after the final treatment, skin elasticity in the plasma plus vitamin C group decreased by 1.2% from the eighth session, whereas no notable change was observed in the control group. The results demonstrate that plasma treatment at 7.4 W combined with vitamin C significantly affects skin elasticity. Specifically, skin elasticity in the control group remained almost unchanged throughout all treatment sessions. However, in the group treated with 7.4 W plasma and vitamin C, there was a slight decrease in skin elasticity immediately after treatment, followed by a significant increase over time, with substantial improvements observed at four and eight weeks post-treatment, as well as six weeks after the final session (Figure 6(d)). The data clearly demonstrate that vitamin C treatment behaves differently from the other three treatments, showing no significant effect on skin elasticity. Among the other treatments, the combination of vitamin C and plasma exhibits the greatest impact on skin elasticity over the course of treatment. This is followed by the 7.4 W plasma treatment and the 3.3 W plasma treatment, respectively, in terms of their effects on skin elasticity. When tissue undergoes treatment with cold atmospheric plasma, its perfusion levels increase. Consequently, this rise in perfusion leads to enhanced oxygen delivery to the skin, ultimately resulting in heightened perfusion and oxygenation. This, in turn, fosters increased fibroblast production and collagen synthesis. The stimulating effects of plasma lead to an excessive accumulation of fluid volume within the interstitial tissue, resulting in edema and an increase in the tissue water index. Tissue oxygen supply (StO2) measures the amount of oxygen in the superficial skin layers, reaching a depth of 1 mm. This parameter was evaluated within 530-600 nm and 730-800 nm spectral ranges. The Oxygen delivery provided by FEDBD plasma was examined at different time intervals, and the mean quantities were recorded (Figures 7 and 8). Oxygen delivery at power levels of 3.3 and 7.4 W and plasma at 7.4 W supplemented with vitamin C immediately post-plasma processing resulted in the heightened presence of generated species and elevated temperature. With a power setting of 3.3 W, a 19.28% increase was noted after one hour, followed by a return to nearly baseline levels after 120 minutes of processing. Observations suggested oxygen delivery rose by about 7% one-day post-processing and reverted to the initial state after three days. The average oxygen delivery values are as follows: 38, 44, 44.77, 45.33, 39.33, 40.44, and 39.88, respectively. At a power of 7.4 W, a 26% increase was observed one hour later, until approximately 120 minutes after processing, when the oxygen delivery returned nearly to its initial state. Additionally, oxygen delivery remained constant one and three days following plasma processing. The mean values in this group are as follows: 40.88, 47.77, 52.11, 51.44, 41.66, 41.66, and 41.44. In 7.4 W plasma with the addition of vitamin C, a 27.15% increase was noted in oxygenation levels 60 minutes following treatment until approximately 120 minutes post-processing, when oxygen delivery returned close to its initial state. Oxygenation levels remained consistent one and three days after plasma treatment, similar to those observed with 7.4 W plasma alone. The average values were 40.11, 46.44, 52, 51, 40.22, 41.22, and 41.55. In the last phase, following the application of vitamin C ointment on the dorsal skin of the hand, no alterations were detected in oxygen delivery. The average values were computed as 43.55, 43.77, 43.33, 43.88, and 44.11, 42.88, 43.33 for the time points before, immediately after, 30, 60, and 120 minutes after, 1 day after, and 3 days after treatment, respectively. In the control group, substantial changes are not anticipated. The average values are as follows: 40.44, 40.55, 39.66, 39.55, 40.88, 40.66, 41.55. Perfusion denotes the process whereby blood is delivered to tissues, organs, or systems within the body, facilitated by blood circulation through the vascular network. This process ensures the provision of oxygen and nutrients to cells while facilitating the removal of waste products. Perfusion is crucial in sustaining tissues and organs' normal function and viability throughout the body. In medical contexts, perfusion evaluates the adequacy of blood flow to specific bodily regions. Techniques like perfusion imaging are employed to analyze blood flow patterns and identify regions experiencing insufficient perfusion, potentially indicating underlying health conditions or diseases. Proper perfusion is vital for overall health, tissue damage, or organ dysfunction prevention. This parameter is measured within the spectral ranges of 650-730 nm and 820-930 nm. Perfusion levels in plasma, both at 3.3 and 7.4 W, and when supplemented with vitamin C immediately post-plasma processing, increased due to the presence of generated species. After 60 minutes, there was a 12.43% increase in the 3.3 W plasma group, a 26% increase in the 7.4 W plasma group, and a 23.59% increase in the 7.4 W plasma group supplemented with vitamin C. These three groups returned almost to baseline perfusion levels approximately 120 minutes after processing. Moreover, perfusion remained stable one and three days after plasma processing in each of the three groups. The mean values at 3.3 W were 41.11, 44.55, 46.66, 46.22, 41.77, 42.33, and 42.66, while at 7.4 W they were 38.66, 47.33, 48.33, 48.66, 39.77, 43.33, and 42.66. In plasma plus vitamin C group, they were 33.44, 39, 39.66, 41.33, 34.55, 32.66, and 32. Based on the calculated average values (37.88, 37.77, 38.22, 38, 38, 38.66, and 37), it can be concluded that perfusion levels remained unchanged in the group treated with vitamin C. The mean variations observed in the control group are equal to 46.33, 46.66, 45.66, 46.22, 46.44, 47.88, 47.77 (Figures 9 and 10). The tissue water index (TWI) quantitatively measures water content in biological tissues using imaging techniques like hyperspectral or near-infrared spectroscopy. By examining the absorption and reflection of light at specific near-infrared wavelengths, the TWI offers insights into tissue hydration and structural health. This index is crucial in medical diagnostics and research, aiding in monitoring conditions such as edema, dehydration, and wound healing, thereby providing a non-invasive method to assess and track tissue water content changes over time. This measurement evaluates the relative water content of the specific tissue using wavelengths between 870 to 900 nm and 950 to 975 nm. The tissue water index, influenced by edema resulting from fluid accumulation within the tissue, showed no notable alterations following plasma treatment at 3.3 and 7.4 W, as well as with 7.4 W supplemented with vitamin C. After 60 minutes, there was an increase of 4.59%, 7.83%, and 5.31% respectively, before gradually reverting to their baseline levels. The average values calculated at 3.3 W are 36.33, 37.22, 38.33, 38, 36.44, 36.44, and 36.33, and similarly, at 7.4 W, the findings show average values of 36.88, 37.11, 38, 39.77, 37.11, 36.77, and 36.22. The plasma plus vitamin C group's average values are 37.66, 38.11, 39.33, 39.66, 37.88, 38.66, and 37.77. Additionally, the tissue water index parameter in the next group (treated only with vitamin C) also remained unchanged, with the examined average values as follows: 42.22, 42.66, 42.22, 40.66, 41, 42.88, and 40.77. Besides, the average changes observed in the control group are 39.77, 39.22, 39.22, 38, 39.11, 38.55, and 39.44 (Figures 11 and 12). Hemoglobin [44–46] is a globular protein in erythrocytes, or red blood cells, that is tasked with transporting oxygen molecules from the lungs to various tissues and organs throughout the body. This protein's molecular structure allows it to bind to oxygen in regions of high oxygen partial pressure, such as the lungs, and release it in regions with lower oxygen partial pressure, aiding in cellular respiration and metabolic processes. Research suggests that exposure to cold plasma may stimulate biological processes that could indirectly influence hemoglobin levels. The Tissue Hemoglobin Index (THI) quantifies the concentration of hemoglobin distributed in the circulatory system using wavelengths ranging from 530 to 590 nm and 730 to 820 nm. Cold plasma enhances hemoglobin levels because of its nitrogen species content. Following plasma treatment at a power of 3.3 W, the THI increased by 37.5% after one hour. It returned to baseline after approximately 120 minutes. The THI has increased in the condition of 7.4 W power output and when supplemented with vitamin C under the same power output. This increase reaches 37.5% and 31.35% after 60 minutes post-processing. Subsequently, in both conditions, approximately 120 minutes after processing, the THI returns almost to its initial state. However, no alterations were observed following the vitamin C group, with average values of 26, 21.88, 21.88, 21.44, 22.44, 22.55, and 21. The mean values determined at 3.3 W are 13.33, 16.33, 21.66, 18.33, 13.88, 14.11, and 13.77. Also, the average values measured at 7.4 W are 16, 19.11, 20.22, 22, 17.66, 17.88, and 17. The average values for the plasma combined with vitamin C group are 34, 37.66, 38.66, 44.66, 36.22, 34, and 34.55, respectively. As expected, the values in the control group did not exhibit significant changes, fluctuating within the range of 12 to 13.55 (Figures 13 and 14). FEDBD plasma can potentially increase skin permeability, facilitating the enhanced absorption of nutrients and antioxidants like vitamin C. This enhancement aids in the improvement and lightening of skin tone. Figure 15 displays images captured under uniform conditions (consistent lighting and distance) with hyperspectral imaging at four intervals: before treatment, 4 and 8 weeks following the initial treatment session, and 6 weeks after the final treatment session. No significant changes were observed in the control group. In the 3.3 W plasma group, the hand skin was gradually lightened. The 7.4 W plasma group also exhibited skin lightening, but to a greater extent than the 3.3 W plasma group. Vitamin C alone did not affect skin color alteration; however, the combination of vitamin C with 7.4 W plasma demonstrated the most pronounced improvement. 3. Conclusion Plasma skin rejuvenation is a novel technique capable of stimulating skin rejuvenation with minimal recovery time and without notable side effects or tissue damage. This study confirms that the FEDBD device significantly enhances skin elasticity and quality. Treatment at 7.4 W yields the most pronounced improvements, particularly when combined with vitamin C. The findings suggest that plasma treatment effectively cleanses the skin by removing impurities while promoting hydration and oxygenation. This research highlights the potential of cold plasma as a powerful tool in dermatological therapies, advocating for further exploration of its synergistic effects with active agents for enhanced skin rejuvenation. Treatment with cold atmospheric plasma significantly enhances tissue perfusion, oxygen delivery, and fibroblast activity. Optimal power levels of 7.4 W, especially when combined with vitamin C, produce the most substantial improvements in oxygenation and skin lightening, showing the synergy of cold plasma and vitamin C. While oxygen delivery and perfusion return to baseline within a few hours, the increased tissue water index and hemoglobin concentrations indicate a temporary physiological response. FEDBD plasma treatment shows promising potential for improving skin health and appearance. 4. Materials and Methods In this study, the skin rejuvenation process employed the FEDBD plasma device developed by Plasma Fanavari Jam Company (Fig. 1 ). FEDBD plasmas are superior to plasma jets because they have restricted capabilities; they do not apply plasma directly but use the by-products formed after radiation. Additionally, the biological surface area exposed to FEDBD plasma is significantly larger than that exposed to plasma jets, enabling the treatment of a more extensive area [ 34 , 35 ]. The apparatus features a mode alteration button for interrupting and restoring current flow and a power switch beneath the mode change button. Furthermore, the device is outfitted with an emergency button to disengage the power supply during critical situations. Adjacent to these buttons, there are screws for adjusting voltage and frequency within specific ranges. The apparatus incorporates a glass probe, generating a plasma flow within a few millimeters of the target surface through the ionization of atmospheric air. To achieve optimal plasma formation, frequencies of 21 kHz and power levels of 3.3 W and 7.4 W were selected. The device's probe was situated at a distance ranging from 3 to 5 millimeters from the target surface, and all experiments were conducted under these specified conditions. A homogeneous plasma is produced within the device's probe to undergo skin processing. The skin of an animal or a human acts as the secondary electrode, and the plasma is directly administered onto the surface. In pursuit of achieving electrical, optical, and thermal safety parameters, diverse characteristics were subject to evaluation. The examination of voltage fluctuations across diverse intensities was undertaken using a high-voltage probe (Tektronix, p6015A, 1:1000) and an oscilloscope apparatus. The oscilloscope utilized is the Tektronix DPO3012 model, with specifications of 100MHz bandwidth and 2.5GS/s sampling rate. In this investigation, the AvaSpec-ULS 3648-USB2 spectrometer by AVANTES, offering a resolution power between 6/0 and 7/0 nanometers, was utilized to identify the type and intensity of active species within the plasma. This spectrometer enables the detection of wavelengths spanning from 200 to 11000 nanometers, facilitating the determination of the electron density of the plasma radiation spectrum. Given that minor changes in the natural temperature of the human body can serve as indicators of potential illnesses, the physiological control mechanism regulates these temperature fluctuations. The temperature parameter holds great importance in assessing the impact of cold plasma on living tissues. In this investigation, variations in the temperature of the targeted tissue during plasma processing at different time intervals were documented using the FLIR thermal camera. The impact of FEDBD generated plasma on oxygenation levels and skin perfusion was analyzed using a hyperspectral imaging system (TIVITA Tissue, Diaspective Vision, Pepelow, Germany). Hyperspectral imaging is an advanced tool that allows for quantitative analysis of tissues beyond the visual capabilities of the human eye. In medical applications, hyperspectral imaging systems utilize the reflective and absorptive properties of substances such as tissue hemoglobin index (THI), tissue oxygen saturation (StO2), infrared perfusion index (IR), and tissue water index (TWI). [ 36 ]. The Elastometer EM 25 apparatus serves to assess skin elasticity. Renowned as the foremost probe globally, EM 25 swiftly and effortlessly gauges the skin's elastic attributes, often equated with its biological age. Additionally, it evaluates the skin's resilience and firmness in contrast to its capacity to revert to its initial state. The probe array is vertically positioned on the skin, yielding results as a percentage, while accompanying graphs aid in correlating these measurements with the individual's age. The Contact Angle Measurement Device, SDC100, was utilized to evaluate the degree of contact angle. This apparatus facilitates the visual computation of liquid contact angles on solid surfaces. Contact angle measurement typically involves water droplets ranging from 4 to 10 microliters in volume. Equipped with a CCD camera and angle measurement software, the system captures images of the droplet and performs angle calculations. The skin was precisely positioned beneath the water syringe on a flat surface to conduct the procedure. Following monitor adjustments, the syringe was gradually brought closer to the skin, allowing the water droplet to come into contact. Subsequently, the measurement was conducted. Twenty-four individuals participated in the study and were distributed into the following groups: Group One received plasma treatment at 3.3 W for 5 minutes and 8 seconds. Group Two received plasma treatment at 7.4 W for 5 minutes and 8 seconds. Group Three underwent a vitamin C treatment regimen involving the application of a vitamin C ointment. Group Four underwent plasma treatment at 7.4 W for 5 minutes and 8 seconds, followed promptly by vitamin C treatment (C prime serum). 24 participants were randomly divided into four groups, undergoing treatment sessions once weekly over an eight-week period. Subsequently, a biometric analysis of the individual's skin was conducted six weeks post-treatment completion to assess the efficacy of the interventions. Vitamin C is commonly used in skin rejuvenation due to its various beneficial properties for skin health. It is a potent antioxidant, which helps neutralize free radicals in the skin, which can otherwise cause damage and lead to signs of aging such as wrinkles and fine lines. Additionally, vitamin C plays a crucial role in collagen synthesis, essential for maintaining skin elasticity and firmness. By promoting collagen production, vitamin C can help improve the overall texture and appearance of the skin, making it look more youthful and radiant. Furthermore, vitamin C has been shown to have brightening effects on the skin, helping to fade dark spots and hyperpigmentation, resulting in a more even skin tone. Overall, including vitamin C in skincare regimens can contribute to healthier, more youthful-looking skin. This investigation was analyzed utilizing the statistical software SPSS. The data were categorized into groups comprising plasma with power levels of 3.3 W, 7.4 W, vitamin C, and plasma combined with vitamin C. Examination intervals encompassed pre-processing, immediate post-processing, the fourth week of processing, the eighth week of processing, and a follow-up period six weeks post-treatment. To establish a consistent baseline across participants, specific eligibility criteria were implemented. Participants were required to be within the age range of 25 to 45 years and to have refrained from smoking for at least six months prior to the study. Additional requirements included the absence of chronic skin conditions, such as eczema or psoriasis, and a lack of recent or current skin treatments on the target area. Only those in generally good health, without underlying conditions impacting skin physiology, and with no known sensitivities to study products or procedures, were included. These criteria were intended to reduce variability and support reliable evaluation of plasma treatment effects. Besides, all experiments were conducted at the Plasma Research Center of Shahid Beheshti University, providing a controlled academic environment for consistent data collection and analysis. Ethics endorsement and informed consent : For any scientific investigation, particularly those involving animals or human subjects, securing ethical approval is a crucial prerequisite. This approval is issued by ethical committees or relevant institutions to ensure that the study conforms to established ethical standards and international protocols, thereby safeguarding the welfare and rights of participants. In this study, all procedures were approved by the National Institute for Medical Research Development (NIMAD) at Tehran University of Medical Sciences, under protocol number IR.TUMS.MEDICINE.REC.1400.1135 [ 37 ]. It should be noted that this study was registered on Iranian Registry of Clinical Trials with Trial Id: 65352, IRCT Id: IRCT20160514027888N1and Registration date: 11-09-2022. Also, the authors confirm that all methods were performed in accordance with the relevant guidelines and regulations [ 47 ]. It should be noted that the authors declare that all subjects and/or their legal guardian(s) have signed an informed consent form for both study participation AND publication of identifying information/images in an online open-access publication. all individuals and/or their legal guardian(s) have signed an informed consent form both to participate in the study and to publish identifying information/images in an open access online publication. for both study participation AND publication of identifying information/images in an online open-access publication Declarations Author Contribution N.E, F.O, M.A.N, and M.R.K. initiated the research and designed the experiments. M.A.N, M.A.A, B.S, and M.R.K. conducted all parts of the experiments. N.E, F.O, M.L, and M.R.K performed biological analyses, and N.E, F.O and M.A.N, carried out plasma treatment and characterization and analyzed the data. N.E, F.O and M.R.K wrote the manuscript. 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Supplementary Files ProtocolforPlasmaSkinRejuvenationUsingFEDBDDevice.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 01 Apr, 2025 Reviews received at journal 28 Mar, 2025 Reviewers agreed at journal 28 Mar, 2025 Reviewers agreed at journal 26 Dec, 2024 Reviewers agreed at journal 26 Dec, 2024 Reviews received at journal 19 Dec, 2024 Reviewers agreed at journal 19 Dec, 2024 Reviewers invited by journal 26 Nov, 2024 Editor assigned by journal 25 Nov, 2024 Editor invited by journal 14 Nov, 2024 Submission checks completed at journal 13 Nov, 2024 First submitted to journal 10 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5237210","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":386510989,"identity":"bcdd9e29-a55e-4549-b09f-959da44a00a9","order_by":0,"name":"Najmeh Eskandari","email":"","orcid":"","institution":"Shahid Beheshti University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Najmeh","middleName":"","lastName":"Eskandari","suffix":""},{"id":386510990,"identity":"6cded673-e75a-4da9-ac3c-6129763dec89","order_by":1,"name":"Farzane Ostovarpour","email":"","orcid":"","institution":"Shahid Beheshti University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Farzane","middleName":"","lastName":"Ostovarpour","suffix":""},{"id":386510991,"identity":"543cfbab-66a8-448b-8a54-02e8d6180f83","order_by":2,"name":"Mohammad Ali Nilfrosh Zadeh","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Ali Nilfrosh","lastName":"Zadeh","suffix":""},{"id":386510992,"identity":"c5ad749f-17c8-431e-903a-c3fe0214c456","order_by":3,"name":"Mohammad Reza Lotfi","email":"","orcid":"","institution":"Shahid Beheshti University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Reza","lastName":"Lotfi","suffix":""},{"id":386510993,"identity":"42436b44-b30d-4218-81cc-81b74904fbd6","order_by":4,"name":"Mohammadreza Khani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYBACgwNsbEBKQo5BAiLAQ1CLJVSLMfFa7CFaGBIbJIh1mNkBtrQHP3dYpM+f3WPA8KOGQca8gbCW44a9ZyRyN9w5Y8DYc4yBR+YAQS3sbRK8bUAtEjkGDLwNDDwEHWgA1CL5t00iXX5GjgHjX+K0sB2TBtqSwHAjx4CZSFvY0qRl2yQMN9w5VnBY5pgEUVrMJN+21cnLz27e+PBNjY094cCWf4BgH2BgIDp2RsEoGAWjYBTgAwAqOTeOrQwOeAAAAABJRU5ErkJggg==","orcid":"","institution":"Shahid Beheshti University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Mohammadreza","middleName":"","lastName":"Khani","suffix":""},{"id":386510994,"identity":"5b58492f-f51b-49ad-8dc5-e8c0f8df482c","order_by":5,"name":"Mohammad Amir Amirkhani","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Amir","lastName":"Amirkhani","suffix":""},{"id":386510995,"identity":"5728aaff-85e2-410a-b810-d2f9bf719469","order_by":6,"name":"Babak Shokri","email":"","orcid":"","institution":"Shahid Beheshti University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Babak","middleName":"","lastName":"Shokri","suffix":""}],"badges":[],"createdAt":"2024-10-10 07:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5237210/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5237210/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70903880,"identity":"9b838c7d-03a5-41af-a7b2-ca0e8a063376","added_by":"auto","created_at":"2024-12-09 06:11:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":367300,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFEDBD device and analysis devices.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5237210/v1/300ed5222a9989fb46445ee0.png"},{"id":70903865,"identity":"3f8caddf-67f2-4253-aef3-313dd61ab744","added_by":"auto","created_at":"2024-12-09 06:11:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":99888,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptical emission spectroscopy of FEDBD plasma.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5237210/v1/38ee11836f079b1673963188.png"},{"id":70905370,"identity":"08b6476f-8e39-456b-8419-d6c434a94258","added_by":"auto","created_at":"2024-12-09 06:27:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2052177,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) and (b). The temperature range of the skin surface at 3.3 and 7.4 W, respectively. (c) The graph of temperature changes.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5237210/v1/f26fa9d621fa2b1fe79d970a.png"},{"id":70903859,"identity":"558c2b0b-19a0-4e41-8e77-8be9d64fca4e","added_by":"auto","created_at":"2024-12-09 06:11:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33764,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a). The HT-9600 dust meter device and Figure. 4(b). 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The temporal fluctuations of voltage and current at power levels of 3.3 and 7.4 W\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5237210/v1/3cf84f53c2432a10ba0d13bb.png"},{"id":70904241,"identity":"d6dcb2cf-af66-4d97-9728-33bd21352248","added_by":"auto","created_at":"2024-12-09 06:19:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":156193,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElasticity mean: Control, 3.3 W, 7.4 W, Vitamin C, and 7.4 W + Vitamin C group.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5237210/v1/a4b70c73dd4baaaa2851630b.png"},{"id":70903888,"identity":"968e41c3-372d-466a-b237-b83ddcc57bc5","added_by":"auto","created_at":"2024-12-09 06:11:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":29332,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMean oxygenation levels measured at different time intervals during the study\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5237210/v1/ac7e4baba090c011aa908104.png"},{"id":70904266,"identity":"f7f28f7e-d209-425a-a160-16c6e1f5e073","added_by":"auto","created_at":"2024-12-09 06:19:45","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1615032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of skin oxygenation levels between (a) the control group and (b) the group treated with 7.4 W of plasma\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5237210/v1/15edf99e31af42d49ab6682e.png"},{"id":70903870,"identity":"24ce1525-43b1-49f8-9eb7-00ae3fcb38da","added_by":"auto","created_at":"2024-12-09 06:11:40","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":32410,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMean perfusion levels measured at different time intervals during the study\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5237210/v1/0019ca27cd62b9a7c9cf13b2.png"},{"id":70903963,"identity":"0f3e58ae-4935-438a-9192-1629ad86e26a","added_by":"auto","created_at":"2024-12-09 06:11:49","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1414390,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of skin perfusion index levels between (a) the control group and (b) the group treated with 7.4 W of plasma\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-5237210/v1/63cd3489d1624a2900c3fe82.png"},{"id":70903900,"identity":"69af307c-a3f7-41ea-a413-662924b6991c","added_by":"auto","created_at":"2024-12-09 06:11:43","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":32118,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMean tissue water levels measured at different time intervals during the study\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-5237210/v1/4814a07f5f5d57b8b5ae012f.png"},{"id":70904250,"identity":"e8c2f3b2-393e-4661-8a00-b10b1fa385f1","added_by":"auto","created_at":"2024-12-09 06:19:42","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":1653273,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of skin tissue water index levels between (a) the control group and (b) the group treated with 7.4 W of plasma\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-5237210/v1/d661553f6429868b57eef92c.png"},{"id":70903868,"identity":"adfa34f4-9415-405e-84a4-e84fd0c50376","added_by":"auto","created_at":"2024-12-09 06:11:40","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":37059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMean tissue hemoglobin levels measured at different time intervals during the study\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-5237210/v1/85616f4b88a27622f4dc00a7.png"},{"id":70903915,"identity":"d71cad01-fadc-4c6d-9be9-869a1eaceeea","added_by":"auto","created_at":"2024-12-09 06:11:44","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":1346732,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of skin tissue hemoglobin index levels between (a) the control group and (b) the group treated with 7.4 W of plasma\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-5237210/v1/c6d9d891494eebb2df9931a4.png"},{"id":70903869,"identity":"ec52e9f7-0776-413b-bc06-bd9e959a0469","added_by":"auto","created_at":"2024-12-09 06:11:40","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":972975,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVisual changes of skin over time under different treatment conditions, demonstrating varying degrees of redness and surface smoothness across different stages of the study\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-5237210/v1/a21f85fa78560eea73ebc0fe.png"},{"id":70905560,"identity":"644a73b9-ab5c-400d-a6a9-bef3236222e2","added_by":"auto","created_at":"2024-12-09 06:35:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10112417,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5237210/v1/a9ed5824-fcba-4276-b91b-a590266afaa5.pdf"},{"id":70903857,"identity":"bd85a903-3f60-4b0f-8410-e299f3273233","added_by":"auto","created_at":"2024-12-09 06:11:39","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":21482,"visible":true,"origin":"","legend":"","description":"","filename":"ProtocolforPlasmaSkinRejuvenationUsingFEDBDDevice.docx","url":"https://assets-eu.researchsquare.com/files/rs-5237210/v1/d402a41a1b71dc190cecefea.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Randomized Trial of Cold Plasma and Vitamin C Synergy: Effects on Skin Hydration and Wrinkle Reduction","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePlasma, often referred to as the fourth state of matter [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], finds versatile applications across various scientific domains owing to its unique properties. Plasma applications extend beyond materials science, medicine, and astrophysics into environmental remediation, energy production, and agriculture. In environmental science, plasma-based technologies offer efficient air and water purification solutions by degrading pollutants and disinfecting contaminants. Diverse applications underscore the interdisciplinary nature of plasma science and its potential to address pressing challenges across multiple fields [\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In the field of medicine, plasma plays a crucial role in sterilization processes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], wound healing therapies [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and plasma-based cancer treatments [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], leveraging its antimicrobial and tissue-regenerative properties.\u003c/p\u003e \u003cp\u003eThe integumentary system, the most conspicuous and susceptible organ in the human body, experiences anatomical and functional alterations as a consequence of the aging process. Maintaining the integrity of the skin initiates a comprehension of its anatomical, physiological, and functional aspects, along with the recognition of conditions and caregiving factors that might influence its structure and performance [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Scientific investigations suggest that implementing appropriate skin cleansing and protective strategies and nutritional and environmental interventions can reduce certain aging-related effects and yield positive psychological and health implications [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe skin, constituting the body's largest organ, spans an average area of around 2 square meters in adults [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The composition of the skin might exhibit quantitative differences contingent upon variables such as location, age, health status, and gender, among other factors [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Moreover, the skin [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] is vital in preventing water and electrolyte loss. From an immunological standpoint, the skin functions as a dynamic sensory and excretory organ, playing a role in regulating body temperature. Daily, the skin is subjected to many factors, such as prolonged exposure to excessive sunlight, rigorous cleanliness practices, overly dry environmental conditions, and environmental impacts during leisure and work activities. Consequently, prioritizing skin care is crucial for maintaining optimal health.\u003c/p\u003e \u003cp\u003eThe aging phenomenon is identified by the build-up of macro-molecular damage, disruptions in tissue regeneration, and the progressive decline in physiological coherence. The aging process [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] arises due to a synergistic interplay of internal factors, including hormone levels, genotypes, endocrine gland metabolism, and external factors, such as ultraviolet radiation, nutritional status, chemical pollutants, etc.\u003c/p\u003e \u003cp\u003eReactive oxygen and nitrogen species (RONS), exemplified by hydrogen peroxide, ozone, and nitric oxide, manifest diverse biological effects, motivating researchers to explore the application of Cold Atmospheric Plasma (CAP) in various biomedical contexts. Cold plasma has shown promising results in cancer treatment by selectively inducing apoptosis in cancer cells, decreasing tumor volume, and inhibiting metastasis [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNevertheless, FEDBD exhibited significantly superior efficacy compared to all the other apparatuses. FEDBD plasma plays a role in enhancing blood coagulation, indicating that plasma can catalyze intricate biochemical processes involved in blood coagulation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo revitalize the skin on the hands and face and address clinical manifestations throughout different stages of the pathogenesis of vulgaris acne, characterized by the simultaneous involvement of multiple follicles in the affected area, employing atmospheric cold plasma emerges as a suitable approach [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Charipour et al. employed the FEDBD plasma device for skin rejuvenation in animal models. Their study's primary accomplishment was the preservation of skin elasticity [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe current study evaluated the effectiveness of a floating electrode dielectric barrier discharge (FEDBD) device in rejuvenating the skin on human hands. Active species were identified using optical spectroscopy, and the thermal impact of the plasma was assessed by measuring skin temperature with FLIR. The Elastometer instrument was used to measure the elastic properties of the skin. The gas analyzer was employed to quantify the levels of various gases present in the environment. Besides, this project employed a hyperspectral imaging system to analyze changes in oxygen levels, perfusion, hemoglobin content, and tissue water.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e\u003cstrong\u003e2.1 Plasma characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne of the most effective techniques for identifying active plasma species and examining their properties is spectroscopy. Before initiating the research, the FEDBD plasma was analyzed at power levels of 3.3 and 7.4 W. The observed peaks (Figure 2) in this analysis fell within the 280-420 nanometer range, with the wavelengths and origins of the primary emission peaks in the spectra specified for each. According to the OES analysis, most species present and produced in the FEDBD plasma are active nitrogen species, likely due to the 78% nitrogen content in the air. Other detected species include OH and CO at 295 and 297 nanometers, respectively, N₂\u0026nbsp;at 313, N\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eand N 337.1, 380.46, and 405 nanometers, N\u003csub\u003e2\u003c/sub\u003e at 315.01 nanometers, and excited N₂⁺ species at 258.08 nanometers, formed through increased collisions with high-energy electrons [38]. The presence of these active species is crucial in dermatological and regenerative medicine due to their impact on skin cell function. Specifically, they enhance the activity of fibroblast cells, which play a key role in maintaining skin structure and integrity. Fibroblasts are responsible for producing collagen, a primary structural protein that provides strength and elasticity to the skin. When these cells are more active, collagen production increases, improving skin firmness and resilience.\u003c/p\u003e\n\u003cp\u003eAdditionally, the regeneration of the epidermis, the outermost layer of the skin, is accelerated, promoting faster healing and rejuvenation. This process is particularly important in wound healing, anti-aging treatments, and therapies to improve skin health and appearance. The enhanced activity of fibroblasts and the resulting increase in collagen synthesis and epidermal regeneration underscore the therapeutic potential of these active species in clinical applications [33]. The relative intensity of the species was compared at power levels of 3.3 and 7.4 W. As the power increases, the particles\u0026apos; energy and collisions also increase, leading to greater ionization. Consequently, the emission intensity of the species increases.\u003c/p\u003e\n\u003cp\u003eAs previously stated, a thermal camera (FLIR) was employed to monitor the skin temperature during plasma processing to prevent skin damage. Skin temperature readings were taken before and after plasma treatment and 3 and 5 minutes during treatment (Figure 3(a) and (b)). This protocol was executed for both 3.3 W and 7.4 W power levels. As illustrated in Figure 3(c), at a power level of 3.3 W prior to plasma treatment, the temperature was recorded at 31.5\u0026deg;C. For 3 minutes of plasma exposure, the temperature rose by 2.4\u0026deg;C, peaking at 36.6\u0026deg;C after 5 minutes of plasma treatment. Following the treatment\u0026apos;s conclusion, a downward temperature trend was observed, with it settling at 32.9\u0026deg;C. This pattern was also evident at a power level of 7.4 W, albeit with slightly higher temperature variations. Initially, before treatment, the temperature was 31.3\u0026deg;C. During 3 minutes of exposure to 7.4 W plasma, the temperature increased by 5.6\u0026deg;C, reaching 36.9\u0026deg;C. Subsequently, after 5 minutes of treatment, the temperature further increased to 38.8\u0026deg;C, eventually decreasing to 32.8\u0026deg;C post-treatment.\u003c/p\u003e\n\u003cp\u003eExposure of the skin to a temperature of 45 degrees Celsius results in the denaturation of proteins, leading to loss of function and structural alterations, ultimately causing skin burns. Conversely, an elevation in temperature up to around 39 degrees Celsius induces the proliferation of keratinocytes, which can be advantageous in tissue repair and regeneration processes. As observed at power levels of 3.3 and 7.4 W, these temperatures are conducive to stimulating and increasing fibroblast cells.\u003c/p\u003e\n\u003cp\u003eThe skin\u0026apos;s surface contains particles such as various pollutants, oils, and dust. To quantify these particles pre- and post-plasma processing, the HT-9600 dust meter device was employed. This apparatus features a silent internal pump designed for ambient air suction. The device functions by conducting a 60-second suction initially, followed by displaying the particle count on its monitor in three ranges: 0.3, 2.5, and 10 micrometers, demonstrating the atmospheric particle levels. In both power settings, the particle count in the specified ranges was initially reported as 0 before processing. However, after plasma processing at 3.3 W, the particle count increased to 3,820,000 in the 2.5-micrometer range and 116,000 in the 10-micrometer range. Furthermore, at 7.4 W, the particle counts were recorded at 4,347,000 in the 2.5-micrometer range and 733,000 in the 10-micrometer range. Figure 4(a) illustrates the performance outcomes of the dust meter device before and after plasma processing at power levels of 3.3 and 7.4 W, highlighting superior cleaning efficiency at 7.4 W.\u003c/p\u003e\n\u003cp\u003eThe PTM600 series portable composite gas analyzer detects diverse gas concentrations in the surrounding environment. It can identify a mixture of one to six gases and includes a sampling pump. The tube connected to the gas analyzer was positioned on the skin during plasma processing for six minutes. The carbon monoxide production at the end of the process was measured and recorded at power levels of 3.3 and 7.4 W. The carbon monoxide generated by the plasma reacts with particles on the skin, effectively removing oils and various contaminants. CO concentration, measured in ppm, is higher at 7.4 W compared to 3.3 W. This suggests that the higher power level leads to more efficient cleaning of oils from the skin surface. The CO levels generated during the six-minute FEDBD plasma processing were 3.2 ppm at 3.3 W and 6.9 ppm at 7.4 W, which is entirely safe. Low concentrations of CO gas can have beneficial effects on health. In addition to cleansing the skin surface, it can increase blood circulation and oxygen delivery to the skin, stimulating fibroblast proliferation and promoting collagen synthesis. The amount of ozone generated by the FEDBD plasma device at power levels of 3.3 and 7.4 W was measured using an ozone detector. This detector, equipped with a suction pump, calculated the ozone gas concentration. The tube connected to the detector was placed on the skin surface, and different ozone concentrations were recorded by moving the plasma device probe. As the probe neared the tube connected to the detector, the amount of ozone produced by the device increased, while moving the probe away from the tube resulted in a lower ozone level. Ozone production was zero at three centimeters from the target surface. Thus, the gas\u0026apos;s effect is confined to the skin surface, posing no respiratory risks and ensuring safety. Ozone gas\u0026nbsp;[39] enhances metabolism, accelerates healing, slow aging, and improves blood circulation and the immune system. Moreover, it has antioxidant effects, reduces inflammation, and stimulates microcirculation. Thanks to its immunological effects, this boosts the tissue\u0026apos;s oxygen delivery capacity and enhances regenerative abilities. Therefore, stimulating and proliferating fibroblasts, the cells responsible for collagen, glycosaminoglycans, and proteoglycans production, helps strengthen the cellular matrix\u0026apos;s main components responsible for skin integrity [40]. The average ozone concentrations measured at power levels of 3.3 and 7.4 W were reported to be 31.67 and 33.5 ppm, respectively.\u003c/p\u003e\n\u003cp\u003eTo examine the results of water absorption, a droplet of water measuring two microliters was applied to the skin, and measurements were taken. This process was repeated after plasma processing for both power levels of 3.3, and 7.4 W. ROS and RNS produced by plasma induce changes in the superficial layer of the skin. Nitrides and nitrates resulting from plasma lead to skin acidification, causing the removal of lipids and existing carbons, thus resulting in skin cleansing. Additionally, bonds with new chemical compounds primarily based on oxygen increase the polar components of the branched surface layer. Besides, UV radiation within plasma prompts the creation of hydroxides and epoxides, the hydrogenation of double bonds, and the fragmentation of carbon chains. As a result, the physical and chemical interactions between plasma components and the branched surface layer result in heightened wettability [41]. A larger contact angle signifies hydrophobicity, while a smaller contact angle indicates greater hydrophilicity and chemical affinity of the surface. As depicted in Figure 4(b), at a power of 7.4 W, the skin surface demonstrates higher hydrophilicity at a power of 3.3 W.\u003c/p\u003e\n\u003cp\u003eIn the realm of plasma physics, the electrical characteristics of plasma are significantly influenced by the temporal progression of voltage and current. Typically visualized graphically, this correlation is represented by a curve delineating alterations in voltage and current with respect to time. Comprehending these fluctuations is essential for scrutinizing the dynamic attributes of plasma. The voltage-current relationship offers critical insights into plasma behavior, facilitating researchers in advancing plasma-based technologies and applications across diverse domains. The FEDBD plasma is a type of glow discharge. We will have high current and voltage peaks when the plasma is ignited, so they will not change significantly. However, they will change over time due to voltage fluctuations. The wavefront within the FEDBD plasma apparatus manifests as sinusoidal, representing the periodic oscillations of the electrical field across the discharge gap, thereby leading to periodic alterations in plasma properties. Figures 5(a) and 5(b) illustrate voltage and current temporal fluctuations at power levels of 3.3 and 7.4 W, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Skin Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSkin elasticity [42] denotes the capability of the skin to stretch and revert to its original form, primarily attributed to the presence of the protein elastin in the dermal layer. Elastin fibers [43] create a network that imparts elastic properties to the skin, enabling it to return to its normal structure after stretching or contracting. To evaluate the Elastometer under various conditions, the variable was measured at five different time points: before treatment, immediately post-treatment, at four and eight weeks during the treatment period, and at the follow-up session, employing the repeated measures method\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e In the 3.3 W plasma group, skin elasticity increased by 3.13% immediately after treatment compared to pre-treatment levels, while no changes were observed in the control group. Furthermore, in the 3.3 W plasma group, skin elasticity increased by approximately 5% four weeks post-treatment compared to immediately after treatment. In the 3.3 W plasma group, skin elasticity increased by 3% after eight weeks of treatment. Furthermore, skin elasticity decreased by 0.08% in this group six weeks after the final treatment compared to the eighth session. It can be concluded that a significant difference in skin elasticity exists between the control group and the 3.3 W plasma treatment group. In the control group, skin elasticity remained relatively stable or occasionally decreased throughout the treatment sessions. Conversely, in the 3.3 W plasma treatment group, skin elasticity gradually increased and sustained over time as the treatment progressed (Figure 6(a)).\u003c/p\u003e\n\u003cp\u003eIn the 7.4 W plasma group, skin elasticity decreased by 5% immediately post-treatment compared to pre-treatment levels, while no changes were observed in the control group. Furthermore, in the 7.4 W plasma group, skin elasticity increased by about 16% four weeks after treatment compared to immediately post-treatment, whereas the control group showed a 2% decrease. The 7.4 W plasma group continued to show an upward trend in skin elasticity after eight weeks of treatment. At the follow-up session (six weeks after the last treatment), skin elasticity decreased by 4% compared to the eighth session, while the control group experienced a 0.7% decrease. In the control group, skin elasticity remained relatively stable before and after treatment but started to decline four weeks post-treatment. Conversely, skin elasticity initially decreased post-treatment in the 7.4 W plasma treatment group but increased over time, with improvements noted at both four and eight weeks post-treatment (Figure 6(b)).\u003c/p\u003e\n\u003cp\u003eIn the vitamin C group, skin elasticity increased by 0.3% immediately after treatment compared to pre-treatment levels, whereas the control group showed no change. After four weeks of treatment, skin elasticity in the vitamin C group decreased by approximately 0.8% relative to the immediate post-treatment measurement, while the control group experienced a 2.4% increase. Additionally, skin elasticity increased by 1.3% over the eight-week treatment period. Furthermore, six weeks after the final treatment, skin elasticity decreased compared to both the eighth session and the pre-treatment levels in both the control and vitamin C groups. The data indicate no significant difference in skin elasticity between the control and vitamin C treatment groups. Skin elasticity levels in both groups are nearly identical. Thus, vitamin C treatment alone does not significantly impact skin elasticity statistically (Figure 6(c)).\u003c/p\u003e\n\u003cp\u003eIn the plasma treatment group, applying 7.4 W of power along with vitamin C resulted in an immediate 4% reduction in skin elasticity relative to baseline measurements, while no significant change was detected in the control group. After four weeks, skin elasticity in the treatment group increased by 19% compared to the immediate post-treatment measurement, whereas the control group experienced a 4.7% decline. The findings demonstrated that the increase in skin elasticity continued in the plasma plus vitamin C group, with a 9% improvement compared to the fourth session after eight weeks of treatment. In contrast, the control group only showed a 2.3% improvement. Six weeks after the final treatment, skin elasticity in the plasma plus vitamin C group decreased by 1.2% from the eighth session, whereas no notable change was observed in the control group. The results demonstrate that plasma treatment at 7.4 W combined with vitamin C significantly affects skin elasticity. Specifically, skin elasticity in the control group remained almost unchanged throughout all treatment sessions. However, in the group treated with 7.4 W plasma and vitamin C, there was a slight decrease in skin elasticity immediately after treatment, followed by a significant increase over time, with substantial improvements observed at four and eight weeks post-treatment, as well as six weeks after the final session (Figure 6(d)).\u003c/p\u003e\n\u003cp\u003eThe data clearly demonstrate that vitamin C treatment behaves differently from the other three treatments, showing no significant effect on skin elasticity. Among the other treatments, the combination of vitamin C and plasma exhibits the greatest impact on skin elasticity over the course of treatment. This is followed by the 7.4 W plasma treatment and the 3.3 W plasma treatment, respectively, in terms of their effects on skin elasticity.\u003c/p\u003e\n\u003cp\u003eWhen tissue undergoes treatment with cold atmospheric plasma, its perfusion levels increase. Consequently, this rise in perfusion leads to enhanced oxygen delivery to the skin, ultimately resulting in heightened perfusion and oxygenation. This, in turn, fosters increased fibroblast production and collagen synthesis. The stimulating effects of plasma lead to an excessive accumulation of fluid volume within the interstitial tissue, resulting in edema and an increase in the tissue water index. Tissue oxygen supply (StO2) measures the amount of oxygen in the superficial skin layers, reaching a depth of 1 mm. This parameter was evaluated within 530-600 nm and 730-800 nm spectral ranges. The Oxygen delivery provided by FEDBD plasma was examined at different time intervals, and the mean quantities were recorded (Figures 7 and 8). Oxygen delivery at power levels of 3.3 and 7.4 W and plasma at 7.4 W supplemented with vitamin C immediately post-plasma processing resulted in the heightened presence of generated species and elevated temperature. With a power setting of 3.3 W, a 19.28% increase was noted after one hour, followed by a return to nearly baseline levels after 120 minutes of processing. Observations suggested oxygen delivery rose by about 7% one-day post-processing and reverted to the initial state after three days. The average oxygen delivery values are as follows: 38, 44, 44.77, 45.33, 39.33, 40.44, and 39.88, respectively. At a power of 7.4 W, a 26% increase was observed one hour later, until approximately 120 minutes after processing, when the oxygen delivery returned nearly to its initial state.\u003c/p\u003e\n\u003cp\u003eAdditionally, oxygen delivery remained constant one and three days following plasma processing. The mean values in this group are as follows: 40.88, 47.77, 52.11, 51.44, 41.66, 41.66, and 41.44. In 7.4 W plasma with the addition of vitamin C, a 27.15% increase was noted in oxygenation levels 60 minutes following treatment until approximately 120 minutes post-processing, when oxygen delivery returned close to its initial state. Oxygenation levels remained consistent one and three days after plasma treatment, similar to those observed with 7.4 W plasma alone. The average values were 40.11, 46.44, 52, 51, 40.22, 41.22, and 41.55. In the last phase, following the application of vitamin C ointment on the dorsal skin of the hand, no alterations were detected in oxygen delivery. The average values were computed as 43.55, 43.77, 43.33, 43.88, and 44.11, 42.88, 43.33 for the time points before, immediately after, 30, 60, and 120 minutes after, 1 day after, and 3 days after treatment, respectively. In the control group, substantial changes are not anticipated. The average values are as follows: 40.44, 40.55, 39.66, 39.55, 40.88, 40.66, 41.55.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePerfusion denotes the process whereby blood is delivered to tissues, organs, or systems within the body, facilitated by blood circulation through the vascular network. This process ensures the provision of oxygen and nutrients to cells while facilitating the removal of waste products. Perfusion is crucial in sustaining tissues and organs\u0026apos; normal function and viability throughout the body.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eIn medical contexts, perfusion evaluates the adequacy of blood flow to specific bodily regions. Techniques like perfusion imaging are employed to analyze blood flow patterns and identify regions experiencing insufficient perfusion, potentially indicating underlying health conditions or diseases. Proper perfusion is vital for overall health, tissue damage, or organ dysfunction prevention. This parameter is measured within the spectral ranges of 650-730 nm and 820-930 nm. Perfusion levels in plasma, both at 3.3 and 7.4 W, and when supplemented with vitamin C immediately post-plasma processing, increased due to the presence of generated species. After 60 minutes, there was a 12.43% increase in the 3.3 W plasma group, a 26% increase in the 7.4 W plasma group, and a 23.59% increase in the 7.4 W plasma group supplemented with vitamin C. These three groups returned almost to baseline perfusion levels approximately 120 minutes after processing. Moreover, perfusion remained stable one and three days after plasma processing in each of the three groups. The mean values at 3.3 W were 41.11, 44.55, 46.66, 46.22, 41.77, 42.33, and 42.66, while at 7.4 W they were 38.66, 47.33, 48.33, 48.66, 39.77, 43.33, and 42.66. In plasma plus vitamin C group, they were 33.44, 39, 39.66, 41.33, 34.55, 32.66, and 32. Based on the calculated average values (37.88, 37.77, 38.22, 38, 38, 38.66, and 37), it can be concluded that perfusion levels remained unchanged in the group treated with vitamin C. The mean variations observed in the control group are equal to 46.33, 46.66, 45.66, 46.22, 46.44, 47.88, 47.77 (Figures 9 and 10).\u003c/p\u003e\n\u003cp\u003eThe tissue water index (TWI) quantitatively measures water content in biological tissues using imaging techniques like hyperspectral or near-infrared spectroscopy. By examining the absorption and reflection of light at specific near-infrared wavelengths, the TWI offers insights into tissue hydration and structural health. This index is crucial in medical diagnostics and research, aiding in monitoring conditions such as edema, dehydration, and wound healing, thereby providing a non-invasive method to assess and track tissue water content changes over time. This measurement evaluates the relative water content of the specific tissue using wavelengths between 870 to 900 nm and 950 to 975 nm. The tissue water index, influenced by edema resulting from fluid accumulation within the tissue, showed no notable alterations following plasma treatment at 3.3 and 7.4 W, as well as with 7.4 W supplemented with vitamin C. After 60 minutes, there was an increase of 4.59%, 7.83%, and 5.31% respectively, before gradually reverting to their baseline levels. The average values calculated at 3.3 W are 36.33, 37.22, 38.33, 38, 36.44, 36.44, and 36.33, and similarly, at 7.4 W, the findings show average values of 36.88, 37.11, 38, 39.77, 37.11, 36.77, and 36.22. The plasma plus vitamin C group\u0026apos;s average values are 37.66, 38.11, 39.33, 39.66, 37.88, 38.66, and 37.77. Additionally, the tissue water index parameter in the next group (treated only with vitamin C) also remained unchanged, with the examined average values as follows: 42.22, 42.66, 42.22, 40.66, 41, 42.88, and 40.77. Besides, the average changes observed in the control group are 39.77, 39.22, 39.22, 38, 39.11, 38.55, and 39.44 (Figures 11 and 12).\u003c/p\u003e\n\u003cp\u003eHemoglobin [44\u0026ndash;46] is a globular protein in erythrocytes, or red blood cells, that is tasked with transporting oxygen molecules from the lungs to various tissues and organs throughout the body. This protein\u0026apos;s molecular structure allows it to bind to oxygen in regions of high oxygen partial pressure, such as the lungs, and release it in regions with lower oxygen partial pressure, aiding in cellular respiration and metabolic processes. Research suggests that exposure to cold plasma may stimulate biological processes that could indirectly influence hemoglobin levels. The Tissue Hemoglobin Index (THI) quantifies the concentration of hemoglobin distributed in the circulatory system using wavelengths ranging from 530 to 590 nm and 730 to 820 nm. Cold plasma enhances hemoglobin levels because of its nitrogen species content. Following plasma treatment at a power of 3.3 W, the THI increased by 37.5% after one hour. It returned to baseline after approximately 120 minutes. The THI has increased in the condition of 7.4 W power output and when supplemented with vitamin C under the same power output. This increase reaches 37.5% and 31.35% after 60 minutes post-processing. Subsequently, in both conditions, approximately 120 minutes after processing, the THI returns almost to its initial state. However, no alterations were observed following the vitamin C group, with average values of 26, 21.88, 21.88, 21.44, 22.44, \u0026nbsp;22.55, and 21. The mean values determined at 3.3 W are 13.33, 16.33, 21.66, 18.33, 13.88, 14.11, and 13.77. Also, the average values measured at 7.4 W are 16, 19.11, 20.22, 22, 17.66, 17.88, and 17. The average values for the plasma combined with vitamin C group are 34, 37.66, 38.66, 44.66, 36.22, 34, and 34.55, respectively. As expected, the values in the control group did not exhibit significant changes, fluctuating within the range of 12 to 13.55 (Figures 13 and 14).\u003c/p\u003e\n\u003cp\u003eFEDBD plasma can potentially increase skin permeability, facilitating the enhanced absorption of nutrients and antioxidants like vitamin C. This enhancement aids in the improvement and lightening of skin tone. Figure 15 displays images captured under uniform conditions (consistent lighting and distance) with hyperspectral imaging at four intervals: before treatment, 4 and 8 weeks following the initial treatment session, and 6 weeks after the final treatment session. No significant changes were observed in the control group. In the 3.3 W plasma group, the hand skin was gradually lightened. The 7.4 W plasma group also exhibited skin lightening, but to a greater extent than the 3.3 W plasma group. Vitamin C alone did not affect skin color alteration; however, the combination of vitamin C with 7.4 W plasma demonstrated the most pronounced improvement.\u003c/p\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003ePlasma skin rejuvenation is a novel technique capable of stimulating skin rejuvenation with minimal recovery time and without notable side effects or tissue damage. This study confirms that the FEDBD device significantly enhances skin elasticity and quality. Treatment at 7.4 W yields the most pronounced improvements, particularly when combined with vitamin C. The findings suggest that plasma treatment effectively cleanses the skin by removing impurities while promoting hydration and oxygenation. This research highlights the potential of cold plasma as a powerful tool in dermatological therapies, advocating for further exploration of its synergistic effects with active agents for enhanced skin rejuvenation. Treatment with cold atmospheric plasma significantly enhances tissue perfusion, oxygen delivery, and fibroblast activity. Optimal power levels of 7.4 W, especially when combined with vitamin C, produce the most substantial improvements in oxygenation and skin lightening, showing the synergy of cold plasma and vitamin C. While oxygen delivery and perfusion return to baseline within a few hours, the increased tissue water index and hemoglobin concentrations indicate a temporary physiological response. FEDBD plasma treatment shows promising potential for improving skin health and appearance.\u003c/p\u003e"},{"header":"4. Materials and Methods","content":"\u003cp\u003eIn this study, the skin rejuvenation process employed the FEDBD plasma device developed by Plasma Fanavari Jam Company (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e1\u003c/span\u003e). FEDBD plasmas are superior to plasma jets because they have restricted capabilities; they do not apply plasma directly but use the by-products formed after radiation. Additionally, the biological surface area exposed to FEDBD plasma is significantly larger than that exposed to plasma jets, enabling the treatment of a more extensive area [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The apparatus features a mode alteration button for interrupting and restoring current flow and a power switch beneath the mode change button. Furthermore, the device is outfitted with an emergency button to disengage the power supply during critical situations. Adjacent to these buttons, there are screws for adjusting voltage and frequency within specific ranges. The apparatus incorporates a glass probe, generating a plasma flow within a few millimeters of the target surface through the ionization of atmospheric air. To achieve optimal plasma formation, frequencies of 21 kHz and power levels of 3.3 W and 7.4 W were selected. The device's probe was situated at a distance ranging from 3 to 5 millimeters from the target surface, and all experiments were conducted under these specified conditions. A homogeneous plasma is produced within the device's probe to undergo skin processing. The skin of an animal or a human acts as the secondary electrode, and the plasma is directly administered onto the surface.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn pursuit of achieving electrical, optical, and thermal safety parameters, diverse characteristics were subject to evaluation. The examination of voltage fluctuations across diverse intensities was undertaken using a high-voltage probe (Tektronix, p6015A, 1:1000) and an oscilloscope apparatus. The oscilloscope utilized is the Tektronix DPO3012 model, with specifications of 100MHz bandwidth and 2.5GS/s sampling rate. In this investigation, the AvaSpec-ULS 3648-USB2 spectrometer by AVANTES, offering a resolution power between 6/0 and 7/0 nanometers, was utilized to identify the type and intensity of active species within the plasma. This spectrometer enables the detection of wavelengths spanning from 200 to 11000 nanometers, facilitating the determination of the electron density of the plasma radiation spectrum.\u003c/p\u003e \u003cp\u003eGiven that minor changes in the natural temperature of the human body can serve as indicators of potential illnesses, the physiological control mechanism regulates these temperature fluctuations. The temperature parameter holds great importance in assessing the impact of cold plasma on living tissues. In this investigation, variations in the temperature of the targeted tissue during plasma processing at different time intervals were documented using the FLIR thermal camera.\u003c/p\u003e \u003cp\u003eThe impact of FEDBD generated plasma on oxygenation levels and skin perfusion was analyzed using a hyperspectral imaging system (TIVITA Tissue, Diaspective Vision, Pepelow, Germany). Hyperspectral imaging is an advanced tool that allows for quantitative analysis of tissues beyond the visual capabilities of the human eye. In medical applications, hyperspectral imaging systems utilize the reflective and absorptive properties of substances such as tissue hemoglobin index (THI), tissue oxygen saturation (StO2), infrared perfusion index (IR), and tissue water index (TWI). [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Elastometer EM 25 apparatus serves to assess skin elasticity. Renowned as the foremost probe globally, EM 25 swiftly and effortlessly gauges the skin's elastic attributes, often equated with its biological age. Additionally, it evaluates the skin's resilience and firmness in contrast to its capacity to revert to its initial state. The probe array is vertically positioned on the skin, yielding results as a percentage, while accompanying graphs aid in correlating these measurements with the individual's age.\u003c/p\u003e \u003cp\u003eThe Contact Angle Measurement Device, SDC100, was utilized to evaluate the degree of contact angle. This apparatus facilitates the visual computation of liquid contact angles on solid surfaces. Contact angle measurement typically involves water droplets ranging from 4 to 10 microliters in volume. Equipped with a CCD camera and angle measurement software, the system captures images of the droplet and performs angle calculations. The skin was precisely positioned beneath the water syringe on a flat surface to conduct the procedure. Following monitor adjustments, the syringe was gradually brought closer to the skin, allowing the water droplet to come into contact. Subsequently, the measurement was conducted.\u003c/p\u003e \u003cp\u003eTwenty-four individuals participated in the study and were distributed into the following groups: Group One received plasma treatment at 3.3 W for 5 minutes and 8 seconds. Group Two received plasma treatment at 7.4 W for 5 minutes and 8 seconds. Group Three underwent a vitamin C treatment regimen involving the application of a vitamin C ointment. Group Four underwent plasma treatment at 7.4 W for 5 minutes and 8 seconds, followed promptly by vitamin C treatment (C prime serum). 24 participants were randomly divided into four groups, undergoing treatment sessions once weekly over an eight-week period. Subsequently, a biometric analysis of the individual's skin was conducted six weeks post-treatment completion to assess the efficacy of the interventions. Vitamin C is commonly used in skin rejuvenation due to its various beneficial properties for skin health. It is a potent antioxidant, which helps neutralize free radicals in the skin, which can otherwise cause damage and lead to signs of aging such as wrinkles and fine lines. Additionally, vitamin C plays a crucial role in collagen synthesis, essential for maintaining skin elasticity and firmness. By promoting collagen production, vitamin C can help improve the overall texture and appearance of the skin, making it look more youthful and radiant. Furthermore, vitamin C has been shown to have brightening effects on the skin, helping to fade dark spots and hyperpigmentation, resulting in a more even skin tone. Overall, including vitamin C in skincare regimens can contribute to healthier, more youthful-looking skin. This investigation was analyzed utilizing the statistical software SPSS. The data were categorized into groups comprising plasma with power levels of 3.3 W, 7.4 W, vitamin C, and plasma combined with vitamin C. Examination intervals encompassed pre-processing, immediate post-processing, the fourth week of processing, the eighth week of processing, and a follow-up period six weeks post-treatment.\u003c/p\u003e \u003cp\u003eTo establish a consistent baseline across participants, specific eligibility criteria were implemented. Participants were required to be within the age range of 25 to 45 years and to have refrained from smoking for at least six months prior to the study. Additional requirements included the absence of chronic skin conditions, such as eczema or psoriasis, and a lack of recent or current skin treatments on the target area. Only those in generally good health, without underlying conditions impacting skin physiology, and with no known sensitivities to study products or procedures, were included. These criteria were intended to reduce variability and support reliable evaluation of plasma treatment effects. Besides, all experiments were conducted at the Plasma Research Center of Shahid Beheshti University, providing a controlled academic environment for consistent data collection and analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEthics endorsement and informed consent\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eFor any scientific investigation, particularly those involving animals or human subjects, securing ethical approval is a crucial prerequisite. This approval is issued by ethical committees or relevant institutions to ensure that the study conforms to established ethical standards and international protocols, thereby safeguarding the welfare and rights of participants. In this study, all procedures were approved by the National Institute for Medical Research Development (NIMAD) at Tehran University of Medical Sciences, under protocol number IR.TUMS.MEDICINE.REC.1400.1135 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. It should be noted that this study was registered on Iranian Registry of Clinical Trials with Trial Id: 65352, IRCT Id: IRCT20160514027888N1and Registration date: 11-09-2022. Also, the authors confirm that all methods were performed in accordance with the relevant guidelines and regulations [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. It should be noted that the authors declare that all subjects and/or their legal guardian(s) have signed an informed consent form for both study participation AND publication of identifying information/images in an online open-access publication.\u003c/p\u003e \u003cp\u003e all individuals and/or their legal guardian(s) have signed an informed consent form both to participate in the study and to publish identifying information/images in an open access online publication.\u003c/p\u003e \u003cp\u003efor both study participation AND publication of identifying information/images in an online open-access publication\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eN.E, F.O, M.A.N, and M.R.K. initiated the research and designed the experiments. M.A.N, M.A.A, B.S, and M.R.K. conducted all parts of the experiments. N.E, F.O, M.L, and M.R.K performed biological analyses, and N.E, F.O and M.A.N, carried out plasma treatment and characterization and analyzed the data. N.E, F.O and M.R.K wrote the manuscript. All authors discussed the result and revised the manuscript\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding author upon request.\u003c/p\u003e\u003ch2\u003eFunding details\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Skin and Stem Cell Research Center of Tehran University of Medical Sciences with Grant/Award Number 1400-1-101-52207 and Plasma Medicine Lab of Laser and Plasma Research Institute, Shahid Beheshti University.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eF.F. Chen, Introduction to plasma physics, Springer Science \u0026amp; Business Media, 2012.\u003c/li\u003e\n\u003cli\u003eJ.A. 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Zhang, H.-P. Li, Characteristics of Portable Air Floating-Electrode Dielectric-Barrier-Discharge Plasmas Used for Biomedicine, Plasma Chem. Plasma Process. 43 (2023) 1567\u0026ndash;1585. doi:https://doi.org/10.1007/s11090-023-10384-1.\u003c/li\u003e\n\u003cli\u003eG. Lu, B. Fei, Medical hyperspectral imaging: a review, J. Biomed. Opt. 19 (2014) 10901. doi:https://doi.org/10.1117/1.JBO.19.1.010901.\u003c/li\u003e\n\u003cli\u003eN. Percie du Sert, V. Hurst, A. Ahluwalia, S. Alam, M.T. Avey, M. Baker, W.J. Browne, A. Clark, I.C. Cuthill, U. Dirnagl, The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research, J. Cereb. Blood Flow Metab. 40 (2020) 1769\u0026ndash;1777. doi:https://doi.org/10.1177/0271678X20943.\u003c/li\u003e\n\u003cli\u003eS.C. Weatherhead, P.M. Farr, D. Jamieson, J.S. Hallinan, J.J. Lloyd, A. Wipat, N.J. Reynolds, Keratinocyte apoptosis in epidermal remodeling and clearance of psoriasis induced by UV radiation, J. Invest. 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Bond., Springer, 2008: pp. 1\u0026ndash;125.\u003c/li\u003e\n\u003cli\u003ehttps://www.nature.com/srep/journal-policies/editorial-policies#experimental-subjects\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cold atmospheric pressure plasma, FEDBD plasma, ambient cold atmospheric plasma, skin rejuvenation, collagen production","lastPublishedDoi":"10.21203/rs.3.rs-5237210/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5237210/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs the skin ages, it naturally becomes drier and loses elasticity, resulting in wrinkles. Over time, the production of collagen and elastin in the skin decreases. Several methods exist for reducing skin wrinkles, including cold plasma technology. Plasma is an advanced treatment employed for skin rejuvenation and aesthetic enhancement. This technique can enhance skin appearance by boosting collagen and elastin production, thereby diminishing wrinkles. This study utilizes a FEDBD device for hand skin rejuvenation. The plasma treatment was administered over 8 sessions spanning 8 weeks. Skin analyses and laboratory tests such as elasticity, oxygenation, perfusion, tissue water index, and hemoglobin were performed before the plasma treatment, immediately after the treatment, at the fourth and eighth weeks of treatment, and six weeks post-final session. The findings of this study suggest that plasma can assist in cleansing the skin surface through the production of CO and OH. Additionally, an increase in the maintenance of skin hydration and enhanced oxygenation and perfusion was reported. The data reveal that, while vitamin C alone does not significantly improve skin elasticity, combining it with plasma treatments, particularly at 7.4 W, leads to a notable enhancement in skin elasticity over time that proves the synergy effect of cold plasma. The combination therapy is the most effective in improving skin elasticity compared to other treatments.\u003c/p\u003e","manuscriptTitle":"Randomized Trial of Cold Plasma and Vitamin C Synergy: Effects on Skin Hydration and Wrinkle Reduction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-09 06:11:30","doi":"10.21203/rs.3.rs-5237210/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-01T06:13:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-28T12:40:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"157780827928147909954098219035073242355","date":"2025-03-28T06:31:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"199155789581660315660625112693264671475","date":"2024-12-26T23:39:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"213559612958036310207229800015888106933","date":"2024-12-26T14:28:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-19T06:16:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31227921520658851552808634904511943862","date":"2024-12-19T06:03:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-26T09:11:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-26T03:24:49+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-11-14T11:24:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-13T07:40:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-10-10T07:01:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b1e98f46-d111-4f2b-ba33-b79d1705715b","owner":[],"postedDate":"December 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":41183720,"name":"Physical sciences/Physics/Plasma physics"},{"id":41183721,"name":"Health sciences/Medical research/Pre clinical studies"}],"tags":[],"updatedAt":"2025-06-16T13:38:41+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-09 06:11:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5237210","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5237210","identity":"rs-5237210","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}