The CAMP-generating device, from Ion Medical Inc. (Bio Stimulation Microwave Plasma v1.0, Gyeonggi-do, South Korea), was used in this study (Figure 1B). The plasma was generated through microwave energy application (30–50 W; 2,450 MHz) with non-ionized argon, used as a carrier gas. After the gas flowed through an electric field, CAMP was ignited inside the nozzle and emitted outside. The intensity of CAMP could be controlled by altering the power consumption and gas velocity. The flow rate of the argon gas could be adjusted to 10–20 L/min. The distance between the CAMP device and the apex point of the cell culture media was 2 cm. The temperature of the surface contact zone was maintained at <40°C.

A Yorkshire Terrier, male, 7-year-old dog was referred to SNU-VMTH (Seoul National University Veterinary Medical Teaching Hospital, Seoul, South Korea) with lameness and footpad ulceration of the left forepaw for 4 weeks. History-taking showed no improvement with prednisolone and cephalexin prescribed by a referring veterinarian. This patient was exposed to CAMP, as described in the figure legend.

The immortalized human keratinocyte cell line (HaCaT) was bought from Cell Line Service (Eppelheim, Germany), and canine progenitor epidermal keratinocytes (CPEK) were bought from CELLnTEC (Bern, Switzerland). For the CPEK cell line, 9–20 passages were used, and there was no sign of senescence of cells throughout the experiment period. Both cell lines were grown in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, United States) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, MA, United States), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco) at 37°C and 5% CO₂ in an incubator with a humidified atmosphere.

HaCaT cells were seeded in a 6-well plate at a density of 6.5 × 10⁵ cells/well and further grown for 18 h. Two evaluated methods, CAMP-activated media (CAM) and direct CAMP treatment (dCAMP), were used to determine cell viability. For CAM, media were exposed to CAMP for indicated times and then transferred to the HaCaT cells. Gas-activated media (GAM) were used as a control. After 24 h and 48 h incubation, the number of viable cells in proliferation and/or cytotoxicity was determined. For the direct CAMP treatment, HaCaT and CPEK cells in six-well plates were directly treated with CAMP or gas only. After treatment for 15 min, culture media were substituted with fresh complete media. The cell viability was evaluated using CellTiter 96^® AQueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, United States), according to the manufacturer’s protocol. Briefly, 200 μL of collected media was added into the 96-well plate after adding one solution and further incubating cells for 2 h. Absorbance at 492 nm was used to detect cell proliferation.

HaCaT or CPEK cells were seeded at a density of 2.5 × 10 6 cells/well in six-well plates and grown for 18 h. After treatment with the CAM or dCAMP regimen, as well as those controls, scratches were developed using a sterile pipette tip. Cells were washed twice using PBS to remove cell debris, and new fresh complete media were added. After 24 h and 48 h of incubation, the area of the wound lesion was observed using a light microscope (Nikon Ti-U; Nikon Instruments, Tokyo, Japan). The wound area was quantified using ImageJ software (Version 1.53k, National Institutes of Health, Bethesda, MD, United States) with the wound-healing tool add-on (Suarez-Arnedo et al., 2020).

The CPEK cells were seeded at a density of 1 × 10 6 cells/well in a six-well plate and grown for 18 h. Cells were then treated with 30-s dCAMP or gas. After cell incubation for 15 min, the culture media were replaced by new fresh complete media. After 24 h of incubation, cells were trypsinized and seeded into a 24-well transwell plate (37224, SPLInsert™ Hanging, SPL Life Sciences, Gyeonggi-do, South Korea) and further incubated for 24 h. Then, cells were washed with ×1 PBS twice and fixed with 4% PFA (Biosesang, Gyeonggi-do, South Korea) for 15 min. Later, the cells were stained using 1% crystal violet (61135, Sigma Aldrich, MO, United States) in 2% ethanol for 15 min, and the membrane was rinsed with 1X PBS thrice. The images of cells on the membrane of transwells were captured using a light microscope, and the number of cells per field was counted using ImageJ software.

CPEK cells were seeded at a density of 2.5 × 10 6 cells/well in a 6-cm cell culture dish. After incubation for 18 h, cells were directly treated with CAMP or gas for 30 s and then incubated for 15 min before replacing the media with a new fresh complete medium. After media replacement, the cells were further incubated for 24 h. Total RNA from CPEK cells was extracted using the RNeasy Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer’s protocol. RNA-Seq was conducted by Macrogen (Seoul, South Korea). Briefly, RNA-Seq libraries were created using a TruSeq Stranded mRNA Library Prep Kit (San Diego, CA, United States) for the Illumina platform, according to the manufacturer’s instructions. After sequencing, the quality of reads was evaluated, and to decrease the bias of the analysis result, preprocessing was conducted to remove artifacts, including adapter sequence, contaminant DNA, and PCR duplicates using FastQC software (version 0.11.7) and Trimmomatic program (version 0.38). After preprocessing, read data were mapped to the reference genome (CanFam3.1) using the HISAT2 program (version 2.1.0), and aligned reads were generated. Using the reference-based aligned read information, transcript assembly was conducted using the StringTie program (version 2.1.3b). The expression profile values were obtained for each sample for the known transcript, and read count, FPKM (fragment per kilobase of transcript per million mapped reads), and transcripts per kilobase million values were summarized according to the transcript/gene. Using this value, differentially expressed gene (DEG) analysis was conducted using DESeq2, and significant results were selected as |fc| ≥ 2 and nbinomWaldTest raw p-value <0.05. Gene Ontology (GO) enrichment was evaluated based on the g:Profiler tool (https://biit.cs.ut.ee/gprofiler/) for a significant list of enriched genes, and gene set enrichment analysis was conducted based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) database (http://www.genome.jp/kegg/) for a significant gene list. To control the false-positive rate, Benjamini and Hochberg’s approach was used to adjust the p-values, and a p-value <0.05 was considered a significant difference.

CPEK cells were seeded at a density of 2.5 × 10 6 cells/dish in 6-cm culture dishes and grown for 18 h. Cells were then directly treated with CAMP or gas, and the culture media were replaced with new fresh media after 15 min of incubation. Following further incubation for 24 h, the cells were collected for analysis. Total RNA was extracted using the TRIzol™ reagent (Thermo Fisher Scientific, MA, United States) following the company’s protocol. Total RNA (1 μg) was reverse-transcribed into cDNA using a Verso cDNA synthesis kit (AB1453B, Thermo Fisher, IL, United States) using a MiniAmp Plus Thermal Cycler (Applied Biosystems, Waltham, MA, United States). The primers used for qRT-PCR are listed in the table.

mRNA expression was measured using qRT-PCR. SYBR Green reagents (PowerUp SYBR Green Master Mix, A25741, Applied Biosystems, Thermo Scientific) in a QuantStudio 1 Real-Time PCR System (Applied Biosystems, Marsiling Industrial Estate, Singapore) were used. The mRNA level of each gene was normalized to that of β-actin (housekeeping gene). The relative gene expression was calculated using the comparative Ct method (ΔΔCT Method).

Cells were seeded at a density of 2.5 × 10 6 cells/dish in 6-cm culture dishes and grown for 18 h. Cells were then treated with dCAMP or gas. After media replacement and further incubation for 24 h, cells were washed twice in ice-cold PBS and lysed with RIPA buffer containing proteinase inhibitors. The concentration of proteins was quantified using the Pierce™ BCA protein assay kit (Thermo Scientific, Abbott Park, IL, United States). Afterward, 80 µg of proteins was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. The membrane was blocked using 5% BSA for 1 hour at RT, and specific antibodies were incubated at 4°C overnight. The primary antibodies used (at a dilution of 1:1000) included rabbit anti-E-cadherin (24E10, #3195S, Cell Signaling, MA, United States), rabbit anti-Snail (C15D3, #3879S, Cell Signaling), rabbit anti-Slug (C19G7, #9585P, Cell Signaling), rabbit anti-vimentin (#A11952, ABclonal, MA, United States), and rabbit anti-YAP/TAZ (D24F4, #8418S, Cell Signaling). The blotted membrane was subsequently incubated with HRP-conjugated anti-GAPDH (G9, #sc-365062, Santa Cruz, TX, United States) used as the internal control. After washing using 1% TBST and blotting using goat anti-rabbit, the membrane was incubated with HRP-conjugated IgG secondary antibody (1:5000 dilution, #31460, Thermo Scientific) in 5% skim milk. Membranes were developed using the ECL substrate (Pierce™ ECL Western Blotting Substrate, 32106, Thermo Scientific) and visualized using Alliance Q9 Mini (Cambridge, United Kingdom) and quantified using ImageJ software.

Data are expressed as mean ± SD or SEM with three independent experiments. Statistical analyses were conducted using Student’s t-test and one-way ANOVA followed by post hoc Dunnett’s test using GraphPad Prism 9 software (version 9.5.0). All comparisons were made relative to its control, and significant differences have been presented as *p < 0.05, **p < 0.01, and ***p < 0.001.

The Yorkshire Terrier dog (male, 7-year-old) was referred to Seoul National University Veterinary Medical Teaching Hospital with lameness and footpad ulceration of the left forepaw for 4 weeks. The physical examination and blood analysis showed no specific finding. During the dermatological examination, the dog suffered edema, ulceration, and moderate erythema on his left forepaw footpad. Cytology showed pyogranulomatous inflammation with cocci, suggesting a foreign body rather than neoplasia. However, no foreign body with a puncture was found, and the lesion was extruded. During treatment using CAMP, discomfort, edema, erythema, and size of the lesion were reduced, and the lesion was resolved on day 8 (Figure 1A). To investigate the appropriate treatment to further experiment, the treatment techniques were set using both CAMP-activated media (CAM) and direct CAMP treatment (dCAMP) (Figure 1B). For the CAM-treated experiment, the HaCaT cells were treated with CAM, the culture media derived from the CAMP-treated medium with various exposure times, and further incubated. At 24 h and 48 h, cell viability was analyzed. The results showed that cells treated with 120-s CAM showed a significant decrease in cell viability (67.79% ± 3.80%), compared with gas-activated media (GAM), while the other doses showed no significant difference at 24 h (Figure 2A). At 48 h time point, cell survival results showed a significant decrease in cell survival in 60- and 120-s CAM-treated HaCaT cells (87.15% ± 2.34% and 40.08% ± 2.49%, respectively), compared with GAM (Figure 2A). Therefore, 60 s CAM was selected for further experiments. However, in a scratch assay, the results exhibited no significant effect of CAM on wound-healing activity (Figure 2C). In case of dCAMP, HaCaT cells were directly treated with CAMP for the indicated exposure times and then further incubated with new fresh complete media for 24 h and 48 h. Cell viability assay results revealed that the percentage of cell proliferation in treatment with dCAMP for 45- and 60 s at 24 h and treatment with dCAMP for 30 s, 45 s, and 60 s at 48 h (for 24 h, 79.81% ± 9.21% and 65.27% ± 6.42%, respectively; for 48 h, 83.84% ± 8.18%, 68.92% ± 9.95%, and 58.38% ± 16.82%, respectively) shows a significant reduction rate in cell proliferation (Figure 2B). According to ISO 10993-5:2009, percentages of cell viability above 80% are considered non-cytotoxic (Iso, 2009); therefore, 30 s of dCAMP was selected for use in the wound-healing assay. Although there was no significant effect of CAM on wound-healing activity, the results of the dCAMP treatment method illustrated that directly treated HaCaT cells with CAMP significantly increased the percentage of wound closure width compared with gas control (77.03% ± 7.73% vs. 52.37% ± 7.66%, Figure 2D). All these results suggested that the dCAMP treatment regimen exhibits a favorable effect on wound healing in HaCaT cells.

Since our data suggested that dCAMP might provide a positive effect on wound healing in dogs (Figure 1A), the molecular mechanisms of those effects on dog keratinocytes were evaluated using the CPEK cell line. As shown in Figure 3A, after treating cells with dCAMP for various exposure times, cell viability assay showed no significant effect on cell survival with all those times of treatment at 24 h after incubation; however, the proliferation rate of dCAMP-treated CPEK cells began to significantly reduce at 48 h after treatment for 30 s, 45 s, and 60 s, compared with gas (80.95% ± 4.22%, 74.48% ± 1.20%, and 59.40% ± 6.51%, respectively). Therefore, dCAMP treatment for 30 s exposure time was selected for further study due to the percentage of cell survival being higher than 80. In addition, there was no cell morphology change before and/or after dCAMP treatment for 30 s exposure time, as shown in Figure 3B.

To evaluate the wound-healing activity, scratch assay and transwell assay were performed. For the scratch assay (Figure 3C), dCAMP-treated cells show a significantly higher acceleration rate of lesion closure than gas control at 24 h (92.32% ± 5.13% vs 72.61% ± 5.12%). Unfortunately, there was no difference between both groups at 48 h; the reason was that the scratch area of the dCAMP-treated cells had been utterly closed before the evaluation time-point (100.00% vs 92.92%). For the transwell assay (Figure 3D), dCAMP-treated CPEK cells demonstrated a significantly higher number of migratory and invasive cells than those treated with gas (749.33 ± 35.92 cells/field vs 556.00 ± 32.42 cells/field). These suggested that dCAMP treatment promotes cell migration and cell invasion of canine keratinocytes.

RNA-Seq data were generated from CPEK cells directly treated with CAMP for 30 s. To ensure comparability between the gene expression profiles of dCAMP-treated cells and gas control, a correlation heatmap was generated (Figure 4A). The correlation coefficient graphical diagram showed a positive correlation between both groups. The cluster analysis, as presented in Figure 4B, was performed and grouped based on the degree of similarity of the expression pattern for each gene in each sample, using FPKM values of each gene. From the MA plot (Figure 4C), genes with at least a two-fold difference and adjusted p < 0.05 were selected to determine DEGs between the two groups. Among those 67,709 genes (protein-coding 63,959 genes, pseudogene 2,576 genes, lncRNA 1,096 genes, and miRNA 42 genes), 327 significantly regulated genes were identified; 300 genes were upregulated; and 27 genes were downregulated in the dCAMP-treated CPEK cells (Figures 4D, E). From DEGs (Supplementary Data), the GO analysis result (Figure 4F) revealed the role of dCAMP treatment in the biological process and molecular function. Subsequently, KEGG analysis showed the associated pathway induced by direct CAMP treatment; including signaling pathways regulating pluripotency of stem cells, metabolic pathway, and Hippo signaling pathway (Figure 4G).

To verify the gene enrichment analysis data from RNA-Seq, shown in the heatmap (Figure 5A), we selected several significantly regulated genes involved in the cell cycle, cell proliferation, angiogenesis, cell adhesion, and wound healing. The selected genes, including DSG4, FERMT2, SPDYA, IL12a, IL18, and TLR4, were significantly upregulated by dCAMP treatment, consistent with the heatmap data. Since collagen genes are involved in the wound-healing process, we examined the expression of collagen genes. Our data demonstrated that there was no change in Col1A1 and Col1A2 mRNA expression; however, Col3A1 mRNA levels were significantly upregulated in dCAMP-treated CPEK cells compared with the control. For the gene expression of collagen IV, which is abundant in the basement membrane, Col4A1 was upregulated in dCAMP-treated cells, but not for Col4A2, compared with the gas control (Figure 5B).

A number of studies have reported that the Hippo pathway plays a role in skin homeostasis and wound-healing process (Wang et al., 2017; Rognoni and Walko, 2019). As shown in Figure 6A, the Western blotting data showed no induction of YAP or TAZ protein after treatment with 30-s dCAMP in CPEK cells without wound lesions. Interestingly, with wound lesions, the results showed significant induction of YAP protein (1.4-fold), but not TAZ protein, which is specific to dCAMP-treated CPEK cells. Subsequently, the mRNA expression showed substantial upregulation in YAP, TEAD4, and its downstream target CTGF/CCN2 mRNA level (Figure 6B). In addition, EMT is a critical process to promote cell migration, which is essential for the wound-healing process. Therefore, EMT markers were evaluated at both the transcription and translation levels. CPEK cells with wound lesions treated with dCAMP for 30 s were used for the investigation. The data revealed the protein downregulation of E-cadherin (0.8-fold) and protein upregulation of vimentin (1.8-fold), Slug (2.3-fold), and Snail (1.4-fold) (Figure 6C). These were confirmed using qRT-PCR to measure mRNA expression levels. The data showed the same pattern as protein-level expression: downregulation of CHD1 and upregulation of vimentin, SNAI1, and SNAI2 mRNA expression (Figure 6D). These results suggested that dCAMP treatment plays roles in different mechanisms involved in wound healing, including the Hippo pathway and EMT process.