Facial aging is a complex process involving changes in facial anatomy.1 2–4 5,6 7–9 10–14
Radiofrequency (RF) causes neocollagenesis in the skin at temperatures of 40 to 45°C.15 16 17
The HIFES modality aims to remodel and tone delicate facial muscles, counter the sagging appearance of skin, and improve the facial contour with increased volume.18
This veterinary study aimed to evaluate the effects of synchronized radiofrequency and HIFES technology on porcine skin, particularly effects of the therapy on collagen and elastin fibers in the dermis, through a noninvasive hands-free applicator.
Materials and Methods
This veterinary study protocol was approved by the Ethics Committee for Animal Protection of the Ministry of Agriculture of the Czech Republic. Before the study, the animals underwent blood tests to assess their condition and health status. Animals were kept at ambient room temperature and fed a cereal diet. The study was conducted on 8 female large white pigs (Sus scrofa domesticus), weighing between 60 and 80 kg. There were 2 study groups, namely, the active group with 6 sows receiving simultaneous RF and HIFES therapy under general settings, and the control group with 2 untreated sows.
The synchronized RF and HIFES treatments were delivered with a study device (EMFACE, BTL Industries Inc, Boston, MA) through noninvasive self-adhesive electrode applicators placed on the ventrolateral part of the abdomen of the sows (Figure 1 ). The area covered by the electrode applicator had a diameter of 15 cm, and the treatment lasted 20 minutes at 100% power setting. The temperature in the dermis and subcutaneous fat layer was measured using a fiber optic temperature probe (LumaSense Flurotropic Thermometer) inserted into the tissue using an 18-G 1.5-inch injection needle. Treatment was done once per week for a total of 4 weeks. Treated sows were kept under full general anesthesia during the introduction to the surgical hall and the entire treatment phase, which lasted up to approximately 1 hour before awakening again.
Figure 1.:
Illustrative set-up of the hands-free, self-adhesive device applicators on the ventrolateral abdomen of a sow that received active simultaneous RF and HIFES treatment. In human subjects, applicator A is intended for the forehead and B for the cheeks.
Skin samples were collected by punch biopsy (6 mm diameter, Kruse Buster) from the treatment area (in the active group) and in the analogous anatomical area of the untreated control animals. Biopsies were obtained pretreatment (baseline) and post-treatment (1-month and 2-months follow-ups) in all animals including the control. After biopsy sampling, the wounds were dressed.
Histologic Analysis
The tissue samples from the punch biopsy were processed and sectioned in preparation for Orcein and Trichrome staining protocol. After staining, slides were mounted and observed. In total, 6 slices were prepared out of every biopsy sample.
Collagen
Masson's trichrome procedure was followed for collagen-specific staining. Collagen fibers were selectively stained and developed a green color. The stained samples were mounted to a microscopy slide.
Elastin
Visualization of elastin fibers was done with Orcein staining protocol. The elastin fibers were selectively stained and developed a brown-dark color.
The slides were observed and photographed using an automated slide scanning microscope (Hitachi Axio Scan.Z1, Carl Zeiss AG, Germany; 20×/0.8NA Plan-Apochromat objective) in a bright field. Quantitative analysis of collagen and elastin was performed with the Image J software based on semi-automatic segmentation in the Hue-Saturation-Brightness color system. The appropriate threshold differentiating the collagen and elastin fibers from the background was identified in the selected regions of interest (ROI = 1800 × 1,200 μm). After the collagen and elastin fibers were selected, their densities were expressed as the occupied area (square micrometers), which the fibers encompassed in the studied images' ROI.
Statistical analysis Student t -test and repeated measures analysis of variance test were performed with significance level set at α = 0.05. Post-hoc Tukey honest significant test was conducted for multiple comparisons.
Results
All the sows were in good health and condition before and during the study duration. The animals recovered from anesthesia without any complications. The dermal temperature was maintained slightly above 42°C, although not exceeding 42.5°C. The measurements in the fat layer showed a temperature elevation to approximately 41.9°C, elucidating the nature of the temperature gradient observed during the treatment (Figure 2 ). There were no side effects or adverse events related to the treatment.
Figure 2.:
Thermoprobe measurement results during the treatment. The threshold temperature of approximately 40 to 42.5°C was reached in the dermis within the first 2 minutes and maintained in the range for the rest of the treatment time. The temperature in the fat was also monitored, showing about half a degree lower temperatures than the dermis.
Collagen
In the active group, the average area occupied by collagen was 1.12 ± 0.09 × 106 μm2 at baseline. The average collagen-occupied area increased to 1.34 ± 0.08 × 106 μm2 and 1.41 ± 0.07 × 106 μm2 at the 2-month follow-up. Compared with baseline, in the active group, the average collagen amount increased (p < .001) at both post-treatment follow-ups. In the control group, there was no significant difference (p > .05) in collagen fibers, because the collagen content fluctuated in the range of 1.08 ± 0.04 to 1.09 ± 0.05 × 106 μm2 throughout the whole study. There was a significant difference (p < .001) in the collagen-occupied sample area at both the 1-month (+19.6%) and 2-month (+26.3%) follow-ups (Figure 3 ) when comparing treated and control samples.
Figure 3.:
In the active group, the collagen fibers (p < 0.001) increased, occupying a greater area after 2 months follow-up compared to baseline. No significant change occurred in the control group.
Figure 4.:
In the active group, the elastin fibers (p < 0.001) increased, occupying a greater area after 2 months follow-up compared to baseline. There was no significant change in the control group.
Elastin
In the active group, the mean elastin-occupied area at baseline was 0.11 ± 0.03 × 106 μm2 . At the 1-month follow-up, the average elastin amount was 0.19 ± 0.02 × 106 μm2 . At the 2-month follow-up, the area encompassed by elastin increased further to 0.22 ± 0.03 × 106 μm2 . Compared with baseline, the average elastin-occupied area increased at both follow-ups (p < .001). At both, the 1-month (+75.9%) and 2-month (+110.8%) follow-ups, the amount of elastin was significantly different (p < .001) comparing the active and control group (Figure 4 ). In the control group, there were no significant changes (p > .05) in elastin density at the follow-up points, elastin content ranged between 0.71 ± 0.04 × 106 to 0.73 ± 0.06 × 106 μm2 .
Exemplary samples of the collagen (Figure 5A ,B) and elastin (Figure 6A ,B) microscopic evaluation results of the active group are shown below.
Figure 5.:
Bright-field visualization of collagen fibers stained by trichrome stain, active group sample. The collagen fibers appear in green color. In the slide on the right (2-months follow-up, B), the collagen fibers are noticeably denser, occupying a greater area when compared to the left (baseline, A).
Figure 6.:
Bright-field visualization of elastin fibers stained by Orcein stain (active group sample). The baseline sample (left, A) has noticeably fewer elastin fibers, observed as dark/brown filaments (indicated by red arrows), than the 2-month follow-up (right, B). The surrounding collagen tissue (pale grey) also appears denser and better organized at 2 months post-treatment.
Discussion
This study evaluated the effects of synchronized RF and HIFES on porcine skin. Histologic analysis of connective tissue structural proteins in the dermis showed a statistically significant increase in collagen and elastin content at 1 month and 2 months post-treatment. All the treated animals in the study recovered well after each treatment and did not experience any complications or adverse events.
Collagen production and remodeling are vital in tissue regeneration and may be beneficial to promote connective tissue fascia repair in the skin of the face. In this study, collagen content increased by +19.6% at 1 month and +26.3% at 2 months post-treatment follow-up. Elastin content increased by +75.9% at 1 month and +110.8% at 2 months after treatment, indicating that combined RF and HIFES enhanced expression of both structural proteins. The HIFES modality, in combination with RF, is intended for noninvasive improvement of skin tissue quality. Therefore, if confirmed in human subjects, this treatment method may be an alternative to current, invasive procedures aimed at improving facial appearance.19–23
To achieve a therapeutic effect on the skin, reaching the desired tissue temperature is key. Sustaining a lower, moderate therapeutic temperature between 40 and 45°C is essential for treatment effectiveness to circumvent cutaneous tissue necrosis and side effects because of overheating.24 Figure 2 , the dermal temperature measured via a thermal probe reached 40°C within the first 2 minutes from the start of the treatment and was maintained in the range 40 to 42.5°C for the duration of the treatment. This temperature is the target therapeutic range, which ensures treatment efficacy without affecting the underlying fat tissue, as intended during facial treatments. However, the effect on the fat should be a subject of further studies to fully rule out any alterations in response to the treatment.
The control group and temperature tracking were objective evaluation methods. Alternatively, immunofluorescence and scanning electron microscopy may be used for comprehensive connective and adipose tissue analysis. Gross examination of subcutaneous adipose tissue would expound the knowledge about the effects of sublipolytic temperatures on the underlying fat tissue structure in the treated area.25,26
Conclusion
The novel RF + HIFES technology for targeting facial skin, fascia, and muscles was investigated with a focus on the changes in the skin tissue using a porcine animal model. The study results showed that the procedure induces a denser network of collagen and elastin fibers in porcine skin after treatment with synchronized radiofrequency and HIFES observed through histologic analysis and skin temperature measurement. The enhanced collagen and elastin expression observed in this study may be beneficial for skin remodeling and revitalization if replicated in human subjects.
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