Abstract
Background/Aim
Various devices for non-invasive body shape correction are being developed along with the growth of the beauty industry. Radiofrequency (RF) can selectively reduce subcutaneous fat without causing skin damage. The efficacy of the procedure can be improved by applying RF to a large area simultaneously with multiple handpieces. This study evaluated the safety and efficacy of a new RF device with multi-channel handpieces.
Materials and Methods
In ex vivo experiments, the RF device was used to treat porcine tissue comprising the skin, subcutaneous, and muscle layers. The device’s safety was evaluated by temperature measurements of porcine tissue and histological analysis. In in vivo experiments, the dorsal skin of pigs was treated with the RF device. The safety and efficacy of the device were evaluated by measuring the skin temperature, subcutaneous fat layer thickness, and conducting histological analysis.
Results
The skin temperature did not exceed the set temperature during treatment, and skin damage was not observed in histologic analysis in both ex vivo and in vivo experiments. In in vivo experiments, the subcutaneous fat layer thickness and subcutaneous lipocyte size were decreased after treatment. In addition, the fibrous tissue between subcutaneous lipocytes was increased in the RF treatment group compared with the non-treatment group.
Conclusion
The RF device used in this study effectively reduced the size of subcutaneous lipocytes and increased fibrous tissue without skin damage. Therefore, the safe and effective use of this device for non-invasive fat reduction may be possible in clinical settings.
Keywords: Subcutaneous fat, radiofrequency, skin temperature, lipocyte size, fibrous tissue
Cellulite is a condition in which subcutaneous fat accumulates in pockets, making the skin appear as a lumpy orange peel. Even a slight accumulation of subcutaneous fat can lead to poor lymphatic and microvascular circulation, facilitating cellulite formation (1). Cellulite can cause body image changes by depositing fat in certain body areas. More than 80% of postpubertal women have cellulite; accordingly, there is a growing demand for cosmetic procedures to remove cellulite to achieve an ideal body image (2). The treatment of cellulite can be categorized into surgical and non-surgical treatments. Surgical treatments, which involve making an incision in the skin to remove fat, can have side effects, such as scarring, uneven skin surface, anesthesia side effects, and pulmonary embolism in severe cases, and the recovery process is painful and requires a long period to return to daily life. Non-surgical treatments include cryolipolysis (3), radiofrequency (RF) application (4), low-level laser therapy (5), and high-intensity focused ultrasound (6). They do not require anesthesia and have a relatively quick recovery period, allowing patients to return to daily life immediately. Various devices for non-invasive body shape correction are being developed along with the growth of the beauty industry (7).
Lipolysis using RF energy is a method that generates deep heat within the human body with a frequency of more than 100,000 Hz. The deep heat generated by RF energy causes the expansion of blood vessels, which increases blood flow and improves oxygen supply. In addition, the warming effect activates the metabolic function of cells, which increases energy consumption and causes lipolysis (8-10). Resistive electric transfer (RET) is a method that applies heat uniformly and continuously to control and maintain the temperature of the treatment area. It can cause selective damage to fat cells without damaging surrounding tissues due to the selective thermal damage mechanism.
Most existing RF devices have only a small number of RF generators and handpieces, leading to inefficiency in treatment. The new RF lipolysis device developed in this study had six handpieces with two generators. The device could treat a wide range of areas in a short time and could treat multiple areas simultaneously. This study aimed to evaluate the safety and efficacy of the RF lipolysis device with multi-channel handpieces in a porcine model.
Materials and Methods
Ethical considerations. This experimental preclinical study examined ex vivo and in vivo specimens/subjects. This approach is more often used due to a greater preference for alternative testing models to reduce the number of animals used in the research and development of innovative technologies.
This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Chonnam National University in Korea (CNU-IACUC-YB-2021-40). A porcine model was used for this experiment because the skin of pigs is highly similar to human skin (11). Therefore, the study design was considered suitable to provide evidence of safety and efficacy applicable to humans.
Experimental device. The device used in this study (NeoSculpt, PolyBioTech Co., Ltd., Gwangju, Republic of Korea) consisted of six handpieces with two RF generators (Figure 1). The temperature of each of the six handpieces could be set and controlled.
Figure 1. NeoSculpt device (PolyBioTech Co., Ltd., Gwangju, Republic of Korea) developed for subcutaneous fat reduction in a porcine model. (A) NeoSculpt consisted of (B) six handpieces with two radiofrequency (RF) generators. This study was conducted to obtain data on the safety and efficacy of the test device for subcutaneous fat reduction.
Ex vivo procedures. The sample was porcine tissue comprising the skin, subcutaneous, and muscle layers from the dorsal region, obtained in its fresh state from animals slaughtered on the same day. The sample was placed on an insulation material at room temperature. RF was applied at 40˚C and 43˚C (15 min each application). The temperature was checked every 5 min with a thermographic camera (E5; FLIR System Inc., Wilsonville, OR, USA) (Figure 2). After 15 min of RF application, the samples were treated with 10% neutral formalin for histological staining following gross examination.
Figure 2. Ex vivo procedure. (A) Radiofrequency (RF) was applied for 15 min. (B) The temperature was examined every 5 min with a thermographic camera. Heat transfer to the tissue was evaluated after generating heat on the surface of the ex vivo porcine skin.
In vivo procedures. The lowest possible number of animals (N=5) was used for ethical reasons. Following the ‘3 Rs’ principle of animal experimentation, the study’s sample size was chosen. Conventional pigs weighing 20-30 kg were purchased from Deoksung Seed Pig Agriculture Co., Ltd. (Damyang, Republic of Korea). The animals were bred in an environment with optimal conditions (temperature: 20±2˚C; humidity: 60±15%; ventilation: 35~300 m3/h/sow), where they remained for 7 days for adaptation purposes.
Before treatment, the animals were anesthetized with 5 mg/kg tiletamine/zolazepam (Zoletil®; Virbac Korea, Seoul, Republic of Korea) and 2 mg/kg xylazine HCl (Rompun®; Bayer Korea Ltd., Seoul, Republic of Korea) intramuscularly. Subsequently, 7.5 mg/kg enrofloxacin (Baytril® 50 Inj.; Bayer Korea Ltd.) was injected subcutaneously for antibiotic treatment, and 2 mg/kg ketoprofen (Eagle Ketoprofen 10%; Eaglevet, Seoul, Republic of Korea) and 5 mg/kg tramadol HCl (Maritrol®; Jeil Pharmaceutical, Seoul, Republic of Korea) were injected intramuscularly for analgesia. The animals were intubated and maintained under respiratory anesthesia with isoflurane 2%. Treatment areas were marked on the dorsal surface (approximately 2.5 in2) (Figure 3). RF was applied to the marked areas for 30 min at a set temperature of 45˚C. The skin temperature during treatment was monitored with a thermographic camera (Figure 4). The subcutaneous fat layer thickness was measured using an ultrasound diagnostic device before, immediately after, and at 1 and 7 days after RF application. Biopsies were performed using an 8 mm-sized biopsy punch before, immediately after, and at 1 and 7 days after RF application. The biopsies were performed perpendicularly to the skin surface; thus, the tissue samples contained the skin and subcutaneous layer. The wounds were sutured with 2-0 Dafilon (B/BRAUN, Seoul, Republic of Korea).
Figure 3. Treatment layout for porcine in vivo experiments. Treatment areas were divided into the radiofrequency (RF) application site (red square) and the non-treatment site (blue square). The samples were collected before RF application (red dot), immediately after RF application (green dot), at one day after RF application (yellow dot), and at seven days after RF application (blue dot) for histological analysis.
Figure 4. In vivo procedure. (A) Treatment areas were marked on the dorsal skin of pigs. (B) Radiofrequency (RF) was applied after the collection of samples at the non-treatment site. (C) The skin temperature was monitored with a thermographic camera during RF treatment.
Data analysis. The samples were fixed with 10% neutral buffered formalin at room temperature for 48 h for histology examination. After fixation, the samples were washed with running water, dehydrated with ethanol, cleared by incubation in xylene, and embedded in paraffin. Serial sections (6 μm-thick) were obtained with a microtome (HistoCore AUTOCUT; Leica, Deer Park, IL, USA). All tissue specimens were stained with the hematoxylin and eosin (H&E) stain for measurement of the lipocyte size and Masson’s trichrome (MT) stain for measurement of the percentage of fibrous tissue using Leica Autostainer XL (ST5010; Leica). Digital images were acquired using a light microscope/digital slide scanner (Axio Scan.Z1; Zeiss, Oberkochen, Germany). Three areas (500×500 μm) were selected per specimen, and the lipocyte size (μm2) and percentage of fibrous tissue (%) in each area were measured using an image analysis program (ImageJ; NIH, Bethesda, MD, USA). The mean values of the three areas were calculated and analyzed.
Statistical analysis. Statistical significance was analyzed by Student’s t-test using SPSS Statistics version 27.0 (SPSS Inc., Chicago, IL, USA). Values with p<0.05 were considered statistically significant.
Results
Ex vivo results.
Gross examination. After RF application, gross examination was conducted by an expert as a safety evaluation to check for changes in or damage to the skin. For each temperature (40˚C and 43˚C), no external damage or change was observed on the skin surface. In addition, an increase in the transparency of subcutaneous fat at the site of RF application was observed, which was more evident at 43˚C than at 40˚C (Figure 5).
Figure 5. Gross examination showing an increase in the transparency of subcutaneous fat at the radiofrequency (RF) application site (red rectangle). (A) RF application at 40°C for 15 min. (B) RF application at 43°C for 15 min.
Temperature measurement. Evaluation of temperature changes in the full skin layer using a thermographic camera revealed that heat was gradually transferred from the skin surface to the fat layer after RF application. The skin temperature did not exceed the set temperature for most of the treatment time (Figure 6).
Figure 6. Ex vivo thermal changes in porcine skin temperature. Radiofrequency (RF) was applied at (A) 40°C and (B) 43°C (15 min each application). Heat was gradually transferred to the tissue, and the skin temperature did not exceed the set temperature for most of the treatment time.
Histology. Histopathologic examination of the skin was performed after RF application at the treatment site. For both temperatures (40˚C and 43˚C), H&E staining showed no skin damage after RF application, and destruction of the fascia was identified.
In vivo results.
Temperature measurement. The average temperature of the skin surface in the RF region was measured as 43.38˚C using a thermographic camera. The skin surface temperature did not exceed the set temperature of 45˚C during treatment, and there were no thermal burns on the skin surface (Figure 7).
Figure 7. In vivo thermal changes in porcine skin temperature. Radiofrequency (RF) transmitted heat only to the skin surface under the applicator, and the skin surface temperature did not exceed the set temperature of 45°C, a safe temperature that does not cause skin damage.
Subcutaneous fat layer thickness. RF energy was delivered to the subcutaneous fat area, and only the subcutaneous fat area was reduced on ultrasound examination. At one day after RF treatment, the subcutaneous fat layer thickness was decreased in the RF treatment group (2.84±0.058 mm) compared with the control group (2.98±0.058 mm). At seven days after RF treatment, there was also a significant decrease in the subcutaneous fat layer thickness in the RF treatment group (2.84±0.068 mm) compared with the control group (3.14±0.121 mm).
Lipocyte size measurement. H&E staining showed sporadic lesions in the subcutaneous fat layer, and the cell membrane of the lipocytes was unstable or destroyed immediately after treatment. The subcutaneous lipocyte size was decreased by 30.46% (p<0.001) immediately after RF application, 33.69% (p<0.001) at one day after RF application, and 41.91% (p<0.001) at seven days after RF application compared with before RF application. Furthermore, the subcutaneous lipocyte size was decreased by 27.87% (p<0.01) immediately after RF treatment, 30.83% (p<0.01) at one day after RF treatment, and 34.81% (p<0.05) at seven days after RF treatment in the RF treatment group compared with the control group (Figure 8).
Figure 8. Comparison of the subcutaneous lipocyte size immediately after, at one day, and at seven days after radiofrequency (RF) treatment. (A) Hematoxylin and eosin (H&E)-stained images of subcutaneous lipocytes over time with and without RF treatment. (B) Graph comparing the subcutaneous lipocyte size over time with and without RF treatment. Data are expressed as the mean±SE. *p<0.05 vs. Control.
Fibrous tissue measurement. MT staining was performed to observe changes in the fibrotic septa between the lipocytes over time after RF treatment. The percentage of fibrous tissue was increased by 2.97 times (p<0.01) immediately after RF treatment, 2.75 times (p<0.001) at one day after RF treatment, and 2.96 times (p<0.001) at seven days after RF treatment compared with before RF treatment. Moreover, the percentage of fibrous tissue was increased by 2.75 times (p<0.001) immediately after RF treatment, 2.78 times (p<0.001) at one day after RF treatment, and 2.41 times (p<0.001) at seven days after RF treatment in the RF treatment group compared with the control group (Figure 9).
Figure 9. Comparison of the percentage of fibrous tissue between subcutaneous fat immediately after, at one day, and at seven days after radiofrequency (RF) application. (A) Masson’s trichrome (MT)-stained images of fibrous tissue over time with and without RF treatment. (B) Graphs comparing the percentage of fibrous tissue over time with and without RF treatment. Data are expressed as the mean±SE. **p<0.01, ***p<0.001 vs. Control.
Discussion
RF lipolysis devices have been developed as medical devices that utilize the heat generated by RF application to the body for subcutaneous fat reduction. To effectively use RF for cosmetic purposes, it is vital to understand not only the skin aging process but also the associated parameters, such as the frequency and temperature suitable for the skin. The effects induced by RF are parameter-dependent; thus, proper parameters must be used to achieve the therapeutic goal of RF (12,13).
According to de Araújo et al. (2015), although there are no standardized reports of the therapeutic effects of RF energy based on the frequency, the most common frequencies used by RF devices range from 1 MHz to 6 MHz (14). The RF penetration depth is an inverse function of its frequency, i.e., lower penetration at higher frequencies and greater penetration at lower frequencies (15). The higher the frequency of RF energy and the less vascularized the tissue, the greater the heat production will be (16). This study evaluated the safety and efficacy of a new RF device at 2 MHz frequency for subcutaneous fat reduction in a porcine model.
Thermoreceptors can be divided into high- and low-threshold receptors. High-threshold receptors are activated mainly by temperatures lower than 15˚C and higher than 45˚C, which are usually painful. Low-threshold receptors respond to temperatures between 15˚C and 45˚C, which are temperatures at which a person does not feel pain, and a brief stimulus duration typically used for assessment does not damage tissue (17). Lipocytes are heat-sensitive to temperatures of 50˚C and 45˚C for one and three min, respectively. Thermal exposure to 43-45˚C for at least 15 min could induce lipocyte cell death in vivo (18). Therefore, an ideal RF lipolysis device must heat subcutaneous fat to 43-45˚C, while limiting the temperature of the skin surface to below 45˚C. In this study, the skin surface temperature was maintained at an average temperature of 43.38˚C during the procedure, never exceeding 45˚C. Therefore, the RF device utilized in the present study could be a safe and effective device for reducing subcutaneous fat.
RF lipolysis devices with RET apply uniform and continuous heat to the skin, which has a powerful effect on fat removal without causing pain and scarring of the skin. Each tissue in the body has its resistance value; the higher the resistance, the greater the response to heat. In comparison with skin and muscle, lipocytes have a high resistance; thus, heat can cause selective damage to lipocytes without damaging the surrounding tissue (19). The temperature of the skin or muscle is relatively low; thus, there is little chance of damage. The RF device used in this study did not cause damage to the skin.
Skin histological biopsies were performed to confirm the effect of subcutaneous fat reduction. Histological changes in the dorsal subcutaneous fat layer were observed and compared using H&E staining, which showed that the size of lipocytes was markedly reduced in the RF treatment group compared with the control group. These results demonstrated that this new RF device could induce lipocyte damage.
RF thermal stimulation is thought to stimulate fibroblasts, leading to the production of new collagen (neo-collagenesis) and new elastin (neo-elastogenesis) (20). This increase in fibrous tissue contributes to increased skin elasticity. Therefore, it is important to aim for lipocyte reduction and fibrous tissue growth when developing RF devices. In this study, MT staining confirmed that fibrous tissue between the lipocytes was increased at the treatment sites. The results indicated that this new RF device could effectively increase skin elasticity.
Earlier generations of RF devices consist of one or two handpieces; thus, the operator needs to stay with the patient during treatment, and the narrow treatment area requires substantial time and labor input from the operator. Recently, several RF devices that can apply RF to large areas simultaneously with multiple handpieces have been developed (21). The RF device developed in this study consisted of six 40 cm2 RF handpieces that could be placed simultaneously over multiple localized fat pockets. These handpieces could cover an area of up to 240 cm2, and treatment could be completed in a timely manner. Therefore, the use of this RF device may be a safe and effective method for simultaneously reducing subcutaneous fat over large areas.
Conclusion
This study was conducted to obtain data on the safety and efficacy of a new RF lipolysis device using RET with multi-channel handpieces developed by PolyBioTech Co., Ltd. for subcutaneous fat reduction. The device used in this study effectively reduced the average size of subcutaneous lipocytes and increased the percentage of fibrous tissue without side effects such as skin damage. Based on the findings, the safe and effective use of this device for non-invasive fat reduction may be possible in clinical settings.
Conflicts of Interest
The Authors have no conflicts of interest to declare in relation to this study.
Authors’ Contributions
Writing – original draft preparation, review, and editing: J.K., Y.C., K.J., and S.S.K.; Conceptualization: J.K., Y.C., K.J., and S.S.K.; Investigation and formal analysis: S.E.K., S.S., and K.M.S. All Authors contributed to the experiments and approved the designed experiments and study protocol.
Acknowledgements
This work was supported by the Technology Innovation Program (or Industrial Strategic Technology Development Program) (20017903, Development of medical combination device for active precise delivery of embolic beads for transcatheter arterial chemoembolization and simulator for embolization training to cure liver tumor) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea).
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