Primary human dermal fibroblast cultures, established by explanting tissue specimens obtained from neonatal foreskin, were utilized in passages.3-8 All cell cultures were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum and 2 mM glutamine.

Human dermal fibroblasts were exposed in vitro to infrared (IR) radiation, and an IR radiation emitting device (MG Care®, Seoul, Korea) was used as the source. This device emits far IR radiation (900 to 1000 µm) with an energy flux of 35 mW/cm². Human dermal fibroblasts were exposed to IR radiation for 1 to 5 hours. Collagen and elastin assays were performed after a 24-hour post-irradiation incubation period. Results were compared with a control group which was not exposed to IR radiation.

Collagen assays were performed using the Sircol collagen assay kit (Biocolor, UK) according to manufacturer's instructions. Briefly, the collected supernatant was centrifuged at 1,500 rpm for 4 min to isolate the extracellular matrix, and 100 µl of the resulting supernatant was mixed with 1 ml of Sircol dye for 30 min and centrifuged at 10,000 rpm for 5 min to isolate the formed collagen-dye complex. After decanting the suspension, droplets were dissolved in 1 ml Sircol alkali reagent and assessed at 513 nm by spectrophotometry. Total soluble collagen was expressed as a concentration (µg/mL) relative to control fibroblasts that did not receive infrared radiation.

Similarly, soluble elastin content was determined according to Fastin Elastin Assay instructions7 and with the use of a commercial kit containing the dye label 5,10,15,20-tetraphenyl-21-23 porphrine sulphonate (Biocolor Ltd, Belfast, England).

Both assays were performed 3 times and results were obtained by averaging the data.

Twenty females (ages 35-61; mean 44.3 years, skin phototypes III-IV) with facial wrinkles and hyperpigmented lesions were enrolled in the study from March to October, 2004. Patients received 15-20 minute daily treatments (on 5 weekdays) of far IR radiation for a period of 6 months. The emitting device, which was made of medical silicon, covered the entire face except for the eyes, mouth, and nostrils; the temperature of the device was approximately 32.0℃ to 35.0℃ and the radiation energy was 35 mW/cm². Photographic documentation was taken after 0, 3, and 6 months of treatment, and clinical improvement scores were determined every 4 weeks. All of the patients completed the six-month treatment session and were able to return for follow-up evaluations. Each patient and a medical observer independently performed clinical assessments using a well-established grading scale of 0 = < 25% (minimal), 1 = 26-50% (fair), 2 = 51-75% (good), 3 = 75-90% (excellent), 4 = 91-100% (clear) improvement. Assessment categories included improvements in small wrinkles, color tone, brown spots, roughness and tightness. Average improvement scores were calculated as the mean of the grading scales of all categories. Categories of the clinical assessment by the medical observer included fine wrinkles, coarse wrinkles, roughness, mottled hyperpigmentation, laxity, and skin tone. Standardized photographs and dermoscopic observations (Coscam CCL-205, Sometech Cosmetic, Seoul, Korea) were reviewed to help the medical observers identify each clinical grade. Skin biopsies with a 2-mm punch were performed on the cheeks for hyperpigmented lesions and on the lateral sides of eyes for wrinkles prior to treatment and 1 month after final treatment. In addition to hematoxylin-eosin stain, special stains such as elastic stain and Masson-trichrome stain were performed to verify the difference in amount of elastic and collagen fibers in the dermis. Side effects were recorded and rated in severity (0 = none, 1 = mild, 2 = moderate, 3 = severe) at each follow-up visit. We performed a Wilcoxon rank sum test (SAS version 8.2) to compare the treatment effects between the two groups and a Shapiro-Wilk test (SAS version 8.2) to compare baseline and post-treatment measurements.

Total soluble collagen was increased 2 hours after IR radiation exposure, relative to controls, and total content increased with the duration of IR radiation exposure (Fig. 1); the results were an average of three measurements. The concentration of total soluble collagen in the control was 4.6 µg/mL, and in treatment groups the concentrations increased to 3.18 µg/mL, 13.37 µg/mL, 19.77 µg/mL, 29.23 µg/mL, and 36.46 µg/mL with 1, 2, 3, 4, and 5 hours of IR treatment, respectively. Soluble elastin also increased 1 hour after infrared radiation compared to the control. Similar to collagen, the content of soluble elastin increased with longer durations of irradiation (Fig. 2). The concentration of soluble elastin in the control was 4.58 µg/mL, and in treatment groups the concentrations of soluble elastin increased to 9.17 µg/mL, 15.1 µg/mL, 17.61 µg/mL, 26.84 µg/mL, and 29.31 µg/mL, with 1, 2, 3, 4, and 5 hours of irradiation treatment, respectively.

The average improvement score, as evaluated by patients, was 1.88, which was statistically significant (p < 0.05). The grading scale in the category of roughness and tightness was 2.55 and 2.45, respectively, indicating a 51-75% improvement. The grading scale of skin color tone was 1.95. The grading scale of small wrinkles was 1.8, indicating fair improvement (26-50%). Unlike the previous categories, however, hyperpigmented lesions showed minimal improvement (Fig. 3). The average grading scale, as evaluated by a medical observer, was 1.31, which was also statistically significant (p < 0.05). Roughness was the most improved category, with a grading scale of 2.45 (Fig. 4). Fine wrinkles were at least fairly improved (26-50%) in all patients with the grading scale of 1.9; however, coarse wrinkle showed minimal improvement (Fig. 5). Skin tone and laxity, with a grading scale of 1.1 and 1.2, respectively, were fairly improved (26-50%) in all 20 patients, and hyperpigmented lesions did not show any statistically significant improvement.

Histopathologic examinations indicated that there was no significant difference in the basal hyperpigmentation after 6 months of treatment relative to the control group. Neither the depth nor the amount of solar elastosis in the dermis was significantly different after 6-months of treatment with IR radiation. There were no differences noted between the two groups using special stains such as elastin stain or Masson-trichrome stain.

In general, any side effects of infrared radiation treatment were minimal and transient. Of the 20 patients treated, 80% developed mild transient erythema, which lasted only a few hours after the treatment and was not a significant problem for the patients. Other complaints included mild dryness (3 patients) and scaling of the face (2 patients). One patient experienced exacerbation of perioral dermatitis, which subsided without any treatment. There were no instances of pigmentary alteration or burns due to treatment, and all of the treatment sessions were generally well tolerated with minimal complaints.