Hair has psychological and social importance across ages in shaping an individual’s personality and general appearance [1]. Significant progress has been made in finding effective and safe methods for hair growth. Low-level laser therapy (LLLT), currently called photobiomodulation therapy (PBMT), due to the photochemical effect of light being absorbed, promotes a chemical change known as photobiostimulation [2,3]. It influences the release of several growth factors involved in the formation of epithelial cells, fibroblasts, collagen, and vascular proliferation, thus promoting wound healing [4] and reducing pain [5], promoting enzyme synthesis [6], and acting on lysosomes [7] and mitochondria [8]. As for the therapeutic mechanism, light emitting diode (LED) mainly affects mitochondria, thus enhancing the generation of reactive oxygen species, adenosine triphosphate (ATP) generation, and transcription factor induction. Today, the use of LEDs in dermatologic applications is well established and includes skin rejuvenation [9], acne treatment [10], scar prevention [11], hair loss treatment (such as androgenetic alopecia) [12], fat reduction [13], and cellulite treatment [14]. Red light penetrates the skin to the deepest extent, transmitting visible light to target dermal structures, such as appendages and fibroblasts. When human fibroblasts were irradiated with red light (628 nm at 0.88 J/cm2), it stimulated cell growth by directly upregulating genes involved in cell proliferation and apoptosis [15]. It can increase type I procollagen in fibroblast cultures and decrease matrix metalloproteinase-1 levels in reconstituted human skin tissue at 660 nm at 44 J/cm2 [16].
LEDs are known to have biological effects, but the optimal lighting parameters for different uses are unknown. Biological effects depend on irradiation parameters such as wavelength, dose (fluence), intensity (power density or irradiance), irradiation time (processing time), continuous wave or pulse mode, and the latter depending on the pulse pattern [3]. Clinically, the frequency, treatment spacing, design, and the total number of treatments should be considered. To improve the light treatment effect and convenience of LED technology-based devices in the future, research on the development of more diverse forms and materials for LED device production should be performed. There is a demand for an easy-to-use, portable, and inexpensive LED device that offers additional benefits to its use, which has driven the discovery and development of many products [17]. The helmet was designed with interchangeable areas for high flexibility and caused the production of more nitrate oxide and ATP in each hair cell [18]. It efficiently stimulates the scalp at the same time as the amount of energy. Due to these interchangeable areas, the LED helmet will have more energy and power to therapeutically increase the follicle-stimulating effect of PBMT for hair restoration. Therefore, we evaluated the efficacy of a similar application of the helmet shape, a dome-shaped laser diode (LD)/LED module was manufactured, and an experiment was conducted on an animal model.
| Ethics statement: All procedures of this study involving animals were conducted following the guidelines of the Institutional Animal Care and Use Committee of CRONEX in Korea (Institutional Review Board no. 2021-05010). |
LDs with emitting wavelengths of 655 ± 5 nm (LD, QL65D5SA; QSI Co. Ltd) and LEDs with wavelengths of 630 ± 20 nm (CTSRZ12A; SEOULVIOSYS) were used as the LLLT sources, with parameters indicated in Table 1. The mean energies per unit of each light source with USB2000 (Spectrometer and optical fiber; Ocean Optics) for LEDs and PM400+S120C (Optical Power and Energy Meter Console + photodiode power sensor; Thorlabs GmbH). The operating currents differed according to the specifications of each LD, but the peak power was 3 mW. The laser beam (continuous to pulse type) was delivered to the mouse dorsal skin region through direct irradiation using a dome-shaped module with an aperture of 20 mm. All procedures were applied to the all groups under the same conditions. The animals were randomly selected into six groups (n = 10 per group): Each group comprised group 1, untreated control; group 2, minoxidil (MXD), topical application (3% MXD; Hyundai Pharm), 0.2 ml, applied 3 times a week; group 3 (G3), LD 3 mW, spacing 15 mm applied 3 times a week; group 4, LD 3 mW, spacing 10 mm applied 3 times a week; group 5 (G5), LD 3 mW, spacing 5 mm applied 3 times a week; group 6 (G6), LD 3 mW, spacing 10 mm applied 5 times a week. The LED operating currents were the same and peak power was 3 mW for the LEDs, spacing 10 mm.
Table 1 . Low-level laser therapy parameters of test modules
| Parameter | Group 3 | Group 4 | Group 5 | Group 6 |
|---|---|---|---|---|
| Laser diode (655 ± 5 nm) | ||||
| Units | 18 | 24 | 54 | 24 |
| Spacing (mm) | 15 | 10 | 5 | 10 |
| Power (mW) | 3 | 3 | 3 | 3 |
| Light emitting diode (630 ± 20 nm) | ||||
| Units | 96 | 96 | 96 | 96 |
| Spacing (mm) | 10 | 10 | 10 | 10 |
| Power (mW) | 3 | 3 | 3 | 3 |
| Treated area (cm2) | 37.5 | |||
| Exposure time per point (min) | 18 | |||
| Period (times a wk) | 3 | 3 | 3 | 5 |
| Energy/session (J/cm2) | 4.3 | 4.8 | 7.4 | 4.8 |
Skin surface measurements of the temperature distribution following treatment were performed on the dorsal skin of mice using an infrared thermography system (FLIR E85; Teledyne FLIR). After hair removal on the dorsal part of the mouse, the test area was set and respiratory anesthesia was performed. Then, the mouse was inserted into the kit (dome-shape; 20 mm height [from LD/LEDs to mouse dorsal skin], 5.0 × 7.5 cm), and the device was operated for a total of 18 minutes under the conditions for each mode (Fig. 1A-C).
Figure 1. The dome-shaped laser diode (LD)/light emitting diode (LED) module for hair growth. Inside each module are the key features of the dome-shaped design. (A) LD 18 units, spacing 15 mm. (B) LD 24 units, spacing 10 mm. (C) LD 54 units, spacing 5 mm. (D) Surface temperature (temp.) changes of the mouse dorsal skin during application of dome-shaped LD/LED module and after device removal immediately after treatment. Thermal imaging, (A-C) representative example of thermal images of the dorsal skin area (spacing 15 mm, E), (spacing 10 mm, F), and (spacing 5 mm, G). This device delivered heat that did not affect surrounding skin tissue.
In total, 60 female C57BL/6J mice, 6 weeks old (Samtako Bio Korea) were randomly divided into 6 groups (10 mice/group). The degree of induction of the growth phase was evaluated by observing the change in skin color while the test module was applied for several weeks after the back hairs of C57BL/6 mice aged 6-7 weeks, in a resting state, were shaved. The dorsal hair of C57BL/6 mice has a time-synchronized growth cycle and stem pigmentation that occurs only in the anagen phase of hair growth.
To record the progress of hair growth, photographs were taken on days 3, 10, 14, and 18. The effect of hair growth in each group was 0%-19% (1 point), 20%-39% (2 points), 40%-59% (3 points), 60%-79% depending on the degree of hair growth by visually observing each animal (4 points), and 80%-100% (5 points). ImageJ (version 1.47; National Institutes of Health) was used to quantify the total hair growth and follicle count on day 18 from the photographs.
At the end of the test, the skin on the back was removed and fixed with 10% formalin. After dehydration with alcohol and xylene (step by step), and embedding in paraffin, tissue sections of 5 μm or less were prepared using a microtome, and paraffin was removed again with alcohol and xylene. The epidermis was observed by staining with hematoxylin and eosin, and the infiltration of inflammatory cells into the skin tissue was observed. In a horizontal cut section with a thickness of 5 μm, the maximum hair follicle cross-sectional area was taken, and the hair follicle per 1 mm2 area was calculated using image analysis software ImageJ.
Immunohistochemical staining was performed using the UltraVision LP Large Volume Detection System HRP Polymer kit (Thermo Scientific). The primary antibodies used were proliferating cell nuclear antigen (PCNA) (ab29; Abcam), β-catenin (ab227499; Abcam), Sonic Hedgehog (Shh) (ab135240; Abcam), and fibroblast growth factor-7 (FGF-7) (NBP1-91898; Novus). Paraffin sections were removed using xylene and immersed in ethanol for rehydration. The sample was heated in 3% hydrogen peroxide for 10 minutes and washed 4 times with 1X TBS to inhibit endogenous peroxidase activity. To suppress non-specific reactions, after reacting with Ultra V Block (Thermo Scientific Lab Vision) at room temperature for 5 minutes, the primary antibody was uniformly added to each tissue and reacted at 4°C for 18 hours in a moist chamber. After washing the slides, the primary antibody enhancer was allowed to react for 10 minutes, followed by the HRP polymer for 15 minutes at room temperature. After washing, the slides were stained with 3,3’-Diaminobenzidine substrate for 10 to 30 seconds, counterstained with Mayer’s hematoxylin, and sealed by dehydration. Two independent blinded pathologists evaluated each serial section. Each pathologist assigned each section a score according to the following scale: 0, negative control; +, moderately increased staining; ++, considerably increased staining, based on the percentage of stained cells in each category [19].
For the analysis of in vivo results, trends were analyzed using figures for each period and group and table organized by mean ± standard error. For comparison between the control group and the test group, the
The test module comprises LD/LED panels that can be adjusted to follow the contour of a curved target, for example, the scalp. When the beams from all the panels intersect, a zone of even higher photon intensity is created to enhance the treatment efficacy. Greater energy is delivered at a distance from the surface of the LD/LEDs in the dome-shaped module than at the surface of the module, that is, directly in front of the LD/LEDs with a 20 mm distance between them and the target tissue. After group separation, photos were taken for each individual on days 3, 10, 14, and 18 after the application of the test modules. No special symptoms or sudden changes in body weight were visibly observed, and no abnormalities such as thermal burns, erythema, or edema were found during the test period. During the treatment period (three or five times a week), no heat damage or erythema was observed on the skin surface. Standard treatment protocols were for treatments of 18 minutes for a 5.0 × 7.5 cm area, keeping the surface temperature at ~37.7°C (Fig. 1D-G). Photographs were taken after anesthesia by injecting isoflurane into a mouse-specific respiratory anesthesia machine to minimize movement.
As shown in Fig. 2, the hair growth area of each group started to increase remarkably from day 14 and the MXD groups increased significantly compared to the control (untreated) group. It was confirmed that the hair extension rate of the LD/LED irradiation group was slower than that of the MXD 3% group. In contrast, on the 18th day, a statistically significant increase was confirmed in all groups except for G3 (LD spacing 15 mm) compared to the control group. The narrower LD spacing induces an even response in the overall treatment area, affecting hair growth. Therefore, it is evaluated that there will be a difference in the effectiveness of hair growth according to the specification change of LD/LED.
Figure 2. Photometric comparison of hair growth in C57BL/6J mouse. (A) Experiment design. After depilation, the dorsal skin of C57BL/6 mice was treated with either with the untreated control, topical 3% minoxidil, or each photobiomodulation (PBM) condition after 3, 10, 14, and 18 days. (B) Hair growth scores were evaluated using a scoring index (0 = no growth, 1 = up to 19% growth, 2 = 20%-39%, 3 = 40%-59%, 4 = 60%-79%, and 5 = 80%-100%). Values shown are mean ± standard error (n = 10/mouse; *In this study, the skin was sectioned horizontally on the skin surface, and the point where the cross-sectional area of the hair follicle was the maximum was calculated. The hair growth phase is characterized by the thickening of the entire skin layer as it grows up to the fat layer. By the 18th day, it was confirmed that the length of the hair follicles and skin thickness increased. In the H&E analysis, skin inflammation due to device irradiation was not observed, confirming that the treatment of the test device was safe. Through this analysis, the measurement values of number of hair follicles were recorded.
The number of hair follicles between groups is shown in Fig. 3. The ratio of anagen to telogen was determined by the shape of hair follicles in vertically sectioned slides [20]. As a result, compared with the control, the ratio of anagen to telogen in the skin of all test groups except for the G3 (LD spacing 15 mm) was significantly increased. Comparing the G5 and G6 to the positive control group, there were more anagen transitions than the positive control group. Importantly, the number and size of hair follicles by PBM were also enlarged, indicating anagen phase induction. These results suggest that PBM promoted hair growth by inducing the anagen phase.
Figure 3. Effects of photobiomodulation therapy on hair follicles in telogen stage in C57BL/6J mice. A representative histology result from ten animals is shown; (A) upper panels show longitudinal sections (H&E, ×200); (B) lower panels show transverse sections (H&E, ×200). (C) Histograms of hair follicle count in transverse sections. Values are mean ± standard error (n = 10/mouse; *To evaluate the signaling mechanism underlying the induction of the anagen phase in the PBM and MXD groups, we performed immunohistochemistry (IHC) analysis. As a result of the overall immunohistochemical staining, it was possible to confirm the tendency of the overall expression of hair growth-related proteins to be lower in the G3 (LD spacing 15 mm). Except for the G3, the MXD group and the rest of the PBM group showed equivalent results (Fig. 4). IHC analysis showed that there is a difference between MXD absorbed from the top of the skin and the way light energy is delivered only to the target point. Furthermore, β-catenin, Shh, FGF-7, and PCNA staining were positive in transitional cells and putative hair-like structures surrounding the cells, which is indicative of the formation of the hair follicle. These results suggest that PBM activates certain functions in the anagen phase and that PBM activates hair follicle development through the β-catenin pathway. Taken together, this suggests a possible role for PBM in the hair-promoting activity related to hair development.
Figure 4. Representative sections of skin tissues of C57BL/6J mice stained for (A) β-catenin, (B) fibroblast growth factor-7 (FGF-7), (C) proliferating cell nuclear antigen (PCNA), and (D) Sonic Hedgehog (Shh). Dorsal skin biopsies were taken after 18 days and immunohistochemically stained, with 3,3’-Diaminobenzidine as chromogen with hematoxylin counterstain. Two independent, blinded pathologists evaluated each serial section. Each pathologist assigned each section a score according to the following scale: 0 = negative control; + = moderately increased staining; ++ = considerably increased staining, based on the percentages of stained cells in each category. Representative images of immunohistochemical staining, ×200; n = 10 mice per group (G).
To date, the medical management of pattern hair loss requires 5α-reductase inhibitors (finasteride and dutasteride) and topical MXD, which require frequent and indefinite use and have limited effectiveness and associated side effects [21]. The need for more sustainable treatment option has led to the emergence of LLLT, which has become widely popular because they are commercially available devices that can be used at home, are inexpensive, easy to operate, and have an excellent safety profile [22]. Fast-emerging areas of light-based therapy include treatment of cellulite and hair loss. Both conditions are very prevalent and lack acceptable treatment options. Recent studies have shown that cellulite can be treated by an anti-cellulite gel combined with red/near infrared radiation LED light exposure [23]. Also, light-based treatments have also been shown to promote hair regrowth and increase hair tensile strength [17].
In this study, the effect of PBM was evaluated in mice. In particular the effect of accelerating the transition from the resting phase to the anagen phase (growth induction effect) and the effect of delaying the transition from the growth phase to the catagen phase (inhibiting the catagen phase or extending the anagen phase) was assessed. Although human hair has different hair cycles for individual hairs, all hairs in mice initially have the same hair cycle, which can be matched using an artificial method. This is useful to experimentally observe changes in the hair cycle.
Several key factors determine clinical outcomes: peak wavelength and distribution range, the power density at the treatment site, duration of treatment, total fluence, and treatment regimen. Although most studies have used commercially available LED devices, differences in light output and power density between manufacturers may contribute to the variability of the results. Some clinical studies that did not achieve the desired results may be using LEDs at suboptimal wavelength, power density, or fluence for the desired therapeutic effect [24]. For example, high power density or low power density light sources can be used for different treatment session lengths to achieve the same fluence. Even if the fluence is the same, differences in power density may alter the findings of the study. Flat-shaped vs. dome-shaped forms of light sources may also be important for clinical outcomes, but there are insufficient data to make recommendations.
Combination with LD/LED increased the number of closed hair follicles in the deep layers of the subcutaneous layer, suggesting a facilitating effect on the development of hair follicles in the anagen phase to the catagen phase. Total hair follicles may increase with higher doses of LD, LED, and combination therapy [18]. Also, the difference in light output is a factor that affects skin thickness (hair length), hair density, and size [12]. The increase in hair follicles in the anagen phase may be due to the induction of the early anagen phase or the transition of hair growth from the telogen to anagen phase. In the latter phase, follicles undergo rapid proliferation of follicular keratinocytes and elongation and thickening of the hair shaft. Here, considering that LD, LED, and combination increased PCNA positive cells in the subcutaneous cells, growth phase-like hair follicles were regenerated by the rapid proliferation of stromal keratinocytes to generate increased hair follicle size for new hair fibers. This suggests that PBM can contribute to the induction of hair follicles in the growth phase and improve changes in the hair growth cycle.
Growth factors including epidermal growth factor, keratinocyte growth factor (KGF), insulin-like growth factor-I, and transforming growth factor, have shown mitotic and motility-inducing effects on keratinocytes. The dome-shaped LD/LED module contributed to improving the hair growth cycle through the upregulation of FGF-7 (such as KGF). FGF-7 has also been shown to inhibit the transition to catagen shapes in hair follicle organ cultures [25]. Several activators must be expressed up to the critical threshold concentrations to trigger anagen onset in hair follicle growth. Among them, the expression of β-catenin and Shh acts as a major regulator of hair follicle growth and circulation, acting as anagen-inducing signaling molecules [26]. Induced β-catenin expression was observed in the dermal papilla at the onset of the anagen phase and was also detected in the stem cell progeny of the stromal throughout the anagen phase. Shh is mainly expressed during the anagen phase, stopping when the hair follicle catagen phase begins and its expression is difficult to detect in telogen hair [27]. To elucidate the molecular mechanism by which PBM induces anagen hair follicles, we investigated the expression levels of β-catenin and Shh in the skin.
The dome-shaped LD/LED module was easily irradiated onto the dorsal skin of depilated mice to enable hair growth photo-stimulation. Hair growth in depilated mice was promoted by periodic irradiation with red light. After irradiating the dome-shaped LD/LED module on mouse skin, hair growth-related Wnt/β-catenin signals were observed in the mouse skin extracted without heat/inflammatory tissue damage. It was confirmed that the main influencing factors were the units of LDs and the spacing, and the total energy transfer was also important considering the effect in the daily irradiation group. Additionally, LD spacing changes were found to a greater depth (to the bulb) and greater extent (beyond the bulge) in those follicles treated with LD. Laser parameters may be important when choosing the ideal laser for achieve satisfactory results.
Finding optimal values for each of these parameters is needed, as well as the appropriate combination of irradiation and treatment times to achieve optimal target tissue effects. Another problem is that most of the current studies are based only on needed animal samples and/or short treatment periods. These limitations complicate the understanding and improvement of LED treatment efficacy. Future studies should clarify the facilitating effect on the growth cycle in appropriate models and related mechanisms.
In conclusion, we report the first use of a dome-shaped LD/LED module to promote hair growth by inducing anagen in telogen C57BL/6 mice. We observed an increase in the number and size of hair follicles, which is evidence of anagen induction in the PBM group. Immunohistochemical analysis showed that β-catenin, Shh, and FGF-7 were expressed earlier in the PBM group than in the control group. Taken together, these results suggest that PBM promotes hair growth by inducing the growth phase of hair follicles, and we demonstrated that a dome-shaped LD/LED module can enhance hair growth capability.
We thank CRONEX (Hwasung, Republic of Korea) for renting its research place in this study.
Conceptualization: TRK. Data curation: TRK. Formal analysis: TRK, DWM. Investigation: SJL (Seong Jae Lee). Methodology: SJL (Sang Joong Lee). Project administration: BHY, JL. Software: JH. Validation: JK. Visualization: SK. Writing–original draft: TRK. Writing–review & editing: all authors.
Tae-Rin Kwon is an editorial board member of the journal but was not involved in the review process of this manuscript. Otherwise, there is no conflict of interest to declare.
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Author ORCID Information Tae-Rin Kwon Dong Wook Moon Byoung Ho Yoon Sang Joong Lee Seong Jae Lee Jaeho Hwang Jungwook Kim Soomin Kim Jungkwan Lee |
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