Anti-melanogenesis Effect of a Dual-delivery Long-pulsed 1,064-nm Neodymium-doped Yttrium Aluminum Garnet and 755-nm Alexandrite Laser on Human Skin Explants

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Long-pulsed (LP) lasers can be applied for pigmentary disorders, with high efficacy and a low risk of side effects, along with a short procedure time1, 2. Recently, a dual-wavelength LP laser system was introduced1. This system seems to be theoretically more effective because it allows targeting of chromophores elaborately. However, there are still no clinical results for human skin. Actual human research on laser application requires skin biopsies at baseline and at subsequent time points; therefore, there are ethical and esthetic problems. To overcome these obstacles, a skin explant model involving culture of skin tissue obtained from excision has been proposed3. Park et al.4 reported the usefulness of skin explants for histologic analysis after fractional laser treatment. They showed re-epithelialization of laser wounded ex-vivo skin cultures. In this study, we investigated the anti-melanogenesis effects of a dual-wavelength LP 1,064-nm Neodymium-doped Yttrium Aluminum Garnet (Nd:YAG)/755-nm Alexandrite laser on human skin explants.

Full-thickness discarded skin tissues were obtained from female patients aged 30~50 years after abdominoplasty at Asan Medical Center. Informed patient consent and institutional approval were obtained (IRB number: 2020-0091). Three samples, each from three different individual were included. Skin tissues were exposed to ultraviolet B (UVB) or laser with target parameters as described in Table 1. Thereafter, each section of skin tissue was separately cut into 2 cm² sections. The skin was washed with 100% EtOH followed by 70% EtOH and maintained moist in a petri dish with PBS. Explants were placed on the bottom of metal grids with the epidermis facing up and exposed to air, and were incubated at 37℃ in 5% CO₂ for 6 days. Culture medium consisted of DMEM supplemented with 10% fetal bovine serum and 10% ascorbic acid, and the medium was replaced daily. A dual-wavelength LP 1,064-nm Nd:YAG/755-nm Alexandrite laser (SANDRO Dual; WONTECH Co., Ltd.) was used (Fig. 1A). Using the hybrid mode in this device, LP Nd:YAG/Alexandrite laser pulses or Alexandrite/Nd:YAG laser pulses can be consecutively delivered to target tissue according to inter-pulse interval settings ranging from 10 to 200 ms. Pulse duration can be regulated from 5 to 50 ms for each of the Alexandrite/Nd:YAG laser pulses. In the “flushing” mode for the treatment of flushing and erythema, the skin was exposed to the laser immediately and harvested in 6 days. In the pigment modes, the skin was pre-exposed to UVB (70 mJ/cm²) for 2 days, exposed to the laser, and then harvested in 2 days, as a previous study noted increase in melanin after 48 hours of UVB irradiation5. Harvested skin tissue was fixed in 10% formalin before embedding in paraffin. Paraffin-embedded human skin tissues were stained with H&E, Fontana-Masson stain (ID Labs), Masson trichrome, and CD31. Image analysis was performed to quantify the amount of melanin, vessel, and collagen, using Image J software (National Institutes of Health). The melanin index was determined by measuring the percent of stained area to total epidermis area in six random areas. Vascularity was also analyzed by measuring the percent of CD31-stained area to total dermis area in eight random areas, as suggested by a previous study6. Collagen content was visualized according to the Masson trichrome-stained area to total area.

Fig. 1
(A) A photo of irradiating dual-wavelength long-pulsed 1,064-nm Nd:YAG/755-nm Alexandrite laser to full-thickness skin tissues. (B) Basal melanin pigment reduction can be confirmed after 1,064 nm-755 nm toning hybrid mode laser irradiation (black arrows) (Fontana-Masson stain, magnification ×100). (C, D) Masson trichrome staining shows dermal collagens in untreated control specimens (C) and treated specimens (D) (Masson trichrome stain, magnification ×40). Compared with controls, the production of collagen is increased after irradiation in 1,064-nm Nd:YAG/755-nm Alexandrite toning hybrid mode laser treatment. (E~H) Immunohistochemical staining for CD31 indicates vascular endothelial cells in untreated control specimens (E, F) and treated specimens (G, H) (E, G: CD31, ×40; F, H: ×100). In flushing hybrid 755-1,064 nm mode, the blood vessel area is significantly reduced compared with the control.

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Table 1
Laser parameters in this study

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All laser-treated tissues showed reduced melanin content compared with UV only-treated tissues. In the toning single mode with 755-nm Alexandrite, the effect of reducing the melanin content had borderline significance (p=0.065). However, in the LP 1,064-nm Nd:YAG/755-nm Alexandrite toning hybrid mode, the effect of melanin reduction was statistically significant (p=0.026) (Fig. 1B). This hybrid mode showed no significant increase or decrease in blood vessel area before and after laser treatment. Moreover, the hybrid mode has the advantage of better collagen production while showing similar effects in terms of vascular damage and induction of inflammation compared with the single mode (Fig. 1C, D). In the flushing hybrid mode with the 755-nm Alexandrite/1,064-nm Nd:YAG laser, blood vessel area significantly reduced compared with the control (Fig. 1E~H).

The LP 1,064-nm Nd:YAG laser has the advantage of penetrating deep into the skin tissue, but has the disadvantage of causing an excessive photothermal effect1. Thus, the LP 1,064-nm Nd:YAG laser has a low therapeutic effect for epidermal pigment lesions7. On the other hand, the LP 755-nm Alexandrite laser has a higher absorption coefficient for melanin and shows superficial penetration compared with the LP 1,064-nm Nd:YAG laser. It shows dominant partial vessel contraction and thus has the advantages of a lower bleeding injury rate and lower collagen damage compared with the LP 1,064-nm Nd:YAG laser8. However, a high risk of post-treatment hypoor hyperpigmentation in patients with darker skin has been reported with the LP 755-nm Alexandrite laser1, 7.

Theoretically, it can be said that the dual wavelengths for irradiating continuously can complement each other’s shortcomings with better photoacoustic and photothermal effects. In fact, this has been proven in bovine serum albuminpolyacrylamide hydrogel (tissue-mimicking phantom)1. In this study, we confirmed that even in human skin explants, the dual-wavelength system removed melanin pigment more effectively while having less effect on surrounding tissues. Furthermore, it was evidenced that the photorejuvenation effect of generating collagen occurred. This is also supported by the report of Lee et al.9, in which 52.3% of patients had clinical improvement in facial pigmentation and/or skin laxity either by using a LP 755-nm alexandrite or 1,064-nm Nd:YAG laser. In our previous randomized controlled trial, the use of an LP 1,064-nm Nd:YAG laser significantly increased the skin elasticity of periorbital wrinkles10.

This study has the limitation of a small sample size. Skin explants have the inherent limitation of difficulty in observation of the later stages of laser-induced changes. However, the results of this study are significant because this is the first trial on human skin explants to prove the efficacy of a dual-wavelength LP laser system.