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Significance of melanin distribution in the epidermis for the protective effect against UV light – Scientific Reports

Reconstructed human pigmented epidermal model (RHE)

Reconstructed human pigmented epidermal models were used to evaluate the effect of melanin after UV irradiation. The epiCS®-M epidermal models (Henkel AG & Co. KGaA, Düsseldorf, Germany) were cultured from normal human primary epidermal keratinocytes and melanocytes from Asian-Caucasian or Afro-American donors. RHE were then classified as tanned and light models, respectively. To assess melanin levels, a total of n = 5 samples of light RHE and n = 16 samples of tanned RHE were evaluated. The effects of UV irradiation were investigated by evaluating n = 36 RHE samples, including n = 18 samples of light RHE and n = 18 samples of tanned RHE.

Upon receipt, the RHE models were cultured for seven days in six-well plates at 37 °C, 5% CO2, and 95% humidity in epiCS ®-M culture medium. The medium was changed every two days. On the eighth day of culture, the melanin content was assessed, and the models were irradiated. The determination of pigmentation of in vitro models by using optical parameters is not feasible due to technical limitations when applying the electrode to the cell culture insert. A method for melanin extraction and quantification was established and validated on ex vivo human skin. Parameters such as ITA° of the RHE could also be estimated from these results.

Ex vivo human skin

Melanin extraction and quantification on ex vivo human skin enables the estimation of the pigmentation type of the models, allowing for their classification based on melanin content. Ex vivo human skin samples were obtained from surgical residues of breast (n = 4) and abdominal (n = 9) reduction surgery from healthy subjects aged 24–63 years, of different ethnicities. All patients had given their informed written consent. All procedures and measurements were approved by the Ethics Committee of the Charité Universitätsmedizin Berlin (EA1/324/19) and complied with the Declaration of Helsinki. In the laboratory, subcutaneous tissue was removed down to the dermis using a scalpel (Aesculap AG, Tuttlingen, Germany), and the skin surface was cleaned using PBS solution (Gibco™, New York, USA). A Skin-Colorimeter CL 400 (Courage & Khazaka electronic GmbH, Cologne, Germany) was used to measure the skin pigmentation (ITA°). Ten consecutive measurements were performed on adjacent skin areas. Values were expressed in degrees [°] and were averaged. According to the ITA°, the skin types of the samples are classified into six skin groups, from very light to dark skin: “very light” > 55° > “light” > 41° > “intermediate” > 28° > “tan” > 10° > “brown” > 30° > “dark”40.

In vivo human skin

Healthy volunteers were enrolled to evaluate the in vivo melanin distribution in different skin types using TPE-FLIM and Fontana–Masson staining. All studies were undertaken with the approval of the Ethics Committee of the Charité—Universitätsmedizin Berlin and the written consent from the volunteers in accordance with the Declaration of Helsinki and are registered in the German Clinical Trials Register (DRKS00028055). A Skin-Colorimeter CL 400 (Courage + Khazaka electronic GmbH, Cologne, Germany) was used to measure the skin pigmentation (ITA°). Three male and three female volunteers aged 26 to 34 years with “very light” (n = 2), “light” (n = 3) and “intermediate” skin (n = 1), were enrolled to evaluate the transversal melanin distribution by TPE-FLIM in the area of the inner forearm. Skin color measured by reflectance spectrophotometers allowed to validate of the FLIM parameters used to assess melanin distribution in the skin. In addition, to evaluate the longitudinal distribution of melanin by Fontana–Masson staining, a skin biopsy was taken from the lower back of four male and one female volunteers aged 21 to 38 years. One subject of each skin type (“very light”, “light”, “intermediate”, “tanned”, and “brown”). The biopsy was fixated in neutral buffered 4% formalin solution (Sigma # HT501128-4L, Merck KGaA, Darmstadt, Germany), embedded in paraffin, and stained with Fontana-Masson for histological detection of melanin.

Melanin content

Ex vivo human skin was prepared and subjected to melanin extraction. The epidermis was first separated by a heat-separation procedure41. 8-mm (ø) punch biopsies (pfm medical, Kai Industries Co. Ltd., Oyana, Japan) of each skin sample were taken and placed on the hot plate at 60 °C for 1 min. The epidermis was then completely separated from the dermis using tweezers. Epidermal samples were dried for 1 h at room temperature and weighed into tubes. Samples (between 3 and 5 mg dry weight) were solubilized in SOLVABLE™ (PerkinEimer Inc., Waltham, USA) at a concentration of 10 mg/mL. The samples were homogenized using a TissueLyser II (QIAGEN, The Netherlands) for 10 min at 30 rs−1 with stainless steel beads and dissolved by subsequent heating in a boiling water bath for 1 h. For spectrophotometric characterization of the skin melanin, the absorption spectra of the resulting solutions were recorded in the range of 450 to 600 nm on a UV/VIS spectrophotometer Lambda 650 S (PerkinElmer LAS GmbH, Rodgau-Jügesheim, Germany). To estimate the total melanin (eumelanin and pheomelanin), the absorbance at 500 nm was analyzed12,40,42, and the melanin content was calculated by interpolation of the results with standard curves, generated by the absorbance of synthetic melanin standards dissolved in SOLVABLE™ (Sigma-Aldrich Chemie GmbH, Steinheim am Albuch, Germany). The results were normalized by the concentration of epidermal samples in SOLVABLE™ (10 mg/mL). For the RHE, the extraction and estimation of the melanin content required some adjustments. The insert membrane with the epidermis equivalent was cut from the cell culture insert and dried for 1 h at room temperature. Each model (approximately 2 mg dry weight) was solubilized in SOLVABLE™ at a concentration of 5 mg/mL. A cell culture insert membrane was used as control. Homogenization and spectrophotometric characterization were performed as previously described for ex vivo skin. Absolute concentrations were also obtained using synthetic melanin as a standard and normalized by the concentration of the epidermal samples (RHE) in SOLVABLE™ (5 mg/mL). For each ex vivo skin sample (n = 13 skin donors) n = 5 epidermis samples were analyzed. For light RHE n = 5 and for tanned RHE n = 16.

UV irradiation

For the irradiation of the RHE, different types of UVR at different doses were investigated. Far UV-C radiation of 233 nm wavelength (0.041 mW/cm2, UV-C LED irradiation source with a short pass optical filter suppressing wavelengths > 240 nm, Ferdinand-Braun-Institute gGmbH, Berlin, Germany) at a dose of 60 mJ/cm2 was applied, which has been shown to be effective in reducing germs on the skin and wounds and is safe for the skin due to its low penetration into the deep layers of the epidermis. A broadband UV lamp containing the UV-B (280–315 nm; 50.69%) and UV-A (315–400 nm; 48.21%) fractions was used in this study (Fig. 6). The lamp used was model TH-1E from Cosmedico®, JW Sales GmbH, Stuttgart, Germany, with an intensity of 41 μW/cm2. The inclusion of UV-B and UV-A wavelengths allowed the assessment of direct DNA damage and free radical formation, respectively, due to their different effects on the skin43. To assess DNA damage, a dose corresponding to 1/10 MED (3 mJ/cm2 for skin type II44), a dose which is considered acceptable for the skin, was applied34. A dose of 150 mJ/cm2 of UV was used to assess UV-induced free radicals. The pursuit of a biological response to UV irradiation comparable to that of far UV-C irradiation in DNA damage and radical formation has led to variations in the doses used for UV irradiation. Non-irradiated RHE samples, served as negative control. Irradiance of radiation for 233 nm was measured with the UV radiometer SXL55 with a SiC UV-C sensor (sglux GmbH, Berlin, Germany), and an ILT 1400 radiometer photometer (International Light Technologies Inc., Peabody, MA, USA) for UV-B (SEL240) lamps. Data of n = 6 biopsies of n = 3 models for each type of RHE.

Figure 6
figure 6

Emission spectra of applied UV light sources. The normalized spectra are shown for 233 nm (green), broadband UV-radiation (blue).

For irradiation, the RHE were placed in PBS previously heated to 37 °C to avoid possible photosensitivity of the culture medium. The negative controls were also transferred to 6-well plates with PBS to exclude any influence of this procedure and stored in parallel in the dark for the duration of the irradiation.

Normalized emission spectra of the used UV light sources are shown in Fig. 6.

Analysis of DNA damage

To assess the effect of far UV-C and UV irradiation on skin cells, CPD and 6-4PP were evaluated in the epidermis. Two 4-mm (ø) biopsies were taken from each model, for a total of n = 6 biopsies per RHE type (n = 3 light RHE and n = 3 tanned RHE). n = 6 biopsies were then evaluated immediately and 24 h after irradiation, in each group: non-irradiated, 233 nm irradiation, and UV irradiation. The biopsies were taken from each model, fixated in neutral buffered 4% formalin solution (Sigma # HT501128-4L, Merck KGaA, Darmstadt, Germany) and embedded in paraffin (paraffin blocks) (Histosec™, Merck Millipore GmbH, Darmstadt, Germany). DNA damage was assessed immediately and 24 h after (reculture of RHE at 37 °C and 5% CO2) irradiation using the immunohistochemical approach described previously45. Tissue slides underwent immunostaining with specific antibodies, including anti-6-4PP (clone 64M-2, Cosmo Bio, USA) and anti-CPD (clone TDM-2, Cosmo Bio, USA). Detection of 6-4PP+ and CPD+ was achieved using Alkaline Phosphatase/RED, Rabbit/Mouse (Agilent Technologies, USA). Negative controls were implemented by omitting the primary antibody. Sections were examined using an AxioImager Z1 microscope (Carl Zeiss MicroImaging, Inc., USA) in a blinded manner. Manual counting of all positive epidermal cells was performed, and the results were expressed as the percentage of positive cells in a given image.

Quantitative radical measurements

Quantitative analysis of UV-induced free radicals in RHE was performed by Electron paramagnetic resonance (EPR) spectroscopy on an X-band EPR spectrometer (Bruker Elexsys E500, BioSpin GmbH, Karlsruhe, Germany) using the spin marker PCA (3-(carboxy)-2,2,5,5-tetramethylpyrrolidin-1-oxyl) (Merck KgaA, Darmstadt Germany). Biopsies were obtained using 3-mm (ø) punch biopsies, treated with PCA, and exposed to the respective radiation sources: 60 mJ/cm2 233 nm and 150 mJ/cm2 UV radiation (refer to “UV irradiation” section). The samples were then immediately analyzed by EPR, as previously described by Zwicker et al.33. Considering that UV-A radiation is primarily responsible for radical formation and that the UV lamp also includes a UV-B fraction46, the radiation dose had to be increased by about 5 MED (150 mJ/cm2) for skin type II to achieve a biological response comparable to that of 233 nm irradiation.

Melanin distribution

Melanin distribution in human skin in vivo and in tanned RHE was assessed longitudinally and transversely by Fontana-Masson staining (see “In vivo human skin” section) and TPE-FLIM, respectively. Imaging by TPE-FLIM was performed using a two-photon microscope (DermaInspect™, JenLab GmbH, Jena, Germany) integrated with a tunable femtosecond Ti:sapphire laser (Mai Tai® XF, Newport Spectra-Physics GmbH, Darmstadt, Germany). The laser power was set to 45 mW. Images were acquired upon excitation at 760 nm and detection at 550 nm. In vitro and in vivo imaging was performed with an NA oil-immersion objective. A drop of saline solution was placed on the area of the skin or the RHE to be imaged. For the measurement, a metal accessory containing a cover glass with a drop of immersion oil was placed directly on the skin of the volunteers (volar side of the forearm) or the RHE. Images sized 200 × 200 µm and 50 × 50 µm were recorded on at least 6 spots per sample in the basal epidermal layer (above the papillary layer and collagen bundles) as well as in the stratum spinosum (10 µm from the stratum basale).

Data analysis

Fluorescence lifetime data analysis was performed using SPCImage software version 8.4 (Becker & Hickl GmbH, Berlin, Germany) assuming a bi-exponential decay and a binning value of 2. Melanin distribution was determined both within the cells of the stratum basale and the global distribution in the stratum basale. In the first case, 50 × 50 µm FLIM images (clearly delineated cells), were evaluated for the mean fluorescence lifetime (τm), with melanin contributing significantly to very short fluorescence lifetimes (τ1). The use of the τm, defined as the weighted average of the short lifetime components (τ1) and long lifetime component 2) in each image pixel and their respective amplitudes (a1and (a2), has been shown to correlate directly with the melanin concentration and was then justified for the evaluation of melanin distribution in the basal cells24,26,29.

$${tau }_{m}= frac{{a}_{1}{tau }_{1}+ {a}_{2}{tau }_{2 }}{{a}_{1}+ {a}_{2}}.$$

(2)

The images were exported in tif format and further processed for melanin distribution analysis using Fiji/ImageJ 1.53q (W. Rasband, NIH, USA). A region of interest of 400 × 400 pixels was defined and the manual segmentation of each of the defined cells was performed. The melanin area in each cell was expressed as the ratio of the number of melanin pixels to the number of epidermal cell pixels (excluding the nucleus) and was calculated using Matlab software (MathWorks, Inc., Natick, MA, USA). Pixels with τm < 480 ps would correspond to melanin pixels, as described before by Pena et al. in a recent publication26.

For the second case, the overview of the melanin distribution in the stratum basale, 200 × 200 µm FLIM images were processed by applying different thresholds to create the ideal melanin mask based on τm as well as τ1 and a1. For τm, pixels with τm < 480 ps would correspond to melanin pixels. For τ1, a mask of τ1 < 150 ps was set, and the amplitude a1 was varied between 80 and 100 until the best correlation model with skin pigmentation was obtained. The melanin area in the stratum basale is defined as the ratio of the number of melanin pixels to the number of total pixels and is calculated using Matlab software.

Statistical analysis

Statistical analyses were performed using IBM SPSS® Statistics 26 software (IBM, Armonk, N.Y., USA). For multiple measurements, a mean with corresponding standard error of the mean was calculated (MW ± SEM) and a test for outliers was performed. The relationship between spectrophotometric melanin-related descriptor (melanin content) and skin color optical parameter by ITA° was described graphically using a scatter plot and a linear regression model. The relationship between melanin area in the stratum basale (measured by TPE-FLIM) and ITA° was described graphically using a scatter plot and a polynomial regression model. The 95% confidence and prediction intervals were presented. The strength of the relationship was characterized by the coefficient of determination R2 (the square of the correlation coefficient) and tested for significance by ANOVA.

The Shapiro–Wilk test was used to test data for normal distribution and the Levene test for equality of variances. Two-tailed Student’s t-tests were performed for mean comparisons of the melanin content. For radical formation results, multiple comparisons of means were performed using a Kruskal–Wallis ANOVA, followed by Bonferroni post hoc tests. Pairwise comparisons for more than three groups (DNA damage) with non-parametric results and a significant difference under Kruskal–Wallis ANOVA were performed using multiple Wilcoxon-Mann–Whitney tests with manual Bonferroni correction. A p-value < 0.05 was chosen to indicate a significant difference. Data visualization as well as determination of regressions was performed using OriginPro® version 2019b (OriginLab Corporation, Northampton, Massachusetts, USA).

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