Human Skin Explants
Human skin explants were obtained, with informed consent from healthy female donors undergoing abdominoplasty, under authorization granted by the French government ethical committee according to French law L.1245 CSP. The explants were sourced from Biopredic, France. Within 2 h of surgery, skin was cut into 0.8 cm2 pieces and placed epidermis side up in 6-well Transwell plates containing 1.5 ml of Dulbecco’s Modified Eagle’s Medium (DMEM; GIBCO, Grand Island, NY, USA), supplemented with 2 mM l-glutamine (GIBCO) and antibiotics (100 IU/ml penicillin G and 100 lU/ml streptomycin; GIBCO). Explants were incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 48 h prior to study initiation.
M. furfur (DSM 6170) was obtained from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Brunswick, Germany) and cultivated in specific modified Dixon medium (m-Dixon; Sigma Aldrich, St. Louis, USA). Fungal cultures were incubated at 30 °C for 42–48 h.
The investigational product tested was an NSFC containing stearyl glycyrrhetinate, piroctone olamine, and biosaccharide gum-2 (full composition in Table 1). NSFC without anti-inflammatory ingredients (NSFC-AI) had a similar composition to NSFC, with the exception of ingredients with known or suspected anti-inflammatory effects (Table 1). Petrolatum (Vaselina pura filante Acofarderm), ketoconazole 2% cream (Fungarest 20 mg/g®), and hydrocortisone (HC) 1% cream (Cortaid Hydrocortisone Maximum Strength Anti-Itch Cream) were purchased at a local pharmacy.
SEBD Model Development and Product Testing
Skin explants were stripped using D-squame tapes (Clinical and Derm LLC, Dallas, USA) to remove approximately 40% of the stratum corneum (confirmed by tape absorbance measurement). Control skin samples were not stripped. At 48 h after stripping, skin explants were inoculated with 1 × 106 colony forming units (CFU) of M. furfur and incubated at 37 °C, 5% CO2, and 95% humidity. At 24 h after inoculation, test products (dose 2 mg/cm2) were applied topically to explants using a micropipette and spread with a microspatula. Control explants were not treated. Two independent studies and at least three replicates for each product were performed.
Determination of Skin Viability
Skin viability was determined using a commercially available lactate dehydrogenase (LDH) assay kit (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega, WI, USA) according to the manufacturer’s specifications. Skin viability was additionally measured using a resazurin assay. Briefly, skin explants were treated with 6 µM of resazurin NaCl solution (Sigma Aldrich, St. Louis, USA) for 1 h, and the concentration of resorufin (RES) formed was quantified in a fluorometer plate reader (530 nm excitation wavelength and 590 nm emission wavelength).
Malassezia furfur Quantification and Visual Analysis
Viable fungal cells were recovered from skin explants by tape stripping. Tape strips were immersed in a solution of physiological buffered saline and 0.1% Triton X-100 and the number of CFUs determined by serial dilution in m-Dixon followed by direct plating. Plates were incubated at 30 °C for 42–48 h. Presence of M. furfur on skin explants was visualized by staining with Crystal Violet (0.5%; Sigma Aldrich, St. Louis, USA).
Cytokine levels (TNF-α, IL-8, and IL-6) in explant culture media were determined using a commercially available ELISA kit according to the manufacturer’s specifications (Thermo Fisher Scientific, MA, USA). Results were expressed as average interleukin concentration (picograms per milliliter).
Results are reported as mean ± standard deviation (SD). The homogeneity of variance was confirmed by the Levene’s test and the normality confirmed by the Anderson–Darling test. Unpaired t tests and one-factor analysis of variance (ANOVA) with Bonferroni–Dunn’s correction were performed to assess differences between groups. A p value less than 0.05 was considered significant.