Strat-M® positioning for skin permeation studies: A comparative study including EpiSkin® RHE, and human skin.

In the development and optimization of dermatological products, In Vitro Permeation Testing (IVPT) is pivotal for controlled study of skin penetration. To enhance standardization and replicate human skin properties reconstructed human skin and synthetic membranes are explored as alternatives. Strat-M® is a membrane designed to mimic the multi-layered structure of human skin for IVPT. For instance, in Strat-M®, the steady-state fluxes (JSS) of resorcinol in formulations free of permeation enhancers were found to be 41 ± 5 µg/cm2·h for the aqueous solution, 42 ± 6 µg/cm2·h for the hydrogel, and 40 ± 6 µg/cm2·h for the oil-in-water emulsion. These results were closer to excised human skin (5 ± 3, 9 ± 2, 13 ± 6 µg/cm2·h) and surpassed the performance of EpiSkin® RHE (138 ± 5, 142 ± 6, and 162 ± 11 µg/cm2·h). While mass spectrometry and Raman microscopy demonstrated the qualitative molecular similarity of EpiSkin® RHE to human skin, it was the porous and hydrophobic polymer nature of Strat-M® that more faithfully reproduced the skin's diffusion-limiting barrier. Further validation through similarity factor analysis (∼80-85%) underscored Strat-M®'s significance as a reliable substitute for human skin, offering a promising approach to enhance realism and reproducibility in dermatological product development.

Stratum corneum ceramide profiles in vitro, ex vivo, and in vivo: characterization of the α-hydroxy double esterified ceramides.

The presence of a new ceramide subclass, the 1-O-acyl omega-linoleoyloxy ceramides [1-O-E (EO) Cer], has been previously highlighted in reconstructed human epidermis (RHE). These ceramides are double esterified on two positions. The first is the 1-O position of the sphingoid base moiety with a long to very long chain of acyl residues (1-O-E), and the second is the position of the ω-hydroxyl group of the fatty acid moiety with linoleic acid (EO). Considering its chemical structure and hydrophobicity, this subclass can contribute to the skin barrier. Thus, it is important to determine whether this subclass is also present in native human stratum corneum (SC). This work compares ceramide structures of this novel subclass between RHE (in vitro) and two sources of human SC (in vivo and ex vivo) using normal-phase high-performance liquid chromatography coupled to high-resolution mass spectrometry (NP-HPLC/HR-MSn). The results confirm the presence of this double esterified ceramide subclass [1-O-E (EO) Cer] in human SC. The molecular profile obtained from the RHE was very close to that found in the human SC (in vivo and ex vivo). In addition, thanks to the targeted MS2/MS3 analysis, a new ceramide subclass was discovered and characterized in the three studied samples. We propose to name it [A-1-O-E (EO) Cer] because in these ceramides species, the fatty acid-esterified with the sphingoid base on the 1-O position-is hydroxylated on the α position. These results highlight the potential of both the analytical method and the characterization approach employed in this study.

Assessment of the skin barrier function in the reconstructed human epidermis using a multimodal approach at molecular, tissue and functional levels.

Reconstructed human epidermis models are used as epidermis alternatives in skin research studies. It is necessary to provide molecular and functional characterization in order to assess these models. Our aim is to establish a link between the barrier function and the structure and composition of the stratum corneum using several complementary techniques. The following three studies were performed on reconstructed human epidermis during the keratinocyte differentiation process: (i) caffeine percutaneous penetration kinetics, (ii) epidermis thickness measurement, stratum corneum formation and lipid organization by Raman microspectroscopy and (iii) lipid composition evolution by liquid chromatography coupled to high-resolution mass spectrometry. The results demonstrated that the caffeine penetration decreased along the differentiation process. Raman in-depth images demonstrated an increase in stratum corneum and RHE thickness accompanied by the evolution of lipid organization. Lipid analysis showed an increase of the ceramide amount and an inverse relationship between ceramide and its precursor levels during the differentiation process. Different behaviors between several ceramide subclasses are highlighted and they relied on the corresponding differentiation stages. The generation of the most important ceramides for the barrier function is closely followed. A period shift between lipid generation and their organization was found. Our analytical data allowed identifying the following 3 groups of maturation days: before day 15, between days 15 and 19, and after day 19. The chemical and physiological states of the barrier function for each group are described thanks to a multimodal approach.

Biomolecular modifications during keratinocyte differentiation: Raman spectroscopy and chromatographic techniques.

From the basal layer until the stratum corneum, lipid and protein biomarkers associated with morphological changes denote keratinocyte differentiation and characterize each epidermis layer. Herein, we followed keratinocyte differentiation in the early stages using HaCaT cells over a period of two weeks by two complementary analytical techniques: Raman microspectroscopy and high-performance liquid chromatography coupled with high resolution mass spectrometry. A high concentration of calcium in the medium induced HaCaT cell differentiation in vitro. The results from both techniques underlined the keratinocyte passage from the granular layer (day 9) to the stratum corneum layer (day 13). After 13 days of differentiation, we observed a strong increase in the lipid content, decrease in proteins, decrease in DNA, and a decrease in glucosylceramides/ceramides and sphingomyelins/ceramides ratios.

Comprehensive characterization and simultaneous analysis of overall lipids in reconstructed human epidermis using NPLC/HR-MSn: 1-O-E (EO) Cer, a new ceramide subclass.

Stratum corneum lipids are responsible for the skin's barrier function. They are the final product of epidermis lipid biosynthesis. During this process, lipids evolve from simple to complex structures in three main levels respectively (stratum basal level, stratum granulosum level, and stratum corneum level). Our aim was to simultaneously analyze and characterize the structure of total epidermis lipids. A powerful analytical method (normal-phase liquid chromatography coupled with high-resolution mass spectrometry (NPLC/HR-MSn)) was developed in order to separate, in a single run, lipid classes with a wide polarity range. Chromatographic conditions were particularly designed to analyze lipids of intermediate polarity such as ceramides. Rich information was obtained about the molecular structure of keratinocyte differentiation biomarkers such as ceramides, glucosylceramides, and sphingomyelins and the microstructures of reconstructed human epidermis lipids using HR-MSn. A new subclass of ceramides, 1-O-Acyl Omega-linoleoyloxy ceramides [1-O-E (EO) Cer] has been highlighted. This class is double esterified on the 1-O-position of sphingoid base with long to very long chain acyl residues (1-O-E) and on the position of ω-hydroxyl group of fatty acid with the linolenic acid (EO). Considering its chemical structure and hydrophobicity, this subclass can contribute to the skin barrier. In addition, we detected a new epidermis sphingomyelins. Our lipidomic approach offers a direct access to epidermis biomarkers.

Impact of simulated biological aging on physicochemical and biocompatibility properties of cyclic olefin copolymers.

We study the effect of simulated biological aging on the properties of cyclic olefin copolymers and particularly their biocompatibility. Already reported as biocompatible polymers according to ISO/EN 10993 guidelines, COC are good candidates for medical devices. The influence of two major additives (antioxidants and lubricants) was investigated and comparison with non-aging COC was done. Four in vitro simulated biological conditions were tested: 2 extreme pH (1 and 9) to simulate digestive tract environment; THP-1-derived macrophages contact and pro-oxidant medium with hypochlorite solution simulating the oxidative attack during the foreign body reaction. After one month of incubation with the different media at 37 °C, surface topography was studied by atomic force microscopy (AFM) and IR spectroscopy. Extracts of incubated media were also analysed in chromatography to investigate potential degradation products. Cytotoxicity (MTT and LDH) of the materials was evaluated using cell culture methods with L929 fibroblasts. Oxidative stress (ROS and SOD analysis) and two inflammatory biomarkers (Il-6 and TNF-α secretion) were explored on THP-1-derived macrophages in direct contact with aged COC. Surface topography of COC was modified by aging conditions with an influence of antioxidant presence and under some conditions. HPLC analysis realized on freeze-dried solutions issued from the different incubations showed the presence of traces of low molecular weight compounds issued from polyphenolic antioxidant and from COC degradation. GC-MS analysis carried out directly on the different incubated COC, showed no detectable leachable molecules. No cytotoxicity has been observed with the different aged COC. However, results show that the pH environment had an influence on the cytotoxicity tests with a protecting effect of antioxidant presence; and pro-oxidant incubating conditions decreased cellular viability on COC. pH 1 and pH 9 conditions also induced an increase of ROS production which was partially reduced for COC containing an antioxidant or a lubricant. Il-6 production was globally more important for aged COC compared with basal condition and particularly for oxidative simulated environment. Those results indicate that physiological factors like pH or oxidant conditions have an impact on surface topography and on COC interaction with the biological environment but without compromising their biocompatibility. Antioxidant or lubricant presence could modulate these variations pointing out the necessity of a thoroughly investigation for biocompatibility assessment of COC as a component of implantable devices. COCs show a good biocompatibility even after accelerated aging under extreme biological conditions.

Origanum essential oils reduce the level of melanin in B16-F1 melanocytes.

BACKGROUND: Hyperpigmentation disorders are considered signs of skin aging and are aesthetically unpleasant. Most active ingredients used against hyperpigmentation disorders predominantly target tyrosinase activity. OBJECTIVES: To study the effect of two Origanum essential oils on the melanogenic activity of B16-F1 murine melanocytes. The main component of these oils, carvacrol, was also investigated and a model for anti-melanogenic activity is proposed. MATERIALS AND METHODS: B16-F1 melanocytes were exposed to different concentrations of essential oils and carvacrol. The level of tyrosinase and melanin was determined using spectrophotometric measurements. RESULTS: Essential oils of Origanum syriacum and Origanum ehrenbergii led to a significant 14% and 17% reduction in melanin level at 40 µg mL-1, respectively. However, neither demonstrated a significant effect on the level of intracellular tyrosinase. The same effects were found for carvacrol which led to a 30% reduction in melanin at 45 µg mL-1. CONCLUSION: Our results indicate that the oils studied are anti-melanogenic. We propose a mechanism, similar to that for hydroquinone, whereby carvacrol functions as a competitive inhibitor of tyrosinase, thus inhibiting oxidation of tyrosine and causing a deregulation of melanogenesis.

Biocompatibility assessment of cyclic olefin copolymers: Impact of two additives on cytotoxicity, oxidative stress, inflammatory reactions, and hemocompatibility.

This work reports the biocompatibility evaluation of cyclic olefin copolymers (COC) as candidates for implantable medical devices. The focus was to establish the influence of two major additives (antioxidant and lubricant) on the overall biocompatibility. The cytotoxicity was evaluated according to ISO 10993-5 guidelines using L929 fibroblasts, HUVEC, and THP-1-derived macrophages. Oxidative stress (ROS, GSH/GSSG, and SOD analysis) and pro-inflammatory cytokines (Il-6 and TNF-α secretion) were quantified using THP-1 cells in direct contact with films. Hemocompatibility was assessed through haemolysis testing, dynamic blood coagulation, platelet adhesion, and activation (membranous P-selectin expression). Results show that the different types of COC have successfully passed the in vitro biocompatibility tests. The presence of antioxidant induces however a slight decrease in ROS production in correlation with a high SOD activity and a modification in blood coagulation profile probably linked to antioxidant recrystallization phenomenon on the surface of COC. The lubricant presence reduced haemolysis, fibrinogen adhesion, and platelet activation. Surface nanotopography of COC highlights different types of needles and globules according to the present additive. Those primary results indicate that COC are promising biomaterial. However, additives influenced some biological parameters pointing out the necessity of a global approach of risk analysis for biocompatibility evaluation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3333-3349, 2017.