Xenobiotic metabolizing enzymes in human skin and SkinEthic reconstructed human skin models

Skin metabolism is becoming a major consideration in the development of new cosmetic ingredients, skin being the first organ exposed to them. In order to replace limited samples of Excised human skin (EHS), in vitro engineered human skins have been developed. 3D models are daily used to develop and evaluate new cosmetic ingredients and have to be characterized and compared with EHS in terms of metabolic capabilities. This work presents the determination of apparent catalytic parameters (apparent Vmax, Km and the ratio Vmax/Km) in 3D models compared with EHS for cytochrome P450 dependent monooxygenase isoforms involved in drug metabolism, esterases, alcohol dehydrogenases, aldehyde dehydrogenases, peroxidases, glutathione S‐transferases, N‐acetyl transferases, uridinyl diphosphate glucuronyl transferases and sulfotransferases. Results show that all these enzymes involved in the metabolism of xenobiotics are expressed and functional in the EHS and 3D models. Also, the Vmax/Km ratios (estimating the intrinsic metabolic clearances) show that the metabolic abilities are the most often comparable between the skin models and EHS. These results indicate that the 3D models can substitute themselves for EHS to select cosmetic ingredients on the basis of their metabolism, efficacy or/and safety.     

Effect of exposure of human skin to a dry environment

Background/aims: Changes in the skin conditions after exposure to low humidity have been generally experienced in everyday life, but there have been few reports to approach it—especially in healthy skin. We have examined the effect of low humidity on healthy human skin by using noninvasive measurement devices. Methods: Skin conditions on the ventral forearm and the cheek before and after 3 or 6 h exposure to low humidity were evaluated by measuring skin surface conductance, skin surface capacitance and transepidermal water loss. Skin surface replicas were also taken before and after exposure and analysed for roughness parameters—Ra (arithmetic mean roughness value), Rz (10-point height), Sm (mean value of the profile element) and VC1 (anisotropy of skin furrows). Results: There was a significant decrease of water content of stratum corneum at both test sites from the time points 0 h to 3 h and 6 h (P < 0.01) and transepidermal water loss from the time point 0 h to 6 h (P < 0.05). Regarding the roughness parameters, a significant increase of Rz in the directions of 45°/225° and 90°/270° to the body axis and Sm in the directions of 0°/180° (P < 0.05) on the forearm and VC1 (P < 0.05) on the cheek. The parameter Rz also showed a tendency to increase in the directions of 45°/225° (P = 0.06) on the cheek. A specific pattern of the changes to be related to the Langer's lines in the surface morphology was observed. The changes of skin surface pattern in our experiment lead us to consider that exposure to low humidity even in such a short period would be related to inducing aggravation of skin texture and the formation of fine wrinkles. Conclusion: A short exposure of skin to a low-humidity environment induced changes in the moisture contents in the stratum corneum and skin surface pattern, which lead us to assume that a dry environment in our daily life would make fine wrinkles related to lack of water in the stratum corneum.     

The relationship between skin function,barrier properties,and body‐dependent factors

Background

Skin is a multilayer interface between the body and the environment, responsible for many important functions, such as temperature regulation, water transport, sensation, and protection from external triggers.

Objectives

This paper provides an overview of principal factors that influence human skin and describes the diversity of skin characteristics, its causes and possible consequences. It also discusses limitations in the barrier function of the skin, describing mechanisms of absorption.

Methods

There are a number of in vivo investigations focusing on the diversity of human skin characteristics with reference to barrier properties and body‐dependent factors.

Results

Skin properties vary among individuals of different age, gender, ethnicity, and skin types. In addition, skin characteristics differ depending on the body site and can be influenced by the body‐mass index and lifestyle. Although one of the main functions of the skin is to act as a barrier, absorption of some substances remains possible.

Conclusions

Various factors can alter human skin properties, which can be reflected in skin function and the quality of everyday life. Skin properties and function are strongly interlinked.

     

Remittance spectroscopy mapping of human skin in vivo

Remittance spectroscopy of human skin may be influenced by probe application pressure and body site.

Methods:

We investigated remittance spectroscopy qualities of human skin in vivo in different areas: a) forearm, b) frontal, c) back, d) back of the hand, e) palms and f) cheek. Twenty volunteers of skin type 2–3 free of inflammatory skin diseases, were enrolled into the study. Spectroscopy readings were performed with a fiber optic spectrometer (Ocean Optics, USA). The readings were taken with standardized force (0 and 100 pont) by applying the probe vertically to the skin surface. The remittance in relation to wavelength was registered. White light with wavelengths from 420 to 750 nm were used. Individual remittance values and their standard deviations were obtained from 20 readings each.

Results/Conclusions:

Spectroscopic patterns of skin are influenced by external force and regional factors. Standardization remains critical for the use of this approach in bioengineering of skin.     

Comparison of structure and organization of cutaneous lipids in a reconstructed skin model and human skin: spectroscopic imaging and chromatographic profiling

The use of animals for scientific research is increasingly restricted by legislation, increasing the demand for human skin models. These constructs present comparable bulk lipid content to human skin. However, their permeability is significantly higher, limiting their applicability as models of barrier function, although the molecular origins of this reduced barrier function remain unclear. This study analyses the stratum corneum (SC) of one such commercially available reconstructed skin model (RSM) compared with human SC by spectroscopic imaging and chromatographic profiling. Total lipid composition was compared by chromatographic analysis (HPLC). Raman spectroscopy was used to evaluate the conformational order, lateral packing and distribution of lipids in the surface and skin/RSM sections. Although HPLC indicates that all SC lipid classes are present, significant differences are observed in ceramide profiles. Raman imaging demonstrated that the RSM lipids are distributed in a non‐continuous matrix, providing a better understanding of the limited barrier function.     

Effects of physical and chemical treatments upon biophysical properties and micro-relief of human skin

The aim of the present study was to determine the attendant effects of physical (tape-stripping) and chemical (three commercial hydrating formulations) treatments upon biophysical and micro-relief properties of human skin. In the first set of experiment, the effects of tape-stripping onto human stratum corneum (SC) biophysical and micro-relief properties were assessed in nine volunteers. Transepidermal water loss (TEWL), skin hydration and micro-relief parameters (including total length of the lines in mm per mm(2); total surface in %; roughness of the skin measured in gray level (Ra); maximum profile valley (Rv) depth; maximum profile peak height (Rp); maximum height (Rt), peak density (Pc) and coefficient of anisotropy) were determined by using SkinEvidence Pro after subsequent tape-stripping of SC. The relevance of roughness determination as gray level by SkinEvidence Pro was confirmed by using surface roughness standards. In the second set of experiment, the effectiveness of three commercial hydrating formulations onto human SC biophysical parameters and micro-relief properties were assessed in six volunteers. TEWL, hydration and micro-relief parameters were assessed onto pre-treated acetone skin and then treated by three commercial hydrating formulations after 2, 4 and 6 h skin exposure. A linear relation between hydration and cutaneous parameters (total length of the lines, Ra and Rp) as function of SC removed was shown. Skin barrier properties evaluated by TEWL measurements, were not modified by topical formulations. However, skin treated by topical formulations showed slightly higher hydration than the one determined in control group, while micro-relief parameters were not modified. In this study was showed that biophysical and micro-relief parameters were closely related in tape-stripping experiment. Efficiency of topical formulations was suggested upon skin hydration but not onto skin micro-relief and barrier function recovering. From both experiments, it appears that different mechanisms relating to skin hydration and potential modification of cutaneous micro-relief were suggested.     

An improved human skin explant culture method for testing and assessing personal care products

Background

Cultured human skin models have been widely used in the evaluation of dermato-cosmetic products as alternatives to animal testing and expensive clinical testing. The most common in vitro skin culture approach is to maintain skin biopsies in an airlifted condition at the interface of the supporting culture medium and the air phase. This type of ex vivo skin explant culture is not, however, adequate for the testing of cleansing products, such as shampoos and body washes. One major deficiency is that cleansing products would not remain confined on top of the epidermis and have a high chance of running off toward the dermal side, thus compromising the experimental procedure and data interpretation.

Materials and Methods

Here, we describe an improved ex vivo method for culturing full-thickness human skin for the effective testing and evaluation of skin care products by topical application.

Results

This newly developed ex vivo human skin culture method has the ability to maintain healthy skin tissues for up to 14 days in culture. Importantly, the model provides a quick and safe way to evaluate skin care products at different time points after single or repetitive topical applications using a combined regimen of leave-on and wash-off. We found that the results obtained using the new skin culture method are reproducible and consistent with the data collected from clinical testing.

Conclusion

Our new ex vivo skin explant method offers a highly efficient and cost-effective system for the evaluation and testing of a variety of personal care products and new formulations.     

Laser induced autofluorescence studies of animal skin used in modeling of human cutaneous tissue spectroscopic measurements

BACKGROUND/PURPOSE: Laser-induced autofluorescence spectroscopy provides excellent possibilities for medical diagnostics of different tissue pathologies including cancer. However, to create the whole picture of pathological changes, investigators collect spectral information from patients in vivo or they study different tumor models to obtain objective information for fluorescent properties of every kind of healthy and diseased tissue. Therefore, it is very important to find the most appropriate, and close to the human skin, animal samples from the fluorescence point of view, which will allow the extrapolation of the animal data to human spectroscopic diagnostics. METHODS: In the present work, we examined the autofluorescence properties of different animal skin tissues, which are considered as the most common skin models. A nitrogen laser was used as an excitation source. Samples of healthy mouse, chicken and pig skin in vivo and/or ex vivo were studied and were compared with results obtained from investigations of healthy human skin in vivo. RESULTS AND CONCLUSION: Specific features of the recorded spectra are discussed and the possible origin of the obtained fluorescence signals is proposed. Quantitative evaluation of data extrapolation for each skin type is also depicted.     

Staphylococci of the normal human skin flora

Summary 352 strains of Staphylococci of the normal human skin flora were sampled from one volunteer by single scrabbing in a ca. 3 cm² measuring area. They were biotyped by the scheme of Pelzer et al. (1973)—a modified Baird-Parker-Scheme (1963)— and the resistance to antibiotics was investigated by the method of Bauer et al. (1966).All the nine biotypes of Staphylococci were found in variable quantities. It seems problematic to call one biotype as the main type. Morphologically identical colonies of Staphylococci from the indigenous flora of the human skin were not identical in their biotypes as previously described by Pelzer (1976).Only the investigation of all Staphylococci colonies from the culture plate can evaluate all biotypes of Staphylococci of the normal human skin flora, and can give the right quantitative correlation. Staphylococci were found to be sensitive and resistant up to four antibiotics, and one biotype did not show one type of antibiogram.     

Comparison of surface free energy between reconstructed human epidermis and in situ human skin

Background/aims: The objectives of this study were to characterise the Episkin^® model of reconstructed epidermis histologically and energetic, and to compare the results with the data obtained by Mavon (9, 10) on different areas of skin on the human body and on different skin types. Methods: Histologically, the two skin systems studied were stained using Goldner's trichome method. Surface free energy was determined by the measurement of contact angles, using the posed drop method. Results: Histologically, the two skin systems demonstrated the same cellular organisation in stratified layers. With regard to surface free energy, Episkin, skin from the forehead, normal and hyper‐seborrheic skins are hydrophilic surfaces, while skin from the forearm or of an oligo‐seborrheic type tends to be hydrophobic. Episkin and skin from the forehead exhibit similar surface free energy profiles. It is impossible to state whether Episkin has characteristics closer to those of normal or hyper‐seborrheic skin, but they differ significantly from those of oligo‐seborrheic skin. Conclusion: The results obtained during this study justify the use of Episkin in a range of tests where the use of in situ human skin raises problems.     

Modulation of skin collagen metabolism by irradiation: collagen synthesis is increased in irradiated human skin

Radiation-induced fibrosis is a common side-effect of cancer treatment. The pathophysiological events leading to fibrosis are not known in detail. We analysed the effect of therapeutic irradiation on human skin collagen synthesis, skin thickness, gelatinases and their inhibitors. Twenty randomly chosen women who had been treated for breast cancer with surgery and radiation therapy participated in the study. In each patient, the irradiated skin area was compared with a corresponding non-treated skin area. Suction blister fluid (SBF) and serum samples were analysed for the aminoterminal propeptides of type I and type III procollagens (PINP and PIIINP), tissue inhibitors of matrix metalloproteinases (MMPs) 1 and 2 (TIMP-1 and TIMP-2) and MMP-9 and MMP-2/TIMP-2 complex. Skin biopsies were analysed for PINP and immunohistochemical staining was used for PIIINP. In irradiated skin, PINP, PIIINP, TIMP-1 and MMP-2/TIMP-2 complex levels in SBF and the number of PINP-positive fibroblasts in tissue sections were significantly higher in comparison with non-treated skin. The levels of TIMP-2 in irradiated and non-irradiated skin were similar. MMP-9 could not be detected in SBF with the assay used. The serum levels of MMP-9 were higher in the treated subjects than the reference values. The serum values of PINP, PIIINP, TIMP-1, TIMP-2 and MMP-2/TIMP-2 complex were not significantly affected. These results indicate increased local collagen synthesis and accumulation of connective tissue in irradiated skin. The marked upregulation of collagen synthesis as a result of irradiation offers a possibility to treat this complication with compounds such as topical steroids which downregulate collagen synthesis.     

Viscoelastic properties of human skin and processed dermis

Background/aims: The purpose of this work is to attempt to determine the elastic spring constant for collagen and elastic fibers (elastin) in skin and to detemine if the values of these elastic constants are similar to those reported for other tissues.
Methods: We studied the viscoelastic mechanical properties of human skin and dermis by measuring the incremental stress-strain behavior. Elastic stress-strain curves were used to obtain the elastic spring constant of elastin and collagen while the collagen fibril length was obtained from the slope of viscous stress-strain curves.
Results: Our results suggest that the elastic spring constant for elastin is about 4.0 MPa while that for collagen is about 4.4 GPa. The former value is similar to that calculated for ligamentum nuchae while the latter value is about 70% of the value found for tendon and self-assembled type I collagen fibers. The differences between the elastic constants for collagen molecules in tendon and skin is hypothesized to reflect the higher molecular tilt angle and lower D period found in skin compared to tendon as well as a shorter fibril length.
Conclusion: The differences in the collagen types present in skin and tendon may influence collagen self-assembly and the resulting viscoelastic properties.