An ex vivo human skin model for studying skin barrier repair

In the studies described in this study, we introduce a novel ex vivo human skin barrier repair model. To develop this, we removed the upper layer of the skin, the stratum corneum (SC) by a reproducible cyanoacrylate stripping technique. After stripping the explants, they were cultured in vitro to allow the regeneration of the SC. We selected two culture temperatures 32°C and 37°C and a period of either 4 or 8 days. After 8 days of culture, the explant generated SC at a similar thickness compared to native human SC. At 37°C, the early and late epidermal differentiation programmes were executed comparably to native human skin with the exception of the barrier protein involucrin. At 32°C, early differentiation was delayed, but the terminal differentiation proteins were expressed as in stripped explants cultured at 37°C. Regarding the barrier properties, the SC lateral lipid organization was mainly hexagonal in the regenerated SC, whereas the lipids in native human SC adopt a more dense orthorhombic organization. In addition, the ceramide levels were higher in the cultured explants at 32°C and 37°C than in native human SC. In conclusion, we selected the stripped ex vivo skin model cultured at 37°C as a candidate model to study skin barrier repair because epidermal and SC characteristics mimic more closely the native human skin than the ex vivo skin model cultured at 32°C. Potentially, this model can be used for testing formulations for skin barrier repair.     

Molecular interactions of plant oil components with stratum corneum lipids correlate with clinical measures of skin barrier function

Plant‐derived oils consisting of triglycerides and small amounts of free fatty acids (FFAs) are commonly used in skincare regimens. FFAs are known to disrupt skin barrier function. The objective of this study was to mechanistically study the effects of FFAs, triglycerides and their mixtures on skin barrier function. The effects of oleic acid (OA), glyceryl trioleate (GT) and OA/GT mixtures on skin barrier were assessed in vivo through measurement of transepidermal water loss (TEWL) and fluorescein dye penetration before and after a single application. OA's effects on stratum corneum (SC) lipid order in vivo were measured with infrared spectroscopy through application of perdeuterated OA (OA‐d₃₄). Studies of the interaction of OA and GT with skin lipids included imaging the distribution of OA‐d₃₄ and GT ex vivo with IR microspectroscopy and thermodynamic analysis of mixtures in aqueous monolayers. The oil mixtures increased both TEWL and fluorescein penetration 24 h after a single application in an OA dose‐dependent manner, with the highest increase from treatment with pure OA. OA‐d₃₄ penetrated into skin and disordered SC lipids. Furthermore, the ex vivo IR imaging studies showed that OA‐d₃₄ permeated to the dermal/epidermal junction while GT remained in the SC. The monolayer experiments showed preferential interspecies interactions between OA and SC lipids, while the mixing between GT and SC lipids was not thermodynamically preferred. The FFA component of plant oils may disrupt skin barrier function. The affinity between plant oil components and SC lipids likely determines the extent of their penetration and clinically measurable effects on skin barrier functions.     

pH in nature,humans and skin

The pH plays an important physiological role in nature and humans. pH varies from 1 to 8 in human organs with tight regulation in blood and epithelia of barrier organs. The physiological pH of the stratum corneum is 4.1–5.8 and several mechanisms contribute to its formation: filaggrin degradation, fatty acid content, sodium‐hydrogen exchanger (NHE1) activation and melanosome release. First, the acidic pH of the stratum corneum was considered to present an antimicrobial barrier preventing colonization (e.g. by Staphylococcus aureus and Malassezia). Later on, it was found that the pH influences skin barrier function, lipid synthesis and aggregation, epidermal differentiation and desquamation. Enzymes of ceramide metabolism (e.g. β‐glucocerebrosidase or acid sphingomyelinase) as well as proteases (e.g. chymotryptic enzyme or cathepsin D linked to epidermal differentiation and desquamation) are regulated by the pH. Experimental disruption of the physical barrier leads to an increase of pH, returning to normal levels only after many hours. Inflammatory skin diseases and diseases with an involvement of the epidermis exhibit a disturbed skin barrier and an increased pH. This is known for atopic dermatitis, irritant contact dermatitis, ichthyosis, rosacea and acne, but also for aged and dry skin. Normalizing the pH by acidification through topical treatment helps to establish a physiological microbiota, to repair skin barrier, to induce epidermal differentiation and to reduce inflammation.     

紫外线照射对表皮屏障功能的影响

 表皮屏障在抵御外界危险因素侵害的过程中发挥重要作用。表皮屏障中各组成部分的结构及功能失调与人类的多种皮肤疾病有关。紫外线辐射等因素可引起表皮屏障功能障碍,紫外线照射使表皮屏障变得更易渗透,经皮水丢失量增加,病原体和过敏原更易进入体内,其机制可能与角质层屏障中角化包膜结构蛋白、细胞外脂质与天然保湿因子的含量降低,以及颗粒层中紧密连接屏障功能的减弱有关。近年来一些研究拓展了紫外线照射对表皮屏障的结构与功能影响的认识,本文综述紫外线照射与表皮屏障功能的影响。     

Repair of acetone- and sodium lauryl sulphate-damaged human skin barrier function using topically applied emulsions containing barrier lipids

BACKGROUND: It is generally acknowledged that well-formulated moisturizing skin care products can restore disturbed barrier function that can be assessed by transepidermal water loss (TEWL) measurements. When ceramides and/or other barrier lipids are incorporated, it is, however, not always clearly demonstrated which ingredients of the formulation exert the beneficial effects. OBJECTIVES: In this study the effects of topically applied ceramide-containing mixtures on the barrier repair of sodium lauryl sulphate (SLS)- and acetone-induced skin damage have been studied in human volunteers. TEWL and stratum corneum hydration measurements were carried out. The emulsions applied contained either a mixture of two types of ceramides, CerIII and CerIIIB (emulsion 1) or a complete mixture of ceramides III, IIIB and VI together with phytosphingosine, cholesterol and the free fatty acid linoleic acid (emulsion 2). RESULTS: After SLS damage, it was observed that barrier recovery was significantly accelerated by topical application (14 days, 2 x/d) of emulsion 2 compared with the results obtained with emulsion 1. Corneometrical results were not relevant due to the occurrence of scaly fissured skin, failing to provide a good skin/probe contact. Although no effect on TEWL could be observed, the improvement of skin hydration after acetone treatment and a single application of the emulsions, was significantly more positive for emulsion 2 than for emulsion 1. CONCLUSIONS: The investigative methods used in this study show that ceramides combined with other skin lipids can improve barrier repair after damage.     

A topical heparinoid‐containing product improves epidermal permeability barrier homeostasis in mice

Because of the importance of epidermal functions, including stratum corneum hydration and maintenance of permeability barrier homeostasis, in the pathogenesis of a variety of cutaneous and systemic disorders, a wide range of products has been developed to improve epidermal functions. However, the underlying mechanisms whereby certain products, including heparinoid‐containing product, are far little understood. In the present study, we assessed the impact of a heparinoid‐containing product, Hirudoid^® cream, on epidermal permeability barrier function and expression levels of a panel of epidermal mRNA related to the formation/maintenance of the permeability barrier in mouse skin. Our results showed that while the baseline levels of transepidermal water rates remained unchanged, treatment with Hirudoid^® cream twice daily for 7 days significantly accelerated permeability barrier recovery and increased stratum corneum hydration. In parallel, expression levels of epidermal mRNA for certain differentiation marker‐related proteins, lipid synthetic enzymes, keratinocyte proliferation and antimicrobial peptides also increased significantly. Together, these results provide the underlying mechanisms by which topical Hirudoid^® cream improves epidermal permeability barrier and antimicrobial function. Because of its benefits for epidermal functions, heparinoid‐containing product could be more useful in the management of skin conditions, characterized by abnormal permeability barrier and antimicrobial function.     

The protein phosphatase 2A regulatory subunit Ppp2r2a is required for Connexin‐43 dephosphorlyation during epidermal barrier acquisition

Epidermal barrier acquisition during late mammalian development is a prerequisite for terrestrial existence. Over a 24‐h period, the epidermis goes from being a barrier‐deficient, dye permeable epithelium to a barrier‐competent epithelium. We have previously shown that Akt signalling is necessary for barrier acquisition in the mouse and that the protein phosphatase 2A regulatory subunit Ppp2r2a causes barrier acquisition by dephosphorylation of cJun. Here, we demonstrate that there is transient interaction between the gap junction protein Connexin 43 (Cx43) and Zonula occludins‐1 (Zo‐1) during epidermal barrier acquisition. Ppp2r2a knockdown prevented plasma membrane co‐localisation and interaction between the two proteins. Ppp2r2a knockdown also increased phosphorylation at Serine 368 of Connexin 43. Cx43 phosphorlyation at Serine368 occurred just prior to the interaction between Connexin 43 and Zo‐1. We therefore propose a model in which Ppp2r2a is required both for the initial interaction between Zo‐1 and Cx43 and the consequent dephosphorylation of Connexin 43, preventing interaction of Zo‐1 and allowing Zo‐1 to initiate tight junction formation and barrier acquisition.     

表皮通透屏障功能对皮肤的调节作用及意义

表皮通透屏障功能除调节水分经表皮进出机体外,还对皮肤的其他生物功能如炎症、表皮增生、pH及离子的分布等也具有重要地调节作用。而且,维持表皮通透屏障功能在最佳水平有利于预防某些皮肤病的发生;改善皮肤屏障功能有助于某些皮肤病的治疗。     

Stress test of the skin: The cutaneous permeability barrier treadmill

Studying skin barrier function is central to our understanding of many skin disorders. The past decade has seen a surge of skin barrier related investigative work. Genetic, biochemical and cell biology experiments have added much evidence to the importance of the barrier in disease pathogenesis of a variety of disorders including ichthyosis, atopic dermatitis and psoriasis. However, functional assays prove ever more important to demonstrate relevance of any of these findings. A paper published by Monash and Blank 60 years ago describes a stress test of the skin barrier, measuring skin barrier recovery, a functional test of tremendous implications. This seminal paper has not been cited for almost 15 years, time to acknowledge its critical importance and to review the relevance of this method today.     

Epidermal barrier lipids in human vernix caseosa: corresponding ceramide pattern in vernix and fetal skin

BACKGROUND: Vernix caseosa is a protective biofilm covering the fetus during the last trimester. Vernix and epidermal barrier lipids (i.e. cholesterol, free fatty acids and ceramides) appear to share protective functions for fetal and neonatal skin. OBJECTIVES: To analyse vernix samples for epidermal barrier lipid content, and to compare lipid profiles of vernix with those of fetal and postnatal epidermis. METHODS: Vernix samples were collected from 21 healthy term neonates. Skin samples were collected from 10 fetuses aborted between gestational week (GW) 16 and 25, nine infants and 11 older children. Lipids were extracted according to standard protocols and analysed by high-performance thin-layer chromatography. RESULTS: Vernix contained 196.5 +/- 70.1 microg barrier lipids mg-1 protein (mean +/- SD). Cholesterol formed the major barrier lipid fraction (52.8%), followed by free fatty acids (27.7%) and ceramides (20.1%). The ceramide composition of vernix resembled that of mid-gestational (GW 23-25) fetal epidermis both qualitatively and quantitatively, while there were major differences from postnatal epidermis. The total epidermal ceramide concentration increased significantly between prenatal and postnatal samples. CONCLUSIONS: The composition pattern of ceramides mirrors that of mid-gestational fetal epidermis. Vernix thus represents a 'homologous' substitute for the immature epidermal barrier in fetal skin. The differential role of individual ceramides in this process remains to be established.     

Nicotinamide increases biosynthesis of ceramides as well as other stratum corneum lipids to improve the epidermal permeability barrier

BACKGROUND: Stratum corneum lipids, particularly ceramides, are important components of the epidermal permeability barrier that are decreased in atopic dermatitis and aged skin. OBJECTIVES: We investigated the effects of nicotinamide, one of the B vitamins, on biosynthesis of sphingolipids, including ceramides and other stratum corneum lipids, in cultured normal human keratinocytes, and on the epidermal permeability barrier in vivo. METHODS: The rate of sphingolipid biosynthesis was measured by the incorporation of [14C]-serine into sphingolipids. RESULTS: When the cells were incubated with 1-30 micromol L-1 nicotinamide for 6 days, the rate of ceramide biosynthesis was increased dose-dependently by 4.1-5. 5-fold on the sixth day compared with control. Nicotinamide also increased the synthesis of glucosylceramide (7.4-fold) and sphingomyelin (3.1-fold) in the same concentration range effective for ceramide synthesis. Furthermore, the activity of serine palmitoyltransferase (SPT), the rate-limiting enzyme in sphingolipid synthesis, was increased in nicotinamide-treated cells. Nicotinamide increased the levels of human LCB1 and LCB2 mRNA, both of which encode subunits of SPT. This suggested that the increase in SPT activity was due to an increase in SPT mRNA. Nicotinamide increased not only ceramide synthesis but also free fatty acid (2.3-fold) and cholesterol synthesis (1.5-fold). Topical application of nicotinamide increased ceramide and free fatty acid levels in the stratum corneum, and decreased transepidermal water loss in dry skin. CONCLUSIONS: Nicotinamide improved the permeability barrier by stimulating de novo synthesis of ceramides, with upregulation of SPT and other intercellular lipids.     

A topical lipophilic niacin derivative increases NAD, epidermal differentiation and barrier function in photodamaged skin

The effects of myristyl nicotinate (MN), a nicotinic acid derivative designed to deliver nicotinic acid to skin without vasodilatation, on subjects with photodamaged skin have been studied. MN increased skin cell nicotinamide adenine dinucleotide (NAD) by 25% (P = 0.001) demonstrating effective delivery of nicotinic acid to skin. Relative to placebo, MN treatment of photodamaged facial skin increased stratum corneum thickness by approximately 70% (P = 0.0001) and increased epidermal thickness by approximately 20% (P = 0.001). In two separate studies, MN treatment increased rates of epidermal renewal by 6% (P = 0.003) to 11% (P = 0.001) and increased the minimal erythemal dose by 8.9 (P = 0.07) and 10% (P = 0.05) relative to placebo. MN treatment resulted in reductions in the rates of transepidermal water loss (TEWL) of approximately 20% relative to placebo on cheeks (P = 0.012) and arms (P = 0.017) of study subjects. Results of a tape stripping challenge before and after MN treatment demonstrated a significant correlation (P = 0.03) between increased skin NAD content and resistance to changes in TEWL for MN treated but not placebo subjects. Rates of TEWL changed more rapidly and to a greater extent in atopic subjects compared with normal subjects. The results indicate that MN enhances epidermal differentiation and barrier function in skin, suggesting that this method of nicotinic acid delivery may prove useful in limiting progression of actinic skin damage and possibly in treating other conditions involving skin barrier impairment.     

Modulation of stratum corneum lipid composition and organization of human skin equivalents by specific medium supplements

Our in‐house human skin equivalents contain all stratum corneum (SC) barrier lipid classes, but have a reduced level of free fatty acids (FAs), of which a part is mono‐unsaturated. These differences lead to an altered SC lipid organization and thereby a reduced barrier function compared to human skin. In this study, we aimed to improve the SC FA composition and, consequently, the SC lipid organization of the Leiden epidermal model (LEM) by specific medium supplements. The standard FA mixture (consisting of palmitic, linoleic and arachidonic acids) supplemented to the medium was modified, by replacing protonated palmitic acid with deuterated palmitic acid or by the addition of deuterated arachidic acid to the mixture, to determine whether FAs are taken up from the medium and are incorporated into SC of LEM. Furthermore, supplementation of the total FA mixture or that of palmitic acid alone was increased four times to examine whether this improves the SC FA composition and lipid organization of LEM. The results demonstrate that the deuterated FAs are taken up into LEMs and are subsequently elongated and incorporated in their SC. However, a fourfold increase in palmitic acid supplementation does not change the SC FA composition or lipid organization of LEM. Increasing the concentration of the total FA mixture in the medium resulted in a decreased level of very long chain FAs and an increased level of mono‐unsaturated FAs, which lead to deteriorated SC lipid properties. These results indicate that SC lipid properties can be modulated by specific medium supplements.     

Barrier recovery rate varies time-dependently in human skin

The recovery in cutaneous barrier functions, assessed in terms of transepidermal water loss, 1 h after tape stripping of volar forearm skin in human volunteers, was investigated at different times over the 24 h day. The barrier recovery rate was significantly lower between 20:00 h and 23:00 h than that at other time points. The skin surface temperature and the basal transepidermal water loss reached their highest values at about 03:00 h (33.6 degrees C and 0.30 mg cm-2 h-1), while the cortisol level in the saliva was highest at 09:00 h (7.8 pmol mL-1). These results suggest significant time-dependent variation in cutaneous barrier repair independent of changes in skin temperature and cortisol level.     

主观皮肤类型与皮肤屏障功能的关系

目的:比较不同主观皮肤类型(油性、中性和干性皮肤)屏障功能指标的差异。方法:利用无创性方法对自评为油性、中性和干性皮肤的20~25岁北京城市女性(各30例)进行皮脂分泌率(SER)、角质层含水量、p H值和经皮肤水分丢失(TEWL)值的检测,并采用胶带连续粘脱后监测TEWL值变化的方法评价角质层完整性,采用角质层取样蛋白定量的方法评价角质层黏合力和丝氨酸蛋白酶活性。结果:主观皮肤类型为中性皮肤者具有最佳的屏障功能;干性皮肤和油性皮肤者面颊部的屏障功能均有不同程度的受损,表现为TEWL值明显升高、p H值升高、丝氨酸蛋白酶活性增加;但二者也有区别,油性皮肤者角质层完整性下降更明显,而干性皮肤主要是角质层黏合力明显减弱。结论:自评为油性和干性皮肤者与中性皮肤者相比,屏障功能均存在一定程度的缺陷,油性皮肤与干性皮肤均具有不同特点,且与其屏障受损的机制不同有关。     

Barium sulphate with a negative zeta potential accelerates skin permeability barrier recovery and prevents epidermal hyperplasia induced by barrier disruption

BACKGROUND: Barium sulphate, a stable inorganic material, has been used in contrast media and cosmetic products because of its stability. As a negative external electric potential accelerates the skin barrier repair after barrier disruption, we hypothesized that topical application of barium sulphate may affect the skin barrier recovery rate depending on its zeta potential. OBJECTIVES: To investigate whether barium sulphate particles in aqueous solution have different zeta potentials depending on their surface structure, and to investigate the possible relation between zeta potential and skin barrier recovery rate. METHODS: Mice were subjected to tape stripping to disrupt barrier function, or were treated with acetone and kept in a dry environment to induce epidermal hyperplasia. They were then treated with different forms of barium sulphate, and barrier recovery was monitored by measurements of transepidermal water loss. RESULTS: There was a significant correlation between the barrier recovery rate and zeta potential of barium sulphate applied topically. Barium sulphate with a negative zeta potential significantly accelerated barrier recovery, but barium sulphate with a positive zeta potential did not accelerate or even delayed barrier repair. Barium sulphate with a negative zeta potential had an X-ray diffraction pattern different from that with a positive potential. The distribution of calcium in the epidermis was also influenced by the polarity of zeta potential. CONCLUSIONS: These findings suggest a new pharmacological approach towards altering barrier function or epidermal hyperplasia with inorganic particles in healthy and diseased skin.