Generation of human skin equivalents under submerged conditions-mimicking the in utero environment.

In this study we generated human skin equivalents (HSEs) under submerged conditions mimicking the aqueous in utero environment and investigated the morphology and differentiation process of the formed epidermis. Further, the skin barrier, which resides in the stratum corneum (SC), was characterized by its lipid content, hydration level, and natural moisturizing factor level. The submerged HSEs showed comparable tissue morphology and similar expression of several differentiation markers and SC lipid composition compared with HSEs grown at the air-liquid interface and native human skin. The SC of the submerged HSEs, however, contained more free water and less natural moisturizing factors compared with the air-exposed counterparts. These results show that the presented cell culture method can be utilized to generate HSEs under submerged conditions to study epidermal formation under aqueous conditions.

Effect of synthetic vernix biofilms on barrier recovery of damaged mouse skin.

The aim of this work was to investigate whether topical application of synthetic biofilms supports and accelerates the recovery of the murine skin barrier, disrupted by sequential tape stripping. Therefore, various biofilms were applied topically on disrupted mouse skin to determine which formulation could improve barrier function, as was observed previously for the natural biofilm vernix caseosa (VC). The biofilms [i.e. particles (synthetic corneocytes) embedded in a synthetic lipid matrix] mimic closely the physicochemical properties and structure of VC. Various formulations were prepared using different particle:lipid ratios, particles with different initial water content and uncoated or lipid-coated particles. It was observed that application of all tested formulations improved the skin barrier recovery rate and reduced crust formation and epidermal hyperproliferation. However, only one of the biofilms [i.e. B1; composed of uncoated particles with 50% (w/w) initial water content and particle:lipid ratio of 2:1] mimicked the effects of native VC most closely. This indicates the importance of the presence of individual components, i.e. barrier lipids and water, as well as the ratio of these components. Consequently, these observations suggest the potential use of this biofilm treatment clinically.

Mimicking vernix caseosa--preparation and characterization of synthetic biofilms.

The multiple protecting and barrier-supporting properties of the creamy, white biofilm vernix caseosa (VC) before and after birth suggest that a VC biomimetic could be an innovative barrier cream for barrier-deficient skin. The aim of this study was the rational design and preparation of synthetic biofilms mimicking the unique composition and properties of natural VC. Hexagonal, highly hydrated hyperbranched polyglycerol microgel particles (30 microm in diameter) were embedded in a synthetic lanolin-based lipid mixture using a micromixer. In these formulations, the water content of the particles (i.e. 50% and 80%), an additional lipid coating of the particles and different particle/lipid ratios were varied. Characterization with confocal laser scanning microscopy (CLSM) showed a homogeneous distribution of the labeled particles in the lipid matrix. Regarding structural appearance, particle density and distribution, the formulations with a high particle/lipid ratio (5:1) resembled native VC very closely. Comparable results between native VC and the synthetic formulations were obtained concerning water handling properties, thermotropic behavior while lower elasticity and lower viscosity were observed for the synthetic biofilms. The biofilm formulations were stable for at least 1 month at 4 degrees C. In conclusion, our formulations mimic natural VC very closely and are promising candidates for in vivo studies.

Skin barrier disruption by acetone: observations in a hairless mouse skin model.

To disrupt the barrier function of the skin, different in vivo methods have been established, e.g., by acetone wiping or tape-stripping. In this study, the acetone-induced barrier disruption of hairless mice was investigated in order to establish a reliable model to study beneficial, long-term effects on barrier recovery after topical application. For both treatments (i.e., acetone treatment and tape-stripping) the transepidermal water loss directly after disruption and the subsequent barrier recovery profile were similar. Histological assessment showed significant lower number of corneocyte layers in acetone-treated and tape-stripped skin compared to untreated skin, while there was no statistical difference between the two treatments. Lipid analysis of acetone-treated skin revealed that only small fraction of lipids were extracted consisting of predominantly nonpolar lipids. Importantly, the ratio of the barrier lipids, i.e., cholesterol, free fatty acids and ceramides, remained similar between control and acetone-treated skin. This reflects the undisrupted lipid organization, as determined by small-angle X-ray diffraction measurements: the long-periodicity lamellar phase was still present after acetone treatment. Our results contradict earlier studies which reported no mechanical stratum corneum removal, a substantial extraction of lipids and disruption in lipid organization. In conclusion, our studies demonstrate that barrier disruption due to acetone treatment is mainly due to removal of corneocytes.

Development of a murine model to evaluate the effect of vernix caseosa on skin barrier recovery.

The aim of this study was twofold, that is the generation of a reliable model for skin barrier disruption and repair and to evaluate recovery of damaged skin after application of vernix caseosa (VC). VC was selected as its wound healing properties were suggested previously, but never clearly demonstrated. Five different levels of barrier disruption in mice, accomplished by tape-stripping, were evaluated. Disruption models such as moderate, severe #1 and #2 (transepidermal water loss (TEWL) of 31 +/- 2, 59 +/- 4 and 66 +/- 3 g/m(2)/h, respectively) showed complete recovery within 72 h. However, not all corneocytes were removed after tape-stripping. Additionally, models such as severe #3 and #4 (TEWL of 73 +/- 5 and 79 +/- 6 g/m(2)/h, respectively) with a more severe disruption were evaluated. After tape-stripping, all corneocytes were removed and the remaining epidermis was intact. However, model #3 still showed complete recovery within 72 h. With model #4, a crust was formed and almost complete recovery (approximately 90%) was obtained within only 8 days. The effect of VC application on recovery of disrupted skin was evaluated with model #3 and #4. Model #3 showed that application of VC predominantly influenced initial recovery and is therefore merely appropriate to study the effect of formulations in the initial recovery period. Topical application of VC on model #4 considerably increased initial and long-term recovery. Moreover, VC application promoted rapid formation of stratum corneum and prevented epidermal thickening. These observations not only confirm the ability of VC to enhance barrier recovery, but also suggest potential use of this treatment clinically.

Long periodicity phase in extracted lipids of vernix caseosa obtained with equilibration at physiological temperature.

The outermost layer of the skin, the stratum corneum (SC), comprises the main barrier function between body and environment. The SC features a highly structured lipid organization: a short periodicity phase and a long periodicity phase (LPP) with a repeat distance of 6 and 13 nm, respectively. Like SC, vernix caseosa (VC), the creamy white skin-surface biofilm of the newborn, also contains barrier lipids, i.e. ceramides, cholesterol and free fatty acids. Aim of this study was to investigate whether isolated VC lipids also form the characteristic LPP. Several preparation methods were examined and only when the solution of the lipid mixture, isolated either from VC or SC, was dried under nitrogen at 37 degrees C and subsequently spread onto a support, the LPP was formed. When VC barrier lipids were first exposed to elevated temperatures and subsequently cooled down, the LPP was formed at around 34 degrees C, which is at a much lower temperature than observed with the lipids in SC. In conclusion, we showed for the first time that depending on the preparation method, (i) VC lipids also form the LPP and (ii) the LPP in VC lipids and SC lipids was obtained at a low equilibration temperature, mimicking the physiological condition.

Two new methods for preparing a unique stratum corneum substitute.

Stratum corneum lipids play an important role in the barrier function of the skin. An in vitro permeation model consisting of synthetic lipids has previously been developed to replace human stratum corneum (SC) in permeation studies. This model is referred to as the stratum corneum substitute (SCS). In order to improve its reproducibility and to increase the efficiency in preparing the SCS, two new preparation methods are developed. Subsequently the properties of the SCS prepared by the various methods, i.e. the manual airbrush method, the rotor airbrush method and the linomat method, are investigated. The results show that the SCS prepared with the various methods share the properties of a uniform lipid composition and lipid distribution. Furthermore, irrespective of the preparation method, the lipids form crystalline lamellar phases, mimicking the lipid organization and orientation in human SC. As a result, permeation profiles of benzoic acid through SCS are very similar to human SC. The rotor method increases the efficiency and reproducibility of the manual airbrush method, while the linomat method reduces the lipid loss during preparation and results in SCS with a more uniform membrane thickness. In conclusion, the linomat method was chosen as the preferred method for preparing the substitute.

Lanolin-derived lipid mixtures mimic closely the lipid composition and organization of vernix caseosa lipids.

The aim of the present study was to use semi-synthetic lipid mixtures to mimic the complex lipid composition, organization and thermotropic behaviour of vernix caseosa (VC) lipids. As VC shows multiple protecting and barrier supporting properties before and after birth, it is suggested that a VC substitute could be an innovative barrier cream for barrier deficient skin. Lanolin was selected as the source of the branched chain sterol esters and wax esters--the main lipid classes of VC. Different lipid fractions were isolated from lanolin and subsequently mixed with squalene, triglycerides, cholesterol, ceramides and fatty acids to generate semi-synthetic lipid mixtures that mimic the lipid composition of VC, as established by high-performance thin-layer chromatography. Differential scanning calorimetry and Fourier transform infrared spectroscopy investigations revealed that triglycerides play an important role in the (lateral) lipid organization and thermotropic behaviour of the synthetic lipid mixtures. Excellent resemblance of VC lipids was obtained when adding unsaturated triglycerides. Moreover, these lipid mixtures showed similar long range ordering as VC. The optimal lipid mixture was evaluated on tape-stripped hairless mouse skin in vivo. The rate of barrier recovery was increased and comparable to VC lipid treatment.

Temperature-induced changes in structural and physicochemical properties of vernix caseosa.

The skin of the third trimester fetus and early newborn exhibits a complex, multifunctional, highly hydrated but viscous skin-surface biofilm called vernix caseosa (VC). During birth, VC undergoes a substantial change from an aqueous and warm surrounding into a gaseous and colder environment postnatally. The aim of this study was to investigate the structural and physicochemical changes in VC, which accompany physiologically relevant variations in environment parameters, such as temperature and humidity. A remarkable difference was observed in water release and uptake properties: dehydration and rehydration processes take place two to four times faster at 37 degrees C than at room temperature (RT). The dehydration was irreversible; rehydration was only possible to a final weight of 55% (37 degrees C) and 46% (RT) of the pre-desiccation weight. Differential scanning calorimetry showed two different overlapping phase transitions within physiological temperature range. Investigation of the lipid organization by Fourier transform infrared spectroscopy and small-angle X-ray diffraction revealed a more disordered state of lipids at 37 degrees C than at RT, which might explain the faster dehydration and rehydration process at 37 degrees C as well as the changes in thermotropic rheological behavior. In conclusion, we demonstrated that VC properties adjust to the fundamental change from the intrauterine to the post-natal environment.

Water distribution and natural moisturizer factor content in human skin equivalents are regulated by environmental relative humidity.

Human skin equivalents (HSEs) show great similarities to human native skin. However, one of the key processes impaired under in vitro conditions is desquamation. Desquamation involves the degradation of the corneodesmosomes, in which various enzymes participate. Activation of these enzymes is affected by several microenvironmental factors such as pH and water level. The water level is assumed to depend on the presence of natural moisturizing factors (NMF). In this study, the levels of water and one of the prominent NMF components--pyrrolidone carboxylic acid (PCA)--were examined. In HSE generated under normal culture conditions (93% relative humidity (RH)), the water level and PCA content appeared to be much lower than in the native counterpart. To increase the water and PCA levels in HSE, a culture method was established in which HSE was reconstructed under reduced RH. Although at 40% RH the PCA levels in reconstructed and native tissue are similar, the hydration levels in reconstructed tissue remain still lower. Only topical application of water induced marked swelling of corneocytes. This clearly shows that the stratum corneum water level in HSE is regulated by other, still unknown, factors, in addition to NMF.

The skin barrier in healthy and diseased state.

The primary function of the skin is to protect the body for unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. The stratum corneum consists of corneocytes surrounded by lipid regions. As most drugs applied onto the skin permeate along the lipid domains, the lipid organization is considered to be very important for the skin barrier function. It is for this reason that the lipid organization has been investigated quite extensively. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid organization is different from that of other biological membranes. In stratum corneum, two lamellar phases are present with repeat distances of approximately 6 and 13 nm. Moreover the lipids in the lamellar phases form predominantly crystalline lateral phases, but most probably a subpopulation of lipids forms a liquid phase. Diseased skin is often characterized by a reduced barrier function and an altered lipid composition and organization. In order to understand the aberrant lipid organization in diseased skin, information on the relation between lipid composition and organization is crucial. However, due to its complexity and inter-individual variability, the use of native stratum corneum does not allow detailed systematic studies. To circumvent this problem, mixtures prepared with stratum corneum lipids can be used. In this paper first the lipid organization in stratum corneum of normal and diseased skin is described. Then the role the various lipid classes play in stratum corneum lipid organization and barrier function has been discussed. Finally, the information on the role various lipid classes play in lipid phase behavior has been used to interpret the changes in lipid organization and barrier properties of diseased skin.

Preparation and characterization of a stratum corneum substitute for in vitro percutaneous penetration studies.

The intercellular stratum corneum (SC) lipids form the main barrier for diffusion of substances through the skin. A porous substrate covered with synthetic SC lipids would be an attractive model to study percutaneous penetration, hereby replacing native human SC. Prerequisite is that this stratum corneum substitute (SCS) is prepared with a uniform lipid composition and layer thickness. Furthermore, the lipid organization and orientation should resemble that in SC. The objective of this study was to investigate the utility of an airbrush spraying device to prepare a SCS composed of cholesterol, ceramides and free fatty acids on a polycarbonate filter. The results demonstrate that a proper choice of solvent mixture and lipid concentration is crucial to achieve a uniform distribution of the applied lipids over the filter surface. A smooth and tightly packed lipid layer is only obtained when the equilibration conditions are appropriately chosen. The SCS possesses two crystalline lamellar phases with periodicities similar to those present in native SC. The orientation of these lamellae is mainly parallel to the surface of the polycarbonate filter, which resembles the orientation of the intercellular SC lipids. In conclusion, the airbrush technique enables generation of a homogeneous SCS, which ultimately may function as a predictive in vitro percutaneous penetration model.

A novel in vitro percutaneous penetration model: evaluation of barrier properties with p-aminobenzoic acid and two of its derivatives.

PURPOSE: The objective of this study was to evaluate the utility of a stratum corneum substitute (SCS) as a novel in vitro percutaneous penetration model. The SCS consists of synthetic stratum corneum (SC) lipids (cholesterol, free fatty acids, and specific ceramides) applied onto a porous substrate. The composition, organization, and orientation of lipids in the SCS bear high resemblance to that of the intercellular barrier lipids in SC. METHODS: The barrier integrity of the SCS was evaluated by means of passive diffusion studies, using three model compounds with different lipophilicities. The effects of lipid layer thickness, permeant lipophilicity, and altered lipid composition on the barrier properties were investigated, using isolated human SC as a control sample. RESULTS: For all three model compounds, the permeability characteristics of the SCS with a 12-mum-thick lipid layer closely resemble those of human SC. Modification of the lipid composition, generating an SCS that lacks the characteristic long periodicity phase as present in SC, was accompanied by a 2-fold increased permeability. CONCLUSIONS: The SCS offers an attractive tool to predict solute permeation through human skin. Moreover, as its lipid composition can be modified, they may also serve as a suitable screening model for diseased skin.

New insights into ultrastructure, lipid composition and organization of vernix caseosa.

The upper layer of the epidermis, the stratum corneum (SC), is very important for skin barrier function. During the last trimester of gestation, the SC of the fetus is protected by a cheesy, white biofilm called vernix caseosa (VC). VC consists of water-containing corneocytes embedded in a lipid matrix and the basic structure shows certain similarities with the SC. This study aimed to characterize VC, with the main focus on an integral analysis of free and (to the corneocytes) bound lipids, on the lipid organization, and on ultrastructure. Free lipids of VC show a wide distribution in polarity; nonpolar lipids such as sterol esters and triglycerides predominate, having a chain length of up to 32 carbon atoms. The profile of fatty acids, omega-hydroxyacids and omega-hydroxyceramides - representing the bound lipids of VC - shows high similarity to that of SC. Morphological studies revealed the presence of highly hydrated corneocytes embedded in lipids, the latter being occasionally accumulated as lipid pools. Freeze fracture electron microscopy showed smooth surfaces of corneocytes and a heterogeneous appearance of intercellular lipids. The results suggest a lower degree of ordering of VC lipids as compared to the SC. A small-angle X-ray diffraction study showed similar results.

Lipid mixtures prepared with well-defined synthetic ceramides closely mimic the unique stratum corneum lipid phase behavior.

Lipid lamellae present in the outermost layer of the skin, the stratum corneum, form the main barrier for the diffusion of molecules through the skin. The presence of a unique 13 nm lamellar phase and its high crystallinity are characteristic for the stratum corneum lipid phase behavior. In the present study, small-angle and wide-angle X-ray diffraction were used to examine the organization in lipid mixtures prepared with a unique set of well-defined synthetic ceramides, varying from each other in head group architecture and acyl chain length. The results show that equimolar mixtures of cholesterol, free fatty acids, and synthetic ceramides (resembling the composition of pig ceramides) closely resemble the lamellar and lateral stratum corneum lipid organization, both at room and higher temperatures. Exclusion of several ceramide classes from the mixture does not affect the lipid organization. However, complete substitution of ceramide 1 (acylceramide with a sphingosine base) with ceramide 9 (acylceramide with a phytosphingosine base) reduces the formation of the long periodicity lamellar phase. This indicates that the head group architecture of acylceramides affects the lipid organization. In conclusion, lipid mixtures prepared with well-defined synthetic ceramides offer an attractive tool with which to unravel the importance of the molecular structure of individual ceramides for proper lipid organization.

Proteomic profiling identifies an UV-induced activation of cofilin-1 and destrin in human epidermis.

The human skin is the only line of defense against UV radiation. A series of responses to protect the skin are induced by UV radiation. In this study, a proteomic approach was used to study these responses. We have performed high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis of (solar simulated) UV-exposed reconstructed skin equivalents as well as native skin. Differentially expressed proteins were processed for mass spectrometric analysis, when consistent differences were observed in all individual human skin equivalents. In addition to proteins known to be involved in UV responses (HSP27, MnSOD, and PDX-2), we identified two novel proteins that were downregulated following UV exposure. Further analysis revealed that these proteins were the phosphorylated forms of the actin cytoskeleton modulators cofilin-1 and destrin. The de-phosphorylation of cofilin-1 was confirmed using western blotting of UV-exposed skin equivalents and ex vivo skin protein extracts. In conclusion, our study indicates the potency of a proteomic approach to study UV-induced changes in a tissue culture system mimicking human skin as well as excised human skin.

Basement membrane reconstruction in human skin equivalents is regulated by fibroblasts and/or exogenously activated keratinocytes.

This study was undertaken to examine the role fibroblasts play in the formation of the basement membrane (BM) in human skin equivalents. For this purpose, keratinocytes were seeded on top of fibroblast-free or fibroblast-populated collagen matrix or de-epidermized dermis and cultured in the absence of serum and exogenous growth factors. The expression of various BM components was analyzed on the protein and mRNA level. Irrespective of the presence or absence of fibroblasts, keratin 14, hemidesmosomal proteins plectin, BP230 and BP180, and integrins alpha1beta1, alpha2beta1, alpha3beta1, and alpha6beta4 were expressed but laminin 1 was absent. Only in the presence of fibroblasts or of various growth factors, laminin 5 and laminin 10/11, nidogen, uncein, type IV and type VII collagen were decorating the dermal/epidermal junction. These findings indicate that the attachment of basal keratinocytes to the dermal matrix is most likely mediated by integrins alpha1beta1 and alpha2beta1, and not by laminins that bind to integrin alpha6beta4 and that the epithelial-mesenchymal cross-talk plays an important role in synthesis and deposition of various BM components.

Acylceramide head group architecture affects lipid organization in synthetic ceramide mixtures.

The lipid organization in the upper layer of the skin, the stratum corneum (SC), is important for the skin barrier function. This lipid organization, including the characteristic 13 nm lamellar phase, can be reproduced in vitro with mixtures based on cholesterol, free fatty acids and natural as well as synthetic ceramides (CER). In human SC, nine CER classes have been identified (CER1-CER9). Detailed studies on the effect of molecular structure of individual ceramides on the SC lipid organization are only possible with synthetic lipid mixtures, as their composition can be accurately chosen and systematically modified. In the present study, small-angle X-ray diffraction was used to examine the organization in synthetic lipid mixtures of which the synthetic ceramide fraction was prepared with sphingosine-based CER1 or phytosphingosine-based CER9. The latter acylceramide contains an additional hydroxyl group at the sphingoid backbone. The results show that a gradual increase in CER1 level consistently promotes the formation of the 13 nm lamellar phase and that partial replacement of CER1 by CER9 does not affect the phase behavior. Interestingly, complete substitution of CER1 with CER9 reduces the formation of the long periodicity phase and results in phase separation of CER9.

Modelling the stratum corneum lipid organisation with synthetic lipid mixtures: the importance of synthetic ceramide composition.

Cholesterol (CHOL), free fatty acids (FFA) and nine classes of ceramides (CER1-CER9) form the main constituents of the intercellular lipid lamellae in stratum corneum (SC), which regulate the skin barrier function. Both the presence of a unique 13-nm lamellar phase, of which the formation depends on the presence of CER1, and its dense lateral packing are characteristic for the SC lipid organisation. The present study focuses on the lipid organisation in mixtures prepared with CHOL, FFA and a limited number of synthetic CER, namely CER1, CER3 and bovine brain CER type IV (SigmaCERIV). The main objective is to determine the optimal molar ratio of CER3 to SigmaCERIV for the formation of the 13-nm lamellar phase. CER3 contains a uniform acyl chain length, whereas SigmaCERIV contains fatty acids with varying chain lengths. Using small angle X-ray diffraction (SAXD), it is demonstrated that the CER3 to SigmaCERIV ratio affects the formation of the 13-nm lamellar phase and that the optimal ratio depends on the presence of FFA. Furthermore, the formation of the 13-nm lamellar phase is not very sensitive to variations in the total CER level, which is similar to the in vivo situation.

Diffusible factors released by fibroblasts support epidermal morphogenesis and deposition of basement membrane components.

Epithelial-mesenchymal interactions play an important role in controlling epidermal morphogenesis and homeostasis but little is known about the mechanisms of these interactions. To examine whether diffusible factors produced by fibroblasts and/or keratinocytes support epidermal morphogenesis and basement membrane formation, organotypic keratinocyte monocultures were established in media collected either from organotypic fibroblast or keratinocyte-monocultures or from keratinocyte-fibroblast cocultures, and the expression of keratin 10, 16, and 17 and basement membrane components (types IV and VII collagen, laminin 5, nidogen, BP 180, LAD-1) were examined. We found that diffusible factors released by keratinocytes were not sufficient to support the establishment of normalized epidermal phenotype and deposition of basement membrane components in contrast to fibroblast- or keratinocyte/fibroblast-derived factors. Keratinocytes appear to affect the spectrum of secreted soluble factors, as keratinocyte/fibroblast-derived factors were more effective to accomplish continuous linear deposition of laminin 5 and of nidogen. The finding that released amounts of keratinocyte growth factor and granulocyte macrophage colony stimulating factor were not sufficient to fully support epidermal morphogenesis and deposition of basement membrane components is suggestive for the involvement of other released diffusible factors. Generation of organotypic keratinocyte monocultures in the presence of fibroblast- or keratinocyte/fibroblast-derived soluble factors resulted in enhanced expression of keratins K16 and K17 and the absence of type IV collagen. This observation indicates that next to paracrine acting factors, epidermal homeostasis is controlled by mutual keratinocyte-fibroblast interaction.