Full-thickness Skin Model Research Articles

Proanthocyanidin-enriched extract from Myrothamnus flabellifolia Welw. exerts antiviral activity against herpes simplex virus type 1 by inhibition of viral adsorption and penetration

Extracts from the aerial parts of the South African resurrection plant Myrothamnus flabellifolia Welw. have been used traditionally against infections of the upper respiratory tract and skin diseases. A polyphenol-enriched extract was investigated for potential antiviral effects against herpes simplex virus type 1 (HSV-1) and adenovirus, and the underlying mode of action was to be studied. Antiviral effects of an acetone-water extract (MF) from Myrothamnus flabellifolia on HSV-1 and adenovirus type 3 were tested in infected Vero cells by plaque reduction assay, MTT test and immunofluorescence. The influence of the extract on the HSV-1 envelope glycoprotein D was shown by Western blot. Organotypic full thickness skin models consisting of multilayer skin equivalents were used for the investigation of MF effects on HSV-1 replication. MF exhibited strong antiviral activity against HSV-1. The HSV-1-specific inhibitory concentration (IC(50)) was determined as 0.4 μg/mL and the cytotoxic concentration (CC(50)) against Vero cells as 50 μg/mL. A selectivity index (SI) (ratio of CC(50) to IC(50)) of approximately 120 was calculated when MF was added to the virus inoculum for 1h at 37°C prior to infection. The replication of adenovirus 3 was not affected by MF. MF abolished virus entry into the host cell by blocking viral attachment to the cell surface. When added after attachment at a concentration of >6 μg/mL, the extract also inhibited penetration of HSV-1 into the host cell. Polyphenolic compounds from MF directly interacted with viral particles, leading to the oligomerisation of envelope proteins as demonstrated for the essential viral glycoprotein D (gD). Using organotypic full thickness tissue cultures, it was shown that treatment of HSV-1 infected cultures with the MF resulted in reduced viral spread. A polyphenol-enriched extract from Myrothamnus flabellifolia strongly acts against HSV-1 by blocking viral entry into the cells.

Integration of Langerhans-like cells into a human skin equivalent

Studies regarding cellular interactions between Langerhans cells and other skin cells are somehow hampered by the difficult cultivation of these cells in vitro. Here, we show that the human MUTZ-3 cell line can be differentiated into Langerhans-like cells in the presence of a cytokine cocktail including GM-CSF, TGF-β1 and TNF-α. We used the expression of langerin, CD1a, CCR6 and the intracellular presence of Birbeck granules to identify the differentiated MUTZ-3 cells (MUTZ-3-LCs). The aim of this study was to integrate MUTZ-3-LCs into a three-dimensional full-thickness skin model. On top of fibroblast-containing collagen matrix a mixture of primary human keratinocytes and MUTZ-3-LCs were seeded and cultured for 24h. Subsequently, the models were lifted up to the air-liquid interface. Histological evaluation featured a fully stratified epidermis with all characteristic epidermal strata. Langerin-positive cells were detected suprabasally within the epidermis indicating that keratinocytes provide environmental conditions for long-time maintenance of MUTZ-3-LCs. These skin models provide a tool to further investigate the interactions between Langerhans-like cells and other skin cells and particularly learn more about the cutaneous immune response.

Simulation of Electron-Beam Irradiation of Skin Tissue Model

Monte Carlo simulation of electrons stopping in liquid water was used to model the penetration and quality of electron-beam irradiation incident on the full-thickness EpiDerm™ skin model (EpiDermFT™ MatTek, Ashland, VA). This 3D tissue model has a fully developed basement membrane separating an epidermal layer of keratinocytes in various stages of differentiation from a dermal layer of fibroblasts embedded in collagen. The simulations were motivated by a desire to selectively expose the epidermal layer to low-linear energy transfer (LET) radiation in the presence of a nonirradiated dermal layer. The variable-energy electron microbeam at the Pacific Northwest National Laboratory (PNNL) was used as a model of device characteristics and irradiation geometry. At the highest beam energy available (90 keV), we estimate that no more than a few percent of the beam energy will be deposited in the dermal layer. Energy deposition spectra were calculated for 10-µm-thick layers near the 10th, 50th and 90th percentiles of penetration by the 90 keV electron beam. Bimodal spectra showed an increasing component of "stoppers" with increasing depth, which increases the probability of large energy deposition events. Nevertheless, screening by tissue above the layer of interest is the main factor determining energy deposited at a given depth.

Penetration enhancement of two topical 5‐aminolaevulinic acid formulations for photodynamic therapy by erbium:YAG laser ablation of the stratum corneum: continuous versus fractional ablation

5-Aminolaevulinic acid (ALA) is used in photodynamic therapy (PDT). Response rates of PDT vary widely, which may be because of the limited uptake of topically applied photosensitisers. We investigated skin penetration and fluorescence induction of protoporphyrin IX (PpIX) after applying either 20% ALA cream or 20% aminolaevulinic acid solution on laser-stripped stratum corneum (SC) in an ex vivo full-thickness porcine skin model. Both formulations are used in clinical practice. To enhance the skin penetration of ALA, we used two different 2940-nm erbium:yttrium-aluminium-garnet (Er:YAG) laser systems to partially ablate the SC: continuous and fractional ablation. Different fluences were applied ranging from 0.5 to 1.5 J/cm(2) (continuous ablation) and from 4 to 24 J/cm(2) (fractional ablation). Fluorescence microscopy was used for detecting PpIX-induced fluorescence. Compared to skin without laser pretreatment, mean fluorescence intensity (MFI) of PpIX was enhanced 13.8-fold after continuous ablation with 1.0 J/cm(2) and 7.3-fold after fractional ablation with 4 J/cm(2); each laser procedure was followed by 4-h incubation with lipophilic ALA cream. Optimal parameters for continuous ablation without damage to the epidermis were 1 J/cm(2) for both formulations, fractional ablation was best with 4 J/cm(2). Histological evaluations of laser-treated skin showed necrosis and apoptosis, depending on light dose. In laser-stripped skin, PpIX fluorescence was detected earlier and reached deeper epidermal layers than in untreated skin. Continuous laser ablation induced higher PpIX fluorescence levels than fractional ablation. This method offers a promising new tool for enhancing ALA penetration in PDT without damaging the underlying tissue.

Effects of ultrasound and sodium lauryl sulfate on the transdermal delivery of hydrophilic permeants: Comparative in vitro studies with full-thickness and split-thickness pig and human skin

The simultaneous application of ultrasound and the surfactant sodium lauryl sulfate (referred to as US/SLS) to skin enhances transdermal drug delivery (TDD) in a synergistic mechanical and chemical manner. Since full-thickness skin (FTS) and split-thickness skin (STS) differ in mechanical strength, US/SLS treatment may have different effects on their transdermal transport pathways. Therefore, we evaluated STS as an alternative to the well-established US/SLS-treated FTS model for TDD studies of hydrophilic permeants. We utilized the aqueous porous pathway model to compare the effects of US/SLS treatment on the skin permeability and the pore radius of pig and human FTS and STS over a range of skin electrical resistivity values. Our findings indicate that the US/SLS-treated pig skin models exhibit similar permeabilities and pore radii, but the human skin models do not. Furthermore, the US/SLS-enhanced delivery of gold nanoparticles and quantum dots (two model hydrophilic macromolecules) is greater through pig STS than through pig FTS, due to the presence of less dermis that acts as an artificial barrier to macromolecules. In spite of greater variability in correlations between STS permeability and resistivity, our findings strongly suggest the use of 700 μm-thick pig STS to investigate the in vitro US/SLS-enhanced delivery of hydrophilic macromolecules.

Identification of Novel in vitro Test Systems for the Determination of Glucocorticoid Receptor Ligand-Induced Skin Atrophy

Topical glucocorticoids (GCs) demonstrate good anti-inflammatory effects but are limited by their side effect potential, with skin atrophy being the most prominent one. Thus, determining the atrophogenic potential of novel compounds is important. The aim of this study was to establish an in vitro skin atrophy model. A screening cascade was applied and GCs with a known atrophogenic potential were used as tool compounds. Five rodent and human cutaneous cell types/cell lines and 2 human skin equivalents were tested. Known and suspected atrophy markers related to collagen metabolism and epidermal thickness were measured. Altogether, a combination of 7 different cellular assays with up to 16 markers each were investigated. A reproducible, more than 2-fold, regulation of the candidate markers by dexamethasone or clobetasol was found for: (a) matrix metalloproteinase (MMP) 1, 2, 3 and 9 expression in human keratinocytes, (b) COL1A1 and COL3A1 expression in 3T3 fibroblasts, and (c) epidermal thickness, collagen and MMP synthesis in the full-thickness skin model (FTSM). These 3 models were further investigated with a panel of 4–5 GCs, demonstrating dose dependency and correlation with the atrophogenic potential of the tool compounds, qualifying them as potentially suitable. Finally, the predictability of these models for the in vivo situation was analyzed, testing a novel selective GC receptor agonist (SEGRA) in comparison to clobetasol. The results from the in vitro models suggested less atrophogenic effects for the SEGRA compound, which indeed was confirmed in the hr/hr rat skin atrophy model. In conclusion, a combination of 3 in vitro models based on 3T3 cells, human keratinocytes and FTSM with several readouts is recommended to determine atrophogenicity of GC receptor ligands. Further experiments are necessary to eventually reduce this panel and to demonstrate the true predictability for the clinic.

The Phenion® Full-Thickness Skin Model for Percutaneous Absorption Testing

In recent years many efforts have been made to replace dermal toxicity testing of chemicals in the animal by in vitro assays. As a member of a German research consortium, we have previously contributed to the validation of an in vitro test protocol for percutaneous absorption studies on the basis of reconstructed human epidermis and both human and pig skin ex vivo. Aiming to assess the barrier properties of a newly developed reconstructed skin model, this protocol has now been transferred to the Phenion® Full-Thickness Skin Model (FT model). The permeation of testosterone and caffeine was quantified in parallel to that of pig skin using Franz-type diffusion cells. In addition, the permeation of benzoic acid and nicotine was studied. As expected, the FT model is more permeable than pig skin, yet its barrier properties are well in accordance with those of reconstructed human epidermis when compared to previous data. In fact, the FT model most efficiently retards testosterone as the compound of highest lipophilicity, which can be explained by an additional uptake by a reservoir formed by the dermis equivalent. Thus, the structure closely parallels human skin. In consequence, the Phenion FT model appears to be suitable for percutaneous absorption studies in hazard analysis and should be subjected to a catch-up validation study.

Microvesicating effects of sulfur mustard on an in vitro human skin model

Bis-(β-chloroethyl) sulfide (SM) is a potent skin vesicant previously used for chemical warfare. Progress in determination of the mechanistic basis of SM pathology, and development of prophylactic and/or therapeutic countermeasures to SM exposure has been hampered by lack of physiologically relevant models of human skin. The current work evaluated a newly developed tissue engineered full-thickness human skin model in a completely in vitro approach to investigation of SM-induced dermal pathology. The model was first characterized with regard to overall morphology, lipid composition, basement membrane (BM) composition and ultrastructural features that are important targets of SM pathologic activity. Well-developed BM ultrastructural features were observed at the dermal–epidermal junction (DEJ), thus demonstrating successful resolution of a primary deficiency of models previously evaluated for SM studies. Studies were then conducted to evaluate histopathological effects of SM on the model. Good replication of in vivo effects was observed, including apoptosis of basal keratinocytes (KC) and microblister formation at the DEJ. Tissue engineered skin models with well-developed basement membrane structures thus appear to be useful tools for in vitro mechanistic studies of SM vesicant activity and development of preventive/therapeutic approaches for SM pathology.

Reconstructed Epidermis and Full-Thickness Skin for Absorption Testing: Influence of the Vehicles used on Steroid Permeation

A protocol for percutaneous absorption studies has been validated, based on the use of reconstructed human epidermis (RHE) and aqueous solutions of test substances. However, it is often the case that it is more-complex formulations of drugs or chemicals which will make contact with the skin surface. To investigate whether RHE and the reconstructed full-thickness skin model (FT-model) can be used to predict uptake from formulations, we compared the permeation of hydrocortisone and testosterone when applied in emulsion form and as a solution containing the penetration enhancer, ethanol. Human and pig skin and a non-cornified alveolar model served as references. The results were compared with steroid release from the formulations. The permeation rates of the steroids were ranked as: alveolar model >> RHE > FT-model, pig skin > human skin. In accordance with the rapid hydrocortisone release from the formulations, the permeation rates of this steroid exceeded those of testosterone. Only minor differences were observed when comparing the testosterone formulations, in terms of release and permeation. However, the ranking of the permeation of the hydrocortisone formulations was: solution > w/o emulsion > o/w emulsion, which permitted the elucidation of penetration enhancing effects, which is not possible with drug release studies. Differences in penetration were most obvious with native skin and reconstructed tissues, which exhibited a well-developed penetration barrier. In conclusion, RHE and skin preparations may be useful in the development of topical dermatics, and in the framework of hazard analysis of toxic compounds and their various formulations.

Towards new aspects of melatonin research in dermato‐endocrinology

Based on extensive investigation of melatonin in human cell models (keratinocytes, fibroblasts), a great variety of melatonin actions, mechanisms, intracellularly localized synthesis and metabolism as well as receptor expression has been unravelled, identifying melatonin as a key player in cutaneous biology and dermato‐endocrinology. Melatonin was reported to act directly protective as a radical scavenger in many conditions of skin‐related stress (ultraviolet radiation, ionizing radiation, thermal injury, ulcer formation, skin flap necrosis) and to modulate cell growth via membrane (MT1, MT2), cytosolic (MT3/NQO2) and nuclear receptors (RORα). Membrane and nuclear melatonin receptor expression is hair cycle dependent in murine hair follicles, whereas estradiol receptor (ERα) expression is down‐regulated by melatonin. Furthermore, the modulation of secondary endocrine signalling (e.g. prolactin release, estrogen receptor‐mediated signalling) in the skin is mediated by melatonin. Melatonin levels of magnitudes higher than in the plasma have been detected in human keratinocytes and murine and human hair follicles, whereby noradrenalin, the classical stimulator of melatonin synthesis, increased melatonin production in hair follicles. Additionally, a melatoninergic antioxidative system (MAS) of the skin has been identified with UV‐enhanced formation of melatonin metabolites, 2‐OH‐melatonin and AFMK, the latter being a strong antioxidant itself. Alternatively, melatonin indirectly protects cells by up‐regulating gene expression and activity of non‐melatonin autonomous protective systems, the intracellular antioxidative enzyme network which is composed of Mn‐SOD, Cu‐Zn‐SOD, CAT and GPx. To further drive melatonin research towards the human organism level, two human organ culture models have been choosen or newly established i) the human hair organ culture model and ii) the human full thickness skin model, respectively. Two crucial stressors of human skin/hair biology are used to investigate their morphology and functional effect expression in these models: ultraviolet radiation and chemotherapy‐induced cytotoxicity. Standardized stress conditions in both models serve to investigate differential expression of stress levels, their time‐dependent dynamics and effect quality. Finally, reduction of UV‐damaged keratinocytes (sun‐burn cells) by melatonin in the full‐skin organ model and anti‐apoptotic effects in chemotherapy‐induced hair follicle damage have been observed.

A novel treatment option for photoaged skin

DNA damage as a result of ultraviolet (UV) exposure plays an important role in the progression of cutaneous aging. Both folic acid and creatine have been linked to the process of DNA protection and repair. This study aims to investigate the effects of a commercially available folic acid- and creatine-containing formulation to fight the clinical signs of premature skin aging. Both in vitro and in vivo home-in-use studies using a folic acid- and creatine-containing formulation were performed aiming to elucidate the efficacy in terms of improvement of skin regeneration, protection from UV-induced DNA damage (Comet assay), reduction of wrinkle volume, and skin visco-elasticity. Furthermore, clinical evaluation and photography were carried out to determine the improvement of clinically graded parameters after treatment. Cultured full-thickness epidermal skin models supplemented with folic acid and creatine after epithelial perturbation showed an accelerated skin regeneration compared to untreated control models. Similarly, application of a folic acid- and creatine-containing formulation significantly improved epidermal turnover in vivo as evidenced by smaller corneocytes derived from the treated sites relative to the vehicle-treated sides. In addition, topical in vivo application of this formulation significantly protected from UV-induced DNA lesions, increased skin firmness, and reduced wrinkle volume compared to untreated control areas. Expert grading confirmed a significant decrease of fine and coarse wrinkles in the periocular region as well as overall wrinkles, tactile roughness, and laxity. Taken together, these results show that the combination of folic acid and creatine significantly accelerates epidermal skin regeneration in vitro and in vivo. Together with the finding of improved biomechanical skin properties, we conclude that the described topical formulation provides an effective treatment option for (photo)-aged skin.

Percutaneous penetration and distribution of VX using in vitro pig or human excised skin : Validation of demeton- S-methyl as adequate simulant for VX skin permeation investigations

The organophosphorus (OP) chemical warfare V agent O-ethyl- S-[2(di-isopropylamino)ethyl] methyl phosphonothioate (VX), is a highly toxic compound which mainly penetrates the body via percutaneous pathways. Hence, the following prerequisite: to ascertain compound absorption and percutaneous profile distribution with a view to further assessing the efficacy of topical skin protectants. We first selected the most appropriate receptor fluid to carry out in vitro VX absorption experiments, namely: Hanks's Balanced Salt Solution (HBSS). After a 24-h topical exposure time lapse, we measured altogether the percentage of applied dose unabsorbed and absorbed, penetration rate, lag time, permeability coefficient ( K p), and dose of VXeq present in skin. To such an end, we used full-thickness and split-thickness pig-ear or human abdominal skin membranes. Further, we scrutinised the potential use of two specific molecules as suitable surrogates for VX percutaneous penetration analyses: thus, we compared the present VX toxicokinetic parameters to earlier findings from our research unit, with respect to OP insecticides demethon- S-methyl (DSM) and paraoxon (POX). Within the framework of our study, we wish to highlight the following evidence: (a) pig-ear skin proves a relevant model to predict in vitro human abdominal skin, taking into account a 2-fold higher skin permeability to VXeq; (b) both full or split-thickness skin membranes may be used indiscriminately to gauge penetration rate and absorbed dose; (c) DSM applied on full-thickness pig-ear skin is the most relevant model to mimic the in vitro VX absorption through full-thickness skin model.

Skin cell monolayer versus Phenion® Full Thickness skin model as tools for identification of skin active natural products

Identification and development of plant derived extracts and natural products for skin regeneration and wound-healing is getting more and more into the focus of dermatological research. Physiological activity of natural products on human skin cells can be investigated in monolayer cell cultures of keratinocytes and dermal fibroblasts. Additionally to that the Phenion® Full Thickness skin model, a three dimensional complex tissue engineered skin substitute, can be used for effective identification of protective and regenerative effects of test compounds.

Evaluation of beneficial and adverse effects of glucocorticoids on a newly developed full-thickness skin model

Glucocorticoids (GCs) are highly effective compounds widely used in the treatment of inflammatory diseases; however, they offer distinct adverse effects such as skin thinning in response to long-term topical treatment. Nevertheless it is difficult to deduce the safety of a newly synthesized compound from its structural formula. Efficient assay systems that measure beneficial and adverse effects are needed. In the present study the applicability of a three-dimensional full-thickness skin model (FTSM) is tested to display GC-induced effects regarding anti-inflammation and atrophy. It is shown that topical application of a commercial GC ointment suppresses the ultraviolet (UV)B induced induction of interleukin (IL)-6 and IL-8. Addition of purified betamethasone-17-valerate, prednicarbate and clobetasol-17-propionate to the culture medium for 14days caused a reduction in the number of epidermal cell-layers corresponding to the atrophic risk found in vivo. Similarly, repeated topical application of five GC creams induced epidermal thinning. Evidence is given that the inhibitory effect on keratinocyte proliferation contributes to this effect. Furthermore, dermal thinning was monitored by measuring type I collagen synthesis; a decreased collagen synthesis similar to the in vivo situation is shown. The present study demonstrates the versatility of this FTSM in the validation of effectiveness and safety of GCs.

Elastin Expression in a Newly Developed Full-Thickness Skin Equivalent

The resilience of the human skin is mediated by elastic fibres mainly consisting of fibrillins and elastin. In order to establish a model system to study the impact of cosmetic and pharmaceutical compounds on the elastic system in vitro, we analyzed the expression of elastin in a newly developed full-thickness skin model. After a 5-week cultivation period the skin model developed a fully differentiated epidermis including a stratum corneum. The dermis contains fibroblasts embedded in extracellular matrix proteins. The models were viable until at least 51 days at the air-liquid interface (ALI) culture. Using immunohistochemistry we detected elastin first on day 7 of ALI. With proceeding culture time, elastin-positive fibres of different lengths and distribution patterns accumulated in the dermal compartment. Elastin mRNA expression started on day 7 of ALI, increased until day 10 and then dropped to a level comparable to that of day 7. Our results demonstrate that in our full-thickness skin model an in vivo-like elastic system, which clearly mimics at least two subsets of dermal elastic fibres, is generated. This physiological property favours the model as a promising animal-free approach to study those processes leading to an environment- and age-dependent decrease in skin elasticity.

154 Determination of phototoxicity properties of different compounds using a full thickness skin model (AST-2000)

154 Determination of phototoxicity properties of different compounds using a full thickness skin model (AST-2000)