Requisite instruments for the establishment of three-dimensional epidermal human skin equivalents-A methods review.

Human skin equivalents (HSEs) are three-dimensional skin organ culture models raised in vitro. This review gives an overview of common techniques for setting up HSEs. The HSE consists of an artificial dermis and epidermis. 3T3-J2 murine fibroblasts, purchased human fibroblasts or freshly isolated and cultured fibroblasts, together with other components, for example, collagen type I, are used to build the scaffold. Freshly isolated and cultured keratinocytes are seeded on top. It is possible to add other cell types, for example, melanocytes, to the HSE-depending on the research question. After several days and further steps, the 3D skin can be harvested. Additionally, we show possible markers and techniques for evaluation of artificial skin. Furthermore, we provide a comparison of HSEs to human skin organ culture, a model which employs human donor skin. We outline advantages and limitations of both models and discuss future perspectives in using HSEs.

An Interleukin-4 and Interleukin-13 Induced Atopic Dermatitis Human Skin Equivalent Model by a Skin-On-A-Chip.

Currently, the mechanism of progression of atopic dermatitis (AD) is not well understood because there is no physiologically appropriate disease model in terms of disease complexity and multifactoriality. Type 2 inflammation, mediated by interleukin (IL)-4 and IL-13, plays an important role in AD. In this study, full-thickness human skin equivalents consisting of human-derived cells were fabricated from pumpless microfluidic chips and stimulated with IL-4 and IL-13. The morphological properties, gene expression, cytokine secretion and protein expression of the stimulated human skin equivalent (HSE) epidermis were investigated. The results showed epidermal and spongy formations similar to those observed in lesions in AD, and decreased expression of barrier-related filaggrin, loricrin and involucrin genes and proteins induced by IL-4Rα signaling. In addition, we induced the expression of carbonic anhydrase II (CAII), a gene specifically expressed in the epidermis of patients with AD. Thus, AD human skin equivalents can be used to mimic the key pathological features of atopic dermatitis, overcoming the limitations of existing studies that rely solely on mouse models and have been unable to translate their effects to humans. Our results will be useful for future research on the development of therapeutic agents for atopic dermatitis.

Enhanced Nrf2 up-regulation by extracellular basic pH in a human skin equivalent system.

Extracellular basic pH regulates cellular processes in wounds, and consequently influenced wound healing. Oxidative defence system modulation in the skin helps heal wounds, inhibits skin ageing and improves the skin condition. Moreover, the role of keratinocyte growth factor (KGF) and nuclear factor erythroid 2-related factor 2 (Nrf2) in antioxidant systems has been reported in various skin models. However, the effects of extracellular basic pH on wound- or skin ageing-related skin damage have not been examined. Thus, we investigated the antioxidant systems affected by extracellular basic pH in a 3D human skin equivalent system (3HSE). Extracellular basic pH decreased KGF expression and enhanced the oxidative defence system, and thus activated Nrf2 in the 3HSE. Additionally, extracellular basic pH and KGF treatment up-regulated Nrf2 activation and its regulation of the oxidative defence system in the 3HSE. This indicates that Nrf2 up-regulation is enhanced by reactive oxygen species production, rather than KGF, and by extracellular basic pH of the skin. The inhibition of skin damage through pH imbalance and KGF regulation suggests that the development of pH-regulating or pH-maintaining materials may provide effective therapeutic strategies for maintaining a healthy skin.

A comparison of the in vitro permeation of 3-O-ethyl-l-ascorbic acid in human skin and in a living skin equivalent (LabSkin™).

OBJECTIVES: The safety assessment of personal care products often entails determining dermal absorption of their ingredients. Such experiments are typically performed in human or animal skin in vitro; however, ethical and safety considerations are associated with obtaining these tissues. Several human skin equivalent models (HSEs) have been developed as alternatives to human tissue. The barrier function of such models however, is normally less developed than human skin. Here, we examine the permeability of the HSE LabSkinTM to a model compound, 3-O-ethyl-l-ascorbic acid (EA) compared with human skin. METHODS: Skin uptake and permeation of EA was investigated in vitro using heat-separated human epidermis and LabSkinTM . Finite dose (5 µL cm-2 ) Franz-diffusion studies were conducted using 2 % (w/w) EA in a ternary solvent mixture comprising propylene glycol (PG), propylene glycol monolaurate (PGML), and isopropyl myristate (IPM). These excipients are commonly used in cosmetic products and they have been reported to promote permeation of EA in a different model, namely porcine skin. RESULTS: Permeation of EA through LabSkinTM was evident from 2 h; however, EA permeation in human skin was not detected until 5 h. Similar amounts of EA permeated through the two membranes at time points 8, 10, 12 and 24 h (p > 0.05). The cumulative amounts of EA delivered through LabSkinTM at 24 h were 41.3 ± 2.0 µg cm-2 , corresponding to 55.1 ± 1.8 % of the applied dose. Similar amounts permeated across human skin, 49.4 ± 4.1 µg cm-2 , accounting for 58.0 ± 4.2 % of the dose applied (p > 0.05). CONCLUSION: The permeation of EA in LabSkinTM compared well with results for human epidermis in terms of the permeation profiles and the cumulative amounts of EA that permeated. The data suggest that the skin barrier of the two models was similar with regard to their overall permeability to the hydrophilic active EA. The findings are promising for the use of LabSkinTM as a surrogate for human skin in permeability testing. Future studies will focus on exploring the reproducibility and robustness of LabSkinTM for delivery of other actives that span a range of physicochemical properties.

OBJECTIFS: L'évaluation de la sécurité des produits de soins personnels implique souvent de déterminer l'absorption cutanée de leurs ingrédients. Ces expérimentations sont généralement réalisées in vitro sur la peau humaine ou animale ; cependant, des considérations éthiques et de sécurité sont associées à l'obtention de ces tissus. Plusieurs modèles équivalents de peau humaine (Human Skin Equivalent, HSE) ont été développés comme alternatives au tissu humain. La fonction barrière de ces modèles est cependant normalement moins développée que la peau humaine. Ici, nous examinons la perméabilité du HSE LabSkin™ à un composé modèle, l'acide 3-O-éthyl-l-ascorbique (EA) en le comparant à la peau humaine. MÉTHODES: L'absorption cutanée et la perméation de l'EA ont été étudiées in vitro à l'aide d'épiderme humain séparé par la chaleur et de LabSkin™. Des études de diffusion de Franz à dose limitée (5 µL cm-2 ) ont été réalisées en utilisant 2 % (p/p) d'EA dans un mélange de solvant ternaire contenant du propylène glycol (PG), du propylène glycol monolaurate (PGML) et du myristate d'isopropyle (IPM). Ces excipients sont fréquemment utilisés dans les produits cosmétiques et il a été rapporté qu'ils favorisent la perméation de l'EA dans un modèle différent, à savoir la peau porcine. RÉSULTATS: La perméation de l'EA par LabSkin™ était évidente dès 2 h ; cependant, la perméation de l'EA dans la peau humaine n'a pas été détectée avant 5 h. Des quantités similaires d'EA ont pénétré les deux membranes aux points temporels 8, 10, 12 et 24 h (p > 0,05). Les quantités cumulées d'EA délivrées par LabSkin™ à 24 h étaient de 41,3 ± 2,0 µg cm-2 , correspondant à 55,1 ± 1,8 % de la dose appliquée. Des quantités similaires ont pénétré la peau humaine, 49,4 ± 4,1 µg cm-2 , représentant 58,0 ± 4,2 % de la dose appliquée (p > 0,05). CONCLUSION: La perméation de l'EA dans LabSkin™ a bien soutenu la comparaison quant aux résultats concernant l'épiderme humain en termes de profils de perméation et de quantités cumulées d'EA qui ont pénétré. Les données suggèrent que la barrière cutanée des deux modèles était similaire en ce qui concerne leur perméabilité globale à l'EA hydrophile actif. Les résultats sont prometteurs pour l'utilisation de LabSkin™ en tant que substitut de la peau humaine dans les tests de perméabilité. Les études futures se concentreront sur l'exploration de la reproductibilité et de la robustesse de LabSkin™ pour la délivrance d'autres principes actifs qui couvrent un éventail de propriétés physicochimiques.

Subacute dermal toxicity of perfluoroalkyl carboxylic acids: comparison with different carbon-chain lengths in human skin equivalents and systemic effects of perfluoroheptanoic acid in Sprague Dawley rats.

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are used in various fields but raise concerns regarding human health and environmental consequences. Among PFASs, perfluorooctanoic acid (PFOA) and short-chain perfluoroalkyl carboxylic acids (SC PFCAs) are detectable in skin-contact consumer products and have dermal absorption potential. Here, we investigated the effects of dermal exposure to PFOA and SC PFCAs using in vitro and in vivo models. Human skin equivalents were topically treated with 0.25 mM and 2.5 mM PFOA and SC PFCAs (perfluoropentanoic acid, PFPeA; perfluorohexanoic acid, PFHxA; and perfluoroheptanoic acid, PFHpA) for 6 days, and cell viability, interleukin (IL)-1α, oxidative stress markers (malondialdehyde, MDA; and 8-hydroxydeoxyguanosine, 8-OHdG), and histopathology were examined. MDA levels were significantly higher in the PFASs groups than in controls. Compared with SC PFCAs, 2.5 mM PFOA caused more IL-1α (p < 0.001) release, decreased skin thickness and microscopic abnormalities. To evaluate systemic effects, Sprague Dawley (SD) rats were dermally treated with 250 and 1000 mg/kg PFHpA for 2 weeks and clinical and anatomic pathology were assessed. At 1000 mg/kg, 83% of the rats died, with severe ulcerative dermatitis at the application site. Adverse PFHpA-treated systemic changes were observed in the kidney, liver and testes, and histopathologic lesions such as renal tubular necrosis, hepatocellular necrosis, and germ cell degeneration were seen at 250 and 1000 mg/kg. Our study suggests that SC PFCAs have fewer effects on the skin than PFOA, but SC PFCAs can have adverse effects on major organs with systemic exposure at high concentrations.

Electric field-induced changes in biomechanical properties in human dermal fibroblasts and a human skin equivalent.

PURPOSE: An electric field (EF) can be used to change the mechanical properties of cells and skin tissues. We demonstrate EF-induced elasticity changes in human dermal fibroblasts (HDFs) and a human skin equivalent and identify the underlying principles related to the changes. METHODS: HDFs and human skin equivalent were stimulated with electric fields of 1.0 V/cm. Change in cellular elasticity was determined by using atomic force microscopy. Effects of EF on the biomechanical and chemical properties of a human skin equivalent were analyzed. In cells and tissues, the effects of EF on biomarkers of cellular elasticity were investigated at the gene and protein levels. RESULTS: In HDFs, the cellular elasticity was increased and the expression of biomarkers of cellular elasticity was regulated by the EF. Expression of the collagen protein in the human skin equivalent was changed by EF stimulation; however, changes in density and microstructure of the collagen fibrils were not significant. The viscoelasticity of the human skin equivalent increased in response to EF stimulation, but molecular changes were not observed in collagen. CONCLUSIONS: Elasticity of cells and human skin equivalent can be regulated by electrical stimulation. Especially, the change in cellular elasticity was dependent on cell age.

Glucose Exerts an Anti-Melanogenic Effect by Indirect Inactivation of Tyrosinase in Melanocytes and a Human Skin Equivalent.

Sugars are ubiquitous in organisms and well-known cosmetic ingredients for moisturizing skin with minimal side-effects. Glucose, a simple sugar used as an energy source by living cells, is often used in skin care products. Several reports have demonstrated that sugar and sugar-related compounds have anti-melanogenic effects on melanocytes. However, the underlying molecular mechanism by which glucose inhibits melanin synthesis is unknown, even though glucose is used as a whitening as well as moisturizing ingredient in cosmetics. Herein, we found that glucose significantly reduced the melanin content of α-melanocyte-stimulating hormone (MSH)-stimulated B16 cells and darkly pigmented normal human melanocytes with no signs of cytotoxicity. Furthermore, topical treatment of glucose clearly demonstrated its whitening efficacy through photography, Fontana-Masson (F&M) staining, and multi-photon microscopy in a pigmented 3D human skin model, MelanoDerm. However, glucose did not alter the gene expression or protein levels of major melanogenic proteins in melanocytes. While glucose potently decreased intracellular tyrosinase activity in melanocytes, it did not reduce mushroom tyrosinase activity in a cell-free experimental system. However, glucose was metabolized into lactic acid, which can powerfully suppress tyrosinase activity. Thus, we concluded that glucose indirectly inhibits tyrosinase activity through conversion into lactic acid, explaining its anti-melanogenic effects in melanocytes.

Tyrosinase-Targeting Gallacetophenone Inhibits Melanogenesis in Melanocytes and Human Skin-Equivalents.

Demands for safe depigmentation compounds are constantly increasing in the pharmaceutical and cosmetic industry, since the numerous relevant compounds reported to date have shown undesirable side effects or low anti-melanogenic effects. In this study, we reported three novel inhibitors of tyrosinase, which is the key enzyme in melanogenesis, identified using docking-based high throughput virtual screening of an in-house natural compound library followed by mushroom tyrosinase inhibition assay. Of the three compounds, gallacetophenone showed high anti-melanogenic effect in both human epidermal melanocytes and a 3D human skin model, MelanoDerm. The inhibitory effect of gallacetophenone on tyrosinase was elucidated by computational molecular modeling at the atomic level. Binding of gallacetophenone to the active site of tyrosinase was found to be stabilized by hydrophobic interactions with His367, Ile368, and Val377; hydrogen bonding with Ser380 and a water molecule bridging the copper ions. Thus, our results strongly suggested gallacetophenone as an anti-melanogenic ingredient that inhibits tyrosinase.

A Comparative Study of Engineered Dermal Templates for Skin Wound Repair in a Mouse Model.

Engineered dermal templates have revolutionised the repair and reconstruction of skin defects. Their interaction with the wound microenvironment and linked molecular mediators of wound repair is still not clear. This study investigated the wound bed and acellular "off the shelf" dermal template interaction in a mouse model. Full-thickness wounds in nude mice were grafted with allogenic skin, and either collagen-based or fully synthetic dermal templates. Changes in the wound bed showed significantly higher vascularisation and fibroblast infiltration in synthetic grafts when compared to collagen-based grafts (P ≤ 0.05). Greater tissue growth was associated with higher prostaglandin-endoperoxide synthase 2 (Ptgs2) RNA and cyclooxygenase-2 (COX-2) protein levels in fully synthetic grafts. Collagen-based grafts had higher levels of collagen III and matrix metallopeptidase 2. To compare the capacity to form a double layer skin substitute, both templates were seeded with human fibroblasts and keratinocytes (so-called human skin equivalent or HSE). Mice were grafted with HSEs to test permanent wound closure with no further treatment required. We found the synthetic dermal template to have a significantly greater capacity to support human epidermal cells. In conclusion, the synthetic template showed advantages over the collagen-based template in a short-term mouse model of wound repair.

[Use of 2D and 3D cell cultures in dermatology]. / Einsatz von 2-D- und 3-D-Kulturen in der Dermatologie.

The skin is a complex organ that performs a number of vital functions, including forming a physical barrier that protects our body from the penetration of pathogens and irritants and from excessive transepidermal water loss. In addition to its passive properties, the skin is also actively involved in the immune process. A complex structure of different cell types and structures allows the skin to fulfil these functions. In vitro research often faces the problem that simple 2D cell cultures are not able to adequately map these functions. Here 3D skin models offer a possible solution. In recent years, there has been significant development in this field; the reproducibility of the method as well as the physiological structure and tissue architecture of the 3D skin models have been improved. Depending on the research question, protocols for 3D skin models have been published, ranging from simple multilayer epidermis models to highly complex vascularized 3D full skin models.

Mannosylerythritol lipids inhibit melanogenesis via suppressing ERK-CREB-MiTF-tyrosinase signalling in normal human melanocytes and a three-dimensional human skin equivalent.

Hyperpigmentation is caused by excessive production of melanin in melanocytes. Mannosylerythritol lipids (MELs) are glycolipid biosurfactants that are abundantly produced by yeasts and used commercially in cosmetics. However, the potential depigmenting efficacy of MELs has not been evaluated. In this study, the depigmentary effect of MELs was tested in primary normal human melanocytes (NHMs), α-melanocyte-stimulating hormone (MSH)-stimulated B16 cells (murine melanoma cells) and a human skin equivalent (MelanoDerm) using photography, Fontana-Masson (F&M) staining and two-photon microscopy. Mannosylerythritol lipids significantly decreased the melanin contents in NHMs and α-MSH-stimulated B16 cells. Consistent with these findings, MELs treatment had a clear whitening effect in a human skin equivalent, brightening the tissue colour and reducing the melanin content. The molecular mechanism underlying the anti-melanogenic effect of MELs treatment was examined by real-time PCR and Western blotting. Mechanistically, MELs clearly suppressed the gene expression levels of representative melanogenic enzymes, including tyrosinase, Tyrp-1 and Tyrp-2, by inhibiting the ERK/CREB/MiTF signalling pathway in NHMs. This work demonstrates for the first time that MELs exert whitening effects on human melanocytes and skin equivalent. Thus, we suggest that MELs could be developed as a potent anti-melanogenic agent for effective whitening, beyond their use as a biosurfactant in cosmetics.

Development of a new diaper dermatitis-like reconstructed skin equivalent for testing children atopic dermatitis relieving cosmetics.

BACKGROUND: Diaper dermatitis (DD) is the most common acute inflammatory skin disease. It has a serious effect on children's and families' quality of life. We aimed to screen and evaluate the efficacy of different formulas for relieving the diaper dermatitis symptoms by developing a kind of diaper dermatitis-like reconstructed human skin equivalent in vitro. MATERIALS AND METHOD: We developed the human skin equivalent for diaper dermatitis with 0.2% Sodium lauryl sulfate (SLS). The diaper dermatitis-like human skin equivalent was characterized by high level of inflammation, such as overexpression of interleukin-1α (IL-1α), and impaired skin barrier. Four formulas with potential of anti-inflammation and promotion of skin barrier function were topically applied on the diaper dermatitis-like human skin equivalent surface. The afterward protection efficacy was evaluated by endpoints of IL-1α, tissue viability, and skin barrier function. RESULTS: The chemical irritant induced high release of IL-1α, impaired tissue viability, and skin barrier function. The cream prepared with potential of anti-inflammation and skin protection could effectively decrease and relive the impact of irritant with decreased level of IL-1α and the higher tissue viability than the placebo exposure. CONCLUSION: The results showed that diaper dermatitis-like human skin equivalent induced by SLS can mimic the skin irritation response of the diaper rash.

Dehydroabietic Acid Induces Regeneration of Collagen Fibers in Ultraviolet B-Irradiated Human Dermal Fibroblasts and Skin Equivalents.

BACKGROUND/AIMS: Dehydroabietic acid (DAA) is a natural phytochemical found in red pine trees and herbal plants. While DAA and its derivatives are known for improving diabetes and hyperlipidemia, the antiaging effect and its underlying mechanisms of DAA on skin have not been fully examined. Here, we assessed the antiaging effects of DAA on human dermal fibroblasts and skin equivalents. METHODS: We investigated the effect of DAA on the secretion of type I procollagen and matrix metalloproteinase-1 (MMP-1) in ultraviolet B (UVB)-irradiated neonatal normal human dermal fibroblasts (NHDFn). Using nonlinear optical imaging techniques, we visualized quantitative and qualitative changes of collagen fibers by DAA treatment in human skin equivalent models. RESULTS: DAA induces increases in type I procollagen secretion when treated on UVB-irradiated NHDFn. DAA also downregulates secretion of MMP-1 through the inhibition of the JNK signaling pathway. In human skin equivalent models, we successfully visualized the spatial distribution of collagen fibers in the dermis and found that quantity, diameter, and arrangement of collagen fibers in the dermis were significantly improved by DAA treatment. CONCLUSION: Our results suggest that DAA could be a useful agent for improving skin photoaging through the protection and regeneration of collagen fibers in skin.

Bryostatin and its synthetic analog, picolog rescue dermal fibroblasts from prolonged stress and contribute to survival and rejuvenation of human skin equivalents.

Skin health is associated with the day-to-day activity of fibroblasts. The primary function of fibroblasts is to synthesize structural proteins, such as collagen, extracellular matrix proteins, and other proteins that support the structural integrity of the skin and are associated with younger, firmer, and more elastic skin that is better able to resist and recover from injury. At sub-nanomolar concentrations (0.03-0.3 nM), bryostatin-1 and its synthetic analog, picolog (0.1-10 nM) sustained the survival and activation of human dermal fibroblasts cultured under the stressful condition of prolonged serum deprivation. Bryostatin-1 treatment stabilized human skin equivalents (HSEs), a bioengineered combination of primary human skin cells (keratinocytes and dermal fibroblasts) on an extracellular matrix composed of mainly collagen. Fibroblasts activated by bryostatin-1 protected the structural integrity of HSEs. Bryostatin-1 and picolog prolonged activation of Erk in fibroblasts to promote cell survival. Chronic stress promotes the progression of apoptosis. Dermal fibroblasts constitutively express all components of Fas associated apoptosis, including caspase-8, an initiator enzyme of apoptosis. Prolong bryostatin-1 treatment reduced apoptosis by decreasing caspase-8 and protected dermal fibroblasts. Our data suggest that bryostatin-1 and picolog could be useful in anti-aging skincare, and could have applications in tissue engineering and regenerative medicine.

Effect of 3,6-anhydro-l-galactose on α-melanocyte stimulating hormone-induced melanogenesis in human melanocytes and a skin-equivalent model.

3,6-Anhydro-l-galactose (l-AHG) is a bioactive sugar that is a major component of agarose. Recently, l-AHG was reported to have anti-melanogenic potential in human epidermal melanocytes (HEMs) and B16F10 melanoma cells; however, its underlying molecular mechanisms remain unknown. At noncytotoxic concentrations, l-AHG has been shown to inhibit alpha-melanocyte-stimulating hormone-induced melanin synthesis in various cell models, including HEMs, melan-a cells, and B16F10 cells. Although l-AHG did not inhibit tyrosinase activity in vitro, reverse transcription-polymerase chain reaction results demonstrated that the anti-melanogenic effect of l-AHG was mediated by transcriptional repression of melanogenesis-related genes, including tyrosinase, tyrosinase-related protein-1 (TRP-1), tyrosinase-related protein-2 (TRP-2), and microphthalmia-associated transcription factor (MITF) in HEMs. Western blot analysis showed that l-AHG effectively attenuated α-melanocyte-stimulating hormone-induced melanogenic proteins by inhibiting cyclic adenosine monophosphate/cyclic adenosine monophosphate-dependent protein kinase, mitogen-activated protein kinase, and Akt signaling pathways in HEMs. Topical application of l-AHG significantly ameliorated melanin production in a 3D pigmented human skin model. Collectively, these results suggest that l-AHG could be utilized as novel cosmetic compounds with skin-whitening efficacy.

Type IV collagen aggregates promote keratinocyte proliferation and formation of epidermal layer in human skin equivalents.

Type IV collagen isolated from lens capsule without enzymatic treatment is known to form a gel under physiological condition and influences cellular activities. In case of human keratinocytes, the suppression of proliferation on reconstituted type IV collagen gels was reported in monolayer culture. In this study, we examined effects of type IV collagen isolated from porcine lens capsule on epidermal formation in human skin equivalents (HSEs). Type IV collagen aggregates were prepared under the culture condition and the aggregates suppressed keratinocyte proliferation in monolayer culture as well as the culture on the gels. In HSEs, type IV collagen aggregates were reconstituted on the surface of contracted collagen gels containing human dermal fibroblasts and the keratinocytes were then cultured on the aggregates for 14 days. Interestingly, in HSEs with type IV collagen aggregates, the BrdU-positive keratinocytes were increased and the thickness of the epidermal layer was around twice than that of control culture. Epidermal differentiation markers were expressed in the upper layer of the epidermis and the defined deposition of human basement membrane components were increased at the dermal-epidermal junction. These results indicate that the type IV collagen aggregates stimulate the proliferation of basal keratinocytes and improve the stratification of epidermal layers in HSEs.

The Ginsenoside Derivative 20(S)-Protopanaxadiol Inhibits Solar Ultraviolet Light-Induced Matrix Metalloproteinase-1 Expression.

Ginsenosides are major pharmacologically active compounds present in ginseng (Panax ginseng). Among the ginsenosides, 20-O-ß-D-glucopyranosyl-20(S)-protopanaxadiol (GPPD) and ginsenoside Rb1 (Rb1) have previously been reported to exhibit anti-wrinkle effects. In this study, 20(S)-protopanaxadiol (20(S)-PPD), an aglycone derivative of the Rb1 metabolite was investigated for its anti-wrinkle benefit and compared to GPPD and Rb1. The anti-wrinkle effect of 20(S)-PPD during solar UV light was investigated using a human skin equivalent model and human keratinocytes. 20(S)-PPD attenuated solar UV-induced matrix metalloproteinase (MMP)-1 expression to a greater extent than GPPD and Rb1. 20(S)-PPD treatment modulated MMP-1 mRNA expression and the transcriptional activity of activator protein (AP)-1, a major transcription factor of MMP-1. Two upstream signaling pathways for AP-1, the MEK1/2-ERK1/2-p90RSK and MEK3/6-p38 pathways, were also suppressed. Taken together, these findings highlight the potential of 20(S)-PPD for further development as a preventative agent for sunlight-induced skin wrinkle. J. Cell. Biochem. 118: 3756-3764, 2017. © 2017 Wiley Periodicals, Inc.

Acceleration of Apoptosis by Extracellular Basic pH in a 3D Human Skin Equivalent System.

Previously, we have shown that extracellular basic pH plays a significant role in both the direct and indirect regulation of cellular processes in a wound; this in turn affects the wound-healing process. Several studies have demonstrated the importance of apoptosis modulation in the wound-healing process, especially in removing inflammatory cells and in inhibiting scar formation. However, the effects of extracellular basic pH on wound healing-related skin damage are yet to be examined. Therefore, we investigated the induction of accelerated apoptosis by extracellular basic pH in skin. Apoptosis-related protein levels were measured using an array kit, target protein expression levels were detected by immunostaining, lactate dehydrogenase was analyzed spectrophotometrically, and Annexin V levels were measured by fluorescence staining. Basic pH (8.40) strongly upregulated extrinsic apoptosis proteins (Fas, high temperature requirement A, and p21) and slightly upregulated intrinsic apoptosis proteins (cytochrome c, B-cell lymphoma 2 [Bcl-2], Bcl-2-associated death promoter, and Bcl-2-like protein 4) in a 3D human skin equivalent system. Moreover, basic pH (8.40) induced heat shock protein (HSP) 60 and 70. In addition, basic pH-exposed Fas- and HSP60-knockdown cells showed significantly decreased levels of apoptosis. Taken together, these results indicate that extracellular basic pH increases early-stage apoptosis through Fas/FasL via modulation of HSP60 and HSP70.

Antimelanogenic Efficacy of Melasolv (3,4,5-Trimethoxycinnamate Thymol Ester) in Melanocytes and Three-Dimensional Human Skin Equivalent.

BACKGROUND/AIMS: Excessive melanogenesis often causes unaesthetic hyperpigmentation. In a previous report, our group introduced a newly synthesized depigmentary agent, Melasolv™ (3,4,5-trimethoxycinnamate thymol ester). In this study, we demonstrated the significant whitening efficacy of Melasolv using various melanocytes and human skin equivalents as in vitro experimental systems. METHODS: The depigmentary effect of Melasolv was tested in melan-a cells (immortalized normal murine melanocytes), α-melanocyte-stimulating hormone (α-MSH)-stimulated B16 murine melanoma cells, primary normal human melanocytes (NHMs), and human skin equivalent (MelanoDerm). The whitening efficacy of Melasolv was further demonstrated by photography, time-lapse microscopy, Fontana-Masson (F&M) staining, and 2-photon microscopy. RESULTS: Melasolv significantly inhibited melanogenesis in the melan-a and α-MSH-stimulated B16 cells. In human systems, Melasolv also clearly showed a whitening effect in NHMs and human skin equivalent, reflecting a decrease in melanin content. F&M staining and 2-photon microscopy revealed that Melasolv suppressed melanin transfer into multiple epidermal layers from melanocytes as well as melanin synthesis in human skin equivalent. CONCLUSION: Our study showed that Melasolv clearly exerts a whitening effect on various melanocytes and human skin equivalent. These results suggest the possibility that Melasolv can be used as a depigmentary agent to treat pigmentary disorders as well as an active ingredient in cosmetics to increase whitening efficacy.

Photoprotection by pistachio bioactives in a 3-dimensional human skin equivalent tissue model.

Reactive oxygen species (ROS) generated during ultraviolet (UV) light exposure can induce skin damage and aging. Antioxidants can provide protection against oxidative injury to skin via "quenching" ROS. Using a validated 3-dimensional (3D) human skin equivalent (HSE) tissue model that closely mimics human skin, we examined whether pistachio antioxidants could protect HSE against UVA-induced damage. Lutein and γ-tocopherol are the predominant lipophilic antioxidants in pistachios; treatment with these compounds prior to UVA exposure protected against morphological changes to the epithelial and connective tissue compartments of HSE. Pistachio antioxidants preserved overall skin thickness and organization, as well as fibroblast morphology, in HSE exposed to UVA irradiation. However, this protection was not substantiated by the analysis of the proliferation of keratinocytes and apoptosis of fibroblasts. Additional studies are warranted to elucidate the basis of these discordant results and extend research into the potential role of pistachio bioactives promoting skin health.