Assessment of the photoprotective potential and structural characterization of secondary metabolites of Antarctic fungus Arthrinium sp.

Interest in Antarctic fungi has grown due to their resilience in harsh environments, suggesting the presence of valuable compounds from its organisms, such as those presenting photoprotective potential, since this environment suffers the most dangerous UV exposure in the world. Therefore, this research aimed to assess the photoprotective potential of compounds from sustainable marine sources, specifically seaweed-derived fungi from Antarctic continent. These studies led to discovery of photoprotective and antioxidant properties of metabolites from Arthrinium sp., an endophytic fungus from Antarctic brown algae Phaeurus antarcticus. From crude extract, fractions A-I were obtained and compounds 1-6 isolated from E and F fractions, namely 3-Hydroxybenzyl alcohol (1), (-)-orthosporin (2), norlichexanthone (3), anomalin B (4), anomalin A (5), and agonodepside B (6). Compounds 1, 2, and 6 were not previously reported in Arthrinium. Fraction F demonstrated excellent absorbance in both UVA and UVB regions, while compound 6 exhibited lower UVB absorbance, possibly due to synergistic effects. Fraction F and compound 6 displayed photostability and were non-phototoxic to HaCaT cells. They also exhibited antioxidant activity by reducing intracellular ROS production induced by UVA in keratinocyte monolayers and reconstructed human skin models (resulting in 34.6% and 30.2% fluorescence reduction) and did not show irritation potential in HET-CAM assay. Thus, both are promising candidates for use in sunscreens. It is noted that Fraction F does not require further purification, making it advantageous, although clinical studies are necessary to confirm its potential applicability for sunscreen formulations.

Standardised Reconstructed Skin Models in Toxicology and Pharmacology: State of the Art and Future Development.

Three-dimensional (3D) reconstructed human skin (RhS) models featuring fully-differentiated characteristics of in vivo human epidermis have been known for almost 40 years. In this chapter, the topic of commercial in vitro tissue models is described, taking RhS models as an example. The need for highly standardised models is evident for regulatory testing purposes, e.g. the classification and labelling of chemicals and formulations, as well as for pharmacology-oriented research and drug development. Following the standardisation of RhS model production by commercial developers, international validation studies and regulatory acceptance, 3D RhS models are now used globally in both industrial and academic research laboratories. Industrial production of standardised 3D RhS models involves GMP-compliant processes together with ISO 9001 documentation in order to control and ensure reproducibility and quality. Key biological, functional, and performance features that are addressed in industrial production include barrier properties, histological and immunohistochemical characterisation, lipid profile characterisation, and tissue viability before and after transport. An up-to-date survey of commercial RhS tissue producers and the regulatory acceptance status of major safety, hazard, and efficacy assays currently available to chemical and pharmaceutical industries is presented in this chapter. Safety and ethical concerns related to the use of human tissue in the industrial production of RhS models are discussed. Finally, innovative approaches to the production of standardised 3D RhS models including automated production, development of more representative 3D RhS models using advanced additive manufacturing tools, microfluidics technologies, and bioprinting are presented. The future outlook for 3D RhS models includes a prevalence of high-quality models which will be fabricated by end-users rather than commercial producers. These will overcome problems with shipments and customs clearance that many users still face when buying RhS from overseas commercial suppliers. Open-source technologies and commercial components for "do-it-yourself" RhS will significantly change the skin model market as well as regulatory acceptance of open-source models during the next decade. All of these developments and improvements will together allow more widespread use of in vitro RhS models for broader application as animal replacements in areas ranging from industrial and regulatory toxicology and pharmacology, to drug development and personalised medicine.

Skin Disease Models In Vitro and Inflammatory Mechanisms: Predictability for Drug Development.

Investigative skin biology, analysis of human skin diseases, and numerous clinical and pharmaceutical applications rely on skin models characterized by reproducibility and predictability. Traditionally, such models include animal models, mainly rodents, and cellular models. While animal models are highly useful in many studies, they are being replaced by human cellular models in more and more approaches amid recent technological development due to ethical considerations. The culture of keratinocytes and fibroblasts has been used in cell biology for many years. However, only the development of co-culture and three-dimensional epidermis and full-skin models have fundamentally contributed to our understanding of cell-cell interaction and cell signalling in the skin, keratinocyte adhesion and differentiation, and mechanisms of skin barrier function. The modelling of skin diseases has highlighted properties of the skin important for its integrity and cutaneous development. Examples of monogenic as well as complex diseases including atopic dermatitis and psoriasis have demonstrated the role of skin models to identify pathomechanisms and drug targets. Recent investigations have indicated that 3D skin models are well suitable for drug testing and preclinical studies of topical therapies. The analysis of skin diseases has recognized the importance of inflammatory mechanisms and immune responses and thus other cell types such as dendritic cells and T cells in the skin. Current developments include the production of more complete skin models comprising a range of different cell types. Organ models and even multi-organ systems are being developed for the analysis of higher levels of cellular interaction and drug responses and are among the most recent innovations in skin modelling. They promise improved robustness and flexibility and aim at a body-on-a-chip solution for comprehensive pharmaceutical in vitro studies.

Assessment of cytotoxicity and sensitization potential of intradermally injected tattoo inks in reconstructed human skin.

BACKGROUND: The number of people within the European population having at least one tattoo has increased notably, and with it the number of tattoo-associated clinical complications. Despite this, safety information and testing regarding tattoo inks remain limited. OBJECTIVE: To assess cytotoxicity and sensitization potential of 16 tattoo inks after intradermal injection into reconstructed human skin (RHS). METHODS: Commercially available tattoo inks were injected intradermally into RHS (reconstructed epidermis on a fibroblast-populated collagen hydrogel) using a permanent makeup device. RHS biopsies, tissue sections, and culture medium were assessed for cytotoxicity (thiazolyl blue tetrazolium bromide assay [MTT assay]), detrimental histological changes (haematoxylin and eosin staining), and the presence of inflammatory and sensitization cytokines (interleukin [IL]-1α, IL-8, IL-18; enzyme-linked immunosorbent assay). RESULTS: Varying degrees of reduced metabolic activity and histopathological cytotoxic effects were observed in RHS after ink injection. Five inks showed significantly reduced metabolic activity and enhanced sensitization potential compared with negative controls. DISCUSSION: Using the RHS model system, four tattoo inks were identified as highly cytotoxic and classified as potential sensitizers, suggesting that allergic contact dermatitis could emerge in individuals carrying these inks. These results indicate that an RHS-based assessment of cytotoxicity and sensitization potential by intradermal tattoo ink injection is a useful analytical tool to determine ink-induced deleterious effects.

Micro-environmental cross-talk in an organotypic human melanoma-in-skin model directs M2-like monocyte differentiation via IL-10.

Preclinical assessment of novel therapies to fight cancer requires models that reflect the human physiology and immune response. Here, we established an in vitro three-dimensional (3D) reconstructed organotypic human melanoma-in-skin (Mel-RhS) model to investigate cellular and molecular features of tumor formation over a period of 6 weeks. Tumor nests developed over time at the epidermal-dermal junction and spread towards the dermis, in places disrupting the basement membrane. This coincided with secretion of matrix metalloproteinase 9 (MMP-9) by melanoma cells. These features resemble the initial stages of invasive melanoma. Interestingly, while the SK-MEL-28 cell line did not secrete detectable levels of interleukin-10 (IL-10) in traditional two-dimensional monolayers, it did express IL-10 in the 3D Mel-RhS, as did the surrounding keratinocytes and fibroblasts. This cellular cross-talk-induced secretion of IL-10 in the Mel-RhS indicated the generation of an immune suppressive microenvironment. Culture supernatants from Mel-RhS interfered with monocyte-to-dendritic-cell differentiation, leading to the development of M2-like macrophages, which was in part prevented by antibody-mediated IL-10 blockade. Indeed, high-dimensional single-cell analysis revealed a shift within the monocyte population away from a CD163+PD-L1+ M2-like phenotype upon IL-10 blockade. Thus, the 3D configuration of the Mel-RhS model revealed a role for IL-10 in immune escape through misdirected myeloid differentiation, which would have been missed in classical monolayer cultures.

Titanium salts tested in reconstructed human skin with integrated MUTZ-3-derived Langerhans cells show an irritant rather than a sensitizing potential.

BACKGROUND: The nature of clinically related adverse reactions to titanium is still unknown. OBJECTIVE: To determine whether titanium salts have irritant or sensitizing potential in a reconstructed human skin (RHS) model with integrated Langerhans cells (LCs). METHODS: RHS-LCs (ie, reconstructed epidermis) containing primary differentiated keratinocytes and CFSE+ CD1a+ -LCs generated from the MUTZ-3 cell line on a primary fibroblast-populated collagen hydrogel (dermis) were topically exposed to titanium(IV) bis(ammonium lactato)dihydroxide (TiALH). LC migration and plasticity were determined. RESULTS: TiALH resulted in CFSE+ CD1a+ -LC migration out of the epidermis. Neutralizing antibodies to CCL5 and CXCL12 showed that LC migration was CCL5 and not CXCL12 mediated. LCs accumulating within the dermis after TiALH exposure were CFSE+ Lang+ CD68+ which is characteristic of a phenotypic switch of MUTZ-LC to a macrophage-like cell. Furthermore, TiALH did not result in increased interleukin (IL)-1ß or CCR7 messenger RNA (mRNA) in the dermis, but did result in increased IL-10 mRNA. In addition, monocultures of MUTZ-LCs failed to increase LC maturation biomarkers CD83, CD86, and CXCL-8 when exposed to noncytotoxic concentrations of four different titanium salts. CONCLUSION: These results classify titanium salts as irritants rather than sensitizers and indicate that titanium implant-related complaints could be due to localized irritant-mediated inflammation arising from leachable agents rather than a titanium metal allergy.

Differential influence of Streptococcus mitis on host response to metals in reconstructed human skin and oral mucosa.

BACKGROUND: Skin and oral mucosa are continuously exposed to potential metal sensitizers while hosting abundant microbes, which may influence the host response to sensitizers. This host response may also be influenced by the route of exposure that is skin or oral mucosa, due to their different immune properties. OBJECTIVE: Determine how commensal Streptococcus mitis influences the host response to nickel sulfate (sensitizer) and titanium(IV) bis(ammonium lactato)dihydroxide (questionable sensitizer) in reconstructed human skin (RHS) and gingiva (RHG). METHODS: RHS/RHG was exposed to nickel or titanium, in the presence or absence of S. mitis for 24 hours. Histology, cytokine secretion, and Toll-like receptors (TLRs) expression were assessed. RESULTS: S. mitis increased interleukin (IL)-6, CXCL8, CCL2, CCL5, and CCL20 secretion in RHS but not in RHG; co-application with nickel further increased cytokine secretion. In contrast, titanium suppressed S. mitis-induced cytokine secretion in RHS and had no influence on RHG. S. mitis and metals differentially regulated TLR1 and TLR4 in RHS, and predominantly TLR4 in RHG. CONCLUSION: Co-exposure of S. mitis and nickel resulted in a more potent innate immune response in RHS than in RHG, whereas titanium remained inert. These results indicate the important influence of commensal microbes and the route of exposure on the host's response to metals.

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.

Improved metal allergen reactivity of artificial skin models by integration of Toll-like receptor 4-positive cells.

BACKGROUND: Reconstructed human epidermis (RhE) is widely used to replace animal models in order to assess the proinflammatory and allergenic effects of chemicals. Unfortunately, RhE lacks proinflammatory responsiveness for metal haptens, which are the most prevalent human contact allergens, raising concerns about its reliability for predicting skin allergens. OBJECTIVES: To investigate whether this limitation of RhE might be attributable to a lack of functional expression of Toll-like receptor 4 (TLR4), which governs proinflammatory sensitivity to nickel and cobalt. MATERIALS AND METHODS: RhE, dendritic cell (DC)-containing RhE and full-thickness skin equivalent (FTSE) were compared regarding their proinflammatory responsiveness to metal allergens. RESULTS: The incorporation of dermal fibroblasts was sufficient to confer metal sensitivity to RhE. Unlike keratinocytes, normal human fibroblasts expressed high levels of TLR4 mRNA and induced interleukin-8 expression upon stimulation with nickel or cobalt. Consistently, dermal isolates from FTSE expressed considerable amounts of TLR4 mRNA, whereas RhE or epidermis isolated from FTSE, normal human epidermis or inflamed human epidermis failed to express TLR4. Similarly, co-culture with TLR4-positive DCs bestowed RhE with proinflammatory responsiveness to metals. CONCLUSION: Our data suggest that FTSE or DC/RhE co-culture models can circumvent the shortcomings of RhE assays, and combine the benefits of complex and monoculture-based test systems in a single assay.

Towards the development of a RNAi-based topical treatment for psoriasis: Proof-of-concept in a 3D psoriasis skin model.

RNA interference has emerged as a powerful tool for therapeutic gene silencing, as it offers the possibility to silence virtually any known pathology-causing gene. However, in vivo delivery of RNAi molecules is hampered by their unfavourable physicochemical characteristics and susceptibility to degradation by endogenous enzymes. To overcome these limitations, we recently developed an elastic liposomal formulation, called DDC642, as topical delivery system of therapeutic RNAi molecules for skin disorders. In this study, we validated the therapeutic efficacy of DDC642-encapsulated RNAi molecules in the treatment of psoriasis using 3 different in vitro models: a standardized keratinocyte monolayer culture, psoriasis-induced keratinocytes and a psoriasis-reconstructed skin model. Four genes (IL22RA1, KRT17, DEFB4 and TSLP), known to be upregulated in psoriatic lesions, and thereby key players in psoriasis pathogenesis were selected. Moreover, the possibility of using a combined siRNA therapy in the topical treatment of psoriasis was explored. Results indicate a successful gene silencing of each different target, both at mRNA and protein levels. Additionally, siRNA-DDC642 treatment resulted in a reduced expression of specific psoriasis markers, indicating their potential in future therapeutic approach. The examined siRNA combination (ie simultaneous knockdown of KRT17, DEFB4 and TSLP) showed an enhanced reduction in TSLP expression, whereas the decrease in K17 protein expression was impaired in psoriatic keratinocytes. Although the here examined siRNA combination could still be further improved, our study proved already in vitro the clinical potential of targeting multiple genes at once, each playing a different role in a complex disease such as psoriasis.

Ultrastructural and Molecular Analysis of Ribose-Induced Glycated Reconstructed Human Skin.

Aging depicts one of the major challenges in pharmacology owing to its complexity and heterogeneity. Thereby, advanced glycated end-products modify extracellular matrix proteins, but the consequences on the skin barrier function remain heavily understudied. Herein, we utilized transmission electron microscopy for the ultrastructural analysis of ribose-induced glycated reconstructed human skin (RHS). Molecular and functional insights substantiated the ultrastructural characterization and proved the relevance of glycated RHS beyond skin aging. In particular, electron microscopy mapped the accumulation and altered spatial orientation of fibrils and filaments in the dermal compartment of glycated RHS. Moreover, the epidermal basement membrane appeared thicker in glycated than in non-glycated RHS, but electron microscopy identified longitudinal clusters of the finest collagen fibrils instead of real thickening. The stratum granulosum contained more cell layers, the morphology of keratohyalin granules decidedly differed, and the stratum corneum lipid order increased in ribose-induced glycated RHS, while the skin barrier function was almost not affected. In conclusion, dermal advanced glycated end-products markedly changed the epidermal morphology, underlining the importance of matrix⁻cell interactions. The phenotype of ribose-induced glycated RHS emulated aged skin in the dermis, while the two to three times increased thickness of the stratum granulosum resembled poorer cornification.

Biotransformation of 2,4-toluenediamine in human skin and reconstructed tissues.

Reconstructed human epidermis (RHE) is used for risk assessment of chemicals and cosmetics and RHE as well as reconstructed human full-thickness skin (RHS) become important for e.g., the pre-clinical development of drugs. Yet, the knowledge regarding their biotransformation capacity is still limited, although the metabolic activity is highly relevant for skin sensitization, genotoxicity, and the efficacy of topical dermatics. The biotransformation of the aromatic amine 2,4-toluenediamine (2,4-TDA) has been compared in two commercially available RHS to normal human skin ex vivo, and in primary epidermal keratinocytes and dermal fibroblasts as well as in vitro generated epidermal Langerhans cells and dermal dendritic cells. The mono N-acetylated derivative N-(3-amino-4-methyl-phenyl)acetamide (M1) was the only metabolite detectable in substantial amounts indicating the predominance of N-acetylation. RHS exceeded human skin ex vivo in N-acetyltransferase activity and in cell cultures metabolite formation ranked as follows: keratinocytes > fibroblasts ~ Langerhans cells ~ dendritic cells. In conclusion, our results underline the principal suitability of RHS as an adequate test matrix for the investigation of N-acetylation of xenobiotics which is most relevant for risk assessment associated with cutaneous exposure to aromatic amines.

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

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

Thermosensitive dendritic polyglycerol-based nanogels for cutaneous delivery of biomacromolecules.

Genetic skin diseases caused by mutations resulting in diminished protein synthesis could benefit from local substitution of the missing protein. Proteins, however, are excluded from topical applications due to their physicochemical properties. We prepared protein-loaded thermoresponsive poly(N-isopropylacrylamide)-polyglycerol-based nanogels exhibiting a thermal trigger point at 35°C, which is favorable for cutaneous applications due to the native thermal gradient of human skin. At≥35°C, the particle size (~200nm) was instantly reduced by 20% and 93% of the protein was released; no alterations of protein structure or activity were detected. Skin penetration experiments demonstrated efficient intraepidermal protein delivery particularly in barrier deficient skin, penetration of the nanogels themselves was not detected. The proof of concept was provided by transglutaminase 1-loaded nanogels which efficiently delivered the protein into transglutaminase 1-deficient skin models resulting in a restoration of skin barrier function. In conclusion, thermoresponsive nanogels are promising topical delivery systems for biomacromolecules. FROM THE CLINICAL EDITOR: Many skin disorders are characterized by an absence of a specific protein due to underlying gene mutation. In this article, the authors described the use of a thermoresponsive PNIPAM-dPG nanogel for cutaneous protein delivery in a gene knock-down model of human skin. The results may have implication for nano-based local delivery of therapeutic agents in skin.

Evaluation of the photoprotective and antioxidant potential of an avobenzone derivative.

Solar radiation can cause damage to the skin, and the use of sunscreens is one of the main protective measures. However, photounstable ultraviolet (UV) filters can generate photoproducts and reactive oxygen species (ROS). Adding antioxidants, such as resveratrol, to enhance the action of UV filters in sunscreens is an interesting strategy for reducing the damage caused by UV radiation exposure. However, new compounds must have their stability, safety and efficacy guaranteed. Avobenzone, a commonly used UV filter, stands out as a promising candidate for structural modification to enhance its stability. Its molecular hybridization with other UV filters and antioxidants can lead to safer and more effective compounds. In this study, the photoprotective and antioxidant potential of a derivative of avobenzone, hybridized with resveratrol's molecule, was evaluated using in vitro models of cells in monolayer and reconstructed human skin (RHS). Phototoxic potential was assessed using fibroblasts, while the antioxidant activity was measured using the DCFH2-DA probe in HaCaT keratinocytes and in-house RHS. The derivative exhibited UV absorption and demonstrated photostability. It did not exhibit any phototoxic nor photoreactivity potential. Additionally, it was able to photo stabilize a combination of photounstable UV filters, avobenzone and octyl methoxycinnamate, and to reduce their phototoxic potential. In terms of antioxidant activity, the derivative successfully protected against UVA-induced ROS production in the HaCaT keratinocytes model, showing statistical equivalence to the antioxidant control, quercetin (10 µg/mL). Furthermore, experiments conducted in the RHS model demonstrated a significant reduction of 30.7% in ROS generation compared to the irradiated control. This study demonstrated that structural modifications of avobenzone can lead to the development of a broad spectrum (absorbing UVB and UVA II radiation, as well as a portion of the UVA I radiation), non-phototoxic, non-photoreactive and photostable derivative for sunscreen and anti-aging formulations. This derivative enhances protection against oxidative stress induced by UV radiation and improves the effectiveness of sun protection. In addition to the monolayer model, the use of a standardized in-house RHS model was highly relevant for evaluating the effects of UV radiation and skin aging. This model closely mimics human physiological conditions and enables the testing of new compounds and the investigation of protective mechanisms against skin damage.

Strategies to make human skin models based on cellular senescence for ageing research.

Human skin ageing is closely related to the ageing of the whole organism, and it's a continuous multisided process that is influenced not only by genetic and physiological factors but also by the cumulative impact of environmental factors. Currently, there is a scientific community need for developing skin models representing ageing processes to (i) enhance understanding on the mechanisms of ageing, (ii) discover new drugs for the treatment of age-related diseases, and (iii) develop effective dermo-cosmetics. Bioengineers worldwide are trying to reproduce skin ageing in the laboratory aiming to better comprehend and mitigate the senescence process. This review provides details on the main ageing molecular mechanisms and procedures to obtain in vitro aged skin models.

The reconstructed skin micronucleus assay in EpiDerm™: reduction of false-positive results - a mechanistic study with epigallocatechin gallate.

The high rate of false-positive or misleading results in in vitro mammalian genotoxicity testing is a hurdle in the development of valuable chemicals, especially those used in cosmetics, for which in vivo testing is banned in the European Union. The reconstructed skin micronucleus (RSMN) assay in EpiDerm™ (MatTek Corporation, USA) has shown promise as a follow-up for positive in vitro mammalian genotoxicity tests. However, few studies have explored its better predictive performance compared with existing in vitro assays. In the present study, we followed the protocol of the RSMN assay and used eight chemicals to compare micronucleus (MN) induction with EpiDerm™ with that in normal human epidermal keratinocytes (NHEKs), both derived from human skin. The assessments of EpiDerm™ conformed to those of in vivo MN assay, whereas those of NHEKs did not. The effect of cell differentiation status on MN induction was further addressed using a model compound, epigallocatechin gallate (EGCG), which is a major component of green tea extract that shows positive results in in vitro mammalian genotoxicity assays via oxidative stress and negative results in in vivo MN studies. RSMN assay in an underdeveloped epidermal model, EpiDerm-201™ (MatTek Corporation), showed a negative result identical to that in EpiDerm™, indicating that the barrier function of keratinocytes has limited impact. Analysis of the gene expression profile of both EpiDerm™ and NHEKs after EGCG treatment for 12h revealed that the expression of genes related to genotoxic response was significantly induced only in NHEKs. Conversely, antioxidative enzyme activities (catalase and glutathione peroxidase) in EpiDerm™ were higher than those in NHEKs. These results indicate that EpiDerm™ has antioxidant properties similar to those of a living body and is capable of eliminating oxidative stress that may be caused by EGCG under in vitro experimental conditions.

A Reconstructed Human Melanoma-in-Skin Model to Study Immune Modulatory and Angiogenic Mechanisms Facilitating Initial Melanoma Growth and Invasion.

Invasion, immune modulation, and angiogenesis are crucial in melanoma progression. Studies based on animals or two-dimensional cultures poorly recapitulate the tumor-microenvironmental cross-talk found in humans. This highlights a need for more physiological human models to better study melanoma features. Here, six melanoma cell lines (A375, COLO829, G361, MeWo, RPMI-7951, and SK-MEL-28) were used to generate an in vitro three-dimensional human melanoma-in-skin (Mel-RhS) model and were compared in terms of dermal invasion and immune modulatory and pro-angiogenic capabilities. A375 displayed the most invasive phenotype by clearly expanding into the dermal compartment, whereas COLO829, G361, MeWo, and SK-MEL-28 recapitulated to different extent the initial stages of melanoma invasion. No nest formation was observed for RPMI-7951. Notably, the integration of A375 and SK-MEL-28 cells into the model resulted in an increased secretion of immune modulatory factors (e.g., M-CSF, IL-10, and TGFß) and pro-angiogenic factors (e.g., Flt-1 and VEGF). Mel-RhS-derived supernatants induced endothelial cell sprouting in vitro. In addition, observed A375-RhS tissue contraction was correlated to increased TGFß release and α-SMA expression, all indicative of differentiation of fibroblasts into cancer-associated fibroblast-like cells and reminiscent of epithelial-to-mesenchymal transition, consistent with A375's most prominent invasive behavior. In conclusion, we successfully generated several Mel-RhS models mimicking different stages of melanoma progression, which can be further tailored for future studies to investigate individual aspects of the disease and serve as three-dimensional models to assess efficacy of therapeutic strategies.

UTP increases wound healing in the self assembled skin substitute (SASS).

The therapeutic potential of purinergic signaling has been explored for a wide variety of diseases, including those related to the skin. In this study, we used the self-assembled skin substitutes (SASS), a highly functional reconstructed human skin model, which shares many properties with normal human skin, to study the impact of purinergic receptors agonists, such as ATP, UTP and a P2Y receptor antagonist, Reactive Blue 2 during wound healing. After treating the wounded skins, we evaluated the wound area, reepithelialization, length of migrating tongues toward the wound, quality of the skins through the cytokeratin 10 and laminin-5 expression, epidermal and dermal cell proliferation. In addition, the expression of the main ectoenzymes capable of hydrolyzing nucleotides were investigated through the wounded SASS regions: unwounded region, wound margin, intermediate region and migrating epidermal tongue. After 3 days, under the UTP treatment, the wounded SASS showed an increase in the reepithelialization and in the proliferation of keratinocytes and fibroblasts, without altering the quality of the skin. We also identified the presence of the ectoenzymes NTPDase1 and NPP1 in the reconstructed human skin model, suggesting their involvement in wound healing. Considering the need for new therapies capable of promoting healing in complex wounds, although these results are still preliminary, they suggest the involvement of extracellular nucleotides in human skin healing and the importance to understand their role in this mechanism. New experiments it will be necessary to determine the mechanisms by which the purinergic signaling is involved in the skin wound healing.

Strat-M® positioning for skin permeation studies: A comparative study including EpiSkin® RHE, and human skin.

In the development and optimization of dermatological products, In Vitro Permeation Testing (IVPT) is pivotal for controlled study of skin penetration. To enhance standardization and replicate human skin properties reconstructed human skin and synthetic membranes are explored as alternatives. Strat-M® is a membrane designed to mimic the multi-layered structure of human skin for IVPT. For instance, in Strat-M®, the steady-state fluxes (JSS) of resorcinol in formulations free of permeation enhancers were found to be 41 ± 5 µg/cm2·h for the aqueous solution, 42 ± 6 µg/cm2·h for the hydrogel, and 40 ± 6 µg/cm2·h for the oil-in-water emulsion. These results were closer to excised human skin (5 ± 3, 9 ± 2, 13 ± 6 µg/cm2·h) and surpassed the performance of EpiSkin® RHE (138 ± 5, 142 ± 6, and 162 ± 11 µg/cm2·h). While mass spectrometry and Raman microscopy demonstrated the qualitative molecular similarity of EpiSkin® RHE to human skin, it was the porous and hydrophobic polymer nature of Strat-M® that more faithfully reproduced the skin's diffusion-limiting barrier. Further validation through similarity factor analysis (∼80-85%) underscored Strat-M®'s significance as a reliable substitute for human skin, offering a promising approach to enhance realism and reproducibility in dermatological product development.