Full-thickness Skin Equivalents Research Articles

Attenuation of fibroblast activation and fibrosis by adropin in systemic sclerosis.

Fibrotic diseases impose a major socioeconomic challenge on modern societies and have limited treatment options. Adropin, a peptide hormone encoded by the energy homeostasis-associated (ENHO) gene, is implicated in metabolism and vascular homeostasis, but its role in the pathogenesis of fibrosis remains enigmatic. Here, we used machine learning approaches in combination with functional in vitro and in vivo experiments to characterize adropin as a potential regulator involved in fibroblast activation and tissue fibrosis in systemic sclerosis (SSc). We demonstrated consistent down-regulation of adropin/ENHO in skin across multiple cohorts of patients with SSc. The prototypical profibrotic cytokine TGFβ reduced adropin/ENHO expression in a JNK-dependent manner. Restoration of adropin signaling by therapeutic application of bioactive adropin34-76 peptides in turn inhibited TGFβ-induced fibroblast activation and fibrotic tissue remodeling in primary human dermal fibroblasts, three-dimensional full-thickness skin equivalents, mouse models of bleomycin-induced pulmonary fibrosis and sclerodermatous chronic graft-versus-host-disease (sclGvHD), and precision-cut human skin slices. Knockdown of GPR19, an adropin receptor, abrogated the antifibrotic effects of adropin in fibroblasts. RNA-seq demonstrated that the antifibrotic effects of adropin34-76 were functionally linked to deactivation of GLI1-dependent profibrotic transcriptional networks, which was experimentally confirmed in vitro, in vivo, and ex vivo using cultured human dermal fibroblasts, a sclGvHD mouse model, and precision-cut human skin slices. ChIP-seq confirmed adropin34-76-induced changes in TGFβ/GLI1 signaling. Our study characterizes the TGFβ-induced down-regulation of adropin/ENHO expression as a potential pathomechanism of SSc as a prototypical systemic fibrotic disease that unleashes uncontrolled activation of profibrotic GLI1 signaling.

Comparison of photodamage in non-pigmented and pigmented human skin equivalents exposed to repeated ultraviolet radiation to investigate the role of melanocytes in skin photoprotection.

Daily solar ultraviolet (UV) radiation has an important impact on skin health. Understanding the initial events of the UV-induced response is critical to prevent deleterious conditions. However, studies in human volunteers have ethical, technical, and economic implications that make skin equivalents a valuable platform to investigate mechanisms related to UV exposure to the skin. In vitro human skin equivalents can recreate the structure and function of in vivo human skin and represent a valuable tool for academic and industrial applications. Previous studies have utilised non-pigmented full-thickness or pigmented epidermal skin equivalents to investigate skin responses to UV exposure. However, these do not recapitulate the dermal-epidermal crosstalk and the melanocyte role in photoprotection that occurs in vivo. In addition, the UV radiation used in these studies is generally not physiologically representative of real-world UV exposure. Well-characterised pigmented and non-pigmented skin equivalents that contain human dermal fibroblasts, endogenous secreted extracellular matrix proteins (ECM) and a well-differentiated and stratified epidermis have been developed. These constructs were exposed to UV radiation for ×5 consecutive days with a physiologically relevant UV dose and subsequently analysed using appropriate end-points to ascertain photodamage to the skin. We have described that repeated irradiation of full-thickness human skin equivalents in a controlled laboratory environment can recreate UV-associated responses in vitro, mirroring those found in photoexposed native human skin: morphological damage, tanning, alterations in epidermal apoptosis, DNA lesions, proliferation, inflammatory response, and ECM-remodelling. We have found a differential response when using the same UV doses in non-pigmented and pigmented full-thickness skin equivalents, emphasising the role of melanocytes in photoprotection.

Human Probiotic Lactobacillus paracasei-Derived Extracellular Vesicles Improve Tumor Necrosis Factor-α-Induced Inflammatory Phenotypes in Human Skin.

Lactic acid bacteria (LAB), a probiotic, provide various health benefits. We recently isolated a new Lactobacillus paracasei strain with strong anti-inflammatory effects under lipopolysaccharide-induced conditions and proposed a new mode of action-augmenting the endoplasmic reticulum stress pathway for anti-inflammatory functions in host cells. The beneficial effects of the L. paracasei strains on the skin have been described; however, the effects of L. paracasei-derived extracellular vesicles (LpEVs) on the skin are poorly understood. Herein, we investigated whether LpEVs can improve inflammation-mediated skin phenotypes by determining their effects on primary human skin cells and a three-dimensional (3D) full-thickness human skin equivalent under tumor necrosis factor (TNF)-α-challenged inflammatory conditions. LpEVs were efficiently taken up by the human skin cells and were much less cytotoxic to host cells than bacterial lysates. Furthermore, low LpEV concentrations efficiently restored TNF-α-induced cellular phenotypes, resulting in increased cell proliferation and collagen synthesis, but decreased inflammatory factor levels (matrix metalloproteinase 1, interleukin 6, and interleukin 8) in the human dermal fibroblasts, which was comparable to that of retinoic acid, a representative antiaging compound. The beneficial effects of LpEVs were validated in a 3D full-thickness human skin equivalent model. LpEV treatment remarkably restored the TNF-α-induced epidermal malformation, abnormal proliferation of keratinocytes in the basal layer, and reduction in dermal collagen synthesis. Additionally, LpEVs penetrated and reached the deepest dermal layer within 24 h when overlaid on top of a 3D full-thickness human skin equivalent. Furthermore, they possessed superior antioxidant capacity compared with the human cell-derived EVs. Taken together, the anti-inflammatory probiotic LpEVs can be attractive antiaging and antioxidant substances for improving inflammation-induced skin phenotypes and disorders.

On-Site Construction of a Full-Thickness Skin Equivalent with Endothelial Tube Networks via Multilayer Electrospinning for Wound Coverage.

Novel full-thickness skin substitutes are of increasing interest due to the inherent limitations of current models lacking capillary networks. Herein, we developed a novel full-thickness skin tissue containing blood capillary networks through a layer-by-layer assembly approach using a handy electrospinning apparatus and evaluated its skin wound coverage potential in vivo. The average diameter and thickness of fabricated poly-ε-caprolactone-cellulose acetate scaffolds were easily tuned in the range of 474 ± 77-758 ± 113 nm and 9.43 ± 2.23-29.96 ± 5.78 μm by varying electrospinning distance and duration, as indicated by FE-SEM. Besides, keratinocytes exhibited homogeneous differentiation throughout the fibrous matrix prepared with electrospinning distance and duration of 9 cm and 1.5 min within five-layer (5L) epidermal tissues with thickness of 135-150 μm. Moreover, coculture of vascular endothelial cells, circulating fibrocytes, and fibroblasts within the 5L dermis displayed network formation in vitro, resulting in reduced inflammatory factor levels and enhanced integration with the host vasculature in vivo. Additionally, the skin equivalent grafts consisting of the epidermal layer, biomimetic basement membrane, and vascularized dermis layer with an elastic modulus of approximately 11.82 MPa exhibited accelerated wound closure effect indicative of re-epithelialization and neovascularization with long-term cell survival into the host, which was confirmed by wound-healing rate, bioluminescence imaging activity, and histological analysis. It is the first report of a full-thickness skin equivalent constructed using a battery-operated electrospinning apparatus, highlighting its tremendous potential in regenerative medicine.

Biological effects of air pollution on the function of human skin equivalents.

The World Health Organization reports that 99% of the global population are exposed to pollution levels higher than the recommended air quality guidelines. Pollution-induced changes in the skin have begun to surface; however, the effects require further investigation so that effective protective strategies can be developed. This study aimed to investigate some of the aging-associated effects caused by ozone and particulate matter (PM) on human skin equivalents. Full-thickness skin equivalents were exposed to 0.01 μg/μL PM, 0.05 μg/μL PM, 0.3 ppm ozone, or a combination of 0.01 μg/μL PM and 0.3 ppm ozone, before skin equivalents and culture medium were harvested for histological/immunohistochemical staining, gene and protein expression analysis using qPCR, Western blotting, and ELISA. Markers include MMP-1, MMP-3, COL1A1, collagen-I, 4-HNE, HMGCR, and PGE2. PM was observed to induce a decrease in epidermal thickness and an enhanced matrix building phenotype, with increases in COL1A1 and an increase in collagen-I protein expression. By contrast, ozone induced an increase in epidermal thickness and was found to induce a matrix-degrading phenotype, with decreases in collagen-I gene/protein expression and increases in MMP-1 and MMP-3 gene/protein expression. Ozone was also found to induce changes in lipid homeostasis and inflammation induction. Some synergistic damage was also observed when combining ozone and 0.01 μg/μL PM. The results presented in this study identify distinct pollutant-induced effects and show how pollutants may act synergistically to augment damage; given individuals are rarely only exposed to one pollutant type, exposure to multiple pollutant types should be considered to develop effective protective interventions.

Effects of Indole-3-Lactic Acid, a Metabolite of Tryptophan, on IL-4 and IL-13-Induced Human Skin-Equivalent Atopic Dermatitis Models.

Indole-3-lactic acid (I3LA) is a well-known metabolite involved in tryptophan metabolism. Indole derivatives are involved in the differentiation of immune cells and the synthesis of cytokines via the aryl hydrocarbon receptors for modulating immunity, and the indole derivatives may be involved in allergic responses. I3LA was selected as a candidate substance for the treatment of atopic dermatitis (AD), and its inhibitory effect on AD progression was investigated. Full-thickness human skin equivalents (HSEs) consisting of human-derived cells were generated on microfluidic chips and stimulated with major AD-inducing factors. The induced AD-HSEs were treated with I3LA for 7 days, and this affected the AD-associated genetic biomarkers and increased the expression of the major constituent proteins of the skin barrier. After the treatment for 14 days, the surface became rough and sloughed off, and there was no significant difference between the increased AD-related mRNA expression and the skin barrier protein expression. Therefore, the short-term use of I3LA for approximately one week is considered to be effective in suppressing AD.

Recombinant Human Collagen-Based Bioinks for the 3D Bioprinting of Full-thickness Human Skin Equivalent.

As a major extracellular matrix component within the skin, collagen has been widely used to engineer human skin tissues. However, most collagen is extracted from animals. Here, we introduced recombinant human type III collagen (rhCol3) as a bioactive component to formulate bioinks for the bioprinting of a full-thickness human skin equivalent. Human dermal fibroblasts were encapsulated in the gelatin methacryloyl-rhCol3 composite bioinks and printed on a transwell to form the dermis layer, on which human epidermal keratinocytes were seeded to perform an air-liquid interface culture for 6 weeks. After optimizing the bioink formulation and bioprinting process, we investigated the effect of rhCol3 on skin tissue formation. The results suggest that a higher concentration of rhCol3 would enhance the growth of both cells, resulting in a more confluent (~100%) spreading of the epidermal keratinocytes at an early stage (3 days), compared to the rhCol3-free counterpart. Moreover, in an in vivo experiment, adding rhCol3 in the hydrogel formulation would contribute to the skin wound healing process. Taken together, we conclude that rhCol3 could act as a functional bioink component to promote basic skin cellular processes for skin tissue engineering.

Design of an Integrated Microvascularized Human Skin-on-a-Chip Tissue Equivalent Model.

Tissue-engineered skin constructs have been under development since the 1980s as a replacement for human skin tissues and animal models for therapeutics and cosmetic testing. These have evolved from simple single-cell assays to increasingly complex models with integrated dermal equivalents and multiple cell types including a dermis, epidermis, and vasculature. The development of micro-engineered platforms and biomaterials has enabled scientists to better recreate and capture the tissue microenvironment in vitro, including the vascularization of tissue models and their integration into microfluidic chips. However, to date, microvascularized human skin equivalents in a microfluidic context have not been reported. Here, we present the design of a novel skin-on-a-chip model integrating human-derived primary and immortalized cells in a full-thickness skin equivalent. The model is housed in a microfluidic device, in which a microvasculature was previously established. We characterize the impact of our chip design on the quality of the microvascular networks formed and evidence that this enables the formation of more homogenous networks. We developed a methodology to harvest tissues from embedded chips, after 14 days of culture, and characterize the impact of culture conditions and vascularization (including with pericyte co-cultures) on the stratification of the epidermis in the resulting skin equivalents. Our results indicate that vascularization enhances stratification and differentiation (thickness, architecture, and expression of terminal differentiation markers such as involucrin and transglutaminase 1), allowing the formation of more mature skin equivalents in microfluidic chips. The skin-on-a-chip tissue equivalents developed, because of their realistic microvasculature, may find applications for testing efficacy and safety of therapeutics delivered systemically, in a human context.

Structural and Functional Validation of a Full-Thickness Self-Assembled Skin Equivalent for Disease Modeling.

Recently, various types of in vitro-reconstructed 3D skin models have been developed for drug testing and disease modeling. Herein, we structurally and functionally validated a self-assembled reconstructed skin equivalent (RSE) and developed an IL-17a-induced in vitro psoriasis-like model using a self-assembled RSE. The tissue engineering approach was used to construct the self-assembled RSE. The dermal layer was generated using fibroblasts secreting their own ECM, and the epidermal layer was reconstructed by seeding keratinocytes on the dermal layer. To generate the psoriatic model, IL-17A was added to the culture medium during the air–liquid interface culture period. Self-assembled RSE resulted in a fully differentiated epidermal layer, a well-established basement membrane, and dermal collagen deposition. In addition, self-assembled RSE was tested for 20 reference chemicals according to the Performance Standard of OECD TG439 and showed overall sensitivity, specificity, and accuracy of 100%, 90%, and 95%, respectively. The IL-17a-treated psoriatic RSE model exhibited psoriatic epidermal characteristics, such as epidermal hyperproliferation, parakeratosis, and increased expression of KRT6, KRT17, hBD2, and S100A9. Thus, our results suggest that a self-assembled RSE that structurally and functionally mimics the human skin has a great potential for testing various drugs or cosmetic ingredients and modeling inflammatory skin diseases.

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.

In vitro and in vivo toxicity assessment of the senotherapeutic Peptide 14

Senotherapeutic molecules decrease cellular senescence burden, constituting promising approaches to combat the accumulation of senescent cells observed in chronological aging and age-related diseases. Numerous molecules have displayed senotherapeutic potential, but toxicity has been frequently observed. Recently, a new senotherapeutic compound, Peptide 14, was developed to modulate cellular senescence in the skin. In order to assess the potential toxic and genotoxic effects of the peptide, we observed the viability of human primary dermal fibroblasts and epidermal keratinocytes with Peptide 14 treatment, and show that it is mostly non-toxic in concentrations up to 100 μM. Cancer lines were also used to investigate its potential of modulating proliferation. Different concentrations of the peptide promoted a discrete reduction in the proliferation of cancerous cells of the MeWo and HeLa lineages. In full-thickness human skin equivalents, topically formulated Peptide 14 also failed to exert any significant irritation, nor cellular toxicity when added to the culture media. Genotoxic assays including the Ames, micronucleus, and karyotyping tests also indicate the safety of the peptide. Finally, the irritative potential of the peptide was assessed in human subjects in a repeated insult patch test executed using 1 mM peptide. No visible skin reactions were observed in any of the 54 participants. Taken together, the present data support that Peptide 14 is a senotherapeutic molecule with a positive safety profile as tested with cruelty-free models, justifying further studies involving the peptide.

Effect of 3D-Fibroblast Dermis Constructed by Layer-by-Layer Cell Coating Technique on Tight Junction Formation and Function in Full-Thickness Skin Equivalent.

Human skin equivalents (HSEs) consisting of an epidermis and dermis have been used as promising tools for drug evaluation and for clinical applications in regenerative medicine. Normal human dermal fibroblasts (NHDFs) are essential for the fabrication of HSEs because they play an important role in the maturation of the epidermis. Recently, epidermal tight junctions (TJs), which are complex cell-cell junctions, have attracted much attention as a second barrier and regulator for other barrier functions. In a previous study, we revealed the expression of TJ-related proteins and the time course of formation of TJ structure in the HSE (layer-by-layer (LbL)-three-dimensional (3D) Skin) constructed by layer-by-layer (LbL) cell coating technique that have a unique dermis consisting of NHDFs only (3D-fibroblast dermis). However, the effect of the 3D-fibroblast dermis on the formation of functional epidermal TJs is unknown. In this study, we investigated the effect of the 3D-fibroblast dermis on the expression of TJ-related proteins and TJ function in LbL-3D Skin. We demonstrated that the 3D-fibroblast dermis affects the long-term expression of TJ-related proteins and the formation of TJ with barrier function in the epidermis. These results show that the 3D-fibroblast dermis in LbL-3D Skin contributes to the formation and maintenance of functional TJs as in native human skin by direct contact with KCs.

Keratinocyte Growth Factor 2 Ameliorates UVB-Induced Skin Damage via Activating the AhR/Nrf2 Signaling Pathway.

Objective: Exposure to ultraviolet B (UVB) can cause skin damage through oxidative stress, DNA damage, and apoptosis. Keratinocyte growth factor (KGF) has been shown to reduce the content of intracellular reactive oxygen species (ROS) following UVB exposure, a role that is crucial for the efficient photoprotection of skin. The present study evaluated the photoprotective effect of KGF-2 on UVB-induced skin damage and explored its potential molecular mechanism. Methods: To evaluate the effect of KGF-2 on UVB-induced damage ex vivo, a human epidermal full-thickness skin equivalent was pretreated without or with KGF-2 and then exposed to UVB and the levels of histopathological changes, DNA damage, inflammation, and apoptosis were then evaluated. The ability of KGF-2 to protect the cells against UVB-inflicted damage and its effect on ROS production, apoptosis, and mitochondrial dysfunction were determined in HaCaT cells. Results: Pretreatment of the epidermis with KGF-2 ameliorated the extent of photodamage. At the cellular level, KGF-2 could attenuate ROS production, apoptosis, DNA damage, and mitochondrial dysfunction caused by UVB exposure. KGF-2 could also activate the aryl hydrocarbon receptor (AhR) to trigger the Nrf2 signaling pathway. Conclusion: Taken together, our findings suggested that KGF-2 could ameliorate UVB-induced skin damage through inhibiting apoptosis, reducing oxidative stress, and preventing DNA damage and mitochondrial dysfunction via regulating AhR/Nrf2 signaling pathway.

A perfusable vascularized full-thickness skin model for potential topical and systemic applications

Vascularization of reconstructed tissues is one of the remaining hurdles to be considered to improve both the functionality and viability of skin grafts and the relevance of in vitro applications. Our study, therefore, sought to develop a perfusable vascularized full-thickness skin equivalent that comprises a more complex blood vasculature compared to existing models. We combined molding, auto-assembly and microfluidics techniques in order to create a vascularized skin equivalent representing (a) a differentiated epidermis with a physiological organization and correctly expressing K14, K10, Involucrin, TGM1 and Filaggrin, (b) three perfusable vascular channels with angiogenic sprouts stained with VE-Caderin and Collagen IV, (c) an adjacent microvascular network created via vasculogenesis and connected to the sprouting macrovessels. Histological analysis and immunostaining of CD31, Collagen IV, Perlecan and Laminin proved the integrity of vascular constructs. In order to validate the vascularized skin potential of topical and systemic applications, caffeine and minoxidil, two compounds with different chemical properties, were topically applied to measure skin permeability and benzo[a]pyrene pollutant was systemically applied to evaluate systemic delivery. Our results demonstrated that perfusion of skin reconstructs and the presence of a complex vascular plexus resulted in a more predictive and reliable model to assess respectively topical and systemic applications. This model is therefore aimed at furthering drug discovery and improving clinical translation in dermatology.

18228 A novel acetyl dipeptide demonstrates pleiotropic gene expression, optimized bioavailability, and perceived anti-aging benefits

Peptides, composed of amino acids, are developed for topical skin benefits due to their similarity to and effect on the skin’s structural components, such as collagen, elastin, and decorin. A novel acetyl dipeptide (ADP) with known anti-aging, anti-inflammatory, and compromised skin benefits, was further evaluated. RNA was isolated in vitro from full-thickness skin equivalents treated twice for 48 hours with ADP in different formulations. Whole-transcriptome analysis identified differentially expressed genes.

Long-Term and Clinically Relevant Full-Thickness Human Skin Equivalent for Psoriasis.

Psoriasis is an incurable, immune-mediated inflammatory disease characterized by the hyperproliferation and abnormal differentiation of keratinocytes. To study in depth the pathogenesis of this disease and possible therapy options suitable, pre-clinical models are required. Three-dimensional skin equivalents are a potential alternative to simplistic monolayer cultures and immunologically different animal models. However, current skin equivalents lack long-term stability, which jeopardizes the possibility to simulate the complex disease-specific phenotype followed by long-term therapeutic treatment. To overcome this limitation, the cell coating technique was used to fabricate full-thickness human skin equivalents (HSEs). This rapid and scaffold-free fabrication method relies on coating cell membranes with nanofilms using layer-by-layer assembly, thereby allowing extended cultivation of HSEs up to 49 days. The advantage in time is exploited to develop a model that not only forms a disease phenotype but can also be used to monitor the effects of topical or systemic treatment. To generate a psoriatic phenotype, the HSEs were stimulated with recombinant human interleukin 17A (rhIL-17A). This was followed by systemic treatment of the HSEs with the anti-IL-17A antibody secukinumab in the presence of rhIL-17A. Microarray and RT-PCR analysis demonstrated that HSEs treated with rhIL-17A showed downregulation of differentiation markers and upregulation of chemokines and cytokines, while treatment with anti-IL-17A antibody reverted these gene regulations. Gene ontology analysis revealed the proinflammatory and chemotactic effects of rhIL-17A on the established HSEs. These data demonstrated, at the molecular level, the effects of anti-IL-17A antibody on rhIL-17A-induced gene regulations. This shows the physiological relevance of the developed HSE and opens venues for its use as an alternative to ex vivo skin explants and animal testing.

A Simple Method for the Production of Human Skin Equivalent in 3D, Multi-Cell Culture.

An important problem for researchers working in the field of dermatology is the preparation of the human skin equivalent (HSE). Here, we describe a simple and reliable protocol for preparing a skin model from the commercially available cell lines: keratinocytes, fibroblasts, and melanocytes. Importantly, in our 3D model, the keratinocytes are diverse that brings this model closer to the natural skin. For the production of HSE, we used available primary PCS-200-010, PCS-201-010, PCS-200-013, and immortalized CRL-4048 and CRL-4001 cell lines. We used genipin, which is necessary for collagen cross-linking and studied its cytotoxicity for keratinocytes and fibroblasts. The addition of 20 μM genipin reduced the shrinkage of the collagen in the constructs from 59% to 24% on day 12 of the culture of the construct. A higher concentration (80–200 µM) of genipin reduced shrinkage by 14% on average. Genipin in concentration 10 μM and below was not cytotoxic to the keratinocytes, and 150 μM and below to the fibroblasts. Hematoxylin and eosin staining showed that the morphology of HSEs was identical to that of native human skin. The immunohistochemical staining of the constructs showed the presence of vimentin-positive fibroblasts in the skin layer, while the melanocytes were in the epidermis and in the basal layer. We observed that the longer differentiation of constructs led to the higher secretion of GM-CSF, IL-10, IL-15, IL-1α, IL-6, IL-7, IL-8, and MCP-1. We also observed that the longer time of differentiation led to a more stable secretion of all analytes, which was reflected in the coefficient of variation. We described here a simple, reliable, and cost-effective production of the full-thickness human skin equivalents that can be used in the research and industry. With the global trend to decrease animal use for the research and testing, our HSE could be a useful testing tool and an alternative research model.

Impact of intercellular crosstalk between epidermal keratinocytes and dermal fibroblasts on skin homeostasis

Dermal fibroblasts seem critical for epidermal maturation and differentiation and recent work demonstrated that diseased fibroblasts may drive pathophysiological processes. Nevertheless, still very little is known about the actual crosstalk between epidermal keratinocytes and dermal fibroblasts and the impact of dermal fibroblasts on epidermal maturation and differentiation. Aiming for a more fundamental understanding of the impact of the cellular crosstalk between keratinocytes and fibroblasts on the skin homeostasis, we generated full-thickness skin equivalents with and without fibroblasts and subsequently analysed them for the expression of skin differentiation markers, their barrier function, skin lipid content and epidermal cell signalling. Skin equivalents without fibroblasts consistently showed an impaired differentiation and dysregulated expression of skin barrier and tight junction proteins, increased skin permeability, and a decreased skin lipid/protein ratio. Most interestingly, impaired Ras/Raf/ERK/MEK signalling was evident in skin equivalents without fibroblasts.Our data clearly indicate that the epidermal-dermal crosstalk between keratinocytes and fibroblasts is critical for adequate skin differentiation and that fibroblasts orchestrate epidermal differentiation processes.

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

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. 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. RhE, dendritic cell (DC)-containing RhE and full-thickness skin equivalent (FTSE) were compared regarding their proinflammatory responsiveness to metal allergens. 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. 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.

Engineered Skin Tissue Equivalents for Product Evaluation and Therapeutic Applications.

The current status of skin tissue equivalents that have emerged as relevant tools in commercial and therapeutic product development applications is reviewed. Due to the rise of animal welfare concerns, numerous companies have designed skin model alternatives to assess the efficacy of pharmaceutical, skincare, and cosmetic products in an in vitro setting, decreasing the dependency on such methods. Skin models have also made an impact in determining the root causes of skin diseases. When designing a skin model, there are various chemical and physical considerations that need to be considered to produce a biomimetic design. This includes designing a structure that mimics the structural characteristics and mechanical strength needed for tribological property measurement and toxicological testing. Recently, various commercial products have made significant progress towards achieving a native skin alternative. Further research involve the development of a functional bilayered model that mimics the constituent properties of the native epidermis and dermis. In this article, the skin models are divided into three categories: in vitro epidermal skin equivalents, in vitro full-thickness skin equivalents, and clinical skin equivalents. A description of skin model characteristics, testing methods, applications, and potential improvements is presented.