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Integration of reconstructed human skin (RhS) into organ-on-chip (OoC) platforms addresses current limitations imposed by static culturing. This innovation, however, is not without challenges. Microfluidic devices, while powerful, often encounter usability, robustness, and gas bubble issues that hinder large-scale high-throughput setups. This study aims to develop a novel re-usable multi-well microfluidic adaptor (MMA) with the objective to provide a flexible tool for biologists implementing complex 3D biological models (e.g., skin) while enabling simultaneous user control over temperature, medium flow, oxigen (O 2 ), nitrogen (N 2 ), and carbon dioxide (CO 2 ) without the need for an incubator. The presented MMA device is designed to be compatible with standard, commercially available 6-well multi-well plates (6MWPs) and 12-well transwells. This MMA-6MWP setup is employed to generate a skin-on-chip (SoC). RhS viability is maintained under flow for three days and their morphology closely resembles that of native human skin. A proof-of-concept study demonstrates the system's potential in toxicology applications by combining endothelialised RhS with flowing immune cells. This dynamic setting activates the monocyte-like MUTZ-3 cells (CD83 and CD86 upregulation) upon topical exposure of RhS to a sensitizer, revealing the MMA-6MWP's unique capabilities compared to static culturing, where such activation is absent., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Background: Commensal bacteria colonizing oral mucosa and skin play an essential role in maintaining host-microbiome homeostasis. It is unknown whether cytotoxicity resulting from metal ions leaching from medical devices may be influenced by commensal microbes., Objective: Determine whether the extent of apoptosis triggered by nickel or titanium ions is influenced by Streptococcus mitis and whether apoptosis occurs via the intrinsic or extrinsic apoptosis pathway., Methods: Reconstructed Human Gingiva (RHG) and Skin (RHS) were topically exposed to titanium or nickel salts in the presence or absence of S. mitis. Cytotoxicity and apoptosis were assessed by histology, immunohistochemistry, TUNEL assay, and Western Blot., Results: S. mitis alone resulted in negligible cytotoxicity. After metal exposure, localized apoptosis was observed in the epithelium and fibroblasts within the lamina propria hydrogel of both RHG and RHS. S. mitis enhanced metal-mediated apoptosis in gingiva but not in skin. Apoptosis was mediated via the extrinsic pathway caspase 8. Activation of the execution phase of apoptosis occurred via caspases 3 and 7, and PARP-1., Conclusion: Our study supports the finding that metals have irritant, cytotoxic properties resulting in apoptosis when leaching into skin or gingiva. Particularly for gingiva, commensal microbes exaggerate this detrimental effect., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: In tissue engineering, real-time monitoring of tumors and of the dynamics of the microenvironment within in vitro models has traditionally been hindered by the need to harvest the cultures to obtain material to analyze. Line-field confocal optical coherence tomography (LC-OCT) has proven to be useful in evaluating in vivo skin conditions, including melanoma, by capturing dynamic, three-dimensional (3D) information without the need for invasive procedures, such as biopsies. Additionally, the M-Duo Technology® developed by IMcoMET presents a unique opportunity for continuous in situ biomarker sampling, providing insights into local cellular behavior and interactions., Objective: This study aimed to validate the non-destructive mapping capabilities of two advanced methodologies (LC-OCT by DAMAE Medical and M-Duo Technology® by IMcoMET) to investigate the living microenvironment of in vitro reconstructed human skin (RhS) and melanoma-RhS (Mel-RhS)., Methods: LC-OCT and M-Duo Technology® were compared to conventional analysis of the RhS and Mel-RhS microenvironments., Results: LC-OCT successfully visualized the distinct layers of the epidermis and tumor structures within the Mel-RhS, identifying keratinocytes, melanocytes, tumor nests, and fibroblasts. The M-Duo Technology® revealed differences in in situ cytokine (IL-6) and chemokine (CCL2, CXCL10, and IL-8) secretion between Mel-RhS and the control RhS. Notably, such differences were not detected through conventional investigation of secreted proteins in culture supernatants., Conclusion: The combination of LC-OCT's high-resolution imaging and M-Duo Technology®'s in situ microenvironmental mapping has the potential to provide a synergistic platform for non-invasive, real-time analysis, allowing for prolonged observation of processes within Mel-RhS models without the need for culture disruption., Competing Interests: Declaration of Competing Interest AM, PO, and VT are founders of IMcoMET, which is involved in the development of the M-Duo Technology® discussed in this article. AM, PO, PT, and SV are currently employed by IMcoMET. JO is currently employed by DAMAE Medical and is listed as inventor on four patent families linked to the LC-OCT technology. The other authors have no conflict of interest to declare., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Dysregulation of skin metabolism is associated with a plethora of diseases such as psoriasis and dermatitis. Until now, reconstructed human skin (RhS) models lack the metabolic potential of native human skin, thereby limiting their relevance to study human healthy and diseased skin. We aimed to determine whether incorporation of an adipocyte-containing hypodermis into RhS improves its metabolic potential and to identify major metabolic pathways up-regulated in adipose-RhS., Methods: Primary human keratinocytes, fibroblasts and differentiated adipose-derived stromal cells were co-cultured in a collagen/fibrin scaffold to create an adipose-RhS. The model was extensively characterized structurally in two- and three-dimensions, by cytokine secretion and RNA-sequencing for metabolic enzyme expression., Results: Adipose-RhS showed increased secretion of adipokines. Both RhS and adipose-RhS expressed 29 of 35 metabolic genes expressed in ex vivo native human skin. Addition of the adipose layer resulted in up-regulation of 286 genes in the dermal-adipose fraction of which 7 were involved in phase I (CYP19A1, CYP4F22, CYP3A5, ALDH3B2, EPHX3) and phase II (SULT2B1, GPX3) metabolism. Vitamin A, D and carotenoid metabolic pathways were enriched. Additionally, pro-inflammatory (IL-1β, IL-18, IL-23, IL-33, IFN-α2, TNF-α) and anti-inflammatory cytokine (IL-10, IL-12p70) secretion was reduced in adipose-RhS., Conclusions: Adipose-RhS mimics healthy native human skin more closely than traditional RhS since it has a less inflamed phenotype and a higher metabolic activity, indicating the contribution of adipocytes to tissue homeostasis. Therefore it is better suited to study onset of skin diseases and the effect of xenobiotics., (© 2024. The Author(s).)

Sensory innervation of the skin is essential for its function, homeostasis, and wound healing mechanisms. Thus, to adequately model the cellular microenvironment and function of native skin, in vitro human skin equivalents (hSE) containing a sensory neuron population began to be researched. In this work, a fully human 3D platform of hSE innervated by induced pluripotent stem cell-derived nociceptor neurospheres (hNNs), mimicking the native mode of innervation, is established. Both the hSE and nociceptor population exhibit morphological and phenotypical characteristics resembling their native counterparts, such as epidermal and dermal layer formation and nociceptor marker exhibition, respectively. In the co-culture platform, neurites develop from the hNNs and navigate in 3D to innervate the hSE from a distance. To probe both skin and nociceptor functionality, a clinically available capsaicin patch (Qutenza) is applied directly over the hSE section and neuron reaction is analyzed. Application of the patch causes an exposure time-dependent neurite regression and degeneration. In platforms absent of hSE, axonal degeneration is further increased, highlighting the role of the skin construct as a barrier. In sum, an in vitro tool of functional innervated skin with high interest for preclinical research is established., (© 2022 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH.)

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., (© 2021 The Authors. Contact Dermatitis published by John Wiley & Sons Ltd.)

Wound healing is an essential process to restore tissue integrity after trauma. Large skin wounds such as burns often heal with hypertrophic scarring and contractures, resulting in disfigurements and reduced joint mobility. Such adverse healing outcomes are less common in the oral mucosa, which generally heals faster compared to skin. Several studies have identified differences between oral and skin wound healing. Most of these studies however focus only on a single stage of wound healing or a single cell type. The aim of this review is to provide an extensive overview of wound healing in skin versus oral mucosa during all stages of wound healing and including all cell types and molecules involved in the process and also taking into account environmental specific factors such as exposure to saliva and the microbiome. Next to intrinsic properties of resident cells and differential expression of cytokines and growth factors, multiple external factors have been identified that contribute to oral wound healing. It can be concluded that faster wound closure, the presence of saliva, a more rapid immune response, and increased extracellular matrix remodeling all contribute to the superior wound healing and reduced scar formation in oral mucosa, compared to skin.

Understanding skin metabolism is important when considering drug discovery and safety assessment. This review compares xenobiotic skin metabolism in ex vivo skin to reconstructed human skin and reconstructed human epidermis models, concentrating on phase I and phase II enzymes. Reports on phase I enzymes are more abundant than for phase II enzymes with mRNA and protein expression far more reported than enzyme activity. Almost all of the xenobiotic metabolizing enzymes detected in human skin are also present in liver. However, in general the relative levels are lower in skin than in liver and fewer enzymes are reported., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Despite continuous exposure to environmental pathogens, injured mucosa within the oral cavity heals faster and almost scar free compared with skin. Saliva is thought to be one of the main contributing factors. Saliva may possibly also stimulate skin wound healing. If so, it would provide a novel therapy for treating skin wounds, for example, burns. This study aims to investigate the therapeutic wound healing potential of human saliva in vitro. Human saliva from healthy volunteers was filter sterilized before use. Two different in vitro wound models were investigated: (a) open wounds represented by 2D skin and gingiva cultures were used to assess fibroblast and keratinocyte migration and proliferation and (b) blister wounds represented by introducing freeze blisters into organotypic reconstructed human skin and gingiva. Re-epithelialization and differentiation (keratin K10, K13, K17 expression) under the blister and inflammatory wound healing mediator secretion was assessed. Saliva-stimulated migration of skin and oral mucosa fibroblasts and keratinocytes, but only fibroblast proliferation. Topical saliva application to the blister wound on reconstructed skin did not stimulate re-epithelization because the blister wound contained a dense impenetrable dead epidermal layer. Saliva did promote an innate inflammatory response (increased CCL20, IL-6, and CXCL-8 secretion) when applied topically to the flanking viable areas of both wounded reconstructed human skin and oral mucosa without altering the skin specific keratin differentiation profile. Our results show that human saliva can stimulate oral and skin wound closure and an inflammatory response. Saliva is therefore a potential novel therapeutic for treating open skin wounds., (© 2019 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.)

Background: During the last decade, the number of people with ≥1 tattoo has increased noticeably within the European population. Despite this, limited safety information is available for tattoo inks., Objectives: To test the skin sensitization potential of 5 tattoo inks in vitro by using reconstructed human skin (RHS) and the contact sensitization biomarker interleukin (IL)-18., Methods: Two red and 3 black tattoo inks, 1 additive (Hamamelis virginiana extract) and 1 irritant control (lactic acid) were tested. The culture medium of RHS (reconstructed epidermis on a fibroblast-populated collagen hydrogel) was supplemented with test substances in a dose-dependent manner for 24 hours, after which cytotoxicity (histology; thiazolyl blue tetrazolium bromide assay) and skin sensitization potential (IL-18 secretion; enzyme-linked immunosorbent assay) were assessed., Results: All but 1 ink showed cytotoxicity. Notably, 1 red ink and 1 black ink were able to cause an inflammatory response, indicated by substantial release of IL-18, suggesting that these inks may be contact sensitizers., Conclusions: The in vitro RHS model showed that 4 tattoo inks were cytotoxic and 2 were able to cause an inflammatory IL-18 response, indicating that an individual may develop allergic contact dermatitis when exposed to these tattoo inks, as they contain contact sensitizers., (© 2018 The Authors. Contact Dermatitis published by John Wiley & Sons Ltd.)

Keloid scars are often described as having an actively growing peripheral margin with a regressing centre. The aim of this study was to examine the possible heterogeneity within keloids and the involvement of different regions within and around keloid scars in the pathogenesis, using an in vitro keloid scar model. In vitro skin models were constructed from keratinocytes and fibroblasts from normal skin and different regions within and around keloid scars: periphery, centre, and (adjacent) surrounding-normal-skin regions. Additionally, fibroblasts were isolated from the superficial-central and deep-central regions of the keloid and combined with central keratinocytes. All keloid regions showed increased contraction compared to normal skin models, particularly in central regions. Myofibroblasts were present in all keloid regions but were more abundant in models containing central-deep keloid fibroblasts. Secretion of anti-fibrotic HGF and extracellular matrix collagen IV gene expression was reduced in the central deep keloid compared to normal skin. No significant differences between peripheral and central regions within keloids were observed for inflammatory cytokine CCL20, CCL27, CXCL8, IL-6 and IL-18 secretion. Parameters for surrounding-normal-skin showed similarities to both non-lesional normal skin and keloids. In conclusion, a simple but elegant method of culturing keloid-derived keratinocytes and fibroblasts in an organotypic 3D scar model was developed, for the dual purpose of studying the underlying pathology and ultimately testing new therapeutics. In this study, these tissue engineered scar models show that the central keloid region shows a more aggressive keloid scar phenotype than the periphery and that the surrounding-normal-skin also shares certain abnormalities characteristic for keloids.

To understand scar pathology, develop new drugs, and provide a platform for personalized medicine, physiologically relevant human scar models are required, which are characteristic of different scar pathologies. Hypertrophic scars and keloids are two types of abnormal scar resulting from unknown abnormalities in the wound healing process. While they display different clinical behavior, differentiation between the two can be difficult-which in turn means that it is difficult to develop optimal therapeutic strategies. The aim of this study was to develop in vitro reconstructed human hypertrophic and keloid scar models and compare these to normotrophic scar and normal skin models to identify distinguishing biomarkers. Keratinocytes and fibroblasts from normal skin and scar types (normotrophic, hypertrophic, keloid) were used to reconstruct skin models. All skin models showed a reconstructed differentiated epidermis on a fibroblast populated collagen-elastin matrix. Both abnormal scar types showed increased contraction, dermal thickness, and myofibroblast staining compared to normal skin and normotrophic scar. Notably, the expression of extracellular matrix associated genes showed distinguishing profiles between all scar types and normal skin (hyaluronan synthase-1, matrix-metalloprotease-3), between keloid and normal skin (collagen type IV), between normal scar and keloid (laminin α1), and between keloid and hypertrophic scar (matrix-metalloprotease-1, integrin α5). Also, inflammatory cytokine and growth factor secretion (CCL5, CXCL1, CXCL8, CCL27, IL-6, HGF) showed differential secretion between scar types. Our results strongly suggest that abnormal scars arise from different pathologies rather than simply being on different ends of the scarring spectrum. Furthermore, such normal skin and scar models together with biomarkers, which distinguish the different scar types, would provide an animal free, physiologically relevant scar diagnostic and drug testing platform for the future.

Skin and oral mucosa substitutes are a therapeutic option for closing hard-to-heal skin and oral wounds. Our aim was to develop bi-layered skin and gingiva substitutes, from 3 mm diameter biopsies, cultured under identical conditions, which are compliant with current European regulations for advanced therapy medicinal products. We present in vitro mode of action methods to (i) determine viability: epithelial expansion, proliferation (Ki-67), metabolic activity (MTT assay); (ii) characterize skin and gingiva substitutes: histology and immunohistochemistry; and (iii) determine potency: soluble wound healing mediator release (enzyme-linked immunosorbent assay). Both skin and gingiva substitutes consist of metabolically active autologous reconstructed differentiated epithelium expanding from the original biopsy sheet on a fibroblast populated connective tissue matrix (donor dermis). Gingival epithelium expanded 1.7-fold more than skin epithelium during the 3 week culture period. The percentage of proliferating Ki-67-positive cells located in the basal layer of the gingiva substitute was >1.5-fold higher than in the skin substitute. Keratins 16 and 17, which are upregulated during normal wound healing, were expressed in both the skin and gingiva substitutes. Notably, the gingiva substitute secreted higher amounts of key cytokines involved in mitogenesis, motogenesis and chemotaxis (interleukin-6 > 23-fold, CXCL8 > 2.5-fold) as well as higher amounts of the anti-fibrotic growth factor, hepatocyte growth factor (>7-fold), compared with the skin substitute. In conclusion, while addressing the viability, characterization and potency of the tissue substitutes, important intrinsic differences between skin and gingiva were discovered that may explain in part the superior quality of wound healing observed in the oral mucosa compared with skin., (Copyright © 2017 The Authors. Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.)

Allergic and irritant skin reactions caused by topical exposure to permanent hair dyes are a common problem. For regulatory and ethnical purposes, it is required to perform chemical safety assessment following the replacement, reduction, and refinement of animal testing (3Rs). Permanent hair dyes are formed by a mixture of ingredients that vary from low to extreme skin sensitizing potency and that inter-react to form unknown by-products. Because of the complex reaction, this cytotoxic mechanism has not yet been elucidated and is the subject of this study. Here, we topically exposed p-phenylenediamine (PPD), Resorcinol (RES), Hydrogen Peroxide (H 2 O 2 ) alone or as a mixture to RhE and evaluated parameters related to skin irritation such as epidermal viability, keratinocytes damage, barrier loss and IL-1 α. Our data indicates that ingredients tested alone did not lead to an increase of cytotoxic parameters related to skin irritation. However, when the mixture of PPD/H 2 O 2 /RES and PPD/H 2 O 2 was applied to the RhE, some of the parameters such as morphological changes including the presence of apoptotic cells, barrier loss and increased IL- 1 α release were observed. The results indicate that the mixture of ingredients used in permanent hair dyes have an irritant effect in RhE while the ingredients alone not., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The development of improved, innovative models for the detection of toxicity of drugs, chemicals, or chemicals in cosmetics is crucial to efficiently bring new products safely to market in a cost-effective and timely manner. In addition, improvement in models to detect toxicity may reduce the incidence of unexpected post-marketing toxicity and reduce or eliminate the need for animal testing. The safety of novel products of the pharmaceutical, chemical, or cosmetics industry must be assured; therefore, toxicological properties need to be assessed. Accepted methods for gathering the information required by law for approval of substances are often animal methods. To reduce, refine, and replace animal testing, innovative organotypic in vitro models have emerged. Such models appear at different levels of complexity ranging from simpler, self-organized three-dimensional (3D) cell cultures up to more advanced scaffold-based co-cultures consisting of multiple cell types. This review provides an overview of recent developments in the field of toxicity testing with in vitro models for three major organ types: heart, skin, and liver. This review also examines regulatory aspects of such models in Europe and the UK, and summarizes best practices to facilitate the acceptance and appropriate use of advanced in vitro models.

Antigen exposure to oral mucosa is generally thought to lead to immune tolerance induction. However, very little is known about the subset composition and function of dendritic cells (DC) migrating from human oral mucosa. Here we show that migratory DC from healthy human gingival explants consist of the same phenotypic subsets in the same frequency distribution as DC migrating from human skin. The gingival CD1a+ Langerhans cell and interstitial DC subsets lacked CXCR4 expression in contrast to their cutaneous counterparts, pointing to different migration mechanisms, consistent with previous observations in constructed skin and gingival equivalents. Remarkably, without any exogenous conditioning, gingival explants released higher levels of inflammatory cytokines than human skin explants, resulting in higher DC migration rates and a superior ability of migrated DC to prime allogeneic T cells and to induce type-1 effector T cell differentiation. From these observations we conclude that rather than an intrinsic ability to induce T cell tolerance, DC migrating from oral mucosa may have a propensity to induce effector T cell immunity and maintain a high state of alert against possible pathogenic intruders in the steady state. These findings may have implications for oral immunization strategies.

Understanding the healthy and diseased state of skin is important in many areas of basic and applied research. Although the field of skin tissue engineering has advanced greatly over the last years, current in vitro skin models still do not mimic the complexity of the human skin. Skin-on-chip and induced pluripotent stem cells (iPSC) might be key technologies to improve in vitro skin models. This review summarizes the state of the art of in vitro skin models with regard to cell sources (primary, cell line, iPSC) and microfluidic devices. It can be concluded that iPSC have the potential to be differentiated into many kinds of immunologically matched cells and skin-on-chip technology might lead to more physiologically relevant skin models due to the controlled environment, possible exchange of immune cells, and an increased barrier function. Therefore the combination of iPSC and skin-on-chip is expected to lead to superior healthy and diseased in vitro skin models.

Both oral mucosa and skin have the capacity to maintain immune homeostasis or regulate immune responses upon environmental assault. Whereas much is known about key innate immune events in skin, little is known about oral mucosa. Comparative studies are limited due to the scarce supply of oral mucosa for ex vivo studies. Therefore, we used organotypic tissue equivalents (reconstructed epithelium on fibroblast-populated collagen hydrogel) to study cross talk between cells. Oral mucosa and skin equivalents were compared regarding secretion of cytokines and chemokines involved in LC migration and general inflammation. Basal secretion, representative of homeostasis, and also secretion after stimulation with TNFα, an allergen (cinnamaldehyde), or an irritant (SDS) were assessed. We found that proinflammatory IL-18 and chemokines CCL2, CCL20, and CXCL12, all involved in LC migration, were predominantly secreted by skin as compared to gingiva. Furthermore, CCL27 was predominantly secreted by skin whereas CCL28 was predominantly secreted by gingiva. In contrast, general inflammatory cytokines IL-6 and CXCL8 were secreted similarly by skin and gingiva. These results indicate that the cytokines and chemokines triggering innate immunity and LC migration are different in skin and gingiva. This differential regulation should be figured into novel therapy or vaccination strategies in the context of skin versus mucosa.

In this report, the construction of a functional, immunocompetent, full-thickness skin equivalent (SE) is described, consisting of an epidermal compartment containing keratinocytes, melanocytes, and human LCs derived from the MUTZ-3 cell line (MUTZ-LC) and a fibroblast-populated dermal compartment. The CD1a(+)Langerin(+)HLA-DR(+) MUTZ-LCs populate the entire epidermis at a similar density to that found in native skin. Exposure of the SE to subtoxic concentrations of the allergens NiSO(4) and resorcinol resulted in LC migration out of the epidermis toward the fibroblast-populated dermal compartment. A significant dose-dependent up-regulation of the DC maturation-related CCR7 and IL-1β transcripts and of CD83 at the protein level upon epidermal exposure to both allergens was observed, indicative of maturation and migration of the epidermally incorporated LC. We have thus successfully developed a reproducible and functional full-thickness SE model containing epidermal MUTZ-LC. This model offers an alternative to animal testing for identifying potential chemical sensitizers and for skin-based vaccination strategies and provides a unique research tool to study human LC biology in situ under controlled in vitro conditions.

The ability of stem cells to self-renew as well as their multilineage differentiation potential makes them ideal candidates for skin regeneration strategies. Mesenchymal stem cells residing in human adult dermis, in contrast to adipose tissue, have not yet been described. The objective of this study was to determine the stemness and chemokine-mediated homing potential of dermal stromal cells (DSC) and to compare this with adipose stem cells (ASC). DSC have a less stellate form than ASC, confirming that DSC and ASC are two different types of mesenchymal cell populations. However, DSC display a mesenchymal stem cell phenotype (CD31(-), CD34(+), CD45(-), CD54(+), CD90(+), CD105(+), and CD166(+) similar to ASC and are also multipotent in their ability to differentiate into adipocytes, chondrocytes, and osteoblasts. Both ASC and DSC display a similar set of chemokine receptors (CCR3, CCR4, CCR6, CCR10, CXCR1, and CXCR2). Several ligands for these receptors, with CCL5/RANTES being the most potent, can induce migration of ASC and DSC in an in vitro wound-healing assay. Taken together, these results show that a population of mesenchymal stem cells resides in the dermis of human adult skin and these dermal-derived stem cells have a phenotypic and chemokine-mediated homing potential similar to adipose stem cells, which to our knowledge is previously unreported.

Irritant contact dermatitis is the result of an innate inflammatory response of the skin to direct injury. It is caused by a single, repeated or continued application of an irritant, with the source most often being a chemical. Therefore, European regulations require strict screening of all ingredients in consumer products. Until recently, identifying a potential irritant has completely relied on animal testing (for example, Draize test). Besides the ethical problems, both the 7th Amendment to the Cosmetics Directive and Registration, Evaluation and Authorization of Chemicals legislation have stimulated the development of alternative tests for the assessment of potential toxicological effects of substances. This review is aimed at describing current in vitro skin irritation models and the biomarkers used to assess the degree of irritancy of a potential irritant. Four models are described: keratinocyte and fibroblast cultures grown under submerged culture conditions, epidermal equivalents, skin equivalents and freshly isolated skin. Biomarkers such as IL-1alpha, IL-6, IL-8, PGE2, SKALP, HSP70 and kinases are described along with changes in metabolic activity (MTT assay) and cytosolic leakage (lactate dehydrogenase assay). Noticeable is the limited number of genomic and proteomic studies. Such studies have the potential to identify novel biomarkers and to elucidate the mechanism of irritant contact dermatitis., (2009 S. Karger AG, Basel.)

The initial step in Langerhans cell (LC) migration from the epidermis to the lymph node involves migration of maturing LC into the dermis. Here, we investigated the migration of LC out of the epidermis after exposure of the skin to contact allergens. Ex vivo intact human skin, epidermal sheets, and LC derived from the MUTZ-3 cell line (MUTZ-LC) were used to determine whether dermal fibroblasts play a role in mediating LC migration towards the dermis. Exposure of epidermal sheets or MUTZ-LC to allergens (nickel sulphate, 2,4-dinitrochlorobenzene, and cinnamaldehyde) or a cytokine maturation cocktail resulted in LC migration towards dermal fibroblasts. This was due to upregulation of CXCR4 on maturing LC and secretion of CXCL12/stromal derived factor-1 chemokine by fibroblasts. Neutralizing antibodies to either CXCL12 or CXCR4 completely blocked migration. Injection of CXCL12 neutralizing antibodies into intact human skin totally inhibited LC migration into the dermis. In contrast, neutralizing antibodies to CCL19/CCL21 did not inhibit migration into the dermis. We describe a novel and essential role of dermis-derived CXCL12 in initiating migration of maturing human LC to the dermis thus permitting their further journey to the draining lymph nodes.

Calcineurin (Cn) is the target of the immunosuppressive drugs cyclosporine A (CsA), tacrolimus (Trl), and pimecrolimus (Prl). Trl and Prl are often used topically for treatment of various skin diseases. The Cn inhibitors CsA and Trl are mostly used for maintenance therapy of transplant patients. Their long-term use, however, causes a dramatic increase in skin cancer risk. By using a newly developed assay for Cn measurement in blood, we were able to demonstrate Cn activity in total skin homogenates. A significantly higher activity was found in epidermis compared to dermis. In skin cell cultures, fibroblasts showed the highest activity as compared to keratinocytes and melanocytes. Of the Cn inhibitors, Trl showed stronger inhibition than CsA and Prl (57 and 55% in fibroblast and keratinocyte cultures, respectively). Also, the lowest IC(50) (the half maximal inhibitory concentration) values were found for Trl (0.5 and 1.3 nM in two different fibroblast cultures). Cn activity and its inhibition can thus be studied in dermatological samples. The effects of Cn inhibition in fibroblasts and keratinocytes may be of influence on the overall functioning of the skin immune system.

In human skin, two main populations of dendritic cells (DC) can be discriminated: dermal DC (DDC) and epidermal Langerhans cells (LC). Although extensively studied, most of the knowledge about DDC and LC phenotype and function is obtained from studying DDC and LC cultured in vitro or DDC and LC migrated from skin explants. These studies have left the exact relationship between steady-state human LC and DDC unclear: in particular, whether CD1a+ DDC represent migrated LC or whether they constitute a separate subset. To gain further insight in the kinship between skin-resident CD1a+ DDC and LC, we analyzed CD1a+ DDC and LC, isolated from steady-state skin samples, by high-density microarray analysis. Results show that the CD1a+ DDC specifically express markers associated with DDC phenotype, such as the macrophage mannose receptor, DC-specific ICAM-grabbing nonintegrin, the scavenger receptor CD36, coagulation factor XIIIa, and chemokine receptor CCR5, whereas LC specifically express Langerin, membrane ATPase (CD39), and CCR6, all hallmarks of the LC lineage. In addition, under steady-state conditions, both DC subsets display a strikingly different activation status, indicative of distinct functional properties. CD1a+ DDC exhibit a more activated, proinflammatory, migratory, and T cell-stimulatory profile, as compared with LC, whereas LC mainly express molecules involved in cell adhesion and DC retention in the epidermis. In conclusion, transcriptional profiling is consistent with the notion that CD1a+ DDC and LC represent two distinct DC subsets but also that under steady-state conditions, CD1a+ DDC and epidermal LC represent opposites of the DC activation spectrum.

In this review, we discuss and compare studies of xenobiotic metabolism in both human skin and 3D human skin reconstructs. In comparison to the liver, the skin is a less studied organ in terms of characterising metabolic capability. While the skin forms the major protective barrier to environmental chemical exposure, it is also a potential target organ for adverse health effects. Occupational, accidental or intended-use exposure to toxic chemicals could result in acute or delayed injury to the skin (e.g. inflammation, allergy, cancer). Skin metabolism may play a role in the manifestation or amelioration of adverse effects via the topical route. Today, we have robust testing strategies to assess the potential for local skin toxicity of chemical exposure. Such methods (e.g. the local lymph node assay for assessing skin sensitisation; skin painting carcinogenicity studies) incorporate skin metabolism implicitly in the in vivo model system used. In light of recent European legislation (i.e. 7(th) Amendment to the Cosmetics Directive and Registration Evaluation and Authorisation of existing Chemicals (REACH)), non-animal approaches will be required to reduce and replace animal experiments for chemical risk assessment. It is expected that new models and approaches will need to account for skin metabolism explicitly, as the mechanisms of adverse effects in the skin are deconvoluted. 3D skin models have been proposed as a tool to use in new in vitro alternative approaches. In order to be able to use 3D skin models in this context, we need to understand their metabolic competency in relation to xenobiotic biotransformation and whether functional activity is representative of that seen in human skin.

The immune system is called into action by alarm signals generated from injured tissues. We examined the nature of these alarm signals after exposure of skin residential cells to contact allergens (nickel sulfate and potassium dichromate) and a contact irritant [sodium dodecyl sulfate (SDS)]. Nickel sulfate, potassium dichromate, and SDS were applied topically to the stratum corneum of human skin equivalents. A similar concentration-dependent increase in chemokine (CCL20, CCL27, and CXCL8) secretion was observed for all three chemicals. Exposure to nickel sulfate and SDS was investigated in more detail: similar to chemokine secretion, no difference was observed in the time- and concentration-dependent increase in pro-inflammatory cytokine [interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha)] secretion. Maximal increase in IL-1alpha secretion occurred within 2 h after exposure to both nickel sulfate and SDS and prior to increased chemokine secretion. TNF-alpha secretion was detectable 8 h after chemical exposure. After allergen or irritant exposure, increased CCL20 and CXCL8, but not CCL27, secretion was inhibited by neutralizing human antibodies to either IL-1alpha or TNF-alpha. Our data show that alarm signals consist of primary and secondary signals. IL-1alpha and TNF-alpha are released as primary alarm signals, which trigger the release of secondary chemokine (CCL20 and CXCL8) alarm signals. However, some chemokines, for example, CCL27 can be secreted in an IL-1alpha and TNF-alpha independent manner. Our data suggest that skin residential cells respond to both allergen and irritant exposure by releasing mediators that initiate infiltration of immune responsive cells into the skin.

Allergic contact dermatitis (ACD) is a T-cell-mediated disease in which expression of a distinct repertoire of chemokines results in the recruitment of effector T cells into the skin. While it is becoming clear which chemokines and receptors determine the development of ACD, the mechanisms involved in the retention of T cells in the skin after resolution of inflammation are still unknown. Unravelling these mechanisms will help us to understand local skin memory as observed in retest reactivity and flare-up reactions. This study was designed to evaluate the role of chemokine-chemokine receptor interactions in local T-cell retention. The results show that expression of the CCR10 targeting ligand CCL27 is not only increased during inflammation, but also remains increased several weeks after clinical responsiveness to patch testing. In parallel with increased CCL27 expression, an increased number of infiltrating cells could still be detected in skin that, clinically, had returned to normal 21 days after patch testing. These persisting cells were characterized as CD4+ cells expressing CCR10, while no CD8+ CCR10+ cells could be detected. The presence of these cells is most likely an allergen-mediated effect, as increased levels of CCL27 and CCR10 could not be detected 21 days after initiating an irritant contact dermatitis reaction. In contrast to CCL27, increased expression of CXCL9, CXCL10, and CXCL11 could only be observed during the clinically inflammatory phase of ACD. In conclusion, local CCL27-mediated retention of CCR10+ CD4+ T cells in sites previously challenged by ACD could be responsible for phenomena such as local skin memory observed in retest reactions and flare-up reactions in which the presence of persisting T cells results in an accelerated inflammatory response upon renewed allergen challenge., (Copyright 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Magnetic resonance microscopy of skin from hairless rats under the influence of electrical fields was conducted for two cases: 1) low voltage constant electrical fields and 2) high-voltage short pulse, electrical fields. Under conditions of the low voltage and low current iontophoresis, i.e., 0 to 20 V, and 0 to 0.5 mA/cm2, it was found that the skin structure, as observed by magnetic resonance microscopy, did not significantly change until 20 Volts were applied across the 0.1 cm thick skin. Under these conditions, the viable epidermis appeared to swell, and this result corresponded to observations from scanning electron microscopy and other research from the literature. High voltage electrical fields, i.e., 220 V 1 ms pulses repeated once per second, appeared to hydrate the stratum corneum as is consistent with published literature on electroporation. In the case of iontophoresis, water self-diffusion coefficients in the epidermis and hair follicle regions at all voltages were affected by the electrical field. Statistical analysis at the 95% confidence level for the comparison of the average differences between diffusion coefficients with the electrical field on and with the electrical field off for pair matched pixels for the viable epidermis show that for 5 V (p = 0.00377), 10 V (p = 0.0108), 20 V (p = 0.0219) regimes there are statistically significant (p < or = 0.05) changes due to the applied electric field. The same analysis for the hair follicle region at 5 V (p = 6.89 x 10(-7)), 10 V (p = 1.42 x 10(-5)), 20 V (p = 3.23 x 10(-3)) also show statistically significant changes (p < or = 0.05). When the electroporation pulse was applied, the water diffusion coefficients increased by about 30% to 6.6 x 10(-6) cm2/s +/- 2.4 x 10(-7) cm2/s and 8.3 x 10(-6) cm2/s +/- 3.7 x 10(-7) cm2/s, for the epidermis and hair follicle regions, respectively. Significant differences were noted between diffusion coefficients in the viable epidermis and the hair follicles for all cases.

For the investigation of the skin irritancy potential of chemicals in an in vitro model, it is necessary to have sensitive end-points that predict the effects on native human skin. Our aim was to investigate whether the induction of the proteinase inhibitor SKALP in reconstructed epidermis can be used as a marker. The influence of culture conditions and the effect of topical application of sodium lauryl sulfate and oleic acid on SKALP expression were evaluated using immunohistochemistry and Northern blotting. SKALP expression was induced by serum, epidermal growth factor and fibroblasts. In the presence of retinoic acid and 1,25-dihydroxyvitamin D3 SKALP expression was inhibited, whereas supplementation with ascorbic acid and a-tocopherol had no effect. Tape-stripping of excised skin and topical treatment with sodium lauryl sulfate induced SKALP protein expression. Application of sodium lauryl sulfate and oleic acid on reconstructed epidermis also induced SKALP at the protein level but no significant effects could be demonstrated at mRNA levels. In conclusion, SKALP expression, which was increased upon application of sodium lauryl sulfate and oleic acid, can be used as an in vitro end-point for skin irritancy, irrespective of the modifying effects of culture conditions.

Abstract: Testing chemicals for their ability to cause skin irritation is required for all ingredients of products that come into contact with the skin. Here, we describe a potential method for determining the irritant potency of a chemical in vitro and apply the method to two different reconstructed epidermis models which exhibit different barrier properties. Two surfactants: sodium dodecyl sulphate, Triton X100 and two non-surfactants: 2-4-di-nitro-chloro-benzene, cinnamaldehyde were applied topically in a dose response for 24h. Biomarkers IL-1alpha, IL-1RA, IL-8 and MTT were assessed and EC50 values determined. Variation in barrier properties between the epidermal models led to variation in the extent of penetration of surfactants, but not of non-surfactants which in turn influenced the EC50 value obtained from surfactants. Furthermore, EC50 values showed that no single biomarker could be classed as the most sensitive biomarker since biomarker sensitivity differed between the different chemicals studied. However, the ranking of the chemicals in order of strong to weak irritant was the same irrespective of the model used and also independent of the biomarker used (Triton X100>DNCB>SDS>CA). This study describes a method which not only distinguishes an irritant from a non-irritant but which may possibly also be used to determine irritant potency. [Copyright &y& Elsevier]