Intraluminal release of citrullinated histone 3 from various cellular origins coincides with microvascular thrombosis in burn wounds.

Loss of perfusion in the burn wound might cause wound deepening and impaired healing. We previously showed persistent microvascular thrombosis coinciding with intraluminal neutrophils extracellular traps in human burned skin. This study investigates the presence of intraluminal citrullinated histone 3 (H3cit) from different cellular origins (neutrophils, monocytes, and lymphocytes) in relation to microvascular thrombosis of burn wounds. Eschar was obtained from burn patients (n = 18) 6-40 days postburn with a mean total burned body surface area of 23%. Microvascular presence of tissue factor (TF), factor XII (FXII) and thrombi was assessed by immunohistochemistry. Intramicrovascular cell death was analyzed via immunofluorescent microscopy, combining antibodies for neutrophils (MPO), monocytes (CD14), and lymphocytes (CD45) with endothelial cell markers CD31 and H3cit. Significantly increased microvascular expression of TF, FXII, and thrombi (CD31+) was found in all eschar samples compared with control uninjured skin. Release of H3cit from different cellular origins was observed in the lumen of the dermal microvasculature in the eschar tissue 7-40 days postburn, with release from neutrophilic origin being 2.7 times more abundant. Intraluminal presence of extracellular H3cit colocalizing with either MPO, CD14, or CD45 is correlated to increased microvascular thrombosis in eschar of burn patients.

Oral host-microbe interactions investigated in 3D organotypic models.

The oral cavity is inhabited by abundant microbes which continuously interact with the host and influence the host's health. Such host-microbe interactions (HMI) are dynamic and complex processes involving e.g. oral tissues, microbial communities and saliva. Due to difficulties in mimicking the in vivo complexity, it is still unclear how exactly HMI influence the transition between healthy status and disease conditions in the oral cavity. As an advanced approach, three-dimensional (3D) organotypic oral tissues (epithelium and mucosa/gingiva) are being increasingly used to study underlying mechanisms. These in vitro models were designed with different complexity depending on the research questions to be answered. In this review, we summarised the existing 3D oral HMI models, comparing designs and readouts, discussing applications as well as future perspectives.

The JAK-STAT pathway in keloid pathogenesis: a systematic review with qualitative synthesis.

Keloid tissues contain inflammatory cells and upregulated pro-inflammatory cytokines. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway mediate cellular responses to these cytokines. We performed a systematic review on the role of the JAK-STAT pathway in keloid pathogenesis and the evidence for JAK-STAT inhibitors in keloid treatment. The search combined the terms (1) keloid and (2) JAK or TYK or STAT and included MeSH terms and synonyms. Two reviewers screened the articles and assessed the full texts on eligibility. Data were collected on the tested drugs and molecules, the type of cells and tissues used in the experiments, and study findings on the association between the JAK-STAT pathway and keloid cells and tissues. A total of twenty preclinical studies were included. Eleven preclinical studies proved that STAT3 expression and phosphorylation are enhanced in keloid tissue and keloid fibroblasts. Thirteen different JAK and/or STAT inhibitors were investigated. Tested drugs inhibited keloid progression as demonstrated by different processes, including reduced collagen production, cell proliferation and migration, increased cell cycle arrest and apoptosis, enhanced antioxidant responses, decreased (paracrine) signalling, and decreased profibrotic gene expression. No clinical studies have been published to date. Preclinical studies indicate a role for the JAK-STAT pathway in keloid pathogenesis and a potential role for JAK-STAT inhibitors in keloid treatment. The effect of these drugs should be further investigated on relevant biomarkers in a human keloid skin model, preferably including immune cells besides keloid fibroblasts and keratinocytes and in clinical studies.

Intralesional Corticosteroid Administration in the Treatment of Keloids: A Scoping Review on Injection Methods.

BACKGROUND: Intralesional corticosteroid administration (ICA) is a first-line treatment for keloids. However, its clinical results are still highly variable and often suboptimal. Treatment results may strongly be influenced by various operator-dependent factors. The aim of this study was to map the details of ICA in keloids described in randomized controlled trials (RCTs), hence presenting the scientific practice of a first-line treatment for keloids in the best available evidence. SUMMARY: A systematic search was performed on PubMed, Ovid MEDLINE, Ovid EMBASE, and CENTRAL. Eligible studies were RCTs including patients with keloids treated with intralesional corticosteroids. Treatment and study design-related data were charted on a predefined form. Thirty-eight RCTs were included for data extraction. Triamcinolone acetonide was used in 37 (97.4%) studies. Dosing per cm2 could only be compared among ten (26%) studies and varied from 1 to 20 mg. The maximum dose per session varied from 20 to 80 mg. Local anesthetics were administered in seven (20%) RCTs. Treatment intervals varied from weekly to monthly, with 4 weeks most frequently (50%) used. Needle size was reported in eleven (29%) studies and varied from 26 to 30-gauge. Syringe size was specified in four (11%) studies, being 1 mL. The injection level was described in eleven (29%) studies. Blanching as endpoint was reported in ten (26%) studies. Outcome measures varied widely, from height, surface area, or volume, to Vancouver Scar Scale, Patient and Observer Scar Assessment Scale, pain and itch scores, patient satisfaction, and different efficacy rates. Only six studies had a follow-up of ≥6 months. Recurrence was identified in two studies with 18 weeks and 1 year of follow-up. Adverse events were reported in 23 (61%) studies.

Current Practice in Keloid Treatment: a Survey of Dutch Dermatologists and Plastic Surgeons.

BACKGROUND: Several therapeutic options are available for the treatment of keloids, but it remains unclear which treatment options are most commonly used by practitioners. OBJECTIVE: To explore the prevailing treatment for different keloid phenotypes among dermatologists and plastic surgeons in the Netherlands. METHODS: Members of the Dutch society for Plastic surgery and the Dutch society for Dermatology and Venereology were asked to participate. Questions elaborated on the treatment for a small and a large keloid on the mandibula and multiple keloids on the chest. RESULTS: One hundred forty-three responses were obtained. Heterogeneity in treatment was extremely high for the small, large, and multiple keloids with 27, 35, and 33 various first choices, respectively. Intralesional corticosteroids were most often chosen for all 3 different keloid phenotypes. These were mostly (61%) administered as monotherapy for the small keloid and mostly combined with other treatments for the large keloid (19%) and multiple keloids (43%). Surgery was chosen regularly (22%) for the large keloid, mostly combined with intralesional corticosteroids (10%) or brachytherapy (8.4%). CONCLUSION: Keloid treatment is very heterogeneous among dermatologists and plastic surgeons, even in a relatively small country as the Netherlands. Moreover, the treatment choice depends on the keloid phenotype.

Nickel allergy is associated with a broad spectrum cytokine response.

BACKGROUND: Nickel-induced proliferation or cytokine release by peripheral blood mononuclear cells may be used for in vitro diagnosis of nickel allergy. OBJECTIVES: Aim of this study was to explore the nickel-specific cytokine profile to further elucidate the pathogenesis of nickel allergic contact dermatitis (ACD) and to identify potential new biomarkers for nickel ACD. METHODS: Peripheral blood mononuclear cells from patients and controls were cultured with T-cell skewing cytokine cocktails and/or nickel. Cytokine and chemokine concentrations were assessed in culture supernatants using validated multiplex assays. Specific cytokine production was related to history of nickel allergy and patch-test results. RESULTS: Twenty-one of the 33 analytes included in the analysis were associated with nickel allergy and included type1 (TNF-α, IFN-γ, TNF-ß), type 2 (IL-3, IL-4, IL-5, IL-13), type 1/2 (IL-2, IL-10), type 9 (IL-9), type 17/1 (IL-17A[F], GM-CSF, IL-21) and type 22 (IL-22) derived cytokines as well as the T-cell/antigen presentation cell derived factors Thymus and activation regulated chemokine (TARC), IL-27 and IP-10. Receiver operator characteristics (ROC) analysis showed that IL-5 was the strongest biomarker for nickel allergy. CONCLUSIONS: A broad spectrum of 33 cytokines and chemokines is involved in the allergen-specific immune response in nickel allergic patients. IL-5 remains, next to the lymphocyte proliferation test, the strongest biomarker for nickel allergy.

Progress on Reconstructed Human Skin Models for Allergy Research and Identifying Contact Sensitizers.

Contact with the skin is inevitable or desirable for daily life products such as cosmetics, hair dyes, perfumes, drugs, household products, and industrial and agricultural products. Whereas the majority of these products are harmless, a number can become metabolized and/or activate the immunological defense via innate and adaptive mechanisms resulting in sensitization and allergic contact dermatitis upon following exposures to the same substance. Therefore, strict safety (hazard) assessment of actives and ingredients in products and drugs applied to the skin is essential to determine I) whether the chemical is a potential sensitizer and if so II) what is the safe concentration for human exposure to prevent sensitization from occurring. Ex vivo skin is a valuable model for skin penetration studies but due to logistical and viability limitations the development of in vitro alternatives is required. The aim of this review is to give a clear overview of the organotypic in vitro skin models (reconstructed human epidermis, reconstructed human skin, immune competent skin models incorporating Langerhans Cells and T-cells, skin-on-chip) that are currently commercially available or which are being used in a laboratory research setting for hazard assessment of potential sensitizers and for investigating the mechanisms (sensitization key events 1-4) related to allergic contact dermatitis. The limitations of the models, their current applications, and their future potential in replacing animals in allergy-related science are discussed.

From simplicity to complexity in current melanoma models.

Despite the recent impressive clinical success of immunotherapy against melanoma, development of primary and adaptive resistance against immune checkpoint inhibitors remains a major issue in a large number of treated patients. This highlights the need for melanoma models that replicate the tumor's intricate dynamics in the tumor microenvironment (TME) and associated immune suppression to study possible resistance mechanisms in order to improve current and test novel therapeutics. While two-dimensional melanoma cell cultures have been widely used to perform functional genomics screens in a high-throughput fashion, they are not suitable to answer more complex scientific questions. Melanoma models have also been established in a variety of experimental (humanized) animals. However, due to differences in physiology, such models do not fully represent human melanoma development. Therefore, fully human three-dimensional in vitro models mimicking melanoma cell interactions with the TME are being developed to address this need for more physiologically relevant models. Such models include melanoma organoids, spheroids, and reconstructed human melanoma-in-skin cultures. Still, while major advances have been made to complement and replace animals, these in vitro systems have yet to fully recapitulate human tumor complexity. Lastly, technical advancements have been made in the organ-on-chip field to replicate functions and microstructures of in vivo human tissues and organs. This review summarizes advancements made in understanding and treating melanoma and specifically aims to discuss the progress made towards developing melanoma models, their applications, limitations, and the advances still needed to further facilitate the development of therapeutics.

Scar formation from the perspective of complexity science: a new look at the biological system as a whole.

A burn wound is a complex systemic disease at multiple levels. Current knowledge of scar formation after burn injury has come from traditional biological and clinical studies. These are normally focused on just a small part of the entire process, which has limited our ability to sufficiently understand the underlying mechanisms and to predict systems behaviour. Scar formation after burn injury is a result of a complex biological system-wound healing. It is a part of a larger whole. In this self-organising system, many components form networks of interactions with each other. These networks of interactions are typically non-linear and change their states dynamically, responding to the environment and showing emergent long-term behaviour. How molecular and cellular data relate to clinical phenomena, especially regarding effective therapies of burn wounds to achieve minimal scarring, is difficult to unravel and comprehend. Complexity science can help bridge this gap by integrating small parts into a larger whole, such that relevant biological mechanisms and data are combined in a computational model to better understand the complexity of the entire biological system. A better understanding of the complex biological system of post-burn scar formation could bring research and treatment regimens to the next level. The aim of this review/position paper is to create more awareness of complexity in scar formation after burn injury by describing the basic principles of complexity science and its potential for burn care professionals.

Application of Microphysiological Systems to Enhance Safety Assessment in Drug Discovery.

Enhancing the early detection of new therapies that are likely to carry a safety liability in the context of the intended patient population would provide a major advance in drug discovery. Microphysiological systems (MPS) technology offers an opportunity to support enhanced preclinical to clinical translation through the generation of higher-quality preclinical physiological data. In this review, we highlight this technological opportunity by focusing on key target organs associated with drug safety and metabolism. By focusing on MPS models that have been developed for these organs, alongside other relevant in vitro models, we review the current state of the art and the challenges that still need to be overcome to ensure application of this technology in enhancing drug discovery.

Hypertrophic scars and keloids: Overview of the evidence and practical guide for differentiating between these abnormal scars.

Although hypertrophic scars and keloids both generate excessive scar tissue, keloids are characterized by their extensive growth beyond the borders of the original wound, which is not observed in hypertrophic scars. Whether or not hypertrophic scars and keloids are two sides of the same coin or in fact distinct entities remains a topic of much debate. However, proper comparison between the two ideally occurs within the same study, but this is the exception rather than the rule. For this reason, the goal of this review was to summarize and evaluate all publications in which both hypertrophic scars and keloids were studied and compared to one another within the same study. The presence of horizontal growth is the mainstay of the keloid diagnosis and remains the strongest argument in support of keloids and hypertrophic scars being distinct entities, and the histopathological distinction is less straightforward. Keloidal collagen remains the strongest keloid parameter, but dermal nodules and α-SMA immunoreactivity are not limited to hypertrophic scars alone. Ultimately, the current hypertrophic scars-keloid differences are mostly quantitative in nature rather than qualitative, and many similar abnormalities exist in both lesions. Nonetheless, the presence of similarities does not equate the absence of fundamental differences, some of which may not yet have been uncovered given how much we still have to learn about the processes involved in normal wound healing. It therefore seems pertinent to continue treating hypertrophic scars and keloids as separate entities, until such a time as new findings more decisively convinces us otherwise.

Prognostic tools for hypertrophic scar formation based on fundamental differences in systemic immunity.

Unpredictable hypertrophic scarring (HS) occurs after approximately 35% of all surgical procedures and causes significant physical and psychological complaints. Parallel to the need to understanding the mechanisms underlying HS formation, a prognostic tool is needed. The objective was to determine whether (systemic) immunological differences exist between patients who develop HS and those who develop normotrophic scars (NS) and to assess whether those differences can be used to identify patients prone to developing HS. A prospective cohort study with NS and HS groups in which (a) cytokine release by peripheral blood mononuclear cells (PBMC) and (b) the irritation threshold (IT) after an irritant (sodium lauryl sulphate) patch test was evaluated. Univariate regression analysis of PBMC cytokine secretion showed that low MCP-1, IL-8, IL-18 and IL-23 levels have a strong correlation with HS (P < .010-0.004; AUC = 0.790-0.883). Notably, combinations of two or three cytokines (TNF-a, MCP-1 and IL-23; AUC: 0.942, Nagelkerke R2 : 0.727) showed an improved AUC indicating a better correlation with HS than single cytokine analysis. These combination models produce good prognostic results over a broad probability range (sensitivity: 93.8%, specificity 86.7%, accuracy 90,25% between probability 0.3 and 0.7). Furthermore, the HS group had a lower IT than the NS group and an accuracy of 68%. In conclusion, very fundamental immunological differences exist between individuals who develop HS and those who do not, whereas the cytokine assay forms the basis of a predictive prognostic test for HS formation, the less invasive, easily performed irritant skin patch test is more accessible for daily practice.

Patch test-relevant concentrations of metal salts cause localized cytotoxicity, including apoptosis, in skin ex vivo.

BACKGROUND: Metal alloys containing contact sensitizers (nickel, palladium, titanium) are extensively used in medical devices, in particular dentistry and orthopaedic surgery. The skin patch test is used to test for metal allergy. OBJECTIVE: To determine whether metal salts, when applied to freshly excised skin at patch test-relevant concentrations and using a method which mimics skin patch testing, cause in changes in the epidermis and dermis. METHODS: Tissue histology, apoptosis, metabolic activity, and inflammatory cytokine release were determined for two nickel salts, two palladium salts, and four titanium salts. RESULTS: Patch test-relevant concentrations of all metal salts caused localized cytotoxicity. This was observed as epidermis separation at the basement membrane zone, formation of vacuoles, apoptotic nuclei, decreased metabolic activity, and (pro)inflammatory cytokine release. Nickel(II) sulfate hexahydrate, nickel(II) chloride hexahydrate, titanium(IV) bis(ammonium lactato)dihydroxide, and calcium titanate were highly cytotoxic. Palladium(II) chloride, sodium tetrachloropalladate(II), titanium(IV) isopropoxide, and titanium(IV) dioxide showed mild cytotoxicity. CONCLUSION: The patch test in itself may be damaging to the skin of the patient being tested. These results need further verification with biopsies obtained during clinical patch testing. The future challenge is to remain above the elicitation threshold at noncytotoxic metal concentrations.

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.

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.

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.

Sensing of latent EBV infection through exosomal transfer of 5'pppRNA.

Complex interactions between DNA herpesviruses and host factors determine the establishment of a life-long asymptomatic latent infection. The lymphotropic Epstein-Barr virus (EBV) seems to avoid recognition by innate sensors despite massive transcription of immunostimulatory small RNAs (EBV-EBERs). Here we demonstrate that in latently infected B cells, EBER1 transcripts interact with the lupus antigen (La) ribonucleoprotein, avoiding cytoplasmic RNA sensors. However, in coculture experiments we observed that latent-infected cells trigger antiviral immunity in dendritic cells (DCs) through selective release and transfer of RNA via exosomes. In ex vivo tonsillar cultures, we observed that EBER1-loaded exosomes are preferentially captured and internalized by human plasmacytoid DCs (pDCs) that express the TIM1 phosphatidylserine receptor, a known viral- and exosomal target. Using an EBER-deficient EBV strain, enzymatic removal of 5'ppp, in vitro transcripts, and coculture experiments, we established that 5'pppEBER1 transfer via exosomes drives antiviral immunity in nonpermissive DCs. Lupus erythematosus patients suffer from elevated EBV load and activated antiviral immunity, in particular in skin lesions that are infiltrated with pDCs. We detected high levels of EBER1 RNA in such skin lesions, as well as EBV-microRNAs, but no intact EBV-DNA, linking non-cell-autonomous EBER1 presence with skin inflammation in predisposed individuals. Collectively, our studies indicate that virus-modified exosomes have a physiological role in the host-pathogen stand-off and may promote inflammatory disease.

Endothelial cells enhance adipose mesenchymal stromal cell-mediated matrix contraction via ALK receptors and reduced follistatin: Potential role of endothelial cells in skin fibrosis.

Abnormal cutaneous wound healing can lead to formation of fibrotic hypertrophic scars. Although several clinical risk factors have been described, the cross-talk between different cell types resulting in hypertrophic scar formation is still poorly understood. The aim of this in vitro study was to investigate whether endothelial cells (EC) may play a role in skin fibrosis, for example, hypertrophic scar formation after full-thickness skin trauma. Using a collagen/elastin matrix, we developed an in vitro fibrosis model to study the interaction between EC and dermal fibroblasts or adipose tissue-derived mesenchymal stromal cells (ASC). Tissue equivalents containing dermal fibroblasts and EC displayed a normal phenotype. In contrast, tissue equivalents containing ASC and EC displayed a fibrotic phenotype indicated by contraction of the matrix, higher gene expression of ACTA2, COL1A, COL3A, and less secretion of follistatin. The contraction was in part mediated via the TGF-ß pathway, as both inhibition of the ALK4/5/7 receptors and the addition of recombinant follistatin resulted in decreased matrix contraction (75 ± 11% and 24 ± 8%, respectively). In conclusion, our study shows that EC may play a critical role in fibrotic events, as seen in hypertrophic scars, by stimulating ASC-mediated matrix contraction via regulation of fibrosis-related proteins.

Comparison of the skin sensitization potential of 3 red and 2 black tattoo inks using interleukin-18 as a biomarker in a reconstructed human skin model.

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.