MUTZ-3 derived Langerhans cells in human skin equivalents show differential migration and phenotypic plasticity after allergen or irritant exposure

Abstract

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.

Authors

Ilona J. Kosten, Sander W. Spiekstra, Tanja D. de Gruijl, Susan Gibbs

Link

https://doi.org/10.1016/j.taap.2015.05.017

Characterization of In Vitro Reconstructed Human Normotrophic, Hypertrophic, and Keloid Scar Models

Abstract

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.

Authors

Grace C. Limandjaja, Lenie J. van den Broek, Melanie Breetveld, Taco Waaijman, Stan Monstrey, Edith M. de Boer, Rik J. Scheper, Frank B. Niessen, and Susan Gibbs

Link

https://doi.org/10.1089/ten.tec.2017.0464

Evaluation of a novel oral mucosa in vitro implantation model for analysis of molecular interactions with dental abutment surfaces

Abstract

Background

Abutment surfaces are being designed to promote gingival soft tissue attachment and integration. This forms a seal around prosthetics and consequently ensures long‐term implant survival. New scalable and reproducible models are necessary to evaluate and quantify the performance of these surfaces.

Purpose

To evaluate a novel implantation model by histomorphometric and immunohistochemical characterization of the interactions between human oral gingival tissue and titanium abutments with either novel anodized or conventional machined surface.

Materials and Methods

Abutments were inserted into an organotypic reconstructed human gingiva (RHG) model consisting of differentiated gingival epithelium cells on a fibroblast populated lamina propria hydrogel following a tissue punch. Epithelial attachment, down‐growth along the abutment surface, and phenotype were assessed via histomorphology, scanning electron microscopy, and immunohistochemistry 10 days after implantation.

Results

The down‐growing epithelium transitioned from a gingival margin to a sulcular and junctional epithelium. The sulcus depth and junctional epithelial length were similar to previously reported pre‐clinical and clinical lengths. A collagen IV/laminin 5 basement membrane formed between the epithelium and the underlying connective tissue. The RHG expanded in thickness approximately 2‐fold at the abutment surface. The model allowed the evaluation of protein expression of adhering soft tissue cells for both tested abutments.

Conclusions

The RHG model is the first in vitro 3D model to enable the assessment of not only human epithelial tissue attachment to dental abutments but also the expression of protein markers involved in soft tissue attachment and integration. The two abutments showed no noticeable difference in epithelial attachment.

Keywords: abutment, dental implant, in vitro, junctional epithelium, model, organotypic, reconstructed human gingiva, reconstructed oral mucosa, soft tissue attachment

Authors

Sanne Roffel, BSc, 1 Gang Wu, PhD, 2 Ivana Nedeljkovic, PhD, 3 Michael Meyer, PhD, 4 Tojo Razafiarison, PhD, 4 and Susan Gibbs, PhD 1 , 5

Link

https://doi.org/10.1111/cid.12750

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

Abstract

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.

Keywords: IL-10; M2 macrophages; Melanoma; Reconstructed human skin; Tumor microenvironment; Tumor progression.

Authors

Elisabetta Michielon, Marta López Gonzålez, Judith L A Burm, Taco Waaijman, Ekaterina S Jordanova, Tanja D de Gruijl, Susan Gibbs

Link

https://doi.org/10.1007/s00262-020-02626-4

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

Abstract

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.

Keywords: allergy; commensal bacteria; host microbiome; in vitro; innate immune response; metals; oral mucosa; reconstructed human gingiva; reconstructed human skin; skin.

Authors

Lin Shang, Dongmei Deng, Sanne Roffel, Susan Gibbs

Link

https://doi.org/10.1111/cod.13668

Reconstructed human skin shows epidermal invagination towards integrated neopapillae indicating early hair follicle formation in vitro

Abstract

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.

Keywords: IL-18; allergic contact dermatitis; allergy; cytotoxicity; in vitro; reconstructed human skin; sensitization; tattoo ink.

Authors

Irit Vahav, Lenie J van den Broek, Maria Thon, Hanneke N Monsuur, Sander W Spiekstra, Beren Atac, Rik J Scheper, Roland Lauster, Gerd Lindner, Uwe Marx, Susan Gibbs

Link

https://doi.org/10.1002/term.3039

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

Abstract

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.

Keywords: IL-18; allergic contact dermatitis; allergy; cytotoxicity; in vitro; reconstructed human skin; sensitization; tattoo ink.

Authors

Joey J J P Karregat, Thomas Rustemeyer, Sebastiaan A S van der Bent, Sander W Spiekstra, Maria Thon, David Fernandez Rivas, Susan Gibbs

Link

https://doi.org/10.1111/cod.13908

Stable reconstructed human gingiva–microbe interaction model: Differential response to commensals and pathogens

Abstract

Background: To investigate human oral health and disease, models are required which represent the interactions between the oral mucosa and microbiome. Our aim was to develop an organotypic model which maintains viability of both host and microbes for an extended period of time.

Methods: Reconstructed Human Gingiva (RHG) were cultured air-lifted with or without penicillin-streptomycin (PS) and topically exposed to Streptococcus gordonii (commensal) or Aggregatibacter actinomycetemcomitans (pathogen) for 72 hours in agar. RHG histology, viability and cytokines (ELISA), and bacterial viability (colony forming units) and location (FISH) were assessed.

Results: The low concentration of topically applied agar did not influence RHG viability. Topically applied bacteria in agar remained localized and viable for 72 hours and did not spill over to infect RHG culture medium. PS in RHG culture medium killed topically applied bacteria. Co-culture with living bacteria did not influence RHG viability (Ki67 expression, MTT assay) or histology (epithelium differentiation, Keratin10 expression). RHG exposed to S. gordonii (with or without PS) did not influence low level of IL-6, IL-8, CCL2, CCL5, CCL20 or CXCL1 secretion. However, all cytokines increased (except CCL2) when RHG were co-cultured with A. actinomycetemcomitans. The effect was significantly more in the presence of living, rather than dead, A. actinomycetemcomitans. Both bacteria resulted in increased expression of RHG antimicrobial peptides (AMPs) Elafin and HBD-2, with S. gordonii exposure resulting in the most Elafin secretion.

Conclusion: This technical advance enables living human oral host–microbe interactions to be investigated during a 72-hour period and shows differences in innate immunology triggered by S. gordonii and A. actinomycetemcomitans.

Authors

Yan Zhang, Lin Shang, Sanne Roffel, Bastiaan P. Krom, Susan Gibbs and Dongmei Deng

Link

https://doi.org/10.3389/fcimb.2022.991128

Proof-of-Concept Organ-on-Chip Study: Topical Cinnamaldehyde Exposure of Reconstructed Human Skin with Integrated Neopapillae Cultured under Dynamic Flow

Abstract

Pharmaceutical and personal care industries require human representative models for testing to ensure the safety of their products. A major route of penetration into our body after substance exposure is via the skin. Our aim was to generate robust culture conditions for a next generation human skin-on-chip model containing neopapillae and to establish proof-of-concept testing with the sensitizer, cinnamaldehyde. Reconstructed human skin consisting of a stratified and differentiated epidermis on a fibroblast populated hydrogel containing neopapillae spheroids (RhS-NP), were cultured air-exposed and under dynamic flow for 10 days. The robustness of three independent experiments, each with up to 21 intra-experiment replicates, was investigated. The epidermis was seen to invaginate into the hydrogel towards the neopapille spheroids. Daily measurements of lactate dehydrogenase (LDH) and glucose levels within the culture medium demonstrated high viability and stable metabolic activity throughout the culture period in all three independent experiments and in the replicates within an experiment. Topical cinnamaldehyde exposure to RhS-NP resulted in dose-dependent cytotoxicity (increased LDH release) and elevated cytokine secretion of contact sensitizer specific IL-18, pro-inflammatory IL-1ÎČ, inflammatory IL-23 and IFN-Îł, as well as anti-inflammatory IL-10 and IL-12p70. This study demonstrates the robustness and feasibility of complex next generation skin models for investigating skin immunotoxicity.

Keywords: 

reconstructed human skinhair folliclein vitroneopapillaeorgan on chiprobustreproduciblesensitizer

Authors

Irit Vahav, Maria Thon, Lenie J. van den Broek, Sander W. Spiekstra, Beren Atac, Gerd Lindner, Katharina Schimek, Uwe Marx and Susan Gibbs

Link

https://doi.org/10.3390/pharmaceutics14081529

A Multi-Organ-on-Chip Approach to Investigate How Oral Exposure to Metals Can Cause Systemic Toxicity Leading to Langerhans Cell Activation in Skin

Abstract

Investigating systemic toxicity in vitro is still a huge challenge. Here, a multi-organ-on-chip approach is presented as a typical case of topical exposure of oral mucosa to metals, which are known to activate the immune system and in turn may result in skin inflammation. Reconstructed human gingiva (RHG) and reconstructed human skin containing MUTZ-3-derived Langerhans cells (MUTZ-LC) in the epidermis (RHS-LC) were incorporated into a HUMIMIC Chip3plus, connected by dynamic flow and cultured for a total period of 72 h. Three independent experiments were performed each with an intra-experiment replicate in order to assess the donor and technical variations. After an initial culture period of 24 h to achieve stable dynamic culture conditions, nickel sulfate was applied topically to RHG for 24 h, and LC activation (maturation and migration) was determined in RHS-LC after an additional 24 h incubation time. A stable dynamic culture of RHG and RHS-LC was achieved as indicated by the assessment of glucose uptake, lactate production, and lactate dehydrogenase release into the microfluidics compartment. Nickel exposure resulted in no major histological changes within RHG or RHS-LC, or cytokine release into the microfluidics compartment, but did result in an increased activation of LC as observed by the increased mRNA levels of CD1a, CD207, HLA-DR, and CD86 in the dermal compartment (hydrogel of RHS-LC (PCR)). This is the first study to describe systemic toxicity and immune cell activation in a multi-organ setting and can provide a framework for studying other organoids in the future.

Keywords: Langerhans cell; nickel; organ on a chip; organotypic; reconstructed human gingiva; reconstructed human skin.

Authors

Jasper J Koning, Charlotte T Rodrigues Neves, Katharina Schimek, Maria Thon, Sander W Spiekstra, Taco Waaijman, Tanja D de Gruijl, Susan Gibbs

Link

https://doi.org/10.3389/ftox.2021.824825