Reconstructed Human Skin with Hypodermis Shows Essential Role of Adipose Tissue in Skin Metabolism.

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-1ß, 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.

A Humanized In Vitro Model of Innervated Skin for Transdermal Analgesic Testing.

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.

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

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-1ß, inflammatory IL-23 and IFN-γ, 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.

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.

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

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.

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

Application of reconstructed human Skin (RhS) is a promising approach for the treatment of extensive wounds and for drug efficacy and safety testing. However, incorporating appendages, such as hair follicles, into RhS still remains a challenge. The hair follicle plays a critical role in thermal regulation, dispersion of sweat and sebum, sensory and tactile functions, skin regeneration, and repigmentation. The aim of this study was to determine whether human neopapilla could be incorporated into RhS (differentiated epidermis on fibroblast and endothelial cell populated dermis) and whether the neopapillae maintain their inductive follicular properties in vitro. Neopapillae spheroids, constructed from expanded and self-aggregating dermal papilla cells, synthesized extracellular matrix typically found in follicular papillae. Compared with dermal fibroblasts, neopapillae showed increased expression of multiple genes (Wnt5a, Wnt10b, and LEF1) known to regulate hair development and also increased secretion of CXCL1, which is a strong keratinocyte chemoattractant. When neopapillae were incorporated into the dermis of RhS, they stimulated epidermal down-growth resulting in engulfment of the neopapillae sphere. Similar to the native hair follicle, the differentiated invaginating epidermis inner side was keratin 10 positive and the undifferentiated outer side keratin 10 negative. The outer side was keratin 15 positive confirming the undifferentiated nature of these keratinocytes aligning a newly formed collagen IV, laminin V positive basement membrane within the hydrogel. In conclusion, we describe a RhS model containing neopapillae with hair follicle-inductive properties. Importantly, epidermal invagination occurred to engulf the neopapillae, thus demonstrating in vitro the first steps towards hair follicle morphogenesis in RhS.

Human saliva stimulates skin and oral wound healing in vitro.

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.

Comparison of advanced therapy medicinal product gingiva and skin substitutes and their in vitro wound healing potentials.

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.

Comparative phenotypic and functional analysis of migratory dendritic cell subsets from human oral mucosa and skin.

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.

SMC is recruited to oriC by ParB and promotes chromosome segregation in Streptococcus pneumoniae.

Segregation of replicated chromosomes is an essential process in all organisms. How bacteria, such as the oval-shaped human pathogen Streptococcus pneumoniae, efficiently segregate their chromosomes is poorly understood. Here we show that the pneumococcal homologue of the DNA-binding protein ParB recruits S. pneumoniae condensin (SMC) to centromere-like DNA sequences (parS) that are located near the origin of replication, in a similar fashion as was shown for the rod-shaped model bacterium Bacillus subtilis. In contrast to B. subtilis, smc is not essential in S. pneumoniae, and Δsmc cells do not show an increased sensitivity to gyrase inhibitors or high temperatures. However, deletion of smc and/or parB results in a mild chromosome segregation defect. Our results show that S. pneumoniae contains a functional chromosome segregation machine that promotes efficient chromosome segregation by recruitment of SMC via ParB. Intriguingly, the data indicate that other, as of yet unknown mechanisms, are at play to ensure proper chromosome segregation in this organism.