Primary Human Epidermal Keratinocytes Research Articles

Isolation of Epidermal Keratinocytes from Human Skin: The Scratch-Wound Assay for Assessment of Epidermal Keratinocyte Migration.

The migration of epidermal keratinocytes is the basis for skin reepithelialization during wound healing. The in vitro scratch-wound assay using monolayers of primary human epidermal keratinocytes is a straightforward and effective method to assess their migratory capacity. The mechanical scratch of a confluent monolayer directly disrupts the adhesion of the keratinocytes to one another and to the underlying matrix, resembling the physical trauma of a wound in an in vitro assay. The keratinocytes will undergo an epithelial-to-mesenchymal transition, which will confer an ability to migrate toward each other to cover the gap by restructuring cell-cell and cell-extracellular matrix connections. However, a good scratch-wound method and protocol to ensure scratch reproducibility is essential, particularly when using primary cell cultures where donor variability may also impact on results.

In vitro antioxidant potential of Nephelium lappaceum L. rind extracts and geraniin on human epidermal keratinocytes

Abstract The aim of this study was to evaluate the anti-aging properties of Nephelium lappaceum L. rind phenolic and polysaccharide extracts and geraniin on human epidermal keratinocytes. The phenolic and polysaccharide extracts of N. lappaceum L. rind and geraniin were evaluated for anti-aging and antioxidant activities in human primary epidermal keratinocytes (HPEKs). The geraniin in the phenolic extract was also quantified by UPLC analysis. The extractive yield of crude phenolic extract (18.37%) was higher than polysaccharide extract (1.99%). The Phenolic extract and geraniin exhibited potential radical scavenging activity, enhanced HPEK's proliferation and reduced HPEK's oxidative stress by increasing SOD activity and suppressing nitric oxide formation. The geraniin content in the phenolic extract was 474.9 mg/g extract. The results indicated that the phenolic compounds of N. lappaceum L. rind, particularly in geraniin, were attributed to antioxidant properties (R = 0.967–0.972). The results suggest that phenolic compounds and geraniin, as the principal constituents of N. lappaceum L. rind, could be further utilized as alternative sources of anti-aging active ingredients in cosmetics and that geraniin could be used as a marker compound for the extract's standardization.

Adipose-derived stromal/stem cells improve epidermal homeostasis

Wound healing is regulated by complex interactions between the keratinocytes and other cell types including fibroblasts. Recently, adipose-derived mesenchymal stromal/stem cells (ASCs) have been reported to influence wound healing positively via paracrine involvement. However, their roles in keratinocytes are still obscure. Therefore, investigation of the precise effects of ASCs on keratinocytes in an in vitro culture system is required. Our recent data indicate that the epidermal equivalents became thicker on a collagen vitrigel membrane co-cultured with human ASCs (hASCs). Co-culturing the human primary epidermal keratinocytes (HPEK) with hASCs on a collagen vitrigel membrane enhanced their abilities for cell proliferation and adhesion to the membrane but suppressed their differentiation suggesting that hASCs could maintain the undifferentiated status of HPEK. Contrarily, the effects of co-culture using polyethylene terephthalate or polycarbonate membranes for HPEK were completely opposite. These differences may depend on the protein permeability and/or structure of the membrane. Taken together, our data demonstrate that hASCs could be used as a substitute for fibroblasts in skin wound repair, aesthetic medicine, or tissue engineering. It is also important to note that a co-culture system using the collagen vitrigel membrane allows better understanding of the interactions between the keratinocytes and ASCs.

Circulatory miR-98-5p levels are deregulated during diabetes and it inhibits proliferation and promotes apoptosis by targeting PPP1R15B in keratinocytes

ABSTRACTAlthough deregulated circulatory miRNA signatures during diabetes have been identified for some years now, the effects of such miRNAs on several target tissues are not yet thoroughly investigated. The skin that is nourished by components present in the circulation exhibits several notable abnormal features during diabetes. We, therefore, hypothesized that such altered circulatory miRNA levels might be critical in the onset and progression of impaired skin health during diabetes. RNA sequencing from blood samples of normal and type 2 diabetic human subjects identified 9 upregulated and 19 downregulated miRNAs. miR-98-5p was significantly downregulated and its overexpression down-regulated PPP1R15B levels in HaCaT cells and this was prevented by the miR-98-5p inhibitor. This was validated in human primary epidermal keratinocytes and further supported by a dual reporter luciferase assay of the PPP1R15B 3ʹUTR where miR-98-5p significantly decreased the luciferase activity which was prevented in the presence of the miRNA inhibitor and by mutation in the miRNA binding site. By targeting PPP1R15B, miR-98-5p increases levels of p-eIF2α, BiP and CHOP. Consequently, there was induction of apoptosis accompanied with decreased proliferation in the presence of miR-98-5p. Conversely, miR-98-5p inhibition alone inhibited apoptosis and promoted proliferation. Taken together, our data suggest that by targeting PPP1R15B, miR-98-5p induces apoptosis and decreases proliferation. As opposed to this since circulatory miR-98-5p levels are decreased in diabetes, we believe that this decrease in the circulation that feeds the skin layers might be a major contributor of hyperproliferation as seen in the skin during diabetes.Abbreviations: miRNAs: MicroRNAs; PPP1R15B: PPP1R15B: Protein Phosphatase 1 Regulatory Subunit 15B; TGFβR1: Transforming Growth Factor Beta Receptor 1; ER: Endoplasmic Reticulum; Bip: Binding Immunoglobulin Protein; Chop: CCAAT-enhancer-binding protein homologous protein; p-eIF2α: Eukaryotic Translation Initiation Factor 2a; Bax: Bcl2-associated X protein; Bcl-2: B-cell CLL/lymphoma 2; PCNA: Proliferating Cell Nuclear Antigen; K5: Cytokeratin 5; qRT-PCR: Quantitative Real-Time PCR; ESCC: Oesophageal squamous cell carcinoma; HCC: Hepatocellular carcinoma; CTHRC1: Collagen triple helix repeat containing 1; SALL4: Sal-like protein 4; TNFα: Tumour Necrosis Factor alpha; PGC-1β: Peroxisome Profilerator-activated receptor-γ coactivator-1β; IGF2BP1: Insulin-like growth factor 2 mRNA binding protein 1.

Wound Healing Potential of Low Temperature Plasma in Human Primary Epidermal Keratinocytes.

Low temperature plasma (LTP) was recently shown to be potentially useful for biomedical applications such as bleeding cessation, cancer treatment, and wound healing, among others. Keratinocytes are a major cell type that migrates directionally into the wound bed, and their proliferation leads to complete wound closure during the cutaneous repair/regeneration process. However, the beneficial effects of LTP on human keratinocytes have not been well studied. Therefore, we investigated migration, growth factor production, and cytokine secretion in primary human keratinocytes after LTP treatment. Primary cultured keratinocytes were obtained from human skin biopsies. Cell viability was measured with the EZ-Cytox cell viability assay, cell migration was evaluated by an in vitro wound healing assay, gene expression was analyzed by quantitative real-time polymerase chain reaction, and protein expression was measured by enzyme-linked immunosorbent assays and western blotting after LTP treatment. Cell migration, the secretion of several cytokines, and gene and protein levels of angiogenic growth factors increased in LTP-treated human keratinocytes without associated cell toxicity. LTP treatment also significantly induced the expression of hypoxia inducible factor-1α (HIF-1α), an upstream regulator of angiogenesis. Further, the inhibition of HIF-1α expression blocked the production of angiogenic growth factors induced by LTP in human keratinocytes. Our results suggest that LTP treatment is an effective approach to modulate wound healing-related molecules in epidermal keratinocytes and might promote angiogenesis, leading to improved wound healing.

556 Specific Staphylococcal species alter the response of primary human epidermal keratinocyte to ultraviolet radiation

556 Specific Staphylococcal species alter the response of primary human epidermal keratinocyte to ultraviolet radiation

An in vitro feasibility investigation considering primary human melanocytes for spray- grafting of freshly isolated autologous skin cells for burn treatment and a clinical case report

A skin cell-spray grafting technique that enables the on-site application of freshly isolated autologous single cell suspensions was already applied in many cases on caucasian patients with low skin coloration. Our project hypothesis is that these suspensions contain keratinocytes and vital melanocytes, that are of particular interest for the treatment of patients of darker skin color. To test this, we applied an in vitro model, wherein the feasibility of i) isolating and ii) spraying of freshly isolated autologous melanocyte-keratinocyte cell suspensions was investigated. Primary human epidermal keratinocytes (HEKs) and melanocytes (MCs) were isolated from skin biopsies (n=8). Biochemical parameter, cell counts, cell morphology, growth behavior and immunofluorescence results were compared in two groups using MC cultures and co-cultures of MCs with HEKs. Case information on using the method clinically with one patient is included. The sprayed mixed cell suspensions proliferated in all groups without measurable loss of viability, and MCs exhibited a regular cell morphology in monoculture up to passage 4°. The sprayed MCs and HEKs demonstrated in vitro glucose and lactate metabolism that was comparable to the pipetted controls. In co-culture, well distributed CK14+ HEKs and NKI/beteb+ MCs could be demonstrated, which interacted in the in vitro model. The ratio of HEKs : MCs in our primary cultures were microscopically counted (n=8 each) as mean +/- SD 1,211,000 (+/- 574,343) HEK : 99,625 (+/- 59,025) MC; i.e., a ratio of approx. 12 : 1. Using the isolation method clinically for a patient with dark skin coloration after suffering severe second-degree burns shows a satisfying re-pigmentation of the resulting wound post healing. Freshly isolated spray-on melanocyte/keratinocyte suspensions provide for a considerable amount of viable HEKs and MCs. Using MCs in spray-grafting suspensions could represent a promising approach for treating severe partial-thickness burns and innovative therapy developments that also aim to address cosmetic aspects.

Rapid treatment of full‐thickness skin loss using ovine tendon collagen typeIscaffold with skin cells

The full-thickness skin wound is a common skin complication affecting millions of people worldwide. Delayed treatment of this condition causes the loss of skin function and integrity that could lead to the development of chronic wounds or even death. This study was aimed to develop a rapid wound treatment modality using ovine tendon collagen type I (OTC-I) bio-scaffold with or without noncultured skin cells. Genipin (GNP) and carbodiimide (EDC) were used to cross-link OTC-I scaffold to improve the mechanical strength of the bio-scaffold. The physicochemical, biomechanical, biodegradation, biocompatibility, and immunogenicity properties of OTC-I scaffolds were investigated. The efficacy of this treatment approach was evaluated in an in vivo skin wound model. The results demonstrated that GNP cross-linked OTC-I scaffold (OTC-I_GNP) had better physicochemical and mechanical properties compared with EDC cross-linked OTC-I scaffold (OTC-I_EDC) and noncross-link OTC-I scaffold (OTC-I_NC). OTC-I_GNP and OTC-I_NC demonstrated no toxic effect on cells as it promoted higher cell attachment and proliferation of both primary human epidermal keratinocytes and human dermal fibroblasts compared with OTC-I_EDC. Both OTC-I_GNP and OTC-I_NC exhibited spontaneous formation of bilayer structure in vitro. Immunogenic evaluation of OTC-I scaffolds, in vitro and in vivo, revealed no sign of immune response. Finally, implantation of OTC-I_NC and OTC-I_GNP scaffolds with noncultured skin cells demonstrated enhanced healing with superior skin maturity and microstructure features, resembling native skin in contrast to other treatment (without noncultured skin cells) and control group. The findings of this study, therefore, suggested that both OTC-I scaffolds with noncultured skin cells could be promising for the rapid treatment of full-thickness skin wound.

Small Molecule and Pooled CRISPR Screens Investigating IL17 Signaling Identify BRD2 as a Novel Contributor to Keratinocyte Inflammatory Responses.

Interleukin-17A (IL17A) plays a critical role in the development of numerous autoimmune diseases, including psoriasis. The clinical success of IL17A neutralizing biologics in psoriasis has underlined its importance as a drug discovery target. While many studies have focused on the differentiation and trafficking of IL17A producing T-helper 17 cells, less is known about IL17A-initiated signaling events in stromal and parenchymal cells leading to psoriatic phenotypes. We sought to discover signaling nodes downstream of IL17A contributing to disease pathogenesis. Using IL17A and tumor necrosis factor α (TNF) to stimulate primary human epidermal keratinocytes, we employed two different phenotypic screening approaches. First, a library of ∼22000 annotated compounds was screened for reduced secretion of the pro-inflammatory chemokine IL8. Second, a library of 729 kinases was screened in a pooled format by utilizing CRISPR-Cas9 and monitoring IL8 intracellular staining. The highest-ranking novel hits identified in both screens were the bromodomain and extra-terminal domain (BET) family proteins and bromodomain-containing protein 2 (BRD2), respectively. Comparison of BRD2, BRD3, and BRD4 silencing with siRNA and CRISPR confirmed that BRD2 was responsible for mediating IL8 production. Pan-BRD inhibitors and BRD2 knockout also reduced IL17A/TNF-mediated CXC motif chemokines 1/2/6 (CXCL1/2/6) and granulocyte colony stimulating factor (G-CSF) production. In RNA-Seq analysis, 438 IL17A/TNF dependent genes were reduced in BRD2-deficient primary keratinocytes. KEGG pathway analysis of these genes showed enrichment in TNF signaling and rheumatoid arthritis relevant genes. Moreover, a number of genes important for keratinocyte homeostasis and cornification were dysregulated in BRD2-deficient keratinocytes. In IL17A/TNF/IL22 stimulated three-dimensional organotypic raft cultures, pan-BRD inhibition reduced inflammatory factor production but elicited aberrant cornification, consistent with RNA-Seq analysis. These studies highlight a novel role for BRDs and BRD2 in particular in IL17A-mediated inflammatory signaling.

Role of TRPV4‐TAZ signaling axis in epithelial‐mesenchymal transition

Epithelial to mesenchymal transition (EMT) is a critical event in development, fibrogenesis, and oncogenesis. Emerging data support a role for both a mechanical signal, and a biochemical signal, e.g., transforming growth factor β1 (TGFβ1) in EMT. Here, we report evidence that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive channel sensitive to both matrix stiffness and TGFβ1 stimuli, is a likely mediator of EMT. We found that antagonism of TRPV4 blocked both matrix stiffness‐ and TGFβ1‐induced EMT in normal human primary epidermal keratinocytes (NHEKs), as determined by changes in morphology and alterations in expression of EMT markers including E‐cadherin, N‐cadherin, and α‐smooth muscle actin (α‐SMA). In a murine skin fibrosis model, TRPV4 deletion resulted in decreased expression of the mesenchymal marker, α‐SMA, and increased expression of epithelial marker, E‐cadherin. Mechanistically, we found that: i) TRPV4 activity was essential for the nuclear translocation of TAZ (transcriptional coactivator with PDZ‐binding motif) in response to matrix stiffness and TGFβ1; ii) Antagonism of TRPV4 activity inhibited both matrix stiffness‐induced and TGFβ1‐induced expression of TAZ proteins; and iii) TRPV4 antagonism suppressed both matrix stiffness‐induced and TGFβ1‐induced activation of Smad2/3, but not of AKT. Altogether, these data identify a novel role for TRPV4 in regulating EMT in response to both matrix stiffness and TGFβ1.Support or Funding InformationAHA (13SDG17310007), Startup grant from University of Maryland, NIH (1R01EB024556‐01), and NSF (CMMI‐1662776) grants to Shaik RahamanThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ginsenoside composition of Panax ginseng flower extracts obtained using different high hydrostatic pressure extraction conditions

Ginsenosides are active constituents of ginseng (Panax ginseng) that have possible anti-aging, physiological and pharmacological activities, such as anti-cancer and anti-inflammatory effects. Although the ginseng root is generally used more often than the aerial parts for medicinal purposes, the flowers also contain numerous ginsenosides, including Rb2, Rc, Rd, Re and Rg1. Therefore, an extract from the flowers of the P. ginseng could have the pharmacological efficacy of bioactive compounds including ginsenosides. The high hydrostatic pressure extraction (HHPE) is a method that is used for the efficient extraction of bioactive compounds from plant materials. In this study, we compared the yield of ginsenosides from ginseng flowers under different conditions of extraction pressure and time of HHPE. The results indicate that the total yield of the ginsenosides improved as the pressure increased from 0.1 to 80 MPa and treatment duration increased to 24 hours. In addition, the ginsenoside extracts from HHPE at 80 MPa, which possessed a higher total ginsenoside concentration, decreased the viability of the primary human epidermal keratinocytes (HEKs) significantly than the ginsenoside extracts from HHPE at 0.1 MPa. Collectively, we found that the method of HHPE that was performed for 24 hours at 80 MPa showed the highest yield of ginsenosides from the flowers of P. ginseng. In addition, our study provides a foundation for the efficient extraction of ginsenosides, which had a potent bioactivity, from flowers of P. ginseng through HHPE.

Apigenin restores impairment of autophagy and downregulation of unfolded protein response regulatory proteins in keratinocytes exposed to ultraviolet B radiation

Ultraviolet (UV)-B radiation is a major environmental risk factor that is responsible for the development and progression of many skin cancers. Apigenin, a type of bioflavonoid, has been reported to inhibit UVB-induced skin cancer. However, how apigenin functions in keratinocytes with UV damage remains unclear. In this study, by lactate dehydrogenase (LDH) release assay, we found that apigenin treatment increased cell death in the primary human epidermal keratinocytes (HEKs) and the cutaneous squamous cell carcinoma cell line COLO-16. Apigenin treatment reduced microtubule-associated protein 1 light chain 3 (LC3)-II turnover, acridine orange staining and GFP-LC3 puncta in both cell types, suggesting autophagy inhibition. However, apigenin treatment restored the inhibition of autophagy in UVB-challenged HEKs. Moreover, apigenin treatment restored the UVB-induced downregulation of ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia, Rad3-related (ATR) and the unfolded protein response (UPR) regulatory proteins, BiP, IRE1α and PERK in HEKs. Apigenin treatment also inhibited UVB-induced apoptosis and cell death in HEKs. In addition, autophagy inhibition by autophagy-related gene (ATG) 5 RNA interference interrupted apigenin-induced restoration of ATR, ATM and BiP, which were downregulated in HEKs exposed to UVB radiation. Our findings indicate that apigenin exhibits a novel protective effect in keratinocytes with UVB damage, suggesting potential application as a photoprotective agent.

Keratinocyte differentiation promotes ER stress-dependent lysosome biogenesis

Keratinocytes maintain epidermal integrity through cellular differentiation. This process enhances intraorganelle digestion in keratinocytes to sustain nutritional and calcium-ionic stresses observed in upper skin layers. However, the molecular mechanisms governing keratinocyte differentiation and concomitant increase in lysosomal function is poorly understood. Here, by using primary neonatal human epidermal keratinocytes, we identified the molecular link between signaling pathways and cellular differentiation/lysosome biogenesis. Incubation of keratinocytes with CaCl2 induces differentiation with increased cell size and early differentiation markers. Further, differentiated keratinocytes display enhanced lysosome biogenesis generated through ATF6-dependent ER stress signaling, but independent of mTOR-MiT/TFE pathway. In contrast, chemical inhibition of mTORC1 accelerates calcium-induced keratinocyte differentiation, suggesting that activation of autophagy promotes the differentiation process. Moreover, differentiation of keratinocytes results in lysosome dispersion and Golgi fragmentation, and the peripheral lysosomes showed colocalization with Golgi-tethering proteins, suggesting that these organelles possibly derived from Golgi. In line, inhibition of Golgi function, but not the depletion of Golgi-tethers or altered lysosomal acidity, abolishes keratinocyte differentiation and lysosome biogenesis. Thus, ER stress regulates lysosome biogenesis and keratinocyte differentiation to maintain epidermal homeostasis.

The Potential Application of Spring Sargassum glaucescens Extracts in the Moisture-Retention of Keratinocytes and Dermal Fibroblast Regeneration after UVA-Irradiation

Sargassum glaucescens is a marine brown alga with high antioxidant activity. To evaluate the potential application of Sargassum glaucescens extracts (SGE) in skincare, we performed in vitro assays in dermal fibroblasts and epidermal keratinocytes. The antioxidant activity of SGE was confirmed by the suppression of H2O2-induced reactive oxygen species (ROS) production in dermal fibroblasts and in vitro 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity. In the wound healing assay, application of 2 mg/ml SGE stimulated the wound closure of CCD-966SK fibroblasts by a 2.95-fold in comparison to the control. Furthermore, treatment with SGE of concentrations ranging from 0.25 to 1 mg/ml promoted CCD-966SK cell regeneration after UVA irradiation. At the molecular level, 1 mg/ml SGE induced expressions of anti-oxidative genes SOD1 (Superoxide dismutase 1) and GPX1 (Glutathione peroxidase 1), and DNA repair regulatory genes XRCC1 (X-ray repair cross-complementing protein 1) and ERCC6 (Excision repair cross-complementation Group 6) in CCD-966SK cells after UVA irradiation. Therefore, SGE displayed beneficial effects on cell regeneration and the protection of dermal cells against UVA irradiation. In epidermal cells, SGE stimulated the cell proliferation of human primary epidermal keratinocytes. Application of 0.03125 mg/ml SGE induced the expressions of skin barrier-related genes TGM1 (Transglutaminase 1), KRT10 (Keratin 10) and KRT14 in keratinocytes. Meanwhile, SGE induced the gene expression of FLG (Filaggrin), which promoted the production of natural moisturizing factor (NMF) for maintaining the moisture and barrier functions of skin.

Understanding the implications of engineered nanoparticle induced autophagy in human epidermal keratinocytes in vitro

Engineered nanoparticles (NPs) such as TiO2 and ZnO are key UV-blocking ingredients in sunscreens. While toxicological risks of applying these materials are generally regarded as low due to minute levels of penetration across the skin, our understanding of the physiological influence of potential cell-nanoparticle interactions in the skin is limited. This study plugs the current knowledge gap by profiling TiO2 and ZnO NP interaction with primary human epidermal keratinocytes, based on potential levels of NP penetration across the skin. Specific attention was given to profiling real-life relevant levels of exposure, and accurate dosimetry measurements in vitro. ZnO was expectedly more cytotoxic than TiO2. Although both NPs generated Reactive Oxygen Species (ROS) and Mitochondrial Superoxide (MSO) within 4 h exposure to sub-lethal concentrations, ZnO induction of these oxidative stress markers increased much more significantly after 24 h exposure. Exposure to increasing NP concentrations increased autophagy induction along with activation of inflammatory responses in the keratinocytes, primarily through the TRAF6-mediated pathway. Sustained induction of autophagy led to degradation of TRAF6 and, only in the case of TiO2, reduced NF-κB activation. This pro-survival mode of autophagy induction provides further insights into the on-going debate on the use of these NPs in consumer products.

Tropisetron via α7 nicotinic acetylcholine receptor suppresses tumor necrosis factor-α-mediated cell responses of human keratinocytes.

Tropisetron is a serotonin receptor (5-HT-R)-modulating agent and approved as an antiemetic for patients undergoing chemotherapy. In the gut, it acts via specific serotonin receptors, 5-HT3 -R, to elicit its beneficial effects against nausea. We investigated whether tropisetron can affect inflammatory cell responses of human primary epidermal keratinocytes (NHK) which are key cells in the regulation of skin homoeostasis. Tropisetron significantly and dose-dependently suppressed tumor necrosis factor (TNF)-α-mediated mRNA expression and protein secretion of interleukin (IL)-6 and IL-8 in these cells. This effect of tropisetron was independent of p65/NF-κB as shown by various NF-κB signal transduction read-outs. Importantly, the anti-inflammatory tropisetron effect on NHK was neither mediated by 5-HT3 -R nor 5-HT4 -R since these receptors were absent in NHK. In contrast, NHK expressed α7 nicotinic acetylcholine receptors (α7nAchR) which previously were found to bind tropisetron. The α7nAchR antagonist α-bungarotoxin neutralized, whereas AR-R17779, a specific α7nAchR agonist, mimicked the suppressive effect of tropisetron on TNF-α-mediated IL-6 and IL-8 expression in NHK. Our findings suggest that tropisetron and probably other α7nAchR-activating agents could be useful for the future therapy of inflammatory skin diseases.

A gelatin/collagen/polycaprolactone scaffold for skin regeneration.

BackgroundA tissue-engineered skin substitute, based on gelatin (“G”), collagen (“C”), and poly(ε-caprolactone) (PCL; “P”), was developed.MethodG/C/P biocomposites were fabricated by impregnation of lyophilized gelatin/collagen (GC) mats with PCL solutions, followed by solvent evaporation. Two different GC:PCL ratios (1:8 and 1:20) were used.ResultsDifferential scanning calorimetry revealed that all G/C/P biocomposites had characteristic melting point of PCL at around 60 °C. Scanning electron microscopy showed that all biocomposites had similar fibrous structures. Good cytocompatibility was present in all G/C/P biocomposites when incubated with primary human epidermal keratinocytes (PHEK), human dermal fibroblasts (PHDF) and human adipose-derived stem cells (ASCs) in vitro. All G/C/P biocomposites exhibited similar cell growth and mechanical characteristics in comparison with C/P biocomposites. G/C/P biocomposites with a lower collagen content showed better cell proliferation than those with a higher collagen content in vitro. Due to reasonable mechanical strength and biocompatibility in vitro, G/C/P with a lower content of collagen and a higher content of PCL (GCLPH) was selected for animal wound healing studies. According to our data, a significant promotion in wound healing and skin regeneration could be observed in GCLPH seeded with adipose-derived stem cells by Gomori’s trichrome staining.ConclusionThis study may provide an effective and low-cost wound dressings to assist skin regeneration for clinical use.

Dynamic Culture Substrates That Mimic the Topography of the Epidermal-Dermal Junction.

The junction between the epidermal and dermal layers of human skin undulates, the width and depth of the undulations varying with age and disease. We previously showed that when primary human epidermal keratinocytes are seeded on collagen-coated undulating static polydimethylsiloxane (PDMS) elastomer substrates, the stem cells, differentiated cells, and proliferating cells become patterned in response to cues from the underlying substrate. To investigate how patterning occurs over time, we have now created a dynamic model, in which a collagen-coated poly(d,l-lactide-co-glycolide) (PLGA) membrane is placed over a polyimide sheet containing circular holes, differing in diameter and spacing. When a vacuum is applied the membrane is induced to undulate, the heights of the undulations depending on the pressure applied and the size of the holes. We observed clustering of cells with high levels of β1 integrin expression, a stem cell marker, in the base of the undulations within 48 h of applying the vacuum. Differentiating involucrin-positive cells did not cluster; however, there was clustering of cells with high E-cadherin expression and nuclear YAP. Rho kinase inhibition resulted in loss of clustering, suggesting a role for Rho family members in the process.Impact StatementIn human skin the junction between the epidermis and dermis undulates. Epidermal stem cells pattern according to their position relative to those undulations. Here we describe a rig in which epidermal cells are cultured on a collagen-coated poly(d,l-lactide-co-glycolide) (PLGA) membrane. When a vacuum is applied the membrane is induced to undulate. Stem cells cluster in response to the vacuum, whereas differentiating cells do not. Rho kinase inhibition results in loss of clustering, suggesting a role for Rho family members in the process. This dynamic platform is a new tool for investigating changes in the skin with age and disease.

Patterning of human epidermal stem cells on undulating elastomer substrates reflects differences in cell stiffness

In human skin the junction between epidermis and dermis undulates, the width and depth of the undulations varying with age and disease. When primary human epidermal keratinocytes are seeded on collagen-coated polydimethylsiloxane (PDMS) elastomer substrates that mimic the epidermal-dermal interface, the stem cells become patterned by 24 h, resembling their organisation in living skin. We found that cell density and nuclear height were higher at the base than the tips of the PDMS features. Cells on the tips not only expressed higher levels of the stem cell marker β1 integrin but also had elevated E-cadherin, Desmoglein 3 and F-actin than cells at the base. In contrast, levels of the transcriptional cofactor MAL were higher at the base. AFM measurements established that the Young’s modulus of cells on the tips was lower than on the base or cells on flat substrates. The differences in cell stiffness were dependent on Rho kinase activity and intercellular adhesion. On flat substrates the Young’s modulus of calcium-dependent intercellular junctions was higher than that of the cell body, again dependent on Rho kinase. Cell patterning was influenced by the angle of the slope on undulating substrates. Our observations are consistent with the concept that epidermal stem cell patterning is dependent on mechanical forces exerted at intercellular junctions in response to undulations in the epidermal-dermal interface. Statement of significanceIn human skin the epidermal-dermal junction undulates and epidermal stem cells are patterned according to their position. We previously created collagen-coated polydimethylsiloxane (PDMS) elastomer substrates that mimic the undulations and provide sufficient topographical information for stem cells to cluster on the tips. Here we show that the stiffness of cells on the tips is lower than cells on the base. The differences in cell stiffness depend on Rho kinase activity and intercellular adhesion. We propose that epidermal stem cell patterning is determined by mechanical forces exerted at intercellular junctions in response to the slope of the undulations.

Mechanistic insight into the activity of a sulfone compound dapsone on Propionibacterium (Newly Reclassified as Cutibacterium) Acnes-mediated cytokine production.

Acne remains as the most common skin disease in the United States even despite multiple approved topical and systemic medications available. These treatments available over the counter and by prescription can be classified based on comedolytic, antibacterial and anti-inflammatory activities and are often used in combination. Therefore, understanding of the mechanism of action is critical to achieving the best clinical outcome and synergy. One of the newer acne medications with historical data suggesting both antibacterial and anti-inflammatory activity is dapsone. In order to gain mechanistic insight into the anti-inflammatory activity of dapsone in Propionibacterium (a former genus name recently reclassified as "Cutibacterium") (P.acnes)-driven inflammation, we used two human in vitro models: primary human neonatal epidermal keratinocytes and human monocytes (THP-1). We demonstrate that dapsone suppresses production of specific cytokine signatures interleukin (IL)1α and IL8 in human epidermal keratinocytes and IL1β, IL6, IL8 and tumor necrosis factor-α in THP-1 cells in response to P.acnes. Using THP-1 cell in vitro model, we show that IL1β and CASP-1 are regulated by dapsone independently of NFκB activity at transcriptional and post-transcriptional levels, respectively.