Skin Equivalent Model Research Articles

Sandwich-structure transferable free-form OLEDs for wearable and disposable skin wound photomedicine

Free-form optoelectronic devices can provide hyper-connectivity over space and time. However, most conformable optoelectronic devices can only be fabricated on flat polymeric materials using low-temperature processes, limiting their application and forms. This paper presents free-form optoelectronic devices that are not dependent on the shape or material. For medical applications, the transferable OLED (10 μm) is formed in a sandwich structure with an ultra-thin transferable barrier (4.8 μm). The results showed that the fabricated sandwich-structure transferable OLED (STOLED) exhibit the same high-efficiency performance on cylindrical-shaped materials and on materials such as textile and paper. Because the neutral axis is freely adjustable using the sandwich structure, the textile-based OLED achieved both folding reliability and washing reliability, as well as a long operating life (>150 h). When keratinocytes were irradiated with red STOLED light, cell proliferation and cell migration increased by 26 and 32%, respectively. In the skin equivalent model, the epidermis thickness was increased by 39%; additionally, in organ culture, not only was the skin area increased by 14%, but also, re-epithelialization was highly induced. Based on the results, the STOLED is expected to be applicable in various wearable and disposable photomedical devices.

Side-effect test of sorafenib using 3-D skin equivalent based on microfluidic skin-on-a-chip

Sorafenib is a neovascular blocker that prevents the formation of new blood vessels, and by consequence, the growth of cancer tissue, through multiple kinase inhibitors that target angiogenesis. However, several side effects have been reported by patients on sorafenib. Therefore, this study investigates the skin side effects of sorafenib as revealed by hematoxylin and eosin (H&E) staining, real-time polymerase chain reaction (RT-PCR), and immunohistochemical staining results using a human skin equivalent model produced from human fibroblast and keratinocyte. Thickening of the granules and the protein expressed through the changes on the granular layer, similar to the tissues of an actual patient, was observed. It is expected that the findings of this study will greatly improve the preclinical evaluations and promote the use of the skin-on-a-chip system for evaluating the side effects of drugs and the efficacy of cosmetics.

IDR-1018 induces cell proliferation, migration, and reparative gene expression in 2D culture and 3D human skin equivalents.

Skin lesions are associated with functional/cosmetic problems for those afflicted. Scarless regeneration is a challenge, not limited to the skin, and focus of active investigation. Recently, the host defense peptide innate defense regulatory peptide 1018 (IDR-1018) has shown exciting regenerative properties. Nevertheless, literature regarding IDR-1018 regenerative potential is scarce and limited to animal models. Here, we evaluated the regenerative potential of IDR-1018 using human 2D and 3D human skin equivalents. First, we investigated IDR-1018 using human cells found in skin-primary fibroblasts, primary keratinocytes, and the MeWo melanocytes cell line. IDR-1018 promoted cell proliferation and expression of marker of proliferation Ki-67, matrix metalloproteinase 1, and hyaluronan synthase 2 by fibroblasts. In keratinocytes, a drastic increase in expression was observed for Ki-67, matrix metalloproteinase 1, C-X-C motif chemokine receptor type 4, C-X-C motif chemokine receptor type 7, fibroblast growth factor 2, hyaluronan synthase 2, vascular endothelial growth factor, and elastin, reflecting an intense stimulation of these cells. In melanocytes, increased migration and proliferation were observed following IDR-1018 treatment. The capacity of IDR-1018 to promote dermal contraction was verified using a dermal model. Finally, using a 3D human skin equivalent lesion model, we revealed that the regenerative potential of IDR1018, previously tested in mice and pigs, is valid for human skin tissue. Lesions closed faster in IDR-1018-treated samples, and the gene expression signature observed in 2D was reproduced in the 3D human skin equivalents. Overall, the present data show the regenerative potential of IDR-1018 in an experimental system comprising human cells, underscoring the potential application for clinical investigation.

Decreased expression of suprabasin induces aberrant differentiation and apoptosis of epidermal keratinocytes: Possible role for atopic dermatitis

BackgroundSuprabasin (SBSN), a secreted protein, is expressed in various epithelial tissues. The role of SBSN in epidermal differentiation and atopic dermatitis (AD) pathology remains largely unknown. ObjectiveTo evaluate the effects of SBSN on epidermal keratinocytes and its role in AD. MethodsWe examined the SBSN expression levels in the stratum corneum and the epidermis by proteome analysis and immunohistochemistry, respectively. The serum SBSN concentration was measured by ELISA. These values were compared between AD and healthy control. Morphological changes in the epidermis were investigated in SBSN-knockdown three-dimensional human living skin equivalent (LSE) model with or without IL-4/IL-13. ResultsEpidermal SBSN expression was decreased in AD lesional skin compared to healthy skin, as assessed by the stratum corneum proteome analysis and immunohistochemistry. The SBSN serum levels were significantly lower in AD patients than in normal subjects (P<0.05). The SBSN-deficient LSE exhibited compact stratum corneum, immature stratum granulosum, and increased keratinocyte apoptosis. Th2 cytokines, IL-4 and IL-13, did not affect SBSN expression in LSE. There were no differentiation-associated makers that were affected by the SBSN knockdown. SBSN deficiency-induced apoptosis of keratinocytes was exaggerated by IL-4/IL-13, and accordingly, the addition of recombinant SBSN induced significant keratinocyte proliferation (P<0.05). ConclusionOur data demonstrated that SBSN regulates normal epidermal barrier. Th2 cytokines unaffect SBSN expression in keratinocytes, but promote SBSN deficiency-induced apoptosis. It is suggested that SBSN has an anti-apoptotic activity, and its deficiency is involved in the pathogenesis of AD.

Attenuation of negative effects of senescence in human skin using an extract from Sphingomonas hydrophobicum: development of new skin care solution.

Intrinsic skin ageing is mainly caused by cellular senescence. p16 and p21 are two important tumour suppressor proteins that play essential roles during cell proliferation and ageing through regulating the expression of several genes. Moreover, physical changes between the ages of 55 and 60years affect one's physical and disrupt self-esteem. The cosmetics industry has focused on bioactive substances derived from natural products such as plants, mushrooms and marine algae to counteract the deleterious effect on skin senescence. Besides these products, compounds produced by bacteria may decelerate individual senescence. In order to evaluate the potential benefits of bacteria extract over skin ageing, we investigated whether a Sphingomonas hydrophobicum (SH) extract is able to protect our skin against senescence mechanisms. We used an ageing full-thickness skin equivalent model, which was treated or not with the bacteria extract in a systemic way for 42days. p21 and p16 and senescence-associated galactosidase activity were used to detect cellular senescence with immunohistology. Using a psychobiological approach, we evaluated in vivo the effect of SH extract on self-esteem, isotropy and suppleness. Sphingomonas extract significantly suppressed senescence associated with β-galactosidase activation. It also significantly inhibited the expression of cell cycle inhibitors (p21 and p16). At the same time, the bacteria extract has a significant positive impact on the issue by increasing the expression of versican and fibrillin-1. Significant improvements of self-esteem were reported after 56days of SH extract application. These psychological benefits were accompanied by a significant improvement in skin suppleness and isotropy. Sphingomonas extract delays intrinsic skin ageing process by inhibiting cellular senescence, and has also the capability to restructure the skin. These beneficial physiological effects induced by SH extract have a positive influence on self-esteem. Because skin ageing causes emotional distress, SH extract can serve as an anti-ageing cosmeceutical agent and help to build a better psychological health, and help individuals to assume ageing.

Improving skin integration around long-term percutaneous devices using fibrous scaffolds in a reconstructed human skin equivalent model.

The interface between synthetic percutaneous devices and skin is a common area for bacterial infection, which may ultimately result in failure of the device. Better integration of percutaneous devices with skin may help reduce infection rates due to the creation of a dermal seal. However, the mismatch in material and chemical properties of devices and skin presents a challenge for closing the dermal gap at the skin-device interface. Here, we have used a tissue engineering approach to tissue integration by creating a highly fibrous poly(ε-caprolactone) scaffold using melt electrowriting and seeding this with dermal fibroblasts, followed by maturation and insertion into a full-thickness defect made in an ex vivo skin model. The integration of seeded scaffolds was compared with controls including a non-seeded scaffold and a polymer tube with a smooth surface. Dermal fibroblast inclusion in the scaffold and epidermal upgrowth versus downgrowth/marsupialization around the device were used as measures of integration. Based on these measures, almost all pre-seeded scaffolds performed better than both the non-seeded scaffolds and smooth tubes. The hypothesis is that the fibroblasts act as a barrier to epithelial downward migration, and provide healthy tissue for nascent epidermal development.

286 SerpinB12 is an intrinsic mesotrypsin inhibitor regulating corneocyte desquamation and denucleation

It is suggested that kallikrein-related peptidases (KLKs) are key players in corneocyte desquamation and this process is regulated by lympho-epithelial Kazal-type inhibitor (LEKTI). However, it is unclear how these proteases are activated and how activated KLKs are released from LEKTI in the upper cornified layer. Previously we reported cloning of a new PRSS3 gene product, keratinocyte-specific mesotrypsin from the cDNA library. It has been reported that mesotrypsin is insusceptible to intrinsic inhibitors. Moreover, we found that mesotrypsin effectively activated pro-KLK5 and pro-KLK7. Although LEKTI strongly inhibited KLKs, mesotrypsin was not suppressed by LEKTI but rather degraded them. Surprisingly, we isolated a novel inhibitor and identified it as serpinB12 (Another name is YUKOPIN). Thin skin, such as the eyelid, showed strong mesotrypsin staining, while serpinB12 staining was barely detectable. In contrast, footpad skin, which has a thick cornified layer, showed strong serpinB12 staining at the granular layer. We constructed skin equivalent models, in which serpinB12 was over-expressed or knocked-down. Cornified layers were increased and the epidermis became thicker in serpinB12 over-expression. When serpinB12 was down-regulated, the epidermal structure became very thin. Furthermore, we found that excessive over-expression of serpinB12 induced parakeratosis. Immunohistochemical study showed that serpinB12 was expressed in parakeratic area of diseased skin, such as psoriasis and atopic dermatitis. Our findings provided a new insight into the desquamation mechanisms, in which mesotrypsin and serpinB12 play critical roles. Moreover, our results suggest that serpinB12 would be involved in controlling the thickness of stratum corneum through mesotrypsin regulation, and excessive serpinB12 expression might be the cause of parakeratotic disease condition.

STAT3 signalling pathway is implicated in keloid pathogenesis by preliminary transcriptome and open chromatin analyses.

Keloids are wounding-induced fibroproliferative human tumor-like skin scars of complex genetic makeup and poorly defined pathogenesis. To reveal dynamic epigenetic and transcriptome changes of keloid fibroblasts, we performed RNA-seq and ATAC-seq analysis on an early passage keloid fibroblast cell strain and its paired normal control fibroblasts. This keloid strain produced keloid-like scars in a plasma clot-based skin equivalent humanized keloid animal model. RNA-seq analysis reveals gene ontology terms including hepatic fibrosis, Wnt-β-catenin, TGF-β, regulation of epithelial-mesenchymal transition (EMT), STAT3 and adherens junction. ATAC-seq analysis suggests STAT3 signalling is the most significantly enriched gene ontology term in keloid fibroblasts, followed by Wnt signalling (Wnt5) and regulation of the EMT pathway. Immunohistochemistry confirms that STAT3 (Tyr705 phospho-STAT3) is activated and β-catenin is up-regulated in the dermis of keloid clinical specimens and keloid skin equivalent implants from the humanized mouse model. A non-linear dose-response of cucurbitacin I, a selective JAK2/STAT3 inhibitor, in collagen type I expression of keloid-derived plasma clot-based skin equivalents implicates a likely role of STAT3 signalling in keloid pathogenesis. This work also demonstrates the utility of the recently established humanized keloid mouse model in exploring the mechanism of keloid formation.

Reduced humidity induces skin barrier dysfunction and secretion of dipeptidyl peptidase-4 (DPP-4) in a skin-equivalent model

Seasonal changes can affect the physiological condition of the skin and cause various cutaneous disorders. The skin barrier function tends to worsen during winter when humidity is lower compared to other seasons. To determine the influence of relative humidity (RH) on the function of the skin barrier, we performed biological and histological assays using skin equivalents that were cultured under reduced humidity in an environmental humidity chamber. We found that reduced humidity led to decreased epidermal thickness and disruption of the skin barrier. Reduced humidity induced the decrease of filaggrin, loricrin and damage to tight junction. In addition, dipeptidyl peptidase-4 (DPP4), which has roles in the immunological process, was upregulated in a skin-equivalent model under reduced humidity. These results suggest that reduced humidity affects the skin barrier function and regulates the secretion of DPP4 in a skin-equivalent model.

The Activation of PPAR-α and Wnt/β-catenin by Luffa cylindrica Supercritical Carbon Dioxide Extract

Luffa cylindrica (LC) is a very fast-growing climber and its fruit have been considered as agricultural wastes. We conducted to check the comparative qualities of ethanol solvent extraction (LCE) and supercritical carbon dioxide extraction (LCS) of L. cylindrica fruit and seed. LCS had higher antioxidant and polyphenol contents than LCE. LCS were significantly increased peroxisome proliferator-activated receptor (PPAR)-a and involucrin expression as epidermal differentiation marker in 3D skin equivalent model. LCS also showed antimicrobial activity against Staphylococcus aureus, a causative bacteria in atopic dermatitis. In addition, LCS inhibited the adipocyte differentiation of 3T3-L1 cells. When treated with the extract at a concentration of 100 μg/mL, the Wnt/β-catenin pathway reporter luciferase activity of HEK 293-TOP cells was increased approximately by 2-folds compared to that of the untreated control group. These results indicate that L. cylindrica supercritical carbon dioxide extract may serve as a cosmeceutical for improving skin barrier function and the treatment of obesity.

The Inhibition of Melanogenesis Via the PKA and ERK Signaling Pathways by Chlamydomonas reinhardtii Extract in B16F10 Melanoma Cells and Artificial Human Skin Equivalents.

Abnormal melanin synthesis results in several hyperpigmentary disorders such as freckles, melanoderma, age spots, and other related conditions. In this study, we investigated the antimelanogenic effects of an extract from the microalgae Chlamydomonas reinhardtii (CE) and potential mechanisms responsible for its inhibitory effect in B16F10, normal human epidermal melanocyte cells, and human skin-equivalent models. The CE extract showed significant dose-dependent inhibitory effects on α-melanocyte-stimulating, hormone-induced melanin synthesis in cells. Additionally, the CE extract exhibited suppressive effects on the mRNA and protein expression of microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2. The CE extract also inhibited the phosphorylation of protein kinase A and extracellular signal-related kinase, which function as upstream regulators of melanogenesis. Using a three-dimensional, reconstructed pigmented epidermis model, the CE-mediated, anti-pigmentation effects were confirmed by Fontana-Masson staining and melanin content assays. Taken together, CE extract can be used as an anti-pigmentation agent.

Ameliorating effect of dipotassium glycyrrhizinate on an IL-4- and IL-13-induced atopic dermatitis-like skin-equivalent model.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that is not fully understood. Defects in skin barrier function and dysregulation of the Th2 immune response are thought to be pivotal in AD pathogenesis. In this study, we used keratinocytes and AD-like skin equivalent models using Th2 cytokines IL-4 and IL-13. The keratinocytes and AD-like skin model were used to investigate the effect of dipotassium glycyrrhizinate (KG), which is widely used as an anti-inflammatory agent for AD treatment. KG decreased AD-related gene expression in keratinocytes stimulated with Th2 cytokines. KG alleviated AD-like phenotypes and gene expression patterns and inhibited release of AD-related cytokines in the AD-like skin equivalent models. These findings indicate KG has potential effectiveness in AD treatment and AD-like skin equivalent models may be useful for understanding AD pathogenesis.

The effect of calcium gluconate with natural extracts on skin toxicity of hydrofluoric acid

Hydrofluoric acid (HF) is a highly corrosive acid. The conventional treatment for HF burn is topical application of calcium gluconate (CG). Our study aimed to assess the synergistic effects of natural extracts (Centella asiatica, Portulaca oleracea, and blueberry extracts) in combination with CG in healing HF induced burn wound. We investigated the effects of different topical CG formulations with natural extracts using cell proliferation assay, western blot, reverse transcription- polymerase chain reaction, human skin equivalent model (HSEM), and mouse model. Topical CG formulations with natural extracts showed better recovery from HF burns shown by greater collagen expression in the dermal fibroblast model, HSEM and mouse model. Our study demonstrated that CG in combination with natural extracts show superior wound healing in treating HF burn compared to CG monotherapy.

LB1611 Development of a 3D full-thickness skin equivalent model containing sebocyte organoids derived from human iPS cells

LB1611 Development of a 3D full-thickness skin equivalent model containing sebocyte organoids derived from human iPS cells

LB1547 Anti-aging effect of EGF-DOTAP complex on human skin

Epidermal growth factor (EGF) is known to stimulate skin cell growth and synthesis of extracellular matrix such as hyaluronan(HA) which is decreased with aging in skin. However, EGF is hard to penetrate the stratum corneum and permeate into skin due to its size and hydrophilicity. To improve the skin delivery of EGF, we have used multi-lamellar complexes consist of EGF and 1,2-dioleoyl-3-tirmethylammonium-propane (DOTAP). EGF-DOTAP complex showed higher permeation rate in human skin and synthesis of hyaluronan in human skin equivalent model than normal liposome. A randomized, double-blind, vehicle-controlled, split-face study was designed to evaluate the anti-aging effects of a cream containing EGF-DOTAP complex on human skin. Twenty–one Korean women completed a 12-weeks study and were applied twice daily to the face. As compared with the vehicle control, EGF-DOTAP cream significantly improved the facial photo-damage score, skin roughness (R1), maximum roughness (R2), average roughness (R3, Ra), arithmetic roughness average (R5), and dermal density at 12 weeks. In conclusion, this study suggests that topical application of EGF-DOTAP complex exhibited significant improvement on skin aging by enhancing the permeation of EGF.

Heterogenity of dermal fibroblasts: skin equivalent models that aim to recreate the cutaneous microenvironment.

Heterogenity of dermal fibroblasts: skin equivalent models that aim to recreate the cutaneous microenvironment.

Quantitative Investigation of Terbinafine Hydrochloride Absorption into a Living Skin Equivalent Model by MALDI-MSI.

The combination of microspotting of analytical and internal standards, matrix sublimation, and recently developed software for quantitative mass spectrometry imaging has been used to develop a high-resolution method for the determination of terbinafine hydrochloride in the epidermal region of a full thickness living skin equivalent model. A quantitative assessment of the effect of the addition of the penetration enhancer (dimethyl isosorbide (DMI)) to the delivery vehicle has also been performed, and data have been compared to those obtained from LC-MS/MS measurements of homogenates of isolated epidermal tissue. At 10% DMI, the levels of signal detected for the drug in the epidermis were 0.20 ± 0.072 mg/g tissue for QMSI and 0.28 ± 0.040 mg/g tissue for LC-MS/MS at 50% DMI 0.69 ± 0.23 mg/g tissue for QMSI and 0.66 ± 0.057 mg/g tissue for LC-MS/MS. Comparison of means and standard deviations indicates no significant difference between the values obtained by the two methods.

Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa

Herlitz junctional epidermolysis bullosa (H-JEB) is an incurable, devastating, and mostly fatal inherited skin disease for which there is only supportive care. H-JEB is caused by loss-of-function mutations in LAMA3, LAMB3, or LAMC2, leading to complete loss of laminin 332, the major component of anchoring filaments, which mediate epidermal-dermal adherence. LAMB3 (laminin β3) mutations account for 80% of patients with H-JEB, and ∼95% of H-JEB-associated LAMB3 mutations are nonsense mutations leading to premature termination codons (PTCs). In this study, we evaluated the ability of gentamicin to induce PTC readthrough in H-JEB laminin β3-null keratinocytes transfected with expression vectors encoding eight different LAMB3 nonsense mutations. We found that gentamicin induced PTC readthrough in all eight nonsense mutations tested. We next used lentiviral vectors to generate stably transduced H-JEB cells with the R635X and C290X nonsense mutations. Incubation of these cell lines with various concentrations of gentamicin resulted in the synthesis and secretion of full-length laminin β3 in a dose-dependent and sustained manner. Importantly, the gentamicin-induced laminin β3 led to the restoration of laminin 332 assembly, secretion, and deposition within the dermal/epidermal junction, as well as proper polarization of α6β4 integrin in basal keratinocytes, as assessed by immunoblot analysis, immunofluorescent microscopy, and an in vitro 3D skin equivalent model. Finally, newly restored laminin 332 corrected the abnormal cellular phenotype of H-JEB cells by reversing abnormal cell morphology, poor growth potential, poor cell-substratum adhesion, and hypermotility. Therefore, gentamicin may offer a therapy for H-JEB and other inherited skin diseases caused by PTC mutations.

Basic Fibroblast Growth Factor Influences Epidermal Homeostasis of Living Skin Equivalents through Affecting Fibroblast Phenotypes and Functions

Aims: To elucidate the possible mechanisms of how basic fibroblast growth factor (bFGF) influences epidermal homeostasis in a living skin equivalent (LSE) model. Methods: Several wound healing-related growth factors were analyzed at protein and mRNA levels for dermal fibroblasts of induced alpha-smooth muscle actin (α-SMA)-positive or α-SMA-negative phenotypes. During culturing an LSE model by seeding normal human keratinocytes on a fibroblast-populated type I collagen gel, bFGF or neutralizing antibody for keratinocyte growth factor (KGF) was added to investigate its effects on fibroblast phenotypes and, subsequently, epidermal homeostasis by histology and immunohistochemistry. Results: The α-SMA-positive phenotype of fibroblasts induced by transforming growth factor beta-1 (TGF-β₁) markedly suppressed the expression of KGF and hepatocyte growth factor (HGF), and slightly upregulated vascular endothelial growth factor (VEGF) and TGF-β₁ at mRNA and protein levels, compared with α-SMA-negative fibroblasts treated with bFGF. α-SMA expression of fibroblasts at the epidermal-mesenchymal junction of the LSEs was suppressed by the addition of bFGF, and a better-differentiated epidermis was presented. The abrogation of KGF from fibroblasts by the addition of the KGF neutralizing antibody disenabled the LSE culturing system to develop an epidermis. Conclusions: bFGF, through affecting the phenotypes and functions of fibroblasts, especially KGF expression, influenced epidermal homeostasis in an LSE model.

1116 The extract of Ganoderma lucidum inhibits MMP-1 expression through suppression of ERK activation in UVB irradiated dermal fibroblast and skin equivalent model

1116 The extract of Ganoderma lucidum inhibits MMP-1 expression through suppression of ERK activation in UVB irradiated dermal fibroblast and skin equivalent model