Artificial Skin Equivalents Research Articles

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.

Tribological behaviour of skin equivalents and ex-vivo human skin against the material components of artificial turf in sliding contact

This research aims to analyse the interaction of three artificial skin equivalents and human skin against the main material components of artificial turf. The tribological performance of Lorica, Silicone Skin L7350 and a recently developed Epidermal Skin Equivalent (ESE) were studied and compared to ex-vivo human skin samples. The surface of the ex-vivo skin was analysed via confocal microscopy and histology. The results indicated a similar performance of the Lorica and ESE samples when compared to the ex-vivo skin whereas the Silicone Skin L7350 showed significant differences. The results suggested also a key role of the intrinsic properties of the NBR material to the friction against human skin, compared to PA 6.6 or PE surfaces in a similar sliding contact.

A novel adamantyl benzylbenzamide derivative, AP736, inhibits melanogenesis in B16F10 mouse melanoma cells via glycogen synthase kinase 3β phosphorylation.

Recently, much effort has been made to develop effective dermatological depigmenting compounds. In this study, we investigated the novel candidate compound, AP736 (an adamantyl benzylbenzamide derivative), and its effects on melanogenesis in B16F10 melanoma cells, as well as the mechanisms involved. AP736 has been reported to exert anti-melanogenic effects in melanocytes in vitro and in artificial skin equivalents through the inhibition of key melanogenic enzymes and the suppression of the cAMP-protein kinase A (PKA)-cAMP response element‑binding protein (CREB) signaling pathway. Thus, we examined another pathway of melanogenesis involving the effects of AP736 on the glycogen synthesis kinase 3β (GSK3β) pathway. Melanin content and tyrosinase activity were measured using a spectrophotometer after the cells were treated with AP736. The AP736-induced activation of signaling pathways was examined by western blot analysis. We confirmed that AP736 decreased melanin production in a dose-dependent manner; however, it did not directly inhibit tyrosinase, the rate-limiting melanogenic enzyme. The expression of microphthalmia-associated transcription factor, tyrosinase, and related signal transduction pathways was also investigated. The Wnt signaling pathway is deeply involved in melanogenesis; therefore, phosphorylation by GSK3β was assessed following treatment with AP736. AP736 induced GSK3β phosphorylation (inactivation), but it did not alter the level of β-catenin. Furthermore, the expression of α-melanocyte-stimulating hormone-induced tyrosinase was downregulated by AP736. Our data suggest that AP736 exerts hypopigmentary effects through the downregulation of tyrosinase via GSK3β phosphorylation.

Non-invasive Quality Control for Production Processes of Artificial Skin Equivalents by Optical Coherence Tomography

The engineering of artificial tissue and organs is widely gaining relevance in biotechnological and biomedical research. Particularly by the law enforced reduction of animal testing, alternative methods need to be developed to simulate and test the effects of chemical substances on human organs. Regarding the branches for pharmaceuticals, chemicals and cosmetics most products need to be tested on penetration of the human skin tissue. As an alternative method to animal testing, skin equivalents (SEs) based on tissue engineering may be used to test the skin barrier function.In the framework of a large scale research project, we built a fully automated production process based on the adaption of manual laboratory protocols to carry out the biological process steps for the manufacturing of SEs. In a first step, keratinocytes are mechanically and enzymatic isolated from human foreskin biopsies. In a second process, the cells are cultivated in MTP format cultivation dishes und undergo a series of passages. In a last process step, the keratinocytes are concentrated and dispensed in 24 well membrane based MTPs. Due to biological fluctuations in cell and tissue growth, the production process faces major challenges to control and process a vast variety in batch duration and timing. This results in a strong impact in the final tissue quality. For quality control in the manual process, histologies of the SEs are prepared followed by staining and microscopic imaging. To overcome this time intensive process, we developed a fully automated optical coherence tomography OCT system. OCT is a non- invasive and non-destructive imaging technique based on the interferometric detection of optical light feedback from the probe tissue in the near infrared light. OCT generates 2D and 3D tomographic images with a micron resolution and a tissue penetration up to 2mm. We developed custom image processing algorithms, to detect the individual layer structure and corresponding layer thicknesses. The OCT results show a high degree of correlation with the histological findings regarding structure and layer thicknesses. Further, we show in a first study a method to use this OCT layer detection technique as a potential substitution measurement to the ET50 test used for analyzing the in vitro skin barrier function.

TNF-like weak inducer of apoptosis (TWEAK) and TNF-α cooperate in the induction of keratinocyte apoptosis

Activation of skin keratinocytes followed by their apoptotic death leads to eczema and spongiosis formations in patients with atopic dermatitis (AD). TNF-like weak inducer of apoptosis (TWEAK) binds to its receptor, fibroblast growth factor-inducible 14 (Fn14), and controls many cellular activities, including proliferation, migration, differentiation, apoptosis, angiogenesis, and inflammation. The aim of the study was to investigate the role of TWEAK and Fn14 in the formation of eczema in patients with AD. Primary keratinocytes were isolated from nonlesional skin from patients with AD and psoriasis and from normal skin of healthy donors. Apoptosis analysis was performed by using annexin V/7-aminoactinomycin D and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. The expression and regulation of TWEAK, TNF-α, Fn14, TNF receptor (TNFR) 1, and TNFR2 were measured by means of RT-PCR, flow cytometric analysis, and ELISA. TWEAK and Fn14 expression of lesional AD and psoriatic skin and normal control skin was analyzed by using immunohistochemistry and immunofluorescence. TWEAK and TNF-α cooperate in the induction of apoptosis in primary keratinocytes obtained from patients with AD, patients with psoriasis, and healthy subjects and in artificial skin equivalents. TNFR1 and Fn14 were the main receptors involved. TWEAK upregulates TNF-α expression in primary keratinocytes, whereas TNF-α did not affect the expression of TWEAK and its receptors. High TWEAK expression was observed in AD lesions but not in psoriatic lesions or normal skin. Fn14 was highly expressed in the lesional skin of patients with AD and patients with psoriasis and in healthy control skin. The high expression of TWEAK in lesional AD skin contributes to the difference in keratinocyte apoptosis and lesional formation between AD and psoriasis.

IL-32 is expressed by human primary keratinocytes and modulates keratinocyte apoptosis in atopic dermatitis

Keratinocyte (KC) apoptosis is an important mechanism of eczema and spongiosis in patients with atopic dermatitis (AD) and is mediated by IFN-gamma, which is secreted by T(H)1 cells. IL-32 is a proinflammatory cytokine that is involved in the inflammatory processes of rheumatoid arthritis, chronic obstructive pulmonary disease, and Crohn disease. Recently, it was shown that upregulation of IL-32 induces apoptosis. The aim of the study was to investigate the expression and function of IL-32 in patients with AD. The expression of IL-32 in KCs was analyzed by means of RT-PCR, ELISA, and flow cytometry. Transfections of small interfering RNA were performed in primary KCs, and apoptosis was analyzed by means of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, annexin-V, and 7-amino actinomycin D stainings. Immunofluorescence stainings were used to detect IL-32 in skin biopsy specimens, and serum levels of IL-32 were analyzed by means of ELISA. We report that IL-32 is expressed in human primary KCs on stimulation with IFN-gamma, TNF-alpha, and T(H)1 cells in contrast to T(H)2, regulatory T (Treg), or T(H)17 cells, which showed no effect. Transfection of primary KCs and artificial skin equivalents with small interfering RNA to IL-32, which resulted in a clear decrease in IL-32 expression, significantly reduced KC apoptosis. Immunofluorescence staining demonstrated that IL-32 was expressed in AD lesional skin, whereas it was present in neither skin biopsy specimens from healthy donors nor in lesional skin from patients with psoriasis. Serum levels of IL-32 from patients with AD correlated with disease severity, but increased serum levels of IL-32 were also detected in asthmatic patients. The present study demonstrates KCs as a source of IL-32, which modulates KC apoptosis and contributes to the pathophysiology of AD.

Use of various models for in vitro percutaneous absorption studies of ultraviolet filters

The percutaneous absorption test aims to estimate the passage of a substance across the skin. The absorption process can be described in three steps: (a) penetration of a substance into the skin layer, followed by (b) penetration from one layer into another (permeation) and finally (c) resorption into the vascular system. In vivo and in vitro models are available but owing to ethical reasons as well as the latter providing greater feasibility, in vitro models are preferred. This present study reviews the natural membranes (human skin and animal models: pig, rabbit, rat, hairless mouse, guinea-pig and mouse), artificial skin equivalents and synthetic membranes that are currently being used for in vitro percutaneous absorption studies of UV filters, in order to provide the researcher with a greater insight when selecting membrane models for given experimental conditions.

Differential expression of p63 isoforms in normal skin and hyperproliferative conditions

The p63 is regarded as a potential stem cell marker. Expression of p63 isoforms was examined in normal skin and hyperproliferative conditions including psoriasis and artificial skin equivalents (SEs). Rapidly adhering (RA) and slowly adhering (SA) cells were isolated, and Western blotting was performed. Expression of p63 (4A4) and p63 (H-137) is similar in all conditions, although there is some variation in psoriasis. However, expression of p63alpha (C-12) is markedly different. In normal skin, p63alpha (C-12)-positive cells were scattered in whole epidermis. But in psoriasis, p63alpha (C-12)-positive cells were observed at the tips of rete ridges. In SEs, p63alpha (C-12)-positive cells were not well observed. Western blot results showed that the RA cells express p63 (4A4) and p63 (H-137) strongly compared with SA or nonadhering (NA) cells. In contrast, SA or NA cells strongly express p63alpha (C-12). Results suggest that both p63 (4A4) and p63 (H-137) can detect epidermal stem cells. But, p63 (H-137) seemed to be a better marker because p63 (H-137)-positive cells were more localized at basal layer. In addition, it can be said that p63alpha (C-12) can detect TAp63, which is important in differentiation of epidermis. Furthermore, it is concluded that molecular control of TAp63 is especially disorganized in hyperproliferative condition including psoriasis and SEs.

Beyond Antimicrobials

Pressure ulcers are a major source of morbidity and mortality for older adults, especially frail elders who may be malnourished and have significant comorbid conditions. Successful treatment of pressure ulcers requires coordinated efforts in wound care, pressure relief, and optimization of medical conditions that impact healing, such as diabetes mellitus and congestive heart failure. The role of newer therapies such as growth factors, artificial skin equivalents, and vacuum-assisted closure is growing. Infectious disease physicians should develop a working knowledge of these treatment modalities and the potential advantages and disadvantages of each.

Absence of T-regulatory cell expression and function in atopic dermatitis skin

The role of regulatory T cells has been widely reported in the suppression of T-cell activation. A dysfunction in CD4(+)CD25(+) T-regulatory cell-specific transcription factor FoxP3 leads to immune dysregulation, polyendocrinopathy, enteropathy X-linked syndrome, often associated with atopic dermatitis. Increasing the number and activity of regulatory T cells in affected organs has been suggested as a remedy in various inflammatory diseases, including allergy. To determine the presence and function of regulatory T cells in atopic dermatitis. Immunohistochemistry of lesional atopic dermatitis skin and control skin conditions was used to demonstrate regulatory cells and cytokines in situ. The role of effector and regulatory T cells as well as their specific cytokines in apoptosis in human keratinocyte cultures and artificial skin equivalents was investigated. Human T-regulatory type 1 cells, their suppressive cytokines, IL-10 and TGF-beta, as well as receptors for these cytokines were significantly expressed, whereas CD4(+)CD25(+)FoxP3(+) T-regulatory cells were not found in lesional and atopy patch test atopic dermatitis or psoriasis skin. Both subsets of regulatory T cells suppress the allergen-specific activation of T(H)1 and T(H)2 cells. In coculture and artificial skin equivalent experiments, subsets of T-regulatory cells neither induced keratinocyte death nor suppressed apoptosis induced by skin T cells, T(H)1 cells, IFN-gamma, or TNF-alpha. A dysregulation of disease-causing effector T cells is observed in atopic dermatitis lesions, in association with an impaired CD4(+)CD25(+)FoxP3(+) T-cell infiltration, despite the expression of type 1 regulatory cells in the dermis.

Proteolytic Processing of the Laminin α3 G Domain Mediates Assembly of Hemidesmosomes but Has No Role on Keratinocyte Migration

Laminin-5 (Lm5), the major adhesion ligand of basal epithelial cells, undergoes complex extracellular proteolytic processing that influences cell adhesion and migration. In tumor cell lines, the proteolytic truncation of the C-terminal G domain of the Lm alpha3 chain induces assembly of hemidesmosomes and downregulates cell migration. To define the biological functions of the alpha3 G domain processing in physiological conditions, we have expressed a series of mutant alpha3 complementary DNA in human primary alpha3-null keratinocytes immortalized by human papillomavirus E6E7 (HKalpha3 cells). Using monolayer and organotypic cell cultures we show that: (1) the hinge region between subdomains G3 and G4 carries the proteolytic cleavage sites; (2) nucleation of the hemidesmosomal proteins is independent of the proteolytic maturation of the alpha3 G domain, whereas formation of mature hemidesmosomes relies on proteolytic cleavage of alpha3; and (3) the proteolytic processing plays no role in cell migration, which suggests that nucleation of hemidesmosomal structures in culture does not reflect the migratory potential of the epithelial cells. Our results also demonstrate that HKalpha3 cells are a unique model system, which will be useful to dissect the functions and molecular interactions of Lm5.

Harnessing wound healing and regeneration for tissue engineering

Biomedical science has made major advances in understanding how cells grow into functioning tissue and the signalling mechanisms used to achieve this are slowly being dissected. Tissue engineering is the application of that knowledge to the building or repairing of organs, including skin, the largest organ in the body. Generally, engineered tissue is a combination of living cells and a supporting matrix. Besides serving as burn coverings, engineered skin substitutes can help patients with diabetic foot ulcers. Today, most of these ulcers are treated with an approach that includes antibiotics, glucose control, special shoes and frequent cleaning and bandaging. The results of such treatments are often disappointing and ineffectual, and scarring remains a major problem, mechanically, cosmetically and psychologically. Within our group we are attempting to address this by investigating novel approaches to skin tissue engineering. We are identifying novel therapeutic manipulations to improve the degree of integration between a tissue engineered dermal construct and the host by both molecular manipulation of growth factors but also by understanding and harnessing mechanisms of regenerative biology. For the purpose of this summary, we will concentrate primarily on the latter of these two approaches in that we have identified a novel mouse mutant that completely and perfectly regenerates skin and cartilaginous components following ear injury. This experimental animal will allow us to characterize not only novel genes involved in the regeneration process but also to utilize cells from such animals in artificial skin equivalents to assess their behaviour compared with normal cells. This approach should allow us to create a tissue-engineered substitute, which more closely resembles the normal regional microanatomy and physiology of the skin, allowing better integration to the host with minimal or no scarring.

Novel and recurrent mutations in the laminin-5 genes causing lethal junctional epidermolysis bullosa: molecular basis and clinical course of Herlitz disease

Herlitz disease (H-JEB), the lethal form of junctional epidermolysis bullosa, is a rare genodermatosis presenting from birth with widespread erosions and blistering of skin and mucosae because of tissue cleavage within the epidermal basement membrane. Mutations in any of the three genes encoding the alpha3, beta3 and gamma2 chains of laminin-5 underlie this recessively inherited disorder. Here, we report the molecular basis and clinical course of H-JEB in 12 patients. Two novel nonsense mutations in the gene LAMA3 (E281X and K1299X) and a novel frame-shift mutation in the gene LAMB3 (1628insG) leading to a premature termination codon were identified by DNA sequencing and confirmed by restriction fragment length polymorphism analysis. In the four patients affected, neither the resulting truncated polypeptide chains nor assembled laminin-5 protein were detectable by immunofluorescence. Three patients were found to be heterozygous for the known hotspot mutation R635X and the recurrent mutations Q373X or 29insC in the gene LAMB3, whereas five others were homozygous for R635X. Significant variations in the disease progression and survival times between 1 and 30 months in this group of H-JEB patients emphasised the impact of modifying factors and the importance of immunostaining or mRNA assessment as parallel diagnostic methods. Interestingly, the only patients who survived for longer than 6 months were four females carrying the mutation R635X homozygously. In one of them, the clinical course may have been improved by treatment with artificial skin equivalents. These data may stimulate further investigation of genotype-phenotype correlations and facilitate mutation analysis and genetic counselling of affected families.

Nicotine-Enhanced Epithelial Differentiation in Reconstructed Human Oral Mucosa in vitro

Oral mucosal keratinocytes represent the cells that first encounter tobacco components. Therefore, tobacco-induced abnormal alteration of the mucosal keratinocytes may contribute to the development of oral white lesions. Nicotine is an ingredient of all tobacco products and pharmacologically the most active component of tobacco smoke. To clarify the effects of nicotine on the keratinization of oral mucosal and epidermal keratinocytes, we reconstructed artificial buccal mucosal and skin equivalents using keratinocytes and fibroblasts from noncornifying buccal mucosa and adult foreskin, respectively. The effect of nicotine on keratinization was assessed with morphology, immunohistochemistry and immunoblotting. Long-term treatment with nicotine for 2 weeks enhanced in a dose-dependent manner the expression of differentiation-specific proteins of oral mucosal keratinocytes on living oral mucosal equivalent and epidermal keratinocytes on living skin equivalent, respectively. The effect of nicotine on the cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Oral mucosal keratinocytes showed a higher resistance to nicotine toxicity than epidermal keratinocytes. Our results suggest that nicotine stimulates differentiation of both mucosal and epidermal keratinocytes, and this nicotine-induced abnormal differentiation may be associated with the development of oral white lesions.