Full-thickness Skin Model Research Articles

Direct biological effects of fractional ultrapulsed CO2 laser irradiation on keratinocytes and fibroblasts in human organotypic full-thickness 3D skin models.

Molecular effects of various ablative and non-ablative laser treatments on human skin cells-especially primary effects on epidermal keratinocytes and dermal fibroblasts-are not yet fully understood. We present the first study addressing molecular effects of fractional non-sequential ultrapulsed CO2 laser treatment using a 3D skin model that allows standardized investigations of time-dependent molecular changes ex vivo. While histological examination was performed to assess morphological changes, we utilized gene expression profiling using microarray and qRT-PCR analyses to identify molecular effects of laser treatment. Irradiated models exhibited dose-dependent morphological changes resulting in an almost complete recovery of the epidermis 5days after irradiation. On day 5 after laser injury with a laser fluence of 100mJ/cm2, gene array analysis identified an upregulation of genes associated with tissue remodeling and wound healing (e.g., COL12A1 and FGF7), genes that are involved in the immune response (e.g., CXCL12 and CCL8) as well as members of the heat shock protein family (e.g., HSPB3). On the other hand, we detected a downregulation of matrix metalloproteinases (e.g., MMP3), differentiation markers (e.g., LOR and S100A7), and the pro-inflammatory cytokine IL1α.Overall, our findings substantiate the understanding of time-dependent molecular changes after CO2 laser treatment. The utilized 3D skin model system proved to be a reliable, accurate, and reproducible tool to explore the effects of various laser settings both on skin morphology and gene expression during wound healing.

Non-thermal near-infrared exposure photobiomodulates cellular responses to ionizing radiation in human full thickness skin models

Ionizing and near-infrared radiation are both part of the therapeutic spectrum in cancer treatment. During cancer therapy ionizing radiation is typically used for non-invasive reduction of malignant tissue, while near-infrared photobiomodulation is utilized in palliative medical approaches, e.g. for pain reduction or impairment of wound healing. Furthermore, near-infrared is part of the solar wavelength spectrum. A combined exposure of these two irradiation qualities – either intentionally during medical treatment or unintentionally due to solar exposure – is therefore presumable for cancer patients. Several studies in different model organisms and cell cultures show a strong impact of near-infrared pretreatment on ionizing radiation-induced stress response. To investigate the risks of non-thermal near-infrared (NIR) pretreatment in patients, a human in vitro full thickness skin models (FTSM) was evaluated for radiation research. FTSM were pretreated with therapy-relevant doses of NIR followed by X-radiation, and then examined for DNA-double-strand break (DSB) repair, cell proliferation and apoptosis. Double-treated FTSM revealed a clear influence of NIR on X-radiation-induced stress responses in cells in their typical tissue environment. Furthermore, over a 24h time period, double-treated FTSM presented a significant persistence of DSBs, as compared to samples exclusively irradiated by X-rays. In addition, NIR pretreatment inhibited apoptosis induction of integrated fibroblasts, and counteracted the radiation-induced proliferation inhibition of basal keratinocytes. Our work suggests that cancer patients treated with X-rays should be prevented from uncontrolled NIR irradiation. On the other hand, controlled double-treatment could provide an alternative therapy approach, exposing the patient to less radiation.

Cross-linked Collagen Hydrogel Matrix Resisting Contraction To Facilitate Full-Thickness Skin Equivalents.

Full-thickness skin equivalents are gathering increased interest as skin grafts for the treatment of large skin defects or chronic wounds or as nonanimal test platforms. However, their fibroblast-mediated contraction and poor mechanical stability lead to disadvantages toward their reproducibility and applicability in vitro and in vivo. To overcome these pitfalls, we aimed to chemically cross-link the dermal layer of a full-thickness skin model composed of a collagen type I hydrogel. Using a noncytotoxic four-arm succinimidyl glutarate polyethylene glycol (PEG-SG), cross-linking could be achieved in cell seeded collagen hydrogels. A concentration of 0.5 mg of PEG-SG/mg of collagen led to a viability comparable to non-cross-linked collagen hydrogels and no increased release of intracellular lactate dehydrogenase. Cross-linked collagen hydrogels were more mechanically stable and less prone to enzymatic degradation via collagenase when compared with non-cross-linked collagen hydrogels. Remarkably, during 21 days, cross-linked collagen hydrogels maintain their initial surface area, whereas standard dermal models contracted up to 50%. Finally, full-thickness skin equivalents were generated by seeding human epidermal keratinocytes on the surface of the equivalents and culturing these equivalents at an air-liquid interface. Immunohistochemical stainings of the cross-linked model revealed well-defined epidermal layers including an intact stratum corneum and a dermal part with homogeneously distributed human dermal fibroblasts. These results indicate that cross-linking of collagen with PEG-SG reduces contraction of collagen hydrogels and thus increases the applicability of these models as an additional tool for efficacy and safety assessment or a new generation of skin grafts.

Construction of Three-Dimensional Dermo-Epidermal Skin Equivalents Using Cell Coating Technology and Their Utilization as Alternative Skin for Permeation Studies and Skin Irritation Tests.

In vitro generated human skin equivalents are generating interest as promising tools in basic study, as alternatives to animal testing, and for clinical applications in regenerative medicine. For prediction of skin irritation and corrosion, three-dimensional human skin equivalents consisting of differentiated human keratinocytes (KCs) have been developed and some models have been internationally accepted. However, more delicate assessments using full-thickness skin models, such as skin sensitization tests, cannot be performed due to the lack of a dermis containing fibroblasts or appendages. In a previous study, we developed dermo-epidermal human skin equivalents (DESEs) using a cell coating technique, which employs cell surface coating by layer-by-layer assembled extracellular matrix (ECM) films. The DESEs with dermis consisting of normal human dermal fibroblasts (NHDFs) and epidermis consisting of human KCs were easily fabricated by using this technology. In this study, the constructed DESEs were evaluated as an alternative skin for skin permeation and irritation tests. A good relationship of permeability coefficient of chemicals was observed between the DESEs and human skin data. We investigated whether the DESEs, a new in vitro skin model, are capable of identifying skin irritant and nonirritant substances among 20 reference chemicals. It was confirmed that the DESEs are applicable to skin irritation testing as defined in the European Centre for the Validation of Alternative Methods (ECVAM) Performance Standard (OECD Test Guideline 439). We further studied the construction of DESEs with density-controlled blood capillary networks using human umbilical vein endothelial cells (HUVECs). The results suggest that DESEs allowing incorporation of skin appendages are more promising alternatives to animal testing and can be applied to the design of physiologically relevant in vitro skin models.

Raman microspectroscopy as a diagnostic tool for the non-invasive analysis of fibrillin-1 deficiency in the skin and in the in vitro skin models.

Mutations in building blocks of the fibrillin microfibril/ elastic fiber network manifest in disease conditions such as aneurysms, emphysema or lax skin. Understanding how structural changes induced by fibrillin-1 mutation impact the architecture of fibrillin microfibrils, which then translates into an altered activation state of targeted growth factors, represents a huge challenge in elucidating the genotype-phenotype correlations in connective tissue disorders such as Marfan syndrome. This study shows that Raman microspectroscopy is able to reveal structural changes in fibrillin-1 microfibrils and elastic fiber networks and to discriminate between normal and diseased networks in vivo and in vitro. Therefore Raman microspectroscopy may be utilized as a non-invasive and sensitive diagnostic tool to identify connective tissue disorders and monitor their disease progression.

Hyaluronan Hybrid Cooperative Complexes as a Novel Frontier for Cellular Bioprocesses Re-Activation.

Hyaluronic Acid (HA)-based dermal formulations have rapidly gained a large consensus in aesthetic medicine and dermatology. HA, highly expressed in the Extracellular Matrix (ECM), acts as an activator of biological cascades, stimulating cell migration and proliferation, and operating as a regulator of the skin immune surveillance, through specific interactions with its receptors. HA may be used in topical formulations, as dermal inducer, for wound healing. Moreover, intradermal HA formulations (injectable HA) provide an attractive tool to counteract skin aging (e.g., facial wrinkles, dryness, and loss of elasticity) and restore normal dermal functions, through simple and minimally invasive procedures. Biological activity of a commercially available hyaluronic acid, Profhilo®, based on NAHYCO™ technology, was compared to H-HA or L-HA alone. The formation of hybrid cooperative complexes was confirmed by the sudden drop in η0 values in the rheological measurements. Besides, hybrid cooperative complexes proved stable to hyaluronidase (BTH) digestion. Using in vitro assays, based on keratinocytes, fibroblasts cells and on the Phenion® Full Thickness Skin Model 3D, hybrid cooperative complexes were compared to H-HA, widely used in biorevitalization procedures, and to L-HA, recently proposed as the most active fraction modulating the inflammatory response. Quantitative real-time PCR analyses were accomplished for the transcript quantification of collagens and elastin. Finally immunofluorescence staining permitted to evaluate the complete biosynthesis of all the molecules investigated. An increase in the expression levels of type I and type III collagen in fibroblasts and type IV and VII collagen in keratinocytes were found with the hybrid cooperative complexes, compared to untreated cells (CTR) and to the H-HA and L-HA treatments. The increase in elastin expression found in both cellular model and in the Phenion® Full Thickness Skin Model 3D also at longer time (up to 7 days), supports the clinically observed improvement of skin elasticity. The biomarkers analyzed suggest an increase of tissue remodeling in the presence of Profhilo®, probably due to the long lasting release and the concurrent action of the two HA components.

Interactions of donor sources and media influence the histo-morphological quality of full-thickness skin models.

Human artificial skin models are increasingly employed as non-animal test platforms for research and medical purposes. However, the overall histopathological quality of such models may vary significantly. Therefore, the effects of manufacturing protocols and donor sources on the quality of skin models built-up from fibroblasts and keratinocytes derived from juvenile foreskins is studied. Histo-morphological parameters such as epidermal thickness, number of epidermal cell layers, dermal thickness, dermo-epidermal adhesion and absence of cellular nuclei in the corneal layer are obtained and scored accordingly. In total, 144 full-thickness skin models derived from 16 different donors, built-up in triplicates using three different culture conditions were successfully generated. In univariate analysis both media and donor age affected the quality of skin models significantly. Both parameters remained statistically significant in multivariate analyses. Performing general linear model analyses we could show that individual medium-donor-interactions influence the quality. These observations suggest that the optimal choice of media may differ from donor to donor and coincides with findings where significant inter-individual variations of growth rates in keratinocytes and fibroblasts have been described. Thus, the consideration of individual medium-donor-interactions may improve the overall quality of human organ models thereby forming a reproducible test platform for sophisticated clinical research.

553 Complex skin models and impedance spectroscopy as new tools for dermatological research

The aim of this study was to improve current tests based on in vitro skin models both regarding a higher in vivo correlation and analytical methods. Despite advances in the development of in-vitro-tissue-models such as reconstructed human epidermis (RHE), the questions in dermatological research, which can be addressed with these models is limited. This is due to a lack of key cellular components and a restricted live time of the models. In addition, the analysis of the models is still dependent on invasive methods such as histological processing or MTT staining. To overcome these pitfalls, we achieved advanced culture systems and biomaterials which allow long term culture of complex tissue-equivalents. Using these technologies, we have developed the first full thickness skin-model with a perfused vascular network. Furthermore, as an alternative for destructive methods, we have established a non-destructive technology to analyze the integrity of the epidermal barrier based on impedance spectroscopy. RHE typically exhibits characteristic impedance spectra in a frequency ranging between 1 Hz and 100 kHz, which is comparable to the spectra of freshly isolated human epidermal biopsies. From these spectra, we extracted electrical parameters of the RHE such as the capacitance and the ohmic resistance. These parameters change significantly during epidermal differentiation and were used to quantify the effects of mechanical and chemical disruption of the epidermal integrity. Most relevant, impedance spectroscopy shows a sufficient sensitivity to detect a transient decreased ohmic resistance caused by 2-propanol, which is classified as a non-irritant by MTT assays. This result indicates that impedance spectroscopy can be employed as an additional method to assess mild irritative effects. In our work we could create new technologies for the generation and analysis of tissue-models which is a vital requirement to value of in-vitro-test-models for dermatological research.

Biophysical and biological characterization of a new line of hyaluronan-based dermal fillers: A scientific rationale to specific clinical indications

Chemico-physical and biological characterization of hyaluronan-based dermal fillers is of key importance to differentiate between numerous available products and to optimize their use. These studies on fillers are nowadays perceived as a reliable approach to predict their performance in vivo. The object of this paper is a recent line of hyaluronic acid (HA)-based dermal fillers, Aliaxin®, available in different formulations that claim a complete facial restoration. The aim of the study is to provide biophysical and biological data that may support the clinical indications and allow to predict performance possibly with respect to similar available products. Aliaxin® formulations were tested for their content in soluble HA, water uptake capacity, rheological behavior, stability to enzymatic degradation, and for in vitro capacity to stimulate extracellular matrix components production. The formulations were found to contain a low amount of soluble HA and were equivalent to each other regarding insoluble hydrogel concentration. The different crosslinking degree declared by the producer was consistent with the trend in water uptake capacity, rigidity, viscosity. No significant differences in stability to enzymatic hydrolysis were found. In vitro experiments, using a full thickness skin model, showed an increase in collagen production in the dermoepidermal junction.Results support the claims of different clinical indications, the classification of products regarding hydro-, lift-action and the specifically suggested needle gauge for the delivery. The biological outcomes also support products effectiveness in skin structure restoration. These data predicted a better performance regarding hydro-action, tissue integration, clinical management during delivery, and a high durability of the aesthetic effect when compared to data on marketed similar products.

345 Cell culture media that are less proliferative and more homeostatic improve differentiation, responsiveness and co-culture of keratinocytes, fibroblasts and melanocytes in 2D and 3D culture

Combinations of highly proliferative cell culture additives such as serum (FCS), pituitary extract (BPE), insulin and EGF/FGF-family members are routinely used to generate rapid in vitro proliferation of a variety of skin cell types. Highly concentrated proliferative signals such as these are ideal for amplifying cells, however they are poorly suited to many other situations where very different cell behaviours are required. We have investigated a range of less proliferative, more homeostatic media for their suitability when inducing differentiation and co-culturing keratinocytes, fibroblasts and melanocytes. Differentiation was improved in all three cell types, with 2x better barrier (3D keratinocytes), 2.5x more melanin (2D melanocytes), and 5x more ECM (2D fibroblasts). Homeostatic media were also found to increase responsiveness of melanocytes and fibroblasts to external stimuli such as tyrosinase inhibitors and vitamin C treatment respectively. Variants of standard proliferation media were also found to deliver balanced 2D co-culture of keratinocytes and fibroblasts, with each cell type delivering similar proliferation rates when seeded at equal density on standard tissue culture plastic. Finally, a fibroblast medium tailored for ECM production was also found to be ideally suited to the fast and simple establishment of full-thickness skin models with fully natural dermal matrix, thereby avoiding the handling challenges and variability associated with traditional collagen scaffolds. In conclusion, less proliferative, more homeostatic culture media were found to deliver a range of improved cell behaviours that complement the single rapid-growth behaviour created by standard proliferation media.

Data integration reveals key homeostatic mechanisms following low dose radiation exposure

The goal of this study was to define pathways regulated by low dose radiation to understand how biological systems respond to subtle perturbations in their environment and prioritize pathways for human health assessment. Using an in vitro 3-D human full thickness skin model, we have examined the temporal response of dermal and epidermal layers to 10cGy X-ray using transcriptomic, proteomic, phosphoproteomic and metabolomic platforms. Bioinformatics analysis of each dataset independently revealed potential signaling mechanisms affected by low dose radiation, and integrating data shed additional insight into the mechanisms regulating low dose responses in human tissue. We examined direct interactions among datasets (top down approach) and defined several hubs as significant regulators, including transcription factors (YY1, MYC and CREB1), kinases (CDK2, PLK1) and a protease (MMP2). These data indicate a shift in response across time — with an increase in DNA repair, tissue remodeling and repression of cell proliferation acutely (24–72h). Pathway-based integration (bottom up approach) identified common molecular and pathway responses to low dose radiation, including oxidative stress, nitric oxide signaling and transcriptional regulation through the SP1 factor that would not have been identified by the individual data sets. Significant regulation of key downstream metabolites of nitrative stress was measured within these pathways. Among the features identified in our study, the regulation of MMP2 and SP1 was experimentally validated. Our results demonstrate the advantage of data integration to broadly define the pathways and networks that represent the mechanisms by which complex biological systems respond to perturbation.

IL-17 Induces an Expanded Range of Downstream Genes in Reconstituted Human Epidermis Model

BackgroundIL-17 is the defining cytokine of the Th17, Tc17, and γδ T cell populations that plays a critical role in mediating inflammation and autoimmunity. Psoriasis vulgaris is an inflammatory skin disease mediated by Th1 and Th17 cytokines with relevant contributions of IFN-γ, TNF-α, and IL-17. Despite the pivotal role IL-17 plays in psoriasis, and in contrast to the other key mediators involved in the psoriasis cytokine cascade that are capable of inducing broad effects on keratinocytes, IL-17 was demonstrated to regulate the expression of a limited number of genes in monolayer keratinocytes cultured in vitro.Methodology/Principal FindingsGiven the clinical efficacy of anti-IL-17 agents is associated with an impressive reduction in a large set of inflammatory genes, we sought a full-thickness skin model that more closely resemble in vivo epidermal architecture. Using a reconstructed human epidermis (RHE), IL-17 was able to upregulate 419 gene probes and downregulate 216 gene probes. As possible explanation for the increased gene induction in the RHE model is that C/CAAT-enhancer-binding proteins (C/EBP) -β, the transcription factor regulating IL-17-responsive genes, is expressed preferentially in differentiated keratinocytes.Conclusions/SignificanceThe genes identified in IL-17-treated RHE are likely relevant to the IL-17 effects in psoriasis, since ixekizumab (anti-IL-17A agent) strongly suppressed the “RHE” genes in psoriasis patients treated in vivo with this IL-17 antagonist.

Human embryonic epidermis contains a diverse Langerhans cell precursor pool

Despite intense efforts, the exact phenotype of the epidermal Langerhans cell (LC) precursors during human ontogeny has not been determined yet. These elusive precursors are believed to migrate into the embryonic skin and to express primitive surface markers, including CD36, but not typical LC markers such as CD1a, CD1c and CD207. The aim of this study was to further characterize the phenotype of LC precursors in human embryonic epidermis and to compare it with that of LCs in healthy adult skin. We found that epidermal leukocytes in first trimester human skin are negative for CD34 and heterogeneous with regard to the expression of CD1c, CD14 and CD36, thus contrasting the phenotypic uniformity of epidermal LCs in adult skin. These data indicate that LC precursors colonize the developing epidermis in an undifferentiated state, where they acquire the definitive LC marker profile with time. Using a human three-dimensional full-thickness skin model to mimic in vivo LC development, we found that FACS-sorted, CD207(-) cord blood-derived haematopoietic precursor cells resembling foetal LC precursors but not CD14(+)CD16(-) blood monocytes integrate into skin equivalents, and without additional exogenous cytokines give rise to cells that morphologically and phenotypically resemble LCs. Overall, it appears that CD14(-) haematopoietic precursors possess a much higher differentiation potential than CD14(+) precursor cells.

Supramolecular elastomer based on polydimethylsiloxanes (SESi) film: synthesis, characterization, biocompatibility, and its application in the context of wound dressing

Supramolecular elastomer based on polydimethylsiloxanes (SESi) is a kind of novel elastomer cross-linked by the multihydrogen bonds supplied by the functional groups linked to the end of the PDMS chains, such as amide, imidazolidone, pending urea (1,1-dialkyl urea), and bridging urea (1,3-dialkyl urea). SESi showed lower glass transition temperature (T g) at about −113 °C because of the softer chain of PDMS, and could show real rubber-like elastic behaviors and acceptable water vapor transmission rate under room temperature. The high biocompatibility of SESi in the form of films was demonstrated by the cytotoxicity evaluation (MTT cytotoxicity assay and direct contact assay), hemolysis assay, and skin irritation evaluation. Based on detailed comparisons between commercial Tegaderm™ film and SESi film using a full-thickness rat skin model experiment, it was found that SESi film showed similar wound contraction rate as that of Tegaderm™ film on day seven, 10, and 14; only on day five, SESi film showed a significant (p < 0.05) lower wound contraction rate. And, the wounds covered with SESi film were filled with new epithelium without any significant adverse reactions, similar with that of Tegaderm™ film.

Human Skin Model Containing Melanocytes: Essential Role of Keratinocyte Growth Factor for Constitutive Pigmentation—Functional Response to α-Melanocyte Stimulating Hormone and Forskolin

To study human skin pigmentation in a physiological in vitro model, we developed a pigmented reconstructed skin reproducing the three-dimensional architecture of the melanocyte environment and the interactions of melanocyte with its cellular partners, keratinocytes, and fibroblasts. Co-seeding melanocytes and keratinocytes onto a fibroblast-populated collagen matrix led to a correct integration of melanocytes within the epidermal basal layer, but melanocytes remained amelanotic even after supplementation with promelanogenic factors. Interestingly, normalization of keratinocyte differentiation using keratinocyte growth factor instead of epidermal growth factor finally allowed an active pigmentary system to develop, as shown by the expression of key melanogenic markers, the production, and transfer of melanosome-containing melanin into keratinocytes. Various degrees of constitutive pigmentation were reproduced using melanocytes from different skin phenotypes. Furthermore, induction of pigmentation was achieved by treatment with known propigmenting molecules, αMSH and forskolin, thus demonstrating the functionality of the pigmentary system. This pigmented full-thickness skin model therefore represents a highly relevant tool to study the role of cell-cell, cell-matrix, and mesenchymal-epithelial interactions in the control of skin pigmentation.

Development of a short-term human full-thickness skin organ culture model in vitro under serum-free conditions

Skin exerts a variety of important functions to maintain its integrity and viability. It can be used as an experimental ex vivo model to study wound healing, oxidative stress, skin aging, carcinogenesis as well as topical or "systemic" therapeutic intervention in vitro. This report aims to validate a serum-free human full-thickness skin organ culture model with regard to dependency of skin viability on culture duration and location of skin cross-section (1-5) from the outer (section 1) to the inner side (section 5) of a skin specimen (5 × 10 mm). Cultured skin was analyzed in time-dependent manner for structural damage (H&E staining) and 'balance' between proliferation (Ki67) and apoptosis [cleavage of caspase-3, lactate dehydrogenase (LDH), TUNEL]. First structural disturbances were observed at 48 h (section 3; middle part), increasing with prolonged culture time. Cleavage of caspase-3 and appearance of apoptotic [TUNEL(+)] cells showed significant increase at 72 h in sections 4 and 5, respectively. This correlated to increasing LDH release. Parallel analysis of proliferating [Ki67(+)] cells revealed simultaneous down-regulation within the first 48 h reaching complete absence of Ki67(+) cells at 72 h. These data define an accurate, standardized and robust serum-free short-term ex vivo human full-thickness skin model which is suitable for experimental studies of up to 48 or 72 h in vitro. This model therefore might be used for research related to, e.g., short-term experimentally induced inflammation, UV-induced structural and functional damage, wound healing and substance penetration.

Test systems for the determination of glucocorticoid receptor ligand induced skin atrophy

Topical glucocorticoids are highly anti-inflammatory effective but limited by their side effect potential, with skin atrophy being the most prominent one. Thus, determining the atrophogenic potential of novel compounds targeting the glucocorticoid receptor is important. Significant progress in the understanding of glucocorticoid receptor mediated molecular action has been made providing the basis for novel glucocorticoid receptor ligands with a potentially superior effect/side effect profile. Such compounds, however, need to be tested. The present gold standard for the reliable prediction of glucocorticoid induced skin atrophy are still in vivo models, however, in vitro models may replace them to some extent in the future. Indeed, advances in technologies to determine the atrophogenic potential of compounds in vitro has been made recently and promising novel test models like the human full thickness skin models are emerging. Their full predictive value, however, needs to be further evaluated. Currently, a screening approach starting with a combination of several in vitro test systems followed by subsequent testing of the most promising compounds in rodent models is recommended prior entering clinical studies with selected development compounds.

Test systems for the determination of glucocorticoid receptor ligand induced skin atrophy.

Topical glucocorticoids are highly anti-inflammatory effective but limited by their side effect potential, with skin atrophy being the most prominent one. Thus, determining the atrophogenic potential of novel compounds targeting the glucocorticoid receptor is important. Significant progress in the understanding of glucocorticoid receptor mediated molecular action has been made providing the basis for novel glucocorticoid receptor ligands with a potentially superior effect/side effect profile. Such compounds, however, need to be tested. The present gold standard for the reliable prediction of glucocorticoid induced skin atrophy are still in vivo models, however, in vitro models may replace them to some extent in the future. Indeed, advances in technologies to determine the atrophogenic potential of compounds in vitro has been made recently and promising novel test models like the human full thickness skin models are emerging. Their full predictive value, however, needs to be further evaluated. Currently, a screening approach starting with a combination of several in vitro test systems followed by subsequent testing of the most promising compounds in rodent models is recommended prior entering clinical studies with selected development compounds.

Confocal microscopy for modeling electron microbeam irradiation of skin

For radiation exposures employing targeted sources such as particle microbeams, the deposition of energy and dose will depend on the spatial heterogeneity of the sample. Although cell structural variations are relatively minor for two-dimensional cell cultures, they can vary significantly for fully differentiated tissues. Employing high-resolution confocal microscopy, we have determined the spatial distribution, size, and shape of epidermal keratinocyte nuclei for the full-thickness EpiDerm™ skin model (MatTek, Ashland, VA). Application of these data to calculate the microdosimetry and microdistribution of energy deposition by an electron microbeam is discussed.

Characterization of enzyme activities of Cytochrome P450 enzymes, Flavin-dependent monooxygenases, N-acetyltransferases and UDP-glucuronyltransferases in human reconstructed epidermis and full-thickness skin models

With the perspective to use human reconstructed skin models for genotoxicity testing which require metabolic activation of xenobiotics, this study aimed to characterize activities of biotransforming enzymes within two human reconstructed skin models, the epidermis model EpiDerm™ (MatTek) and the Phenion® Full-Thickness skin model Phenion®FT (Henkel). According to existing gene expression profiles, Cytochrome P450 (CYP) enzymes, Flavin-dependent monooxygenases (FMO), N-acetyltransferases (NAT) and UDP-glucuronyltransferases (UDP-GT) were investigated in S9 or microsomal fractions. CYP-catalyzed monooxygenation was assayed using 7-ethoxyresorufin, pentoxyresorufin and benzyloxyresorufin as substrates. FMO activity was tested using benzydamine. Conjugating activities of NAT and UDP-GT were determined by acetylation of p-aminobenzoic acid or glucuronation of 4-methylumbelliferone, respectively. Although CYPs were detected by expression profiling, no CYP activity was detected in either the epidermal nor the full-thickness reconstructed skin model while expression and activity of FMO, UDP-GT and NAT were demonstrated in both.