Charles University Faculty of Pharmacy in Hradec Králové Department of pharmeceutical technology Candidate: Oksana Yanok Supervisor: Pharm.Dr. Andrej Kováčik, Ph.D. Title of Diploma Thesis: The effects of glucosyl sphingosine on barrier function of skin and complex skin model. The skin barrier, which provides protection from water loss and harmful environmental influences is located in the stratum corneum. The dominant group of lipids within the stratum corneum are ceramides (Cer), which also have the most important role in ensuring the barrier properties of the skin. The enzymes sphingomyelin deacylase and glucosylceramide deacylase hydrolyze the amide bond of Cer precursors, which leads to highly polar metabolites, called lysolipids. The increased activity of these enzymes is considered to be one of the major factors leading to the development of a number of skin diseases characterized by a skin barrier disorder (for example atopic dermatitis). In this study we prepared model membranes mimicking a healthy skin barrier as an equimolar mixtures of human Cer, cholesterol, free fatty acids with the addition of 5 % cholesterol sulfate. We also prepared models in which the amount of Cer was gradually reduced and replaced by the hydrophilic lysolipid glucosylsphingosine. The permeability was measured...

La géométrie d'une pièce fabriquée réelle diffère de la pièce virtuelle de CAO (Conception Assistée par Ordinateur. Cette différence est due à la somme des écarts inhérents à la fabrication. L'objectif de ce travail est d’introduire des pièces virtuelles ayant des défauts de forme (Skin Model Shape) dans les applications d'ingénierie afin de répondre aux exigences croissantes de l'industrie en matière de gestion de la qualité de la géométrie des produits. Les travaux traitent de divers aspects, particulièrement de la génération de défauts de forme, de la simulation d'assemblage et de la métrologie virtuelle.Les méthodes permettant de générer des défauts de forme sur des surfaces simples sont analysés et classées. En raison des défauts de forme, la combinaison de surfaces simples pour générer une pièce entière induit une incohérence géométrique au niveau des arêtes. Une méthode globale basée sur les éléments finis et une méthode locale basée sur le lissage local de maillage sont utilisées pour résoudre ce problème.Pour prédire l'écart des caractéristiques fonctionnelles, la simulation d'assemblage est effectuée en utilisant des surfaces avec défauts de forme. Une approche est développée sur la base de la condition de complémentarité linéaire et du torseur de petits déplacements pour prendre en compte les conditions aux limites de l'assemblage, telles que les déplacements et les charges.Des méthodes pour évaluer les écarts sur les modèles de surfaces avec défauts de forme sont également étudiées. Les spécifications sur le produit sont exprimées avec GeoSpelling et évaluées à l'aide du torseur de petits déplacements. Les méthodes développées sont intégrées dans un laboratoire virtuel pour l'apprentissage en ligne.Les études susmentionnées complètent et étendent les méthodes de gestion des tolérances basées sur GeoSpelling et le « skin » modèle. / The geometry of a real manufactured part differs from the virtual workpieces designed in Computer Aided Design (CAD) systems. This difference is due to the accumulation of unavoidable manufacturing deviations. The objective of this work is to implement virtual workpieces with form defects (Skin Model Shape) in engineering applications to meet the industry’s increasing demands in product geometry quality management. Various aspects are covered here, in particular form defect generation, assembly simulation and virtual metrology.Methods to generate form defects on simple surfaces are reviewed and classified. Due to form defects, the combination of simple surfaces to generate a whole part led to inconsistency on the edges. A global FEA-based method and a local mesh smoothing based method are used to overcome this issue.To predict the deviation of functional characteristics, assembly simulation is conducted using skin model shapes. An approach is developed based on the Linear Complementarity Condition and the Small Displacement Torsor to take into account assembly boundary conditions, such as displacements and loads.Methods to evaluate deviation values on skin model shapes are also studied. Product specifications are expressed with GeoSpelling, and evaluated using the Small Displacement Torsor method. The developed methods are integrated into an online Virtual Laboratory for e-learning.The above-mentioned studies complement and extend the tolerance management methods based on GeoSpelling and skin models.

Wound infection is a major clinical burden that can significantly impede the healing process and cause severe pain. Prolonged wound infection can lead to long-term hospitalization or death. Pre-clinical research to evaluate new drugs or treatment strategies relies on animal studies. However, animal studies have several challenges including interspecies variations, cost, and, ethics question the success of these models. Recent advances in tissue engineering have enabled the development of in vitro human skin models for wound infection modeling and drug testing. The existing skin models are mostly representative of the healthy human skin and its normal functions. However, to study the wound healing process and the response of skin to the infection, there is still a need to develop a skin model mimicking the wound infection. This work presents a simplified functional infected epidermis model, fabricated with enzymatically crosslinked gelatin hydrogel. The immortalized human keratinocytes, HaCaT cells, was successfully cultured and differentiated to a multilayer epidermis structure at the air-liquid interface, and expressed terminal differentiation marker, filaggrin, in the outer layer. The barrier function of the epidermis model was studied by measuring the electrical resistance and tissue permeability across the layer. The results showed that the developed epidermis model offered a higher electrical resistance and a lower drug permeability compared to the cell monolayer on gelatin and cell-free gelatin. To show the capability of the developed epidermis model in wound modeling and drug, the model was infected with Escherichia coli and the inflammatory response of keratinocytes was studied by measuring the level of proinflammatory cytokines, including IL-1β and TNF-α. The results demonstrated the proinflammatory response of the epidermis model to infection by producing a higher level of TNF-α and IL-1β compared to the control group. While treating with antibiotic ciprofloxacin terminated the proinflammatory response and reduced the level of TNF-α and IL-1β. The robust fabrication procedure and functionality of this model suggest that this model has great potential for wound modeling and high throughput drug testing. / Graduate

The management and the control of product geometrical variations during the whole development process is an important issue for cost reduction, quality improvement and company competitiveness in the global manufacturing era. During the design phase, geometric functional requirements and tolerances are derived from the design intent. Geometric modeling tools, now largely support the modeling of product shapes and dimensions. However, permissible geometrical variations cannot be intuitively assessed using existing modeling tools. In addition, the manufacturing and measurement stages are two main geometrical variations generators according to the two well know axioms of manufacturing imprecision and measurement uncertainty. A comprehensive view of Geometrical Product Specifications should consider not only the complete tolerancing process, tolerance modeling and tolerance representation but also shape geometric representations, and suitable processing techniques and algorithms. GeoSpelling as the basis of the GPS standard enables a comprehensive modeling framework and an unambiguous language to describe geometrical variations covering the overall product life cycle thanks to a set of concepts and operations based on the fundamental concept of the "Skin Model". However, the "operationalization" of GeoSpelling has not been successfully completed and few research studies have focused on the skin model simulation. The skin model as a discrete shape model is the main focus of this dissertation. We investigate here discrete geometry fundamentals of GeoSpelling, Monte Carlo Simulation Techniques and Statistical Shape Analysis methods to simulate and analyze "realistic shapes" when considering geometrical constraints requirements (derived from functional specifications and manufacturing considerations). In addition to mapping fundamental concepts and operations to discrete geometry one's, the work presented here investigates a discrete shape model for both random and systematic errors when taking into account second order approximation of shapes. The concept of a mean skin model and its robust statistics are also developed. The results of the skin model simulations and visualizations are reported. By means of a case study based on a cross-shaped sheet metal part where the manufacturing process is simulated using Finite Element Analysis considering stochastic variations, the results of the skin model simulations are shown, and the performances of the method are described.

Les différentes étapes et processus de la fabrication additive (FA) induisent des erreurs de sources multiples et complexes qui soulèvent des problèmes majeurs au niveau de la qualité géométrique du produit fabriqué. Par conséquent, une modélisation effective des écarts géométriques est essentielle pour la FA. Le paradigme Skin Model Shapes (SMS) offre un cadre intégral pour la modélisation des écarts géométriques des produits manufacturés et constitue ainsi une solution efficace pour la modélisation des écarts géométriques en FA.Dans cette thèse, compte tenu de la spécificité de fabrication par couche en FA, un nouveau cadre de modélisation à base de SMS est proposé pour caractériser les écarts géométriques en FA en combinant une approche dans le plan et une approche hors plan. La modélisation des écarts dans le plan vise à capturer la variabilité de la forme 2D de chaque couche. Une méthode de transformation des formes est proposée et qui consiste à représenter les effets de variations sous la forme de transformations affines appliquées à la forme nominale. Un modèle paramétrique des écarts est alors établi dans un système de coordonnées polaires, quelle que soit la complexité de la forme. Ce modèle est par la suite enrichi par un apprentissage statistique permettant la collecte simultanée de données des écarts de formes multiples et l'amélioration des performances de la méthode.La modélisation des écarts hors plan est réalisée par la déformation de la couche dans la direction de fabrication. La modélisation des écarts hors plan est effectuée à l'aide d'une méthode orientée données. Sur la base des données des écarts obtenues à partir de simulations par éléments finis, deux méthodes d'analyse modale: la transformée en cosinus discrète (DCT) et l'analyse statistique des formes (SSA) sont exploitées. De plus, les effets des paramètres des pièces et des procédés sur les modes identifiés sont caractérisés par le biais d'un modèle à base de processus Gaussien.Les méthodes présentées sont finalement utilisées pour obtenir des SMSs haute-fidélité pour la fabrication additive en déformant les contours de la couche nominale avec les écarts prédits et en reconstruisant le modèle de surface non idéale complet à partir de ces contours déformés. Une toolbox est développée dans l'environnement MATLAB pour démontrer l'efficacité des méthodes proposées. / The intricate error sources within different stages of the Additive Manufacturing (AM) process have brought about major issues regarding the dimensional and geometrical accuracy of the manufactured product. Therefore, effective modeling of the geometric deviations is critical for AM. The Skin Model Shapes (SMS) paradigm offers a comprehensive framework aiming at addressing the deviation modeling problem at different stages of product lifecycle, and is thus a promising solution for deviation modeling in AM. In this thesis, considering the layer-wise characteristic of AM, a new SMS framework is proposed which characterizes the deviations in AM with in-plane and out-of-plane perspectives. The modeling of in-plane deviation aims at capturing the variability of the 2D shape of each layer. A shape transformation perspective is proposed which maps the variational effects of deviation sources into affine transformations of the nominal shape. With this assumption, a parametric deviation model is established based on the Polar Coordinate System which manages to capture deviation patterns regardless of the shape complexity. This model is further enhanced with a statistical learning capability to simultaneously learn from deviation data of multiple shapes and improve the performance on all shapes.Out-of-plane deviation is defined as the deformation of layer in the build direction. A layer-level investigation of out-of-plane deviation is conducted with a data-driven method. Based on the deviation data collected from a number of Finite Element simulations, two modal analysis methods, Discrete Cosine Transform (DCT) and Statistical Shape Analysis (SSA), are adopted to identify the most significant deviation modes in the layer-wise data. The effect of part and process parameters on the identified modes is further characterized with a Gaussian Process (GP) model. The discussed methods are finally used to obtain high-fidelity SMSs of AM products by deforming the nominal layer contours with predicted deviations and rebuilding the complete non-ideal surface model from the deformed contours. A toolbox is developed in the MATLAB environment to demonstrate the effectiveness of the proposed methods.

La gestion et le contrôle des variations géométriques des produits pendant les processus de développement représentent une préoccupation importante pour la réduction des coûts, l’amélioration de la qualité et la compétitivité des entreprises dans un contexte de mondialisation. Pendant la phase de conception, les exigences fonctionnelles et les tolérances géométriques sont issues de l'intention de conception. La modélisation des formes et le dimensionnement des produits sont aujourd'hui largement supportés par des outils de modélisation géométrique. Toutefois, les variations géométriques ne peuvent pas être évaluées en utilisant intuitivement les outils de modélisation existants. En outre, les étapes de fabrication et de mesure sont les deux principaux générateurs de variations géométriques desquels découlent les deux axiomes bien connus de l'imprécision de la fabrication et de l'incertitude de la mesure. Une vision globale des spécifications géométriques des produits (GPS) devrait considérer non seulement le processus complet de tolérancement, la modélisation des tolérances, et la représentation des tolérances mais aussi les représentations des formes géométriques et les techniques de traitement appropriées ainsi que les algorithmes associés. GeoSpelling, solution considérée comme fondement des normes ISO GPS, offre un langage non ambigu et un cadre complet pour la modélisation et la description des variations géométriques sur le cycle de vie des produits. GeoSpelling s’appuie sur un ensemble de concepts forts dont celui du "Skin Model". Cependant, l’«opérationnalisation» de GeoSpelling n'a pas été réalisée et peu de recherches ont porté sur la génération du Skin Model. Le Skin Model, vu comme un modèle de forme discrète est l'objectif principal de cette thèse. Dans ce travail, les fondamentaux de la géométrie discrète sont appliqués à GeoSpelling, les techniques de simulation de Monte Carlo et les méthodes statistiques d'analyse de formes sont développées pour simuler et analyser les "formes réalistes" prenant en compte, les contraintes géométriques dérivées de spécifications fonctionnelles et de considérations de fabrication. En plus de cartographier les concepts fondamentaux et les opérations de GeoSpelling avec la géométrie discrète, ce travail propose un modèle de forme discrète intégrant les erreurs aléatoires et systématiques approchées du second ordre. Le concept d'un Skin Model moyen et ses statistiques robustes sont également développés. Une étude de cas plus complète, basée sur un embouti de tôle en forme de croix pour lequel le processus de fabrication est simulé avec des variations stochastiques, permet d’illustrer les résultats des simulations du skin model. Les performances de la méthode sont ensuite évaluées. / The management and the control of product geometrical variations during the whole development process is an important issue for cost reduction, quality improvement and company competitiveness in the global manufacturing era. During the design phase, geometric functional requirements and tolerances are derived from the design intent. Geometric modeling tools, now largely support the modeling of product shapes and dimensions. However, permissible geometrical variations cannot be intuitively assessed using existing modeling tools. In addition, the manufacturing and measurement stages are two main geometrical variations generators according to the two well know axioms of manufacturing imprecision and measurement uncertainty. A comprehensive view of Geometrical Product Specifications should consider not only the complete tolerancing process, tolerance modeling and tolerance representation but also shape geometric representations, and suitable processing techniques and algorithms. GeoSpelling as the basis of the GPS standard enables a comprehensive modeling framework and an unambiguous language to describe geometrical variations covering the overall product life cycle thanks to a set of concepts and operations based on the fundamental concept of the “Skin Model”. However, the “operationalization” of GeoSpelling has not been successfully completed and few research studies have focused on the skin model simulation. The skin model as a discrete shape model is the main focus of this dissertation. We investigate here discrete geometry fundamentals of GeoSpelling, Monte Carlo Simulation Techniques and Statistical Shape Analysis methods to simulate and analyze “realistic shapes” when considering geometrical constraints requirements (derived from functional specifications and manufacturing considerations). In addition to mapping fundamental concepts and operations to discrete geometry one’s, the work presented here investigates a discrete shape model for both random and systematic errors when taking into account second order approximation of shapes. The concept of a mean skin model and its robust statistics are also developed. The results of the skin model simulations and visualizations are reported. By means of a case study based on a cross-shaped sheet metal part where the manufacturing process is simulated using Finite Element Analysis considering stochastic variations, the results of the skin model simulations are shown, and the performances of the method are described.

Rapporten innefattar en jämförande studie mellan olika typer av membran och beläggningar.Främst sker en jämförelse mellan materialens förmåga att andas. Vattentäthet testas på nyamaterial och efter olika sorters nötning så som martindale, flexing och tvätt för att få en ökadförståelse för materialen. Verktyget som används i studien för att mätaånggenomsläppligheten är hudmodellen. Resultatet presenteras med ett Ret-värde vilket är enförkortning på Evaporative resistance of a textile. Metoden används för att på ett bra sättsimulera hur huden svettas.Projektet har utförts på Swerea IVF som är ett forskningsinstitut beläget i Mölndal.Hudmodellen är en av de senaste stora investeringarna på Swereas textil och plast avdelning.Resultatet av studien visar att laminat andas bättre än beläggningar, dock har bärarmaterialetstor inverkan på resultaten. Ett tydligt samband mellan grövre material och sämre andning harobserverats. Många av de material som testats i studien uppvisar god förmåga att andas, därbåde flera av de mikroporösa och hydrofila materialen uppvisar Ret-värden under 13, vilketinnebär mycket god andning. En delstudie har varit att testa hur materialens andande förmågaförändras vid lägre relativ fuktighet. Resultatet blev att mikroporösa material inte påverkaslika mycket som de hydrofila materialen som då får en sämre andning.Efter de resultat studien har visat kan slutsatsen dras att tunna laminat är att föredra då högånggenomsläpplighet är ett krav. Behövs däremot ett högt motstånd mot nötning kan etttjockare material med fördel användas, vilket dock kan leda till högre ångmotstånd.This report is a comparative study between different types of membranes and coatings, thebreathability of the fabrics being the main focus of research. The fabrics' waterproofness wastested on new materials and by abrasion including martindale, flexing and washing. The toolthat has been used to measure water-vapour resistance is the skin model. The result ispresented by a Ret-value, which is short for evaporative resistance of a textile. The method isused to simulate the sweating body in a realistic way. Swerea IVF is the research institutelocated in Mölndal where the project has been carried out. The skin model is one of the latestbig investments at Swereas textile and plastic department.The results of the study shows that laminates breath better than coatings. It is important topoint out, however, that the carrier has great influence on the fabric in question. In addition,the results indicate a relation between thick fabrics and less breathability. Many of thematerials that have been tested show good permeability to breath, including bothmicropourous and hydrophilic materials. Most of them demonstrate a Ret-value less than 13,which means very good breathability. Further tests also show how the breathability changeswith lower relative humidity, indicating that microporous materials are less affected thanhydrophilic materials, thus having a higher resistance to water vapour.The conclusion of the study is that thin laminates is to prefer when high breathability isrequired. If the demand is high resistance to abrasion, a thicker material is prefered, whichalso yields a higher resistance to water permability. / Program: Textilingenjörsutbildningen

Bovine Dermatitis digitalis (DD) ist eine weltweit verbreitete Infektionskrankheit bei Rindern, die primär die plantare Haut über dem Kronenrand nahe des Zwischenzehenspalts der Hinterklauen betrifft. Schmerzhafte ulzero-proliferative Läsionen mit akuten und chronischen Erscheinungsformen führen zu Verhaltensänderungen und Lahmheit der Tiere. DD hat damit einen erheblichen Einfluss auf deren Wohl und ihre Leistungen. Zahlreiche Untersuchungen zur Ätiologie der Krankheit ergaben, dass es sich um das Zusammenspiel verschiedener Ursachen handelt. Einer synergistischen multifaktoriellen Infektion mit starker Beteiligung von Bakterien der Gattung Treponema kommt dabei besondere Bedeutung zu. Aspekte wie Tierhaltung, Hygienestandards und genetische Prädispositionen wurden ebenfalls intensiv untersucht. Nichtsdestotrotz bleiben Infektionsherde, Transmissionsrouten und Pathomechanismen weitgehend unklar. Zum besseren Verständnis der Ereignisse, die zu DD-Läsionen führen, sollte im Zuge dieser Arbeit ein organotypisches Zellkulturmodell der bovinen Haut erstellt werden, welches in späteren Versuchen mit dem Krankheitserreger zum Einsatz kommen soll. Verlässliche und reproduzierbare Techniken zur Isolation und Kultur von bovinen primären Keratinozyten und Fibroblasten wurden etabliert; geeignete Zellkulturmedien für die Langzeitkultivierung und –aufbewahrung der Hautzellen wurden identifiziert. Zur Erstellung des Hautmodells wurden zwei verschiedene Ansätze miteinander verglichen. Der zweite Ansatz, bei dem Keratinozyten direkt auf ein dermales Äquivalent, d.h. ein Pad aus bovinem Kollagen I mit eingesäten post-mitotischen Fibroblasten, gesät wurden, brachte ein vielversprechendes Hautmodell hervor. Die inkorporierten post-mitotischen Fibroblasten wiesen eine charakteristische Zellmorphologie mit intakten Nuklei auf. Die terminale Differenzierung der Keratinozyten auf dem dermalen Äquivalent wurde mittels Immunfluoreszenzfärbungen mit Antikörpern gegen die Markerproteine Keratin 14 und Desmoglein 1 gezeigt. Die Ergebnisse erster Experimente mit Treponema spp. verdeutlichen, dass das Hautäquivalent ein geeignetes Modell zur Untersuchung der Pathogenese der DD darstellt. / Bovine digital dermatitis (DD) is a worldwide occurring, infectious disease in cattle primarily affecting the plantar skin above the coronary band near the interdigital cleft on hind feet. Painful ulceroproliferative lesions with acute and chronic appearances lead to behavioral changes and lameness. Hence, DD has a major impact on animal welfare and performance. Substantial efforts in investigating the etiology of the disease revealed a synergistic origin with evidence for a multibacterial infection and the strong involvement of bacteria from the genus Treponema. As the interaction between host, pathogen and environment is not negligible, surrounding circumstances such as housing, general hygiene and genetic predispositions have been investigated intensively. Nevertheless, infection reservoirs, transmission routes and pathomechanisms remain widely unclear. To better understand the cellular and molecular events during Treponema-infection of bovine skin, it was the specific aim of this study to establish an organotypic in vitro skin model, which could be challenged with the causative agent of the disease. A technique to reliably and reproducibly isolate primary keratinocytes and fibroblasts from the site of infection was established. Appropriate cell culture media for the long-term cultivation and storage of bovine skin cells were identified. Two different methods to develop the skin model were compared. The second strategy in which keratinocytes were directly seeded on top of a dermal equivalent, i.e. a bovine collagen type I pad with embedded post-mitotic fibroblasts, gave rise to a promising organotypic skin equivalent. The incorporated post-mitotic fibroblasts showed a characteristic cell morphology with intact nuclei. The terminal differentiation of the keratinocytes on top of the dermal equivalent was shown with anti-K14 and anti-Dsg1 immunofluorescence stainings. The results of initial Treponema-experiments proved that the skin equivalent is a suitable model to investigate the underlying mechanisms during Treponema-infection of bovine skin and hence, the pathogenesis of DD.

Topische Glukokortikoide (GCs) sind wirksam bei Therapie von entzündlichen Hauterkrankungen. Durch ihr Nebenwirkungspotential (z.B. Induktion von Hautatrophie) ist ihr Einsatz jedoch begrenzt. Für die Medikamentenentwicklung ist die Bestimmung des atrophogenen Potenzials neuer Verbindungen daher von großer Bedeutung. Derzeit stehen dafür keine prädiktiven in vitro Modelle zur Verfügung. Ziel dieser Arbeit war daher die Etablierung solcher Modelle. Es wurden kutane Zelltypen (3T3-Zellen, Rattenfibroblasten, HaCaT-Zellen, humane Keratinozyten [NHEK] und Fibroblasten) und Vollhautmodelle (CellSystems AST-2000 und Phenions FTSM) untersucht. Atrophie-Marker, die Proliferation, Kollagen-Metabolismus und Epidermisdicke betreffend, wurden auf mRNA-, Protein- bzw. zellulärer Ebene gemessen. Außerdem wurden mittels Genexpressionsanalysen von GC-behandelter Nagerhaut neue potenzielle Marker identifiziert, deren Regulation in vitro jedoch nicht bestätigt werden konnte. Nach Pilotexperimenten wurden 3 Modelle ausgewählt und für Evaluierungsexperimente mit Referenz-GCs behandelt: 1). MMP1, -2, -3 und -9 mRNA-Expression in NHEK, 2). COL1A1 und COL3A1 mRNA-Expression in 3T3-Zellen, 3.) Epidermisdicke, Kollagen- und MMP-Synthese in FTSM. Die Messparameter der 3 Modelle erwiesen sich als dosisabhängig reguliert und korrelierten mit dem atrophogenen Potenzial der GCs. Schließlich wurde die Prädiktabilität der 3 in vitro Modelle für die in vivo Situation im Nager analysiert. In allen 3 in vitro Systemen induzierte die Behandlung mit einem selektiven GC-Rezeptor-Agonisten weniger atrophogene Effekte als das Referenz-GC. Ähnliche Ergebnisse wurden auch in vivo im Rattenhautatrophie-Modell gefunden. Zusammenfassend wird eine Kaskade von 3 in vitro Modellen empfohlen, um das atrophogene Potential von GC-Rezeptor-Liganden zu bestimmen. Der tatsächliche prädiktive Wert für die klinische Situation sollte in weiteren Studien untersucht werden. / Topical glucocorticoids (GCs) are effective for the therapy of inflammatory skin diseases. However, their use is limited by their side effect potential, with skin atrophy being the most prominent one. Thus, determining the atrophogenic potential of novel compounds is of importance for drug development. Currently, there are no according predictive in vitro models available. The aim of this study was to establish such atrophy models. Rodent and human cutaneous cell types (3T3 cells, rat fibroblasts, HaCaT cells, human keratinocytes [NHEK] and fibroblasts) and human full-thickness skin equivalents (CellSystems AST-2000 and Phenions FTSM) were investigated. Atrophy markers related to proliferation, collagen metabolism and epidermal thickness were measured on mRNA, protein and cellular level, respectively. Additionally, by gene expression profiling of GC-treated rodent skin novel potential markers were identified, but subsequently not confirmed in vitro. After pilot studies 3 models were selected and treated with reference GCs for evaluation experiments: 1.) MMP1, -2, -3 and -9 mRNA expression in NHEK, 2.) COL1A1 and COL3A1 mRNA expression in 3T3 cells, 3.) epidermal thickness, collagen and MMP synthesis in FTSM. The read out parameters of all 3 test systems turned out to be regulated dose-dependently and correlated with the atrophogenic potential of the GCs. Finally, the predictability of the 3 recommended in vitro test system for the rodent in vivo situation was analyzed. In all 3 in vitro test systems, the treatment with a novel selective GC receptor agonist induced less atrophogenic effects than the reference GC clobetasol. Indeed, similar results were found in the hr/hr rat skin atrophy model. In summary, a cascade of 3 in vitro models is recommended to be applied for the characterization of the atrophogenicity of GC receptor ligands. Further experiments are necessary to eventually demonstrate the true predictability of these models for the clinical situation.