Multilayered Epidermis Research Articles

Mathematical model for constructing a matrix of coefficients thermal conductivity under low-temperature influence on the multilayer epidermis of biological tissue

Objective. The purpose of the study is to develop a mathematical model for constructing a matrix of thermal conductivity coefficients under low-temperature exposure to the multilayer epidermis of biological tissue. Method. The research is based on methods of thermo dynamic analysis, full-scale and computational modeling of processes under low-temperature influence. Result. A mathematical model of the matrix of thermal conductivity coefficients under low-temperature effects on biological tissue has been developed. Heat transfer in a multilayer medium under low-temperature influence on biological tissue is presented as a process with a discrete step in time and coordinate. Thermal properties are evenly distributed throughout the volume of layers of biological tissue. For exposure to cold, layers of the epidermis of biological tissue were used, divided into uniform identical cells, where the cell area of the biological tissue was taken to be equal to one. Conclusion. The results obtained in this work can be useful to specialists who deal with modeling problems with free boundaries.

Retromer Chaperones: Potential Therapeutics for Treatment of Skin Disease?

Retromer Chaperones: Potential Therapeutics for Treatment of Skin Disease?

Morpho-structural adaptations of the integument in different aquatic organisms

The integument acts as a barrier to protect the body from harmful pathogenic infectious agents, parasites, UV rays, trauma, and germs. The integument of invertebrates and vertebrates are structurally different: while invertebrates usually have a simple monolayer epidermis frequently covered by mucus, cuticles, or mineralized structures, vertebrates possess a multilayered epidermis with several specialized cells. This study aims to describe by morphological, histological, and immunohistochemical analyses, the morpho-structural adaptations throughout evolution of the integument of gastropod Aplysia depilans (Gmelin, 1791), ascidian Styela plicata (Lesuer, 1823), myxine hagfish Eptatretus cirrhatus (Forster, 1801) and teleost Heteropneustes fossilis (Bloch, 1794) for the first time, with special reference to sensory epidermal cells. Different types of cells could be identified that varied according to the species; including mucous cells, serous glandular cells, clavate cells, club cells, thread cells, and support cells. In all integuments of the specimens analyzed, sensory solitary cells were identified in the epidermis, immunoreactive to serotonin and calbindin. Our study provided an essential comparison of integuments, adding new information about sensory epidermal cells phylogenetic conservation and on the structural changes that invertebrates and vertebrates have undergone during evolution.

Skin penetration of active folate salts and their effects on wound healing in vitro: new approaches for dermal folate application

# Objectives The skin has a high demand for folates because of its constant renewal and growth. Folates play an important role in the metabolism of healthy skin and in the regeneration of irritated or injured skin. The high proliferation rate supports wound healing in chronic and degenerative skin diseases. In contrast to systemic folate administration, there is little known on skin penetration of folates after topical application. Therefore, skin penetration with L-formyltetrahydrofolate di-arginine salt (L-FTHF-di-arginine), L-formyltetrahydrofolate calcium gluconate mixture (L-FTHF-Ca) and L-methyltetrahydrofolate dicholine salt (L-MTHF-dicholine) was studied. # Methods We investigated the epidermal penetration of 2.5% aqueous solution of L-FTHF di-arginine, L-FTHF Ca and L-MTHF dicholine using *in vitro* reconstructed epidermal skin models. These multi-layered epidermis models closely emulate the barrier of human skin. Aqueous solutions of - L-FTHF di-arginine, L-FTHF Ca and L-MTHF dicholine were applied topically to the keratinized layer. The folate concentration below the multi-layer was analyzed after 24 hours by high-performance liquid chromatography (HPLC)-analysis to detect penetration. In order to assess an effect on wound healing *in vitro*, the selected folates were tested in a keratinocyte scratch assay. # Results Analysis of the examined folates in the cell culture medium located opposite of the application site showed their penetration through the reconstructed epidermal model. In epidermal models to which L-FTHF di-arginine was added, L-MTHF dicholine was detected on the opposite site. L-FTHF salts were partially converted to L-MTHF. Improved wound healing was shown in keratinocyte scratch assay when the selected folates were added to the medium. # Conclusion The *in vitro* experiments on an epidermal skin model have revealed the following new findings: a) the different reduced folates investigated (L-FTHF di-arginine, L-FTHF Ca and L-MTHF dicholine) penetrate well into and through the skin, in contrast to folic acid, which is commonly used; b) they neither disturb the viability of skin cells nor their barrier function, but even improve it; c) the proliferation and migration of epidermal cells, which is important for wound healing, is promoted; d) epidermal cells are able to convert L-FTHF locally into L-MTHF, i.e., to the dominant biologically active form in the circulation. This creates important prerequisites for a future topical application of folates for the treatment of certain skin diseases and the prophylaxis of skin aging processes.

177 The Striatin-Interacting Phosphatase And Kinases complex is important for skin epidermal differentiation and barrier formation

Several human skin diseases, such as atopic dermatitis, psoriasis as well as regeneration processes such as wound healing are associated with epidermal barrier defects. The multi-layered epidermis is composed of proliferating basal keratinocytes that initiate upward differentiation through migration and intercalation, which ultimately lead to tight junction formation and cornification to serve as essential barriers against environmental insults and dehydration. The assembly of functional tight junctions at the interfaces of the granular layer is largely controlled by cytoskeletal rearrangements, including filamentous (F)-actin organization.

098 A burst of progenitor proliferation drives epidermal stratification

The formation of complex stratified epithelial barriers in mammals is essential for animal survival. During mouse embryonic development the skin epithelium is generated from a simple single-layered epithelium which stratifies and differentiates to form a multi-layered epidermis just before birth. But the mechanisms underlying epidermal stratification and how cells switch their cell fate from basal to suprabasal cells are still unknown. Recently, we proposed a two-phase model of epidermal stratification, with the first major phase starting around embryonic day E13.5 when the epithelium gets multi-layered.

Nano- and Micro-Porous Chitosan Membranes for Human Epidermal Stratification and Differentiation.

The creation of partial or complete human epidermis represents a critical aspect and the major challenge of skin tissue engineering. This work was aimed at investigating the effect of nano- and micro-structured CHT membranes on human keratinocyte stratification and differentiation. To this end, nanoporous and microporous membranes of chitosan (CHT) were prepared by phase inversion technique tailoring the operational parameters in order to obtain nano- and micro-structured flat membranes with specific surface properties. Microporous structures with different mean pore diameters were created by adding and dissolving, in the polymeric solution, polyethylene glycol (PEG Mw 10,000 Da) as porogen, with a different CHT/PEG ratio. The developed membranes were characterized and assessed for epidermal construction by culturing human keratinocytes on them for up to 21 days. The overall results demonstrated that the membrane surface properties strongly affect the stratification and terminal differentiation of human keratinocytes. In particular, human keratinocytes adhered on nanoporous CHT membranes, developing the structure of the corneum epidermal top layer, characterized by low thickness and low cell proliferation. On the microporous CHT membrane, keratinocytes formed an epidermal basal lamina, with high proliferating cells that stratified and differentiated over time, migrating along the z axis and forming a multilayered epidermis. This strategy represents an attractive tissue engineering approach for the creation of specific human epidermal strata for testing the effects and toxicity of drugs, cosmetics and pollutants.

Novel leukocyte-depleted platelet-rich plasma-based skin equivalent as an in vitro model of chronic wounds: a preliminary study

BackgroundChronic leg ulcerations are associated with Haemoglobin disorders, Type2 Diabetes Mellitus, and long-term venous insufficiency, where poor perfusion and altered metabolism develop into a chronic inflammation that impairs wound closure. Skin equivalent organotypic cultures can be engineered in vitro to study skin biology and wound closure by modelling the specific cellular components of the skin. This study aimed to develop a novel bioactive platelet-rich plasma (PRP) leukocyte depleted scaffold to facilitate the study of common clinical skin wounds in patients with poor chronic skin perfusion and low leukocyte infiltration. A scratch assay was performed on the skin model to mimic two skin wound conditions, an untreated condition and a condition treated with recombinant tumour necrotic factor (rTNF) to imitate the stimulation of an inflammatory state. Gene expression of IL8 and TGFA was analysed in both conditions. Statistical analysis was done through ANOVA and paired student t-test. P < 0.05 was considered significant.ResultsA skin model that consisted of a leukocyte-depleted, platelet-rich plasma scaffold was setup with embedded fibroblasts as dermal equivalents and seeded keratinocytes as multi-layered epidermis. Gene expression levels of IL8 and TGFA were significantly different between the control and scratched conditions (p < 0.001 and p < 0.01 respectively), as well as between the control and treated conditions (p < 0.01 and p < 0.001 respectively). The scratch assay induced IL8 upregulation after 3 h (p < 0.05) which continued to increase up to day 1 (p < 0.05). On the other hand, the administration of TNF led to the downregulation of IL8 (p < 0.01), followed by an upregulation on day 2. IL8 gene expression decreased in the scratched condition after day 1 as the natural healing process took place and was lower than in the treated condition on day 8 (p < 0.05).Both untreated and treated conditions showed a downregulation of TGFA 3 h after scratch when compared with the control condition (p < 0.01). Administration of rTNF showed significant downregulation of TGFA after 24 h when compared with the control (p < 0.01) and treated conditions (p < 0.05).ConclusionThis study suggests that a leukocyte-depleted PRP-based skin equivalent can be a useful model for the in vitro study of chronic skin wounds related to poor skin perfusion.

Using 3D-bioprinting scaffold loaded with adipose-derived stem cells to burns wound healing.

Three dimensional (3D) printing has recently expanded in popularity and has become an effective approach for tissue engineering. Advances in tissue engineering have increased the effectiveness of cell-based therapies. Indeed, the ultimate goal of such treatment is the development of conditions similar to fetal wound regeneration. In this context, technology of 3D printing also allows researchers to more effectively compose multi-material and cell-laden scaffolds with less effort. In this study, we explored a synthetic gel scaffold derived from 3D bioprinter with or without stem cells to accelerate wound healing and skin defects. Adipose-derived stem cells (ADSCs) were isolated and seeded into 3D bioprinter derived-gel scaffold. Morphological and cell adherence properties of 3D scaffold were assessed by hemotoxylin& eosin (H&E) staining and scanning electron microscopy and cell viability was determined by methylthiazolyldiphenyl-tetrazolium bromide assay. In vivo assessment of the scaffold was done using H&E staining in the full-thickness burn rat model. The experimental groups included; (a) untreated (control), (b) 3D bioprinter derived-gel scaffold (Trial 1), and (c) 3D bioprinter derived-gel scaffold loaded with ADSC (Trial 2). Our results represented 3D bioprinter derived-gel scaffold with or without ADSCs accelerated wound contraction and healing compared to control groups. Epithelization was completed until 21days after operation in scaffold alone. In scaffold with ADSCs group, epithelization was faster and formed a multi-layered epidermis with the onset of cornification. In conclusion, 3D bioprinter derived-gel scaffold with or without ADSCs has the potential to be used as a wound graft material in skin regenerative medicine.

Water conservation strategies through anatomical traits in the endangered arid zone species Salvadora oleoides Decne.

Salvadora oleoides Decne. (Salvadoraceae) is a facultative and mesomorphic xerophyte, well adapted to arid and semi-arid regions. Twelve populations were selected from different ecological regions of Punjab to investigate structural modifications that are essential for water conservation, by either improving water-storing capacity or preventing water loss from the plant body, hence, enabling this species to cope with environmental extremities. We observed significant plasticity in structural features, which ensures ecological success in a variety of environmental conditions. Huge variation was seen in various tissue systems, i.e., epidermis and epidermal appendages, collenchyma, size of internal oil glands, storage parenchyma, shape, size and arrangement of vascular tissue, intensity of sclerification, and stomatal size, shape and orientation. The desert populations showed additional parenchymatous layer under multilayered epidermis, xylem completely surrounded by phloem, broad metaxylem vessels, intensive sclerification in and outside vascular tissue, and numerous and small sized stomata on abaxial leaf surface. Population from salt-affected areas showed greater stem cross-sectional area, enlarge palisade parenchymatous cells, multilayered epidermis, large storage parenchyma, and sclerified vascular bundles. Roadside populations possessed sclerified vascular bundles and dense hairiness on leaf surface.

An Engineered Infected Epidermis Model for In Vitro Study of the Skin's Pro-Inflammatory Response.

Wound infection is a major clinical challenge that can significantly delay the healing process, can create pain, and requires prolonged hospital stays. Pre-clinical research to evaluate new drugs normally involves animals. However, ethical concerns, cost, and the challenges associated with interspecies variation remain major obstacles. Tissue engineering enables the development of in vitro human skin models for drug testing. However, existing engineered skin models are representative of healthy human skin and its normal functions. This paper presents a functional infected epidermis model that consists of a multilayer epidermis structure formed at an air-liquid interface on a hydrogel matrix and a three-dimensionally (3D) printed vascular-like network. The function of the engineered epidermis is evaluated by the expression of the terminal differentiation marker, filaggrin, and the barrier function of the epidermis model using the electrical resistance and permeability across the epidermal layer. The results showed that the multilayer structure enhances the electrical resistance by 40% and decreased the drug permeation by 16.9% in the epidermis model compared to the monolayer cell culture on gelatin. We infect the model with Escherichia coli to study the inflammatory response of keratinocytes by measuring the expression level of pro-inflammatory cytokines (interleukin 1 beta and tumor necrosis factor alpha). After 24 h of exposure to Escherichia coli, the level of IL-1β and TNF-α in control samples were 125 ± 78 and 920 ± 187 pg/mL respectively, while in infected samples, they were 1429 ± 101 and 2155.5 ± 279 pg/mL respectively. However, in ciprofloxacin-treated samples the levels of IL-1β and TNF-α without significant difference with respect to the control reached to 246 ± 87 and 1141.5 ± 97 pg/mL respectively. The robust fabrication procedure and functionality of this model suggest that the model has great potential for modeling wound infections and drug testing.

Use of 2D and 3D cell cultures in dermatology

The skin is acomplex organ that performs anumber of vital functions, including forming aphysical barrier that protects our body from the penetration of pathogens and irritants and from excessive transepidermal water loss. In addition to its passive properties, the skin is also actively involved in the immune process. Acomplex structure of different cell types and structures allows the skin to fulfil these functions. In vitro research often faces the problem that simple 2D cell cultures are not able to adequately map these functions. Here 3D skin models offer apossible solution. In recent years, there has been significant development in this field; the reproducibility of the method as well as the physiological structure and tissue architecture of the 3D skin models have been improved. Depending on the research question, protocols for 3D skin models have been published, ranging from simple multilayer epidermis models to highly complex vascularized 3D full skin models.

Re-epithelialization and immune cell behaviour in an ex vivo human skin model

A large body of literature is available on wound healing in humans. Nonetheless, a standardized ex vivo wound model without disruption of the dermal compartment has not been put forward with compelling justification. Here, we present a novel wound model based on application of negative pressure and its effects for epidermal regeneration and immune cell behaviour. Importantly, the basement membrane remained intact after blister roof removal and keratinocytes were absent in the wounded area. Upon six days of culture, the wound was covered with one to three-cell thick K14+Ki67+ keratinocyte layers, indicating that proliferation and migration were involved in wound closure. After eight to twelve days, a multi-layered epidermis was formed expressing epidermal differentiation markers (K10, filaggrin, DSG-1, CDSN). Investigations about immune cell-specific manners revealed more T cells in the blister roof epidermis compared to normal epidermis. We identified several cell populations in blister roof epidermis and suction blister fluid that are absent in normal epidermis which correlated with their decrease in the dermis, indicating a dermal efflux upon negative pressure. Together, our model recapitulates the main features of epithelial wound regeneration, and can be applied for testing wound healing therapies and investigating underlying mechanisms.

647 Investigating re-epithelialization in a novel ex vivo human skin model

Although numerous wound healing models have been established, a model to study re-epithelialization in vitro, without disrupting the underlying dermal compartment, is lacking. Therefore, we established a new standardized human ex vivo model based on application of negative pressure, inducing blistering and consequently dermal epidermal separation. Effects of this treatment for epidermal regeneration and immune cells were investigated. Immunostaining revealed type IV collagen expression on the remaining dermal compartment, indicating that the basement membrane and thus the underlying dermis remained intact after removal of the blister roof epidermis. Keratinocytes were absent in the wounded area as evidenced by a lack of K14 staining. Four to six days after removal of the epidermis and culture, the wound was completely covered again with one to three-cell thick K14+ keratinocyte layers. Keratinocytes (∼20%) covering the initial wound bed expressed the endogenous cell proliferation marker Ki67, indicating that proliferation and migration were involved in wound closure. After eight to twelve days, a multi-layered epidermis was formed expressing several epidermal differentiation markers (e.g., K10, filaggrin, DSG-1, CDSN). While application of negative pressure did not affect the morphology, phenotype and density of Langerhans cells, we observed more T cells in the blister roof epidermis as compared to normal epidermis. Additionally, we identified several populations in blister roof epidermis and suction blister fluid that are absent in normal epidermis which correlated with their decrease in the dermis, indicating an influx upon pressure. Whether and how this contributes to the wound healing process is currently under investigation. This model recapitulates the main features of epithelial wound regeneration, and thus can be applied for testing wound healing therapies and investigating the underlying mechanisms in the future.

Changes in defense traits of young leaves in subtropical forests succession

Plants develop diverse adaptive traits in changeable environments, yet whether plant defense traits change during succession remains unclear. In this study, we investigated the young leaf physical traits (i.e., upright orientation of leaves, trichomes, an enhanced cuticle, and a multilayered epidermis) and leaf color trait (i.e., red pigmentation) of dominant plants in three subtropical forests. These forests included a pioneer forest, a mixed coniferous-broadleaved forest, and a monsoon-evergreen broadleaved forest representing early, middle, and later successional stages, respectively. Our results show that the red color trait in young leaves is related to anti-herbivory defense, and the percentage of species with red young leaves is higher in later than in early succession. Physical defense tends to be weaker for red young leaves than for green young leaves in early and middle successions. In addition, the number of defense traits of young leaves increases with succession. We speculate that young leaves in subtropical forests depend increasingly on multiple defense traits during succession because of the increased biotic stresses and environmental complexity in later succession.

Morpho-anatomy of the inflorescence of Musa × paradisiaca

Bananas and plantains are herbaceous monocotyledonous plants belonging to the genus Musa, Musaceae, which has a widespread distribution around the world. Various parts of banana plant are commonly used in traditional medicines. Several species of Musa are reported to possess anti-inflammatory, anti-hyperglycemic and antidiabetic properties. This work is aimed at studying the morphological and anatomical characteristics of the inflorescences of Musa×paradisiaca L., that could contribute to the characterization of these species cultivated in Brazil. Plant materials were collected and prepared in accordance with standard optical microscopy techniques. Morphological characterizations were conducted using morphological descriptors for inflorescences, including some descriptors from International Plant Genetic Resources Institute for Musa spp. Microscope slides were prepared using glycol-methacrylate and were stained in toluidine blue. Main features observed for M.×paradisiaca inflorescence were amphistomatic bracts with tetracytic stomata, fiber caps next to the phloem, adaxial and abaxial uniseriate epidermis, and papillose on the abaxial face. Outer tepals have multilayer epidermis and vascular bundles aligned next to the abaxial face. Free tepal has unilayeredepidermis. Anthers are tetrasporangiate and the locules are separated by the septum. Ovary is inferior and trilocular with external unilayered and internal epidermis. The main morpho-anatomical characteristics of inflorescence of Musa×paradisiaca are highlighted in this study, contributing to provide more information about the characterization of this species cultivated in Brazil.

1352 Basal layer divisions in epidermis over the human lifespan

While proliferation is decreased in aged tissues, no change in SC number was detected by our group or Bickenbachs. Lechler et al. examined types of SC divisions. Divisions perpendicular to the basement membrane were 8% in single-layered embryonic epidermis, 66% in multi-layer epidermis and 85% in adult mice. In humans there is a decrease in cells positive for the proliferation marker Ki67. Our aim was to quantify mitoses of basal keratinocytes in vivo, and examine the types of division, over the human lifespan. We quantified divisions in the basal layer in neonatal (0-7 days), adult (30-45 years) and aged (70-80 years) skin. Asymmetric segregation of polarity proteins is a defining characteristic of SCs. We visualized division orientation in the basal layer using gtubulin (spindle) and NUMA (polarity protein) in biopsies. Because of the limited divisions seen in vivo, we also quantified division type in keratinocytes from freshly obtained biopsies, in vitro, using Numb segregation to identify asymmetric SC divisions. The number of divisions/10cm of basal layer was significantly decreased with age [neonatal 7.6±2 (n=5) vs. adult 3.2±0.5 (n=4) vs. aged 1.5± 0.4 (n=8), p=0.001]. Both perpendicular (asymmetric) and parallel (symmetric) divisions were decreased with age. No significant change in the proportion of perpendicular (asymmetric) SC divisions was detected (neonatal 41.7±14.4% vs. adult 27.5±15.9% vs. aged 36.8±8%). Similar results were found in vitro [neonatal 29.1±7.4% (n=11) vs. adult 32.2% (n= 2) vs. aged 31.2±11.6% (n= 7)]. We conclude that epidermal basal cell divisions decrease over the human lifespan such that the number of divisions in 70+ year olds is 20% of that in neonates. However, the ratio of asymmetric/symmetric divisions remains unchanged. These findings could reflect a decrease in SC number, an increase in SC quiescence, and/or longer cell cycle duration in human epidermis.

In vitro skin models to study epithelial regeneration from the hair follicle.

The development of dermal equivalents (DEs) for the treatment of burns has contributed toward efficient wound closure. A collagen-glycosaminoglycan DE (C-GAG) grafted with hair follicles converted a full-thickness wound to partial-thickness resulting in complete wound closure and restored hair. In this study we compared the ability of both intact pilosebaceous units (PSU) or truncated hair follicles (THF) to regenerate a multilayered epidermis in vitro when implanted into de-epidermalized dermis (DED) or C-GAG with the epidermis generated in vivo using C-CAG. Keratinocytes explanted from the outer root sheath of PSU and THF in both DED and C-GAG but only formed a multilayered epidermis with PSU in DED. PSU were more effective at forming multilayered epidermis in DED than THF. Both DED and C-GAG skin expressed proliferation (PCNA), differentiation (K1, K10), hyperproliferation (K6, K16), basal (K14), putative stem cell (p63), extracellular matrix protein (Collagen IV), mesenchymal (vimentin) and adherens junction (β-catenin) markers. These data suggest DEs supported initial maintenance of the implanted hair follicles, in particular PSU, and provide an excellent model with which to investigate the regulation of hair follicle progenitor epithelial cells during epidermal regeneration. Although neither PSU nor THF formed multilayered epidermis in C-CAG in vitro, hair follicles implanted into engrafted C-GAG on a burns patient resulted in epithelial regeneration and expression of proliferation and differentiation markers in a similar manner to that seen in vitro. However, the failure of C-GAG to support epidermal regeneration in vitro suggests in vivo factors are essential for full epidermal regeneration using C-GAG.

Photocrosslinkable Gelatin Hydrogel for Epidermal Tissue Engineering.

Natural hydrogels are promising scaffolds to engineer epidermis. Currently, natural hydrogels used to support epidermal regeneration are mainly collagen- or gelatin-based, which mimic the natural dermal extracellular matrix but often suffer from insufficient and uncontrollable mechanical and degradation properties. In this study, a photocrosslinkable gelatin (i.e., gelatin methacrylamide (GelMA)) with tunable mechanical, degradation, and biological properties is used to engineer the epidermis for skin tissue engineering applications. The results reveal that the mechanical and degradation properties of the developed hydrogels can be readily modified by varying the hydrogel concentration, with elastic and compressive moduli tuned from a few kPa to a few hundred kPa, and the degradation times varied from a few days to several months. Additionally, hydrogels of all concentrations displayed excellent cell viability (>90%) with increasing cell adhesion and proliferation corresponding to increases in hydrogel concentrations. Furthermore, the hydrogels are found to support keratinocyte growth, differentiation, and stratification into a reconstructed multilayered epidermis with adequate barrier functions. The robust and tunable properties of GelMA hydrogels suggest that the keratinocyte laden hydrogels can be used as epidermal substitutes, wound dressings, or substrates to construct various in vitro skin models.

Generation of a three-dimensional full thickness skin equivalent and automated wounding.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.