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The extracellular matrix (ECM) of soft tissues in vivo has remarkable biological and structural properties. Thereby, the ECM provides mechanical stability while it still can be rearranged via cellular remodeling during tissue maturation or healing processes. However, modern synthetic alternatives fail to provide these key features among basic properties. Synthetic matrices are usually completely degraded or are inert regarding cellular remodeling. Based on a refined electrospinning process, a method is developed to generate synthetic scaffolds with highly porous fibrous structures and enhanced fiber-to-fiber distances. Since this approach allows for cell migration, matrix remodeling, and ECM synthesis, the scaffold provides an ideal platform for the generation of soft tissue equivalents. Using this matrix, an electrospun-based multilayered skin equivalent composed of a stratified epidermis, a dermal compartment, and a subcutis is able to be generated without the use of animal matrix components. The extension of classical dense electrospun scaffolds with high porosities and motile fibers generates a fully synthetic and defined alternative to collagen-gel-based tissue models and is a promising system for the construction of tissue equivalents as in vitro models or in vivo implants.

3D printing is a rapidly evolving field for biological (bioprinting) and non-biological applications. Due to a high degree of freedom for geometrical parameters in 3D printing, prototype printing of bioreactors is a promising approach in the field of Tissue Engineering. The variety of printers, materials, printing parameters and device settings is difficult to overview both for beginners as well as for most professionals. In order to address this problem, we designed a guidance including test bodies to elucidate the real printing performance for a given printer system. Therefore, performance parameters such as accuracy or mechanical stability of the test bodies are systematically analysed. Moreover, post processing steps such as sterilisation or cleaning are considered in the test procedure. The guidance presented here is also applicable to optimise the printer settings for a given printer device. As proof of concept, we compared fused filament fabrication, stereolithography and selective laser sintering as the three most used printing methods. We determined fused filament fabrication printing as the most economical solution, while stereolithography is most accurate and features the highest surface quality. Finally, we tested the applicability of our guidance by identifying a printer solution to manufacture a complex bioreactor for a perfused tissue construct. Due to its design, the manufacture via subtractive mechanical methods would be 21-fold more expensive than additive manufacturing and therefore, would result in three times the number of parts to be assembled subsequently. Using this bioreactor we showed a successful 14-day-culture of a biofabricated collagen-based tissue construct containing human dermal fibroblasts as the stromal part and a perfusable central channel with human microvascular endothelial cells. Our study indicates how the full potential of biofabrication can be exploited, as most printed tissues exhibit individual shapes and require storage under physiological conditions, after the bioprinting process.

Background Bacterial nanocellulose (BNC) is considered a promising carrier for various substances and novel approaches using BNC in combination with antiseptics are well documented. However, the difference in the molecular weight of these molecules influences their uptake by and release from BNC. Analysing the diffusion of standard molecules with different weight, e.g. dextrans, offers the possibility to investigate the mobility of various molecules. We aimed to test the use of BNC regarding uptake and release of different standard molecules as well as two commercially available antiseptics for possible applications in future wound dressings. Material and methods Diffusion profiles, uptake and release of three FITC-dextran molecules differing in weight as well as octenidine (Octenisept®) and povidone-iodine (Betaisodona®)-based antiseptics were tested with BNC-based wound dressings. Furthermore, the antiseptic efficacy of BNC in combination with antiseptics against Staphylococcus aureus was tested. Results Uptake and release capacity for FITC-dextran molecules showed a molecular weight-dependent mobility from BNC into an agarose gel. The loading capacity of BNC was also inversely proportional to the molecular weight of the antiseptics. The release test for octenidine showed a sustained and prolonged delivery into a solid matrix, whereas povidone-iodine was released faster. Both antiseptic solutions combined with BNC showed a good dose-dependent efficacy against S. aureus. Conclusion Results obtained from the mobility of FITC-dextran molecules in the BNC matrix could open possible applications for the combination of BNC with other molecules for medical applications. Combination of both tested antiseptics with BNC showed to be an efficient approach to control bacterial infections.

Due to the rapidly increasing development and use of cellular products, there is a rising demand for non-animal-based test platforms to predict, study and treat undesired immunity. Here, we generated human organotypic skin models from human biopsies by isolating and expanding keratinocytes, fibroblasts and microvascular endothelial cells and seeding these components on a collagen matrix or a biological vascularized scaffold matrix in a bioreactor. We then were able to induce inflammation-mediated tissue damage by adding pre-stimulated, mismatched allogeneic lymphocytes and/or inflammatory cytokine-containing supernatants histomorphologically mimicking severe graft versus host disease (GvHD) of the skin. This could be prevented by the addition of immunosuppressants to the models. Consequently, these models harbor a promising potential to serve as a test platform for the prediction, prevention and treatment of GvHD. They also allow functional studies of immune effectors and suppressors including but not limited to allodepleted lymphocytes, gamma-delta T cells, regulatory T cells and mesenchymal stromal cells, which would otherwise be limited to animal models. Thus, the current test platform, developed with the limitation that no professional antigen presenting cells are in place, could greatly reduce animal testing for investigation of novel immune therapies.

Einleitung: Bei der konservativen Behandlung von Verbrennungen kommen sehr häufig Verbandsmaterialien, die eine antibakterielle Wirkung aufweisen, zum Einsatz. Bakterielle Zellulose (BZ) ist chemisch identisch mit pflanzlicher Zellulose, aber frei von Nebenprodukten. Sie weist aber isoliert nur[zum vollständigen Text gelangen Sie über die oben angegebene URL], 9. Mitteldeutscher Wundkongress

Objective: To establish individually expandable primary fibroblast and keratinocyte cultures from 3-mm skin punch biopsies for patient-derived in vitro skin models to investigate of small fiber pathology. Methods: We obtained 6-mm skin punch biopsies from the calf of two patients with small fiber neuropathy (SFN) and two healthy controls. One half (3 mm) was used for diagnostic intraepidermal nerve fiber density (IENFD). From the second half, we isolated and cultured fibroblasts and keratinocytes. Cells were used to generate patient-derived full-thickness three-dimensional (3D) skin models containing a dermal and epidermal component. Cells and skin models were characterized morphologically, immunocyto- and -histochemically (vimentin, cytokeratin (CK)-10, CK 14, ki67, collagen1, and procollagen), and by electrical impedance. Results: Distal IENFD was reduced in the SFN patients (2 fibers/mm each), while IENFD was normal in the controls (8 fibers/mm, 7 fibers/mm). Two-dimensional (2D) cultured skin cells showed normal morphology, adequate viability, and proliferation, and expressed cell-specific markers without relevant difference between SFN patient and healthy control. Using 2D cultured fibroblasts and keratinocytes, we obtained subject-derived 3D skin models. Morphology of the 3D model was analogous to the respective skin biopsy specimens. Both, the dermal and the epidermal layer carried cell-specific markers and showed a homogenous expression of extracellular matrix proteins. Interpretation: Our protocol allows the generation of disease-specific 2D and 3D skin models, which can be used to investigate the cross-talk between skin cells and sensory neurons in small fiber pathology.

Allergic contact dermatitis is critically determined by the ability of haptens to mount an innate immune signal additionally to a directed T cell response. Reconstructed human epidermis (RhE) is widely employed as replacement assay to avoid animal experiments for safety evaluation of proinflammatory effects of new ingredients in industrial products. Unfortunately, RhE lacks responsiveness for metal haptens, the most relevant human contact allergens, raising concerns about their suitability as adequate assay system for allergen testing. Here we investigated whether the defect of RhE might rely on a lack of functional TLR4 expression, which critically governs proinflammatory sensitivity to nickel and cobalt. By comparing RhE to reconstructed human full skin (RhS) we demonstrate that addition of dermal fibroblasts is sufficient to confer proinflammatory responsiveness for those metals. Unlike cultured epidermal keratinocytes, normal human fibroblasts expressed high levels of TLR4 mRNA and triggered production of IL‐8 upon stimulation with nickel, cobalt or the TLR4 agonist LPS. Consistently, dermal isolates from RhS expressed considerable amounts of TLR4 mRNA whereas RhE or epidermis isolated from RhS or normal or inflammatory activated donor skin failed to express substantial TLR4 mRNA. Co‐culture with human DCs similarly was able to license metal responsiveness of RhE, suggesting that integration of naturally TLR4 positive cells can compensate the defect of RhE. Our data suggest the use of RhS or RhE/DC co‐culture models could circumvent current shortcomings of RhE concerning allergen risk assessment and may allow to combine benefits of complex and monoculture‐based test systems in a single assay.

Background: Reconstructed human epidermis (RhE) is widely used to replace animal models in order to assess the proinflammatory and allergenic effects of chemicals. Unfortunately, RhE lacks proinflammatory responsiveness for metal haptens, which are the most prevalent human contact allergens, raising concerns about its reliability for predicting skin allergens. Objectives: To investigate whether this limitation of RhE might be attributable to a lack of functional expression of Toll-like receptor 4 (TLR4), which governs proinflammatory sensitivity to nickel and cobalt. Materials and Methods: RhE, dendritic cell (DC)-containing RhE and full-thickness skin equivalent (FTSE) were compared regarding their proinflammatory responsiveness to metal allergens. Results: The incorporation of dermal fibroblasts was sufficient to confer metal sensitivity to RhE. Unlike keratinocytes, normal human fibroblasts expressed high levels of TLR4 mRNA and induced interleukin-8 expression upon stimulation with nickel or cobalt. Consistently, dermal isolates from FTSE expressed considerable amounts of TLR4 mRNA, whereas RhE or epidermis isolated from FTSE, normal human epidermis or inflamed human epidermis failed to express TLR4. Similarly, co-culture with TLR4-positive DCs bestowed RhE with proinflammatory responsiveness to metals. Conclusion: Our data suggest that FTSE or DC/RhE co-culture models can circumvent the shortcomings of RhE assays, and combine the benefits of complex and monoculture-based test systems in a single assay.

Background: Up to today, animal studies are the gold standard for preclinical testing of new therapeutics for cutaneous wound healing. Alternative in vitro studies are mostly restricted to 2D cell cultures and thus only poorly reflect the complex physiological wound situation. Methods: To overcome this limitation, we present two 3D models based on human primary cells for studying different stages of cutaneous wound healing in vitro. For observation of the reepithelialization, human epidermal models are wounded locally using a biopsy punch and wound healing is monitored. We introduce impedance spectroscopy as a method to determine the efficacy of wound healing non‐destructively via the skin barrier. Results are compared to phase‐contrast microscopy, laser scanning and histology images as well as metabolic activity. For allowing the integration and analysis of deeper wounds, we expanded the models to full thickness skin equivalents with a collagen‐based extracellular matrix including fibroblasts. Results: Upon wounding, the impedance of epidermal models with initial values between 6.3 and 11.4kOcm2 dropped significantly by ‐95%. During on‐line monitoring over fourteen days the barrier recovered, indicated by significant increases in the impedance back to levels between 7.9 and 14.8kOcm2. For full thickness skin equivalents, too, this proved to be a promising screening technique with impedances starting at 276Ocm2 and dropping by ‐63% following wounding. With ongoing wound closure, the barrier recovered to 310Ocm2. Reepithelialization appeared to be donor dependent for both models, correlating well with results from imaging techniques and metabolic activity. Conclusions: Altogether, impedance analysis allows for rapid screening and evaluation of test substances and wound therapies, whereby only a small set of models is needed, cutting the costs of preclinical studies. Finally, with the help of these in vitro models, the amount of animals used in preclinical studies could be reduced.

Background: Two‐dimensional (2D) cell culture and animal ‐models show only limited comparability to the in vivo situation of burn wounds. Therefore, a human burn wound model, which is based on a reconstructed human epidermis (RHE), was developed. Methods: Burn injuries were introduced to RHE (based on an open source protocol developed by Pumay et al.; and refined by Groeber et al.). via contact with an 83°C heated metal rod and examined for up to 14 days subsequently. The properties of models were characterized by viability testing, electrical impedance measurement, measurement of secreted factors in the supernatant, glucose consumption and histological examination. Results: Burnt areas in wound models showed significantly lower viability in MTT tests, compared to surrounding tissue at the wound edges. Furthermore, burning led to significant differences concerning secretion of LDH, glucose consumption and barrier integrity over the course of 14 days in three different donors. Histological examination showed excessive damage of basal and suprabasal cells in the burn area, and ingrowing cells from the wound edges. Treatment of injuries with commercially available products showed promising results regarding wound healing, which were comparable to the in vivo situation. Moreover, in comparison to a non‐thermal wound model significant differences between the different wound models were found, comparable to those of the in vivo situation. Conclusions: Burned RHE models represent a reproducible in vitro test model for burn wounds and their treatment. The models showed symptoms of injury and healing comparable to clinical observation in histological examination, as well as in molecular and physical measurements. They therefore hold the potential to replace, or at least reduce, animal based test procedures.

To protect the human skin from extensive solar radiation, melanocytes produce melanin and disperse it via mela­nosomes to keratinocytes in the basal and suprabasal layers of the human epidermis. Moreover, melanocytes are associated with pathological skin conditions such as vitiligo and psoriasis. Thus, an in vitro skin model that comprises a defined cutaneous pigmentation system is highly relevant in cosmetic, pharmaceutical and medical research. Here, we describe how the epidermal melanin unit can be established in vitro. Primary human melanocytes were introduced into an open source reconstructed epidermis. Following 14 days at the air liquid interface, a differentiated epidermis had formed and the melanocytes were located in the basal layer. The functionality of the epidermal melanin unit could be shown by the transfer of melanin to the surrounding keratinocytes, and a significantly increased melanin content of models stimulated with either UV-radiation or the melanin precursor dihydroxyphenylalanine (DOPA). An UV50 assay was developed to test the protective effect of melanin. In analogy to the IC50 value in risk assessment, the UV50 value facilitates a quantitative investigation of harmful effects of natural UV-radiation to the skin in vitro. Employing this test, we could demonstrate that the melanin content correlates with the resilience against simulated sunlight, which comprises 2.5% UVB and 97.5% UVA. Besides demonstrating the protective effect of melanin in vitro, the assay was used to determine the protective effect of a consumer product in a highly standardized setup.

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 collage n 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.

Despite advances in the development of in-vitro-tissue-models such as reconstructed human epidermis (RHE), the number of endpoints in toxicity-testing, 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 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (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 increase the success of in-vitro-test-methods.