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Gelatin is one of the most prominent biopolymers in biomedical material research and development. It is frequently used in hybrid hydrogels, which combine the advantageous properties of bio‐based and synthetic polymers. To prevent the biological component from leaching out of the hydrogel, the biomolecules can be equipped with azides. Those groups can be used to immobilize gelatin covalently in hydrogels by the highly selective and specific azide-alkyne cycloaddition. In this contribution, we functionalized gelatin with azides at its lysine residues by diazo transfer, which offers the great advantage of only minimal side‐chain extension. Approximately 84-90% of the amino groups are modified as shown by 1H‐NMR spectroscopy, 2,4,6‐trinitrobenzenesulfonic acid assay as well as Fourier‐transform infrared spectroscopy, rheology, and the determination of the isoelectric point. Furthermore, the azido‐functional gelatin is incorporated into hydrogels based on poly(ethylene glycol) diacrylate (PEG‐DA) at different concentrations (0.6, 3.0, and 5.5%). All hydrogels were classified as noncyctotoxic with significantly enhanced cell adhesion of human fibroblasts on their surfaces compared to pure PEG‐DA hydrogels. Thus, the new gelatin derivative is found to be a very promising building block for tailoring the bioactivity of materials.

Human adipose-derived stem cells (hASCs) have become an important cell source for the use in tissue engineering and other medical applications. Not every biomaterial is suitable for human cell culture and requires surface modifications to enable cell adhesion and proliferation. Our hypothesis is that chemical surface modifications introduced by low-discharge plasma enhance the adhesion and proliferation of hASCs. Polystyrene (PS) surfaces were modified either by ammonia (NH3), carbon dioxide (CO2) or acrylic acid (AAc) plasma. The results show that the initial cell adhesion is significantly higher on all modified surfaces than on unmodified material as evaluated by bright field microscopy, live/dead staining, total DNA amount and scanning electron microscopy. The formation of focal adhesions was well pronounced on the Tissue Culture PS, NH3-, and CO2-plasma modified samples. The number of matured fibrillar adhesions was significantly higher on NH3-plasma modified surfaces than on all other surfaces. Our study validates the suitability of chemical plasma activation and represents a method to enhance hASCs adhesion and improved cell expansion. All chemical modification promoted hASCs adhesion and can therefore be used for the modification of different scaffold materials whereby NH3-plasma modified surfaces resulted in the best outcome concerning hASCs adhesion and proliferation.

In recent years, the development and application of decellularized extracellular matrices (ECMs) for use as biomaterials have grown rapidly. These cell-derived matrices (CDMs) represent highly bioactive and biocompatible materials consisting of a complex assembly of biomolecules. Even though CDMs mimic the natural microenvironment of cells in vivo very closely, they still lack specifically addressable functional groups, which are often required to tailor a biomaterial functionality by bioconjugation. To overcome this limitation, metabolic glycoengineering has emerged as a powerful tool to equip CDMs with chemical groups such as azides. These small chemical handles are known for their ability to undergo bioorthogonal click reactions, which represent a desirable reaction type for bioconjugation. However, ECM insolubility makes its processing very challenging. In this contribution, we isolated both the unmodified ECM and azide-modified clickECM by osmotic lysis. In a first step, these matrices were concentrated to remove excessive water from the decellularization step. Next, the hydrogel-like ECM and clickECM films were mechanically fragmentized, resulting in easy to pipette suspensions with fragment sizes ranging from 7.62 to 31.29 mm (as indicated by the mean d90 and d10 values). The biomolecular composition was not impaired as proven by immunohistochemistry. The suspensions were used for the reproducible generation of surface coatings, which proved to be homogeneous in terms of ECM fragment sizes and coating thicknesses (the mean coating thickness was found to be 33.2 ± 7.3 mm). Furthermore, they were stable against fluid-mechanical abrasion in a laminar flow cell. When primary human fibroblasts were cultured on the coated substrates, an increased bioactivity was observed. By conjugating the azides within the clickECM coatings with alkyne-coupled biotin molecules, a bioconjugation platform was obtained, where the biotin-streptavidin interaction could be used. Its applicability was demonstrated by equipping the bioactive clickECM coatings with horseradish peroxidase as a model enzyme.

Although state-of-the-art treatments of respiratory failure clearly have made some progress in terms of survival in patients suffering from severe respiratory system disorders, such as acute respiratory distress syndrome (ARDS), they failed to significantly improve the quality of life in patients with acute or chronic lung failure, including severe acute exacerbations of chronic obstructive pulmonary disease or ARDS as well. Limitations of standard treatment modalities, which largely rely on conventional mechanical ventilation, emphasize the urgent, unmet clinical need for developing novel (bio)artificial respiratory assist devices that provide extracorporeal gas exchange with a focus on direct extracorporeal CO2 removal from the blood. In this review, we discuss some of the novel concepts and critical prerequisites for such respiratory lung assist devices that can be used with an adequate safety profile, in the intensive care setting, as well as for long-term domiciliary therapy in patients with chronic ventilatory failure. Specifically, we describe some of the pivotal steps, such as device miniaturization, passivation of the blood-contacting surfaces by chemical surface modifications, or endothelial cell seeding, all of which are required for converting current lung assist devices into ambulatory lung assist device for long-term use in critically ill patients. Finally, we also discuss some of the risks and challenges for the long-term use of ambulatory miniaturized bioartificial lungs.

Size and function of bioartificial tissue models are still limited due to the lack of blood vessels and dynamic perfusion for nutrient supply. In this study, we evaluated the use of cytocompatible methacryl-modified gelatin for the fabrication of a hydrogel-based tube by dip-coating and subsequent photo-initiated cross-linking. The wall thickness of the tubes and the diameter were tuned by the degree of gelatin methacryl-modification and the number of dipping cycles. The dipping temperature of the gelatin solution was adjusted to achieve low viscous fluids of approximately 0.1 Pa s and was different for gelatin derivatives with different modification degrees. A versatile perfusion bioreactor for the supply of surrounding tissue models was developed, which can be adapted to several geometries and sizes of blood-vessel mimicking tubes. The manufactured bendable gelatin tubes were permeable for water and dissolved substances, like Nile Blue and serum albumin. As a proof of concept, human fibroblasts in a three-dimensional collagen tissue model were successfully supplied with nutrients via the central gelatin tube under dynamic conditions for 2 days. Moreover, the tubes could be used as scaffolds to build-up a functional and viable endothelial layer. Hence, the presented tools can contribute to solving current challenges in tissue engineering.

Artificial adipose tissue (AT) constructs are urgently needed to treat severe wounds, to replace removed tissue, or for the use as in vitro model to screen for potential drugs or study metabolic pathways. The clinical translation of products is mostly prevented by the absence of a vascular component that would allow a sustainable maintenance and an extension of the construct to a relevant size. With this study, we aimed to evaluate the suitability of a novel material based on bacterial cellulose (CBM) on the defined adipogenic differentiation of human adipose-derived stem cells (ASCs) and the maintenance of the received adipocytes (diffASCs) and human microvascular endothelial cells (mvECs) in mono- and coculture. A slight acceleration of adipogenic differentiation over regular tissue culture polystyrene (TCPS) was seen on CBM under defined conditions, whereas on the maintenance of the generated adipocytes, comparable effects were detected for both materials. CBM facilitated the formation of vascular-like structures in monoculture of mvECs, which was not observed on TCPS. By contrast, vascular-like structures were detected in CBM and TCPS in coculture by the presence of diffASCs. Concluding, CBM represents a promising material in vascularized AT engineering with the potential to speed up and simplify the in vitro setup of engineered products.

Vascularized adipose tissue models are highly demanded as alternative to existing animal models to elucidate the mechanisms of widespread diseases, screen for new drugs or asses corresponding safety levels. Standardly used animal-derived sera therein, are associated to ethical concerns, the risk of contaminations and many uncertainties in their composition and impact on cells. Therefore their use should be completely omitted. In this study we developed a serum-free, defined co-culture medium and implemented it to set up an adipocyte-endothelial cell (EC) co-culture model. Human adipose-derived stem cells were differentiated under defined conditions (diffASCs) and, like human microvascular ECs (mvECs), cultured in a developed defined co-culture medium in mono-, indirect or direct co-culture for 14 days. The developed defined co-culture medium was superior to compared mono-culture media and facilitated the functional maintenance and maturation of diffASCs including perilipin A expression, lipid accumulation and glycerol and leptin release. The medium equally allowed mvEC maintenance, confirmed by the expression of CD31 and vWF and acLDL uptake. Thereby mvECs showed a strong dependency on EC-specific factors. Additionally the development of vascular structures by mvECs was facilitated when directly co-cultured with diffASCs. The completely defined co-culture system allows for the serum-free setup of adipocyte/EC co-cultures and thereby represents a valuable and ethically acceptable tool for the setup of vascularized adipose tissue models.

To date, special interest has been paid to composite scaffolds based on polymers enriched with hydroxyapatite (HA). However, the role of HA containing different trace elements such as silicate in the structure of a polymer scaffold has not yet been fully explored. Here, we report the potential use of silicate-containing hydroxyapatite (SiHA) microparticles and microparticle aggregates in the predominant range from 2.23 to 12.40 µm in combination with polycaprolactone (PCL) as a hybrid scaffold with randomly oriented and well-aligned microfibers for regeneration of bone tissue. Chemical and mechanical properties of the developed 3D scaffolds were investigated with XRD, FTIR, EDX and tensile testing. Furthermore, the internal structure and surface morphology of the scaffolds were analyzed using synchrotron X-ray µCT and SEM. Upon culturing human mesenchymal stem cells (hMSC) on PCL-SiHA scaffolds, we found that both SiHA inclusion and microfiber orientation affected cell adhesion. The best hMSCs viability was revealed at 10 day for the PCL-SiHA scaffolds with well-aligned structure (~82%). It is expected that novel hybrid scaffolds of PCL will improve tissue ingrowth in vivo due to hydrophilic SiHA microparticles in combination with randomly oriented and well-aligned PCL microfibers, which mimic the structure of extracellular matrix of bone tissue.

The coculture of osteogenic and angiogenic cells and the resulting paracrine signaling via soluble factors are supposed to be crucial for successfully engineering vascularized bone tissue equivalents. In this study, a coculture system combining primary human adipose-derived stem cells (hASCs) and primary human dermal microvascular endothelial cells (HDMECs) within two types of hydrogels based on methacryloyl-modified gelatin (GM) as three-dimensional scaffolds was examined for its support of tissue specific cell functions. HDMECs, together with hASCs as supporting cells, were encapsulated in soft GM gels and were indirectly cocultured with hASCs encapsulated in stiffer GM hydrogels additionally containing methacrylate-modified hyaluronic acid and hydroxyapatite particles. After 14 days, the hASC in the stiffer gels (constituting the ""bone gels"") expressed matrix proteins like collagen type I and fibronectin, as well as bone-specific proteins osteopontin and alkaline phosphatase. After 14 days of coculture with HDMEC-laden hydrogels, the viscoelastic properties of the bone gels were significantly higher compared with the gels in monoculture. Within the soft vascularization gels, the formed capillary-like networks were significantly longer after 14 days of coculture than the structures in the control gels. In addition, the stability as well as the complexity of the vascular networks was significantly increased by coculture. We discussed and concluded that osteogenic and angiogenic signals from the culture media as well as from cocultured cell types, and tissue-specific hydrogel composition all contribute to stimulate the interplay between osteogenesis and angiogenesis in vitro and are a basis for engineering vascularized bone.

Though bioprinting is a forward-looking approach in bone tissue engineering, the development of bioinks which are on the one hand processable with the chosen printing technique, and on the other hand possess the relevant mechanical as well as osteoconductive features remains a challenge. In the present study, polymer solutions based on methacrylated gelatin and methacrylated hyaluronic acid modified with hydroxyapatite (HAp) particles (5 wt%) were prepared. Encapsulation of primary human adipose-derived stem cells in the HAp-containing gels and culture for 28 d resulted in a storage moduli significantly increased to 126% ± 9.6% compared to the value on day 1 by the sole influence of the HAp. Additional use of osteogenic media components resulted in an increase of storage module up to 199% ± 27.8%. Similarly, the loss moduli was increased to 370% ± 122.1% under the influence of osteogenic media components and HAp. Those changes in rheological material characteristics indicate a distinct change in elastic and viscous hydrogel properties, and are attributed to extensive matrix production in the hydrogels by the encapsulated cells, what could also be proven by staining of bone matrix components like collagen I, fibronectin, alkaline phosphatase and osteopontin. When using the cell-laden polymer solutions as bioinks to build up relevant geometries, the ink showed excellent printability and the printed grid structure's integrity remained intact over a culture time of 28 d. Again, an intense matrix formation as well as upregulation of osteogenic markers by the encapsulated cells could be shown. In conclusion, we demonstrated that our HAp-containing bioinks and hydrogels on basis of methacrylated gelatin and hyaluronic acid are on the one hand highly suitable for the build-up of relevant three-dimensional geometries with microextrusion bioprinting, and on the other hand exhibit a significant positive effect on bone matrix development and remodeling in the hydrogels, as indicated by rheological measurements and staining of bone components. This makes the developed composite hydrogels an excellent material for bone bioprinting approaches.

In vitro cultured cells produce a complex extracellular matrix (ECM) that remains intact after decellularization. The biological complexity derived from the variety of distinct ECM molecules makes these matrices ideal candidates for biomaterials. Biomaterials with the ability to guide cell function are a topic of high interest in biomaterial development. However, these matrices lack specific addressable functional groups, which are often required for their use as a biomaterial. Due to the biological complexity of the cell-derived ECM, it is a challenge to incorporate such functional groups without affecting the integrity of the biomolecules within the ECM. The azide-alkyne cycloaddition (click reaction, Huisgen-reaction) is an efficient and specific ligation reaction that is known to be biocompatible when strained alkynes are used to avoid the use of copper (I) as a catalyst. In our work, the ubiquitous modification of a fibroblast cell-derived ECM with azides was achieved through metabolic oligosaccharide engineering by adding the azide-modified monosaccharide Ac4GalNAz (1,3,4,6-tetra-O-acetyl-N-azidoacetylgalactosamine) to the cell culture medium. The resulting azide-modified network remained intact after removing the cells by lysis and the molecular structure of the ECM proteins was unimpaired after a gentle homogenization process. The biological composition was characterized in order to show that the functionalization does not impair the complexity and integrity of the ECM. The azides within this ""clickECM"" could be accessed by small molecules (such as an alkyne-modified fluorophore) or by surface-bound cyclooctynes to achieve a covalent coating with clickECM.

In vitro composed vascularized adipose tissue is and will continue to be in great demand e.g. for the treatment of extensive high-graded burns or the replacement of tissue after tumor removal. Up to date, the lack of adequate culture conditions, mainly a culture medium, decelerates further achievements. In our study, we evaluated the influence of epidermal growth factor (EGF) and hydrocortisone (HC), often supplemented in endothelial cell (EC) specific media, on the co-culture of adipogenic differentiated adipose-derived stem cells (ASCs) and microvascular endothelial cells (mvECs). In ASCs, EGF and HC are thought to inhibit adipogenic differentiation and have lipolytic activities. Our results showed that in indirect co-culture for 14 days, adipogenic differentiated ASCs further incorporated lipids and partly gained an univacuolar morphology when kept in media with low levels of EGF and HC. In media with high EGF and HC levels, cells did not incorporate further lipids, on the contrary, cells without lipid droplets appeared. Glycerol release, to measure lipolysis, also increased with elevated amounts of EGF and HC in the culture medium. Adipogenic differentiated ASCs were able to release leptin in all setups. MvECs were functional and expressed the cell specific markers, CD31 and von Willebrand factor (vWF), independent of the EGF and HC content as long as further EC specific factors were present. Taken together, our study demonstrates that adipogenic differentiated ASCs can be successfully co-cultured with mvECs in a culture medium containing low or no amounts of EGF and HC, as long as further endothelial cell and adipocyte specific factors are available.

Blood vessel reconstruction is still an elusive goal for the development of in vitro models as well as artificial vascular grafts. In this study, we used a novel photo-curable cytocompatible polyacrylate material (PA) for freeform generation of synthetic vessels. We applied stereolithography for the fabrication of arbitrary 3D tubular structures with total dimensions in the centimeter range, 300 µm wall thickness, inner diameters of 1 to 2 mm and defined pores with a constant diameter of approximately 100 µm or 200 µm. We established a rinsing protocol to remove remaining cytotoxic substances from the photo-cured PA and applied thio-modified heparin and RGDC-peptides to functionalize the PA surface for enhanced endothelial cell adhesion. A rotating seeding procedure was introduced to ensure homogenous endothelial monolayer formation at the inner luminal tube wall. We showed that endothelial cells stayed viable and adherent and aligned along the medium flow under fluid-flow conditions comparable to native capillaries. The combined technology approach comprising of freeform additive manufacturing (AM), biomimetic design, cytocompatible materials which are applicable to AM, and biofunctionalization of AM constructs has been introduced as BioRap(®) technology by the authors.

The composition of vascularized adipose tissue is still an ongoing challenge as no culture medium is available to supply adipocytes and endothelial cells appropriately. Endothelial cell medium is typically supplemented with epidermal growth factor (EGF) as well as hydrocortisone (HC). The effect of EGF on adipocytes is discussed controversially. Some studies say it inhibits adipocyte differentiation while others reported of improved adipocyte lipogenesis. HC is known to have lipolytic activities, which might result in mature adipocyte dedifferentiation. In this study, we evaluated the influence of EGF and HC on the co-culture of endothelial cells and mature adipocytes regarding their cell morphology and functionality. We showed in mono-culture that high levels of HC promoted dedifferentiation and proliferation of mature adipocytes, whereas EGF seemed to have no negative influence. Endothelial cells kept their typical cobblestone morphology and showed a proliferation rate comparable to the control independent of EGF and HC concentration. In co-culture, HC promoted dedifferentiation of mature adipocytes, which was shown by a higher glycerol release. EGF had no negative impact on adipocyte morphology. No negative impact on endothelial cell morphology and functionality could be seen with reduced EGF and HC supplementation in co-culture with mature adipocytes. Taken together, our results demonstrate that reduced levels of HC are needed for co-culturing mature adipocytes and endothelial cells. In co-culture, EGF had no influence on mature adipocytes. Therefore, for the composition of vascularized adipose tissue constructs, the media with low levels of HC and high or low levels of EGF can be used.

The extracellular matrix (ECM) is a complex three-dimensional network of biomolecules that surround the cells within a human tissue. The ECM is tissue-specific and is considered the natural environment of the cells. Due to the high biological activity, various types of ECM are able to promote cell adhesion, proliferation and differentiation in a tissue specific manner [1]. Primary cells are capable of producing an ECM in vitro, which can be isolated after several days of culture [2]. Due to its diverse functions, the isolated ECM is a promising biomaterial for use in tissue engineering and regenerative medicine. However, if this isolated ECM is used as a biomaterial for surface coatings, a major limitation is the lack of mechanical stability, since physisorption is the primary source of adhesion between the molecules [3]. To overcome this limitation our approach was to covalently immobilize the human ECM on artificial surfaces. Therefore, we performed metabolic oligosaccharide engineering (MOE) to introduce click groups into the glycan structures of the ECM. Here we demonstrate that this method is suitable to fabricate a ""clickable"" human ECM (clickECM) which can be covalently immobilized on click-functionalized artificial surfaces via copper-free click reaction [4] (Fig. 1). We cultured primary human dermal fibroblasts over a period of six days to generate a human dermal ECM. By treating the cells with an azidemodified monosaccharide (Ac4GalNAz), we were able to introduce click functionalities into the glycans of the ECM. Histochemical and immunofluorescence analysis were performed to characterize the biological composition of the resulting clickECM in comparison with untreated ECM and human skin dermis. Then, artificial surfaces were functionalized with activated alkynes for the covalent immobilization of clickECM. Coating of unmodified ECM via physisorption served as a control. The stability of these coatings was compared by calculating the percentage area covered with ECM before and after an intensive washing step in detergent-supplemented buffer. The bioactive properties of these coatings were evaluated by quantifying the cell proliferation of HaCaT cells seeded on either clickECM-coated or uncoated glass substrates. We could show that MOE can be used to introduce click groups into the ECM of human dermal fibroblasts. This clickECM is comparable to the unmodified ECM and to human skin dermis in terms of the distribution of glycans and typical ECM components. The clickECM can be covalently immobilized on alkyne-modified surfaces resulting in a significant increase in coating stability compared to a conventional ECM coating via physisorption (Fig. 2). Cell proliferation of HaCaT cells was significantly enhanced on the clickECM-coated surfaces compared to uncoated glass substrates. These results demonstrate that the covalent immobilization provides high stability, while also preserving the biological activity of the human ECM. We therefore propose that clickECM is a promising biomaterial for the use in in tissue engineering and regenerative medicine e. g. as a tissue-specific coating material to mimic the natural environment of cells.

In vitro engineering of autologous fatty tissue constructs is still a major challenge for the treatment of congenital deformities, tumor resections or high-graded burns. In this study, we evaluated the suitability of photo-crosslinkable methacrylated gelatin (GM) and mature adipocytes as components for the composition of three-dimensional fatty tissue constructs. Cytocompatibility evaluations of the GM and the photoinitiator Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) showed no cytotoxicity in the relevant range of concentrations. Matrix stiffness of cell-laden hydrogels was adjusted to native fatty tissue by tuning the degree of crosslinking and was shown to be comparable to that of native fatty tissue. Mature adipocytes were then cultured for 14 days within the GM resulting in a fatty tissue construct loaded with viable cells expressing cell markers perilipin A and laminin. This work demonstrates that mature adipocytes are a highly valuable cell source for the composition of fatty tissue equivalents in vitro. Photo-crosslinkable methacrylated gelatin is an excellent tissue scaffold and a promising bioink for new printing techniques due to its biocompatibility and tunable properties.

For the fabrication of bone tissue equivalents via tissue engineering and bioprinting approaches, one crucial element is the used biomaterial which on the one hand should support and enhance the function of the used cells, and on the other hand guarantee processability with the chosen manufacturing technique. The use of gelatin-based hydrogels for cell encapsulation provides the positive properties of the base material collagen, and offers the possibility of adjusting composition as well as characteristics like viscosity and gelling behavior [1,2]. Further modification of the material with components like hydroxyapatite (HAp) was shown to have a positive impact on processability with bioprinting approaches and properties of the material as well as its bioactivity [3,4,5]. In the current study, bioinks based on methacrylated gelatin (GM) and modified with HAp particles were used and analyzed for said parameters. The viscosity of the bioinks was measured, as well as water uptake by the hydrogels and the gels' mechanical properties. Human mesenchymal stem cells from adipose tissue (hASCs) of three donors were encapsulated in differently modified GM hydrogels, and matrix development during culture for 28 days in standard and osteogenic media was examined. To prove the development of a bone matrix, the mechanical properties of the hydrogels were analyzed, and the hydrogels were stained for bone matrix markers like collagen type I and osteocalcin. Further analysis of matrix composition was conducted via Raman spectroscopy. The modification with HAp led to improved properties of the bioinks as well as the resulting hydrogels, as being shown by increased ink viscosity and mechanical properties of the hydrogels. The degree of water uptake, however, was not influenced. The staining of the hASC-laden hydrogels cultured under osteogenic conditions showed an increased deposition of bone components like collagen and non-collagenous proteins like osteocalcin in the modified hydrogels compared to the control GM gels after 28 days of culture. Surprisingly, this increase was also seen in the HAp-containing gels cultured under standard conditions (Fig. 1A), what illustrated the significant positive impact of the added HAp onto osteogenesis. Similarly, the spectroscopic analysis of said hydrogels on day 1 and day 28 of culture revealed peaks resulting from typical bone components like the collagen-related amino-acid proline (853/869/1067 cm-1) or carbonate which is found in the biological mineral phase of bone (1074 cm-1) (Fig. 1B). The developing bone matrix could also be detected by a rising storage module of the gels, resulting in an increase between 25% and 100% compared to day 1 (Fig. 1C) and representing the mechanical strength of the newly formed matrix. In the current study, we could show the suitability of a GM-based material for the use in bone tissue engineering. The properties of the analyzed inks and resulting hydrogels could be modified by the addition of HAp, and the osteoinductive impact of the added mineral phase leading to a significant increase in osteogenesis in the hydrogels was shown. We therefore anticipate that the material will be highly suitable as a bioink for the bioprinting of bone tissue equivalents.

White adipose tissue (WAT) comprises about one fourth of the human body, interacts with many other organs via paracrine and endocrine signals and sequestrates lipophilic substances. Thereby it impacts distribution and levels of drugs [1]. Additionally WAT is the origin of different diseases and thus target of several drugs addressing those [2]. Therefore an adipose tissue testsystem is highly needed to analyze fundamental biological issues in physiological or diseased state, screen for potential drugs or create affiliated safety profiles. Additionally adipose tissue substitutes are highly desired to treat lost, deformed or burned subcutaneous fatty tissue [3]. In several promising attempts substantial progress was achieved e.g. by the encapsulation of adipocytes in a 3D environment with native matrix components like gelatine [4] or the optimization of media composition [5]. However, regardless of its potential in vivo or in vitro application, an adipose tissue model has to fulfill two main requirements which could not be implemented so far. First, the model has to show in vitro stability for a period of several weeks to enable its maturation and time-dependent investigations. Second, culture conditions have to be free of animal derived components or preferably completely defined to exclude possible impairments through unknown constituents and allow for GMP-compliant production. In this approach a novel cellulose based matrix and a specific media supplementation are combined to achieve defined adipocyte differentiation and long term maintenance. Human primary adipose-derived stem cells (ASCs) were isolated, characterized and expanded xenofree. ASCs were next to tissue culture polystyrol applied to a collagen coated surface and the cellulose-based matrix. Adipogenic differentiation of ASCs was induced by addition of a composed defined differentiation medium and continued for 14 days. On day 14 medium was switched to a composed defined adipocyte maintenance medium. Obtained adipocytes were subsequently cultured for additional 28 days. Preservation of adipocyte characteristics were evaluated by immunofluorescence staining of specific markers like perilipin A and the quantitative analysis of stored lipids. Retained adipocyte metabolism was determined by released leptin, lipolysis by released glycerol levels. Our results classify the cellulose-based matrix as a useful tool to realize and optimize defined adipogenic differentiation of ASCs and the subsequent adipocyte maintenance. The matrix constitution enables strong cell matrix interaction and thereby strengthens cell adherence which is often diminished under defined culture conditions. Due to matrix-induced cell quiescence adipocytes' long-term stability was enhanced. Based on matrix-derived support successful differentiation and maintenance were confirmed via the composed media under completely defined conditions. Therefore the cellulose-based matrix is a promising biomaterial for stable long-term culture of quiescent cells with preservation of cell specific functions and characteristics which could as well be applied in other setups. Consecutive defined adipose tissue models could be further expanded e.g. to co-culture models and used as testsystems or tissue substitutes.

Perivascular cells are multilineage cells located around the vessel wall and important for wall stabilization. In this study, we evaluated a stem cell media and a perivascular cell-specific media for the culture of primary perivascular cells regarding their cell morphology, doubling time, stem cell properties, and expression of cell type-specific markers. When the two cell culture media were compared to each other, perivascular cells cultured in the stem cell medium had a more elongated morphology and a faster doubling rate and cells cultured in the pericyte medium had a more typical morphology, with several filopodia, and a slower doubling rate. To evaluate stem cell properties, perivascular cells, CD146− cells, and mesenchymal stem cells (MSCs) were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. It was seen that perivascular cells, as well as CD146− cells and MSCs, cultured in stem cell medium showed greater differentiation than cells cultured in pericyte-specific medium. The expression of pericyte-specific markers CD146, neural/glial antigen 2 (NG2), platelet-derived growth factor receptor-v (PDGFR-v), myosin, and a-smooth muscle actin (a-SMA) could be found in both pericyte cultures, as well as to varying amounts in CD146− cells, MSCs, and endothelial cells. The here presented work shows that perivascular cells can adapt to their in vitro environment and cell culture conditions influence cell functionality, such as doubling rate or differentiation behavior. Pericyte-specific markers were shown to be expressed also from cells other than perivascular cells. We can further conclude that CD146+ perivascular cells are inhomogeneous cell population probably containing stem cell subpopulations, which are located perivascular around capillaries.

Einleitung: Patienten mit schweren Lungenschäden, wie sie beispielsweise bei der chronisch-obstruktiven Lungenerkrankung auftreten, sind auf den Einsatz von Membranoxygenatoren zur Oxygenierung und Kohlenstoffdioxidentfernung angewiesen. Standardmäßig werden hierzu Hohlfasermembranen aus Polymeren, z.B. Polymethylpenten (PMP), eingesetzt, an deren Oberfläche der Gasaustausch stattfindet. Aufgrund der fehlenden antithrombogenen Eigenschaften des Fasermaterials kommt es jedoch zur Thrombenbildung, welche wiederum die Gasaustauschrate und damit die Lebensdauer der Membran verringert. Eine Endothelialisierung der Faseroberfläche soll daher antithrombogene Eigenschaften vermitteln. Materialien und Methoden: Aus Hautbiopsaten isolierte humane dermale mikrovaskuläre Endothelzellen (HDMECs) wurden auf die beschichteten PMP-Fasermatten ausgesät. Im Anschluss an die Adhäsion der Zellen, wurden die Fasermatten in einem Flussreaktor unter dynamischen Bedingungen kultiviert. Die Besiedlung der Fasern, sowie die Viabilität der Zellen, die Integrität des Zellmonolayers und die Expression endothelzell-spezifischer Marker wurde analysiert. Hierzu fand eine Lebend-Tot-Färbung mittels Fluoresceindiacetat und Propidiumiodid (FDA/PI), sowie Immunfluoreszenzfärbungen von vaskulärem endothelialem Cadherin (VE-Cadherin) und PECAM-1 statt. Ergebnisse und Diskussion: Mittels FDA/PI-Färbung wurde direkt im Anschluss an die Adhäsionszeit sowie nach 7- bis 14-tägiger dynamischer Kultur nachgewiesen, dass die Bedeckung großer Teile der Faseroberfläche mit viablen Zellen erreicht werden konnte. Die Zellen zeigten die Expression der Proteine VECadherin und PECAM-1, welche wichtige Bestandteile der Zell-Zell-Kontakte innerhalb des Endothels darstellen und damit dessen Integrität - die Voraussetzung für die Antithrombogenität - widerspiegeln. Im Gegensatz zu früheren Studien konnte in den vorliegenden Versuchen die dynamische Kultivierung primärer humaner Endothelzellen auf beschichteten PMP-Fasern verbessert werden. Eine dynamische Kultivierung unter physiologischen Flussbedingungen war ebenfalls möglich.

The establishment of adipose tissue test systems is still a major challenge in the investigation of cellular and molecular interactions responsible for the pathogenesis of inflammatory diseases involving adipose tissue. Mature adipocytes are mainly involved in these pathologies, but rarely used in vitro, due to the lack of an appropriate culture medium which inhibits dedifferentiation and maintains adipocyte functionality. In our study, we showed that Dulbecco's Modified Eagle's Medium/Ham's F-12 with 10% fetal calf serum (FCS) reported for the culture of mature adipocytes favors dedifferentiation, which was accompanied by a high glycerol release, a decreasing release of leptin, and a low expression of the adipocyte marker perilipin A, but high expression of CD73 after 21 days. Optimized media containing FCS, biotin, pantothenate, insulin, and dexamethasone decelerated the dedifferentiation process. These cells showed a lower lipolysis rate, a high level of leptin release, as well as a high expression of perilipin A. CD73-positive dedifferentiated fat cells were only found in low quantity. In this work, we showed that mature adipocytes when cultured under optimized conditions could be highly valuable for adipose tissue engineering in vitro.

Introduction: COPD is the 4th leading cause of death worldwide. Extracorporeal lung assist devices that promote oxygenation and /or CO2 removal can improve lung protection and increase quality of life. As a drawback these systems limit patient mobility. In addition, long-term use of these devices is frequently accompanied by thromboembolic complications and device fouling due to inappropriate material properties of the blood contacting surfaces. Our goal is to develop the first wearable miniaturized lung assist device with improved hemocompatible surfaces. Methods: We designed prototypes of new, miniaturized hardware components with an optimized geometry to minimize hemolysis and thrombogenicity. To improve the hemocompatibility of the gas exchange membranes polymethlypentene (PMP), we seeded human dermal endothelial cells onto PMP hollow fibers, either coated with heparin/albumin, or following covalent functionalization with heparin/ REDV. Seeding efficiency and cell coverage were quantified microscopically. Results: We successfully miniaturized all hardware components and developed a new design of the gas exchanger, yielding improved blood distribution concomitant with adequate gas exchange in vitro and in vivo. Following seeding of stacked PMP fiber mats, the endothelial cells formed a confluent monolayer on the fibers, which was maintained for 5 days on the heparin/REDV coated fibers under both static and dynamic in vitro testing conditions in a novel perfusion bioreactor system. Conclusion: We successfully designed and implemented an advanced concept of a miniaturized wearable lung assist device, which is currently being tested in a porcine model. Future studies will test these devices in a first-in-man trial.

Engineering of large vascularized adipose tissue constructs is still a challenge for the treatment of extensive high-graded burns or the replacement of tissue after tumor removal. Communication between mature adipocytes and endothelial cells is important for homeostasis and the maintenance of adipose tissue mass but, to date, is mainly neglected in tissue engineering strategies. Thus, new co-culture strategies are needed to integrate adipocytes and endothelial cells successfully into a functional construct. This review focuses on the cross-talk of mature adipocytes and endothelial cells and considers their influence on fatty acid metabolism and vascular tone. In addition, the properties and challenges with regard to these two cell types for vascularized tissue engineering are highlighted.

Bone homeostasis is maintained by osteoblasts (bone formation) and osteoclasts (bone resorption). While there have been numerous studies investigating mesenchymal stem cells and their potential to differentiate into osteoblasts as well as their interaction with different bone substitute materials, there is only limited knowledge concerning in vitro generated osteoclasts. Due to the increasing development of degradable bone-grafting materials and the need of sophisticated in vitro test methods, it is essential to gain deeper insight into the process of osteoclastogenesis and the resorption functionality of human osteoclasts. Therefore, we focused on the comparison of osteoclastogenesis and resorption activity on tissue culture polystyrene (TCPS) and bovine extracellular bone matrices (BMs). Cortical bone slices were used as two-dimensional (2D) substrates, whereas a thermally treated cancellous bone matrix was used for three-dimensional (3D) experiments. We isolated primary human monocytes and induced osteoclastogenesis by medium supplementation. Subsequently, the expression of the vitronectin receptor (V3) and cathepsin K as well as the characteristic actin formation on TCPS and the two BMs were examined. The cell area of human osteoclasts was analyzed on TCPS and on BMs, whereas significantly larger osteoclasts could be detected on BMs. Additionally, we compared the diameter of the sealing zones with the measured diameter of the resorption pits on the BMs and revealed similar diameters of the sealing zones and the resorption pits. We conclude that using TCPS as culture substrate does not affect the expression of osteoclast-specific markers. The analysis of resorption activity can successfully be conducted on cortical as well as on cancellous bone matrices. For new in vitro test systems concerning bone resorption, we suggest the establishment of a 2D assay for high throughput screening of new degradable bone substitute materials with osteoclasts.

Sunlight has various effects on human health. Several important metabolic processes are only enabled by sunlight. But longtime sun bathing and extended outdoor activities can cause skin irritation, inflammation or even skin cancer due to high radiation dose. We developed in vitro skin models of different complexity to investigate UV-light associated skin damage. Substances and their phototoxic, sun protective or photo-sensitizing potential can be analyzed to prevent white skin cancer.

Introduction: In the field of orthopaedics and traumatology, the improvement of implant materials plays a crucial role for a better treatment of bone defects. Newly developed biodegradable materials have the potential to provide enhanced mechanical properties and reduce the need for implant removal surgery. The initial evaluation of newly designed materials in vitro to predict the outcome in vivo is strongly desired. Therefore, to approach the in vivo situation we established co-culture conditions for primary human mesenchymal stem cells (hMSCs) and monocytes (MCs) to address the bone remodelling process. Our aim of this study was the evaluation of the osteoclastogenesis of monocytes in consequence to the interaction and cross talk with hMSCs without any differentiation factors by media supplementation. Materials and Methods: Human monocytes were isolated of buffy coats and were characterized by FACS analysis, histochemical staining and differentiation studies. For co-culture experiments, hMSCs were seeded either on tissue culture plates (TCPS) or on bovine cortical bone slices. Two set ups were considered: proliferative media and the induction of the osteogenic differentiation for 14 days. Subsequently, monocytes were seeded on top of the pre-cultured hMSCs and were evaluated after another 14 days for an osteoclastic phenotype by immunofluorescence staining and REM analysis. Results and Discussion / References: MCs were successfully differentiated to osteoclasts by media supplementation including RANKL and M-CSF. Co-culture of hMSCs and MCs led to the expression of an osteoclastic phenotype after 14 days. Cathepsin K, VNR and Actin/Vinculin were expressed and analyzed by immunofluorescence staining revealing multinucleated cells within the co-culture set up. The co-culture of hMSCs and hMCs lead to an osteoclastic phenotype after 14 days. The interaction of both cell types is therefore sufficient to generate osteoclast like cells. The bone remodelling process can be simulated in vitro by using monocytes and mesenchymal stem cells. However, the activity of the osteoclasts must be further investigated e.g. by the detection of resorption pits in order to test efficiently newly designed materials. The coculture could further be transferred into a three-dimensional setting to consider the influence of a multilayer culture. This work was supported by the Fraunhofer Internal Programs under Grant No. MAVO 824682 (DegraLast).

Introduction: Patienten mit schweren Lungenschäden, wie sie beispielsweise bei der chronisch-obstruktiven Lungenerkrankung auftreten, sind auf den Einsatz von Membranoxygenatoren zur Oxygenierung und Kohlenstoffdioxidentfernung angewiesen. Standardmäßig werden hierzu Hohlfasermembranen aus Polymeren, z.B. Polymethylpenten (PMP), eingesetzt, an deren Oberfläche der Gasaustausch stattfindet. Aufgrund der fehlenden antithrombogenen Eigenschaften des Fasermaterials kommt es jedoch zur Thrombenbildung, welche wiederum die Gasaustauschrate und damit die Lebensdauer der Membran verringert. Eine Endothelialisierung der Faseroberfläche soll daher antithrombogene Eigenschaften vermitteln1. Materials and Methods: Aus Hautbiopsaten isolierte humane dermale mikrovaskuläre Endothelzellen (HDMECs) wurden auf der Ober- und der Unterseite einer unmodifizierten PMP-Fasermatte ausgesät. Im Anschluss an die Adhäsion der Zellen, welche unter statischen Zellkulturbedingungen stattfand, wurde die Fasermatte in einen Flussreaktor eingespannt, und eine dynamische Kultivierung der Zellen für zwei bis sieben Tage fand statt. Eine weitere besiedelte Fasermatte wurde parallel als Kontrolle statisch kultiviert. Anschließend wurde die Besiedlung der Fasern, sowie die Viabilität der Zellen, ihre Proliferation, die metabolische Aktivität, die Integrität des Zellmonolayers und die Expression endothelzell-spezifischer Marker analysiert. Hierzu fand eine Lebend-Tot-Färbung mittels Fluoresceindiacetat und Propidiumiodid (FDA/PI), die Färbung des Proliferationsmarkers Ki-67, die Analyse der Metabolisierung des Tetrazoliums MTT, sowie Immunfluoreszenzfärbungen von vaskulärem endothelialem Cadherin (VE-Cadherin) und PECAM-1 statt. Der Anteil der besiedelten Faseroberfläche an der Gesamtoberfläche der Fasermatte wurde mittels einer FDA/PI-Färbung und anschließender Auswertung mit ImageJ bestimmt. Results and Discussion / References: Mittels FDA/PI-Färbung und MTT-Assay wurde direkt im Anschluss an die Adhäsionszeit nachgewiesen, dass die Bedeckung großer Teile der Faseroberfläche mit viablen Zellen erreicht werden konnte. Nach der dynamischen Kultivierung war die Zelldichte auf den Fasern reduziert, was auf die mechanische Beanspruchung der Zellen durch den Medienfluss zurückzuführen ist. Der Vergleich beider Seiten der Fasermatte nach FDA/PI-Färbung zeigte jedoch, dass ein Ablösen der Zellen lediglich von der durch Zellkulturmedium direkt angeströmten Seite der Fasern stattfand, während es auf der entgegengesetzten Seite der Fasern über die Kultivierungszeit hinweg zu einer Proliferation der Zellen kam. Sowohl nach statischer, als auch nach dynamischer Kultivierung zeigten die Zellen die Expression der Proteine VE-Cadherin und PECAM-1, welche wichtige Bestandteile der Zell-Zell-Kontakte innerhalb des Endothels darstellen und damit dessen Integrität - die Voraussetzung für die Antithrombogenität - widerspiegeln. Im Gegensatz zu früheren Studien2-4 konnte in den vorliegenden Versuchen die erfolgreiche statische Kultivierung primärer humaner Endothelzellen auf PMP-Fasern ohne vorherige Oberflächenmodifikation gezeigt werden. Auch eine dynamische Kultivierung war möglich, es zeigte sich im Vergleich zur statischen Kultur jedoch eine signifikant reduzierte Zellzahl, was auf eine eingeschränkte Zelladhäsion auf dem Fasermaterial schließen lässt. Dieser Umstand macht eine Oberflächenfunktionalisierung der PMP-Fasern notwendig, welche auch unter dynamischen Flussbedingungen die Ausbildung eines antithrombogenen Endothelzell-Monolayers erlaubt.

Introduction: Ther is an urgent need of large fatty tissue constructs for clinical applications. They are necessary if the subcutis is damaged or lacking due to high-graded burns, congenital deformities, tumor resection or trauma. To date, no suitable replacement is available; autologous fatty tissue transfer results in a high shrinkage of the tissue1. Also the use of adipogen differentiated stem cells has not shown the requested long term functionality and differentiation2. Our strategy is to build up a subcutis equivalent by using mature adipocytes in a chemically modified gelatin matrix, which allows an easy and fast tissue composition Materials and Methods: For the generation of 3D constructs, bovine gelatin was methacrylated3. Cytotoxicity of methacrylated gelatin and the non-cured and cured photoinitiator lithium acylphosphinate (LAP) was determined. An isolation method for mature adipocytes from human fatty tissue was established. These mature adipocytes were characterized in a 2D culture by comparison to differentiated adipose-derived stem cells (ASCs) regarding cell morphology, size and lipid accumulation. Adipocytes were encapsulated into methacrylated gelatin and cultured for several weeks. The elasticity of the constructs was adapted to that of native fatty tissue which was evaluated performing rheological measurements. Viable cells were detected by staining of lipid droplets with Nile Red, nuclei with Hoechst 33342 and cytoplasm with fluorescein diacetate (FDA). Furthermore, H&E staining and the staining of perilipin A and collagen IV were performed on paraffin sections. Cell functionality was determined by glycerol and leptin release. Results and Discussion / References: Methacrylated gelatin as well as the non-cured and cured photoinitiator LAP showed no cytotoxicity tested with adipocytes. Isolated mature adipocytes showed a different morphology, cell size and lipid accumulation pattern compared to differentiated ASCs. A method for the encapsulation of mature adipocytes into methacrylated gelatin was successfully established. The elasticity of the subcutis equivalents was comparable to that of native fatty tissue which was seen in rheological measurements. Cells stayed viable and functional for up to 14 days, which was shown by FDA staining, glycerol and leptin release as well as perilipin A and collagen IV stainings. Conclusion: We were able to show that human adipocytes are a suitable cell type for buildup 3D subcutis equivalents. Methacrylated gelatin is an excellent matrix due to its biocompatibility and tunable properties. Our long-term goal is the composition of large subcutis constructs supplied by a vascular system.

Introduction: Patients with third degree burn wounds would benefit from a three-layer skin graft consisting of the epidermis, dermis and hypodermis. As part of the ArtiVasc 3D EU project we are trying to come closer to this goal and to generate a vascularized 3D skin substitute. Materials and methods: To generate the hypodermis a construct of hydrogels and electrospun matrices will be produced and then seeded with adipose derived stem cells (ASCs) and adipocytes. Results: ASCs adhered and proliferated on the matrices. After 21 days fat vacuoles could be detected as a sign of successful differentiation of ASCs. The use of bioreactors additionally improved the viability of cells within the hydrogel. Discussion and conclusions: The combination of hydrogels and electrospun matrices represents a promising approach for the generation of adipose tissue. Acknowledgments: The research was funded by the European Union Seventh Framework Programme (FP7) through the ArtiVasc 3D project under grant agreement no. 263416. Disclosures: The authors have nothing to disclose.

Introduction: The development and improvement of new materials for bone tissue engineering is desired due to the restricted availability of autologous material and improvable material characteristics of available alloplastic materials1. To test the potential of newly designed materials and/or modifications, the adhesion, proliferation and differentiation of human mesenchymal stem cells (hMSCs) can be used to perform in vitro studies. In this study we evaluated the adhesion velocity of hMSCs on different modified materials as titan, iron composites and polystyrene to estimate the modification strategy. Materials and Methods: hMSCs were isolated of bone marrow and were characterized thoroughly by FACS analysis, histochemical staining and qRT-PCR studies concerning their osteogenic differentiation potential. Cell adhesion on the different modified materials was evaluated by immunological staining for actin and vinculin at different time points. The osteogenic differentiation of the cells on selected modifications was detected by quantitative measurements of the alkaline phosphatase as well as determining the total DNA volume of adherent cells. Results and Discussion / References: Modified surfaces revealed an influence on cell adhesion. An increase of adherent cells was detected after 24 hours and 48 hours of cell culture. Furthermore, an increase of ALP was seen in differentiated cells after 14 days of osteogenic induction. The results of this study indicate the importance of appropriate surface characteristics for cell adhesion and differentiation. In vitro testing of newly developed materials/surfaces is an important tool to reduce animal testing and must be further developed to acheive reproducible and significant test methods with primary cells.

The future vision of implants comprises individually tailored prostheses and the generation of artificial tissue and organs generated from the patient's own cells. In order to develop artificial, biomimetic structures which perform as well as natural ones, we need fabrication processes that do not set any limits to the generation of shapes, and materials that allow for tailoring of their physical, chemical, and biological properties. We introduce new biocompatible materials for the manufacturing of flexible structures by freeform fabrication.

The control of surface properties is a substantial step in the development and improvement of biomaterials for clinical applications as well as for their use in tissue engineering. Interaction of the substrate surface with the biochemical or biological environment is crucial for the outcome of the applied biomaterial and therefore should meet specific requirements regarding the chemical composition, wettability, elasticity, and charge. In this study, we examined the effect of chemical groups introduced by low pressure plasma treatments of polystyrene surfaces on the cell behavior of primary human mesenchymal stem cells (hMSCs) and dermal microvascular endothelial cells (hDMECs). X-ray photoelectron spectroscopy analysis and contact angle measurements were employed to evaluate ammonia-, carbon dioxide-, and acrylic acid-plasma modifications to substrate surfaces. HMSCs and hDMECs were analyzed simultaneously to identify the most suitable surface functionalization for each cell type. Significantly higher cell proliferation was detected on ammonia plasma-treated surfaces. Cell-material interaction could be shown on all created interfaces as well as the expression of typical cell markers. Hence, the applied plasma treatment presents a suitable tool to improve culture condition on polystyrene for two important cell types (hMSCs and hDMECs) in the field of tissue engineering.