TY - JOUR A1 - Hauptstein, Julia A1 - Forster, Leonard A1 - Nadernezhad, Ali A1 - Horder, Hannes A1 - Stahlhut, Philipp A1 - Groll, Jürgen A1 - Blunk, Torsten A1 - Teßmar, Jörg T1 - Bioink Platform Utilizing Dual-Stage Crosslinking of Hyaluronic Acid Tailored for Chondrogenic Differentiation of Mesenchymal Stromal Cells JF - Macromolecular Bioscience N2 - 3D bioprinting often involves application of highly concentrated polymeric bioinks to enable fabrication of stable cell-hydrogel constructs, although poor cell survival, compromised stem cell differentiation, and an inhomogeneous distribution of newly produced extracellular matrix (ECM) are frequently observed. Therefore, this study presents a bioink platform using a new versatile dual-stage crosslinking approach based on thiolated hyaluronic acid (HA-SH), which not only provides stand-alone 3D printability but also facilitates effective chondrogenic differentiation of mesenchymal stromal cells. A range of HA-SH with different molecular weights is synthesized and crosslinked with acrylated (PEG-diacryl) and allylated (PEG-diallyl) polyethylene glycol in a two-step reaction scheme. The initial Michael addition is used to achieve ink printability, followed by UV-mediated thiol–ene reaction to stabilize the printed bioink for long-term cell culture. Bioinks with high molecular weight HA-SH (>200 kDa) require comparably low polymer content to facilitate bioprinting. This leads to superior quality of cartilaginous constructs which possess a coherent ECM and a strongly increased stiffness of long-term cultured constructs. The dual-stage system may serve as an example to design platforms using two independent crosslinking reactions at one functional group, which allows adjusting printability as well as material and biological properties of bioinks. KW - hyaluronic acid KW - biofabrication KW - chondrogenic differentiation KW - dual-stage crosslinking KW - extracellular matrix Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-257556 VL - 22 IS - 2 ER - TY - JOUR A1 - Hauptstein, Julia A1 - Forster, Leonard A1 - Nadernezhad, Ali A1 - Groll, Jürgen A1 - Teßmar, Jörg A1 - Blunk, Torsten T1 - Tethered TGF-β1 in a hyaluronic acid-based bioink for bioprinting cartilaginous tissues JF - International Journal of Molecular Sciences N2 - In 3D bioprinting for cartilage regeneration, bioinks that support chondrogenic development are of key importance. Growth factors covalently bound in non-printable hydrogels have been shown to effectively promote chondrogenesis. However, studies that investigate the functionality of tethered growth factors within 3D printable bioinks are still lacking. Therefore, in this study, we established a dual-stage crosslinked hyaluronic acid-based bioink that enabled covalent tethering of transforming growth factor-beta 1 (TGF-β1). Bone marrow-derived mesenchymal stromal cells (MSCs) were cultured over three weeks in vitro, and chondrogenic differentiation of MSCs within bioink constructs with tethered TGF-β1 was markedly enhanced, as compared to constructs with non-covalently incorporated TGF-β1. This was substantiated with regard to early TGF-β1 signaling, chondrogenic gene expression, qualitative and quantitative ECM deposition and distribution, and resulting construct stiffness. Furthermore, it was successfully demonstrated, in a comparative analysis of cast and printed bioinks, that covalently tethered TGF-β1 maintained its functionality after 3D printing. Taken together, the presented ink composition enabled the generation of high-quality cartilaginous tissues without the need for continuous exogenous growth factor supply and, thus, bears great potential for future investigation towards cartilage regeneration. Furthermore, growth factor tethering within bioinks, potentially leading to superior tissue development, may also be explored for other biofabrication applications. KW - biofabrication KW - bioink KW - chondrogenic differentiation KW - dual-stage crosslinking KW - hyaluronic acid KW - tethering KW - transforming growth factor-beta 1 Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284239 SN - 1422-0067 VL - 23 IS - 2 ER - TY - JOUR A1 - Brand, Jessica S. A1 - Forster, Leonard A1 - Böck, Thomas A1 - Stahlhut, Philipp A1 - Teßmar, Jörg A1 - Groll, Jürgen A1 - Albrecht, Krystyna T1 - Covalently Cross-Linked Pig Gastric Mucin Hydrogels Prepared by Radical-Based Chain-Growth and Thiol-ene Mechanisms JF - Macromolecular Bioscience N2 - Mucin, a high molecular mass hydrophilic glycoprotein, is the main component of mucus that coats every wet epithelium in animals. It is thus intrinsically biocompatible, and with its protein backbone and the o-glycosidic bound oligosaccharides, it contains a plethora of functional groups which can be used for further chemical modifications. Here, chain-growth and step-growth (thiol-ene) free-radical cross-linked hydrogels prepared from commercially available pig gastric mucin (PGM) are introduced and compared as cost-efficient and easily accessible alternative to the more broadly applied bovine submaxillary gland mucin. For this, PGM is functionalized with photoreactive acrylate groups or allyl ether moieties, respectively. Whereas homopolymerization of acrylate-functionalized polymers is performed, for thiol-ene cross-linking, the allyl-ether-functionalized PGM is cross-linked with thiol-functionalized hyaluronic acid. Morphology, mechanical properties, and cell compatibility of both kinds of PGM hydrogels are characterized and compared. Furthermore, the biocompatibility of these hydrogels can be evaluated in cell culture experiments. KW - click chemistry KW - photopolymerization KW - hydrogels KW - mucin KW - thiol-ene Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-318453 VL - 22 IS - 4 ER - TY - JOUR A1 - Ryma, Matthias A1 - Genç, Hatice A1 - Nadernezhad, Ali A1 - Paulus, Ilona A1 - Schneidereit, Dominik A1 - Friedrich, Oliver A1 - Andelovic, Kristina A1 - Lyer, Stefan A1 - Alexiou, Christoph A1 - Cicha, Iwona A1 - Groll, Jürgen T1 - A Print-and-Fuse Strategy for Sacrificial Filaments Enables Biomimetically Structured Perfusable Microvascular Networks with Functional Endothelium Inside 3D Hydrogels JF - Advanced Materials N2 - A facile and flexible approach for the integration of biomimetically branched microvasculature within bulk hydrogels is presented. For this, sacrificial scaffolds of thermoresponsive poly(2-cyclopropyl-2-oxazoline) (PcycloPrOx) are created using melt electrowriting (MEW) in an optimized and predictable way and subsequently placed into a customized bioreactor system, which is then filled with a hydrogel precursor solution. The aqueous environment above the lower critical solution temperature (LCST) of PcycloPrOx at 25 °C swells the polymer without dissolving it, resulting in fusion of filaments that are deposited onto each other (print-and-fuse approach). Accordingly, an adequate printing pathway design results in generating physiological-like branchings and channel volumes that approximate Murray's law in the geometrical ratio between parent and daughter vessels. After gel formation, a temperature decrease below the LCST produces interconnected microchannels with distinct inlet and outlet regions. Initial placement of the sacrificial scaffolds in the bioreactors in a pre-defined manner directly yields perfusable structures via leakage-free fluid connections in a reproducible one-step procedure. Using this approach, rapid formation of a tight and biologically functional endothelial layer, as assessed not only through fluorescent dye diffusion, but also by tumor necrosis factor alpha (TNF-α) stimulation, is obtained within three days. KW - hydrogels KW - microvasculature KW - melt electrowriting Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-318532 VL - 34 IS - 28 ER - TY - JOUR A1 - Weigl, Franziska A1 - Blum, Carina A1 - Sancho, Ana A1 - Groll, Jürgen T1 - Correlative Analysis of Intra– Versus Extracellular Cell Detachment Events via the Alignment of Optical Imaging and Detachment Force Quantification JF - Advanced Materials Technologies N2 - In recent decades, hybrid characterization systems have become pillars in the study of cellular biomechanics. Especially, Atomic Force Microscopy (AFM) is combined with a variety of optical microscopy techniques to discover new aspects of cell adhesion. AFM, however, is limited to the early-stage of cell adhesion, so that the forces of mature cell contacts cannot be addressed. Even though the invention of Fluidic Force Microscopy (FluidFM) overcomes these limitations by combining the precise force-control of AFM with microfluidics, the correlative investigation of detachment forces arising from spread mammalian cells has been barely achieved. Here, a novel multifunctional device integrating Fluorescence Microscopy (FL) into FluidFM technology (FL-FluidFM) is introduced, enabling real-time optical tracking of entire cell detachment processes in parallel to the undisturbed acquisition of force-distance curves. This setup, thus, allows for entailing two pieces of information at once. As proof-of-principle experiment, this method is applied to fluorescently labeled rat embryonic fibroblast (REF52) cells, demonstrating a precise matching between identified force-jumps and visualized cellular unbinding steps. This study, thus, presents a novel characterization tool for the correlated evaluation of mature cell adhesion, which has great relevance, for instance, in the development of biomaterials or the fight against diseases such as cancer. KW - Fluorescence Microscopy KW - FluidFM technology KW - detachment force quantification Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-318544 SN - 2365-709X VL - 7 IS - 11 ER - TY - JOUR A1 - Pereira, Ana Rita A1 - Trivanović, Drenka A1 - Stahlhut, Philipp A1 - Rudert, Maximilian A1 - Groll, Jürgen A1 - Herrmann, Marietta T1 - Preservation of the naïve features of mesenchymal stromal cells in vitro: Comparison of cell- and bone-derived decellularized extracellular matrix JF - Journal of Tissue Engineering N2 - The fate and behavior of bone marrow mesenchymal stem/stromal cells (BM-MSC) is bidirectionally influenced by their microenvironment, the stem cell niche, where a magnitude of biochemical and physical cues communicate in an extremely orchestrated way. It is known that simplified 2D in vitro systems for BM-MSC culture do not represent their naïve physiological environment. Here, we developed four different 2D cell-based decellularized matrices (dECM) and a 3D decellularized human trabecular-bone scaffold (dBone) to evaluate BM-MSC behavior. The obtained cell-derived matrices provided a reliable tool for cell shape-based analyses of typical features associated with osteogenic differentiation at high-throughput level. On the other hand, exploratory proteomics analysis identified native bone-specific proteins selectively expressed in dBone but not in dECM models. Together with its architectural complexity, the physico-chemical properties of dBone triggered the upregulation of stemness associated genes and niche-related protein expression, proving in vitro conservation of the naïve features of BM-MSC. KW - decellularization KW - bone model KW - stem cell niche KW - stemness KW - osteogenesis KW - 3D models Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-268835 VL - 13 ER - TY - JOUR A1 - Shan, Junwen A1 - Böck, Thomas A1 - Keller, Thorsten A1 - Forster, Leonard A1 - Blunk, Torsten A1 - Groll, Jürgen A1 - Teßmar, Jörg T1 - TEMPO/TCC as a Chemo Selective Alternative for the Oxidation of Hyaluronic Acid JF - Molecules N2 - Hyaluronic acid (HA)-based hydrogels are very commonly applied as cell carriers for different approaches in regenerative medicine. HA itself is a well-studied biomolecule that originates from the physiological extracellular matrix (ECM) of mammalians and, due to its acidic polysaccharide structure, offers many different possibilities for suitable chemical modifications which are necessary to control, for example, network formation. Most of these chemical modifications are performed using the free acid function of the polymer and, additionally, lead to an undesirable breakdown of the biopolymer’s backbone. An alternative modification of the vicinal diol of the glucuronic acid is oxidation with sodium periodate to generate dialdehydes via a ring opening mechanism that can subsequently be further modified or crosslinked via Schiff base chemistry. Since this oxidation causes a structural destruction of the polysaccharide backbone, it was our intention to study a novel synthesis protocol frequently applied to selectively oxidize the C6 hydroxyl group of saccharides. On the basis of this TEMPO/TCC oxidation, we studied an alternative hydrogel platform based on oxidized HA crosslinked using adipic acid dihydrazide as the crosslinker. KW - hyaluronic acid KW - oxidation KW - hydrogel formation KW - Schiff base chemistry Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-248362 SN - 1420-3049 VL - 26 IS - 19 ER - TY - JOUR A1 - Horder, Hannes A1 - Guaza Lasheras, Mar A1 - Grummel, Nadine A1 - Nadernezhad, Ali A1 - Herbig, Johannes A1 - Ergün, Süleyman A1 - Teßmar, Jörg A1 - Groll, Jürgen A1 - Fabry, Ben A1 - Bauer-Kreisel, Petra A1 - Blunk, Torsten T1 - Bioprinting and differentiation of adipose-derived stromal cell spheroids for a 3D breast cancer-adipose tissue model JF - Cells N2 - Biofabrication, including printing technologies, has emerged as a powerful approach to the design of disease models, such as in cancer research. In breast cancer, adipose tissue has been acknowledged as an important part of the tumor microenvironment favoring tumor progression. Therefore, in this study, a 3D-printed breast cancer model for facilitating investigations into cancer cell-adipocyte interaction was developed. First, we focused on the printability of human adipose-derived stromal cell (ASC) spheroids in an extrusion-based bioprinting setup and the adipogenic differentiation within printed spheroids into adipose microtissues. The printing process was optimized in terms of spheroid viability and homogeneous spheroid distribution in a hyaluronic acid-based bioink. Adipogenic differentiation after printing was demonstrated by lipid accumulation, expression of adipogenic marker genes, and an adipogenic ECM profile. Subsequently, a breast cancer cell (MDA-MB-231) compartment was printed onto the adipose tissue constructs. After nine days of co-culture, we observed a cancer cell-induced reduction of the lipid content and a remodeling of the ECM within the adipose tissues, with increased fibronectin, collagen I and collagen VI expression. Together, our data demonstrate that 3D-printed breast cancer-adipose tissue models can recapitulate important aspects of the complex cell–cell and cell–matrix interplay within the tumor-stroma microenvironment KW - adipose-derived stromal cells KW - adipose tissue KW - bioprinting KW - breast cancer model KW - extracellular matrix KW - hyaluronic acid KW - spheroids Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236496 VL - 10 IS - 4 ER - TY - JOUR A1 - Haider, Malik Salman A1 - Ahmad, Taufiq A1 - Yang, Mengshi A1 - Hu, Chen A1 - Hahn, Lukas A1 - Stahlhut, Philipp A1 - Groll, Jürgen A1 - Luxenhofer, Robert T1 - Tuning the thermogelation and rheology of poly(2-oxazoline)/poly(2-oxazine)s based thermosensitive hydrogels for 3D bioprinting JF - Gels N2 - As one kind of “smart” material, thermogelling polymers find applications in biofabrication, drug delivery and regenerative medicine. In this work, we report a thermosensitive poly(2-oxazoline)/poly(2-oxazine) based diblock copolymer comprising thermosensitive/moderately hydrophobic poly(2-N-propyl-2-oxazine) (pPrOzi) and thermosensitive/moderately hydrophilic poly(2-ethyl-2-oxazoline) (pEtOx). Hydrogels were only formed when block length exceeded certain length (≈100 repeat units). The tube inversion and rheological tests showed that the material has then a reversible sol-gel transition above 25 wt.% concentration. Rheological tests further revealed a gel strength around 3 kPa, high shear thinning property and rapid shear recovery after stress, which are highly desirable properties for extrusion based three-dimensional (3D) (bio) printing. Attributed to the rheology profile, well resolved printability and high stackability (with added laponite) was also possible. (Cryo) scanning electron microscopy exhibited a highly porous, interconnected, 3D network. The sol-state at lower temperatures (in ice bath) facilitated the homogeneous distribution of (fluorescently labelled) human adipose derived stem cells (hADSCs) in the hydrogel matrix. Post-printing live/dead assays revealed that the hADSCs encapsulated within the hydrogel remained viable (≈97%). This thermoreversible and (bio) printable hydrogel demonstrated promising properties for use in tissue engineering applications. KW - poly(2-ethyl-2-oxazoline) KW - shear thinning KW - shape fidelity KW - cyto-compatibility KW - bio-printability Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-241781 SN - 2310-2861 VL - 7 IS - 3 ER - TY - JOUR A1 - Seifert, Annika A1 - Gruber, Julia A1 - Gbureck, Uwe A1 - Groll, Jürgen T1 - Morphological control of freeze‐structured scaffolds by selective temperature and material control in the ice‐templating process JF - Advanced Engineering Materials N2 - Herein, it is aimed to highlight the importance of the process parameter choice during directional solidification of polymer solutions, as they have a significant influence on the pore structure and orientation. Biopolymer solutions (alginate and chitosan) are directionally frozen, while systematically varying parameters such as the external temperature gradient, the temperature of the overall system, and the temperatures of the cooling surfaces. In addition, the effect of material properties such as molecular weight, solution concentration, or viscosity on the sample morphology is investigated. By selecting appropriate temperature gradients and cooling surface temperatures, aligned pores ranging in size between (50 ± 22) μm and (144 ± 56) μm are observed in the alginate samples, whereas the pore orientation is influenced by altering the external temperature gradient. As this gradient increases, the pores are increasingly oriented perpendicular to the sample surface. This is also observed in the chitosan samples. However, if the overall system is too cold, that is, using temperatures of the lower cooling surface down to −60 °C combined with low temperatures of the upper cooling surface, control over pore orientation is lost. This is also found when viscosity of chitosan solutions is above ≈5 Pas near the freezing point. KW - unidirectional freezing KW - anisotropic porous structures KW - morphology controls KW - systematic investigations Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-256330 VL - 24 IS - 3 ER -