@article{HauptsteinForsterNadernezhadetal.2022, author = {Hauptstein, Julia and Forster, Leonard and Nadernezhad, Ali and Horder, Hannes and Stahlhut, Philipp and Groll, J{\"u}rgen and Blunk, Torsten and Teßmar, J{\"o}rg}, title = {Bioink Platform Utilizing Dual-Stage Crosslinking of Hyaluronic Acid Tailored for Chondrogenic Differentiation of Mesenchymal Stromal Cells}, series = {Macromolecular Bioscience}, volume = {22}, journal = {Macromolecular Bioscience}, number = {2}, doi = {10.1002/mabi.202100331}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-257556}, pages = {2100331}, year = {2022}, abstract = {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.}, language = {en} } @article{HauptsteinForsterNadernezhadetal.2022, author = {Hauptstein, Julia and Forster, Leonard and Nadernezhad, Ali and Groll, J{\"u}rgen and Teßmar, J{\"o}rg and Blunk, Torsten}, title = {Tethered TGF-β1 in a hyaluronic acid-based bioink for bioprinting cartilaginous tissues}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {2}, issn = {1422-0067}, doi = {10.3390/ijms23020924}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284239}, year = {2022}, abstract = {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.}, language = {en} } @article{RymaGencNadernezhadetal.2022, author = {Ryma, Matthias and Gen{\c{c}}, Hatice and Nadernezhad, Ali and Paulus, Ilona and Schneidereit, Dominik and Friedrich, Oliver and Andelovic, Kristina and Lyer, Stefan and Alexiou, Christoph and Cicha, Iwona and Groll, J{\"u}rgen}, title = {A Print-and-Fuse Strategy for Sacrificial Filaments Enables Biomimetically Structured Perfusable Microvascular Networks with Functional Endothelium Inside 3D Hydrogels}, series = {Advanced Materials}, volume = {34}, journal = {Advanced Materials}, number = {28}, doi = {10.1002/adma.202200653}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-318532}, year = {2022}, abstract = {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.}, language = {en} } @article{WeiglBlumSanchoetal.2022, author = {Weigl, Franziska and Blum, Carina and Sancho, Ana and Groll, J{\"u}rgen}, title = {Correlative Analysis of Intra- Versus Extracellular Cell Detachment Events via the Alignment of Optical Imaging and Detachment Force Quantification}, series = {Advanced Materials Technologies}, volume = {7}, journal = {Advanced Materials Technologies}, number = {11}, issn = {2365-709X}, doi = {10.1002/admt.202200195}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-318544}, year = {2022}, abstract = {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.}, language = {en} } @article{PereiraTrivanovićStahlhutetal.2022, author = {Pereira, Ana Rita and Trivanović, Drenka and Stahlhut, Philipp and Rudert, Maximilian and Groll, J{\"u}rgen and Herrmann, Marietta}, title = {Preservation of the na{\"i}ve features of mesenchymal stromal cells in vitro: Comparison of cell- and bone-derived decellularized extracellular matrix}, series = {Journal of Tissue Engineering}, volume = {13}, journal = {Journal of Tissue Engineering}, doi = {10.1177/20417314221074453}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-268835}, pages = {1-12}, year = {2022}, abstract = {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 na{\"i}ve 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 na{\"i}ve features of BM-MSC.}, language = {en} } @article{BrandForsterBoecketal.2022, author = {Brand, Jessica S. and Forster, Leonard and B{\"o}ck, Thomas and Stahlhut, Philipp and Teßmar, J{\"o}rg and Groll, J{\"u}rgen and Albrecht, Krystyna}, title = {Covalently Cross-Linked Pig Gastric Mucin Hydrogels Prepared by Radical-Based Chain-Growth and Thiol-ene Mechanisms}, series = {Macromolecular Bioscience}, volume = {22}, journal = {Macromolecular Bioscience}, number = {4}, doi = {10.1002/mabi.202100274}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-318453}, year = {2022}, abstract = {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.}, language = {en} } @article{ShanBoeckKelleretal.2021, author = {Shan, Junwen and B{\"o}ck, Thomas and Keller, Thorsten and Forster, Leonard and Blunk, Torsten and Groll, J{\"u}rgen and Teßmar, J{\"o}rg}, title = {TEMPO/TCC as a Chemo Selective Alternative for the Oxidation of Hyaluronic Acid}, series = {Molecules}, volume = {26}, journal = {Molecules}, number = {19}, issn = {1420-3049}, doi = {10.3390/molecules26195963}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-248362}, year = {2021}, abstract = {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.}, language = {en} } @article{HorderGuazaLasherasGrummeletal.2021, author = {Horder, Hannes and Guaza Lasheras, Mar and Grummel, Nadine and Nadernezhad, Ali and Herbig, Johannes and Erg{\"u}n, S{\"u}leyman and Teßmar, J{\"o}rg and Groll, J{\"u}rgen and Fabry, Ben and Bauer-Kreisel, Petra and Blunk, Torsten}, title = {Bioprinting and differentiation of adipose-derived stromal cell spheroids for a 3D breast cancer-adipose tissue model}, series = {Cells}, volume = {10}, journal = {Cells}, number = {4}, doi = {10.3390/cells10040803}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-236496}, year = {2021}, abstract = {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}, language = {en} } @article{DoryabTaskinStahlhutetal.2021, author = {Doryab, Ali and Taskin, Mehmet Berat and Stahlhut, Philipp and Schr{\"o}ppel, Andreas and Orak, Sezer and Voss, Carola and Ahluwalia, Arti and Rehberg, Markus and Hilgendorff, Anne and St{\"o}ger, Tobias and Groll, J{\"u}rgen and Schmid, Otmar}, title = {A Bioinspired in vitro Lung Model to Study Particokinetics of Nano-/Microparticles Under Cyclic Stretch and Air-Liquid Interface Conditions}, series = {Frontiers in Bioengineering and Biotechnology}, volume = {9}, journal = {Frontiers in Bioengineering and Biotechnology}, issn = {2296-4185}, doi = {10.3389/fbioe.2021.616830}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-223830}, year = {2021}, abstract = {Evolution has endowed the lung with exceptional design providing a large surface area for gas exchange area (ca. 100 m\(^{2}\)) in a relatively small tissue volume (ca. 6 L). This is possible due to a complex tissue architecture that has resulted in one of the most challenging organs to be recreated in the lab. The need for realistic and robust in vitro lung models becomes even more evident as causal therapies, especially for chronic respiratory diseases, are lacking. Here, we describe the Cyclic In VItro Cell-stretch (CIVIC) "breathing" lung bioreactor for pulmonary epithelial cells at the air-liquid interface (ALI) experiencing cyclic stretch while monitoring stretch-related parameters (amplitude, frequency, and membrane elastic modulus) under real-time conditions. The previously described biomimetic copolymeric BETA membrane (5 μm thick, bioactive, porous, and elastic) was attempted to be improved for even more biomimetic permeability, elasticity (elastic modulus and stretchability), and bioactivity by changing its chemical composition. This biphasic membrane supports both the initial formation of a tight monolayer of pulmonary epithelial cells (A549 and 16HBE14o\(^{-}\)) under submerged conditions and the subsequent cell-stretch experiments at the ALI without preconditioning of the membrane. The newly manufactured versions of the BETA membrane did not improve the characteristics of the previously determined optimum BETA membrane (9.35\% PCL and 6.34\% gelatin [w/v solvent]). Hence, the optimum BETA membrane was used to investigate quantitatively the role of physiologic cyclic mechanical stretch (10\% linear stretch; 0.33 Hz: light exercise conditions) on size-dependent cellular uptake and transepithelial transport of nanoparticles (100 nm) and microparticles (1,000 nm) for alveolar epithelial cells (A549) under ALI conditions. Our results show that physiologic stretch enhances cellular uptake of 100 nm nanoparticles across the epithelial cell barrier, but the barrier becomes permeable for both nano- and micron-sized particles (100 and 1,000 nm). This suggests that currently used static in vitro assays may underestimate cellular uptake and transbarrier transport of nanoparticles in the lung.}, language = {en} } @article{SeifertGrollWeichholdetal.2021, author = {Seifert, Annika and Groll, J{\"u}rgen and Weichhold, Jan and Boehm, Anne V. and M{\"u}ller, Frank A. and Gbureck, Uwe}, title = {Phase Conversion of Ice-Templated α-Tricalcium Phosphate Scaffolds into Low-Temperature Calcium Phosphates with Anisotropic Open Porosity}, series = {Advanced Engineering Materials}, volume = {23}, journal = {Advanced Engineering Materials}, number = {5}, doi = {10.1002/adem.202001417}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-256311}, year = {2021}, abstract = {The current study aims to extend the material platform for anisotropically structured calcium phosphates to low-temperature phases such as calcium-deficient hydroxyapatite (CDHA) or the secondary phosphates monetite and brushite. This is achieved by the phase conversion of highly porous α-tricalcium phosphate (α-TCP) scaffolds fabricated by ice-templating into the aforementioned phases by hydrothermal treatment or incubation in phosphoric acid. Prior to these steps, α-TCP scaffolds are either sintered for 8 h at 1400 °C or remain in their original state. Both nonsintered and sintered α-TCP specimens are converted into CDHA by hydrothermal treatment, while a transformation into monetite and brushite is achieved by incubation in phosphoric acid. Hydrothermal treatment for 72 h at 175 °C increases the porosity in nonsintered samples from 85\% to 88\% and from 75\% to 88\% in the sintered ones. An increase in the specific surface area from (1.102 ± 0.005) to (9.17 ± 0.01) m2 g-1 and from (0.190 ± 0.004) to (2.809 ± 0.002) m2 g-1 due to the phase conversion is visible for both the nonsintered and sintered samples. Compressive strength of the nonsintered samples increases significantly from (0.76 ± 0.11) to (5.29 ± 0.94) MPa due to incubation in phosphoric acid.}, language = {en} }