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- Item3D Quantification of Vascular-Like Structures in z Stack Confocal Images(Amsterdam : Elsevier, 2020) Bonda, Ulrich; Jaeschke, Anna; Lighterness, Anthony; Baldwin, Jeremy; Werner, Carsten; De-Juan-Pardo, Elena M.; Bray, Laura J.Optical slice microscopy is commonly used to characterize the morphometric features of 3D cellular cultures, such as in vitro vascularization. However, the quantitative analysis of those structures is often performed on a single 2D maximum intensity projection image, limiting the accuracy of data obtained from 3D cultures. Here, we present a protocol for the quantitative analysis of z stack images, utilizing Fiji, Amira, and WinFiber3D. This protocol facilitates the in-depth examination of vascular-like structures within 3D cell culture models.
- ItemEncapsulation of bacteria in bilayer Pluronic thin film hydrogels: A safe format for engineered living materials(Amsterdam : Elsevier, 2023) Bhusari, Shardul; Kim, Juhyun; Polizzi, Karen; Sankaran, Shrikrishnan; del Campo, AránzazuIn engineered living materials (ELMs) non-living matrices encapsulate microorganisms to acquire capabilities like sensing or biosynthesis. The confinement of the organisms to the matrix and the prevention of overgrowth and escape during the lifetime of the material is necessary for the application of ELMs into real devices. In this study, a bilayer thin film hydrogel of Pluronic F127 and Pluronic F127 acrylate polymers supported on a solid substrate is introduced. The inner hydrogel layer contains genetically engineered bacteria and supports their growth, while the outer layer acts as an envelope and does not allow leakage of the living organisms outside of the film for at least 15 days. Due to the flat and transparent nature of the construct, the thin layer is suited for microscopy and spectroscopy-based analyses. The composition and properties of the inner and outer layer are adjusted independently to fulfil viability and confinement requirements. We demonstrate that bacterial growth and light-induced protein production are possible in the inner layer and their extent is influenced by the crosslinking degree of the used hydrogel. Bacteria inside the hydrogel are viable long term, they can act as lactate-sensors and remain active after storage in phosphate buffer at room temperature for at least 3 weeks. The versatility of bilayer bacteria thin-films is attractive for fundamental studies and for the development of application-oriented ELMs.
- ItemIn situ detection of cracks during laser powder bed fusion using acoustic emission monitoring(Amsterdam : Elsevier, 2022) Seleznev, Mikhail; Gustmann, Tobias; Friebel, Judith Miriam; Peuker, Urs Alexander; Kühn, Uta; Hufenbach, Julia Kristin; Biermann, Horst; Weidner, AnjaDespite rapid development of laser powder bed fusion (L-PBF) and its monitoring techniques, there is still a lack of in situ crack detection methods, among which acoustic emission (AE) is one of the most sensitive. To elaborate on this topic, in situ AE monitoring was applied to L-PBF manufacturing of a high-strength Al92Mn6Ce2 (at. %) alloy and combined with subsequent X-ray computed tomography. By using a structure borne high-frequency sensor, even a simple threshold-based monitoring was able to detect AE activity associated with cracking, which occurred not only during L-PBF itself, but also after the build job was completed, i.e. in the cooling phase. AE data analysis revealed that crack-related signals can easily be separated from the background noise (e.g. inert gas circulation pump) through their specific shape of a waveform, as well as their energy, skewness and kurtosis. Thus, AE was verified to be a promising method for L-PBF monitoring, enabling to detect formation of cracks regardless of their spatial and temporal occurrence.
- ItemDevelopment and characterization of a metastable Al-Mn-Ce alloy produced by laser powder bed fusion(Amsterdam : Elsevier, 2021) Gabrysiak, Katharina; Gustmann, Tobias; Freudenberger, Jens; Neufeld, Kai; Giebeler, Lars; Leyens, Christoph; Kühn, UtaLaser powder bed fusion (LPBF) can help to overcome two challenges occurring by casting of metastable Al alloys: (1) the high amount of casting defects and (2) the limited part size while maintaining rapid solidification of the whole cross-section. In this study, an Al92Mn6Ce2 alloy was processed crack-free without baseplate heating by LPBF. The high cooling rate during fabrication has a significant impact on the microstructure, which was characterized by SEM, TEM and XRD. The processing through LPBF causes a high amount and a strong refinement of the intermetallic Al20Mn2Ce precipitates. This leads, compared to suction-cast specimens, to a higher hardness (180 HV 5) and a higher tolerable compressive stress (>1200 MPa) associated with a pronounced plasticity without failure up to a strain of 40%. The extraordinary mechanical properties of additively manufactured Al92Mn6Ce2 can extend the possibilities of producing novel LPBF lightweight structures for potential applications under harsh conditions.
- ItemDevelopment of bioactive catechol functionalized nanoparticles applicable for 3D bioprinting(Amsterdam : Elsevier, 2021) Puertas-Bartolomé, María; Włodarczyk-Biegun, Małgorzata K.; del Campo, Aránzazu; Vázquez-Lasa, Blanca; San Román, JulioEfficient wound treatments to target specific events in the healing process of chronic wounds constitute a significant aim in regenerative medicine. In this sense, nanomedicine can offer new opportunities to improve the effectiveness of existing wound therapies. The aim of this study was to develop catechol bearing polymeric nanoparticles (NPs) and to evaluate their potential in the field of wound healing. Thus, NPs wound healing promoting activities, potential for drug encapsulation and controlled release, and further incorporation in a hydrogel bioink formulation to fabricate cell-laden 3D scaffolds are studied. NPs with 2 and 29 M % catechol contents (named NP2 and NP29) were obtained by nanoprecipitation and presented hydrodynamic diameters of 100 and 75 nm respectively. These nanocarriers encapsulated the hydrophobic compound coumarin-6 with 70% encapsulation efficiency values. In cell culture studies, the NPs had a protective effect in RAW 264.7 macrophages against oxidative stress damage induced by radical oxygen species (ROS). They also presented a regulatory effect on the inflammatory response of stimulated macrophages and promoted upregulation of the vascular endothelial growth factor (VEGF) in fibroblasts and endothelial cells. In particular, NP29 were used in a hydrogel bioink formulation using carboxymethyl chitosan and hyaluronic acid as polymeric matrices. Using a reactive mixing bioprinting approach, NP-loaded hydrogel scaffolds with good structural integrity, shape fidelity and homogeneous NPs dispersion, were obtained. The in vitro catechol NPs release profile of the printed scaffolds revealed a sustained delivery. The bioprinted scaffolds supported viability and proliferation of encapsulated L929 fibroblasts over 14 days. We envision that the catechol functionalized NPs and resulting bioactive bioink presented in this work offer promising advantages for wound healing applications, as they: 1) support controlled release of bioactive catechol NPs to the wound site; 2) can incorporate additional therapeutic functions by co-encapsulating drugs; 3) can be printed into 3D scaffolds with tailored geometries based on patient requirements.
- ItemMolecular stiffness cues of an interpenetrating network hydrogel for cell adhesion(Amsterdam : Elsevier, 2022) Li, Bin; Çolak, Arzu; Blass, Johanna; Han, Mitchell; Zhang, Jingnan; Zheng, Yijun; Jiang, Qiyang; Bennewitz, Roland; del Campo, AránzazuUnderstanding cells' response to the macroscopic and nanoscale properties of biomaterials requires studies in model systems with the possibility to tailor their mechanical properties and different length scales. Here, we describe an interpenetrating network (IPN) design based on a stiff PEGDA host network interlaced within a soft 4-arm PEG-Maleimide/thiol (guest) network. We quantify the nano- and bulk mechanical behavior of the IPN and the single network hydrogels by single-molecule force spectroscopy and rheological measurements. The IPN presents different mechanical cues at the molecular scale, depending on which network is linked to the probe, but the same mechanical properties at the macroscopic length scale as the individual host network. Cells attached to the interpenetrating (guest) network of the IPN or to the single network (SN) PEGDA hydrogel modified with RGD adhesive ligands showed comparable attachment and spreading areas, but cells attached to the guest network of the IPN, with lower molecular stiffness, showed a larger number and size of focal adhesion complexes and a higher concentration of the Hippo pathway effector Yes-associated protein (YAP) than cells linked to the PEGDA single network. The observations indicate that cell adhesion to the IPN hydrogel through the network with lower molecular stiffness proceeds effectively as if a higher ligand density is offered. We claim that IPNs can be used to decipher how changes in ECM design and connectivity at the local scale affect the fate of cells cultured on biomaterials.
- ItemTailoring biocompatible Ti-Zr-Nb-Hf-Si metallic glasses based on high-entropy alloys design approach(Amsterdam : Elsevier, 2020) Calin, Mariana; Vishnu, Jithin; Thirathipviwat, Pramote; Popa, Monica-Mihaela; Krautz, Maria; Manivasagam, Geetha; Gebert, AnnettPresent work unveils novel magnetic resonance imaging (MRI) compatible glassy Ti-Zr-Nb-Hf-Si alloys designed based on a high entropy alloys approach, by exploring the central region of multi-component alloy phase space. Phase analysis has revealed the amorphous structure of developed alloys, with a higher thermal stability than the conventional metallic glasses. The alloys exhibit excellent corrosion properties in simulated body fluid. Most importantly, the weak paramagnetic nature (ultralow magnetic susceptibility) and superior radiopacity (high X-ray attenuation coefficients) offer compatibility with medical diagnostic imaging systems thereby opening unexplored realms for biomedical applications.
- ItemSolving the puzzle of hierarchical martensitic microstructures in NiTi by (111)-oriented epitaxial films(Amsterdam : Elsevier, 2023) Lünser, Klara; Undisz, Andreas; Wagner, Martin F.-X.; Nielsch, Kornelius; Fähler, SebastianThe martensitic microstructure decides on the functional properties of shape memory alloys. However, for the most commonly used alloy, NiTi, it is still unclear how its microstructure is built up because the analysis is hampered by grain boundaries of polycrystalline samples. Here, we eliminate grain boundaries by using epitaxially grown films in (111)B2 orientation. By combining scale-bridging microscopy with integral inverse pole figures, we solve the puzzle of the hierarchical martensitic microstructure. We identify two martensite clusters as building blocks and three kinds of twin boundaries. Nesting them at different length scales explains why habit plane variants with ⟨011⟩B19' twin boundaries and {942} habit planes are dominant; but also some incompatible interfaces occur. Though the observed hierarchical microstructure agrees with the phenomenological theory of martensite, the transformation path decides which microstructure forms. The combination of local and global measurements with theory allows solving the scale bridging 3D puzzle of the martensitic microstructure in NiTi exemplarily for epitaxial films.
- ItemElectrodeposition of Sr-substituted hydroxyapatite on low modulus beta-type Ti-45Nb and effect on in vitro Sr release and cell response(Amsterdam : Elsevier, 2019) Schmidt, Romy; Gebert, Annett; Schumacher, Matthias; Hoffmann, Volker; Voss, Andrea; Pilz, Stefan; Uhlemann, Margitta; Lode, Anja; Gelinsky, MichaelBeta-type Ti-based alloys are promising new materials for bone implants owing to their excellent mechanical biofunctionality and biocompatibility. For treatment of fractures in case of systemic diseases like osteoporosis the generation of implant surfaces which actively support the problematic bone healing is a most important aspect. This work aimed at developing suitable approaches for electrodeposition of Sr-substituted hydroxyapatite (Srx-HAp) coatings onto Ti-45Nb. Potentiodynamic polarization measurements in electrolytes with 1.67 mmol/L Ca(NO3)2, which was substituted by 0, 10, 50 and 100% Sr(NO3)2, and 1 mmol/L NH4H2PO4 at 333 K revealed the basic reaction steps for OH– and PO4 3− formation needed for the chemical precipitation of Srx-HAp. Studies under potentiostatic control confirmed that partial or complete substitution of Ca2+- by Sr2+-ions in solution has a significant effect on the complex reaction process. High Sr2+-ion contents yield intermediate phases and a subsequent growth of more refined Srx-HAp coatings. Upon galvanostatic pulse-deposition higher reaction rates are controlled and in all electrolytes very fine needle-like crystalline coatings are obtained. With XRD the incorporation of Sr-species in the hexagonal HAp lattice is evidenced. Coatings formed in electrolytes with 10 and 50% Sr-nitrate were chemically analyzed with EDX mapping and GD-OES depth profiling. Only a fraction of the Sr-ions in solution is incorporated into the Srx-HAp coatings. Therein, the Sr-distribution is laterally homogeneous but non-homogeneous along the cross-section. Increasing Sr-content retards the coating thickness growth. Most promising coatings formed in the electrolyte with 10% Sr-nitrate were employed for Ca, P and Sr release analysis in Tris-Buffered Saline (150 mM NaCl, pH 7.6) at 310 K. At a sample surface: solution volume ratio of 1:200, after 24 h the amount of released Sr-ions was about 30–35% of that determined in the deposited Srx-HAp coating. In vitro studies with human bone marrow stromal cells (hBMSC) revealed that the released Sr-ions led to a significantly enhanced cell proliferation and osteogenic differentiation and that the Sr-HAp surface supported cell adhesion indicating its excellent cytocompatibility. © 2019 The Authors
- ItemAlF3-assisted flux growth of mullite whiskers and their application in fabrication of porous mullite-alumina monoliths(Amsterdam : Elsevier, 2021) Abdullayev, Amanmyrat; Klimm, Detlef; Kamutzki, Franz; Gurlo, Aleksander; Bekheet, Maged F.Mullite is a promising material with its competitive thermochemical and mechanical properties. Although mullite could be obtained by several synthesis methods, the flux method emerges with its advantages over other methods. However, obtaining mullite whiskers with a high aspect ratio and length for ceramic reinforcements is still challenging. In this work, mullite whiskers were grown from AlF3-assisted flux. The addition of AlF3 to flux salt not only decreases the formation temperature of mullite to as low as 700 °C and suppresses the formation of corundum side phase, but also increases the length and aspect ratio of the whiskers. The obtained mullite whiskers were used as reinforcement for porous alumina monoliths prepared by the freeze casting route and subsequent sintering at 1500 °C. The fabricated mullite-alumina monoliths show competitive compressive strength of 25.7 MPa while having as high as 70.6% porosity, which makes them a potential candidate for membrane applications.