A Mild Method for the Activation of Cation Exchange Membranes Used in Tubular PEM Electrolyzers.

Co-extrusion of both half-cells in tubular PEM water electrolyzers can lower the costs for hydrogen production, since the number of components is reduced and the production process is simplified. However, after co-extrusion of the inner half-cell and the ion exchange membrane, the membrane is in its fluoride sulfonyl form and must be hydrolyzed to achieve the proton conductive sulfonic acid to be ready for use. Common practice is the hydrolysis using concentrated alkaline solutions, which causes a corrosion of the laminated anode electrode. We developed a less corrosive method using triethylsilanol as reactant. Tubular membranes hydrolyzed with this new procedure were characterized and tested in an electrolyzer laboratory test setup.

Maintenance of a bone collagen phenotype by osteoblast-like cells in 3D periodic porous titanium (Ti-6Al-4 V) structures fabricated by selective electron beam melting.

Regular 3D periodic porous Ti-6Al-4 V structures were fabricated by the selective electron beam melting method (EBM) over a range of relative densities (0.17-0.40) and pore sizes (500-1500 µm). Structures were seeded with human osteoblast-like cells (SAOS-2) and cultured for four weeks. Cells multiplied within these structures and extracellular matrix collagen content increased. Type I and type V collagens typically synthesized by osteoblasts were deposited in the newly formed matrix with time in culture. High magnification scanning electron microscopy revealed cells attached to surfaces on the interior of the structures with an increasingly fibrous matrix. The in-vitro results demonstrate that the novel EBM-processed porous structures, designed to address the effect of stress-shielding, are conducive to osteoblast attachment, proliferation and deposition of a collagenous matrix characteristic of bone.

Structure Design of Soft Magnetic Materials Using Electron-Beam-Based Additive Manufacturing.

Fe93.5 Si6.5 (wt%) soft magnetic materials in toroidal shape are additively manufactured by means of electron beam powder bed fusion (PBF-EB). Different hatching strategies are applied to realize specific patterns of molten material alternating with non-molten powder particles. The specimens produced using different hatching strategies show identical relative densities but various structural features resulting in different magnetic properties. The magnetic performance of the specimens is characterized by determining hysteresis loops (B-H curves), power losses, and maximum magnetic flux density at frequencies between 50 and 1000 Hz. At constant mass, the different structures induced by using various hatching strategies have a strong influence on the hysteresis losses. These losses can be significantly reduced by applying a targeted structure design. The modified specimens show superior magnetic properties at sub-kHz compared to some soft magnetic materials fabricated by means of conventional methods and laser powder bed fusion (PBF-L).

In Situ Inclusion Detection and Material Characterization in an Electron Beam Powder Bed Fusion Process Using Electron Optical Imaging.

Electron Beam Powder Bed Fusion (PBF-EB) is an Additive Manufacturing (AM) method that utilizes an electron beam to melt and consolidate metal powder. The beam, combined with a backscattered electron detector, enables advanced process monitoring, a method termed Electron Optical Imaging (ELO). ELO is already known to provide great topographical information, but its capabilities regarding material contrast are less studied. In this article the extents of material contrast using ELO are investigated, focusing mainly on identifying powder contamination. It will be shown that an ELO detector is capable of distinguishing a single 100 µm foreign powder particle, during an PBF-EB process, if the backscattering coefficient of the inclusion is sufficiently higher than its surroundings. Additionally, it is investigated how the material contrast can be used for material characterization. A mathematical framework is provided to describe the relationship between the signal intensity in the detector and the effective atomic number Zeff of the imaged alloy. The approach is verified with empirical data from twelve different materials, demonstrating that the effective atomic number of an alloy can be predicted to within one atomic number from its ELO intensity.

Impact of Particle Size Distribution in the Preform on Thermal Conductivity, Vickers Hardness and Tensile Strength of Copper-Infiltrated AISI H11 Tool Steel.

Spontaneous infiltration of a porous preform by a metallic melt provides the potential of generating metal matrix composites (MMCs) with tailored combinations of material properties at low cost. The bulk of tool inserts for injection molding must sustain high mechanical and thermal loads and simultaneously exhibit high thermal conductivity for efficient temperature control of the mold insert. To fulfill these contradictory requirements, AISI H11 tool steel preforms were infiltrated by liquid copper. The impact of the fine powder fraction (0 wt.% to 15 wt.%) blended to a coarse H11 powder in the preform on thermal conductivity, Vickers hardness and tensile strength was elucidated. The thermal conductivity of the composites could be enhanced by a factor of 1.84 (15 wt.% fine powder) and 2.67 (0 wt.% fine powder) with respect to the sintered H11 tool steel. By adding 15 wt.% fine powder to the coarse host powder, the tensile strength and Vickers hardness of the copper-infiltrated steel were 1066.3 ± 108.7 MPa and 366 ± 24 HV1, respectively, whereas the H11 tool steel yielded 1368.5 ± 89.3 MPa and 403 ± 17 HV1, respectively. Based on the results obtained, an appropriate particle size distribution (PSD) may be selected for preform preparation according with the requirements of a future mold insert.

A Novel Window into Angiogenesis-Intravital Microscopy in the AV-Loop-Model.

Due to the limitations of current in vivo experimental designs, our comprehensive knowledge of vascular development and its implications for the development of large-scale engineered tissue constructs is very limited. Therefore, the purpose of this study was to develop unique in vivo imaging chambers that allow the live visualization of cellular processes in the arteriovenous (AV) loop model in rats. We have developed two different types of chambers. Chamber A is installed in the skin using the purse sting fixing method, while chamber B is installed subcutaneously under the skin. Both chambers are filled with modified gelatin hydrogel as a matrix. Intravital microscopy (IVM) was performed after the injection of fluorescein isothiocyanate (FITC)-labeled dextran and rhodamine 6G dye. The AV loop was functional for two weeks in chamber A and allowed visualization of the leukocyte trafficking. In chamber B, microvascular development in the AV loop could be examined for 21 days. Quantification of the microvascular outgrowth was performed using Fiji-ImageJ. Overall, by combining these two IVM chambers, we can comprehensively understand vascular development in the AV loop tissue engineering model¯.

Structure of the ceramide-bound SPOTS complex.

Sphingolipids are structural membrane components that also function in cellular stress responses. The serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in sphingolipid biogenesis. Its activity is tightly regulated through multiple binding partners, including Tsc3, Orm proteins, ceramides, and the phosphatidylinositol-4-phosphate (PI4P) phosphatase Sac1. The structural organization and regulatory mechanisms of this complex are not yet understood. Here, we report the high-resolution cryo-EM structures of the yeast SPT in complex with Tsc3 and Orm1 (SPOT) as dimers and monomers and a monomeric complex further carrying Sac1 (SPOTS). In all complexes, the tight interaction of the downstream metabolite ceramide and Orm1 reveals the ceramide-dependent inhibition. Additionally, observation of ceramide and ergosterol binding suggests a co-regulation of sphingolipid biogenesis and sterol metabolism within the SPOTS complex.

MiniMelt: An instrument for real-time tracking of electron beam additive manufacturing using synchrotron x-ray techniques.

The development of a sample environment for in situ x-ray characterization during metal Electron Beam Powder Bed Fusion (PBF-EB), called MiniMelt, is presented. The design considerations, the features of the equipment, and its implementation at the synchrotron facility PETRA III at Deutsches Elektronen-Synchrotron, Hamburg, Germany, are described. The equipment is based on the commercially available Freemelt ONE PBF-EB system but has been customized with a unique process chamber to enable real-time synchrotron measurements during the additive manufacturing process. Furthermore, a new unconfined powder bed design to replicate the conditions of the full-scale PBF-EB process is introduced. The first radiography (15 kHz) and diffraction (1 kHz) measurements of PBF-EB with a hot-work tool steel and a Ni-base superalloy, as well as bulk metal melting with the CMSX-4 alloy, using the sample environment are presented. MiniMelt enables time-resolved investigations of the dynamic phenomena taking place during multi-layer PBF-EB, facilitating process understanding and development of advanced process strategies and materials for PBF-EB.

Yeast Svf1 binds ceramides and contributes to sphingolipid metabolism at the ER cis-Golgi interface.

Ceramides are essential precursors of complex sphingolipids and act as potent signaling molecules. Ceramides are synthesized in the endoplasmic reticulum (ER) and receive their head-groups in the Golgi apparatus, yielding complex sphingolipids (SPs). Transport of ceramides between the ER and the Golgi is executed by the essential ceramide transport protein (CERT) in mammalian cells. However, yeast cells lack a CERT homolog, and the mechanism of ER to Golgi ceramide transport remains largely elusive. Here, we identified a role for yeast Svf1 in ceramide transport between the ER and the Golgi. Svf1 is dynamically targeted to membranes via an N-terminal amphipathic helix (AH). Svf1 binds ceramide via a hydrophobic binding pocket that is located in between two lipocalin domains. We showed that Svf1 membrane-targeting is important to maintain flux of ceramides into complex SPs. Together, our results show that Svf1 is a ceramide binding protein that contributes to sphingolipid metabolism at Golgi compartments.

Compartmentation and functions of sphingolipids.

Sphingolipids (SLs) are one of the three major lipid classes in all eukaryotic cells. They function as structural molecules of membranes and can also act as highly active signaling molecules. SL biosynthesis is mainly occurring at the endoplasmic reticulum and the Golgi apparatus. However, SL intermediates are also generated at other organelles such as the plasma membrane and the lysosome. SL biosynthesis is therefore highly compartmentalized. Maintaining SL levels is necessary for the function of multiple trafficking pathways. One major challenge is to decipher the complex regulatory networks controlling SL biosynthesis, the coordination of vesicular and non-vesicular SL transport as well as their role in trafficking. Recent investigations have shed new light on the regulation of SL biosynthesis. Here, we review how SL biosynthesis is coordinated, how SLs are transported and how their levels affect trafficking pathways. Finally, we discuss recently developed methods to study SL metabolism with spatio-temporal resolution.

Basic Mechanism of Surface Topography Evolution in Electron Beam Based Additive Manufacturing.

This study introduces and verifies a basic mechanism of surface topography evolution in electron beam additive manufacturing (E-PBF). A semi-analytical heat conduction model is used to examine the spatio-temporal evolution of the meltpool and segment the build surface according to the emerging persistent meltpool domains. Each persistent domain is directly compared with the corresponding melt surface, and exhibits a characteristic surface morphology and topography. The proposed underlying mechanism of topography evolution is based on different forms of material transport in each distinct persistent domain, driven by evaporation and thermocapillary convection along the temperature gradient of the emerging meltpool. This effect is shown to be responsible for the upper bound of the standard process window in E-PBF, where surface bulges form. Based on this mechanism, process strategies to prevent the formation of surface bulges for complex geometries are proposed.

Microvascular development in the rat arteriovenous loop model in vivo-A step by step intravital microscopy analysis.

The arteriovenous (AV) loop model is a key technique to solve one of the major problems of tissue engineering-providing adequate vascular support for a tissue construct of significant size. However, the molecular and cellular mechanisms of vascularization and factors influencing the generation of new tissue in the AV loop are still poorly understood. We previously established a novel intravital microscopy approach to study these events. In this study, we implanted our observation chamber filled with two types of hydrogels such as fibrin and methacrylate gelatin (GelMA) and performed intravital microscopy (IVM) on days 7, 14, and 21. Initial microvessel formation was observed in GelMA on day 14, while the vessel network showed clear indicators of network rearrangement and maturation on day 21. No visible microvessels were observed in fibrin. The chambers were explanted on day 21. Histological examination revealed higher numbers of microvessels in GelMA compared to fibrin, while the AV loop was thrombosed in all fibrin constructs, possibly due to matrix degradation. GelMA proved to be an ideal matrix for IVM studies in the AV loop model due to its slow degradation and transparency. This IVM model can be employed as a novel tool for live and thus faster comprehension of crucial events in the tissue regeneration process, which can improve tissue engineering application.

Revealing dynamic processes in laser powder bed fusion with in situ X-ray diffraction at PETRA III.

The high flux combined with the high energy of the monochromatic synchrotron radiation available at modern synchrotron facilities offers vast possibilities for fundamental research on metal processing technologies. Especially in the case of laser powder bed fusion (LPBF), an additive manufacturing technology for the manufacturing of complex-shaped metallic parts, in situ methods are necessary to understand the highly dynamic thermal, mechanical, and metallurgical processes involved in the creation of the parts. At PETRA III, Deutsches Elektronen-Synchrotron, a customized LPBF system featuring all essential functions of an industrial LPBF system, is used for in situ x-ray diffraction research. Three use cases with different experimental setups and research questions are presented to demonstrate research opportunities. First, the influence of substrate pre-heating and a complex scan pattern on the strain and internal stress progression during the manufacturing of Inconel 625 parts is investigated. Second, a study on the nickel-base superalloy CMSX-4 reveals the formation and dissolution of γ' precipitates depending on the scan pattern in different part locations. Third, phase transitions during melting and solidification of an intermetallic γ-TiAl based alloy are examined, and the advantages of using thin platelet-shaped specimens to resolve the phase components are discussed. The presented cases give an overview of in situ x-ray diffraction experiments at PETRA III for research on the LPBF technology and provide information on specific experimental procedures.

Impact of Endothelial Progenitor Cells in the Vascularization of Osteogenic Scaffolds.

The microvascular endothelial network plays an important role in osteogenesis, bone regeneration and bone tissue engineering. Endothelial progenitor cells (EPCs) display a high angiogenic and vasculogenic potential. The endothelialization of scaffolds with endothelial progenitor cells supports vascularization and tissue formation. In addition, EPCs enhance the osteogenic differentiation and bone formation of mesenchymal stem cells (MSCs). This study aimed to investigate the impact of EPCs on vascularization and bone formation of a hydroxyapatite (HA) and beta-tricalcium phosphate (ß-TCP)-fibrin scaffold. Three groups were designed: a scaffold-only group (A), a scaffold and EPC group (B), and a scaffold and EPC/MSC group (C). The HA/ß-TCP-fibrin scaffolds were placed in a porous titanium chamber permitting extrinsic vascularization from the surrounding tissue. Additionally, intrinsic vascularization was achieved by means of an arteriovenous loop (AV loop). After 12 weeks, the specimens were explanted and investigated by histology and CT. We were able to prove a strong scaffold vascularization in all groups. No differences regarding the vessel number and density were detected between the groups. Moreover, we were able to prove bone formation in the coimplantation group. Taken together, the AV loop is a powerful tool for vascularization which is independent from scaffold cellularization with endothelial progenitor cells' prior implantation.

A Single Crystal Process Window for Electron Beam Powder Bed Fusion Additive Manufacturing of a CMSX-4 Type Ni-Based Superalloy.

Using suitable scanning strategies, even single crystals can emerge from powder during additive manufacturing. In this paper, a full microstructure map for additive manufacturing of technical single crystals is presented using the conventional single crystal Ni-based superalloy CMSX-4. The correlation between process parameters, melt pool size and shape, as well as single crystal fraction, is investigated through a high number of experiments supported by numerical simulations. Based on these results, a strategy for the fabrication of high fraction single crystals in powder bed fusion additive manufacturing is deduced.

Electron-Optical In Situ Imaging for the Assessment of Accuracy in Electron Beam Powder Bed Fusion.

The current study evaluates the capabilities of electron-optical (ELO) in situ imaging with respect to monitoring and prediction of manufacturing precision in electron beam powder bed fusion. Post-process X-ray computed tomography of two different as-built parts is used to quantitatively evaluate the accuracy and limitations of ELO imaging. Additionally, a thermodynamic simulation is performed to improve the understanding of ELO data and to assess the feasibility of predicting dimensional accuracy numerically. It is demonstrated that ELO imaging captures the molten layers accurately (deviations <100 µm) and indicates the creation of surface roughness. However, some geometrical features of the as-built parts exhibit local inaccuracies associated with thermal stress-induced deformation (deviations up to 500 µm) which cannot be captured by ELO imaging. It is shown that the comparison between in situ and post-process data enables a quantification of these effects which might provide the possibility for developing effective countermeasures in the future.

New Grain Formation Mechanisms during Powder Bed Fusion.

Tailoring the mechanical properties of parts by influencing the solidification conditions is a key topic of powder bed fusion. Depending on the application, single crystalline, columnar, or equiaxed microstructures are desirable. To produce single crystals or equiaxed microstructures, the control of nucleation is of outstanding importance. Either it should be avoided or provoked. There are also applications, such as turbine blades, where both microstructures at different locations are required. Here, we investigate nucleation at the melt-pool border during the remelting of CMSX-4® samples built using powder bed fusion. We studied the difference between remelting as-built and homogenized microstructures. We identified two new mechanisms that led to grain formation at the beginning of solidification. Both mechanisms involved a change in the solidification microstructure from the former remelted and newly forming material. For the as-built samples, a discrepancy between the former and new dendrite arm spacing led to increased interdentritic undercooling at the beginning of solidification. For the heat-treated samples, the collapse of a planar front led to new grains. To identify these mechanisms, we conducted experimental and numerical investigations. The identification of such mechanisms during powder bed fusion is a fundamental prerequisite to controlling the solidification conditions to produce single crystalline and equiaxed microstructures.

Comparison of Transmission Measurement Methods of Elastic Waves in Phononic Band Gap Materials.

Periodic cellular structures can exhibit metamaterial properties, such as phononic band gaps. In order to detect these frequency bands of strong wave attenuation experimentally, several devices for wave excitation and measurement can be applied. In this work, piezoelectric transducers are utilized to excite two additively manufactured three-dimensional cellular structures. For the measurement of the transmission factor, we compare two methods. First, the transmitted waves are measured with the same kind of piezoelectric transducer. Second, a laser Doppler vibrometer is employed to scan the mechanical vibrations of the sample on both the emitting and receiving surfaces. The additional comparison of two different methods of spatial averaging of the vibrometer data, that is, the quadratic mean and arithmetic mean, provides insight into the way the piezoelectric transducers convert the transmitted signal. Experimental results are supported by numerical simulations of the dispersion relation and a simplified transmission simulation.

Free Transplantation of a Tissue Engineered Bone Graft into an Irradiated, Critical-Size Femoral Defect in Rats.

Healing of large bone defects remains a challenge in reconstructive surgery, especially with impaired healing potential due to severe trauma, infection or irradiation. In vivo studies are often performed in healthy animals, which might not accurately reflect the situation in clinical cases. In the present study, we successfully combined a critical-sized femoral defect model with an ionizing radiation protocol in rats. To support bone healing, tissue-engineered constructs were transferred into the defect after ectopic preossification and prevascularization. The combination of SiHA, MSCs and BMP-2 resulted in the significant ectopic formation of bone tissue, which can easily be transferred by means of our custom-made titanium chamber. Implanted osteogenic MSCs survived in vivo for a total of 18 weeks. The use of SiHA alone did not lead to bone formation after ectopic implantation. Analysis of gene expression showed early osteoblast differentiation and a hypoxic and inflammatory environment in implanted constructs. Irradiation led to impaired bone healing, decreased vascularization and lower short-term survival of implanted cells. We conclude that our model is highly valuable for the investigation of bone healing and tissue engineering in pre-damaged tissue and that healing of bone defects can be substantially supported by combining SiHA, MSCs and BMP-2.

Personalized medicine for reconstruction of critical-size bone defects - a translational approach with customizable vascularized bone tissue.

Tissue engineering principles allow the generation of functional tissues for biomedical applications. Reconstruction of large-scale bone defects with tissue-engineered bone has still not entered the clinical routine. In the present study, a bone substitute in combination with mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC) with or without growth factors BMP-2 and VEGF-A was prevascularized by an arteriovenous (AV) loop and transplanted into a critical-size tibia defect in the sheep model. With 3D imaging and immunohistochemistry, we could show that this approach is a feasible and simple alternative to the current clinical therapeutic option. This study serves as proof of concept for using large-scale transplantable, vascularized, and customizable bone, generated in a living organism for the reconstruction of load-bearing bone defects, individually tailored to the patient's needs. With this approach in personalized medicine for the reconstruction of critical-size bone defects, regeneration of parts of the human body will become possible in the near future.