Collagen IV assembly is influenced by fluid flow in kidney cell-derived matrices.

Kidney podocytes and endothelial cells assemble a complex and dynamic basement membrane that is essential for kidney filtration. Whilst many components of this specialised matrix are known, the influence of fluid flow on its assembly and organisation remains poorly understood. Using the coculture of podocytes and glomerular endothelial cells in a low-shear stress, high-flow bioreactor, we investigated the effect of laminar fluid flow on the composition and assembly of cell-derived matrix. With immunofluorescence and matrix image analysis we found flow-mediated remodelling of collagen IV. Using proteomic analysis of the cell-derived matrix we identified changes in both abundance and composition of matrix proteins under flow, including the collagen-modifying enzyme, prolyl 4-hydroxylase (P4HA1). To track collagen IV assembly, we used CRISPR-Cas9 to knock in the luminescent marker HiBiT to the endogenous COL4A2 gene in podocytes. With this system, we found that collagen IV was secreted and accumulated consistently under both static and flow conditions. However knockdown of P4HA1 in podocytes led to a reduction in the secretion of collagen IV and this was more pronounced under flow. Together, this work demonstrates the effect of fluid flow on the composition, modification, and organisation of kidney cell-derived matrix and provides an in vitro system for investigating flow-induced matrix alteration in the context of kidney development and disease.

Graphene-Based Transparent Flexible Strain Gauges with Tunable Sensitivity and Strain Range.

Monolayers of graphene oxide, assembled into densely packed sheets at an immiscible hexane/water interface, form transparent conducting films on polydimethylsiloxane membranes after reduction in hydroiodic acid (HI) vapor to reduced graphene oxide (rGO). Prestraining and relaxing the membranes introduces cracks in the rGO film. Subsequent straining opens these cracks and induces piezoresistivity, enabling their application as transparent strain gauges. The sensitivity and strain range of these gauges is controlled by the cracked film structure that is determined by the reducing conditions used in manufacture. Reduction for 30 s in HI vapor leads to an array of parallel cracks that do not individually span the membrane. These cracks do not extend on subsequent straining, leading to a gauge with a usable strain range >0.2 and gauge factor (GF) at low strains ranging from 20 to 100, depending on the prestrain applied. The GF reduces with increasing applied strain and asymptotes to about 3, for all prestrains. Reduction for 60 s leads to cracks spanning the entire membrane and an increased film resistance but a highly sensitive strain gauge, with GF ranging from 800 to 16,000. However, the usable strain range reduces to <0.01. A simple equivalent resistor model is proposed to describe the behavior of both gauge types. The gauges show a repeatable and stable response with loading frequencies >1 kHz and have been used to detect human body strains in a simple e-skin demonstration.

Stability Bounds for Micron Scale Ag Conductor Lines Produced by Electrohydrodynamic Inkjet Printing.

Continuous conducting lines of width 5-20 µm have been printed with a Ag nanoparticle ink using drop-on-demand (DOD) electrohydrodynamic (EHD) inkjet printing on Si and PDMS substrates, with advancing contact angles of 11° and 35°, respectively, and a zero receding contact angle. It is only possible to achieve stable parallel sided lines within a limited range of drop spacings, and this limiting range for stable line printing decreases as the contact angle of the ink on the substrate increases. The upper bound drop spacing for stable line formation is determined by a minimum drop overlap required to prevent contact line retraction, and the lower bound is governed by competing flows for drop spreading onto an unwetted substrate and a return flow driven by a Laplace pressure difference between the newly deposited drops and the fluid some distance from the growing tip. The upper and lower bounds are shown to be consistent with those predicted using existing models for the stability of inkjet printed lines produced using piezoelectric droplet generators. A comparison with literature data for EHD printed lines finds that these limiting bounds apply with printed line widths as small as 200 nm using subfemtoliter drop volumes. When a fine grid pattern is printed, local differences in Laplace pressure lead to the line width retracting to the minimum stable width and excess ink being transported to the nodes of the grid. After printing and sintering, the printed tracks have a conductivity of about 15%-20% of bulk Ag on the Si substrate, which correlates with a porosity of about 60%.

Stability of Lines with Zero Receding Contact Angle Produced by Inkjet Printing at Small Drop Volume.

We present an experimental study of the maximum and minimum bounding drop spacing for a parallel-sided liquid line produced by inkjet printing with drop volumes of 1.5 and 8.5 pL, on substrates with advancing contact angles of 46 and 54°, and zero receding contact angle. The results are used to validate models of the limiting bounds for the formation of stable parallel-sided lines as a function of drop spacing and transverse printing speed. The model for the maximum drop spacing bound (minimum line width) shows a good agreement with our results, but, when used to predict the stable line width, there is an influence of printing speed not captured by the model. This is probably because of a coupling between printed drop volume and ejection velocity outside the scope of the model. The minimum drop spacing bound (maximum stable line width) is limited by a bulging instability, and our results agree with the existing model, except for printing with the largest drop volumes at low temperature. It is shown that under these conditions, there is a different mechanism for bulging that occurs after printing over a period of minutes, if the liquid bead is present on the surface for a significant period of time before drying. Our results suggest that this mechanism is possibly triggered by imperfections on the substrate.

Fabrication of microvascular constructs using high resolution electrohydrodynamic inkjet printing.

Fabrication of the intricate anatomy of vasculature within engineered tissue remains one of the key challenges facing the field of tissue engineering. We report the use of electrohydrodynamic (EHD) inkjet printing to create hydrogel-based microvascular tissues with hierarchical and branching channels, whose minimum feature size of 30µm approaches the physical scale of native capillary blood vessels. The principle relies on the use of complementary thermoreversible gelling properties of Pluronic F127 (PF-127) and gelatin methacryloyl, which served as sacrificial templates and permanent matrices respectively. Human dermal fibroblasts and human umbilical vein endothelial cells were successfully co-cultured within the engineered microvascular tissue constructs for up to 21 days, and attained high cell viability. Tissue specific morphology was maintained on perfusion. The ability to create cellularised, vascularised proto-tissues with high spatial resolution using EHD inkjet printing, provides a new strategy for developing advanced vascular models with the potential to impact upon an extensive range of biomedical applications.

Nanoindentation of Molecular Crystals: Lessons Learned from Aspirin.

Nanoindentation enables the measurement of mechanical properties from single crystals with dimensions of a few micrometers. This experimental technique, however, has only recently been applied to molecular crystals. Key differences between the application of this technique to molecular crystals and metals and other inorganics are identified. From this, protocols for the measurement of hardness and elastic modulus of molecular crystals of pharmaceutical interest are proposed. Using form I aspirin as a model system, the impact of single crystal sample surface preparation (washing and cleaving) on the surface roughness is explored. We show the importance of using a calibration sample with hardness and stiffness close to that of molecular crystals for the acquisition of more accurate data. The issue of solvent occlusions formed during crystal growth is discussed as a source of material property variation as well as tip contamination. It is proposed that this in part explains the significantly larger variation of the measured mechanical properties among different single crystals compared to those performed on a unique sample. Because both the indentation modulus and the hardness can vary significantly for low depth indents, samples were tested over a wide range of depths, which revealed that a minimum depth of penetration is required for the acquisition of data. This experiment is crucial and needs to be carried out for every system under study since it allows for the determination of the minimum-working load. Post-indentation imaging combined with crystallographic analysis and molecular simulations allows for the characterization and rationalization of the material plastic deformation mechanisms.

Atmospheric Pressure Catalytic Vapor Deposition of Graphene on Liquid Sn and Cu-Sn Alloy Substrates.

The chemical vapor deposition (CVD) of graphene on liquid substrates produces high quality graphene films due to the defect-free and atomically flat surfaces of the liquids. Through the detailed study of graphene growth on liquid Sn using atmospheric pressure CVD (APCVD), the quality of graphene has been found to have a close relationship with hydrogen flow rate that reflects on hydrogen partial pressure inside the reactor (PH2) and hydrogen solubility of the growth substrates. The role of PH2 was found to be crucial, with a low defect density monolayer graphene being obtained in low PH2 (90.4 mbar), while partial graphene coverage occurred at high PH2 (137.3 mbar). To further understand the role of substrate's composition, binary alloy with compositions of 20, 30, 50, 60 and 80 wt.% tin in copper were made by arc-melting. Graphene quality was found to decrease with increasing the content of copper in the Cu-Sn alloys when grown using the conditions optimised for Sn substrates and this was related to the change in hydrogen solubility and the high catalytic activity of Cu compared to Sn. This shall provide a tool to help optimising CVD conditions for graphene growth based on the properties of the used catalytic substrate.

Fluid/Fiber Interactions and the Conductivity of Inkjet Printed Ag on Textile Substrates.

X-ray tomographic reconstruction reveals that the distribution of Ag after inkjet printing and sintering a nanoparticle conducting ink on a woven polyester textile substrate is strongly controlled by the fiber surface properties and fabric architecture. Capillarity confines the transport of the ink predominantly within the warp or weft yarns of the fabric and there is little transport of ink between the yarns. Changing the fiber surface energy through the Scotchgard treatment leads to an increase in the contact angle, reducing ink transport along the fibers and an increase in conductance. A similar effect is seen when printed drop spacing is reduced, increasing the local Ag concentration. Electrical conductivity is strongly influenced by the fiber density in each yarn direction and, in this case, the different densities lead to different electrical conductance values. Through the use of image segmentation, it is possible to identify a low level of electrical interconnection between the warp and weft yarns. Conductance within a yarn is shown to depend on Ag concentration via a percolation mechanism and this is confirmed by a simple model relating the volume of the largest interconnected Ag object present to the measured conductance. These results illustrate the complexity of the interaction between conductive inks and fibrous substrates and that concepts, such as sheet resistance, used to characterize printed conductors on solid substrates are not applicable to textile substrates.

Aberrant Differentiation of Human Pluripotent Stem Cell-Derived Kidney Precursor Cells inside Mouse Vascularized Bioreactors.

BACKGROUND: Numerous studies have documented the in vitro differentiation of human pluripotent stem cells (hPSCs) into kidney cells. Fewer studies have followed the fates of such kidney precursor cells (KPCs) inside animals, a more life-like setting. Here, we tested the hypothesis that implanting hPSC-derived KPCs into an in vivo milieu surgically engineered to be highly vascular would enhance their maturation into kidney tissues. METHODS: 3D printed chambers containing KPCs were implanted into the thighs of adult immunodeficient mice. In some chambers, an arterial and venous flow-through (AVFT) was surgically fashioned. After 3 weeks and 3 months, implants were studied by histology, using qualitative and quantitative methods. RESULTS: After 3 weeks, chambers containing AVFTs were richer in small vessels than contralateral chambers without AVFTs. Glomeruli with capillary loops and diverse types of tubules were detected in all chambers. At 3 months, chambers contained only rudimentary tubules and glomeruli that appeared avascular. In chambers with AVFTs, prominent areas of muscle-like cells were also detected near tubules and the abnormal tissues immunostained for transforming growth factor ß1. These features have similarities to renal dysplasia, a typical histological signature of human congenital kidney malformations. CONCLUSIONS: This study urges a note of caution regarding the in vivo fates of hPSC-derived kidney precursors, with pathological differentiation appearing to follow a period of increased vascularity.

Chemical vapour deposition of graphene on copper-nickel alloys: the simulation of a thermodynamic and kinetic approach.

Chemical vapour deposition (CVD) of graphene on transition metals is generally believed to be the fabrication route best suited for the production of high-quality large-area graphene sheets. The mechanism of CVD graphene growth is governed by interactions in both the gas phase and at the surface. Here we present a simulation of the CVD graphene growth mechanism which includes thermodynamics, gas phase kinetics and the surface reaction in a sequential manner. The thermodynamic simulation shows that the deposition driving force is the greatest for high carbon to hydrogen ratios and reaches a maximum at around 850 °C. No graphene growth is observed below this temperature. The surface kinetic model also shows that below this temperature, the carbon surface concentration is less than the solubility limit, thus no film can grow. The effect of the reaction chamber geometry on the product concentrations was clear from the gas phase decomposition reactions. The gas residence times studied here (around 0.07 s) show that the optimum gas phase composition is far from that expected at thermodynamic equilibrium. The surface kinetics of CH4 reactions on Ni, Cu and Cu-Ni surfaces shows good agreement with the experimental results for different growth pressures (0.1 to 0.7 mbar), temperatures (600 to 1200 °C) and different Ni thicknesses (25-500 µm). Also, the model works well when substrates with various C solubilities are used. The thermodynamic and kinetic models described here can be used for the design of improved reactors to optimise the production of graphene with differing qualities, either single or multi-layer and sizes. More importantly, the transfer to a continuous process with a moving substrate should also be possible using the model if it is extended from 2D to 3D.

Probing Ink-Powder Interactions during 3D Binder Jet Printing Using Time-Resolved X-ray Imaging.

Capillary-driven ink infiltration through a porous powder bed in three-dimensional (3D) binder jet printing (inkjet printing onto a powder bed) controls the printing resolution and as-printed "green" strength of the resulting object. However, a full understanding of the factors controlling the kinetics of the infiltration remains incomplete. Here, high-resolution in situ synchrotron radiography provides time-resolved imaging of the penetration of an aqueous solution of eythylene glycol through a porous alumina powder bed, used as a model system. A static drop-on-demand inkjet printer was used to dispense liquid droplets onto a powder surface. The subsequent migration of the liquid front and its interactions with powder particles were tracked using fast synchrotron X-radiography in the Diamond Synchrotron, with phase-contrast imaging at a frame rate of 500 Hz. Image processing and analysis reveal that both the time-dependent increment in the wetting area and the propagation of the "interface leading edge" exhibit heterogeneous behavior in both temporal and spatial domains. However, mean infiltration kinetics are shown to be consistent with existing infiltration models based on the Washburn equation modified to account for the spreading of the liquid drop on the powder surface and using a modified term for the bed porosity.

Direct 3D printing of graphene using capillary suspensions.

Conventional 3D printing of graphene requires either a complex formulation of the ink with large quantities of polymers or essential post-processing steps such as freeze drying to allow printability. Here we present a graphene capillary suspension (GCS) containing 16.67 wt% graphene nanoparticles in aqueous suspension with 3.97 wt% carboxymethyl cellulose (CMC) as a stabiliser and a small quantity of the immiscible liquid octanol. This is shown to have the appropriate rheological properties for 3D printing, which is demonstrated through the fabrication of a simple lattice structure by direct writing and air drying at room temperature. The printed structure has a porosity of 81%, is robust for handling with a compression strength of 1.3 MPa and has an electrical conductivity of 250 S m-1. After heat treatment at 350 °C conductivity is 2370 S m-1 but the strength reduces to 0.4 MPa. X-Ray tomography of the internal architecture after printing shows the formation of the capillary suspension eliminates ordering of the 2D materials during extrusion through the printer nozzle. Thus capillary suspensions can be used to direct write graphene 3D structures without the necessity of complicated drying steps or burn-out of large quantities of polymer additions, facilitating shape retention and property control as compared to current 2D material ink formulations used for 3D printing.

Tiled Monolayer Films of 2D Molybdenum Disulfide Nanoflakes Assembled at Liquid/Liquid Interfaces.

Thin films of MoS2 bilayer nanoflakes, which are predominantly a single flake thick and with flakes in edge-to-edge contact, have been produced via self-assembled tiling at the planar interface between two immiscible liquids. Films of several square centimeters extent can be produced with a total covered area approaching 90% and over 70% of the film covered by single flakes without overlap. Films produced through liquid/liquid assembly are shown to produce a lower uncovered area fraction and more uniform thickness when compared with films of similar areal coverage produced by the "top-down" techniques of spin coating and spray coating. Statistical analysis of flake coverage data, measured by atomic force microscopy (AFM), shows that liquid/liquid assembly produces a distinctly different variation in film thickness than conventional top-down deposition. This supports the hypothesis that the two-dimensional (2D) confinement of liquid/liquid assembly produces more uniform films. Demonstrator field-effect transistors (FETs) manufactured from the films exhibit mobility and on/off current ratios of 0.73 cm2 V-1 s-1 and 105, respectively, comparable to FETs of similar layout and chemical vapor deposition (CVD)-grown or mechanically cleaved single-crystal MoS2 channel material. This work demonstrates the use of liquid/liquid interfaces as a useful tool for the self-assembly of high-performance thin-film devices made from dispersions of 2D materials.

The systemic influence of chronic smoking on skin structure and mechanical function.

One of the major functions of human skin is to provide protection from the environment. Although we cannot entirely avoid, for example, sun exposure, it is likely that exposure to other environmental factors could affect cutaneous function. A number of studies have identified smoking as one such factor that leads to both facial wrinkle formation and a decline in skin function. In addition to the direct physical effects of tobacco smoke on skin, its inhalation has additional profound systemic effects for the smoker. The adverse effects on the respiratory and cardiovascular systems from smoking are well known. Central to the pathological changes associated with smoking is the elastic fibre, a key component of the extracellular matrices of lungs. In this study we examined the systemic effect of chronic smoking (>40 cigarettes/day; >5 years) on the histology of the cutaneous elastic fibre system, the nanostructure and mechanics of one of its key components, the fibrillin-rich microfibril, and the micromechanical stiffness of the dermis and epidermis. We show that photoprotected skin of chronic smokers exhibits significant remodelling of the elastic fibre network (both elastin and fibrillin-rich microfibrils) as compared to the skin of age- and sex-matched non-smokers. This remodelling is not associated with increased gelatinase activity (as identified by in situ zymography). Histological remodelling is accompanied by significant ultrastructural changes to extracted fibrillin-rich microfibrils. Finally, using scanning acoustic microscopy, we demonstrated that chronic smoking significantly increases the stiffness of both the dermis and the epidermis. Taken together, these data suggest an unappreciated systemic effect of chronic inhalation of tobacco smoke on the cutaneous elastic fibre network. Such changes may in part underlie the skin wrinkling and loss of skin elasticity associated with smoking. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The formation mechanism of hexagonal Mo2C defects in CVD graphene grown on liquid copper.

Thin Mo2C hexagonal defects precipitate in CVD graphene when Mo crucibles are engaged to hold the liquid copper substrate, while these defects disappear when W crucibles are present. These defects have been identified as the thin precipitates of Mo2C. The growth mechanism of the Mo2C defects is demonstrated through thermodynamic calculations. This can be beneficial in graphene defect engineering through the vapour phase transport of the volatile MoO3 phase.

Patterned, morphing composites via maskless photo-click lithography.

Morphing materials, also known as smart materials are attracting increasing attention as sensors, actuators and in soft robotic applications. In this work bilayered morphing composites were created by exploiting the thiol-ene photoclick reaction via maskless digital light processing (DLP). This technique allows for gradients and patterns of near infrared (nIR)-triggered materials to be efficiently crosslinked to substrates, with suitable interfacial adhesion to realise complex morphing. Photo-thermally responsive composites are produced by DLP patterning of reduced graphene oxide-filled chitosan-methacrylamide (rGO-chitosan-MA) on thiolated polydimethylsiloxane substrates via thiol-ene photoclick reaction. Morphing composites with parallel striped patterns and box-like hinges were printed via DLP to realise self-rolling and self-folding behaviours. Bilayered structures, with gradient rGO-chitosan-MA thicknesses (2-8 µm), were produced by controlling the light intensity from the DLP device. These gradient bilayered structures enable photothermal-triggered gradient bending and morphing exemplified here by a "walking worm" and a kirigami-inspired "opening flower". Thermo-mechanical calculations were performed to estimate bending angles, and finite element analysis applied to simulate self-folding and bending. The difference between simulation and measurements is in the range 0.4-7.6%, giving confidence to the assumptions and simplifications applied in design.

Acoustic Poration and Dynamic Healing of Mammalian Cell Membranes during Inkjet Printing.

We have investigated the effect of piezoelectric actuating voltage on cell behavior after drop on demand inkjet printing using mouse 3T3 cells as a model cell line. Cell viability after printing was assessed using a live/dead assay, Alamar Blue as an assay for cell proliferation, and propidium iodide (PI) and Texas Red labeled dextran molecular probes to assess cell membrane integrity. No significant difference was found for the cell death rate compared between an unprinted control population and after printing at 80, 90, and 100 V, respectively. However, cell proliferation was lower than that of the control population at all time points postprinting. Cell membrane integrity was quantified using PI and dextran probes of mean molecular weight of 3, 10, 40, and 70 kDa. Total membrane damage (assessed by PI) increased with increasing piezoelectric actuator driving voltage, and this was always greater than the unprinted control cells. The uptake of the labeled dextran only occurs after inkjet printing and was never observed with the control cells. The largest dextran molecular probe of 70 kDa was only taken up by cells after printing using the lower printing voltages of 80 and 90 V and was absent after printing at 100 V. At the two lower printing voltages, the membrane damage is recovered, and no dextran molecule penetrated the cells 2 h after printing. However, printing at 100 V leads to an increased uptake of 3 and 10 kDa dextran molecules, the retention of membrane porosity, and continued uptake of these 3 and 10 kDa dextran for greater than 2 h postprinting. We hypothesize that the change in membrane porosity with increasing actuation voltage can be explained by distinct nucleation and growth stages for pore formation in response to printing stress.

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins.

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the cells themselves. Here, we present the method of Light-Induced Molecular Adsorption of Proteins (LIMAP) using the PRIMO system as a patterning technique to produce micro-patterned extracellular matrix (ECM) substrates using a single or combination of proteins. The method enables printing of ECM patterns in micron resolution with excellent reproducibility. We provide a step-by-step protocol and demonstrate how this can be applied to study the processes of neuronal pathfinding. LIMAP has significant advantages over existing micro-printing methods in terms of the ease of patterning more than one component and the ability to generate a pattern with any geometry or gradient. The protocol can easily be adapted to study the contribution of almost any chemical component towards cell fate and cell behavior. Finally, we discuss common issues that can arise and how these can be avoided.

Screen-Printing of a Highly Conductive Graphene Ink for Flexible Printed Electronics.

Conductive inks for the future printed electronics should have the following merits: high conductivity, flexibility, low cost, and compatibility with wide range of substrates. However, the state-of-the-art conductive inks based on metal nanoparticles are high in cost and poor in flexibility. Herein, we reported a highly conductive, low cost, and super flexible ink based on graphene nanoplatelets. The graphene ink has been screen-printed on plastic and paper substrates. Combined with postprinting treatments including thermal annealing and compression rolling, the printed graphene pattern shows a high conductivity of 8.81 × 104 S m-1 and good flexibility without significant conductivity loss after 1000 bending cycles. We further demonstrate that the printed highly conductive graphene patterns can act as current collectors for supercapacitors. The supercapacitor with the printed graphene pattern as the current collector and printed activated carbon as the active material shows a good rate capability of up to 200 mV s-1. This work potentially provides a promising route toward the large-scale fabrication of low cost yet flexible printed electronic devices.

Angiogenesis and tissue formation driven by an arteriovenous loop in the mouse.

The rapid vascularisation of biomaterials and artificial tissues is a key determinant for their in vivo viability and ultimately for their integration in a host; therefore promoting angiogenesis and maintaining the newly formed vascular beds has become a major goal of tissue engineering. The arteriovenous loop (AVL) has been an extensively studied platform which integrates microsurgery with cells scaffolds and growth factors to form neotissues. Most AVL studies to date are limited to larger animal models, which are surgically easier to perform, but have inherent limits for the understanding and interrogation of the underlying in vivo mechanisms due the paucity of transgenic models. Here, we demonstrate for the first time in a mouse model the utility of the AVL in the de novo production of vascularized tissue. We also present the combined use of the model with 3D printed chambers, which allow us to dictate size and shape of the tissues formed. This novel platform will allow for an understanding of the fundamental mechanisms involved in tissue generation de novo.