The DIAD Approach to Correlative Synchrotron X-ray Imaging and Diffraction Analysis of Human Enamel.

The Dual Imaging and Diffraction (DIAD) beamline at Diamond Light Source (Didcot, U.K.) implements a correlative approach to the dynamic study of materials based on concurrent analysis of identical sample locations using complementary X-ray modalities to reveal structural detail at various length scales. Namely, the underlying beamline principle and its practical implementation allow the collocation of chosen regions within the sample and their interrogation using real-space imaging (radiography and tomography) and reciprocal space scattering (diffraction). The switching between the two principal modes is made smooth and rapid by design, so that the data collected is interlaced to obtain near-simultaneous multimodal characterization. Different specific photon energies are used for each mode, and the interlacing of acquisition steps allows conducting static and dynamic experiments. Building on the demonstrated realization of this state-of-the-art approach requires further refining of the experimental practice, namely, the methods for gauge volume collocation under different modes of beam-sample interaction. To address this challenge, experiments were conducted at DIAD devoted to the study of human dental enamel, a hierarchical structure composed of hydroxyapatite mineral nanocrystals, as a static sample previously affected by dental caries (tooth decay) as well as under dynamic conditions simulating the process of acid demineralization. Collocation and correlation were achieved between WAXS (wide-angle X-ray scattering), 2D (radiographic), and 3D (tomographic) imaging. While X-ray imaging in 2D or 3D modes reveals real-space details of the sample microstructure, X-ray scattering data for each gauge volume provided statistical nanoscale and ultrastructural polycrystal reciprocal-space information such as phase and preferred orientation (texture). Careful registration of the gauge volume positions recorded during the scans allowed direct covisualization of the data from two modalities. Diffraction gauge volumes were identified and visualized within the tomographic data sets, revealing the underlying local information to support the interpretation of the diffraction patterns. The present implementation of the 4D microscopy paradigm allowed following the progression of demineralization and its correlation with time-dependent WAXS pattern evolution in an approach that is transferable to other material systems.

Advanced Time-Stepping Interpretation of Fly-Scan Continuous Rotation Synchrotron Tomography of Dental Enamel Demineralization.

High-resolution spatial and temporal analysis and 3D visualization of time-dependent processes, such as human dental enamel acid demineralization, often present a challenging task. Overcoming this challenge often requires the development of special methods. Dental caries remains one of the most important oral diseases that involves the demineralization of hard dental tissues as a consequence of acid production by oral bacteria. Enamel has a hierarchically organized architecture that extends down to the nanostructural level and requires high resolution to study its evolution in detail. Enamel demineralization is a dynamic process that is best investigated with the help of in situ experiments. In previous studies, synchrotron tomography was applied to study the 3D enamel structure at certain time points (time-lapse tomography). Here, another distinct approach to time-evolving tomography studies is presented, whereby the sample image is reconstructed as it undergoes continuous rotation over a virtually unlimited angular range. The resulting (single) data set contains the data for multiple (potentially overlapping) intermediate tomograms that can be extracted and analyzed as desired using time-stepping selection of data subsets from the continuous fly-scan recording. One of the advantages of this approach is that it reduces the amount of time required to collect an equivalent number of single tomograms. Another advantage is that the nominal time step between successive reconstructions can be significantly reduced. We applied this approach to the study of acidic enamel demineralization and observed the progression of demineralization over time steps significantly smaller than the total acquisition time of a single tomogram, with a voxel size smaller than 0.5 µm. It is expected that the approach presented in this paper can be useful for high-resolution studies of other dynamic processes and for assessing small structural modifications in evolving hierarchical materials.

Residual Stresses in Surgical Growing Rods.

The treatment of early onset scoliosis using surgical growing rods suffers from high failure rate. Fatigue resistance can be improved by inducing compressive residual stresses within the near surface region. An in-depth investigation of the residual stresses profile evolution is performed through the sequence of material processing steps followed by surgeons handling operations, in connection to material properties. The final goal is to guide further improvements of growing rod lifetime. Residual stress evaluation was carried out on Ti-6Al-4V rods using digital image correlation applied to microbeam ring-core milling by focused ion beam. This provided experimental stress profiles in shot-peened rods before and after bending and demonstrated that compressive residual stresses are maintained at both concave and convex rod sides. A finite element model using different core and skin conditions was validated by comparison to experiments. The combination of an initial shot peening profile associated with a significant level of backstress was found to primarily control the generation of compressive stresses at the rod surface after bending. Guidelines to promote larger compressive stresses at the surface were formulated based on a parametric analysis. The analysis revealed the first order impact of the initial yield strength, kinematic hardening parameters and intensity of the shot peening operation, while the bending angle and the depth of shot peening stresses were found to be of minor importance. Materials exhibiting large kinematic hardening and low yield strength should be selected in order to induce compressive residual stresses at key fatigue initiation site.

Revealing the origins of vortex cavitation in a Venturi tube by high speed X-ray imaging.

Hydrodynamic cavitation is useful in many processing applications, for example, in chemical reactors, water treatment and biochemical engineering. An important type of hydrodynamic cavitation that occurs in a Venturi tube is vortex cavitation known to cause luminescence whose intensity is closely related to the size and number of cavitation events. However, the mechanistic origins of bubbles constituting vortex cavitation remains unclear, although it has been concluded that the pressure fields generated by the cavitation collapse strongly depends on the bubble geometry. The common view is that vortex cavitation consists of numerous small spherical bubbles. In the present paper, aspects of vortex cavitation arising in a Venturi tube were visualized using high-speed X-ray imaging at SPring-8 and European XFEL. It was discovered that vortex cavitation in a Venturi tube consisted of angulated rather than spherical bubbles. The tangential velocity of the surface of vortex cavitation was assessed considering the Rankine vortex model.

Hair-Reinforced Elastomer Matrix Composites: Formulation, Mechanical Testing, and Advanced Microstructural Characterization.

Epoxy matrix composites reinforced with high-performance fibers, such as carbon, Kevlar, and glass, exhibit excellent specific stiffness and strength in many mechanical applications. However, these composites are disappointingly non-recyclable and are usually disposed of in landfill sites, with no realistic prospect for biodegradation in a reasonable time. In contrast, moldable composites with carbonized elastomeric matrices developed in the last decades possess attractive mechanical properties in final net-shape products and can also be incinerated or recycled. Many carbon and inorganic fillers have recently been evaluated to adjust the properties of carbonized elastomeric composites. Renewable organic fillers, such as human or animal hair, offer an attractive fibrous material with substantial potential for reinforcing composites with elastomeric matrices. Samples of unidirectional fiber composites (with hair volume fractions up to 7%) and quasi-isotropic short fiber composites (with hair volume fractions up to 20%) of human hair-reinforced nitrile butadiene rubbers (HH-NBRs) were produced in the peroxide-cured and carbonized states. The samples were characterized using scanning electron microscopy (SEM), Raman spectroscopy, and photoacoustic microscopy. Mechanical tests were performed under tension using a miniature universal testing machine. The expected effect of fiber reinforcement on the overall mechanical performance was demonstrated for both cured and carbonized composites. Considerable enhancement of the elastic modulus (up to ten times), ultimate tensile strength (up to three times), and damage tolerance was achieved. The evidence of satisfactory interfacial bonding between hair and rubber was confirmed via SEM imaging of fracture surfaces. The suitability of photoacoustic microscopy was assessed for 3D reconstructions of the fiber sub-system's spatial distribution and non-destructive testing.

Multi-resolution Correlative Ultrastructural and Chemical Analysis of Carious Enamel by Scanning Microscopy and Tomographic Imaging.

Caries, a major global disease associated with dental enamel demineralization, remains insufficiently understood to devise effective prevention or minimally invasive treatment. Understanding the ultrastructural changes in enamel is hampered by a lack of nanoscale characterization of the chemical spatial distributions within the dental tissue. This leads to the requirement to develop techniques based on various characterization methods. The purpose of the present study is to demonstrate the strength of analytic methods using a correlative technique on a single sample of human dental enamel as a specific case study to test the accuracy of techniques to compare regions in enamel. The science of the different techniques is integrated to genuinely study the enamel. The hierarchical structures within carious tissue were mapped using the combination of focused ion beam scanning electron microscopy with synchrotron X-ray tomography. The chemical changes were studied using scanning X-ray fluorescence (XRF) and X-ray wide-angle and small-angle scattering using a beam size below 80 nm for ångström and nanometer length scales. The analysis of XRF intensity gradients revealed subtle variations of Ca intensity in carious samples in comparison with those of normal mature enamel. In addition, the pathways for enamel rod demineralization were studied using X-ray ptychography. The results show the chemical and structural modification in carious enamel with differing locations. These results reinforce the need for multi-modal approaches to nanoscale analysis in complex hierarchically structured materials to interpret the changes of materials. The approach establishes a meticulous correlative characterization platform for the analysis of biomineralized tissues at the nanoscale, which adds confidence in the interpretation of the results and time-saving imaging techniques. The protocol demonstrated here using the dental tissue sample can be applied to other samples for statistical study and the investigation of nanoscale structural changes. The information gathered from the combination of methods could not be obtained with traditional individual techniques.

Time-Lapse In Situ 3D Imaging Analysis of Human Enamel Demineralisation Using X-ray Synchrotron Tomography.

Caries is a chronic disease that causes the alteration of the structure of dental tissues by acid dissolution (in enamel, dentine and cementum) and proteolytic degradation (dentine and cementum) and generates an important cost of care. There is a need to visualise and characterise the acid dissolution process on enamel due to its hierarchical structure leading to complex structural modifications. The process starts at the enamel surface and progresses into depth, which necessitates the study of the internal enamel structure. Artificial demineralisation is usually employed to simulate the process experimentally. In the present study, the demineralisation of human enamel was studied using surface analysis carried out with atomic force microscopy as well as 3D internal analysis using synchrotron X-ray tomography during acid exposure with repeated scans to generate a time-lapse visualisation sequence. Two-dimensional analysis from projections and virtual slices and 3D analysis of the enamel mass provided details of tissue changes at the level of the rods and inter-rod substance. In addition to the visualisation of structural modifications, the rate of dissolution was determined, which demonstrated the feasibility and usefulness of these techniques. The temporal analysis of enamel demineralisation is not limited to dissolution and can be applied to other experimental conditions for the analysis of treated enamel or remineralisation.

Synchrotron X-ray Studies of the Structural and Functional Hierarchies in Mineralised Human Dental Enamel: A State-of-the-Art Review.

Hard dental tissues possess a complex hierarchical structure that is particularly evident in enamel, the most mineralised substance in the human body. Its complex and interlinked organisation at the Ångstrom (crystal lattice), nano-, micro-, and macro-scales is the result of evolutionary optimisation for mechanical and functional performance: hardness and stiffness, fracture toughness, thermal, and chemical resistance. Understanding the physical-chemical-structural relationships at each scale requires the application of appropriately sensitive and resolving probes. Synchrotron X-ray techniques offer the possibility to progress significantly beyond the capabilities of conventional laboratory instruments, i.e., X-ray diffractometers, and electron and atomic force microscopes. The last few decades have witnessed the accumulation of results obtained from X-ray scattering (diffraction), spectroscopy (including polarisation analysis), and imaging (including ptychography and tomography). The current article presents a multi-disciplinary review of nearly 40 years of discoveries and advancements, primarily pertaining to the study of enamel and its demineralisation (caries), but also linked to the investigations of other mineralised tissues such as dentine, bone, etc. The modelling approaches informed by these observations are also overviewed. The strategic aim of the present review was to identify and evaluate prospective avenues for analysing dental tissues and developing treatments and prophylaxis for improved dental health.

Hydrogel-Inducing Graphene-Oxide-Derived Core-Shell Fiber Composite for Antibacterial Wound Dressing.

The study reveals the polymer-crosslinker interactions and functionality of hydrophilic nanofibers for antibacterial wound coatings. Coaxial electrospinning leverages a drug encapsulation protocol for a core-shell fiber composite with a core derived from polyvinyl alcohol and polyethylene glycol with amorphous silica (PVA-PEG-SiO2), and a shell originating from polyvinyl alcohol and graphene oxide (PVA-GO). Crosslinking with GO and SiO2 initiates the hydrogel transition for the fiber composite upon contact with moisture, which aims to optimize the drug release. The effect of hydrogel-inducing additives on the drug kinetics is evaluated in the case of chlorhexidine digluconate (CHX) encapsulation in the core of core-shell fiber composite PVA-PEG-SiO2-1x-CHX@PVA-GO. The release rate is assessed with the zero, first-order, Higuchi, and Korsmeyer-Peppas kinetic models, where the inclusion of crosslinking silica provides a longer degradation and release rate. CHX medicated core-shell composite provides sustainable antibacterial activity against Staphylococcus aureus.

Revealing the static and dynamic nanomechanical properties of diatom frustules-Nature's glass lace.

Diatoms are single cell microalgae enclosed in silica exoskeletons (frustules) that provide inspiration for advanced hybrid nanostructure designs mimicking multi-scale porosity to achieve outstanding mechanical and optical properties. Interrogating the structure and properties of diatoms down to nanometer scale leads to breakthrough advances reported here in the nanomechanical characterization of Coscinodiscus oculus-iridis diatom pure silica frustules, as well as of air-dried and wet cells with organic content. Static and dynamic mode Atomic Force Microscopy (AFM) and in-SEM nanoindentation revealed the peculiarities of diatom response with separate contributions from material nanoscale behavior and membrane deformation of the entire valve. Significant differences in the nanomechanical properties of the different frustule layers were observed. Furthermore, the deformation response depends strongly on silica hydration and on the support from the internal organic content. The cyclic loading revealed that the average compliance of the silica frustule is 0.019 m/N and increases with increasing number of cycles. The structure-mechanical properties relationship has a direct impact on the vibrational properties of the frustule as a complex micrometer-sized mechanical system. Lessons from Nature's nanostructuring of diatoms open up pathways to new generations of nano- and microdevices for electronic, electromechanical, photonic, liquid, energy storage, and other applications.

An In Vivo Rat Study of Bioresorbable Mg-2Zn-2Ga Alloy Implants.

In the present study, pins made from the novel Mg-2Zn-2Ga alloy were installed within the femoral bones of six Wistar rats. The level of bioresorption was assessed after 1, 3, and 6 months by radiography, histology, SEM, and EDX. Significant bioresorption was evident after 3 months, and complete dissolution of the pins occurred at 6 months after the installation. No pronounced gas cavities could be found at the pin installation sites throughout the postoperative period. The animals' blood parameters showed no signs of inflammation or toxication. These findings are sufficiently encouraging to motivate further research to broaden the experimental coverage to increase the number of observed animals and to conduct tests involving other, larger animals.

A concept of "materials" diffraction and imaging beamline for SKIF: Siberian circular photon source.

Over the next decade, the extremely brilliant fourth generation synchrotron radiation sources are set to become a key driving force in materials characterization and technology development. In this study, we present a conceptual design of a versatile "Materia" diffraction and imaging beamline for a low-emittance synchrotron radiation facility. The beamline was optimized for operation with three main principal delivery regimes: parallel collimated beam ∼1 mm beam size, micro-focus regime with ∼10 µm beam spot size on the sample, and nano-focus regime with <100 nm focus. All regimes will operate in the photon energy range of 10-30 keV with the key feature of the beamline being fast switching between them, as well as between the various realizations of diffraction and imaging operation modes while maintaining the target beam position at the sample, and with both spectrally narrow and spectrally broad beams up to the energy band ΔE/E of 5 × 10-2. The manuscript presents the details of the principal characteristics selected for the insertion device and beamline optics, the materials characterization techniques, including the simulations of thermal load impact on the critical beamline optics components. Significant efforts were made to design the monochromators to mitigate the very high beam power load produced by a superconducting undulator source. The manuscript will be of interest to research groups involved in the design of new synchrotron beamlines.

Analysis of Stress Relaxation in Bulk and Porous Ultra-High Molecular Weight Polyethylene (UHMWPE).

The reported study was devoted to the investigation of viscoelastic behavior for solid and porous ultra-high molecular weight polyethylene (UHMWPE) under compression. The obtained experimental stress curves were interpreted using a two-term Prony series to represent the superposition of two coexisting activation processes corresponding to long molecular (~160 s) and short structural (~20 s) time scales, respectively, leading to good statistical correlation with the observations. In the case of porous polymer, the internal strain redistribution during relaxation was quantified using digital image correlation (DIC) analysis. The strongly inhomogeneous deformation of the porous polymer was found not to affect the relaxation times. To illustrate the possibility of generalizing the results to three dimensions, X-ray tomography was used to examine the porous structure relaxation at the macro- and micro-scale levels. DIC analysis revealed positive correlation between the applied force and relative density. The apparent stiffness variation for UHMWPE foams with mixed open and closed cells was described using a newly proposed three-term expression. Furthermore, in situ tensile loading and X-ray scattering study was applied for isotropic solid UHMWPE specimens to investigate the evolution of internal structure and orientation during drawing and stress relaxation in another loading mode.

Two-dimensional wavefront characterization of adaptable corrective optics and Kirkpatrick-Baez mirror system using ptychography.

Aberrations introduced during fabrication degrade the performance of X-ray optics and their ability to achieve diffraction limited focusing. Corrective optics can counteract these errors by introducing wavefront perturbations prior to the optic which cancel out the distortions. Here we demonstrate two-dimensional wavefront correction of an aberrated Kirkpatrick-Baez mirror pair using adaptable refractive structures. The resulting two-dimensional wavefront is measured using hard X-ray ptychography to recover the complex probe wavefield with high spatial resolution and model the optical performance under coherent conditions. The optical performance including the beam caustic, focal profile and wavefront error is examined before and after correction with both mirrors found to be diffraction limited after correcting. The results will be applicable to a wide variety of high numerical aperture X-ray optics aiming to achieve diffraction limited focussing using low emittance sources.

Inhibiting weld cracking in high-strength aluminium alloys.

Cracking from a fine equiaxed zone (FQZ), often just tens of microns across, plagues the welding of 7000 series aluminum alloys. Using a multiscale correlative methodology, from the millimeter scale to the nanoscale, we shed light on the strengthening mechanisms and the resulting intergranular failure at the FQZ. We show that intergranular AlCuMg phases give rise to cracking by micro-void nucleation and subsequent link-up due to the plastic incompatibility between the hard phases and soft (low precipitate density) grain interiors in the FQZ. To mitigate this, we propose a hybrid welding strategy exploiting laser beam oscillation and a pulsed magnetic field. This achieves a wavy and interrupted FQZ along with a higher precipitate density, thereby considerably increasing tensile strength over conventionally hybrid welded butt joints, and even friction stir welds.

Grain Structure Engineering of NiTi Shape Memory Alloys by Intensive Plastic Deformation.

To explore an effective route of customizing the superelasticity (SE) of NiTi shape memory alloys via modifying the grain structure, binary Ni55Ti45 (wt) alloys were fabricated in as-cast, hot swaged, and hot-rolled conditions, presenting contrasting grain sizes and grain boundary types. In situ synchrotron X-ray Laue microdiffraction and in situ synchrotron X-ray powder diffraction techniques were employed to unravel the underlying grain structure mechanisms that cause the diversity of SE performance among the three materials. The evolution of lattice rotation, strain field, and phase transformation has been revealed at the micro- and mesoscale, and the effect of grain structure on SE performance has been quantified. It was found that (i) the Ni4Ti3 and NiTi2 precipitates are similar among the three materials in terms of morphology, size, and orientation distribution; (ii) phase transformation happens preferentially near high-angle grain boundary (HAGB) yet randomly in low-angle grain boundary (LAGB) structures; (iii) the smaller the grain size, the higher the phase transformation nucleation kinetics, and the lower the propagation kinetics; (iv) stress concentration happens near HAGBs, while no obvious stress concentration can be observed in the LAGB grain structure during loading; (v) the statistical distribution of strain in the three materials becomes asymmetric during loading; (vi) three grain lattice rotation modes are identified and termed for the first time, namely, multi-extension rotation, rigid rotation, and nondispersive rotation; and (vii) the texture evolution of B2 austenite and B19' martensite is not strongly dependent on the grain structure.

Fracture Toughness of Moldable Low-Temperature Carbonized Elastomer-Based Composites Filled with Shungite and Short Carbon Fibers.

This work evaluated the fracture toughness of the low-temperature carbonized elastomer-based composites filled with shungite and short carbon fibers. The effects of the carbonization temperature and filler content on the critical stress intensity factor (K1c) were examined. The K1c parameter was obtained using three-point bending tests for specimens with different l/b ratio (notch depth to sample thickness) ranging from 0.2 to 0.4. Reliable detection of the initiation and propagation of cracks was achieved using an acoustic sensor was attached to the samples during the bending test. The critical stress intensity factor was found to decrease linearly with increasing carbonization temperature. As the temperature increased from 280 to 380 °C, the K1c parameter was drastically reduced from about 5 to 1 MPa·m1/2 and was associated with intense outgassing during the carbonization step that resulted in sample porosity. The carbon fiber addition led to some incremental toughening; however, it reduced the statistical dispersion of the K1c values.

A SERS platform based on diatomite modified by gold nanoparticles using a combination of layer-by-layer assembly and a freezing-induced loading method.

Siliceous diatom frustules represent an up-and-coming platform for a range of bio-assisted nanofabrication processes able to overcome the complexity and high cost of current engineering technology solutions in terms of negligibly small power consumption and environmentally friendly processing combined with unique highly porous structures and properties. Herein, the modification of diatomite - a soft, loose, and fine-grained siliceous sedimentary rock composed of the remains of fossilized diatoms - with gold nanoparticles using layer-by-layer technology in combination with a freezing-induced loading approach is demonstrated. The obtained composite structures are characterized by dynamic light scattering, extinction spectroscopy, scanning (SEM) and transmission electron microscopy (TEM), and photoacoustic imaging techniques, and tested as a platform for surface-enhanced Raman scattering (SERS) using Rhodamine 6G. SEM, TEM, and energy dispersive X-ray spectroscopy (EDX) confirmed a dense coating of gold nanoparticles with an average size of 19 nm on the surface of the diatomite and within the pores. The photoacoustic signal excited at a wavelength of 532 nm increases with increasing loading cycles of up to three polyelectrolyte-gold nanoparticle bilayers. The hybrid materials based on diatomite modified with gold nanoparticles can be used as SERS substrates, but also as biosensors, catalysts, and platforms for advanced bioimaging.

Effect of Graphene Oxide and Nanosilica Modifications on Electrospun Core-Shell PVA-PEG-SiO2@PVA-GO Fiber Mats.

Electrospinning is a well-established method for the fabrication of polymer biomaterials, including those with core-shell nanofibers. The variability of structures presents a great range of opportunities in tissue engineering and drug delivery by incorporating biologically active molecules such as drugs, proteins, and growth factors and subsequent control of their release into the target microenvironment to achieve therapeutic effect. The object of study is non-woven core-shell PVA-PEG-SiO2@PVA-GO fiber mats assembled by the technology of coaxial electrospinning. The task of the core-shell fiber development was set to regulate the degradation process under external factors. The dual structure was modified with silica nanoparticles and graphene oxide to ensure the fiber integrity and stability. The influence of the nano additives and crosslinking conditions for the composite was investigated as a function of fiber diameter, hydrolysis, and mechanical properties. Tensile mechanical tests and water degradation tests were used to reveal the fracture and dissolution behavior of the fiber mats and bundles. The obtained fibers were visualized by confocal fluorescence microscopy to confirm the continuous core-shell structure and encapsulation feasibility for biologically active components, selectively in the fiber core and shell. The results provide a firm basis to draw the conclusion that electrospun core-shell fiber mats have tremendous potential for biomedical applications as drug carriers, photocatalysts, and wound dressings.

Increased connectivity of hiPSC-derived neural networks in multiphase granular hydrogel scaffolds.

To reflect human development, it is critical to create a substrate that can support long-term cell survival, differentiation, and maturation. Hydrogels are promising materials for 3D cultures. However, a bulk structure consisting of dense polymer networks often leads to suboptimal microenvironments that impedes nutrient exchange and cell-to-cell interaction. Herein, granular hydrogel-based scaffolds were used to support 3D human induced pluripotent stem cell (hiPSC)-derived neural networks. A custom designed 3D printed toolset was developed to extrude hyaluronic acid hydrogel through a porous nylon fabric to generate hydrogel granules. Cells and hydrogel granules were combined using a weaker secondary gelation step, forming self-supporting cell laden scaffolds. At three and seven days, granular scaffolds supported higher cell viability compared to bulk hydrogels, whereas granular scaffolds supported more neurite bearing cells and longer neurite extensions (65.52 ± 11.59 µm) after seven days compared to bulk hydrogels (22.90 ± 4.70 µm). Long-term (three-month) cultures of clinically relevant hiPSC-derived neural cells in granular hydrogels supported well established neuronal and astrocytic colonies and a high level of neurite extension both inside and beyond the scaffold. This approach is significant as it provides a simple, rapid and efficient way to achieve a tissue-relevant granular structure within hydrogel cultures.