The interplay between nucleation and patterning during shear-induced crystallization from solution in a parallel plate geometry.

Cooling crystallization of small organic molecules from solution is an important operation for the separation and purification of drug products. In this research, shear-induced nucleation from a supersaturated solution is studied in a parallel plate geometry. Under conditions of shear and small gap sizes, narrow mesoscale circular bands of small crystals appeared spontaneously and reproducibly on the plate's surface. We have investigated the connection between nucleation and the emergence of these circular patterns. Our results show that nucleation occurs preferably in zones with high local shear rate (located at the outer edges of the plates), compared to zones with low local shear rate (at the center of the plates). The time before nucleation occurs decreases significantly for increasing mean shear rate and time. The circular crystalline patterns appear at the plate's surface, where heterogeneous nucleation first occurs. Multiple hypotheses are explored to understand the pattern formation in crystallization. Since no satisfactory explanation is found, a new mechanism is proposed. This hypothesis involves crystals initially forming on the surface of the plates and undergoing stick-slip motion, which influences the local nucleation kinetics. This results in an interplay between (secondary) nucleation and stick-slip motion at the start of the crystallization process. By modifying the surface of the plates, their ability to act as a heterogeneous nucleation site can be altered, allowing control over the formation of patterns.

Large-scale perfused tissues via synthetic 3D soft microfluidics.

The vascularization of engineered tissues and organoids has remained a major unresolved challenge in regenerative medicine. While multiple approaches have been developed to vascularize in vitro tissues, it has thus far not been possible to generate sufficiently dense networks of small-scale vessels to perfuse large de novo tissues. Here, we achieve the perfusion of multi-mm3 tissue constructs by generating networks of synthetic capillary-scale 3D vessels. Our 3D soft microfluidic strategy is uniquely enabled by a 3D-printable 2-photon-polymerizable hydrogel formulation, which allows for precise microvessel printing at scales below the diffusion limit of living tissues. We demonstrate that these large-scale engineered tissues are viable, proliferative and exhibit complex morphogenesis during long-term in-vitro culture, while avoiding hypoxia and necrosis. We show by scRNAseq and immunohistochemistry that neural differentiation is significantly accelerated in perfused neural constructs. Additionally, we illustrate the versatility of this platform by demonstrating long-term perfusion of developing neural and liver tissue. This fully synthetic vascularization platform opens the door to the generation of human tissue models at unprecedented scale and complexity.

Stress-controlled shear flow alignment of collagen type I hydrogel systems.

Disease research and drug screening platforms require in vitro model systems with cellular cues resembling those of natural tissues. Fibrillar alignment, occurring naturally in extracellular matrices, is one of the crucial attributes in tissue development. Obtaining fiber alignment in 3D, in vitro remains an important challenge due to non-linear material characteristics. Here, we report a cell-compatible, shear stress-based method allowing to obtain 3D homogeneously aligned fibrillar collagen hydrogels. Controlling the shear-stress during gelation results in low strain rates, with negligible effects on the viability of embedded SH-SY5Y cells. Our approach offers reproducibility and tunability through a paradigm shift: The shear-stress initiation moment, being the critical optimization parameter in the process, is related to the modulus of the developing gel, whereas state of the art methods often rely on a predefined time to initiate the alignment procedure. After curing, the induced 3D alignment is maintained after the release of stress, with a linear relation between the total acquired strain and the fiber alignment. This method is generally applicable to 3D fibrillar materials and stress/pressure-controlled setups, making it a valuable addition to the fast-growing field of tissue engineering. STATEMENT OF SIGNIFICANCE: Controlling fiber alignment in vitro 3D hydrogels is crucial for developing physiologically relevant model systems. However, it remains challenging due to the non-linear material characteristics of fibrillar hydrogels, limiting the scalability and repeatability. Our approach tackles these challenges by utilizing a stress-controlled rheometer allowing us to monitor structural changes in situ and determine the optimal moment for applying a shear-stress inducing alignment. By careful parameter control, we infer the relationship between time, induced strain, alignment and biocompatibility. This tunable and reproducible method is both scalable and generally applicable to any fibrillar hydrogel, therefore, we believe it is useful for research investigating the link between matrix anisotropy and cell behavior in 3D systems, organ-on-chip technologies and drug research.

Glycyrrhizin-Based Hydrogels Accelerate Wound Healing of Normoglycemic and Diabetic Mouse Skin.

Efficient wound repair is crucial for mammalian survival. Healing of skin wounds is severely hampered in diabetic patients, resulting in chronic non-healing wounds that are difficult to treat. High-mobility group box 1 (HMGB1) is an important signaling molecule that is released during wounding, thereby delaying regenerative responses in the skin. Here, we show that dissolving glycyrrhizin, a potent HMGB1 inhibitor, in water results in the formation of a hydrogel with remarkable rheological properties. We demonstrate that these glycyrrhizin-based hydrogels accelerate cutaneous wound closure in normoglycemic and diabetic mice by influencing keratinocyte migration. To facilitate topical application of glycyrrhizin hydrogels on cutaneous wounds, several concentrations of glycyrrhizinic acid in water were tested for their rheological, structural, and biological properties. By varying the concentration of glycyrrhizin, these hydrogel properties can be readily tuned, enabling customized wound care.

Biofilm-induced changes to the composite surface.

OBJECTIVES: Composites may undergo biodegradation in the oral cavity. The objective was to investigate the effect of single- and multi-species biofilms on the surface roughness and topography of two composites. METHODS: Disk-shaped specimens of a paste-like, Bis-GMA-free (Gradia Direct Anterior, GC), and a flowable, Bis-GMA-based composite (Tetric EvoFlow, Ivoclar-Vivadent) were prepared. After ethylene-oxide sterilization (38°C), specimens (n=3) were incubated with Streptococcus mutans or mixed bacterial culture (Streptococcus mutans, Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nucleatum). As negative controls, unexposed specimens and specimens exposed to sterile medium (BHI) were used. Specimens exposed to acidified BHI medium (pH=5) and enzymatic solution of cholesterol esterase served as positive control. Following 6-week incubation, the attached biofilms were collected for real-time PCR assessment, after which the surface roughness and topography of the specimens were analyzed with atomic force microscopy. Surface hydrophilicity/hydrophobicity was determined by contact angle measurements. Biofilm structure was analyzed with scanning electron microscopy. RESULTS: Even though multi-species biofilms were thicker, with more cells attached, they did not significantly affect the surface roughness of the composites. On the other hand, S. mutans alone significantly increased the roughness of Tetric by 40.3%, while its effect on Gradia was lower (12%). The total amount of attached bacteria, however, did not differ between the composites. CONCLUSIONS: S. mutans can increase the surface roughness of composites, depending on their composition. This ability of S. mutans is, however, mitigated in co-culture with other species. In particular, bacterial esterases seem to be responsible for the increased composite surface roughness upon biofilms exposure. CLINICAL SIGNIFICANCE: Cariogenic bacteria can degrade composites, thereby increasing the surface roughness. Increased roughness and subsequent improved bacterial accumulation may facilitate the development of secondary caries around composites, which is the most common reason for the restoration failure.

Interfacial Rheology of Sterically Stabilized Colloids at Liquid Interfaces and Its Effect on the Stability of Pickering Emulsions.

Particle-laden interfaces can be used to stabilize a variety of high-interface systems, from foams over emulsions to polymer blends. The relation between the particle interactions, the structure and rheology of the interface, and the stability of the system remains unclear. In the present work, we experimentally investigate how micron-sized, near-hard-sphere-like particles affect the mechanical properties of liquid interfaces. In particular, by comparing dried and undried samples, we investigate the effect of aggregation state on the properties of the particle-laden liquid interface and its relation to the stability of the corresponding Pickering emulsions. Partially aggregated suspensions give rise to a soft-solid-like response under shear, whereas for stable PMMA particulate layers a liquid-like behavior is observed. For interfacial creep-recovery measurements, we present an empirical method to correct for the combined effect of the subphase drag and the compliance of the double-wall ring geometry, which makes a significant contribution to the apparent elasticity of weak interfaces. We further demonstrate that both undried and dried PMMA particles can stabilize emulsions for months, dispelling the notion that particle aggregation, in bulk or at the interface, is required to create stable Pickering emulsions. Our results indicate that shear rheology is a sensitive probe of colloidal interactions but is not necessarily a predictor of the stability of interfaces, e.g., in quiescent Pickering emulsions, as in the latter the response to dilatational deformations can be of prime importance.

Nanoscale Study of Polymer Dynamics.

The thermal motion of polymer chains in a crowded environment is anisotropic and highly confined. Whereas theoretical and experimental progress has been made, typically only indirect evidence of polymer dynamics is obtained either from scattering or mechanical response. Toward a complete understanding of the complicated polymer dynamics in crowded media such as biological cells, it is of great importance to unravel the role of heterogeneity and molecular individualism. In the present work, we investigate the dynamics of synthetic polymers and the tube-like motion of individual chains using time-resolved fluorescence microscopy. A single fluorescently labeled polymer molecule is observed in a sea of unlabeled polymers, giving access to not only the dynamics of the probe chain itself but also to that of the surrounding network. We demonstrate that it is possible to extract the characteristic time constants and length scales in one experiment, providing a detailed understanding of polymer dynamics at the single chain level. The quantitative agreement with bulk rheology measurements is promising for using local probes to study heterogeneity in complex, crowded systems.

Effect of confinement on droplet breakup in sheared emulsions.

The breakup of Newtonian droplets in a Newtonian matrix during shear flow is investigated in a counterrotating parallel plate device. For bulk conditions, the critical capillary number for breakup is known to be only determined by the viscosity ratio. Here, we show that the critical capillary number is also affected by the degree of confinement: for low viscosity ratios, confinement suppresses breakup, whereas for high viscosity ratios, confinement promotes breakup. This way, above a critical value for the degree of confinement, even droplets with a viscosity ratio larger than 4, which are unbreakable by shear in a bulk situation, can be broken in a simple shear flow field.

Structure development in confined polymer blends: steady-state shear flow and relaxation.

In this work, the structure development in immiscible polymer blends in confined geometries is systematically investigated. Poly(dimethylsiloxane)/poly(isobutylene) blends with a droplet-matrix structure are subjected to simple shear flows. The confined environment is created by using a Linkam shearing cell in which the gap is systematically decreased to investigate the transition from "bulk" behavior toward "confined" behavior. Small-angle light scattering experiments in a confinement, which have not yet been reported in the literature, and also microscopy are used to observe the morphology development during steady-state shearing and relaxation. These experiments indicate that the size and relaxation of single droplets in a confined environment are still governed by the relations that describe the structure development in bulk situations. Yet, depending on the applied shear rates and blend concentrations, the droplets organize in superstructures such as pearl necklaces or extended superstrings in a single layer between the plates. These structures are stable under flow. To observe a single layer, a critical ratio of droplet size to gap spacing is required, but this ratio is clearly below the one already reported in the literature.