Microrheology imaging of fiber suspensions – a case study for lyophilized collagen I in HCl solutions

In fiber suspensions with low optical contrast, the in situ characterization of structural properties with conventional microscopy methods fails. However, overlaying subsequent images of multiple particle tracking (MPT) videos including short trajectories usually discarded in MPT analysis allowed for direct visualization of individual fibers and the network structure of lyophilized collagen I (Coll) distributed in hydrochloric acid solutions. MPT yielded a broad distribution of mean square displacements (MSDs). Freely diffusing tracer particles yielded viscosities indicating that, irrespective of concentration, a constant amount of Coll is dissolved in the aqueous phase. Particles found elastically trapped within fibrous Coll structures exhibited a broad range of time-independent MSDs and we propose a structure comprising multiple fiber bundles with dense regions inaccessible to tracers and elastic regions of different stiffness in between. Bulky aggregates inaccessible to the 0.2 μm tracers exist even at low Coll concentrations, a network of slender fibers evolves above the sol–gel transition and these fibers densify with increasing Coll concentration. This novel MPT-based imaging technique possesses great potential to characterize the fiber distribution in and structural properties of a broad range of biological and technical suspensions showing low contrast when imaged with conventional techniques. Thus, MPT imaging and microrheology will help to better understand the effect of fiber distribution and network structure on the viscoelastic properties of complex suspensions

Imaging of the microstructure of Carbopol dispersions and correlation with their macroelasticity: A micro- and macrorheological study

We developed a new data analysis strategy, the so-called micro-rheo-mapping technique, based on multiparticle tracking experiments to obtain an accurate and direct visualization of the microstructure of commercial acrylate thickeners of Carbopol-type with high (Ultrez 10), intermediate (ETD 2020), and low (ETD 2050) degree of crosslinking. At low polymer concentration, aggregates made of several primary Carbopol particles are formed with an average diameter of 43 ± 11, 56 ± 14, and 10 ± 2.5 μm for Ultrez 10, ETD 2020, and ETD 2050, respectively. For ETD 2050, the least crosslinked thickener, the shell of dangling polymer chains covering the aggregate surface is thicker than for ETD 2020 and Ultrez 10. At technically relevant polymer concentrations, our results indicate, for all three thickeners, that the microstructure is highly heterogeneous with regions of different crosslink densities. One region inaccessible for tracer particles corresponding to a mixture of polydisperse aggregates and individual primary particles with a core mesh size less than 200 nm and a second, diluted enough to be accessible and which exhibits both elastic and viscous characteristics. The study of the impact of pH, polymer concentration, and crosslink density on these local structural and viscoelastic heterogeneities as well as macrorheological properties allowed us to establish a correlation between microstructure and macroelasticity. In particular, we found that the bulk shear modulus strongly depends on the fraction of inaccessible areas, making this microscopic parameter most relevant for describing the macroelasticity of Carbopol gels, whereas the local elasticity of the interstitial regions is of minor importance

The Influence of Rheological and Wetting Properties of Hydrogel-based Bio-Inks on Extrusion-based Bioprinting

The wall slip and flow behavior of alginate as well as gelatin based hydrogels with respect to the impact of these rheological and wetting properties on extrusion-based bioprinting (EBB) was investigated. Capillary rheometry and printing tests indicate that slip is negligible at high stresses relevant for EBB, i.e., well above the hydrogels yield stress. On the contrary, rotational rheometry performed at low shear stresses revealed that alginate hydrogels present much stronger slip than gelatin gels, irrespective of crosslinker and polymer concentration. This result is presumably due to the formation of a heterogeneous microstructure for alginate gels and has an unfavorable impact on the printing quality with the production of large fluctuations in line width and higher line spread ratio

Targeted Micro-Phase separation – A generic design concept to control the elasticity of extrudable hydrogels

Hydrogels are ubiquitous in nature and technology. Controlling their mechanical properties and under-standing their complex microstructure is critical e.g. for 3D bioprinting or tissue engineering applications. Here a generic design concept for tuning the elasticity of extrudable gels at given polymer or particle concentration is presented. Targeted micro-phase separation leading to micro-heterogeneities (1–100 µm) yields high gel strength allowing for extrusion of uniform filaments with high shape accuracy as long as the heterogeneity length scale is small compared to the extruded filament diameter (>500 μm). Micro-mechanical and structural heterogeneity was enhanced in alginate hydrogels by accelerating crosslinking kinetics, corresponding to gel elasticity variation of more than two orders of magnitude (17 Pa to 2300 Pa), enabling filament extrusion (1046 µm) with high shape fidelity. Introducing poly(vinylalcohol) into gelatin gels resulted in more heterogeneous materials with a 2-fold increase in elasticity (951 Pa to 1993 Pa) and thinner filaments (908 µm to 590 µm). Higher ionic strength in Laponite-based hydrogels induced nanoparticle aggregation, leading to higher elasticity (857 Pa to 2316 Pa) enabling smooth filament extrusion. Eliminating the often tacitly assumed hydrogel uniformity on the microscale provides additional degrees of freedom to achieve high gel strength without increasing polymer, particle or crosslinker concentration

Targeted micro-heterogeneity in bioinks allows for 3D printing of complex constructs with improved resolution and cell viability

Three-dimensional bioprinting is an evolving versatile technique for biomedical applications. Ideal bioinks have complex micro-environment that mimic human tissue, allow for good printing quality and provide high cell viability after printing. Here we present two strategies for enhancing gelatin-based bioinks heterogeneity on a 1–100 µm length scale resulting in superior printing quality and high cell viability. A thorough spatial and micro-mechanical characterization of swollen hydrogel heterogeneity was done using multiple particle tracking microrheology. When poly(vinyl alcohol) is added to homogeneous gelatin gels, viscous inclusions are formed due to micro-phase separation. This phenomenon leads to pronounced slip and superior printing quality of complex 3D constructs as well as high human hepatocellular carcinoma (HepG2) and normal human dermal fibroblast (NHDF) cell viability due to reduced shear damage during extrusion. Similar printability and cell viability results are obtained with gelatin/nanoclay composites. The formation of polymer/nanoclay clusters reduces the critical stress of gel fracture, which facilitates extrusion, thus enhancing printing quality and cell viability. Targeted introduction of micro-heterogeneities in bioinks through micro-phase separation is an effective technique for high resolution 3D printing of complex constructs with high cell viability. The size of the heterogeneities, however, has to be substantially smaller than the desired feature size in order to achieve good printing quality

Accurate quantification of DNA content in DNA hydrogels prepared by rolling circle amplification

Accurate quantification of polymerized DNA in rolling circle amplification (RCA)-based hydrogels is challenging due to the high viscosity of these materials, however, it can be achieved with a photometric nucleotide depletion assay or qPCR. We show that the DNA content strongly depends on the template sequence and correlates with the mechanical properties of the hydrogels