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.

Bottom-Up Assembly of DNA-Silica Nanocomposites into Micrometer-Sized Hollow Spheres.

Although DNA nanotechnology has developed into a highly innovative and lively field of research at the interface between chemistry, materials science, and biotechnology, there is still a great need for methodological approaches for bridging the size regime of DNA nanostructures with that of micrometer- and millimeter-sized units for practical applications. We report on novel hierarchically structured composite materials from silica nanoparticles and DNA polymers that can be obtained by self-assembly through the clamped hybridization chain reaction. The nanocomposite materials can be assembled into thin layers within microfluidically generated water-in-oil droplets to produce mechanically stabilized hollow spheres with uniform size distributions at high throughput rates. The fact that cells can be encapsulated in these microcontainers suggests that our concept not only contributes to the further development of supramolecular bottom-up manufacturing, but can also be exploited for applications in the life sciences.

Self-Assembling All-Enzyme Hydrogels for Flow Biocatalysis.

Continuous flow biocatalysis is an emerging field of industrial biotechnology that uses enzymes immobilized in flow channels for the production of value-added chemicals. We describe the construction of self-assembling all-enzyme hydrogels that are comprised of two tetrameric enzymes. The stereoselective dehydrogenase LbADH and the cofactor-regenerating glucose 1-dehydrogenase GDH were genetically fused with a SpyTag or SpyCatcher domain, respectively, to generate two complementary homo-tetrameric building blocks that polymerize under physiological conditions into porous hydrogels. Mounted in microfluidic reactors, the gels show excellent stereoselectivity with near quantitative conversion in the reduction of prochiral ketones along with high robustness under process and storage conditions. The gels function as compartment that retains intermediates thus enabling high total turnover numbers of the expensive cofactor NADP(H).

Squeeze flow rheometry as a novel tool for the characterization of highly concentrated protein solutions.

This study aims at defining rheological parameters for the characterization of highly concentrated protein solutions. As a basis for comparing rheological behavior with protein solution characteristics the protein phase behavior of Lysozyme from chicken egg white with concentrations up to 225 mg/mL, changing pH values and additive concentrations was studied in a microbatch scale format. The prepared phase diagrams, scored after 40 days (t40) give insights into the kind and kinetics of the phase transitions that occur. Oscillatory frequency sweep measurements of samples with exactly the same conditions were conducted immediately after preparation (t0). The protein solutions behave viscoelastic and show a characteristic curve shape of the storage modulus (G') and the loss modulus (G″). The graphs provide information about the cross-linking degree of the respective sample. The measured rheological parameters were sensitive concerning solution composition, protein concentration and solution inner structure. The rheological moduli G' and G″ and especially the ratio of these parameters over a frequency range from 100 to 40000 rad/sec give information about the aggregation tendency of the protein under tested conditions. We succeeded to correlate protein phase behavior with the defined rheological key parameter ωCO. This point represents the frequency value of the intersection point from G' and G″. In our study Lysozyme expressed a ωCO threshold value of 20000 rad/sec as a lower limit for stable protein solutions. The predictability of lysozyme aggregation tendency and crystallization by means of squeeze flow rheometry is shown.

Micromechanical Indentation Platform for Rapid Analysis of Viscoelastic Biomolecular Hydrogels.

The advent of biomedical applications of soft bioinspired materials has entailed an increasing demand for streamlined and expedient characterization methods meant for both research and quality control objectives. Here, a novel measurement system for the characterization of biological hydrogels with volumes as low as 75 µL was developed. The system is based on an indentation platform equipped with micrometer drive actuators that allow the determination of both the fracture points and Young's moduli of relatively stiff polymers, including agarose, as well as the measurements of viscosity for exceptionally soft and viscous hydrogels, such as DNA hydrogels. The sensitivity of the method allows differentiation between DNA hydrogels produced by rolling circle amplification based on different template sequences and synthesis protocols. In addition, the polymerization kinetics of the hydrogels can be determined by time-resolved measurements, and the apparent viscosities of even more complex DNA-based nanocomposites can be measured. The platform presented here thus offers the possibility to characterize a broad variety of soft biomaterials in a targeted, fast, and cost-effective manner, holding promises for applications in fundamental materials science and ensuring reproducibility in the handling of complex materials.

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 1-100µm 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.

Miniaturized high-throughput synthesis and screening of responsive hydrogels using nanoliter compartments.

The traditional pipeline of hydrogel development includes individual one-by-one synthesis and characterization of hydrogels. This approach is associated with the disadvantages of low-throughput and high cost. As an alternative approach to classical one-by-one synthesis, high-throughput development of hydrogels is still tremendously under-represented in the field of responsive material development, despite the urgent requirement for such techniques. Here, we report a platform that combines highly miniaturized hydrogel synthesis with screening for responsive properties in a high-throughput manner. The platform comprises a standard glass slide patterned with 1 â€‹× â€‹1 â€‹mm hydrophilic regions separated by superhydrophobic liquid-impermeable barriers, thus allowing deposition of various precursor solutions onto the hydrophilic spots without cross-contamination. The confinement of these solutions provided by the hydrophilic/superhydrophobic pattern allows encapsulation of cells within the hydrogel, and enables variation in hydrogel height and width. We have also proved the proper mixing of chemicals within the nanoliter-sized droplets. We have successfully implemented this platform for the synthesis of hydrogels, constructing 53 unique hydrogels, to demonstrate the versatility and utility of the platform. Photodegradation studies were performed on 20 hydrogels, revealing structure/function relationships between the hydrogel composition and photodegradability, and covering the range of degradability from non-degradable to rapidly degradable materials.

Monitoring matrix remodeling in the cellular microenvironment using microrheology for complex cellular systems.

Multiple particle tracking (MPT) microrheology was employed for monitoring the development of extracellular matrix (ECM) mechanical properties in the direct microenvironment of living cells. A customized setup enabled us to overcome current limitations: (i) Continuous measurements were enabled using a cell culture chamber, with this, matrix remodeling by fibroblasts in the heterogeneous environment of macroporous scaffolds was monitored continuously. (ii) Employing tracer laden porous scaffolds for seeding human mesenchymal stem cells (hMSCs), we followed conventional differentiation protocols. Thus, we were, for the first time able to study the massive alterations in ECM elasticity during hMSC differentiation. (iii) MPT measurements in 2D cell cultures were enabled using a long distance objective. Exemplarily, local mechanical properties of the ECM in human umbilical vein endothelial cell (HUVEC) cultures, that naturally form 2D layers, were investigated scaffold-free. Using our advanced setup, we measured local, apparent elastic moduli G0,app in a range between 0.08 and 60 Pa. For fibroblasts grown in collagen-based scaffolds, a continuous decrease of local matrix elasticity resulted during the first 10 hours after seeding. The osteogenic differentiation of hMSC cells cultivated in similar scaffolds, led to an increase of G0,app by 100 %, whereas after adipogenic differentiation it was reduced by 80 %. The local elasticity of ECM that was newly secreted by HUVECs increased significantly upon addition of protease inhibitor and in high glucose conditions even a twofold increase in G0,app was observed. The combination of these advanced methods opens up new avenues for a broad range of investigations regarding cell-matrix interactions and the propagation of ECM mechanical properties in complex biological systems. STATEMENT OF SIGNIFICANCE: Cells sense the elasticity of their environment on a micrometer length scale. For studying the local elasticity of extracellular matrix (ECM) in the direct environment of living cells, we employed an advanced multipleparticle tracking microrheology setup. MPT is based on monitoring the Brownian motion oftracer particles, which is restricted by the surrounding network. Network elasticity can thusbe quantified. Overcoming current limitations, we realized continuous investigations of ECM elasticityduring fibroblast growth. Furthermore, MPT measurements of stem cell ECM showed ECMstiffening during osteogenic differentiation and softening during adipogenic differentiation.Finally, we characterized small amounts of delicate ECM newly secreted in scaffold-freecultures of endothelial cells, that naturally form 2D layers.

A Phenolic Acid Decarboxylase-Based All-Enzyme Hydrogel for Flow Reactor Technology.

Carrier-free enzyme immobilization techniques are an important development in the field of efficient and streamlined continuous synthetic processes using microreactors. Here, the use of monolithic, self-assembling all-enzyme hydrogels is expanded to phenolic acid decarboxylases. This provides access to the continuous flow production of p-hydroxystyrene from p-coumaric acid for more than 10 h with conversions ≥98% and space time yields of 57.7 g·(d·L)-1. Furthermore, modulation of the degree of crosslinking in the hydrogels resulted in a defined variation of the rheological behavior in terms of elasticity and mesh size of the corresponding materials. This work is addressing the demand of sustainable strategies for defunctionalization of renewable feedstocks.

Valency engineering of monomeric enzymes for self-assembling biocatalytic hydrogels.

All-enzyme hydrogels are efficient reagents for continuous flow biocatalysis. These materials can be obtained by self-assembly of two oligomeric enzymes, modified with the complementary SpyTag and SpyCatcher units. To facilitate access to the large proportion of biocatalytically relevant monomeric enzymes, we demonstrate that the tagging valency of the monomeric (S)-stereoselective ketoreductase Gre2p from Saccharomyces cerevisiae can be designed to assemble stable, active hydrogels with the cofactor-regenerating glucose 1-dehydrogenase GDH from Bacillus subtilis. Mounted in microfluidic reactors, these gels revealed high conversion rates and stereoselectivity in the reduction of prochiral methylketones under continuous flow for more than 8 days. The sequential use as well as parallelization by 'numbering up' of the flow reactor modules demonstrate that this approach is suitable for syntheses on the semipreparative scale.

Carbon-nanotube reinforcement of DNA-silica nanocomposites yields programmable and cell-instructive biocoatings.

Biomedical applications require substrata that allow for the grafting, colonization and control of eukaryotic cells. Currently available materials are often limited by insufficient possibilities for the integration of biological functions and means for tuning the mechanical properties. We report on tailorable nanocomposite materials in which silica nanoparticles are interwoven with carbon nanotubes by DNA polymerization. The modular, well controllable and scalable synthesis yields materials whose composition can be gradually adjusted to produce synergistic, non-linear mechanical stiffness and viscosity properties. The materials were exploited as substrata that outperform conventional culture surfaces in the ability to control cellular adhesion, proliferation and transmigration through the hydrogel matrix. The composite materials also enable the construction of layered cell architectures, the expansion of embryonic stem cells by simplified cultivation methods and the on-demand release of uniformly sized stem cell spheroids.

Structural, microrheological and kinetic properties of a ternary silica-Pluronic F127-starch thermosensitive system.

HYPOTHESIS: The sol-gel transition in aqueous suspensions consisting of silica particles and thermosensitive polymer is controlled by inter-particle forces and solution properties of the polymer. Addition of a second non-thermosensitive polymer may affect the transition. The purpose of this work was to characterize the kinetics of the sol-gel transition and to understand the effects of a second non-thermosensitive polymer on the microstructure, using a combination of classical rheology and microrheology. EXPERIMENTS: Classical rotational rheology as well as two microrheology methods, Multiple Particle Tracking (MPT) and Diffusing Wave Spectroscopy (DWS), were used to investigate the sol-gel transition of a ternary silica-Pluronic F127-starch thermosensitive system. FINDINGS: Classical rheometry and DWS indicated sol-gel transition temperature ∼25 °C at 1 wt% Pluronic, independently of the concentration of the other components. DWS showed a fast gelation process, less than two minutes for all samples, beside a second slow kinetic process. In the gel state, MPT indicated micro-structural and micro-viscoelastic differences compared to rotational rheology. This was explained by formation of an elastic matrix of silica and polymers in combination with assembly of silica particles in large macroporous agglomerates. Presence of starch led to breakdown of the macroporous network, leaving the homogeneous elastic network left.

Microstructure, local viscoelasticity and cell culture suitability of 3D hybrid HA/collagen scaffolds.

As mechanical properties of cell culture substrates matter, methods for mechanical characterization of scaffolds on a relevant length scale are required. We used multiple particle tracking microrheology to close the gap between elasticity determined from bulk measurements and elastic properties sensed by cells. Structure and elasticity of macroporous, three-dimensional cryogel scaffolds from mixtures of hyaluronic acid (HA) and collagen (Coll) were characterized. Both one-component gels formed homogeneous networks, whereas hybrid gels were heterogeneous in terms of elasticity. Most strikingly, local elastic moduli were significantly lower than bulk moduli presumably due to non-equilibrium chain conformations between crosslinks. This was more pronounced in Coll and hybrid gels than in pure HA gels. Local elastic moduli were similar for all gels, irrespective of their different swelling ratio and bulk moduli. Fibroblast cell culture proved the biocompatibility of all investigated compositions. Coll containing gels enabled cell migration, adhesion and proliferation inside the gels.

Microstructure, local dynamics, and flow behavior of colloidal suspensions with weak attractive interactions.

We present a comprehensive micro- and macrorheological study of the effect of weak depletion attraction (Ψdep ≈ 1-10 kBT) on dense colloidal suspensions stabilized by short-range repulsive interactions. We used aqueous polymer dispersions as model system and demonstrated the unique capabilities of multiple particle tracking (MPT) to disclose structural changes in such technically important systems exhibiting many characteristic features of hard sphere systems. Below the hard sphere freezing point ϕc, viscosity increases monotonically with increasing Ψdep due to the transition from a fluid to a fluid/crystalline and finally to a gel state. Above ϕc, increasing attraction strength first results in a viscosity reduction corresponding to the formation of large, permeable crystals and then in a viscosity increase when a network of dense, small crystals forms. The fraction of the fluid and crystal phase, particle concentration in each phase as well as the modulus of the micro-crystals are obtained, the latter decreases with Ψdep. Above the colloidal glass transition strong heterogeneities and different local particle mobility in the repulsive and attractive arrested states are found. Particles are trapped in the cage of neighboring particles rather than in an attractive potential well. The intermediate ergodic state exhibits uniform tracer diffusivity.

Microrheology with fluorescence correlation spectroscopy.

We show that fluorescence correlation spectroscopy (FCS) using a commercial spectrometer can be applied to passive microrheological (MR) experiments. The method probes the local rheological properties of materials on length scales of the focus dimension of the confocal microscope. For a feasibility study, we performed measurements on a high molecular weight poly(ethylene oxide)-water solution to allow direct comparison of the results to previous studies using diffusing wave spectroscopy, quasielastic light scattering, and particle tracking methods. We were able to detect mean-square center-of-mass displacements ranging from somewhat better than [SYMBOL: SEE TEXT]2(t) approximately 100 nm2 up to above Deltar2(t) approximately 10(6) nm2. Thus, we were able to derive the bulk rheological shear moduli covering more than five decades in frequency (from omega