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.
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.
Abstract 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.
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 × 1 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.
The frequency‐dependent shear modulus of aqueous wormlike micellar solutions of cetylpyridinium chloride (CPyCl) and sodium salicylate (NaSal) has been measured in a broad frequency range from 10−2 to 106 rad/s using DWS based tracer microrheology as well as mechanical techniques including rotational rheometry, oscillatory squeeze flow and torsional resonance. Good agreement between mechanical and optical techniques has been found in the frequency range from 10−1 to 105 rad/s [1]. At intermediate frequencies between 10 and 104 rad/s squeeze flow provides most accurate data and is used to determine the plateau modulus G0, which is related to the crosslink density or mesh size of the entanglement network as well as the scission energy Esciss, which is deduced form the temperature dependence of the shear moduli in the plateau zone. In the frequency range above 104 rad/s DWS including a new inertia correction is most reliable and is used to determine the persistence length lp. This quantity is calculated from the loss modulus G″, which exhibits a ω3/4‐scaling in this frequency range as expected for semiflexible objects like wormlike micelles, according to the statistical mechanical theory of Gittes and MacKintosh [2]. The system CPyCl/NaSal is known to exhibit two maxima in zero‐shear viscosity and terminal relaxation time as the salt/surfactant ratio R varies [3]. The first maximum is attributed to a transition from linear to branched micelles [4], the second one is accompanied by a charge reversal due to strongly binding counterions. Recently, the changes in micellar length, branching and branching point density as well as formation of rings has been documented in a comprehensive TEM‐study [5]. Here we discuss the variation of G0, Esciss and lp with salt/surfactant ratio R at constant surfactant concentration of 100 mM CPyCl. G0 increases at the linear‐to‐branched micelles transition, and this is attributed to the additional contribution of branching points to the crosslink density. Esciss exhibits two maxima analogous the zero‐shear viscosity, which can be understood in terms of the variation of micellar length and variation of the amount of branched micelles and contour length between branching points consistent with [5]. The persistence length decreases slightly with increasing R. This is considered to be an ionic strength effect, the linear‐to‐branched transition obviously does not have an effect on lp.
We have studied the effect of counterion binding efficiency on the linear viscoelastic properties of wormlike micelles formed from hexadecyltrimethylammonium bromide (CTAB) in the presence of different nonpenetrating inorganic salts: potassium bromide (KBr), sodium nitrate (NaNO3), and sodium chlorate (NaClO3). We have varied the salt/surfactant ratio R at fixed surfactant concentration of 350 mM. Results are compared to data for the system cetylpyridinium chloride (CPyCl) and the penetrating counterion sodium salicylate (NaSal) (Oelschlaeger, C.; Schopferer, M.; Scheffold, F.; Willenbacher, N. Langmuir 2009, 25, 716−723). Mechanical high-frequency rheology and diffusing wave spectroscopy (DWS) based tracer microrheology are used to determine the shear moduli G′ and G′′ in the frequency range from 0.1 Hz up to 1 MHz (Willenbacher, N.; Oelschlaeger, C.; Schopferer, M.; Fischer, P.; Cardinaux, F.; Scheffold, F. Phys. Rev. Lett. 2007, 99, 068302, 1−4). This enables us to determine the plateau modulus G0, which is related to the cross-link density or mesh size of the entanglement network, the bending stiffness κ (also expressed as persistence length lp = κ/kBT) corresponding to the semiflexible nature of the micelles, and the scission energy Esciss, which is related to their contour length. The viscosity maximum shifts to higher R values, and the variation of viscosity with R is less pronounced as the binding strength decreases. The plateau modulus increases with R at low ionic strength and is constant around the viscosity maximum; the increase in G0 at high R, which is presumably due to branching, is weak compared to the system with penetrating counterion. The scission energy Esciss ≈ 20 kBT is independent of counterion binding efficiency irrespective of R and is slightly higher compared to the system CPyCl/NaSal, indicating that branching may be significant already at the viscosity maximum in this latter case. The micellar flexibility increases with increasing binding efficiency of counterions according to the Hofmeister series. The persistence length values for systems CTAB/KBr, CTAB/NaNO3, and CTAB/NaClO3 are 40, 34, and 29 nm, respectively, independent of R, and are significantly higher than in the case of CPyCl/NaSal.
In this work, we present new insights related to a debate on the morphological structure of hydroxyethyl cellulose (HEC) molecules when dissolved in water, i.e., whether HEC adopts a linear-flexible or a rod-like fibrillar configuration. We have employed "seven" rheological techniques to explore the viscoelastic properties of HEC solutions at different time and length scales. This work demonstrates an excellent convergence between various rheological techniques over a broad range of frequencies and concentrations, allowing us to derive microstructural information for aqueous HEC solutions without the use of complex optical imaging techniques. We find that when dissolved in water unmodified HEC behaves like a linear uncharged polymer, with an entangled mass concentration of ce = 0.3 wt%. Moreover, for the first time we provide the concentration scaling laws (across ce) for the longest relaxation time λ of HEC solutions, obtained from direct readings and not inferred from fitting procedures of fluids shear flow curves.
The next wave of biocatalysis will be enabled by machine-assisted flow processes that require tailor-made materials to achieve efficient product conversion. In their Communication on page 17028 ff., C. M. Niemeyer and co-workers describe self-assembling all-enzyme hydrogels that are made from two cooperating enzymes and can be produced in macroscopic quantities (blue background image). Mounted in microfluidic reactors, the gels show excellent stereoselectivity with near quantitative conversion in the reduction of ketones along with extraordinary high turnover of expensive cofactors.
We present a macro- and microrheological study of the effect of ionic strength on the phase behavior, structure and flow properties of colloidal dispersions stabilized by short-range repulsive interactions inferred via co-polymerization of weak acid groups. We investigated the impact of ionic strength, by varying the concentration of natrium chloride (NaCl), on dispersions with only repulsive interactions, but also when additional attractive depletion forces are present due to added non-absorbent polymer, namely polyethyleneoxide (PEO). For dispersions with only repulsive electrosteric interactions, at low particle volume fractions (ϕ < 0.4), increasing ionic strength hardly affects the relative viscosity whereas at higher particle volume fractions, a decrease in viscosity is observed due to a reduced range of electrosteric repulsion between particles, corresponding to a reduction of the effective particle volume fraction ϕeff. For dispersions including attractive interactions, at volume fractions ϕ = 0.45 below the hard sphere freezing point (ϕc=0.5), independently of the ionic strength, the bulk viscosity increases monotonically with increasing PEO concentration due to the transition from a fluid to a fluid/crystalline and finally to a gel state. However, the increase in ionic strength shifts the concentrations of both phase transitions to lower polymer concentrations, indicating that in presence of salt a weaker attraction is required to induce these transitions. At ϕ=0.52, just above ϕc, for dispersions without added salt, broadening of the fluid-crystalline coexistence regime, due to added PEO, results in a viscosity minimum corresponding to different size and packing density of the crystalline regions as observed earlier in Weis et al. [1]. When salt is added, the initial state for dispersions without PEO is a fluid state due to the reduction of ϕeff and the addition of a small amount of PEO leads to an increase in bulk viscosity due to the formation of large crystals for dispersions containing 10 mM NaCl and a network of small, dense crystals of size ≈10 µm when 100 mM NaCl is added. At ϕ=0.54, a result similar to ϕ=0.52 is obtained for dispersions without added salt, whereas in presence of salt, we find that the fluid/crystalline coexistence regime narrows down as the range of electrosteric repulsion decreases and the addition of PEO results in gel formation and gels become more uniform with increasing attraction strength. These investigations demonstrate how superimposed short range electrosteric repulsion and weak depletion attraction affect microstructure and flow behavior of colloidal dispersions.
