Synthesis, Structural and Micromechanical Properties of 3D Hyaluronic Acid-Based Cryogel Scaffolds.

In this study, macroporous, elastic, three-dimensional scaffolds formed of hyaluronic acid mixed with ethylene glycol diglycidyl ether as a chemical cross-linker have been prepared by cryogelation for application in tissue engineering. These cryogels are characterized by large interconnected pores of size ∼50-300 µm and pore wall thickness of ∼5-30 µm as determined from confocal microscopy images. Variation of pH, freezing temperature, and polymerization time allows for control of pore size and shape as well as matrix thickness. These structural properties then determine mechanical strength as well as swelling capacity. Furthermore, increasing hyaluronic acid concentration decreases cryogel pore size, reduces swelling properties, and reinforces mechanical properties. On the other hand, decreasing cross-linker concentration, at a constant hyaluronic acid concentration, increases pore size and swelling capacity but provides less rigidity. Additionally, for the first time, local elastic properties of the polymer matrix and viscous properties of the pores have been characterized using multiple particle tracking microrheology. Local matrix elasticity, relaxation time of hyaluronic acid chains, and the degree of heterogeneity are discussed in detail. These latter properties are crucial for the development of new tissue engineering constructs and will help to understand how local matrix viscoelasticity affects cell cultivation. Finally, elastic moduli obtained in bulk rheology are much higher than corresponding values deduced from microrheology. This discrepancy might be explained by the formation of very highly cross-linked cores in the network where no tracer particle can penetrate.

Relating foam and interfacial rheological properties of ß-lactoglobulin solutions.

We have determined bulk rheology of ß-lactoglobulin (BLG) foams and surface viscoelasticity of corresponding protein solutions by varying pH as well as type, valency and concentration of the added salt in a wide range. Foam rheology was characterized by the storage modulus G0, the apparent yield stress τy, and the critical strain γc,foam defining the cessation of the linear viscoelastic response. These quantities were determined at gas volume fractions ϕ between 82% and 96%. Surface viscoelasticity was characterized in shear and dilation, corresponding shear and dilational moduli G, E' as well as the critical stress τc,surface and strain γc,surface marking the onset of non-linear response in oscillatory surface shear experiments were determined at fixed frequency. Beyond the widely accepted assumption that G0 and τy are solely determined by the Laplace pressure within the droplets and the gas volume fraction we have found that both quantities strongly depend on corresponding interfacial properties. G0 increases linearly with G and even stronger with E', τy varies proportional to τc,surface and γc,foam scales linearly with γc,surface. Furthermore, deviations from these simple scaling laws with significantly higher reduced G0 and τy values are observed only for foams at pH 5 and when a trivalent salt was added. Then also the dependence of these quantities on ϕ is unusually weak and we attribute these findings to protein aggregation and structure formation across the lamellae than the dominating bulk rheology.

Chain flexibility and dynamics of polysaccharide hyaluronan in entangled solutions: a high frequency rheology and diffusing wave spectroscopy study.

We have investigated the linear viscoelastic properties of high molecular weight hyaluronan in aqueous solution using an experimental approach combining mechanical rheometry and optical microrheology. The complex shear modulus has been measured over a broad frequency range from 10(-1) to 10(7) rad/s. Chain flexibility is characterized by the persistence length lpand this parameter has been determined for the first time in the entangled regime here from high frequency modulus data. At NaHA concentrations above the entanglement concentration ce, lp is essentially independent of polymer concentration, temperature, and ionic strength. The latter is consistent with the Odijk-Skolnick-Fixman theory. The scaling exponent describing the concentration dependence of the plateau modulus G0 agrees well with predictions for polymers in good solvents. The scaling exponents for the specific viscosity ηsp and relaxation time TR are slightly higher than theoretically predicted for polyelectrolytes in the high salt limit, indicating, that molecular aggregation occurs at higher polymer concentrations.

Clogging of microchannels by nano-particles due to hetero-coagulation in elongational flow.

We have investigated the phenomenon of flow-induced aggregation in highly concentrated colloidal dispersions exposed to strongly converging flow fields. This phenomenon is relevant not only for classical technical operations like coating, pumping or filtration, but also for the application of concentrated suspensions in upcoming processing technologies based on microfluidic devices. A ring-slit device (gap height 10-25 µm), which allows for a variation of flow kinematics in a wide range, has been developed in order to investigate this phenomenon. Various polymer dispersions with different particle surface properties have been used as model systems. Our experiments exclude, that channel clogging is due to retention of pre-existing aggregates, fouling or hydrodynamic bridging. Instead, we demonstrate that clogging of the microchannel is induced by hetero-coagulation between primary colloidal particles and micron-sized impurities present at concentrations on the order of 100-1000 ppm. Clogging can occur even if the diameter of these impurities is less than a tenth of the gap height. Aggregation takes place in the converging flow field at the channel entrance, but not in the shear field within the slit. It can be suppressed by appropriate stabilization of the primary particles.

Effect of counterion binding efficiency on structure and dynamics of wormlike micelles.

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 (NaNO(3)), and sodium chlorate (NaClO(3)). 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 G(0), which is related to the cross-link density or mesh size of the entanglement network, the bending stiffness kappa (also expressed as persistence length l(p) = kappa/k(B)T) corresponding to the semiflexible nature of the micelles, and the scission energy E(sciss), 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 G(0) at high R, which is presumably due to branching, is weak compared to the system with penetrating counterion. The scission energy E(sciss) approximately = 20 k(B)T 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/NaNO(3), and CTAB/NaClO(3) are 40, 34, and 29 nm, respectively, independent of R, and are significantly higher than in the case of CPyCl/NaSal.

Linear-to-branched micelles transition: a rheometry and diffusing wave spectroscopy (DWS) study.

The frequency-dependent shear modulus of aqueous wormlike micellar solutions of cetylpyridinium chloride (CPyCl) and sodium salicylate (NaSal) has been measured over a broad frequency range from 10(-2) to 10(6) rad/s using diffusing wave spectroscopy (DWS) based tracer microrheology as well as mechanical techniques including rotational rheometry and oscillatory squeeze flow. Good agreement between mechanical and optical techniques is found in the frequency range from 10(-1) to 10(5) rad/s (Willenbacher, N.; Oelschlaeger, C.; Schopferer, M.; Fischer, P.; Cardinaux, F.; Scheffold, F. Phys. Rev. Lett. 2007, 99 (6), 068302). At intermediate frequencies between 10 and 10(4) rad/s, squeeze flow provides most accurate data and is used to determine the plateau modulus G(0), which is related to the cross-link density or mesh size of the entanglement network, as well as the scission energy E(sciss), which is deduced from the temperature dependence of the shear moduli in the plateau zone. In the frequency range above 10(4) rad/s, DWS including a new inertia correction is most reliable and is used to determine the persistence length l(p). The system CPyCl/NaSal is known to exhibit two maxima in zero-shear viscosity and terminal relaxation time as the salt/surfactant ratio R is varied (Rehage, H.; Hoffman, H. J. Phys. Chem. 1988, 92 (16), 4712-4719). The first maximum is attributed to a transition from linear to branched micelles (Lequeux, F. Europhys. Lett. 1992, 19 (8), 675-681), and the second one is accompanied by a charge reversal due to strongly binding counterions. Here, we discuss the variation of G(0), E(sciss), and l(p) with salt/surfactant ratio R at constant surfactant concentration of 100 mM CPyCl. G(0) increases at the linear-to-branched micelles transition, and this is attributed to the additional contribution of branching points to the cross-link density. E(sciss) exhibits two maxima analogous to 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 the results of a comprehensive cryo-transmission electron microscopy (TEM) study (Abezgauz, L.; Ramon, O.; Danino, D. Department of Biotechnology and Food Engineering, Technion, Haifa, Israel. European Colloid and Interface Society, Geneva, 2007). The persistence length decreases with increasing R. This decrease is stronger than expected from the decrease of Debye length according to the Odijk-Skolnick-Fixman (OSF) theory and is attributed to the penetration of salicylate ions into the micelles; the linear-to-branched transition obviously does not have an effect on l(p).

Broad bandwidth optical and mechanical rheometry of wormlike micelle solutions.

We characterize the linear viscoelastic shear properties of an aqueous wormlike micellar solution using diffusing wave spectroscopy (DWS) based tracer microrheology as well as various mechanical techniques such as rotational rheometry, oscillatory squeeze flow, and torsional resonance oscillation covering the frequency range from 10(-1) to 10(6) rad/s. Since DWS as well as mechanical oscillatory squeeze flow and torsional resonance oscillation cover a sufficiently high frequency range, the persistence length of wormlike micelles could be determined directly from rheological measurements for the first time.

Hydrodynamic and Colloidal Interactions in Concentrated Charge-Stabilized Polymer Dispersions.

Hydrodynamic and colloidal interactions are explored in concentrated, charge-stabilized colloidal dispersions by measuring the dependence of rheology (e.g., low and high-shear viscosity, high-frequency viscosity, and modulus) and self-diffusivity on salt content, particle size, and concentration. Model, sulfonated polystyrene lactices of varying diameter are prepared and investigated by shear rheology, high-frequency torsional resonance, electrophoresis, titration, and dynamic light scattering. The high-frequency and high-shear viscosity both are dominated by hydrodynamic interactions, but are shown not to be identical, due to the microstructure distortion resulting from high shear rates. The short-time self-diffusion is also shown to be insensitive to direct particle interactions, but has a different concentration dependence than the high-frequency viscosity, further illustrating a predicted violation of a generalized Stokes-Einstein relationship for these properties. The apparent colloidal surface charge is extracted from the high-frequency elastic modulus measurements on concentrated dispersions. The surface charge is in good agreement with results from critical coagulation concentration measurements and perturbation theories, but disagrees with electrophoretic mobility experiments. This indicates that the effective surface charge determined by torsional high-frequency measurements is a more reliable predicter of the salt stability of charge-stabilized dispersions, in comparison to zeta-potentials determined from electrophoretic mobilities. Further, we demonstrate by direct comparison that measurements of the apparent plateau modulus by rotational rheometry underestimate the true, high-frequency modulus and provide unreliable estimates for the surface charge. Copyright 2000 Academic Press.

High-Frequency Viscosity and Shear Modulus of Sterically Stabilized Colloid Particles as Probed by Torsional Resonance Oscillation.

A study of the high-frequency viscosity etainfinity' and of the high-frequency shear modulus Ginfinity' of a sterically stabilized latex is presented. The experimental measurements have been done using a torsional resonator described recently (J. Bergenholtz et al., J. Colloid Interface Sci. 202, 430 (1998)). The data of etainfinity' compare favorably with recent theoretical predictions and point to a partial draining of the steric layer by the solvent as expected. The high-shear modulus, however, is much lower than predicted by theory. The pair potential calculated from Ginfinity' by neglect of hydrodynamic interaction has a considerably longer range than expected from the structure of the steric layer. This points to the fact that hydrodynamic interactions are not negligible for these systems and must be included in a quantitative analysis of the high-frequency shear modulus Ginfinity'. Copyright 1999 Academic Press.