Transition from fractal to spherical aggregates of globular proteins: Brownian-like activation and/or hydrodynamic stress?

We report on the structure of whey protein aggregates formed by a short heating coupled to shear at high temperatures (80-120) and neutral pH in scale-up processing conditions, using gel filtration chromatography, light scattering, small angle neutron scattering, and cryogenic transmission electron microscopy. The results are interpreted in terms of coexistence of residual non-aggregated proteins and aggregates. The characteristics of aggregates such as the size, the aggregation number and the shape evidence two different morphologies. Whereas aggregates formed at 80 °C show a selfsimilar structure down to a length scale of the monomer with a fractal dimension typical for reaction limited cluster aggregation (D~2.2), aggregates formed at higher temperature show a spherical morphology, with the structure from small angle neutron scattering data best modelled with the form factor of a polydisperse sphere. We compare the structure of these aggregates to that of aggregates formed in quiescent conditions at lab scale. The structure transition is interpreted in terms of a non-trivial interplay between three perturbation factors: interparticle interaction, temperature and shear.

Injection of calcium phosphate pastes: prediction of injection force and comparison with experiments.

Calcium phosphate ceramics suspensions (ICPCS) are used in bone and dental surgery as injectable bone substitutes. This ICPCS biomaterial associates biphasic calcium phosphate (BCP) granules with hydroxypropylmethylcellulose (HPMC) polymer. Different ICPCS were prepared and their rheological properties were evaluated in parallel disks geometry as a function of the BCP weight ratio (35, 40, 45 and 50 %). The suspensions show a strongly increased viscosity as compared to the suspending fluid and the high shear rate part of the flow curve can be fitted with a power law model (Ostwald-de Waele model). The fitting parameters depend on the composition of the suspension. A simple device has been used to characterize extrusion of the paste using a disposable syringe fitted with a needle. The injection pressure of four ICPCS formulations was studied under various conditions (needle length and radius and volumetric flow rate), yielding an important set of data. A theoretical approach based on the capillary flow of non-Newtonian fluids was used to predict the necessary pressure for injection, on the basis of flow curves and extrusion conditions. The extrusion pressure calculated from rheological data shows a quantitative agreement with the experimental one for model fluids (Newtonian and HPMC solution) but also for the suspension, when needles with sufficiently large diameters as compared to the size of particles, are used. Depletion and possibly wall slip is encountered in the suspensions when narrower diameters are used, so that the injection pressure is less than that anticipated. However a constant proportionality factor exists between theory and injection experiments. The approach developed in this study can be used to correlate the rheological parameters to the necessary pressure for injection and defines the pertinent experimental conditions to obtain a quantitative agreement between theory and experiments.

The stability mechanisms of an injectable calcium phosphate ceramic suspension.

Calcium phosphate ceramics are widely used as bone substitutes in dentistry and orthopedic applications. For minimally invasive surgery an injectable calcium phosphate ceramic suspension (ICPCS) was developed. It consists in a biopolymer (hydroxypropylmethylcellulose: HPMC) as matrix and bioactive calcium phosphate ceramics (biphasic calcium phosphate: BCP) as fillers. The stability of the suspension is essential to this generation of "ready to use" injectable biomaterial. But, during storage, the particles settle down. The engineering sciences have long been interested in models describing the settling (or sedimentation) of particles in viscous fluids. Our work is dedicated to the comprehension of the effect of the formulation on the stability of calcium phosphate suspension before and after steam sterilization. The rheological characterization revealed the macromolecular behavior of the suspending medium. The investigations of settling kinetics showed the influence of the BCP particle size and the HPMC concentration on the settling velocity and sediment compactness before and after sterilization. To decrease the sedimentation process, the granule size has to be smaller and the polymer concentration has to increase. A much lower sedimentation velocity, as compared to Stokes law, is observed and interpreted in terms of interactions between the polymer network in solution and the particles. This experimentation highlights the granules spacer property of hydrophilic macromolecules that is a key issue for interconnection control, one of the better ways to improve osteoconduction and bioactivity.

Self-similar assemblies of globular whey proteins at the air-water interface: effect of the structure.

We investigated the structure of heat-induced assemblies of whey globular proteins using small angle neutron scattering (SANS), static and dynamic light scattering (SLS and DLS), and cryogenic transmission electron microscopy (Cryo-TEM). Whey protein molecules self-assemble in fractal aggregates with a structure density depending on the electrostatic interactions. We determined the static and dynamic properties of interfacial layer formed by the protein assemblies, upon adsorption and spreading at the air-water interface using surface film balance and interfacial dilatational rheology. Upon spreading, all whey protein systems show a power-law scaling behavior of the surface pressure versus concentration in the semi-dilute surface concentration regime, with an exponent ranging from 5.5 to 9 depending on the electrostatic interactions and the aggregation state. The dilatational modulus derived from surface pressure isotherms shows a main peak at 6-8 mN/m, generally considered to be the onset of a conformational change in the monolayer, and a second peak or a shoulder at 15 mN/m. Long-time adsorption kinetics give similar results for both the native whey proteins and the corresponding self-similar assemblies, with a systematic effect of the ionic strength.

Beta-lactoglobulin aggregates in foam films: effect of the concentration and size of the protein aggregates.

Single foam films made from mixtures of nonaggregated proteins and protein aggregates have been studied using a thin film balance apparatus. Their features (heterogeneity, stability and resistance to pressure change) are dependent on the aggregate size and on the ratio between nonaggregated proteins and protein aggregates. A phase diagram of these foam films has been drawn and a correlation of the structural properties of foam film and stability of real foams has been found. In particular, the formation of a gel-like network within the foam film coincides with the stability of the corresponding 3-D foams.

Gelation studies of a cellulose-based biohydrogel: the influence of pH, temperature and sterilization.

The present paper investigates the rheological properties of silated hydroxypropylmethylcellulose (Si-HPMC) biohydrogel used for biomaterials and tissue engineering applications. The general property of this modified cellulose ether is the occurrence of self-hardening due to silanol condensation subsequent to a decrease in pH (from 12.4 to nearly 7.4). The behavior of unsterilized and sterilized Si-HPMC solutions in diluted and concentrated domains is first described and compared. In addition, the influence of physiological parameters such as pH and temperature on the rate of the gelation process is studied. In dilute solution, the intrinsic viscosity ([eta]) of different pre-steam sterilization Si-HPMC solutions indicates that macromolecular chains occupy a larger hydrodynamic volume than the post-steam sterilization Si-HPMC solutions. Although the unsterilized Si-HPMC solutions demonstrate no detectable influence of pH upon the rheological behavior, a decrease in the limiting viscosities (eta(0)) of solutions with increasing pH is observed following steam sterilization. This effect can be explained by the formation of intra- and intermolecular associations during the sterilization stage originating from the temperature-induced phase separation. The formation of Si-HPMC hydrogels from injectable aqueous solution is studied after neutralization by different acid buffers leading to various final pHs. Gelation time (t(gel)) decreases when pH increases (t(gel) varies from 872 to 11s at pH 7.4 and 11.8, respectively). The same effect is observed by increasing the temperature from 20 to 45 degrees C. This is a consequence of the synergistic effect of the increased reaction rate and acid buffer diffusion. pH and temperature are important parameters in the gelation process and their influence is a key factor in controlling gelation time. By adapting the gel parameters one could propose hydrogels with cross-linking properties adapted to clinical applications by controlling the amount of pH of neutralization and temperature.

Beta-lactoglobulin aggregates in foam films: correlation between foam films and foaming properties.

Single foam films made from aqueous protein solutions containing aggregates have been studied using a thin film balance apparatus. When the solutions are free from non-aggregated proteins, the films are either fluid or gelified, depending on the aggregate size. When non-aggregated proteins are present, they adsorb at the film surfaces, acting as anchors for the aggregates, and ultimately leading to stable foam films if the amount of non-aggregated proteins is sufficient. In all cases, gel-like films are more stable than fluid films and there is a good correlation between foam and film stability.

The rheological properties of silated hydroxypropylmethylcellulose tissue engineering matrices.

This paper describes the rheological properties of silated hydroxypropylmethylcellulose (HPMC-Si) used in biomaterials domain as a three-dimensional synthetic matrix for tissue engineering. The HPMC-Si is an HPMC grafted with 3-glycidoxypropyltrimethoxysilane (GPTMS). HPMC and HPMC-Si were studied. It is shown that although silanization reduces the hydrodynamic volume in dilute solution, it does not affect significantly the rheological behavior of the concentrated solutions. The HPMC-Si viscous solution (pH 12.8) cross-links by decreasing the pH using an acid buffer, since HPMC-Si solution transforms into an elastic state. The kinetics of cross-linking and final elastic properties is influenced by several parameters such as polymer concentration, pH and temperature. pH and temperature play an important role in the silanol condensation, mainly responsible for network formation. The study of the gelation process revealed the dependence of the final concentration of HPMC-Si hydrogel on cross-linking kinetics and viscoelastic properties. The percolation theory was applied to determine gel point and to discuss the dependence of storage (G') and loss (G'') moduli on frequency. Results showed that both G' and G'' exhibit a power-law behavior with an exponent (0.68) which extends over the entire frequency range. This method is the only one to characterize the time where a liquid viscous phase shifts to hydrogel with elastic properties. In this case it was about 23 min for a final pH of 7.4.

Combined rheological and ultrasonic study of alginate and pectin gels near the sol-gel transition.

The sol-gel transition of biopolymer mixtures has been investigated by rheological and ultrasonic measurements. A scaling analysis of the data was performed for both types of measurements. A gel time was determined from rheology for the pure pectin samples, and the data could be fitted to a universal scaling form near the transition point. Its critical exponents are in good agreement with the predictions of scalar percolation theory. In addition, the ultrasonic signal of the pectin samples close to the transition was analyzed in terms of a high-frequency scaling approach for the attenuation and the velocity. For the alginate samples and the mixtures, for which the gel point cannot be determined reliably from rheology, the ultrasonic measurements were analyzed using the same scaling form as for the pectin sample, thus providing a method for estimating the gel point, even in the absence of rheological data.

In situ investigations on organic foam films using neutron and synchrotron radiation.

We report on small-angle neutron scattering (SANS) and X-ray scattering (SAXS) investigations of foam films stabilized by sodium dodecyl sulfate. Previous measurements on dry foams (Axelos, M. A. V.; Boue, B. Langmuir 2003, 19, 6598) have shown the presence of spikes in the two-dimensional scattering data which suggest that the incident beam is reflected on some film surfaces. The latter interpretation is confirmed by new neutron studies performed on ordered ("bamboo") foams which allow selection of single films. In the first case, we show that the spikes of the scattered intensity can be obtained by reflection on two parts of the foam, namely, the films and the Plateau borders. With synchrotron radiation, first observations of distinct interference fringes have allowed an accurate determination of the film thickness. A comparison with X-ray and neutron data is made, opening a general discussion about the capabilities of small-angle scattering techniques for studying the microscopic properties of foam films.

Oleosins of Arabidopsis thaliana: expression in Escherichia coli, purification, and functional properties.

The interfacial behavior of oleosins, the most abundant proteins from seeds oil bodies, was investigated using the pendant drop method at water/oil interfaces and compared to the behavior of beta-casein and lysozyme, proteins with contrasted emulsifying properties. Recombined high (rS3) and low (rS4) molecular weight oleosins comprising N-terminal histidine tags were purified to electrophoretic homogeneity. rS3 decreased the interfacial tension at the oil/water interface better than rS4, oleosins being more efficient than beta-casein. Oleosins formed aggregates when spread on noncompressed phospholipid (PL) films at the air/water interface as observed using a Langmuir-Blodgett balance equipped with a Brewster angle microscope. Oleosin spread at the surface of a compressed PL monolayer (5-20 mN/m) did not aggregate. Pressure increased immediately and proportionally to the amount of protein spread on the monolayer. The results stress the capacity of oleosins to be inserted in oil and in PL monolayers, which is of particular relevancy to their potential uses as water/oil emulsifiers.