Powdered Cross-Linked Gelatin Methacryloyl as an Injectable Hydrogel for Adipose Tissue Engineering.

The tissue engineering field is currently advancing towards minimally invasive procedures to reconstruct soft tissue defects. In this regard, injectable hydrogels are viewed as excellent scaffold candidates to support and promote the growth of encapsulated cells. Cross-linked gelatin methacryloyl (GelMA) gels have received substantial attention due to their extracellular matrix-mimicking properties. In particular, GelMA microgels were recently identified as interesting scaffold materials since the pores in between the microgel particles allow good cell movement and nutrient diffusion. The current work reports on a novel microgel preparation procedure in which a bulk GelMA hydrogel is ground into powder particles. These particles can be easily transformed into a microgel by swelling them in a suitable solvent. The rheological properties of the microgel are independent of the particle size and remain stable at body temperature, with only a minor reversible reduction in elastic modulus correlated to the unfolding of physical cross-links at elevated temperatures. Salts reduce the elastic modulus of the microgel network due to a deswelling of the particles, in addition to triple helix denaturation. The microgels are suited for clinical use, as proven by their excellent cytocompatibility. The latter is confirmed by the superior proliferation of encapsulated adipose tissue-derived stem cells in the microgel compared to the bulk hydrogel. Moreover, microgels made from the smallest particles are easily injected through a 20G needle, allowing a minimally invasive delivery. Hence, the current work reveals that powdered cross-linked GelMA is an excellent candidate to serve as an injectable hydrogel for adipose tissue engineering.

Peptide Sequence Variations Govern Hydrogel Stiffness: Insights from a Multi-Scale Structural Analysis of H-FQFQFK-NH2 Peptide Derivatives.

Throughout the past decades, amphipathic peptide-based hydrogels have proven to be promising materials for biomedical applications. Amphipathic peptides are known to adopt ß-sheet configurations that self-assemble into fibers that then interact to form a hydrogel network. A fundamental understanding of how the peptide sequence alters the structural properties of the hydrogels would allow for a more rational design of novel peptides for a variety of biomedical applications in the future. Therefore, the current work investigates how changing the type of amino acid, the amphipathic pattern, and the peptide length affects the secondary structure, fiber characteristics, and stiffness of peptide-based hydrogels. Hereto, seven amphipathic peptides of different sequence and length, four of which have not been previously reported, based on and including the hexapeptide H-Phe-Gln-Phe-Gln-Phe-Lys-NH2, are synthesized and thoroughly characterized by circular dichroism (CD), Fourier Transform Infrared (FTIR) spectroscopy, Wide Angle X-ray Scattering (WAXS), Small Angle X-ray Scattering (SAXS), Transmission Electron Microscopy (TEM), and Thioflavin T (ThT) fibrillization assays. The results show that a high amount of regularly spaced ß-sheets, a high amount of fibers, and fiber bundling contribute to the stiffness of the hydrogel. Furthermore, a study of the time-dependent fibril formation process reveals complex transient dynamics. The peptide strands structure through an intermediate helical state prior to ß-sheet formation, which is found to be concentration- and time-dependent.

Phase behavior of medium-length hydrophobically associating PEO-PPO multiblock copolymers in aqueous media.

HYPOTHESIS: The micellization of block copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) is driven by the dehydration of PPO at elevated temperatures. At low concentrations, a viscous solution of isolated micelles is obtained, whereas at higher concentrations, crowding of micelles results in an elastic gel. Alternating PEO-PPO multiblock copolymers are expected to exhibit different phase behavior, with altered phase boundaries and thermodynamics, as compared to PEO-PPO-PEO triblock copolymers (Pluronics®) with equal hydrophobicity, thereby proving the pivotal role of copolymer architecture and molecular weight. EXPERIMENTS: Multiple characterization techniques were used to map the phase behavior as a function of temperature and concentration of PEO-PPO multiblock copolymers (ExpertGel®) in aqueous solution. These techniques include shear rheology, differential and adiabatic scanning calorimetry, isothermal titration calorimetry and light transmittance. The micellar size and topology were studied by dynamic light scattering. FINDINGS: Multiblocks have lower transition temperatures and higher thermodynamic driving forces for micellization as compared to triblocks due to the presence of more than one PPO block per chain. With increasing concentration, the multiblock copolymers in solution gradually evolve into a viscoelastic network formed by soluble bridges in between micellar nodes, whereas hairy triblock micelles jam into liquid crystalline phases resembling an elastic colloidal crystal.

Production and molecular characterization of tailored citrus pectin-derived compounds.

In this study, tailored-made citrus pectin-derived compounds were produced through controlled enzymatic and/or chemical modifications of commercial citrus pectin with different degrees of methylesterification (DM) and similar average molecular weight (MW). In the first treatment, degradation of the citrus pectin (CP) materials by endo-polygalacturonase (EPG) yielded pectins with average Mw's (between 2 and 60 kDa). Separation and identification of the oligosaccharide fraction present in these samples, revealed the presence of non-methylesterified galacturonic acid oligomers with degree of polymerization (DP) 1-5. In the second treatment, exploiting the combined effect of EPG and pectin lyase, compounds with MW between 2 and 21 kDa, containing methylesterified and non-methylesterified polygalacturonans (DP 1-6), were generated. Finally, CP was sequentially modified by chemical saponification and the action of EPG. A sample of DM 11% and MW 2.7 kDa, containing POS (DP 1-5), was produced. Diverse pectin-derived compounds were successfully generated for further studies exploring their functionality.

Modified Rhamnogalacturonan-Rich Apple Pectin-Derived Structures: The Relation between Their Structural Characteristics and Emulsifying and Emulsion-Stabilizing Properties.

In the context of the increasing interest in natural food ingredients, the emulsifying and emulsion-stabilizing properties of three rhamnogalacturonan-rich apple pectin-derived samples were assessed by evaluating a range of physicochemical properties. An apple pectin (AP74) was structurally modified by a ß-eliminative reaction to obtain a RG-I-rich pectin sample (AP-RG). Subsequent acid hydrolysis of AP-RG led to the generation of pectin material with partially removed side chains (in particular arabinose depleted) (AP-RG-hydrolyzed), thus exhibiting differences in rhamnose, arabinose, and galactose in comparison to AP-RG. All samples exhibited surface activity to some extent, especially under acidic conditions (pH 2.5). Furthermore, the viscosity of the samples was assessed in relation to their emulsion-stabilizing properties. In a stability study, it was observed that the non-degraded AP74 sample at pH 2.5 exhibited the best performance among all the apple pectin-derived samples evaluated. This emulsion presented relatively small oil droplets upon emulsion production and was less prone to creaming than the emulsions stabilized by the (lower molecular weight) RG-I-rich materials. The AP-RG and AP-RG-hydrolyzed samples presented a slightly better emulsion stability at pH 6.0 than at pH 2.5. Yet, neither pectin sample was considered having good emulsifying and emulsion-stabilizing properties, indicated by the presence of coalesced and flocculated oil droplets.

The role of lipids in determining the air-water interfacial properties of wheat, rye, and oat dough liquor constituents.

Bread is mainly made from wheat but also from other cereals such as rye and oats. We here report on the role of dough liquor (DL) proteins and lipids in determining the stability of gas cell air-water (A-W) interfaces in wheat, rye, and oat bread making. Surprisingly, most lipids in DLs of these cereals are nonpolar. Their main polar DL lipids are phospholipids. Lipids at wheat and rye DL stabilized A-W interfaces impair interactions between its proteins, as reflected by an increased A-W interfacial shear viscosity of the adsorbed film upon defatting. In contrast, removing most lipids from oat DL pronouncedly increased the A-W interface surface tension, demonstrating that lipids are the prominent adsorbed species.

Impact of different sequences of mechanical and thermal processing on the rheological properties of Porphyridium cruentum and Chlorella vulgaris as functional food ingredients.

Microalgae are a promising and sustainable source for enhancing the nutritional value of food products. Moreover, incorporation of the total biomass might contribute to the structural properties of the enriched food product. Our previous study demonstrated the potential of Porphyridium cruentum and Chlorella vulgaris as multifunctional food ingredients, as they displayed interesting rheological properties after applying a specific combination of mechanical and thermal processing. The aim of the current study was to investigate the impact of a different sequence of high pressure homogenization (HPH) and thermal processing on the thickening and gelling potential of these microalgal biomasses in aqueous suspensions. Thermal processing largely increased the gel strength and viscosity of both microalgae, which was ascribed to larger and stronger aggregates as a result of partial solubilization of polymers, while subsequent HPH generally reduced the rheological properties. Interestingly, large amounts of intact cells were still observed for both microalgae when HPH was performed after a thermal treatment, irrespective of the applied homogenization pressure, implying that cell disruption was hindered by the preceding thermal treatment. Although thermal processing was regarded as the most effective processing technique to obtain increased rheological properties, the combination with a preceding HPH treatment should still be considered when cell disruption is desired, for instance to increase the bioavailability of intracellular components. Finally, biomass of P. cruentum showed the largest potential for use as a structuring agent, as the gel strength and viscosity in thermally treated suspensions of this microalga were about 10 times higher than for C. vulgaris.

Effect of the Helix-Coil Transition in Bovine Skin Gelatin on Its Associative Phase Separation with Lysozyme.

It is known that the formation of electrostatic polyelectrolyte complexes can induce conformational changes in the interacting macromolecules. However, the opposite effect, namely, that of the helix-coil transition of one of the interacting polyelectrolytes on its associative phase separation with another polyelectrolyte and the possible phase transitions in such systems, has not been determined. Atomic force and confocal laser scanning microscopy, phase analysis, dynamic and electrophoretic light scattering, turbidimetry, absorption, and fluorescence measurements as well as differential scanning calorimetry and rheology were used to study the effect of the helix-coil transition in bovine skin gelatin (Gel) on its associative phase separation with hen egg white lysozyme (Lys) at different temperatures (18-40 °C) and various Lys/Gel weight ratios (0.01-100) at low ionic strength (0.01) and pH 7.0. The effects of the main variables on the phase state, the phase diagram, and the main complexation and binding parameters as well as the temperature and enthalpy of the helix-coil transition of Gel within the complexes were investigated. Associative phase separation is observed only for the system with Gel in the helix state. Effective charge and structure and the solution and rheological behavior of the formed complexes proved to be dependent on the [An-]/[Cat+] charge ratio. The localization of Lys within the complex particles has irregular character without the formation of a single center of binding. The binding of Lys with Gel does not lead to the disruption of its tertiary structure or to an appreciable change in the thermodynamic parameters of the thermal transitions of Lys. Gel in the coil state interacts only weakly with Lys, forming water-soluble complex associates. It is suggested that the Voorn-Overbeek model could potentially describe the stronger binding and phase separation in the case of Gel in the helix state.

Adsorption of Ellipsoidal Particles at Liquid-Liquid Interfaces.

The adsorption of particles at liquid-liquid interfaces is of great scientific and technological importance. In particular, for nonspherical particles, the capillary forces that drive adsorption vary with position and orientation, and complex adsorption pathways have been predicted by simulations. On the basis of the latter, it has been suggested that the timescales of adsorption are determined by a balance between capillary and viscous forces. However, several recent experimental results point out the role of contact line pinning in the adsorption of particles to interfaces and even suggest that the adsorption dynamics and pathways are completely determined by the latter, with the timescales of adsorption being determined solely by particle characteristics. In the present work, the adsorption trajectories of model ellipsoidal particles are investigated experimentally using cryo-SEM and by monitoring the altitudinal orientation angle using high-speed confocal microscopy. By varying the viscosity and the viscosity jump across the interfaces, we specifically interrogate the role of viscous forces.

Effect of the GO Reduction Method on the Dielectric Properties, Electrical Conductivity and Crystalline Behavior of PEO/rGO Nanocomposites.

The effect of the reduction method to prepare reduced graphene oxide (rGO) on the melt linear viscoelastic properties, electrical conductivity, polymer matrix crystalline behavior and dielectric properties of PEO-rGO nanocomposites was investigated. Reduction was performed chemically with either sodium borohydride (NaBH4) or hydrazine monohydrate (N2H4·H2O) or both reduction agents consecutively as well as thermally at 1000 °C. The different reduction methods resulted in exfoliated rGO sheets with different types and amounts of remaining functional groups, as indicated by FT-IR, Raman, TGA and XRD characterization. Moreover, their electrical conductivity ranged between 10-4 and 10-1 S/cm, with the consecutive use of both chemical reduction agents being far superior. PEO nanocomposites with filler loadings of 0.5 wt %, 1 wt % and 2 wt % were prepared by solvent mixing. The rGO fillers affected the melt linear viscoelastic and crystalline behavior of the PEO matrix and resulted in nanocomposites with a substantially increased electrical conductivity. Despite the wide variability in filler conductivity, the effects on the polymer nanocomposite properties were less distinctive. A correlation was obtained between the reduction of the mobility of the polymer chains (evaluated by the glass transition temperature) and the dielectric strength of the interfacial polarisation originating from the effective entrapment of GO/rGO filler charges at the interface with the less conductive PEO. Thus, favorable interactions of the polar PEO with the filler led to reduced mobility of the PEO chains and thereby a more effective entrapment of the filler charges at the PEO interface.

Dynamics of particle-covered droplets in shear flow: unusual breakup and deformation hysteresis.

The dynamics of droplets exhibiting an elastic interface generated by a percolated network of particle aggregates at the interface is microscopically investigated in a counter rotating shear flow device. The droplet deformation is significantly suppressed by interfacially localized nanoparticles, even at high Ca numbers, resulting in suspension-like behavior at high particle coverage. When the Ca number surpasses a critical value, the particle network locally breaks up, resulting in localized deformability of the interface and breakup dynamics characterized by extremely irregular shapes. Finally, the destruction of the interfacial network results in hysteresis effects in the droplet deformation.

Simple microfluidic stagnation point flow geometries.

A geometrically simple flow cell is proposed to generate different types of stagnation flows, using a separation flow and small variations of the geometric parameters. Flows with high local deformation rates can be changed from purely rotational, over simple shear flow, to extensional flow in a region surrounding a stagnation point. Computational fluid dynamic calculations are used to analyse how variations of the geometrical parameters affect the flow field. These numerical calculations are compared to the experimentally obtained streamlines of different designs, which have been determined by high speed confocal microscopy. As the flow type is dictated predominantly by the geometrical parameters, such simple separating flow devices may alleviate the requirements for flow control, while offering good stability for a wide variety of flow types.

Effect of Compression on the Molecular Arrangement of Itraconazole-Soluplus Solid Dispersions: Induction of Liquid Crystals or Exacerbation of Phase Separation?

Predensification and compression are unit operations imperative to the manufacture of tablets and capsules. Such stress-inducing steps can cause destabilization of solid dispersions which can alter their molecular arrangement and ultimately affect dissolution rate and bioavailability. In this study, itraconazole-Soluplus solid dispersions with 50% (w/w) drug loading prepared by hot-melt extrusion (HME) were investigated. Compression was performed at both pharmaceutically relevant and extreme compression pressures and dwell times. The starting materials, powder, and compressed solid dispersions were analyzed using modulated differential scanning calorimetry (MDSC), X-ray diffraction (XRD), small- and wide-angle X-ray scattering (SWAXS), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), and broadband dielectric spectroscopy (BDS). MDSC analysis revealed that compression promotes phase separation of solid dispersions as indicated by an increase in glass transition width, occurrence of a peak in the nonreversing heat flow signal, and an increase in the net heat of fusion indicating crystallinity in the systems. SWAXS analysis ruled out the presence of mesophases. BDS measurements elucidated an increase in the Soluplus-rich regions of the solid dispersion upon compression. FTIR indicated changes in the spatiotemporal architecture of the solid dispersions mediated via disruption in hydrogen bonding and ultimately altered dynamics. These changes can have significant consequences on the final stability and performance of the solid dispersions.

Contact angles of microellipsoids at fluid interfaces.

The wetting of anisotropic colloidal particles is of great importance in several applications, including Pickering emulsions, filled foams, and membrane transduction by particles. However, the combined effect of shape and surface chemistry on the three-phase contact angle of anisotropic micrometer and submicrometer colloids has been poorly investigated to date, due to the lack of a suitable experimental technique to resolve individual particles. In the present work, we investigate the variation of the contact angle of prolate ellipsoidal colloids at a liquid-liquid interface as a function of surface chemistry and aspect ratio using freeze-fracture shadow-casting cryo-SEM. The method, initially demonstrated for spherical colloids, is extended here to the more general case of ellipsoids. The prolate ellipsoidal particles are prepared from polystyrene and poly(methyl methacrylate) spheres using a film stretching technique, in which cleaning steps are needed to remove all film material from the particle surface. The effects of the preparation protocol are reported, and wrinkling of the three-phase contact line is observed when the particle surface is insufficiently cleaned. For identically prepared ellipsoids, the cosine of the measured contact angle is, in a first approximation, a linearly decreasing function of the contact line length and thus a decreasing function of the aspect ratio. Such a trend violates Young-Laplace's equation and can be rationalized by adding a correction term to the ideal Young-Laplace contact angle that expresses the relative importance of line effects relative to surface effects. From this term the contribution of an effective line tension can be extracted. This contribution includes the effects that both surface chemical and topographical heterogeneities have on the contact line and which become increasingly more important for ellipsoids with higher aspect ratios, where the contact line length to contact area ratio increases.

A Review on the Relationships between Processing, Food Structure, and Rheological Properties of Plant-Tissue-Based Food Suspensions.

Nowadays, there is much interest in controlling the functional properties of processed fruit- and vegetable-derived products, which has stimulated renewed research interest in process-structure-function relations. In this review, we focus on rheology as a functional property because of its importance during the entire production chain up to the moment of consumption and digestion. This review covers the literature of the past decade with respect to process-structure-rheology relations in plant-tissue-based food suspensions. It became clear that the structure of plant-tissue-based food suspensions, consisting of plant-tissue-based particles in an aqueous serum phase, is affected by many unit operations (for example, heat treatment) and that also the sequence of unit operations can have an effect on the final structural properties. Furthermore, particle concentration, particle size, and particle morphology were found to be key structural elements determining the rheological properties of these suspensions comprising low amounts of starch and serum pectin. Since the structure of plant-tissue-based products was shown to be changed during processing, rheological parameters of these products were simultaneously altered. Therefore, this review also comprises a discussion of the effect on rheological properties of the most relevant processing steps in the production of plant-tissue-based products. Linking changes in rheology due to processing with process-induced alterations in structural characteristics turned out to be quite intricate. The current knowledge on process-structure-function relations can form the basis for future improved and novel food process and product design.

The effect of geometrical confinement on coalescence efficiency of droplet pairs in shear flow.

Droplet coalescence is determined by the combined effect of the collision frequency and the coalescence efficiency of colliding droplets. In the present work, the effect of geometrical confinement on coalescence efficiency in shear flow is experimentally investigated by means of a counter rotating parallel plate device, equipped with a microscope. The model system consisted of Newtonian droplets in a Newtonian matrix. The ratio of droplet diameter to plate spacing (2R/H) is varied between 0.06 and 0.42, thus covering bulk as well as confined conditions. Droplet interactions are investigated for the complete range of offsets between the droplet centers in the velocity gradient direction. It is observed that due to confinement, coalescence is possible up to higher initial offsets. On the other hand, confinement also induces a lower boundary for the initial offset, below which the droplets reverse during their interaction, thus rendering coalescence impossible. Numerical simulations in 2D show that the latter phenomenon is caused by recirculation flows at the front and rear of confined droplet pairs. The lower boundary is independent of Ca, but increases with increasing confinement ratio 2R/H and droplet size. The overall coalescence efficiency is significantly larger in confined conditions as compared to bulk conditions.

Stiffness of Ca(2+)-pectin gels: combined effects of degree and pattern of methylesterification for various Ca(2+) concentrations.

The influence of the degree and pattern of methylesterification (DM and PM, respectively) on the stiffness of Ca(2+)-pectin gels is extensively examined, at various Ca(2+) concentrations. Accordingly, a highly methyl-esterified pectin was selectively de-esterified using NaOH, plant or fungal pectin methylesterase in order to produce series of pectins with varied pattern and broad ranges of methylesterification. The PM was quantified as absolute degree of blockiness (DB(abs)). Ca(2+)-pectin gels were prepared at various Ca(2+) concentrations. Gel stiffness (G' at 1rad/s) was determined and mapped out as a function of DM, DB(abs) and Ca(2+) concentration. At low Ca(2+) concentrations, G' depends on polymer's DM and DB(abs). At high Ca(2+) concentrations, a master curve is obtained over a wide range of DM, irrespective of DB(abs). Depending on methylesterification pattern, increase of G' is related not only to an increase in the number of junction zones per pectin chain, but also to an increase in the size of junction zones and the number of dimerised chains occurring in the gels. These results provide a detailed insight into the occurrence of junction zones in Ca(2+)-pectin gels.

Effect of thermally reduced graphene sheets on the phase behavior, morphology, and electrical conductivity in poly[(α-methyl styrene)-co-(acrylonitrile)/poly(methyl-methacrylate) blends.

The effects of thermally reduced graphene sheets (TRG) on the phase separation in poly[(α-methyl styrene)-co-(acrylonitrile)]/poly(methyl-methacrylate) blends were monitored using melt rheology, conductivity spectroscopy, and electron microscopic techniques. The TRG were incorporated in the single-phase material by solution mixing. The composite samples were then allowed to phase separate in situ. The thermodynamics of phase separation have been investigated by monitoring the evolution of the storage modulus (G') as a function of temperature as the system passes through the binodal and the spinodal lines of the phase diagram. The phase separation kinetics were probed by monitoring the evolution of G' as a function of time at a quench depth well in the spinodal region. It was observed that TRG significantly influenced the phase separation temperature, the shape of the phase diagram and the rate of phase separation. The state of dispersion of TRG in the blends was assessed using electron microscopy and conductivity spectroscopy measurements. Interestingly, the composite samples (monophasic) were virtually insulators at room temperature, whereas highly conducting materials were obtained as a result of phase separation in the biphasic materials.

Study of the flow field in the magnetic rod interfacial stress rheometer.

Several technological applications, consumer products, and biological systems derive their functioning from the presence of a complex fluid interface with viscoelastic interfacial rheological properties. Measurements of the "excess" rheological properties of such an interface are complicated by the intimate coupling of the bulk and interfacial flows. In the present work, analytical, numerical, and experimental results of the interfacial flow fields in a magnetic rod interfacial stress rheometer (ISR) are presented. Mathematical solutions are required to correct the experimentally determined apparent interfacial shear moduli and phase angles for the drag exerted by the surrounding phases, especially at low Boussinesq numbers. Starting from the Navier-Stokes equations and using the generalized Boussinesq-Scriven equation as a suitable boundary condition, the problem is solved both analytically and numerically. In addition, experimental data of the interfacial flow field are reported, obtained by following the trajectories of tracer particles at the interface with time. Good agreement is found between the three methods, indicating that both the analytical solution and the numerical simulations give an adequate description of the flow field and the resulting local interfacial shear rate at the rod. Based on these results, an algorithm to correct the experimental data of the ISR is proposed and evaluated, which can be extended to different types of interfacial shear rheometers and geometries. An increased accuracy is obtained and the measurement range of the ISR is expanded toward viscosities and elastic moduli of smaller magnitude.

A sliding plate microgap rheometer for the simultaneous measurement of shear stress and first normal stress difference.

A new generation of the "flexure-based microgap rheometer" (the N-FMR) has been developed which is also capable of measuring, in addition to the shear stress, the first normal stress difference of micrometer thin fluid films. This microgap rheometer with a translation system based on compound spring flexures measures the rheological properties of microliter samples of complex fluids confined in a plane couette configuration with gap distances of h = 1-400 µm up to shear rates of γ = 3000 s(-1). Feed back loop controlled precise positioning of the shearing surfaces with response times <1 ms enables to control the parallelism within 1.5 µrad and to maintain the gap distance within 20 nm. This precise gap control minimizes squeeze flow effects and allows therefore to measure the first normal stress difference N(1) of the thin film down to a micrometer gap distance, with a lower limit of N(1)/γ = 9.375×10(-11) η/h(2) that depends on the shear viscosity η and the squared inverse gap. Structural development of complex fluids in the confinement can be visualized by using a beam splitter on the shearing surface and a long working distance microscope. In summary, this new instrument allows to investigate the confinement dependent rheological and morphological evolution of micrometer thin films.