Self-healing fish gelatin/sodium montmorillonite biohybrid coacervates: structural and rheological characterization

Complex coacervation driven by associative electrostatic interactions was studied in mixtures of exfoliated sodium-montmorillonite (Na(+)-MMT) nanoplatelets and fish gelatin, at a specific mixing ratio and room temperature. Structural and viscoelastic properties of the coacervate phase were investigated as a function of pH by means of different complementary techniques. Independent of the technique used, the results consistently showed that there is an optimum pH value at which the coacervate phase shows the tightest structure with highest elasticity. The solid-like coacervates showed an obvious shear-thinning behavior and network fracture but immediately recovered back into their original elastic character upon removal of the shear strain. The nonlinear mechanical response characterized by single step stress relaxation experiments revealed the same trend for the yield stress and isochronal shear modulus of the coacervates as a function of pH with a maximum at pH 3.0 and lower values at 2.5 and 3.5 pHs, followed by a very sharp drop at pH 4.0. Finally, small-angle X-ray scattering (SAXS) data confirmed that at pHs lower than 4.0 the coacervate phases were dense and structured with a characteristic length scale (ξ(SAXS)) of ~7-9 nm. Comparing the ξ(SAXS) with rheological characteristic length (ξ(rheol)) estimated from low-frequency linear viscoelastic data and network theory, it was concluded that both the strength of the electrostatic interactions and the conformation of the gelatin chains before and during of the coacervation process are responsible for the structure and rigidity of the coacervates.     

The mechanical properties of simple epithelial keratins 8 and 18: discriminating between interfacial and bulk elasticities

The abundance and cytoplasmic organization of keratin filaments enables them to contribute to the maintenance of structural integrity in epithelial tissues. Co-polymers of the type II keratin 8 and type I keratin 18 form the major intermediate filament network in simple epithelia. We investigated the mechanical properties of K8-K18 filament suspensions using rheological assays in conjunction with light and electron microscopy. Suspensions of K8-K18 filaments behave like a viscoelastic solid under standard assembly conditions. Bulk elasticity is weakly dependent on deformation frequency but is very sensitive to the concentration (G' approximately C1.5) and size of individual keratin polymers, in agreement with recent models of semiflexible-polymer physics. K8-K18 filaments can self-organize to form a bundled network that exhibits gel-like mechanical properties. In all cases the mechanical properties of the suspensions correlate with the structural features of individual polymers, as seen under light and electron microscopy. Importantly, these bulk viscoelastic properties of K8-K18 filaments are revealed only when interfacial elastic effects are minimized by the application of phospholipids at the air-liquid interface. Suspensions of K5-K14 and vimentin filaments also exhibit interfacial elasticity, which distorts the interpretation of the viscoelastic moduli as determined by standard rheometry. The potential for modulation of mechanical properties through self-organization may be a general property of keratin polymers and contribute to their organization and function in vivo.     

Linear viscoelastic properties of bovine brain tissue in shear

We report the results from a series of rheological tests of fresh bovine brain tissue. Using a standard Bohlin VOR shear rheometer, shear relaxation and oscillating strain sweep experiments were performed on disks of brain tissue 30 mm in diameter, with a thickness of 1.5–2 mm. The strain sweep experiment showed that the viscoelastic strain limit is of the order of 0.1% strain. Shear relaxation data do not indicate the presence of a long-term elastic modulus, indicating fluid-like behavior. A relaxation spectrum was calculated by inverting the experimental data and used to predict oscillatory response, which agreed well with measured data.     

Biodegradable polymers from renewable sources: rheological characterization of hemicellulose-based hydrogels

Hemicellulose-based hydrogels were prepared by radical polymerization of 2-hydroxyethyl methacrylate or poly(ethylene glycol) dimethacrylate with oligomeric hydrosoluble hemicellulose modified with well-defined amounts of methacrylic functions. The polymerization reaction was carried out in water at 40 degrees C using a redox initiator system. The hydrogels were in general elastic, soft, and easily swellable in water. Their viscoelastic properties were determined by oscillatory shear measurements on 2 mm thick hydrogels under a slight compression to avoid slip, over the frequency range 10(-1) to 10(2). The rheological characterization indicated that the elastic response of the hydrogels was stronger than the viscous response, leading to the conclusion that the hydrogel systems displayed a predominantly solid-like behavior. The curves showed an increase in shear storage modulus with increasing cross-linking density. The nature of the synthetic comonomer in the hemicellulose-based hydrogels also influenced the shear storage modulus. Comparison of hemicellulose-based hydrogels with pure poly(2-hydroxyethyl methacrylate) hydrogels showed that their behaviors were rather similar, demonstrating that the synthetic procedure made it possible to prepare hemicellulose-based hydrogels with properties similar to those of pure poly(2-hydroxyethyl methacrylate) hydrogels.     

Power-law rheology analysis of cells undergoing micropipette aspiration

Accurate quantification of the mechanical properties of living cells requires the combined use of experimental techniques and theoretical models. In this paper, we investigate the viscoelastic response of suspended NIH 3T3 fibroblasts undergoing micropipette aspiration using power-law rheology model. As an important first step, we examine the pipette size effect on cell deformation and find that pipettes larger than ~7 μm are more suitable for bulk rheological measurements than smaller ones and the cell can be treated as effectively continuum. When the large pipettes are used to apply a constant pressure to a cell, the creep deformation is better fitted with the power-law rheology model than with the liquid drop or spring-dashpot models; magnetic twisting cytometry measurement on the rounded cell confirms the power-law behavior. This finding is further extended to suspended cells treated with drugs targeting their cytoskeleton. As such, our results suggest that the application of relatively large pipettes can provide more effective assessment of the bulk material properties as well as support application of power-law rheology to cells in suspension.     

A Multiscale Red Blood Cell Model with Accurate Mechanics, Rheology, and Dynamics

Red blood cells (RBCs) have highly deformable viscoelastic membranes exhibiting complex rheological response and rich hydrodynamic behavior governed by special elastic and bending properties and by the external/internal fluid and membrane viscosities. We present a multiscale RBC model that is able to predict RBC mechanics, rheology, and dynamics in agreement with experiments. Based on an analytic theory, the modeled membrane properties can be uniquely related to the experimentally established RBC macroscopic properties without any adjustment of parameters. The RBC linear and nonlinear elastic deformations match those obtained in optical-tweezers experiments. The rheological properties of the membrane are compared with those obtained in optical magnetic twisting cytometry, membrane thermal fluctuations, and creep followed by cell recovery. The dynamics of RBCs in shear and Poiseuille flows is tested against experiments and theoretical predictions, and the applicability of the latter is discussed. Our findings clearly indicate that a purely elastic model for the membrane cannot accurately represent the RBC's rheological properties and its dynamics, and therefore accurate modeling of a viscoelastic membrane is necessary.     

Nonlinear Viscoelasticity of Actin Transiently Cross-linked with Mutant α-Actinin-4

Filamentous actin and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. They can also play a critical role in disease; for example, mutations in α-actinin-4 (Actn4), a dynamic actin cross-linking protein, cause proteinuric disease in humans and mice. Amino acid substitutions strongly affect the binding affinity and protein structure of Actn4. To study the physical impact of such substitutions on the underlying cytoskeletal network, we examine the bulk mechanical behavior of in vitro actin networks cross-linked with wild-type and mutant Actn4. These networks exhibit a complex viscoelastic response and are characterized by fluid-like behavior at the longest timescales, a feature that can be quantitatively accounted for through a model governed by dynamic cross-linking. The elastic behavior of the network is highly nonlinear, becoming much stiffer with applied stress. This nonlinear elastic response is also highly sensitive to the mutations of Actn4. In particular, we observe that actin networks cross-linked with Actn4 bearing the disease-causing K255E mutation are more brittle, with a lower breaking stress in comparison to networks cross-linked with wild-type Actn4. Furthermore, a mutation that ablates the first actin binding site (ABS1) in Actn4 abrogates the network's ability to stress-stiffen is standard nomenclature. These changes in the mechanical properties of actin networks cross-linked with mutant Actn4 may represent physical determinants of the underlying disease mechanism in inherited focal segmental glomerulosclerosis.     

Effects of biofilm heterogeneity on the apparent mechanical properties obtained by shear rheometry

Rheometry is an experimental technique widely used to determine the mechanical properties of biofilms. However, it characterizes the bulk mechanical behavior of the whole biofilm. The effects of biofilm mechanical heterogeneity on rheometry measurements are not known. We used laboratory experiments and computer modeling to explore the effects of biofilm mechanical heterogeneity on the results obtained by rheometry. A synthetic biofilm with layered mechanical properties was studied, and a viscoelastic biofilm theory was employed using the Kelvin–Voigt model. Agar gels with different concentrations were used to prepare the layered, heterogeneous biofilm, which was characterized for mechanical properties in shear mode with a rheometer. Both experiments and simulations indicated that the biofilm properties from rheometry were strongly biased by the weakest portion of the biofilm. The simulation results using linearly stratified mechanical properties from a previous study also showed that the weaker portions of the biofilm dominated the mechanical properties in creep tests. We note that the model can be used as a predictive tool to explore the mechanical behavior of complex biofilm structures beyond those accessible to experiments. Since most biofilms display some degree of mechanical heterogeneity, our results suggest caution should be used in the interpretation of rheometry data. It does not necessarily provide the “average” mechanical properties of the entire biofilm if the mechanical properties are stratified.     

Investigation of rheological properties and conformation of silk fibroin in the solution of AmimCl

The conformation and eventual morphology of silk fibroin (SF) chains are crucial for the mechanical properties of SF materials, and are strongly related to the solvation step as a key stage in their processing conditions. In this work, a novel SF/AmimCl (1-allyl-3-methylimidazolium chloride) solution with unique properties is reported and compared with conventional regenerated SF aqueous solutions, based on an investigation of its rheological properties. The steady shearing behavior suggested that AmimCl is a good solvent for SF molecules, and shear thinning of semidiluted SF/AmimCl solution at high shear rates showed behavior similar to that in native spinning, which is due to the rearrangement and orientation of SF molecular chains. Fitting of experimental dynamic viscoelastic data to the Rouse model provided an effective method to estimate the molecular weight of SF. We believe that this work not only provides a better understanding of the relationship between properties of silk protein and aggregation states of their molecular chains, but also provides tools to fabricate high-performance SF-based materials.     

Nonlinear viscoelastic biomaterials: meaningful characterization and engineering inspiration

Nonlinear mechanical properties play an important role in numerous biological functions, for instance the locomotion strategy used by terrestrial gastropods. We discuss the progress made toward bioinspired snail-like locomotion and the pursuit of an engineered fluid that imitates the nonlinear viscoelastic properties of native gastropod pedal mucus. The rheological behavior of native pedal mucus is characterized using an oscillatory deformation protocol known as large amplitude oscillatory shear, and we review recently developed techniques for appropriately describing nonlinear viscoelastic behavior. Although materials that exhibit purely elastic and purely viscous nonlinearities are amenable to standard techniques for characterization, pedal mucus samples (and biomaterials in general) are viscoelastic, exhibiting both elastic and viscous nonlinear responses simultaneously and requiring advanced techniques for characterization. We reveal the utility of these new methods by examining trail mucus from the terrestrial slug Limax maximus using oscillatory shear rheology. Material responses which previously could only be described mathematically, with little physical insight, can now be interpreted with familiar language such as strain-stiffening/softening and shear-thickening/thinning. The new methodology is applicable to any complex material that can be tested using imposed oscillatory deformations. We have developed data-analysis software to enable wider use of this framework within and beyond the biomaterials community. The functionality of this software is outlined here.     

Fast fluorescence laser tracking microrheometry. I: instrument development

To gain insight into cellular mechanotransduction pathways, we have developed a fluorescence laser tracking microrheometer (FLTM) to measure material rheological features on micrometer length scales using fluorescent microspheres as tracer particles. The statistical analysis of the Brownian motion of a particle quantifies the viscoelastic properties of the probe's environment, parameterized by the frequency-dependent complex shear modulus G*(ω). This FLTM has nanometer spatial resolution over a frequency range extending from 1 Hz to 50 kHz. In this work, we first describe the consecutive stages of instrument design, development, and optimization. We subsequently demonstrate the accuracy of the FLTM by reproducing satisfactorily the known rheological characteristics of purely viscous glycerol solutions and cross-linked polyacrylamide polymer networks. An upcoming companion article will illustrate the use of FLTM in studying the solid-like versus liquid-like rheological properties of fibroblast cytoskeletons in living biological samples.     

Complex rheological properties of a water-soluble extract from the fronds of the black tree fern, Cyathea medullaris

A water-soluble extract was obtained from the fronds of a New Zealand native black tree fern (Cyathea medullaris or Mamaku in Māori). The extract exhibited complex rheological behavior. Newtonian, shear-thinning, shear-thickening, thixotropic, antithixotropic, and viscoelastic behaviors were observed depending on polymer concentration, shear rate, and shear history. The extract also displayed rod-climbing and self-siphoning properties typical of viscoelastic fluids. Such complex rheological properties have been reported in synthetic or chemically modified polymers but are less frequent in unmodified biopolymers. Although Mamaku extract obtained from the pith of the fern has been traditionally used by the Māori in New Zealand for treating wounds and diarrhea among other ailments, this material has never been characterized before. This study reports on the chemical composition of the extract and on its viscoelastic properties through rotational and oscillatory rheological measurements. Explanations of the mechanism behind the rheological properties were based on transient network models for associating polymers.     

A rheological network model for the continuum anisotropic and viscoelastic behavior of soft tissue

The mechanical behavior of soft tissue demonstrates a number of complex features including nonlinearity, anisotropy, viscoelasticity, and growth. Characteristic features of the time-dependent and anisotropic behavior are related to the properties of various components of the tissue such as fibrous collagen and elastin networks, large proteins and sugars attached to these networks, and interstitial fluid. Attempts to model the elastic behavior of these tissues based on assumptions about the behavior of the underlying constituents have been reasonably successful, but the essential addition of viscoelasticity to these models has been met with varying success. Here, a new rheological network model is proposed using, as its basis, an orthotropic hyperelastic constitutive model for fibrous tissue and a viscoelastic reptation model for soft materials. The resulting model has been incorporated into numerical and computational models, and is shown to capture the mechanical behavior of soft tissue in various modes of deformation including uniaxial and biaxial tension and simple shear.     

Effect of dehydration on the viscoelastic behavior of red cells.

Dehydration of red cells alters their rheological behavior and may contribute to the pathology of disorders such as sickle cell disease. We have measured the viscoelastic properties of individual human HbAA red cells after graded dehydration induced by incubation with valinomycin at different external K levels. With dehydration, the cells underwent progressive reduction in their rate of extensional shape recovery (i.e., after elongation by micropipette manipulation). Their rigidity remained unaffected until the mean cell hemoglobin concentration (MCHC) rose above 50 g/dl, but then increased about 100% as judged from the response of membrane tongues drawn into micropipettes. There was also a marked reduction in the shape recovery rate at this level of dehydration, and the cells no longer behaved elastically but rather showed unrecoverable residual deformation. Additionally, the cytoplasm took on solid-like properties. Changes in cell rigidity and shape recovery rate have been previously demonstrated for dense sickle cells; our results indicate that normal red cells can be induced to behave similarly, but that a greater degree of dehydration is required.     

Mechanical behavior of an individual adherent MLO-Y4 osteocyte under shear flow

Mechanical properties of a single cell and its mechanical response under stimulation play an important role in regulating interactions between cell and extracellular matrix and affecting mechanotransduction. Osteocytes exhibit solid-like viscoelastic behavior in response to the interstitial fluid shear resulting from tissue matrix deformation. This study intends to quantitatively describe the mechanical behavior of osteocytes combining in vitro experiment and fluid–structure interaction (FSI) finite element (FE) model. The cell is configured in the FSI FE model using the observed data from quasi-3D images. Instead of simply assigning the cellular viscoelastic parameters by statistical data, the mechanical parameters are determined by an iterative algorithm comparing the experimental and the computational results from the FE model. The viscoelastic parameters of osteocytes are obtained as: the equilibrium elasticity modulus \(k_{1}=0.15\pm 0.038\,\hbox {kPa}\), instantaneous elasticity modulus \((k_{1}+k_{2})=0.77\pm 0.23\,\hbox {kPa}\), viscosity coefficient \(\eta =1.38\pm 0.33\,\hbox {kPa}\,\hbox {s}\). A novel index to quantify the cell adhesion is also put forward. In addition, an interesting competition phenomenon is revealed on the cell surface concerning stress and strain, i.e., the place with high stress has low strain and that with low stress has high strain. The proposed method provides a novel technique to study the mechanical behavior of individual adherent cell in vitro. It is believed that this quantitative technique not only determines cell mechanical behavior but also helps elucidate the mechanism of mechanotransduction in various types of cells.     

Large deformation shear properties of liver tissue

Characterization of the mechanical properties of soft biological tissues is important for establishing the mechanical tolerances of the tissues, and for input to computational models. In this work, the viscoelastic properties of bovine liver tissue in shear loading have been measured using relaxation and constant shear rate loading. The tissue is nonlinearly viscoelastic for strains greater than 0.2%, has a yield strain of approximately 10, and shows moderate strain-rate sensitivity. The response can be modelled using a nonlinear viscoelastic differential model previously developed for brain tissue.     

A visco-hyperelastic constitutive approach for modeling polyvinyl alcohol sponge

This study proposes the quasi-linear viscoelastic (QLV) model to characterize the time dependent mechanical behavior of poly(vinyl alcohol) (PVA) sponges. The PVA sponges have implications in many viscoelastic soft tissues, including cartilage, liver, and kidney as an implant. However, a critical barrier to the use of the PVA sponge as tissue replacement material is a lack of sufficient study on its viscoelastic mechanical properties. In this study, the nonlinear mechanical behavior of a fabricated PVA sponge is investigated experimentally and computationally using relaxation and stress failure tests as well as finite element (FE) modeling. Hyperelastic strain energy density functions, such as Yeoh and Neo-Hookean, are used to capture the mechanical behavior of PVA sponge at ramp part, and viscoelastic model is used to describe the viscose behavior at hold part. Hyperelastic material constants are obtained and their general prediction ability is verified using FE simulations of PVA tensile experiments. The results of relaxation and stress failure tests revealed that Yeoh material model can define the mechanical behavior of PVA sponge properly compared with Neo-Hookean one. FE modeling results are also affirmed the appropriateness of Yeoh model to characterize the mechanical behavior of PVA sponge. Thus, the Yeoh model can be used in future biomechanical simulations of the spongy biomaterials. These results can be utilized to understand the viscoelastic behavior of PVA sponges and has implications for tissue engineering as scaffold.     

Influence of Extraction Method on the Rheological Properties of Jackfruit (<Emphasis Type="Italic">Artocarpus heterophyllus</Emphasis>) Seed Starch Dispersions

Starches are widely used in food processing, and their rheological properties are affected by thermal conditions imposed during the process. The objective of the present study was to assess the rheological behavior of jackfruit seed starch (JSS) dispersions, with a particular interest on the effects of the starch extraction techniques using either water or an alkali solution (0.1 M sodium hydroxide) as solvents. The analyses on the starches were performed using small amplitude oscillatory shear tests, including frequency, temperature, and time sweeps, and determining flow curves at different temperatures (10–40 °C). JSS dispersions were classified as weak-intermediary gels, and those prepared with JSS extracted with water showed higher values of G’ and G” in all of the tested samples. All gelatinized samples exhibited viscoelastic behavior, and with increasing temperatures the storage and loss moduli increased until a maximum value before reaching a plateau. Gelatinization temperatures were slightly lower for dispersions prepared with JSS extracted with water. Gelatinization kinetics showed that increasing temperatures caused a reduction in the time to achieve gelatinization. The JSS dispersions displayed thixotropic behavior, which was influenced by their concentration and the extraction method employed. The flow curves exhibited pseudoplastic behavior. The Power Law model was used to describe the JSS dispersions rheological behavior, which enabled to assess the effects of starch concentration and extraction method on their rheological properties.     

Elastic contributions dominate the viscoelastic properties of sputum from cystic fibrosis patients

Sputum samples from cystic fibrosis (CF) patients were investigated by oscillatory, creep and steady shear rheological techniques over a range of time scales from 10(-3) to 10(6) s. The viscoelastic changes obtained by mixing sputa with the actin-filament-severing protein gelsolin and with the thiol-reducing agent dithiothreitol (DTT) were also investigated. At small strains sputum behaves like a viscoelastic solid rather than a liquid. A nearly constant steady shear viscosity at low shear rates is only observed after long shearing times which cause irreversible changes in the samples. Creep-recovery tests confirm that sputa exhibit viscoelastic properties, with a significant elastic recovery. The results suggest that measurements of elastic moduli, rather than viscosities are more closely related to the mechanical properties of sputum in situ. Severing of actin filaments lowers the elastic modulus by 30-40%, but maintains viscoelastic integrity, while reduction of thiols in the glycoproteins nearly completely fluidizes the samples.     

Characterization of a hyper-viscoelastic phantom mimicking biological soft tissue using an abdominal pneumatic driver with magnetic resonance elastography (MRE)

The purpose of this study was to create a polymer phantom mimicking the mechanical properties of soft tissues using experimental tests and rheological models. Multifrequency Magnetic Resonance Elastography (MMRE) tests were performed on the present phantom with a pneumatic driver to characterize the viscoelastic (μ, η) properties using Voigt, Maxwell, Zener and Springpot models. To optimize the MMRE protocol, the driver behavior was analyzed with a vibrometer. Moreover, the hyperelastic properties of the phantom were determined using compressive tests and Mooney-Rivlin model. The range of frequency to be used with the round driver was found between 60 Hz and 100 Hz as it exhibits one type of vibration mode for the membrane. MRE analysis revealed an increase in the shear modulus with frequency reflecting the viscoelastic properties of the phantom showing similar characteristic of soft tissues. Rheological results demonstrated that Springpot model better revealed the viscoelastic properties (μ=3.45 kPa, η=6.17 Pas) of the phantom and the Mooney-Rivlin coefficients were C(10)=1.09.10(-2) MPa and C(01)=-8.96.10(-3) MPa corresponding to μ=3.95 kPa. These studies suggest that the phantom, mimicking soft tissue, could be used for preliminary MRE tests to identify the optimal parameters necessary for in vivo investigations. Further developments of the phantom may allow clinicians to more accurately mimic healthy and pathological soft tissues using MRE.