Local rheology of human neutrophils investigated using atomic force microscopy

During the immune response, neutrophils display localized mechanical events by interacting with their environment through the micro-vascular transit, trans-endothelial, and trans-epithelial migration. Nano-mechanical studies of human neutrophils on localized nano-domains could provide the essential information for understanding their immune responsive functions. Using the Atomic Force Microscopy (AFM)-based micro-rheology, we have investigated rheological properties of the adherent human neutrophils on local nano-domains. We have applied the modified Hertz model to obtain the viscoelastic moduli from the relatively thick body regions of the neutrophils. In addition, by using more advanced models to account for the substrate effects, we have successfully characterized the rheological properties of the thin leading and tail regions as well. We found a regional difference in the mechanical compliances of the adherent neutrophils. The central regions of neutrophils were significantly stiffer (1,548 ± 871 Pa) than the regions closer to the leading edge (686 ± 801 Pa), while the leading edge and the tail (494 ± 537 Pa) regions were mechanically indistinguishable. The frequency-dependent elastic and viscous moduli also display a similar regional difference. Over the studied frequency range (100 to 300 Hz), the complex viscoelastic moduli display the partial rubber plateau behavior where the elastic moduli are greater than the viscous moduli for a given frequency. The non-disparaging viscous modulus indicates that the neutrophils display a viscoelastic dynamic behavior rather than a perfect elastic behavior like polymer gels. In addition, we found no regional difference in the structural damping coefficient between the leading edge and the cell body. Thus, we conclude that despite the lower loss and storage moduli, the leading edges of the human neutrophils display partially elastic properties similar to the cell body. These results suggest that the lower elastic moduli in the leading edges are more favorable for the elastic fluctuation of actin filaments, which supports the polymerization of the actin filaments leading to the active protrusion during the immune response.     

Determination of viscoelastic moduli of arteries

A model for the arterial wall, motivated by wave propagation findings, is developed. The wall is taken to be a viscoelastic, orthotropic, prestressed shell which is materially characterized at any prestress level by ten incremental moduli. By using the momentum equations and the wave propagation characteristics for three axisymmetric modes, the ten moduli are found in terms of the three wave speeds, the three attenuation coefficients and the prestresses.     

Mechanical pulse wave propagation in gel, normal and oedematous tissues.

The velocity, attenuation and frequency content of the mechanical pulse wave propagation in gels of various water contents, in normal tissues from various sites and in oedematous tissues from different patients were investigated. The properties of the propagated pulse wave depend on the water content of the gel and the viscoelastic properties of the tissues. From the dependence of the pulse wave propagation velocity on elasticity, viscosity and density, information may be obtained concerning the effects of oedema on the mechanical properties of tissue.     

Evaluation of a three point pressure method for the determination of arterial transmission characteristics

The mathematical expressions required to analyse wave transmission characteristics (frequency dependent phase velocity, attenuation, and reflection) in arteries, under in vivo conditions, are summarized. In addition, a three point pressure method, which theoretically permits experimental determination of the propagation constant (phase velocity and attenuation), was tested under in vitro conditions. It is found to be a potentially powerful tool for in vivo studies if used with the appropriate constraints.     

Viscoelastic behavior and in vivo release study of microgel dispersions with inverse thermoreversible gelation

The dispersion of microgels with two interpenetrating polymer networks of poly( N-isopropylacrylamide) and poly(acrylic acid) (PNIPAM-IPN-PAAc) has been studied for its viscoelastic behavior, biocompatibility, and in vivo release properties. The IPN microgels in water had an average hydrodynamic radius of about 85 nm at 21 degrees C, measured by dynamic light scattering method. The atomic force microscope image showed that the particles were much smaller after they were dried but remained spherical shape. The storage and loss moduli ( G' and G') of dispersions of IPN microgels were measured in the linear stress regime as functions of temperature and frequency at various polymer concentrations using a stress-controlled rheometer. For dispersions with polymer concentrations of 3.0 and 6.0 wt % above 33 degrees C, the samples behave as viscoelastic solids and the storage modulus was larger than the loss modulus over the entire frequency range. The loss tangent was measured at various frequencies as a function of temperature. The gelation temperature was determined to be 33 degrees C at the point where a frequency-independent value of the loss tangent was first observed. At pH 2.5, when heated above the gelation temperature, IPN microgels flocculate by pumping a large amount of water from the gel. When the pH value was adjusted to neutral, deprotonation of -COOH groups on PAAc made the microgel keep water even above the gelation temperature. Using an animal implantation model, the biocompatibility and drug release properties of the IPN microgel dispersion were evaluated. Fluorescein as a model drug was mixed into an aqueous microgel dispersion at ambient temperature. This drug-loaded liquid was then injected subcutaneously in Balb/C mice from Taconic Farms. The test results have shown that the IPN microgels did not adversely promote foreign body reactions in this acute implantation model and the presence of gelled microgel dispersion substantially slowed the release of fluorescein.     

Material properties of the human lumbar facet joint capsule

The human facet joint capsule is one of the structures in the lumbar spine that constrains motions of vertebrae during global spine loading (e.g., physiological flexion). Computational models of the spine have not been able to include accurate nonlinear and viscoelastic material properties, as they have not previously been measured. Capsules were tested using a uniaxial ramp-hold protocol or a haversine displacement protocol using a commercially available materials testing device. Plane strain was measured optically. Capsules were tested both parallel and perpendicular to the dominant orientation of the collagen fibers in the capsules. Viscoelastic material properties were determined. Parallel to the dominant orientation of the collagen fibers, the complex modulus of elasticity was E*=1.63MPa, with a storage modulus of E'=1.25MPa and a loss modulus of: E" =0.39MPa. The mean stress relaxation rates for static and dynamic loading were best fit with first-order polynomials: B(epsilon) = 0.1110epsilon-0.0733 and B(epsilon)= -0.1249epsilon + 0.0190, respectively. Perpendicular to the collagen fiber orientation, the viscous and elastic secant moduli were 1.81 and 1.00 MPa, respectively. The mean stress relaxation rate for static loading was best fit with a first-order polynomial: B (epsilon) = -0.04epsilon - 0.06. Capsule strength parallel and perpendicular to collagen fiber orientation was 1.90 and 0.95 MPa, respectively, and extensibility was 0.65 and 0.60, respectively. Poisson's ratio parallel and perpendicular to fiber orientation was 0.299 and 0.488, respectively. The elasticity moduli were nonlinear and anisotropic, and capsule strength was larger aligned parallel to the collagen fibers. The phase lag between stress and strain increased with haversine frequency, but the storage modulus remained large relative to the complex modulus. The stress relaxation rate was strain dependent parallel to the collagen fibers, but was strain independent perpendicularly.     

Explicit finite element modelling of heel pad mechanics in running: inclusion of body dynamics and application of physiological impact loads

Many heel pathologies including plantar heel pain may result from micro tears/trauma in the subcutaneous tissues, in which internal tissue deformation/stresses within the heel pad play an important role. Previously, many finite element models have been proposed to evaluate stresses inside the heel pad, but the majority of these models only focus on static loading boundary conditions. This study explored a dynamics modelling approach to the heel pad subjected to realistic impact loads during running. In this model, the inertial property and action of the body are described by a lumped parameter model, while the heel/shoe interactions are modelled using a viscoelastic heel pad model with contact properties. The impact force pattern, dynamic heel pad deformation and stress states predicted by the model were compared with published experimental data. Further parametrical studies revealed the model responses, in terms of internal stresses in the skin and fatty tissue, change nonlinearly when body dynamics changes. A reduction in foot's touchdown velocity resulted in a less severe impact landing and stress relief inside the heel pad, for example peak von-Mises stress in fatty tissue, was reduced by 11.3%. Applications of the model may be extendable to perform iterative analyses to further understand the complex relationships between body dynamics and stress distributions in the soft tissue of heel pad during running. This may open new opportunities to study the mechanical aetiology of plantar heel pain in runners.     

Swelling and viscoelastic properties of osmotically stressed elastin

The swelling and viscoelastic properties of purified elastin were studied in aqueous solutions of superswelling agents or osmotic deswelling agents to develop models to study the behavior of elastin at frequencies not easily accessible by direct measurement. Increasing the concentration of any of the deswelling solutes (glucose, sucrose, sodium chloride, ammonium sulphate, dextran, and polyethylene glycol) increased the tensile storage and loss moduli. The viscoelastic behavior was independent of solute when compared on the basis of swelling behavior. The data collected at various solute concentrations at 37°C could be reduced to one master curve, and the master curves for elastin in each of the deswelling solutes were themselves superposable. The ability to reduce the data indicates that dehydration can be used to model elastin's viscoelastic behavior at high frequencies or over short times. The viscoelastic behavior of elastin in the superswelling agents [potassium thiocyanate (KSCN), dimethyl sulfoxide (DMSO), and ethylene glycol (EG)] depended on the solute and was independent of swelling behavior. In KSCN the behavior of elastin seemed to be a continuation of the pattern established by the deswelling agents in that an increase in swelling was accompanied by a decrease in both moduli, and the viscoelastic spectra were reducible to one master curve. In high concentrations of DMSO and EG the spectra were not reducible. KSCN appears a suitable superswelling solute to model elastin's viscoelastic behavior at low frequencies or over long times. © 1996 John Wiley & Sons, Inc.     

On the viscoelastic character of liver tissue: experiments and modelling of the linear behaviour

This paper presents the results of recent investigations into the viscoelastic character of liver tissue, especially the linear response. The linear domain of liver tissue has been determined by a strain sweep oscillation test. The fluid-like viscoelastic behaviour of liver tissue was confirmed by stress relaxation experiments. To demonstrate the consistency and quality of the experimental data, the relaxation spectrum was used to reconstruct the relaxation modulus, as well as the storage and loss moduli. The generalised Maxwell model was successfully employed to model the mechanical behaviour in the linear viscoelastic domain.     

A torsion pendulum for measurement of the viscoelasticity of biopolymers and its application to actin networks

This report describes the design, construction, and method of operation of a torsion pendulum which is specifically designed for the measurement of soft and fragile biopolymer gels. The pendulum can be assembled and employed in a standard biological laboratory and provides data that currently require access to specialized equipment usually limited to physics or material science laboratories. This instrument measures the shear moduli of viscoelastic materials by applying either steady or oscillating shear forces to a disc-shaped sample and measuring the resulting angular displacement of a pendulum attached to one face of the sample. The device is easily constructed using commercially available materials and no specialized machinery. Shear stresses as low as 0.03 Pa and shear rates as low as 0.00003 s-1 can be measured in steady shear experiments, and dynamic shear moduli from 1 to 2500 Pa measured by oscillatory measurements with sample volumes as low as 0.5 ml. The use of the torsion pendulum is illustrated by measuring the effects of two different actin binding proteins on the viscoelasticity of actin filament networks.     

A new method for measuring the yield stress in thin layers of sedimenting blood.

A new method is presented to describe the low shear rate behavior of blood. We observed the response of a thin layer of sedimenting blood to a graded shear stress in a wedge-shaped chamber. The method allows quantitation of the degree of phase separation between red cells and plasma, and extracts the yield stress of the cell phase as a function of hematocrit. Our studies showed that the behavior of normal human blood underwent a transition from a solid-like gel to a Casson fluid. This transition began at the Casson predicted yield stress. The viscoelastic properties of blood were examined at shear stresses below the yield stress. The measured Young's elastic moduli were in good agreement with published data. The yield stress of blood showed a linear dependence on hematocrit up to 60%, and increased more rapidly at higher hematocrit.     

Viscoelasticity of biological materials - measurement and practical impact on biomedicine

Mechanical behavior of biological structures under dynamic loading generally depends on elastic as well as viscous properties of biological materials. The significance of "viscous" parameters in real situations remains to be elucidated. Behavior of rheological models consisting of a combination of inertial body and two Voigt's bodies were described mathematically with respect to inverse problem solution, and behavior in impulse and harmonic loadings was analyzed. Samples of walls of porcine and human aorta thoracica in transverse direction and samples of human bone (caput femoris, substantia compacta) were measured. Deformation responses of human skin in vivo were also measured. Values of elastic moduli of porcine aorta walls were in the interval from 10(2)kPa to 10(3) kPa, values of viscous coefficients were in the interval from 10(2) Pa.s to 10(3) Pa.s. The value of shear stress moduli of human caput femoris, substantia compacta range from 52.7 to 161.1 MPa, and viscous coefficients were in the interval from 27.3 to 98.9 kPa.s. The role of viscous coefficients is significant for relatively high loading frequencies - in our materials above 8 Hz in aorta walls and 5 Hz for bones. In bones, the viscosity reduced maximum deformation corresponding to short rectangular stress.     

Critical stresses for cancer cell detachment in microchannels

We present experiments involving cancer cells adhering to microchannels, subjected to increasing shear stresses (0.1–30 Pa). Morphological studies were carried out at different shear stresses. Cells exhibit spreading patterns similar to those observed under static conditions, as long as the shear stress is not too high. At critical wall shear stresses (around 2−5 Pa), cell-substrate contact area decreases until detachment at the larger stresses. Critical shear stresses are found to be lower for higher confinements (i.e. smaller cell height to channel height ratio). Fluorescent techniques were used to locate focal adhesions (typically 1 μm² in size) under various shearing conditions, showing that cells increase the number of focal contacts in the region facing the flow. To analyze such data, we propose a model to determine the critical stress, resulting from the competition between hydrodynamic forces and the adhesive cell resistance. With this model, typical adhesive stresses exerted at each focal contact can be determined and are in agreement with previous works.     

Tissue 4

The finding that there are considerable tissue stresses (TSs)in the hypocotyl of Helianthus annuus L. prompts the question:how are the stresses generated? Here, a one-dimensional modelis formulated which, based on (i) symplastic, turgor-inducedextensions of tissues which differ in moduli of elasticity,and (ii) static equilibrium, predicts the occurrence of longitudinalTSs in stem-like organs, and gives their dependence on turgorpressure. To calculate the longitudinal forces which generatethe TSs in a stem, the moduli of elasticity of the tissues needto be known. The moduli were determined for uniaxial and multiaxialstresses for the outer tissue (OT) and the inner tissue (IT)of the hypocotyl. In the OT, the moduli were strongly dependenton applied uniaxial stress. The magnitudes of the calculatedlongitudinal forces (tensile and compressive) in the hypocotyl,were comparable to those measured. It follows that the TSs mayarise without differential growth of the tissues. Key words: Epidermis, model, moduli of elasticity, turgor, sunflower hypocotyl, tissue stresses     

Effect of vascular volume and edema on wave propagation in canine lungs

The velocities of longitudinal and transverse stress waves transmitted through inflated lung parenchyma depend on the lung stiffness, as defined by the bulk and shear moduli, and the lung density. We examined the relationship between stress wave velocities and lung density. A saline-filled reservoir was connected to the vessels of caudal dog lobes held inflated at 5 cmH2O transpulmonary pressure, and vascular volume and extravascular lung water were increased in steps by increasing vascular pressure. At each step, we measured the transmitted signals at locations 2 and 7 cm from an impulse surface distortion by means of microphones embedded in the lung surface. Longitudinal and transverse wave velocities were computed by using cross-correlation analysis of microphone signal pairs. Both wave velocities decreased as lung density increased: as a first approximation, wave velocities were inversely proportional to the square root of lung density. This behavior is consistent with the propagation of small-amplitude stress waves through an elastic continuum. Estimated bulk and shear moduli were 26 and 3.5 cmH2O, respectively, and were consistent with results from quasi-static deformation tests.     

Hydraulic input impedance measurements in physical models of the arterial wall

A white noise method was used to measure the hydraulic input impedance and transmission characteristics in physical models of an arterial system made of single, unbranched latex tubes. The experimentally obtained impedance curves show a rise in modulus and a positive phase at high frequencies in the absence of wave reflections. Using the impedance moduli in the presence of wave reflections, wave velocity and attenuation were calculated. The influence of wall nonlinearity on hydraulic impedance was also examined. It is concluded that, in the model used neither wave reflections nor wall nonlinearity can account for the deviations of the experimental impedance curves from the theoretically predicted ones. Impedance moduli in the presence of reflections may be used to study transmission characteristics (wave velocity and attenuation) of the model.     

Design and validation of a bioreactor for engineering vocal fold tissues under combined tensile and vibrational stresses

Criteria are outlined for the design of a bioreactor that can simulate the vibrational stresses in vocal fold movement during speech. Requirements are 0-1 mm amplitudes in the 20-200 Hz frequency range, a variable on-off stress regime, and maintenance of tissue viability over several days. The bioreactor uses dual drivers, one for low frequency (or static) strains, and another for high-frequencies vibrational strains. Response is linear at the driving end for an input of 0-5 V. The amplitude decreases linearly with frequency at constant input voltage, and the phase changes by nearly 180 degrees over the 20-200 Hz range. Human vocal fold fibroblasts were cultured in a polymer substrate and subjected to static and vibrational forces. The results indicate that vibratory strain alters the expression levels of many extracellular matrix-related genes, as well as the spatial distribution of cells and matrix.     

Viscoelastic properties and antimicrobial activity of cellulose fiber sheets impregnated with Ag nanoparticles

A silver nanoparticle colloid was prepared by a modified Tollens method using d-glucose as the reduction agent. The obtained nanoparticles were used for the modification of pine, linter and recycled cellulose fibers. Although the silver contents were relatively low (0.05-0.13wt.%), the cellulose-sheets prepared from the modified fibers show improved mechanical and viscoelastic properties. The tensile index (strength) increased with up to 30% in comparison to the index of the sheets obtained from the untreated fibers. The influence of the nanoparticles on the viscoelastic properties of the cellulose sheets was investigated by dynamic mechanical analysis (DMA) in the temperature range from -120 to 20°C and with a force frequency of 100Hz. A broad relaxation transition positioned at -80°C was observed in the loss modulus spectrum of all the cellulose sheets, while the Ag-modified sheets exhibited higher storage moduli values in the whole temperature range. The antimicrobial activity tests show that the pine, silver and recycled cellulose fiber sheets with silver nanoparticles can be successfully employed to prevent the viability and growth of the common pathogens Staphylococcus aureus, Escherichia coli and Candida albicans.