Mechanical properties of cultured human airway smooth muscle cells from 0.05 to 0.4 Hz.

We investigated the rheological properties of living human airway smooth muscle cells in culture and monitored the changes in rheological properties induced by exogenous stimuli. We oscillated small magnetic microbeads bound specifically to integrin receptors and computed the storage modulus (G') and loss modulus (G") from the applied torque and the resulting rotational motion of the beads as determined from their remanent magnetic field. Under baseline conditions, G' increased weakly with frequency, whereas G" was independent of the frequency. The cell was predominantly elastic, with the ratio of G" to G' (defined as eta) being approximately 0. 35 at all frequencies. G' and G" increased together after contractile activation and decreased together after deactivation, whereas eta remained unaltered in each case. Thus elastic and dissipative stresses were coupled during changes in contractile activation. G' and G" decreased with disruption of the actin fibers by cytochalasin D, but eta increased. These results imply that the mechanisms for frictional energy loss and elastic energy storage in the living cell are coupled and reside within the cytoskeleton.     

Microrheology of human lung epithelial cells measured by atomic force microscopy

Lung epithelial cells are subjected to large cyclic forces from breathing. However, their response to dynamic stresses is poorly defined. We measured the complex shear modulus (G(*)(omega)) of human alveolar (A549) and bronchial (BEAS-2B) epithelial cells over three frequency decades (0.1-100 Hz) and at different loading forces (0.1-0.9 nN) with atomic force microscopy. G(*)(omega) was computed by correcting force-indentation oscillatory data for the tip-cell contact geometry and for the hydrodynamic viscous drag. Both cell types displayed similar viscoelastic properties. The storage modulus G'(omega) increased with frequency following a power law with exponent approximately 0.2. The loss modulus G"(omega) was approximately 2/3 lower and increased similarly to G'(omega) up to approximately 10 Hz, but exhibited a steeper rise at higher frequencies. The cells showed a weak force dependence of G'(omega) and G"(omega). G(*)(omega) conformed to the power-law model with a structural damping coefficient of approximately 0.3, indicating a coupling of elastic and dissipative processes within the cell. Power-law behavior implies a continuum distribution of stress relaxation time constants. This complex dynamics is consistent with the rheology of soft glassy materials close to a glass transition, thereby suggesting that structural disorder and metastability may be fundamental features of cell architecture.     

Rheology of passive and adhesion-activated neutrophils probed by atomic force microscopy

The rheology of neutrophils in their passive and activated states plays a key role in determining their function in response to inflammatory stimuli. Atomic force microscopy was used to study neutrophil rheology by measuring the complex shear modulus G*(omega) of passive nonadhered rat neutrophils on poly(HEMA) and neutrophils activated through adhesion to glass. G*(omega) was measured over three frequency decades (0.1-102.4 Hz) by indenting the cells 500 nm with a spherical tip and then applying a 50-nm amplitude multi-frequency signal. G*(omega) of both passive and adhered neutrophils increased as a power law with frequency, with a coupling between elastic (G') and loss (G') moduli. For passive neutrophils at 1.6 Hz, G' = 380 +/- 121 Pa, whereas G' was fourfold smaller and the power law coefficient was of x = 1.184. Adhered neutrophils were over twofold stiffer with a lower slope (x = 1.148). This behavior was adequately described by the power law structural damping model but not by liquid droplet and Kelvin models. The increase in stiffness with frequency may modulate neutrophil transit, arrest, and transmigration in vascular microcirculation.     

Rheological evidence of the gelation behavior of hyaluronan-gellan mixtures

It was found that solutions of calcium hyaluronate (CaHA) (0.1 to approximately 0.5 wt%) could form a gel by mixing with solutions of sodium type gellan (0.1 to approximately 0.5 wt%), although neither polymer by itself forms a gel at low concentrations (0.1 to approximately 0.5 wt% in this experiment). The rheological properties of CaHA-gellan mixtures were investigated by dynamic and steady shear measurements. Both storage shear modulus G' and loss shear modulus G' for CaHA-gellan mixtures increased with increasing time, and tended to an equilibrium value after 1 h. After reaching steady values of G' and G", the frequency dependence of G' and G' was observed. G' was always larger than G' in the accessible frequency range from 10(-2) to 10(2) rad/s. The effects of pH and calcium ions were examined. Gel formation of the mixtures was promoted by decreasing pH and adding from 0.01 to 0.1 M calcium ions, but excessive calcium ions weakened the gel.     

Viscoelasticity of human alveolar epithelial cells subjected to stretch

Alveolar epithelial cells undergo stretching during breathing and mechanical ventilation. Stretch can modify cell viscoelastic properties, which may compromise the balance of forces in the alveolar epithelium. We studied the viscoelasticity of alveolar epithelial cells (A549) subjected to equibiaxial distention with a novel experimental approach. Cells were cultured on flexible substrates and subjected to stepwise deformations of up to 17% with a device built on an inverted microscope. Simultaneously, cell storage (G') and loss (G') moduli were measured (0.1-100 Hz) with optical magnetic twisting cytometry. G' and G' increased with strain up to 64 and 30%, respectively, resulting in a decrease in G'/G' (15%). This stretch-induced response was inhibited by disruption of the actin cytoskeleton with latrunculin A. G' increased with frequency following a power law with exponent alpha = 0.197. G' increased proportionally to G' but exhibited a more marked frequency dependence at high frequencies. Stretching (14%) caused a fall in alpha (13%). At high stretching amplitudes, actual cell strain (14.4%) was lower than the applied substrate strain (17.3%), which could indicate a partial cell detachment. These data suggest that cytoskeletal prestress modulates the elastic and frictional properties of alveolar epithelial cells in a coupled manner, according to soft glassy rheology. Stretch-induced cell stiffening could compromise the balance of forces at the cell-cell and cell-matrix adhesions.     

Measurement of cell microrheology by magnetic twisting cytometry with frequency domain demodulation.

Magnetic twisting cytometry (MTC) (Wang N, Butler JP, and Ingber DE, Science 260: 1124-1127, 1993) is a useful technique for probing cell micromechanics. The technique is based on twisting ligand-coated magnetic microbeads bound to membrane receptors and measuring the resulting bead rotation with a magnetometer. Owing to the low signal-to-noise ratio, however, the magnetic signal must be modulated, which is accomplished by spinning the sample at approximately 10 Hz. Present demodulation approaches limit the MTC range to frequencies <0.5 Hz. We propose a novel demodulation algorithm to expand the frequency range of MTC measurements to higher frequencies. The algorithm is based on coherent demodulation in the frequency domain, and its frequency range is limited only by the dynamic response of the magnetometer. Using the new algorithm, we measured the complex modulus of elasticity (G*) of cultured human bronchial epithelial cells (BEAS-2B) from 0.03 to 16 Hz. Cells were cultured in supplemented RPMI medium, and ferromagnetic beads (approximately 5 microm) coated with an RGD peptide were bound to the cell membrane. Both the storage (G', real part of G*) and loss (G", imaginary part of G*) moduli increased with frequency as omega(alpha) (2 pi x frequency) with alpha approximately equal to 1/4. The ratio G"/G' was approximately 0.5 and varied little with frequency. Thus the cells exhibited a predominantly elastic behavior with a weak power law of frequency and a nearly constant proportion of elastic vs. frictional stresses, implying that the mechanical behavior conformed to the so-called structural damping (or constant-phase) law (Maksym GN, Fabry B, Butler JP, Navajas D, Tschumperlin DJ, LaPorte JD, and Fredberg JJ, J Appl Physiol 89: 1619-1632, 2000). We conclude that frequency domain demodulation dramatically increases the frequency range that can be probed with MTC and reveals that the mechanics of these cells conforms to constant-phase behavior over a range of frequencies approaching three decades.     

Mechanical properties of actin filament networks depend on preparation, polymerization conditions, and storage of actin monomers.

This study investigates possible sources for the variance of more than two orders of magnitude in the published values for the shear moduli of purified actin filaments. Two types of forced oscillatory rheometers used in some of our previous work agree within a factor of three for identical samples. Polymers assembled in EGTA and Mg2+ from fresh, gel-filtered ATP-actin at 1 mg/ml typically have an elastic storage modulus (G') of approximately 1 Pa at a deformation frequency of 0.1-1 Hz. G' is slightly higher when actin is polymerized in KCl with Ca2+ and Mg2+. Gel filtration removes minor contaminants from actin but has little effect on G' for most preparations of actin from acetone powder. Storage of actin monomers without frequent changes of buffer containing fresh ATP and dithiothreitol can result in changes that increase the G' of filaments by more than a factor of 10. Frozen storage can preserve the properties of monomeric actin, but care is necessary to prevent protein denaturation or aggregation due to freezing or thawing.     

Alpha and beta mechanical dispersions in high sugar/acyl gellan mixtures

The real (G') and imaginary (G") components of the complex modulus have been measured between 0.1 and 100 rad/s in the temperature range of 70--55 degrees C for a mixture of 1% high acyl gellan with 79% glucose syrup, and 79% glucose syrup. The method of reduced variables gave superposed curves of G' and G" as a function of timescale of measurement, which matched the thermal profiles of shear modulus obtained by scanning at the constant rate of 1 degrees C/min. Data of the gellan/co-solute mixture could be analysed in terms of two distinct mechanisms. For the alpha dispersion, G' and G" superposed with the horizontal reduction factor a(T) whose temperature dependence followed an equation of the Williams-Landel-Ferry form. Mechanical spectra of the beta dispersion also superposed with the factor a(T) whose temperature dependence, however, corresponded to a constant energy of activation. Relaxation spectra have been calculated for both dispersions. Those for the alpha mechanism were attributed to the chain backbone motions and the friction coefficient per tetrasaccharide repeat unit in backbone motion was calculated from the extended Rouse theory. When the contribution of the solvent alone was studied, no spectra for the beta dispersion were observed supporting the hypothesis of the dispersion being attributed to the side-chain motions of the acyl groups. The spectra of the beta mechanism were relatively broader than for the alpha dispersion. The relative location of the beta dispersion on the time scale or temperature range was found to be between the alpha dispersion (glass transition region) and the glassy state.     

Disentangling alpha from beta mechanical relaxations in the rubber-to-glass transition of high-sugar-chitosan mixtures

The occurrence of molecular motions in addition to those of the glass-transition region (alpha mechanism) were investigated in chitosan and a branched derivative substituted with alkyl chains having eight carbon atoms. Once hydrophobic interactions of the alkyl groups in aqueous solution were demonstrated, polymers were mixed with glucose syrup at high levels of solids. The real (G') and imaginary (G") components of the complex dynamic modulus in high-solid mixtures were measured between 0.1 and 100 rad s(-1) in the temperature range from -55 to 50 degrees C. The method of reduced variables gave superposed curves of G' and G", which unveiled an anomaly in the dispersion of the alkylated derivative both in terms of higher modulus values and dominant elastic component of the polymeric network, as compared with the glass-transition region of chitosan. It was proposed that the new mechanical feature was due to beta mechanism, and master curves of viscoelastic functions and relaxation processes were constructed to rationalize it.     

Study of the release mechanism of diltiazem hydrochloride from matrices based on chitosan-alginate and chitosan-carrageenan mixtures

The aim of this work was to establish the diltiazem hydrochloride release mechanism from the chitosan-alginate matrix tablet (MCB/AS) and chitosan-carrageenan matrix tablet (MCS/CSI). The weight loss for MCS/CSI is mainly due to the weight loss of the matrix while for MCB/AS it is mainly due to the diltiazem hydrochloride released from the tablet. Using the Peppa's model the release order for MCS/CSI was n = 1.07 +/- 0.13 and for MCB/AS was n = 0.76 +/- 0.02. Thus, MCS/CSI has a transport mechanism, and for MCB/AS the drug release mechanism is a combined process of diffusion and relaxation. MCB/AS has an elastic modulus (G' = 10(5) Pa) one order of magnitude higher than MCS/CSI (G' = 10(4) Pa). MCB/AS is able to uptake solvent without disrupting the microstructure due to its high elastic modulus. Instead MCS/CSI showed a quick erosion process, which conducted to the tablet disintegration due to a fast solvent uptake process.     

Selected contribution: time course and heterogeneity of contractile responses in cultured human airway smooth muscle cells.

We measured the time course and heterogeneity of responses to contractile and relaxing agonists in individual human airway smooth muscle (HASM) cells in culture. To this end, we developed a microrheometer based on magnetic twisting cytometry adapted with a novel optical detection system. Ferromagnetic beads (4.5 microm) coated with Arg-Gly-Asp peptide were bound to integrins on the cell surface. The beads were twisted in a sinusoidally varying magnetic field at 0.75 Hz. Oscillatory bead displacements were recorded using a phase-synchronized video camera. The storage modulus (cell stiffness; G'), loss modulus (friction; G"), and hysteresivity (eta; ratio of G" to G') could be determined with a time resolution of 1.3 s. Within 5 s after addition of histamine (100 microM), G' increased by 2.2-fold, G" increased by 3.0-fold, and eta increased transiently from 0.27 to 0.34. By 20 s, eta decreased to 0.25, whereas G' and G" remained above baseline. Comparable results were obtained with bradykinin (1 microM). These changes in G', G", and eta measured in cells were similar to but smaller than those reported for intact muscle strips. When we ablated baseline tone by adding the relaxing agonist dibutyryl cAMP (1 mM), G' decreased within 5 min by 3.3-fold. With relaxing and contracting agonists, G' could be manipulated through a contractile range of 7.3-fold. Cell populations exhibited a log-normal distribution of baseline stiffness (geometric SD = 2.8) and a heterogeneous response to both contractile and relaxing agonists, partly attributable to variability of baseline tone between cells. The total contractile range of the cells (from maximally relaxed to maximally stimulated), however, was independent of baseline stiffness. We conclude that HASM cells in culture exhibit a clear, although heterogeneous, response to contractile and relaxing agonists and express the essential mechanical features characteristic of the contractile response observed at the tissue level.     

A covalently cross-linked gel derived from the epidermis of the pilot whale Globicephala melas

The rheological properties of the stratum corneum of the pilot whale (Globicephala melas) were investigated with emphasis on their significance to the self-cleaning abilities of the skin surface smoothed by a jelly material enriched with various hydrolytic enzymes. The gel formation of the collected fluid was monitored by applying periodic-harmonic oscillating loads using a stress-controlled rheometer. In the mechanical spectrum of the gel, the plateau region of the storage modulus G' (<1200 Pa) and the loss modulus G" (>120 Pa) were independent of frequency (omega = 43.98 to 0.13 rad x s(-1), tau = 15 Pa, T = 20 degrees C), indicating high elastic performance of a covalently cross-linked viscoelastic solid. In addition, multi-angle laser light scattering experiments (MALLS) were performed to analyse the potential time-dependent changes in the weight-average molar mass of the samples. The observed increase showed that the gel formation is based on the assembly of covalently cross-linked aggregates. The viscoelastic properties and the shear resistance of the gel assure that the enzyme-containing jelly material smoothing the skin surface is not removed from the stratum corneum by shear regimes during dolphin jumping. The even skin surface is considered to be most important for the self-cleaning abilities of the dolphin skin against biofouling.     

Thrombin and histamine induce stiffening of alveolar epithelial cells.

The mechanical properties of alveolar epithelial cells play a central role in maintaining the physical integrity of the alveolar epithelium. We studied the viscoelastic properties of alveolar epithelial cells (A549) in response to thrombin and histamine with optical magnetic twisting cytometry. Ferrimagnetic beads coated with Arg-Gly-Asp (RGD)-peptide or acetylated low-density lipoprotein were bound to cell surface receptors and subsequently twisted in an oscillatory magnetic field (0.1-100 Hz). The cell storage (G') and loss (G') moduli were computed from twisting torque and bead displacement. In measurements with RGD-coated beads, thrombin (0.5 U/ml) induced a rapid and sustained threefold increase in G' and G' at approximately 100 s after challenge. Histamine (100 microM) induced a rapid but transient twofold increase in G' and G' with maximum values 60 s after challenge. Posttreatment with cytochalasin D abolished thrombin-induced cell stiffening. G' increased with frequency following a power law with exponent 0.214. G' increased proportionally to G' up to 10 Hz but showed a steeper rise at higher frequencies. Thrombin caused a fall in the power-law exponent (0.164). In measurements with acetylated low-density lipoprotein-coated beads, minor changes (<20%) were observed in G' and G' after the addition of thrombin and histamine. F-actin staining revealed that thrombin and histamine induced a profound reorganization of the actin cytoskeleton at the cell periphery and formation of actin bundles. In the mechanically dynamic environment of the lung, cell stiffening induced by thrombin and histamine increases centripetal tension, which could contribute to alveolar barrier dysfunction.     

Designing polyHEMA substrates that mimic the viscoelastic response of soft tissue

Matching the mechanical properties of a biomaterial to soft tissue is often overlooked despite the fact that it is well known that cells respond to and are capable of changing their mechanical environment. In this paper, we used NaCl and alginate beads as porogens to make a series of micro- and macro-porous pHEMA substrates (poly(2-hydroxyethly methacrylate)) and quantified their mechanical behavior under low-magnitude shear loads over physiologically relevant frequencies. Using a stress-controlled rheometer, we performed isothermal (37°C) frequency response experiments between 0.628 and 75.4rad/s (0.01-12Hz) at 0.1% strain. Both micro- and macro-porous pHEMA substrates were predominately elastic in nature with a narrow range of G' and G″ values that mimicked the response of human skin. The magnitude of the G' and G″ values of the macro-porous substrates were designed to closely match human skin. To determine how cell growth might alter their mechanical properties, pHEMA substrates were functionalized and human skin fibroblasts grown on them for fourteen days. As a result of cell growth, the magnitude of G' and G″ increased at low frequencies while also altering the degree of high frequency dependence, indicating that cellular interactions with the micro-pore infrastructure has a profound effect on the viscoelastic behavior of the substrates. These data could be fit to a mathematical model describing a soft-solid. A quantitative understanding of the mechanical behavior of biomaterials in regimes that are physiologically relevant and how these mechanics may change after implantation may aid in the design of new materials.     

Measurements of mechanical properties of the blastula wall reveal which hypothesized mechanisms of primary invagination are physically plausible in the sea urchin Strongylocentrotus purpuratus.

Computer simulations showed that the elastic modulus of the cell layer relative to the elastic modulus of the extracellular layers predicted the effectiveness of different force-generating mechanisms for sea urchin primary invagination [L. A. Davidson, M. A. R. Koehl, R. Keller, and G. F. Oster (1995) Development 121, 2005-2018]. Here, we measured the composite elastic modulus of the cellular and extracellular matrix layers in the blastula wall of Strongylocentrotus purpuratus embryos at the mesenchyme blastula stage. Combined, these two layers exhibit a viscoelastic response with an initial stiffness ranging from 600 to 2300 Pa. To identify the cellular structures responsible for this stiffness we disrupted these structures and correlated the resulting lesions to changes in the elastic modulus. We treated embryos with cytochalasin D to disrupt the actin-based cytoskeleton, nocodazole to disrupt the microtubule-based cytoskeleton, and a gentle glycine extraction to disrupt the apical extracellular matrix (ECM). Embryos treated less than 60 min in cytochalasin D showed no change in their time-dependent elastic modulus even though F-actin was severely disrupted. Similarly, nocodazole had no effect on the elastic modulus even as the microtubules were severely disrupted. However, glycine extraction resulted in a 40 to 50% decrease in the elastic modulus along with a dramatic reduction in the hyalin protein at the apical ECM, thus implicating the apical ECM as a major mechanical component of the blastula wall. This finding bears on the mechanical plausibility of several models for primary invagination.     

The role of prestress and architecture of the cytoskeleton and deformability of cytoskeletal filaments in mechanics of adherent cells: a quantitative analysis.

Mechanical properties of adherent cells were investigated using methods of engineering mechanics. The cytoskeleton (CSK) was modeled as a filamentous network and key mechanisms and corresponding molecular structures which determine cell elastic behavior were identified. Three models of the CSK were considered: open-cell foam networks, prestressed cable nets, and a tensegrity model as a special case of the latter. For each model, the modulus of elasticity (i.e. an index of resistance to small deformation) was given as a function of mechanical and geometrical properties of CSK filaments whose values were determined from the data in the literature. Quantitative predictions for the elastic modulus were compared with data obtained previously from mechanical tests on adherent cells. The open-cell foam model yielded the elastic modulus (10(3)-10(4)Pa) which was consistent with measurements which apply a large compressive stress to the cell. This suggests that bending of CSK filaments is the key mechanism for resisting large compression. The prestressed cable net and tensegrity model yielded much lower elastic moduli (10(1)-10(2)Pa) which were consistent with values determined from equilibrium measurements at low applied stress. This suggests that CSK prestress and architecture are the primary determinants of the cell elastic response. The tensegrity model revealed the possibility that buckling of microtubules of the CSK also contributed to cell elasticity.     

Structural changes during heat-induced gelation of globular protein dispersions

Macroscopic and molecular structural changes during heat-induced gelation of beta-lactoglobulin, bovine serum albumin, ovalbumin, and alpha-lactalbumin aqueous dispersions were probed by the mechanical and CD spectroscopy, respectively. Aqueous solutions of the native globular proteins, except for alpha-lactalbumin, exhibited solid-like mechanical spectra-namely, the predominant storage modulus G' over the loss modulus G" in the entire frequency range examined (0.1-100 rad/s), suggesting that these protein solutions were highly structured even before gelation, possibly due to strong repulsions among protein molecules. Such solid-like structures were susceptible to nonlinearly large shear but recovered almost immediately at rest. During gelation by isothermal heating, major changes in the secondary structure of the globular proteins completed within a few minutes, while values of the modulus continued to develop for hours with maintaining values of tandelta (= G"/G') less than unity. As a result, a conventional criterion for mechanically defining the gelation point, such as a crossover between G' and G", was inapplicable to these globular protein systems. beta-Lactoglobulin gels that had passed the gelation point satisfied power laws (G' approximately G" approximately omega(n)) believed to be valid only at the gelation point, suggesting that fractal gel networks, similar to those of critical gels (i.e., gels at the gelation point), were formed.     

The elastic modulus of fibrin clots and fibrinogen gels: the effect of fibronectin and dithiothreitol

The elastic modulus (G') of factor XIIIa induced fibrinogen gels was found to be substantially lower than the G' of fibrin gels that were formed by clotting fibrinogen with thrombin. The addition of fibronectin and/or the reducing reagent dithiothreitol (DTT) to the factor XIIIa coagulation mixture led to the formation of a weaker gel structure, while the rigidity of thrombin induced clots was not appreciably affected by the inclusion of the DTT but increased somewhat in the presence of fibronectin. The reasons for the differing clot rigidities are discussed in terms of biochemical mechanisms.     

Effects of monoamine neuromediators on the growth-related variables of <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12

The monoamine neuromediators serotonin (5-HT), histamine, dopamine (DA), and norepinephrine (NE), added to an Escherichia coli K-12 strain MC 4100 culture upon inoculation, stimulate cell proliferation (determined from CFU formation) and biomass accumulation (monitored nephelometrically) during the late lag phase and the early exponential growth phase. These effects are less significant in the late exponential and stationary phase cultures. According to the concentration dependence of the stimulatory effects, the neuromediators can be classified into two groups: (i) the catecholamines DA and NE, whose effects increase almost linearly with increasing concentrations within the range of 0.1–100 μM, and (ii) histamine and 5-HT, which are characterized by bell-shaped concentration dependence curves with maxima at 0.1 (histamine) and 1 μM (5-HT). On an agar-containing medium, the growing E. coli population includes solitary cells and compact cell groups (microcolonies). In this system, both tested catecholamines exert a relatively weak stimulatory influence that manifests itself as an increase in the number of both solitary cells and cell groups, and occurs at concentrations of 10 μM and higher. In analogy to the culture grown on the liquid medium, 5-HT and histamine are distinguished by nonlinear concentration dependence curves: their effects peak at 0.1 μM (histamine) or 1 μM (5-HT); an increase in the neuromediator concentrations results in a decrease in effects that are enhanced by further increasing the concentrations to the submillimolar range. DA increases the percentage of solitary cells, whereas the other tested amines promote cell group formation. The results are interpreted in terms of specific (probably receptor-dependent) mechanisms of action in the neuromediators involved.