Oscillatory shear alters endothelial hydraulic conductivity and nitric oxide levels

This study addresses the role of nitric oxide (NO) and downstream signaling pathways in mediating the influences of oscillatory shear stress on the hydraulic conductivity (L(p)) of bovine aortic endothelial cell (BAEC) monolayers. Exposure of BAEC monolayers to 20 dyne/cm2 steady shear stress for 3 h induced a 3.3-fold increase in L(p). When an oscillatory shear amplitude of 10 dyne/cm2 was superimposed on a steady shear of 10 dyne/cm2 to produce a non-reversing oscillatory shear pattern (10+/-10 dyne/cm2), L(p) increased by 3.0-fold within 90 min. When the amplitude was increased to 15 dyne/cm2, resulting in a reversing oscillatory shear pattern (10+/-15 dyne/cm2), the increase in L(p) over 3 h was completely suppressed. Twenty and 10+/-10 dyne/cm2 induced 2.9- and 2.6-fold increases in NO production above non-sheared controls, respectively, whereas 10+/-15 dyne/cm2 stimulated a 14-fold increase in NO production. The inhibition of L(p) with reversing oscillatory shear may be associated with alterations in cyclic guanosine monophosphate (cGMP) production downstream of NO which is up-regulated by reversing oscillatory shear, but is unaffected by steady shear.     

The mechanics of F-actin microenvironments depend on the chemistry of probing surfaces

To understand the microscopic mechanical properties of actin networks, we monitor the motion of embedded particles with controlled surface properties. The highly resolved Brownian motions of these particles reveal the viscoelastic character of the microenvironments around them. In both non-cross-linked and highly cross-linked actin networks, particles that bind F-actin report viscoelastic moduli comparable to those determined by macroscopic rheology experiments. By contrast, particles modified to prevent actin binding have weak microenvironments that are surprisingly insensitive to the introduction of filament cross-links. Even when adjacent in the same cross-linked gel, actin-binding and nonbinding particles report viscoelastic moduli that differ by two orders of magnitude at low frequencies (0.5-1.5 rad/s) but converge at high frequencies (> 10(4) rad/s). For all particle chemistries, electron and light microscopies show no F-actin recruitment or depletion, so F-actin microheterogeneities cannot explain the deep penetration (approximately 100 nm) of nonbinding particles. Instead, we hypothesize that a local depletion of cross-linking around nonbinding particles explains the phenomena. With implications for organelle mobility in cells, our results show that actin binding is required for microenvironments to reflect macroscopic properties, and conversely, releasing actin enhances particle mobility beyond the effects of mere biochemical untethering.     

Localized activation of m-calpain in migrating human umbilical vein endothelial cells stimulated by shear stress

Using a parallel-plate flow-chamber and confocal laser scanning microscopy (CLSM), we studied the mode of cytoskeletal reorganization in migrating HUVECs stimulated by shear stress. Activation of m-calpain associated with a change in the spatial distribution of cytoplasmic ionized Ca2+ concentration ([Ca2+](i)) was studied. Shear stress (10 dyne/cm(2)) caused migration and decrease in the F-actin content of HUVECs. Migrating individual HUVECs showed the lamellipodium formed in the direction of cell migration, in which [Ca2+](i) elevated to 148 +/- 12 nM in a localized fashion. We found the appearance of activated m-calpain in the local area of the migrating HUVECs, which was associated with a decrease in the amounts of pp125FAK and ezrin. The localized rise in [Ca2+](i) might be closely related to morphological change to regulate the direction of cell migration induced by shear stress through localized activation of m-calpain.     

Local measurements of viscoelastic moduli of entangled actin networks using an oscillating magnetic bead micro-rheometer.

A magnetically driven bead micro-rheometer for local quantitative measurements of the viscoelastic moduli in soft macromolecular networks such as an entangled F-actin solution is described. The viscoelastic response of paramagnetic latex beads to external magnetic forces is analyzed by optical particle tracking and fast image processing. Several modes of operation are possible, including analysis of bead motion after pulse-like or oscillatory excitations, or after application of a constant force. The frequency dependencies of the storage modulus, G'(omega), and the loss modulus, G'(omega), were measured for frequencies from 10(-1) Hz to 5 Hz. For low actin concentrations (mesh sizes epsilon > 0.1 micron) we found that both G'(omega) and G'(omega) scale with omega 1/2. This scaling law and the absolute values of G' and G' agree with conventional rheological measurements, demonstrating that the magnetic bead micro-rheometer allows quantitative measurements of the viscoelastic moduli. Local variations of the viscoelastic moduli (and thus of the network density and mesh size) can be probed in several ways: 1) by measurement of G' and G' at different sites within the network; 2) by the simultaneous analysis of several embedded beads; and 3) by evaluation of the bead trajectories over macroscopic distances. The latter mode yields absolute values and local fluctuations of the apparent viscosity eta(x) of the network.     

Ultrastructural study of periodic lamellar granules in human neutrophils

Granules consisting of periodically arranged membranous lamellae and amorphous electron-opaque material, i.e., periodic lamellar granules, are present in human neutrophils. To date, no extensive ultrastructural studies have been carried out on these granules because of their infrequent presence in neutrophils. The bone marrow of 18 cases of chronic myeloproliferative disorders, including one case of chronic neutrophilic leukemia in which periodic lamellar granules were frequently seen in neutrophils, was investigated by electron microscopy. Periodic lamellar granules were seen in neutrophils in 12 of the 18 cases at varying frequencies. They were preferentially seen in immature neutrophils. The transverse profiles of these granules revealed concentric complete/incomplete rings or periodic parallel straight lines, i.e., various patterns of lamellar arrangement were present. Periodic lamellar granules were positive for myeloperoxidase and lysozyme at the electron-microscopic level. These results suggest that these granules represent a primary neutrophil granule subtype. However, their functional and pathologic significance remains unknown.     

Diffusing wave spectroscopy microrheology of actin filament networks.

Filamentous actin (F-actin), one of the constituents of the cytoskeleton, is believed to be the most important participant in the motion and mechanical integrity of eukaryotic cells. Traditionally, the viscoelastic moduli of F-actin networks have been measured by imposing a small mechanical strain and quantifying the resulting stress. The magnitude of the viscoelastic moduli, their concentration dependence and strain dependence, as well as the viscoelastic nature (solid-like or liquid-like) of networks of uncross-linked F-actin, have been the subjects of debate. Although this paper helps to resolve the debate and establishes the extent of the linear regime of F-actin networks' rheology, we report novel measurements of the high-frequency behavior of networks of F-actin, using a noninvasive light-scattering based technique, diffusing wave spectroscopy (DWS). Because no external strain is applied, our optical assay generates measurements of the mechanical properties of F-actin networks that avoid many ambiguities inherent in mechanical measurements. We observe that the elastic modulus has a small magnitude, no strain dependence, and a weak concentration dependence. Therefore, F-actin alone is not sufficient to generate the elastic modulus necessary to sustain the structural rigidity of most cells or support new cellular protrusions. Unlike previous studies, our measurements show that the mechanical properties of F-actin are highly dependent on the frequency content of the deformation. We show that the loss modulus unexpectedly dominates the elastic modulus at high frequencies, which are key for fast transitions. Finally, the measured mean square displacement of the optical probes, which is also generated by DWS measurements, offers new insight into the local bending fluctuations of the individual actin filaments and shows how they generate enhanced dissipation at short time scales.     

ULTRASTRUCTURE OF CARPOSPOROGENESIS IN THE PARASITIC RED ALGA FAUCHEOCOLAX ATTENUATA SETCH. (RHODYMENIALES,RHODYMENIACEAE)

Carpospore differentiation in Faucheocolax attenuata Setch. can be separated into three developmental stages. Immediately after cleaving from the multinucleate gonimoblast cell, young carpospores are embedded within confluent mucilage produced by gonimoblast cells. These carpospores contain a large nucleus, few starch grains, concentric lamellae, as well as proplastids with a peripheral thylakoid and occasionally some internal (photosynthetic) thylakoids. Proplastids also contain concentric lamellar bodies. Mucilage with a reticulate fibrous substructure is formed within cytoplasmic concentric membranes, thus giving rise to mucilage sacs. Subsequently, these mucilage sacs release their contents, forming an initial reticulate deposition of carpospore wall material. Dictyosome vesicles with large, single dark-staining granules also contribute to wall formation and may create a separating layer between the mucilage and carpospore wall. During the latter stages of young carpospores, starch is polymerized in the perinuclear cytoplasmic area and is in close contact with endoplasmic reticulum. Intermediate-aged carpospores continue their starch polymerization. Dictyosomes deposit more wall material, in addition to forming fibrous vacuoles. Proplastids form thylakoids from concentric lamellar bodies. Mature carpospores are surrounded by a two-layered carpospore wall. Cytoplasmic constituents include large floridean starch granules, peripheral fibrous vacuoles, mature chloroplasts and curved dictyosomes that produce cored vesicles which in turn are transformed into adhesive vesicles. Pit connections remain intact between carpospores but begin to degenerate. This degeneration appears to be mediated by microtubules.     

Single lamellar mechanics of the human lumbar anulus fibrosus

The mechanical behavior of the entire anulus fibrosus is determined essentially by the tensile properties of its lamellae, their fiber orientations, and the regional variation of these quantities. Corresponding data are rare in the literature. The paper deals with an in vitro study of single lamellar anulus lamellae and aims to determine (i) their tensile response and regional variation, and (ii) the orientation of lamellar collagen fibers and their regional variation. Fresh human body-disc-body units (L1–L2, n=11) from cadavers were cut midsagittally producing two hemidisc units. One hemidisc was used for the preparation of single lamellar anulus specimens for tensile testing, while the other one was used for the investigation of the lamellar fiber orientation. Single lamellar anulus specimens with adjacent bone fragments were isolated from four anatomical regions: superficial and deep lamellae (3.9±0.21 mm, mean ± SD, apart from the outer boundary surface of the anulus fibrosus) at ventro-lateral and dorsal positions. The specimens underwent cyclic uniaxial tensile tests at three different strain rates in 0.15 mol/l NaCl solution at 37°C, whereby the lamellar fiber direction was aligned with the load axis. For the characterization of the tensile behavior three moduli were calculated: E_low (0–0.1 MPa), E_medium (0.1–0.5 MPa) and E_high (0.5–1 MPa). Additionally, specimens were tested with the load axis transverse to the fiber direction. From the second hemidisc fiber angles with respect to the horizontal plane were determined photogrammetrically from images taken at six circumferential positions from ventral to dorsal and at three depth levels. Tensile moduli along the fiber direction were in the range of 28–78 MPa (regional mean values). Superficial lamellae have larger E_medium (p=0.017) and E_high (p=0.012) than internal lamellae, and the mean value of superficial lamellae is about three times higher than that of deep lamellae. Tensile moduli of ventro-lateral lamellae do not differ significantly from the tensile moduli of dorsal lamellae, and E_low is generally indifferent with respect to the anatomical region. Tensile moduli transverse to the fiber direction were about two orders of magnitude smaller (0.22±0.2 MPa, mean ± SD, n=5). Tensile properties are not correlated significantly with donor age. Only small viscoelastic effects were observed. The regional variation of lamellar fiber angle is described appropriately by a regression line ||=23.2+0.130× (r²=0.55, p<0.001), where is the polar angle associated with the circumferential position. The single anulus lamella may be seen as the elementary structural unit of the anulus fibrosus, and exhibits marked anisotropy and distinct regional variation of tensile properties and fiber angles. These features must be considered for appropriate physical and numerical modeling of the anulus fibrosus.     

Assessment of composition and anisotropic elastic properties of secondary osteon lamellae

Measurement of the elastic properties of single osteon lamellae is still one of the most demanding tasks in bone mechanics to be solved. By means of site-matched Raman microspectroscopy, acoustic microscopy and nanoindentation the structure, chemical composition and anisotropic elasticity of individual lamellae in secondary osteons were investigated. Acoustic impedance images (911-MHz) and two-dimensional Raman spectra were acquired in sections of human femoral bone. The samples were prepared with orientations at various observation angles theta relative to the femoral long axis. Nanoindentations provided local estimations of the elastic modulus and landmarks necessary for spatial fusion of the acoustic and spectral Raman images. Phosphate nu(1) (961 cm(-1)) and amide I (1665 cm(-1)) band images representing spatial distributions of mineral and collagen were fused with the acoustic images. Acoustic impedance was correlated with the indentation elastic modulus E(IT) (R(2)=0.61). Both parameters are sensitive to elastic tissue anisotropy. The lowest values were obtained in the direction perpendicular to the femoral long axis. Acoustic images exhibit a characteristic bimodal lamellar pattern of alternating high and low impedance values. Since this undulation was not associated with a variation of the phosphate nu(1)-band intensity in the Raman images, it was attributed to variations of the lamellar orientation. After threshold segmentation and conversion to elastic modulus the orientation and transverse isotropic elastic constants were derived for individual ensembles of apparent thin and thick lamellae. Our results suggest that this model represents the effective anisotropic properties of an asymmetric twisted plywood structure made of transverse isotropic fibrils. This is the first report that proves experimentally the ability of acoustic microscopy to map tissue elasticity in two dimensions with micrometer resolution. The combination with Raman microspectroscopy provides a unique way to study bone and mineral metabolism and the relation with mechanical function at the ultrastructural tissue level.     

Reconstituting the barrier properties of a water-tight epithelial membrane by design of leaflet-specific liposomes

To define aspects of lipid composition and bilayer asymmetry critical to barrier function, we examined the permeabilities of liposomes that model individual leaflets of the apical membrane of a barrier epithelium, Madin-Darby canine kidney type 1 cells. Using published lipid compositions we prepared exofacial liposomes containing phosphatidylcholine, sphingomyelin, glycosphingolipids, and cholesterol; and cytoplasmic liposomes containing phosphatidylethanolamine, phosphatidylserine, and cholesterol. The osmotic permeability of cytoplasmic liposomes to water (P(f)), solutes, and NH(3) was 18-90-fold higher than for the exofacial liposomes (P(f(ex)) = 2.4 +/- 0.4 x 10(-4) cm/s, P(f(cy)) = 4.4 +/- 0.3 x 10(-3) cm/s; P(glycerol(ex)) = 2.5 +/- 0.3 x 10(-8) cm/s, P(glycerol(cy)) = 2.2 +/- 0.02 x 10(-6) cm/s; P(NH3(ex)) = 0. 13 +/- 0.4 x 10(-4) cm/s, P(NH3(cy)) = 7.9 +/- 1.0 x 10(-3) cm/s). By contrast, the apparent proton permeability of exofacial liposomes was 4-fold higher than cytoplasmic liposomes (P(H+(ex)) = 1.1 +/- 0. 1 x 10(-2) cm/s, P(H+(cy)) = 2.7 +/- 0.6 x 10(-3) cm/s). By adding single leaflet permeabilities, we calculated a theoretical P(f) for a Madin-Darby canine kidney apical membrane of 4.6 x 10(-4) cm/s, which compares favorably with experimentally determined values. In exofacial liposomes lacking glycosphingolipids or sphingomyelin, permeabilities were 2-7-fold higher, indicating that both species play a role in barrier function. Removal of cholesterol resulted in 40-280-fold increases in permeability. We conclude: 1) that we have reconstituted the biophysical properties of a barrier membrane, 2) that the barrier resides in the exofacial leaflet, 3) that both sphingomyelin and glycosphingolipids play a role in reducing membrane permeability but that there is an absolute requirement for cholesterol to mediate this effect, 4) that these results further validate the hypothesis that each leaflet offers an independent resistance to permeation, and 5) that proton permeation was enhanced by sphingolipid/cholesterol interactions.     

Evidence for a gelsolin-rich, labile F-actin pool in human polymorphonuclear leukocytes.

Filamentous (F) actin is a major cytoskeletal element in polymorphonuclear leukocytes (PMNs) and other non-muscle cells. Exposure of PMNs to agonists causes polymerization of monomeric (G) actin to F-actin and activates motile responses. In vitro, all purified F-actin is identical. However, in vivo, the presence of multiple, diverse actin regulatory and binding proteins suggests that all F-actin within cells may not be identical. Typically, F-actin in cells is measured by either NBDphallacidin binding or as cytoskeletal associated actin in Triton-extracted cells. To determine whether the two measures of F-actin in PMNs, NBDphallacidin binding and cytoskeletal associated actin, are equivalent, a qualitative and quantitative comparison of the F-actin in basal, non-adherent endotoxin-free PMNs measured by both techniques was performed. F-actin as NBDphallacidin binding and cytoskeletal associated actin was measured in cells fixed with formaldehyde prior to cell lysis and fluorescent staining (PreFix), or in cells lysed with Triton prior to fixation (PostFix). By both techniques, F-actin in PreFix cells is higher than in PostFix cells (54.25 +/- 3.77 vs. 23.5 +/- 3.7 measured as mean fluorescent channel by NBDphallacidin binding and 70.3 +/- 3.5% vs. 47.2 +/- 3.6% of total cellular actin measured as cytoskeletal associated actin). These results show that in PMNs, Triton exposure releases a labile F-actin pool from basal cells while a stable F-actin pool is resistant to Triton exposure. Further characterizations of the distinct labile and stable F-actin pools utilizing NBDphallacidin binding, ultracentrifugation, and electron microscopy demonstrate the actin released with the labile pool is lost as filament. The subcellular localization of F-actin in the two pools is documented by fluorescent microscopy, while the distribution of the actin regulatory protein gelsolin is characterized by immunoblots with anti-gelsolin. Our studies show that at least two distinct F-actin pools coexist in endotoxin-free, basal PMNs in suspension: 1) a stable F-actin pool which is a minority of total cellular F-actin, Triton insoluble, resistant to depolymerization at 4 degrees C, gelsolin-poor, and localized to submembranous areas of the cell; and 2) a labile F-actin pool which is the majority of total cellular F-actin, Triton soluble, depolymerizes at 4 degrees C, is gelsolin-rich, and distributed diffusely throughout the cell. The results suggest that the two pools may subserve unique cytoskeletal functions within PMNs, and should be carefully considered in efforts to elucidate the mechanisms which regulate actin polymerization and depolymerization in non-muscle cells.     

Elastic modulus and hardness of cortical and trabecular bone lamellae measured by nanoindentation in the human femur.

The mechanical properties of bone tissue are determined by composition as well as structural, microstructural and nanostructural organization. The aim of this study was to quantify the elastic properties of bone at the lamellar level and compare these properties among osteonal, interstitial and trabecular microstructures from the diaphysis and the neck of the human femur. A nanoindentation technique with a custom irrigation system was used for simultaneously measuring force and displacement of a diamond tip pressed 500 nm into the moist bone tissue. An isotropic elastic modulus was calculated from the unloading curve with an assumed Poisson ratio of 0.3, while hardness was defined as the maximal force divided by the corresponding contact area. The elastic moduli ranged from 6.9 +/- 4.3 GPa in trabecular tissue from the femoral neck of a 74 yr old female up to 25.0 +/- 4.3 GPa in interstitial tissue from the diaphyseal cortex of a 69 yr old female. The mean elastic modulus was found to be significantly influenced by the type of lamella (p < 10(-6)) and by donor (p < 10(-6)). The interaction between the type of lamella and the donor was also highly significant (p < 10(-6)). Hardness followed a similar distribution as elastic modulus among types of lamellae and donor, but with lower statistical contrast. It is concluded that the nanostructure of bone tissue must differ substantially among lamellar types, anatomical sites and individuals and suggests that tissue heterogeneity is of potential importance in bone fragility and adaptation.     

Isolation and characterization of differentiated alveolar type II cells from fetal human lung

A method has been developed for isolating differentiated type II cells from human lung of 18-24-week gestation. The procedure involves an initial 4-day culture of lung explants in the presence of dexamethasone (10 nM) and triiodothyronine (2 nM). Type II cells (and fibroblasts) are isolated by trypsin digestion of the explants, two differential adherence steps and incubation overnight in primary culture. This method provides a high yield of type II cells ((50 +/- 15) X 10(6) cells/g wet weight of explant) with a purity of 85 +/- 5% in 16 experiments. The type II cells contain numerous perinuclear granules which stain darkly with toluidine blue and Papanicolaou stain; electron microscopy showed these inclusions to be lamellar bodies with tightly stacked, well defined lamellae. Type II cells, but not fibroblasts, were positive by immunofluorescence histology for surfactant apoprotein and binding of Maclura pomifera lectin which binds to the surface of type II but not type I cells in vivo. The rate of both [3H]acetate and [3H]choline incorporation into phosphatidylcholine (PC) was several-fold greater in type II cells than fibroblasts; the saturation of PC was 36.2 and 25.9%, respectively. Release of saturated PC was stimulated by terbutaline, the ionophore A23187, and tetradecanoyl phorbol acetate in type II cells but not fibroblasts. We conclude that differentiated type II cells can be isolated in relatively high yield and purity from hormone-treated explants of fetal human lung.     

Digital image analysis of cytoskeletal F-actin disintegration in renal microvascular endothelium following ischemia/reperfusion.

BACKGROUND: Damaged and/or dysfunctional microvascular endothelium has been implicated in the pathogenesis of ischemic acute renal failure (ARF). Rapidly occurring changes in the endothelial F-actin cytoskeleton as observed in vitro might be responsible, but have been proven difficult to measure accurately in situ. Therefore, the purpose of this study was to examine several methods of digital image analysis in order to quantify the alterations of endothelial F-actin after renal ischemia and reperfusion (I/R), and to relate these to deterioration of renal function. METHODS: Frozen sections of Sham and I/R rat kidneys were fixed in 4% formaldehyde and stained with rhodamine-phallo?din. Microvascular structures were captured using a 3i Marianastrade mark digital imaging fluorescence microscope workstation. Images were analyzed using 3i SlideBooktrade mark software, employing several masking techniques and line-scans. RESULTS: Digital image analysis demonstrated a decrease in the mean intensity of rhodamine-phallo?din fluorescence after I/R from 1030 +/- 187 to 735 +/- 121 a.u. (arbitrary units, mean +/- SD, n = 7). The number of F-actin fragments per pixel increased from (15.8 +/- 4.9) x 10(-5) to (20.7 +/- 3.5) x 10(-5) (n = 7), indicating cytoskeletal fragmentation. In addition, line-scan analysis demonstrated a disturbed spatial orientation of the F-actin cytoskeleton after I/R. Finally, the loss of F-actin correlated with a rise in plasma creatinine. CONCLUSIONS: The methods of digital image analysis described in the present study demonstrate that renal I/R induces profound changes in the F-actin cytoskeletal structure of microvascular endothelial cells, implicating an injured and dysfunctional microvascular endothelium, which may contribute to acute renal failure (ARF).     

Effects of fluid shear stress on mRNA expression of carbonic anhydrase II in polarized rat osteoclasts

The present study was designed to determine the effects of fluid shear stress on the mRNA expression of carbonic anhydrase II (CAII) in polarized rat osteoclasts. Cellular morphology of the polarized osteoclasts generated by a mechanical anatomical technique was examined by tartrate-resistant acid phosphatase (TRAP) staining and the osteoclastic resorption of dentine slices. The polarized osteoclasts were then stress-loaded by using a flow shear stress device newly developed by the osteoclast research group (patent number 200420034438; China), at 9 dyne/cm(2) for various time periods [0 (control group), 15, 30, 60, and 120 min], or at various stress levels [0 (control), 0.9, 2.9, 8.7, and 26.3 dyne/cm(2)] for 30 min. The mRNA expression of CAII was quantified using real-time fluorescent quantitative PCR (RT-PCR) and the data were analyzed with SPSS 12.0 software. The polarized osteoclasts were larger than regular monocytes (about 30 microm diameter) with irregular configuration, and the majority of polarized osteoclasts appeared to be spherical and had approximately 2-20 nuclei. The TRAP positive polarized osteoclasts showed asymmetrical red staining in the cytoplasm, and had many filaments and vacuoles. These cells formed resorptive pits in dentine slices. The levels of CAII mRNA expression were shown to be time-dependent, with the E+5 copy numbers being 7.88+/-0.09, 11.14+/-0.12, 15.83+/-0.18, 1.94+/-0.02, and 1.37+/-0.01 in cells treated at 9 dyne/cm(2) for 0, 15, 30, 60 and 120 min, respectively (P < 0.05). The levels of CAII mRNA expression (E+5 copy numbers) in cells treated with the stress levels of 0, 0.9, 2.9, 8.7 and 26.3 dyne/cm(2) were 7.97+/-0.201, 11.26+/-0.688, 15.94+/-0.201, 31.88+/-1.496, and 45.08+/-2.639, respectively (P < 0.05). These results indicate that there is a relationship between the fluid shear stress and the mRNA expression of CAII in polarized rat osteoclasts.     

Hyaluronan, hydration and flow conductivity of rat dermis.

We have measured the flow conductivity, kappa/eta, of discs of rat dermis with and without digestion with hyaluronidase. We found no significant difference between the flow conductivity of hyaluronan digested (kappa/eta = 5.56 +/- 2.74 cm4/dyne.s (n = 13)) and untreated tissue (kappa/eta = 6.03 +/- 3.15 cm4/dyne.s (n = 13)). For the first time in such experiments the overall tissue hyaluronan content as well as the difference in concentration of this material across tissues subjected to conditions of flow was measured. Similarly, the overall hydration and the difference in fluid content across the tissue is also reported. We have demonstrated that approximately 99% of the tissue hyaluronan was digested as a result of activity of the enzyme. No difference in hyaluronan across the tissue was found in the flow experiments (i.e., with or without digestion). We found significant change in the overall tissue hydration for controls and for either of the two types of flow experiments performed. Likewise, we found significant hydration differences across the tissues under both types of flow conditions. A trend in decreasing hydration associated with digestion of hyaluronan in the flow experiments was found.     

Properties of intermediate filament networks assembled from keratin 8 and 18 in the presence of mg(2+)

The mechanical properties of epithelial cells are modulated by structural changes in keratin intermediate filament networks. To investigate the relationship between network architecture and viscoelasticity, we assembled keratin filaments from recombinant keratin proteins 8 (K8) and 18 (K18) in the presence of divalent ions (Mg²⁺). We probed the viscoelastic modulus of the network by tracking the movement of microspheres embedded in the network during assembly, and studied the network architecture using scanning electron microscopy. Addition of Mg²⁺ at physiological concentrations (<1 mM) resulted in networks whose structure was similar to that of keratin networks in epithelial cells. Moreover, the elastic moduli of networks assembled in vitro were found to be within the same magnitude as those measured in keratin networks of detergent-extracted epithelial cells. These findings suggest that Mg²⁺-induced filament cross-linking represents a valid model for studying the cytoskeletal mechanics of keratin networks.     

An application of nanoindentation technique to measure bone tissue Lamellae properties

Measuring the microscopic mechanical properties of bone tissue is important in support of understanding the etiology and pathogenesis of many bone diseases. Knowledge about these properties provides a context for estimating the local mechanical environment of bone related cells thait coordinate the adaptation to loads experienced at the whole organ level. The objective of this study was to determine the effects of experimental testing parameters on nanoindentation measures of lamellar-level bone mechanical properties. Specifically, we examined the effect of specimen preparation condition, indentation depth, repetitive loading, time delay, and displacement rate. The nanoindentation experiments produced measures of lamellar elastic moduli for human cortical bone (average value of 17.7 +/- 4.0 GPa for osteons and 19.3 +/- 4.7 GPa for interstitial bone tissue). In addition, the hardness measurements produced results consistent with data in the literature (average 0.52 +/- 0.15 GPa for osteons and 0.59 +/- 0.20 GPa for interstitial bone tissue). Consistent modulus values can be obtained from a 500-nm-deep indent. The results also indicated that the moduli and hardnesses of the dry specimens are significantly greater (22.6% and 56.9%, respectively) than those of the wet and wet and embedded specimens. The latter two groups were not different. The moduli obtained at a 5-nm/s loading rate were significantly lower than the values at the 10- and 20-nm/s loading rates while the 10- and 20-nm/s rates were not significantly different. The hardness measurements showed similar rate-dependent results. The preliminary results indicated that interstitial bone tissue has significantly higher modulus and hardness than osteonal bone tissue. In addition, a significant correlation between hardness and elastic modulus was observed.     

Red cell extensional recovery and the determination of membrane viscosity.

A theory of membrane viscoelasticity developed by Evans and Hochmuth in 1976 is used to analyze the time-dependent recovery of an elongated cell. Before release, the elongated cell is the static equilibrium where external forces are balanced by membrane elastic force resultants. Upon release, the cell recovers its initial shape with a time-dependent exponential behavior characteristic of the viscoelastic solid model. It is shown that the model describes the time-dependent recovery process very well for a time constant in the range of 0.1-0.13 s. The time constant is the ratio membrane surface viscosity eta:membrane surface elasticity mu. Measurements for the shear modulus mu of 0.006 dyne/cm give a value for the surface viscosity of red cell membrane as a viscoelastic solid material of eta = mu tc = (6-8) X 10(-4) poise . cm.