Micromechanics of smooth adhesive organs in stick insects: pads are mechanically anisotropic and softer towards the adhesive surface

Animals have evolved adhesive structures on their legs to cling to the substrate during locomotion. Here we characterise the ultrastructure and mechanical properties of adhesive pads in Carausius morosus (Phasmatodea) using atomic force microscopy (AFM) as well as transmission and scanning electron microscopy (TEM, SEM). The smooth adhesive arolium has a soft cuticle consisting of principal rods, which branch into finer fibres near the surface. Indentation experiments showed that the pad material consists of distinct layers with different mechanical properties. The 100–300 nm thick outermost layer consisting of the cuticulin envelope and the epicuticle is extremely soft and resilient (mean effective Young’s modulus 12 kPa), while the subjacent procuticle is a much stiffer material (mean effective Young’s modulus 625 kPa). AFM contact mode imaging revealed that the cuticle is mechanically anisotropic, which can be explained by its fibrillar inner structure. We propose that the described layered structure of smooth adhesive pads, consisting of materials decreasing in stiffness towards the outer surface, represents a superior design to conform and adhere to substrates with roughnesses at different length scales. This design principle could be easily implemented in technical adhesives, and thus has a potential to inspire biomimetic applications.     

Depth-sensing analysis of cytoskeleton organization based on AFM data

Atomic force microscopy is a common technique used to determine the elastic properties of living cells. It furnishes the relative Young’s modulus, which is typically determined for indentation depths within the range 300–500 nm. Here, we present the results of depth-sensing analysis of the mechanical properties of living fibroblasts measured under physiological conditions. Distributions of the Young’s moduli were obtained for all studied cells and for every cell. The results show that for small indentation depths, histograms of the relative values of the Young’s modulus described the regions rich in the network of actin filaments. For large indentation depths, the overall stiffness of a whole cell was obtained, which was accompanied by a decrease of the modulus value. In conclusion, the results enable us to describe the non-homogeneity of the cell cytoskeleton, particularly, its contribution linked to actin filaments located beneath the cell membrane. Preliminary results showing a potential application to improve the detection of cancerous cells, have been presented for melanoma cell lines.     

Development of platelet contractile force as a research and clinical measure of platelet function

This article reviews work performed at the Medical College of Virginia of Virginia Commonwealth University during the development of a whole-blood assay of platelet function. The new assay is capable of assessing platelet function during clotting and thus allows measurement of the contribution of platelets to thrombin generation. Because platelets are monitored in the presence of thrombin, the test gages platelets under conditions of maximal activation. Three parameters are simultaneously assessed on one 700-μL sample of citrated whole blood. Platelet contractile force (PCF), the force produced by platelets during clot retraction, is directly measured as a function of time. This parameter is sensitive to platelet number, platelet metabolic status, glycoprotein IIb/IIIa status, and the presence of antithrombin activities. Clot elastic modulus (CEM), also measured as a function of time, is sensitive to fibrinogen concentration, platelet concentration, the rate of thrombin generation, the flexibility of red cells, and the production of force by platelets. The third parameter, the thrombin generation time (TGT) is determined from the PCF kinetics curve. Because PCF is absolutely thrombin dependent (no thrombin—no force), the initial upswing in PCF occurs at the moment of thrombin production. TGT is sensitive to clotting factor deficiencies, clotting factor inhibitors, and the presence of antithrombins, all of which prolong the TGT and are known to be hemophilic states. Treatment of hemophilic states with hemostatic agents shortens, the TGT toward normal. TGT has been demonstrated to be shorter and PCF to be increased in coronary artery disease, diabetes mellitus, and several other thrombophilic states. Treatment of thrombophilic states with a variety of heparin and nonheparin anticoagulants prolongs the TGT toward normal. The combination of PCF, CEM, and TGT measured on the same sample may allow rapid assessment of global hemostasis and the response to a variety of procoagulant and anticoagulant medications.     

Motion of polymerized albumin particles in a model arteriole in the presence of red blood cells

Polymerized albumin particles (poly Alb) with recombinant glycoprotein Ibα (rGPIbα-poly Alb) are a promising candidate for a platelet substitute. Thus, we focused on the lateral motion of poly Alb in the presence of red blood cells, because the lateral motion plays an important role in aggregate formation. We visualized the microscopic motion of poly Alb toward the immobilized ligand (von Willebrand factor, VWF) surface in a model arteriole with red blood cells with a high-speed camera. At a higher shear rate of 1,500 s⁻¹, the concentration profile of poly Alb appeared to peak near the wall. This profile enhances the interaction between the particles and wall. Particularly the migration angle, being the angle of the poly Alb velocity vector, was enlarged near the wall and contributed to transfer of poly Alb toward the immobilized VWF surface. This tendency is desirable to achieve the adhesion of particles on the wall.     

The application of atomic force microscopy to topographical studies and force measurements on the secreted adhesive of the green alga Enteromorpha

Atomic force microscopy (AFM) enables the topographical structure of cells and biological materials to be resolved under natural (physiological) conditions, without fixation and dehydration artefacts associated with imaging methods in vacuo. It also provides a means of measuring interaction forces and the mechanical properties of biomaterials. In the present study, AFM has been applied for the first time to the study of the mechanical properties of a natural adhesive produced by a green plant cell. Swimming spores of the green alga Enteromorpha linza (L.) J. Ag. (7–10 μm) secrete an adhesive glycoprotein which provides firm anchorage to the substratum. Imaging of the adhesive in its hydrated state revealed a swollen gel-like pad, approximately 1 μm thick, surrounding the spore body. Force measurements revealed that freshly released adhesive has an adhesion strength of 173 ± 1.7 mN m⁻¹ (mean ± SE; n=90) with a maximum value for a single adhesion force curve of 458 mN m⁻¹. The adhesive had a compressibility (equivalent to Young's modulus) of 0.54 × 10⁶ ± 0.05 × 10⁶ N m⁻² (mean ± SE; n=30). Within minutes of release the adhesive underwent a progressive `curing' process with a 65% reduction in mean adhesive strength within an hour of settlement, which was also reflected in a reduction in the average length of the adhesive polymer strands (polymer extension) and a 10-fold increase in Young's modulus. Measurements on the spore surface itself revealed considerably lower adhesion-strength values but higher polymer-extension values than the adhesive pad, which may reflect the deposition of different polymers on this surface as a new cell wall is formed. The study demonstrates the value of AFM to the imaging of plant cells in the absence of fixation and dehydration artefacts and to the characterisation of the mechanical properties of plant glycoproteins that have potential utility as adhesives. Received: 22 February 2000 / Accepted: 20 April 2000     

Collagen Network of Articular Cartilage Modulates Fluid Flow and Mechanical Stresses in Chondrocyte

The extracellular matrix of articular cartilage modulates the mechanical signals sensed by the chondrocytes. In the present study, a finite element model (FEM) of the chondrocyte and its microenvironment was reconstructed using the information from fourier transform infrared imaging spectroscopy. This environment consisted of pericellular, territorial (mainly proteoglycans), and inter-territorial (mainly collagen) matrices. The chondrocyte, pericellular, and territorial matrix were assumedto be mechanically isotropic and poroelastic, whereas the inter-territorial matrix, due to its high collagen content, was assumed to be transversely isotropic and poroelastic. Under instantaneous strain-controlled compression, the FEM indicated that the fluid pressure within the chondrocyte increased nonlinearly as a function of the in-plane Young’s modulus of the collagen network. Under instantaneous force-controlled compression, the chondrocyte experienced the highest fluid pressure when the in-plane Young’s modulus of the collagen network was ~4 MPa. Based on the present results, the mechanical characteristics of the collagen network of articular cartilage can modify fluid flow and stresses in chondrocytes. Therefore, the integrity of the collagen network may be an important determinant in cell stimulation and in the control of the matrix maintenance.     

Thermal, Mechanical and Microstructures Properties of Cellulose Derivatives Films: A Comparative Study

The proposal in this study was to evaluate the physical properties of different biopolymers films. The materials used were: pectin, carboxyl methylcellulose, methylcellulose, hydroxyl propylcellulose, hydroxypropyl-methylcellulose, and corn waxy starch; from these polysaccharides aqueous dispersions were prepared to 3% (w/v) for obtained films. In these biopolymer films, the thermal diffusivities (α) was evaluated by the Open Photoacoustic Cell method; also, their mechanical properties as tensile strength, elongation, and Young’s modulus were measured, their crystallinity percentage was evaluated by X-ray diffraction and microstructure through atomic force microscopy in contact mode. From the polysaccharide films, it was observed that most of them were flexible and transparent. In the case of the films, mechanical properties were found that the highest value of tensile strength and Young’s modulus corresponded to carboxyl methylcellulose with 69.17 and 1,912.20 MPa values, respectively. Also, Open Photoacoustic Cell method and X-ray diffraction measurements showed that there exist a correlation between the thermal diffusivity values and the crystallinity measured in the biopolymer films. It was also observed that α values of cellulose derived was affected by the substitution group in the molecule, reaching the highest α value, the films of carboxyl methylcellulose. Regarding the microstructural of the films, starch showed the highest roughness value (88.6 nm) whereas hydroxypropyl-methylcellulose resulted with the lowest roughness value (7.67 nm).     

Young’s modulus of elasticity of Schlemm’s canal endothelial cells

Schlemm’s canal (SC) endothelial cells are likely important in the physiology and pathophysiology of the aqueous drainage system of the eye, particularly in glaucoma. The mechanical stiffness of these cells determines, in part, the extent to which they can support a pressure gradient and thus can be used to place limits on the flow resistance that this layer can generate in the eye. However, little is known about the biomechanical properties of SC endothelial cells. Our goal in this study was to estimate the effective Young’s modulus of elasticity of normal SC cells. To do so, we combined magnetic pulling cytometry of isolated cultured human SC cells with finite element modeling of the mechanical response of the cell to traction forces applied by adherent beads. Preliminary work showed that the immersion angles of beads attached to the SC cells had a major influence on bead response; therefore, we also measured bead immersion angle by confocal microscopy, using an empirical technique to correct for axial distortion of the confocal images. Our results showed that the upper bound for the effective Young’s modulus of elasticity of the cultured SC cells examined in this study, in central, non-nuclear regions, ranged between 1,007 and 3,053 Pa, which is similar to, although somewhat larger than values that have been measured for other endothelial cell types. We compared these values to estimates of the modulus of primate SC cells in vivo, based on images of these cells under pressure loading, and found good agreement at low intraocular pressure (8–15 mm Hg). However, increasing intraocular pressure (22–30 mm Hg) appeared to cause a significant increase in the modulus of these cells. These moduli can be used to estimate the extent to which SC cells deform in response to the pressure drop across the inner wall endothelium and thereby estimate the extent to which they can generate outflow resistance.     

Prolyl hydroxylation of collagen type I is required for efficient binding to integrin alpha 1 beta 1 and platelet glycoprotein VI but not to alpha 2 beta 1

Collagen is a potent adhesive substrate for cells, an event essentially mediated by the integrins alpha 1 beta 1 and alpha 2 beta 1. Collagen fibrils also bind to the integrin alpha 2 beta 1 and the platelet receptor glycoprotein VI to activate and aggregate platelets. The distinct triple helical recognition motifs for these receptors, GXOGER and (GPO)n, respectively, all contain hydroxyproline. Using unhydroxylated collagen I produced in transgenic plants, we investigated the role of hydroxyproline in the receptor-binding properties of collagen. We show that alpha 2 beta 1 but not alpha 1 beta 1 mediates cell adhesion to unhydroxylated collagen. Soluble recombinant alpha 1 beta 1 binding to unhydroxylated collagen is considerably reduced compared with bovine collagens, but binding can be restored by prolyl hydroxylation of recombinant collagen. We also show that platelets use alpha 2 beta 1 to adhere to the unhydroxylated recombinant molecules, but the adhesion is weaker than on fully hydroxylated collagen, and the unhydroxylated collagen fibrils fail to aggregate platelets. Prolyl hydroxylation is thus required for binding of collagen to platelet glycoprotein VI and to cells by alpha 1 beta 1. These observations give new insights into the molecular basis of collagen-receptor interactions and offer new selective applications for the recombinant unhydroxylated collagen I.     

Hemostatic effects of polymerized albumin particles bearing rGPIa/IIa in thrombocytopenic mice

The recombinant fragment of the platelet membrane glycoprotein Ia/IIa (rGPIa/IIa) was conjugated to the polymerized albumin particles (polyAlb) with the average diameter of 180 nm. The intravenous administration of rGPIa/IIa-polyAlb to thrombocytopenic mice ([platelet] = 2.1+/-0.3 x 10(5) particles/ microL) with three doses of ca. 2.4 x 10(10), 7.2 x 10(10), and 2.4 x 10(11)particles/kg, respectively, significantly reduced their bleeding time to 426+/-71, 378+/-101, and 337+/-46 s, respectively, whereas that of the control groups (PBS) was 730+/-198 s. The injection of rGPIa/IIa-polyAlb (2.4 x 10(11)particles/kg) was approximately equal to the effect of the injection of the mouse platelets at a dose of 2.0 x 10(10) particles/kg. It was confirmed that rGPIa/IIa-polyAlb had a recognition ability against collagen and could contribute to the hemostasis in the thrombocytopenic mice as a platelet substitute.     

An Atomic Force Microscope Study Revealed Two Mechanisms in the Effect of Anticancer Drugs on Rate-Dependent Young’s Modulus of Human Prostate Cancer Cells

Mechanical properties of cells have been recognized as a biomarker for cellular cytoskeletal organization. As chemical treatments lead to cell cytoskeletal rearrangements, thereby, modifications of cellular mechanical properties, investigating cellular mechanical property variations provides insightful knowledge to effects of chemical treatments on cancer cells. In this study, the effects of eight different anticancer drugs on the mechanical properties of human prostate cancer cell (PC-3) are investigated using a recently developed control-based nanoindentation measurement (CNM) protocol on atomic force microscope (AFM). The CNM protocol overcomes the limits of other existing methods to in-liquid nanoindentation measurement of live cells on AFM, particularly for measuring mechanical properties of live cells. The Young’s modulus of PC-3 cells treated by the eight drugs was measured by varying force loading rates over three orders of magnitude, and compared to the values of the control. The results showed that the Young’s modulus of the PC-3 cells increased substantially by the eight drugs tested, and became much more pronounced as the force load rate increased. Moreover, two distinct trends were clearly expressed, where under the treatment of Disulfiram, paclitaxel, and MK-2206, the exponent coefficient of the frequency- modulus function remained almost unchanged, while with Celebrex, BAY, Totamine, TPA, and Vaproic acid, the exponential rate was significantly increased.     

Atomic force microscopy measurements. Measurements of binding strength between a single pair of molecules in physiological solutions

Atomic force microscopy (AFM) measurements of intermolecular binding strength between a single pair of complementary cell adhesion molecules in physiological solutions provided the first quantitative evidence for their cohesive function. This novel AFM based nanobiotechnology opens a molecular mechanic approach for studying structure to function related properties of any type of individual biological macromolecules. The presented example of Porifera cell adhesion glyconectin proteoglycans showed that homotypic carbohydrate to carbohydrate interactions between two primordial proteogylycans can hold the weight of 1600 cells. Thus, glyconectin type carbohydrates, as the most peripheral cell surface molecules of sponges (today’s simplest living Metazoa), are proposed to the primary cell adhesive molecules essential for the evolution of the multicellularity.     

Interaction of human platelet membrane glycoproteins with collagen and lectins

The binding of platelets to collagen is the first step in hemostasis. We attempted three approaches for elucidation of the chemical nature of receptors of human platelets for collagen. First, we examined the effect of platelet surface alteration by chymotrypsin treatment. On increasing the concentration of chymotrypsin, collagen-induced platelet aggregation and the release reaction decreased, and in parallel with this change, remarkable decrease of membrane glycoproteins IIb and V, as well as 400 kDa and 300 kDa membrane proteins, was observed. Secondly, effects of several lectins on the platelet-collagen interaction were examined. Lens culinaris agglutinin was found to specifically inhibit the platelet aggregation and release reaction induced by collagen. This inhibition appeared to be caused mainly by blocking of the collagen receptors on platelets by Lens culinaris agglutinin. Furthermore, Lens culinaris agglutinin was found to bind preferentially to glycoprotein IIb as identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of platelet membranes followed by staining with ¹²⁵I-Lens culinaris agglutinin. In addition, a polymerized preparation of Lens culinaris agglutinin induced platelet aggregation. Thirdly, the membrane component which could bind to collagen-Sepharose 4B was determined. Analysis by SDS-polyacrylamide gel electrophoresis combined with autoradiography or fluorography revealed that glycoprotein IIb was most enriched in the bound fraction to collagen. From these results, glycoprotein IIb is most likely a receptor for collagen on human platelet membranes.     

Isolation and characterization of a platelet surface collagen binding complex related to VLA-2

A heterodimeric, Mg++-dependent, collagen binding protein has been isolated from platelet membranes. Electrophoretic properties and monoclonal antibody reactivity indicate that the heavy chain of the complex is platelet membrane glycoprotein Ia and that the light chain is glycoprotein IIa. Furthermore, the receptor appears to be identical with the recently defined VLA-2 complex found on activated T-lymphocytes, platelets and other cells. When incorporated into liposomes, the purified complex mediates the Mg++-dependent adhesion of the liposomes to collagen substrates. These observations suggest that the VLA-2 complex mediates cellular adhesion to collagen in platelets and possibly in other cells.     

Atomic force microscopy study of the rabbit skeletal muscle ryanodine receptors in different functional states

Atomic force microscope was applied to investigate the effect of extrinsic phospholipid on the structure of rabbit skeletal muscle ryanodine receptor/calcium release channel (RyR1). In addition, in the presence of extrinsic phospholipid, the height and elasticity of the RyR1s in different functional states were also measured. The results indicate: (i) most of the RyR1s showed a normal structure only in the presence of extrinsic phospholipid; (ii) treatment of the RyR1s with AMP and Ca²⁺ together could increase their Young’s Modulus but not change their apparent height; (iii) no detectable change in either height or Young’s Modulus of the RyR1s appeared, if the RyR1s were treated with other activators or inhibitors.     

Probing elasticity and adhesion of live cells by atomic force microscopy indentation

Atomic force microscopy (AFM) indentation has become an important technique for quantifying the mechanical properties of live cells at nanoscale. However, determination of cell elasticity modulus from the force–displacement curves measured in the AFM indentations is not a trivial task. The present work shows that these force–displacement curves are affected by indenter-cell adhesion force, while the use of an appropriate indentation model may provide information on the cell elasticity and the work of adhesion of the cell membrane to the surface of the AFM probes. A recently proposed indentation model (Sirghi, Rossi in Appl Phys Lett 89:243118, 2006), which accounts for the effect of the adhesion force in nanoscale indentation, is applied to the AFM indentation experiments performed on live cells with pyramidal indenters. The model considers that the indentation force equilibrates the elastic force of the cell cytoskeleton and the adhesion force of the cell membrane. It is assumed that the indenter-cell contact area and the adhesion force decrease continuously during the unloading part of the indentation (peeling model). Force–displacement curves measured in indentation experiments performed with silicon nitride AFM probes with pyramidal tips on live cells (mouse fibroblast Balb/c3T3 clone A31-1-1) in physiological medium at 37°C agree well with the theoretical prediction and are used to determine the cell elasticity modulus and indenter-cell work of adhesion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Changes induced by hyperosmotic mannitol in cerebral endothelial cells: an atomic force microscopic study

Understanding the reaction of living cells in response to different extracellular stimuli, such as hyperosmotic stress, is of primordial importance. Mannitol, a cell-impermeable non-toxic alcohol, has been used successfully for reversible opening of the blood–brain barrier in hyperosmotic concentrations. In this study we analyzed the effect of hyperosmotic mannitol on the shape and surface structure of living cerebral endothelial cells by atomic force microscope imaging technique. Addition of clinically relevant concentrations of mannitol to the culture medium of the confluent cells induced a decrease of about 40% in the observed height of the cells. This change was consistent both at the nuclear and peripheral region of the cells. After mannitol treatment even a close examination of the contact surface between the cells did not reveal gap between them. We could observe the appearance of surface protrusions of about 100 nm. By force measurements the elasticity of the cells were estimated. While the Young’s modulus of the control cells appeared to be 8.04 ± 0.12 kPa, for the mannitol-treated cells it decreased to an estimated value of 0.93 ± 0.04 kPa which points to large structural changes inside the cell.     

Quantification of biomechanical properties of human corneal scar using acoustic radiation force optical coherence elastography

Biomechanical properties of corneal scar are strongly correlated with many corneal diseases and some types of corneal surgery, however, there is no elasticity information available about corneal scar to date. Here, we proposed an acoustic radiation force optical coherence elastography system to evaluate corneal scar elasticity. Elasticity quantification was first conducted on ex vivo rabbit corneas, and the results validate the efficacy of our system. Then, experiments were performed on an ex vivo human scarred cornea, where the structural features, the elastic wave propagations, and the corresponding Young’s modulus of both the scarred region and the normal region were achieved and based on this, 2D spatial distribution of Young’s modulus of the scarred cornea was depicted. Up to our knowledge, we realized the first elasticity quantification of corneal scar, which may provide a potent tool to promote clinical research on the disorders and surgery of the cornea.     

Photoinactivation effects of hematoporphyrin monomethyl ether on Gram-positive and -negative bacteria detected by atomic force microscopy

The photodynamic antimicrobial chemotherapy as a promising approach for efficiently killing pathogenic microbes is attracting increasing interest. In this study, the cytotoxic and phototoxic effects of hematoporphyrin monomethyl ether (HMME) on the Gram-positive and Gram-negative bacteria were investigated. The cell viability was assessed by colony-forming unit method, and the results indicated that there was no significant cytotoxicity but high phototoxicity in the examined concentrations. Notably, the Gram-positive bacteria were more sensitive to HMME in phototoxicity. Simultaneously, an atomic force microscope (AFM) was used to detect the changes in morphological and nanomechanical properties of bacteria before and after HMME treatment. AFM images indicate that upon photoinactivation, the bacterial surface changed from a smooth, homogeneous architecture to a heterogenous, crackled morphology. The force spectroscopy measurements reveal that the cell wall became less rigid and the Young’s modulus decreased about 50%, whereas the tip-cell-surface adhesion forces increased significantly compared to those of native cells. It was speculated that the photodynamic effects of HMME induced the changes in the chemical composition of the outer membrane and exposure of some proteins inside the envelope. AFM can be utilized as a powerful and sensitive method for studying the interaction between bacteria and drugs.     

Spider silk softening by water uptake: an AFM study

We have investigated the mechanical properties of spider dragline fibers of three Nephila species under varied relative humidity. Force maps have been collected by atomic force microscopy. The Young’s modulus E was derived from the indentation curves of each pixel by the modified Hertz model. An average decrease in E by an order of magnitude was observed upon immersion of the fiber in water. Single fiber stretching experiments were carried out for comparison, and also showed a strong dependence on relative humidity. However, the absolute values of E are significantly higher than those obtained by indentation. The results of this work thus show that the elastic properties of spider silk are highly anisotropic, and that the silk softens significantly for both tensile and compressional strain (indentation) upon water uptake. In addition, the force maps indicate a surface structure on the sub-micron scale.