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PubMed2004

Probing surface adhesion forces of Enterococcus faecalis to medical-grade polymers using atomic force microscopy.

The aim of this study was to compare the initial adhesion forces of the uropathogen Enterococcus faecalis with the medical-grade polymers polyurethane (PU), polyamide (PA), and poly(tetrafluoroethylene) (PTFE). To quantify the cell-substrate adhesion forces, a method was developed using atomic force microscopy (AFM) in liquid that allows for the detachment of individual live cells from a polymeric surface through the application of increasing force using unmodified cantilever tips. Results show that the lateral force required to detach E. faecalis cells from a substrate differed depending on the nature of the polymeric surface: a force of 19 +/- 4 nN was required to detach cells from PU, 6 +/- 4 nN from PA, and 0.7 +/- 0.3 nN from PTFE. Among the unfluorinated polymers (PU and PA), surface wettability was inversely proportional to the strength of adhesion. AFM images also demonstrated qualitative differences in bacterial adhesion; PU was covered by clusters of cells with few cell singlets present, whereas PA was predominantly covered by individual cells. Moreover, extracellular material could be observed on some clusters of PU-adhered cells as well as in the adjacent region surrounding cells adhered on PA. E. faecalis adhesion to the fluorinated polymer (PTFE) showed different characteristics; only a few individual cells were found, and bacteria were easily damaged, and thus detached, by the tip. This work demonstrates the utility of AFM for measurement of cell-substrate lateral adhesion forces and the contribution these forces make toward understanding the initial stages of bacterial adhesion. Further, it suggests that initial adhesion can be controlled, through appropriate biomaterial design, to prevent subsequent formation of aggregates and biofilms.

Bacterial Adhesion↗

PubMed2005

Atomic force microscopy study on specificity and non-specificity of interaction forces between Enterococcus faecalis cells with and without aggregation substance.

Enterococcus faecalis is one of the leading causes of hospital-acquired infections, and indwelling medical devices are especially prone to infection. E. faecalis expressing aggregation substance (Agg) adheres to biomaterial surfaces by means of positive cooperativity, i.e. the ability of one adhering organism to stimulate adhesion of other organisms in its immediate vicinity. In this study, atomic force microscopy (AFM) was used to measure the specificity and non-specificity of interaction forces between E. faecalis cells with and without Agg. Bacteria were attached to a substratum surface and a tip-less cantilever. Two E. faecalis strains expressing different forms of Agg showed nearly twofold higher interaction forces between bacterial cells than a strain lacking Agg [adhesive force (F(adh)), -1.3 nN]. The strong interaction forces between the strains with Agg were reduced after adsorption of antibodies against Agg from -2.6 and -2.3 nN to -1.2 and -1.3 nN, respectively. This suggests that the non-specific interaction force between the enterococci amounts to approximately 1.2 nN, while the specific force component is only twofold stronger. Comparison of the results of the AFM interaction forces with the positive cooperativity after adhesion to a biomaterial in a parallel-plate flow chamber showed that in the absence of strong interaction forces between the cells, positive cooperativity was also absent. In conclusion, this is believed to be the first time that the influence of specific antibodies on interaction forces between E. faecalis cells has been demonstrated by AFM, thereby experimentally distinguishing between specific and non-specific force components.

Adhesins, Bacterial↗

PubMedDate not supplied

Effect of water on the surface molecular mobility of poly(lactide) thin films: an atomic force microscopy study.

Physical properties associated with molecular mobility on the surface of thin films with 300 nm thickness for poly(lactide)s (PLAs) were studied under vacuum conditions as well as under aqueous conditions by using friction force mode atomic force microscopy (AFM). Two types of PLAs were applied for the experimental samples as uncrystallizable PLA (uc-PLA) and crystallizable PLA (c-PLA). The friction force on the surface of thin films was measured as a function of temperature to assess the surface molecular mobility both under vacuum and under aqueous conditions. A lower glass-transition temperature of the uc-PLA surface in water was detected than that under vacuum conditions. In the case of the c-PLA thin film, change in friction force was detected at a lower temperature under aqueous conditions than in vacuo. A morphological change was observed in the c-PLA thin film during heating process from room temperature to 100 degrees C by temperature-controlled AFM. The surface of the c-PLA thin film became rough due to the cold crystallization, and the crystallization of c-PLA molecules in water took place at a lower temperature than in vacuo. These friction force measurements and AFM observations suggest that molecular motion on the surface of the both uc- and c-PLA thin films is enhanced in the presence of water molecules. In addition, in situ AFM observation of the enzymatic degradation process for the c-PLA thin film crystallized at 160 degrees C was carried out in buffer solution containing proteinase K at room temperature. The amorphous region around the hexagonal crystal was eroded within 15 min. It has been suggested that the adsorption of water molecules on the PLA film surface enhances the surface molecular mobility of the glassy amorphous region of PLA and induces the enzymatic hydrolysis by proteinase K.

Adsorption↗

PubMed2006

Unbinding of the streptavidin-biotin complex by atomic force microscopy: a hybrid simulation study.

A hybrid molecular simulation technique, which combines molecular dynamics and continuum mechanics, was used to study the single-molecule unbinding force of a streptavidin-biotin complex. The hybrid method enables atomistic simulations of unbinding events at the millisecond time scale of atomic force microscopy (AFM) experiments. The logarithmic relationship between the unbinding force of the streptavidin-biotin complex and the loading rate (the product of cantilever spring constant and pulling velocity) in AFM experiments was confirmed by hybrid simulations. The unbinding forces, cantilever and tip positions, locations of energy barriers, and unbinding pathway were analyzed. Hybrid simulation results from this work not only interpret unbinding AFM experiments but also provide detailed molecular information not available in AFM experiments.

Biotin↗

PubMed2004

In-situ atomic force microscopy observation of enzymatic degradation in poly(hydroxyalkanoic acid) thin films: normal and constrained conditions.

The enzymatic degradation of lamellar crystals in poly(hydroxyalkanoic acid) thin films has been visualized by using in-situ dynamic force mode (tapping mode) atomic force microscopy (AFM) in buffer solution. It was found that poly(hydroxybutyric acid) (PHB) depolymerase from Ralstonia pickettii T1 degraded the thin surface layers formed at room temperature first, and that lamellar crystals formed at the crystallization temperature (110 degrees C) were eroded from the crystallographic a-axis to show splintered morphologies at the tips of the crystals. In some cases, lamellar crystals were hydrolyzed from the crystallographic b-axis, resulting in the formation of small crevices. These results suggest that disordered molecular chain-packing regions exist in the crystal along the crystallographic a- and b-axes, and that enzymatic degradation predominantly occurs from these defective regions. In addition, cantilever-tip-induced enzymatic degradation was carried out in the presence of PHB depolymerase. A concave area was artificially formed on the stacked lamellar crystals by the AFM tip. In-situ AFM observation has revealed that enzymatic degradation proceeds along both the longitudinal and lateral directions of the lamellae. At the same time, the PHB depolymerase preferentially eroded the concave area along the crystallographic c-axis. These results demonstrated that the PHB depolymerase predominantly degrades the less-ordered molecular chain-packing regions in the crystals.

3-Hydroxybutyric Acid↗

PubMed2006

Functional atomic force microscopy investigation of osteopontin affinity for silicon stabilized tricalcium phosphate bioceramic surfaces.

Resorbable silicon stabilized tricalcium phosphate (Si-TCP)-based bioceramics are characterized from a biological perspective by measuring the intermolecular interaction force between osteopontin (OPN) protein and the material surface using atomic force microscopy (AFM). OPN protein was covalently bound to silicon nitride AFM tips and adsorption and adhesion forces were measured in an electrolyte with a composition similar to that of physiological fluids. A strong relationship exists between the adhesion force of OPN on the material surface, the number of adherent osteoclasts (OC) and the resorption of the material. OPN adhesion is strongest on hydroxyapatite (HA) surfaces, or in samples that induce a HA-like surface through a precipitation reaction in electrolytic media. It is proposed that the increased biological response of the Si-TCP phase can be attributed in part to its reactivity in a physiological electrolyte, which involves a rapid conversion to a calcium deficient HA phase with a corresponding increase in the adhesion strength of OPN to the material, with a consequentially higher OC resorption response.

Adsorption↗

PubMed2006

Adhesion forces between functionalized latex microspheres and protein-coated surfaces evaluated using colloid probe atomic force microscopy.

Proteins are important in bacterial adhesion, but interactions at molecular-scales between proteins and specific functional groups are not well understood. The adhesion forces between four proteins [bovine serum albumin (BSA), protein A, lysozyme, and poly-d-lysine] and COOH, NH2 and OH-functionalized (latex) colloids were examined using colloid probe atomic force microscopy (AFM) as the function of colloid residence time (T) and solution ionic strength (IS). For three of the proteins, OH-functionalized colloids produced higher adhesion forces to proteins (2.6-30.5 nN; IS=1 mM, T=10s) than COOH- and NH2-functionalized colloids (1.6-6.8 nN). However, protein A produced the largest adhesion force (8.1+/-1.0 nN, T=10 s) with the COOH-functionalized colloid, demonstrating the importance of specific and unanticipated protein-functional group interactions. The NH2-functionalized colloid typically produced the lowest adhesion forces with all proteins, likely due to repulsive electrostatic forces and weak bonds for NH2-NH2 interactions. The adhesion force (F) between functionalized colloids and proteins consistently increased with residence time (T), and data was well fitted by F=ATn. The constant value of n=0.21+/-0.07 for all combinations of proteins and functionalized colloids indicated that water exclusion and protein rearrangement were the primary factors affecting adhesion over time. Adhesion forces decreased inversely with IS for all functional groups interacting with surface proteins, consistent with previous findings. These results demonstrate the importance of specific molecular-scale interactions between functional groups and proteins that will help us to better understand factors colloidal adhesion to surfaces.

Adsorption↗

PubMed1996

Comparative scanning, transmission and atomic force microscopy of the microtubular cytoskeleton in fenestrated liver endothelial cells.

Endothelial fenestrae control the exchange of fluids, solutes and particles between the sinusoidal lumen and the microvillous surface of the parenchymal cells. Fenestrae have a critical dimension in the order of 150-200 nm, making it necessary to use microscopes with a resolution better than the light microscope. Comparative whole-mount preparations of isolated, purified and cultured rat liver sinusoidal endothelial cells (LEC) were studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). Examination of detergent-extracted LEC by SEM and TEM shows an integral cytoskeleton: sieve plates are delineated by a sieve plate-associated cytoskeleton ring and fenestrae by a fenestrae-associated cytoskeleton ring. By using microtubule altering agents we could demonstrate: (1) the architectural role of microtubules in arranging fenestrae, (2) the existence of a population of microtubules resistant against low temperature and colchicine, (3) the ability of LEC to shift the microtubule assembly-disassembly steady state under various conditions, (4) and the necessity of an intact microtubular cytoskeleton to support the increase in the number of fenestrae after cytochalasin B. Topographical examinations of AFM images revealed that sieve plates are delineated by elevated borders, probably projections of the underlying tubular cytoskeleton.

Animals↗

PubMed2000

Atomic force microscopy and electron microscopy analysis of retrovirus Gag proteins assembled in vitro on lipid bilayers.

We have used an in vitro system that mimics the assembly of immature Moloney murine leukemia virus (M-MuLV) particles to examine how viral structural (Gag) proteins oligomerize at membrane interfaces. Ordered arrays of histidine-tagged Moloney capsid protein (his-MoCA) were obtained on membrane bilayers composed of phosphatidylcholine (PC) and the nickel-chelating lipid 1, 2-di-O-hexadecyl-sn-glycero-3-(1'-2"-R-hydroxy-3'N-(5-amino-1-carboxy pentyl)iminodiacetic acid)propyl ether (DHGN). The membrane-bound arrays were analyzed by electron microscopy (EM) and atomic force microscopy (AFM). Two-dimensional projection images obtained by EM showed that bilayer-bound his-MoCA proteins formed cages surrounding different types of protein-free cage holes with similar cage holes spaced at 81.5-A distances and distances between dissimilar cage holes of 45.5 A. AFM images, showing topological features viewed near the membrane-proximal domain of the his-MoCA protein, revealed a cage network of only symmetrical hexamers spaced at 79-A distances. These results are consistent with a model in which dimers constitute structural building blocks and where membrane-proximal and distal his-MoCA regions interact with different partners in membrane-bound arrays.

Capsid↗

PubMed2005

Surface charge heterogeneities measured by atomic force microscopy.

Unfavorable aggregation and deposition of colloidal particles in natural and engineered systems is still a subject of debate. Complicating factors such as surface roughness, secondary minimum aggregation, and the nature of discrete surface charge and surface potential make it difficult to attribute a specific cause to these phenomena. The presence of surface charge heterogeneity and its influence on interaction forces, which are responsible for aggregation and deposition, are studied in this work through the application of atomic force microscopy (AFM). Force-volume-mode AFM was used to map interaction forces on a surface and relate them to surface charge heterogeneities. The experimental system consisted of a silica plate and a standard silicon nitride AFM tip. Copper ions were used for sorption on the silica surface in order to modify the surface charge and cause charge reversal. Different concentrations of copper ions were selected to identify conditions of partial coverage of the silica surface. The pH and ionic strength of the solutions were varied, and the extension of the surface charge modification and its influence on the resulting interaction forces were monitored via AFM force measurements. Depending on the pH and ionic strength, the interaction force was found to change at certain regions on the surface from attraction to either weak or strong repulsion. Force imaging allowed the visual localization of zones of strong repulsive interaction that diminished in size with increasing ionic strength. X-ray photoelectron spectroscopy analysis was used to confirm the presence of copper on the surface. Local charge differences on a surface result in local differences in surface forces, not only in magnitude but also in direction. This behavior may explain the aggregation, deposition, and transport of colloidal particles under unfavorable chemical conditions.

Colloids↗

PubMed2005

Experimentally derived sticking efficiencies of microparticles using atomic force microscopy.

The sticking efficiencies (alpha) of colloidal particles have been derived from the intersurface potential energy between 2 microm carboxylated polystyrene microspheres and a silica glass plate using the interaction force boundary layer model. The intersurface potential energies were calculated from force-distance data measured using atomic force microscopy (AFM) and from calculations based on Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. AFM forces were measured in aqueous solutions over a range of pH and ionic strength conditions, and DLVO calculations were performed on identical systems. In most conditions, sticking efficiencies that were calculated from AFM data are considerably largerthan values calculated from DLVO predictions. Sticking efficiencies vary between 0 and 1 and are strongly dependent upon solution chemistry. AFM-derived sticking efficiencies are consistent with measured microsphere and collector zeta-potentials; sticking efficiencies are lower for more negatively charged surfaces. These results provide the first alpha estimates of a microparticle-collector system that are calculated directly from physically measured interfacial nanoforces. This study clearly demonstrates that significant differences exist between DLVO- and AFM-derived sticking efficiencies.

Colloids↗

PubMed2002

Affinity scale between a carrier and a drug in DPI studied by atomic force microscopy.

The dry powder inhalers (DPIs) consist, in the most cases, of ordered mixture where the particles adhesion results of interactions between the drug and the carrier. Generally, one step of production process is the micronization of the drug particles in order to reduce the size for ordered mixing optimization. But this operation is known to partially create an amorphous surface. In this case, surrounding storage conditions, like relative humidity (RH), are able to modify the percentage of amorphous drug surface. The aim of this study was to investigate surface reactivity, surface energy and direct force measurements by atomic force microscopy (AFM) between lactose (carrier) and zanamivir (drug) crystals references in various conditions of RH. Secondly, an amorphization of the drug surface was induced by humidity relative treatment in order to evaluate the consequences of the transition from crystal to amorphous phase. The study demonstrated that the amorphization of drug surface induces an increase of drug affinity with the carrier surface. Ex situ and in situ amorphization of zanamivir tend to reach the affinity measured between raw materials: carrier and micronized drug particles. AFM allowed adhesion force discrimination between the different forms of the drug particles and demonstrated the potential for investigating adhesion properties in DPI formulation.

Drug Carriers↗

PubMed2000

Electrostatic forces on the surface of metals as measured by atomic force microscopy.

Electrostatic forces play an important role in modulating the interaction of plasma proteins and blood cellular components with the surface of the vascular endothelium. Based on the concept that electrostatic forces residing on the surface of metal intravascular prostheses, such as the stent, also are critical in influencing blood interactions with those surfaces and the vascular wall, these studies were designed to measure these forces on 4 metals using atomic force microscopy (AFM). AFM measurements performed in a low saline aqueous medium at physiological pH indicate a similar net electronegative surface charge level for gold and 316l stainless steel that is significantly higher than the level measured on an electropolished Nitinol surface. Heat oxidation of the Nitinol surface increased the overall electronegativity and created a more homogeneous surface charge distribution. This study demonstrates that AFM force measurements can be a valuable approach to understanding the electrostatic surface of metallic as well as other biomaterials that may be important in understanding how these surfaces influence vascular healing at intravascular interventional sites.

Biocompatible Materials↗

PubMed2002

Combining constitutive materials modeling with atomic force microscopy to understand the mechanical properties of living cells.

The goal of this work is to study the properties of living cells and cell membranes by using atomic force microscopy. During atomic force microscopy (AFM) measurement, there is a strong mechanical coupling between the AFM tip and the cell. The purpose of this paper is to present a model of the overall mechanical response of the cell that allows us to separate out the mechanical response of the cell from the local surface interactions we wish to quantify. These local interactions include recognition (or binding) events between molecules bound to an AFM tip (e.g., an antibody) and molecules or receptors on the cell surface (e.g., the respective antigen). The computational model differs from traditional Hertzian contact models by explicitly taking into account the mechanics of the biomembrane and cytoskeleton. The model also accounts for the mechanical response of the living cell during arbitrary deformation. The indentation of a bovine sperm cell is used to test the validity of this model, and further experiments are proposed to fully parameterize the model.

Animals↗

PubMed2003

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.

Cell Line↗

PubMed1999

Single integrin molecule adhesion forces in intact cells measured by atomic force microscopy.

Cross-talk between cells and the extracellular matrix is critically influenced by the mechanical properties of cell surface receptor-ligand interactions; these interactions are especially well defined and regulated in cells capable of dynamically modifying their matrix environment. In this study, attention was focused on osteoclasts, which are absolutely dependent on integrin extracellular matrix receptors in order to degrade bone; other bone cells, osteoblasts, were used for comparison. Integrin binding forces were measured in intact cells by atomic force microscopy (AFM) for several RGD-containing (Arg-Gly-Asp) ligands and ranged from 32 to 97 picoNewtons (pN); they were found to be cell and amino acid sequence specific, saturatable and sensitive to the pH and divalent cation composition of the cellular culture medium. In contrast to short linear RGD hexapeptides, larger peptides and proteins containing the RGD sequence, such as osteopontin (a major non-collagenous bone protein) and echistatin (a high affinity RGD sequence containing antagonist snake venom protein), showed different binding affinities. This demonstrates that the context of the RGD sequence within a protein has considerable influence upon the final binding force for receptor interaction. These data also demonstrate that AFM, as a methodological approach, can be adapted to cell biology studies wherever cell-matrix interactions play a critical role, and, moreover, may have applicability to the analysis of receptor-ligand interactions in cell membranes in general.

Animals↗

PubMed2005

Mechanical properties of the interaction between fibronectin and alpha5beta1-integrin on vascular smooth muscle cells studied using atomic force microscopy.

The mechanical properties of integrin-extracellular matrix (ECM) interactions are important for the mechanotransduction of vascular smooth muscle cells (VSMC), a process that is associated with focal adhesions, and can be of particular significance in cardiovascular disease. In this study, we characterized the unbinding force and binding activity of the initial fibronectin (FN)-alpha5beta1 interaction on the surface of VSMC using atomic force microscopy (AFM). It is postulated that these initial binding events are important to the subsequent focal adhesion assembly. FN-VSMC adhesions were selectively blocked by antibodies against alpha5- and beta1-integrins as well as RGD-containing peptides but not by antibodies against alpha4- and beta3-integrins, indicating that FN primarily bound to alpha5beta1. A characteristic unbinding force of 39 +/- 8 pN was observed and interpreted to represent the FN-alpha5beta1 single-bond strength. The ability of FN to adhere to VSMC (binding probability) was significantly reduced by integrin antagonists, serum starvation, and platelet-derived growth factor (PDGF)-BB, whereas lysophosphatidic acid (LPA) increased FN binding. However, no significant change in the resolved unbinding force was observed. After engagement, the force required to dislodge the FN-coated bead from VSMC increased with increasing of contact time, suggesting a time-dependent increase in number of adhesions and/or altered binding affinity. LPA enhanced this process, whereas PDGF reduced it, suggesting that these factors also affect the multimolecular process of focal contact assembly. Thus AFM is a powerful tool for the characterization of the mechanical properties of integrin-ECM interactions and their regulation. Our results indicate that the functional activity of alpha5beta1 and focal contact assembly can be rapidly regulated.

Animals↗

PubMed2005

Resolving the nanoscale adhesion of individual gecko spatulae by atomic force microscopy.

Animals that cling to walls and walk on ceilings owe this ability to micrometre and nanoscale attachment elements. The highest adhesion forces are encountered in geckoes, which have developed intricate hierarchical structures consisting of toes (millimetre dimensions), lamella (400-600microm size), setae (micrometre dimensions) and spatulae ( approximately 200nm size). Adhesion forces of setae on different substrates have previously been measured by a micro-electromechanical system technique. Here we report the first successful experiments in which the force-displacement curves were determined for individual spatulae by atomic force microscopy. The adhesion force for these smallest elements of the gecko's attachment system is reproducibly found to be about 10nN. This method sheds new light on the nanomechanisms of attachment and will help in the rational design of artificial attachment systems.

Adhesiveness↗