Adhesion behaviors of human trophoblast cells by contact with endothelial cells

Although it is still not clear whether migratory trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by a mechanism of directional cell division/proliferation, this process involves the attachment and adhesion of trophoblasts to endothelial cells lining the blood vessel walls. This raises the possibility that the cell–cell contact with endothelial cells may regulate trophoblast cell adhesion behaviors according to the surrounding flow condition. To test this, the adhesion forces of early gestation human trophoblast cells (TCs) cultured on glass slides coated with type I rat collagen or cultured with human umbilical vein endothelial cells (HUVECs) were measured quantitatively using a micropipette aspiration technique. Then, the resistance of TCs co-cultured with HUVECs to flow-induced shear stress was assessed with a flow chamber technique. The results showed that the adhesion force of TCs to glass slides coated with collagen was positively correlated with the concentration of collagen. By contact with endothelial cells, the adhesion force and the resistance to shear stress for the TCs were significantly enhanced. The interdiction of integrin β1 interaction remarkably reduced the adhesion forces of TCs to endothelial cells, hence their resistance to shear stress. The results therefore suggest that the contacts of TCs with endothelial cells enhance the adhesion forces of human TCs, partially by regulating with the integrin β1 according to the flow condition (i.e., the shear stress) in such a way to prevent the TCs from being carried downstream by flowing blood.     

Quantifying nanoparticle adhesion mediated by specific molecular interactions

Receptor-mediated targeting of nanometric contrast agents or drug carriers holds great potential for treating cardiovascular and vascular-associated diseases. However, predicting the ability of these vectors to adhere to diseased cells under dynamic conditions is complex due to the interplay of transport, hydrodynamic force, and multivalent bond formation dynamics. Therefore, we sought to determine the effects of adhesion molecule density and flow rate on adhesion of 210 nm particles, with the goal of identifying criteria to optimize binding efficiency and selectivity. Our system employed a physiologically relevant ligand, the vascular adhesion molecule ICAM-1, and an ICAM-1 specific antibody tethered to the nanoparticle using avidin-biotin chemistry. We measured binding and dissociation of these particles in a flow chamber as a function of antibody density, ligand density, and flow rate, and using a transport-reaction model we distilled overall kinetic rate constants for adhesion and detachment from the binding data. We demonstrate that both attachment and detachment of 210 nm particles can be correlated with receptor and ligand valency and are minimally affected by shear rate. Furthermore, we uncovered a time-dependent mechanism governing particle detachment, in which the rate of detachment decreases with contact time according to a power law. Finally, we use our results to illustrate how to engineer adhesion selectivity for specific molecular targeting applications. These results establish basic principles dictating nanoparticle adhesion and dissociation and can be used as a framework for the rational design of targeted nanoparticle therapeutics that possess optimum adhesive characteristics.     

A new micropatterning method of soft substrates reveals that different tumorigenic signals can promote or reduce cell contraction levels

In tissues, cell microenvironment geometry and mechanics strongly impact on cell physiology. Surface micropatterning allows the control of geometry while deformable substrates of tunable stiffness are well suited for the control of the mechanics. We developed a new method to micropattern extracellular matrix proteins on poly-acrylamide gels in order to simultaneously control cell geometry and mechanics. Microenvironment geometry and mechanics impinge on cell functions by regulating the development of intra-cellular forces. We measured these forces in micropatterned cells. Micropattern geometry was streamlined to orient forces and place cells in comparable conditions. Thereby force measurement method could be simplified and applied to large-scale experiment on chip. We applied this method to mammary epithelial cells with traction force measurements in various conditions to mimic tumoral transformation. We found that, contrary to the current view, all transformation phenotypes were not always associated to an increased level of cell contractility.     

Quantitative method for determining the lateral strength of bacterial adhesion and application for characterizing adhesion kinetics

A quantitative method for measuring the shear force required to detach individual adhered bacteria using atomic force microscopy (AFM) was developed. By determining the total compression of the cantilever during cell detachment events, a more accurate means of calculating the applied lateral force necessary to remove individual cells was achieved compared to previous methods. In addition, a tunable assay for monitoring the dynamics of Pseudomonas aeruginosa and Staphylococcus aureus adhesion strength was employed. The accumulation of force measurements over time allowed for the characterization of adhesion strength kinetics. P. aeruginosa reinforced its adhesion to the surface at a rate 7-fold faster than for S. aureus; the average adhesion strength of P. aeruginosa was larger than that of S. aureus at corresponding time points. Adhered cells of the same species and strain demonstrated a range of adhesion forces that broadened with time, indicating that the change in adhesion strength does not proceed uniformly.     

Improved mesenchymal stem cell seeding on RGD-modified poly(L-lactic acid) scaffolds using flow perfusion

Arg-Gly-Asp (RGD) has been widely utilized to increase cell adhesion to three-dimensional scaffolds for tissue engineering. However, cell seeding on these scaffolds has only been carried out statically, which yields low cell seeding efficiencies. We have characterized, for the first time, the seeding of rat mesenchymal stem cells on RGD-modified poly(L-lactic acid) (PLLA) foams using oscillatory flow perfusion. The incorporation of RGD on the PLLA foams improves scaffold cellularity in a dose-dependent manner under oscillatory flow perfusion seeding. When compared to static seeding, oscillatory flow perfusion is the most efficient seeding technique. Cell detachment studies show that cell adhesion is dependent on the applied flow rate, and that cell attachment is strengthened at higher levels of RGD modification.     

Experimental Study of Fibrin/Fibrin-Specific Molecular Interactions Using a Sphere/Plane Adhesion Model

Fibrin, the biopolymer produced in the final step of the coagulation cascade, is involved in the resistance of arterial thrombi to fragmentation under shear flow. However, the nature and strength of specific interactions between fibrin monomers are unknown. Thus, the shear-induced detachment of spherical monodispersed fibrin-coated latex particles in adhesive contact with a plane fibrin-coated glass surface has been experimentally studied, using an especially designed shear stress flow chamber. A complete series of experiments for measuring the shear stress necessary to release individual particles under various conditions (various number of fibrin layers involved in the adhesive contact, absence or presence of plasmin, the main physiological fibrinolytic enzyme) has been performed. The nonspecific DLVO interactions have been shown to be negligible compared to the interactions between fibrin monomers. A simple adhesion model based on the balance of forces and torque on particles, assuming an elastic behavior of the fibrin polymer bonds, to analyze the experimental data in terms of elastic force at rupture of an elementary intermonomeric fibrin bond has been used. The results suggested that this force (of order 400 pN) is an intrinsic quantity, independent of the number of fibrin layers involved in the adhesive contact. Copyright 2001 Academic Press.     

Targeted particulate adhesion to cellulose surfaces mediated by bifunctional fusion proteins

The adhesion of particles to surfaces is an integral element in many commercial and biological applications. In this article, we report on the direct measurements of protein-mediated deposition and binding of particles to model cellulose surfaces. This system involves a family of heterobifunctional fusion proteins that bind specifically to both a red dye and cellulose. Amine-coated particles were labeled with a red dye, and a fusion protein was attached to these particles at various number densities. The strength of adhesion of a single particle to a cellulose fiber was measured using micropipette manipulation as a function of the specificity of the protein and its surface density and contact time. The frequency and force of adhesion were seen to increase with contact time in fiber experiments. The dynamics of adhesion of the functionalized particles to cellulose-coated glass slides under controlled hydrodynamic flow was explored using a flow chamber for two scenarios: detachment of bound particles and attachment of particles in suspension as a function of the shear rate and surface density of protein. Highly specific adhesion was observed. The critical shear rate for particle detachment was an increasing function of cellulose binding domain (CBD) density on particle surface. A rapid irreversible attachment of particles to cellulose was observed under flow. Using a family of proteins that were divalent for binding either the red dye or cellulose, we found that particle detachment occurred because of the failure of the cellulose-CBD bond. A comparison of fiber binding and particle detachment results suggests that forces of adhesion of particles to cellulose of up to 2 nN can be obtained with this molecular system through multiple interactions. This study, along with the adhesion simulations currently under development, forms the basis of particulate design for specific adhesion applications.     

Control of cell detachment in a microfluidic device using a thermo-responsive copolymer on a gold substrate

The control of cell adhesion is crucial in many procedures in cellular biotechnology. A thermo-responsive poly(N-isopropylacrylamide)-poly(ethylene glycol)-thiol (PNIPAAm-PEG-thiol) copolymer was synthesized for the formation of self-assembled monolayers (SAM) that allow the control of adhesion of cells on gold substrates. The contact angle of water on these layers varies between 65 degrees at a temperature of 45 degrees C and 54 degrees at 25 degrees C. This behaviour is consistent with a transition of the polymer chains from an extended and highly hydrated to a collapsed coil-like state. At 37 degrees C, cultivated fibroblasts adhere and spread normally on this surface and detach by reducing the temperature below the lower critical solution temperature (LCST). Layers can repeatedly be used without loss of their functionality. In order to quantify the capability of the copolymer layer to induce cell detachment, defined shear forces are applied to the cells. For this purpose, the laminar flow in a microfluidic device is used. Our approach provides a strategy for the optimization of layer properties that is based on establishing a correlation between a functional parameter and molecular details of the layers.     

Contact mechanics of nanoparticles

We perform molecular dynamics simulations on the detachment of nanoparticles from a substrate. The critical detachment force, f*, is obtained as a function of the nanoparticle radius, R(p), shear modulus, G, surface energy, γ(p), and work of adhesion, W. The magnitude of the detachment force is shown to increase from πWR(p) to 2.2πWR(p) with increasing nanoparticle shear modulus and nanoparticle size. This variation of the detachment force is a manifestation of neck formation upon nanoparticle detachment. Using scaling analysis, we show that the magnitude of the detachment force is controlled by the balance of the nanoparticle elastic energy, neck surface energy, and energy of nanoparticle adhesion to a substrate. It is a function of the dimensionless parameter δ ∝ γ(p)(GR(p))(-1/3)W(-2/3), which is proportional to the ratio of the surface energy of a neck and the elastic energy of a deformed nanoparticle. In the case of small values of the parameter δ ? 1, the critical detachment force approaches a critical Johnson, Kendall, and Roberts force, f* ≈ 1.5πWR(p), as is usually the case for strongly cross-linked, large nanoparticles. However, in the opposite limit, corresponding to soft small nanoparticles for which δ?1, the critical detachment force, f*, scales as f*∝ γ(p)(3/2)R(p)(1/2)G(-1/2). Simulation data are described by a scaling function f*∝ γ(p)(3/2)R(p)(1/2)G(-1/2)δ(-1.89).     

Mechanics of cell spreading within 3D-micropatterned environments

Most tissue cells evolve in vivo in a three-dimensional (3D) microenvironment including complex topographical patterns. Cells exert contractile forces to adhere and migrate through the extracellular matrix (ECM). Although cell mechanics has been extensively studied on 2D surfaces, there are too few approaches that give access to the traction forces that are exerted in 3D environments. Here, we describe an approach to measure dynamically the contractile forces exerted by fibroblasts while they spread within arrays of large flexible micropillars coated with ECM proteins. Contrary to very dense arrays of microposts, the density of the micropillars has been chosen to promote cell adhesion in between the pillars. Cells progressively impale onto the micropatterned substrate. They first adhere on the top of the pillars without applying any detectable forces. Then, they spread along the pillar sides, spanning between the elastic micropillars and applying large forces on the substrate. Interestingly, the architecture of the actin cytoskeleton and the adhesion complexes vary over time as cells pull on the pillars. In particular, we observed less stress fibers than for cells spread on flat surfaces. However, prominent actin stress fibers are observed at cell edges surrounding the micropillars. They generate increasing contractile forces during cell spreading. Cells treated with blebbistatin, a myosin II inhibitor, relax their internal tension, as observed by the release of pillar deformations. Moreover, cell spreading on pillars coated with ECM proteins only on their tops are not able to generate significant traction forces. Taken together, these findings highlight the dynamic relationship between cellular forces and acto-myosin contractility in 3D environments, the influence of cytoskeletal network mechanics on cell shape, as well as the importance of cell-ECM contact area in the generation of traction forces.     

Capillary force required to detach micron-sized particles from solid surfaces—Validation with bubbles circulating in water and 2 μm-diameter latex spheres

The adhesion forces holding micron-sized particles to solid surfaces can be studied through the detachment forces developed by the transit of an air–liquid interface in a capillary. Two key variables affect the direction and magnitude of the capillary detachment force: (i) the thickness of the liquid film between the bubble and the capillary walls, and (ii) the effective angle of the triple phase contact between the particles and the interface. Variations in film thickness were calculated using a two-phase flow model. Film thickness was used to determine the time-variation of the capillary force during transit of the bubble. The curve for particle detachment was predicted from the calculated force. This curve proved to be non-linear and gave in situ information on the effective contact angle developing at the particle–bubble interface during detachment. This approach allowed an accurate determination of the detachment force. This theoretical approach was validated using latex particles 2 μm in diameter.     

High‐Frequency Mechanostimulation of Cell Adhesion

Cell adhesion is regulated by molecularly defined protein interactions and by mechanical forces, which can activate a dynamic restructuring of adhesion sites. Previous attempts to explore the response of cell adhesion to forces have been limited to applying mechanical stimuli that involve the cytoskeleton. In contrast, we here apply a new, oscillatory type of stimulus through push–pull azobenzenes. Push–pull azobenzenes perform a high‐frequency, molecular oscillation upon irradiation with visible light that has frequently been applied in polymer surface relief grating. We here use these oscillations to address single adhesion receptors. The effect of molecular oscillatory forces on cell adhesion has been analyzed using single‐cell force spectroscopy and gene expression studies. Our experiments demonstrate a reinforcement of cell adhesion as well as upregulated expression levels of adhesion‐associated genes as a result of the nanoscale “tickling” of integrins. This novel type of mechanical stimulus provides a previously unprecedented molecular control of cellular mechanosensing.     

Detachment of Particles from Surfaces: An AFM Study

We have measured interactions between hydrophilic and hydrophobic surfaces in an aqueous medium at various pH and ionic strengths as well as in some organic solvents using atomic force microscopy and analyzed them in terms of particle adhesion and detachment from surfaces. In hydrophilic systems the forces observed were found to be well described by DLVO theory at large separation distances. Very long range hydrophobic forces were not observed in hydrophilic-hydrophobic systems. Nevertheless, the jump into contact was found to occur at distances greater that those predicted by just van der Waals attraction. The interaction between two hydrophobic surfaces was dominated by the long-range attraction due to hydrophobic forces. This interaction was found to be sensitive to the type of substrate as well as to the pH and electrolyte concentration. Measured pull-off forces showed poor reproducibility. However, average values showed clear trends and were used to estimate interfacial energies or work of adhesion for all systems studied by means of the Derjaguin approximation. These values were compared to those calculated by the surface tension component theory using the acid-base approach. Good qualitative agreement was obtained, giving support for the usefulness of this approach in estimating interfacial energies between surfaces in liquid media. A comparison of the measured adhesion force with hydrodynamic detachment experiments showed good qualitative agreement. Copyright 2001 Academic Press.     

Effect of flow and surface conditions on human lymphocyte isolation using microfluidic chambers

Phenotypically pure subpopulations of lymphocytes can provide valuable insights into the immune response to injury and disease. The isolation of these subpopulations presents unique challenges, particularly when preprocessing incubation to attach fluorescent or antibody tags is to be minimized. This paper examines the separation of T and B lymphocytes from mixtures using microfluidic chambers coated with antibodies, focusing on flow conditions and surface chemistry. The adhesion of both cell types decreases as shear stress increases irrespective of the surface chemistry. The incorporation of poly(ethylene glycol) chains along with the antibodies on the chamber surface is shown to significantly improve the reproducibility of cell adhesion and is thus an important part of the overall system design. Furthermore, this technique is shown to be an effective way of isolating highly pure subpopulations of lymphocytes from model mixtures, even when the target cell concentration is low.     

Stress-induced polymer deformation in shear flows

By means of dynamic Monte Carlo simulation of bulk lattice polymers in Couette shear flow, it was demonstrated that in addition to velocity gradient the constant driving forces acting as the activation aspect of shear stresses can also raise polymer deformation. Moreover, enhancing driving forces in a flow without any velocity gradient can reproduce nonNewtonian fluid behaviors of long-chain polymers. The simulations of Poiseuille shear flow with a gradient of shear stresses show that, the velocity gradient dominates small deformation in the flow layers of low shear stresses, while the shear stress dominates large deformation in the flow layers of high shear stresses. This result implies that the stress-induced deformation could be mainly responsible for the occurrence of non-Newtonian fluid behaviors of real polymers at high shear rates.     

A computational study of the hydrodynamically assisted organization of DNA-functionalized colloids in 2D

We study computationally the self-organization of DNA-functionalized colloidal particles confined to two dimensions and subjected to a linear shear force. We show that hydrodynamic forces allow a more thorough sampling of phase space than thermal or Brownian forces alone. Two particle types are present in each of our dynamic simulations each signifying its own specific oligonucleotide sequence grafted to the particle surface: A-type and B-type. Particles are modeled as interacting via a type-specific DNA attraction where unlike-types have affinities for each other while like-types do not. The particles are small enough to feel Brownian motion while the shear adds motion to the particles. We find the formation of lines of A-type and B-type particles in simulations with an imposed shear. Simulations without imposed shear form a frustrated network with little or no linear order. An orientational distribution function, g2(r), quantifies the degree of linear order. A phase diagram is constructed, finding a linear dependence of the minimum DNA force necessary for line formation on the dimensionless shear rate. A force analysis performed on the structures shows that the lines orient perpendicular to the axis of the elongation component of the shear because it is this orientation that allows the DNA attraction to resist the shear.     

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.     

Molecular understanding of the adhesive interactions between silica surface and epoxy resin: Effects of interfacial water

The molecular mechanism of the adhesion between silica surface and epoxy resin under atmospheric conditions is investigated by periodic density-functional-theory (DFT) calculations. Slab models of the adhesion interface were built by integrating a fragment of epoxy resin and hydroxylated (0 0 1) surface of α-cristobalite in the presence of adsorbed water molecules. Effects of adsorbed water on the adhesion interaction are evaluated on the basis of geometry-optimized structures, adhesion energies, and forces. Calculated results demonstrate that adsorbed water molecules significantly reduce both the adhesion energies and forces of the silica surface–epoxy resin interface. The reduction of adhesion properties can be associated with structural deformation of water molecules confined in the tight space between the adhesive and adherend as well as structural flexibility of the hydrogen-bonding network in the interfacial region during detachment of the epoxy resin from the hydrophilic silica surface. © 2018 Wiley Periodicals, Inc.     

Mechanical response analysis and power generation by single-cell stretching.

To harvest useful information about cell response due to mechanical perturbations under physiological conditions, a cantilever-based technique was designed, which allowed precise application of arbitrary forces or deformation histories on a single cell in vitro. Essential requirements for these investigations are a mechanism for applying an automated cell force and an induced-deformation detection system based on fiber-optical force sensing and closed loop control. The required mechanical stability of the setup can persist for several hours since mechanical drifts due to thermal gradients can be eliminated sufficiently (these gradients are caused by local heating of the cell observation chamber to 37 degrees C). During mechanical characterization, the cell is visualized with an optical microscope, which enables the simultaneous observation of cell shape and intracellular morphological changes. Either the cell elongation is observed as a reaction against a constant load or the cell force is measured as a response to constant deformation. Passive viscoelastic deformation and active cell response can be discriminated. The active power generated during contraction is in the range of Pmax= 10(-16) Watts, which corresponds to 2500 ATP molecules s(-1) at 10 k(B)T/molecule. The ratio of contractive to dissipative power is estimated to be in the range of 10(-2). The highest forces supported by the cell suggest that about 10(4) molecular motors must be involved in contraction. This indicates an energy-conversion efficiency of approximately 0.5. Our findings propose that, in addition to the recruitment of cell-contractile elements upon mechanical stimulation, the cell cytoskeleton becomes increasingly crosslinked in response to a mechanical pull. Quantitative stress-strain data, such as those presented here, may be employed to test physical models that describe cellular responses to mechanical stimuli.     

Stick slip contact mechanics between dissimilar materials: effect of charging and large friction

Measurements of the contact radius as a function of applied force between a mica surface and a silica surface (mica/silica) in air are reported. The load/unload results show that the contact radius generally increases with applied force. Because of the presence of charging due to contact electrification, both a short-range van der Waals adhesion force and longer-range electrostatic adhesive interaction contribute to the measured force. The results indicate that approximately 20% of the pull-off force is due to van der Waals forces. The contact radius versus applied force results can be fit to Johnson-Kendall-Roberts (JKR) theory by considering that only the short-range van der Waals forces contribute to the work of adhesion and subtracting a constant longer-range electrostatic force. Also, an additional and unexpected step function is superimposed on the contact radius versus applied force curve. Thus, the contact diameter increases in a stepped dependence with increasing force. The stepped contact behavior is seen only for increasing force and is not observed when symmetric mica/mica or silica/silica contacts are measured. In humid conditions, the contact diameter of the mica/silica contact increases monotonically with applied force. Friction forces between the surfaces are also measured and the shear stress of a mica/silica interface is 100 times greater than the shear stress of a mica/mica interface. This large shear stress retards the increase in contact area as the force is increased and leads to the observed stepped contact mechanics behavior.