Critical fluid shear stress analysis for cell–polymer adhesion

A simple methodology to assess cell adhesion on materials was developed. We demonstrated that the cell adhesion strength could be quantified. Using this method, we were able to compare the NIH/3T3 Swiss mouse fibroblasts adhesion strength to poly(methyl methacrylate) and polycarbonate. A controlled fluid shear stress was applied to cells using a parallel plate rotational system. Cells detached from the surface in the radial direction. Results showed that there was a critical radius where the shear stress experienced by the cells equaled the cell adhesion strength. The cells outside this radius were removed while those inside maintained initial confluency. The quantitative evaluation of cell adhesion is beneficial for development of biomaterials.     

Fluid-flow-induced mesenchymal stem cell migration: role of focal adhesion kinase and RhoA kinase sensors

The study of mesenchymal stem cell (MSC) migration under flow conditions with investigation of the underlying molecular mechanism could lead to a better understanding and outcome in stem-cell-based cell therapy and regenerative medicine. We used peer-reviewed open source software to develop methods for efficiently and accurately tracking, measuring and processing cell migration as well as morphology. Using these tools, we investigated MSC migration under flow-induced shear and tested the molecular mechanism with stable knockdown of focal adhesion kinase (FAK) and RhoA kinase (ROCK). Under steady flow, MSCs migrated following the flow direction in a shear stress magnitude-dependent manner, as assessed by root mean square displacement and mean square displacement, motility coefficient and confinement ratio. Silencing FAK in MSCs suppressed morphology adaptation capability and reduced cellular motility for both static and flow conditions. Interestingly, ROCK silencing significantly increased migration tendency especially under flow. Blocking ROCK, which is known to reduce cytoskeletal tension, may lower the resistance to skeletal remodelling during the flow-induced migration. Our data thus propose a potentially differential role of focal adhesion and cytoskeletal tension signalling elements in MSC migration under flow shear.     

Quantitative measurements of the strength of adhesion of human neutrophils to a substratum in a microfluidic device

We describe a quantitative assay of the strength of adhesion of activated and nonactivated human neutrophils to a substratum, which is carried out in a custom-made microfluidic device. The strength of adhesion is quantified by the fraction of cells remaining adherent (ACF) after a given time of exposure to shear stress in a test microchannel. The microfluidic device is made of two layers of poly(dimethylsiloxane) with integrated membrane valves. This construction allows concurrent testing of two different populations of cells, as well as setting well-defined times of exposure of cells to stress and of their incubation prior to the exposure. The test microchannels have a tapered profile, exposing cells to nearly an order of magnitude range of shear stress. ACF is measured periodically by computer-controlled videomicroscopy scans of the device, with up to 60,000 individual cells identified within a 90 seconds scan. The high throughput of the scans allows reliable quantitative assessment of the ACF. Adhesion of untreated neutrophils and neutrophils activated with formyl-Met-Leu-Phe was tested concurrently in a series of experiments with a fibrinogen-coated glass substratum. At optimized testing conditions, the ACF of activated cells was consistently found to be three times higher than that of nonactivated cells. An adhesion assay could be completed within 11 min from the loading of cells into the device without any intervention by the operator. The proposed device and assay could be used to assess the state of activation of neutrophils in human blood with a potential application to diagnostics of inflammation.     

Measuring bone cell adhesion on implant surfaces using a spinning disc device

The adhesion behaviour of osteoblastic cells on implant surfaces is a main focus during the development of osteoconductive implant surfaces. Therefore, besides cell spreading and proliferation on surfaces the adhesion strength of cells to the substrate is of high interest. There are different approaches to determine cell adhesion but only few quantitative methods. For this purpose, we have developed an adhesion device based on the spinning disc principle in conjunction with an inverse confocal laser scanning microscope (LSM). Mirror polished disc‐shaped test samples made of titanium‐ (Ti6Al4V) and cobalt‐alloys (Co28Cr6Mo), as well as stainless steel (316L), were seeded with osteoblasts, stained with a fluorescent dye, at defined radial positions and were incubated for 18 h in cell culture medium (DMEM). After incubation the test samples were placed into the adhesion chamber filled with DMEM. By means of a computer controlled motor the test samples were rotated for 3 min. Using the LSM the detachment of the cells at defined radial positions was determined and the cell count was recorded before and after rotation with the help of imaging software. An average shear stress of 47.1 N/m², 53.2 N/m² and 49.4 N/m² was assessed for the mirror polished Ti6Al4V, Co28Cr6Mo and 316L surfaces respectively. The technique is suitable for studying bone cell adhesion strength on orthopaedic implant materials. Future investigations will focus on different bioactive and anti‐infectious implant surfaces, as well as soluble bioactive factors.     

Monitoring cell adhesion by piezoresonators: impact of increasing oscillation amplitudes

In recent years, the quartz crystal microbalance (QCM) has been established as a sensitive analytical tool to monitor the attachment and spreading of mammalian cells to in vitro surfaces. Due to its superior time resolution, the device is capable of reading even subtle differences in cell adhesion kinetics. However, thickness shear mode piezoresonators, which are the core component of the QCM approach, can be used not only as a sensor but also as an actuator when the oscillation amplitude of the crystal is increased so that molecular recognition at the solid-liquid interface is disturbed. In this study, we have addressed the impact of elevated lateral oscillation amplitudes on the adhesion kinetics of three mammalian cell lines. We used AT-cut piezoresonators with a fundamental resonance frequency of 5 MHz, and the analytical readout was performed by impedance analysis. Formation of stable cell-substrate contacts is retarded or entirely blocked when the lateral oscillation amplitude (in the center of the resonator) exceeds values higher than 20 nm. Shear oscillations of similar amplitude were, however, not sufficient to displace attached cells from the surface. Moreover, the experimental data prove that the normal QCM readout with oscillation amplitudes smaller than 1 nm is, indeed, non-invasive with respect to mammalian cells.     

Microwell device for targeting single cells to electrochemical microelectrodes for high-throughput amperometric detection of quantal exocytosis

Electrochemical microelectrodes are commonly used to detect spikes of amperometric current that correspond to exocytosis of oxidizable transmitter from individual vesicles, i.e., quantal exocytosis. We are developing transparent multielectrochemical electrode arrays on microchips in order to automate measurement of quantal exocytosis. Here, we report development of an improved device to target individual cells to each microelectrode in an array. Efficient targeting (~75%) is achieved using cell-sized microwell traps fabricated in SU-8 photoresist together with patterning of poly(l-lysine) in register with electrodes to promote cell adhesion. The surface between electrodes is made resistant to cell adhesion using poly(ethylene glycol) in order to facilitate movement of cells to electrode "docking sites". We demonstrate the activity of the electrodes using the test analyte ferricyanide and perform recordings of quantal exocytosis from bovine adrenal chromaffin cells on the device. Multiple cell recordings on a single device demonstrate the consistency of spike measurements, and multiple recordings from the same electrodes demonstrate that the device can be cleaned and reused without degradation of performance. The new device will enable high-throughput studies of quantal exocytosis and may also find application in rapidly screening drugs or toxins for effects on exocytosis.     

Adhesive interactions of geckos with wet and dry fluoropolymer substrates

Fluorinated substrates like Teflon^® (poly(tetrafluoroethylene); PTFE) are well known for their role in creating non-stick surfaces. We showed previously that even geckos, which can stick to most surfaces under a wide variety of conditions, slip on PTFE. Surprisingly, however, geckos can stick reasonably well to PTFE if it is wet. In an effort to explain this effect, we have turned our attention to the role of substrate surface energy and roughness when shear adhesion occurs in media other than air. In this study, we removed the roughness component inherent to commercially available PTFE and tested geckos on relatively smooth wet and dry fluoropolymer substrates. We found that roughness had very little effect on shear adhesion in air or in water and that the level of fluorination was most important for shear adhesion, particularly in air. Surface energy calculations of the two fluorinated substrates and one control substrate using the Tabor–Winterton approximation and the Young–Dupré equation were used to determine the interfacial energy of the substrates. Using these interfacial energies we estimated the ratio of wet and dry normal adhesion for geckos clinging to the three substrates. Consistent with the results for rough PTFE, our predictions show a qualitative trend in shear adhesion based on fluorination, and the quantitative experimental differences highlight the unusually low shear adhesion of geckos on dry smooth fluorinated substrates, which is not captured by surface energy calculations. Our work has implications for bioinspired design of synthetics that can preferentially stick in water but not in air.     

Fibrin-mediated endothelial cell adhesion to vascular biomaterials resists shear stress due to flow

In vitro endothelial cell (EC) seeding onto biomaterials for blood-contacting applications can improve the blood compatibility of materials. Adhesive proteins adsorbed from serum that is supplemented with the culture medium intercede the initial cell adhesion and subsequent spreading on material surface during culture. Nevertheless, physical and chemical properties of vascular biomaterial surface fluctuate widely between materials resulting in dissimilarity in protein adsorption characteristics. Thus, a variation is expected in cell adhesion, growth and the ability of cell to resist shear stress when tissue engineering on to vascular biomaterials is attempted. This study was carried out with an objective to determine the significance of a matrix coating on cell adhesion and shear stress resistance when cells are cultured on materials such as polytetrafluoroethylene (PTFE, Teflon) and polyethyleneterephthalate (Dacron), ultra high molecular weight polyethylene (UHMWPE) and titanium (Ti), that are used for prosthetic devices. The study illustrates the distinction of EC attachment and proliferation between uncoated and matrix-coated surfaces. The cell attachment and proliferation on uncoated UHMWPE and titanium surfaces were not significantly different from matrix-coated surfaces. However, shear stress resistance of the cells grown on composite coated surfaces appeared superior compared to the cells grown on uncoated surface. On uncoated vascular graft materials, the cell adhesion was not supported by serum alone and proliferation was scanty as compared to matrix-coated surface. Therefore, coating of implant devices with a composite of adhesive proteins and growth factors can improve EC attachment and resistance of the cells to the forces of flow.     

Combined atomic force microscopy and optical microscopy measurements as a method to investigate particle uptake by cells

We propose a combination of atomic force microscopy (AFM) and optical microscopy for the investigation of particle uptake by cells. Positively and negatively charged polymer microcapsules were chosen as model particles, because their interaction with cells had already been investigated in detail. AFM measurements allowed the recording of adhesion forces on a single-molecule level. Due to the micrometer size of the capsules, the number of ingested capsules could be counted by optical microscopy. The combination of both methods allowed combined measurement of the adhesion forces and the uptake rate for the same model particle. As a demonstration of this system, the correlation between the adhesion of positively or negatively charged polymer microcapsules onto cell surfaces and the uptake of these microcapsules by cells has been investigated for several cell lines. As is to be expected, we find a correlation between both processes, which is in agreement with adsorption-dependent uptake of the polymer microcapsules by cells.     

Shear assay measurements of cell adhesion on biomaterials surfaces

The paper examines the adhesion of human osterosarcoma (HOS) cells to selected biomaterials surfaces that are relevant to implantable biomedical systems and bio-micro-electro-mechanical systems (BioMEMS). The four biomaterials that were explored include: silicon, silicon coated with a nanoscale layer of titanium, Ti–6Al–4V, and poly-di-methy-siloxane (PDMS). The interfacial strengths between the HOS cells and the biomaterials surfaces were determined using a shear assay technique. The adhesion forces were determined using a combination of confocal microscopy images of the three-dimensional cell structure, and computational fluid dynamics (CFD) simulations that coupled actual cell morphologies and non-Newtonian fluid properties in the computation of the adhesion forces. After cell detachment by the shear assay, immunofluorescence staining of the biomedical surfaces was used to reveal the proteins associated with cell detachment. These revealed that the nano-scale Ti coating increases the cell/surface adhesion strength. Silicon with Ti coating has the strongest adhesion strength, while the other surfaces had similar adhesion strength. The measured strengths are shown to be largely associated with the detachment of focal adhesion proteins from extra-cellular matrix (ECM) proteins.     

Effect of the interface stiffness and skin–core adhesion efficiency on the interfacial stress distribution of sandwich structures

This paper investigates the load distribution along the skin–core interface length of FRP-metal laminates subjected to in-plane tension, using an improved shear lag model. The model elucidates the significance of the interface stiffness concept on the skin–core interface load distribution in FRP-metal laminate beams and introduces a basic hypothesis according which, the interface stiffness depends on the difference in shear moduli of the constituent materials as well as on the degree of adhesion which, in turn, depends on the abrupt jump in shear moduli at the skin–core boundary. Details of an experimental procedure carried out and of experimental results obtained are also presented. From the combined experimental and theoretical investigation it can be concluded that for a given FRP-metal laminate structure with known core and facing properties, it is possible to evaluate the skin–core degree of adhesion as well as the interface stiffness and predict the interfacial stress distribution by simply conducting a single tensile test. Thus, the proposed method can be considered as a tool for the structural performance and reliability control of FRP-metal laminate structures.     

Polyelectrolyte Multilayer-Treated Electrodes for Real-Time Electronic Sensing of Cell Proliferation

We report on the use of polyelectrolyte multilayer (PEM) coatings as a non-biological surface preparation to facilitate uniform cell attachment and growth on patterned thin-film gold (Au) electrodes on glass for impedance-based measurements. Extracellular matrix (ECM) proteins are commonly utilized as cell adhesion promoters for electrodes; however, they exhibit degradation over time, thereby imposing limitations on the duration of conductance-based biosensor experiments. The motivation for the use of PEM coatings arises from their long-term surface stability as promoters for cell attachment, patterning, and culture. In this work, a cell proliferation monitoring device was fabricated. It consisted of thin-film Au electrodes deposited with a titanium-tungsten (TiW) adhesion layer that were patterned on a glass substrate and passivated to create active electrode areas. The electrode surfaces were then treated with a poly(ethyleneimine) (PEI) anchoring layer and subsequent bilayers of sodium poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). NIH-3T3 mouse embryonic fibroblast cells were cultured on the device, observed by optical microscopy, and showed uniform growth characteristics similar to those observed on a traditional polystyrene cell culture dish. The optical observations were correlated to electrical measurements on the PEM-treated electrodes, which exhibited a rise in impedance with cell proliferation and stabilized to an approximate 15 % increase as the culture approached confluency. In conclusion, cells proliferate uniformly over gold and glass PEM-treated surfaces, making them useful for continuous impedance-based, real-time monitoring of cell proliferation and for the determination of cell growth rate in cellular assays.     

Superhydrophobicity vs. Ice Adhesion: The Quandary of Robust Icephobic Surface Design

The mesoscale, multitier texture of the lotus leaf has served as an inspiration to fabricate surface designs with controllable superhydrophobic properties, targeting a broad range of applications. The choice of material for such designs is directly related to surface performance, in particular under adverse and realistic conditions. Due to its importance in many applications, here aluminium is employed as a material platform and identify key porous hierarchical textures, yielding extraordinary impalement‐resistant behavior: Droplet repellency is demonstrated consistently for water impact velocities up to 12 m s⁻¹ (extreme Weber number, We ≈ 3500). Despite impressive superhydrophobic behavior, if ice forms on such surfaces, ice adhesion is markedly stronger than on less hydrophobic alumina nanotube array structures. In a departure from the findings of the well‐accepted shear stress‐based ice adhesion criterion, a deviation between decreasing ice adhesion strength and increasing hydrophobicity is observed. This is explained with ice adhesion mechanism, depending strongly on the applied stress field orientation and the respective effective ice–substrate contact area. Our results indicate that ice adhesion criteria for the performance of icephobic surfaces should account for the simultaneous presence of shear and tensile stresses, instead of shear stresses alone.     

Validation of pure shear test device using finite element method and experimental methods

In this paper, we will validate the Iosipescu shear test (IST) and the Hawong Iosipescu shear test (HIST) used for the pure shear test device; the HIST is modified from the IST. This validation uses finite element method and experimental method. In the IST the loads are applied on the outside edges of the specimen, while in the HIST the loads are applied on the neutral surface of the specimen. It has been certified that HIST is more effective than IST because of the simplicity of the loading device, loading method and the distribution of pure shear stress, etc. We know that the specimen with a 110° V-notched angle is more effective than the specimen with a 90° notched angle for a pure shear test device. In specimens with a 90° notched angle, the maximum shear stress occurs at the end of V-notch. While in specimens with a 110° notched angle, it is produced at the center of specimen. In both HIST and IST the most ideal ratio of a/b is 0.3 although the ratios of 0.3, 0.4, 0.5 of a/b are also useful, except for 0.2. When HIST or IST is under mode fracture, HIST or IST with β=90° is more effective than those with β=0°, as a pure shear test device.     

Granule attrition by coupled particle impact and shearing

A novel device has been developed for continuous shearing and repeated impact of granules in order to simulate granule attrition and dust formation under realistic plant conditions of mechanical stresses, shear strains and strain rates. The device subjects the granules to multiple impacts at a range of velocities prevailing in typical process plants, and to shear deformations using two rollers with an adjustable gap to simulate the level of shear stresses and strains experienced during bulk motion, e.g. discharge from silos onto conveyor belts, etc. In this paper, the device operation and tests carried out to determine the settings required for attaining a desired impact velocity and shear strain rate are described. Subsequently, the extent of breakage of the granules is determined for the specified settings and the results are compared with data obtained by more established methods, e.g. annular shear cell and single particle impact tests.     

Sliding-induced adhesion of stiff polymer microfibre arrays. II. Microscale behaviour.

The adhesive pads of geckos provide control of normal adhesive force by controlling the applied shear force. This frictional adhesion effect is one of the key principles used for rapid detachment in animals running up vertical surfaces. We developed polypropylene microfibre arrays composed of vertical, 0.3 microm radius fibres with elastic modulus of 1 GPa which show this effect for the first time using a stiff polymer. In the absence of shear forces, these fibres show minimal normal adhesion. However, sliding parallel to the substrate with a spherical probe produces a frictional adhesion effect which is not seen in the flat control. A cantilever model for the fibres and the spherical probe indicates a strong dependence on the initial fibre angle. A novel feature of the microfibre arrays is that adhesion improves with use. Repeated shearing of fibres temporarily increases maximum shear and pull-off forces.     

Interactions between multiple cell types in parallel microfluidic channels: monitoring platelet adhesion to an endothelium in the presence of an anti-adhesion drug

A simple method for immobilizing endothelial cells in the channels of a microfluidic device fabricated with soft lithography is presented that requires no surface oxidation of the substrate material used in conjunction with the microfluidic device and is operable even with a reversible seal. Specifically, optimal conditions for culturing bovine pulmonary artery endothelial cells (bPAECs) to the surface of a Petri dish were investigated. The parameters investigated included fibronectin concentration, temperature, seeding density, and immobilization time. To enhance the utility of the device, all optimization studies, and studies involving platelet adhesion to the immobilized endothelium, were performed in parallel channels, thereby enabling improved throughput over a single channel device. The optimal conditions for cell immobilization included coating the Petri dish with 100 microg/mL fibronectin, a seeding cell density of 1.00 x 10(5) cells mL(-1), and an immobilization time of 90 min at 37 degrees C. The device was then employed to monitor the physical interaction (adhesion) of platelets to the immobilized endothelium in the presence of a known platelet activator (ADP) and a drug inhibitor of platelet activation. The number of platelets adhering to the endothelial cells in the channels increased from 17.0 +/- 2.3 in the absence of ADP to 63.2 +/- 2.4 in the presence of 5.00 microM ADP. Moreover, the data presented here also shows that inhibition of endothelium nitric oxide (NO) production, a recognized inhibitor of platelet adhesion to the endothelium, increased the number of platelets adhering to the surface to 35.4 +/- 1.0. In the presence of NO inhibition and 5.00 microM ADP, the affect on platelet adhesion was further increased to 127 +/- 5.2. Finally, this device was employed to investigate the effect of a drug known to inhibit platelet adhesion (clopidogrel) and, in the presence of the drug, the platelet adhesion due to activation by 5.00 microM ADP decreased to 24.0 +/- 3.8. This work is the first representation of multiple cell types physically interacting in the channels of a microfluidic device and further demonstrates the potential of these devices in the drug discovery process and drug efficacy studies.     

Channel surface patterning of alternating biomimetic protein combinations for enhanced microfluidic tumor cell isolation

Here, we report a new method for multicomponent protein patterning in a microchannel and also a technique for improving immunoaffinity-based circulating tumor cell (CTC) capture by patterning regions of alternating adhesive proteins using the new method. The first of two proteins, antiepithelial cell adhesion molecule (anti-EpCAM), provides the specificity for CTC capture. The second, E-selectin, increases CTC capture under shear. Patterning regions with and without E-selectin allows captured leukocytes, which also bind E-selectin and are unwanted impurities in CTC isolation, to roll a short distance and detach from the capture surface. This reduces leukocyte capture by up to 82%. The patterning is combined with a leukocyte elution step in which a calcium chelating buffer effectively deactivates E-selectin so that leukocytes may be rinsed away 60% more efficiently than with a buffer containing calcium. The alternating patterning of this biomimetic protein combination, used in conjunction with the elution step, reduces capture of leukocytes while maintaining a high tumor cell capture efficiency that is up to 1.9 times higher than the tumor cell capture efficiency of a surface with only anti-EpCAM. The new patterning technique described here does not require mask alignment and can be used to spatially control the immobilization of any two proteins or protein mixtures inside a sealed microfluidic channel.     

Quantitative comparison of adhesion in metal-to-plastic systems

A peel test device was used to monitor metal-polymer adhesion. This technique showed variations in the resulting force within 1%, and enabled the ranking of many systems. A change in the 50 nm-adhesion layer resulted in a variation of the fracture energy by a factor 2.3. A change of the substrate led to a change in adhesion by a factor 40. The peeling results were also combined with attenuated total reflectance Fourier transform infra-red measurements to provide qualitative insight of the bonds at the substrate surface. Differences were also visible after surface pretreatment by plasma or sputter etching with different gases, as changes in interaction bonds as well as in adhesion were observed.     

Characterization of the interface in a carbon-epoxy composite using transmission electron microscopy

Two commercial kinds of unsized pan-based carbon fibres (high strength and high modulus) were subjected to electrochemical treatments (oxidative and non-oxidative) and to a nitrogen plasma. After embedding in an epoxy resin, they were sectioned by ultramicrotomy (diamond knife), and the interface between the resin matrix and the fibre was studied by the lattice fringes method (TEM). The adhesion of the fibre to the matrix depends on the surface treatment and the external microtexture of the fibre. High-modulus fibres (plasma etching) show a good adhesion only when the carbon layers are perpendicular to the interface. High-strength fibres exhibit two types of adhesion. Some treatments yield a surface which mainly presents carbon layers parallel to the interface with a rugosity of about 1–2 nm. The second type of adhesion consists of fibre-matrix interpenetration. In this case carbon layers are slightly exfoliated and perpendicular to the interface. For most treatments, interfacial shear stress was determined by the pull-out test. We found no correlation between TEM observations and shear stress data. Consequently, we were unable to discuss adhesion from the transmission electron microscopy results. It would probably be more reliable to consider the problem with a concept based on interface toughness. However, as a first step, we have defined a new parameter, the contact index, and have shown the relations between the contact index, the morphology of the interface and the interfacial shear stress determined by the pull-out test.