The role of van der Waals forces in adhesion of micromachined surfaces

Interfacial adhesion and friction are important factors in determining the performance and reliability of microelectromechanical systems. We demonstrate that the adhesion of micromachined surfaces is in a regime not considered by standard rough surface adhesion models. At small roughness values, our experiments and models show unambiguously that the adhesion is mainly due to van der Waals dispersion forces acting across extensive non-contacting areas and that it is related to 1/Dave2, where Dave is the average surface separation. These contributions must be considered because of the close proximity of the surfaces, which is a result of the planar deposition technology. At large roughness values, van der Waals forces at contacting asperities become the dominating contributor to the adhesion. In this regime our model calculations converge with standard models in which the real contact area determines the adhesion. We further suggest that topographic correlations between the upper and lower surfaces must be considered to understand adhesion completely.     

Controlling cell destruction using dielectrophoretic forces

Measurements are reported of the main factors, namely the AC voltage frequency and magnitude, that were observed to influence the number of cells destroyed during dielectrophoresis (DEP) experiments on Jurkat T cells and HL60 leukemia cells. Microelectrodes of interdigitated and quadrupolar geometries were used. A field-frequency window has been identified that should be either avoided or utilised, depending on whether or not cell damage is to be minimised or is a desired objective. The width and location of this frequency window depends on the cell type, as defined by cell size, morphology and dielectric properties, and is bounded by two characteristic frequencies. These frequencies are the DEP cross-over frequency, where a cell makes the transition from negative to positive DEP, and a frequency determined by the time constant that controls the frequency dependence of the field induced across the cell membrane. When operating in this frequency window, and for the microelectrode designs used in this work, cell destruction can be minimised by ensuring that cells are not directed by positive DEP to electrode edges where fields exceeding 30-40 kV/m are generated. Alternatively, this field-frequency window can be exploited to selectively destroy specific cell types in a cell mixture.     

Collective cell guidance by cooperative intercellular forces

Cells comprising a tissue migrate as part of a collective. How collective processes are coordinated over large multi-cellular assemblies has remained unclear, however, because mechanical stresses exerted at cell-cell junctions have not been accessible experimentally. We report here maps of these stresses within and between cells comprising a monolayer. Within the cell sheet there arise unanticipated fluctuations of mechanical stress that are severe, emerge spontaneously, and ripple across the monolayer. Within that stress landscape, local cellular migrations follow local orientations of maximal principal stress. Migrations of both endothelial and epithelial monolayers conform to this behaviour, as do breast cancer cell lines before but not after the epithelial-mesenchymal transition. Collective migration in these diverse systems is seen to be governed by a simple but unifying physiological principle: neighbouring cells join forces to transmit appreciable normal stress across the cell-cell junction, but migrate along orientations of minimal intercellular shear stress.     

Steric stabilization and cell adhesion

We present theoretical and experimental arguments supporting the hypothesis that the cell surface glycocalyx may negatively regulate adhesive phenomena. First, it is recalled that a repulsive interaction of several thousands of piconewtons may be generated on a contact area of about 1/100 m² by a combination of electrostatic and entropic forces (steric stabilization). Second, electron microscopical data are reported to provide an estimate of the thickness of the cell coats of murine macrophages and sheep erythrocytes made phagocytable by exposure to glutaraldehyde or specific antibodies. Using conventional carbohydrate staining procedures, it is shown that the total thickness of the electron-dark regions in areas of intercellular contact is lower than the sum of the thicknesses of electron-dark regions on free cell areas. Further, removing negative charges with neuraminidase or neutralizing these charges with polylysine may reduce intermembrane distance in contact areas. Third, it is shown that a decrease of erythrocyte surface charges with neuraminidase increases their adhesion to murine phagocytes under dynamic, not static conditions. It is concluded that a major determinant of steric stabilization is the relative length of adhesion molecules and surface repeller elements, and that repulsion is particularly important under dynamic conditions. Thus, dynamic effects must be included in models of steric stabilization.     

Cellular unbinding forces of initial adhesion processes on nanopatterned surfaces probed with magnetic tweezers

To study the dependence of unbinding forces on the distance of molecularly defined and integrin specific c(-RGDfK-) ligand patches in initial cellular adhesion processes, we developed a magnetic tweezers setup for applying vertical forces of up to 200 pN to rat embryonic fibroblasts. The ligand patch distance is controlled with a hexagonally close packed pattern of biofunctionalized gold nanoparticles prepared by block-copolymer micelle nanolithography. Each gold nanoparticle potentially activates up to one alpha(v)beta(3)-integrin. The distances between the gold nanoparticles determine the separation of individual integrins and thus the assembly of integrin clusters. The results show an increase in cellular unbinding forces from approximately 6 to more than 200 pN for a decreasing ligand distance of 145 to 58 nm after 5 min of cell adhesion. Furthermore, we observe a strong dependence on adhesion time during the first 10 min of cell surface contact suggesting an active, cooperative cell response that is controlled by the spacing between individually activated integrins.     

Endothelial cell adhesion on bioerodable polymers

This paper presents the results of a preliminary screening of a new class of bioerodable polymers, partial esters of alternating copolymers of maleic anhydride and mono-methoxyoligoethyleneglycol vinyl ethers (PAM) for use in engineered vascular tissue. Different initial concentrations of PAM and human serum albumin (HSA) were spin-coated onto glass substrates and the surface properties of the resulting films and their relationship to endothelial cell adhesion was examined. An optimum PAM/HSA blend for use as the cell contact surface of a bioerodable scaffold was identified. © 2001 Kluwer Academic Publishers     

可降解海藻酸钠-壳聚糖支架用于细胞黏附生长的研究

通过调节海藻酸钠与壳聚糖的比例制备了具有不同降解速度的组织工程支架,并以HepG2为细胞模型考察可降解支架对细胞黏附生长的影响.研究表明:壳聚糖含量越高,支架被溶菌酶降解的速度越快,但支架在培养液中的稳定性越高;MTT结果显示细胞在壳聚糖含量为100%和67%的支架中培养时活性较高,但活死染色显示细胞多以分散状态黏附在支架上,降低壳聚糖含量时细胞活性较低且多以聚集形式存在于支架空隙内部.通过调节海藻酸钠与壳聚糖的比例制备出可降解的组织工程支架,可以控制细胞的生长速度及黏附状态,有望为细胞的生长及功能发挥提供更适宜的环境.     

Influence of tableting forces and lubricant concentration on the adhesion strength in complex layer tablets

The strength of adhesion in complex two-layer tablets is assessed using statistical methods with respect to the applied tableting forces for the first layer and for applying the second layer on the first, as well as regarding the fraction of the lubricant. These results, obtained on a single-punch tablet press, are compared with the results for three-layer tablets produced on a rotary press at production scale. The strongest negative influence on adhesion strength was exerted by the amount of lubricant in the central layer. As expected, compression forces for central-layer tableting also had a negative effect, whereas the compression forces for complex layer tableting exerted a positive effect on layer adhesion. The validity of the derived model equation was proved by experiments: It was shown that the adhesion strength in complex layer tablets produced in production scale can be predicted from laboratory-scale experiments. This makes optimization of the formulation and parameter settings at an early stage of development possible.     

Comparison of mesenchymal stem cell and osteosarcoma cell adhesion to hydroxyapatite

Immortalized cells are often used to model the behavior of osteogenic cells on orthopaedic and dental biomaterials. In the current study we compared the adhesive behavior of two osteosarcoma cell lines, MG-63 and Saos-2, with that of mesenchymal stem cells (MSCs) on hydroxyapatite (HA). It was found that osteosarcoma cells demonstrated maximal binding to fibronectin-coated HA, while MSCs alternately preferred HA coated with collagen-I. Interesting, the binding of MG-63 and Saos-2 cells to fibronectin was mediated by both α5 and αv-containing integrin heterodimers, whereas only αv integrins were used by MSCs. Cell spreading was also markedly different for the three cell types. Osteosarcoma cells exhibited optimal spreading on fibronectin, but poor spreading on HA disks coated with fetal bovine serum. In contrast, MSCs spread very well on serum-coated surfaces, but less extensively on fibronectin. Finally, we evaluated integrin expression and found that MSCs have higher levels of α2 integrin subunits relative to MG-63 or Saos-2 cells, which may explain the enhanced adhesion of MSCs on collagen-coated HA. Collectively our results suggest that osteosarcoma cells utilize different mechanisms than MSCs during initial attachment to protein-coated HA, thereby calling into question the suitability of these cell lines as in vitro models for cell/biomaterial interactions.     

Observations of cellular responses to diamagnetic forces acting on cell components

Abstract

The magnetooptical measurements of the properties of living cells have a potentially large impact on cellular engineering and biotechnology because the noninvasive approach to applying magnetic fields on cells enables the detection of the dynamics of intracellular components under natural conditions. In this study, we examine a magnetooptical response in smooth muscle cells exposed to a vertical magnetic field of 5 T. The time course of the linearly polarized light transmittance of cells showed both a gradual decrease and fluctuations during exposure at 5 T. Real-time observations of smooth muscle cells and giant rodlike vesicles revealed that magnetic fields cause morphological changes in the cells and vesicles. In addition, results of the optical transmittance measurement of a fish scale indicate that cellular or tissue components are diamagnetically reoriented by magnetic fields.     

Observations of cellular responses to diamagnetic forces acting on cell components

The magnetooptical measurements of the properties of living cells have a potentially large impact on cellular engineering and biotechnology because the noninvasive approach to applying magnetic fields on cells enables the detection of the dynamics of intracellular components under natural conditions. In this study, we examine a magnetooptical response in smooth muscle cells exposed to a vertical magnetic field of 5 T. The time course of the linearly polarized light transmittance of cells showed both a gradual decrease and fluctuations during exposure at 5 T. Real-time observations of smooth muscle cells and giant rodlike vesicles revealed that magnetic fields cause morphological changes in the cells and vesicles. In addition, results of the optical transmittance measurement of a fish scale indicate that cellular or tissue components are diamagnetically reoriented by magnetic fields.     

Microfluidic-based cell sorting of Francisella tularensis infected macrophages using optical forces

We have extended the principle of optical tweezers as a noninvasive technique to actively sort hydrodynamically focused cells based on their fluorescence signal in a microfluidic device. This micro fluorescence-activated cell sorter (microFACS) uses an infrared laser to laterally deflect cells into a collection channel. Green-labeled macrophages were sorted from a 40/60 ratio mixture at a throughput of 22 cells/s over 30 min achieving a 93% sorting purity and a 60% recovery yield. To rule out potential photoinduced cell damage during optical deflection, we investigated the response of mouse macrophage to brief exposures (<4 ms) of focused 1064-nm laser light (9.6 W at the sample). We found no significant difference in viability, cell proliferation, activation state, and functionality between infrared-exposed and unexposed cells. Activation state was measured by the phosphorylation of ERK and nuclear translocation of NF-kappaB, while functionality was assessed in a similar manner, but after a lipopolysaccharide challenge. To demonstrate the selective nature of optical sorting, we isolated a subpopulation of macrophages highly infected with the fluorescently labeled pathogen Francisella tularensis subsp. novicida. A total of 10,738 infected cells were sorted at a throughput of 11 cells/s with 93% purity and 39% recovery.     

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.     

Microfluidic shear devices for quantitative analysis of cell adhesion

We describe the design, construction, and characterization of microfluidic devices for studying cell adhesion and cell mechanics. The method offers multiple advantages over previous approaches, including a wide range of distractive forces, high-throughput performance, simplicity in experimental setup and control, and potential for integration with other microanalytic modules. By manipulating the geometry and surface chemistry of the microdevices, we are able to vary the shear force and the biochemistry during an experiment. The dynamics of cell detachment under different conditions can be captured simultaneously using time-lapse videomicroscopy. We demonstrate assessment of cell adhesion to fibronectin-coated substrates as a function of the shear stress or fibronectin concentration in microchannels. Furthermore, a combined perfusion-shear device is designed to maintain cell viability for long-term culture as well as to introduce exogenous reagents for biochemical studies of cell adhesion regulation. In agreement with established literature, we show that fibroblasts cultured in the combined device reduced their adhesion strength to the substrate in response to epidermal growth factor stimulation.     

Micro- and nano-patterned substrates to manipulate cell adhesion

Cell adhesion to material surfaces regulates host responses to implanted biomaterials and the performance of cell arrays and biotechnological cell culture supports. Therefore, the engineering of substrates that control cell adhesive interactions is critical to the development of bio-interactive interfaces and biotechnological culture supports. We describe the application of advanced fabrication techniques to engineer substrates with well defined chemistry and topography to manipulate cell adhesive interactions. Microcontact printing of self-assembled monolayers and hot embossing imprint lithography approaches were integrated to manipulate focal adhesion assembly, cell adhesion, and cellular spreading and alignment. These micro- and nanopatterned substrates provide useful tools for the analyses of structure-function relationships in adhesive interactions.     

Topographic cell instructive patterns to control cell adhesion,polarization and migration

Topographic patterns are known to affect cellular processes such as adhesion, migration and differentiation. However, the optimal way to deliver topographic signals to provide cells with precise instructions has not been defined yet. In this work, we hypothesize that topographic patterns may be able to control the sensing and adhesion machinery of cells when their interval features are tuned on the characteristic lengths of filopodial probing and focal adhesions (FAs). Features separated by distance beyond the length of filopodia cannot be readily perceived; therefore, the formation of new adhesions is discouraged. If, however, topographic features are separated by a distance within the reach of filopodia extension, cells can establish contact between adjacent topographic islands. In the latter case, cell adhesion and polarization rely upon the growth of FAs occurring on a specific length scale that depends on the chemical properties of the surface. Topographic patterns and chemical properties may interfere with the growth of FAs, thus making adhesions unstable. To test this hypothesis, we fabricated different micropatterned surfaces displaying feature dimensions and adhesive properties able to interfere with the filopodial sensing and the adhesion maturation, selectively. Our data demonstrate that it is possible to exert a potent control on cell adhesion, elongation and migration by tuning topographic features’ dimensions and surface chemistry.     

Human osteoprogenitor responses to orthopaedic implant: mechanism of cell attachment and cell adhesion

Cell culture models are becoming prevalent in the investigation of tissue responses to implant materials. Cellular attachment and cell adhesion studies can aid in the development of more effective orthopaedic and dental implants. Cell attachment was studied on extracellular matrix proteins (type I, IV collagen, peptide solubilized elastin (PSE), fibronectin laminin). Human osteoprogenitor cells responded differently to these collagenous and non-collagenous proteins. PSE and type I or type IV collagen are the most effective proteins in cellular attachment and cell spreading. Cell behaviour was measured in the presence of macroporous materials (Porites astreoïdes from the West Indies and a bovine hydroxyapatite ceramic ENDOBON^®) and bioartificial connective matrices comprising hydroxyapatite, peptide solubilized elastin, collagen, fibronectin and chondroïtin-6-sulfate, components of the extracellular matrix (ECM). Human osteoprogenitor cells responded differently to the materials tested according to the content of components of ECM. About 40% of attached cells were obtained on the composite materials PSE, collagen, fibronectin and chondroïtin-6-sulfate, and about 10% on the macroporous materials, whatever their porosity and their chemical components. These results demonstrate a need for more effective surface treatment to promote cell attachment, cell spreading and cell growth.     

Non-viral gene delivery regulated by stiffness of cell adhesion substrates

Non-viral gene vectors are commonly used for gene therapy owing to safety concerns with viral vectors. However, non-viral vectors are plagued by low levels of gene transfection and cellular expression. Current efforts to improve the efficiency of non-viral gene delivery are focused on manipulations of the delivery vector, whereas the influence of the cellular environment in DNA uptake is often ignored. The mechanical properties (for example, rigidity) of the substrate to which a cell adheres have been found to mediate many aspects of cell function including proliferation, migration and differentiation, and this suggests that the mechanics of the adhesion substrate may regulate a cell's ability to uptake exogeneous signalling molecules. In this report, we present a critical role for the rigidity of the cell adhesion substrate on the level of gene transfer and expression. The mechanism relates to material control over cell proliferation, and was investigated using a fluorescent resonance energy transfer (FRET) technique. This study provides a new material-based control point for non-viral gene therapy.     

Mesenchymal stem cell adhesion and spreading on nanostructured biomaterials

Bone marrow-derived human mesenchymal stem cells were seeded in serum-free media onto ion beam-deposited nanostructured metalloceramic (Ti-Cr-N) films and plasma-nitrided titanium disks, which were left uncoated as well as precoated with fetal bovine serum. Precoating the disks with serum appears to stimulate cell spreading on both the titanium nitride and metalloceramic materials for as little as 1 hour incubation time. The implication is that both of these materials can adsorb serum proteins in amounts sufficient to influence cell adhesion and spreading for potentially improved in vivo response of orthopedic and dental implants. The materials in this study may prove to exhibit enhanced biological and mechanical properties when compared to conventional micron-scale implant materials such as titanium or cobalt-chrome alloys.