Cell Adhesion Strength Research Articles

Maggots and wound healing: an investigation of the effects of secretions from Lucilia sericata larvae upon the migration of human dermal fibroblasts over a fibronectin‐coated surface

Lucilia sericata larvae, or greenbottle fly maggots, placed within chronic wounds have been observed to remove necrotic tissue and infection. They are also believed to actively promote granulation tissue formation. Interactions between fibroblasts and the surrounding extracellular matrix play a crucial role in tissue formation, influencing fibroblast proliferation, migration, and tissue remodeling. For example, the strength of cell adhesion to surfaces coated with extracellular matrix influences cell motility. L. sericata larval excretory/secretory products having previously been shown to modify fibroblast adhesion to collagen and particularly fibronectin, it was hypothesized that these products would alter fibroblast migration. This was investigated using a two-dimensional in vitro wound assay, time-lapse digital photography, enzyme class-specific substrates and inhibitors, and gel electrophoresis. Results showed that L. sericata excretory/secretory products promoted fibroblast migration upon a fibronectin-coated surface. This was related to the degradation of fibronectin by serine proteinases within maggot excretion/secretions. The presence of a metalloproteinase activity may also have played a role. Thus, a possible mechanism by which maggots enhance tissue formation within wounds may be via the promotion of fibroblast motility, providing for a wider distribution of viable fibroblasts.

A Live Bioprobe for Studying Diatom-Surface Interactions

Atomic force microscopy has been employed to compare the adhesion of Navicula species I diatoms to surfaces of a hydrophobic elastomer, Intersleek, and a hydrophilic mineral, mica. This was accomplished using tipless atomic force microscopy cantilevers functionalized with live diatom cells. Both surfaces were tested with the same diatom bioprobe. Force versus distance curves generated during these experiments revealed comparable cell adhesion strengths on Intersleek and mica, indicating that Navicula diatoms secrete extracellular polymeric substances with hydrophobic and hydrophilic properties. A statistical analysis of force curves was carried out and the average values of works of detachment of a diatom from Intersleek and mica surfaces were determined.

Corneal epithelial adhesion strength to tethered‐protein/peptide modified hydrogel surfaces

In this study, we investigated the suitability of microjet impingement for use on hydrogel materials to determine the cellular adhesion strength of corneal epithelial cells grown on novel hydrogels with extracellular matrix proteins (laminin and/or fibronectin) or a peptide sequence (fibronectin adhesion promoting peptide, FAP) tethered to their surface with poly(ethylene glycol) chains. The deformation of the hydrogel surface in response to the force of the microjet was analyzed both visually and mathematically. After the results of these experiments and calculations determined that no deformation occurred and that the pressure required for indentation (1.25 x 10(6) Pa) was three factors of 10 greater than the maximum pressure of the microjet, the relative mean adhesion strength of primary rabbit corneal epithelial cells grown on the novel poly(2-hydroxyethyl methacrylate-co-methacrylic acid) hydrogels was determined and compared with that of the same type of cells grown on control glass surfaces. Only confluent cell layers were tested. Cells grown on control glass surfaces adhered with a mean relative adhesion strength of 488 +/- 28 dynes/cm2. Under identical conditions, cells grown on laminin- and FAP-tethered hydrogel surfaces were unable to be removed, indicating an adhesion strength greater than 516 dynes/cm2. Cells grown on fibronectin- and fibronectin/laminin (1:1)-tethered surfaces showed significantly lower relative adhesion strengths (201 +/- 50 and 189 +/- 11 dynes/cm2, respectively), compared with laminin- and FAP-tethered surfaces (p = 0.001). Our results demonstrate that the microjet impingement method of cell adhesion analysis is applicable to hydrogel substrates. Additionally, analysis of our test surfaces indicates that fibronectin tethered to this hydrogel in the quantity and by the method used here does not induce stable ligand/receptor bonding to the epithelial cell membrane to the same degree as does laminin or FAP.

The changes of cell adhesion to collagen-coated Al 2O 3 by ion bombardment

Immobilization of protein to artificial materials by ion bombardment to enhance cell adhesion has been studied. Type-I collagen was coated onto alumina and bombarded with 50 keV He + ions at doses between 2×10 12 and 1×10 15 ions/cm 2. The collagen-coated surfaces were mounted on parallel-plate flow chambers, with a flowing shear stress of 2 Pa for 1 h, in a flow system prepared for tests of collagen adhesive strength. After bovine aorta endothelial cells (BAEC) were cultured onto ion-bombarded specimens in a culture medium, a cell adhesive strength test was carried out under the same experimental conditions as the collagen adhesive strength test. The result was that the collagen adhesion strength decreased for specimens bombarded with less than 1×10 13 ions/cm 2. As the dose increased, the collagen adhesive strength also increased. The cell adhesive strength of He +-ion-bombarded collagen-coated alumina increased at a dose of 1×10 14 ions/cm 2 and decreased at doses of 1×10 13 and 1×10 15 ions/cm 2.

Tissue and cell engineering

The extracellular environment (ECM) comprises multiple physical and chemical cues that are integrated into adhesion signaling pathways and modulate cell responses fundamental for tissue regeneration. In this chapter, we will outline the importance of ECM molecule and ECM-synthetic ligand presentations in biomaterial design. In particular, we will discuss the use of different chemical linkers that allow the control over surface immobilization, orientation, and release of such molecules and ligands. We will next present different strategies for the delivery of adhesive ligands and growth factors on material surfaces, with the aim of enhancing cell adhesion strength and signaling. A final discussion will be centered on challenges in the design of surfaces to precisely guide cell responses during tissue growth and wound healing.

The effect of non-specific interactions on cellular adhesion using model surfaces

The contribution of non-specific interactions between cells and model functional surfaces was measured using a spinning disc apparatus. These model functional surfaces were created using self-assembled monolayers (SAM) of alkylsilanes terminated with epoxide, carboxyl (COOH), amine (NH2), and methyl (CH3) groups. These SAMs were characterized using ellipsometry, atomic force microscopy, contact angle goniometry, and X-ray photoelectron spectroscopy to confirm the presence of well-formed monolayers of expected physicochemical characteristics. All substrates also demonstrated excellent stability under prolonged exposure (up to 18h) to aqueous conditions. The adhesion strength of K100 erythroleukemia cells to the functional substrates followed the trend: CH3 < COOH ≈ epoxide ≪ NH2. The NH2 SAM surface exhibited nearly an order of magnitude greater adhesion strength than the other SAMs and this non-specific effect exceeded the adhesion measured when RGD tri-peptides were also immobilized on the surface. These findings illustrate the importance of substrate selection in quantitative studies of peptide-mediated cellular adhesion.

Quantifying cell-adhesion strength with micropipette manipulation: principle and application.

Quantifying cell-adhesion strength is of great importance in biology and medicine. Cell-adhesion strength can be characterized by separating two adherent cells and determining the force required to do so, or by measuring the lifetime of a receptor-ligand bond that mediates cell adhesion. To this end, several micropipette-based experimental techniques that operate at both cellular and molecular levels have been developed over the past few decades. In this review, we provide an overview of three of these techniques, i.e., the step-pressure technique (SPT), the biomembrane-force probe (BFP), and the micropipette-aspiration technique (MAT). More detailed discussion will be given about the requirements and applications of the MAT.

Hollow-fiber assay for ligand-mediated cell adhesion.

The investigation of receptor-ligand interactions in the cellular context presents significant technical challenges, first, to immobilize the ligand in a manner that preserves functional properties and, second, to relate ligand properties to cell adhesion and other cellular processes. Ligand-mediated cell adhesion was characterized by the development of a cellulose hollow-fiber adhesion assay in which ligand (protein A) was immobilized onto the cellulose membrane as a recombinant fusion protein containing a cellulose-binding domain affinity tag. Modules containing single cellulose hollow fibers were connected to a micro-flow system for cell deposition and detachment with fluid shear stress. The cell adhesion process that occurred inside a segment of hollow fiber was observed in real time by using an inverted microscope equipped with a CCD camera and digital frame grabber. Image analysis software was developed to count cells and record digital images. Cell adhesion strength was characterized by counting the number of cells that were detached by application of fluid shear stress with values that ranged from 2.3 to 185 dyne/cm2. The median shear stress of detachment of KG1a cells was directly related to the duration of membrane contact and the amount of immobilized monoclonal antibody (anti-CD34). The hollow-fiber assay provides a general method to determine functional properties of molecular domains that interact with cell surface receptors and markers.

The conditional inactivation of the beta-catenin gene in endothelial cells causes a defective vascular pattern and increased vascular fragility.

Using the Cre/loxP system we conditionally inactivated β-catenin in endothelial cells. We found that early phases of vasculogenesis and angiogenesis were not affected in mutant embryos; however, vascular patterning in the head, vitelline, umbilical vessels, and the placenta was altered. In addition, in many regions, the vascular lumen was irregular with the formation of lacunae at bifurcations, vessels were frequently hemorrhagic, and fluid extravasation in the pericardial cavity was observed. Cultured β-catenin −/− endothelial cells showed a different organization of intercellular junctions with a decrease in α-catenin in favor of desmoplakin and marked changes in actin cytoskeleton. These changes paralleled a decrease in cell–cell adhesion strength and an increase in paracellular permeability. We conclude that in vivo, the absence of β-catenin significantly reduces the capacity of endothelial cells to maintain intercellular contacts. This may become more marked when the vessels are exposed to high or turbulent flow, such as at bifurcations or in the beating heart, leading to fluid leakage or hemorrhages.

Thermo-Responsive PNiPAAm-g-PEG Films for Controlled Cell Detachment

A series of graft copolymers consisting of either poly(N-isopropylacrylamide) (PNiPAAm) or poly(N,N-diethylacrylamide) (PDEAAm) as a thermo-responsive component in the polymer backbone and poly(ethyleneglycol) (PEG) were immobilized as thin films and cross-linked on a fluoropolymer substrate using low-pressure argon plasma treatment. The surface-immobilized hydrogels exhibit a transition from partially collapsed to completely swollen, which is in the range of 32-35 degrees C and corresponds to the lower critical solution temperature of the soluble polymers. The hydrogels were used as cell carriers in culture experiments with L929 mouse fibroblast cells to probe for cell adhesion, proliferation, and temperature-dependent detachment of cell layers. The fibroblast cells adhere, spread, and proliferate on the hydrogel layers at 37 degrees C and become completely detached after reducing the temperature by 3 K. The cell release characteristics were further correlated to the swelling and collapsing behavior of the hydrogel films and the polymer solutions as measured in PBS solution and RPMI cell cultivation medium. It could be shown that, long before the swelling has completed upon temperature reduction, the cells detach. This can be attributed to the large content of PEG present in the hydrogel, which weaken the cell adhesion strength to the hydrogel layers.

Adsorption-Induced Conformational Changes in Fibronectin Due to Interactions with Well-Defined Surface Chemistries

Protein adsorption onto synthetic materials influences cell adhesion and signaling events that direct cell function in numerous biomedical applications. Adsorption of fibronectin (FN) to different surfaces alters protein structure and modulates α5β1 integrin binding, cell adhesion, cell spreading, and cell migration. In the present study, self-assembled monolayers of alkanethiols on Au were used to analyze the effects of surface chemistry (CH3, OH, NH2, and COOH) on the adsorption of a recombinant fragment of FN, FNIII7-10, that incorporates both the synergy and RGD cell binding motifs. Surface chemistry potentiated differential FNIII7-10 adsorption kinetics and adsorbed structure as determined by surface plasmon resonance spectroscopy and antibody binding assays. FNIII7-10 functional activity, determined by cell adhesion strength, was modulated in a fashion consistent with these structural changes (OH = NH2 > COOH > CH3). However, these changes in protein parameters did not correlate simply to differences in surface hydrophobicity, indicating that additional surface parameters influence protein adsorption. These results demonstrate that surface chemistry modulates adsorbed protein structure and activity and establish a relationship between surface-dependent changes in structural domains of FNIII7-10 and functional activity.

Surface chemistry modulates fibronectin conformation and directs integrin binding and specificity to control cell adhesion.

Integrin-mediated cell adhesion to proteins adsorbed onto synthetic surfaces anchors cells and triggers signals that direct cell function. In the case of fibronectin (Fn), adsorption onto substrates of varying properties alters its conformation/structure and its ability to support cell adhesion. In the present study, self-assembled monolayers (SAMs) of alkanethiols on gold were used as model surfaces to investigate the effects of surface chemistry on Fn adsorption, integrin binding, and cell adhesion. SAMs presenting terminal CH(3), OH, COOH, and NH(2) functionalities modulated adsorbed Fn conformation as determined through differences in the binding affinities of monoclonal antibodies raised against the central cell-binding domain (OH > COOH = NH(2) > CH(3)). Binding of alpha(5)beta(1) integrin to adsorbed Fn was controlled by SAM surface chemistry in a manner consistent with antibody binding (OH > COOH = NH(2) > CH(3)), whereas alpha(V) integrin binding followed the trend: COOH >> OH = NH(2) = CH(3), demonstrating alpha(5)beta(1) integrin specificity for Fn adsorbed onto the NH(2) and OH SAMs. Cell adhesion strength to Fn-coated SAMs correlated with alpha(5)beta(1) integrin binding (OH > COOH = NH(2) > CH(3)), and experiments with function-perturbing antibodies demonstrated that this receptor provides the dominant adhesion mechanism in this cell model. This work establishes an experimental framework to analyze adhesive mechanisms controlling cell-surface interactions and provides a general strategy of surface-directed control of adsorbed protein activity to manipulate cell function in biomaterial and biotechnological applications.

Biophysical methods for monitoring cell-substrate interactions in drug discovery.

Cell-substrate interactions are implicated in a number of relevant pathways for drug targets such as angiogenesis, arteriosclerosis, chronic inflammatory diseases, and carcinogenesis. Moreover, cell adhesion and cytoskeletal activity have served as valuable indicators for cytotoxicity, cell density, and cell morphology. This review focuses on impedance, capacitance, resonant frequency, and refractive index measurements for monitoring cell adhesion in real time and without the use of cell labeling. ECIS, QCM, and OWLS deliver information about the cell-substrate interactions, cell-cell contact, and the strength of cell adhesion. Because of high sensitivity of these assays, events down to the single cell level have been observed, and resolutions at the nanometer level of cell-substrate distances have been achieved. The physical principles, including assay sensitivity and selectivity, are discussed in the context of cellular pathways of cell adhesion and migration. With the miniaturization of these types of sensors, cell migration and adhesion measurements in combination with specific fluorescent assays might thus deliver a high-content platform for drug development.

Stick and grip: measurement systems and quantitative analyses of integrin-mediated cell adhesion strength.

Cell adhesion to extracellular matrix components involves integrin receptor-ligand binding and adhesion strengthening, comprising receptor clustering, cytoskeletal interactions, and cell spreading. Although elucidation of the biochemical events in adhesive interactions is rapidly advancing, the mechanical processes and mechanisms of adhesion strengthening remain poorly understood. Because the biochemical and biophysical processes in adhesive interactions are tightly coupled, mechanical analyses of adhesion strength provide critical information on structure-function relationships. This review focuses on (a) measurement systems for cell adhesion strength and (b) quantitative analyses of integrin-mediated strengthening to extracellular matrix components.

ビーズをAFMプローブとして用いた細胞接着強度の定量的測定法

ビーズをAFMプローブとして用いた細胞接着強度の定量的測定法

High CO2 atmosphere modulating the phenolic response associated with cell adhesion and hardening of Annona cherimola fruit stored at chilling temperature.

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5.) activity, tanning ability, and polyphenols levels were measured in cherimoya (Annona cherimola Mill.) fruit treated with 20% CO(2) + 20% O(2) + 60% N(2) for 1, 3, or 6 days during chilling temperature (6 degrees C) storage. The residual effect of CO(2) after transfer to air was also studied. These observations were correlated with texture and cellular characteristics, visualized by cryo-SEM. Tanning ability and the early increase in tannin polyphenols induced by chilling temperature were reduced by CO(2) treatment. Conversely, high CO(2) atmosphere enhanced the nontannin polyphenol fraction as compared with fruit stored in air. Lignin accumulation and PAL activation observed in untreated fruit after prolonged storage at chilling temperature were prevented by high CO(2). Moreover, the restraining effect on lignification was less effective when the CO(2) treatment was prolonged for 6 days. In addition, fruits held at these conditions had greater firmness and the histological characterization of the separation between cells was similar to that in untreated fruits. We conclude that CO(2) treatment modulates the phenolic response that seems to regulate the strength of cell adhesion and so to prevent hardening caused by chilling temperature storage.

Effect of adsorbed fibronectin concentration on cell adhesion and deformation under shear on hydrophobic surfaces.

To facilitate tissue integration with biomaterials proteins and peptides frequently are immobilized on the biomaterial surface. In particular, extracellular matrix proteins--which interact specifically with integrin adhesion receptors on the cell surface--can stimulate initial cell attachment by serving both as a ligand for receptor-mediated attachment and as a stimulant of focal adhesion formation and cytoskeletal reorganization. Consequently, the strength of cell adhesion should depend both on the strength of cell/surface contacts and cytoskeleton-dependent properties of the cell (i.e., morphology, compliance). To examine this dual role of extracellular matrix proteins, murine fibroblasts were seeded onto self-assembled monolayers (SAMs) of dodecanethiolate coated with 0 to 0.45 microg/cm(2) of fibronectin (Fn) and then detached by hydrodynamic shear using a radial-flow chamber (RFC). Cell adhesion was characterized in terms of the critical wall shear stress for detachment (tau(wc)), and the compliance was evaluated from measurements of cell displacement and elongation as a function of the fibronectin concentration. Critical wall shear stress and cell displacement were found to be insensitive to Fn at concentrations below 0.23 microg/cm(2) while above this threshold tau(wc) increased and displacement decreased with increasing Fn concentration. Elongation of the cells in the direction of flow was independent of Fn concentration, but correlated linearly with tau(wc) for Fn densities below 0.23 microg/cm(2). These studies show that Fn concentration affects both tau(wc) and cell displacement under shear, and that tau(wc) is sensitive to cell compliance. In addition, they suggest that the dominant mechanism of cell detachment from hydrophobic substrates involves cell displacement.

Quantification of fibronectin and cell surface interactions by AFM

The cell adhesion strength between FN and the living cell was successfully examined using an AFM cantilever having a FN-coated spherical microbead as a probe. The microbead was used to increase (by some 55-fold) the area of contact with the cell surface. Fibronectin (FN) was chosen as the ligand and immobilized on the bead. The area under the force-extension curve obtained during retraction of the FN-coated tip from the cell surface after contact was defined as ‘separation work’ and used as a parameter to evaluate the strength of the interaction. In previous studies, final rupture forces were taken to represent the strength of interactions, but in the present study using a large contact area, separation work was expected to reflect the overall adhesion strength in the contact area. The mean separation work between a FN-coated bead and mouse fibroblast cell was 0.69±0.45×10 −17 J/μm 2. In the presence of 0.1 mg/ml FN in the scanning solution, this value decreased to 0.10±0.08×10 −17 J/μm 2. The separation work between an unmodified microbead and cell was calculated to be 0.15±0.13×10 −17 J/μm 2. Mapping of the separation work over a limited area of the cell surface was found to be reproducible for six repeated scans and the distribution of FN-binding sites on the cell surface was investigated by mapping over a large area. The results showed that separation work is a good measure of specific interactions between FN and the cell surface, and the system developed here is a promising quantitative method to evaluate cell adhesion force.

PR-39 coordinates changes in vascular smooth muscle cell adhesive strength and locomotion by modulating cell surface heparan sulfate-matrix interactions.

PR-39 is proline-rich peptide produced at sites of tissue injury. While the functional properties of this peptide have not been fully defined, PR-39 may be an important regulator of processes related to cell-matrix adhesion since it reportedly upregulates syndecan-4, which is a critical determinant of focal adhesion formation. The ability of PR-39 to modulate the adhesion and chemokinetic migration behavior of arterial smooth muscle cells (SMCs) in a fashion coordinated with syndecan-4 expression was investigated. Treatment of SMCs with PR-39 did not alter syndecan-1 mRNA, but did induce a two-fold increase in syndecan-4 mRNA (P < 0.0001) and significantly enhanced cell surface expression of both syndecan-4 (P < 0.01) and heparan sulfate (HS) (P < 0.05). These observations were consistent with an observed increase in cell-matrix adhesive strength (P < 0.05) and a reduction in cell speed (P < 0.01) on fibronectin-coated substrates. Incubation of PR-39 treated cells with a soluble fibronectin derived heparin-binding peptide, as a competitive inhibitor of heparan sulfate/matrix interactions, abolished these effects. These data suggest that PR-39 mediated alterations of cell adhesion and motility may be related, in part, to the increased expression of heparan sulfate glycosaminoglycans (GAGs) that accompany the upregulation of cell surface syndecan-4. Furthermore, this investigation supports the notion that factors which control syndecan-4 expression may play an important role in regulating adhesion related cell processes.

Crystal structure of the M-fragment of alpha-catenin: implications for modulation of cell adhesion.

The cytoskeletal protein alpha-catenin, which shares structural similarity with vinculin, is required for cadherin-mediated cell adhesion, and functions to modulate cell adhesive strength and to link the cadherins to the actin-based cytoskeleton. Here we describe the crystal structure of a region of alpha-catenin (residues 377-633) termed the M-fragment. The M-fragment is composed of a tandem repeat of two antiparallel four-helix bundles of virtually identical architectures that are related in structure to the dimerization domain of alpha-catenin and the tail region of vinculin. These results suggest that alpha-catenin is composed of repeating antiparallel helical domains. The region of alpha-catenin previously defined as an adhesion modulation domain corresponds to the C-terminal four-helix bundle of the M-fragment, and in the crystal lattice these domains exist as dimers. Evidence for dimerization of the M-fragment of alpha-catenin in solution was detected by chemical cross-linking experiments. The tendency of the adhesion modulation domain to form dimers may explain its biological activity of promoting cell-cell adhesiveness by inducing lateral dimerization of the associated cadherin molecule.