The strength of the protein-material interaction determines cell fate (original) (raw)
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Microscopy Research and Technique, 2017
Fibronectin is an extracellular matrix protein that is involved in cell adhesion, growth, migration, differentiation, and wound healing. Fibronectin coatings are currently used in many laboratories for biomedical and biotechnology purposes. In this study we have investigated the adhesion and mechanical properties of fibronectin coatings. The coatings were also used to study the role of the residence time and the influence of the loading rate in nonspecific interactions. The results showed that the adhesion force between silica and fibronectin increased with loading rate delivering similar values for residence times of 1 and 2 s. Further analysis indicated that the distance to the transition state was about 0.5 nm. Moreover, the adhesion force did not vary with the loading rate for contact time of 0 s. The unfolding of fibronectin domains also depended of the Dwell time (no unfolding events were observed for zero residence time). Applied loads of 2 nN were able to stretch the fibrone...
Cell Adhesion Strength Increases Linearly with Adsorbed Fibronectin Surface Density
Tissue Engineering, 1997
Cell adhesion is involved in numerous physiological processes and is important to biotechnological applications, including tissue engineering and development of artificial organs. The relationship between cell adhesion strength and fibronectin (Fn) surface density was analyzed using a spinning disk device that applied a linear range of forces to attached cells under uniform surface chemical conditions. ROS 17/2.8 cells were seeded onto Fn-coated glass substrates for 15 min. Cells were then subjected to detachment forces for 10 min and adherent cells were counted at different radial distances. The fraction of adherent cells decreased non-linearly with applied force, and the resulting detachment profile was accurately described by a sigmoidal curve. Analysis of detachment profiles for different Fn concentrations and quantitative Fn adsorption measurements revealed that, for short attachment times, cell adhesion strength increased linearly with Fn surface density. This linear dependence of attachment strength on adsorbed Fn was observed for two different glasses, a non-reactive substrate and a surface-active glass, suggesting that this relationship is not substrate specific. The increases in adhesion strength were effectively blocked by a monoclonal antibody directed against the RGD cell binding domain. The linear relationship between attachment strength and ligand density is consistent with theoretical models for initial receptormediated adhesion and suggests the absence of cooperative receptor-ligand binding during the initial phases of cell adhesion.
Subtle variations in polymer chemistry modulate substrate stiffness and fibronectin activity
Soft Matter, 2010
A family of polymer substrates which consists of a vinyl backbone chain with the side groups -COO(CH 2 ) x CH 3 , with x ¼ 0, 1, 3, 5 was prepared. Substrates with decreasing stiffness, characterised by the elastic modulus at 37 C, and similar chemical groups were obtained. Firstly, we have investigated whether these minute variations in polymer chemistry lead to differences in fibronectin (FN) adsorption: the same FN density was obtained on every substrate (450 ng cm À2 ) but the supramolecular organisation of the protein at the material interface, as obtained with AFM, was different for x ¼ 0 and the other surfaces (x ¼ 1, 3, 5). Consequently, this allows one to use a set of substrates (x ¼ 1, 3, 5) to investigate the effect of substrate stiffness on cell behavior as the unique physical parameter, i.e. after ruling out any influence of the length of the side group on protein conformation. Moreover, the importance of investigating the intermediate layer of proteins at the cellmaterial interface is stressed: the effect of x ¼ 0 and x ¼ 1 on cell behavior cannot be ascribed to the different stiffness of the substrate anymore, since the biological activity of the protein on the material surface was also different. Afterwards, initial cellular interaction was investigated using MC3T3-E1 osteoblasts-like cells and focusing on actin cytoskeleton development, focal adhesion formation and the ability of cells to reorganize the adsorbed FN layer on the different substrates. Image analysis was used to quantify the frequency distribution of the focal plaques, which revealed broader distributions on the stiffer substrates, with formation of larger focal plaques revealing that cells exert higher forces on stiffer substrates.
Colloids and Surfaces B-biointerfaces, 2010
Fibronectin (FN) fibrillogenesis is a cell-mediated process involving integrin activation that results in conformational changes of FN molecules and the organization of actin cytoskeleton. A similar process can be induced by some particular chemistries in the absence of cells, e.g., poly(ethyl acrylate) (PEA), which enhance FN-FN interactions leading to the formation of a biologically active network on the material surface. We have investigated the organization of a recombinant fragment of fibronectin (FNIII 7-10 ) upon adsorption on this particular chemistry, PEA. Atomic force microscopy (AFM) was used to identify individual molecules of the fragment after adsorption, as well as the evolution of the distribution of adsorbed molecules on the surface of the material as the concentration of the adsorbing solution increased. Globular molecules that turn into small aggregates were found as a function of solution concentration. Above a threshold concentration of the adsorbing solution (50 g/mL) an interconnected network of the FNIII 7-10 fragment is obtained on the material surface. The bioavailability of specific cell adhesion domains, including RGD, within the molecules was higher on PEA than on the control glass. The biological activity of the fragment was further investigated by evaluating focal adhesion formation and actin cytoskeleton for MC3T3-E1 osteoblast-like cells. Well-developed focal adhesion complexes and insertions of actin stress fibers were found on PEA in a similar way as it happens in the control SAM-OH. Moreover, increasing the hydrophilicity of the surface by incorporating -OH groups led to globular molecules of the fragment homogeneously distributed throughout the surface; and the cell-material interaction is reduced as depicted by the lack of well-developed focal plaques and actin cytoskeleton. (A.J. García), masalsan@fis.upv.es (M. Salmerón-Sánchez).
Colloids and Surfaces B: Biointerfaces, 2012
Using single-cell force spectroscopy, we compared the initial adhesion of L929 fibroblasts to planar and nanostructured silicon substrates as a function of fibronectin concentration. The nanostructures were periodically grooved with a symmetric groove-summit period of 180 nm and a groove depth of 120 nm. Cell adhesion strength to the bare nanostructure was lower (79% ± 13%) than to the planar substrate, which we attribute to reduced contact area. After pre-incubation with a low fibronectin concentration (5 g/ml) the adhesion strengths to both surfaces increased, with adhesion strength on the nanostructure outweighing that of the planar substrate by 133% ± 14%. At a high fibronectin concentration (25 g/ml) the adhesion strengths on both surfaces further increased and showed wide variations. In parallel, the nanostructure lost its clear advantage over the planar substrate. Our results demonstrate that cell adhesion is influenced by substrate topography and fibronectin, which mediate the interplay between specific interactions, non-specific interactions, and cell mechanics. Two parallel processes govern the initial adhesion strength: the detachment of the cell body from the substrate and the extraction of tethers from the cell membrane. The duration of the latter process is determined by tether lifetimes, and is a major contributor to the overall work required for cell-substrate detachment. Cell body detachment and tether lifetimes are affected by surface topography and may be strongly modulated by the presence of adsorbed proteins, whereas the tether extraction forces remained unchanged by these factors.
Transmembrane protein integrins play a key role in cell adhesion. Cell−biomaterial interactions are affected by integrin expression and conformation, which are actively controlled by cells. Although integrin structure and function have been studied in detail, quantitative analyses of integrin-mediated cell−biomaterial interactions are still scarce. Here, we have used atomic force spectroscopy to study how integrin distribution and activation (via intracellular mechanisms in living cells or by divalent cations) affect the interaction of human pluripotent stem cells (WA07) and human hepatocarcinoma cells (HepG2) with promising biomaterials human recombinant laminin-521 (LN-521) and cellulose nanofibrils (CNF). Cell adhesion to LN-521-coated probes was remarkably influenced by cell viability, divalent cations, and integrin density in WA07 colonies, indicating that specific bonds between LN-521 and activated integrins play a significant role in the interactions between LN-521 and HepG2 and WA07 cells. In contrast, the interactions between CNF and cells were nonspecific and not influenced by cell viability or the presence of divalent cations. These results shed light on the underlying mechanisms of cell adhesion, with direct impact on cell culture and tissue engineering applications.
Journal of Biomedical Materials Research, 2003
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 Ͼ COOH ϭ NH 2 Ͼ CH 3 ). Binding of ␣ 5  1 integrin to adsorbed Fn was controlled by SAM surface chemistry in a manner consistent with antibody binding (OH Ͼ COOH ϭ NH 2 Ͼ CH 3 ), whereas ␣ V integrin binding followed the trend:
Journal of Biomedical Materials Research Part A, 2009
The effect of polystyrene surface polarity on the conformation of adsorbed fibronectin (FN) has been studied with atomic force microscopy. We demonstrated that bare sulfonated and nonsulfonated polystyrene surfaces featured similar topographies. After the FN adsorption, direct comparison of both types of substrata revealed drastically different topographies, roughness values, and also cell-adhesive properties. This was interpreted in terms of FN conformational changes induced by the surface polarity. At high-solute FN concentrations the multilayer FN adsorption took place resulting, for the sulfonated substratum, in an increase of surface roughness, whereas for the nonsulfonated one the roughness was approximately stable. Conversely, the FN conformation characteristic for the first saturative layer tended to be conserved in the consecutive layers, as evidenced by height histograms. The height of individual FN molecules indicated, consonantly with the derived thickness of the adsorbed protein layer (the latter value being 1.4 nm and 0.6 nm, respectively, for an unmodified and sulfonated polystyrene surface), that molecules are flattened on polar surfaces and more compact on nonsulfonated ones. It was also demonstrated that the FN adsorption and conformation on polymeric substrata, and hence the resultant cell-adhesive properties, depended on the chemistry of the original surface rather than on its topography. Our results also demonstrated the ability of surface polarity to influence the protein conformation and its associated biological activity.
Proceedings of the National Academy of Sciences, 2005
The mechanical properties of cell adhesion substrates regulate cell phenotype, but the mechanism of this relation is currently unclear. It may involve the magnitude of traction force applied by the cell, and͞or the ability of the cells to rearrange the cell adhesion molecules presented from the material. In this study, we describe a FRET technique that can be used to evaluate the mechanics of cell-material interactions at the molecular level and simultaneously quantify the cell-based nanoscale rearrangement of the material itself. We found that these events depended on the mechanical rigidity of the adhesion substrate. Furthermore, both the proliferation and differentiation of preosteoblasts (MC3T3-E1) correlated to the magnitude of force that cells generate to cluster the cell adhesion ligands, but not the extent of ligand clustering. Together, these data demonstrate the utility of FRET in analyzing cell-material interactions, and suggest that regulation of phenotype with substrate stiffness is related to alterations in cellular traction forces.
Biomaterials, 2006
The ability of fibronectin (Fn) to mediate cell adhesion through binding to a 5 b 1 integrins is dependent on the conditions of its adsorption to the surface. Using a model system of alkylsilane SAMs with different functional groups (X ¼ OH, COOH, NH 2 and CH 3 ) and an erythroleukemia cell line expressing a single integrin (a 5 b 1 ), the effect of surface properties on the cellular adhesion with adsorbed Fn layers was investigated. 125 I-labeled Fn, a modified biochemical cross-linking/extraction technique and a spinning disc apparatus were combined to quantify the Fn adsorption, integrin binding and adhesion strength, respectively. This methodology allows for a binding equilibrium analysis that more closely reflects cellular adhesion found in stable tissue constructs in vivo. Differences in detachment strength and integrin binding were explained in terms of changes in the adhesion constant (c, related to affinity) and binding efficiency of the adsorbed Fn for the a 5 b 1 integrins (CH 3 ENH 2 oCOOHEOH) and the resulting average bond strength. Fn interacted more strongly with a 5 b 1 integrins when adsorbed on COOH vs. OH surfaces suggesting that negative charge may be a critical component of inducing efficient cellular adhesion. As evident by the low c values, Fn adsorbed on NH 2 and CH 3 surfaces interacted inefficiently with a 5 b 1 integrins and also possessed significant non-specific components to adhesion. Lastly, comparison of cellular adhesion to Fn adsorbed onto smooth and rough surfaces showed that nano-scale roughness altered cellular adhesion by increasing the surface density of adsorbed Fn. r