Role of fibrinogen conformation in platelet activation

Platelet adhesion and activation induced by fibrinogen (Fbg) coating on polysaccharide layers of hyaluronic acid (Hyal) and its sulfated derivative (HyalS) were analyzed. Hyal or HyalS was coated and grafted on the glass substrate using a photolithographic method. The Fbg coating was achieved by two different routes: the immobilization of Fbg by means of covalent bond to the polysaccharide layers and the mere adsorption of Fbg to Hyal and HyalS surfaces. Platelet adhesion and activation to the surfaces were evaluated using, respectively, scanning electron microscopy (SEM) and quantifying the release of Platelet Factor 4 by ELISA. The method used for the coating of the surfaces with the Fbg influenced the platelet response. In fact, platelet adhesion and activation took place on surfaces covered by bound Fbg but not on those containing adsorbed Fbg. To explain this difference, the molecular mechanism involved in the Fbg--platelet interaction was investigated blocking platelet membrane receptors by monoclonal antibodies. Because the interaction between Fbg and the GPIIb/IIIa platelet membrane receptor was the only molecular pathway involved, Fbg conformation after the interaction (adsorption or binding) with the Hyal and the HyalS chains and the role of serum proteins adsorbed on the Fbg containing surfaces were accurately analyzed. Both adsorbed and bound Fbg prevented the adsorption of further serum proteins; consequently, a direct interaction between Fbg and platelets was supposed and the different platelet behavior was ascribed to the different conformational changes that occurred after the adsorption and the chemical binding of the Fbg to the Hyal and HyalS surfaces.     

A novel strategy to obtain a hyaluronan monolayer on solid substrates

The aim of this study was to find a novel simple method to obtain polysaccharide ultrathin layers on solid substrates to investigate the interaction between the surface and the biological environment. A Hyaluronan (Hyal) monolayer with a well-defined chemistry was obtained by exploiting the capability of organosilanes to spontaneously adhere onto glass surfaces. A silane alkylic chain was conjugated with Hyal, and the derivatized polysaccharide was allowed to spontaneously adhere onto a glass surface. The elemental analysis of the modified polysaccharide demonstrated that one out of five disaccharide units was conjugated with the alkyl silane chain, corresponding to a substitution degree of the carboxylate groups of approximately 20%. The film of the modified polysaccharide was characterized by means of X-ray photoelectron spectroscopy (XPS), water contact angle, and atomic force microscopy (AFM) measurements. XPS analysis demonstrated that we obtained a Hyal layer with a thickness of about 2.0 nm corresponding to a Hyal monolayer. The Hyal-coated surfaces appeared to be rather smooth and highly hydrophilic and showed significant resistance to nonspecific cell adhesion.     

Staphylococcus aureus-Fibronectin Interactions with and without Fibronectin-Binding Proteins and Their Role in Adhesion and Desorption

Adhesion and residence-time-dependent desorption of two Staphylococcus aureus strains with and without fibronectin (Fn) binding proteins (FnBPs) on Fn-coated glass were compared under flow conditions. To obtain a better understanding of the role of Fn-FnBP binding, the adsorption enthalpies of Fn with staphylococcal cell surfaces were determined using isothermal titration calorimetry (ITC). Interaction forces between staphylococci and Fn coatings were measured using atomic force microscopy (AFM). The strain with FnBPs adhered faster and initially stronger to an Fn coating than the strain without FnBPs, and its Fn adsorption enthalpies were higher. The initial desorption was high for both strains but decreased substantially within 2 s. These time scales of staphylococcal bond ageing were confirmed by AFM adhesion force measurement. After exposure of either Fn coating or staphylococcal cell surfaces to bovine serum albumin (BSA), the adhesion of both strains to Fn coatings was reduced, suggesting that BSA suppresses not only nonspecific but also specific Fn-FnBP interactions. Adhesion forces and adsorption enthalpies were only slightly affected by BSA adsorption. This implies that under the mild contact conditions of convective diffusion in a flow chamber, adsorbed BSA prevents specific interactions but does allow forced Fn-FnBP binding during AFM or stirring in ITC. The bond strength energies calculated from retraction force-distance curves from AFM were orders of magnitude higher than those calculated from desorption data, confirming that a penetrating Fn-coated AFM tip probes multiple adhesins in the outermost cell surface that remain hidden during mild landing of an organism on an Fn-coated substratum, like that during convective diffusional flow.     

Tailoring of the titanium surface by immobilization of heparin/fibronectin complexes for improving blood compatibility and endothelialization: an in vitro study

To improve the blood compatibility and endothelialization simultaneously and to ensure the long-term effectiveness of the cardiovascular implants, we developed a surface modification method, enabling the coimmobilization of biomolecules to metal surfaces. In the present study, a heparin and fibronectin mixture (Hep/Fn) covalently immobilized on a titanium (Ti) substrate for biocompatibility was investigated. Different systems [N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N-hydroxysuccinimide, electrostatic] were used for the formation of Hep/Fn layers. Atomic force microscopy (AFM) showed that the roughness of the silanized Ti surface decreased after the immobilization of Hep/Fn. Fourier transform infrared spectroscopy (FTIR), Toluidine Blue O (TBO) test, and immunochemistry assay showed that Hep/Fn mixture was successfully immobilized on Ti surface. Blood compatibility tests (hemolysis rate, APTT, platelet adhesion, fibrinogen conformational change) showed that the coimmobilized films of Hep/Fn mixture reduced blood hemolysis rate, prolonged blood coagulation time, reduced platelets activation and aggregation, and induced less fibrinogen conformational change compared with a bare Ti surface. Endothelial cell (EC) seeding showed more EC with better morphology on pH 4 samples than on pH 7 and EDC/NHS samples, which showed rounded and aggregated cells. Systematic evaluation showed that the pH 4 samples also had much better blood compatibility. All results suggest that the coimmobilized films of Hep/Fn can confer excellent antithrombotic properties and with good endothelialization. We envisage that this method will provide a potential and effective solution for the surface modification of cardiovascular implant materials.     

Immobilisation of sulphated hyaluronan for improved biocompatibility

Hyaluronan (Hyal) was modified by the insertion of sulphate to hydroxyl groups. A series of heparin-like compounds with controllable properties was obtained. The physicochemical and biological behaviours of all these sulphated hyaluronan acids (HyalSx) and their complexes with heavy metal ions (Cu2+ and Zn2+) were investigated. HyalS, derivatives showed a good anticoagulant activity and low platelet aggregation which increased with increasing degree of sulphation. Moreover HyalSx and their Cu2+ complexes were demonstrated to favour the growth of human endothelial cells. However, the utilisation of HyalSx as a material is hindered by its high solubility in physiological solution. Our approach to improve its stability was directed to the synthesis of new HyalSx-based hydrogels and on the preparation of new biocompatible polymeric surfaces obtained through covalent photoimmobilisation of HyalSx. The reaction of primary ovine chondrocytes and B10D2 endothelial cells was studied on both matrices in terms of cell number, F-actin and CD44 receptor immunostaining. Analysis of cell movement showed that the cells respond to HyalSx showing good adhesion and spreading. These results suggest that HyalSx containing materials could be used as biomaterials to aid cartilage repair and vessel endothelisation.     

Effect of receptor-ligand affinity on the strength of endothelial cell adhesion.

The objective of this study was to determine the effect of receptor-ligand affinity on the strength of endothelial cell adhesion. Linear and cyclic forms of the fibronectin (Fn) cell-binding domain peptide Arg-Gly-Asp (RGD) were covalently immobilized to glass, and Fn was adsorbed onto glass slides. Bovine aortic endothelial cells attached to the surfaces for 15 min. The critical wall shear stress at which 50% of the cells detached increased nonlinearly with ligand density and was greater with immobilized cyclic RGD than with immobilized linear RGD or adsorbed Fn. To directly compare results for the different ligand densities, the receptor-ligand dissociation constant and force per bond were estimated from data for the critical shear stress and contact area. Total internal reflection fluorescence microscopy was used to measure the contact area as a function of separation distance. Contact area increased with increasing ligand density. Contact areas were similar for the immobilized peptides but were greater on surfaces with adsorbed Fn. The dissociation constant was determined by nonlinear regression of the net force on the cells to models that assumed that bonds were either uniformly stressed or that only bonds on the periphery of the contact region were stressed (peeling model). Both models provided equally good fits for cells attached to immobilized peptides whereas the peeling model produced a better fit of data for cells attached to adsorbed Fn. Cyclic RGD and linear RGD both bind to the integrin alpha v beta 3, but immobilized cyclic RGD exhibited a greater affinity than did linear RGD. Receptor affinities of Fn adsorbed to glycophase glass and Fn adsorbed to glass were similar. The number of bonds was calculated assuming binding equilibrium. The peeling model produced good linear fits between bond force and number of bonds. Results of this study indicate that 1) bovine aortic endothelial cells are more adherent on immobilized cyclic RGD peptide than linear RGD or adsorbed Fn, 2) increased adhesion is due to a greater affinity between cyclic RGD and its receptor, and 3) the affinity of RGD peptides and adsorbed Fn for their receptors is increased after immobilization.     

Cu(II) and Zn(II) complexes with hyaluronic acid and its sulphated derivative. Effect on the motility of vascular endothelial cells

With the aim of improving the compatibility of biomaterials to be used for the construction of cardiovascular prosthesis, we have designed bioactive macromolecules resulting from chemical modifications of hyaluronic acid (Hyal). The stability constants of Cu(II) and Zn(II) complexes with the sulphated derivative of hyaluronic acid (HyalS3.5) were evaluated. Two different complexes have been found for each metal ion, CuL, Cu(OH)2L and ZnL, Zn(OH)2L (L means the disaccharide unit of the ligands) in aqueous solution at 37 degrees C. The dihydroxo Cu(II) complex was present in high percentage at pH=7.4. On the contrary, the Zn(II) ion was present with a relatively low percentage of both complexes. The ability to stimulate endothelial cell adhesion and migration was evaluated for Hyal, HyalS3.5 and their complexes with Cu(II) and Zn(II) ions. The results revealed that Hyal and [Cu(OH)2HyalS3.5](4.5)- induced cell adhesion, while [ZnHyalS3.5](2.5)- and [Zn(OH)2HyalS3.5](4.5)- inhibited the process. The chemotactic activity of increasing concentrations of the above complexes was also evaluated, demonstrating that [Cu(OH)2HyalS3.5](4.5)- complex at 1 microM concentration was the most active in inducing cell migration. These results have been also strengthened by analysing adherent cell migration in agarose. In conclusion, sulphated hyaluronic acid coordinated to Cu(II) seems to be a promising matrix molecule for the construction of cardiovascular prosthesis.     

Effect of the covalently linked fatty acid 18-MEA on the nanotribology of hair's outermost surface

Highly ordered lipids adsorbed or grafted on surfaces are known to provide protection and lubrication custom engineered surfaces. We have used atomic force microscopy (AFM) to measure adhesion and frictional properties of the outermost surfaces of a variety of human hairs with the aim of both understanding the role of 18-methyleicosanoic acid (18-MEA), an unusual branched-chain fatty acid covalently bound to the cuticle surface, and investigating how treatments or the ethnic origin affect this layer. Results show that an unmodified silicon nitride AFM tip is able to detect changes at the hair surface that can be related to the absence or presence of this layer due to treatment conditions and in particular that this monolayer has a lubricant effect.     

In situ immobilization of proteins and RGD peptide on polyurethane surfaces via poly(ethylene oxide) coupling polymers for human endothelial cell growth

A "CBABC"-type pentablock coupling polymer, mesylMPEO, was designed and synthesized to promote human endothelial cell growth on the surfaces of polyurethane biomaterials. The polymer was composed of a central 4,4'-methylenediphenyl diisocyanate (MDI) coupling unit and poly(ethylene oxide) (PEO) spacer arms with methanesulfonyl (mesyl) end groups pendent on both ends. As the presurface modifying additive (pre-SMA), the mesylMPEO was noncovalently introduced onto the poly(ether urethane) (PEU) surfaces by dip coating, upon which the protein/peptide factors (gelatin, albumin, and arginine-glycine-aspartic acid tripeptide [RGD]) were covalently immobilized in situ by cleavage of the original mesyl end groups. The pre-SMA synthesis and PEU surface modification were characterized using nuclear magnetic resonance spectroscopy ((1)H NMR), attenuated total reflection infrared spectroscopy (ATR-FTIR), and X-ray photoelectron spectroscopy (XPS). Human umbilical vein endothelial cells (HUVEC) were harvested manually by collagenase digestion and seeded on the modified PEU surfaces. Cell adhesion ratios (CAR) and cell proliferation ratios (CPR) were measured using flow cytometry, and the individual cell viability (ICV) was determined by MTT assay. The cell morphologies were investigated by optical inverted microscopy (OIM) and scanning electrical microscopy (SEM). The gelatin- and RGD-modified surfaces were HUVEC-compatible and promoted HUVEC growth. The albumin-modified surfaces were compatible but inhibited cell adhesion. The results also indicated that, for HUVEC in vitro cultivation, the cell adhesion stage was of particular importance and had a significant impact on the cell responses to the modified surfaces.     

Covalent surface immobilization of Arg-Gly-Asp- and Tyr-Ile-Gly-Ser-Arg-containing peptides to obtain well-defined cell-adhesive substrates

The synthetic peptides Gly-Arg-Gly-Asp-Tyr and Gly-Tyr-Ile-Gly-Ser-Arg-Tyr, which contain Arg-Gly-Asp (RGD) and Tyr-Ile-Gly-Ser-Arg (YIGSR), the ligands for two important classes of cell adhesion receptors, were covalently coupled to a nonadhesive modified glass surface by the N-terminal Gly. The N-terminal Gly served as a spacer, and the C-terminal Y served as a site for radioiodination. These modified substrates supported the adhesion and spreading of cultured human foreskin fibroblasts (HFFs) independently of adsorbed proteins and, it was demonstrated that a covalently immobilized YIGSR-containing peptide has biological activity. The surface concentration of grafted peptide on the glass was measured by 125I radio-labeling and was 12.1 pmol/cm2. HFFs spread on both immobilized peptide substrates, but at much slower rates on grafted YIGSR glass surfaces than on the RGD-containing substrates. Cells formed focal contacts on the RGD-derivatized substrates in the presence or absence of serum. Focal contacts formed on the YIGSR-grafted surfaces only when serum was present in the medium and had morphologies different from those observed on the RGD-containing substrates. Serum influenced the organization of microfilaments and the extent of spreading of adherent cells, although adsorption of adhesion proteins was minimal on all substrates. This derivatization method produced chemically stable substrates which may be useful in studying receptor-mediated cell adhesion, as the quantity of peptide available at the surface may be precisely measured and controlled.     

Top-down grafting of xyloglucan to gold monitored by QCM-D and AFM: enzymatic activity and interactions with cellulose

This study focuses on the manufacture and characterization of model surfaces consisting of end-grafted xyloglucan (XG), a naturally occurring polysaccharide, onto a gold substrate. The now well-established XET-technology was utilized for enzymatic incorporation of a thiol moiety at one end of the xyloglucan backbone. This functionalized macromolecule was subsequently top-down grafted to gold, forming a thiol-bonded xyloglucan brushlike layer. The grafting was monitored in situ with QCM-D, and a significant difference in the adsorbed/grafted amount between unmodified xyloglucan and the thiol-functionalized polymer was observed. The grafted surface was demonstrated to be accessible to enzyme digestion using the plant endo-xyloglucanase TmNXG1. The nanotribological properties toward cellulose of the untreated crystal, brush-modified surface, and enzyme-exposed surfaces were compared with a view to understanding the role of xyloglucan in friction reduction. Friction coefficients obtained by the AFM colloidal probe technique using a cellulose functionalized probe on the xyloglucan brush showed an increase of a factor of 2 after the enzyme digestion, and this result is interpreted in terms of surface roughness. Finally, the brush is shown to exhibit binding to cellulose despite its highly oriented nature.     

Substrate-independent approach for the generation of functional protein resistant surfaces

A new route for coating various substrates with antifouling polymer layers was developed. It consisted in deposition of an amino-rich adhesion layer by means of RF magnetron sputtering of Nylon 6,6 followed by the well-controlled, surface-initiated atom transfer radical polymerization of antifouling polymer brushes initiated by bromoisobutyrate covalently attached to amino groups present in the adhesion layer. Polymer brushes of hydroxy- and methoxy-capped oligoethyleneglycol methacrylate and carboxybetaine acrylamide were grafted from bromoisobutyrate initiator attached to a 15 nm thick amino-rich adhesion layer deposited on gold, silicon, polypropylene, and titanium-aluminum-vanadium alloy surfaces. Well-controlled polymerization kinetics made it possible to control the thickness of the brushes at a nanometer scale. Zero fouling from single protein solutions and a reduction of more than 90% in the fouling from blood plasma observed on the uncoated surfaces was achieved. The feasibility of functionalization with bioactive compounds was tested by covalent attachment of streptavidin onto poly(oligoethylene glycol methacrylate) brush and subsequent immobilization of model antibodies and oligonucleotides. The procedure is nondestructive and does not require any chemical preactivation or the presence of reactive groups on the substrate surface. Contrary to current antifouling modifications, the developed coating can be built on various classes of substrates and preserves its antifouling properties even in undiluted blood plasma. The new technique might be used for fabrication of biotechnological and biomedical devices with tailor-made functions that will not be impaired by fouling from ambient biological media.     

Collagen adsorption on quercetin loaded polycaprolactone microspheres: approach for "stealth" implant

We recently experimented with collagen coating on the surface of quercetin loaded polycaprolactone microspheres by simple adsorption technique to mimic extra cellular matrix and reduce immune or inflammatory responses at the site of implants. The collagen immobilization on polymeric scaffold surfaces through various surface modification techniques was the current scenario to improve bio-integration of the polymers with the in vivo system. Nevertheless, it requires other chemicals or processing methods to modify the surface of polymers to immobilize the collagen covalently. Here protein adsorption principle is used for the coating of collagen onto the surface of solid microspheres and characterized. Optical, ATR-FTIR, SEM analysis confirm collagen coating. The reduction in burst release of the quercetin from the PCL microspheres further confirms its presence and role in the controlled release. The results indicate that the adsorption technique can be the simple strategy to coat collagen on the surface of polyester implants to develop stealth implant in shorter time with low cost technology.     

Effect of adsorbed fibronectin on the differential adhesion of osteoblast-like cells and Staphylococcus aureus with and without fibronectin-binding proteins

The influence of fibronectin (Fn) coated surfaces patterned with poly(ethylene glycol) microgels having inter-gel spacings between 0.5 and 3.0 μm on the adhesion of Staphylococcus aureus strains with and without Fn-binding proteins and cellular adhesion/spreading was investigated. Quantitative force measurements between a S. aureus cell and a patterned surface showed that the adhesion force between the bacterium and the patterned surface increased substantially after Fn adsorption, regardless of the strain used, but decreased with decreasing inter-gel spacing. In flow-chamber experiments, the Fn-binding strain adhered at a higher rate after Fn adsorption than the strain lacking Fn-binding proteins. In both cases, the adhesion rates decreased with decreasing inter-gel spacing. Osteoblast-like cells could bind to patterned surfaces despite the microgels, and adsorbed Fn substantially amplified this effect. Even under highly non-adhesive conditions associated with closely spaced microgels, adsorbed Fn preserves a window of inter-gel spacing around 1 μm where the adhesion of staphylococcal cells is hindered while cells can still adhere and spread.     

Inhibition de l'adhérence de Porphyromonas gingivalis sur la surface de titane greffé de poly(styrène sulfonate de sodium)

Dental implant-associated infections as peri-implantitis represent one of the major causes of osteointegration failures of oral implants. Adhesion of Porphyromonas gingivalis, one of the bacterial strains mainly involved in such infections, is tightly dependent on the topographical and/or physico-chemical properties of the implant surfaces. As a matter of fact, we showed that the grafting of one bioactive polymer such as poly(sodium styrene sulfonate) onto titanium implant surfaces allowed a sensitive decrease of Staphylococcus aureus adhesion (> 40%). The aim of the study consists in evaluating the adhesion of P. gingivalis onto titanium surfaces grafted with poly(sodium stryrene sulfonate) in order to elaborate implants exhibiting appropriate inhibiting properties towards the adhesion of periodontal pathogens. The grafting of poly(sodium stryrene sulfonate) onto titanium surfaces is carried out in two steps: chemical oxydation of titanium to initiate radical species then grafting of poly(sodium stryrene sulfonate) by radical polymerization. Chemical characterization of the surfaces is achieved by Fourier transformed infrared spectroscopy (FTIR). Bacterial adhesion was studied on grafted and non grafted (control) titanium surfaces, preadsorbed or not by plasmatic proteins. Protein adsorption as well as bacteria adhesion is followed by fluorescence spectroscopy by using proteins or bacteria previously labelled with fluorescence probes; the quantification of adsorption and bacteria adhesion are performed by image analysis. Results showed that protein adsorption is more important (~3 times) and that P. gingivalis adhesion is strongly inhibited (~73%) onto poly(sodium styrene sulfonate) grafted surfaces when compared to titanium control. Moreover, the inhibition of bacterial adhesion on grafted surfaces preadsorbed with plasma proteins is comparable to that observed on grafted surfaces preadsorbed with fibronectin. In conclusion, the obtained results evidenced that the grafting of titanium surface by poly(sodium styrene sulfonate) led to significant inhibition of P. gingivalis adhesion and that this inhibitory activity involved adsorbed proteins. Poly(sodium styrene sulfonate) grafted titanium surfaces present a high interest for the elaboration of oral implants in various clinical dental applications.     

Modulation of cell proliferation and differentiation through substrate-dependent changes in fibronectin conformation

Integrin-mediated cell adhesion to extracellular matrices provides signals essential for cell cycle progression and differentiation. We demonstrate that substrate-dependent changes in the conformation of adsorbed fibronectin (Fn) modulated integrin binding and controlled switching between proliferation and differentiation. Adsorption of Fn onto bacterial polystyrene (B), tissue culture polystyrene (T), and collagen (C) resulted in differences in Fn conformation as indicated by antibody binding. Using a biochemical method to quantify bound integrins in cultured cells, we found that differences in Fn conformation altered the quantity of bound alpha5 and beta1 integrin subunits but not alphav or beta3. C2C12 myoblasts grown on these Fn-coated substrates proliferated to different levels (B > T > C). Immunostaining for muscle-specific myosin revealed minimal differentiation on B, significant levels on T, and extensive differentiation on C. Differentiation required binding to the RGD cell binding site in Fn and was blocked by antibodies specific for this site. Switching between proliferation and differentiation was controlled by the levels of alpha5beta1 integrin bound to Fn, and differentiation was inhibited by anti-alpha5, but not anti-alphav, antibodies, suggesting distinct integrin-mediated signaling pathways. Control of cell proliferation and differentiation through conformational changes in extracellular matrix proteins represents a versatile mechanism to elicit specific cellular responses for biological and biotechnological applications.     

Doubly biomimetic catecholic phosphorylcholine copolymer: a platform strategy for fabricating antifouling surfaces

A doubly biomimetic PMNC polymer bearing cell antifouling phosphorylcholine and mussel adhesive protein catechol groups is synthesized. The polymer can be deposited onto a variety of substrates by dip-coating in an aqueous solution, adhering to surfaces via the catechol functional group while at the same time forming a cell outer membrane mimetic antifouling surface. Contact angle, ATR-FTIR and XPS measurements confirm polymer coating formation on a variety of inorganic and organic substrates. BSA and bovine plasma fibrinogen protein adsorption on PMNC coated surfaces are reduced significantly compared to unmodified substrates, and platelet adhesion from human serum onto the PMNC coated substrate surfaces is highly suppressed in this study.     

Bioactive polymer fibers to direct endothelial cell growth in a three-dimensional environment

This study reports the fabrication of bioactive polymer fibers onto which signaling molecules can control and direct cell responses. To encourage and control directional biological responses, GRGDS peptides were immobilized onto the surface of 100 microm diameter poly(ethylene terephtalate) (PET) fibers (monofilaments). PET fiber surfaces were first coated with a thin polymeric interfacial bonding layer bearing amine groups by plasma polymerization. Carboxy-methyl-dextran (CMD) was covalently grafted onto the surface amine groups using water-soluble carbodiimide chemistry. GRGDS were covalently immobilized onto CMD-coated fiber surfaces. X-ray photoelectron spectroscopy (XPS) analyses enabled characterization of the multilayer fabrication steps. Human umbilical vein endothelial cells were seeded and grown on fibers to investigate cell patterning behavior (i.e., adhesion, spreading, cytoskeleton organization, and cell orientation). Cell adhesion was reduced on CMD-coated fibers, whereas amine- and GRGDS-coated fibers promoted cell adhesion and spreading. Cell adhesion was enhanced as the GRGDS concentration increased. Epifluorescence microscopic visualization of cells on RGD-coated substrates showed well-defined stress fibers and sharp spots of vinculin, typical of focal adhesions. In comparison to plasticware commonly used in cell cultures, fiber curvature promoted cell orientation along the fiber axis.     

Urea potentiometric enzymatic biosensor based on charged biopolymers and electrodeposited polyaniline

A potentiometric biosensor based on urease was developed for the quantitative determination of urea concentration in aqueous solutions for biomedical applications. The urease was either physisorbed onto an electrodeposited polyaniline film (PANI), or immobilized on a layer-by-layer film (LbL) assembled over the PANI film, that was obtained by the alternate deposition of charged polysaccharides (carboxymethylpullulan (CMP) and chitosan (CHI)). In the latter case, the urease (Urs) enzyme was either physically adsorbed or covalently grafted to the LbL film using carbodiimide coupling reaction. Potentiometric responses of the enzymatic biosensors were measured as a function of the urea concentration in aqueous solutions (from 10(-6) to 10(-1) mol L(-1) urea). Very high sensitivity and short response time were observed for the present biosensor. Moreover, a stability study showed a higher stability over time for the potentiometric response of the sensor with the enzyme-grafted LbL film, testifying for the protective nature of the polysaccharide coating and the interest of covalent grafting.     

Focal adhesion sites and the removal of substratum-bound fibronectin

Fibronectin was not removed from the substratum beneath focal adhesion sites when fibroblasts spread in serum-free medium on adsorbed fibronectin substrata, or when fibroblasts spread in serum-containing medium on covalently cross-linked fibronectin substrata. Under these conditions, there was colocalization between 140-kD fibronectin receptors and focal adhesion sites. It was concluded that removal of adsorbed fibronectin from beneath focal adhesion sites was a mechanical process that required serum. The effect of serum was nonspecific since serum could be replaced by equivalent concentrations of serum albumin, ovalbumin, or gamma globulins. Quantitative measurements indicated that the presence of proteins in the incubation medium weakens the interaction of fibronectin with the substratum, thereby allowing the adsorbed protein to be removed from the substratum at sites of high stress. After removing fibronectin from the substratum, cells reorganized this material into patches and fibrils beneath cells, and the reorganized fibronectin colocalized with fibronectin receptors. Some of the patches of fibronectin were phagocytosed. The fibronectin fibrils were observed to be in register with actin filament bundles and sometimes translocated to the upper cell surfaces. It is proposed that removal of fibronectin from beneath focal adhesion sites is an example of how cells can modify their extracellular matrices through contractile activity.