Emmanuel Pauthe

Kinetics of conformational changes of fibronectin adsorbed onto model surfaces.

Fibronectin (FN), a large glycoprotein found in body fluids and in the extracellular matrix, plays a key role in numerous cellular behaviours. We investigate FN adsorption onto hydrophilic bare silica and hydrophobic polystyrene (PS) surfaces using Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) in aqueous medium. Adsorption kinetics using different bulk concentrations of FN were followed for 2h and the surface density of adsorbed FN and its time-dependent conformational changes were determined. When adsorption occurs onto the hydrophilic surface, FN molecules keep their native conformation independent of the adsorption conditions, but the amount of adsorbed FN increases with time and the bulk concentration. Although the protein surface density is the same on the hydrophobic PS surface, this has a strong impact on the average conformation of the adsorbed FN layer. Indeed, interfacial hydration changes induced by adsorption onto the hydrophobic surface lead to a decrease in unhydrated beta-sheet content and cause an increase in hydrated beta-strand and hydrated random domain content of adsorbed FN. This conformational change is mainly dependent on the bulk concentration. Indeed, at low bulk concentrations, the secondary structures of adsorbed FN molecules undergo strong unfolding, allowing an extended and hydrated conformation of the protein. At high bulk concentrations, the molecular packing reduces the unfolding of the stereoregular structures of the FN molecules, preventing stronger spreading of the protein.

Transmigration of human ovarian adenocarcinoma cells through endothelial extracellular matrix involves αv integrins and the participation of MMP2

The growth of ovarian carcinoma is dependent upon their vascularistion, but the interaction of ovarian cancer cells with the endothelium and their invasion through an endothelial environment remain poorly understood at the molecular level. To investigate adhesive events underlying this process with focusing on the role of αv integrins and MT1MMP‐MMP2 proteinases, we used in vitro models of cocultures of human ovarian adenocarcinoma cell lines (IGROV1 and SKOV3) with human umbilical vein endothelial cells (HUVECs). Immunostaining of HUVECs revealed the network organisation of fibrillar fibronectin (Fn) and pericellular vitronectin (Vn). During coculture, IGROV1 and SKOV3 cells gain access to subendothelial basement membrane of HUVECs and dislocated endothelial Fn without affecting endothelial Vn. Transmigration assays revealed that tumour cells invade Vn and, with an higher efficiency, Fn. Our data also highlighted that ovarian carcinoma cells migrated through the Fn‐rich HUVEC‐ECM. The expression of MMP2 and MT1‐MMP was revealed in tumour cells within an endothelial environment. Furthermore, we found that cell migration through the endothelial ECM was almost totally dependent on αv integrin function, whereas β1 integrins were not solicited. In addition, inhibitors of MMP2 activity (alone or combined with anti‐αv integrin MAb) or TSRI265 (which blocks MMP2‐αvβ3 association) were found to impede this process. Finally, αv integrins, MT1‐MMP and MMP2 were found in ovarian carcinoma cells within the 3‐dimensional architecture of intraperitoneal tumour nodes collected from nude mice xenografted with IGROV1 or SKOV3 cell lines or within human tumour tissues. αv integrins therefore appear as essential to the migration properties of human ovarian carcinoma cells, especially in an endothelial environment, with MMP2 participating to this process.

Carbon nanotube-based antimicrobial biomaterials formed via layer-by-layer assembly with polypeptides.

Biomaterials capable of suppressing microbial infection are of clear importance in various health care applications, e.g. implantable devices. In this study, we investigate the antimicrobial activity of single-walled carbon nanotubes (SWNT) layer-by-layer (LbL) assembled with the polyelectrolytes poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA). SWNT dispersion in aqueous solution is achieved through the biocompatible nonionic surfactant polyoxyethylene(20) sorbitan monolaurate (Tween 20), and the amphiphilic polymer phospholipid-poly(ethylene glycol) (PL-PEG). Absorbance spectroscopy and transmission electron microscopy (TEM) show SWNT with either Tween 20 or PL-PEG in aqueous solution to be well dispersed, at about the level of SWNT in chloroform. Quartz crystal microgravimetry with dissipation (QCMD) measurements show both SWNT-Tween and SWNT-PL-PEG to LbL assemble with PLL and PGA into multilayer films, with the PL-PEG system yielding the greater final SWNT content. Escherichia coli and Staphylococcus epidermidis inactivation rates are significantly higher (up to 90%) upon 24h incubation with SWNT containing films, compared to control films (ca. 20%). This study demonstrates the potential usefulness of SWNT/PLL/PGA thin films as antimicrobial biomaterials.

Nanofilm Biomaterials: Localized Cross-Linking To Optimize Mechanical Rigidity and Bioactivity

Nanofilm biomaterials, formed by the layer-by-layer assembly of charged macromolecules, are important systems for a variety of cell-contacting biomedical and biotechnological applications. Mechanical rigidity and bioactivity are two key film properties influencing the behavior of contacting cells. Increased rigidity tends to improve cells attachment, and films may be rendered bioactive through the incorporation of proteins, peptides, or drugs. A key challenge is to realize films that are simultaneously rigid and bioactive. Chemical cross-linking of the polymer framework--the standard means of increasing a films rigidity--can diminish bioactivity through deactivation or isolation of embedded biomolecules or inhibition of film biodegradation. We present here a strategy to decouple mechanical rigidity and bioactivity, potentially enabling nanofilm biomaterials that are both mechanically rigid and bioactive. Our idea is to selectively cross-link the outer region of the film, resulting in a rigid outer skin to promote cell attachment, while leaving the film interior (with any embedded bioactive species) unaffected. We propose an approach whereby an N-hydroxysulfosuccinimide (sulfo-NHS) activated poly(L-glutamic acid) is added as the terminal layer of a multilayer film and forms (covalent) amide bonds with amino groups of poly(L-lysine) placed previously within the film. We characterize film assembly and cross-linking extent via quartz crystal microbalance with dissipation monitoring (QCMD), Fourier transform infrared spectroscopy in attenuated total reflection mode (FTIR-ATR), and laser scanning confocal microscopy (LSCM) and measure the attachment and metabolic activity of preosteoblastic MC3T3-E1 cells. We show cross-linking to occur primarily at the film surface and the subsequent cell attachment and metabolic activity to be enhanced compared to native films. Our method appears promising as a means to realize films that are simultaneously mechanically rigid and bioactive.

Fibronectin layers by matrix-assisted pulsed laser evaporation from saline buffer-based cryogenic targets

The deposition of fibronectin (FN) from saline buffer-based cryogenic targets by matrix-assisted pulsed laser evaporation (MAPLE) onto silicon substrates is reported. A uniform distribution of FN was revealed by Ponceau staining after control experiments on nitrocellulose paper. Well-organized particulates with heights from hundreds of nanometers up to more than 1 μm packed in homogeneous layers were evidenced by optical microscopy and profilometry on Si substrates. Atomic force microscopy images showed regions composed of buffer and FN aggregates forming a compact film. Comparison of infrared spectra of drop-cast and MAPLE-deposited FN confirmed the preservation of composition and showed no degradation of the protein. The protein deposition on Si was confirmed by antibody staining. Small aggregates and fluorescent fibrils were visualized by fluorescence microscopy. Superior attachment of human osteoprogenitor cells cultivated for 3 h proved the presence of stable and intact FN molecules after transfer.

Genipin-Cross-Linked Layer-by-Layer Assemblies: Biocompatible Microenvironments To Direct Bone Cell Fate

The design of biomimetic coatings capable of improving the osseointegration of bone biomaterials is a current challenge in the field of bone repair. Toward this end, layer-by-layer (LbL) films composed of natural components are suitable candidates. Chondroitin sulfate A (CSA), a natural glycosaminoglycan (GAG), was used as the polyanionic component because it promotes osteoblast maturation in vivo. In their native state, GAG-containing LbL films are generally cytophobic because of their low stiffness. To stiffen our CSA-based LbL films, genipin (GnP) was used as a natural cross-linking agent, which is much less cytotoxic than conventional chemical cross-linkers. GnP-cross-linked films display an original combination of microscale topography and tunable mechanical properties. Structural characterization was partly based on a novel donor/acceptor Förster resonance energy transfer (FRET) couple, namely, FITC/GnP, which is a promising approach for further inspection of any GnP-cross-linked system. GnP-cross-linked films significantly promote adhesion, proliferation, and early and late differentiation of preosteoblasts.

Pre-osteoblasts on poly(l-lactic acid) and silicon oxide: Influence of fibronectin and albumin adsorption

Cell adhesion and subsequent viability are critical initial steps in biomaterial-tissue integration and are strongly dependent on the material properties and the presence of matrix proteins. In the present study MC3T3-E1 osteoblast-like cell behavior on silicon oxide (SO) and poly(L-lactic acid) (PLLA) substrates has been examined, with a focus on the influence of the adhesive protein fibronectin and the non-adhesive protein albumin adsorbed on the substrates. Quartz crystal microgravimetry showed adsorption of fibronectin and albumin to be nearly identical on SO and PLLA. Subsequent exposure a previously adsorbed fibronectin layer to albumin decreased the rigidity of the adsorbed layer without any measurable increase in adsorbed mass. Cell adhesion and spreading were significantly enhanced on both SO and PLLA substrates coated with fibronectin or with fibronectin and albumin, compared with uncoated or albumin-coated substrates. The only statistically significant difference between the two substrates in these assays was increased spreading on PLLA compared with SO in the presence of fibronectin and albumin. Cell proliferation was significantly higher on SO compared with PLLA after 7 days culture, but depended on the presence of fibronectin only in the PLLA system. In contrast, mitochondrial activity was higher on PLLA than on SO, and was enhanced by fibronectin on both substrates. PLLA substrates coated with fibronectin and subsequently exposed to albumin exhibited the highest level of cell differentiation, as assayed via alkaline phosphatase activity. These results demonstrate the importance of adsorbed proteins on osteoblast-like cell-surface interactions.

Carbon nanotube bundling: influence on layer-by-layer assembly and antimicrobial activity

Antimicrobial surfaces are needed for many health care applications. Single walled carbon nanotubes (SWNT) have shown promise as antimicrobial agents, but important questions persist concerning the effects of tube bundling, a common phenomenon owing to strong hydrophobicity. We investigate here the influence of bundling on the layer-by-layer (LbL) assembly of SWNT with charged polymers, and on the antimicrobial properties of the resultant films. We employ a poly(ethylene glycol) functionalized phospholipid (PL-PEG) to disperse SWNT in aqueous solution, and consider cases where SWNT are dispersed (i) as essentially isolated objects and (ii) as small bundles. Quartz crystal microgravimetry with dissipation (QCMD) and ellipsometry measurements show the bundled SWNT system to adsorb in an unusually strong fashion – with layers twice (when hydrated) and three times (when dried) as thick as those of isolated SWNT. Molecular dynamics simulation reveals a lower PL-PEG density and degree of solution extension on bundled versus isolated SWNT, suggesting thicker adsorbed layers may result from suppressed steric repulsion between bundled nanotubes. Enhanced van der Waals attraction in the bundled system may also play a role. Scanning electron micrographs reveal Escherichia coli on films with bundled SWNT to be essentially engulfed by the nanotubes, whereas the bacteria rest upon films with isolated SWNT. While both systems inactivate 90% of bacteria in 24 h, the bundled SWNT system is “fast-acting,” reaching this inactivation rate in 1 h. This study demonstrates the significant impact of SWNT bundling on LbL assembly and antimicrobial activity, explores the molecular basis of nanotube–nanotube interactions, and demonstrates the possibility of bacteria-engulfing, fast-acting, SWNT-based antimicrobial coatings.

On the potential for fibronectin/phosphorylcholine coatings on PTFE substrates to jointly modulate endothelial cell adhesion and hemocompatibility properties

The use of biomolecules as coatings on biomaterials is recognized to constitute a promising approach to modulate the biological response of the host. In this work, we propose a coating composed by 2 biomolecules susceptible to provide complementary properties for cardiovascular applications: fibronectin (FN) to enhance endothelialization, and phosphorylcholine (PRC) for its non thrombogenic properties. Polytetrafluoroethylene (PTFE) was selected as model substrate mainly because it is largely used in cardiovascular applications. Two approaches were investigated: 1) a sequential adsorption of the 2 biomolecules and 2) an adsorption of the protein followed by the grafting of phosphorylcholine via chemical activation. All coatings were characterized by immunofluorescence staining, X-Ray Photoelectron Spectroscopy and Scanning Electron Microscopy analyses. Assays with endothelial cells showed improvement on cell adhesion, spreading and metabolic activity on FN-PRC coatings compared with the uncoated PTFE. Platelets adhesion and activation were both reduced on the coated surfaces when compared with uncoated PTFE. Moreover, clotting time tests exhibited better hemocompatibility properties of the surfaces after a sequential adsorption of FN and PRC. In conclusion, FN-PRC coating improves cell adhesion and non-thrombogenic properties, thus revealing a certain potential for the development of this combined deposition strategy in cardiovascular applications.

Inorganic–Organic Thin Implant Coatings Deposited by Lasers

The lifetime of bone implants inside the human body is directly related to their osseointegration. Ideally, future materials should be inspired by human tissues and provide the material structure-function relationship from which synthetic advanced biomimetic materials capable of replacing, repairing, or regenerating human tissues can be produced. This work describes the development of biomimetic thin coatings on titanium implants to improve implant osseointegration. The assembly of an inorganic-organic biomimetic structure by UV laser pulses is reported. The structure consists of a hydroxyapatite (HA) film grown onto a titanium substrate by pulsed-laser deposition (PLD) and activated by a top fibronectin (FN) coating deposited by matrix-assisted pulsed laser evaporation (MAPLE). A pulsed KrF* laser source (λ = 248 nm, τ = 25 ns) was employed at fluences of 7 and 0.7J/cm(2) for HA and FN transfer, respectively. Films approximately 1500 and 450 nm thick were obtained for HA and FN, respectively. A new cryogenic temperature-programmed desorption mass spectrometry analysis method was employed to accurately measure the quantity of immobilized protein. We determined that less than 7 μg FN per cm(2) HA surface is adequate to improve adhesion, spreading, and differentiation of osteoprogenitor cells. We believe that the proposed fabrication method opens the door to combining and immobilizing two or more inorganic and organic materials on a solid substrate in a well-defined manner. The flexibility of this method enables the synthesis of new hybrid materials by simply tailoring the irradiation conditions according to the thermo-physical properties of the starting materials.