Pierre Descouts

Physical and biological effects of a surface coating procedure on polyurethane catheters

Central venous catheters are widely used in clinical practice; however, complications such as venous thrombosis or infection are frequent. The physical and biological effects of a coating procedure designed to improve the blood-contacting properties of polyurethane central venous catheters (CVCs) were studied. The surface atomic composition of poly(vinyl pyrrolidone) (PVP)-coated or uncoated Pellethane single lumen CVCs was characterized by electron spectroscopy for chemical analysis (ESCA), which confirmed the presence of an oxygen-rich PVP layer on the former material. Topological analysis of both single and triple lumen CVCs by scanning force microscopy (SFM) revealed a very smooth surface in PVP-coated catheters compared to the more frequent surface irregularities found either in uncoated Pellethane or in four additional randomly selected, commercially available triple lumen polyurethane CVCs. The PVP-coated Pellethane showed a strong reduction in either fibrinogen or fibronectin adsorption compared to all other PVP-free polyurethane CVCs. This decreased protein adsorption led to a proportional reduction in protein-mediated adhesion of either Staphylococcus aureus or Staphylococcus epidermidis and in the binding of a monoclonal antibody directed against the cell-binding domain of fibronectin. Increased surface smoothness and hydrophilic properties of polyurethane CVCs might decrease the risk of bacterial colonization and infection.

Adhesion of Pseudomonas aeruginosa strains to untreated and oxygen-plasma treated poly(vinyl chloride) (PVC) from endotracheal intubation devices

Pseudomonas aeruginosa pneumonia is a life threatening complication in mechanically ventilated patients that requires the ability of the bacteria to adhere to, and colonize the endotracheal intubation device. New strategies to prevent or reduce these nosocomial infections are greatly needed. We report here the study of a set of P. aeruginosa clinical isolates, together with specific mutants, regarding their adhesion on native and chemically modified poly(vinyl chloride) (PVC) surfaces from endotracheal intubation devices. The adhesion of the different strains to untreated PVC varied widely, correlating with several physico-chemical characteristics known to influence the attachment of bacteria to inert surfaces. The adhesion patterns were compared to the calculations obtained with the DLVO theory of colloidal stability. These results illustrate the importance of testing different clinical isolates when investigating bacterial adhesion. Oxygen plasma treatment of the PVC pieces yielded a hydrophilic surface and reduced the number of adhering bacteria by as much as 70%. This reduction is however unlikely to be sufficient to prevent P. aeruginosa colonization of endotracheal intubation devices.

Surface properties of a specifically modified high-grade medical polyurethane

A high-grade medical polyurethane (PUR) was specifically modified to mimic the vascular vessel lumen. Since vascular endothelium represents a unique non-thrombogenic surface, we developed a surface modification process to design a new PUR surface which promotes endothelial cell adhesion. Biologically active synthetic RGD-containing peptide has been covalently coupled on the PUR surface. In order to optimise the RGD coupling, intermediate steps of PUR surface modification, such as plasma functionalisation and spacer polysaccharide grafting were investigated. Surface topography and friction images, chemistry and wettability differences of individual modification steps were controlled using atomic and lateral force microscopy, angle-resolved X-ray photoelectron spectroscopy and static contact angle measurements. Human umbilical vein endothelial cells adhesion tests were performed in vitro on all the samples. Only the RGD-containing peptide-grafted PUR has shown the endothelial cell attachment with an almost entire coverage of the surface substrate.

Temperature-responsive size-exclusion chromatography using poly(N-isopropylacrylamide) grafted silica

Silica-based packing materials induce non-specific interactions with proteins in aqueous media because of the nature of their surface, mainly silanol groups. Therefore, the silica surface has to be modified in order to be used as stationary phase for the High Performance Size-Exclusion Chromatography (HPSEC) of proteins. For this purpose, porous silica beads were coated with hydrophilic polymer gels (dextrans of different molecular weights) carrying a calculated amount of diethyl-aminoethyl groups (DEAE). Actually, as shown by HPSEC, these dextran modified supports minimize non-specific adsorption for proteins and pullulans in aqueous solution. Then, in order to change the pore size in response to temperature, temperature responsive polymer of poly(N-isopropylacrylamide) (PIPAAm) was introduced into the surface of dextran-DEAE on porous silica beads. The structure of these supports before and after modification was alternately studied by Scanning Electronic Microscopy (SEM) and Scanning Force Microscopy (SFM). An adsorption of radiolabelled albumin was performed to complete our study. Silica modifications by dextran-DEAE and PIPAAm improve the neutrality of the support and minimize the non-specific interactions between the solid support and proteins in solution. At low temperature, the support having PIPAAm exhibits a high resolution domain in HPSEC and finally permits a better resolution of proteins and pullulans. At higher temperature, hydrophobic properties of PIPAAm produce interactions with some proteins and trigger off a slight delay of their elution time.

Hydrogen desorption from sand-blasted and acid-etched titanium surfaces after glow-discharge treatment

Hydrogen desorption from argon plasma-treated titanium implants with a high surface roughness was studied. Implants with a high surface roughness have shown an increase in mechanical stability in bone tissue and a different behavior of osteoblasts in vitro. High surface roughness was produced by grit blasting and acid etching, resulting in an increase of the sub-surface hydrogen concentration and the formation of a titanium hydride. After an argon plasma treatment the surface oxide, which always covers titanum surfaces exposed to an oxygen-containing environment, and some of the hydrogen were sputtered away, decreasing the hydrogen concentration in the sub-surface region. Nuclear reaction analysis was used to determine the hydrogen concentration as a function of depth. The total amount of sub-surface (down to a depth of < or = 2 microm) hydrogen remaining after plasma treatment decreased with increasing plasma intensity to below the levels observed in non-acid-etched samples (approximately 1-2%). Thermal desorption spectroscopy was used for desorption studies and investigation of H(2) desorption activation energies. With a surface oxide present, the onset of hydrogen desorption is at ca 400 degrees C, which is the oxide decomposition temperature in vacuum, with an activation energy of ca 2 eV/molecule of H(2). After plasma treatment, that is, without surface oxide present, the onset of desorption was observed at ca 300 degrees C and with an activation energy of ca 0.8 eV/molecule of H(2), indicating a bulk diffusion-limited desorption.

Influence of surface and protein modification on immunoglobulin G adsorption observed by scanning force microscopy

Scanning force microscopy has been used successfully to produce images of individual protein molecules. However, one of the problems with this approach has been the high mobility of the proteins caused by the interaction between the sample and the scanning tip. To stabilize the proteins we have modified the adsorption properties of immunoglobulin G on graphite and mica surfaces. We have used two approaches: first, we applied glow discharge treatment to the surface to increase the hydrophilicity, favoring adhesion of hydrophilic protein molecules; second, we used the arginine modifying reagent phenylglyoxal to increase the protein hydrophobicity and thus enhance its adherence to hydrophobic surfaces. We used scanning force microscopy to show that the glow discharge treatment favors a more homogeneous distribution and stronger adherence of the protein molecules to the graphite surface. Chemical modification of the immunoglobulin caused increased aggregation of the proteins on the surface but did not improve the adherence to graphite. On mica, clusters of modified immunoglobulins were also observed and their adsorption was reduced. These results underline the importance of the surface hydrophobicity and charge in controlling the distribution of proteins on the surface.

Morphological difference between fibronectin sprayed on mica and on PMMA.

We have imaged with scanning force microscopy in air fibronectin (Fn) molecules sprayed on mica and on polymethylmetacrylate (PMMA), the latter being extensively used as biomaterial for implants. On mica we can observe small aggregates as well as individual molecules whose shape is influenced by the tip interaction during the scanning process, most of the isolated molecules showing a V-shape oriented in the scan direction. This indicates that the arms of the molecules are relatively free to move and the binding to the mica substrate is located near the disulfide bridge between the two subunits of the molecule. On the other side, when Fn molecules are sprayed on PMMA under the same conditions as for mica, we observe a thin network which we interpret as Fn molecules bound to each other. We relate our observation to the fact that mica is known to be strongly hydrophilic, which could reduce the Fn binding properties by interacting relatively strongly with molecules. On the other side, PMMA being hydrophobic, would interact less with molecules, leaving more binding sites for inter-molecular attachment.

Scanning force microscopy and cryo-electron microscopy of tobacco mosaic virus as a test specimen

In this study, tobacco mosaic virus (TMV) provides a resolution criterion for specimen preparation methods as well as for imaging parameters of the scanning force microscope (SFM). We present scanning force microscopic images of the virus embedded in 0.5% buffered phosphotungstic acid solution adsorbed on a freshly cleaved mica surface, and imaged under atmospheric conditions. Individual TMV particles were clearly identified with a characteristic shape of long rods of about 300 nm long and 60-70 nm in apparent width due to the geometric parameters of the tip. The structure of the virus was compared with cryo-electron microscopic data of vitrified suspensions observed to a resolution of 1.15 nm. Uncoated TMV particles were also deposited on evaporated titanium thin films and imaged by SFM.

Surface properties of electropolished titanium and vanadium

Abstract The surface topography, the chemical composition and the hydroxylation state are the surface properties playing a key role in the first stage of the biocompatibility process, namely the adsorption of water and proteins on the implant surface. To understand the very different tissue response to titanium and vanadium, we have measured the above-mentioned surface properties on similarly prepared Ti and V electropolished samples. Scanning force microscopy shows granular and homogeneous surfaces in both Ti and V samples, but with roughness twice as small in the case of V and with a lateral grain size of the order of 20–30 nm for Ti and of 80–100 nm for V. The surface chemical composition is strongly affected by thermal treatments, as revealed by Auger electron spectroscopy. On electropolished Ti, the surface segregation of Cl (originating from the electropolishing bath) occurs at 720 K and is well described by a purely diffusive model, i.e. Ficks law. For the segregation of S on Ti at higher temperature, we have extracted the energy of segregation and observed a rather strong influence of sulphur diffusion depending on the presence of chlorine on the surface. Finally, thermal desorption spectroscopy measurements indicate that water is mainly dissociated on hydroxyl groups on both Ti and V; the large amount of detected water indicates that it is deeply trapped inside the sample and not only chemisorbed on its surface.

Covalent immobilization of immunoglobulins G and Fab′ fragments on gold substrates for scanning force microscopy imaging in liquids

Scanning force microscopy offers the possibility of observing protein molecules under liquid environment. The main difficulty in obtaining reproducible images is given by the low adhesion of the molecules to the substrate. Physisorbed molecules are displaced by the scanning tip or are resuspended in the medium. We have therefore performed a covalent immobilization of immunoglobulin G (IgG) or its monovalent Fab′ fragment on gold surfaces thanks to thiol groups. For this purpose, multiple thiol groups were chemically introduced into the IgG molecule by treatment with Traut’s reagent. As an alternative, for a Fab′ fragment, we prepared molecules with a single thiol group located close to the C terminus of the truncated heavy chain. Both immobilization techniques enable us to observe clearly discernable individual molecules in liquid media. The grafting of Fab′ fragments on gold surface open new opportunities to study protein interactions.