Yannis Missirlis

Interactions of bacteria with specific biomaterial surface chemistries under flow conditions.

The effect of specific chemical functionalities on the adhesion of two Staphylococcus epidermidis strains under flow was investigated by using surfaces prepared by self-assembly of alkyl silane monolayers on glass. Terminal methyl (CH(3)) and amino (NH(2)) groups were formed in solution and by chemical vapor deposition of silanes, at elevated temperature. Hydroxyl (OH)-terminated glass was used as control. Surface modification was verified by contact angle and zeta potential measurements, atomic force microscopy and X-ray photoelectron spectroscopy. A parallel plate flow chamber was used to evaluate bacterial adhesion at various shear rates. The effect of the solutions ionic strength on adhesion was also studied. Adhesion was found to be dependent on the monolayers terminal functionality. It was higher on the CH(3) followed by the NH(2) and minimal on the OH-terminated glass for both strains. The increase in the ionic strength significantly enhanced adhesion to the various substrates, in accordance with the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. The extended DLVO theory explained well the combined effects of surface and solution properties on bacterial adhesion under low shear rates. However, the increase in the shear rate restricted the predictability of the theory and revealed macromolecular interactions between bacteria and NH(2)-terminated surfaces.

Comparison of haemocompatibility improvement of four polymeric biomaterials by two heparinization techniques.

Two surface heparinization procedures, introduced by Bamford and Al-Lamee (Polymer 22 (1996) 4885; 13 (1994) 2844) and Seifert et al. (J. Mater. Sci.: Mater. Med. 7 (1996) 465), respectively, were applied to four commercially available biomaterials (silicone rubber, polyethylene, polypropylene and polyvinylchloride) in order to compare their efficiency in improving haemocompatibility. The indirect method (Bamford and Al-Lamee) produced a much better heparinization yield-10.5% maximum, compared to the direct one (Seifert et al.), of only 0.20% maximum. Both methods provided a better response of the heparinized biomaterials compared to the uncoated ones in terms of platelet retention and a significantly better response in terms of activation of the coagulation system, suggesting that heparin molecules remained biologically functional in both cases. The results were particularly interesting in the case of polyvinylchloride where the maximum immobilization yield was obtained by the indirect method resulting also to a pronounced haemocompatibility improvement. Scanning electron microscopy studies confirmed adhered platelet morphology whereas atomic force microscopy was used to examine surface morphology of heparinized and reference materials surface.

An approach to the optimization of preparation of bioprosthetic heart valves

The stress and strain states of the valve leaflets during fixation with glutaraldehyde affect their final mechanical parameters. Comparative studies of the stress-strain relationships of aortic valve leaflet strips from fresh, statically and dynamically fixed porcine and human valves were made. Static pressures of 5 mmHg, 16 mmHg, and 95 mmHg result in stress-strain relationships which are in a region between that of fresh porcine and fresh human leaflet strips in the circumferential direction, while they are far from that of fresh porcine tissue (larger strains) in the radial direction. Leaflet strips, fixed under dynamic loading between zero and a predefined maximum load, set at an early post-transition state, give parameters not significantly different from those of human valves.

Staphylococcus epidermidis adhesion to He, He/O2 plasma treated PET films and aged materials: Contributions of surface free energy and shear rate

Adhesion studies of bacteria (Staphylococcus epidermidis) to plasma modified PET films were conducted in order to determine the role of the surface free energy under static and dynamic conditions. In particular, we investigated the effect of the ageing time on the physicochemical surface properties of helium (He) and 20% of oxygen in helium (He/O(2)) plasma treated polyethylene terephthalate (PET) as well as on the bacterial adhesion. Treatment conditions especially known to result in ageing sensitive hydrophilicity (hydrophobic recovery) were intentionally chosen in an effort to obtain the widest possible range of surface energy specimens and also to avoid strong changes in the morphological properties of the surface. Both plasma treatments are shown to significantly reduce bacterial adhesion in comparison to the untreated PET. However, the ageing effect and the subsequent decrease in the surface free energy of the substratum surfaces with time - especially in the case of He treated samples - seem to favor bacterial adhesion and aggregation. The dispersion-polar and the Lifshitz-van der Waals (LW) acid-base (AB) thermodynamic approaches were applied to calculate the Gibbs free energy changes of adhesion (DeltaG(adh)) of S. epidermidis interacting with the substrates. There was a strong correlation between the thermodynamic predictions and the measured values of bacterial adhesion, when adhesion was performed under static conditions. By decoupling the (DeltaG(adh)) values into their components, we observed that polar/acid-base interactions dominated the interactions of bacteria with the substrates in aqueous media. However, under flow conditions, the increase in the shear rate restricted the predictability of the thermodynamic models.

STRESS RELAXATION BEHAVIOUR OF TRABECULAR BONE SPECIMENS

The present study defines several conditions under which stress relaxation tests can be performed and investigates the viscoelastic behaviour of trabecular bone in compression through a series of stress relaxation tests at three strain levels and in three loading directions of each cubic specimen. A viscoelastic model is proposed to characterize the behaviour of trabecular bone and a spectrum of relaxation times is determined. Trabecular bone from the femoral head is non-linearly viscoelastic and displays anisotropic behaviour, which cannot be more symmetric elastically than orthotropic.

Bacterial adhesion onto materials with specific surface chemistries under flow conditions

Staphylococcus epidermidis adhesion onto materials with specific chemical functionalities, under flow, was investigated by using surfaces prepared by self-assembly of alkyl silane monolayers on glass. Terminal methyl (CH3) and amino (NH2) groups were formed by chemical vapor deposition of silanes, at elevated temperature. Carboxyl (COOH) terminated groups were prepared by further modification of NH2 groups with succide anhydride and positively charged NH2 groups by adsorption of poly-l-lysine hydrobromide. Hydroxyl (OH) terminated glass was used as control. Surface modification was verified by contact angle measurements, atomic force microscopy and X-ray photoelectron spectroscopy. A parallel plate flow chamber was used to evaluate bacterial adhesion at various shear rates. Adhesion was found to be depended on the monolayer’s terminal functionality. It was higher on the CH3 followed by the positively charged NH2, the non-charged NH2 groups, the COOH and minimal on the OH-terminated glass. The increase in the material surface free energy significantly reduced the adhesion of a hydrophilic bacterial strain, and this is in accordance with the predictions of the thermodynamic theory. However, the increase in the shear rate restricted the predictability of the theory and revealed macromolecular interactions between bacteria and NH2- and COOH-terminated surfaces.

Tuning Cell Adhesion and Growth on Biomimetic Polyelectrolyte Multilayers by Variation of pH During Layer-by-Layer Assembly

Polyelectrolyte multilayers of chitosan and heparin are assembled on glass where heparin is applied at pH = 4, 9 and 4 during the formation of the first layers followed by pH = 9 at the last steps (denoted pH 4 + 9). Measurements of wetting properties, layer mass, and topography show that multilayers formed at pH = 4 are thicker, contain more water and have a smoother surface compared to those prepared at pH = 9 while the pH = 4 + 9 multilayers expressed intermediate properties. pH = 9 multilayers are more cell adhesive and support growth of C2C12 cells better than pH = 4 ones. However, pH 4 + 9 conditions improve the bioactivity to a similar level of pH = 9 layers. Multilayers prepared using pH 4 + 9 conditions form thick enough layers that may allow efficient loading of bioactive molecules.

Measurement of interaction forces between fibrinogen coated probes and mica surface with the atomic force microscope: The pH and ionic strength effect

The study of protein-surface interactions is of great significance in the design of biomaterials and the evaluation of molecular processes in tissue engineering. The authors have used atomic force microscopy (AFM) to directly measure the force of attraction/adhesion of fibrinogen coated tips to mica surfaces and reveal the effect of the surrounding solution pH and ionic strength on this interaction. Silica colloid spheres were attached to the AFM cantilevers and, after plasma deposition of poly(acrylic acid), fibrinogen molecules were covalently bound on them with the help of the cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in the presence of N-hydroxysulfosuccinimide (sulfo-NHS(. The measurements suggest that fibrinogen adsorption is controlled by the screening of electrostatic repulsion as the salt concentration increases from 15 to 150 mM, whereas at higher ionic strength (500 mM) the hydration forces and the compact molecular conformation become crucial, restricting adsorption. The protein attraction to the surface increases at the isoelectric point of fibrinogen (pH 5.8), compared with the physiological pH. At pH 3.5, apart from fibrinogen attraction to the surface, evidence of fibrinogen conformational changes is observed, as the pH and the ionic strength are set back and forth, and these changes may account for fibrinogen aggregation in the protein solution at this pH.

Standardized Nanomechanical Atomic Force Microscopy Procedure (SNAP) for Measuring Soft and Biological Samples

We present a procedure that allows a reliable determination of the elastic (Young’s) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever’s spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions.

Haemocompatibility improvement of metallic surfaces by covalent immobilization of heparin-liposomes.

Stainless steel surfaces were processed by means of plasma enhanced chemical vapor deposition (PE-CVD) fed with acrylic acid vapors in order to functionalize them with carboxyl groups, which were subsequently activated for covalent immobilization of heparin-loaded (HEP) NH(2) group-functionalized (Fun) nanoliposomes (NLs). Empty Fun or HEP non-functionalized (control) NLs were used as controls. NLs were characterized for mean diameter, surface charge and heparin encapsulation/release. Different lipid compositions were used for NL construction; PC/Chol (2:1mol/mol) or PC/Chol (4:1mol/mol) (fluid type vesicles) [which allow gradual release of heparin] and DSPC/Chol (2:1mol/mol) (rigid type vesicles). Surface haemocompatibility was tested by measuring blood clotting time. Platelet adhesion on surfaces was evaluated morphologically by SEM and CLSM. The haemocompatibility of plasma-processed surfaces was improved (compared to untreated surfaces); Fun-HEP NL-coated surfaces demonstrated highest coagulation times. For short surface/blood incubation periods, surfaces coated with Fun-HEP NLs consisting of PC/Chol (2:1) had higher coagulation times (compared to DSPC/Chol NLs) due to faster release of heparin. Heparin release rate from the various NL types and surface platelet adhesion results were in agreement with the corresponding blood coagulation times. Concluding, covalent immobilization of drug entrapping NLs on plasma processed surfaces is a potential method for preparation of controlled-rate drug-eluting metallic stents or devices.