Marcus Textor

A comparative study of protein adsorption on titanium oxide surfaces using in situ ellipsometry, optical waveguide lightmode spectroscopy, and quartz crystal microbalance/dissipation

Abstract The adsorption kinetics of three model proteins—human serum albumin, fibrinogen and hemoglobin—has been measured and compared using three different experimental techniques: optical waveguide lightmode spectroscopy (OWLS), ellipsometry (ELM) and quartz crystal microbalance (QCM-D). The studies were complemented by also monitoring the corresponding antibody interactions with the pre-adsorbed protein layer. All measurements were performed with identically prepared titanium oxide coated substrates. All three techniques are suitable to follow in-situ kinetics of protein–surface and protein–antibody interactions, and provide quantitative values of the adsorbed adlayer mass. The results have, however, different physical contents. The optical techniques OWLS and ELM provide in most cases consistent and comparable results, which can be straightforwardly converted to adsorbed protein molar (‘dry’) mass. QCM-D, on the other hand, produces measured values that are generally higher in terms of mass. This, in turn, provides valuable, complementary information in two respects: (i) the mass calculated from the resonance frequency shift includes both protein mass and water that binds or hydrodynamically couples to the protein adlayer; and (ii) analysis of the energy dissipation in the adlayer and its magnitude in relation to the frequency shift (c.f. adsorbed mass) provides insight about the mechanical/structural properties such as viscoelasticity.

Optical grating coupler biosensors.

By incorporating a grating in a planar optical waveguide one creates a device with which the spectrum of guided lightmodes can he measured. When the surface of the waveguide is exposed to different solutions, the peaks in the spectrum shift due to molecular interactions with the surface. Optical waveguide lightmode spectroscopy (OWLS) is a highly sensitive technique that is capable of real-time monitoring of these interactions. Since this integrated optical method is based on the measurement of the polarizability density (i.e., refractive index) in the vicinity of the waveguide surface, radioactive, fluorescent or other kinds of labeling are not required. In addition, measurement of at least two guided modes enables the absolute mass of adsorbed molecules to be determined. In this article, the technique will be described in some detail, and applications from different areas will be discussed. Selected examples will be presented to demonstrate how monitoring the modification of different metal oxides with polymers and the response of the coated oxides to biofluids help in the design of novel biomaterials; how OWLS is useful for accurate bioaffinity sensing, which is a key issue in the development of new drugs; and how the quantitative study of protein-DNA/RNA and cell surface interactions can enhance the understanding of processes in molecular and cellular biology.

Immobilization of the cell-adhesive peptide Arg-Gly-Asp-Cys (RGDC) on titanium surfaces by covalent chemical attachment

Surface modification of acid-pretreated titanium with 3-aminopropyltriethoxylsilane (APTES) in dry toluene resulted in covalently bonded siloxane films with surface coverage that was relatively controllable by regulating the reaction conditions. A hetero-bifunctional cross-linker, N-succinimidyl-3-maleimidopropionate (SMP), reacted with the terminal amino groups, forming the exposed maleimide groups. Finally, a model cell-binding peptide, Arg–Gly–Asp–Cys (RGDC), was immobilized on the surface through covalent addition of the cysteine thiol groups to the maleimide groups. X-ray photoelectron spectroscopy, radiolabelling techniques, and ellipsometry were used to quantify and characterize the modified surfaces.

Anodic plasma-chemical treatment of CP titanium surfaces for biomedical applications

The anodic plasma-chemical (APC) process was used to modify CP titanium surfaces for biomedical applications. This technique allows for the combined chemical and morphological modification of titanium surfaces in a single process step. The resulting conversion coatings, typically several micrometer thick, consist mainly of titanium oxide and significant amounts of electrolyte constituents. In this study, a new electrolyte was developed containing both calcium-stabilized by complexation with EDTA-and phosphate ions at pH 14. The presence of the Ca-EDTA complex, negatively charged at high pH, favors incorporation of high amounts of calcium into the APC coatings during the anodic (positive) polarization. The coating properties were evaluated as a function of the process variables by XPS, GD-OES, Raman spectroscopy, SEM and tensile testing, and compared to those of calcium-free APC coatings and uncoated CP titanium surfaces. The maximal Ca/P atomic ratio in the coating produced with the new APC electrolyte was approximately 1.3, with higher Ca concentrations than reported in conventional APC coatings. The dissolution behavior of the incorporated, amorphous CaP phases was investigated by exposure to a diluted EDTA solution. The coatings produced in the new electrolyte system exhibit favorable mechanical stability. The new APC technology is believed to be a versatile and cost-effective coating technique to render titanium implant surfaces bioactive.

Properties and Biological Significance of Natural Oxide Films on Titanium and Its Alloys

This chapter covers information on the composition, microstructure and physico-chemical properties of thin oxide films on titanium and titanium alloys. The focus is on thin layers in the sense of ‘natural’ oxide films grown at ambient or higher temperatures with emphasis on titanium oxide, with some selected additional information on oxides related to metals commonly used as alloying elements in titanium alloys for biomedical applications. This chapter does not, however, include thicker oxide films such as those produced by electrochemical or plasma techniques, which are covered in Chap. 8.

Peptide functionalized poly(l-lysine)-g-poly(ethylene glycol) on titanium: resistance to protein adsorption in full heparinized human blood plasma

The graft copolymer poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and its RGD- and RDG-functionalized derivatives (PLL-g-PEG/PEG-peptide) were assembled from aqueous solutions on titanium (oxide) surfaces. The polymers were characterized by NMR in order to determine quantitatively the grafting ratio, g (Lys monomer units/PEG side chains), and the fraction of the PEG side chains carrying the terminal peptide group. The titanium surfaces modified with the polymeric monomolecular adlayers were exposed to full heparinized blood plasma. The adsorbed masses were measured by in situ ellipsometry. The different PLL-g-PEG-coated surfaces showed, within the detection limit of the ellipsometric technique, no statistically significant protein adsorption during exposure to plasma for 30 min at 22 degrees C or 37 degrees C, whereas clean, uncoated titanium surfaces adsorbed approximately 350 ng/cm2 of plasma proteins. The high degree of resistance of the PEGylated surface to non-specific adsorption makes peptide-modified PLL-g-PEG a useful candidate for the surface modification of biomedical devices such as implants that are capable of eliciting specific interactions with integrin-type cell receptors even in the presence of full blood plasma. The results refer to short-term blood plasma exposure that cannot be extrapolated a priori to long-term clinical performance.

Comparative investigation of the surface properties of commercial titanium dental implants. Part I: chemical composition.

The surfaces of five commercially available titanium implants (Brånemark Nobel Biocare, 3i ICE, 3i OSSEOTITE, ITI-TPS, and ITI-SLA) were compared by scanning electron microscopy, X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectroscopy. All five implant types were screw-shaped and fabricated from commercially pure (cp) titanium, but their surface properties differed both as regards surface morphology and surface chemical composition. The macro- and microstructure of the implant surfaces were investigated by scanning electron microscopy. The surfaces chemical composition was determined using the surface-sensitive analytical techniques of X-ray photoelectron spectroscopy and time-of-flight secondary ion spectrometry. Surface topographies were found to reflect the type of mechanical/chemical fabrication procedures applied by the manufacturers. The titanium oxide (passive) layer thickness was similar (5–6 nm) and typical for oxide films grown at or near room temperature. A variety of elements and chemical compounds not related to the metal composition were found on some implant types. They ranged from inorganic material such as sodium chloride to specific organic compounds believed to be due to contamination during fabrication or storage. The experimental findings are believed to make a contribution to a better understanding of the interplay between industrial fabrication procedure and physico-chemical implant surface properties.

CHARACTERIZATION OF ANODIC SPARK-CONVERTED TITANIUM SURFACES FOR BIOMEDICAL APPLICATIONS

The aim of the present study was to characterize the surface morphology, microstructure and the chemical composition of anodic spark-converted titanium surfaces. The coatings were prepared in an electrochemical cell by the anodic spark deposition technique in an aqueous solution of CaH2PO4)2. The coatings were characterized by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), electron probe microanalysis (EPMA) and X-ray diffraction (XRD). The properties of the coatings are described in terms of morphology.

Polyoxazolines for Nonfouling Surface Coatings — A Direct Comparison to the Gold Standard PEG

The prevention of surface fouling is becoming increasingly important for the development of anti-infective medical implants, biosensors with improved signal-to-noise ratios, and low-fouling membranes to name a few examples. We review a direct comparison of poly(ethylene glycol), the gold standard polymer to impart surfaces with nonfouling properties, to an alternative polymer, poly(2-methyl-2-oxazoline) (PMOXA), and show that both polymers are equally excellent in rendering surfaces nonfouling while PMOXA coatings are more stable in oxidative environments. We discuss prerequisites for the fabrication of nonfouling surface coatings and implications for the polymer choice according to application requirements.

Chemically patterned, metal-oxide-based surfaces produced by photolithographic techniques for studying protein- and cell-interactions. II: Protein adsorption and early cell interactions.

Protein adsorption and adhesion of primary human osteoblasts on chemically patterned, metal-oxide-based surfaces comprising combinations of titanium, aluminium, vanadium and niobium were investigated. Single metal samples with a homogeneous surface and bimetal samples with a surface pattern produced by photolithographic techniques were used. The physical and chemical properties of the samples have been extensively characterised and are presented in a companion paper. Here, we describe their properties in terms of cell responses during the initial 24h of cell culture. Regarding the cell number and activity there was no significant difference between any of the single metal surfaces. However the morphology of cells on vanadium surfaces became spindle-like. In contrast to the behaviour on single metal samples, cells exhibited a pronounced reaction on bimetallic surfaces that contained aluminium. Cells tended to stay away from aluminium, which was the least favoured metal in all two-metal combinations. An initial cell alignment relative to the pattern geometry was detectable after 2h and was fully developed after 18h of incubation. The organisation of f-actin and microtubules as well as the localisation of vinculin were all more pronounced on non-aluminium regions. We hypothesised that the differences in cell response could be associated with differences in the adsorption of serum proteins onto the various metal oxides. Protein adsorption experiments were performed using microscopy in conjunction with immunofluorescent stains. They indicated that both fibronectin and albumin adsorption were significantly greater on the non-aluminium regions, suggesting that differences in cellular response correlate with a modulation of the concentration of serum proteins on the surface.