Claudia Sperling

Current strategies towards hemocompatible coatings

A wide range of biomedical devices is applied clinically in contact with blood. Tailoring the surface properties of the involved biomaterials is a common approach to enhance performance and to limit adverse reactions. This review summarizes current trends in coating technologies developed for that purpose. Inorganic coatings were shown to substantially improve the durability and inertness of biomaterials while a number of advanced polymer coatings were demonstrated to be very effective by targeting specific biochemical pathways. However, to fully utilize the power of these bioactive coatings safety issues need to be thoroughly addressed in future studies.

Blood coagulation on biomaterials requires the combination of distinct activation processes

The rational design of hemocompatible materials requires a mechanistic understanding of activation processes induced at the blood-material interface. Binary self-assembled monolayers of alkyl thiols (SAMs) with various ratios of -CH3 and -COOH terminations were used to study the relevance of hydrophobic and negatively charged surfaces for the initiation of blood coagulation. Platelet adhesion and activation of the intrinsic coagulation pathway scaled with the surface composition: the numbers of adherent platelets were highest on the 100%-CH3 surface whereas the greatest contact activation was seen on 100%-COOH surfaces. In vitro whole blood incubation assays showed, however, that the surfaces exposing either -CH3 or -COOH groups induced comparably low levels of thrombin formation while the surfaces with intermediate contents of both terminating groups had significantly higher values. These results reveal that contact activation and platelet adhesion have a strong synergistic effect on coagulation on blood-contacting materials even though these events in isolation are not sufficient to induce substantial thrombin formation. Successful surface design strategies for hemocompatible materials therefore need to carefully consider the interplay of both processes.

Quantitative analysis of immobilized proteins and protein mixtures by amino acid analysis

Biomolecular surface engineering of materials often requires precise, versatile and efficient quantification of immobilized proteins at solid surfaces. Acidic hydrolysis of surface-bound proteins and subsequent HPLC analysis of fluorescence-derivatized amino acids were adapted and critically evaluated for that purpose. Contaminations and concentration-dependent amino acid retrieval during HPLC were found to influence the accuracy of the method. In addition to the choice of adequate conditions for hydrolysis, derivatization and chromatographic separation extensions of the data evaluation were suggested to improve the accuracy of the approach when applied to single protein systems: comparing the experimentally obtained amino acid ratio to the protein constitution enabled to identify the properly separated and detected amino acids. Those amino acids were selected for a more precise calculation of the amount of immobilized protein. To further increase the accuracy of the method, the retrieval of amino acids corresponding to protein amounts in the range between 0.5 and 4.0 microg was analyzed for a variety of proteins of interest to derive protein-specific correction factors. The evaluation of amino acid data was furthermore applied to quantify binary protein mixtures at similar settings. This method was proven useful to detect the composition of protein mixtures throughout a wide range of absolute and relative concentrations.

Plasma modification of polytetrafluoroethylene for immobilization of the fibrinolytic protein urokinase

Abstract The modification of surface properties is necessary to improve the blood compatibility of polytetrafluoroethylene (PTFE). This paper describes the concept of a modification of PTFE surfaces. It consists of three steps: first, water vapour microwave plasma modification; second, acrylic acid vapour-phase post-grafting; and third, immobilization of the fibrinolytic protein urokinase. During or after each step, the modification results were characterized by zeta potential, ellipsometry, wetting measurements and X-ray photoelectron spectroscopy.

Immobilization of human thrombomodulin onto PTFE.

Human thrombomodulin (hTM) is an endothelial cell-surface glycoprotein and has effective anticoagulant properties. This protein was immobilized onto polytetrafluorethylene (PTFE) surfaces to create biomaterials with enhanced haemocompatibility. The PTFE surface was functionalized by CO2 plasma activation and subsequent vapour-phase graft polymerization of acrylic acid. Surface characterization after plasma treatment, grafting and hTM immobilization was achieved by attenuated total reflection-Fourier transform–infrared spectroscopy, X-ray photoelectron spectroscopy, zeta potential and wetting measurements. The activity of immobilized hTM was estimated using the protein C activation test.

The ability of surface characteristics of materials to trigger leukocyte tissue factor expression.

Biomaterial-induced thrombosis is usually attributed to blood coagulation initiated by contact phase and platelet-related reactions. Considering the major role of extrinsic initiation in blood coagulation in vivo, we studied the material related-induction of this pathway by investigating the relevance of surface properties for the expression of Tissue Factor (TF), a critical initiator of the extrinsic pathway of coagulation. We incubated materials with self-assembled monolayers of alkylthiols (SAMs) displaying various ratios of -CH(3), -OH, and -COOH terminations with fresh heparinized whole human blood in vitro. The transcription of TF-mRNA in leukocytes showed clear differences in relation to surface properties and increased over time. In addition, a positive correlation between TF transcription and its presence on leukocytes, granulocyte activation, and complement activation was found. Cells displaying the highest TF expression after material contact had significantly lower intracellular TF, pointing to previous TF release. Yet under the conditions of our whole blood incubation set-up within the limited time frame the observed initiation of the extrinsic pathway did not trigger blood coagulation.

Surface endotoxin contamination and hemocompatibility evaluation of materials

To evaluate the blood compatibility of new materials, a clear distinction between properties of the materials and effects due to surface contamination by adsorbed endotoxins is essential. This study compares direct contact approaches and elution methods with water, organic solvents, nonionic, and zwitterionic detergents for determination of surface-adsorbed endotoxin by the limulus amoebocyte lysate (LAL) test and determines the blood compatibility of various surfaces with controlled endotoxin contamination in vitro. The LAL test in direct contact with an endotoxin-contaminated surface was concluded to be not practicable for most devices and its sensitivity showed a high dependence on surface characteristics. Among the elution methods, 0.2% Tween-20 showed most stable elution characteristics and appears therefore preferable. Biological reactions at in vitro blood exposure were found to be only minimally influenced by adsorbed endotoxin during the time window of 2 h, allowing for a straightforward discrimination between materials and endotoxin-dependent reactions.

Synergistic effect of hydrophobic and anionic surface groups triggers blood coagulation in vitro

Biomaterial induced coagulation encompasses plasmatic and cellular processes. The functional loss of biomedical devices possibly resulting from these thrombotic reactions motivates the need for a better understanding of processes occurring at blood–biomaterial interfaces. Well defined model surfaces providing specific chemical–physical properties (self assembled monolayers (SAMs)) displaying hydrophobic or/and acidic terminal groups were used to uncover initial mechanisms of biomaterial induced coagulation. We investigated the influence of electrical charge and wettability on platelet- and contact activation, the two main actors of blood coagulation, which are often considered as separate mechanisms in biomaterials research. Our results show a dependence of contact activation on acidic surface groups and a correlation of platelet adhesion to surface hydrophobicity. Clot formation resulting from the interplay of blood platelets and contact activation was only found on surfaces combining both acidic and hydrophobic surface groups but not on monolayers displaying extreme hydrophobic/acidic properties.

Immobilization of the irreversible thrombin inhibitor D-Phe-Pro-Arg-chloromethylketone: A concept for hemocompatible surfaces?

The irreversible thrombin inhibitor D-Phe-Pro-Arg-chloromethylketone (PPACK) was covalently immobilized to PEGylated polymer thin films at its primary alpha-amino group. Activity assays and capture of radioconjugated thrombin reveal that the PPACK-decorated surfaces could bind thrombin forming up to 30% of a monolayer density. Back-calculation of this high thrombin-inhibiting capacity indicated that the surface immobilization of the inhibitor was still associated with more than two orders of magnitude of loss of activity; increasing activity was observed at higher surface densities. PPACK-containing polymer films almost duplicated the plasma coagulation time when compared with the reference substrate without inhibitor. In whole blood, however, the anticoagulant properties were below those previously found for benzamidine-type reversible thrombin inhibitors; in addition, the surface exhibited inflammatory properties. It is concluded that immobilized reversible thrombin inhibitors are more effective by passivating higher amounts of thrombin in a cooperative action with antithrombin III.

The effect of octadecyl chain immobilization on the hemocompatibility of poly (2-hydroxyethyl methacrylate)

Albumin-scavenging surfaces decorated with n-alkyl chains represent an established strategy for blood-contacting applications. To evaluate this concept, a set of poly (2-hydroxyethyl methacrylate) (pHEMA) films modified with different amounts of octadecyl isocyanate (C18) was investigated in an in vitro hemocompatibility assay using freshly drawn human whole blood. In addition, the hydrogel materials were thoroughly characterized with respect to changes in wettability and elasticity, which accompanied the gradual chemical modification of pHEMA. An increase of the surface C18 content induced enhanced hydrophobicity and stiffness. Immobilization of C18 chains was found to substantially reduce the coagulation activation and the complement activation by the pHEMA films. Platelet adhesion and degranulation (PF4 release) were similar on the modified and the unmodified pHEMA. Platelet adhesion to pHEMA hydrogels was lower than the polytetrafluoroethylene reference. We conclude that the immobilization of octadecyl chains improved the hemocompatibility of pHEMA materials under conditions that might be encountered in low shear blood flow.