Surface Modification Of Ti-O Film By Antithrombotic Biomolecule Immobilization And Evaluation Of Its Antithrombotic Properties

It is a significant work to improve the blood compatibility of blood-contacting biomaterials. A promising method is by antithrombotic biomolecules immobilization on the biomaterial surfaces. In this paper, Ti-O thin films were used as the substrate and were modified by various biomolecule immobilization through three methods. Firstly, the influence of the Ti-O film structure on the surface H₃PO₄ chemisorption on the Ti-O surface was studied, and biomolecule immobilization on Ti-O film was achieved by further silanization via chemisorption of H₃PO₄ interface. Sencondly, in order to obtain a stable surface, a self-assembling monolayer of alkylphosphonic acid on Ti-O film was prepared and different biomolecules were then further immobilized by photochemical methods, e.g., heparin was immobilized to obtain an active antithrombotic surface, bovine serum albumin (BSA) was immobilized to obtain an inert surface and gelatin was used to obtain a biomimetic surface. Finally, it was tried to construct heparinylated monolayer or multilayer on the Ti-O films through biotin-avidin biorecognition. The chemical composition and surface property of Ti-O film and the biomolecule immobilized Ti-O film were qualitatively and quantitatively characterized using Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), water contact angle analysis, fluorescence labeling method, surface profile analysis and staining methods. The blood compatibility and cell compatibility were investigated using in vitro platelet adhesion experiment, APTT test, evaluation of the denatured fibrinogen on surfaces, cell culture and in vivo implantation test. The following conclusions are obtained according to the results coming from the research mentioned above.1. The structure of Ti-O thin film has significant influence on the chemisorption of H₃PO₄ on the Ti-O surface. It is beneficial for the H3PO4 chemisorption on the surface when anatase Ti-O film acts as the substrate compared with rutile Ti-O film. The results reveal the dominating monodentate coordination of phosphoric acid to anatase Ti-O film and bidentate coordination to rutile Ti-O film. Since the middle layer of silane hydrolyzes continuously, immobilized biomolecules on the Ti-O film are unstable by the method of silanization via chemisorption of H3PO4 interface.2. A stable organic monolayer can be obtained by 3-aminopropylphosphonic acid (APP) self assembling on Ti-O film. Biomolecules were immobilized further by photochemical method on the APP modified surface. Some biomolecules were released in the first 3 days when incubation in phosphate buffer solution (PBS), and after that it was stable. The effective surface density of immobilized heparin is 1.2μg/cm², and that of immobilized gelatin is 2.3μg/cm². It showed active antithrombotic surface by immobilization of heparin and inert surface by immobilization of BSA both have the effects on inhibiting fibrinogen denaturation and decreasing platelet adhesion and aggregation on the surfaces. Biomolecule patterned surface was obtained by using a photomask when irradiating. Platelet adhesion on the patterned surface displayed a patterned distribution, and it mainly aggregated on the non-biomolecule immobilized region which identifies the immobilized heparin or BSA indeed has the ability of inhibiting platelet from adhering on the surface. In addition, the preliminary in vivo evaluation also showed the modified surfaces have good anticoagulant properties. Gelatin immobilized biomimetic surface has good cell compatibility, while it increases the fibrinogen denaturation and platelet adhesion on the surface.3. Heparinylated monolayer or multilayer on the Ti-O films was constructed through biotin-avidin biorecognition. The activity of the biotinylated heparin decreases with the increase of biotinylated modification ratio. B-hepI has 72% activity of the virgin heparin, and B-hepII has only 60%. Heparinylated multilayer has the properties of inhibiting platelet adhesion and aggregation. APTT increases with the first several layers of heparin and then it trends to be stable with the following increase of layers.