Molecular level interactions between blood components and model biomaterials studied by atomic force microscopy.

摘要

A complete understanding of blood-biomaterial interactions is necessary for the development of blood-compatible biomaterials that can be used for making long-term implants. To this end, in this in vitro study, the interactions of purified blood components with model biomaterials have been studied with an objective of establishing relationships between the surface properties and the biological response. The principal technique that was used in this study is atomic force microscopy (AFM), which allows both visualization of biomolecules in physiologically relevant environments and the measurement of interaction forces from the muN to pN range.; Tapping mode atomic force microscopy was used to study the time-dependent changes in the structure of fibrinogen under aqueous conditions following adsorption on two model surfaces: hydrophobic graphite and hydrophilic mica. Fibrinogen is a key plasma protein involved in initiation of thrombosis on synthetic surfaces, and its adsorption to the biomaterial surface and subsequent interactions with the blood platelets are of fundamental interest. Fibrinogen was observed in the characteristic trinodular form. Based on the differences in the relative heights of the D and the E domains, four initial orientation states were observed for fibrinogen adsorbed on both surfaces. On graphite, the initial asymmetric orientation states disappeared with spreading over time. Spreading kinetics of fibrinogen on the two surfaces was determined by measuring the heights of the D and E domains over a time-period of ∼2 hours. The spreading of the D and E domains on graphite was analyzed using an 'exponential-decay-of-height' model and a two-step spreading model is proposed.; With the objective of relating the observed post-adsorption structural changes in fibrinogen to the surface availability of active epitopes and extending AFM imaging studies to complex multicomponent protein films, the adhesion mapping mode of AFM was developed for biologically sensitive imaging. AFM probes were functionalized by covalently linking polyclonal antibodies against fibrinogen. Adhesion mapping mode of AFM was used to generate both topographic images and adhesion images. The efficacy of the functionalized probes was first established by performing adhesion mapping on patterned dual-component protein films formed by microcontact printing bovine serum albumin (BSA) on a mica surface and then backfilling with fibrinogen. Next, adhesion mapping was done on randomly distributed two-component protein monolayers generated by sequential adsorption of submonolayer amounts of fibrinogen followed by backfilling with bovine serum albumin. (Abstract shortened by UMI.)