Thrombosis at the blood-polymer interface confounds biomaterials design. This proposal investigates the biocompatibility of an exciting family of biomaterials, polydimethylsiloxanes end-functionalized with poly(ethylene oxide) (PEO siloxanes), to further such design. The properties of the PEO siloxanes will be tailored to optimize blood compatibility. These include the molecular weight of the poly(ethylene oxide), its tethering density, and the surface energetics of the PEO substrate. Platelet behavior and RGD protein adsorption elucidate the hemocompatibility of these biomaterials and will be examined. Macrophage adhesion and activation on the PEO siloxanes will also be examined in order to appraise their behavior as long-term implants. We will investigate the role of vitronectin adsorbed to these biomaterials because it should further characterize their biocompatibility. Adsorbed vitronectin is unique among adhesive blood proteins since the platelet deposition profiles to vitronectin-preadsorbed biomaterials resemble those to bare counterparts in acute thrombosis. The contact angle of bare and protein-preadsorbed biomedical polymers will be measured to hopefully provide insight into the ability of PEO siloxanes to resist protein adsorption. The adsorption of another RGD protein, fibrinogen, will be studied analogous to that of vitronectin because different adsorption tendencies are expected from fibrinogen due to different size, plasma concentration, and so forth. The incubation time and bulk concentration for adsorption will be selected to maximize the biological activity of adsorbed proteins. Fibrinogen and vitronectin will be isolated from citrated human blood plasma. Human serum albumin will be used as a control. Protein adsorption will be quantified and the kinetics determined using radioiodinated proteins. Radioiodination will be conducted using the chloramine-T method. A computerized goniometer will be used to measure the contact angles of bare and protein-preadsorbed PEO siloxanes in order to determine the free energies of these surfaces. For adhesion and adsorption experiments, the PEO siloxanes will be spin cast onto clean glass coverslips that have been pretreated with trichlorosilane to make the coverslips hydrophobic. Platelets will be isolated in an active form and suspended in Tyrodes' solution using gel-filtration chromatography of human platelet-rich plasma. The adhesion of gel-filtered platelets will be appraised, radiolabelling them for quantification of adhesion in vitro to bare and protein-preadsorbed polymers. Scanning electron microscopy will be used to assess platelet circularity, spread area and thus, activation. The PEO siloxanes will also be characterized with chemiluminescence using lucigenin to appraise the release of reactive oxygen species by macrophages in contact with these biomaterials. The human monocyte- derived macrophages will be obtained by elutriation. Macrophages will also be incubated with surfaces of the PEO siloxanes, bare or protein- preadsorbed, fixed, and imaged with scanning electron microscopy to appraise morphology and adhesion.
