With any blood contacting artificial surface, whether for long-term or short-term application, the biological response has its basis in the molecular and cellular interactions occurring at the material surface. This is the case whether the material is designed to be biologically """"""""inert"""""""" and shed blood proteins and/or cells or is developed to be biologically """"""""active"""""""" and provide a substrate for protein attachment and cell growth. Our studies have been, and continue to be, directed toward elucidating the basic cellular mechanisms involved in adhesion and the subsequent interactions of adherent platelets and cells with the substrate. The work is also designed to provide information on how the """"""""pioneer"""""""" proteins and cells/platelets interact in static and in flow conditions to subsequently modify the surface through protein rearrangement, selective protein deletion or addition, cell spreading, ligand receptor interactions, and protein or cell detachment. An understanding of these primary events relative to material (polymer) structure and relative to secondary events such as thrombus formation, embolization, cell attachment and growth is a key goal of the proposed studies. To this end correlative light and electron microscopic techniques are used in conjunction with various immunolabeling and ligand labeling procedures and with biochemical, physiological and pharmacological assessments. This approach permits visualization of bulk and surface polymer structure at the nanometer level of resolution and allows material structure to be directly correlated with protein attachment and orientation including protein-protein interactions. This in turn can be correlated to subsequent platelet adhesion, spreading, activation, and platelet-platelet interactions leading, or not leading, to thrombus formation and embolization. Specifically the structure of several classes of phase separated polyurethanes with differing soft segment/hard segment ratios within each class are to be determined with stereo high voltage (1 MeV) TEM and with low voltage (1-5 kV) high resolution SEM. Conventional ESCA and contact angle studies on these polymers are also to be performed. Adsorption, conformation, and epitope expression of various serum proteins, to these polymers, is to be observed by direct imaging of proteins and with colloidal gold immunolabeling or direct ligand labeling procedures. Spatial resolution is on the order of 2-5 nm (well below the size of individual polymer domains and the size of the proteins). Platelet attachment, spreading, Ca++ mobilization, secretion, cytoskeletal reorganization, receptor availability and motility are to be evaluated relative to polymer ultrastructure, surface characteristics, and protein adsorption/orientation. This is to be done on bare as well as protein exposed polymer and in static and flow conditions. Platelet-platelet interactions are also be to be studied as are surface modifications such as protein rearrangement, or selective protein addition or protein removal by platelets. These studies will enhance the understanding of blood-materials interactions at the molecular and cellular level and will aid in the design of """"""""biocompatible"""""""" materials either in the sense of inert, cell """"""""shedding"""""""" surfaces or surfaces designed for cell adhesion and growth. Additionally these in vitro studies with human proteins/cells facilitate comparisons with in vivo, and in vitro studies using other species.
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