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In vivo protein adsorption on polymers: visualization of adsorbed proteins on vascular implants in dogs

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The absorption of plasma proteins is an important event at the blood-material interface, and strongly affects subsequent cellular interaction and thrombus formation. Although considerable efforts have been expended to elucidate the mechanism of protein adsorption and the role of absorbed protein layer at the blood-material interface, there has been little knowledge of how the initial adsorbed proteins are maintained or changed in a time-variant process in in vivo long-term implantation. In this study, we described detailed analyses concerning the characterization of adsorbed proteins on HEMA-styrene block copolymer surfaces (HEMA-st) and poly(ethylene oxide)(PEO) grafted BiomerĀ® (B-PE04K) for in vivo long-term canine vascular graft implants as well as in vitro short-term experiments. Biomer vascular grafts (6 mm I.D., 7 cm in length) were fabricated by a dip coating and the luminal surface was modified with PEO grafting, HEMA-st coating, or Biomer coating (control). These surface modified grafts were recirculated for different time intervals (5, 15, 30, 60 and 120 min) using citrated canine whole blood. The grafts were then implanted in the abdominal aortas of dogs and evaluated for graft patency and protein adsorption. The adsorbed proteins (albumin, IgG and fibrinogen) were quantified using an in situ radioimmunoassay. Surface protein layer thickness was measured by transmission electron microscopy (TEM). Visualization of absorbed plasma proteins (albumin, IgG and fibrinogen) was performed with TEM using an immunoperoxidase double antibody technique. In in vitro recirculation systems, albumin and IgG showed similar Langmuir type pattern onto all test surfaces. On B-PEO4K surfaces, fibrinogen adsorption kinetics demonstrated 'Vroman effect'. The Biomer and B-PE04K grafts occluded within 1 month, while HEMA-st grafts were patent for over 3 months. Biomer and B-PE04K showed thick multilayers of adsorbed proteins, and the thickness increased with implantation periods and the composition altered with time. In contrast, HEMA-st showed a monolayer-like adsorbed protein pattern, and the composition and thickness were consistent regardless of implantation time including in vitro short-time experiments, which may attribute to less conformational change of adsorbed proteins on HEMA-st surfaces. In terms of nonthrombogenicity, the stable monolayer-like adsorbed protein layer on HEMA-st surfaces exhibited improved blood compatibility over thick multilayered adsorbed proteins on Biomer and B-PE04K surfaces.

Keywords: Hydrophilic/hydrophobic block copolymer; immunoperoxidase method; protein adsorption; transmission electron microscopy (TEM)

Document Type: Research Article


Affiliations: 1: Institute of Biomedical Science, Terumo Corporation R&D Center, 1500 Inokuchi, Nakai-machi, Ashigarakami-gun, Kanagawa 259-01, Japan, The Heart Institute of Japan 2: Institute of Biomedical Engineering, Tokyo Women's Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162, Japan 3: The Heart Institute of Japan 4: Department of Polymer Chemistry, Tokyo Institute of Technology, 2-12-1 Ohokayama, Meguro-ku, Tokyo 152, Japan 5: Department of Pharmaceutics/Center for Controlled Chemical Delivery, University of Utah, 421 Wakara Way, Salt Lake City, Utah 84108, U.S.A.

Publication date: 1993-01-01

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