Cholesterol-Modified Polyurethane Valve Cusps Demonstrate Blood Outgrowth Endothelial Cell Adhesion Post-Seeding In Vitro and In Vivo Stanley J. Stachelek,

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Presentation transcript:

Cholesterol-Modified Polyurethane Valve Cusps Demonstrate Blood Outgrowth Endothelial Cell Adhesion Post-Seeding In Vitro and In Vivo Stanley J. Stachelek, PhD, Ivan Alferiev, PhD, Jeanne M. Connolly, MS, Michael Sacks, PhD, Robert P. Hebbel, MD, Richard Bianco, BA, Robert J. Levy, MD The Annals of Thoracic Surgery Volume 81, Issue 1, Pages 47-55 (January 2006) DOI: 10.1016/j.athoracsur.2005.07.061 Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Implantation of cell-seeded polyurethane valve leaflets. (A) Valve leaflet mounted in holder (arrow), which is used for manipulation both during cell culture and surgery. (B) Holder and trimmed leaflet shown with handle in place. (C) Sutures prepared on valve sewing annulus while still in holder, resulting in (D) implantation with minimal handling of seeded valve. The Annals of Thoracic Surgery 2006 81, 47-55DOI: (10.1016/j.athoracsur.2005.07.061) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Biomechanics: stress–strain behavior comparing cholesterol-modified polyurethane (two lower gray lines) with unmodified polyurethane (two upper black lines) films, demonstrating comparable elastic properties as indicated in plots of the elastic modulus versus applied strain. The Annals of Thoracic Surgery 2006 81, 47-55DOI: (10.1016/j.athoracsur.2005.07.061) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Characterization of sheep blood outgrowth endothelial cells in vitro. (A) Western blot showing the presence of PECAM-1, Flk-1, and Tie1 proteins. (MW = molecular weight.) (B) Cobblestone morphology of blood outgrowth endothelial cells grown on polyurethane-cholesterol (hematoxylin and eosin stain, magnification ×200). (C) Immunohistochemistry demonstrating von Willebrand’s factor (brown stain; magnification ×200), compared with (D) negative immunoglobin G control slide. The Annals of Thoracic Surgery 2006 81, 47-55DOI: (10.1016/j.athoracsur.2005.07.061) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Increased collagen synthesis by blood outgrowth endothelial cells grown on polyurethane-cholesterol (PU-Chol) compared with blood outgrowth endothelial cells grown on unmodified polyurethane (PU). *p = 0.006. (cpm = counts per minute.) The Annals of Thoracic Surgery 2006 81, 47-55DOI: (10.1016/j.athoracsur.2005.07.061) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 Adhesion of blood outgrowth endothelial cells exposed to constant flow. (A) Graphical representation of blood outgrowth endothelial cells retention after a 2-hour exposure to simulated valvular levels of shear flow (75 dynes/cm2). *p = 0.006. (B) Phase-contrast photomicrographs comparing representative starting fields of cells versus cells retained after 2 hours of shear flow: (a–c) time zero (t0); (d–f) 2 hours after flow (t2h) for blood outgrowth endothelial cells grown on polyurethane (PU: a, d), glass (b, e), and polyurethane-cholesterol (PU-Chol: c, f). Magnification ×200. The Annals of Thoracic Surgery 2006 81, 47-55DOI: (10.1016/j.athoracsur.2005.07.061) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 6 Appearance of pulmonary polyurethane valve leaflet explants. (A) Gross examination of explants of blood outgrowth endothelial cell–seeded polyurethane-cholesterol leaflets (30 days), showing translucent surfaces with no gross abnormalities, and of (B) 30-day explants of unseeded polyurethane leaflets, showing gross organized thrombi extending to the cusp surface. Immunostaining for CD41/CD61, a marker of activated platelets, showed no immunoreactivity on the intact endothelium of seeded polyurethane-cholesterol 30-day explant (C), but highly positive areas (brown) on unseeded explants (D). (E through G) Fluorescent micrographs of explanted polyurethane leaflets. Nuclei shown by en face 4,6-diamidino-2-phenylindole mount of representative 30-day blood outgrowth endothelial cell–seeded polyurethane-cholesterol (E) and 30-day polyurethane-cholesterol leaflets (F). Phase-contrast bright-field examination of blood outgrowth endothelial cell–seeded explant (G), shown merged with 4,6-diamidino-2-phenylindole fluorescent micrograph of nuclei, showing an organized flow-related orientation of retained blood outgrowth endothelial cells as indicated by white arrow. (H) Hematoxylin and eosin stain of 30-day unseeded polyurethane explant showing only inflammatory cells, including monocyte/macrophages and giant cells. Magnifications of C and D are ×650, E–G are ×200, and H is ×400. The Annals of Thoracic Surgery 2006 81, 47-55DOI: (10.1016/j.athoracsur.2005.07.061) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 7 Quantitation of cells adhering to pulmonary valve leaflet replacement explants, compared with seeded handling control. Explanted leaflets were 4,6-diamidino-2-phenylindole mounted en face, and the number of nuclei found in representative ×200 fields was counted. Blood outgrowth endothelial cell–seeded polyurethane-cholesterol (Chol-PU) leaflets had significantly more cells than unseeded leaflets of any configuration (*p ≤ 0.005), and not significantly fewer than did unimplanted handling controls. (PU = polyurethane.) The Annals of Thoracic Surgery 2006 81, 47-55DOI: (10.1016/j.athoracsur.2005.07.061) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions

Fig 8 Characterization of cells adhering to blood outgrowth endothelial cell–seeded polyurethane-cholesterol pulmonary valve leaflet explants. Comparison of cross section of unimplanted blood outgrowth endothelial cell-seeded polyurethane-cholesterol leaflet (A) versus en face 30-day explant (B) and cross section of 90-day explant (C), using immunostaining (brown) for von Willebrand’s factor, demonstrating a persistent endothelial phenotype. (Aa), (B), and (Ca) show strongly positive results, compared with negative immunoglobin G controls (Ab and Cb). Magnification ×400. The Annals of Thoracic Surgery 2006 81, 47-55DOI: (10.1016/j.athoracsur.2005.07.061) Copyright © 2006 The Society of Thoracic Surgeons Terms and Conditions