Immobilization of heparin to EDC/NHS-crosslinked collagen: Characterization and in vitro evaluation

M.J.B. Wissink, R. Beernink, J.S. Pieper, A.A. Poot, G.H.M. Engbers, T. Beugeling, W.G. van Aken, J. Feijen

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In the present study, heparin immobilization to a non-cytotoxic crosslinked collagen substrate for endothelial cell seeding was investigated. Crosslinking of collagen using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) resulted in a material containing 14 free primary amino groups per 1000 amino acid residues (E/N14C). At a fixed molar ratio NHS : EDC of 0.6, the amount of heparin covalently immobilized to E/N14C increased with increasing molar ratios of EDC to heparin carboxylic acid groups (Hep-COOH), to a maximum of approximately 5–5.5 wt% at a ratio of 2. Upon incubation in cell culture medium of endothelial cells, 4 to 7% of the immobilized heparin was released during 11 days. Immobilization of increasing amounts of heparin to E/N14C progressively reduced activation of contact activation proteases. Optimal anticoagulant activity, as measured by thrombin inhibition, was obtained after heparin immobilization using a ratio of EDC to Hep-COOH of 0.2–0.4 (14–20 mg heparin immobilized per gram of collagen). Platelets deposited to (heparinized) E/N14C showed only minor spreading and aggregation, although heparin immobilization slightly increased the number of adherent platelets. The results of this study suggest that heparin immobilization to EDC/NHS-crosslinked collagen may improve the in vivo blood compatibility of this material.
Original languageEnglish
Pages (from-to)151-163
Issue number2
Publication statusPublished - 2001


  • Vascular grafts
  • METIS-204772
  • IR-74443
  • Contact activation
  • Collagen coating
  • Heparin immobilization
  • Platelet interaction
  • Thrombin inactivation


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