Immobilization of heparin to EDC/NHS-crosslinked collagen. Characterization and in vitro evaluation.
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.
Wissink
M J
MJ
Institute for Biomedical Technology, Department of Chemical Technology, University of Twente, Enschede, The Netherlands.
Beernink
R
R
Pieper
J S
JS
Poot
A A
AA
Engbers
G H
GH
Beugeling
T
T
van Aken
W G
WG
Feijen
J
J
D001672:Biocompatible Materials / Q000737:chemistry* / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D001807:Blood Vessel Prosthesis; D003094:Collagen; D003432:Cross-Linking Reagents; D005022:Ethyldimethylaminopropyl Carbodiimide; D006493:Heparin; D006801:Humans; D008855:Microscopy, Electron, Scanning; D013388:Succinimides; D013917:Thrombin / Q000378:metabolism
D001672:Biocompatible Materials; D003432:Cross-Linking Reagents; D013388:Succinimides; D006493:Heparin; D003094:Collagen; D013917:Thrombin; D005022:Ethyldimethylaminopropyl Carbodiimide