Binding and release of basic fibroblast growth factor from heparinized collagen matrices

Endothelial cell seeding is a promising method to improve the performance of small-diameter vascular grafts. Growth of endothelial cells seeded on the luminal surface of synthetic vascular grafts, coated with a matrix suitable for cell seeding (e.g. collagen), can be accelerated by local, sustained release of basic fibroblast growth factor (bFGF). In this study two potential matrices for in vivo endothelial cell seeding were studied with respect to bFGF binding and release: collagen crosslinked using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), as well as heparinized EDC/NHS-crosslinked collagen. bFGF binding was determined after incubation of circular samples (10 mm diameter) with 0.25 ml bFGF solution for 90 min. Immobilization of increasing amounts of heparin, also using EDC and NHS, to crosslinked collagen containing 14 free primary amino groups per 1000 amino acid residues (E/N14C) resulted in binding of increasing amounts of bFGF. A plateau in bFGF binding was observed for heparinized E/N14C containing approximately 2.0-3.0 wt% of immobilized heparin which was obtained using a molar ratio of EDC to heparin-carboxylic acid groups of 0.4 during heparin immobilization (E/N14C-H(0.4)). At concentrations up to 840 ng bFGF/ml, 10% of the added bFGF bound to E/N14C, while binding of bFGF to E/N14C-H(0.4) amounted to 22%. Both E/N14C and E/N14C-H(0.4) pre-loaded with bFGF showed sustained bFGF release. A burst release of 30% in endothelial cell culture medium (CM) was observed for E/N14C during the first 6 h, compared to 2% release from E/N14C-H(0.4). After 28 days, the bFGF release from E/N14C and E/N14C-H(0.4) in CM amounted to 100 and 65%, respectively. Combined results of binding and release of bFGF indicate that compared to E/N14C, E/N14C-H(0.4) is the substrate of choice for bFGF pre-loading and subsequent endothelial cell seeding.