title: Improved endothelialization of vascular grafts by local release of growth factor from heparinized collagen matrices.
abstract: Endothelial cell seeding, a promising method to improve the performance of small-diameter vascular grafts, requires a suitable substrate, e.g. crosslinked collagen. In addition, the growth of seeded endothelial cells can be improved by local release of a heparin-binding protein, basic fibroblast growth factor (bFGF). In this study, the influence of immobilization of heparin to collagen, crosslinked using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS), on the binding and release of bFGF was determined. Heparin was immobilized also using EDC and NHS. Furthermore, the effects of the release of bFGF from (heparinized) EDC/NHS-crosslinked collagen on the proliferation of seeded endothelial cells was studied in vitro. Immobilization of increasing amounts of heparin to EDC/NHS-crosslinked collagen (containing 14 free epsilon-amino groups per 1000 amino acid residues, E/N14C) resulted in binding of increasing amounts of bFGF to the material. Maximal bFGF binding was observed for E/N14C containing 20-30 mg heparin immobilized per gram of collagen which was obtained using a molar ratio of EDC to heparin-carboxylic acid groups of 0.4 for heparin immobilization (E/N14C-H(0.4)). Up to concentrations of 320 ng bFGF/ml, 10% of the added bFGF bound to E/N14C, while binding of bFGF to E/N14C-H(0.4) was 22%. The initial release rate of bFGF bound to E/N14C was much higher compared to bFGF bound to E/N14C-H(0.4): respectively, 30 vs. 2% in the first 6 h. After 10 days, the bFGF release from E/N14C and E/N14C-H(0.4) amounted to 83 vs. 42%, respectively. Binding of increasing amounts of bFGF resulted in increased growth of human umbilical vein endothelial cells (HUVECs) seeded on both E/N14C and E/N14C-H(0.4). Nevertheless, after 6 and 10 days of proliferation cell numbers on E/N14C-H(0.4) where higher than cell numbers on E/N14C, irrespective of the bFGF concentration used for loading of the matrix. It is concluded that heparinized, EDC/NHS-crosslinked collagen is a good synthetic vascular graft coating for in vivo endothelial cell seeding.
authors.affiliation: Institute for Biomedical Technology, Polymer Chemistry and Biomaterials Group, Department of Chemical Technology, University of Twente, Enschede, The Netherlands.
authors.forename: M J
authors.initials: MJ
authors.lastname: Wissink
authors.forename: R
authors.initials: R
authors.lastname: Beernink
authors.forename: A A
authors.initials: AA
authors.lastname: Poot
authors.forename: G H
authors.initials: GH
authors.lastname: Engbers
authors.forename: T
authors.initials: T
authors.lastname: Beugeling
authors.forename: W G
authors.initials: WG
authors.lastname: van Aken
authors.forename: J
authors.initials: J
authors.lastname: Feijen
mesh_terms: D000287:Administration, Topical; D001807:Blood Vessel Prosthesis; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry*; D003432:Cross-Linking Reagents; D003692:Delayed-Action Preparations; D004727:Endothelium / Q000502:physiology*; D016222:Fibroblast Growth Factor 2 / Q000493:pharmacokinetics*; D006493:Heparin / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D011485:Protein Binding; D014471:Umbilical Veins / Q000502:physiology
chemical_list: D003432:Cross-Linking Reagents; D003692:Delayed-Action Preparations; D016222:Fibroblast Growth Factor 2; D006493:Heparin; D003094:Collagen