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Biocompatible hydrogels composed of covalently crosslinked, chemically modified hyaluronan (HA), gelatin (Gtn), and heparin (Hp) were synthesized for controlled release of human vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF). We hypothesized that inclusion of small quantities of heparin in these gels would result in regulated growth factor release over an extended period, while still maintaining the in vivo bioactivity of released GFs. To test this hypothesis, HA (200 kDa), Gtn (50 kDa) and heparin (15 kDa) were modified with pendant thiol groups using hydrazide-EDC chemistry. The thiolated biopolymers were then co-crosslinked with poly (ethylene glycol) diacrylate (PEGDA, 3400 Da) to form hydrogels composed of HA or HA and Gtn (50:50 w/w) with 0-3% heparin (w/w relative to total HA or HA-Gtn content). Either VEGF or bFGF was incorporated into the hydrogels before crosslinking with PEGDA. Release of these growth factors in vitro could be sustained over 42 days by less than 1% heparin content, and was found to decrease monotonically with increasing heparin concentration. As little as 0.03% Hp in the gels reduced the released VEGF fraction from 30% to 21%, while 3% heparin reduced it to 19%. Since the minimum heparin concentration capable of effective controlled growth factor release in vitro was found to be 0.3% (w/w), this concentration was selected for subsequent in vivo experiments. To evaluate the bioactivity of released growth factors in vivo, gel samples were implanted into the ear pinnas of Balb/c mice and the resulting neovascularization response measured at 7, 14, and 28 days post-implantation. In the presence of heparin, vascularization was sustained over 28 days. Growth factor release was more rapid in vitro from gels containing gelatin than from gels lacking gelatin, though unexpectedly, the in vivo neovascularization response to gelatin-containing gels was decreased. Nevertheless significant numbers of neovessels were generated. The ability to stimulate localized microvessel growth at controlled rates for extended times through the release of growth factors from covalently-linked, heparin-supplemented hydrogels will ultimately provide a powerful therapeutic tool. |
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