Microvascular maturity elicited in tissue treated with cytokine-loaded hyaluronan-based hydrogels

Hydrogels composed of crosslinked, chemically modified hyaluronic acid (HA), gelatin (Gtn) and heparin (Hp) were preloaded with vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), keratinocyte growth factor (KGF) or platelet derived growth factor (PDGF) either individually or in combination with VEGF and implanted into the Balb/c mouse ear pinna. At 7 and 14 days post-surgery, elicited vascular maturity levels were quantified using immunohistochemical (IHC) staining techniques and reported as a vascular maturity index (VMI). At both time points, it was discovered that the dual cytokine combinations elicited greater maturity levels than that of cytokine administered individually. For example, VEGF and KGF-containing HA:Hp implants at day 7 yielded VMI values of -0.1375 and -0.092, respectively, whereas their combination resulted in a VMI of 0.176 (p<0.007). At day 7, only one of the seven HA:Hp experimental cases yielded a positive VMI (VEGF+KGF), whereas four of the seven HA:Hp cases yielded positive VMI values at day 14, indicating a sustained maturity response. The same general trends were found to exist in tissue treated with HA:Hp:Gtn experimental implants. Differences in elicited maturity also existed between tissue treated with HA:Hp and HA-containing hydrogels (VMI=0.176 for HA:Hp-VEGF+KGF vs. -0.064 for HA-VEGF+KGF, p<0.012), and these differences are thought to result from differences in characteristic cytokine release rates. This result also suggests that the presentation of multiple growth factors (GFs) on immobilized Hp may actively contribute to cytokine related signal transduction, a characteristic that may be exploited in the future to improve the efficacy of cytokine-loaded implants towards tissue regeneration therapeutic strategies.