Determining cytokine induced vascular maturity through\ud immunohistochemical double-staining

Graduation date: 2008As a major natural component of the extracellular matrix (ECM),\ud hyaluronic acid (HA) is an excellent choice for biomimetic, biocompatible\ud therapeutic materials. Furthermore, thiol-modified forms of HA are capable of\ud forming macroporous hydrogels that allow for both controlled cytokine release and\ud extensive vessel in-growth. It has previously been shown that this HA controlled\ud cytokine release may be extended significantly by including thiol-modified heparin\ud (Hp) in the hydrogel matrix. We therefore hypothesized that that the inclusion of\ud minute quantities of Hp in both HA and HA+Gelatin cytokine-loaded hydrogels\ud would, in turn, result in extended levels of elicited microvascular stability and\ud maturity in vivo. To test this hypothesis we formulated several single and dual\ud cytokine-loaded hydrogels and implanted them into the Balb/c mouse ear pinna.\ud These preloaded hydrogels contained vascular endothelial growth factor (VEGF),\ud angiopoietin-1 (Ang-1), keratinocyte growth factor (KGF) or platelet derived growth\ud factor (PDGF) either individually or in combination with VEGF. At 7 and 14 days\ud post surgery, elicited vascular maturity levels were quantified using\ud immunohistochemical (IHC) staining techniques. The degree of circumferential\ud pericyte ensheathement as indicated by alpha smooth muscle actin (α-SMA) IHC\ud signal, together with basement membrane morphology, allowed for a graded\ud determination of elicited microvasculature maturity. The progress of maturation was\ud separated from the natural wound response (sham surgery) and then normalized to the\ud steady state (contralateral ear), and reported as a vascular maturity index (VMI). It\ud was discovered that in both gel types at both time points, the dual cytokine\ud combination elicited greater maturity levels than either cytokine administered\ud individually. For example, VEGF and KGF-containing implants at day 7 yielded\ud VMI values of -0.1375 and -0.092, respectively, whereas their combination resulted\ud in a VMI of 0.176 (p < 0.007). At day 7, only one of the seven HA:Hp experimental\ud cases yielded a positive VMI (VEGF+KGF), whereas four of the seven HA:Hp cases\ud yielded positive VMI values at day 14. This shift from predominantly negative to\ud predominantly positive VMI values suggested not only a sustained microvascular\ud maturity response, but also a gradual improvement in microvessel maturity relative to\ud the sham tissue. Although sustained, and in some cases elevated from 7 to 14 days\ud post film implantation, the microvascular maturity response elicited by HA:Hp films\ud was not invariably higher than that elicited by the HA controls. The levels of elicited\ud microvascular maturity in tissue treated with dual cytokine loaded HA:Hp films were\ud instead found to depend on the identity of the late stage cytokine, suggesting specific\ud heparin-cytokine interactions were responsible for the observed differences