Neo-vessel formation in ischemic tissues relies on numerous growth factors and cell fractions for the formation of mature, stable, functional vasculature. However, the efforts to regenerate tissues typically rely on the administration of a single growth factor or cells alone. Conversely, polymeric matrices have been investigated extensively to deliver multiple growth factors at pre-determined rates to form stable blood vessels in ischemic tissues. We report on a novel sequential delivery system of a fibrin hydrogel containing ionic-albumin microspheres that allows for the controlled release of two growth factors. The use of this system was investigated in the context of therapeutic angiogenesis. Material properties were determined based on degree of swelling measurements and degradation characteristics. Release kinetics of model angiogenic polypeptides FGF-2 and G-CSF were determined using ELISA and the bioactivity of released protein was evaluated in human endothelial cell cultures. The release of growth factors from ionic-albumin microspheres was significantly delayed compared to the growth factor released from fibrin matrices in the absence of spheres. The scaffolds were implanted in a murine critical limb ischemia model at two concentrations, 40 ng (low) and 400 ng (high), restoring 92% of the blood flow in a normally perfused limb using a fibrin hydrogel releasing FGF-2 containing albumin-PLL microspheres releasing G-CSF (measured by LDPI at the high concentration), a 3.2-fold increase compared to untreated limbs. The extent of neo-vessel formation was delineated by immunohistochemical staining for capillary density (CD-31+) and mature vessel formation (α-SMA+). In conclusion, our study demonstrated that the release kinetics from our scaffold have distinct kinetics previously unpublished and the delivery of these factors resulted in hindlimb reperfusion, and robust capillary and mature vessel formation after 8 weeks compared to either growth factor alone or bolus administration of growth factor.

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