Investigating the Role of Matrix Architecture on Vascularization in MMP-Sensitive PEG Hydrogels.

Abstract

The formation of functional blood vessels in engineered or ischemic tissues remains a significant scientific and clinical hurdle. Cell delivery, scaffold design, and growth factor delivery have been investigated to support neovascularization. This thesis focuses on a hybrid approach wherein cells are seeded within a biosynthetic scaffold. Our approach is motivated by the relatively poor performance of cells alone; cell engraftment is minimal (10%) in scaffold-free approaches. Natural and synthetic materials have been utilized to improve engraftment, but the biosynthetic scaffold presented here offers unique advantages to overcome limitations of natural materials and offers tunability of matrix properties and biological response. A PEG hydrogel platform was adapted to investigate the roles of network crosslinking density and susceptibility to proteolysis on vascularization. Four-arm PEG vinyl sulfone (PEGVS) was polymerized by Michael-type addition with reactive cysteine groups on a slowly degraded matrix metalloprotease (MMP) susceptible peptide, GPQG↓IWGQ, or a peptide that is cleaved more rapidly, VPMS↓MRGG. Vascular networks formed in vitro from encapsulated endothelial cells and supportive stromal fibroblasts. Morphogenesis was robust to changes in cross-linking peptide identity, but significantly attenuated in more crosslinked gels. All gel types supported the de novo formation of perfused vasculature from transplanted cells in subcutaneous implants in vivo; however, unlike the in vitro findings, vascularization was not decreased in the more cross-linked gels. A mouse model of hindlimb ischemia was used to further assess the ability of PEG hydrogels to support revascularization in a model relevant for clinical translation. Cell-laden PEG hydrogel precursors and fibrin controls were delivered to SCID mice after femoral artery ligation. PEG hydrogels supported the formation of perfused vasculature irrespective of crosslinking-peptide identity. Hydrogel delivery improved reperfusion to the ischemic limb. Substantial loss of gel mechanical integrity and vessel regression were evident in fibrin gels, but not in PEG gels, 2 weeks post-implantation, suggesting PEG hydrogels are superior to fibrin with regards to vessel persistence. In sum, these findings demonstrate that structurally stable biomimetic PEG-based hydrogels direct vascularization in ischemic tissues via cell transplantation and hold promise in tissue regeneration and therapeutic angiogenesis.