Developing Pre-Vascularized Modular Microenvironments for Ischemic Applications

Abstract

Critical limb ischemia (CLI), the end-stage of lower limb peripheral arterial disease, is characterized by a lack of oxygen/nutrient supply and subsequent tissue death. CLI patients with comorbidities are often ineligible for open bypass surgery. As an alternative approach, we developed pre-vascularized microbeads to jump-start inosculation with host vasculature after minimally invasive delivery and quickly restore blood flow to the ischemic tissue. Endothelial cells and fibroblasts were encapsulated in two microbead types – fibrin (FIB) and collagen-fibrin (COL-FIB) – and embedded immediately in FIB hydrogels to determine which initiated endothelial sprout formation to a greater extent. Cell viability in both microbead types was high (≥75%). Two weeks after embedding, cells in FIB microbeads produced a higher network length than those in COL-FIB microbeads. Cells pre-cultured in FIB microbeads for three days prior to embedding in larger hydrogels exhibited the most extensive endothelial tube area coverage in vitro. Fibrin microbead formulations were also evaluated for their ability to initiate vessel formation in vivo by injection into subcutaneous pockets on the dorsal surface of immuno-compromised mice. Implants containing microbeads pre-cultured for 3 days had greater vessel coverage, higher total number of vessels, and less implant compaction at D3, and more mature vessels at D7 in situ, compared to other conditions. The improved distribution of mature vessels generated by the implants containing pre-cultured microbeads was attributed to the preservation of implant volume, which was confirmed with ultrasound imaging of model implants in vitro. Furthermore, implants containing pre-cultured microbeads displayed lower bulk elastic moduli compared to controls, but higher local stiffness near the microbeads, suggesting the distribution of cells within the implants plays a role in construct compaction and volume maintenance. Collectively, these findings show cellular microbeads pre-cultured for 3 days in vitro are more effectively able to nucleate vascularization in vivo. FIB microbead aggregation increased with pre-culture time. Agarose-based microbeads reduced aggregation and provided the most distributed endothelial network post-embedding when made with hydroxyapatite and fibrinogen. These microbeads supported pre-culture, which helped to increase the network length post-embedding. Overall, this work demonstrates the therapeutic potential of vascularized microbeads for ischemic pathologies.