Cell-Encapsulating Microbeads for Therapeutic Vascularization of Ischemic Tissues
Creators: name: Friend, Nicole
Publisher: name: My University
Year: 2023
Subjects: subject: Vascularization
subject: Microbeads
subject: Fibrin
subject: Poly(ethylene glycol)
subject: Cell-based therapies
subject: Biomedical Engineering
subject: FOS: Medical engineering
subject: Engineering

description: Restoring blood flow to ischemic regions following tissue damage remains a significant challenge in treating a variety of diseases and injuries. Efforts to revascularize ischemic tissues utilizing growth factor (GF) delivery have had minimal clinical success. Cell-based approaches have been explored as an alternative to GF delivery but have been plagued by low cell retention at the target site and reduced cell viability due to the harsh ischemic environment. Prevascularization of tissue constructs may promote more rapid inosculation with host vasculature, potentially enhancing tissue survival and improving therapeutic efficacy; however, implantation of these constructs often requires invasive surgery. This dissertation focuses on the development of injectable, vascularizing tissue modules using both natural and synthetic matrices to address the overarching hypothesis that prevascularized microbeads can jump-start inosculation with host vasculature upon implantation to rapidly revascularize ischemic tissues. Fibrin-based microbeads, containing endothelial (EC) and stromal cells (SC), were pre-cultured in suspension to allow for the formation of primitive microvascular networks within the microbead matrix. In vitro, vessel-like structures sprouted from microbeads to vascularize the surrounding hydrogel. Microbeads pre-cultured for 3 days were implanted into dorsal subcutaneous pockets and within intramuscular pockets in a mouse model of hindlimb ischemia (HLI). In both models, vessels originating from microbeads formed functional connections to host vasculature within 3 days and exhibited extensive, mature microvascular networks after 7 days. The total number of human-derived vessels decreased over time as networks were remodeled and an increase in mature, pericyte-supported vascular structures was observed. In the HLI model, animals treated with microbeads showed increased macroscopic reperfusion of ischemic foot pads 14 days after surgery and improved limb salvage compared to cellular controls. Despite these promising findings, cell-laden fibrin-based microbeads tended to aggregate with extended pre-culture, limiting injectability of prevascularized modules. Degradable, cell-adhesive poly(ethylene glycol)-norbornene (PEGNB) was investigated as an alternative material platform to support the formation of injectable, non-aggregating, prevascularized microbeads. Bulk PEGNB hydrogel formulations that supported vessel morphogenesis were first identified. Enhanced cell-mediated remodeling of PEGNB hydrogels, achieved either by reduced crosslinking or increased degradability, led to more rapid vessel formation and higher degrees of cell-mediated stiffening. EC and SC were then encapsulated in PEGNB microbeads using microfluidic flow-focusing biofabrication. These cell-laden PEGNB microbeads nucleated the formation of robust, well-distributed microvascular networks in vitro. However, while extended pre-culture of PEGNB microbeads enabled vessel morphogenesis within the beads without aggregation, increased pre-culture time led to decreased angiogenic sprouting. In vivo data revealed human-derived capillary-like structures as well as some evidence of lumen formation within PEGNB microbeads implanted in subcutaneous pockets, though in most cases these structures failed to extensively vascularize the surrounding implant region. This dissertation developed vascularizing microbeads using two different material platforms. Fibrin-based microbeads catalyzed the formation of functional microvascular networks in vivo, successfully restoring perfusion to ischemic tissues; however, their tendency to aggregate limited their injectability and ability to be cultured in suspension as discrete units. PEGNB-based microbeads overcame this issue, initiating the formation of well-distributed microvascular networks in vitro and permitting extended pre-culture without microbead aggregation; however, strategies to encourage sprouting from prevascularized PEGNB microbeads need to be explored to more fully realize the potential of this approach. Overall, these findings demonstrate the potential of modular, prevascularized microbeads as a minimally invasive therapeutic for restoring blood flow to ischemic tissues.