The development of a facile polymer microbead-based approach to promoting angiogenesis in dense epithelial tissue

摘要

Introduction: 3D in vitro tissue and organ cultures are of increasing interest as models for human disease and drug development. An enduring need is accomplishing vascularization of such tissues with perfusable capillary vessels. Insufficient vasculature within cultured tissue results in hypoxic conditions due to an oxygen transport limitation, hindering their survival, normal phenotypic outputs, and physiological function. Further, within the vascular niche of an epithelial system, the endothelial cells are surrounded by a vascular basement membrane, which provides mechanical stability, as well as various cytokines that support adjacent cell phenotypes. Epithelial systems such as liver, pancreas and other glands are particularly challenging, as these tissues are cell-dense with relatively little extracellular matrix; hence, successful vascularization approaches based on cell encapsulation or invasion into fibrin or other types of gels are difficult to adapt for these applications. Here, we harness concepts from the bead-based angiogenesis assays to design hydrogel microbeads with cell interaction properties tailored to promoting endothelial invasion into dense tissues. We use microfluidic-based polymer microbead synthesis, which combines single cell-based emergence with patterning techniques in order to actively vascularize dense cell tissue. Modulating the chemical and mechanical properties of the microbeads dictates cell adhesion, sprouting dynamics, and vascular density. Materials and Methods: Norbornene end-functionalized poly(ethylene) glycol was synthesized through the DCM-activated end-capping of a multi-arm PEG with norbornene, as described in literature. Cross-linker peptide motifs representing key binding sites within the extracellular matrix were custom synthesized by Boston Open Labs and incorporated into the crosslinking matrix of the bulk polymer. UV-initiated polymerization via microfluidic-based approaches was used to synthesize polymer microparticles with appropriate chemical and mechanical modifications. Deriving design principles from the classic fibrin bead angiogenesis model, a monolayer of endothelial cells was adsorbed to the surface of the polymer microbeads, and vascular sprouting was subsequently induced within a gel matrix. Both human umbilical vein endothelial cells as well as induced-pluripotent stem cells purchased from Cellular Dynamics International were used in the vascular sprouting experiments. Sprouting dynamics and density were evaluated across various polymer moduli and matrix gel densities. The F-actin filaments of the sprouts were labeled with phalloidin and imaged through confocal microscopy. The images were analyzed using ImageJ software. Results and Discussion: The GFOGER peptide motif mimicking the binding site of collagen was identified and incorporated into the polymer crosslinking network of the polymer. HUVECs successfully adsorbed to this formulation of the polymer microbeads (Figure 1). Upon seeding into a fibrin gel, vascular sprouts of varying densities grew from the microbead into the surrounding matrix (Figure 2). Conclusions: We have started to establish an understanding of how surface functionalities and mechanical properties of polymer microbeads influences sprouting angiogenesis into a dense tissue. Further microbead degradation studies will follow concurrent work being done in the lab; a sortase mutant enzyme that targets an LPRXG peptide motif within the hydrogel crosslinks, exchanging the site for short soluble GGG monomers, rendering the hydrogel degraded.