Introduction: Generating vascularized tissues by seeding endothelial cells (ECs) within microchannels formed in hydrogel scaffolds has the potential to bring high viability tissue constructs to fruition ex vivo. However, solely relying on EC-seeded channels results in tissues with only small artery/vein-like vessels and no capillaries. To avoid non-physiological tissue architectures and instead produce appropriate vessel hierarchies, we are exploring the strategy of eliciting sprouting from EC-seeded channels with the inclusion of angiogenesis-stimulating mesenchymal cells in the hydrogel. This strategy requires a hydrogel optimized to maintain mechanically stable tissues and vasculature while enabling EC sprouting. To this end, we investigated fibrin, collagen, and fibrin-collagen co-gels, as fibrin has the capacity to elicit EC capillary morphogenesis ex vivo and collagen has favorable mechanical properties advantageous for generating stable tissues.
Materials and Methods: Scanning electron microscopy of critical point dried hydrogels was performed to assess fibril microstructure. EC-seeded microcarrier beads were encapsulated into hydrogels and co-cultured with bone marrow stromal cells (BMSCs) to assess biomaterial angiogenicity. Since minimizing gel contraction is critical for maintaining channel patency, contraction was assessed under unrestrained conditions using 200,000 BMSC/mL gel. Microchannels were formed by polymerizing gels around a nylon line threaded between needles within a silicone chamber. Once the line was removed, channels could then be seeded with ECs and perfused using syringe or peristaltic pumps.
Results and Discussion: Co-gel microstructure resembled both collagen and fibrin, with collagen fibril diameters in the range of ~178-290 nm (Fig.1). Fibrin and co-gels elicited EC sprouting, whereas collagen alone elicited independent EC migration. Both fibrin and co-gels (2.5 mg/mL fibrin + 0.5-3.0 mg/mL collagen) were relatively resistant to cell-mediated contraction compared to collagen alone (p ≤ 0.001) (Fig. 2). However, fibrin alone could not support the formation of smooth, mechanically-robust channels, whereas collagen-fibrin co-gels produced smooth channels that withstood flow rates >100 mL/hr without channel warping, provided that collagen was ≥1.0 mg/mL. Finally, ECs were found to sprout outwards from the channels into the co-gels.
Conclusion: The promising features of collagen-fibrin uncovered by this study suggest that such co-gels may be of value for generating vascularized constructs. To further investigate their potential, we have fabricated a custom 3D printer to produce collagen-fibrin constructs with branching channels. Currently, we are optimizing the 3D printing process in conjunction with examining the roles that pulsatile flow and shear stress have in vessel muscularization, with the aim of generating thick tissues with high viability and physiological vascular architectures.
