The means by which extracellular matrix density regulates three-dimensional capillary morphogenesis is unclear. To study this phenomenon, we utilized a fibrin-based in vitro assay in which a fibroblast monolayer is plated atop a fibrin gel approximately 2.5 mm away from endothelial cell-coated beads within the matrix. Increasing fibrin density from 2.5 to 10 mg/ml resulted in a threefold reduction in capillary network formation. However, distributing fibroblasts throughout the matrix completely eliminated this inhibitory effect, resulting in robustly vascularized matrices suitable for in vivo applications, as functional anastomoses formed between the implanted tissues and host vasculature when implanted into immune-compromised mice. Dense matrices did not stimulate fibroblast-mediated matrix remodeling: differentiation into myofibroblasts, matrix production, and protease secretion were not enhanced by the dense condition. Instead, quantifying diffusivity of FITC-dextran (molecular mass 10, 40, 70, and 150 kDa) through fibrin revealed a two- to threefold decrease within the 10 mg/ml matrices. Thus, distributing a proangiogenic source (fibroblasts) throughout the matrix stimulates capillary network formation by overcoming this diffusion restriction due to significantly reduced diffusion distances. Although roles for matrix stiffness and ligand binding density have previously been identified, our results emphasize the importance of diffusion restrictions in limiting capillary morphogenesis.

## PubMed Commons