Sprouting angiogenesis induces significant mechanical heterogeneities and ECM stiffening across length scales in fibrin hydrogels

Matrix stiffness is a well-established instructive cue in two-dimensional cell cultures. Its roles in morphogenesis in 3-dimensional (3D) cultures, and the converse effects of cells on the mechanics of their surrounding microenvironment, have been more elusive given the absence of suitable methods to quantify stiffness on a length-scale relevant for individual cell-extracellular matrix (ECM) interactions. In this study, we applied traditional bulk rheology and laser tweezers-based active microrheology to probe mechanics across length scales during the complex multicellular process of capillary morphogenesis in 3D, and further characterized the relative contributions of neovessels and supportive stromal cells to dynamic changes in stiffness over time. Our data show local ECM stiffness was highly heterogeneous around sprouting capillaries, and the variation progressively increased with time. Both endothelial cells and stromal support cells progressively stiffened the ECM, with the changes in bulk properties dominated by the latter. Interestingly, regions with high micro-stiffness did not necessarily correlate with remodeled regions of high ECM density as shown by confocal reflectance microscopy. Collectively, these findings, especially the large spatiotemporal variations in local stiffness around cells during morphogenesis in soft 3D fibrin gels, underscore that characterizing ECM mechanics across length scales. provides an opportunity to attain a deeper mechanobiological understanding of the microenvironment's roles in cell fate and tissue patterning.