Active Microrheology Reveals Distinct ECM Mechanical Signatures Induced by Stromal Cells of Different Tissue Origins during Vascular Morphogenesis.

Stromal cells (SCs) provide important instructive cues for endothelial cells (ECs) during both normal and neoplastic vascularization. While the tissue-specific origins of ECs are important for function, the impact of SC identity on microvascular function and concurrent changes in tissue mechanical properties remains unclear. We previously showed robust microvasculature forms by codelivery of ECs and supportive SCs, and that SC identity regulates the rate of neovascularization and vessel functionality. Here, we used active microrheology (AMR) and traditional macrorheology to evaluate the dynamics of both local and global ECM mechanics in a 3D EC-SC co-culture model of vascular morphogenesis. Human umbilical vein ECs were co-embedded with either highly contractile lung fibroblasts (LFs) or significantly less contractile bone marrow-derived mesenchymal stromal cells (MSCs) within fibrin gels across various cell-seeding densities. By day 14, interconnected vascular networks developed, with rates of capillary morphogenesis higher in EC-LF than in EC-MSC co-cultures. Vascularization in EC-LF co-cultures was accompanied by ECM stiffening across length scales, in part due to cell contractility. AMR revealed highly heterogeneous local stiffness, with values ranging over 2 orders of magnitude in the same construct. AMR also identified the emergence of local stiffness anisotropy in the direction of capillary growth for EC-LF but not EC-MSC co-cultures by day 14, which was accompanied by significant matrix remodeling and local degradation. Together, these data suggest that different SC populations, through active cell contractility-dependent stiffening and matrix degradation, induce local mechanical cues that differentially influence vascular development. These results highlight the importance of the mechanobiological effects of SCs on the ECM in vascularized engineered tissues.