Dynamic stiffening to improve vasculogenesis of hiPSC-Derived endothelial progenitors.

Multiple groups have reported on the impact of hydrogel stiffness on vascular network formation in vitro, with overall findings indicating that hydrogels with lower stiffness better support vasculogenesis. However, most of this research utilizes hydrogels with static stiffness, even though vasculogenesis occurs in tandem with changes in extracellular matrix stiffness. To that end, we hypothesized that dynamic modulation of hydrogel stiffness during vasculogenesis would improve vascular network formation. Using a Collagen I/Norbornene-modified hyaluronic acid hydrogel system, we diffused additional crosslinker and photoinitiator into the hydrogel and initiated further UV crosslinking at predetermined time points, while maintaining high cell viability (>90%). We observed that in situ stiffening at early time points, prior to the completion of cell elongation, increased vascular network connectivity and volume fraction relative to unstiffened controls, while stiffening at later time points was associated with reduced network formation. Consistent with these findings, cell-laden hydrogels stiffened at early time points implanted subcutaneously in nude mice showed increased blood perfusion, while late-stiffened hydrogels did not improve perfusion relative to controls. Mechanistically, we found that these time-dependent differences were associated with changes in vinculin volume fraction, suggesting differential mechanotransductive signaling. Consistent with this, Rho kinase inhibition suppressed vinculin volume fraction in stiffened hydrogels and partially rescued vascular network formation following in situ stiffening. These findings indicate that the timing of stiffness modulation is a critical factor in determining vasculogenic outcome, and that early-stage stiffening can enhance vascular network formation beyond what is achieved in static hydrogel systems.