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Neural progenitor cells (NPCs) hold immense potential as therapeutic candidates for neural regeneration, and materials-based strategies have emerged as attractive options for NPC expansion. However, maintaining NPC stemness has proven challenging in vitro, due to their propensity to form cell-dense neurospheres. While neurospheres promote cell-cell interactions required for NPC stem maintenance, they also restrict oxygen transport, leading to hypoxia and limited cell expansion. To overcome these limitations, we investigate two materials-based approaches to maintain NPC stemness: 1) physical matrix remodeling within a viscoelastic, stress-relaxing hydrogel and 2) matrix-induced N-cadherin-like signaling through a cell-instructive peptide. While viscoelasticity alone is sufficient to maintain NPC stemness compared to an elastic environment, NPCs still preferentially form neurospheres. The addition of N-cadherin-like peptides promotes a distributed culture of NPCs while maintaining their stemness through cadherin-mediated signaling, ultimately exhibiting improved long-term expansion and neural differentiation. Thus, our findings reveal matrix viscoelasticity and engineered N-cadherin-like interactions as having a synergistic effect on NPC expansion and differentiation within 3D matrices. 

Abstract Mechanical cues from the extracellular matrix (ECM) regulate vascular endothelial cell (EC) morphology and function. Since naturally derived ECMs are viscoelastic, cells respond to viscoelastic matrices that exhibit stress relaxation, in which a cell‐applied force results in matrix remodeling. To decouple the effects of stress relaxation rate from substrate stiffness on EC behavior, we engineered elastin‐like protein (ELP) hydrogels in which dynamic covalent chemistry (DCC) was used to crosslink hydrazine‐modified ELP (ELP‐HYD) and aldehyde/benzaldehyde‐modified polyethylene glycol (PEG‐ALD/PEG‐BZA). The reversible DCC crosslinks in ELP‐PEG hydrogels create a matrix with independently tunable stiffness and stress relaxation rate. By formulating fast‐relaxing or slow‐relaxing hydrogels with a range of stiffness (500–3300 Pa), we examined the effect of these mechanical properties on EC spreading, proliferation, vascular sprouting, and vascularization. The results show that both stress relaxation rate and stiffness modulate endothelial spreading on two‐dimensional substrates, on which ECs exhibited greater cell spreading on fast‐relaxing hydrogels up through 3 days, compared with slow‐relaxing hydrogels at the same stiffness. In three‐dimensional hydrogels encapsulating ECs and fibroblasts in coculture, the fast‐relaxing, low‐stiffness hydrogels produced the widest vascular sprouts, a measure of vessel maturity. This finding was validated in a murine subcutaneous implantation model, in which the fast‐relaxing, low‐stiffness hydrogel produced significantly more vascularization compared with the slow‐relaxing, low‐stiffness hydrogel. Together, these results suggest that both stress relaxation rate and stiffness modulate endothelial behavior, and that the fast‐relaxing, low‐stiffness hydrogels supported the highest capillary density in vivo.