Bio-inspired micropatterned hydrogel to direct and deconstruct hierarchical processing of geometry-force signals by human mesenchymal stem cells during smooth muscle cell differentiation

Micropatterned biomaterial-based hydrogel platforms allow the recapitulation of in vivo-like microstructural and biochemical features that are critical physiological regulators of stem cell development. Herein, we report the use of muscle mimicking geometries patterned on polyacrylamide hydrogels as an effective strategy to induce smooth muscle cell (SMC) differentiation of human mesenchymal stem cells (hMSCs). hMSCs were systemically coerced to elongate with varying aspect ratios (AR) (that is, 1:1, 5:1, 10:1 and 15:1) at a fixed projection area of ∼ 7000 μm². The results showed engineered cellular anisotropy with an intermediate AR 5:1 and AR 10:1, promoting the expression of alpha smooth muscle actin (α-SMA) and enhancement of contractile output. Further mechanistic studies indicated that a threshold cell traction force of ∼ 3.5 μN was required for SMC differentiation. Beyond the critical cytoskeleton tension, hMSCs respond to higher intracellular architectural cues such as the stress fiber (SF) alignment, SF subtype expression and diphosphorylated myosin regulatory light-chain activity to promote the expression and incorporation of α-SMA to the SF scaffold. These findings underscore the importance of exploiting biomimetic geometrical cues as an effective strategy to guide hMSC differentiation and are expected to guide the rational design of advanced tissue-engineered vascular grafts.