3D Mechanical Confinement Directs Muscle Stem Cell Fate and Function

Abstract Muscle stem cells (MuSCs) play a crucial role in skeletal muscle regeneration, residing in a niche that undergoes dimensional and mechanical changes throughout the regeneration process. This study investigates how 3D confinement and stiffness encountered by MuSCs during the later stages of regeneration regulate their function, including stemness, activation, proliferation, and differentiation. An asymmetric 3D hydrogel bilayer platform is engineered with tunable physical constraints to mimic the regenerating MuSC niche. These results demonstrate that increased 3D confinement maintains Pax7 expression, reduces MuSC activation and proliferation, inhibits differentiation, and is associated with smaller nuclear size and decreased H4K16ac levels, suggesting that mechanical confinement modulates both nuclear architecture and epigenetic regulation. MuSCs in unconfined 2D environments exhibit larger nuclei and higher H4K16ac expression compared to those in more confined 3D conditions, leading to progressive activation, expansion, and myogenic commitment. This study highlights the importance of 3D mechanical cues in MuSC fate regulation, with 3D confinement acting as a mechanical brake on myogenic commitment, offering novel insights into the mechano‐epigenetic mechanisms that govern MuSC behavior during muscle regeneration.