3D matrix stiffness drives energy metabolism to orchestrate stem cell osteogenesis via microtubule acetylation and mitochondrial dynamics

Hydrogels are widely recognized as promising materials for bone regeneration. However, how their biophysical properties, particularly stiffness, affect stem cell behavior in three-dimensional (3D) environments remains poorly understood. It is also unclear whether energy metabolism and mitochondrial dynamics play a role in mediating stiffness-regulated stem cell differentiation. Our study demonstrates that a soft extracellular matrix (ECM) enhances cytoskeletal polymerization and cell elongation. In vitro, a soft ECM promoted osteogenic differentiation, while in vivo it facilitated bone regeneration by regulating the formation of a uniform mitochondrial network and promoting mitochondrial fusion. Additionally, a soft matrix increased ATP production by enhancing both glycolysis and oxidative phosphorylation (OXPHOS), indicating a metabolic shift. Microtubule acetylation was upregulated in the soft ECM through the activity of αTAT1, accompanied by increased expression of Kinesin 1, which contributed to mitochondrial network formation and dynamic remodeling. These findings highlight the critical role of microtubule acetylation in mitochondrial organization and dynamics during stiffness-mediated osteogenesis in 3D environments. This work provides valuable insights for the rational design of biomaterials aimed at improving bone regeneration.