A Modular Bioinstructive Platform Reveals Mechanistic Insights into Additive‐Free, Topography‐Driven Osteogenesis

Developing physiologically relevant bone models is critical for drug discovery, disease modeling, and regenerative medicine, yet reproducing osteogenesis in vitro without biochemical induction remains a longstanding challenge. We present a scalable, bioinstructive microparticle platform in which engineered 3D surface topographies induce mesenchymal stem cell osteogenesis through topography-mediated mechanotransduction in the absence of exogenous additives. RNA-Seq and signaling analyses revealed a mechanistic sequence in which cytoskeletal reorganization activates canonical Hedgehog signaling, triggering early upregulation of cytoskeletal components and osteochondral transcription factors, including RUNX2 and SOX9, followed by IGF-II activation and osteogenic commitment. To demonstrate the potential of precision-engineered biomaterials for in vitro modeling, two-photon polymerization lithography was employed to engineer precisely-patterned 3D topographies with tunable dimensions, which elicited graded GLI1 expression without exogenous soluble factors. By decoupling mechanical microenvironments from chemical signaling, this establishes a scalable and modular strategy for reproducible control of cell fate, presenting a broadly applicable strategy for bioinstructive regenerative materials and standardized, additive-free bone models.