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.