Biomimetic Woven Bone Organoids with Multifocal Centers for Retaining Stemness and Prolonging Osteogenic Activity

ABSTRACT Woven bone is the initial form of bone matrix during embryonic skeletal development and early stages of fracture healing. It develops through the fusion of ossification centers, which are dense clusters of human mesenchymal stem cells (hMSCs) with high osteogenic activity. Inspired by this natural process, we engineered biomimetic woven bone organoids (B‐MSC‐S organoids) by embedding multiple MSC spheroids within a single Matrigel droplet and facilitating self‐organization to mimic the fusion of ossification centers. These spheroids function as focal points by simultaneously maintaining stemness and promoting osteogenic differentiation. The resulting biomimetic organoids exhibited enhanced osteogenic activity and prolonged organoid longevity compared to the conventional fabrication method derived from the assembly and differentiation of homogenously dispersed hMSCs. B‐MSC‐S organoids demonstrated biomimetic properties including spatial organization, cell‐cell interactions, and osteogenic transition, and provided an extended testing window, making them an ideal in vitro platform for downstream diagnostic applications. Additionally, following their implantation into calvarial defects, B‐MSC‐S organoids retained viability, sustained osteogenic activity, and contributed to host tissue bone regeneration, highlighting their potential as a therapeutic construct.