A Scaffold-Based 3D Culture Model Including Selected Osteoprogenitors for Bone Regeneration With Controlled Morphology.

We previously identified "highly purified osteoprogenitors" (HipOPs) as a promising model for studying bone organ regeneration in murine systems. However, achieving proper integration of the bone formed by HipOPs into the well-organized host bone requires better morphological control. In this study, we developed a 3D culture model by culturing a HipOP-seeded gelatin sponge on an osteoblast (OB) layer to regulate the morphology of regenerated bone. In vitro studies revealed significant upregulation of genes associated with bone formation, response to stimuli, cell communication, and vascularization in the HipOP population after 48 h of coculture. Moreover, the HipOPs exhibited concentration-dependent responses, with stronger effects observed in regions closer to the OB layer. By day 7, the sponge areas in contact with the OB layer contained more cells with enhanced osteogenic differentiation capacity. When transplanted subcutaneously into immunodeficient mice for 8 weeks, the 3D culture samples formed bone organs comprising bone and bone marrow tissue. Notably, cortical bone appeared only in regions where the sponge had contacted the OB layer, with a gradual decrease in bone density observed outward from that area. Our 3D culture model successfully generated hierarchically structured bone organs, offering significant potential for reconstructing well-integrated bone in defective regions.