Bone regeneration using three‐dimensional spheroids derived from dedifferentiated fat cells

Abstract Aim Dedifferentiated fat (DFAT) cells, similar to mesenchymal stem cells (MSCs), are gaining attention owing to their minimally invasive nature. We aimed to evaluate the effects of 3D DFAT spheroids on bone regeneration by implantation into rat skull defects. Methods Adipose tissue was harvested from 8‐week‐old Sprague–Dawley rats, and DFAT was prepared using the ceiling culture method. DFAT was seeded onto three‐dimensional (3D) culture plates to produce spheroids, enhance cellular interactions, and mimic the in vivo environment. Bone defects were created on the skulls of 15‐week‐old rats using a 6.0‐mm trephine burr. Collagen sponges were transplanted as controls, and 3D DFAT spheroid‐seeded sponges were used in the experimental group. Results DFAT showed a fibroblast‐like morphology. Flow cytometry revealed that DFAT expressed MSC markers but not hematopoietic stem cell markers. Bone analysis revealed increased bone mineral density, volume, and trabecular thickness in the experimental group. Hematoxylin–eosin staining in the control group revealed only a thin layer of fibrous tissue, whereas the experimental group showed new bone tissue formation. The control group lacked Von Willebrand factor (vWf) expression, whereas the experimental group exhibited vWf expression, indicating the development of new blood vessels. Von Kossa staining revealed calcification only in the experimental group. Conclusion Spheroids alone are difficult to retain in bone defects, making their use in combination with collagen sponges more effective for bone regeneration. The study findings suggest that 3D DFAT spheroids may help regenerate rat skull defects, with potential implications for human bone regeneration.