Adipose-derived stem cells (ADSCs), due to their regenerative ability, have beneficial effects on bone and cartilage defects. In addition, spheroid formation of ADSCs obtained using three-dimensional (3D) culture accelerates the regenerative ability of ADSCs. The study investigated the regenerative effect of 3D-cultured small size ADSC spheroids without a scaffold in rats with defects in the critical-sized calvarial bone. ADSC-single cells, ADSC-spheroids, or PBS (as control) were implanted in rats, and radiological and histological assessment of bone regeneration was performed. Bone defects were significantly regenerated in the ADSC-spheroid group compared to that in the control group. ADSC-spheroids also showed the most significant bone regeneration in histological assessment. Immunohistochemistry assessment showed that ADSC-spheroids could survive 12 weeks after cell implantation. In vitro, cell apoptosis in ADSC-spheroids was significantly suppressed compared to that in ADSC-single cells. In addition, gene expression related to bone morphogenesis, angiogenesis, and stemness in ADSC-spheroids was elevated. The scaffold-free 3D-cultured small ADSC-spheroids survived in in vitro and in vivo conditions and promoted bone regeneration. Therefore, injectable small size ADSC-spheroids are a novel and less-invasive therapeutic option for treating bone defects.
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