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Background Mesenchymal stem cells (MSCs) are a favored cell source for regenerative medicine because of their multilinage potential. However, the conventional monolayer technique used to culture MSCs, inadequately overcomes their low differentiation capacity. Culture of MSCs in multicellular spheroids, more accurately mimics the in-vivo microenvironment; thus, resolving this problem. In this study, we assessed whether the osteoregeneartive potential of MSC spheroids is greater than that of monolayer MSCs. Results MSC spheroids were generated from rat MSCs (rMSCs) using low-binding plates. Real-time reverse transcription-polymerase chain reaction and immunocytochemical analysis indicated that osteogenic properties were accelerated in MSC spheroids compared with monolayer rMSCs when treated with an osteoblast-inducer reagent for 7 days. Moreover, increased calcium deposition was visualized in MSC spheroids using Alizarin red staining. In a rat calvarial defect model, micro-computed tomography and histological assays showed that MSC spheroid-engrafted defects experienced enhanced bone regeneration. Conclusions Our in-vitro and in-vivo results reveal that MSCs in the spheroid culture exhibit enhanced osteoregenerative efficiency compared with monolayer MSCs. [ABSTRACT FROM AUTHOR]