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Background: Aiming to construct an osteochondral-like structure, the combination of a β-tricalcium phosphate (βTCP) block with a scaffold-free sheet formed using mesenchymal stem cells (MSCs) was investigated. Methods: Human bone marrow MSCs in a cell culture insert that was set in a 24-well plate were cultivated using a chondrogenic medium containing dexamethasone, IGF-1, and TGFβ3 for 3 weeks during which a cylindrical βTCP block was put on the sheet at day 1, and the cell sheet construct was harvested. In other experiments, at day 14, the construct was put on a cell sheet that was prepared the day before and cultivated for 3 weeks. Results: The addition of a βTCP block resulted in a combined osteochondral-like construct and comparable staining intensity by Alcian blue, while the expression levels of the aggrecan and type II collagen genes decreased a little. During the culture with the βTCP block, the expression levels of the aggrecan gene increased monotonically. The increase in the inoculum cell number from 1.86 to 3.72 × 10 cells resulted in marked increases in the thickness of cell sheet parts in the βTCP block and expression levels of the aggrecan and type II collagen genes, while the thickness of cell sheet parts on the βTCP block scarcely changed. On the other hand, the addition of a cell sheet that was prepared a day before to the construct at day 14 resulted in the marked increase in thickness of the cell sheet part on the βTCP block, while the thickness of that in the βTCP block did not increase. Conclusion: A combined osteochondral-like structure was produced by putting a βTCP block on the sheet of MSC. The thickness of the cell sheet parts in and on the βTCP block could be increased by the increase in inoculum cell number and by providing an additional cell sheet, respectively. [ABSTRACT FROM AUTHOR]