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To establish a large regenerative cartilage without any scaffolds, we cultured chondrocytes on NIsopropylacrylamide (NIPPAM)-coated dishes with 3 × 3 mm2 grids, producing many cell sheets. We then attempted to have the cell sheets form into pellets, which we term cartilage elements, and have the cartilage elements fuse with each other. Finally, we regenerated the large cartilaginous constructs with some stiffness. The NIPPAM coating on culture dishes, which enables to detach cultured chondrocytes only by the decrease in incubation temperature, was useful for preparing many chondrocyte sheets with a homogenous size. The cell sheets could provide effective matrix production and become spherical to form the cartilage elements. For their fusion, those elements were jammed into an agarose mold and were cultured for 3 weeks. The regenerative constructs made by fusing the cartilage elements derived from human or beagle chondrocytes were subcutaneously transplanted into nude mice and the cell-donor beagles, respectively, showing fair cartilage regeneration 2 months after transplantation. They could also prevent severe foreign-body reactions that often occur when using some scaffolds. Thus, by mass production of homogenous cartilage elements using the NIPPAM-coated dishes and their mutual fusion in the agarose mold, mature cartilage was three-dimensionally regenerated without any scaffolds.

Regenerative Cartilage made by Fusion of Cartilage Elements derived from Chondrocyte Sheets prepared in Temperature-Responsive Culture Dishes