Spheroid culture for chondrocytes triggers the initial stage of endochondral ossification.

Endochondral ossification is the process of bone formation derived from growing cartilage duringskeletal development. In previous studies, we provoked the osteocyte differentiation of osteoblast precursor cells under a three-dimensional (3D) culture model. To recapitulate the endochondral ossification, the present study utilized the self-organized scaffold-free spheroid model reconstructed by pre-chondrocyte cells. Within 2-day cultivation in the absence of the chemically induced chondrogenesis supplements, the chondrocyte marker was greatly expressed in the inner region of the spheroid, whereas the hypertrophic chondrocyte marker was strongly detected in the surface region of the spheroid. Notably, we found out that the gene expression levels of osteocyte markers were also greatly upregulated compared to the conventional 2D monolayer. Moreover, after long-term cultivation for 28 days, it induced morphological changes in the spheroid, such as cellular hypertrophy and death. In this study, in order to recapitulate the initial stage of the endochondral ossification, we highlighted the potentials of the 3D culture method to drive the hypertrophic chondrocyte differentiation of the pre-chondrocyte cells.