Chondrogenic differentiation of human periosteum-derived cells in spheroids, HAMA hydrogels, and bioprinted constructs: comparison of kartogenin and TGF- β 1

Abstract Kartogenin (KGN) is a small molecule reported to promote chondrogenesis and inhibit hypertrophic differentiation in mesenchymal stromal cells, but its effect on human periosteum-derived cells (hPDCs) remains unexplored. This study investigated whether KGN can induce chondrogenic differentiation and prevent hypertrophy in hPDC spheroids, either alone or in combination with transforming growth factor-beta 1 (TGF- β 1), when cultured in a microwell system or incorporated into methacrylated hyaluronic acid (HAMA)-based encapsulated or bioprinted constructs. Results showed that TGF- β 1 consistently promoted cartilage matrix production, inducing deposition of collagen type II (COL II), and aggrecan, and upregulating the expression of the early differentiation markers COL2A1, ACAN , and SOX9 , as well as the hypertrophic markers COL10A1 and metalloproteinase 13. In contrast, KGN alone had no effect on spheroid morphology, matrix deposition, protein expression, or gene regulation. When combined with TGF- β 1, HAMA-encapsulated spheroids showed enhanced chondrogenesis, as evidenced by stronger collagen fiber organization, increased glycosaminoglycan deposition, and positive Safranin O staining, absent in material-free conditions. This effect likely reflects HAMA’s supportive microenvironment, which facilitates matrix retention and cellular remodeling. However, the supplement of KGN did not provide any added benefit. These findings demonstrate the limited effect of KGN in hPDC-based cartilage tissue engineering and highlight the importance of cell- and context-specific validation of small molecule modulators.