Fibroblast Growth Factor 2-Immobilized Biointerfaces Drive Exogenous Transforming Growth Factor β1-Independent Chondrogenesis of Human Mesenchymal Stem Cell and Ectopic Cartilage Tissue Formation.

Articular cartilage regeneration remains challenging due to limited intrinsic repair capacity and the tendency of current human mesenchymal stem cell (hMSC)-based strategies to form fibrocartilage or undergo hypertrophy. Here, we present a defined biomaterial platform in which maltose-binding protein-fibroblast growth factor 2 (MBP-FGF2) is surface-immobilized to create a bioinstructive interface that directs in vitro hMSC chondrogenesis without exogenous transforming growth factor-β1 (TGF-β1). The immobilized FGF2 interface promotes spontaneous, scaffold-free spheroid assembly via FGFR1-heparan sulfate proteoglycan-mediated adhesion, providing localized and sustained signaling. Transcriptomic and molecular analyses revealed early activation of FGF- and TGF-β-related pathways, with up-regulation of SOX9, COL2A1, and aggrecan and suppression of COL1A1 and COL10A1. These effects were consistently observed in hMSCs derived from both human embryonic stem cells and human tonsil tissue. Upon subcutaneous implantation into immunodeficient mice, pre-differentiated spheroids generated ectopic cartilage tissue rich in type II collagen and aggrecan, even in the absence of TGF-β1. Taken together, these findings establish FGF2-immobilized biointerfaces as bioinstructive matrices that enable exogenous TGF-β-independent chondrogenesis and ectopic cartilage tissue formation, providing a defined and reproducible route for generating chondrogenic constructs for regenerative medicine.