Sequential Modeling of Fibrotic and Mineralizing Osteoarthritis‐Like Remodeling in hDPSC‐Derived Cartilage Microtissues

Osteoarthritis (OA) involves alterations in cartilage mechanics, matrix composition, and mineralization, yet in vitro models that resolve remodeling transitions remain limited. Scaffold-free cartilage microtissues derived from human dental pulp stem cells (hDPSCs) are subjected to sequential dynamic rocking culture and triiodothyronine (T3) treatment. Dynamic rocking produces superficial Col I-positive fibrotic and matrix-degrading changes, whereas T3 induces proteoglycan loss, increased Col I and MMP13, irregular mineral deposition, and altered nanomechanical properties, defining a mixed terminal catabolic and mineralizing OA-like state. Transcriptomic profiling shows stage-associated reorganization rather than uniform pathway activation. In T3-treated microtissues, ATF6-associated ER-stress abnormalities include ER cisternal dilation and increased ATF6 signal. Targeted ATF6 knockdown reduces ATF6 signal, ER dilation, extracellular-matrix disorganization, and ER-stress/proteostasis and inflammatory/catabolic transcriptional module scores, supporting ATF6-associated signaling as a functionally relevant component of this degenerative state. In mouse temporomandibular joint and knee OA models, intra-articular Ad-shATF6 is associated with partial cartilage preservation and subchondral-bone indices shifted toward control ranges.