Metformin inhibits heterotopic ossification of mouse Achilles tendon by inhibiting the Nr4a1/Wnt/β-catenin signaling pathway.

Heterotopic ossification is a pathological process characterized by aberrant bone formation in soft tissues, leading to joint pain, stiffness, functional impairment, and poor quality of life. We used a mouse Achilles tendon heterotopic ossification model and in vitro tendon-derived stem cells (TDSCs) assays to identify the molecular mechanism of metformin in prevention of heterotopic ossification. Metformin significantly attenuated heterotopic ossification, reducing ectopic bone volume and osteogenic gene expression. In vitro, metformin inhibited TDSCs osteogenic differentiation in a dose-dependent manner, decreasing calcium nodule deposition and osteogenic marker expression. Transcriptomic analysis revealed downregulated Nr4a1 expression in metformin-treated heterotopic ossification samples; in vitro experiments confirmed that Nr4a1 activation enhances TDSCs osteogenesis, and Nr4a1 knockdown suppresses osteogenesis. Metformin also reduced Wnt4 and β-catenin expression, suggesting that Nr4a1 promotes heterotopic ossification by positively regulating Wnt/β-catenin signaling. In sum, metformin downregulates expression of Nr4a1 in TDSCs, which suppresses osteogenic differentiation by inhibiting Wnt/β-catenin signaling. As a mediator of TDSC osteogenic differentiation, Nr4a1 may be a therapeutic target for heterotopic ossification.