Heterotopic ossification (HO) is a pathological process characterized by the abnormal formation of bone in soft tissues, such as muscle and tendons, typically secondary to trauma, surgery, or neurological injury (Hwang et al., 2022, Markes et al., 2023, Lawand et al., 2023). This ectopic bone formation can lead to pain, joint stiffness, and restricted mobility, significantly affecting patients' quality of life (Hwang et al., 2022). The development of HO involves the recruitment and differentiation of mesenchymal stromal/progenitor cells (MSPCs) into osteoblasts, ultimately resulting ectopic bone formation (Markes et al., 2023, Xu et al., 2022). In the process, transforming growth factor-beta (TGF-β) has been identified as a crucial factor, as it not only enhances the recruitment of MSPCs but also promoting their differentiation into osteoblasts (Wang et al., 2018a, Mao et al., 2020). Previous study had shown that excessive activation of TGF-β signaling is closely associated with HO formation (Tu et al., 2023, Liang et al., 2022). Further, TGF-β antibody can effectively block HO progression at inflammatory, chondrogenic or osteogenic stages (Wang et al., 2018b). These remind us that TGF-β is a potential therapeutic target for preventing this pathological condition

Moreover, in bone regeneration, macrophages play a central role in the inflammatory response and subsequent tissue repair processes (Wang et al., 2022, Chen et al., 2023, Schlundt et al., 2021). Depending on the microenvironment, macrophages can polarize into either pro-inflammatory M1 macrophages or anti-inflammatory, pro-repair M2 macrophages (Wang et al., 2019, Shapouri-Moghaddam et al., 2018). Previous studies showed that M2 macrophages can produce high levels of TGF-β, thereby supporting the recruitment and osteogenic differentiation of MSPCs, and promoting HO formation (Wang et al., 2018a, Patel et al., 2022). Inhibiting M2 macrophage polarization can effectively reduce pathological bone formation (Du et al., 2022). Interestingly, the inhibitory effects of different phenotypes of macrophages on bone formation and bone osteoinhibition were different in different studies (Yan et al., 2024). In injured skeletal muscle, macrophage depletion leads to endochondral differentiation of endothelial cells and leads to ectopic bone formation (Tirone et al., 2019). Macrophages treated with lipopolysaccharide (LPS)/Interferon gamma (IFNγ) (promoting an M1-like phenotype) have been shown to decrease TGF-β levels in adipose tissue-derived mesenchymal stem cells more effectively than those treated with IL-4 (promoting an M2-like phenotype) (Grotenhuis et al., 2016). Therefore, it is important to explore the complete landscape of the molecular and cellular mechanisms of macrophages in HO.

Boldine, a naturally occurring alkaloid, has demonstrated anti-inflammatory properties in various disease models, including experimental colitis, where it effectively controls the infiltration of macrophages/monocytes (Pandurangan et al., 2016, Gotteland et al., 1997, Lamba et al., 2024). In addition, boldine has been shown to decrease TGF-β levels in models of kidney injury (Gomez and Velarde, 2018), suggesting its potential in modulating the TGF-β pathway. Further, Boldine has anti-proliferation effect on neural progenitor cells (Jiménez-Madrona et al., 2023). Given these properties, boldine may have therapeutic potential in reducing HO by regulating macrophage polarization and subsequently decreasing TGF-β-mediated MSPC recruitment and differentiation.

In this study, we investigated the effects of boldine on HO formation in an elbow joint trauma model, focusing on its ability to inhibit M2 macrophage-mediated mesenchymal stem cell (MSC) migration. We hypothesized that boldine treatment would attenuate HO by modulating macrophage polarization and reducing TGF-β signaling, thereby interfering with the recruitment and differentiation of MSPCs into osteoblasts. Furthermore, we aimed to explore the broader implications of boldine in regulating macrophage phenotypes and its potential role in preventing pathological bone formation. Our findings provide new insights into the therapeutic potential of boldine in managing HO and contribute to the understanding of macrophage-mediated pathways in pathological bone formation.