Inhibition of M2 macrophage-mediated mesenchymal stem cell migration: Boldine attenuates elbow heterotopic ossification

Abstract

Background

Heterotopic ossification (HO) is characterized by abnormal bone formation in soft tissues, often following trauma or surgery. Transforming growth factor-beta (TGF-β) signaling and M2 macrophage polarization play critical roles in the recruitment and differentiation of mesenchymal stromal/progenitor cells (MSPCs), promoting HO.

Methods

An elbow joint trauma-induced HO mouse model was established, where model mice were treated with dichloromethylene-bisphosphonate (Cl2MBP) liposomes or PBS liposomes to deplete macrophages. In addition, boldine was administered to evaluate its therapeutic effect on HO formation. Bone marrow mesenchymal stem cells (BMSCs) were also extracted for in vitro experiments. Quantitative real-time PCR (qRT-PCR) and Western blot were conducted to assess gene and protein expression. In vivo methods included Micro-Computed Tomography (Micro-CT) to assess bone formation, histological staining to evaluate tissue changes, immunohistochemistry (IHC) and immunofluorescence to analyze macrophage, CD73⁺ and CD105⁺ cells infiltration. In vitro, BMSCs were identified by flow cytometry and treated with interleukin-10 (IL-10) and/or boldine, and assays such as cell viability (Cell Counting Kit 8 (CCK8)), migration (Transwell), immunofluorescence, ALP staining, and Alizarin Red S staining, were conducted to assess osteogenic differentiation.

Results

Boldine treatment significantly reduced HO formation, decreased collagen deposition, and inhibited M2 macrophage infiltration (P < 0.05). In vitro, boldine reduced IL-10-induced cell activity, migration, and osteogenic differentiation of BMSCs and inhibited TGF-β and pSmad2/3/Smad2/3 protein (P < 0.05).

Conclusion

Boldine attenuates HO by inhibiting M2 macrophage-mediated MSPC migration and might involve the TGF-β signaling, suggesting its potential as a therapeutic approach for managing HO.