TGF-131 derived from macrophages contributes to load- induced tendon- bone healing in the murine rotator cuff repair model by promoting chondrogenesis

Aims It has been established that mechanical stimulation benefits tendon -bone (T -B) healing, and macrophage phenotype can be regulated by mechanical cues; moreover, the interaction be-tween macrophages and mesenchymal stem cells (MSCs) plays a fundamental role in tissue repair. This study aimed to investigate the role of macrophage-mediated MSC chondrogene-sis in load-induced T -B healing in depth. Methods C57BL/6 mice rotator cuff (RC) repair model was established to explore the effects of me-chanical stimulation on macrophage polarization, transforming growth factor (TGF)-131 gen-eration, and MSC chondrogenesis within T -B enthesis by immunofluorescence and enzyme -linked immunosorbent assay (ELISA). Macrophage depletion was performed by clodronate liposomes, and T -B healing quality was evaluated by histology and biomechanics. In vitro, bone marrow-derived macrophages (BMDMs) were stretched with CELLOAD- 300 load sys-tem and macrophage polarization was identified by flow cytometry and quantitative real-time polymerase chain reaction (qRT- PCR). MSC chondrogenic differentiation was measured by histochemical analysis and qRT- PCR. ELISA and qRT- PCR were performed to screen the candidate molecules that mediated the pro-chondrogenic function of mechanical stimulated BMDMs. Results Mechanical stimulation promoted macrophage M2 polarization in vivo and in vitro. The conditioned media from mechanically stimulated BMDMs (MS -CM) enhanced MSC chondro-genic differentiation, and mechanically stimulated BMDMs generated more TGF-131. Further, neutralizing TGF-131 in MS -CM can attenuate its pro-chondrogenic effect. In vivo, mechani-cal stimulation promoted TGF-131 generation, MSC chondrogenesis, and T -B healing, which were abolished following macrophage depletion. Conclusion Macrophages subjected to appropriate mechanical stimulation could polarize toward the M2 phenotype and secrete TGF-131 to promote MSC chondrogenesis, which subsequently augments T -B healing.