M2 exosomes modified by hydrogen sulfide promoted bone regeneration by moesin mediated endocytosis

Bone defects caused by trauma or tumor led to high medical costs and poor life quality for patients. The exosomes, micro vesicles of 30-150 nm in diameter, derived from macrophages manipulated bone regeneration. However, the role of hydrogen sulfide (H₂S) in the biogenesis and function of exosomes and its effects on bone regeneration remains elusive. In this study, we used H₂S slow releasing donor GYY4137 to stimulate macrophages and found that H₂S promoted the polarization of M2 macrophages to increase bone regeneration of MSCs in vitro and in vivo. Moreover, we developed the H₂S pre-treated M2 macrophage exosomes and found these exosomes displayed significantly higher capacity to promote bone regeneration in calvarial bone defects by re-establishing the local immune microenvironment. Mechanically, H₂S treatment altered the protein profile of exosomes derived from M2 macrophages. One of the significantly enriched exosomal proteins stimulated by H₂S, moesin protein, facilitated the exosomes endocytosis into MSCs, leading to activated the β-catenin signaling pathway to promote osteogenic differentiation of MSCs. In summary, H₂S pretreated M2 exosomes promoted the bone regeneration of MSCs via facilitating exosomes uptake by MSCs and activate β-catenin signaling pathway. This study not only provides new strategies for promoting bone regeneration, but also provides new insights for the effect and mechanism of exosomes internalization.