Abstract
Bone healing is intricately associated with dynamic macrophage polarization. Modulating macrophages toward the M2 phenotype has emerged as a promising strategy in bone tissue engineering. Calcium silicate, known for its superior osteoconductivity, is widely used as a bone substitute and is particularly effective in promoting bone tissue regeneration when incorporated with bioactive ions. Recent studies have highlighted lithium's immunomodulatory effects, with extracellular vesicles (EVs) identified as potential mediators of these actions. Although M2 macrophage-derived EVs (M2-EVs) have been shown to promote bone regeneration, the underlying mechanisms through which biomaterial-stimulated M2-EVs regulate bone regeneration remain unclear. This study investigated the immunomodulatory effects of lithium-doped calcium silicate (LiCS) scaffolds on macrophages and associated inflammatory cytokine profiles. Notably, miR-145-5p was significantly upregulated in both LiCS-stimulated macrophages and their secreted EVs, suggesting a potential regulatory role. Characterization of these miR-145-5p-enriched EVs revealed enhanced anti-inflammatory responses, stimulation of angiogenesis, and upregulation of osteogenic markers in relation to M1 macrophages, mesenchymal stem cells, and endothelial cells. These findings elucidate the molecular basis of LiCS-stimulated M2-EV-regulated bone regeneration via miR-145-5p, providing new insights into developing immunomodulatory biomaterials in regenerative medicine.