Dicalcium silicate-induced mitochondrial dysfunction and autophagy-mediated macrophagic inflammation promotes osteogenic differentiation of BMSCs.

Dicalcium silicate (Ca 2 SiO 4 , C 2 S) has osteogenic potential but induces macrophagic inflammation. Mitochondrial function plays a vital role in macrophage polarization and macrophagic inflammation. The mitochondrial function of C 2 S-treated macrophages is still unclear. This study hypothesized: (i) the C 2 S modulates mitochondrial function and autophagy in macrophages to regulate macrophagic inflammation, and (ii) C 2 S-induced macrophagic inflammation regulates osteogenesis. We used RAW264.7 cells as a model of macrophage. The C 2 S (75-150 μg/ml) extract was used to analyze the macrophagic mitochondrial function and macrophage-mediated effect on osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (BMSCs). The results showed that C 2 S extract (150 μg/ml) induced TNF-α, IL-1β and IL-6 production in macrophages. C 2 S extract (150 μg/ml) enhanced reactive oxygen species level and intracellular calcium level but reduced mitochondrial membrane potential and ATP production. TEM images showed reduced mitochondrial abundance and altered the mitochondrial morphology in C 2 S (150 μg/ml)-treated macrophages. Protein level expression of PINK1, Parkin, Beclin1 and LC3 was upregulated but TOMM20 was downregulated. mRNA sequencing and KEGG analysis showed that C 2 S-induced differentially expressed mRNAs in macrophages were mainly distributed in the essential signaling pathways involved in mitochondrial function and autophagy. The conditioned medium from C 2 S-treated macrophage robustly promoted osteogenic differentiation in BMSCs. In conclusion, our results indicate mitochondrial dysfunction and autophagy as the possible mechanism of C 2 S-induced macrophagic inflammation. The promotion of osteogenic differentiation of BMSCs by the C 2 S-induced macrophagic inflammation suggests the potential application of C 2 S in developing immunomodulatory bone grafts.