Abstract
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.