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 macro-phagic 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(75–150μg/ml)extract was used to analyze the macrophagic mitochondrial function and macrophagemediated effect on osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells(BMSCs).The results showed that C_(2)S extract(150μg/ml)induced TNF-α,IL-1βand IL-6 production in macrophages.C_(2)S extract(150μg/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(150μg/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.