ABSTRACT
Dicalcium silicate (Ca2SiO4, C2S) has osteogenic potential but induces macrophagic inflammation. Mitochondrial function plays a vital role in macrophage polarization and macrophagic inflammation. The mitochondrial function of C2S-treated macrophages is still unclear. This study hypothesized: (i) the C2S modulates mitochondrial function and autophagy in macrophages to regulate macrophagic inflammation, and (ii) C2S-induced macrophagic inflammation regulates osteogenesis. We used RAW264.7 cells as a model of macrophage. The C2S (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 C2S extract (150 µg/ml) induced TNF-α, IL-1ß and IL-6 production in macrophages. C2S 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 C2S (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 C2S-induced differentially expressed mRNAs in macrophages were mainly distributed in the essential signaling pathways involved in mitochondrial function and autophagy. The conditioned medium from C2S-treated macrophage robustly promoted osteogenic differentiation in BMSCs. In conclusion, our results indicate mitochondrial dysfunction and autophagy as the possible mechanism of C2S-induced macrophagic inflammation. The promotion of osteogenic differentiation of BMSCs by the C2S-induced macrophagic inflammation suggests the potential application of C2S in developing immunomodulatory bone grafts.
ABSTRACT
Dicalcium silicate microparticle (C2S)-based biomaterials have a potential for bone and dental tissue regenerative applications. The C2S-mediated transcriptome level mechanism in mesenchymal stem cells (MSCs) during bone-defect healing has not been investigated yet. In this study, we elucidated the differential expression pattern of messenger RNAs (mRNAs) and circular RNAs (circRNAs) in C2S-treated MSCs and their involvement in the osteogenesis process. C2S robustly enhanced the osteogenic differentiation of MSCs and cranial bone defect healing. C2S-treatment modulated the differential expression of mRNAs and circRNAs in MSCs. Differentially expressed circRNAs and mRNAs were involved in competing endogenous RNA (ceRNA-interaction networks). These ceRNA-interaction networks regulated the signaling pathways associated with osteogenesis, e.g., Wnt, PI3K-Akt, MAPK, and JAK/STAT signaling. C2S-treatment upregulated the expression of circ_1983, Gas7, and Runx2 in BMSCs. RNase R and luciferase activity assay confirmed the stability and miR-6931 sponging property of circ_1983, respectively. Knockdown of circ_1983 enhanced miR-6931 expression but inhibited Gas7 and Runx2 expression and osteogenic differentiation in C2S-treated MSCs. In conclusion, for the first time, we report the role of cicr_1983-miR-6931-Gas7 ceRNA-interaction in C2S-induced osteogenic differentiation of MSCs and bone defect healing. This study opens a new research stream "the role of circRNAs-mediated ceRNA-interaction in biomaterials and stem cell-based bone tissue engineering".
