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

Qianting Luo; Xingyang Li; Wenchao Zhong; Wei Cao; Mingjing Zhu; Antong Wu; Wanyi Chen; Zhitong Ye; Qiao Han; Duraipandy Natarajan; Janak L Pathak; Qingbin Zhang

doi:10.1093/rb/rbab075

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

Regen Biomater. 2021 Dec 13:9:rbab075. doi: 10.1093/rb/rbab075. eCollection 2022.

Authors

Qianting Luo^{1

2}, Xingyang Li¹, Wenchao Zhong¹, Wei Cao^{1

3}, Mingjing Zhu¹, Antong Wu¹, Wanyi Chen¹, Zhitong Ye⁴, Qiao Han¹, Duraipandy Natarajan⁴, Janak L Pathak⁴, Qingbin Zhang¹

Affiliations

¹ Department of Temporomandibular Joint, Affiliated Stomatology Hospital of Guangzhou Medical University, Guangdong Engineering Research Center of Oral Restoration and Reconstruction, Guangzhou Key Laboratory of Basic and Applied Research of Oral Regenerative Medicine, Guangzhou 510182, China.
² Jiangmen Central Hospital, Affiliated Jiangmen Hospital of Sun Yat-sen University, Jiangmen 529030, China.
³ Department of Oral Cell Biology, Academic Centre of Dentistry Amsterdam (ACTA), Vrije Universiteit Amsterdam and University of Amsterdam, Amsterdam 1081LA, The Netherlands.
⁴ Affiliated Stomatology Hospital of Guangzhou Medical University, Guangdong Engineering Research Center of Oral Restoration and Reconstruction, Guangzhou Key Laboratory of Basic and Applied Research of Oral Regenerative Medicine, Guangzhou 510182, China.

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-1β 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.

Keywords: autophagy; dicalcium silicate; macrophagic inflammation; mitochondrial function; osteogenesis.