The mTOR/ULK1 signaling pathway mediates the autophagy-promoting and osteogenic effects of dicalcium silicate nanoparticles

A novel bioactive inorganic material containing silicon, calcium and oxygen, calcium silicate (Ca(2)SiO(4), C(2)S) with a CaO-SiO(2) ingredient, has been identified as a potential candidate for artificial bone. Autophagy has an essential function in adult tissue homoeostasis and tumorigenesis. However, little is known about whether silicate nanoparticles (C(2)S NPs) promote osteoblastic differentiation by inducing autophagy. Here we investigated the effects of C(2)S NPs on bone marrow mesenchymal stem cell differentiation (BMSCs) in osteoblasts. Furthermore, we identified the osteogenic gene and protein expression in BMSCs treated with C(2)S NPs. We found that autophagy is important for the ability of C(2)S NPs to induce osteoblastic differentiation of BMSCs. Our results showed that treatment with C(2)S NPs upregulated the expression of BMP2, UNX2, and OSX in BMSCs, and significantly promoted the expression of LC3 and Beclin, while P62 (an autophagy substrate) was downregulated. C(2)S NP treatment could also enhance Alizarin red S dye (ARS), although alkaline phosphatase (ALP) activity was not significantly changed. However, all these effects could be partially reversed by 3-MA. We then detected potential signaling pathways involved in this biological effect and found that C(2)S NPs could activate autophagy by suppressing mTOR and facilitating ULK1 expression. Autophagy further activated β-catenin expression and promoted osteogenic differentiation. In conclusion, C(2)S NPs promote bone formation and osteogenic differentiation in BMSCs by activating autophagy. They achieve this effect by activating mTOR/ULK1, inducing autophagy, and subsequently triggering the WNT/β-catenin pathway to boost the differentiation and biomineralization of osteoblasts.