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. 2020 Aug 31;18(1):119.
doi: 10.1186/s12951-020-00663-w.

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

Wang Ruolan  1   2 , Chen Liangjiao  3 , Shao Longquan  4   5
Affiliations

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

Wang Ruolan et al. J Nanobiotechnology. .

Abstract

A novel bioactive inorganic material containing silicon, calcium and oxygen, calcium silicate (Ca2SiO4, C2S) with a CaO-SiO2 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 (C2S NPs) promote osteoblastic differentiation by inducing autophagy. Here we investigated the effects of C2S NPs on bone marrow mesenchymal stem cell differentiation (BMSCs) in osteoblasts. Furthermore, we identified the osteogenic gene and protein expression in BMSCs treated with C2S NPs. We found that autophagy is important for the ability of C2S NPs to induce osteoblastic differentiation of BMSCs. Our results showed that treatment with C2S 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. C2S 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 C2S NPs could activate autophagy by suppressing mTOR and facilitating ULK1 expression. Autophagy further activated β-catenin expression and promoted osteogenic differentiation. In conclusion, C2S 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.

Keywords: Autophagy; Dicalcium silicate; MTOR/ULK1; Osteogenesis.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Physicochemical characterization of C2S NPs. a Representative TEM image of the C2S NP morphology and size. b Representative SEM image of C2S NP morphology. c Particle size of C2S powder ranged from 40-150 nm in diameter by laser diffraction. d XRD analyses indicated that the spectrum of sample corresponded to Ca2SiO4. e Representative AFM image and thickness analysis of C2S NPs. f EDS analyses detected Ca, Si, and O corresponding to Ca2SiO4. g Functional groups of C2S NPs identified by the FTIR spectrum showed the presence of C-O, Si–O and unbound water
Fig. 2
Fig. 2
Biocompatibility evaluation of BMSCs exposed to C2S NPs (0, 10, 50, 100 μg/mL). a ICP-MS exploration of ion levels released from C2S NPs in extracellular fluid for 24 h. b Uptake of C2S NPs by BMSCs was observed via TEM at 6 h. c Cellular morphology and structures of cell organelles were observed by rhodamine-phalloidin (Green) and DAPI (Blue) staining of BMSCs at different times points (6, 12, and 24 h), scale bar 20 µm. d Cell viability was evaluated using CCK-8 assays at different times points (6, 12, and 24 h). e The levels of LDH release were detected in BMSCs post-treatment with C2S NPs for 6, 12, and 24 h; f Cells were stained with Annexin V-FITC and PI and analyzed by flow cytometry. Quantification of the cell early and total apoptosis ratio is shown below on the right. Values are expressed as the mean ± SEM n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control group
Fig. 3
Fig. 3
Osteogenic effect of C2S NP (0, 10, 50, 100 μg/mL) nanoparticles in vitro. a The cell cycle was detected by flow cytometry. BMSCs were incubated with 50 and 100 μg/mL C2S NPs for 24 h. b Cell proliferation was evaluated using CCK-8 assays at different times points (2, 3, and 7 days). ALP activity (c) and the formation of mineralized nodules (d) were detected by BCIP/NBT and Alizarin red S staining accompanied by increasing concentrations of C2S NPs at different time points (7, 14, 21 days). e The total mRNA levels of COLI, OSX (Osterix), RUNX2, and BMP2 in BMSCs treated with C2S NPs (0, 10, 50, 100 μg/mL) at different time points (3, 7, 14, 21 days) was detected by RT-PCR. f The total protein levels of β-catenin, RUNX2, BMP2, OPN and AXIN1 were detected by western blotting, and the cells were treated as in (e). Densitometry data were normalized to GAPDH. The relative optical density was analyzed using ImageJ software (below). Values are expressed as the mean ± SEM. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. Compared with the control group
Fig. 4
Fig. 4
C2S NPs stimulate autophagy in BMSCs (a) TEM observations of BMSCs following treatment with 50 μg/mL C2S NPs for 24 h. Distinct autophagic vacuoles (black arrows) were denoted in the enlarged images (lower) from the dash line squares. b–d BMSCs were treated with C2S NPs (0, 10, 50, 100 µg/mL) for 3, 6, and 12 h. FITC-labeled LC3 was observed under a fluorescence microscope. e The fluorescence intensity ratio was analyzed using ImageJ software (below). f total protein levels of Beclin, P62, and LC3(LC3II/LC3I) detected by western blot analysis, treating the cells as in (d). g Densitometry data were normalized to GAPDH. The relative optical density was analyzed using ImageJ software. Values are expressed as the mean ± SEM, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group
Fig. 5
Fig. 5
C2S NPs promote osteogenesis through an autophagy effect in vitro. a The total protein levels of mTOR, p-mTOR, ULK1, p-ULK1, Beclin, P62, and LC3 in BMSCs treated with C2S NPs (0, 10, 50, 100 μg/mL) at different time points (7 and 14 days) were detected by western blot analysis, and densitometry data were normalized to GAPDH. The relative optical density was analyzed using ImageJ software (below). b BMSCs were treated with 3-MA, rapamycin, C2S NPs (50 μg/mL), C2S NPs + 3-MA, and C2S NPs + rapamycin for 6 h. FITC-labeled LC3 was observed under a fluorescence microscope. The fluorescence intensity ratio was analyzed using ImageJ software (below). ALP activity (c) and the formation of mineralized nodules (d) were detected by BCIP/NBT and Alizarin red S staining, accompanied by 3-MA, rapamycin, C2S NPs (50 μg/mL), C2S NPs + 3-MA, and C2S NPs + rapamycin at different time points (7, 14 days). f Total protein levels of mTOR, p-mTOR, ULK1, p-ULK1, Beclin, P62, LC3 β-catenin, RUNX2, BMP2, OPN, and Axin1 in BMSCs were detected by western blot analysis. Inhabitor + C2S group compared with C2S group. f Densitometry data were normalized to GAPDH. The relative optical density was analyzed using ImageJ software. Values are expressed as the mean ± SEM. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. compared with the control group
Fig. 6
Fig. 6
Signaling pathway by which C2S NPs promote osteogenesis through an autophagy effect. C2S NPs can enter the cell and be trafficked to autophagosomes to activate autophagy and further promote the osteogenic differentiation of MSCs in vitro and in vivo. C2S NPs can activate the mTOR/ULK1 signaling pathway to induce autophagy and activate the Wnt/β-catenin pathway to promote osteogenesis. C2S NPs can promote osteogenesis by activating autophagocytosis via the mTOR/ULK1 pathway
Fig. 7
Fig. 7
C2S NPs promote osteogenesis through an autophagy effect in vivo. a Micro-computed tomography (micro CT) reconstruction images revealed new bone formation in the defects implanted with C2S NPs at 4, 8, and 12 weeks. b The relative optical density of new bone formation in the defects was analyzed using ImageJ software. c Histological images of newly formed bone with C2S NPs at 4, 8, and 12 weeks after surgery. d The area between the centers of the red band (tetracycline) and the green band (calcein) in the defects was analyzed using ImageJ software. e Immunohistochemical analysis of the protein expression of BMP2, LC3, ULK1, and p-ULK1in rat calvarial defects after implantation of C2S NPs at 4, 8, and 12 weeks. f The relative optical area of new bone formation in the defects was analyzed using ImageJ software. Values are expressed as the mean ± S.D. n = 3. * p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group
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