Fig 2: BMP9 activated PINK1/Drp1-mediated mitophagy, leading to the reversal of high glucose-induced osteoblast differentiation impairment in MC3T3-E1 cellsThe concentration of glucose in the medium of the L-Glu group was 5.5 mmol/L, and that in the medium of the H-Glu, H-Glu+BMP9, Drp1 siRNA and PINK1 siRNA groups was 25 mmol/L. The final concentration of BMP9 in H-Glu+BMP9, Drp1 siRNA and PINK1 siRNA groups was 100 ng/ml.(A) MC3T3-E1 cells were cultured in osteogenic medium for 14 days after siRNA transfection. Expressions of PINK1, Drp1, Runx2, and Osx were determined by WB.(B) MC3T3-E1 cells were cultured in osteogenic medium for 21 days after siRNA transfection. Alizarin red S staining was performed. Scale bars, 500 μm.(C) MC3T3-E1 cells were cultured in osteogenic medium for 14 days after siRNA transfection. MMP of living cells was assessed by the Image-iT TMRM Reagent. Scale bars, 20 μm.(D) MC3T3-E1 cells were cultured in osteogenic medium for 14 days after siRNA transfection. Mitochondrial reactive oxygen species of living cells were assessed by the MitoSox Reagent. Scale bars, 20 μm.(E) MC3T3E1 cells were transfected with mito-Keima plasmid and siRNA and cultured in osteogenic medium for 14 days. The fluorescent dots of mito-Keima were observed by confocal microscopy. Scale bars, 20 μm.(F) Representative electron micrographs show mitochondria and mitophagosomes (arrowhead) in MC3T3-E1 cells of different groups. Scale bars, 500 nm. Data presented as mean ± SD. A t test was used for comparison between two groups. One-way ANOVA was used for comparison among multiple groups. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Ns, no significance.

Fig 3: BMP9 reversed both the osteoblastogenic differentiation impairment and mitochondrial dysfunction induced by high glucose in MC3T3-E1 cellsThe concentration of glucose in the medium of the L-Glu group was 5.5 mmol/L, and that in the medium of the H-Glu and H-Glu+BMP9 group was 25 mmol/L. The final concentration of BMP9 was 100 ng/ml.(A) Alizarin red S staining was performed in MC3T3-E1 cells after 21 days of osteogenic induction. Scale bars, 500 μm.(B) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Expressions of osteogenic markers were determined by RT-qPCR.(C) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Expressions of Runx2 and Osx were determined by WB.(D and E) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Mitochondrial oxidative capacity was measured in real time. Basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity were calculated by WAVE software.(F and G) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. MMP of living cells was assessed by the Image-iT TMRM Reagent. Scale bars, 50 μm.(H and I) MC3T3-E1 cells were cultured in osteogenic medium for 14 days. Mitochondrial reactive oxygen species of living cells were assessed by the MitoSox Reagent. Scale bars, 50 μm. Data presented as mean ± SD. n = 3 biological replicates. One-way ANOVA was used for comparison among multiple groups. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Ns, no significance.

Fig 4: BMP9 promoted mitophagy markers to the control levels in diabetic mice in vivo(A) Representative images derived from micro-CT analysis, including 2D image construction of distal femur, 3D images reconstruction of trabecular bone of distal femur, and 3D image reconstruction of the femoral midshaft corticoid bone.(B–D) Quantitative analysis of the vBMD, BV/TV and Ct.Th of corticoid bone by micro-CT.(E–I) Quantitative analysis of the BV/TV, Tb.N, Tb.Sp, Tb.Th, and vBMD of trabecular by micro-CT.(J) Representative images of HE-stained decalcified femur sections. Scale bars, 200 μm.(K–M) The right femur was isolated and subjected to biomechanical properties analysis. The maximum load, elastic modulus and SMI were evaluated for each group.(N) Representative images derived from micro-CT analysis, including 2D image construction and 3D image reconstruction of L3 lumbar vertebra.(O–S) Quantitative analysis of the vBMD, BV/TV, Tb.N, Tb.Th, and Tb.Sp of L3 by micro-CT.(T and U) The levels of serum bone turnover parameters PINP and CTX-I were detected by ELISA.(V) WB analysis of protein levels of mitophagy marker in skull.(W) Expressions of ALP, Osx, Drp1, and PINK1 in skull were determined by RT-qPCR.(X) Immunofluorescence analysis of Drp1 and PINK1 expression in femur sections. Scale bars, 10 μm.(Y) Immunofluorescence analysis of PINK1 expression in femur sections. Scale bars, 20 μm.(Z) Immunofluorescence results of double labeling of osteoblasts with PINK1 and Drp1 in femur sections. Scale bars, 20 μm. Data presented as mean ± SD. n = 8 biological replicates. One-way ANOVA was used for comparison among multiple groups. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Ns, no significance.

Fig 5: BMP9 activated PINK1/Drp1-mediated mitophagy, leading to the reversal of high glucose-induced osteoblast differentiation impairment in BMSCsThe concentration of glucose in the medium of the L-Glu group was 5.5 mmol/L, and that in the medium of the H-Glu, H-Glu+BMP9, Drp1 siRNA and PINK1 siRNA groups was 25 mmol/L. The final concentration of BMP9 in H-Glu+BMP9, Drp1 siRNA and PINK1 siRNA groups was 100 ng/ml.(A) BMSCs cells were cultured in osteogenic medium for 14 days after siRNA transfection. Expressions of PINK1, Drp1, Runx2, and Osx were determined by WB.(B) BMSCs cells were cultured in osteogenic medium for 21 days after siRNA transfection. Alizarin red S staining was performed. Scale bars, 1 mm.(C) BMSCs cells were cultured in osteogenic medium for 14 days after siRNA transfection. MMP of living cells was assessed by the Image-iT TMRM Reagent. Scale bars, 20 μm.(D) BMSCs cells were transfected with mito-Keima plasmid and siRNA and cultured in osteogenic medium for 14 days. The fluorescent dots of mito-Keima were observed by confocal microscopy. Scale bars, 20 μm.