Fig 2: AU suppressed the receptor activator of nuclear factor-?B ligand (RANKL)-induced differentiation of RAW264.7 cells to osteoclasts. (A) AU decreased the percentage of numbers and area of TRAP-positive RANKL-exposed RAW264.7 cells (200×) (Scale Bar: 50 µm). Data are expressed as the means ± SEMs (n = 6) and were analysed using a one-way ANOVA. ###p < 0.001 versus control cells; **p < 0.01 and ***p < 0.001 versus RANKL-induced differentiated cells. (B) In RANKL-exposed RAW264.7 cells, AU increased the expression levels of COL I, OCN, OPG, Nrf2, CAT, HO-2, SOD-1 and SOD-2, and decreased the expression levels of TRAP5, NFATc1 and CTSK. The quantitative expression of each protein was normalised to ß-actin and is presented as the fold-expression compared with the expression in control cells (n = 6). CTRL: control; AU: aucubin.

Fig 3: Osteogenesis of BMSCs in vivo.a Magnetic resonance imaging (MRI) examination of the ONFH model. b H&E staining of the ONFH model. Lipopolysaccharide (LPS) and methylprednisolone were used to build the model; saline was used as control. c Observation of XACB and XACB/BMSCs via scanning electron microscopy (SEM). d BMSCs repair of ONFH. Control was treated with lesion debridement (LD) without BMSCs implantation. e H&E and Masson staining to detect new-bone formation and maturation. f–i Western blot analysis of OPG, OCN, and Runx2 expression in osteonecrosis (n = 3). In (g–i), data are presented as means ± standard deviation (SD). Statistical significances were calculated by Student’s t test. BMSCs = bone marrow mesenchymal stem cells, ONFH = osteonecrosis of the femoral head, XACB = xenogeneic antigen-extracted cancellous bone, XACB/BMSCs = tissue-engineered bone, OPG = osteoprotegerin, OCN = osteocalcin, Runx2 = runt-related transcription factor 2.

Fig 4: P53 and Parkin co-regulated mitophagy in BMSCs to promote the repair of early steroid-induced ONFH.a Gross specimens in which we observed repair of osteonecrosis (n = 3). b Masson staining for evaluating new-bone maturation (n = 3). c H&E staining for evaluating new-bone formation (n = 3). d–g Western blot analysis of OCN, Runx2, and OPG expression (n = 3). Control was treated with lesion debridement (LD) without transplantation. In (e–g), data are presented as means ± SD. Statistical significances were calculated by ANOVA. Data were compared with the control, XACB, and XACB/BMSCs groups: *P < 0.05. Data were compared with the XACB/BMSCs/Parkin and XACB/BMSCs/shP53 groups: #P < 0.05.

Fig 5: Lnc Tmem235 inhibits hypoxia-induced apoptosis of BMSCs and promotes repair of early SONFH.a At 2 days after operation, the expression levels of GFP and BIRC5 in the femoral head necrotic area were detected by immunofluorescence (n = 6); GFP green fluorescence labeled BMSCs, which could not penetrate bone tissue, green fluorescent protein (GFP), negative control (NC), overexpression (OE), short hairpin (Sh); b At 2 days after surgery, the fluorescence intensity of DiR in the transplantation area was detected by live imaging of small animals (n = 8); DiR red fluorescence labeled BMSCs, which can penetrate bone tissue; c At 2 days after surgery, TUNEL was used to detect apoptosis in the femoral head necrotic area (n = 6); d Quantitative analysis of DiR fluorescence intensity in the transplanted area as shown in b (n = 8); e Quantitative analysis of the proportion of TUNEL positive cells in the transplanted area as shown in c (n = 6); f At 12 weeks post-surgery, H&E staining and Masson staining were used to evaluate the repair of the necrotic area (n = 6); g At 12 weeks after surgery, micro-CT was used to analyze the repair of necrotic area of femoral head (n = 6); h Quantitative analysis of the number of trabeculae as shown in g (n = 6); i Quantitative analysis of the trabecular thickness as shown in g (n = 6); j Quantitative analysis of the volume of new bone tissue as shown in g (n = 6); k Quantitative analysis of the volume fraction of new bone tissue as shown in g (n = 6); l. At 12 weeks post-surgery, the levels of osteogenic markers (e.g., Runx2, OPN, OCN, and OPG) were detected by western blotting (n = 6); osteopontin (OPN); m Quantitative analysis of OPG expression as shown in l (n = 6); n Quantitative analysis of Runx2 expression as shown in l (n = 6); o Quantitative analysis of OCN expression as shown in l (n = 6); p Quantitative analysis of OPN expression as shown in l (n = 6). In (d, e, h–k, m–p), the data are normally distributed, and the variance is homogeneous. Data are presented as the means ± SDs; differences were tested using one-way ANOVA with Tukey’s post-hoc test; *P < 0.05.