Fig 2: Activation of the endothelial cell (EC)‐perivascular cell axis improves angiogenesis and osteogenesis. (A) Representative platelet‐derived growth factor (PDGF)‐BB (green) staining images of the metaphysis regions of tibial sections from the irradiated Cdh5;Td mice with or without deferoxamine mesylate (DFM) treatment. Nuclei, DAPI (blue). Quantification of fluorescence intensity of PDGF‐BB and vessel density. Scale bars, 100 μm (n = 3). (B) Representative PDGF receptor β (PDGFRβ; green) staining images and quantification of vessel‐related PDGFRβ+ cells of the metaphysis regions of tibial sections from the irradiated Cdh5;Td mice with or without DFM treatment. Nuclei, DAPI (blue). The white arrows represent detached PDGFRβ+ cells. Scale bars, 50 μm (n = 3). (C) Representative fluorescence images of human microvascular endothelial cell line‐1 (HMEC‐1) cells (green) and PDGFRβ;Td+ cells (red) on Matrigel with quantification of EC‐attached PDGFRβ;Td+ cells and tube formation parameters. Scale bars, 50 μm (n = 4). (D) Representative micro‐computed tomography (micro‐CT) images and quantitative analysis of the irradiated tibiae with DFM and/or imatinib mesylate (IM) treatment (n = 4). (E) Representative haematoxylin and eosin (H&E) staining images and quantification of adipocytes of the irradiated tibiae with DFM and/or IM treatment. Scale bars, 500 μm (n = 4). (F) Representative RUNX2 or Adipoq staining images and quantification of the double positive cells in total Td+ cells of tibial sections from the PDGFRβ;Td mice. DFM or IM was administered via intra‐peritoneal injection. The white and yellow arrows represent RUNX2+/PDGFRβ;Td+ cells and Adipoq+/PDGFRβ;Td+ cells, respectively. Scale bars, 25 μm (n = 3). Data are represented as the mean ± SD The p value was calculated by unpaired, two‐tailed Student's t‐test when comparing two treatment groups and by one‐way analysis of variance with Tukey's post hoc test when comparing multiple groups

Fig 3: Autophagy is required for the osteogenic differentiation of hASCs. (A, B): Depletion of ATG7 decreased ALP staining (A) and activity (B) on the seventh day after induction of osteogenic differentiation of hASCs. (C, D): Extracellular matrix mineralization (C) and ARS quantification (D) were decreased in ATG7 knockdown cells after 14 days of osteogenic induction. (E): Protein levels of RUNX2, p62, and LC3B are indicated as shown. GAPDH was used as internal loading control. (F–M): Treatment with 5 mM 3‐MA decreased the osteogenic capacity of PCK2‐expressing cells. Relative mRNA expression of RUNX2 (F) and ALP (G) on the seventh day of osteogenic induction are shown. Relative mRNA expression of RUNX2 (H) and OCN (I) after 14 days of osteogenic induction is shown. ALP staining (J) and activity (K) on the seventh day of osteogenic induction are shown. Extracellular matrix mineralization (L) and ARS quantification (M) on the 14th day of osteogenic induction are presented. (N): Protein expression patterns of PCK2, RUNX2, p62, and LC3B are shown. The control vector cells and PCK2‐expressing cells were cultured in PM, PM containing 5 mM 3‐MA, OM, or OM containing 5 mM 3‐MA for 5 days. GAPDH was used as internal loading control. Data are represented as mean ± SD. **, p ≤ .01; *, p ≤ .05, Student's t test. Abbreviations: 3‐MA, 3‐methyladenine; ALP, alkaline phosphatase; ARS, alizarin red S; ATG7, autophagy‐related‐gene‐7; LC3B, microtubule associated protein 1 light chain 3 β; OCN, osteocalcin; NC, negative control for sh‐ATG7; NS, not significant; OM, osteogenic media; p62, p62/SQSTM1; PCK2, mitochondrial phosphoenolpyruvate carboxykinase; PM, proliferation media; RUNX2, runt‐related transcription factor 2.

Fig 4: Enhanced osteogenic differentiation of BMSCs by MEL treatment. a Relative mRNA levels of osteogenic marker genes in human BMSCs treated with MEL. The ALP, RUNX2, and OPN mRNA levels in BMSCs were measured by qRT-PCR. b ALP, RUNX2, and OPN protein levels in human BMSCs treated with MEL. Western blot analysis was used to detect protein levels using GAPDH as the internal standard. c Elevated ALP expression in human BMSCs caused by MEL treatment. ALP expression in BMSCs was measured by the ALP staining method. Left are gross scanning images (scale bar: 1 mm), the middle are enlarged images (magnification: × 250, scale bar: 160 μm), and the right is quantification of the left gross scanning images. d MEL promotes osteogenic differentiation of human BMSCs. The osteogenic differentiation of BMSCs was evaluated by Alizarin Red staining. Left are gross scanning images (scale bar: 1 mm), the middle are enlarged images (magnification: × 250, scale bar: 160 μm), and the right is quantification of the left gross scanning images. CON: control; MEL: melatonin; ALP: alkaline phosphatase; RUNX2: runt-related transcription factor 2; OPN: osteopontin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; BMSCs: bone marrow mesenchymal stem cells; *P < 0.05, ***P < 0.001

Fig 5: miR-3653-3p reverses circ_0003865-regulated BMSC osteogenic differentiation by directly binding circ_0003865 and the GAS1 gene. a Relative expression of circ_0003865, miR-3653-3p, GAS1, and osteogenic marker genes in BMSCs as measured by qRT-PCR. b GAS1, ALP, RUNX2, and OPN protein levels in human BMSCs as quantitated by western blot analysis using GAPDH as the internal standard. c The proliferation in human BMSCs was tested by CCK-8 assay. d The expression of ALP in human BMSCs was analyzed by ALP staining. The top left are gross scanning images (scale bar: 1 mm), the lower left are enlarged images (magnification: 250×, scale bar: 160 μm), and the right is quantification of the left gross scanning images. e miR-3653-3p inhibitors repressed the osteogenic differentiation of human BMSCs transfected with circ_0003865 siRNAs compared with the si-CIRC group. The Alizarin Red staining was done to evaluate BMSC osteogenic differentiation. The top left are gross scanning images (scale bar: 1 mm), the lower left are enlarged images (magnification: × 250, scale bar: 160 μm), and the right is quantification of the left gross scanning images. f The association of circ_0003865 with miR-3653-3p as detected by an RNA pull-down assay. g, h The direct binding of miR-3653-3p with circ_0003865 and GAS1 gene as measured by the dual-luciferase reporter assay. Between miR-3653-3p and circ_0003865 (g). Between miR-3653-3p and GAS1 gene (h). si-CIRC: circ_0003865 siRNAs; miR-3653-3p In: miR-3653-3p inhibitors; GAS1: growth arrest-specific gene 1; CCK-8: cell counting kit-8; ALP: alkaline phosphatase; RUNX2: runt-related transcription factor 2; OPN: osteopontin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; NC: negative control; *P < 0.05