Table 1.
Sequences of primers for qRT-PCR.
Fig 1.
Analyses of collagenase-released extracellular vesicles isolated from mouse osteoblasts (CREVs).
(A) A schema of a preparation of CREVs. (B) The particle size and concentration of CREVs in NanoSight meter analyses. Results are expressed as the mean of 5 measurements within one experiment in each group. (C) Western blotting analysis of KDM1/LSD1, β-actin, CD9, and Annexin A5 (ANXA5) in mouse osteoblast cell lysates and CREVs. (D) ALP activities of mouse osteoblast cell lysates and CREVs. Results are expressed as the mean ± SEM of 4 independent experiments. Statistical analyses were performed using the unpaired t-test.
Fig 2.
Effects of the local administration of CREVs on bone repair after a femoral bone defect.
(A) Three-dimensional qCT images of damaged sites 7 days after a femoral bone defect. The scale bar indicates 1 mm. (B) The bone defect area at the damaged site 7 days after the femoral bone defect. (C) The bone volume/tissue volume (BV/TV, %) of mineralized bone that formed in the hole region 7 days after the femoral bone defect. Results are expressed as the means ± SEM of 8 mice per group. Statistical analyses were performed using the Mann–Whitney U test (B, C). (D) Images of HE-stained sections of damaged sites 7 days after the femoral bone defect. Scale bars indicate 200 μm.
Fig 3.
Effects of the local administration of CREVs on the osteogenic marker at damaged sites.
Images of ALP-positive cells and the number of ALP-positive cells per area (mm2) (A), bone surface (mm) (B), and the number of DAPI-positive nucleus (C) at the damaged site 7 days after the femoral bone defect. Results are expressed as the means ± SEM of 8 mice per group. Scale bars indicate 100 μm. The dotted line indicates the boundary with cortical bone. (D) The number of TRAP-positive multinucleated cells (MNCs) per bone surface (mm) at the damaged site 7 days after the femoral bone defect. Results are expressed as the means ± SEM of 6 (Vehicle) or 8 (CREVs) mice per group, respectively. Statistical analyses were performed using the Mann–Whitney U test.
Fig 4.
Effects of the local administration of CREVs on the chondrogenic marker at damaged sites.
(A) Images of Toluidine blue-stained sections and the area of the metachromatic-stained region at the damaged site 7 days after the femoral bone defect. Results are expressed as the means ± SEM of 8 mice per group. (B) Images of Alcian blue-stained sections and the area of the Alcian blue-stained region at the damaged site 7 days after the femoral bone defect. Results are expressed as the means ± SEM of 6 (Vehicle) or 8 (CREVs) mice per group, respectively. Scale bars indicate 200 μm. Statistical analyses were performed using the Mann–Whitney U test.
Fig 5.
Effects of CREVs on the osteoblastic differentiation of mouse mesenchymal ST2 cells.
The mRNA expression of osteogenic markers in ST2 cells cultured with or without 200 ng/mL BMP-2 and/or 4 μg/mL CREVs for 72 h. Results are expressed relative to Gapdh mRNA levels and are shown as the means ± SEM of 4 independent experiments. Statistical analyses were performed using a one-way ANOVA followed by the Tukey–Kramer post hoc test.
Fig 6.
Effects of CREVs on the phenotypes of mouse osteoblasts.
(A) The mRNA expression of osteogenic markers in mouse osteoblasts cultured with or without 200 ng/mL BMP-2 and/or 4 μg/mL CREVs for 72 h. Results are expressed relative to Gapdh mRNA levels and are shown as the means ± SEM of 4 independent experiments. (B) The ALP activity of mouse osteoblasts cultured with or without 4 μg/mL CREVs for 72 h. Results are expressed as the mean ± SEM of 4 independent experiments. (C) Images of Alizarin red-stained mineralized mouse osteoblasts cultured with 50 μg/mL ascorbic acid and 10 mM β-glycerophosphate in the presence or absence of 4 μg/mL CREVs. The absorbance of Alizarin red extracted with cetylpyridinium chloride solution (570 nm) is expressed as the mean ± SEM of 8 independent experiments. Statistical analyses were performed using the unpaired t-test.