Figure 5.

CGM‐EVs and Akk‐EVs can be transported to the mouse bone tissues and directly promote osteogenesis and inhibit osteoclastogenesis in vitro. A) Morphological analysis of CGM1‐EVs, EGM1‐EVs, and Akk‐EVs by transmission electron microscopy. Scale bar: 50 nm. B) Particle size distribution of different EVs measured by DLS. C) Ex vivo fluorescent imaging of the femur and tibia from mice treated with vehicle or the DIR‐labeled EVs for 1 h and D) quantification of the fluorescent signals. PO: per os; PR: per rectal; IV: intravenous. Scale bar: 6 mm. n = 3 per group. E) Confocal microscopy analysis of the femoral sections from mice treated with the PKH67‐labeled EVs for 1 h by oral route. CB: cortical bone; TB: trabecular bone; BM: bone marrow. Scale bar: 20 µm. F) Quantification of the fluorescent signals in (E). n = 3 per group. G) Representative images of the Akk‐EVs antibody (Ab)‐stained femoral sections with quantification of the H) positive signals. Scale bar: 10 µm. n = 3 per group. I) Representative ARS staining images of BMSCs receiving different treatments and J) quantitation of the percentage of ARS‐positive areas per well in a 48‐well plate. Scale bar: 100 µm. n = 3 per group. K) Representative TRAP staining images of RAW264.7 cells receiving different treatments and L) quantification of osteoclast number per well in a 48‐well plate. Scale bar: 100 µm. n = 3 per group. Data are presented as mean ± SD. For panels (D), (F), (J), and (L): Red dots indicate CGM1‐EVs or EGM1‐EVs, green dots indicate CGM2‐EVs or EGM2‐EVs, and blue dots indicate CGM3‐EVs or EGM3‐EVs. For panels (D) and (F): * P < 0.05 versus Vehicle group. For panel (H): * P < 0.05 versus Vehicle + Akk‐EVs Ab group and ^# P < 0.05 versus Akk‐EVs + Akk‐EVs Ab group. For panels (J) and (L): * P < 0.05 versus Vehicle group, ^# P < 0.05 versus CGM‐EVs group, ^▲ P < 0.05 versus EGM‐EVs group, and ^▼ P < 0.05 versus Akk‐EVs group. ^** P < 0.01 and ^{***/###/▲▲▲/▼▼▼} P < 0.001.