Young and aged mice underwent tibial fracture surgery. (A) The expression of Tgfb1 in fracture callus at indicated time points was measured by qPCR. n = 3. Relative mRNA expression is the fold-change versus young mice as 1. (B) The concentration of active TGF-β1 protein in fracture callus at indicated time points was measured by ELISA. n = 4. *P < 0.05, for aged versus young; ^#P < 0.05, for young versus young 0 dpf; ^P < 0.05, for aged versus aged 0 dpf, by 2-way ANOVA followed by Tukey’s post hoc test (A and B). (C) Outline of the experimental design. Aged mice were given 2 μg in 10 μL TGF-β Ab, 1D11, or isotype IgG vehicle by intra-callus injection on 1, 3, 5, and 7 dpf and sacrificed on 10 dpf (D–F and H–J) or 28 dpf (G). n = 4–5. (D) Callus volume was measured by micro-CT. *P < 0.05, by unpaired, 2-tailed Student’s t test. (E) Representative images of ABH-stained sections showing more woven bone and callus areas in the anti–TGF-β Ab–treated mice. Scale bar: 1 mm. (F) Woven bone and cartilage areas were analyzed using Visiopharm software. (G) Bone stiffness, strength and toughness were assessed by biomechanical testing at 28 dpf. (H) The percentage and number of MPCs identified as CD45^–CD31^–CD105⁺ cells in fracture callus were determined by flow cytometry. (I) Representative paraffin sections of callus immunostained with anti-Ki67 Ab to detect proliferating cells (arrowheads). External callus is indicated by the dashed lines. Scale bars: 500 μm. Original magnification, ×4 (enlarged insets). (J) The percentage of Ki67⁺ cells was quantified by ImageJ. *P < 0.05, by unpaired, 2-tailed Student’s t test (F, G, H, and J).