Figure 5. While 0.1 μg VEGF enhances, 1 μg recombinant VEGF fails to affect intramembranous bone formation in cortical defects of Vegfa^fl/fl Osx-Cre/ZsG mice at PSD10.

(A) 3D reconstruction of mineralized bone in hole region of Osx-Cre/ZsG mice treated with PBS or 0.1 μg VEGF and Vegfa^fl/fl Osx-Cre/ZsG mice treated with PBS, 0.1 μg or 1 μg VEGF. (B and C) BV/TV, based on μCT of bone formed in hole region and wounded BM (red area in diagram). (D) Density of aniline blue staining and mineralization/collagen ratio in the hole region (yellow stippled rectangles) of Vegfa^fl/fl Osx-Cre/ZsG mice treated with PBS (6.3% ± 1.8% and 47.2% ± 9.0%, respectively) significantly reduced (P < 0.01) compared with Osx-Cre/ZsG mice treated with PBS (24.2% ± 3.2% and 88.5% ± 7.2%, respectively). Administration of 0.1 μg VEGF increases density of aniline blue staining in hole region of Vegfa^fl/fl Osx-Cre/ZsG mice (18.6% ± 3.5%; P < 0.05 vs. Vegfa^fl/fl Osx-Cre/ZsG + PBS), but fails to significantly enhance mineralization/collagen ratio (54.5% ± 10.8% when compared with Vegfa^fl/fl Osx-Cre/ZsG mice treated with PBS). Compared with PBS, 1 μg VEGF has little effect on aniline blue staining and mineralization/collagen ratio (8.2% ± 1.6% and 41.6% ± 9.5%) in Vegfa^fl/fl Osx-Cre/ZsG mice. (E) VEGF (0.1 μg) enhances anti-BSP staining with or without normalization to ZsG⁺ cells in hole region of Vegfa^fl/fl Osx-Cre/ZsG mice (8.9% ± 2.3% and 7.3% ± 1.4%) compared with Vegfa^fl/fl Osx-Cre/ZsG mice treated with PBS (1.8% ± 0.6% and 1.2% ± 0.4%); P < 0.01 with and P < 0.05 without normalization. VEGF (0.1 μg) has no significant effect (1.9% ± 0.9% and 1.0% ± 0.4%). (F) Density of ZsG⁺ cells and total cell number in hole region of Vegfa^fl/fl Osx-Cre/ZsG (CKO) mice not significantly altered by VEGF treatment. Scale bars: 100 μm (A), 200 μm (D), 50 μm (E); n= 5–6 mice for each group. Unpaired 2-tailed Student’s t test used. *P < 0.01; **,^##P < 0.05. *,** vs. Osx-Cre/ZsG + PBS. ^## vs. Vegfa^fl/fl Osx-Cre/ZsG + PBS.