Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair
Kai Hu, Bjorn R. Olsen
Kai Hu, Bjorn R. Olsen
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Research Article Bone biology

Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair

Abstract

Osteoblast-derived VEGF is important for bone development and postnatal bone homeostasis. Previous studies have demonstrated that VEGF affects bone repair and regeneration; however, the cellular mechanisms by which it works are not fully understood. In this study, we investigated the functions of osteoblast-derived VEGF in healing of a bone defect. The results indicate that osteoblast-derived VEGF plays critical roles at several stages in the repair process. Using transgenic mice with osteoblast-specific deletion of Vegfa, we demonstrated that VEGF promoted macrophage recruitment and angiogenic responses in the inflammation phase, and optimal levels of VEGF were required for coupling of angiogenesis and osteogenesis in areas where repair occurs by intramembranous ossification. VEGF likely functions as a paracrine factor in this process because deletion of Vegfr2 in osteoblastic lineage cells enhanced osteoblastic maturation and mineralization. Furthermore, osteoblast- and hypertrophic chondrocyte–derived VEGF stimulated recruitment of blood vessels and osteoclasts and promoted cartilage resorption at the repair site during the periosteal endochondral ossification stage. Finally, osteoblast-derived VEGF stimulated osteoclast formation in the final remodeling phase of the repair process. These findings provide a basis for clinical strategies to improve bone regeneration and treat defects in bone healing.

Authors

Kai Hu, Bjorn R. Olsen

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Figure 9

Deletion of Vegfr2 in osteoblastic cells increases their maturation and mineralization during bone repair at PSD7.

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Deletion of Vegfr2 in osteoblastic cells increases their maturation and ...
(A) 3D reconstruction of injured tibiae (top panel; scale bar: 500 μm) and mineralized bone in hole region (lower panel; scale bar: 100 μm). Representative images of 6–7 mice for each genotype. BVs in hole of Flk1fl/fl (0.0120 ± 0.0024 mm3) and Flk1fl/+ Osx-Cre mice (0.0082 ± 0.0016 mm3) not statistically different, but increased (0.0330 ± 0.0077 mm3) in Flk1fl/fl Osx-Cre mice; P < 0.05 vs. Flk1fl/fl. Tb.N (Tb.N/mm) increased (13.36/mm ± 1.32/mm) and trabecular spacing (Tb.Sp) decreased (0.079 ± 0.011 mm), in Flk1fl/fl Osx-Cre compared with Flk1fl/fl mice (7.08/mm ± 0.77/mm and 0.160 ± 0.023 mm); P < 0.01 (Tb.N), P < 0.05 (Tb.Sp). (B) BV/TV of mineralized bone formed in hole and wounded marrow of control and Vegfr2-deficient mice. (C) Reduced aniline blue staining in hole region (yellow rectangles) of Flk1fl/+ Osx-Cre (21.86% ± 1.53%) compared with Flk1fl/fl (36.70% ± 5.47%) mice; P < 0.05, but increased in Flk1fl/fl Osx-Cre (37.75% ± 10.99%) compared with Flk1fl/+ Osx-Cre mice; P < 0.05. Right: Increased mineralization/collagen ratio in Flk1fl/fl Osx-Cre mice. (D and E) Increased anti-OCN (2.8% ± 0.6%) and anti-ALP staining (20.0% ± 3.4%) in hole region of Flk1fl/fl Osx-Cre compared with Flk1fl/fl mice (0.6% ± 0.4% and 6.7% ± 1.8%); P<0.05 (OCN) and P < 0.01 (ALP). (F) Similar density of ZsG+ cells in hole region of Osx-Cre/ZsG, Flk1fl/+ Osx-Cre/ZsG, and Flk1fl/fl Osx-Cre/ZsG mice. (G) Von Kossa staining of osteoblasts cultured for 21 days in mineralization medium with or without BMP2. (H) Von Kossa staining of MC3T3-1E cells cultured for 21 days in mineralization medium containing 100 μM VEGFR2 kinase inhibitor or vehicle (Veh). Data are representative of 3 independent experiments (G and H). Scale bars: 200 μm (C), 50 μm (D–F); n= 6–7 (B–E); n= 4–6 (F). ANOVA with Tukey’s post-hoc test (A–C and F) and unpaired 2-tailed Student’s t test (D and E) were used. *, #P < 0.01; ##P < 0.05. *, vs. Flk1fl/fl. #,##vs. Flk1fl/+ Osx-Cre.