Synergistic enhancement of bone formation and healing by stem cell-expressed VEGF and bone morphogenetic protein-4

Abstract

We investigated the interaction between angiogenic and osteogenic factors in bone formation and bone healing with ex vivo gene therapy using muscle-derived stem cells genetically engineered to express human bone morphogenetic protein-4 (BMP4), VEGF, or VEGF-specific antagonist (soluble Flt1). Our results show that although VEGF alone did not improve bone regeneration, it acted synergistically with BMP4 to increase recruitment of mesenchymal stem cells, to enhance cell survival, and to augment cartilage formation in the early stages of endochondral bone formation. These early effects, coupled with accelerated cartilage resorption, eventually led to a significant enhancement of bone formation and bone healing. The beneficial effect of VEGF on bone healing elicited by BMP4 depends critically on the ratio of VEGF to BMP4, with an improper ratio leading to detrimental effects on bone healing. Finally, we show that soluble Flt1 inhibits bone formation elicited by BMP4. Thus, VEGF plays an important role in bone formation elicited by BMP4, and it can significantly enhance BMP4-elicited bone formation and regeneration through multiple mechanisms. This study has important implications for the formulation of new strategies to improve bone healing through increasing mesenchymal stem cell recruitment and survival, in combination with muscle-derived stem cell-based gene therapy.

Figure 1

VEGF enhances endochondral bone formation elicited by MDSCs expressing BMP4. (a) Radiograph shows augmented bone formation in the BMP4+VEGF (B4+Ve) site compared with the BMP4 (B4) site 4 weeks PI. (b) Quantitative analysis shows that the BMP4+VEGF group produced significantly more bone than the BMP4 group (*P < 0.01, n = 4). (c–f) Alcian Blue staining shows enhanced cartilage formation (purple) at 7 days and accelerated cartilage resorption at 10 days in the BMP4+VEGF group compared with the BMP4 group. (g–j) von Kossa staining shows more extensive cartilage mineralization (black) at 10 days and increased mineralized bone formation (black) at 14 days in the BMP4+VEGF group compared with the BMP4 group. Magnification: c, d, and g–j, ×100; e and f, ×200.

Figure 2

VEGF enhances angiogenesis during bone formation elicited by MDSCs expressing BMP4. (a and b) CD31 immune staining shows more active capillaries (arrows) branching from the surrounding muscle and growing toward the scaffolds (S) in the BMP4+VEGF group than in the BMP4 group at 4 days PI. (c and d) An extensive network of capillaries (arrows) formed inside the scaffolds of the BMP4+VEGF group, while only a modest level of capillaries formed in the scaffolds of the BMP4 group, at 10 days. (e) Histomorphometry demonstrates that the relative capillary density was significantly higher in the BMP4+VEGF group than in the BMP4 group (*P < 0.01) at 10 days. Magnification: a and b, ×400; c and d, ×100.

Figure 3

VEGF enhances healing of critical-sized calvarial defects elicited by MDSCs expressing BMP4. (a–c) Radiography shows complete bone healing of the defects in both the BMP4 and the BMP4+VEGF groups and no bone healing in the LacZ+VEGF group at 3 weeks PI. Arrows mark the edge of defects or newly formed bone. (d and e) Quantitative analysis shows a significant enhancement of bone regeneration in the BMP4+VEGF groups relative to the BMP4 group (*P < 0.05, n = 4). (f–h) von Kossa staining reveals coarser mineralized bone trabeculae in the BMP4+VEGF group than in the BMP4 group. No mineralized bone formed in the defect of the LacZ+VEGF group. Magnification: f–h, ×40.

Figure 4

Dose response in bone healing induced by BMP4+VEGF-expressing MDSCs. (a–c) Radiography shows complete bone regeneration induced by two different doses of BMP4+VEGF-expressing cells (B4+Ve). No bone formation occurred in the defects implanted with LacZ+VEGF-expressing cells (Lac+Ve). The ratio of BMP4- to VEGF-expressing cells was kept at 5:1. Arrows mark the edge of the defects or the regenerated bone. (d and e) Quantitative analysis shows the correlation between the amount of bone regenerated and the quantity of transduced cells implanted (*P < 0.05, n = 4).

Figure 5

Critical-sized calvarial defects healed through the endochondral ossification pathway in both BMP4 and BMP4+VEGF groups. (a–h) Alcian Blue staining shows cartilage formation in the defects implanted with transduced MDSCs. The edges of the bone defects are marked by arrowheads. Mesenchymal cell infiltration was more abundant in the BMP4+VEGF group (b and d) than in the BMP4 group (a and c) at 7 days PI. c and d display local magnifications of a and b, respectively. At 10 days, cartilage bridging the defects was mostly resorbed, leaving traces of hypertrophic chondrocytes (e–h). g and h display local magnifications of e and f, respectively. (i–l) von Kossa staining demonstrates coupled bone mineralization at 10 days and increased mineralized bone formation at 14 days in the BMP4+VEGF group (j and l) compared with the BMP4 group (i and k). Magnification: a, b, e, f, and i–l, ×40; c, d, g, and h, ×200.

Figure 6

VEGF increases cell survival in the bone regeneration site of critical-sized calvarial defects. TUNEL assay shows a reduction in apoptotic cells (with brown-stained nuclei as indicated by arrows) in the BMP4+VEGF group compared with the BMP4 group at 7 days (a–d) and, more strikingly, at 10 days PI (e–h). c, d, g, and h display higher magnifications of a, b, e, and f, respectively, at the area indicated by arrowheads. Magnification: a, b, e, and f, ×200; c, d, g, and h, ×1,200.

Figure 7

The proper ratio of VEGF to BMP4 is critical to ensure a beneficial effect on bone healing. In groups 1–3, the ratios of VEGF- to BMP4-expressing cells were 0.2, 1, and 5, respectively; in group 4, only VEGF-expressing cells were used. Arrows mark the edge of the bone defects and regenerated bone. (a–d) Radiography shows complete bone healing in groups 1 and 2, partial bone healing in group 3, and no bone healing in group 4. (e–h) von Kossa staining shows regeneration of cortical bone–like structure (arrow) in group 1 (e) but not in other groups, although bone union also occurred in group 2 (f). Bone regeneration was significantly reduced in group 3 as compared with groups 1 and 2 (e–g). Only fibrous tissue fills the defect in group 4 (h). Arrowheads mark the edge of the critical-sized bone defects and the regenerated bone. Magnification: ×40. (i and j) Quantitative analysis shows a significant decrease in bone regeneration in group 3 as compared with groups 1 and 2, while no bone formation was detected in the group containing only VEGF-expressing cells. Group 1 vs. group 2, P > 0.05, n = 4; group 1 vs. group 3, P < 0.01, n = 4; *P < 0.01 when compared to group 1.

Figure 8

VEGF antagonist inhibits bone formation elicited by MDSCs expressing BMP4. (a) Radiography shows decreased bone formation in the BMP4+sFlt1 (B4+sFlt) site compared with the BMP4 (B4) site 4 weeks PI. (b) Quantitative analysis shows a significant difference in relative bone area between the BMP4+sFlt and BMP4 groups (*P < 0.05). (c–h) Alcian Blue staining shows reduced cartilage formation at 7 days and delayed cartilage resorption at 10 and 14 days in the BMP4+sFlt group compared with the BMP4 group. Magnification: ×200.