Synergistic enhancement of bone formation and healing by stem cell–expressed VEGF and bone morphogenetic protein-4 (original) (raw)
Expression of BMP4 or VEGF from transduced MDSCs. MDSCs were able to synthesize, process, and secrete active human BMP4 following transduction with retroBMP4. The level of BMP4 reached 115 ± 20 ng/106 cells/24 hours at 1 week after transduction and persisted for at least 4 weeks in vitro. The secreted BMP4 was biologically active as evidenced by its ability to stimulate alkaline phosphatase activity in C2C12 cells (data not shown). MDSCs transduced with retroVEGF secreted VEGF at an average level of 214 ± 35 ng/106 cells/24 hours over a 4-week period, whereas cells transduced with retroLacZ did not secrete a detectable level of VEGF (data not shown). RetrosFlt-transduced MDSCs secreted sFlt at a level of 20 ± 5 ng/106 cells/24 hours.
VEGF enhanced bone formation elicited by MDSCs expressing BMP4. To determine the effects that supplying extra VEGF would have on BMP4-elicited bone formation, we compared bone formation elicited by BMP4-expressing cells (BMP4) with that elicited by a mixture of BMP4- and VEGF-expressing cells (BMP4+VEGF), with the same number of cells being implanted in the same animals to rule out variations. We chose a ratio of 5:1 (BMP4-/VEGF-expressing cells) based on the rationale that a high dose of VEGF might lead to detrimental effects due to the formation of hemangioma, as previously described (18–20). Radiography showed that the BMP4+VEGF produced significantly more bone than the BMP4 did at 4 weeks PI (Figure 1, a and b). These results were confirmed by biochemical analysis of calcium content and alkaline phosphatase activity of the newly formed bone (not shown).
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.
VEGF enhanced cartilage formation and accelerated cartilage resorption in endochondral bone formation induced by BMP4. Histologic analysis of the stages of bone formation showed increased cartilage formation in the BMP4+VEGF group compared with the BMP4 group at 7 days PI (Figure 1, c and d), with a significant difference in the relative cartilage areas (399 ± 65 vs. 186 ± 29, P = 0.01, n = 4). Interestingly, cartilage resorption was accelerated in the BMP4+VEGF group, leading to more extensive mineralization than in the BMP4 group (Figure 1, e–h) at 10 days PI. At 14 days PI, trabecular bone formation was increased in the BMP4+VEGF group, compared with the BMP4 group (Figure 1, i and j). Notably, the bone formed in the BMP4+VEGF group consisted of denser and better-organized trabeculae, indicating bone of higher quality.
VEGF enhanced angiogenesis in bone formation induced by MDSCs expressing BMP4. Angiogenesis during bone formation induced by MDSCs expressing BMP4 with or without VEGF was investigated using immunohistochemistry for CD31, a specific tissue marker of endothelial cells (21). Angiogenesis was more active in the tissues of the BMP4+VEGF group than in those of the BMP4 group; there were significantly more capillaries branching from the surrounding muscle and growing toward the scaffolds at 4 days PI (Figure 2, a and b), as well as increased mesenchymal cell infiltration and enhanced capillary ingrowth at 7 days PI (not shown). By 10 days PI, a much more extensive capillary network was formed in the BMP+VEGF group than in the BMP4 group (Figure 2, c and d), with a fivefold increase in the relative capillary density (Figure 2e; P < 0.01). Angiogenesis in both groups subsided at 14 days PI (not shown).
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.
VEGF enhanced healing of skull defects induced by MDSCs expressing BMP4. To determine the effects of VEGF on BMP4-induced bone healing, critical-sized calvarial defects were treated with collagen disks impregnated with BMP4-expressing cells alone (6 × 105), both BMP4-expressing and VEGF-expressing cells (5 × 105:1 × 105), or LacZ-expressing and VEGF-expressing cells (5 × 105:1 × 105). The defects were completely healed at 3 weeks PI in all eight animals in the BMP4 and BMP4+VEGF groups; in contrast, no bone healing occurred in the LacZ+VEGF group (Figure 3, a–c). The bone formed in the BMP4+VEGF group was significantly larger and denser than the bone formed in the BMP4 group (Figure 3, d and e) at 3 weeks PI. A similar trend was observed at 6 weeks PI (not shown). These radiographic results were confirmed by histologic analysis, which showed complete bone union of the defects in the BMP4 and BMP4+VEGF groups, with no bone healing in the LacZ+VEGF group (Figure 3, f–h).
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.
We next explored whether it is possible to heal the critical-sized calvarial defects with a reduced number of transduced MDSCs. As shown in Figure 4, complete bone healing was elicited even with a fourfold reduction in the number of transduced cells. Furthermore, the amount of bone formed correlated positively with the number of cells implanted (Figure 4, d and e). It also was noted that the degree of bone healing elicited with 1.5 × 105 BMP4+VEGF-expressing cells was comparable to that elicited by a fourfold greater number of cells expressing BMP4 alone (compare Figure 4b with Figure 3a).
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).
Healing of critical-sized calvarial defects elicited by BMP4 or BMP4+VEGF was mediated through endochondral ossification pathway. Calvarial bone is developed through the intramembranous pathway; non–critical-sized calvarial defects are normally healed through the same pathway. Surprisingly, however, the healing of critical-sized calvarial defects elicited by both BMP4- and BMP4+VEGF-expressing MDSCs was mediated primarily through the endochondral bone formation pathway (Figure 5, a–l), a process mimicking that of ectopic bone formation in skeletal muscle described previously (Figure 1).
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.
VEGF increased cell recruitment and cell survival in bone regeneration elicited by MDSCs expressing BMP4. To explore mechanisms that may account for the increased cartilage formation induced by VEGF during endochondral bone formation elicited by MDSCs expressing BMP4, we assessed the relative number of cells recruited to the bone healing site 7 days PI, when the distribution of the cells in the bone regeneration site is relatively even and cells can be counted accurately. A significant increase in mesenchymal cell recruitment was noted in the BMP4+VEGF group, as compared with the BMP4 group (Table 1). We then determined whether VEGF can enhance cell survival at the bone regeneration sites. Apoptosis assay showed that the number of apoptotic cells was reduced slightly at 7 days and significantly at 10 days PI in the BMP4+VEGF group compared with the BMP4 group (Figure 6, a–h; Table 1).
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.
VEGF enhances cell recruitment and cell survival in bone regeneration elicited by MDSCs expressing BMP4
Proper ratio of VEGF to BMP4 is critical to ensure synergistic effects in bone healing. To determine the optimal ratio of VEGF to BMP4 for improving the healing of critical-sized defects, such defects were implanted with a fixed number of BMP4-expressing cells (1 × 105) supplemented with variable numbers of VEGF-expressing cells (2 × 104, 1 × 105, and 5 × 105 in groups 1, 2, and 3, respectively). The ratios of VEGF to BMP4 in these three groups were thus 0.2, 1, and 5, respectively. The total cell number in each group was brought to 6 × 105 by adding LacZ-expressing cells to rule out variation secondary to cell number differences. Group 4 was implanted with VEGF-expressing cells only (5 × 105). Three weeks PI, the bone defects were healed in all mice of groups 1 and 2, partially healed in group 3 mice, and unhealed in group 4 mice (Figure 7, a–d, i, and j). These radiographic results were confirmed by histologic analysis (Figure 7, e–h), which showed complete bone union in groups 1 and 2, with the quality of healing being superior in group 1 due to better bony bridging of the defect. In contrast, only small patches of mineralized bone formed in group 3. The rest of the tissue filling the defects consisted primarily of soft tissue rich in capillaries (not shown). There was no mineralized bone formation in the control group implanted with only a high dose of VEGF-expressing cells. Thus, neither high nor low doses of VEGF alone improved healing of critical-sized bone defects.
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.
VEGF antagonist inhibited bone formation elicited by MDSCs expressing BMP4. To determine whether intrinsic VEGF activity is important for bone formation elicited by MDSCs expressing BMP4, we examined the effect of a VEGF-specific antagonist (sFlt1). Bone formation was significantly inhibited in all five animals at the sites implanted with 1 × 105 MDSCs expres-sing BMP4 and 2 × 105 MDSCs expressing sFlt1 (BMP4+sFlt), compared with the sites implanted with the same number of MDSCs expressing BMP4 (BMP4) plus 2 × 105 MDSCs expressing LacZ (Figure 8, a and b). Histologic analysis revealed that sFlt1 reduced cartilage formation and delayed cartilage resorption during endochondral bone formation elicited by MDSCs expressing BMP4 (Figure 8, c–h).
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.