Effects of vascular endothelial growth factor 165 on bone tissue engineering

Abstract

To study the relationship between vascular endothelial growth factor (VEGF) and formation and repair of engineering bone, second-generation bone marrow stromal cells (BMSCs) of New Zealand white rabbits that were separated in vitro were transfected with VEGF 165 gene vectors by adenovirus to detect gene expressions. Transfected BMSCs and β-tricalcium phosphate material were complexed and implanted at the femoral injury sites of the study group (n = 12), and the control group (n = 12) were implanted with engineering bones that were not transfected with VEGF. Femoral recoveries of the two groups were observed on the 15th, 30th, 45th and 60th days, and their vascularization and ossification statuses were observed by immunohistochemical methods. The BMSCs transfected with VEGF highly expressed VEGF genes and excreted VEGF. The two groups both experienced increased vascularization and bone volume after implantation (t = 7.92, P<0.05), and the increases of the study group were significantly higher than those of the control group (t = 6.92, P<0.05). VEGF is clinically applicable because it can accelerate the formation and repair of engineering bone by promoting vascularization and ossification.

Figure 1. Design of experimental procedure.

Figure 2. Vascular casting method.

(A) BMSCs, ×200; (B) tricalcium phosphate material; (C) tricalcium phosphate under electron microscope, ×100; (D) tricalcium phosphate under electron microscope, ×40; (E) tricalcium phosphate-complexed BMSCs under electron microscope, ×100; (F) postoperative healing.

Figure 3. VEGF 165 gene expression after transfection.

(A) Positive expression of VEGF in study group (10×10); (B) negative expression of VEGF in control group (10×10).

Figure 4. Relative area ratios of new bones (%).

Figure 5. Ossification results on the 30th, 60th and 90th days (×100).

Left: study group; right: control group. NB: new bone; BM: bone marrow; Arrowheads indicate blood vessels, thick arrows indicate osteoblasts, and thin arrows indicate osteocyte in bone lacuna.

Figure 6. Real-time quantitative PCR and RT-PCR results.

Type I collagen (A) and ALP (B) levels of the BMSCs in the two groups detected on the 14th day using real-time quantitative PCR (n = 6, mean ± SD), *P<0.05; (C) type I collagen, ALP and OCN mRNA levels of the BMSCs in both groups detected on the 14th day using RT-PCR.

Figure 7. Numbers of microvessels at each time point.

Figure 8. Cross sections of undecalcified ground bones 60 days after vascular casting.

Blank arrows indicate cross sections of the blood vessels filled with casting agents. (A) Study group; (B) control group.

Figure 9. CD31 immunohistochemical staining results on the 45th and 90th days.

Upper two: study group; lower two: control group.