Vascularization Of Porous Silk Fibroin Scaffold With Loading Of VEGF165 And Ang-1 Mediated By RGD-modified Adenovirus

Improving the vascularization of artificial dermis is the key problem of enhancing the survival rate of transplanted scaffold, leading leather regeneration and repairing full-thickness skin defect. Vascular endothelial growth factor(VEGF165) and angiopoietin-1(Ang-1) are identified as two important angiogenesis growth factors. Combination of VEGF165 and Ang-1 can not only enhance angiogenesis but also obtain stable blood vessels. In this study, the transfection of VEGF165 and Ang-1 mediated by RGD-modified adenovirus to EA.hy926 cells was studied. Then VEGF165 and Ang-1 mediated by RGD-modified adenovirus was loaded into silk fibroin scaffold. EA.hy926 cells were seeded into the bioactive scaffold and the bioactive scaffold were used to study the promoting the formation of blood vessels of chicken embryo villus allantois membrane(CAM) in vitro. Later, we planted the silk fibroin scaffold loaded with Ad.RGD-VEGF165-Ang-1 in SD rats’ dorsal dermal defects. We studied the Ad.RGD-VEGF165-Ang-1 in scaffold tranfect the host cells and express target gene and its influence in vascularization and dermal regeneration in vivo.First, p Ad.VEGF165-Ang-1 transfer vectors and p Ad.RGD were used for homologous recombination to get homologous recombination plasmids p Ad.RGD-VEGF165-Ang-1. Then the homologous recombination plasmids were linearized and transfected into the human embryonic kidney(QBI-293A) cells by Lipofectamine2000, leading to formation of the recombinant adenoviruses Ad.RGD-VEGF165-Ang-1. Adenovirus vector Ad.RGD-VEGF165-Ang-1 with different multiphcity of infection(MOI=10, 20, 30, 40, 50, 100, 150) were used to transfect EA.hy926 cells. Transfection observed by confocal laser scan microscopy and transfection efficiency which was detected in the flow cytometry showed that the fluorescent expression was enhanced and the transfection efficiency was increased with the increasing of MOI. When the MOI was 50, the transfection efficiency was 81%.Second, Adenovirus vector Ad.RGD-VEGF165-Ang-1 with MOI=50 was used to transfect EA.hy926 cells. After incubation 1、3、5、7 d, the proliferation of EA.hy926 cells was detected by CCK-8. The protein expression of VEGF165 and Ang-1 was texted by enzyme 1inked immunosorbent assay(ELISA). The results showed that it was beneficial to promotes cell proliferation after transfected by adenovirus vector Ad.RGD-VEGF165-Ang-1. ELISA indicated that the concentration of VEGF165 and Ang-1 in transfected groups was significantly higher than non-transfected groups(p<0.05). And the the concentration of VEGF165 and Ang-1 was increasing gradually in 7 days. Ad.RGD-VEGF165-Ang-1 was loaded into silk fibroin scaffold. Virus particle was distributed homogeneously in the hole-wall of silk fibroin scaffold observed by scanning electron microscopy. EA.hy926 cells were eeded in the silk fibroin scaffold loaded with Ad.RGD-VEGF165-Ang-1. The cells can be transfected by adenoviruses in the silk fibroin scaffold. The result indicated that adenoviruses in the silk fibroin scaffolds had active. EA.hy926 cells can fully spread and proliferate in the surface of silk fibroin scaffold. The observed results of CAM indicated that silk fibroin scaffolds loaded with Ad.RGD-VEGF165-Ang-1 can promoted the formation of blood vessels.Further, we studied dermal reconstruction behavior in vivo by planting silk fibroin scaffold loaded with Ad.RGD-VEGF165-Ang-1 in SD rats’ dorsal dermal defects. Hyphology observation of HE and Masson indicated that silk fibroin scaffold loaded with Ad.RGD-VEGF165-Ang-1 can significantly promote the formation of blood vessels in scaffold, produce collagen fibers and dermal tissue regeneration than that of blank control. The analysis of immunohistochemistry showed that the expression of VEGF, Ang-1, CD31 and PDGF was significantly higher in groups of silk fibroin scaffold loaded with Ad.RGD-VEGF165-Ang-1 than that of the blank control and the peak of expression appeared on days 14. These results indicated the expression of target genes VEGF and Ang-1 increased neovascularization in scaffold. On the other hand, they could stimulate the expression of PDGF on wound, accelerating regeneration of dermal.