Enhanced Capillary Formation by Transplanted Ad-VEGF165 Transgenic Epithelial Cells on Silk Fibroin Films

Vascular endothelial growth factors (VEGF) regulate multiple physiological and pathological processes including angiogenesis, i.e. growth of new blood vessels, a hallmark of wound healing. To improve VEGF blood life-time for enhanced angiogenesis in clinical applications, we investigate the possibility of cell transplant therapy using implanting silk protein film substrates loaded with VEGF-transfected THCE cells into a rabbit corneal model. Significantly enhanced corneal neovascularization is observed. THCE cells were cultivated on silk protein films (SF) and transfected with the Ad-VEGF₁₆₅ for 48 hours. In vitro ELISA testing showed that Ad-VEGF₁₆₅ transfected THCE cells expressed significantly higher levels of VEGF then self-secreted VEGF level in untransfected THCE cells. Interestingly, Ang1 and EGF levels were also higher in VEGF-transfected cells. After the transfection, the SF materials (4 mm × 3 mm) were implanted into corneal stromal. Images of the corneal vasculature showed significant angiogenesis induced by VEGF-transfected THCE cells. One month after the implantation, corneal neovasculars extended to the margin of the pupil, with bifurcation and loops formation and the mean area of neovascularization was drastically increased. Immunostaining specimens all showed positive staining for CD34 on corneal stromal. It is concluded that cornea neovascularization was successfully induced by implanting SF/THCE-Ad- VEGF₁₆₅ in rabbit corneal stroma. The regenerated silk fibroin films had good biological compatibility with THCE. Not only the expression level of the target VEGF gene was elevated, but also related genes such as Ang1 and EGF were also activated. These results indicate that regenerated silk fibroin films are a practical biomaterial platform that, once modified with transgenic seed cells, can provide multiple functions such as induced angiogenesis and enhanced tissue wound healing.