PAPER 002: EFFECT OF CELL-BASED VEGF TRANSFER ON RABBIT OSTEOBLASTS IN VITRO

Abstract

Purpose: Vascular Endothelial Growth Factor (VEGF) is vital for both angiogenesis and osteogenesis. The aim of this study was to investigate the effect of cell based VEGF gene delivery on the proliferation and mineralization of rabbit osteoblasts in vitro.

Method: Primary cultured rabbit osteoblasts were divided into four groups (each n=6). In Group I, osteoblasts were transfected with pcDNA3.1-VEGF; in Group II, osteoblasts were transfected with pcDNA-Efficiency Green Fluorescent Protein (EGFP); in Group III, osteoblasts were treated with the supernatant of fibroblasts that were transfected with VEGF genes; and in Group IV, osteoblasts were treated with the supernatant of fibroblasts that were transfected with EGFP. The cells were cultured in a-EME with 10% FBS, 2% penicillin/streptomycin with or without 10-^7 M dexamethasone and 50μg/ml L-ascorbic acid for 28 days. In the last 4 days, the cells were stimulated to initiate calcium mineralized nodule formation by adding 10 mM B-glycerophosphate. They were stained by the Von Kossa technique so that the number and the area of the nodules could be assessed by an imaging analysis system.

Results: The cells transfected by VEGF were indicated by the EGFP marked cells under a fluorescent microscope. There was a significant difference in the total nodule area (mean 18.38 mm² SE 3.73 and 5.07 mm² SE 0.55, p< 0.05) and count (mean 18.67 SE 3.22 and 2.17 SE 0.40, p< 0.001) between Group I and Group II (ANOVA, SPSS). More unmineralized and smaller nodules were found in Group III and Group IV. However, the nodules in Group III covered greater areas with dark brown staining in the cell culture dishes when compared with Group IV.

Conclusion: The observations indicate that cell based VEGF gene delivery has a positive effect on the proliferation and mineralization of osteoblasts. The greatest effect is seen with direct transfection of osteoblast cells. Cell-based VEGF gene therapy may be used to promote fracture healing.