CELL-BASED GENE THERAPY TO PROMOTE FRACTURE HEALING

Abstract

The purpose of this study was to develop a cell-based VEGF gene therapy in order to accelerate fracture healing and investigate the effect of VEGF on bone repair in vivo.

Twenty-one rabbits were studied. A ten millimeter segmental bone defect was created after twelve millimeter periosteal excision in the middle one third of each tibia and each tibia was plated. Primary cultured rabbit fibroblasts were transfected by use of SuperFect (Qiagen Inc) with pcDNA-VEGF. 5.0 X 106 cells in 1ml PBS were delivered via impregnated gelfoam into the fracture site. Experimental groups were:

1. Transfected fibroblasts with VEGF (n=7),

2. Fibroblasts alone (n=7), and

3. PBS only (n=7). The animals were sacrificed and fracture healing specimens collected at ten weeks post surgery

Radiology: Fracture healing was defined as those with bone bridging of the fracture defect. After ten weeks, fourteen tibial fractures were healed in total including six in group one, four in group two and four in group three. The VEGF group had an earlier initial sufficient volume of bridging new bone formation. Histological evaluation demonstrated ossification across the entire defect in response to the VEGF gene therapy, whereas the defects were predominantly fibrotic and sparsely ossified in groups two and three. Numerous positively stained (CD31) vessels were shown in the VEGF group. MicroCT evaluation showed complete bridging for the VEGF group, but incomplete healing for groups two and three. Micro-CT evaluation of the new bone structural parameters showed that the amount of new bone (volume of bone (VolB) x bone mineral density (BMD)), bone volume fractions (BVF), bone volume/tissues (BV/TV), trabecular thickness (Tb.Th), number (Tb.N) and connectivity density (Euler number) were higher; while structure model index (SMI), bone surface/bone volume (BS/BV), and trabecular separations (Tb.Sp) were lower in the VEGF group than the other groups. P-Values < 0.05 indicated statistical significance (ANOVA, SPSS) in all parameters except for SMI (0.089) and VolBx-BMD (0.197).

These results indicate that cell-based VEGF gene delivery has significant osteogenic and angiogenic effects and demonstrates the ability of cell based VEGF gene therapy to enhance healing of a critical sized defect in a long bone in rabbits.