one morphogenetic protein (BMP) adenoviral vectors for the induction of osteogenesis are being developed for the treatment of bone pathology. However, it is still unknown which BMP adenoviral vector has the highest potential to stimulate bone formation in vivo. In this study, the osteogenic activities of recombinant human BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 adenoviruses were compared in vitro, in athymic nude rats, and in Sprague-Dawley rats. In vitro osteogenic activity was assessed by measuring the alkaline phosphatase activity in C2C12 cells transduced by the various BMP vectors. The alkaline phosphatase activity induced by 2 Â 10 5 PFU/well of BMP viral vector was 4890 Â 10 À12 U/well for ADCMVBMP-9, 302 Â 10 À12 U/well for ADCMVBMP-4, 220 Â 10 À12 U/well for ADCMVBMP-6, 45 Â 10 À12 U/well for ADCMVBMP-2, and 0.43 Â 10 À12 U/ well for ADCMVBMP-7. The average volume of new bone induced by 10 7 PFU of BMP vector in athymic nude rats was 0.3770.03 cm 3 for ADCMVBMP-2, 0.8970.07 cm 3 for ADCMVBMP-4, 1.0270.07 cm 3 for ADCMVBMP-6, 0.2470.05 cm 3 for ADCMVBMP-7, and 0.6370.07 cm 3 for ADCMVBMP-9. In immunocompetent Sprague-Dawley rats, no bone formation was demonstrated in the ADCMVBMP-2, ADCMVBMP-4, and ADCMVBMP-7 groups. ADCMVBMP-6 at a viral dose of 10 8 PFU induced 0.1070.03 cm 3 of new bone, whereas ADCMVBMP-9 at a lower viral dose of 10 7 PFU induced more bone, with an average volume of 0.2970.01 cm 3 .

This study was designed to see if immunosuppression achieved using local application of cyclosporine A (Cs. A) or CD4 and CD8 antibodies would improve bone formation following intramuscular injections of human BMP-4 and BMP-9 adenoviral vectors (ADhBMP4 and ADhBMP9) in Sprague-Dawley rats. Cs. A was injected into the thigh muscle. After 2 days, ADhBMP4, ADhBMP9, and the antibodies were separately injected into the left and right rear legs. At this time, the number of CD4+/CD3+ cells was significantly lower and the number of CD8+/CD3+ cells higher in the Cs. A group than in the control group (Po0.01). The total number of white blood cells 3 days following injection of CD4 and CD8 antibodies was significantly lower than that before the injection (Po0.01). At 4 weeks after the viral and antibody injections, mean bone volumes at the ADhBMP9 treatment sites were 0.2970.01 cm 3 in the viral control group, 0.1770.03 cm 3 in the Cs. A-ADhBMPs group, and 0.5970.07 cm 3 in the antibodies-ADhBMPs group. ADhBMP4 did not induce new bone formation in any group. This study demonstrates that local immunomodulation may improve the osteogenic potential of bone morphogenetic protein gene therapy in the clinical setting.