Background: Bone regeneration approaches that mimic the natural processes of bone repair have generated significant attention. We hypothesized that early delivery of an angiogenic factor combined with sustained exposure to an osteogenic factor would recapitulate the critical aspects of natural bone repair. Materials and methods: Basic fibroblast growth factor (bFGF) and sonic hedgehog (Shh) were constructed to the recombinant adeno-associated virus, respectively (rAAV2-tet-off-bFGF and rAAV2-Shh). The previous viral vector allowed for regulation of the bFGF expression by the addition of doxycycline, a tetracycline analogue. These two viral vectors were used to cotransduce bone marrow-derived mesenchymal stem cells (BMSCs). Several osteogenic markers such as core-binding factor a-1, alkaline phosphatase and osteocalcin were detected by quantitative real-time reverse transcriptase polymerase chain reaction. Meanwhile, protein expressions of transgenes were measured by western blot. Furthermore, these cotransduced BMSCs were seeded on beta-tricalcium phosphate (beta-TCP) granules and then were implanted into the calvarium defect in a rat model. A sample of 30 Sprague-Dawley rats was divided into six groups (n = 5); an 8-mm critical-sized bone defect was made in calvarium of all subjects. Each group was treated with various transgenic BMSCs and beta-TCP composites; and the sixth group is the negative control which was implanted with nothing. At 4 weeks after treatment, the samples were evaluated with histological staining. Results: The expression of osteogenic marker mRNA had an increased tendency after two genes transduction (p < 0.05). In addition, dramatically enhanced regeneration of critical-sized calvarial defects was observed in the groups which were implanted with two transgenic BMSCs and beta-TCP composites. And in these experimental groups, bone areas and vascular densities were increased significantly (p < 0.05) than other groups. Conclusion: Sequential delivery of angiogenic and osteogenic factors likely has a synergistic effect, mimicking the molecular events of natural bone regeneration. (C) 2011 Elsevier Ltd. All rights reserved.