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Bone marrow mesenchymal stem cells (BMSCs) are considered the most important seed cells in bone tissue engineering. The present study aimed to investigate the potential of rabbit BMSCs in osteogenesis after the transfection of human BMP-2 and EGFP recombinant adenovirus. Rabbit BMSCs were isolated and the surface stem cell makers, including CD29, CD44 and CD45 were detected by flow cytometry. The expression of BMP-2 mRNA and protein in BMSCs were detected by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. After an induction with osteogenic medium, the alkaline phosphatase (ALK) activity at 7 days, the type I collagen at 14 days, and the calcium nodules at 21 days were performed using an ALK activity kit, immunohistochemical staining and alizarin red S staining, respectively. The expression levels of proteins related to the Wnt signaling pathway were detected by western blot analysis. The positive rates of CD29, CD44 and CD45 were 62.92±1.99, 93.55±0.99 and 0.21±0.12%. The expression of BMP-2 mRNA and protein was significantly upregulated in Ad-BMP-2/EGFP transfected BMSCs. Furthermore, Ad-BMP-2/EGFP induced ALP activity, promoted the production of type I collagen and calcium nodule formation in rabbit BMSCs. The levels of β-catenin, cyclin D1, Runx2 and c-myc were upregulated in Ad-hBMP-2/EGFP transfected BMSCs, while the level of GSK3β was significantly decreased. Results also indicated that the overexpression of BMP-2 by Ad-hBMP-2/EGFP enhanced the osteogenic differentiation ability of cultured rabbit BMSCs via stimulating the Wnt signaling pathway with the accumulation of β-catenin and suppression of GSK3β. The Ad-hBMP-2/EGFP transfected rabbit BMSCs are expected to be a good seed cell in bone tissue engineering. [ABSTRACT FROM AUTHOR]