Bone morphogenetic protein-9 is one of potential growth factor of bone morphogenetic protein family that can promote bone differentiation. Presently, there are few studies addressing bone morphogenetic protein-9 in osteogenic aspects.OBJECTIVE:
To investigate the possibility of adipose-derived stem cells (ADSCs) transfected by adenovirus-mediated bone morphogenetic protein-9 in rabbits, and the osteogenesis following transfection.DESIGN, TIME AND SETTING:
The cytogene in vitro study was performed at the Laboratory of Institute of Pediatrics, Children's Hospital, Chongqing Medical University from August 2007 to June 2008.MATERIALS:
A total of 5 New Zealand rabbits were supplied by Experimental Animal Center, Chongqing Medical University. Adenovirus vectors carrying human total length bone morphogenetic protein-9 was constructed and presented by Luo Qing, an investigator, from Children's Hospital, Chongqing Medical University.METHODS:
ADSCs were obtained from trachel-back fat tissue of rabbits, isolated and purified in vitro. ADSCs at passage 3 were incubated in a 6-well plate at a density of 1×108/L. ADSCs were transfected with adenovirus carrying human bone morphogenetic protein-9 (multiplicity of infection=100) in the transfection group. A blank control group was set.MAIN OUTCOME MEASURES:
Changes in cell morphology and growth curve were measured. Human bone morphogenetic protein-9 mRNA expression was detected by using fluorescence microscope and RT-PCR following transfection. Osteogenic activity was determined by alkaline phosphatase and calcium-alizarin red staining.RESULTS:
Cell confluency showed slabstone shape at 1 week, and calcified nodule formed at 2 weeks following transfection. No significant difference was detected in cell morphology or calcified nodule in the blank control group. Compared with the blank control group, cell stationary phase prolonged, number was slightly decreased, and doubling time prolonged in the transfection group. Fluorescence expression of human bone morphogenetic protein-9 was found at 12 hours following transfection. Human bone morphogenetic protein-9 mRNA expression showed positive reaction and became increased at 3, 6, 9, 12 days following transfection, but negative reaction in the blank control group. The activity of alkaline phosphatase had an increased tendency with prolonged transfection time. Calcium-alizarin red staining showed calcified nodule. Cells in the blank control group were weakly positive for alkaline phosphatase and calcium-alizarin red staining.CONCLUSION:
Adenovirus carrying human bone morphogenetic protein-9 can successfully transfect rabbit ADSCs. Following transfection, ADSCs highly express human bone morphogenetic protein-9, and have evidence osteogenesis.