| Objective To observe in vitro cultured goat bone marrow mesenchmal stem cells (BMSCs) and bone morphogenetic protein-9 (BMP-9) gene transfection goat BMSCs and planted on the absorbable gelatin sponge, then formed the engineering bone in vitro. To understand their biological properties. To explore the effect in vivo when the complexes were implanted in the goat bone defect model.Methods Culture the goat BMSCs and transfected by Adenovirus-BMP-9 gene in vitro. Changes in cell morphology were observed using an inverted microscope. Cell growth curves were measured using MTT assay. The efficiency of transfection was observed using a fluorescent microscope. Analyse the ALP activity and the changing of Von Kossa's calcium nodus. Co-culture of the transfected and untransfected BMSCs with absorbable gelatin sponge. They were transplanted in the 4 bone defected goat after a short time culture in vitro. The bone formation was observed on 4th week and 12th weeks using X-rays and HE staining.Results Goat BMSCs transfected and untransfected by Ad-BMP-9 were detected slight difference in cell morphology. Growth cycles were no significant difference in transfected and untransfected BMSCs. BMSCs entered lag phase at days 2 and 3, logarithmic phase at day 3, and platform phase at days 6 and 7, and then growth speed was slow. The efficiency of transfected goat BMSCs was 80%. The ALP Reading was much higher in transfected group than the control group (p<0.05). The Von Kossa's calcium nodus was obviously larger in transfected group than the control group. The observing of fluorescent microscope showed the transfected group growed well. On the 4th week, the X-ray indicated that the experiment group showed the sign of bone growth, while the control group showed nothing.On the 12th week, the sign of experiment group were large while the control group become angmetation. The HE staining showed the same results: there was much more new bone formed in the experiment group.Conclusion Culturing the BMSCs like goats was much difficult than the small animals. Cultured goat BMSCs showed strong genetic stability and proliferation ability using the whole bone marrow method. BMP-9 could promote the osteogenic differentiation. The transfered cells growed well on absorbable gelatin sponge. These results shows BMSCs transfected BMP-9 combined with absorbable gelatin sponge could be suitable for bone tissue engineering seed cells. |
