Osteogenic Effects of Bone Morphogenetic Protein-2 Plasmid Gene Transfer

Objective: The aim of this study is to determine the osteogenic effects of BMP-2 gene transfer in bone marrow stromal cells (BMSCs) and rabbit calvarial defects. Methods: BMP-2/pcDNA3.1 plasmid was constructed by subcloning human BMP-2 cDNA into the pcDNA3.1. BMSCs were transfected with the BMP-2/pcDNA3.1. Transfected cells were observed a localization of BMP-2 coding plasmid. And, the level of BMP-2 in the culture media of tranafected cells was examined. The culture media was collected and reused to test that non-transfected cells cultured with this conditioned media could express alkaline phosphatase, and osteocalcin. The BMP-2/pcDNA3.1 plasmids were incorporated into the collagen scaffold. And, the plasmid loaded collagen scaffolds were grafted into rabbit calvarial defects. After two weeks, granulation tissue at the grafted site was obtained and BMP-2 mRNA was examined via RT-PCR. After 4, 8 weeks, animals were sacrificed and calvarial tissue was excised. After specimen preparation, light microscopical examination was done to evaluate formation of bone. Results: Transfected cells were able to incorporate BMP-2 gene in nucleus. And, the level of expressed and secreted BMP-2 was significantly higher than that of without transfection (P<.01). Secreted BMP-2 was able to induce expression of alkaline phosphatase and osteocalcin in the non-transfected cells. BMP-2/pcDNA3.1 released until 14, 28 days from the collagen sponge in vitro, and released plasmid maintained original size until 14 days. hBMP-2 mRNA was detected at the granulation tissue of experimental animals, but not at control animals after two weeks. At both 4 and 8 weeks, experimental groups showed significantly more newly formed bone area than that of control group (P<.01). Conclusions: BMP-2/pcDNA3.1 plasmid gene delivery could induce BMSCs into osteoblastic phenotype cells, and enhance bone regeneration of rabbit calvarial bone defect. (This study was supported by a grant from the ministry of Health and Welfare, Republic of Korea (00-PJ1-PG1-CH10-0002)).