SEGAMI Natsuki Kanazawa Medical University, Department of Oral and Maxillofacial Surgery, Professor, 医学部, 教授 (40148721)
KANEYAMA Keiseki Kanazawa Medical University, Department of Oral and Maxillofacial Surgery, Assistant Professor, 医学部, 助手 (50329380)
|Budget Amount *help
¥3,400,000 (Direct Cost : ¥3,400,000)
Fiscal Year 2003 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 2002 : ¥1,900,000 (Direct Cost : ¥1,900,000)
1.To examine the effectiveness of a gene transfer procedure for the delivery of BMP-2 to the distraction osteogenesis, we constructed a human BMP-2--expressing replication-deficient adenoviral vector, AxCAOBMP-2, and evaluated its osteoinductive activity in vitro and in vivo.
2.C2C12 myoblasts were infected in vitro with this viral vector or an Escherichia coil LacZ gene-expressing control adenovirus vector (AxCALacZ). Twenty-four hours after the infection, indirect immunofluorescence was performed. On day 5 after the infection, alkaline phosphatase (ALP) in the cells and osteocalcin in the culture medium were measured. Furthermore, to examine the effectiveness of gene transfer of BMP-2 in vivo, we evaluated osteoinduction by AxCAOBMP-2, under transient immunosuppression with cyclophosphamide, given at a dose of 125 mg/kg intraperitoneally the day before injection of the adenoviral vector. Twenty-five microliters of AxCAOBMP-2 (8.75 x 10^8 plaque-forming units [pfu],'Group I) and AxCALa
cZ (1.75 x 10^8 pfu, control group) and 5 microl of AxCAOBMP-2 (1.75x 10^8 pfu, Group II) were injected into a right calf muscle of Wistar rats. On day 21, bone formation in each group was investigated radiologically and histologically.
3.Abundant BMP-2 expression in C2C12 cells infected with this viral vector was confirmed by immunofluorescence. C2C12 cellstransferred with the BMP-2 gene by this vector produced ALP in the cells and also produced and secreted osteocalcin in the culture medium. Osteoinduction was found only in the AxCAOBMP-2 treated groups with immunosuppression. Osteoinduction activity was higher in Group I than in Group II.
3.Twelve animals, performed distraction, were treated with AxCAOBMP-2 (8.75 x 10^8 pfu) 25μg in each, during distraction osteogenesis at 7 days and 14 days. In results, Bone formation and ALP activity were increased significantly in comparison with non-treatment groups. It was suggested that Gene transfer procedure for the delivery of BMP-2 effected to the distraction osteogenesis and had a potential shorting distraction period. Less