In vitro and in vivo studies of a bone morphogenetic protein-2 expressing adenoviral vector.
BACKGROUND
Bone morphogenetic proteins (BMPs) play important roles in the migration of osteoblast progenitor cells, the proliferation of mesenchymal cells, and their differentiation into chondrogenic and osteogenic cells. However, the optimum procedure to deliver BMPs remains unknown. To examine the effectiveness of a gene transfer procedure for the delivery of BMP-2, we constructed a human BMP-2-expressing replication-deficient adenoviral vector, AxCAOBMP-2, and evaluated its osteoinductive activity in vitro and in vivo.


METHODS
C2C12 myoblasts were infected in vitro with this viral vector or an Escherichia coli 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 AxCALacZ (1.75 x 10(8) pfu, control group) and 5 microl of AxCAOBMP-2 (1.75 x 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.


RESULTS
Abundant BMP-2 expression in C2C12 cells infected with this viral vector was confirmed by immunofluorescence. C2C12 cells transferred 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.


CONCLUSIONS
This study demonstrated the osteoinductive activity in vitro and in vivo by an adenoviral vector carrying the BMP-2 gene.


CONCLUSIONS
Gene therapy with AxCAOBMP-2 under transient immunosuppression may be useful for bone reconstruction.
Okubo
Y
Y
Graduate School of Medicine, Kyoto University, Japan. okubo@kuhp.kyoto-u.ac.jp
Bessho
K
K
Fujimura
K
K
Iizuka
T
T
Miyatake
S I
SI
D000256:Adenoviridae; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000096:biosynthesis / Q000235:genetics*; D002460:Cell Line; D002478:Cells, Cultured; D019084:Fluorescent Antibody Technique, Indirect; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D066298:In Vitro Techniques; D008297:Male; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D015675:Osteocalcin / Q000096:biosynthesis; D010012:Osteogenesis; D051381:Rats; D017208:Rats, Wistar; D016212:Transforming Growth Factor beta
C525718:BMP2 protein, human; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D016212:Transforming Growth Factor beta; D015675:Osteocalcin; D000469:Alkaline Phosphatase