Combined bone morphogenetic protein-2 and -7 gene transfer enhances osteoblastic differentiation and spine fusion in a rodent model.
To enhance the osteogenic activity of BMP, combination BMP2 and BMP7 gene transfer was performed. This approach led to a significant increase in osteoblastic differentiation of mesenchymal precursors compared with single BMP gene transfer in vitro. When tested in 78 rats, combination gene transfer enhanced mechanically stable spine fusion and bone formation rate versus single BMP gene transfer.


BACKGROUND
Although clinical bone morphogenetic protein (BMP) therapy is effective, required doses are very high. Previous studies have suggested that the co-expression of two different BMP genes can result in the production of heterodimeric BMPs that may be more potent than homodimers. In this study, combined BMP2 and BMP7 gene transfer was performed to test whether this approach improves osteoblastic differentiation and bone formation compared with single BMP gene transfer.


METHODS
A producer cell (A549) was co-transfected with adenovirus vectors encoding BMP2 (AdBMP2) and BMP7 (AdBMP7) or, as controls, each vector alone, AdNull (with no transgene) or no virus. Supernatants were compared for their ability to stimulate osteoblastic differentiation of C2C12 myoblasts and MC3T3-E1 pre-osteoblasts. In a rat posterolateral spine fusion model, co-administration of AdBMP2 and AdBMP7 was compared with treatment with each vector alone, AdNull or no virus in 78 rats. The spines were assessed 8 weeks after surgery for radiographic and mechanical fusion, bone formation, and mineralization.


RESULTS
BMP2 and BMP7 were co-precipitated from supernatants of cells co-transfected with AdBMP2 and AdBMP7, indicating the presence of BMP2/7 heterodimer. Supernatants of co-transfected cells containing relatively low doses (7-140 ng/ml) of BMPs induced osteocalcin expression and alkaline phosphatase activity in both C2C12 and MC3T3-E1 cells, that were up to 6- and 40-fold higher, respectively, than levels induced by maximal doses (200-1000 ng/ml) of either BMP2 or BMP7 alone. In the spine fusion model, co-administration of AdBMP2 and AdBMP7 resulted in a significantly greater number of mechanically stable fusions and also 2-fold higher mineralization rate and bone volume in the fusion mass versus single BMP gene transfer (p < 0.02, all comparisons).


CONCLUSIONS
Combined BMP2 and BMP7 gene transfer is significantly more effective in inducing osteoblastic differentiation and spine fusion than individual BMP gene transfer.
Zhu
Wei
W
Musculoskeletal Integrity Program, Hospital for Special Surgery, 535 East 70th Street, New York, NY 10021, USA.
Rawlins
Bernard A
BA
Boachie-Adjei
Oheneba
O
Myers
Elizabeth R
ER
Arimizu
Jun
J
Choi
Eugene
E
Lieberman
Jay R
JR
Crystal
Ronald G
RG
Hidaka
Chisa
C
D016475:3T3 Cells; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D055396:Bone Morphogenetic Protein 2; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins / Q000235:genetics*; D001842:Bone and Bones / Q000378:metabolism*; D002454:Cell Differentiation; D002460:Cell Line; D045744:Cell Line, Tumor; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000166:cytology; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism*; D010012:Osteogenesis / Q000502:physiology*; D051381:Rats; D013997:Time Factors; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics*
C525718:BMP2 protein, human; C525819:BMP7 protein, human; C525719:Bmp2 protein, mouse; C525720:Bmp2 protein, rat; C525821:Bmp7 protein, rat; D055396:Bone Morphogenetic Protein 2; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins; D016212:Transforming Growth Factor beta