Simultaneous gene transfer of bone morphogenetic protein (BMP) -2 and BMP-7 by in vivo electroporation induces rapid bone formation and BMP-4 expression.
BACKGROUND
Transcutaneous in vivo electroporation is expected to be an effective gene-transfer method for promoting bone regeneration using the BMP-2 plasmid vector. To promote enhanced osteoinduction using this method, we simultaneously transferred cDNAs for BMP-2 and BMP-7, as inserts in the non-viral vector pCAGGS.


METHODS
First, an in vitro study was carried out to confirm the expression of BMP-2 and BMP-7 following the double-gene transfer. Next, the individual BMP-2 and BMP-7 plasmids or both together were injected into rat calf muscles, and transcutaneous electroporation was applied 8 times at 100 V, 50 msec.


RESULTS
In the culture system, the simultaneous transfer of the BMP-2 and BMP-7 genes led to a much higher ALP activity in C2C12 cells than did the transfer of either gene alone. In vivo, ten days after the treatment, soft X-ray analysis showed that muscles that received both pCAGGS-BMP-2 and pCAGGS-BMP-7 had better-defined opacities than those receiving a single gene. Histological examination showed advanced ossification in calf muscles that received the double-gene transfer. BMP-4 mRNA was also expressed, and RT-PCR showed that its level increased for 3 days in a time-dependent manner in the double-gene transfer group. Immunohistochemistry confirmed that BMP-4-expressing cells resided in the matrix between muscle fibers.


CONCLUSIONS
The simultaneous transfer of BMP-2 and BMP-7 genes using in vivo electroporation induces more rapid bone formation than the transfer of either gene alone, and the increased expression of endogenous BMP-4 suggests that the rapid ossification is related to the induction of BMP-4.
Kawai
Mariko
M
Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8525, Japan. marikoka@md.okayama-u.ac.jp
Bessho
Kazuhisa
K
Maruyama
Hiroki
H
Miyazaki
Jun-ichi
J
Yamamoto
Toshio
T
D000818:Animals; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins / Q000096:biosynthesis* / Q000235:genetics*; D001861:Bone Regeneration / Q000235:genetics; D045744:Cell Line, Tumor; D018274:Electroporation / Q000379:methods*; D005786:Gene Expression Regulation / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism / Q000601:surgery; D009999:Ossification, Heterotopic / Q000235:genetics; D010012:Osteogenesis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000235:genetics*; D015854:Up-Regulation / Q000235:genetics
C525718:BMP2 protein, human; C525809:BMP4 protein, human; C525819:BMP7 protein, human; C525719:Bmp2 protein, mouse; C525720:Bmp2 protein, rat; C525810:Bmp4 protein, mouse; C525811:Bmp4 protein, rat; C525821:Bmp7 protein, rat; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins; D016212:Transforming Growth Factor beta