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Open Access Research article

Simultaneous gene transfer of bone morphogenetic protein (BMP) -2 and BMP-7 by in vivo electroporation induces rapid bone formation and BMP-4 expression

Mariko Kawai1*, Kazuhisa Bessho2, Hiroki Maruyama3, Jun-ichi Miyazaki4 and Toshio Yamamoto1

Author Affiliations

1 Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8525, Japan

2 Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan

3 Division of Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8120, Japan

4 Division of Stem Cell Regulation Research, Osaka University Medical School, Osaka 565-0871, Japan

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BMC Musculoskeletal Disorders 2006, 7:62  doi:10.1186/1471-2474-7-62

Published: 3 August 2006

Abstract

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.

Conclusion

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.