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

BMC Musculoskelet Disord. 2006 Aug 3;7:62. doi: 10.1186/1471-2474-7-62.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bone Morphogenetic Protein 2
  • Bone Morphogenetic Protein 4
  • Bone Morphogenetic Protein 7
  • Bone Morphogenetic Proteins / biosynthesis*
  • Bone Morphogenetic Proteins / genetics*
  • Bone Regeneration / genetics
  • Cell Line, Tumor
  • Electroporation / methods*
  • Gene Expression Regulation / genetics
  • Gene Transfer Techniques*
  • Genetic Vectors / genetics
  • Humans
  • Male
  • Mice
  • Muscle, Skeletal / cytology
  • Muscle, Skeletal / metabolism
  • Muscle, Skeletal / surgery
  • Ossification, Heterotopic / genetics
  • Osteogenesis / genetics*
  • Plasmids / genetics
  • Rats
  • Rats, Wistar
  • Time Factors
  • Transforming Growth Factor beta / genetics*
  • Up-Regulation / genetics

Substances

  • BMP2 protein, human
  • BMP4 protein, human
  • BMP7 protein, human
  • Bmp2 protein, mouse
  • Bmp2 protein, rat
  • Bmp4 protein, mouse
  • Bmp4 protein, rat
  • Bmp7 protein, rat
  • Bone Morphogenetic Protein 2
  • Bone Morphogenetic Protein 4
  • Bone Morphogenetic Protein 7
  • Bone Morphogenetic Proteins
  • Transforming Growth Factor beta