Human BMP-2 gene transfer using transcutaneous in vivo electroporation induced both intramembranous and endochondral ossification.
It has been generally accepted that bone morphogenetic protein-2 (BMP-2) can induce osteogenesis in skeletal muscles via endochondral ossification. However, it is not clear how the ossification process occurs after the BMP-2 gene transfer to skeletal muscles in rats using in vivo electroporation. In this study, we evaluated the ossification process by BMP-2 gene transfer using in vivo electroporation. The gastrocnemius muscles of Wistar rats were injected with human BMP-2 gene expression vector (pCAGGS-BMP-2), followed by electroporation under the condition of 100 V, 50 msec per 1 sec, x8. Light and electron microscopic and radiographic analyses were performed at 1, 3, 5, 7, and 10 days after treatment. At 7 days, no sign of cartilage and/or bone formation was detected. However, at 10 days after in vivo electroporation, soft X-ray analysis revealed small lucent areas around the plasmid-injected region. Clusters of both cartilage tissues, leading to endochondral ossification and intramembranous bones of various sizes, were observed between muscle fibers. RT-PCR detected osteocalcin mRNA, showing bone formation at 10 days. Our findings strongly suggest that BMP-2 gene transfer using in vivo electroporation induces not only endochondral ossification but also intramembranous ossification.
Kawai
Mariko
M
Department of Oral Morphology, Okayama University Graduate School of Medicine and Dentistry, Okayama, 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; D019485:Bone Morphogenetic Proteins / Q000235:genetics* / Q000378:metabolism; D018274:Electroporation / Q000379:methods*; D018014:Gene Transfer Techniques; D006801:Humans; D008297:Male; D018482:Muscle, Skeletal / Q000000981:diagnostic imaging / Q000378:metabolism / Q000648:ultrastructure; D009999:Ossification, Heterotopic / Q000235:genetics* / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D010012:Osteogenesis / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D011859:Radiography; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013047:Specific Pathogen-Free Organisms; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism
C525718:BMP2 protein, human; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D015675:Osteocalcin