TY - JOUR
T1 - Ectopic bone formation by electroporatic transfer of bone morphogenetic protein-4 gene
AU - Kishimoto, K. N.
AU - Watanabe, Y. Y.
AU - Nakamura, H. H.
AU - Kokubun, S. S.
N1 - Funding Information:
The authors thank Brigid L. M. Hogan for supplying the mouse BMP-4 cDNA. We also thank Shishido for caring for the mice, and Matsuda, Fukuyama, Shoji, and Horaguchi for their technical assistance. This work was supported by a grant from the Sugawara Medical Foundation. We also thank K. Shishido for caring for mice and H. Matsuda, M. Fukuyama, K. Shoji, and K. Horaguchi for their technical assistance.
PY - 2002
Y1 - 2002
N2 - Orthopedic surgeons have long awaited the clinical application of bone morphogenetic proteins (BMPs) for bone regeneration. However, such possible applications involving proteins or genes transferred with virus vectors have encountered many problems, including high cost, immunological reactions, viral infection, etc. We adopted a new gene transfer system of in vivo electroporation with a plasmid expression vector. A solution of plasmid DNA containing mouse BMP-4 (pMiw-BMP4) was injected into the gastrocnemius of BALB/cA mice, and electric pulses were applied through paired-needle electrodes inserted percutaneously. As a control plasmid, LacZ-containing plasmid (pMiwZ) was transferred by electroporation. A control group in which pMiw-BMP4 was injected and not electroporated was also introduced. In these groups, the gastrocnemius was harvested at 7, 14, 21, and 28 days after electroporation (n = 6 in each). As nonplasmid controls, electroporation with saline injection (n = 6), electroporation without injection (n = 6), and saline injection only (n = 3) were prepared. In these groups, the mice were killed 7 days after experimentation. Ectopic calcification or ossification was examined by histology as well as soft X-ray. In all electroporated groups (pMiwZ, pMiw-BMP4, saline injection, and without injection), dystrophic calcification of muscle bundles and infiltration of mesenchymal cells were observed histologically. Ectopic bone formation was observed only in the pMiw-BMP4 electroporation group. At 7 days after pMiw-BMP4 electroporation, extracellular eosinophilic matrix in a collection of mesenchymal cells was observed. Between 14 and 28 days after electroporation, ectopic bone was observed in 44% of mice, and bone marrow-like cells observed in 22%. The newly formed bone was woven. Injection of pMiw-BMP4 or saline induced neither calcification nor ossification. Our findings indicate that BMP-4 transferred by electroporation can induce in vivo and in situ ectopic bone formation in skeletal muscle.
AB - Orthopedic surgeons have long awaited the clinical application of bone morphogenetic proteins (BMPs) for bone regeneration. However, such possible applications involving proteins or genes transferred with virus vectors have encountered many problems, including high cost, immunological reactions, viral infection, etc. We adopted a new gene transfer system of in vivo electroporation with a plasmid expression vector. A solution of plasmid DNA containing mouse BMP-4 (pMiw-BMP4) was injected into the gastrocnemius of BALB/cA mice, and electric pulses were applied through paired-needle electrodes inserted percutaneously. As a control plasmid, LacZ-containing plasmid (pMiwZ) was transferred by electroporation. A control group in which pMiw-BMP4 was injected and not electroporated was also introduced. In these groups, the gastrocnemius was harvested at 7, 14, 21, and 28 days after electroporation (n = 6 in each). As nonplasmid controls, electroporation with saline injection (n = 6), electroporation without injection (n = 6), and saline injection only (n = 3) were prepared. In these groups, the mice were killed 7 days after experimentation. Ectopic calcification or ossification was examined by histology as well as soft X-ray. In all electroporated groups (pMiwZ, pMiw-BMP4, saline injection, and without injection), dystrophic calcification of muscle bundles and infiltration of mesenchymal cells were observed histologically. Ectopic bone formation was observed only in the pMiw-BMP4 electroporation group. At 7 days after pMiw-BMP4 electroporation, extracellular eosinophilic matrix in a collection of mesenchymal cells was observed. Between 14 and 28 days after electroporation, ectopic bone was observed in 44% of mice, and bone marrow-like cells observed in 22%. The newly formed bone was woven. Injection of pMiw-BMP4 or saline induced neither calcification nor ossification. Our findings indicate that BMP-4 transferred by electroporation can induce in vivo and in situ ectopic bone formation in skeletal muscle.
KW - Bone formation
KW - Bone morphogenetic protein (BMP)
KW - Electroporation
KW - Gene therapy
KW - Gene transfer
KW - Tissue engineering
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U2 - 10.1016/S8756-3282(02)00825-6
DO - 10.1016/S8756-3282(02)00825-6
M3 - Article
C2 - 12151088
AN - SCOPUS:0036021057
VL - 31
SP - 340
EP - 347
JO - Bone
JF - Bone
SN - 8756-3282
IS - 2
ER -