Gene expression analysis of ectopic bone formation induced by electroporatic gene transfer of BMP4

Implantation of bone morphogenetic protein (BMP) using a carrier or by BMP gene transfer into rodent muscle can induce bone formation. Implanted BMP becomes bioactive immediately after implantation. In BMP gene transfer, there is a time-lag between the secretion of gene products and bone formation. We analyzed the gene expression of chondrogenic and osteogenic specific markers in the process of ectopic bone formation by using semi-quantitative RT-PCR. A plasmid vector containing mouse BMP4 gene (pCAGGS-BMP4) was transferred into the gastrocnemius muscles of mice using electroporation. Histological examination revealed the process of endochondral bone formation in the pCAGGS-BMP4 transferred muscles. As chondrogenic markers, aggrecan gene maximal expression was detected on day 7 and decreased by day 14, and for collagen X the gene maximal expression was on day 10. As osteogenic markers, osteocalcin (OCN), bone sialoprotein (BSP) and osteopontin (OPN) gene expressions were clearly detected from day 10 and then increased by day 14. In conclusion, the present study proved that ectopic bone formation by BMP4 gene transfer into the muscle induced endochondral ossification that corresponded well with that to that by implantation of demineralized bone matrix.