In vivo endochondral bone formation using a bone morphogenetic protein 2 adenoviral vector

Bone morphogenetic proteins (BMPs) are polypeptides that induce ectopic bon e formation in standard rat in vivo assay systems. Previous studies have de monstrated the clinical utility of these proteins in spinal fusion, fractur e healing, and prosthetic joint stabilization. Gene therapy is also a theor etically attractive technique to express BMPs clinically, since long-term, regulatable gene expression and systemic delivery with tissue-specific expr ession may be possible in future. This study was performed to determine whe ther an adenoviral vector containing the BMP-2 gene can be used to express BMP-2 in vitro and promote endochondral bone formation in vivo. In vitro, U 87 MG cells transduced per cell with 20 MOI of an adenoviral construct cont aining the BMP-2 gene under the control of the universal CMV promoter (Ad-B MP-2) showed positive antibody staining for the BMP-2 protein at posttransf ection day 2. The synthesis and secretion of active BMP-2 into the conditio ned medium of Ad-BMP-2-transduced 293 cells were confirmed by Western blot analysis and the induction of alkaline phosphatase activity in a W-20 strom al cell assay. bn vivo, Sprague-Dawley rats and athymic nude rats were inje cted with Ad-BMP-2 in the thigh musculature and were sacrificed on day 3, 6 , 9, 12, 16, 21, 60, and 110 for histological analysis. The Sprague-Dawley rats showed evidence of acute inflammation, without ectopic bone formation, at the injection sites. In the athymic nude rats, BMP-2 gene therapy induc ed mesenchymal stem cell chemotaxis and proliferation, with subsequent diff erentiation to chondrocytes. The chondrocytes secreted a cartilaginous matr ix, which then mineralized and was replaced by mature bone. This study demo nstrates that a BMP-2 adenoviral vector can be utilized to produce BMP-2 by striated muscle cells in athymic nude rats, leading to endochondral bone f ormation. However, in immunocompetent animals the endochondral response is attenuated, secondary to the massive immune response elicited by the first- generation adenoviral construct.