Local Direct Injection of BMP2-Modified Fibroblasts Induces Ectopic Bone Formation in Immunocompetent Mice

Previous studies have demonstrated that the Bone Morphogenetic Protein-2 (BMP-2) induces bone formation in several animal models. In this study we have evaluated the capability of murine fibroblasts modified with BMP-2 to induce ectopic bone formation in vivo. First generation adenoviruses encoding either human bone morphogenetic protein-2 (BMP-2) or, as a negative control, enhanced green fluorescent protein (eGFP), have been used as vectors to introduce BMP-2 and eGFP genes in coltures of NIH3T3 fibroblast cells. The efficency of infection was evaluated by the fluorescence. Twenty-four hours after the infection the modified cells were pelleted, suspended in PBS and directly injected in the triceps muscle of immunocompetent mice. Development of ectopic bone formation was monitored radiographically for 3 weeks and representative specimens were evaluated histologically after 10, 15, 20 days by alizarin and von kossa staining. By radiological criteria, some of the animals receiving the BMP-2-modified fibroblasts developed ectopic bone formation already after 10 days, whereas all those receiving the eGFP-modified were negative. T 3 weeks half of the animals treated with BMP-2-modified fibroblasts developed bone in muscles. Histologic examination of representative mice at 10 days showed ossification in 50% of the muscles treated with the BMP-2- modified cells, as confirmed with the bone-specific staining. Instead, at the end of the 3-weeks experiment, the control muscles showed no radiological or histological signs of bone formation. This study suggests that direct, local delivery of BMP-2-modified fibroblasts could be a safe and powerful method to induce bone formation in vivo. These promising data encourage the further development of ex-vivo genetic approaches to enhancing bone formation in vivo.