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Aims. Gene therapy research in the field of orthopaedics and traumatology have evolved during the last decade, leading to possible applications for the treatment of pathological conditions, such as bone fractures and cartilage defects. In particular, several gene transfer techniques have been employed so far for inducing bone formation in animal models of bone defects. Cell-based approaches, using in vitro and ex vivo genetically modified cells to be implanted in the animal, produced promising results as they enable the production of physiologic doses of an osteoinductive gene product into selected anatomical sites. In this study we used autologous skin fibroblasts, which are very simple to harvest and propagate in culture, transduced ex vivo with the new osteo-genic factor Lim Mineralization Factor-3 (Ad-LMP-3). These engineered cells produced successful bone healing when implanted by the use of a scaffold in rats, validating the in vivo osteoinductive properties of hLMP-3. Methods. Primary dermal fibroblasts cultures were established using a 1cm 2 biopsy of shaved skin obtained from the abdomen of each rat after anesthesia. Semi-confluent primary fibroblasts were infected with either AdBMP-2 or AdhLMP-3 or both, using a overall multiplicity of infection (MOI) of 100 viral particles per cell. Cells transduced with Ad-eGFP at the same MOI were used as a viral infection control, while untreated cells served as a negative control. The transduced cells were harvested 24 hours after viral infection, resuspended in sterile PBS, let adsorbed on a Hydroxyapatite/Collagen scaffold and then implanted in a bone defect surgically performed in the mandible of immunocompetent rats. The animals were divided in 4 groups: 9 rats were treated with cells infected with AdLMP-3, 9 rats with cells infected with AdBMP-2 (positive controls), 9 rats with cells transduced with Ad-eGFP and 9 rats with untreated cells (controls). 3 Rats from each group were sacrified at 1, 2 and 3 months after the treatment and studied by x-rays, Micro-CT and histology (Von kossa and Alizarin staining). Results. All the animals treated with LMP-3 showed healing of the bone defect after 3 months, as confirmed his-tologically and radiographically. On the contrary none of the controls showed bone formation at latest time point. Conclusions. Recently, Lim Mineralization Proteins (LMP), coded by three different splice variants (LMP-1, LMP-2, LMP-3) of the same gene, have been identified as regulators of the osteoblast differentiation program. We have previously demonstrated that human LMP-3 (hLMP-3) contributes actively to bone formation, acting at least in part, through the BMP-2 signaling pathway, being capable of inducing differentiation of cells of mes-enchymal derivation towards the osteoblastic lineage, through the up-regulation of bone-specific genes, along with ectopic bone formation in vivo and mineralization in vitro. In this study we have analyzed the efficacy of an ex-vivo approach using autologous dermal fibroblasts infected with AdLMP-3. Engineered cells produced bone healing when implanted by the use of a scaffold in a rodent model, validating the in vivo osteoinductive properties of hLMP-3.

Top of pageAbstract 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.