Ex Vivo Gene Therapy Approach Using Human Lim Mineralization Protein-3 To Induce Bone Healing in a Rodent Model

Introduction. Gene therapy research in the field of orthopaedics have evolved during the last decade, leading to possible applications for the treatment of pathological conditions, such as bone fractures and defects. Several gene transfer techniques have been employed so far for inducing bone formation in animal models of bone defects. Cell-based approaches, such as the implantation in animals of ex vivo genetically modified cells, produced promising results. In this study we used autologous skin fibroblasts, which are very simple to harvest and propagate in culture, transduced ex vivo with the osteogenic factor Lim Mineralization Protein-3 (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. Materials and Methods. Primary dermal fibroblasts cultures were established using a biopsy of shaved skin obtained from the abdomen of each rat. Semi-confluent primary fibroblasts were infected with either AdBMP-2 or AdhLMP-3, using a overall multiplicity of infection (MOI) of 100 viral particles per cell. Cells transduced with Ad-eGFP 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, 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: rats treated with cells infected with AdLMP-3, rats treated with cells infected with AdBMP-2, rats treated with cells infected with Ad-eGFP and rats treated with untreated cells. Rats from each group were sacrified at 1, 2 and 3 months after the treatment and studied by x-rays, Micro-CT and histology. Results. All the animals treated with LMP-3 showed healing of the bone defect after 3 months, as confirmed histologically and radiographically. On the contrary none of the controls showed bone formation at the latest time point. Discussion. Recently, Lim Mineralization Proteins (LMP) have been identified as regulators of the osteoblast differentiation program. We have previously demonstrated that human LMP-3 contributes actively to bone formation, acting through the BMP-2 signaling pathway, being capable of inducing differentiation of cells of mesenchymal 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 tested the efficacy of an ex-vivo approach using autologous dermal fibroblasts infected with AdLMP-3. Our results show that it s possible to induce a complete bone healing using this method, and confirm the in vivo osteoinductive properties of hLMP-3.