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To enhance the osteogenic activity of BMP, combination BMP2 and BMP7 gene transfer was performed. This approach led to a significant increase in osteoblastic differentiation of mesenchymal precursors compared with single BMP gene transfer in vitro. When tested in 78 rats, combination gene transfer enhanced mechanically stable spine fusion and bone formation rate versus single BMP gene transfer.Introduction: Although clinical bone morphogenetic protein (BMP) therapy is effective, required doses are very high. Previous studies have suggested that the co-expression of two different BMP genes can result in the production of heterodimeric BMPs that may be more potent than homodimers. In this study, combined BMP2 and BMP7 gene transfer was performed to test whether this approach improves osteoblastic differentiation and bone formation compared with single BMP gene transfer. Materials and Methods: A producer cell (A549) was co-transfected with adenovirus vectors encoding BMP2 (AdBMP2) and BMP7 (AdBMP7) or, as controls, each vector alone, AdNull (with no transgene) or no virus. Supernatants were compared for their ability to stimulate osteoblastic differentiation of C2C12 myoblasts and MC3T3-E1 pre-osteoblasts. In a rat posterolateral spine fusion model, co-administration of AdBMP2 and AdBMP7 was compared with treatment with each vector alone, AdNull or no virus in 78 rats. The spines were assessed 8 weeks after surgery for radiographic and mechanical fusion, bone formation, and mineralization. Results: BMP2 and BMP7 were co-precipitated from supernatants of cells co-transfected with AdBMP2 and AdBMP7, indicating the presence of BMP2/7 heterodimer. Supernatants of co-transfected cells containing relatively low doses (7-140 ng/ml) of BMPs induced osteocalcin expression and alkaline phosphatase activity in both C2C12 and MC3T3-E1 cells, that were up to 6-and 40-fold higher, respectively, than levels induced by maximal doses (200 -1000 ng/ml) of either BMP2 or BMP7 alone. In the spine fusion model, co-administration of AdBMP2 and AdBMP7 resulted in a significantly greater number of mechanically stable fusions and also 2-fold higher mineralization rate and bone volume in the fusion mass versus single BMP gene transfer (p Ͻ 0.02, all comparisons). Conclusion: Combined BMP2 and BMP7 gene transfer is significantly more effective in inducing osteoblastic differentiation and spine fusion than individual BMP gene transfer.

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Abstract. Buc,kgrouiit/ Cartilage has a limited capacity to heal. Although chondrocyte transplantation is a useful therapeutic strategy, the repair process can be lengthy. Previously we have shown that over expression of bone morphogenetic protein-7 (BMP-7) in chondrocytes by adenovirus-mediated gene transfer leads to increased matrix synthesis and cartilage-like tissue formation in vitro. In this context we hypothesized that implantation of genetically modified chondrocytes expressing BMP-7 would accelerate the formation of hyaline-like repair tissue in an equine model of cartilage defect repair.Met/io(l.s: Chondrocytes treated with adenovirus vector encoding BMP-7 (AdBMP-7) or as control. an adenovirus vector encoding an irrelevant gene (Esdierichiu coli cytosine deaminase, AdCD) were implanted into extensive ( I5 mm diameter) articular cartilage defects in the patellofemoral joints of 10 horses. Biopsies were performed to evaluate early healing at 4 weeks. At the terminal time point of 8 months, repairs were assessed for morphology, MRI appearance, compressive strength, biochemical composition and persistence of implanted cells.Residts: Four weeks after surgery AdBMP-7-treated repairs showed an increased level of BMP-7 expression and accelerated healing, with markedly more hyaline-like morphology than control. Quantitative real-time polymerase chain reaction (PCR) analysis of the repair tissue 8 months after surgery showed that few implanted cells persisted. By this time, the controls had healed similarly to the AdBMP-7-treated defects, and no difference was detected in the morphologic, biochemical or biomechanical properties of the repair tissues from the two treatment groups.Conc/ir.siorzs: Implantation of genetically modified chondrocytes expressing BMP-7 accelerates the appearance of hyaline-like repair tissue in experimental cartilage defects.Clinical rele[wxx>: Rehabilitation after cell-based cartilage repair can be prolonged, leading to decreased patient productivity and quality of life. This study shows the feasibility of using genetically modified chondrocytes to accelerate cartilage healing.

Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model. A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 x 10(7) AdIGF-1 modified chondrocytes and the contralateral joint received 2 x 10(7) naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated. Genetic modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and correlated with increased collagen type II immunoreaction up to eight months. Genetic modification of chondrocytes with AdIGF-1 prior to transplantation improved early (four to nine weeks), and to a lesser degree long-term, cartilage healing in the equine model. The equine model of cartilage healing closely resembles human clinical cartilage repair. The results of this study suggest that cartilage healing can be enhanced through genetic modification of chondrocytes prior to transplantation.