Zhu Wei, Rawlins Bernard A, Boachie-Adjei Oheneba, Myers Elizabeth R, Arimizu Jun, Choi Eugene, Lieberman Jay R, Crystal Ronald G, Hidaka Chisa
Musculoskeletal Integrity Program, Hospital for Special Surgery, 535 East 70th Street, New York, NY 10021, USA.
J Bone Miner Res. 2004 Dec;19(12):2021-32. doi: 10.1359/JBMR.040821. Epub 2004 Aug 30.
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.
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.
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.
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 < 0.02, all comparisons).
Combined BMP2 and BMP7 gene transfer is significantly more effective in inducing osteoblastic differentiation and spine fusion than individual BMP gene transfer.
为增强骨形态发生蛋白（BMP）的成骨活性，进行了BMP2和BMP7基因联合转染。与体外单一BMP基因转染相比，这种方法导致间充质前体细胞的成骨分化显著增加。在78只大鼠中进行测试时，与单一BMP基因转染相比，联合基因转染增强了机械稳定的脊柱融合和骨形成率。
尽管临床骨形态发生蛋白（BMP）治疗有效，但所需剂量非常高。先前的研究表明，两种不同BMP基因的共表达可导致产生可能比同二聚体更有效的异二聚体BMP。在本研究中，进行了BMP2和BMP7基因联合转染，以测试与单一BMP基因转染相比，这种方法是否能改善成骨分化和骨形成。
将编码BMP2（AdBMP2）和BMP7（AdBMP7）的腺病毒载体与产生细胞（A549）共转染，或者作为对照，单独转染每个载体、AdNull（无转基因）或不转染病毒。比较上清液刺激C2C12成肌细胞和MC3T3-E1前成骨细胞成骨分化的能力。在大鼠后外侧脊柱融合模型中，将AdBMP2和AdBMP7联合给药与单独使用每个载体、AdNull或不转染病毒在78只大鼠中的治疗效果进行比较。术后8周对脊柱进行评估，以观察影像学和机械融合、骨形成和矿化情况。
从用AdBMP2和AdBMP7共转染的细胞上清液中共同沉淀出BMP2和BMP7，表明存在BMP2/7异二聚体。含有相对低剂量（7-140 ng/ml）BMP的共转染细胞上清液在C2C12和MC3T3-E1细胞中均诱导了骨钙素表达和碱性磷酸酶活性，分别比单独使用最大剂量（200-1000 ng/ml）的BMP2或BMP7诱导的水平高6倍和40倍。在脊柱融合模型中，与单一BMP基因转染相比，AdBMP2和AdBMP7联合给药导致机械稳定融合的数量显著增加，融合块中的矿化率和骨体积也高出2倍（所有比较，p<0.02）。
与单独的BMP基因转染相比，BMP2和BMP7基因联合转染在诱导成骨分化和脊柱融合方面明显更有效。
