The experimental Study for the Production of the Ectopic Bone Formation with Vascularized Flap

我们已经做了和去年相似的实验。采用大鼠模型，将带血管的小肌肉瓣置于硅模腔中含有hBMP-2 cDNA的胶原蛋白片上。我们成功的一个很好的方法，因为我们不需要考虑由于BMP的扩散而引起的意外的全身副作用，我们防止了局部高浓度的BMP的分布和/或泄漏，并且它本身具有确定的大小和形状。通过改变硅胶模具内腔的形状，可以获得理想的骨形态。但是，我们很遗憾，使用质粒DNA直接输送系统的肌瓣造成了坏死。我们尝试了各种缓冲液和载体等方法，但都没有成功。我们在体外诱导裸DNA进入培养细胞系，然后进行皮下异位成骨实验。这一次，一些小的骨被创造出来。一方面，我们也尝试直接应用裸质粒。将含有hBMP-2cDNA的质粒载体（Gene Activates Matrix，GAM）植入钙离子缺乏的大鼠胫骨中。制备不含磷酸钙的组作为对照。结果，在使用GAM和磷酸钙的组中，4周后通过放射学观察到骨桥。此外，6周后证实完全骨形成。此外，不含磷酸钙的GAM产生较少程度的骨形成。作为阴性对照，仅在胶原中，或在未安装hBMP-2cDNA的质粒中，未观察到骨形成。据认为，在这种新系统中的基因导入效率明显提高，与以前使用的GAM相比。

We have tried the similar experiment as last year. It is that a small-vascularized muscle flap was put on the collagen sheet including hBMP-2 cDNA in the lumen of silicon mold using rat model. We successes an excellent method, because that we need not consider unexpected systemic side effects due to the diffusion of BMP, that we prevent locally high concentration of BMP from distribution and/or leaking, and that it has definite size, shape by itself. And it is possible to produce the ideal bone shape by changing the shape of lumen of the mold of silicon. But, we are sorry that the muscle flap using direct delivery system of plasmid DNA had caused necrosis. We could not get success, although we tried various kinds the buffer and the carrier etc. We induced naked DNA into cultured cell line in vitro before subcutaneous ectopic bone formation assay in opposition. This time some small bone was created.On the one hand, we tried also the direct application of the naked plasmid. We installed plasmid vector containing hBMP-2cDNA that was included in GAM (Gene Activates Matrix) into bone deficiency in the tibia of rat with the calcium. The groups in which calcium phosphate is not included were prepared as a control. As a result, in the group that used GAM with the calcium phosphate, the bone bridge was observed Radiologcally 4 weeks later. Moreover complete bone formation was verified 6 weeks later. In addition, GAM without calcium phosphate creates less extent of bone formation. As negative control, in only the collagen, or in the plasmid that has not been installed hBMP-2cDNA, bone formation was not observed. It was thought that the efficiency of gene introduction in this new system rises clearly in comparison with GAM previously used.