Application of newly-developed gene transfer to regeneration of bone and periodontal tissue

作为骨和牙周组织的再生疗法，已经开发了几种方法并在临床上应用，但需要一种简单、安全和经济的方法来满足公众的需求。尽管用质粒DNA进行基因转移是安全和临床应用的，但这种基因转移的效率极低是一个问题。在本研究中，我们得到了以下结果。将载体DNA与钙磷小颗粒结合后移植到组织缺损处，可提高基因转移效率。细小的钙磷颗粒结合在一起，并将其保存在局部。此外，与颗粒的结合增加了对DNA酶的抗性。这些现象将有助于基因转移的增加。利用这种方法，我们将编码人骨形成蛋白2的质粒DNA应用于大鼠胫骨5 mm节段性骨缺损。术后6周，骨缺损完全愈合，8周时骨的机械强度与正常胫骨相当。虽然贴敷部位出现结节状骨，但逐渐吸收，骨形态恢复正常。同样，将编码人骨形成蛋白12的质粒DNA应用于大鼠股骨缺损处。原缺损区至周围组织形成丰富的胶原纤维，形成韧带样组织，缺损区充填骨量。该方法在韧带重建中具有较好的应用前景。体外培养骨膜细胞，将BMP2和/或血管内皮生长因子基因导入骨膜细胞，移植到裸鼠体内。当两个基因都被转移时，形成了最大数量的骨骼。

As regenerative therapies for bone and periodontal tissues several methods have been developed and clinically applied ; however, a method, which is simple, safe and economical for public society, is required. Although gene transfer with plasmid DNA is safe and clinically applied, it is a problem that efficiency of this gene transfer is extremely low. In the present study, we obtained the following results.1. When plasmid DNA was combined with small Ca-phosphate particles and transplanted to the tissue defect, the efficiency of gene transfer increased. Small Ca-phosphate particles bound plasmid DNA and kept it locally. Furthermore, the binding to the particles increased the resistance to DNase. These phenomena would contribute to the increase of gene transfer.2. Utilizing this method, we applied plasmid DNA encoding human BMP2 to 5 mm segmental bone defects of rat tibias. Six weeks the defects completely healed and the mechanical strength of the bone was comparable to the normal tibia at 8 weeks. Although knot-like bone appeared at the applied site, it was gradually absorbed and the bone shape became normal.3. Similarly, plasmid DNA encoding human BMP12 was applied to the bone defects of rat femurs. Ligament-like tissue abundant in collagen fibers was formed from the previous defect to the surrounding tissue whereas the defect was filled with bone. This method would be promising for ligament reconstruction.4. Periosteum cells were cultured and these cells were transferred with BMP2 and/or VEGF gene, which were then transplanted to nude mice. When both genes were transferred, the largest amount of bone was formed.