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，已有几种方法被开发出来并应用于临床；然而，需要一种对公共社会来说简单、安全、经济的方法。虽然质粒DNA基因转移是安全的，并在临床上得到应用，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：1。将质粒DNA与磷酸钙小颗粒结合，移植到组织缺损处，提高了基因转移效率。小的钙磷酸盐颗粒结合质粒DNA并使其保持在局部。此外，与颗粒的结合增加了对dna酶的抵抗力。这些现象将导致基因转移的增加。利用这种方法，我们将编码人BMP2的质粒DNA应用于大鼠胫骨5 mm节段性骨缺损。6周缺损完全愈合，8周时骨的机械强度与正常胫骨相当。应用部位虽出现骨结样，但逐渐被吸收，骨形态恢复正常。同样，将编码人BMP12的质粒DNA应用于大鼠股骨骨缺损。先前的缺损与周围组织形成了富含胶原纤维的韧带样组织，而缺损被骨填充。该方法在韧带重建中具有较好的应用前景。培养骨膜细胞，转染BMP2和/或VEGF基因，移植至裸鼠体内。当这两种基因都被转移时，形成的骨骼数量最多。

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.