Assessment of gene transfer method for induction of osseo integration on dental implant

将CTGF基因转移到成骨细胞中可能是一种很有前途的加速骨形成的策略。本研究探讨了重组腺病毒体外将CTGF基因转染小鼠成骨细胞（MC3T3-E1）的可能性，以及拔牙后的骨再生过程。将编码LacZ基因的重组腺病毒（Ax1CALacZ）以5、10和50倍感染倍数（MOI）转染MC3T3-E1细胞。转染后1天，细胞用X-gal染色。结果moi50转染剂量下，细胞X-gal染色率几乎达到80%，未见明显细胞损伤。制备了CAG启动子编码人CTGF基因的重组腺病毒（Ax1CACTGF），并以moi50剂量转染MC3T3-E1细胞。转染后1、3和7天，采用RT-PCR和western blotting检测转染细胞的CTGF mRNA翻译和蛋白产生情况。转染Ax1CACTGF后，CTGF mRNA和CTGF蛋白的表达在转染后7天均显著上调。拔牙后2 ~ 14天的组织学检查清楚地观察到拔牙创面愈合的顺序期表现。拔牙后第7天，观察到从窝缘处有编织骨填充，小梁骨向窝中心放射，14天后，拔牙窝几乎被新生骨完全填充，并进行了重塑。拔牙创面观察到的任何愈合过程中，通过S-0染色测定，窝内未见软骨样细胞

CTGF gene transfer to the osteoblasts could be a promising strategy to accelerate bone formation. In this study, we investigated the possibility of recombinant adenovirus to transfer CTGF genes to a mouse osteoblastic (MC3T3-E1) cell line in vitro, and bone regeneration process after tooth extraction.Rrecombinant adenovirus encoding the LacZ gene (Ax1CALacZ) were applied to the MC3T3-E1 cells with 5, 10 and 50 multiplicity of infection (MOI). At one day after transfection, those cells were stained with X-gal. As the result, the MOI 50 transfection dosage produced almost 80% X-gal staining of the cells without any obvious cell damages. Recombinant adenovirus encoding the human CTGF gene with CAG promotor (Ax1CACTGF) were fabricated and applied to the MC3T3-E1 cells in the MOI 50 dose. CTGF mRNA translation and protein production by the transfected cells were assessed by RT-PCR and western blotting of the harvested cells using a prepared primer set and an anti-human-CTGF antibody at 1, 3 and 7 days after transfection. Ax1CACTGF transfection caused a marked up-regulation in CTGF mRNA expression and CTGF protein even 7 days after transfection.The sequential phase appearances of extraction wound healing were clearly observed by histological examination from 2 to 14 days after extraction. At 7 days after extraction, infilling with woven bone was observed from the socket margin with trabecular bone radiating toward the center of the socket, and at 14 days after, the extraction socket was almost fully filled by newly generated bone that underwent remodeling During any healing processes observed in the extraction wound, no cartilage-like cells were evident in the socket as determined by S-0 staining