Ex Vivo Gene Delivery Using an Adenovirus Vector in Treatment for Dental Implant

本研究的目的是通过使用腺病毒载体的离体基因递送，为缩短牙种植体的愈合期和扩大其应用做出贡献。结缔组织生长因子（CTGF）在体内骨折愈合过程中表达上调，是体外成骨细胞增殖和分化的有效刺激因子。因此，CTGF基因转染成骨细胞可能是一种有前途的促进骨形成的策略。首先，我们研究了重组腺病毒在体外将CTGF基因转移到小鼠成骨细胞系（MC 3 T3-E1）中的可能性。将编码LacZ基因和具有CAG启动子的人CTGF基因的重组腺病毒（Ax 1CACTGF）以5、10和50的感染复数（MOI）应用于MC 3 T3-E1细胞。结果显示，MOI 50转染剂量下，X-gal染色率达80%，无明显细胞损伤; Ax 1CACTGF转染后7 d，CTGF mRNA表达和CTGF蛋白表达明显上调。第二，我们研究了CTGF在大鼠拔牙窝中的时空表达，以了解新牙槽骨形成的机制。结果显示，拔牙后4天内，CTGF在迁移到牙槽肉芽组织的内皮细胞中表达。拔牙后4、7、10、14 d，成骨样细胞在牙槽窝内大量增殖，CTGF表达。最后，我们尝试了体外基因转染，但不能从大鼠牙槽骨缺损中分离出成骨细胞，基于这些发现，腺病毒介导的CTGF基因转导到培养的成骨样细胞是非常成功的。然而，为了进行体外基因转移，需要发展新的成骨细胞分离方法。

The purpose of this study was to contribute to shortening the healing period of the dental implant and expanding its application by ex vivo gene delivery using an adenovirus vector. It has been well-documented that connective tissue growth factor (CTGF) is up-regulated in the healing process at the bone fracture site in vivo and known as a potent stimulator for the proliferation and differentiation of osteoblasts in vitro. Therefore, CTGF gene transfer to the osteoblasts could be a promising strategy to accelerate bone formation. First, we investigated the possibility of recombinant adenovirus to transfer CTGF genes to a mouse osteoblastic (MC3T3-E1) cell line in vitro. Recombinant adenovirus encoding the LacZ gene and the human CTGF gene with CAG promotor (Ax1CACTGF) were applied to the MC3T3-E1 cells with 5, 10 and 50 multiplicity of infection (MOI). As the result, the MOI 50 transfection dosage produced almost 80% X-gal staining of the cells without any obvious cell damages and Ax1CACTGF transfection caused a marked up-regulation in CTGF mRNA expression and CTGF protein even 7 days after transfection. Second, we investigated the spatial and temporal expression of CTGF in the rat tooth extraction sockets to know the mechanisms of new alveolar bone formation. As the result, CTGF was expressed in the endothelial cells migrating into the granulation tissue at the sockets during 4 days after tooth extraction. Osteoblast-like cells proliferated in the sockets with CTGF expression at 4, 7, 10 and 14 days after extraction. Finally, we tried the ex vivo gene delivery, but we could not isolate osteoblasts from the defects of rat alveolar bone.Based on these findings, adenovirus-mediated CTGF gene transduction to the cultured osteoblast-like cells was highly successful. However, in order to do the ex vivo gene transfer, it is necessary to develop the new methods of isolating osteoblasts.