Honda M J, Shinohara Y, Hata K I, Ueda M
Tooth Regeneration, Division of Stem Cell Engineering, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639 Japan.
Cell Transplant. 2007;16(8):833-47. doi: 10.3727/000000007783465208.
We showed in a previous study that odontogenic epithelial cells can be selectively cultured from the enamel organ in serum-free medium and expanded using feeder layers of 3T3-J2 cells. The subcultured odontogenic epithelial cells retain the capacity for ameloblast-related gene expression, as shown by semiquantitative RT-PCR. The purpose of the present study was to evaluate the potential of subcultured odontogenic epithelial cells to form tooth structures in cell-polymer constructs maintained in vivo. Enamel organs from 6-month-old porcine third molars were dissociated into single odontogenic epithelial cells and subcultured on feeder layers of 3T3-J2 cells. Amelogenin expression was detected in the subcultured odontogenic epithelial cells by immunostaining and Western blotting. The subcultured odontogenic epithelial cells were seeded onto collagen sponge scaffolds in combination with fresh dental mesenchymal cells, and transplanted into athymic rats. After 4 weeks, enamel-dentin-like complex structures were present in the implanted constructs. These results show that our culture system produced differentiating ameloblast-like cells that were able to secrete amelogenin proteins and form enamel-like tissues in vivo. This application of the subculturing technique provides a foundation for further tooth-tissue engineering and for improving our understanding of ameloblast biology.
我们在先前的研究中表明，牙源性上皮细胞可以在无血清培养基中从釉质器官中选择性培养，并使用3T3-J2细胞饲养层进行扩增。如半定量逆转录聚合酶链反应所示，传代培养的牙源性上皮细胞保留了与成釉细胞相关基因表达的能力。本研究的目的是评估传代培养的牙源性上皮细胞在体内维持的细胞-聚合物构建物中形成牙齿结构的潜力。将6个月大猪的第三磨牙的釉质器官解离为单个牙源性上皮细胞，并在3T3-J2细胞饲养层上传代培养。通过免疫染色和蛋白质印迹法在传代培养的牙源性上皮细胞中检测到釉原蛋白的表达。将传代培养的牙源性上皮细胞与新鲜的牙间充质细胞一起接种到胶原海绵支架上，并移植到无胸腺大鼠体内。4周后，植入的构建物中出现了釉质-牙本质样复合结构。这些结果表明，我们的培养系统产生了能够分泌釉原蛋白并在体内形成釉质样组织的分化成釉细胞样细胞。这种传代培养技术的应用为进一步的牙齿组织工程和增进我们对成釉细胞生物学的理解奠定了基础。
