Analysis of gingival epithelial cells and fibroblast using three-dimensional culture system mimic to periodontal pockets

为了探讨牙周病细菌对牙周组织破坏的机制，我们用PIPAAm建立了三维牙龈上皮细胞和成纤维细胞共培养系统。将牙龈上皮细胞接种于Rep细胞板（CellSeed, Tokyo），孵育至融合。然后，上皮细胞在32℃下孵育30 min。孵育后，将上皮细胞作为细胞片取出。然后将上皮细胞片置于1型胶原培养的成纤维细胞上。上皮细胞与成纤维细胞共培养1周后，获得牙周袋三维牙龈重建模拟物。我们比较了三维牙龈重建培养与正常牙龈中claudin、cadherin、JAM-1、紧密连接成分和粘附连接成分的表达。claudin、cadherin和JAM-1在三维牙龈重建培养中的表达模式与正常牙龈相同。然后，我们检测了claudins在人牙周袋中的表达模式。在人牙周袋中，claudins-1、4、7、12组成表达。此外，在三维牙龈重建培养中检测到这些claudin。这些数据表明，我们的三维牙龈重建培养是研究牙周袋与牙周病细菌相互作用的有用工具。在炎症组织中，claudin-10的表达水平明显高于正常组织。虽然这一现象的机制尚不清楚，但这些数据表明，claudin-10在牙周组织破坏的发生中发挥了重要作用。

To investigate the mechanisms that periodontopathic bacteria cause periodontal tissue destruction we established the three-dimensional co-culture gingival epithelial cell and fibroblast) system using PIPAAm. Gingival epithelial cells were seeded to Rep Cell plate (CellSeed, Tokyo) and were incubated to confluent. Then, epithelial cells were incubated at 32℃ for 30 min. After the incubation epithelial cells were removed as a cell sheet. Then the epithelial cell sheet was placed onto fibroblasts cultured in type 1 collagen. After co-culture of epithelial cells and fibroblasts for a week, we obtained the three-dimensional gingiva-reconstructive culture mimic to periodontal pockets. We compared the expression of claudin, cadherin, JAM-1, component of tight junction and adherence junction, in normal gingival to that of the three-dimensional gingiva-reconstructive culture. The expression pattern of claudin, cadherin, and JAM-1 in the three-dimensional gingiva-reconstructive culture was identical to that of normal gingival.Then, we examined the expression pattern of claudins in human periodontal pockets. In human periodontal pockets claudins-1, 4, 7, 12 were expressed constitutively. Moreover, those claudins were detected in three-dimensional gingiva-reconstructive culture. This data suggested that our three-dimensional gingiva-reconstructive culture was a useful tool to investigate the interaction between periodontal pockets and perioidontopathic bacteria. In inflamed tissues, the expression level of claudin-10 was much increased than in normal tissues. Though the mechanism of this phenomenon is unclear, this data suggested that claudin-10 played important role in developing periodontal tissue destruction.