Study of differentiation and proliferation of epithelium of radicular cyst model using three-dimensional culture

本研究的目的是研究胶原凝胶三维培养后牙周韧带上皮细胞的分化和增殖情况。从猪乳牙PDL中获得上皮细胞和成纤维细胞。先培养中间第3根的PDL，再传代第2 ~ 3根的外生上皮细胞和成纤维细胞。将含有IxlO5成纤维细胞/ml的胶原凝胶在5% CO_2室温下37℃培养，第二天加入上皮细胞（8 × 10^5/cm^2表面）。凝胶被抬升在金属网格上，在空气/介质界面下孵育。培养1、2、3、4周后收集组织，包埋OCT复合物或石蜡。石蜡包埋切片用H-E或Masson-Trichrome染色。冷冻切片进行角蛋白免疫染色。将标本与猪口腔牙龈进行对照。培养组织的厚度呈垂直下降趋势，但面积基本不变。随着时间的推移，颜色从透明变为不透明。组织学标本显示，1周后增殖的上皮细胞呈层状结构，而对照组未见层状鳞状上皮，呈4层分化。2周后观察上皮细胞的分散趋势。免疫组化结果显示，培养组织的上皮细胞表达角蛋白，但上皮细胞不能像对照组那样分类。PDL细胞在三维培养组织中可识别成层状上皮层，但从免疫组化表达和组织学表现上看，培养组织与口腔牙龈不同。

The purpose of this study was to investigate the differentiations and proliferations of epithelial cells of the periodontal ligament (PDL) after three-dimensional culture using collagen gel.The epithelial cells and fibroblasts were obtained from PDL of porcine primary teeth. The PDL at the middle third root were primarily cultured, and then the outgrowth epithelial cells and fibroblasts were passaged at second to third. The collagen gel with IxlO5 fibroblast/ml was incubated at 37℃ in 5% CO_2 chamber and added the epithelial cells (8x10^5/cm^2 surfaces) on the following day. The gels were lifted up on the metal grids and incubated under the air/medium interface. The cultured tissues were collected after 1, 2, 3 and 4 weeks, and embedded in OCT compound or paraffin. The paraffin-embedded sections were stained with H-E, or Masson-Trichrome. The cryo-sections were immunostained for keratin. The specimens were compared with the porcine oral gingiva for control.The thickness of the cultured tissues decreased vertically, but the area was almost same during the experimental term. The color visually changed from clear to opaque with passage time. The histological specimens showed that the proliferated epithelial cells had a stratified structure after 1 week, but there was no layer such as the stratified squamous epithelium with four differentiated layers in the control. The dispersing tendency of the cells in the epithelial layer was seen after 2-week incubation. The immunohistochemical findings showed that the epithelium of the cultured tissue were expressed keratin, but the epithelial cells could not be classified as was observed in the control.The stratified epithelial layer was recognized in the three-dimensional cultured tissues by PDL cells, but the cultured tissues were not same as oral gingiva from the immunohistochemical expressions and histological findings.