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

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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.