Methods: Human periodontal ligaments (hPDLs) were isolated from third molar teeth. The periodontal tissues from middle one third of roots were separated by surgical blades. Tissues were minced in small pieces and deriving cells were cultured and resuspended in growth medium (GM). DMEM/F12 supplemented with 15% Fetal Bovine Serum were used for culturing as a GM. Cells were isolated by using a filtered paper soaked with digestive solution, 0.1% Trypsin and 0.02% EDTA/PBS (-), which was modified method from cloning ring and only the epithelial cells like colonies were isolated and observed under the phase contrast microscope from day 1 to 3 weeks. Specialized cells with tight cluster colonies were characterized by immunohistochemistry for determining the specific epithelial and endothelial markers. RT-PCR was also used for characterization the tight junction gene markers.
Results: The specialized cells were observed remarkably from epithelial colonies with the tight cluster arrangement and each colony were formed dependently. From day 1 to 3 weeks, cells were gradually changed in shape and arrangement with circular dimension appearances. Immunohistochemisty demonstrated that specialized cells shown the epithelial markers, which was a Cytokeratin but in the contrast to the endothelial markers, VE-cadherin, which was not detect. RT-PCR demonstrated distinguishably expression of the tight junction genes, which were Claudin-1, 2, 3, ZO-1, and Occludins.
Conclusions: Specialized cells derived from the restricted isolation method were characterized the remarkably expression in tight junction markers. These cells have been established through cell lines by specific tight cluster development. Thus, specialized cells may play the pivotal role and participate with transportation of ion exchange in periodontal tissue.