Characteristic changes of periodontal ligament‐derived cells during passage

Abstract

Although periodontal ligament-derived cells are expected to be a useful source of cells for periodontal tissue engineering, the characteristic changes of primary cultured cells have not been well studied. Therefore, the aim of this study was to investigate the characteristics of periodontal ligament-derived cells and their changes during passage. Human periodontal ligament tissue was obtained from extracted third molars. Cells were subcultured until passage 6 and the cell characteristics from early to late passages were evaluated using immunofluorescence microscopy, alkaline phosphatase activity analyses, reverse transcription-polymerase chain reaction and quantitative real-time polymerase chain reaction. To examine the function of periodontal ligament-derived cells further, cells were transplanted into the renal subcapsule of an immunocompromised rat. Immunofluorescence results showed relatively uniform expression of MSX-2 and osteonectin from passage 1 until passage 6. The STRO-1-positive fraction was 33.5% at passage 0, which was reduced to 14.7% at passage 3. Cultured cells at passage 1 expressed mRNA for collagen type I, collagen type XII, Runx2, alkaline phosphatase, osteonectin, osteopontin, scleraxis, tenomodulin, Msx2, GDF5 and GDF7 genes, but not for bone sialoprotein. The level of mRNA expression from tenomodulin and collagen type XII genes decreased after passage 3. Alkaline phosphatase activity decreased in cells at later passages. Osteogenic induction of periodontal ligament-derived cells resulted in a down-regulation of the tenomodulin gene. Transplanted cells from both early and late passages produced dense collagen fiber bundles without calcified tissue. Cultured periodontal ligament-derived cells were a morphologically homogeneous population, although expression of STRO-1 was limited in primary culture. Cultured cells showed de-differentiation during passage for both osteogenesis- and tendo/ligamentogenesis-related genes.