INVITRO FORMATION OF MINERALIZED NODULES BY PERIODONTAL-LIGAMENT CELLS FROM THE RAT

The purposes of this study were to determine whether periodontal ligament (PDL) cells are capable of producing mineralized nodules in vitro and to analyze ultra-structural features of the nodules. Rat PDL cells were obtained from coagulum in the socket at 2 days after tooth extraction and cultured at confluence in standard medium containing Dulbecco's Modified Eagle's Medium supplemented with 10% FBS and antibiotics. To test mineralized nodule formation, cells were further cultured for an additional 3 weeks in the standard medium containing (1) ascorbic acid (50-mu-g/ml) and sodium beta-glycerophosphate (10 mM), (2) ascorbic acid, sodium beta-glycerophosphate, and dexamethasone (5-mu-M), or (3) ascorbic acid alone. Cells were then fixed in 2.5% glutaraldehyde, postfixed in 1% OsO4, and prepared for light and electron microscopy. Three-dimensional nodules containing mineralized matrices were formed only when the cells were cultured in the presence of ascorbic acid and dexamethasone. They were composed of multilayered fibroblasts (up to 13 layers), and highly organized collagen fibrils with 64 nm cross-banding patterns between the cell layers. The fibroblasts in the nodules exhibited an elongated shape with a high degree of cytoplasmic polarity throughout the nodule, and have the morphological features of PDL fibroblasts as seen in vivo. Mineral deposition with needle-like crystals was initiated on collagen fibrils located in intercellular spaces of the upper cell layers and became increasingly heavier towards the bottom half of the nodules. X-ray microanalysis and electron diffraction analysis confirmed that mineral deposition contained calcium and phosphate in the form of immature hydroxyapatite. These nodules contained neither osteoblasts nor osteocytes, and have their own morphological organization and characteristics which differ from those formed by bone cells in culture. Therefore, these data suggest that PDL cells are capable of forming mineralized tissue in vitro with the morphological characteristics different from bone mineralized nodules.