EXPRESSION OF EXTRACELLULAR-MATRIX PROTEINS IN HUMAN PERIODONTAL-LIGAMENT CELLS DURING MINERALIZATION IN-VITRO

PERIODONTAL REGENERATION IS A COMPLEX PROCESS that requires coordinate d responses from several cell types within the periodontium. It is gen erally accepted that the periodontal ligament (PDL) has a heterogeneou s cell population, where some of the cells may be capable of different iating into either cementoblasts or osteoblasts. Thus, it has been hyp othesized that PDL cells play a role in promoting periodontal regenera tion. However, definitive evidence to support this concept is lacking. Previously, we reported that PDL cells induce biomineralization as de termined by Von Kossa histochemistry and transmission electron microsc opy. To further determine the osteoblast-like properties of PDL cells, human PDL cells were exposed to dexamethasone (DEX) in order to promo te an osteoblast phenotype, and then cell activity monitored during mi neral nodule formation in vitro. For mineralization studies, cells wer e cultured in DMEM containing 10% FBS and a) vehicle only; b) ascorbic acid (50 mu g/ml) and beta-glycerophosphate (10 mM); or c) ascorbic a cid, beta-glycerophosphate and DEX (100 nM) for 30 days. In addition, the effects of DEX on PDL cells in non-mineralizing media were determi ned. Cells were stained weekly to evaluate mineral-like nodules, using the Von Kossa method. Northern blot analyses for mRNA steady state le vels for several bone-associated proteins, i.e., osteopontin (OPN), bo ne sialoprotein (BSP), alkaline phosphatase (ALP), osteocalcin (OCN), alpha(2)(1)(type 1) collagen and osteonectin (ON), were performed. DNA levels were also determined during the 30-day mineralization period. Under phase contrast microscopy, PDL cells in non-mineralizing media t reated with DEX exhibited a more spindle-shaped morphology when compar ed with similar cells not exposed to DEX. Mineralizing conditions were required to induce mineral nodule formation. However, in this situati on, mineral induction was independent of DEX; and furthermore, DEX-tre ated cells did not exhibit a different morphological pattern when comp ared with non-DEX treated cells. Mineral-like nodules wen first seen a l day 15, in concert with an increase followed by a decrease in expres sion of type I collagen and ON mRNA in both DEX-treated and non-treate d cultures. Using Northern blot analysis for detection of specific pro teins, we found that PDL cells did not express OPN, BSP, OCN, or ALP u nder any of the conditions used in this study. DEX did not alter DNA c ontent in the cultures during the mineralization period. These results confirm that human periodontal Ligament cells can be induced to miner alize in vitro and indicate that dexamethasone does not significantly alter the extent and pattern of mineralization.