Synthesis of noncollagenous extracellular matrix proteins during development of mineralized nodules by rat periodontal ligament cells in vitro.

To characterize the mineralized nodules produced by rat periodontal ligament (PDL) cells in vitro, we have studied the synthesis and distribution of mineralized tissue proteins at various stages of nodule formation. PDL cells were obtained from coagulum in the socket at 2 days after tooth extraction and cultured in Dulbecco's Modified Eagles Medium (DMEM) containing 10% fetal bovine serum and antibiotics. Confluent cells were grown in the presence of ascorbic acid (50 micrograms/ml), dexamethasone (5 microM), and beta-glycerophosphate (10 mM) for 3 weeks. Four stages showing distinct morphological characteristics during development of mineralized nodules were identified. Protein synthesis and deposition of proteins into the matrix were studied during these stages by metabolic labeling with [35S]methionine for 24 hours. Large quantities of SPARC (secreted protein, acidic and rich in cysteine) were synthesized by confluent cells but decreased during the progress of mineralized nodule formation. Two forms of osteopontin (OPN) (67 kDa and 61 kDa) were synthesized in comparable quantities by confluent cells; OPN and bone sialoprotein (BSP) were induced by dexamethasone and represented the major proteins in the mineralized matrix. The 67 kDa form of OPN was the predominant species in the mineralized matrix. Both OPN and BSP were localized by immunogold electron microscopy on globular as well as fused electron-dense structures at sites of tissue mineralization.