GROWTH AND DIFFERENTIATION OF PERIODONTAL LIGAMENT-DERIVED CELLS IN SERUM-FREE DEFINED CULTURE

We have developed a serum-free medium for the growth and differentiati on of periodontal ligament-derived cells (PLC). In addition, the expre ssion of both fibroblast growth factor (FGF) and FGF receptor (FGFR) i n the PLC was investigated by immunohistochemical examination, heparin affinity chromatography (HAC), and reverse transcription-polymerase c hain reaction (RT-PCR) analysis. Optimal growth of the cells was achie ved in Iscove's modified Dulbecco's medium supplemented with insulin, transferrin, 2-mercaptoethanol, 2-ethanolamine, sodium selenite, and o leic acid in type-I collagen-coated dishes. Both FGF-1 and FGF-2 stimu lated cell growth and inhibited differentiation as measured by inhibit ion of alkaline phosphatase activity of the cells. An immunohistochemi cal analysis of FGF-1 and FGF-2 revealed that immunoreactive FGF-1 and FGF-2 were detected predominantly in the cytoplasm of growing cells. In addition, perinuclear FGF-1 staining and nuclear FGF-2 staining wer e observed in the same growing cells. In contrast, a faint diffuse sta ining of FGF-1 and FGF-2 was detected in cytoplasm of the confluent di fferentiated cells. The 2.15 M NaCl eluate from HAC of the cell extrac ts exhibited growth-promoting activities for the PLC, and it also stim ulated the growth of human umbilical vein-derived endothelial cells an d inhibited binding of [I-125]-FGF to its receptors, indicating the ce lls produced FGFs or FGF-like growth factors. RT-PCR analysis revealed that the cells expressed FGFR-1 mRNA but not mRNAs for FGFR-2, FGFR-3 and FGFR-4 mRNA. These results suggest that the FGF-FGFR-1 system pla ys an important role in the growth and differentiation of periodontal ligament-derived cells.