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In regenerating periodontal ligament (PDL) around the root of an artificial tooth, an important role is played by some physiologically active substance that promotes adhesion of the cells to the surface of the tooth root and induces cell proliferation and differentiation. In this study, the supernatant of the conditioned medium (CM) of dog periodontal ligament fibroblast (DPLF) was fractionated using an ion exchange chromatography-diethylaminoethyl (IEC-DEAE) column. DPLFs were cultured on hydrophobic dishes coated with each fraction. Cell proliferative activity and alkaline phosphatase (ALPase) activity, including electron microscopic features of the contact surface between the cells and the dish, were investigated. The DPLF-CM was separated by IEC-DEAE column into six fractions. Each fraction promoted an increase in DNA content and ALPase activity of the cultured DPLF, and especially remarkable were fractions 2 and 3. Fraction 2 at a molecular weight (Mw) of 210, 160, 85, 50 and 22 kD, and fraction 3 at Mw = 21 and 23 kD contained the type of proteins not found in other fractions. Electron microscopic analysis revealed that the cells in the coating group were in close contact with the surface of the dishes and that fine fibers protruding from the cell membrane clinged to the dishes. In the control group, a wide gap between the cells and the dishes was observed. These findings suggest that the DPLF-CM fractions contain specific physiological activating factors that induce proliferation and differentiation as well as cell adhesion of the DPLF cells.