Isolation and partial characterization of mitogenic factors from cementum

Cementum is the mineralized structure through which soft connective tissues are attached to the teeth. It is a unique calcified tissue characterized by a low metabolic turnover, lack of blood supply, and presence of very few cells. However, it contains substances that influence the biological activities of fibroblasts of adjacent soft tissues. We have partially characterized cementum proteins that have mitogenic activity toward fibroblasts. Cementum was harvested from bovine teeth, and mitogenic factors were extracted in 0.5 M CH3COOH. Heparin-Sepharose chromatography separated the mitogenic activity into a major and a minor fraction eluted by 0.5 and 2.0 M NaCl, respectively. The distribution of cementum mitogens in heparin-Sepharose fractions was different from that of alveolar bone and other bones. The cementum mitogenic factor eluting with 2.0 M NaCl from a heparin-Sepharose column was shown to be basic fibroblast growth factor (bFGF) on the basis of inhibition by anti-bFGF antibody and Western blots. The 0.5 M NaCl fraction was purified by HPLC with use of a combination of a DEAE-3W column followed by TSK-250 and C18 columns. NaDodSO4-polyacrylamide gel electrophoresis revealed that the purified fraction contained two protein bands with Mr 22,000 and 19,000, and mitogenic activity was associated with the Mr 22,000 species. The activity of this mitogen, designated as CGF, was potentiated by small quantities of plasma-derived serum or epidermal growth factor. It was heat resistant, but was destroyed by reduction. Assays of CGF preparations revealed that they contained no detectable platelet-derived growth factor.