[Biochemical study on the TGF-beta like growth factor derived from bovine periodontal ligament].

Abstract

The effect of matrix components extracted Bovine periodontal ligament (PDL) on cell proliferation and alkaline phosphatase activity of cultured human periodontal ligament fibroblast (HPLF) were examined in order to understand the cell-tissue interaction of periodontal ligament. Bovine PDL tissue was sequentially extracted with 0.05 M Tris HCl buffer, pH 7.4, containing 1M NaCl or 4M GdmCl. After seeding 24 hours, the cultured HPLF were exposed to the extracts for two through eight days. Nine days after seeding, HPLF indicated four times high activity on ALP and 1.6 times the amount of total protein than those of control (without extract), while DNA synthesis increased only 1.2 times. On the contrary, the NaCl extract depressed the ALP activity of HPLF. The GdmCl extract enhanced both the total protein and ALP activity in dose-dependently. The ALP increasing activity of GdmCl extract on HPLF is stable to heat (78 degrees C, 20 min) and collagenase treatment but partially inactivated by trypsin digestion. Since the GdmCl extract also induced colony formation of NRK-49F cell in soft agarose, it was suggested that the extract contain EGF and TGF-beta like factor. Molecular size of this factor was estimated as 20-50Kd using Sepharose CL-6B gel chromatography. Furthermore, this factor from Sepharose CL-6B were separated into two forms by ion exchange CM-Sepharose column chromatography. Purified preparation from reversed phase column chromatography contained 14Kd, 15Kd, 17Kd, 20Kd, 28Kd40Kd, and 46Kd components on SDS-PAGE. This factor may accumulate in extracellular matrix, and may play a role of cell-tissue interaction and homeostasis in periodontal ligament.