A NOVEL ORGAN-CULTURE METHOD TO STUDY THE FUNCTION OF HUMAN ODONTOBLASTS IN-VIVO - GELATINASE EXPRESSION BY ODONTOBLASTS IS DIFFERENTIALLY REGULATED BY TGF-BETA-1

Odontoblasts cannot be cultured by traditional cell culture methods, t hus restricting in vitro studies. Here we present an organ culture met hod for human odonto-blasts that utilizes the pulp chamber as a cultur e crucible. Crowns of human third molars were dissected, pulp was gent ly removed, and the odontoblasts attached to and in the walls of the p ulp chambers were cultured in serum-free OPTIMEM medium, or DMEM/Ham's F12 medium containing 10% serum. Pulp tissues were cultured separatel y. Cell content and morphology were analyzed by SEM, and the removed p ulps were examined by light microscopy. Proteins secreted into the med ium with or without TGF-beta 1 supplementation were metabolically labe led with [S-35]methionine, and the total protein content was assessed by TCA precipitation and SDS-PAGE/fluorography. To assess the role of gelatinolytic enzymes on dentin matrix remodeling, we used enzymograph y to analyze the effect of TGF-beta 1 on gelatinase A and B expression . SEM revealed odontoblasts in pulp chambers after 5 days of culture, with only few or no fibroblasts, and no alterations in the odontoblast cell morphology or differences between the cells cultured in serum-fr ee and serum-containing media. Rarely were any odontoblasts present in pulp tissue. Radiolabeling revealed protein synthesis and secretion u ntil day 6 in both the odontoblast and pulp cultures, with no marked d ifferences between TGF-beta 1-treated and control cultures. The level of gelatinase A remained constant up to 7 days, while gelatinase B exp ression was always low and decreased with time in culture. However, ge latinase B levels were markedly increased upon TGF-beta 1 treatment of cells and remained high to day 7. The results suggest that this metho d provides a novel technique for the study of human odontoblasts in vi tro and that odontoblasts can be cultured even in serum-free condition s.