Mineralized Matrix Synthesis by Isolated Mouse Odontoblast-like Cells In Vitro

First mandibular molar tooth germs were dissected from 17 day mouse embryos. The dental papilla was isolated using both mechanical separation and enzymatic digestion. The cells of the papilla were then enzymatically disaggregated and cultured in 35 mm polystyrene dishes containing alpha minimum essential medium supplemented with 15 % fetal calf serum, 50 μglml ascorbic acid, 1 o-8 M dexamethasone and 10 mM Na-13-glycerophosphate. The cultures were maintained for 23 days. The cultured cells initially appeared as large flat cells having numerous cell processes. Multilayered cell nodules, distributed randomly in the cultures, were apparent after 5 days. Matrix was visible within these nodules after 15 days. At day 23, histochemical staining identified alkaline phosphatase activity and von Kossa stained mineralized matrix localized to the cell nodules. Ultrastructurally, the cultured cells displayed classic characteristics associated with the odontoblast phenotype. Electron diffraction pattern analysis and energy dispersive X-ray microanalysis, of the mineralized matrix, identified the co-localization of Ca and P in the form of hydroxyapatite crystallites. These results indicate that it is possible to grow mouse embryonic dental papilla cells, in vitro, and that these cells will form multilayered nodules of odontoblast-like cells which produce a mineralized matrix. This reproducible method of cell culture lends itself not only to the study of odontoblast differentiation, but also the development of in vitro methods of assessing the biological effects of both restorative materials and other chemical agents