The isolation and primary culture of putative human root odontoblasts

The use of procedures adapted from a routinely successful method of culturing bovine bone has led to the first system for the study of dentinogenesis in vitro. Two types of cells have been grown from pulp obtained from the growing root tips of impacted third molars extracted from 14- to 19-years olds: (1) epithelial-like cells that are probably derived from fragments of the epithelial root sheath and (2) odontoblast-like cells. The cultured epithelial-like cells grow out in distinctive rounded plaques while the odontoblast-like cells are tethered to and/or grow on top of the epithelial-like cells. The odontoblast-like cells produce mineralized matrix by 10 days when cultured on a defined mineralization formula containing conditioned medium obtained from fetal bovine bone cell cultures. Growth factors in this conditioned medium are important to cell proliferation and growth and to the synthesis of mineralized matrix. Sequential enzyme digestion in dispase and dispase/collagenase in serum-free Dulbecco's Modified Eagle's Medium is essential to obtaining adequate cell yields from the apical 3-5 mm of the developing root. Reduction of the number of fibroblasts by treating cultures with dispase in Tyrode's solution midway through the initial growth period enhances the purity of these cell cultures.