Metabolic expression of intrinsic developmental programs for dentine and enamel biomineralization in serumless, chemically-defined, organotypic culture

Abstract

Biomineralization was investigated using embryonic mouse mandibular first molars (M1) cultured in the presence or absence of fetal calf serum. Metabolic features including cell division and Ca2+ and phosphate incorporation into dentine and enamel extracellular matrices were analyzed. The relative timing and magnitude of DNA synthesis for serumless cultures was comparable to in vivo controls. Isotopic calcium and phosphate incorporation into the mineral phase of dentine and enamel matrices, in the absence of serum, fluctuated during development. Molar tooth morphogenesis, cytodifferentiation, and extracellular matrix formation approximated late crown-stage development in serumless cultures. Von Kossa histochemical staining indicated calcium phosphate salt formation in serumless cultures. Analysis of anhydrous fixation-prepared enamel and dentine representing serumless cultured explants indicated that crystal size and orientation were comparable to in vivo enamel and dentine. In contrast, serum-supplemented cultures showed atypical crystal size and orientation. Calcium/phosphorous (Ca/P) ratio values for serumless cultures after 21 days showed Ca/P enamel values of 2.03 (SD +/- 0.04, p less than 0.025) and dentine values of 1.89 (SD +/- 0.01, p less than 0.025). Electron diffraction patterns of enamel and dentine formed in serumless cultures were principally those of highly-ordered crystalline hydroxyapatite. Our results suggest that tissue-specific dentine and enamel biomineralization is regulated by endogenous factors intrinsic to the developmental program of embryonic tooth organs during serumless culture.