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Considerable amount of research has been focused on dentin mineralization, odontoblast differentiation, and their application in dental tissue engineering. However, very little is known about the differential role of functionally and spatially distinct types of dental epithelium during odontoblast development. Here we show morphological and functional differences in dentin located in the crown and roots of mouse molar and analogous parts of continuously growing incisors. Using a reporter (DSPP‐cerulean/DMP1‐cherry) mouse strain and mice with ectopic enamel (Spry2 +/− ;Spry4 −/− ), we show that the different microstructure of dentin is initiated in the very beginning of dentin matrix production and is maintained throughout the whole duration of dentin growth. This phenomenon is regulated by the different inductive role of the adjacent epithelium. Thus, based on the type of interacting epithelium, we introduce more generalized terms for two distinct types of dentins: cementum versus enamel‐facing dentin. In the odontoblasts, which produce enamel‐facing dentin, we identified uniquely expressed genes ( Dkk1 ,  Wisp1 , and  Sall1 ) that were either absent or downregulated in odontoblasts, which form cementum‐facing dentin. This suggests the potential role of Wnt signalling on the dentin structure patterning. Finally, we show the distribution of calcium and magnesium composition in the two developmentally different types of dentins by utilizing spatial element composition analysis (LIBS). Therefore, variations in dentin inner structure and element composition are the outcome of different developmental history initiated from the very beginning of tooth development. Taken together, our results elucidate the different effects of dental epithelium, during crown and root formation on adjacent odontoblasts and the possible role of Wnt signalling which together results in formation of dentin of different quality. © 2021 The Authors.  Journal of Bone and Mineral Research  published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

The Development of Dentin Microstructure Is Controlled by the Type of Adjacent Epithelium