Despite robust and meritorious effort, tooth regeneration is yet to be realized in patients. One of the crucial barriers of tooth regeneration is our insufficient understanding of cell communication between dental epithelium and mesenchyme. Failure of postnatal dentin regeneration is attributed to absent epithelium signaling that no longer exists with ameloblasts undergoing apoptosis following the completion of enamel formation and tooth eruption. In a series of experiments, we first discovered that exosomes were secreted by both dental epithelium and mesenchyme, and diffused through the basement membrane. Exosomes not only were endocytosed by epithelium or mesenchyme stem cells, but also evoked due cellular functions reciprocally in lieu of their source cells, leading to amelogenesis and dentinogenesis. Attenuated exosomal secretion by Rab27a/b knockdown disrupted basement membrane formation, and reduced dentin formation in reconstituted tooth organs, as in Rab27aash/ash mutant mice. We then profiled exosomal constituents by microRNA array and mass spectrometry, and identified several pivotal targets that regulate dentinogenesis. The overall objectives of this proposal, in response to PAR-16-242 (Bioengineering Research Grants, BRG), are to understand the roles of exosomes in epithelium-mesenchyme crosstalk and to formulate strategies towards delivery of exosomes or selected exosomal constituents that enable dentin regeneration. Not only is dentin regeneration of scholarly interest, but also has immense clinical impact. Despite advances in dental care, a total of 33 million individuals in the United States will be without any teeth in either the maxilla or mandible by 2020. Many teeth in children and adults can be saved if dentin regenerates.