The Secretome of the Inductive Tooth Germ Exhibits Signals Required for Tooth Development.

Teeth develop from reciprocal signaling between inductive and receptive cells. The inductive signals for tooth development are initially in the epithelium of the developing branchial arch, but later shift to the underlying mesenchyme of a developing tooth germ. The inductive signals that are needed for tooth development have not yet been fully identified. Our lab previously provided a basis for bioengineering new teeth by separating the tooth germ's epithelial and mesenchyme cells into a single cell population and recombing them. This approach, however, is not clinically applicable as the cells lose their inductive ability when expanded in vitro. In this study, we investigate whether the secretome and small extracellular vehicles (sEV) derived from inductive tooth germ mesenchyme can contribute to inductive signals required for tooth development. To address this, small extracellular vesicles and secretome were purified from inductive tooth germ mesenchyme and characterized. We investigated the proteome of sEV and proteome of inductive tooth germ mesenchyme and the impact of the culture condition and duration on the proteome. Additionally, we investigated the transcriptomic changes in tooth germ epithelium after treatment with sEV from inductive tooth germ mesenchyme. We show that culture duration of inductive tooth germ mesenchyme has an impact on the proteome of sEV purified from these cells. Similarly, culturing these cells in 2D and 3D environments results in different protein content. Proteome unique to sEV derived from inductive shows an association with multiple signaling pathways related to tooth development. Our RNASeq results show that treatment of tooth germ epithelial cells with small extracellular vesicles derived from inductive tooth germ mesenchyme results in an increased expression of some of the known odontogenic genes. Whilst further analysis is required to harvest the full potential of these sEV, our results suggests that extracellular vehicles contribute to signals required during tooth development, potentially through modulation of cellular metabolism and ECM organization.