Tooth-on-a-chip to engineer early dental epithelial-mesenchymal interaction

Tooth enamel-the highly mineralized outer layer shielding teeth from mechanical and chemical wear-is produced during development by specialized dental epithelial (DE) cells called ameloblasts. Functional enamel is challenging to regenerate because the enamel-forming cells-DE-derived ameloblasts-disappear even before tooth eruption. To achieve regeneration, DE and dental mesenchymal (DM) need to interact. Current in-vitro models encompass only limited DE-DM interaction as they offer little control over the interface geometry and usually force both lineages to share a single, sub-optimal medium, leading to variable outcomes. Hence, herein we developed a micro-engineered "tooth-on-a-chip" that shapes the DE-DM interface while independently perfusing lineage-specific media. A three-channel polydimethylsiloxane (PDMS) device, fabricated by rapid 3-D printing and soft lithography, traps DM cells in a fibrin hydrogel between two fluid channels. Within 3 days the DM cells condensed into a papilla-like core and secreted collagen, creating a signaling niche for sequential seeding of DE cells. Quantitative PCR (qPCR) of DM cells showed progressive up-regulation of odontogenic markers, while immunofluorescence confirmed ameloblast differentiation of the DE layer through AMELX expression. Alizarin Red staining, calcium assays, SEM confirmed progressive, spatially confined mineralization at the engineered interface. Although this platform now is in its proof-of-concept stage, it provides the first controllable, dual-medium microenvironment that recapitulates early odontogenesis, amelogenesis, and mineral deposition in a single device, offering a versatile tool for dissecting tooth morphogenesis and accelerating true enamel-regeneration strategies.