Generating Tooth Organoids Using Defined Bioorthogonally Cross-Linked Hydrogels

Generating teeth in vitro requires mimicking tooth developmental processes. Biomaterials are essential to support 3D tooth organoid formation, but their properties must be finely tuned to achieve the required biomimicry for tooth development. For the first time, we used bioorthogonally cross-linked hydrogels as defined 3D matrixes for tooth developmental engineering, and we highlighted how their properties play a pivotal role in enabling 3D tooth organoid formation in vitro. We prepared hydrogels by mixing gelatin precursors modified either with tetrazine (Tz) or norbornene (Nb) moieties. We tuned the hydrogel properties (E = 2–7 kPa; G′ = 500–1500 Pa) by varying the gelatin concentration (8% vs 12% w/V) and stoichiometric ratio (Tz:Nb = 1 vs 0.5). We encapsulated dental epithelial-mesenchymal cell pellets in a library of hydrogels and identified a hydrogel formulation that enabled successful growth kinetics and morphogenesis of tooth germs, introducing a defined tunable platform for tooth organoid engineering and modeling.