Generating\nTooth Organoids Using Defined Bioorthogonally\nCross-Linked Hydrogels

Generating teeth <i>in vitro</i> requires mimicking\ntooth\ndevelopmental processes. Biomaterials are essential to support 3D\ntooth organoid formation, but their properties must be finely tuned\nto achieve the required biomimicry for tooth development. For the\nfirst time, we used bioorthogonally cross-linked hydrogels as defined\n3D matrixes for tooth developmental engineering, and we highlighted\nhow their properties play a pivotal role in enabling 3D tooth organoid\nformation <i>in vitro</i>. We prepared hydrogels by mixing\ngelatin precursors modified either with tetrazine (Tz) or norbornene\n(Nb) moieties. We tuned the hydrogel properties (<i>E</i> = 2–7 kPa; <i>G</i>′ = 500–1500 Pa)\nby varying the gelatin concentration (8% vs 12% w/V) and stoichiometric\nratio (Tz:Nb = 1 vs 0.5). We encapsulated dental epithelial-mesenchymal\ncell pellets in a library of hydrogels and identified a hydrogel formulation\nthat enabled successful growth kinetics and morphogenesis of tooth\ngerms, introducing a defined tunable platform for tooth organoid engineering\nand modeling.