Three-dimensional Micro-culture System for Tooth Tissue Engineering

The arrangement of cells within a tissue plays an essential role in organogenesis, including tooth development. Progress is being made to regenerate teeth by reassociating dissociated embryonic dental cells and implanting them in vivo. In the present study, we tested the hanging drop method to study mixed epithelial-mesenchymal cell reorganization in a liquid instead of semisolid medium to see whether it could lead to tooth histogenesis and organogenesis. This method allowed the control of the proportion and number of cells to be used, and the forming microtissues showed homogeneous size. The liquid environment favored cell migrations as compared with collagen gels. Three protocols were compared. The one that sequentially combined the hanging drop and semisolid medium cultures prior to in vivo implantation gave the best results. Indeed, after implantation, teeth developed, showing a well-formed crown, mineralization of dentin and enamel, and the initiation of root formation. Vascularization and the cellular heterogeneity in the mesenchyme were similar to what was observed in developing molars. Finally, after coimplantation with a trigeminal ganglion, the dental mesenchyme, including the odontoblast layer, became innervated. The real advantage of this technique is the small number of cells required to make a tooth. This experimental model can be employed to study the development, physiology, metabolism, or toxicology in forming teeth and test other cell sources.