Characterization of Biomimetic Dental Cell Sheet GelMA Tooth Bud Mineralization

Objectives: Our long-term goal is to create functional biomimetic tooth buds for eventual tooth replacement in humans. The objective of this study was to characterize mineralized dental tissue formation in a novel 3D biomimetic tooth bud model consisting of dental epithelial (DE), dental mesenchymal (DM) and HUVEC cell sheets (DE-HUVEC, DM-HUVEC), combined with dental cell-HUVEC encapsulated gelatin methacrylate (GelMA) hydrogel scaffolds.
Methods: Porcine DM or DE cells were seeded onto UpCell thermo-responsive tissue culture plates at cell densities of 0.114 and 0.228 cell 10⁶/cm², and HUVECs were seeded on top of each after 12 days in vitro culture. DE-HUVEC and DM-HUVEC cell sheets were harvested after 14 days. Biomimetic tooth buds were created from stacked DE-HUVEC/DM-HUVEC cell sheets sandwiched between GelMA encapsulated DE-HUVEC and DM-HUVEC cells (CS), and cultured in Osteogenic Media for 7 days prior to implantation in vivo for 1, 3 and 6 weeks. Acellular GelMA (G), GelMA encapsulated DE-DM cell constructs (GC), GelMA encapsulated DM cells (GM), and DM cell sheets + GelMA encapsulated DM cells (CSMG) constructs were also examined. In vivo mineralization was assessed and quantified using microCT (Bruker). Dental cell differentiation and neovascularization were assessed using histological and immunohistochemical analyses.
Results: In vivo implanted 3D tooth bud constructs exhibited mineralized tissue formation of specified size and shape, and mineralized tissue density increased over in vivo implantation time. The greatest volumes and highest density mineralized tissue formed in the CS-GelMA constructs after 6 weeks, as compared to other construct types. Evaluations of paraffin embedded and serial sectioned biomimetic tooth bud constructs are being performed using to characterize dental cell differentiation marker and neo-vasculature formation.
Conclusions: We propose our biomimetic 3D Gelma-CS tooth buds as models for dental cell differentiation and mineralized dental tissue formation leading to the formation of functional biomimetic replacement teeth.