Methods: Human DM and Porcine DE cells were cultured according to previous publications. DM cells were mixed with 5% GelMA (Dr. Ali Khademhosseini, Harvard Univ.) solution with 0.1% Irgacure2959, and injected into a 6 mm diameter mold. UV light was used to cross link the gels. DE cells were mixed with ExtracelTM hydrogel solution (Glycosan Biosystems, Almeda, CA), and injected on top of the cross-linked DM-GelMa layer. Extracel gelation occurred within 20 minutes. Three sample groups – 1) DM-DE co-culture, 2) DM-only and 3) DE-only - were cultured in vitro for 3 weeks. Hematoxyline and Eosin (H&E), Immunohistochemical (IHC) and Immunofluorescent (IF) staining were performed to evaluate cell attachment, polarization, and differentiation. Real time PCR (qRT-PCR) analyses was used to measure the expression of DE-DM cell differentiation markers. DM and DE cells were stained with fluorescent Cell-Tracker, and monitored via Confocal microscopy to characterize the cell polarization and proliferation.
Results: Cell tracker stained DE and DM cells both exhibited fluorescent signals after being seeded into hydrogel and GelMA materials. H&E staining showed cell interactions at the junction of two gel layers. qRT-PCR analyses are ongoing.
Conclusions: In conclusion, we report the use of novel hydrogel-Gelma constructs of potential use for dental tissue engineering applications.