Methods: All experiments in this study complied with the guidelines of the Ethical Review Board of Tokyo Medical and Dental University. Human primary dental pulp cells (P3-P5) were obtained from collagenase-digested pulp tissues, which were derived from extracted third molars under informed consent. Mouse dental papilla strain cells (MDP), which were clonal mouse dental papilla cells immortalized by human papillomaviruses type 16 E6 gene deleted with PDZ domain binding motif, were also used. These dental pulp cells were seeded to 96-well flat plates (Greiner; 2-D culture) or 96 well PrimeSurface plates (Sumilon; 3-D spheroid culture) at a concentration of 3×104 cells/well. After 4 days, total RNA was extracted (QuickGene, FujiFilm), and cDNA was synthesized (RevertAid, Fermentas). Expression of odontoblastic and osteoblastic markers was evaluated by real-time PCR (GoTaq® qPCR Master Mix, Promega: CFX96, BioRad). Statistical significance was evaluated by one-way analysis of variance and Tukey-Kramer (p<0.05). Alkaline phosphatase (Alp) activity was also detected by enzyme histochemistry on 4% PFA fixed samples.
Results: Compared to 2-D culture, odontoblastic (Dspp) and osteoblastic (Alp and Bsp) marker expressions of human primary dental pulp cells and MDP were significantly up-regulated by 3-D spheroid culture. Alp activity of these pulp cells was also up-regulated by the 3-D spheroid culture.
Conclusion: 3-D spheroid culture enhanced the odontoblastic and osteoblastic properties of dental pulp cells.