Effects of Wnt10a on dentinogenesis of dental pulp cells in a 3-dimensional culture system using atelocollagen gel.

The culture systems for dental pulp cells are contributing to the development of vital pulp therapy. In general, 2-dimensional (2D) monolayer culture systems are typically adopted to analyze the mechanisms underlying dental pulp cell differentiation. However, several limitations exist to reproducing the natural micro-environment of dental pulp tissue using such 2D culture systems. The aim of the present study was to establish a 3-dimensional (3D) culture system for dental pulp cells using atelocollagen gel and to determine whether the resulting 3D culture system can be used examine the effects of Wnt10a on dentinogenesis. The dental pulp cells were isolated from rat lower incisors and mixed into atelocollagen gel with recombinant Wnt10a (Wnt group) or phosphate-buffered saline (Cont group) for the 3D culture system. Cells added into atelocollagen gel were cultured for 15 days and mineralized nodule formation and mRNA expressions (Dentin sialophosphoprotein [Dspp], Osteocalcin [Otc], and Wnt10a) were examined. The dental pulp cells differentiated into odontoblasts forming mineralized nodules with matrix containing OTC. Real-time quantitative polymerase chain reaction revealed that 3D-cultured dental pulp cells expressed Dspp, Otc, and Wnt10a mRNAs on day 15 and Wnt group cells expressed these mRNAs more strongly than Cont group cells. We confirmed that 3D-cultured dental pulp cells differentiated into odontoblasts forming mineralized dentin nodules, and Wnt10a stimulated the formation of mineralized dentin nodules. These findings suggest that 3D culture systems are likely to prove useful for analysis of dentinogenesis and can contribute to the development of vital pulp therapy.