2021 年 1 巻 1 号 p. 22-29
Purpose: The purpose of this study was to analyze the calcification characteristics of three-dimensional (3D) culture of multi-layered dental pulp cell (DPC) sheets that have potential application in pulp regeneration and evaluation of dental materials and pharmaceuticals.
Methods: DPC were isolated from third molars extracted from three healthy patients aged 18-30 years old. DPC sheets were constructed using a temperature-responsive culture dish (UpCell) and a gelatin stamp (Cell Stamp for UpCell). Histological examination of three-layer and six-layer DPC sheets was performed using hematoxylin and eosin staining (HE). DPC sheets on mineral trioxide aggregates (MTA) were observed using a scanning electron microscope (SEM) to examine the microstructure. The LOX-1 probe was used to examine the effects of hypoxia. Calcification ability was analyzed by real-time PCR and Alizarin Red S staining (ARS). In this study, two-dimensional (2D) culture (monolayer) served as a control.
Results: Thick multilayered cell sheets were confirmed under the macroscopic view and with HE. The SEM images showed that the DPC sheets covered the MTA. Higher osteo/odontogenic differentiation was seen in the multi-layered cell sheets than in the monolayer cell sheets. The expression levels of alkaline phosphatase (ALP), type Ⅰ collagen (COL1A1), osteocalcin (OCN), and dentin sialoprotein (DSPP) genes were higher in the DPC sheets. With ARS, characteristic nodules of mineralization were observed in the cell sheets.
Conclusion: DPC sheets possessed higher mineralizing ability than the monolayer sheets. Cell sheets may be useful for comparative examination of pharmaceuticals, materials, and pulp regeneration in a 3D environment.
