Aggregation of human dental pulp cells into 3D spheroids enhances their migration ability after reseeding

Multicellular three‐dimensional (3D) spheroids allow intimate cell–cell communication and cell–extracellular matrix interaction. Thus, 3D cell spheroids better mimic microenvironment in vivo than two‐dimensional (2D) monolayer cultures. The purpose of this study was to evaluate the behaviors of human dental pulp cells (DPCs) cultured on chitosan and polyvinyl alcohol (PVA) membranes. The protein expression of hypoxia‐inducible factor 1‐α (HIF‐1α) and vascular endothelial growth factor (VEGF), and the migration ability of the DPCs from 2D versus 3D environments were investigated. The results showed that both chitosan and PVA membranes support DPCs aggregation to form multicellular spheroids. In comparison to 2D cultures on tissue culture polystyrene, DPC spheroids exhibited higher protein expression of HIF‐1α and VEGF. The treatment with YC‐1 (inhibitor to HIF‐1α) blocked the upregulation of VEGF, indicating a downstream event to HIF‐1α expression. When DPC spheroids were collected and subjected to the transwell assay, the cells growing outward from 3D spheroids showed greater migration ability than those from 2D cultures. Moreover, DPCs aggregation and spheroid formation on chitosan membrane were abolished by Y‐27632 (inhibitor to Rho‐associated kinases), whereas the inhibitory effect did not exist on PVA membrane. This suggests that the mechanism regulating DPCs aggregation and spheroid formation on chitosan membrane is involved with the Rho‐associated kinase signaling pathway. In summary, the multicellular spheroid structure was beneficial to the protein expression of HIF‐1α and VEGF in DPCs and enhanced the migration ability of the cells climbing from spheroids. This study showed a new perspective in exploring novel strategies for DPC‐based research and application. Our results demonstrate that chitosan and poly(vinyl alcohol) membranes support human dental pulp progenitor cells aggregation to form multicellular spheroids. Confocal immunofluorescence images show the expression of HIF‐1α in the cells throughout the spheroids. The 3D spheroids formation on chitosan membrane and subsequently enhanced cell migration ability are involved with RhoA‐mediated signaling pathway.