Background  Dental pulp stem cells, as high-quality seed cells in tissue engineering, have strong immunomodulatory ability. In the inflammatory microenvironment, the biological functions of dental pulp stem cells will change in different degrees.

  Objective  To study the functional changes of human pulp stem cells (DPSCs) co-cultured with CD3+T cells under the stimulation of tumor necrosis factor-α (TNF-α).

  Methods  DPSCs were isolated by crown splitting method and subcultured. Different concentrations of exogenous TNF-α were added for inducing inflammation in P3 cells. The proliferations of cells were detected by CCK8 assay. The messenger RNA (mRNA) expression levels of TNF-α and interleukin-1β (IL-1β) in control DPSCs obtained from healthy microenvironment (HDPSCs) group and DPSCs obtained from inflammatory microenvironment (IDPSCs) group were detected by real-time PCR (RT-PCR). The mRNA levels of osteogenic genes Runt-related gene 2 (Runx2) and alkaline phosphatase (ALP) before and after conventional culture (undiff) and osteogenic induction (diff) were detected by RT-PCR. CD3⁺ T cells were isolated from human peripheral blood by immunomagnetic beads method. CD3⁺ T cells and DPSCs were co-cultured with or without TNF-α stimulation at the ratio of 1:10, 1:20, 1:50 and 1:100, respectively. The mRNA expression levels of TNF-α, IL-1β, β-catenin and LEF-1 in different groups were detected by RT-PCR.

  Results  At 7 d, compared with the control group, the blank group, the 5 ng/mL TNF-α group and 15 ng/mL TNF-α group, the DPSCs treated by 10 ng/mL TNF-α showed higher proliferation rate (P＜0.05). After osteogenic induction, the mRNA expression levels of Runx2 and ALP were significantly higher in the TNF-α stimulated group than those in the control group. Under the osteogenic differentiation culturing condition, the expressions of osteogenic genes in the control group were lower than those in the TNF-α stimulated group (P＜0.05). The inflammatory factors mRNA levels of IL-1β and TNF-α in DPSCs stimulated by TNF-α were significantly higher than those in control group (P＜0.05). DPSCs were co-cultured with CD3⁺ T cells at different ratio from 1∶10 to 1∶100. In the control group, mRNA expressions of IL-1β, LEF-1 and β-catenin showed the lowest level at the co-culture ratio of 1∶100, and the mRNA expression of TNF-α was the lowest at 1∶20. In the TNF-α stimulated group, the mRNA expression of IL-1β and TNF-α were the lowest when the co-culture ratio was 1∶20, but the mRNA expression levels of β-catenin and LEF-1 were the highest compared with other co-culturing conditions.

  Conclusion  Under TNF-α stimulation, the expression of inflammatory cytokines and osteogenic ability in DPSCs increases. In the immune environment, when co-cultured with CD3⁺ T cells, the development of inflammation is inhibited and the Wnt/β-catenin classical pathway is activated.