Effect of human umbilical cord mesenchymal stem cells treated with Toll-like receptor 3 and human dental pulp cells co-cultured on cell mineralization and anti-inflammatory repair ability

Objective: To explore the effect of human umbilical cord mesenchymal stem cells(hUCMSCs)treated with Toll-like receptor 3 (TLR3) and human dental pulp cells (hDPCs) co-cultured on cell mineralization and anti-inflammatory repair ability.Methods: hUCMSCs were isolated and cultured from the umbilical cord tissues of healthy newborns, and the expressions of cell surface markers CD34, CD45, CD90 and CD105 were detected by flow cytometry and immunofluorescence staining so as to identify hUCMSCs.hUCMSCs were treated with TLR3 agonist Poly (I:C) (10 μg/mL) and co-cultured with hDPCs.The alkaline phosphatase (ALP) level of each group was determined and the cell mineralization in each group was observed by alizarin red staining and Von Kossa staining.LPS (10 μg/mL) was used to stimulate hDPCs to simulate dental pulp inflammation.MTT assay was used to detect the proliferative activity of cells in each group and the mRNA expressions of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), interleukin 10 (IL-10), dentin sialophosphate protein (DSPP), dentin matrix protein-1 (DMP-1), osteocalcin(OCN) and osteopontin (OPN) in each group were detected by RT-qPCR.Results: The isolated hUCMSCs showed negative expressions of CD34 and CD45, and positive expressions of CD90 and CD105, indicating hUCMSCs were successfully isolated.Compared with hUCMSCs and hDPCs cultured separately, the ALP staining of cells in the co-cultured system of hUCMSCs and hDPCs was deepened, ALP activity increased, and many mineralized nodules were formed (P＜0.05).Compared with co-cultured hUCMSCs and hDPCs, the ALP activity of co-cultured hUCMSCs treated with Poly (I:C) and hDPCs further increased and more mineralized nodules were formed (P＜0.05).Compared with cells without LPS stimulation, the proliferative activity of cells in LPS stimulated groups decreased, the mRNA relative expressions of TNF-α, IL-6 and IL-10 increased, and the mRNA relative expressions of DSPP, DMP-1, OCN and OPN decreased (all P＜0.05).Under LPS stimulation, compared with co-cultured hUCMSCs and hDPCs, the cell proliferation activity of co-cultured hUCMSCs treated with Poly(I:C)and hDPCs was higher, the mRNA relative expressions of TNF-α and IL-6 decreased, the mRNA relative expressions of IL-10 further increased and meanwhile the mRNA relative expressions of DSPP, DMP-1, OCN and OPN also increased(all P＜0.05).Conclusion: hUCMSCs treated with TLR3 agonist Poly(I:C)co-cultured with hDPCs can enhance the mineralization ability of cells, inhibit the levels of inflammatory factors under LPS stimulation, and promote the expression of genes related to dentin formation under inflammation, which may be beneficial to the anti-inflammatory repair in pulpitis.