Background   In the chronic inflammatory microenvironment, impaired human periodontal ligament stem cells (hPDLSCs) biological function in patients with periodontitis tissue regeneration is the main reason of treatment difficulties. As a secretory ligand of non-classical Wnt/Ca^{2 +} signaling pathway, Wnt5a plays a key role in the growth and development of stem cells and inflammatory diseases. However, the effect of Wnt5a on the proliferation and osteogenic differentiation of hPDLSCs in inflammatory microenvironments remains unclear.

  Objective   To investigate the effect of Wnt5a overexpression on the proliferation and osteogenic differentiation of human periodontal ligament stem cells in inflammatory microenvironment.

  Methods   Primary human periodontal stem cells were isolated and cultured by enzyme digestion method. Tumor necrosis factor⁃α (TNF⁃α) was used to stimulate hPDLSCs for constructing an inflammatory microenvironment in vitro, labeled as normal group and inflammatory group, and the interleukin (IL)-1β, IL-6, TNF-α, recombinant runt related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), collagen Ⅰ (COL-1) and Wnt5a signaling molecules mRNA expression changes were detected by real-time quantitative PCR under osteogenic induction conditions. Lentivirus transfection technique was used to obtain hPDLSCs overexpressing Wnt5a, then the cells were divided into negative control group, overexpression group, TNF-α + negative control group and TNF-α + overexpression group. The effect of Wnt5a on the proliferation of hPDLSCs was determined by CCK-8 method. After osteogenic induction culture, ALP staining and alizarin red staining (ARS) were used to evaluate the effect of Wnt5a overexpression on the osteogenic differentiation ability of hPDLSCs. The mRNA and protein expression levels of RUNX2 and COL-1 in hPDLSCs after Wnt5a overexpression were detected by real-time quantitative PCR and Western blot.

  Results   The mRNA expression of IL-1β, IL-6, TNF-α (P<0.01) and Wnt5a (P<0.01) increased in the inflammatory group compared with the normal group. However, the mRNA expressions of osteogenic genes RUNX2 (P=0.031), ALP (P<0.01) and COL-1 (P=0.014) decreased significantly. At 48 hours after lentivirus infection, the cells showed GFP green fluorescence under fluorescence microscope. Compared with the negative control group, the mRNA and protein expressions of Wnt5a increased significantly in the overexpression group (all P<0.01). The results of CCK-8 showed that the cell viability of overexpression group was increased compared with that of negative control group (P<0.01), but the cell proliferation viability of TNF-α + overexpression group was decreased compared with that of TNF-α + negative control group (P<0.01). Under osteogenic induction conditions, the mRNA levels and protein expression levels of COL-1 and RUNX2 in early osteogenic markers and the amount of calcium nodules formation decreased in the overexpression group (allP<0.01). However, in the inflammatory microenvironment, the mRNA levels (P=0.022; P=0.016) and the protein expression levels (P<0.01;P<0.01) of COL-1 and RUNX2 increased in the TNF-α + overexpression group compared with those of TNF-α + negative control group. Alizarin red staining showed increased coloring depth and calcium nodules formation, ALP staining showed enhanced in vitro mineralization ability.

  Conclusion   Inflammatory microenvironment up-regulates the expression of Wnt5a in hPDLSCs. After overexpression of Wnt5a under normal culture conditions, the proliferation ability of hPDLSCs is enhanced and the osteogenic differentiation ability is decreased. However, overexpression of Wnt5a inhibits the proliferation of hPDLSCs and promotes the osteogenic differentiation of hPDLSCs in the inflammatory microenvironment stimulated by TNF-α. In the course of chronic periodontitis, human periodontal membrane stem cells may regulate osteogenesis and proliferation through the non-classical Wnt5a signaling pathway.