Abstract: Objective： To investigate the effects of Toll like receptors on the osteogenesis of human pe-riodontal ligament stem cells (hPDLSCs) and probable molecular mechanism. Methods: Real-time PCR and flow cytometry were applied to test the expression of TLRs in hPDLSCs and the positive cell percentage of TLR. hPDLSCs were cultured in osteogenic medium for 7 to 14 days with different TLR agonists at various concentrations . The effect of different TLR on osteogenic differentiation of hPDLSCs was evaluated by alizarin red S staining, alkaline phosphatase (ALP) staining and ALP activity assay. Western blotting was used to analyze the phosphorylation levels of extracellular regulated protein kinases (ERK), c-Jun N-terminal protein kinase (JNK), P38, AKT and expression of Runx2 an osteogenic related gene after treatment with TLR agonists, compared with the effect of inhibitors of mitogen activated protein kinase (MAPK) or protein kinase B (PKB or AKT) on Runx2 expression of hPDLSCs cultured in osteogenic medium. Results: Higher expressions of TLR1,3,4,6 were found in hPDLSCs through real-time PCR. Positive cell percentage of TLR was determined by flow cytometry and described as TLR1: 2.82%±0.68%; TLR2: 1.26%±0.09%; TLR3: 13.23%±2.05%; TLR4: 3.64%±0.79%; TLR6: 3.21%±1.64%, whose tendency was comparable to their mRNA expression in hPDLSCs. Most TLR ligands had no effect on the ALP staining, activity and mineralization of hPDLSCs at lower concentration except for 0.1 mg/L PolyI:C could induce the osteogenic ability of hPDLSCs. On the contrary, Higher concentration of TLR ligands (PolyI:C: 10 mg/L, LPS: 10 mg/L , Pam3CSK4: 1 mg/L, FSL-1: 50 μg/L) had obviously inhibitory effect on osteogenic differentiation of hPDLSCs. Activation of TLR using higher concentration of TLR ligands could downregulate the phosphorylation levels of ERK, P38, JNK and AKT, and also reduced the expression of Runx2, compared with the untreated control. The inhibitors of MAPK (U0126, SP600125，SB203580) and inhibitor of AKT (perifosine) could also inhibit Runx2 expression. Conclusion: Higher concentration of TLR ligands could inhibit osteogenic differentiation of hPDLSCs. This inhibitory effect seemed to be related to decreased phosphorylation of MAPK and AKT.

Key words: Toll like receptor, Human periodontal ligament stem cell (hPDLSCs), Osteogenic dif-ferentiation, Mitogen activated protein kinase (MAPK), Protein kinase B