Objective:To investigate the effect of continuous pulsed-tumor necrosis factor(TNF-α) treatment on nuclear factor-κB(NF-κB) pathway and osteogentic potentials of bone marrow mesenchymal stem cells (BMMSCs). Methods:There were three groups in this study:Group A:negtive control;Group B:BMMSCs treated by TNF-α once;Group C:BMMSCs continuously pulsed-treated by TNF-α for seven days. After the treatment,IκBα and phospho-IκBα (p-IκBα) were detected by Western blot. The mRNA expression of EPHB4,IGF-1,OPG,IL-7 and MMP-1 in BMMSCs were measured by Real-time PCR. Alizarin red staining and alkaline phosphatase(ALP) staining were employed for the measurement of osteogenic differentiation. This experiment also investigated the effect of the addition of NF-κB inhibitor (pyrrolidine dithiocarbamate,PDTC) on TNF-α action. Results:Compared with group A,IκBα protein levels were lower,while phospho-IκBα(p-IκBα) were higher in both group B and C(P < 0.05). Besides,the mRNA expression levels of EPHB4,IGF-1,OPG were up-regulated but IL-7 and MMP-1 down-regulated after TNF-α priming (P < 0.05). The osteogenic potential of TNF-α-primed BMMSCs was enhanced and the pro-osteogenic differentiation effect of TNF-α was reversed in the presence of NF-κB pathway inhibitor PDTC. Conclusion:Our in vitro results indicated that continuously pulsed TNF-α treatment significantly enhances the osteogenic differentiation of BMMSCs and this effect may be mediated at least partially via NF-κB pathway.