Pyrophosphate inhibits periodontal ligament stem cell differentiation and mineralization through MAPK signaling pathways

Background and objective Periodontal ligament stem cells (PDLSCs) are the primary cell source for the regeneration and remodeling of periodontal ligament (PDL). It is crucial to prevent PDLSCs from mineralization when using the PDLSCs for PDL regeneration. At present, little is known about how to inhibit PDLSC mineralization. This study investigates the effects of pyrophosphate (PPi) on inhibiting PDLSC osteogenic differentiation and mineralization as well as the underlying mechanism. Materials and methods Human PDLSCs were cultured in an osteogenic differentiation medium with different PPi concentrations (0, 10, or 100 μM). The effects of PPi on osteogenic differentiation were assessed by ALP activity and the expressions of osteogenic related proteins (OPN, RUNX2, OSX, and DMP1). The mineralization formation was detected by alizarin red staining. The activation of MAPK signaling pathways (ERK1/2, JNK, and p38) was determined by western blotting and pathway blockade assays. The gene expressions of PPi's regulators (Ank, Enpp1, and Alpl) were assessed by real-time PCR. Results Both low and high concentrations (10 μM and 100 μM) of PPi inhibited the mineralization of PDLSCs. The addition of PPi (10 μM or 100 μM) decreased the ALP activity of the PDLSCs to approximately two-thirds of the control group on day 3. PPi reduced the expressions of RUNX2, OSX, and DMP1 on days 7, 14, and 21, while it increased the expression of OPN at the three time points. PPi enhanced the phosphorylation of MAPK pathways, and the application of corresponding MAPK pathway inhibitors reversed the osteogenic inhibition effects of PPi. Conclusion PPi inhibits the osteogenic differentiation and mineralization of PDLSCs in vitro through activating ERK1/2, JNK, and p38 signaling pathways.