Potassium dihydrogen phosphate promotes the proliferation and differentiation of human periodontal ligament stem cells via nuclear factor kappa B pathway

Introduction: Periodontal ligament stem cells (PDLSCs) are vital for the regeneration of periodontal tissues. Potassium dihydrogen phosphate (KH2PO4) has recently been applied as a component of the mineralization inducing medium (MM), which can be used to induce osteogenic differentiation of dental stem cells. However, whether KH2PO4 has effects on PDLSCs has not been studied. Materials and methods: PDLSCs were isolated by magnetic activated cell sorting and cultured. Alkaline phosphatase (ALP) activity and ALP protein expression of PDLSCs treated with different concentrations of KH2PO4 were examined to make sure the optimal concentration of KH2PO4 for the following experiments. The effects of KH2PO4 on the proliferation and differentiation of PDLSCs were investigated by flow cytometry, cell counting kit-8 assay, alizarin red staining, real-time RT-PCR, and Western blot. The involvement of nuclear factor kappa B (NF-kappa B) pathway in KH2PO4-treated PDLSCs was analyzed by Western blot and alizarin red staining. Results: ALP activity assay and ALP protein expression examination revealed that 1.8 mmol/L KH2PO4 was the optimal concentration for the induction of hPDLSCs by KH2PO4. The proliferation and mineralization capacity of PDLSCs treated with KH2PO4 were enhanced as compared with the control group. PDLSCs treated with KH2PO4 showed an improved proliferation capacity in logarithmic growth phase at day 7. As PDLSCs were treated with KH2PO4, the expression of odonto/osteogenic markers (OCN /OCN, DSP/DSPP, OSX/OSX, RUNX2/RUNX2, and ALP/ALP) in cells were up-regulated at day 3 or 7. Moreover, the expression of I kappa B alpha in cytoplasm was down-regulated, along with an increased expression of p-P65 in cytoplasm and an up-regulated expression of P65 in nucleus. When treated with BMS345541 (the specific NF-kappa B inhibitor), the odonto/osteogenic differentiation of KH2PO4-treated PDLSCs was significantly attenuated. Conclusion: KH2PO4 can improve the proliferation and odonto/osteogenic differentiation capacity of PDLSCs via NF-kappa B pathway, and thus represents a potential target involved in the regeneration of periodontium for clinical treatments.