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-κB) 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 IκBα 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-κB 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-κB pathway, and thus represents a potential target involved in the regeneration of periodontium for clinical treatments.