ObjectiveThis study aims to explore the effect of inward rectifier potassium (Kir) 2.1 channel on the osteogenic differentiation of human dental follicle cells (hDFCs) and its mechanism.
MethodshDFCs were isolated and cultured, and their source was verified by flow cytometry. Osteogenic differentiation ability of hDFCs was evaluated by osteogenic induction. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to detect the gene expression of Kir2.1 gene (KCNJ2) in hDFCs. Real-time quantitative PCR (RT-qPCR) was performed to detect the expression of the Kir2.1 gene (KCNJ2) in hDFCs before and after osteogenic induction. Patch clamp technique was conducted to record the membrane potential changes of hDFCs before and after osteogenic induction. Moreover, the effect on the osteogenic differentiation of hDFCs was confirmed by increasing the concentration of extracellular potassium ions (50 mmol·L-1). Kir2.1 channel blockers cesium chloride (CsCl) and C19H20CINO (ML133) were applied to determine the effect of the Kir2.1 potassium channel on the osteogenic differentiation of hDFCs. At the same time, RT-qPCR was used to observe the expression changes of osteogenic differentiation related genes Runx related transcription factor 2 (Runx2) and osteocalcin (OCN) before and after the two intervention measures. Calcium imaging was performed to observe the effect of membrane potential hyperpolarization caused by decreased extracellular potassium level (2 mmol·L-1) on intracellular calcium concentration.
ResultsRT-PCR results showed that hDFCs expressed the Kir2.1 channel gene (KCNJ2). The RT-qPCR results showed that the KCNJ2 gene expression in hDFCs was upregulated 7 days after osteogenic induction. The patch clamp results showed that the membrane potential of hDFCs hyperpolarized to (-47±5.2) mV from (-12±3.2) mV. Alizarin red and alkaline phosphatase staining results showed that increasing the concentration of the extracellular potassium or blocking the function of the Kir2.1 channel significantly inhibited the osteogenic mineralization ability of hDFCs. The membrane potential hyperpolarization increased the intracellular calcium concentration in hDFCs.
ConclusionMembrane potential hyperpolarization mediated by the Kir2.1 channel plays an important role in the osteogenic differentiation of hDFCs.
