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

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. 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. 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. 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.

Xu Y ，Wang Y ，Pang X ，Li Z ，Wu J ，Zhou Z ，Xu T ，Gobin Beharee R ，Jin L ，Yu J ... -  《-》

Mineral trioxide aggregate enhances the osteogenic capacity of periodontal ligament stem cells via NF-κB and MAPK signaling pathways.

Mineral trioxide aggregate (MTA), as a bioactive material, has a widespread application in clinical practice. To date, the effects of MTA on the proliferation and differentiation of human periodontal ligament stem cells (hPDLSCs) remain unclear. hPDLSCs were isolated from human periodontal ligament tissues and cultured with MTA conditioned media. Cell counting kit-8 (CCK-8) assay was performed to assess the proliferation capacity of MTA-treated hPDLSCs. Immunofluorescence assay, alkaline phosphatase (ALP) activity, alizarin red staining, real-time RT-PCR, and western blot analyses were used to investigate the odonto/osteogenic capacity of hPDLSCs as well as the involvement of NF-κB and MAPK pathways. ALP activity assay revealed that 2 mg/ml was the optimal concentration for the induction of hPDLSCs by MTA. The protein expression of DSP, RUNX2, OCN, OSX, OPN, DMP1, ALP, and COL-I in MTA-treated hPDLSCs was significantly higher than those in control group (p < 0.01). When hPDLSCs were treated with the inhibitors of NF-κB and MAPK pathways (U0126, SP600125, SB203580, and BMS345541), the effects of MTA on the differentiation of hPDLSCs were suppressed. Mechanistically, P65 was detected to transfer from cytoplasm to nuclei, as indicated by western blot and immunofluorescence assay. Moreover, MAPK-related proteins and its downstream transcription factors were also upregulated in MTA-treated hPDLSCs. Together, mineral trioxide aggregate can promote the odonto/osteogenic capacity of hPDLSCs via activating the NF-κB and MAPK pathways.

Wang Y ，Zhou Y ，Jin L ，Pang X ，Lu Y ，Wang Z ，Yu Y ，Yu J ... -  《-》

Oxytocin facilitates the proliferation, migration and osteogenic differentiation of human periodontal stem cells in vitro.

To explore the effect of oxytocin (OT) on the proliferation, migration, and osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in vitro. PDLSCs were obtained by limiting dilution method. Immunofluorescence (IF), cell-counting kit-8 (CCK8), cell migration assay, Alizarin Red S staining, cetylpyridinium chloride (CPC) colorimetry, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis were used to examine the effect of OT on oxytocin receptor (OTR) expression, cell proliferation, migration and osteogenic differentiation of PDLSCs. Our study showed that PDLSCs expressed OTR. One hundred nanomolar OT exhibited the maximal effect on migration, while only 50 nM OT significantly promoted proliferation of PDLSCs, as well as mineralized nodule formation and calcium deposition (p < 0.05). Furthermore, 50 nM OT significantly up-regulated expression of osteogenesis-related genes-alkaline phosphatase (ALP), Collagen I (Col I), runt related transcription factor 2 (Runx 2), osteopontin (OPN) and osteocalcin (OCN)-at specific time points compared with osteogenic inductive medium (p < 0.05). In addition, western blot analysis demonstrated that 50 nM OT enhanced protein levels of ALP, Col I, and Runx 2 at day 7 and day 14 (p < 0.01), as well as activating the phosphorylation of extracellular-signal-regulated kinase (ERK) and protein kinase B (AKT) pathway; notably, 50 nM OT inhibited phosphorylation of the phosphatidylinositol 3-kinase (PI3K) pathway (p < 0.05). OT promoted proliferation, migration, and osteogenic differentiation of PDLSCs in vitro. Furthermore, the effect of OT on osteogenic differentiation was mediated through ERK and AKT pathway. Thus, OT may have potential for use in periodontal regeneration.

Ge B ，Liu H ，Liang Q ，Shang L ，Wang T ，Ge S ... -  《-》

10(-7)  m 17β-oestradiol enhances odonto/osteogenic potency of human dental pulp stem cells by activation of the NF-κB pathway.

Oestrogen has been proven to significantly enhance osteogenic potency, while oestrogen deficiency usually leads to impaired osteogenic differentiation of mesenchymal stem cells. However, little is known concerning direct effects of oestrogen on differentiation of human dental pulp stem cells (DPSCs). In this study, human DPSCs were isolated and treated with 10(-7)  m 17β-oestradiol (E2). Alkaline phosphatase (ALP) assay and alizarin red staining were performed. Alkaline phosphatase and alizarin red showed that E2 treatment significantly enhanced ALP activity and mineralization ability of DPSCs, but had no effect on cell proliferation. Real-time RT-PCR and western blot assay demonstrated that odonto/osteogenic markers (ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN and DSPP/DSP) were significantly upregulated in the cells after E2 treatment. Moreover, phosphorylation of cytoplasmic IκBα/P65 and expression of nuclear P65 were enhanced in a time-dependent manner following E2 treatment, suggesting activation of NF-κB signaling. Conversely, inhibition of the NF-κB pathway suppressed E2-mediated upregulation of odonto/osteogenic markers, indicating that the NF-κB pathway was pivotal for E2-mediated differentiation. These findings provide evidence that 10(-7)  m 17β-oestradiol promoted odonto/osteogenic differentiation of human DPSCs via activation of the NF-κB signaling pathway.

Wang Y ，Zheng Y ，Wang Z ，Li J ，Wang Z ，Zhang G ，Yu J ... -  《-》

Yunnan Baiyao Conditioned Medium Promotes the Odonto/Osteogenic Capacity of Stem Cells from Apical Papilla via Nuclear Factor Kappa B Signaling Pathway.

Pang X ，Wang Y ，Wu J ，Zhou Z ，Xu T ，Jin L ，Yu Y ，Li Z ，Gobin R ，Xue C ，Yu J ... -  《-》