CTP-CM enhances osteogenic differentiation of hPDLSCs via NF-κB pathway

Abstract

Objective: The conditioned medium of calcined tooth powder (CTP-CM) is a type of biomimetic mineralized material and well contributing to bone healing and bone formation in vivo. However, little is known about the effect of CTP-CM on human periodontal ligament stem cells (hPDLSCs) as well as the underlying mechanisms.

Methods: ALP activity assay was conducted to select the concentration with the highest ALP level, which was used for the following experiments. Cell proliferation was measured by cell counting kit-8 assay and flow cytometry analysis. Expression levels of osteogenic markers in CTP-CM-induced hPDLSCs were evaluated with real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunofluorescence staining, and Western blot. Mineralization of CTP-CM-induced hPDLSCs was evaluated by alizarin red staining. Furthermore, the involvement of NF-κB pathway was examined by immunofluorescence staining and Western blot.

Results: 20 μg/ml was selected for the further experiments. Functional studies demonstrated that CTP-CM exerted almost no influence on the proliferation of hPDLSCs and CTP-CM increased the osteogenic differentiation of hPDLSCs. Mechanistically, CTP-CM leads to activation of NF-κB signaling pathway. When treated with BMS345541, the osteogenic differentiation of CTP-CM-treated hPDLSCs was significantly attenuated.

Conclusion: CTP-CM can promote the osteogenic differentiation of hPDLSCs via activating NF-κB pathway.

Keywords: osteogenic differentiation; periodontal ligament stem cells; proliferation; the conditioned medium of calcined tooth powder.