Vitamin K2 promotes the osteogenic differentiation of periodontal ligament stem cells via the Wnt/β-catenin signaling pathway

Objective: Vitamin K2 (MK-4, menaquinone 4) plays an important role in osteoprotection. The present study aimed to examine the effect of MK-4 on the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in vitro and probed the potential signaling pathway. Design: PDLSCs were isolated from extracted premolars by tissue block culture method and were identified by flow cytometry. Cell Counting Kit-8 (CCK-8) and colony formation assays were used to determine the effect of MK-4 on the proliferation of PDLSCs. Alkaline phosphatase (ALP) activity was analyzed quantitatively, and extracellular matrix mineralization was examined by Alizarin Red S staining. The mRNA and protein expression levels of ALP, Runx Family Transcription Factor 2 (Runx2), osteocalcin (OCN), and Sp7 Transcription Factor (SP7; Osterix) were measured by qRT-PCR and Western blot. In addition, after adding the inhibitor XAV-939, Western blot was used to assess the correlation with the Wnt/beta-catenin signaling pathway. The above results were obtained by observing at least three fields randomly, and each experiment was repeated at least three times. Results: This study found that 10 5 M MK-4 significantly promoted the osteogenic differentiation of PDLSCs. Gene and protein expression levels of ALP, Runx2, OCN, and Osterix were all upregulated compared with control. Remarkably, after blocking the Wnt/beta-catenin signaling pathway with XAV-939, the effect of MK-4 was apparently reversed. Conclusion: These results demonstrate that MK-4 can promote the osteogenic differentiation of PDLSCs, which is likely related to the activation of the Wnt/beta-catenin signaling pathway.