2022, Vol. 26 ›› Issue (24): 3886-3891

Osteogenic differentiation of human periodontal ligament stem cells under estrogen and crosstalk between the two pathways

Jin Ke1, Wu Xiaoling2, Luo Xiaoling1, Xu Pengfei1, Xu Xiaomei1   

Abstract: BACKGROUND: Periodontal tissue regeneration engineering based on stem cells provides a broad prospect for the treatment of periodontal diseases. Estrogen-mediated osteogenic differentiation in human periodontal ligament stem cells is closely related to the Wnt signaling pathway.
OBJECTIVE: To study the regulation of Wnt/β-catenin and Wnt/Ca2+ signaling pathways on estrogen-mediated osteogenic differentiation in human periodontal ligament stem cells, as well as the crosstalk between the two pathways.  
METHODS: Premolars with healthy periodontal condition were collected to isolate and culture human periodontal ligament stem cells. Surface markers of the cells were detected by flow cytometry. The osteogenic/adipogenic differentiation potential was detected by Alizarin red S and Oil red O staining. The third-generation human periodontal ligament stem cells were randomly divided into blank group, control group, estrogen group, XAV939 (Wnt/β-catenin pathway inhibitor) group, L-690,330 (Wnt/Ca2+ pathway inhibitor) group, and XAV939+L-690,330 group. Except for the blank group, all other groups were cultured for osteogenic induction. qRT-PCR and western blot assay were performed to measure expression of osteogenic markers (Runx2, OCN) and key signal molecules of Wnt/β-catenin and Wnt/Ca2+ pathway. Intracellular Ca2+ concentration was evaluated by Fluo-4 fluorescent probe. Alkaline phosphatase assay was conducted to measure the alkaline phosphatase activity. Osteogenic differentiation potential was detected by Alizarin red S staining.
RESULTS AND CONCLUSION: (1) The human periodontal ligament stem cells isolated and purified by collagenase digestion were spindle shaped and polygonal. Flow cytometry proved that human periodontal ligament stem cells were of mesenchymal origin, and they also demonstrated the capacity to differentiate into osteogenic/adipogenic lineages. (2) Estrogen at 10-7 mol/L in the osteogenic medium can upregulate intracellular Ca2+ concentration, CaMKII and NLK gene expression and CaMKII protein expression of human periodontal ligament stem cells. (3) The usage of Wnt/β-catenin and Wnt/Ca2+ signaling pathway inhibitors can minimize osteogenic differentiation. (4) Inhibiting either of the two signaling pathways can increase crucial protein expression of the other pathway, which had no significant effect on the osteogenic differentiation of the cells. (5) In conclusion, estrogen through Wnt/Ca2+ and Wnt/β-catenin signaling pathways induces osteogenic differentiation of human periodontal ligament stem cells. In addition, there is mutual compensation between the two pathways, thus working together to regulate osteogenic differentiation.  
Key words: stem cells, human periodontal ligament stem cells, estrogen, osteogenic differentiation, Wnt/β-catenin, Wnt/Ca2+, crosstalk, pathways