Differentiation of human amniotic epithelial cells into osteoblasts is induced by mechanical stretch via the Wnt/-catenin signalling pathway

Human amniotic epithelial cells (hAECs) have recently been recognized as a potential source of stem cells. The present study was designed to investigate the effects of mechanical stretch on the osteogenic differentiation of hAECs. As it has been previously reported that the physical environment is an important factor in maintaining the phenotype and functionality of differentiated cells, mechanical stretch was use to mimic the mechanical environment in the present study, with the following parameters: 5% elongation of the hAECs at a frequency of 0.5 Hz, with evaluation at 2, 6, 12 and 24 h time points. The osteogenic differentiation process of the hAECs followed by mechanical stimulation was evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting and immunocytochemistry. Additionally, in a parallel study, a runt-related transcription factor 2 (Runx2)/core binding factor 1 (Cbfa1)-specific short hairpin RNA (shRNA) plasmid vector and a scrambled shRNA control plasmid was constructed for transfection into the hAECs prior to mechanical stimulation. The cultured hAECs exhibited a cobblestone-shaped epithelial-like phenotype and were positive for stage-specific embryonic antigen-4, cytokeratin-19, cluster of differentiation 44 and octamer-binding protein 4, as detected by flow cytometry, western blotting or confocal microscopy. The qPCR and western blotting data demonstrated that the mRNA and protein expression levels of Runx2/Cbfa1, alkaline phosphatase and osteocalcin were upregulated compared with the control group following stretching and they peaked at 12 h. These results indicated that the osteogenic differentiation of the hAECs was induced by mechanical stimuli. Additionally, the mRNA and protein expression levels of -catenin and cyclin D were increased significantly following stretching; however, they were decreased following Runx2/Cbfa1-shRNA transfection as observed by RT-qPCR and western blotting. These results suggested that the Wnt/-catenin pathway may have an important role in mechanical stretch-induced osteogenic differentiation of the hAECs. Furthermore, the combination of stretch and osteogenic induction medium had synergistic effects on the osteogenic differentiation. The results of the present study demonstrated that mechanical stimuli have an important role in osteogenic differentiation of hAECs via the Wnt/-catenin signalling pathway, which may be a potential therapeutic strategy in bone regenerative medicine.