After the cell-scaffold structures were cultured for 4 days under the OM with or without EMF stimulation, the medium was discarded and the cell-seeded scaffolds were washed twice with PBS. Subsequently, the α-MEM medium was used to incubate cells seeded on scaffolds for another 4 days in a normal environment. Then, the medium was collected and 10% FBS was added to make a conditioned medium for the culture of the undifferentiated BMSCs. In addition, the undifferentiated BMSCs cultured with α-MEM served as a blank group. After 1 week of culture, the expression of osteogenic genes [Runt-related gene 2 (Runx2), type 1 collagen (COL1), and osteopontin (OPN)] were analyzed by qPCR and the osteogenic proteins (Runx2, OPN) were detected by immunofluorescence.

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