Alizarin red

The third generation of the adherent cells was cultured in an osteogenic medium (Cyagen Biosciences, Guangzhou, China) for 21 days or in an adipogenic medium (Cyagen Biosciences, Guangzhou, China) for 14 days. After the cultivation, the osteogenesis‐induced cells were stained in alizarin red (Yuanye Bio‐Technology Co., Ltd., Shanghai, China) and the adipogenesis‐induced cells were stained in oil red O (Yuanye Bio‐Technology Co., Ltd., Shanghai, China). The cells were observed and photographed under a microscope. The expressions of CD34 and CD90 were measured by flow cytometry for identification of the stemness of these cells.

The cells (1x10⁵/cm²) suspensions were incubated with antibodies against CD44-phycoerythrin (PE), CD34-PE, CD105-fluorescein isothiocyanate (FITC), and CD45-FITC, respectively (all from BD Bio-Sciences, San Jose, CA, USA), in the dark for 30 min at 4 °C. The fluorescence intensity was measured using flow cytometry. To evaluate the differentiation potential, the stem cells were inoculated into 12-well plates at a density of 5×10³/cm² according to the manufacturer’s protocol for the differentiation-inducing medium. Osteogenesis, adipogenesis, and chondrogenesis media (differentiation kits from Gibco, Thermo Fisher Scientific, Waltham, MA, USA), were used to culture the cells for 21 days (osteogenesis) and 28 days (adipogenesis, chondrogenesis). The osteogenesis, adipogenesis, and chondrogenic differentiation abilities of stem cells loaded with or without SPION@PDA NPs were detected by Alizarin Red, Oil Red O, and Alcian blue staining kits, respectively (Shanghai Yuanye Bio-Technology Co., Ltd, Shanghai, China).