P nitrophenyl phosphate

The BMSCs were cultured in 24-well plates, and then the medium was discarded. Afterward, the cells were washed with phosphate-buffered saline (PBS), fixed with 95% ethanol, stained with ALP solution and cultured for 4 hours in an incubator at 37° C. Then, 2% cobalt sulfide and ammonium sulfide (Tianjin Tianli Chemical Reagent Co., Ltd, Tianjin, China) were added. Subsequently, the cells were incubated with 10 mM p-nitrophenyl phosphate (Meilunbio, Dalian, China) for 30 min for quantitative analysis. Finally, the release of p-nitrophenol was determined by the ALP colorimetric kit (BioVision, USA) to evaluate the ALP activity. The absorbance was measured at 420 nm with a microplate reader (Tecan, Mannedorf, Zurich, Switzerland).

The hBMSCs were inoculated in 24-well plates. When cell confluence reached 80%, the medium was changed to osteogenic differentiation medium (Cyagen, USA). The medium was changed every 3 days and when the medium was changed, exosomes were added to the medium. Alkaline phosphatase (ALP) staining and Alizarin Red S staining were done to evaluate the level of osteogenic differentiation. After the hBMSCs were cultured in osteogenic differentiation medium for 7 days, ALP staining was conducted according to the manufacturer’s instructions (Beyotime, China). The cells were incubated with 10 mM p-nitrophenyl phosphate (Meilunbio, China) as the substrate at 37 °C for 15 min. Afterward, ALP activity was quantified at 420 nm by a microplate reader [34 (link)]. After cultured in osteogenic differentiation medium for 3 weeks, the hBMSCs were fixed, stained, and cleared according to the instructions of the Alizarin Red S staining kit (Solarbio, China). Based on the previously reported methods, the stained mineralization nodules were dissolved with 10% cetylpyridinium chloride at 37 °C for 30 min [35 (link)]. The solution was added to a 96-well plate, and a microplate reader was used detect the OD value at 562 nm for quantitative analysis.

The medium was removed after BMSCs in a 24-well plate and was washed with phosphate buffer saline (PBS). Cells were then fixed with 95% ethanol and stained with ALP solution, followed by 4-h incubation in a 37 °C incubator. Then, 2% cobalt nitrate (Tianli Chemical Reagents, Tianjin, China) and ammonium sulfide (Tianli Fuyu Fine Chemical, China) were added. BMSCs were observed under an optical microscope (Nikon, Tokyo, Japan). The cells then underwent incubation with 10 mM p-nitrophenyl phosphate (Meilunbio, Dalian, China) for 30 min for quantitative analysis. Finally, the absorbance value (420 nm) was determined by a spectrophotometry reader.