Alkaline phosphatase assay kit

To investigate bone ALP activity, an Alkaline Phosphatase Assay Kit (Biyuntian Biotechnology, China) was used. The osteogenesis was induced by the osteogenic differentiation medium (Oricell Therapeutics, China) containing basal medium mixed with 200 μM ascorbic acid 10 mM β-glycerophosphate, 100 nM dexamethasone. Different DDM-FG compounds; DDM-FG1 representing ratio of 1 g DDM with 0.1 ml FG and DDM-FG2 representing ratio of 1 g DDM with 0.5 ml FG, were prepared and placed in 6-well plates until FG was completely gelatinized. MC3T3-E1 cells with a density of 1 × 10⁴/ml were inoculated to the surface of different DDM-FG compounds directly and cultured in 37 °C incubator. At 3 and 7 days, the cell lysis buffer was centrifuged at 1200 rpm/min at 4 °C for 25 min, and the supernatant was taken for testing. After 100 μl reaction termination solution was added to each well, the absorbance was measured at 405 nm with a microplate reader (Muliskan, Thermo, USA). The amount of ALP required for hydrolysis of para-nitrophenyl phosphate to produce 1 μmol of p-nitrophenol per minute was defined as an enzyme activity unit, alkaline phosphatase activity was calculated. The test was independently repeated three times.

Hesperidin and icariin were purchased from Shanghai Yuan Ye Technology Co., Ltd. (batch number B20182, B21576 Shanghai, China), with a purity greater than 98%. DMEM culture medium (batch number 11965092), fetal bovine serum (batch number 12483020), and penicillin (100 U/mL) and streptomycin (100 µg/mL) (batch number 15140122) were obtained from GIBCO (New York, NY, USA). Dimethyl sulfoxide (DMSO) (batch number 472301-500ML), dexamethasone (batch number D8041), ascorbic acid (batch number A103533), β-glycerophosphate (batch number G5422-1), Calcein (batch number C302986), AAPH (batch number ajci23234), and 2,7-difluorofluorescein diacetate (DCFH-DA) (batch number D6470), prednisolone (batch number P0180), etidronate disodium (batch number B27158) were purchased from Beijing Chemical Factory Co., Ltd. (Beijing, China). The alkaline phosphatase assay kit and alkaline phosphatase chromogenic kit were obtained from Biyun Tian Biotechnology Co., Ltd. (batch number P0321S, C3206 Shanghai, China). The NO, TNF-α, and IL-6 enzyme-linked immunosorbent assay kits were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. (batch number Shanghai, China).

Mineralized nodules were detected by alkaline phosphatase (ALP) staining, according to the manufacturer's instructions (Beyotime Institute of Biotechnology, Haimen, China). The ALP activity of individual groups of cells was determined by enzymatic assay using the Alkaline Phosphatase Assay Kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Briefly, the individual groups of cells were stained with black cobaltous sulphide staining for ALP and counterstained with nuclear fast red, followed by imaging under a light microscope (magnification, x100; Nikon Eclipse TC 100; Nikon Corporation, Tokyo, Japan).