Alkaline phosphatase activity and staining

Alkaline phosphatase (ALP) activity in the co-culture supernatant (“Cell Contact” and “No Cell Contact”) was measured on day 7 after challenge with Ti particles. In preparation for this assay, medium was collected and centrifuged twice at 4000×g for 10 min to remove cell debris and Ti particles. ALP activity was assayed using an Alkaline Phosphatase Assay Kit (Sigma-Aldrich, St. Louis, MO). In brief, the assay mixtures contained 2-amino-2-methyl-1-propanol, MgCl₂, p-nitrophenyl phosphate disodium, and cell homogenates. After incubation, the reaction was stopped with NaOH, and the absorbance was read at 405 nm.

The “Cell Contact” and “No Cell Contact” co-cultures were maintained as described above. Similarly, ALP staining was performed on day 7 after challenge with Ti particles. Osteoblasts were washed three times with PBS prior to staining with an Alkaline Phosphatase Stain Kit (Jiancheng, Jiangsu, China). In brief, cells were fixed in methanol and overlaid with 5-bromo-4-chloro-3-indolyl phosphate plus nitroblue tetrazolium chloride in Tris-HCl, NaOH, and MgCl₂, followed by incubation at room temperature for 2 h in the dark.