2.4.1. Alkaline Phosphatase (ALP) Activity

To confirm the effects of immobilized rhPDBF-BB, rhBMP-2, or rhPDGF-BB/rhBMP-2 on osteogenic differentiation, we evaluated the ALP activities and calcium contents, as early and late differentiation of MG-63 osteoblast-like cells, were evaluated, respectively. ALP activities were measured after culture for 3, 7, or 10 days. In brief, cells (1 × 105 cells/mL) were seeded on the surfaces of each Ti disc (*n* = 5). At predesignated times, cells and Ti sample were washed with PBS. Then, RIPA buffer (1×) containing protease and phosphatase inhibitor was added to cells. Cells were then lysed with RIPA (1×) buffer and centrifuged at 13,500 rpm for 1 min to remove cell debris. P-nitrophenyl phosphate solution was then added to supernatants and incubated for 30 min at 37 ◦C and 1 N NaOH was added to stop reactions. Optical densities of ALP were determined using a Flash Multimode Reader at 405 nm.
