Cells were pre-cultivated in normal growth medium for four weeks and then either continued to be cultured in the same medium (reference) or switched to osteogenic medium. To measure osteogenic differentiation by alkaline phosphatase (ALP) activity, cell-seeded scaffolds were collected on days 0, 7, 14, and 21 after the point at which they were transferred to the osteogenic medium. The scaffolds were incubated for 1 h in serum-free medium, washed three times with PBS, and incubated in 2 mL lysis buffer for 20 min on ice. The lysis buffer consisted of PBS with 0.5% Triton® X-100 (#30513, Carl Roth, Karlsruhe, Germany), 25 mM of TRIS (#10601, Carl Roth), 1 mM of MgCl2 (#21892, Carl Roth), and 0.1 mM of ZnCl2 (#Z015250G, Merck, Darmstadt, Germany). To ensure full lysis, the samples were subjected to three cycles of shock freezing (liquid nitrogen) and thawing and were then kept on ice. Special attention was paid to achieving lysis of all cells in the scaffolds. ALP activity was determined using an assay (0.4 mM CSPD™ substrate with Sapphire-II enhancer, #T2210, Thermo Fisher, Darmstadt, Germany) as a measure to determine the degree of cell differentiation to osteoblasts. A total of 20 µL of the lysate was mixed with 100 µL of the reagent in a non-transparent 96-well plate (triplicate measurement). After 20 min at room temperature (RT), chemiluminescence was measured for 0.5 s (Victor X4 multiplate reader, Perkin Elmer). Lysis buffer mixed with the ALP-reagent was used as negative control. A standard curve was generated for reference. Increased protein concentrations (0 to 200 µg/mL in 20 µg/mL steps) were generated from an albumin stock solution (fraction V, 1 mg/mL, #28341, Carl Roth) by dilution in lysis buffer (triplicate measurement). To normalize ALP activity to total protein content, protein concentration was determined using the Bradford assay (5× Roti®Quant, #K015.2, Carl Roth) in a 96-well plate (triplicate measurement). After 5 min at RT, the absorption was measured (λ = 595 nm, 3 s).
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