5.9. ALP Activity and Alizarin Red S Staining

ALP activity was assayed by measuring the release of p-nitrophenol from p-nitrophenyl phosphate (pNPP). Cultured scaffolds must be preconditioned to acquire accurate ALP results. Cultured scaffolds were rinsed twice with PBS solution and equilibrated with ALP buffer (100 mM Tris-Cl, pH 9.5, 100 mM NaCl and 10 mM MgCl₂). Then, samples were immersed in BCIP/NBT solution (Sigma, St. Louis, MO, USA) for 30 min. Enzymatic activity of the scaffolds were terminated by treatment of the scaffolds with 20 mM PBS solution containing EDTA. This was gently washed off with PBS solution and the scaffolds were then incubated in Tris buffer (10 mM, pH 7.5) containing 0.1% Triton X-100 surfactant for 10 min. Scaffolds were then assessed for ALP activity using an ALP kit (ALP-10, Sigma-Aldrich) and 100 µL of lysate was added to a 24-well tissue culture plate containing 100 µL pNPP solution. Stained samples were evaluated under an optical microscope and the quantity of pNPP transformed into p-nitrophenol and inorganic phosphate was measured by analyzing the absorbance at 405 nm using a microplate reader (Spectra III, SLT Lab Instruments, Salzburg, Austria).

Mineralization of osteoblastic cells was quantified and evaluated via alizarin red S (ARS) staining in 24-well plates. Samples were washed three times with PBS solution and immersed in 70% ethanol at 4 °C for 1 h. After samples were air-dried, they were stained with 40 mM ARS solution (pH 4.2) for 1 h and rinsed with distilled water. Samples were de-stained via treatment with 10% cetylpyridinium chloride in 10 mM sodium phosphate buffer (pH 7.0) for 15 min. Samples were evaluated under an optical microscope and optical density was measured at 562 nm using a microplate reader. ALP activity and calcium deposition were normalized to the total protein content. All values are reported as means ± SDs (n = 5).