Nitro blue tetrazolium

To determine whether the early osteogenic differentiation of BMSCs was induced by BMP-2, ALP staining was performed, as previously reported (9 (link)). Following 3, 7, 14 and 21 days of culture, the cells were fixed in 10% formalin, washed with PBS and then incubated in staining solution, containing a mixture of 0.02% 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Santa Cruz Biotechnology, Inc.) and 0.03% nitro blue tetrazolium (NBT; Santa Cruz Biotechnology, Inc.) in 0.1 M TBS, which was added into 5 ml AP buffer (100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl₂ and 0.05% Tween 20, pH 9.5) and incubated for 1 h at room temperature. Furthermore, the activity of ALP and protein content of the three groups were measured on days 3, 7, 14 and 21. The cells lysates were prepared, and the activity of ALP in the lysates was determined using a Lab-Assay-ALP colorimetric assay kit (Wako Pure Chemicals, Osaka, Japan), according to the manufacturer's instructions. The total protein concentrations were determined using a commercial BCA Protein Assay kit (Beyotime Institute of Biotechnology, Shanghai, China). The activity of ALP was calculated as nmol/h phosphorylated Nitrophenol (p-NP) release and was further normalized to the cell protein content.

Dimethyl sulfoxide, diphenylamine, 5,5′‐dithiobis(2‐nitrobenzoic acid), epinephrine, guanidine hydrochloride, hydrogen peroxide, N‐ethylmaleimide, sodium chloride, thiobarbituric acid were procured from Sigma‐Aldrich (St. Louis, MO). Nitroblue tetrazolium, 2,2′‐dipyridyl, l‐glutathione (reduced), and protocatechuic acid are products of Santa Cruz Biotechnology. All other reagents are products of Sigma‐Aldrich (St. Louis, MO).