Cell differentiation, Alizarin Red S staining, and
quantification.
Here, 5 × 104 MC3T3-E1cells/ml in α-MEM were seeded
overnight with SF or TiBP-SF. The medium was removed the next day,
replaced with OIM, and incubated at 37 °C for 3 weeks in a 5%
CO2 incubator. Day 0 is the date the differentiation
medium was added to cells. The entire medium was replaced every 3 days.
Alizarin red S staining was performed to analyze calcium deposits. Cells
were washed twice with Hanks balanced salt solution (HBSS) (Gibco 14025,
Thermo Fisher Scientific, Waltham, MA, USA) and then fixed with 4%
paraformaldehyde for 15 min. The cells were stained with 1% Alizarin
Red S solution for 3 min at 37 °C and rinsed with distilled water to
exclude non-specific staining. For quantitation, cells stained with
Alizarin red were de-stained with 10% cetylpyridinium chloride
(Sigma-Aldrich, Saint Louis, MO, USA) for 15 min, after which the
extracted stain was transferred to 96-well plates, and the absorbance at
562 nm was measured using a microplate reader. After 2 and 3 weeks of
osteogenic induction, the level of OC in the supernatant was directly
measured using an ELISA kit (USCN Life Science, Wu Han, China) according
to the manufacturer’s instructions and a microplate reader at 450 nm.
The assays were performed in triplicate.