In Vitro Mineralization Assay: A Colorimetric Method to Quantify Osteoblast Calcium Deposits on Bone Graft Substitute by Alizarin Red S Staining and Extraction

1. Osteoblast mineralization assay

1. Pre-incubate 14 mm diameter β-tricalcium phosphate (β-TCP) disks in 1 mL of bone growth medium (BGM) in a 24-well suspension culture plate at 37 °C and 5% CO₂ for 24 h before adding the osteoblasts (OBs). Use standard tissue culture plates for controls and suspension culture plates for the biomaterial samples to avoid cell attachment to the plastic.
2. Aspirate the pre-incubation medium and then resuspend primary mouse OBs in BGM. Add 8.8 x 10⁴ OBs/cm² onto the pre-incubated β-TCP disks in a 24-well suspension culture plate and for the control group, into a 24-well tissue culture plate (1 mL/well). OBs will attach to the tissue culture plate or the biomaterial sample.
3. Replace the BGM with 1 mL of osteogenic mineralization medium (MM) containing BGM, 50 µg/mL ascorbic acid, and 5 mM β-glycerophosphate 24 h after the addition of the OBs and incubate for 14 days at 37 °C and 5% CO₂. Change the medium every 2–3 days with 1 mL of freshly prepared MM to each well.

2. Osteoblast mineralization assessed by staining with Alizarin Red S (ARS)

1. Aspirate the culture medium from the wells containing primary OBs 14 days after the addition of MM and rinse twice with 0.5 mL of 1x PBS at RT. Aspirate the PBS and fix the cells by adding 0.5 mL of the 10% buffered formalin solution at RT for 10 min.
2. Aspirate the 10% buffered formalin with a single-use pipet and wash twice with 0.5 mL of ultrapure water.
3. To the fixed OBs, add 0.25 mL of 40 mM ARS staining solution (pH 4.2) dissolved in ultrapure water and incubate the plate at RT for 10 min on a shaker at 100 shakes/min.
4. Aspirate the staining solution with a single-use pipet and rinse with 1 mL of ultrapure water. Repeat this step 5–10 times to remove unspecific staining.
   NOTE: The rinsing solution should be without color.
5. Aspirate the ultrapure water and add 1 mL of cold PBS. Incubate the plate at RT for 10 min on a shaker at 100 shakes/min.
6. Aspirate the PBS, transfer the stained β-TCP disks to a new well, and scan the plates with a flatbed scanner to record mineralization.
7. Add 0.25 mL of 10% cetylpyridinium chloride solution and incubate the plate at RT for 15 min on a shaker at 100 shakes/min to extract the ARS dye.
8. Transfer the supernatants to a 1.5 mL tube and centrifuge at 17,000 x g for 5 min at RT.
9. Add 10% cetylpyridinium chloride solution to the extracts with a dilution ratio of 1:10–1:20 and add the samples (300 µL) into a 96-well plate including two blank wells containing only 10% cetylpyridinium chloride.
10. Prepare 7 ARS reference standards ranging from 4 to 400 µM by diluting the 40 mM ARS staining solution (pH 4.2) with 10% cetylpyridinium chloride solution to generate a standard curve.
11. Add 300 µL of the reference standard in duplicates to the 96-well plate.
12. Read the absorbance of the samples, blanks, and reference standards at 520 nm.
13. Subtract the blank readings from the reference standards and sample readings.