2.12. Matrix Mineralization Assay (Alizarin Red S Staining)

The iMADs cells were infected with Ad-BMP9 or Ad-GFP for 20 h. Approximately 10⁴ infected iMADs cells were seeded into each well of 24 well plates for 2 h, followed by adding 50 μL of freshly prepared CMCh-ACP hybrid gel to each well. After 1 h, the mineralization medium was added and incubated in the 37 °C incubator with 5% CO₂ as described.^{27,53,61−63} At 7 days after coincubation, mineralized matrix nodules were visualized by Alizarin Red S staining as described.^{27,53,61−63} The stained calcium mineral deposits were documented under a bright field microscope.