Alizarin red S quantification

Alizarin Red S staining was performed to assess mineralization of MSCs on SPAs, TPAs and BPXs. After 7 and 14 days of cultivation MSC-seeded constructs were fixed in 4% PFA for 1 hour, washed thrice with PBS and consecutively incubated in 500 μL Alizarin Red S staining solution (40 mM, Merck, Darmstadt, Germany) for 60 min at room temperature on an orbital shaker. After washing MSC-seeded constructs with distilled water Alizarin Red S was extracted using a 10% (w/v) cetylpyridinium chloride (CPC) solution (Carl Roth, Karlsruhe, Germany) for 48 hours. Finally, Alizarin Red S was quantified by measuring the optical density at 560 nm and calculated in accordance with the standard curve. At each time point Alizarin Red S quantification was performed for two independent grafts and technical replicates for 3 donors.