hMSCs were cultured with continuous treatment with osteoblast differentiation reagents contained media. After 21 days, the cell-loaded hydrogels were fixed with 70% ice-cold ethanol for 1 h at 4 °C. After the ethanol was removed, calcium accumulation was measured by staining with 40 mM Alizarin Red-sulfate (AR-S; Sigma-Aldrich, St. Louis, MO, USA) solution, and normalized with non-cultured scaffold, respectively. The stained portions were photographed by digital camera. The deposited stain was then dissolved using 10% cetylpyridinium chloride solution and the absorbance was read at 562 nm by ELISA reader.