The degree of osteoblast differentiation in the cells was evaluated by determining the alkaline phosphatase (ALP) activity. After 7 days of culture using osteogenic induction medium, the adherent cells were removed from the hydrogel by homogenization in PBS with 1% Triton X-100. Then, the suspension was mixed with 0.1 M glycine NaOH buffer (pH 10.4) and 15 mM p-nitrophenyl phosphate (p-NPP; Sigma, St. Louis, MO, USA). After 30 min incubation at 37 °C, the reaction was terminated by adding 0.1 N NaOH, and the p-NPP hydrolysis was determined by ELISA reader (Spectra MAX M3) at 410 nm. Protein concentrations were measured by bicinchoninic acid (BCA) protein assay reagent kit (Pierce, Rockford, IL, USA), and normalized. To determine the hydroxyapatite nucleation on the surface of hydrogel, simulated body fluid (SBF) was used. Briefly, the fabricated hydrogels were immersed in 1× SBF (Biosesang, Sungnam, Korea), and maintained at 37 °C. After immersion period of 7 days, the hydrogels were removed from the fluid, gently rinsed with distilled water, and dehydrated with a graded ethanol series. After the sample was sputter-coated with gold, the behavior of hydroxyapatite crystal growth was observed by SEM (EM-30).