Mineralization and Sterilization of Hydrogel Discs

The hydrogel discs were mineralized as described elsewhere.^{31 (link)} Briefly, hydrogel discs were incubated in deionized (DI) water for 6 h and subsequently immersed in modified simulated body fluid (m-SBF; pH 7.4) for 6 h. The main ionic components of m-SBF are 142.0 mM Na⁺, 5.0 mM K⁺, 1.5 mM Mg²⁺, 2.5 mM Ca²⁺, 103.0 mM Cl⁻, 10.0 mM HCO₃⁻, 1.0 mM HPO₄²⁻ , and 0.5 mM SO₄^2−.33 The hydrogels were briefly rinsed in DI water and soaked in a solution containing 40 mM Ca²⁺ and 24 mM HPO₄²⁻ (pH 5.2) at 25 °C for 45 min while using a rotating shaker (VWR mini-shaker; catalog no. 12620-938) at 200 rpm. The hydrogels were briefly rinsed in DI water and further immersed in m-SBF at 37 °C for 48 h with the daily change of m-SBF. The hydrogels were then equilibrated in PBS for 6 h. The mineralized and nonmineralized PEGDA-co-A6ACA hydrogels were sterilized in 70% ethanol (EtOH) for 6 h. The EtOH-treated matrices were washed in PBS for 5 days with three daily changes of PBS prior to cell culture.