Prior to biological assays, the printed scaffolds were immersed in 70 % ethanol for 5 min; then, the scaffolds were dried in lint-free tissue paper (KIMTECH) and irradiated with UV light inside a biological safety cabinet for 60 min (30 min, per side). After sterilization, the scaffolds were placed in a 48-well cell culture plate and washed with 0.4 mL of sterile PBS to remove any possible debris and remaining ethanol.
Pre-osteoblastic mouse cells MC3T3-E4 (ATCC CRL-2593) were cultured in a medium containing 90 %v/v of α-MEM (Gibco) supplemented with 10 % fetal bovine serum (FBS, Vitrocell), in an incubator (series II 3110, Thermo Fisher Scientific) at 37 °C, humidified and containing 5 % CO2. For cell proliferation assays using Alamar BlueTM Cell Viability Reagent (Invitrogen, Thermo Fisher Scientific), 5 × 103 cells were added to each well and cultured for 1 and 7 days at 37 °C, changing the media every 48 h (N=3). After each time point, the medium was removed, and 0.4 mL of a working solution (1:9 dilution of Alamar Blue in α-MEM) was added. The plate was incubated at 37 °C for 4 h in dark conditions, and, in sequence, the fluorescence (560 nm/590 nm) was recorded using a microplate reader (SpectraMax i3, Molecular Devices). PLA scaffold was the negative control, and its fluorescence on day 1 was considered 100 % of cell viability.
Pre-osteoblastic mouse cells MC3T3-E4 (ATCC CRL-2593) were cultured in a medium containing 90 %v/v of α-MEM (Gibco) supplemented with 10 % fetal bovine serum (FBS, Vitrocell), in an incubator (series II 3110, Thermo Fisher Scientific) at 37 °C, humidified and containing 5 % CO2. For cell proliferation assays using Alamar BlueTM Cell Viability Reagent (Invitrogen, Thermo Fisher Scientific), 5 × 103 cells were added to each well and cultured for 1 and 7 days at 37 °C, changing the media every 48 h (N=3). After each time point, the medium was removed, and 0.4 mL of a working solution (1:9 dilution of Alamar Blue in α-MEM) was added. The plate was incubated at 37 °C for 4 h in dark conditions, and, in sequence, the fluorescence (560 nm/590 nm) was recorded using a microplate reader (SpectraMax i3, Molecular Devices). PLA scaffold was the negative control, and its fluorescence on day 1 was considered 100 % of cell viability.