Model 8

Fibers and inserts were weighed (4 mg) and added in separate to 1 mL of culture medium. The tubes were vortexed until specimen dissolution and kept at 37 °C. BalB/3T3 cells (CCL-163; ATCC, USA) were cultured at 37 °C in 75 cm² flasks using complete culture medium (DMEM supplemented with 10% v/v fetal bovine serum and 1% v/v penicillin/streptomycin/fungizone (PSF)) until 80% confluence was reached. Then, cells were trypsinized using TripLE solution (Sigma-Aldrich, MO, USA) and seeded in wells of 96 well plates (5000 cells/well). After 24 h, culture medium was removed and 100 μL of the sample solutions (4 mg/mL) and 100 μL of fresh culture medium were added to each well. Culture medium was used as negative control. Cells were again incubated at 37 °C for 24 or 48 h. At each timepoint, the medium was discarded and the cells were washed with PBS. Immediately after, freshly prepared CCK8 working solution (100 μL) consisting of 90 μL of culture medium and 10 μL of CCK-8 reagent (Sigma-Aldrich, MO, USA) was added to each well and then the cells incubated for 2 h at 37 °C. Finally, absorbance (450 nm) was recorded using a microplate reader (Model 8; BioRad, CA, USA).

BalB/3T3 cells (CCL-163; ATCC, Manassas, VA, USA) were used to evaluate the cytotoxicity of different 3D printed discs (6 mm diameter × 1 mm height) prepared with the drug-loaded formulations (D10, D20, D10PEG, D20PEG) and the control formulations (C-PEGDA and C-PEGDA:PEG). Cells were cultured at 37 °C in 75 cm² flasks using complete culture medium (DMEM supplemented with 10% v/v fetal bovine serum (FBS) and 1% v/v penicillin/streptomycin/fungizone (PSF)) until 80% confluence was reached. Cells were then trypsinized using TripLE solution (Sigma-Aldrich, St. Louis, MO, USA) and seeded in wells of 24 well plates at a concentration of 3 × 10⁴ cells per well. After 24 h, the discs (n = 5) were placed individually in wells, and plates were returned for incubation at 37 °C for 24 or 48 h. At each timepoint, medium was discarded and cells were washed with PBS. Freshly prepared CCK8 working solution (400 μL) consisting of culture medium (360 μL) and CCK-8 reagent (40 μL) (Dojindo; Tokyo, Japan) was added to each well and incubated for 2 h at 37 °C. Absorbance was recorded at 450 nm using a microplate reader (Model 8; BioRad, Hercules, CA, USA).