Caso4

Chitosan (low molecular weight) with 85% deacetylation (CS), Lignosulfonic acid sodium salt (LS), MgSO₄, sodium citrate, Absolute ethanol, CH₃CH₂OH (≥99.8%), CaSO₄, NH₄Cl, NaCl, Na₂SO₄, KCl, SrCl₂, KBr, K₂HPO₄, HCl, NaOH, hexamethylenetetramine, sodium lactate, and yeast extract were obtained from Sigma-Aldrich (St. Louis, MO, USA). All analytical grade chemicals were used as received. Carbon steel (SS400) rods of 10 mm diameter were obtained locally. The chemical composition of SS400 is 99.25–100% Fe, 0–0.4% Si, 0–0.26% C, 0–0.05% S, and 0–0.04% P. PhenoCure^TM (phenolic resin) was procured from Buehler, Lake Bluff, IL, USA.

We slightly modified the protocol proposed by Hinton and colleagues [11 (link)]. Briefly, we prepared the gelatin slurry support bath by dissolving 10 g of gelatin (Sigma-Aldrich, Stockholm, Sweden) into 250 mL of 11 mM CaSO₄ (Sigma-Aldrich, Stockholm, Sweden), at 45 °C. The slurry was stored at 4 °C, overnight. We added 350 mL of 11 mM CaSO₄ to the gelatin, and, after placing it at −20 °C for 45 min, we blended it for 60 s. We placed 40 mL of blended gelatin into a 50 mL conical tube and centrifuged it for 7 min, at 4100 rpm; the supernatant was then removed, and new gelatin slurry was added, to get a volume of 40 mL. This step was repeated three times, to make all the gelatin blended. The tubes were centrifuged at 225 g for 5 min, at 4 °C, prior to 3D printing; the supernatant was removed, and the slurry was placed into a petri dish. We bioprinted our conductive ink, using Inkredible+ through a 21G needle at a pressure of about 15 kPa. The STL file was created by using Thinkercad (www.thinkercad.com), and it was converted into G-Code, using Slic3r software (https://slic3r.org/). After bioprinting, we placed the Petri dish into an incubator, at 37 °C, for 60 min. The melted gelatin was removed, and after drying, printed scaffolds were sterilized by placing them into 70% ethanol. Finally, the cells were seeded onto the printed scaffolds, for further studies.