Alginic acid sodium salt from brown algae

Methacrylated alginate (MeALG) was synthesized as described previously [85 (link),94 (link)]. Briefly, 0.5% (w/v) was prepared by dissolving 5 g of medium viscosity alginate (alginic acid sodium salt from brown algae, Sigma-Aldrich Inc., St. Louis, MO, USA) in 1 L of DI water. The solution was kept under magnetic stirring at 1–4 °C. Once the alginate was fully dissolved, 10 mL of methacrylate anhydride (MA, Sigma-Aldrich Inc., St. Louis, MO, USA) was added dropwise into the solution within a span of 1.5–2 h. 2 M NaOH solution (Sigma-Aldrich Inc., St. Louis, MO, USA) was simultaneously added dropwise to adjust the pH of the solution to 8–9. After the addition of the MA, pH of the mixture was maintained by gradually dripping 2 M NaOH solution for 8 h using an automated pH controller. The solution was kept at 4 °C overnight. The reaction was resumed the following day by adding 5 mL of MA while maintaining the pH at 8–9. The material was then dialyzed (Spectra/Por^®1 dialysis membrane, 6–8 kDa, Fisher Scientific, Pittsburgh, PA, USA) against DI water for 5 days and lyophilized using a benchtop freeze dryer (Labconco FreeZone 4.5 L, Fisher Scientific, Pittsburgh, PA, USA). ¹H NMR (Bruker Advance III HD 500 MHz, Bruker Scientific, Billerica, MA, USA) was used to confirm the methacrylate percentage as described previously [85 (link)].

Alginic acid sodium salt from brown algae (medium viscosity), piperazine (99%), calcium chloride (<99%), and sodium hydroxide (98%) were purchased from Sigma Chemicals (Madrid, Spain). Barium chloride dehydrate (>99%), reagent grade was supplied by Scharlau. Hydrochloric acid (37%) was purchased from Panreac (Castellar del Valles, Barcelona, Spain). Air for the atomization system was provided by Air Liquid (99.99%).

Alginic acid sodium salt from brown algae (guluronic/mannuronic acid ratio of 70/30) was supplied by Sigma Life Science (Irvine, UK). Vancomycin hydrochloride (C₆₆H₇₅Cl₂N₉O₂₄·HCl, 94.3% purity) was supplied by Guinama (Valencia, Spain). Calcium chloride anhydrous (CaCl₂, >99% purity) was supplied by Scharlab (Barcelona, Spain). Absolute ethanol (EtOH, >99.9% purity) and CO₂ (99.8% purity) were purchased from VWR Chemicals (Fontenay-sous-Bois, France) and Nippon Gases (Madrid, Spain), respectively. Tetraethylorthosilicate (TEOS, 98% purity), ammonium aqueous solution 30–33% and 1H,1H,2H,2H-perfluorodecyltriethoxisilane (PFDTS, 97% purity) were from Sigma–Aldrich (Darmstadt, Germany). Water was purified using reverse osmosis (resistivity > 18 MΩ·cm, Milli-Q, Millipore^®, Madrid, Spain).
Ultrapure nitrogen (N₂ (g), >99% purity) supplied by Praxair (Madrid, Spain) was used for the adsorption-desorption textural analysis. Phosphate buffered saline (PBS) pH 7.4, and potassium dihydrogen phosphate (KH₂PO_4, 98.0–100.5% purity) were both supplied by ITW Reagents (Barcelona, Spain). Sodium hydroxide (NaOH, 99% purity) and acetonitrile (CH₃CN, ≥99.9% purity) were both purchased from VWR Chemicals (Barcelona, Spain).

Composite (bio)inks were prepared by adding MBG in already established 3–6 algMC blend (Guduric et al., 2021 (link)). In brief, a 3 wt% alg solution was prepared by dissolving alginic acid sodium salt from brown algae (Sigma-Aldrich, Steinheim, Germany) in phosphate-buffered saline (PBS), stirred overnight, and autoclaved. Autoclaved methylcellulose (MC) powder (Sigma-Aldrich, Steinheim, Germany) was added into the alg solution to obtain a final concentration of 6 wt%. After mixing with a spatula, the blend was left for 30 min to allow swelling of MC. CaMBG or MgMBG were added into the algMC blend prior to printing to obtain a final concentration of 7 wt%, which was established as a suitable one for printing and biological properties of composite bioinks (Guduric et al., 2021 (link)).

Salts and other ingredients for buffers and
bacteria culture media,
dopamine hydrochloride, fetal bovine serum (FBS), l-glutamine,
sodium l-(+)-lactate, kanamycin sulfate, β-d-1-thiogalactopyranoside (IPTG), and phosphate-buffered saline (PBS)
were obtained from Sigma-Aldrich (St. Louis, MO). Alginic acid sodium
salt from brown algae (ref 71238), poly-l-lysine hydrochloride
(MW 15 000–30 000 Da), and low-molecular-weight
chitosan (ref 448869) were also supplied by Sigma-Aldrich. Chemically
competent E. coli NEB5α cells
were purchased from New England Biolabs (NEB, MA).
M9 culture
medium was prepared with 33.7 mM Na₂HPO₄, 22
mM KH₂PO₄, 8.55 mM NaCl, and 9.35
mM NH₄Cl as 10× stock and autoclaved. It was supplemented
with 0.4% d-glucose (or glycerol), 1 mM MgSO₄,
0.3 mM CaCl₂, and 1 mg/L thiamine from stocks that were
prepared separately and filter-sterilized. Krebs–Ringer N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic
acid (HEPES) (KRH buffer) buffer was prepared with 20 mM HEPES, 135
mM NaCl, 5 mM KCl, 0.4 mM K₂HPO₄, adjusted to
pH 7.4, autoclaved, and supplemented with 1 mM MgSO₄, and
1 mM CaCl₂ from stocks prepared separately and filter-sterilized.
Incubation buffer (buffer A) consisted of 10 mM HEPES, 150 mM NaCl,
20 mM CaCl₂, and disruption buffer (buffer B) of 0.1 M
ethylenediaminetetraacetic acid (EDTA) and 0.2 M potassium citrate;
both were filter-sterilized.

Alginic acid sodium salt from brown algae (Sigma-Aldrich, Steinheim, Germany) was dissolved at a concentration of 3 wt.% in phosphate buffered saline (PBS) and stirred overnight. The resulting alg solution and MC powder (Sigma-Aldrich, Steinheim, Germany) were autoclaved and stored (alg at 4 °C and MC at room temperature) until ink preparation for 3D printing. To prepare algMC blends, MC powder was added to alg solution to obtain final concentrations of algMC of 3 wt.%: 6 wt.% (later called 3–6 blend) or 3 wt.%: 9 wt.% (later called 3–9 blend). The mixtures were stirred thoroughly with a spatula and left for 30 min to allow the swelling of MC. The resulting blends were then used for printing in their native state or mixed with MBG.