Methacrylic anhydride maa

Gelatine from porcine skin (Sigma Aldrich, St. Louis, MO, USA) was added to warm Dulbecco’s phosphate buffer saline (DPBS) (Thermo Fischer, Waltham, MA, USA) at a concentration of 100 mg mL⁻¹ and stirred until fully dissolved at 50 °C. When the gelatine solution was clear, methacrylic anhydride (MAA, Sigma Aldrich) at a concentration of 8% (v/v) was added dropwise. After this, the solution was left stirring for 3 more h at 50 °C. The solution was then diluted 1:5 in DBPS to reach a final gelatine concentration of 20 mg mL⁻¹. Gelatine methacrylate (GelMa) solution was placed in 2000 MWCO dialysis tubes (Sigma Aldrich) and dialysed in ultrapure water at 45–50 °C. Water was changed three times a day for 1 week. Once GelMa solution was dialysed, it was placed at −80 °C for 24 h, lyophilised and stored until further used.

All reagents and solvents were commercially available and used as received unless otherwise indicated. The following reagents and solvents were used:
For gelatin methacrylate synthesis and porous polymerized biomaterials; Gelatin from porcine skin (type A) (Sigma-Aldrich, Darmstadt, Germany, 99%), sodium carbonate (Sigma-Aldrich, 99%), sodium bicarbonate decahydrate (Sigma-Aldrich, 99%), sodium hydroxide (Sigma-Aldrich, 99%), methacrylic anhydride (MAA) (Sigma-Aldrich, ≥94%), milliQ water, deuterium oxide (D₂O), dialysis tubing cellulose membrane (D9777-100FT, 14 KDa), 1-Vinyl-2-pyrrolidone (VP) (Sigma-Aldrich, ≥99%), 2-hydroxyethyl methacrylate (HEMA) (Sigma-Aldrich, ≥99%), Irgacure 2959 (PI) (Sigma-Aldrich, 98%). Gibco™ (Dublin, Ireland) Collagenase, Type I, powder (205 U/mg).
In relation with the cell line used, the mouse osteoblastic cell line (MC3T3-E1) was obtained from Sigma-Aldrich (Mouse C57BL/6 calvaria, Phenotype: Adherent, Karyotype: Not specified, Morphology: Fibroblast-like).

Poly(ε-caprolactone) diol (PCL, Mn 2000 Da, Sigma-Aldrich, St. Louis, MO, USA), isophorone diisocyanate (IPDI, Acros, Geel, Belgium), Sn(Oct)₂ (Alfa Aesar, Haverhill, MA, USA), 2,2-bis(hydroxymethyl) propionic acid (DMPA, Sigma-Aldrich, St. Louis, MO, USA), triethylamine (TEA, J.T. Baker, Phillipsburg, NJ, USA), ethylenediamine (EDA, Tedia, Fairfield, OH, USA), glyoxal (Alfa Aesar, Haverhill, MA, USA), chitosan (170 kDa, 97% deacetylation, Kiotec, Hsinchu, Taiwan), acrylic acid (Showa, Gyoda, Japan), NaOH (Sigma-Aldrich, St. Louis, MO, USA), methacrylic anhydride (MAA, Sigma-Aldrich, St. Louis, MO, USA), acetic acid (Fluka, Buchs, Switzerland), pyrrole (Alfa Aesar, Haverhill, MA, USA), oxidant ammonium persulfate (APS, Sigma-Aldrich, St. Louis, MO, USA), HCl (Showa, Gyoda, Japan), high glucose Dulbecco’s modified Eagle’s medium (HG-DMEM, Gibco, Waltham, MA, USA), fetal bovine serum (FBS, Gibco, Waltham, MA, USA), Ham’s F-12 (Gibco, Waltham, MA, USA), G418 (Invitrogen, Waltham, MA, USA), penicillin-streptomycin-amphotericin (PSA, Caisson Laboratories, Inc., Smithfield, UT, USA), cell counting Kit-8 (CCK-8, Sigma-Aldrich, St. Louis, MO, USA), and KAPA SYBR Green qPCR kit (Kapa Biosystems, Inc., Wilmington, MA, USA) were applied in commercial form.