Gelatin a

Gelatin A with gel strength 90–110 (low-molecular-weight gelatin (LWGA), 20–25 kDa on average), Gelatin A with gel strength 300 (high-molecular-weight gelatin (HWGA), 50–100 kDa on average), and citric acid were purchased from Sigma–Aldrich (St Louis, MO, USA). Sodium alginate (SA, JUNSEI, Tokyo, Japan) and acetic acid were purchased from Dae Jung (Siheung-si, Gyeonggi-do, Korea); 0.25% trypsin-EDTA and fetal bovine serum (FBS) were purchased from Gibco (Paisley, UK). Other reagents and their suppliers were as follows: fluorescein-5-isothiocyanate (FITC) from TCI (Tokyo, Japan), silver staining kit from Thermo Scientific, basic FGF (bFGF) from CHA Meditech (Seongnam-si, Gyeonggi-do, Korea), Dulbecco’s phosphate-buffered saline (DPBS) from Welgene (Gyeongsan-si, Gyeongsangbuk-do, Korea), Dulbecco’s Modified Eagle Medium (DMEM) from Hyclone (Logan, UT, USA), the Cell Counting kit-8 (CCK-8) from Dojindo (Kumamoto, Japan), Procollagen I C-terminal propeptide (PICP) ELISA kit from Aviva systems biology (San Diego, CA, USA), HDF cells from Lonza (Walkersville, MD, USA), and the primary antibody of anti-CD31 (ab28364) and rabbit anti-mouse horseradish peroxidase (HRP) antibody as the secondary antibody from Abcam (Cambridge, UK).

Cells were seeded in six-well tissue culture plates and allowed to adhere in the presence of serum. Then the serum was withdrawn for 24 h. The medium was centrifuged to remove cellular debris. Concentrated samples with equal amounts of proteins were mixed with SDS sample buffer without reducing agent and subjected to 8% SDS–PAGE containing 0.1% gelatin A (Sigma). After electrophoresis, the gels were washed several times in 2.5% Triton X-100 for 1 h at room temperature to remove the SDS, and then incubated for 24–48 h at 37 °C in buffer containing 5 mM CaCl₂ and 1 mM ZnCl₂. Thereafter, the gels were fixed and stained with 0.25% Coomassie Blue R-250 for 4 h, and then destained in 45% methanol and 10% acetic acid. The molecular weights were estimated by reference to prestained SDS–PAGE markers.

Gelatin A (300 bloom grade, from porcine skin) and methacrylic anhydride were purchased from Sigma-Aldrich (USA). GelMA was synthesized as previously described^{60 (link)}. Briefly, a 10% w/v gelatin solution was prepared in PBS at pH 7.4. methacrylic anhydride (8 mL) was added dropwise to methacrylate amine groups along the gelatin backbones. The polymeric mixture was stirred vigorously and maintained at 60 °C for two hours after dilution with PBS. The polymeric solution was then transferred to dialysis membranes made of regenerated cellulose (~12–14 kDa cutoff) and dialyzed with deionized water for one week with two daily water changes. The GelMA solution was then frozen at −80 °C and lyophilized for 72 hours.
To fabricate GelMA-ND nanocomposite hydrogels, NDs were sonicated for 30 minutes (20 kHz, 2 sec on, 1 sec off) in deionized water at the concentration of 0.4% w/v prior addition to the GelMA solution in PBS. Nanocomposite hydrogels composition was modified varying the amount of NDs from 0.05% up to 0.2% w/v keeping constant gelatin concentration at 7% w/v and the amount of photoinitiator Irgacure 2959 (Sigma-Aldrich, USA) at 0.1% w/w. Photochemical hydrogels were then obtained after UV irradiation at 350–400 nm (Omnicure S200, Lumen Dynamics, Canada) for 6 minutes at an intensity of 7 mW/cm².