Tnbsa

The degree of crosslinking was determined using 2,4,6-trinitrobenzensulfonic acid (TNBSA; Thermo Scientific, USA) by calculating the primary amine content of crosslinked and uncrosslinked gelatin samples. Briefly, 2–4 mg of gelatin was placed in a conical tube with 1.0 mL of 4% (w/v) sodium bicarbonate solution (NaHCO₃, pH 8.5) and 1.0 mL of 0.5% (w/v) in methanol. After incubation at 40 °C for 2 h, the solution was treated with 3 mL of 6 M HCl, and the temperature was increased to 60 °C for 1.5 h to solubilize gelatin samples. The absorbance of the resulting solution was measured at 345 nm using a spectrophotometer (Optizen 3220UV; Mecasys Co., Ltd., Korea) after diluting with 4 volumes of deionized water. The degree of crosslinking was calculated using the following equation [9 (link)]: $$\text{Crosslinking degree}\left(\%\right)=\left[1-\left(\frac{{\mathit{\text{absorbance}}}_c/{\mathit{\text{mass}}}_c}{{\mathit{\text{absorbance}}}_u/{\mathit{\text{mass}}}_u}\right)\right]\times 100$$
where the subscripts c and u stand for the crosslinked and uncrosslinked gelatin, respectively (n = 3).

Vitronectin (VN, Peprotech, 1 mg) was PEGylated by modifying a previously published procedure (Almany and Seliktar, 2005 (link)). VN was dissolved in NaHCO₃ 0.1 M (pH 8.5). Subsequently, Maleimide-PEG-Succinimidyl Valerate (MAL-PEG-SVA, 3.4 kDa, LaysanBio) was mixed at a mass ratio VN:SVA 1:4 and incubated for 1 h at room temperature (RT) (Figure 1). The product of the reaction was dialyzed (Mini-A-Lyzer, MWCO 10 kDa, ThermoFisher) against PBS for 1 h at RT and stored at −20°C or immediately used. The degree of PEGylation for different VN:SVA ratios was measured by tracking the reduction in primary amines after the reaction using a 2,4,6-trinitrobenzene sulfonic acid (TNBSA, ThermoFisher) assay, which indicated an increase in the VN degree of modification as the VN:SVA ratio increased (Supplementary Figure S1).