Basic fibroblast growth factor (bfgf)

bFGF (Fujifilm Wako pure Chemical Corp., Japan) was desalted and fluorescently labeled using the Alexa Fluor 594 Microscale Protein Labeling Kit (Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. The absorption spectrum of Alexa Fluor 594 is completely different from that of the hydrogel, meaning that they do not interfere with each other during fluorescence imaging. Female ICR mice (~ 30 g) were used to study in vivo degradation and bFGF sustained release from a hydrogel. A 1 cm incision was made in the mediodorsal skin of mice, and a lateral subcutaneous pocket was prepared. Hydrogel with 5 μg bFGF samples (10 × 1 mm cylinders) was implanted under sterile conditions. Alexa Fluor 594-labeled bFGF aqueous solution was used as a control group (10 μg in PBS, pH 7.4) and was subcutaneously injected. At designated time intervals (days 1, 3, 7, and 14), mice (n = 5 for each day) were sacrificed. Fluorescence of the remaining hydrogel and bFGF on mouse subcutaneous tissue were imaged using the IVIS Lumina XR (excitation, 460 nm and emission, 620 nm for hydrogel imaging; excitation, 580 nm and emission, 620 nm for Alexa Fluor 594-labeled bFGF imaging), and the intensity of fluorescence was analyzed using Living Image Software (PerkinElmer Inc., MA).