*2.4. Preparation of Hydrogels*

IGF-1-free composite solutions were prepared using ALG–POL, SF solution and blank BG NPs and they were used to construct blank BG/ALG–POL/SF gels for their compositional and structural optimization in order to save costly IGF-1. Some composite solutions containing IGF-1 were also prepared by directly adding IGF-1 or incorporating IGF-1-loaded BG NPs into the aqueous ALG–POL/SF mixture. These solutions were further processed into gels by incubating them in a water bath at 37 ◦C. The major parameters for them are summarized in Tables 3 and 4, respectively.


**Table 3.** Parameters for BG/alginate–poloxamer (ALG–POL)/silk fibroin (SF) hydrogels without factor loading (a).

(a) The full volume of solutions: 2mL. (b) See Table 1 for their parameters. (c) Concentration of H2O2: 500 mmol/L. (d) Concentration of HRP: 1000 U/mL. (e) Gelation time was determined by inverting vial every 1 min.


**Table 4.** Parameters for IGF-loaded BG/ALG–POL/SF hydrogels.

(a) GEL-1 and GEL-2 were directly loaded with prescribed amounts of IGF-1 for making comparisons. (b) IGF-1 load inside MBG-2 NPs was regulated by changing the IGF-1feed amount. (c) and (d) See Table 3 for their concentrations.

Gelation time was assessed using the inverted tube testing method. Typically, one of the IGF-1-free composite solutions (2.0 mL) was introduced into a glass vial and it was stirred in an ice/water bath for 5 min before being gelled. Fluidity of the composite solution was checked by regularly inverting the vial, and gelation time was recorded starting from the beginning of vial incubation in the water bath and ending at the point when the solution stopped flowing.
