*2.7. Release of IGF-1*

In vitro IGF-1 release profiles for IGF-1-loaded MBG-1 and MBG-2 NPs were first tested to find out their di fference in release rates. Measurements were conducted using several sets of eppendorf tubes, each filled with 500 μL PBS containing 1% ( *w*/*v*) bovine serum albumin. To each eppendorf tube, 5 mg of IGF-I-loaded MBG-1 or MBG-2 NPs was added, and all sets of tubes were incubated in a shaking water bath at 37 ◦C and 60 rpm for various periods up to 6 days. At predetermined time intervals, eppendorf tubes were withdrawn by group and they were centrifuged for 5 min at around 1000 g to collect supernatants. The release amount of IGF-I was determined using IGF-1 ELISA kit.

In the case of IGF-1 release from gel samples, some cylindrical gel samples were first produced. Each (0.5 mL) of IGF-1-loaded composite solutions (see Table 4) was filled into a cylindrical mold (diameter: 10 mm) and incubated at 37 ◦C for 20 min for gel formation. The gel samples were then introduced into di fferent vials filled with 3 mL of PBS, and the vials were vortexed on a shaking table at 37 ◦C and 60 rpm. At prescribed time points, 1 mL of medium was withdrawn with replenishing the same volume of fresh bu ffer. The released amount of IGF-1 was measured using IGF-1 ELISA Kit.

## *2.8. Bioactivity of Released IGF-1*

The IGF-1's ability to promote the proliferation of osteoblasts was tested to access the bioactivity of released IGF-1 [30–32]. MC3T3-E1 cells (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) were used as testing cells. Cells were expanded in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin in a 5% CO2 humidified atmosphere at 37 ◦C. The expanded cells were resuspended in PBS for further use.

Cells were cultured in 96-well plates at a density of 5 × 10<sup>4</sup> cells/well in complete culture medium at 37 ◦C for 24 h. The cells were serum-starved for 24 h, after which the media was replaced with either serum-free media (denoted as control, 0 ng/mL), serum-free media with free IGF-1 (5 or 50 ng/mL), or serum-free media with released IGF-1 (5 or 50 ng/mL). The cells were cultured for varied durations up to 72 h, and their proliferation was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT, Dojindo, Japan) essay. Briefly, the media was aspirated from the 96-well plates after the prescribed culture period, and a 20 μL aliquot of MTT stock solution and 200 μL of serum-free medium was added to each well. After being further cultured for 4 h, the media was removed from the wells, and 150 μL of DMSO was added to each well with shaking at 37 ◦C for 15 min. After that, the optical density (OD) was determined at 590 nm using a microplate reader (PerkinElmer Inc, USA).

Cells were also cultured on the surface of gels for making further comparison. Briefly, two kinds of IGF-1-contained composite solutions with their compositions respectively matching with GEL-2 and GEL-4 gels (see Table 4), 200 μL apiece, were pipetted into wells of 24-well plates, and cultured at 37 ◦C for gel formation. Subsequently, volume of MC3T3-E1 cell suspension (100 μL) was added to each well (5 × 10<sup>4</sup> cells/well), and cells were then cultured with serum-free media (500 μL) at 37 ◦C for varied periods up to 72 h. Cell proliferation was assessed by OD measurement using above described MTT assay. Cell cultured under monolayer condition in serum-free media (0 ng/mL) without exposing to gels were used as control.
