Scheme 1. The preparation process of the DFO-loaded SFH-HA composite hydrogel.

Fig. 1. Morphology and crystal structure of different hydrogels: (A) SEM images and (B) XRD curves of the hydrogels. (a, a') SFH, pure silk nanofiber hydrogel; (b, b') SFH-DFO, DFO-loaded silk nanofiber hydrogel; (c, c') SFH-HA, silk nanofiber-hydroxyapatite nanocomposite hydrogel; and (d, d') DFO-loaded silk nanofiber-hydroxyapatite nanocomposite hydrogel.

Fig. 2. Characterization of different hydrogels: (A) Flow curves of the hydrogels; (B) Injectability of the different hydrogels through a syringe without a needle (a, b, c, and d) and with a 25 G needle (a', b', c', and d'): (a and a') SFH, (b and b') SFH-DFO, (c and c') SFH-HA, and (d and d') SFH-HA-DFO; (C) Inversion test of the hydrogels at different time points. The samples are as follows: a, SFH, b, SFH-DFO, c, SFH-HA, d, SFH-HA-DFO; (D) Frequency sweep of the hydrogels; (E) Complex modulus (G*) vs temperature of the hydrogels and (F) The release behavior of DFO from the SFH-DFO and SFH-HA-DFO hydrogels in PBS.

Fig. 3. In vitro angiogenesis and osteogenesis of the BMSCs cultured on different hydrogels: (A) Representative confocal images of HUVEC network when BMSCs were cultured on different hydrogels for 12 and 24 h; (B) The total tube length of the networks when BMSCs were cultured on different hydrogels for 12 and 24 h; (C-F) The expression of RUNX2, ALP, OCN and OPN when BMSCs were cultured on the different hydrogels for 7, 14 and 21 days; (G) Immunofluorescence staining of ALP when BMBCs were cultured on different hydrogels for 7 days; (H, I) Immunofluorescence staining of OCN and OPN when BMBCs were cultured on different hydrogels for 21 days. The scale bars were 200 μm. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Fig. 4. Neovascularization and bone regeneration of the defects treated with different hydrogels: (A) the neovascularization in the defects after hydrogel implantation for 2 and 4 weeks. CD31 was stained with red while α-smooth muscle actin positive cells were stained with green. The scale bars were 100 μm; (B) Micro CT images of the defects when treated with different hydrogels for 4, 8 and 12 weeks; (C-F) Bone volume, bone volume/total volume ratio (BV/TV), trabucular number (Tb. N) and trabecular thickness (Tb. Th) analyses of the defects when treated with different hydrogels for 4, 8 and 12 weeks. *p ≤ 0.05;**p ≤ 0.01; ***p ≤ 0.001.

Fig. 5. HE staining (A) and Masson staining (B) of the regenerated bone tissues at 12 weeks post-implantation. HB indicates host bone while NB means now bone. The black arrows points to the new formed blood vessels. The blue stained areas are the formed mature bones.