Dual mode gelation behavior of silk fibroin microgel embedded poly(ethylene glycol) hydrogels

Abstract

Hydrogel formation by more than two cross-linking mechanisms is preferred for the sophisticated manipulation of hydrogel properties. Both chemical and physical crosslinks are often utilized for fabricating stimuli-responsive hydrogels or for compensating the drawbacks of the single crosslinking method. In this study, silk fibroin (SF) microgel embedded poly(ethylene glycol) (PEG) hydrogels were fabricated by dual mode cross-linking based on thiol–ene photo-click chemistry and β-sheet formation of SF. Norbornene-functionalized SF (SF–NB) was incorporated into PEG hydrogels by photo-cross-linking. The equilibrium shear modulus of SF–PEG hybrid hydrogels decreased with increasing SF–NB content. However, the incorporation of SF–NB caused stiffening of SF–PEG hybrid hydrogels gradually over 5 days and the gel modulus was maintained for 2 weeks. In contrast, the modulus of pure PEG hydrogels decreased continuously owing to hydrolytic degradation of ester bonds in the PEGNB macromers. Structural analysis revealed that such a post-gelation stiffening effect was caused by β-sheet transition in SF microgels embedded in the PEG hydrogel matrix. PEG hydrogels incorporated with 4 wt% SF microgels exhibited about 2-fold increase in shear modulus compared with the modulus on day 1 post-gelation. To evaluate the compatibility of these hydrogels as cell culture matrices, the cytotoxicity of the hydrogel was examined using in situ encapsulated A549 cells. SF–PEG hybrid hydrogels showed no apparent cytotoxicity and promoted the proliferation of encapsulated A549 cells even at a higher gel modulus compared with cells in pure PEG hydrogels. These results suggest that SF–PEG hybrid hydrogels fabricated by dual mode crosslinking serve as good candidates for three-dimensional cell culture requiring temporal control of hydrogel stiffness.