Different tissues have complex anisotropic structures to support
biological functions. Mimicking these complex structures in vitro remains a challenge in
biomaterials designs in support of tissue regeneration. Here, inspired by different types of
silk nanofibers, a composite materials strategy was pursued towards this challenge. A combination of fabrication methods was utilized to achieve separate control of amorphous and beta-sheet rich
silk nanofibers in the same
solution. Aqueous solutions containing these two structural types of
silk nanofibers were then simultaneously treated with an electric field and with
ethylene glycol diglycidyl ether (EGDE). Under these conditions, the beta-sheet rich
silk nanofibers in the mixture responded to the electric field while the amorphous nanofibers were active in the crosslinking process with the EGDE. As a result,
cryogels with anisotropic structures were prepared, including mimics for cortical- and cancellous-like bone
biomaterials as a complex osteoinductive niche. In vitro studies revealed that mechanical cues of the
cryogels induced osteodifferentiation of stem cells while the anisotropy inside the
cryogels influenced immune reactions of macrophages. These bioactive
cryogels also stimulated improved bone regeneration in vivo through modulation of
inflammation, angiogenesis and osteogenesis responses, suggesting an effective strategy to develop bioactive matrices with complex anisotropic structures beneficial to tissue regeneration.