A Bio-inspired Multifunctionalized Silk Fibroin Sofia Santi, Ines Mancini, Sandra Dire,̀ Emanuela Callone, Giorgio Speranza, Nicola Pugno, Claudio Migliaresi, and Antonella Motta* ABSTRACT: A bio-inspired multifunctionalized silk fibroin (BMS) was synthesized in order to mimic the interaction of nidogen with the type IV collagen and laminin of basement membranes. The designed BMS consists of a motif of laminin αchain-derived, called IK peptide, and type IV collagen covalently bound to the silk fibroin (SF) by using EDC/NHS coupling and a Cu-free click chemistry reaction, respectively. Silk fibroin was chosen as the main component of the BMS because it is versatile and biocompatible, induces an in vivo favorable bioresponse, and moreover can be functionalized with di fferent methods. The chemical structure of BMS was analyzed by using X-ray photoelectron spectroscopy, attenuated total reflection−Fourier transform infrared, cross-polarization magic angle spinning nuclear magnetic resonance techniques, and colorimetric assay. The SF and BMS solutions were cross-linked by sonication to form hydrogels or casted to make films in order to evaluate and compare the early adhesion and viability of MRC5 cells. BMS hydrogels were also characterized by rheological and thermal analyses. KEYWORDS: silk fibroin, laminin peptide, chemical modification, tissue regeneration, biomimetic ■ INTRODUCTION molecules. It has several actions: providing biochemical and The use of silk fibroin for the fabrication of biomedical mechanical signaling to cells and facilitating intercellular and prostheses and tissue engineering scaffolds has been widely intracellular interactions, modulating cell behavior, 6,7 and investigated in the past years. The molecular structure of the regulating tissue development, function, and repair. silk fibroin (SF), i.e., the protein composing the core part of The BM marks the paths for cell migration and works as a the silkworm filament, consists of a light chain (Mw ≈ 26 kDa) charge-selective filtration barrier due to its net negative charge, and a heavy chain (Mw ≈ 350 kDa) linked together by a regulating the passage of negative molecules from the blood disul fide bond. Hydrophilic groups are mainly present in the stream to tissues. 8,9 It is also a structural support for tissue and light chain, whereas the heavy chain has a primary structure body cavity wall development and performs an important role formed by repetitive sequences of GAGAGS, GAGAGY, and during the neural tissue remodeling, promoting synaptogenesis GAGAGVGY, mainly hydrophobic, that interact with each and myelination of nerves. 10,11 is in terms of composition, morphology, and properties to the extracellular matrix, the faster and more effective the regeneration process will be. The basement membrane (BM) is a sheet-like thin extracellular matrix that is essential for animal development. The BM mainly consists of laminin and type IV collagen, Col(IV), interrelated with nidogen, perlecan, and other other. 1 The amino acid sequence of fibroin and the presence along the chain of RGD-like sequences are considered the main reasons for the favorable interactions that fibroin materials possess with cells and biological environments. 2−5 Moreover, fibroin can be chemically conjugated with active peptides in order to achieve specific biological performances for selected applications. Tissue engineering fixes the objective to produce cell supportive matrices in order to induce the regeneration of damaged tissues and organs. In general, the closer the scaffold The mimesis of the composition, structure, and functionality of the BM is still a challenge. Previous studies reported the conjugation of laminin in different hydrogels based on collagen type I, hyaluronic acid, alginate, or polyacrylamide gels, failing in inducing the cooperation with Col(IV). 12−14 In other cases, 15,16 blends of laminin and Col(IV) have been made. Worth nothing is the fact that changes in the hydrogel’s composition could negatively affect the cell behavior. For example, the loss of