Abstract
Previously, we have described the preparation of a novel fibroin methacrylamide (FbMA), a polymer network with improved functionality, capable of photocrosslinking into Fb hydrogels with elevated stiffness. However, it was unclear how this new functionality affects the structure of the material and its beta-sheet-associated crystallinity. Here, we show that the proposed method of Fb methacrylation does not disturb the protein’s ability to self-aggregate into the stable beta-sheet-based crystalline domains. Fourier transform infrared spectroscopy (FTIR) shows that, although the precursor ethanol-untreated Fb films exhibited a slightly higher degree of beta-sheet content than the FbMA films (46.9% for Fb-F-aq and 41.5% for FbMA-F-aq), both materials could equally achieve the highest possible beta-sheet content after ethanol treatment (49.8% for Fb-F-et and 49.0% for FbMA-F-et). The elasticity modulus for the FbMA-F-et films was twofold higher than that of the Fb-F-et as measured by the uniaxial tension (130 ± 1 MPa vs. 64 ± 6 MPa), and 1.4 times higher (51 ± 11 MPa vs. 36 ± 4 MPa) as measured by atomic force microscopy. The culturing of human MG63 osteoblast-like cells on Fb-F-et, FbMA-F-et-w/oUV, and FbMA-F-et substrates revealed that the photocrosslinking-induced increment of stiffness increases the area covered by the cells, rearrangement of actin cytoskeleton, and vinculin distribution in focal contacts, altogether enhancing the osteoinductive activity of the substrate.
Highlights
The mechanical characteristics of scaffolds for tissue engineering are of the utmost importance for cell attachment and differentiation
Pristine Fb as a lyophilisate was1.used for synthesis of (Fb) its methacrylated derivative fibroin methacrylamide (FbMA) [18]
Our study showed that the MG63 cells can attach, proliferate, and survive on FbMA-F-et films, indicating a good cytocompatibility of the scaffold (Figure 6A–C) [18,39]
Summary
The mechanical characteristics of scaffolds for tissue engineering are of the utmost importance for cell attachment and differentiation. The roles of the charge and hydrophobicity of the substrate in ECM mineralization and ALP activity, as well as vinculin distribution in osteoblastic MC3T3-E1 cells on self-assembled monolayers of alkanethiols on gold with surface functionalization by –CH3, –OH, –COOH, and –NH2 moieties, have been addressed in [49,50]. Genes specific for osteogenesis were upregulated compared to the cells cultured on the substrates functionalized by alkyl and carboxylic groups [36]. Both negative and positive charges promote the recruitment of vinculin during focal adhesion assembly in comparison with neutral and hydrophobic surfaces. Hydrophobicity and the lowered charge can be compensated by the ability of Fb to adsorb positively charged hydrophilic molecules in the plasma, e.g., fibronectin and laminin, thereby altering the physico-chemical properties of the surface and countering the effects of hydrophobicity and negative charge on cellular functions [51]
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