The molecular conformation of silk fibroin regulates osteogenic cell behavior by modulating the stability of the adsorbed protein-material interface

Abstract

Silk fibroin (SF) can be used to construct various stiff material interfaces to support bone formation. An essential preparatory step is to partially transform SF molecules from random coils to β-sheets to render the material water insoluble. However, the influence of the SF conformation on osteogenic cell behavior at the material interface remains unknown. Herein, three stiff SF substrates were prepared by varying the β-sheet content (high, medium, and low). The substrates had a comparable chemical composition, surface topography, and wettability. When adsorbed fibronectin was used as a model cellular adhesive protein, the stability of the adsorbed protein-material interface, in terms of the surface stability of the SF substrates and the accompanying fibronectin detachment resistance, increased with the increasing β-sheet content of the SF substrates. Furthermore, (i) larger areas of cytoskeleton-associated focal adhesions, (ii) higher orders of cytoskeletal organization and (iii) more elongated cell spreading were observed for bone marrow-derived mesenchymal stromal cells (BMSCs) cultured on SF substrates with high vs. low β-sheet contents, along with enhanced nuclear translocation and activation of YAP/TAZ and RUNX2. Consequently, osteogenic differentiation of BMSCs was stimulated on high β-sheet substrates. These results indicated that the β-sheet content influences osteogenic differentiation of BMSCs on SF materials in vitro by modulating the stability of the adsorbed protein-material interface, which proceeds via protein-focal adhesion-cytoskeleton links and subsequent intracellular mechanotransduction. Our findings emphasize the role of the stability of the adsorbed protein-material interface in cellular mechanotransduction and the perception of stiff SF substrates with different β-sheet contents, which should not be overlooked when engineering stiff biomaterials.

Crimped nanofiber scaffold mimicking tendon-to-bone interface for fatty-infiltrated massive rotator cuff repair

“…Furthermore, the mechanical properties of crosslinked scaffolds were significantly improved compared with those of non-crosslinked scaffolds [ 51 , 52 ]. As suggested, the osteogenic induction of osteoblasts was increased on the stiff scaffolds and more bone matrix was generated at the defect site [ [53] , [54] , [55] ]. Tendon defects and fatty infiltration of the muscle contribute to poorer outcomes for fi-MRCTs than for smaller RCTs.…”

Bioinspired silk fibroin materials: From silk building blocks extraction and reconstruction to advanced biomedical applications

“…In this topic, Long et al. have fabricated three kinds of RSF substrates with high, medium, and low stiffness by varying the β -sheet content [ 133 ], which were named as SFH, SFM, and SFL groups respectively, as shown in Fig. 10 A–B.…”

“…When adsorbed fibronectin, the stability of the adsorbed protein-material interface increased with the increasing of stiffness of the RSF substrates. Finally, larger areas of cytoskeleton-associated focal adhesions, higher orders of cytoskeletal organization and more elongated cell spreading were all observed for BMSCs cultured on RSF substrates with high stiffness than lower ones, along with enhanced nuclear translocation and activation of YAP/TAZ [ 133 ]. The stiff RSF substrate with higher β -sheet content further enhanced the osteogenesis of BMSCs, evidenced by higher expression of ALP and collagen Ι, and also denser deposition of calcium [ 133 ], as presented in Fig.…”

“…The RSF substrate with high, medium, and low contents of β -sheets were denoted as SFH, SFM, and SFL group, respectively. Reproduced with permission [ 133 ]. Copyright 2021, Springer Nature.…”

Genetic hybridization of highly active exogenous functional proteins into silk-based materials using “light-clothing” strategy

“…After air drying, the films were annealed by water vapor at room temperature overnight. 46 Finally, they were punched into discs and observed by scanning electron microscope (Sigma-300, Zeiss, German) and digital camera. Nanofiber meshes were prepared by a commonly used electrospinning method.…”