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.
Highlights
Silk fibroin (SF), a natural protein derived from Bombyx mori silk cocoons, is a millennium-old material that has been recently widely adopted in biomedical engineering.[1]
Various groups observed variation with respect to the in vitro osteogenic performance of stiff SF materials with different β-sheet contents.[13,14,15,16]. These studies indicated that this variation might be partially due to a difference in SF conformation, they failed to exclude the role of other influential factors on cells, such as chemical composition or surface topography
(14.8–120.3 MPa) in stiffness as a function of β-sheet content (Supplementary Table 1) was in accordance with the literature.[38] β-sheet content. This finding suggests that more adsorbed FN was detached with the removal of the loosened components from the the variation in stiffness within the Pa to kPa range can be sensed by cells and notably determines stem cell fate and osteogenic differentiation[25], the variation in stiffness of 2D stiff low β-sheet content material surfaces by the application of the external force.[22]. These results indicate that a high content of the β-sheets stabilized the protein-material interface, and a low material substrates for values higher than ~2 MPa does not significantly influence the osteogenic behavior of stem cells.[39,40] content of the β-sheets might compromise the detachment resistance of adsorbed FN via the reduced surface stability of the we do not expect that the stiffness of stiff SF substrates SF materials
Summary
Silk fibroin (SF), a natural protein derived from Bombyx mori silk cocoons, is a millennium-old material that has been recently widely adopted in biomedical engineering.[1]. From the underlying material substrate.[21] In turn, the interfacial instability, especially the detachment of the adsorbed proteinac- Stability of material-protein interfaces with different β-sheet eous layer induced by cells, has been found to largely disturb contents intracellular tension via the cytoskeleton.[22] the After being immersed in phosphate-buffered saline (PBS) for 24 h, adsorbed protein-FA-cytoskeleton link plays an important role in the samples were removed and gently rinsed with Milli-Q water. At the end subcellular localizations of YAP/TAZ and RUNX2 have been of immersion for 14 days, the β-sheet contents of SFL and SFM considered rapid and reliable proxies applied to evaluate material-mediated mechanotransduction by cytoskeletal cues.[25,32] reached 22.8% and 47.6%, respectively (Fig. 2c). The immunofluorescence staining images of FN on the substrates without BMSCs showed a relatively even
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