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.

丝素蛋白 (SF) 可用于构建各种硬质材料界面以支持骨形成。一个重要的准备步骤是将 SF 分子从无规卷曲部分转变为 β 折叠，使材料不溶于水。然而，SF 构象对材料界面成骨细胞行为的影响仍然未知。在此，通过改变 β-折叠含量（高、中和低）制备三种硬 SF 基板。基板具有可比较的化学成分、表面形貌和润湿性。当吸附的纤连蛋白用作模型细胞粘附蛋白时，吸附蛋白-材料界面的稳定性，就 SF 底物的表面稳定性和伴随的纤连蛋白脱离阻力而言，随着 SF 底物的 β-折叠含量的增加而增加。此外，(i) 更大面积的细胞骨架相关粘着斑，(ii) 更高阶的细胞骨架组织和 (iii) 在 SF 基质上培养的骨髓间充质基质细胞 (BMSC) . 低 β-片层含量，以及增强的核易位和 YAP/TAZ 和 RUNX2 的激活。因此，BMSCs 的成骨分化在高 β-折叠底物上受到刺激。这些结果表明，β-折叠含量通过调节吸附的蛋白质-材料界面的稳定性，影响 BMSCs 在体外 SF 材料上的成骨分化，这通过蛋白质-粘着斑-细胞骨架连接和随后的细胞内机械转导进行。