Mechanical interactions between a cell and its environment regulate migration, contractility, gene expression, and cell fate. We integrated micropatterned substrates to engineer adhesive area and a hydrodynamic assay to analyze fibroblast adhesion strengthening on fibronectin. Independently of cell spreading, integrin binding and focal adhesion assembly resulted in rapid sevenfold increases in adhesion strength to steady-state levels. Adhesive area strongly modulated adhesion strength, integrin binding, and vinculin and talin recruitment, exhibiting linear increases for small areas. However, above a threshold area, adhesion strength and focal adhesion assembly reached a saturation limit, whereas integrin binding transitioned from a uniform distribution to discrete complexes. Adhesion strength exhibited exponential increases with bound integrin numbers as well as vinculin and talin recruitment, and the relationship between adhesion strength and these biochemical events was accurately described by a simple mechanical model. Furthermore, adhesion strength was regulated by the position of an adhesive patch, comprised of bound integrins and cytoskeletal elements, which generated a constant 200-nN adhesive force. Unexpectedly, focal adhesion assembly, in particular vinculin recruitment, contributed only 30% of the adhesion strength. This work elucidates the roles of adhesive complex size and position in the generation of cell-extracellular matrix forces.

Protein adsorption onto synthetic materials influences cell adhesion and signaling events that direct cell function in numerous biomedical applications. Adsorption of fibronectin (FN) to different surfaces alters protein structure and modulates α5β1 integrin binding, cell adhesion, cell spreading, and cell migration. In the present study, self-assembled monolayers of alkanethiols on Au were used to analyze the effects of surface chemistry (CH3, OH, NH2, and COOH) on the adsorption of a recombinant fragment of FN, FNIII7 - 10, that incorporates both the synergy and RGD cell binding motifs. Surface chemistry potentiated differential FNIII7 - 10 adsorption kinetics and adsorbed structure as determined by surface plasmon resonance spectroscopy and antibody binding assays. FNIII7 - 10 functional activity, determined by cell adhesion strength, was modulated in a fashion consistent with these structural changes (OH = NH2 > COOH > CH3). However, these changes in protein parameters did not correlate simply to differences in surface hydrophobicity, indicating that additional surface parameters influence protein adsorption. These results demonstrate that surface chemistry modulates adsorbed protein structure and activity and establish a relationship between surface-dependent changes in structural domains of FNIII7 - 10 and functional activity.

Focal adhesion kinase (FAK) is an essential nonreceptor tyrosine kinase regulating cell migration, adhesive signaling, and mechanosensing. Using FAK-null cells expressing FAK under an inducible promoter, we demonstrate that FAK regulates the time-dependent generation of adhesive forces. During the early stages of adhesion, FAK expression in FAK-null cells enhances integrin activation to promote integrin binding and, hence, the adhesion strengthening rate. Importantly, FAK expression regulated integrin activation, and talin was required for the FAK-dependent effects. A role for FAK in integrin activation was confirmed in human fibroblasts with knocked-down FAK expression. The FAK autophosphorylation Y397 site was required for the enhancements in adhesion strengthening and integrin-binding responses. This work demonstrates a novel role for FAK in integrin activation and the time-dependent generation of cell–ECM forces.

The overall objective of the present study was to investigate the mechanotransduction of bovine bone marrow stromal cells (BMSCs) through the interactions between transforming growth factor ␤1 (TGF-␤1), dexamethasone, and dynamic compressive loading. Overall, the addition of TGF-␤1 increased cell viability, extracellular matrix (ECM) gene expression, matrix synthesis, and sulfated glycosaminoglycan content over basal construct medium. The addition of dexamethasone further enhanced extracellular matrix gene expression and protein synthesis. There was little stimulation of ECM gene expression or matrix synthesis in any medium group by mechanical loading introduced on day 8. In contrast, there was significant stimulation of ECM gene expression and matrix synthesis in chondrogenic media by dynamic loading introduced on day 16. The level of stimulation was also dependent on the medium supplements, with the samples treated with basal medium being the least responsive and the samples treated with TGF-␤1 and dexamethasone being the most responsive at day 16. Both collagen I and collagen II gene expressions were more responsive to dynamic loading than aggrecan gene expression. Dynamic compression upregulated Smad2/3 phosphorylation in samples treated with basal and TGF-␤1 media. These findings suggest that interactions between mechanical stimuli and TGF-␤ signaling may be an important mechanotransduction pathway for BMSCs, and they indicate that mechanosensitivity may vary during the process of chondrogenesis.

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