Materials engineered to elicit targeted cellular responses in regenerative medicine must display bioligands with precise spatial and temporal control. Although materials with temporally regulated presentation of bioadhesive ligands using external triggers, such as light and electric fields, have been recently realized for cells in culture, the impact of in vivo temporal ligand presentation on cell-material responses is unknown. Here, we present a general strategy to temporally and spatially control the in vivo presentation of bioligands using cell adhesive peptides with a protecting group that can be easily removed via transdermal light exposure to render the peptide fully active. We demonstrate that non-invasive, transdermal time-regulated activation of cell-adhesive RGD peptide on implanted biomaterials regulates in vivo cell adhesion, inflammation, fibrous encapsulation, and vascularization of the material. This work shows that triggered in vivo presentation of bioligands can be harnessed to direct tissue reparative responses associated with implanted biomaterials.

Focal adhesions mediate force transfer between ECM-integrin complexes and the cytoskeleton. Although vinculin has been implicated in force transmission, few direct measurements have been made, and there is little mechanistic insight. Using vinculinnull cells expressing vinculin mutants, we demonstrate that vinculin is not required for transmission of adhesive and traction forces but is necessary for myosin contractility-dependent adhesion strength and traction force and for the coupling of cell area and traction force. Adhesion strength and traction forces depend differentially on vinculin head (V H ) and tail domains. V H enhances adhesion strength by increasing ECM-bound integrin-talin complexes, independently from interactions with vinculin tail ligands and contractility. A full-length, autoinhibition-deficient mutant (T12) increases adhesion strength compared with V H , implying roles for both vinculin activation and the actin-binding tail. In contrast to adhesion strength, vinculin-dependent traction forces absolutely require a full-length and activated molecule; V H has no effect. Physical linkage of the head and tail domains is required for maximal force responses. Residence times of vinculin in focal adhesions, but not T12 or V H , correlate with applied force, supporting a mechanosensitive model for vinculin activation in which forces stabilize vinculin's active conformation to promote force transfer.cell adhesion | fibronectin I ntegrin-mediated adhesion to ECM provides mechanical anchorage and signals that direct cell migration, proliferation, and differentiation (1, 2), processes central to tissue organization, maintenance, and repair. After ligand binding, integrins cluster into focal adhesion (FA) complexes that transmit adhesive and traction forces (3-6). FAs consist of integrins and actins separated by a ∼40 nm core that includes cytoskeleton (CSK) elements, such as vinculin and talin, and signaling molecules, including focal adhesion kinase and paxillin (7). FAs mediate responses to internal and external stresses by modulating force transfer between integrins and the CSK (8-10). This function has been likened to a "mechanical clutch" between an engine and transmission (11).On the basis of its structure and binding partners, vinculin represents an attractive candidate for orchestrator of clutch function. Vinculin consists of a globular head (V H ) linked to a tail domain (V T ) by a proline-rich strap (12). V H contains talin, α-actinin, and α-and β-catenin binding sites; actin, paxillin, and phosphatidylinositol 4,5-bisphosphate (PIP2) binding sites are in V T ; and vasodilator-stimulated phosphoprotein (VASP), actinrelated protein 2/3 (Arp2/3), and vinexin binding sites reside in the proline-rich region. Interactions with these partners are regulated by an autoinhibited conformation arising from high-affinity intramolecular head-tail binding (13,14). Activation of vinculin can occur by simultaneous binding to talin and actin or α-catenin and actin (15,16). Vinculin is activated when localized t...

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Engineered biointerfaces covered with biomimetic motifs, including short bioadhesive ligands, are a promising material-based strategy for tissue repair in regenerative medicine. Potentially useful coating molecules are ligands for the integrins, major extracellular matrix receptors that require both ligand binding and nanoscale clustering for maximal signaling efficiency. We prepared coatings consisting of well-defined multimer constructs with a precise number of recombinant fragments of fibronectin (monomer, dimer, tetramer, and pentamer) to assess how nanoscale ligand clustering affects integrin binding, stem cell responses, tissue healing, and biomaterial integration. Clinical-grade titanium was grafted with polymer brushes that presented monomers, dimers, trimers, or pentamers of the α5β1 integrin–specific fibronectin III (7 to 10) domain (FNIII7–10). Coatings consisting of trimers and pentamers enhanced integrin-mediated adhesion in vitro, osteogenic signaling, and differentiation in human mesenchymal stem cells more than did surfaces presenting monomers and dimers. Furthermore, ligand clustering promoted bone formation and functional integration of the implant into bone in rat tibiae. This study establishes that a material-based strategy in which implants are coated with clustered bioadhesive ligands can promote robust implant-tissue integration.