David M. Collard

Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation

Biomaterial surface chemistry has profound consequences on cellular and host responses, but the underlying molecular mechanisms remain poorly understood. Using self-assembled monolayers as model biomaterial surfaces presenting well defined chemistries, we demonstrate that surface chemistry modulates osteoblastic differentiation and matrix mineralization independently from alterations in cell proliferation. Surfaces were precoated with equal densities of fibronectin (FN), and surface chemistry modulated FN structure to alter integrin adhesion receptor binding. OH- and NH2-terminated surfaces up-regulated osteoblast-specific gene expression, alkaline phosphatase enzymatic activity, and matrix mineralization compared with surfaces presenting COOH and CH3 groups. These surface chemistry-dependent differences in cell differentiation were controlled by binding of specific integrins to adsorbed FN. Function-perturbing antibodies against the central cell binding domain of FN completely inhibited matrix mineralization. Furthermore, blocking antibodies against β1 integrin inhibited matrix mineralization on the OH and NH2 surfaces, whereas function-perturbing antibodies specific for β3 integrin increased mineralization on the COOH substrate. These results establish surface-dependent differences in integrin binding as a mechanism regulating differential cellular responses to biomaterial surfaces. This mechanism could be exploited to engineer materials that control integrin binding specificity to elicit desired cellular activities to enhance the integration of biomaterials and improve the performance of biotechnological culture supports.

The effect of integrin-specific bioactive coatings on tissue healing and implant osseointegration

Implant osseointegration, defined as bone apposition and functional fixation, is a requisite for clinical success in orthopaedic and dental applications, many of which are restricted by implant loosening. Modification of implants to present bioactive motifs such as the RGD cell-adhesive sequence from fibronectin (FN) represents a promising approach in regenerative medicine. However, these biomimetic strategies have yielded only marginal enhancements in tissue healing in vivo. In this study, clinical-grade titanium implants were grafted with a non-fouling oligo(ethylene glycol)-substituted polymer coating functionalized with controlled densities of ligands of varying specificity for target integrin receptors. Biomaterials presenting the alpha5beta1-integrin-specific FN fragment FNIII 7-10 enhanced osteoblastic differentiation in bone marrow stromal cells compared to unmodified titanium and RGD-presenting surfaces. Importantly, FNIII 7-10-functionalized titanium significantly improved functional implant osseointegration compared to RGD-functionalized and unmodified titanium in vivo. This study demonstrates that bioactive coatings that promote integrin binding specificity regulate marrow-derived progenitor osteoblastic differentiation and enhance healing responses and functional integration of biomedical implants. This work identifies an innovative strategy for the rational design of biomaterials for regenerative medicine.

Polymer brushes and self-assembled monolayers: Versatile platforms to control cell adhesion to biomaterials (Review)

This review focuses on the surface modification of substrates with self-assembled monolayers (SAMs) and polymer brushes to tailor interactions with biological systems and to thereby enhance their performance in bioapplications. Surface modification of biomedical implants promotes improved biocompatibility and enhanced implant integration with the host. While SAMs of alkanethiols on gold substrates successfully prevent nonspecific protein adsorption in vitro and can further be modified to tether ligands to control in vitro cell adhesion, extracellular matrix assembly, and cellular differentiation, this model system suffers from lack of stability in vivo. To overcome this limitation, highly tuned polymer brushes have been used as more robust coatings on a greater variety of biologically relevant substrates, including titanium, the current orthopedic clinical standard. In order to improve implant-bone integration, the authors modified titanium implants with a robust SAM on which surface-initiated atom transfer radical polymerization was performed, yielding oligo(ethylene glycol) methacrylate brushes. These brushes afforded the ability to tether bioactive ligands, which effectively promoted bone cell differentiation in vitro and supported significantly better in vivo functional implant integration.

Multivalent Integrin-Specific Ligands Enhance Tissue Healing and Biomaterial Integration

Titanium implants coated with nanoclustered ligands for integrin adhesion receptors are tightly integrated into bone for orthopedic applications. Forming Bonds with Strangers Like a clique of teenagers, cells get nervous if they sense a stranger in their midst. This cellular stranger anxiety works against the surgeon who uses implants to repair broken bones or tissues. If human cells cannot bond with implanted foreign material, the implant will not be integrated into the existing tissue or, worse, will fall out. One solution is to find materials that can be disguised so as to fool human cells into acceptance. By coating titanium plugs with precisely configured bits of a common extracellular matrix protein, fibronectin, Petrie et al. have deceived surrounding cells into accepting their disguised device. To accomplish this con, they found the optimal configuration of fibronectin that binds a cell surface adhesion receptor, thereby enhancing tissue healing and integration of the titanium implant into bone. Cells can bind to fibronectin, part of the surrounding extracellular matrix, through adhesion receptors called integrins—dimeric transmembrane proteins that come in assorted varieties. This binding confers more than a physical link: It triggers signaling events in the cell that activate motility and metabolic changes, as well as locking the cell’s cytoplasm to the extracellular matrix through the membrane. The authors constructed an artificial extracellular matrix by securing a critical piece of fibronectin (FNIII7–10) to a customizable coiled-coil protein sequence via a flexible protein linker. By altering the coiled coils, these constructs could be assembled to present one, two, three, or five clustered integrin ligands from the fibronectin fragment. The flexible linker allowed the ligands 10 to 50 nm in which to move. The authors fixed the constructs to a titanium surface with a polymer coating and added cells with integrin on their surfaces. The trimeric and pentameric ligands bound and activated twice as much integrin as did the monomeric and dimeric ligands and were more effective at promoting osteoblastic differentiation from stem cells. To see whether the three- and five-ligand clusters improved integration into tissue, the authors implanted titanium plugs coated with the various constructs into holes in rat leg bones. Microscopy revealed that the three- and five-ligand coated implants had 50% more contact with the surrounding bone than did implants coated with monomers or dimers. Even more encouraging, these implants were 250% more securely fixed in place than the one- and two-ligand constructs and 400% more than polymer-coated titanium plugs. By using a life-like disguise to coat the surface, Petrie et al. have improved incorporation of titanium implants into bone. This result can be used by dental and orthopedic surgeons, who routinely use titanium implants in tooth and joint replacement. If coated with clustered fibronectin fragments, these implants may coax the surrounding cells into making firm contacts with their surfaces, securing their acceptance by their neighbors. 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.

Synthesis and modification of functional poly(lactide) copolymers: toward biofunctional materials.

A polylactide copolymer with pendant benzyloxy groups has been synthesized by the copolymerization of a benzyl-ether substituted monomer with lactide. Debenzylation of the polymer to provide pendant hydroxyl groups followed by modification with succinic anhydride affords the corresponding carboxylic acid functionalized copolymer that is amenable to standard carbodiimide coupling conditions to attach amine-containing biological molecules. An amino-substituted biotin derivative was coupled to the carboxyl functional groups of copolymer films as proof-of-concept. In a demonstration of the function of these new materials, an RGD-containing peptide sequence was tethered to copolymer films at various densities and was shown to enhance the adhesion of epithelial cells. This strategy provides the opportunity for the attachment of a variety of ligands, allowing for the fabrication of a versatile class of biodegradable, biocompatible materials.

Closely stacked oligo(phenylene ethynylene)s: effect of π-stacking on the electronic properties of conjugated chromophores.

In this work, a bicyclo[4.4.1]undecane scaffold is used to hold oligo(phenylene ethynylene) units in a cofacially stacked arrangement along the entire length of the conjugated units. We study the impact that the resulting strong interchain interactions have on the photophysical properties. The length of the individual oligomer branches was varied from three to five rings to investigate the effect of conjugation on the electronic properties of the stacked segments. Absorption and fluorescence spectra were recorded and compared to those of the corresponding unstacked analogues. Time-dependent density functional theory calculations were carried out and helped to rationalize the low-energy features present in the fluorescence spectra of the stacked systems. The calculations indicate that the low-energy emissions are due to the presence of excimer-like states. The stronger intensity of the low-energy fluorescence band observed in the five-ring stacked system compared to the three-ring analogue is attributed to the smaller activation barrier that separates the local intrachain state and the excimer-like state in the former compound.

Saccharide Polymer Brushes To Control Protein and Cell Adhesion to Titanium

Attaining control over the surface chemistry of titanium is critical to its use in medical implants, especially to address complications such as infection and loosening of implants over time, which still present significant challenges. The surface-initiated atom transfer radical polymerization (SI-ATRP) of a saccharide-substituted methacrylate, 2-gluconamidoethyl methacrylate (GAMA), affords dense polymer brushes that resist protein adsorption and cell adhesion. We further tailored the nature of the surfaces by covalent attachment of an adhesion peptide to afford control over cell adhesion. Whereas unmodified poly(GAMA) brushes prevent cell adhesion, brushes with a tethered GFOGER-containing peptide sequence promote the deposition of confluent well-spread cells. The presentation of adhesion proteins on a robust bioresistive background in this fashion constitutes a versatile approach to the development of new biomaterials.

Deposition of polyaniline on micro-contact printed self-assembledmonolayersof ω-functionalized alkanethiols

Electrooxidative polymerization of aniline on electrodes modified with microcontract printed self-assembled monolayers of 12-aminododecane-1-thiol and alkanethiols allows for facile production of micron-scale patterns of polyaniline whereas patterns of two alkanethiols do not allow for selective deposition. The effect of terminal functionality on the interfacial properties of monolayers must be considered in addition to the length of the alkyl chain in the selection of adsorbates for spatially resolved electrochemical polymerization.

Synthesis, polymerization and characterization of substituted dithieno[3,4-b:3′,4′-d]thiophenes

Chemical or electrochemical oxidation of substituted dithieno[3,4-b:3′,4′-d]thiophenes provides polymers with defined regiochemical structures. These materials have lower bandgaps (0.7-0.9 eV) than the unsubstituted fused heteroarene. Potential cycling of the 1,3-dimethyl substituted polymer film shows repetitive p- and n-dopability. The chemically-prepared dioctyl analog is soluble in common solvents such as chloroform, dichloromethane and THF. However, overoxidation of the polymers at an electrode surface presents a limitation to the polymerization of substituted analogs of the parent fused heteroarene.

Micron-Scale Patterning of Conjugated Polymers on Microcontact Printed Patterns of Self-Assembled Monolayers

Self-assembled monolayers of alkanethiols on gold inhibit the electrooxidative deposition of polypyrrole and polyaniline. Oxidation of aniline at an electrode modified with μm-scale regions of ethanethiol and 12-aminododecanethiol results in selective deposition of polyaniline on the hydrophobic short chain alkanethiol.