Timothy A. Petrie

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.

Human mesenchymal stem cell differentiation on self-assembled monolayers presenting different surface chemistries

Human mesenchymal stem cells (hMSCs) have tremendous potential as a cell source for regenerative medicine due to their capacity for differentiation into a wide range of connective tissue cell types. Although significant progress has been made in the identification of defined growth factor conditions to induce lineage commitment, the effect of underlying biomaterial properties on functional differentiation is far less understood. Here we conduct a systematic assessment of the role for surface chemistry on cell growth, morphology, gene expression and function during hMSC commitment along osteogenic, chondrogenic and adipogenic lineages. Using self-assembled monolayers of omega-functionalized alkanethiols on gold as model substrates, we demonstrate that biomaterial surface chemistry differentially modulates hMSC differentiation in a lineage-dependent manner. These results highlight the importance of initial biomaterial surface chemistry on long-term functional differentiation of adult stem cells, and suggest that surface properties are a critical parameter that must be considered in the design of biomaterials for stem cell-based regenerative medicine strategies.

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.

Simple application of fibronectin-mimetic coating enhances osseointegration of titanium implants.

Integrin‐mediated cell adhesion to biomolecules adsorbed onto biomedical devices regulates device integration and performance. Because of the central role of integrin‐fibronectin (FN) interactions in osteoblastic function and bone formation, we evaluated the ability of FN‐inspired biomolecular coatings to promote osteoblastic differentiation and implant osseointegration. Notably, these biomolecular coatings relied on physical adsorption of FN‐based ligands onto biomedical‐grade titanium as a simple, clinically translatable strategy to functionalize medical implants. Surfaces coated with a recombinant fragment of FN spanning the central cell binding domain enhanced osteoblastic differentiation and mineralization in bone marrow stromal cell cultures and increased implant osseointegration in a rat cortical bone model compared to passively adsorbed arginine–glycine–aspartic acid peptides, serum proteins and full‐length FN. Differences in biological responses correlated with integrin binding specificity and signalling among surface coatings. This work validates a simple, clinically translatable, surface biofunctionalization strategy to enhance biomedical device integration.

Mixed extracellular matrix ligands synergistically modulate integrin adhesion and signaling

Cell adhesion to extracellular matrix (ECM) components through cell‐surface integrin receptors is essential to the formation, maintenance and repair of numerous tissues, and therefore represents a central theme in the design of bioactive materials that successfully interface with the body. While the adhesive responses associated with a single ligand have been extensively analyzed, the effects of multiple integrin subtypes binding to multivalent ECM signals remain poorly understood. In the present study, we generated a high throughput platform of non‐adhesive surfaces presenting well‐defined, independent densities of two integrin‐specific engineered ligands for the type I collagen (COL‐I) receptor α2β1 and the fibronectin (FN) receptor α5β1 to evaluate the effects of integrin cross‐talk on adhesive responses. Engineered surfaces displayed ligand density‐dependent adhesive effects, and mixed ligand surfaces significantly enhanced cell adhesion strength and focal adhesion assembly compared to single FN and COL‐I ligand surfaces. Moreover, surfaces presenting mixed COL‐I/FN ligands synergistically enhanced FAK activation compared to the single ligand substrates. The enhanced adhesive activities of the mixed ligand surfaces also promoted elevated proliferation rates. Our results demonstrate interplay between multivalent ECM ligands in adhesive responses and downstream cellular signaling. J. Cell. Physiol. 217: 450–458, 2008.

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.

Dendritic cell responses to self-assembled monolayers of defined chemistries.

Biomaterial contact triggers dendritic cell (DC) maturation, to an extent depending on the biomaterial, ultimately enhancing an immune response toward associated antigens, implying a role for biomaterials as adjuvants. Self-assembled monolayers (SAM) of alkanethiols on titanium/gold-coated surfaces presenting different chemistries were used to study effects of biomaterial surface chemistry on DC maturation. Although DCs treated with OH, COOH, or NH(2) SAMs showed modest maturation, those treated with CH(3) SAMs were least mature, all based on cytospins, allostimulatory capacity, or maturation marker expression. Surprisingly, DCs treated with CH(3) SAMs secreted highest levels of proinflammatory tumor necrosis factor-alpha (TNF-alpha) or interleukin-6 (IL-6) but were least mature. Secretion of anti-inflammatory mediators by DCs treated with CH(3) SAMs was not responsible for mitigating DC maturation under these conditions. Interestingly, elevated levels of apoptotic markers were measured associated with DCs and T cells upon CH(3) SAMs contact. Since phagocytosis of apoptotic DCs has strong immunosuppressive effects on DCs, more apoptotic DCs on CH(3) SAMs may account for lower DC maturation. Finally, higher expression of cytotoxic T lymphocyte associated antigen receptor-4 (CTLA-4) on T cells may imply a mechanism of T cell inhibition on CH(3) SAMs.

Molecular assembly and biological activity of a recombinant fragment of fibronectin (FNIII7–10) on poly(ethyl acrylate)

Fibronectin (FN) fibrillogenesis is a cell-mediated process involving integrin activation that results in conformational changes of FN molecules and the organization of actin cytoskeleton. A similar process can be induced by some particular chemistries in the absence of cells, e.g., poly(ethyl acrylate) (PEA), which enhance FN-FN interactions leading to the formation of a biologically active network on the material surface. We have investigated the organization of a recombinant fragment of fibronectin (FNIII(7-10)) upon adsorption on this particular chemistry, PEA. Atomic force microscopy (AFM) was used to identify individual molecules of the fragment after adsorption, as well as the evolution of the distribution of adsorbed molecules on the surface of the material as the concentration of the adsorbing solution increased. Globular molecules that turn into small aggregates were found as a function of solution concentration. Above a threshold concentration of the adsorbing solution (50 microg/mL) an interconnected network of the FNIII(7-10) fragment is obtained on the material surface. The bioavailability of specific cell adhesion domains, including RGD, within the molecules was higher on PEA than on the control glass. The biological activity of the fragment was further investigated by evaluating focal adhesion formation and actin cytoskeleton for MC3T3-E1 osteoblast-like cells. Well-developed focal adhesion complexes and insertions of actin stress fibers were found on PEA in a similar way as it happens in the control SAM-OH. Moreover, increasing the hydrophilicity of the surface by incorporating -OH groups led to globular molecules of the fragment homogeneously distributed throughout the surface; and the cell-material interaction is reduced as depicted by the lack of well-developed focal plaques and actin cytoskeleton.