Tropoelastin

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Anthony S. Weiss - One of the best experts on this subject based on the ideXlab platform.

  • fuzzy binding model of molecular interactions between Tropoelastin and integrin alphavbeta3
    Biophysical Journal, 2021
    Co-Authors: Jazmin Ozsvar, Richard Wang, Anna Tarakanova, Markus J Buehler, Anthony S. Weiss
    Abstract:

    Abstract Tropoelastin is the highly flexible monomer subunit of elastin, required for the resilience of the extracellular matrix in elastic tissues. To elicit biological signalling, multiple sites on Tropoelastin bind to cell surface integrins in a poorly understood multifactorial process. Here, we constructed a full-atomistic molecular model of the interactions between Tropoelastin and integrin αvβ3 using ensemble-based computational methodologies. Conformational changes of integrin αvβ3 associated with outside-in signaling were more frequently facilitated in an ensemble where tropoleastin bound the integrin’s a1 helix rather than the upstream canonical binding site. Our findings support a model of fuzzy binding, whereby many Tropoelastin conformations and defined sites cooperatively interact with multiple αvβ3 regions. This model explains prior experimental binding to distinct Tropoelastin regions, domains 17 and 36, and points to the cooperative participation of domain 20. Our study highlights the utility of ensemble-based approached in helping to understand the interactive mechanisms of functionally significant flexible proteins.

  • domains 12 to 16 of Tropoelastin promote cell attachment and spreading through interactions with glycosaminoglycan and integrins alphav and alpha5beta1
    FEBS Journal, 2021
    Co-Authors: Brigida Bochicchio, Giselle C Yeo, Antonietta Pepe, Daniela Quaglino, Pearl Lee, Deniz Emul, Antonio Laezza, Nicola Ciarfaglia, Anthony S. Weiss
    Abstract:

    Elastin is an extracellular matrix component with key structural and biological roles in elastic tissues. Interactions between resident cells and Tropoelastin, the monomer of elastin, underpin elastin's regulation of cellular processes. However, the nature of Tropoelastin-cell interactions and the contributions of individual Tropoelastin domains to these interactions are only partly elucidated. In this study, we identified and characterized novel cell-adhesive sites in the Tropoelastin N-terminal region between domains 12 and 16. We found that this region interacts with αV and α5β1 integrin receptors, which mediate cell attachment and spreading. A peptide sequence from within this region, spanning domains 14 to mid-domain 16, binds heparan sulfate through electrostatic interactions with peptide lysine residues and induces conformational ordering of the peptide. We propose that domains 14-16 direct initial cell attachment through cell-surface heparan sulfate glycosaminoglycans, followed by αV and α5β1 integrin-promoted attachment and spreading on domains 12-16 of Tropoelastin. These findings advance our mechanistic understanding of elastin matrix biology, with the potential to enhance tissue regenerative outcomes of elastin-based materials.

  • Tropoelastin and elastin assembly
    Frontiers in Bioengineering and Biotechnology, 2021
    Co-Authors: Jazmin Ozsvar, Stuart A Cain, Clair Baldock, Anna Tarakanova, Chengeng Yang, Anthony S. Weiss
    Abstract:

    Elastic fibers are an important component of the extracellular matrix, providing stretch, resilience, and cell interactivity to a broad range of elastic tissues. Elastin makes up the majority of elastic fibers and is formed by the hierarchical assembly of its monomer, Tropoelastin. Our understanding of key aspects of the assembly process have been unclear due to the intrinsic properties of elastin and Tropoelastin that render them difficult to study. This review focuses on recent developments that have shaped our current knowledge of elastin assembly through understanding the relationship between Tropoelastin's structure and function.

  • transglutaminase mediated cross linking of Tropoelastin to fibrillin stabilises the elastin precursor prior to elastic fibre assembly
    Journal of Molecular Biology, 2020
    Co-Authors: Michael P Lockhartcairns, Anthony S. Weiss, Clair Baldock, Helena Newandee, Jennifer Thomson, Anna Tarakanova
    Abstract:

    Elastic fibres are essential components of all mammalian elastic tissues such as blood vessels, lung and skin, and are critically important for the mechanical properties they endow. The main components of elastic fibres are elastin and fibrillin, where correct formation of elastic fibres requires a fibrillin microfibril scaffold for the deposition of elastin. It has been demonstrated previously that the interaction between fibrillin and Tropoelastin, the elastin precursor, increases the rate of assembly of Tropoelastin. Furthermore, Tropoelastin and fibrillin can be cross-linked by transglutaminase-2, but the function of cross-linking on their elastic properties is yet to be elucidated. Here we show that transglutaminase cross-linking supports formation of a 1:1 stoichiometric fibrillin-Tropoelastin complex. SAXS data show that the complex retains features of the individual proteins but is elongated supporting end-to-end assembly. Elastic network models were constructed to compare the dynamics of Tropoelastin and fibrillin individually as well as in the cross-linked complex. Normal mode analysis was performed to determine the structures' most energetically favourable, biologically accessible motions which show that within the complex, Tropoelastin is less mobile and this molecular stabilisation extends along the length of the Tropoelastin molecule to regions remote from the cross-linking site. Together, these data suggest a long-range stabilising effect of cross-linking that occurs due to the covalent linkage of fibrillin to Tropoelastin. This work provides insight into the interactions of Tropoelastin and fibrillin and how cross-link formation stabilises the elastin precursor so it is primed for elastic fibre assembly.

  • tubular fibrous scaffolds functionalized with Tropoelastin as a small diameter vascular graft
    Biomacromolecules, 2020
    Co-Authors: Sofia Oliveira, Anthony S. Weiss, Suzanne M Mithieux, Rui L. Reis, Tatiana Felizardo, Sara Amorim, Ricardo A Pires, Albino Martins, Nuno M Neves
    Abstract:

    Cardiovascular disorders are a healthcare problem in today's society. The clinically available synthetic vascular grafts are thrombogenic and could induce intimal hyperplasia. Rapid endothelialization and matched mechanical properties are two major requirements to be considered when designing functional vascular grafts. Herein, an electrospun tubular fibrous (eTF) scaffold was biofunctionalized with Tropoelastin at the luminal surface. The luminal surface functionalization was confirmed by an increase of the zeta potential and by the insertion of NH2 groups. Tropoelastin was immobilized via its -NH2 or -COOH groups at the activated or aminolysed eTF scaffolds, respectively, to study the effect of exposed functional groups on human endothelial cells (ECs) behavior. Tensile properties demonstrated that functionalized eTF scaffolds presented strength and stiffness within the range of those of native blood vessels. Tropoelastin immobilized on activated eTF scaffolds promoted higher metabolic activity and proliferation of ECs, whereas when immobilized on aminolysed eTF scaffolds, significantly higher protein synthesis was observed. These biofunctional eTF scaffolds are a promising small-diameter vascular graft that promote rapid endothelialization and have compatible mechanical properties.

Robert P Mecham - One of the best experts on this subject based on the ideXlab platform.

  • elastic fiber ultrastructure and assembly
    Matrix Biology, 2019
    Co-Authors: Beth A Kozel, Robert P Mecham
    Abstract:

    Abstract Studies over the years have described a filamentous structure to mature elastin that suggests a complicated packing arrangement of Tropoelastin subunits. The currently accepted mechanism for Tropoelastin assembly requires microfibrils to serve as a physical extracellular scaffold for alignment of Tropoelastin monomers during and before crosslinking. However, recent evidence suggests that the initial stages of Tropoelastin assembly occur within the cell or at unique assembly sites on the plasma membrane where Tropoelastin self assembles to form elastin aggregates. Outside the cell, elastin aggregates transfer to growing elastic fibers in the extracellular matrix where tensional forces on microfibrils generated through cell movement help shape the growing fiber. Overall, these observations challenge the widely held idea that interaction between monomeric Tropoelastin and microfibrils is a requirement for elastin assembly, and point to self-assembly of Tropoelastin as a driving force in elastin maturation.

  • Tropoelastin and fibulin overexpression in the subepithelial connective tissue of human pterygium
    American Journal of Ophthalmology, 2011
    Co-Authors: Consuelo Perezrico, Robert P Mecham, Takako Sasaki, Gemma Pascual, Sandra Sotomayor, M A Montesmollon, Cynthia Trejo, Juan M Bellon, Julia Bujan
    Abstract:

    Purpose To evaluate possible changes in the collagen and elastic components of the subepithelial connective tissue of human pterygium. Design Immunohistochemical study. Methods Immunohistochemical staining using antiTropoelastin, anti-fibulin-2, and anti-fibulin-3 antibodies was performed in 10 normal conjunctival and 20 pterygium specimens. Masson trichome staining also was performed to study subepithelial connective tissue. Sirius red staining was used to identify collagen type I and III components. Tropoelastin, fibulin-2, and fibulin-3 messenger ribonucleic acid (mRNA) expressions were analyzed in 9 conjunctival and 12 pterygium specimens by quantitative real-time polymerase chain reaction assay. Results The subepithelial connective tissue and vessels were more predominant in pterygium compared with the normal conjunctival tissue. Amorphous subepithelial zones were observed in the areas of the pterygium tissue, but not in normal conjunctiva. Increased Tropoelastin staining was seen in the pterygium tissue with areas of degenerative changes or immature formation of elastic fibers, as well an increase in Tropoelastin mRNA, in contrast with fibulin-2 and fibulin-3 messenger levels. Fibulin-2 and fibulin-3 expression was colocalized in the subepithelial connective tissue and was distributed along blood and lymphatic vessels. Collagen type III, an immature form of collagen, was increased in the pathologic samples in association with a tissue remodeling process. Conclusions Elastin metabolism is dysregulated in the pathogenesis of human pterygium with Tropoelastin, fibulin-2, and fibulin-3 overexpression in the subepithelial connective tissue.

  • methods in elastic tissue biology elastin isolation and purification
    Methods, 2008
    Co-Authors: Robert P Mecham
    Abstract:

    Elastin provides recoil to tissues subjected to repeated stretch, such as blood vessels and the lung. It is encoded by a single gene in mammals and is secreted as a 60-70 kDa monomer called Tropoelastin. The functional form of the protein is that of a large, highly crosslinked polymer that organizes as sheets or fibers in the extracellular matrix. Purification of mature, crosslinked elastin is problematic because its insolubility precludes its isolation using standard wet-chemistry techniques. Instead, relatively harsh experimental approaches designed to remove non-elastin 'contaminates' are employed to generate an insoluble product that has the amino acid composition expected of elastin. Although soluble, Tropoelastin also presents problems for isolation and purification. The protein's extreme stickiness and susceptibility to proteolysis requires careful attention during purification and in Tropoelastin-based assays. This article describes the most common approaches for purification of insoluble elastin and Tropoelastin. It also addresses key aspects of studying Tropoelastin production in cultured cells, where elastin expression is highly dependent upon cell type, culture conditions, and passage number.

  • methods in elastic tissue biology elastin isolation and purification
    Methods, 2008
    Co-Authors: Robert P Mecham
    Abstract:

    Elastin provides recoil to tissues subjected to repeated stretch, such as blood vessels and the lung. It is encoded by a single gene in mammals and is secreted as a 60–70 kDa monomer call Tropoelastin. The functional form of the protein is that of a large, highly crosslinked polymer that organizes as sheets or fibers in the extracellular matrix. Purification of mature, crosslinked elastin is problematic because its insolubility precludes its isolation using standard wet-chemistry techniques. Instead, relatively harsh experimental approaches designed to remove non-elastin ‘contaminates’ are employed to generate an insoluble product that has the amino acid composition expected of elastin. Although soluble, Tropoelastin also presents problems for isolation and purification. The protein’s extreme stickiness and susceptibility to proteolysis requires careful attention during purification and in Tropoelastin-based assays. This article describes the most common approaches for purification of insoluble elastin and Tropoelastin. It also addresses key aspects of studying Tropoelastin production in cultured cells, where elastin expression is highly dependent upon cell type, culture conditions, and passage number.

  • Tropoelastin interacts with cell surface glycosaminoglycans via its cooh terminal domain
    Journal of Biological Chemistry, 2005
    Co-Authors: Thomas J Broekelmann, Beth A Kozel, Fred W Keeley, Hideaki Ishibashi, Claudio C Werneck, Lijuan Zhang, Robert P Mecham
    Abstract:

    Using a biochemical and cell biological approach, we have identified a cell interaction site at the carboxyl terminus of Tropoelastin. Cell interactions with the COOH-terminal sequence are not through the elastin-binding protein (EBP67) because neither VGVAPG-like peptides nor galactoside sugars altered adhesion. Our results also show that cell adhesion to Tropoelastin is not promoted by integrins. Through the use of mutant Chinese hamster ovary cell lines defective in glycosaminoglycan biosynthesis, as well as competition studies and enzymatic removal of specific cell-surface glycosaminoglycans, the Tropoelastin-binding moieties on the cell surface were identified as heparan and chondroitin sulfate-containing glycosaminoglycans, with heparan sulfate being greatly preferred. Heparin affinity chromatography combined with cell adhesion assays identified the last 17 amino acids as the sequence element at the carboxyl terminus of Tropoelastin responsible for the adhesive activity.

Steven G Wise - One of the best experts on this subject based on the ideXlab platform.

  • targeted modulation of Tropoelastin structure and assembly
    ACS Biomaterials Science & Engineering, 2017
    Co-Authors: Giselle C Yeo, Clair Baldock, Steven G Wise, Anthony S. Weiss
    Abstract:

    Tropoelastin, as the monomer unit of elastin, assembles into elastic fibers that impart strength and resilience to elastic tissues. Tropoelastin is also widely used to manufacture versatile materials with specific mechanical and biological properties. The assembly of Tropoelastin into elastic fibers or biomaterials is crucially influenced by key submolecular regions and specific residues within these domains. In this work, we identify the functional contributions of two rarely occurring negatively charged residues, glutamate 345 in domain 19 and glutamate 414 in domain 21, in jointly maintaining the native conformation of the Tropoelastin hinge, bridge and foot regions. Alanine substitution of E345 and/or E414 variably alters the positioning and interactive accessibility of these regions, as illustrated by nanostructural studies and detected by antibody and cell probes. These structural changes are associated with a lower propensity for monomer coacervation, cross-linking into morphologically and function...

  • plasma ion activated expanded polytetrafluoroethylene vascular grafts with a covalently immobilized recombinant human Tropoelastin coating reducing neointimal hyperplasia
    ACS Biomaterials Science & Engineering, 2016
    Co-Authors: Steven G Wise, Anthony S. Weiss, Hongjuan Liu, Shisan Bao, Alexey Kondyurin, M Byrom, Paul G Bannon, Glenn A Edwards, M M M Bilek
    Abstract:

    Expanded polytetrafluoroethylene (ePTFE) vascular conduits with less than or equal to 6 mm internal diameter typically occlude due to a combination of thrombus formation and neointimal hyperplasia. We hypothesized that by layering the polymerized elastin precursor, human Tropoelastin, in the synthetic vessel lumen we could mimic the internal elastic lamina and so maintain low thrombogenicity while significantly reducing smooth muscle cell proliferation. The luminal surfaces of ePTFE conduits were activated with plasma immersion ion implantation (PIII) treatment to facilitate covalent attachment of Tropoelastin. Multilayered Tropoelastin vessels (2TE) enhanced endothelial cell attachment and proliferation in vitro and were superior to materials lacking the protein. In an ovine carotid interposition model of graft compatibility, partially Tropoelastin coated vessels (1TE) thrombosed at a greater rate than control ePTFE, but 2TE maintained the same patency as controls. 2TE showed a significant reduction in n...

  • Tropoelastin enhances nitric oxide production by endothelial cells
    Nanomedicine: Nanotechnology Biology and Medicine, 2016
    Co-Authors: Matti A Hiob, Steven G Wise, Andy Trane, Pascal Bernatchez, Anthony S. Weiss
    Abstract:

    Aims: This study aimed to characterize the role of Tropoelastin in eliciting a nitric oxide response in endothelial cells. Materials and methods: Nitric oxide production in cells was quantified following the addition of known nitric oxide synthase pathway inhibitors such as LNAME and 1400W. The effect of eNOS siRNA knockdowns was studied using western blotting and assessed in the presence of PI3K-inhibitor, wortmannin. Results: Tropoelastin-induced nitric oxide production was LNAME and wortmannin sensitive, while being unaffected by treatment with 1400W. Conclusion: Tropoelastin acts through a PI3K-specific pathway that leads to the phosphorylation of eNOS to enhance nitric oxide production in endothelial cells. This result points to the benefit of the use of Tropoelastin in vascular applications, where NO production is a characteristic marker of vascular health.

  • subtle balance of Tropoelastin molecular shape and flexibility regulates dynamics and hierarchical assembly
    Science Advances, 2016
    Co-Authors: Giselle C Yeo, Clair Baldock, Steven G Wise, Anna Tarakanova, Markus J Buehler, Anthony S. Weiss
    Abstract:

    The assembly of the Tropoelastin monomer into elastin is vital for conferring elasticity on blood vessels, skin, and lungs. Tropoelastin has dual needs for flexibility and structure in self-assembly. We explore the structure-dynamics-function interplay, consider the duality of molecular order and disorder, and identify equally significant functional contributions by local and global structures. To study these organizational stratifications, we perturb a key hinge region by expressing an exon that is universally spliced out in human Tropoelastins. We find a herniated nanostructure with a displaced C terminus and explain by molecular modeling that flexible helices are replaced with substantial β sheets. We see atypical higher-order cross-linking and inefficient assembly into discontinuous, thick elastic fibers. We explain this dysfunction by correlating local and global structural effects with changes in the molecule’s assembly dynamics. This work has general implications for our understanding of elastomeric proteins, which balance disordered regions with defined structural modules at multiple scales for functional assembly.

  • a negatively charged residue stabilizes the Tropoelastin n terminal region for elastic fiber assembly
    Journal of Biological Chemistry, 2014
    Co-Authors: Giselle C Yeo, Clair Baldock, Steven G Wise, Anthony S. Weiss
    Abstract:

    Tropoelastin is an extracellular matrix protein that assembles into elastic fibers that provide elasticity and strength to vertebrate tissues. Although the contributions of specific Tropoelastin regions during each stage of elastogenesis are still not fully understood, studies predominantly recognize the central hinge/bridge and C-terminal foot as the major participants in Tropoelastin assembly, with a number of interactions mediated by the abundant positively charged residues within these regions. However, much less is known about the importance of the rarely occurring negatively charged residues and the N-terminal coil region in Tropoelastin assembly. The sole negatively charged residue in the first half of human Tropoelastin is aspartate 72. In contrast, the same region comprises 17 positively charged residues. We mutated this aspartate residue to alanine and assessed the elastogenic capacity of this novel construct. We found that D72A Tropoelastin has a decreased propensity for initial self-association, and it cross-links aberrantly into denser, less porous hydrogels with reduced swelling properties. Although the mutant can bind cells normally, it does not form elastic fibers with human dermal fibroblasts and forms fewer atypical fibers with human retinal pigmented epithelial cells. This impaired functionality is associated with conformational changes in the N-terminal region. Our results strongly point to the role of the Asp-72 site in stabilizing the N-terminal segment of human Tropoelastin and the importance of this region in facilitating elastic fiber assembly.

David L Kaplan - One of the best experts on this subject based on the ideXlab platform.

  • silk Tropoelastin protein films for nerve guidance
    Acta Biomaterialia, 2015
    Co-Authors: James D White, Anthony S. Weiss, Siran Wang, David L Kaplan
    Abstract:

    Abstract Peripheral nerve regeneration may be enhanced through the use of biodegradable thin film biomaterials as highly tuned inner nerve conduit liners. Dorsal root ganglion neuron and Schwann cell responses were studied on protein films comprising silk fibroin blended with recombinant human Tropoelastin protein. Tropoelastin significantly improved neurite extension and enhanced Schwann cell process length and cell area, while the silk provided a robust biomaterial template. Silk–Tropoelastin blends afforded a 2.4-fold increase in neurite extension, when compared to silk films coated with poly- d -lysine. When patterned by drying on grooved polydimethylsiloxane (3.5 μm groove width, 0.5 μm groove depth), these protein blends induced both neurite and Schwann cell process alignment. Neurons were functional as assessed using patch-clamping, and displayed action potentials similar to those cultured on poly(lysine)-coated glass. Taken together, silk–Tropoelastin films offer useful biomaterial interfacial platforms for nerve cell control, which can be considered for neurite guidance, disease models for neuropathies and surgical peripheral nerve repairs.

  • biocompatibility of silk Tropoelastin protein polymers
    Biomaterials, 2014
    Co-Authors: Hongjuan Liu, Anthony S. Weiss, Steven G Wise, M M M Bilek, Jelena Rnjakkovacina, David L Kaplan, Jian Fei, Shisan Bao
    Abstract:

    Blended polymers are used extensively in many critical medical conditions as components of permanently implanted devices. Hybrid protein polymers containing recombinant human Tropoelastin and silk fibroin have favorable characteristics as implantable scaffolds in terms of mechanical and biological properties. A firefly luciferase transgenic mouse model was used to monitor real-time IL-1β production localized to the site of biomaterial implantation, to observe the acute immune response (up to 5 days) to these materials. Significantly reduced levels of IL-1β were observed in silk/Tropoelastin implants compared to control silk only implants at 1, 2 and 3 days post-surgery. Subsequently, mice (n = 9) were euthanized at 10 days (10D) and 3 weeks (3W) post-surgery to assess inflammatory cell infiltration and collagen deposition, using histopathology and immunohistochemistry. Compared to control silk only implants, fewer total inflammatory cells were found in silk/Tropoelastin (∼29% at 10D and ∼47% at 3W). Also fewer ingrowth cells (∼42% at 10D and ∼63% at 3W) were observed within the silk/Tropoelastin implants compared to silk only. Lower IL-6 (∼52%) and MMP-2 (∼84%) (pro-inflammatory) were also detected for silk/Tropoelastin at 10 days. After 3 weeks implantation, reduced neovascularization (vWF ∼43%), fewer proliferating cells (Ki67 ∼58% and PCNA ∼41%), macrophages (F4/80 ∼64%), lower IL-10 (∼47%) and MMP-9 (∼55%) were also observed in silk/Tropoelastin materials compared to silk only. Together, these results suggest that incorporation of Tropoelastin improves on the established biocompatibility of silk fibroin, uniquely measured here as a reduced foreign body inflammatory response.

  • Tropoelastin a versatile bioactive assembly module
    Acta Biomaterialia, 2014
    Co-Authors: Steven G Wise, Giselle C Yeo, Jelena Rnjakkovacina, David L Kaplan, Matti A Hiob, Anthony S. Weiss
    Abstract:

    Elastin provides structural integrity, biological cues and persistent elasticity to a range of important tissues, including the vasculature and lungs. Its critical importance to normal physiology makes it a desirable component of biomaterials that seek to repair or replace these tissues. The recent availability of large quantities of the highly purified elastin monomer, Tropoelastin, has allowed for a thorough characterization of the mechanical and biological mechanisms underpinning the benefits of mature elastin. While Tropoelastin is a flexible molecule, a combination of optical and structural analyses has defined key regions of the molecule that directly contribute to the elastomeric properties and control the cell interactions of the protein. Insights into the structure and behavior of Tropoelastin have translated into increasingly sophisticated elastin-like biomaterials, evolving from classically manufactured hydrogels and fibers to new forms, stabilized in the absence of incorporated cross-linkers. Tropoelastin is also compatible with synthetic and natural co-polymers, expanding the applications of its potential use beyond traditional elastin-rich tissues and facilitating finer control of biomaterial properties and the design of next-generation tailored bioactive materials.

  • multifunctional silk Tropoelastin biomaterial systems
    Israel Journal of Chemistry, 2013
    Co-Authors: Chiara E Ghezzi, Anthony S. Weiss, Jelena Rnjakkovacina, David L Kaplan
    Abstract:

    New multifunctional, degradable, polymeric biomaterial systems would provide versatile platforms to address cell and tissue needs in both in vitro and in vivo environments. While protein-based composites or alloys are the building blocks of biological organisms, similar systems have not been largely exploited to dates to generate ad hoc biomaterials able to control and direct biological functions, by recapitulating their inherent structural and mechanical complexities. Therefore, we have recently proposed silk-Tropoelastin material platforms able to conjugate a mechanically robust and durable protein, silk, to a highly flexible and biologically active protein, Tropoelastin. This review focuses on the elucidation of the interactions between silk and Tropoelastin in order to control material structure, properties, and ultimately functions. In addition, an approach is provided for novel material designs to provide tools to control biological outcomes via surface roughness, elasticity, and net charge for neuronal and mesenchymal stem cell-based tissue engineering.

  • charge tunable autoclaved silk Tropoelastin protein alloys that control neuron cell responses
    Advanced Functional Materials, 2013
    Co-Authors: Min D Tangschomer, Anthony S. Weiss, Wenwen Huang, Xiaoxia Xia, David L Kaplan
    Abstract:

    Tunable protein composites are important for constructing extracellular matrix mimics of human tissues with control of biochemical, structural, and mechanical properties. Molecular interaction mechanisms between silk fibroin protein and recombinant human Tropoelastin, based on charge, are utilized to generate a new group of multifunctional protein alloys (mixtures of silk and Tropoelastin) with different net charges. These new biomaterials are then utilized as a biomaterial platform to control neuron cell response. With a +38 net charge in water, Tropoelastin molecules provide extraordinary elasticity and selective interactions with cell surface integrins. In contrast, negatively charged silk fibroin protein (net charge −36) provides remarkable toughness and stiffness with morphologic stability in material formats via autoclaving-induced beta-sheet crystal physical crosslinks. The combination of these properties in alloy format extends the versatility of both structural proteins, providing a new biomaterial platform. The alloys with weak positive charges (silk/Tropoelastin mass ratio 75/25, net charge around +16) significantly improved the formation of neuronal networks and maintained cell viability of rat cortical neurons after 10 days in vitro. The data point to these protein alloys as an alternative to commonly used poly-L-lysine (PLL) coatings or other charged synthetic polymers, particularly with regard to the versatility of material formats (e.g., gels, sponges, films, fibers). The results also provide a practical example of physically designed protein materials with control of net charge to direct biological outcomes, in this case for neuronal tissue engineering.

M M M Bilek - One of the best experts on this subject based on the ideXlab platform.

  • plasma ion implantation enabled bio functionalization of peek improves osteoblastic activity
    APL bioengineering, 2018
    Co-Authors: Edgar A Wakelin, M M M Bilek, D R Mckenzie, Anthony S. Weiss
    Abstract:

    Slow appositional growth of bone in vivo is a major problem associated with polyether ether ketone (PEEK) based orthopaedic implants. Early stage promotion of osteoblast activity, particularly bone nodule formation, would help to improve contact between PEEK implantable materials and the surrounding bone tissue. To improve interactions with bone cells, we explored here the use of plasma immersion ion implantation (PIII) treatment of PEEK to covalently immobilize biomolecules to the surface. In this study, a single step process was used to covalently immobilize Tropoelastin on the surface of PIII modified PEEK through reactions with radicals generated by the treatment. Improved bioactivity was observed using the human osteoblast-like cell line, SAOS-2. Cells on surfaces that were PIII-treated or Tropoelastin-coated exhibited improved attachment, spreading, proliferation, and bone nodule formation compared to cells on untreated samples. Surfaces that were both PIII-treated and Tropoelastin-coated triggered the most favorable osteoblast-like responses. Surface treatment or Tropoelastin coating did not alter alkaline phosphatase gene expression and activity of bound cells but did influence the expression of other bone markers including osteocalcin, osteonectin, and collagen I. We conclude that the surface modification of PEEK improves osteoblast interactions, particularly with respect to bone apposition, and enhances the orthopedic utility of PEEK.Slow appositional growth of bone in vivo is a major problem associated with polyether ether ketone (PEEK) based orthopaedic implants. Early stage promotion of osteoblast activity, particularly bone nodule formation, would help to improve contact between PEEK implantable materials and the surrounding bone tissue. To improve interactions with bone cells, we explored here the use of plasma immersion ion implantation (PIII) treatment of PEEK to covalently immobilize biomolecules to the surface. In this study, a single step process was used to covalently immobilize Tropoelastin on the surface of PIII modified PEEK through reactions with radicals generated by the treatment. Improved bioactivity was observed using the human osteoblast-like cell line, SAOS-2. Cells on surfaces that were PIII-treated or Tropoelastin-coated exhibited improved attachment, spreading, proliferation, and bone nodule formation compared to cells on untreated samples. Surfaces that were both PIII-treated and Tropoelastin-coated triggered ...

  • plasma ion activated expanded polytetrafluoroethylene vascular grafts with a covalently immobilized recombinant human Tropoelastin coating reducing neointimal hyperplasia
    ACS Biomaterials Science & Engineering, 2016
    Co-Authors: Steven G Wise, Anthony S. Weiss, Hongjuan Liu, Shisan Bao, Alexey Kondyurin, M Byrom, Paul G Bannon, Glenn A Edwards, M M M Bilek
    Abstract:

    Expanded polytetrafluoroethylene (ePTFE) vascular conduits with less than or equal to 6 mm internal diameter typically occlude due to a combination of thrombus formation and neointimal hyperplasia. We hypothesized that by layering the polymerized elastin precursor, human Tropoelastin, in the synthetic vessel lumen we could mimic the internal elastic lamina and so maintain low thrombogenicity while significantly reducing smooth muscle cell proliferation. The luminal surfaces of ePTFE conduits were activated with plasma immersion ion implantation (PIII) treatment to facilitate covalent attachment of Tropoelastin. Multilayered Tropoelastin vessels (2TE) enhanced endothelial cell attachment and proliferation in vitro and were superior to materials lacking the protein. In an ovine carotid interposition model of graft compatibility, partially Tropoelastin coated vessels (1TE) thrombosed at a greater rate than control ePTFE, but 2TE maintained the same patency as controls. 2TE showed a significant reduction in n...

  • surface plasma modification and Tropoelastin coating of a polyurethane co polymer for enhanced cell attachment and reduced thrombogenicity
    Biomaterials, 2014
    Co-Authors: Daniel V Bax, Anthony S. Weiss, D R Mckenzie, Anna Waterhouse, Alexey Kondyurin, M M M Bilek
    Abstract:

    Polymers currently utilized for dermal and vascular applications possess sub-optimal biocompatibility which reduces their efficacy. Improving the cell-binding and blood-contacting properties of these polymers would substantially improve their clinical utility. Tropoelastin is a highly extensible extracellular matrix protein with beneficial cell interactive and low thrombogenic properties. We transferred these benefits to the polyurethane block copolymer Elast-Eon E2A through a specific combination of surface plasma modifications and coating with human Tropoelastin. The cell-binding activity of bound Tropoelastin was modulated by ion implantation of the underlying polymer, and correlated with surface hydrophobicity, carbon and oxygen content. This combined treatment enhanced human dermal fibroblast (HDF) and human umbilical vein endothelial cell (HUVEC) attachment, cytoskeletal assembly and viability, combined with elevated PECAM-1 staining of HUVEC cell junctions. The thrombogenicity of the polymer was ameliorated by Tropoelastin coating. We propose that a combination of metered plasma treatment and Tropoelastin coating of Elast-Eon can serve to improve the biological performance of implantable devices such as vascular conduits.

  • biocompatibility of silk Tropoelastin protein polymers
    Biomaterials, 2014
    Co-Authors: Hongjuan Liu, Anthony S. Weiss, Steven G Wise, M M M Bilek, Jelena Rnjakkovacina, David L Kaplan, Jian Fei, Shisan Bao
    Abstract:

    Blended polymers are used extensively in many critical medical conditions as components of permanently implanted devices. Hybrid protein polymers containing recombinant human Tropoelastin and silk fibroin have favorable characteristics as implantable scaffolds in terms of mechanical and biological properties. A firefly luciferase transgenic mouse model was used to monitor real-time IL-1β production localized to the site of biomaterial implantation, to observe the acute immune response (up to 5 days) to these materials. Significantly reduced levels of IL-1β were observed in silk/Tropoelastin implants compared to control silk only implants at 1, 2 and 3 days post-surgery. Subsequently, mice (n = 9) were euthanized at 10 days (10D) and 3 weeks (3W) post-surgery to assess inflammatory cell infiltration and collagen deposition, using histopathology and immunohistochemistry. Compared to control silk only implants, fewer total inflammatory cells were found in silk/Tropoelastin (∼29% at 10D and ∼47% at 3W). Also fewer ingrowth cells (∼42% at 10D and ∼63% at 3W) were observed within the silk/Tropoelastin implants compared to silk only. Lower IL-6 (∼52%) and MMP-2 (∼84%) (pro-inflammatory) were also detected for silk/Tropoelastin at 10 days. After 3 weeks implantation, reduced neovascularization (vWF ∼43%), fewer proliferating cells (Ki67 ∼58% and PCNA ∼41%), macrophages (F4/80 ∼64%), lower IL-10 (∼47%) and MMP-9 (∼55%) were also observed in silk/Tropoelastin materials compared to silk only. Together, these results suggest that incorporation of Tropoelastin improves on the established biocompatibility of silk fibroin, uniquely measured here as a reduced foreign body inflammatory response.

  • a novel cell adhesion region in Tropoelastin mediates attachment to integrin αvβ5
    Journal of Biological Chemistry, 2014
    Co-Authors: Pearl Lee, M M M Bilek, Daniel V Bax, Anthony S. Weiss
    Abstract:

    Tropoelastin protein monomers assemble to form elastin. Cellular integrin αVβ3 binds RKRK at the C-terminal tail of Tropoelastin. We probed cell interactions with Tropoelastin by deleting the RKRK sequence to identify other cell-binding interactions within Tropoelastin. We found a novel human dermal fibroblast attachment and spreading site on Tropoelastin that is located centrally in the molecule. Inhibition studies demonstrated that this cell adhesion was not mediated by either elastin-binding protein or glycosaminoglycans. Cell interactions were divalent cation-dependent, indicating integrin dependence. Function-blocking monoclonal antibodies revealed that αV integrin(s) and integrin αVβ5 specifically were critical for cell adhesion to this part of Tropoelastin. These data reveal a common αV integrin-binding theme for Tropoelastin: αVβ3 at the C terminus and αVβ5 at the central region of Tropoelastin. Each αV region contributes to fibroblast attachment and spreading, but they differ in their effects on cytoskeletal assembly.