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Wound healing is facilitated by biomaterials-based grafts and substantially impacted by orchestrated inflammatory responses that are essential to the normal repair process. Tropoelastin (TE) based materials are known to shorten the period for wound repair but the mechanism of anti-inflammatory performance is not known. To explore this, we compared the performance of the gold standard Integra Dermal Regeneration Template (Integra), polyglycerol sebacate (PGS), and TE blended with PGS, in a murine full-thickness cutaneous wound healing study. Systemically, blending with TE favorably increased the F4/80 + macrophage population by day 7 in the spleen and contemporaneously induced elevated plasma levels of anti-inflammatory IL-10. In contrast, the PGS graft without TE prompted prolonged inflammation, as evidenced by splenomegaly and greater splenic granulocyte and monocyte fractions at day 14. Locally, the inclusion of TE in the graft led to increased anti-inflammatory M2 macrophages and CD4 + T cells at the wound site, and a rise in Foxp3 + regulatory T cells in the wound bed by day 7. We conclude that the TE-incorporated skin graft delivers a pro-healing environment by modulating systemic and local tissue responses. STATEMENT OF SIGNIFICANCE: Tropoelastin (TE) has shown significant benefits in promoting the repair and regeneration of damaged human tissues. In this study, we show that TE promotes an anti-inflammatory environment that facilitates cutaneous wound healing. In a mouse model, we find that inserting a TE-containing material into a full-thickness wound results in defined, pro-healing local and systemic tissue responses. These findings advance our understanding of TE's restorative value in tissue engineering and regenerative medicine, and pave the way for clinical applications., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A.S.W. is the founding scientist of Elastagen Pty. Ltd., now sold to Allergan, Inc., an AbbVie company., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Elastin is a key elastomeric protein responsible for the elasticity of many organs, including heart, skin, and blood vessels. Due to its intrinsic long life and low turnover rate, damage in elastin induced by pathophysiological conditions, such as hypercalcemia and hyperglycemia, accumulates during biological aging and in aging-associated diseases, such as diabetes mellitus and atherosclerosis. Prior studies have shown that calcification induced by hypercalcemia deteriorates the function of aortic tissues. Glycation of elastin is triggered by hyperglycemia and associated with elastic tissue damage and loss of mechanical functions via the accumulation of advanced glycation end products. To evaluate the effects on elastin's structural conformations and elasticity by hypercalcemia and hyperglycemia at the molecular scale, we perform classical atomistic and steered molecular dynamics simulations on tropoelastin, the soluble precursor of elastin, under different conditions. We characterize the interaction sites of glucose and calcium and associated structural conformational changes. Additionally, we find that elevated levels of calcium ions and glucose hinder the extensibility of tropoelastin by rearranging structural domains and altering hydrogen bonding patterns, respectively. Overall, our investigation helps to reveal the behavior of tropoelastin and the biomechanics of elastin biomaterials in these physiological environments. STATEMENT OF SIGNIFICANCE: Elastin is a key component of elastic fibers which endow many important tissues and organs, from arteries and veins, to skin and heart, with strength and elasticity. During aging and aging-associated diseases, such as diabetes mellitus and atherosclerosis, physicochemical stressors, including hypercalcemia and hyperglycemia, induce accumulated irreversible damage in elastin, and consequently alter mechanical function. Yet, molecular mechanisms associated with these processes are still poorly understood. Here, we present the first study on how these changes in elastin structure and extensibility are induced by hypercalcemia and hyperglycemia at the molecular scale, revealing the essential roles that calcium and glucose play in triggering structural alterations and mechanical stiffness. Our findings yield critical insights into the first steps of hypercalcemia- and hyperglycemia-mediated aging., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2022. Published by Elsevier Ltd.)

Mesenchymal stem cells (MSCs) have emerged as promising cell-based therapies in the treatment of degenerative and inflammatory conditions. However, despite accumulating evidence of the breadth of MSC functional potency, their broad clinical translation is hampered by inconsistencies in therapeutic efficacy, which is at least partly due to the phenotypic and functional heterogeneity of MSC populations as they progress towards senescence in vitro. MSC senescence, a natural response to aging and stress, gives rise to altered cellular responses and functional decline. This review describes the key regenerative properties of MSCs; summarises the main triggers, mechanisms, and consequences of MSC senescence; and discusses current cellular and extracellular strategies to delay the onset or progression of senescence, or to rejuvenate biological functions lost to senescence., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Elastic fibers are an essential part of the pulmonary extracellular matrix (ECM). Intact elastin is required for normal function and its damage contributes profoundly to the etiology and pathology of lung disease. This highlights the need for novel lung-specific imaging methodology that enables high-resolution 3D visualization of the ECM. We consider elastin's involvement in chronic respiratory disease and examine recent methods for imaging and modeling of the lung in the context of advances in lung tissue engineering for research and clinical application., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Recreating the cell niche of virtually all tissues requires composite materials fabricated from multiple extracellular matrix (ECM) macromolecules. Due to their wide tissue distribution, physical attributes and purity, collagen, and more recently, tropoelastin, represent two appealing ECM components for biomaterials development. Here we blend tropoelastin and collagen, harnessing the cell-modulatory properties of each biomolecule. Tropoelastin was stably co-blended into collagen biomaterials and was retained after EDC-crosslinking. We found that human dermal fibroblasts (HDF), rat glial cells (Rugli) and HT1080 fibrosarcoma cells ligate to tropoelastin via EDTA-sensitive and EDTA-insensitive receptors or do not ligate with tropoelastin, respectively. These differing elastin-binding properties allowed us to probe the cellular response to the tropoelastin-collagen composites assigning specific bioactivity to the collagen and tropoelastin component of the composite material. Tropoelastin addition to collagen increased total Rugli cell adhesion, spreading and proliferation. This persisted with EDC-crosslinking of the tropoelastin-collagen composite. Tropoelastin addition did not affect total HDF and HT1080 cell adhesion; however, it increased the contribution of cation-independent adhesion, without affecting the cell morphology or, for HT1080 cells, proliferation. Instead, EDC-crosslinking dictated the HDF and HT1080 cellular response. These data show that a tropoelastin component dominates the response of cells that possess non-integrin based tropoelastin receptors. EDC modification of the collagen component directs cell function when non-integrin tropoelastin receptors are not crucial for cell activity. Using this approach, we have assigned the biological contribution of each component of tropoelastin-collagen composites, allowing informed biomaterial design for directed cell function via more physiologically relevant mechanisms. STATEMENT OF SIGNIFICANCE: Biomaterials fabricated from multiple extracellular matrix (ECM) macromolecules are required to fully recreate the native tissue niche where each ECM macromolecule engages with a specific repertoire of cell-surface receptors. Here we investigate combining tropoelastin with collagen as they interact with cells via different receptors. We identified specific cell lines, which associate with tropoelastin via distinct classes of cell-surface receptor. These showed that tropoelastin, when combined with collagen, altered the cell behaviour in a receptor-usage dependent manner. Integrin-mediated tropoelastin interactions influenced cell proliferation and non-integrin receptors influenced cell spreading and proliferation. These data shed light on the interplay between biomaterial macromolecular composition, cell surface receptors and cell behaviour, advancing bespoke materials design and providing functionality to specific cell populations., Competing Interests: Declaration of Competing Interest Anthony S Weiss is the Scientific Founder of Elastagen Pty Ltd. now sold to Allergan and AbbVie. All other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Ltd.)

Elastic fibres play a key role in bodily functions where fatigue resistance and elastic recovery are necessary while regulating phenotype, proliferation and migration in cells. While in vivo elastic fibres are created at a late foetal stage, a major obstacle in the development of engineered tissue is that human vascular smooth muscle cells (hVSMCs), one of the principal elastogenic cells, are unable to spontaneously promote elastogenesis in vitro. Therefore, the overall aim of this study was to activate elastogenesis in vitro by hVSMCs seeded in fibrin, collagen, glycosaminoglycan (FCG) scaffolds, following the addition of recombinant human tropoelastin. This combination of scaffold, tropoelastin and cells induced the deposition of elastin and formation of lamellar maturing elastic fibres, similar to those found in skin, blood vessels and heart valves. Furthermore, higher numbers of maturing branched elastic fibres were synthesised when a higher cell density was used and by drop-loading tropoelastin onto cell-seeded FCG scaffolds prior to adding growth medium. The addition of tropoelastin showed no effect on cell proliferation or mechanical properties of the scaffold which remained dimensionally stable throughout. With these results, we have established a natural biomaterial scaffold that can undergo controlled elastogenesis on demand, suitable for tissue engineering applications., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Elastic fibers are an essential component of the extracellular matrix where they provide structural integrity and elastic recoil in a number of important tissues. A major constituent of these fibers is elastin, an insoluble metabolically stable polymer formed via extensive crosslinking of the monomeric precursor tropoelastin. Research over the past few decades has shown that tropoelastin possesses unique structural features that differ from both intrinsically disordered and globular proteins. This review details the advances in our understanding of tropoelastin's structural properties and illustrates how these dictate its biological function., (Copyright © 2019 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Electrospun yarns offer substantial opportunities for the fabrication of elastic scaffolds for flexible tissue engineering applications. Currently available yarns are predominantly made of synthetic elastic materials. Thus scaffolds made from these yarns typically lack cell signaling cues. This can result in poor integration or even rejection on implantation, which drive demands for a new generation of yarns made from natural biologically compatible materials. Here, we present a new type of cell-attractive, highly twisted protein-based yarns made from blended tropoelastin and silk fibroin. These yarns combine physical and biological benefits by being rendered elastic and bioactive through the incorporation of tropoelastin and strengthened through the presence of silk fibroin. Remarkably, the process delivered multi-meter long yarns of tropoelastin-silk mixture that were conducive to fabrication of meshes on hand-made frames. The resulting hydrated meshes are elastic and cell interactive. Furthermore, subcutaneous implantation of the meshes in mice demonstrates their tolerance and persistence over 8 weeks. This combination of mechanical properties, biocompatibility and processability into diverse shapes and patterns underscores the value of these materials and platform technology for tissue engineering applications. STATEMENT OF SIGNIFICANCE: Synthetic yarns are used to fabricate textile materials for various applications such as surgical meshes for hernia repair and pelvic organ prolapse. However, synthetic materials lack the attractive biological and physical cues characteristic of extracellular matrix and there is a demand for materials that can minimize postoperative complications. To address this need, we made yarns from a combination of recombinant human tropoelastin and silk fibroin using a modified electrospinning approach that blended these proteins into functional yarns. Prior to this study, no protein-based yarns using tropoelastin were available for the fabrication of functional textile materials. Multimeter-long, uniform and highly twisted yarns based on these proteins were elastic and cell interactive and demonstrated processing to yield textile fabrics. By using these yarns to weave fabrics, we demonstrate that an elastic human matrix protein blend can deliver a versatile platform technology to make textiles that can be explored for efficacy in tissue repair., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

We demonstrate a novel approach for the production of tunable quantities of elastic fibers. We also show that exogenous tropoelastin is rate-limiting for elastin synthesis regardless of the age of the dermal fibroblast donor. Additionally, we provide a strategy to further enhance synthesis by older cells through the application of conditioned media. We show that this approach delivers an elastin layer on one side of the leading dermal repair template for contact with the deep dermis in order to deliver prefabricated elastic fibers to a physiologically appropriate site during subsequent surgery. This system is attractive because it provides for the first time a viable path for sufficient, histologically detectable levels of patient elastin into full-thickness wound sites that have until now lacked this elastic underlayer., Statement of Significance: The scars of full thickness wounds typically lack elasticity. Elastin is essential for skin elasticity and is enriched in the deep dermis. This paper is significant because it shows that: (1) we can generate elastic fibers in tunable quantities, (2) tropoelastin is the rate-limiting component in elastin synthesis in vitro, (3) we can generate elastin fibers regardless of donor age, (4) we describe a novel approach to further increase the numbers and thickness of elastic fibers for older donors, (5) we improve on Integra Dermal Regeneration Template and generate a new hybrid biomaterial intended to subsequently surgically deliver these elastic fibers, (6) the elastic fiber layer is presented on the side of Integra that is intended for delivery into its physiologically appropriate site i.e. the deep dermis., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Neointimal hyperplasia, which results from the activation, proliferation and migration of vascular smooth muscle cells (SMCs), is a detrimental condition for vascular stents or vascular grafts that leads to stenosis. Preventing neointimal hyperplasia of vascular grafts is critically important for the success of arterial vascular grafts. We hypothesized that tropoelastin seeding onto the luminal surface of the graft would prevent neointimal hyperplasia through suppressing neointimal smooth muscle cell proliferation. In this study, we investigated the efficacy of tropoelastin seeding in preventing neointimal hyperplasia of bioresorbable arterial vascular grafts. Poly (glycolic acid) (PGA) fiber mesh coated with poly (l-lactic-co-ε-caprolactone) (PLCL) scaffolds reinforced by poly (l-lactic acid) (PLA) nano-fibers were prepared as bioresorbable arterial grafts. Tropoelastin was then seeded onto the luminal surface of the grafts. Tropoelastin significantly reduced the thickness of the intimal layer. This effect was mainly due to a substantial reduction the number of cells that stained positive for SMC (α-SMA) and PCNA in the vessel walls. Mature elastin and collagen type I and III were unchanged with tropoelastin treatment. This study demonstrates that tropoelastin seeding is beneficial in preventing SMC proliferation and neointimal hyperplasia in bioresorbable arterial vascular grafts., Statement of Significance: Small resorbable vascular grafts can block due to the over-proliferation of smooth muscle cells in neointimal hyperplasia. We show here that the proliferation of these cells is restricted in this type of graft. This is achieved with a simple dip, non-covalent coating of tropoelastin. It is in principle amendable to other grafts and is therefore an attractive process. This study is particularly significant because: (1) it shows that smooth muscle cell proliferation can be reduced while still accommodating the growth of endothelial cells, (2) small vascular grafts with an internal diameter of less than 1mm are amenable to this process, and (3) this process works for resorbable grafts., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Elastomers are popular in vascular engineering applications, as they offer the ability to design implants that match the compliance of native tissue. By mimicking the natural tissue environment, elastic materials are able to integrate within the body to promote repair and avoid the adverse physiological responses seen in rigid alternatives that often disrupt tissue function. The design of elastomers has continued to evolve, moving from a focus on long term implants to temporary resorbable implants that support tissue regeneration. This has been achieved through designing chemistries and processing methodologies that control material behavior and bioactivity, while maintaining biocompatibility in vivo. Here we review the latest developments in synthetic and natural elastomers and their application in cardiovascular treatments., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The elastomeric proteins elastin and resilin have been used extensively in the fabrication of biomaterials for tissue engineering applications due to their unique mechanical and biological properties. Tropoelastin is the soluble monomer component of elastin. Tropoelastin and resilin are both highly elastic with high resilience, substantial extensibility, high durability and low energy loss, which makes them excellent candidates for the fabrication of elastic tissues that demand regular and repetitive movement like the skin, lung, blood vessels, muscles and vocal folds. Combinations of these proteins with silk fibroin further enhance their biomechanical and biological properties leading to a new class of protein alloy materials with versatile properties. In this review, the properties of tropoelastin-based and resilin-based biomaterials with and without silk are described in concert with examples of their applications in tissue engineering., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

This study aimed to characterize the structures of two elastin-like constructs, one composed of a cross-linked elastin-like polypeptide and the other one of cross-linked tropoelastin, and native aortic elastin. The structures of the insoluble materials and human aortic elastin were investigated using scanning electron microscopy. Additionally, all samples were digested with enzymes of different specificities, and the resultant peptide mixtures were characterized by ESI mass spectrometry and MALDI mass spectrometry. The MS(2) data was used to sequence linear peptides, and cross-linked species were analyzed with the recently developed software PolyLinX. This enabled the identification of two intramolecularly cross-linked peptides containing allysine aldols in the two constructs. The presence of the tetrafunctional cross-link desmosine was shown for all analyzed materials and its quantification revealed that the cross-linking degree of the two in vitro cross-linked materials was significantly lower than that of native elastin. Molecular dynamics simulations were performed, based on molecular species identified in the samples, to follow the formation of elastin cross-links. The results provide evidence for the significance of the GVGTP hinge region of domain 23 for the formation of elastin cross-links. Overall, this work provides important insight into structural similarities and differences between elastin-like constructs and native elastin. Furthermore, it represents a step toward the elucidation of the complex cross-linking pattern of mature elastin., (Copyright © 2014. Published by Elsevier B.V.)

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., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Current vascular biomaterials exhibit poor biocompatibility characterised by failure to promote endothelialisation, predisposition to neoinitmal hyperplasia and excessive thrombogenicity. Fibrillin-1, a major constituent of microfibrils is associated with elastic fibres in the arterial wall. Fibrillin-1 binds to endothelial cells through an RGD cell adhesion motif in the fourth TB module. The RGD motif is present in PF8, a recombinant fibrillin-1 fragment. We investigated the potential of PF8 to improve the biocompatibility of PTFE. PF8 enhanced endothelial cell attachment and cell proliferation to a greater extent than fibronectin (p<0.01). PF8 immobilised on PTFE using plasma immersion ion implantation (PIII), retained these favourable cell interactive properties, again promoting endothelial cell attachment and proliferation. The thrombogenicity of covalently bound PF8 on PTFE was assessed in both static and dynamic conditions. In static conditions, uncoated PIII treated PTFE was more thrombogenic than untreated PTFE, while PF8 coating reduced thrombogenicity. Under flow, there was no difference in the thrombogenicity of PF8 coated PTFE and untreated PTFE. Immobilised PF8 shows a striking ability to promote attachment and growth of endothelial cells on PTFE, while providing a non-thrombogenic surface. These features make PF8 a promising candidate to improve the biocompatibility of current synthetic vascular grafts., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

Elastin is predominantly comprised of crosslinked tropoelastin. For many years elastin was considered to serve a solely structural role but is now being increasingly identified as causal in cell signaling, development and repair. We introduced tropoelastin into an in vitro model in which airway smooth muscle cells (ASMCs) were stimulated with transforming growth factor (TGF)-β1 to examine the modulatory effect of this modular elastin sequence on release of angiogenic factors and matrix metalloproteinases (MMPs). Human ASMCs were presented to surfaces coated with tropoelastin or collagen and controls, then stimulated with TGF-β1. Transcript levels of vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) were quantified 4 and 24 h after TGF-β1 stimulation. Protein VEGF release from cells and CTGF sequestered at cell surfaces were measured by ELISA at 24 and 48 h. TGF-β1 increased VEGF mRNA 2.4 fold at 4 h and 5 fold at 24 h, accompanied by elevated cognate protein release 3 fold at 24 h and 2.5 fold at 48 h. TGF-β1 stimulation increased CTGF mRNA 6.9 fold at 4 h and 11.8 fold at 24 h, accompanied by increased sequestering of its protein counterpart 1.2 fold at 24 h and 1.4 fold at 48 h. Pre-incubation of cells with tropoelastin did not modulate VEGF or CTGF mRNA expression, but combined with TGF-β1 stimulation it led to enhanced VEGF release 5.1-fold at 24h and 4.4-fold at 48 h. Pre-incubation with tropoelastin decreased CTGF sequestering 0.6-fold at 24 and 48 h, and increased MMP-2 production. Collagen pre-incubation under the same conditions displayed no effect on TGF-β1 stimulation apart from a slightly decreased (0.9 fold) sequestered CTGF at 48 h. As CTGF is known to anchor VEGF to the matrix and inhibit its angiogenic activity, a process which can be reversed by digestion with MMP-2, these findings reveal that elastin sequences can disrupt the balance of angiogenic factors, with implications for aberrant angiogenesis. The results suggest a model of molecular crosstalk and support an active role for elastin in vascular remodeling., (Copyright © 2013 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Elastomeric protein-based biomaterials, produced from elastin derivatives, are widely investigated as promising tissue engineering scaffolds due to their remarkable properties including substantial extensibility, long-term stability, self-assembly, high resilience upon stretching, low energy loss, and excellent biological activity. These elastomers are processed from different sources of soluble elastin such as animal-derived soluble elastin, recombinant human tropoelastin, and elastin-like polypeptides into various forms including three dimensional (3D) porous hydrogels, elastomeric films, and fibrous electrospun scaffolds. Elastin-based biomaterials have shown great potential for the engineering of elastic tissues such as skin, lung and vasculature. In this review, the synthesis and properties of various elastin-based elastomers with their applications in tissue engineering are described.

We present an electrospun synthetic human elastin:collagen composite scaffold aimed at dermal tissue engineering. The panel of electrospun human tropoelastin and ovine type I collagen blends comprised 80% tropoelastin+20% collagen, 60% tropoelastin+40% collagen and 50% tropoelastin+50% collagen. Electrospinning efficiency decreased with increasing collagen content under the conditions used. Physical and mechanical characterization encompassed fiber morphology, porosity, pore size and modulus, which were prioritized to identify the optimal candidate for dermal tissue regeneration. Scaffolds containing 80% tropoelastin and 20% collagen (80T20C) were selected on this basis for further cell interaction and animal implantation studies. 80T20C enhanced proliferation and migration rates of dermal fibroblasts in vitro and were well tolerated in a mouse subcutaneous implantation study where they persisted over 6 weeks. The 80T20C scaffolds supported fibroblast infiltration, de novo collagen deposition and new capillary formation., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The interaction of proteins and cells with polymers is critical to their use in scientific and medical applications. In this study, plasma immersion ion implantation (PIII) was used to modify the surface of the conducting polymer, polypyrrole, which possesses electrical properties. PIII treatment enabled persistent, covalent binding of the cell adhesive protein, tropoelastin, without employing chemical linking molecules. In contrast tropoelastin was readily eluted from the untreated surface. Through this differential persistence of binding, surface bound tropoelastin supported cell adhesion and spreading on the PIII treated but not the untreated polypyrrole surface. The application of a steel shadow mask during PIII treatment allowed for spatial definition of tropoelastin exclusively to PIII treated regions. The general applicability of this approach to other extracellular matrix proteins was illustrated using collagen I, which displayed similar results to tropoelastin but required extended washing conditions. This approach allowed fine patterning of cell adhesion and spreading to tropoelastin and collagen, specifically on PIII treated polypyrrole regions. We therefore present a methodology to alter the functionality of polypyrrole surfaces, generating surfaces that can spatially control cellular interactions through protein functionalization with the potential for electrical stimulation., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The coacervation of tropoelastin represents the first major stage of elastic fiber assembly. The process has been modeled in vitro by numerous studies, initially with mixtures of solubilized elastin, and subsequently with synthetic elastin peptides that represent hydrophobic repeat units, isolated hydrophobic domains, segments of alternating hydrophobic and cross-linking domains, or the full-length monomer. Tropoelastin coacervation in vitro is characterized by two stages: an initial phase separation, which involves a reversible inverse temperature transition of monomer to n-mer; and maturation, which is defined by the irreversible coalescence of coacervates into large species with fibrillar structures. Coacervation is an intrinsic ability of tropoelastin. It is primarily influenced by the number, sequence, and contextual arrangement of hydrophobic domains, although hydrophilic sequences can also affect the behavior of the hydrophobic domains and thus affect coacervation. External conditions including ionic strength, pH, and temperature also directly influence the propensity of tropoelastin to self-associate. Coacervation is an endothermic, entropically-driven process driven by the cooperative interactions of hydrophobic domains following destabilization of the clathrate-like water shielding these regions. The formation of such assemblies is believed to follow a helical nucleation model of polymerization. Coacervation is closely associated with conformational transitions of the monomer, such as increased β-structures in hydrophobic domains and α-helices in cross-linking domains. Tropoelastin coacervation in vivo is thought to mainly involve the central hydrophobic domains. In addition, cell-surface glycosaminoglycans and microfibrillar proteins may regulate the process. Coacervation is essential for progression to downstream elastogenic stages, and impairment of the process can result in elastin haploinsufficiency disorders such as supravalvular aortic stenosis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Small-diameter synthetic vascular graft materials fail to match the patency of human tissue conduits used in vascular bypass surgery. The foreign surface retards endothelialization and is highly thrombogenic, while the mismatch in mechanical properties induces intimal hyperplasia. Using recombinant human tropoelastin, we have developed a synthetic vascular conduit for small-diameter applications. We show that tropoelastin enhances endothelial cell attachment (threefold vs. control) and proliferation by 54.7 ± 1.1% (3 days vs. control). Tropoelastin, when presented as a monomer and when cross-linked into synthetic elastin for biomaterials applications, had low thrombogenicity. Activation of the intrinsic pathway of coagulation, measured by plasma clotting time, was reduced for tropoelastin (60.4 ± 8.2% vs. control). Platelet attachment was also reduced compared to collagen. Reductions in platelet interactions were mirrored on cross-linked synthetic elastin scaffolds. Tropoelastin was subsequently incorporated into a synthetic elastin/polycaprolactone conduit with mechanical properties optimized to mimic the human internal mammary artery, including permeability, compliance, elastic modulus and burst pressure. Further, this multilayered conduit presented a synthetic elastin internal lamina to circulating blood and demonstrated suturability and mechanical durability in a small scale rabbit carotid interposition model., (Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Tropoelastin assembly is a key step in the formation of elastin. We consider how nanoscale intracellular assemblies of tropoelastin can congregate in an extracellular environment to give microscale aggregates. We describe novel 200-300 nm spherical particles that serve as intermediates in the formation of the coacervate. Their aggregation gives 800 nm to 1 microm species. This process is facilitated by heparan sulfate and dermatan sulfate interactions which effectively lower the critical concentration to facilitate this transition. This coacervation process was examined using a panel of heparin chains of various lengths and showed greatest efficacy for the decasaccharide, followed by the octasaccharide, while the hexasaccharide displayed the shortest efficacious length. We propose that these oligosaccharide interactions enable the charge-mediated aggregation of positively charged tropoelastin. This biochemistry models glycosaminoglycan interactions on the cell surface during elastogenesis which is characterized by the clustering of nascent tropoelastin aggregates to form micron-sized spherules., (2009 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Elastin is a key extracellular matrix protein in a range of tissues and a viable candidate for elastic tissue engineering. Elastin is not typically incorporated into engineered scaffolds because of lack of access to pure, homogeneous human elastin. Recombinant human tropoelastin, the monomer precursor of elastin, can be chemically cross-linked to form a polymer and used to generate biomaterials with physical properties similar to native elastin. In this study, we use electrospinning to generate versatile tropoelastin microfibers. Tropoelastin retained structural and biological properties, including secondary structure and coacervation temperature, after fiber formation but was solubilized on exposure to an aqueous environment prior to cross-linking. Two cross-linking methods were utilized to generate synthetic elastin microfibers that were stable in aqueous environments. Microfibers stably persisted for up to 180 days at 37 degrees C. Three primary human cell types derived from elastic tissues were assessed and found to attach and proliferate on both types of microfibers.

Polymers are used for the fabrication of many prosthetic implants. It is desirable for these polymers to promote biological function by promoting the adhesion, differentiation and viability of cells. Here we have used plasma immersion ion implantation (PIII) treatment of polystyrene to modify the polymer surface, and so modulate the binding of the extracellular matrix protein tropoelastin. PIII treated, but not untreated polystyrene, bound tropoelastin in a sodium dodecyl sulfate (SDS)-resistant manner, consistent with previous enzyme-binding data that demonstrated the capability of these surfaces to covalently attach proteins without employing chemical linking molecules. Furthermore sulfo-NHS acetate (SNA) blocking of tropoelastin lysine side chains eliminated the SDS-resistant binding of tropoelastin to PIII-treated polystyrene. This implies tropoelastin is covalently attached to the PIII-treated surface via its lysine side chains. Cell spreading was only observed on tropoelastin coated, PIII-treated polystyrene surfaces, indicating that tropoelastin was more biologically active on the PIII-treated surface compared to the untreated surface. A contact mask was used to pattern the PIII treatment. Following tropoelastin attachment, cells spread preferentially on the PIII-treated sections of the polystyrene surface. This demonstrates that PIII treatment of polystyrene improves the polymer's tropoelastin binding properties, with advantages for tissue engineering and prosthetic design.

Tropoelastin is the monomer building block of the biopolymer elastin, which is responsible for elasticity in arteries, lung and skin. Previous studies have shown that, in contrast to predictions made based on primary sequence, tropoelastin has little regular secondary structure in aqueous solution and displays considerable flexibility. This investigation defines the level of residual structure present in tropoelastin and uses the naturally-occurring structure-inducing osmolyte trimethylamine N-oxide to examine the potential for regular structure in tropoelastin. Tropoelastin is defined as a thermodynamically unfolded premolten globule, which can account for its ability to elastically deform.

Tropoelastin is a 60-72 kDa alternatively spliced extracellular matrix protein and a key component of elastic fibres. It is found in all vertebrates except for cyclostomes. Secreted tropoelastin is tethered to the cell surface, where it aggregates into organised spheres for cross-linking and incorporation into growing elastic fibres. Tropoelastin is characterised by alternating hydrophobic and hydrophilic domains and is highly flexible. The conserved C-terminus is an area of the molecule of particular biological importance in that it is required for both incorporation into elastin and for cellular interactions. Mature cross-linked tropoelastin gives elastin, which confers resilience and elasticity on a diverse range of tissues. Elastin gene disruptions in disease states and knockout mice emphasise the importance of proper tropoelastin production and assembly, particularly in vascular tissue. Tropoelastin constructs hold promise as biomaterials as they mimic many of elastin's physical and biological properties with the capacity to replace damaged elastin-rich tissue.

Degradation of elastic fibers in tissues can result in the development of disorders that include aneurysms, atherosclerosis, and loss of skin elasticity. Tropoelastin is the precursor of the cross-linked elastin and its expression is triggered by elastin-degrading factors as a response to damage. Factors like UV radiation not only increase the expression of tropoelastin but also potent metalloelastases such as macrophage elastase (MMP-12). The development of elastin-degrading diseases, moreover, is a chronic process during which elastin and tropoelastin are repeatedly exposed to attacks by MMP-12. Hence, in this work we report the in vitro susceptibility of tropoelastin and the potential of MMP-12 to generate matrikines. This work provides evidence that tropoelastin is substantially and rapidly degraded by MMP-12 even at very dilute enzyme concentrations. MMP-12 cleaves at least 86 sites in tropoelastin. Analysis of the generated peptides revealed that some small peptides contained the motif GXXPG that may enable them to bind with the elastin binding protein (EBP). Furthermore, using synthesized peptides it was confirmed that several sites in the sequence encoded by exon 24 which contains repetitive units of biologically active VGVAPG domains are susceptible to attack by MMP-12, provided that the active subsites in MMP-12 (S(4) to S(4)') are occupied. Such cleavage events have lead to the generation of ligands that may bind to EBP.

Macrophage elastase (MMP-12) is a member of the family of matrix metalloproteinases (MMPs) and is active against multiple extracellular protein substrates such as elastin. Its effect on elastin is central to emphysema in the lung and photoaging of skin. Its expression in the skin increases on photodamaged skin and upon aging. Detecting and characterizing peptides cleaved in elastin, therefore, helps to understand such degradative disease processes in the skin and is also needed to assist in the rational design of agents that specifically inhibit the degradation. In this study, cleavage sites of MMP-12 in human skin elastin were extensively investigated. The peptides formed as a result of cleavages by this enzyme in the human skin elastin were characterized using mass spectrometry. A total of 41 peptides ranging from 4 to 41 amino acids were identified and 36 cleavage sites were determined. Amino acids encoded by exons 5, 6, 26, 28-31 were particularly susceptible to cleavages by MMP-12 and none or very few cleavages were detected from domains encoded by the remaining exons. The amino acid preferences of the different subsites on the catalytic domain of MMP-12 were analyzed.

Synaptic core complex formation between syntaxin and synaptosome-associated protein of 25 kDa (SNAP25) on the plasma membrane and synaptobrevin on the vesicle membrane is responsible for membrane fusion and neurotransmitter release. A radiolabeled protein binding assay for synaptic core complex formation was developed. The components of this assay included recombinant glutathione S-transferase (GST)-syntaxin immobilized on glutathione agarose beads, SNAP25 and (125)I-labeled synaptobrevin. Reactions were performed in tubes containing filter inserts to facilitate removal of unbound protein. The radiolabeled protein bound was then quantified by gamma counter. A K(d) of 1.6 microM was determined for the GST-syntaxin/SNAP25/synaptobrevin complex, and a K(d) of 12 microM was determined for the GST-syntaxin/synaptobrevin complex. The assay was used to screen 14 herbal extracts for effectors of core complex formation. Herbs traditionally used to treat neurological conditions such as depression, anxiety, and stress were chosen. A Hypericum perforatum extract was found to have a nonspecific effect via protein complexation, whereas an Albizzia julibrissin extract was found to reduce the level of core complex formation. The assay was used to further investigate the effect of the A. julibrissin extract. The discovery of an inhibitor of core complex formation demonstrates the efficacy of the assay in screening natural products for substances that affect core complex formation.

Elastin is a key structural component of the extracellular matrix. Tropoelastin is the soluble precursor of elastin. In addition to providing elastic recoil to various tissues such as the aorta and lung, elastin, tropoelastin and elastin degradation products are able to influence cell function and promote cellular responses. These responses include chemotaxis, proliferation and cell adhesion. The interaction of elastin products with cells has been attributed to the elastin receptor. However, additional cell-surface receptors have also been identified. These include G protein-coupled receptors and integrins. The potential roles of these receptors in cell-elastin interactions, with particular focus on elastin formation are discussed.

A number of reports point to the presence of proteoglycans and/or glycosaminoglycans within elastic fibers in normal and in pathological conditions. We present data that heparan sulphate (HS)-containing proteoglycans are associated with normal elastic fibers in human dermis and that isolated HS chains interact in vitro with recombinant tropoelastin and with peptides encoded by distinct exons of the human tropoelastin gene (EDPs). By immunocytochemistry, HS chains were identified as associated with the amorphous elastin component in the human dermis and remained associated with the residual elastin in the partially degenerated fibers of old subjects. HS appeared particularly concentrated in the mineralization front of elastic fibers in the dermis of patients affected by pseudoxanthoma elasticum (PXE). In in vitro experiments, HS induced substantial changes in the coacervation temperature and in the aggregation properties of recombinant tropoelastin and of synthetic peptides (EDPs) corresponding to sequences encoded by exons 18, 20, 24 and 30 of the human tropoelastin gene. In particular, HS modified the coacervation temperature and favoured the aggregation into ordered structures of tropoelastin molecules and of EDPs 18, 20 and 24, but not of EDP30. These data strongly indicate that HS-elastin interactions may play a role in tissue elastin fibrogenesis as well as modulating elastin stability with time and in diseases.

Circular dichroism studies of tropoelastin secondary structure show 4+/-1% alpha-helix in aqueous solutions. This is in contrast to the substantially higher amounts (up to 23+/-7%) of alpha-helix predicted by computer algorithms, which propose that regions of alpha-helix are limited to the alanine-rich cross-linking domains. Through the addition of trifluoroethanol, the amount of alpha-helix increased to 17+/-1%, equivalent to that expected on the basis of primary structure. The physiological ability of the protein to coacervate and the critical concentration of monomer required for coacervation were unaffected by levels of alpha-helix. However, the temperature required for coacervation decreased linearly with increasing alpha-helical structure, which correlates with the participation of alpha-helices in association. We propose that the alanine-rich cross-linking domains exist as nascent helices in tropoelastin in aqueous solution. We further suggest a novel mechanism for coacervation whereby formation of alpha-helices and subsequent helical side chain interactions limit the conformational flexibility of the polypeptide, to facilitate associations between hydrophobic domains during elastogenesis.

Abnormal production of matrix metalloproteinases (MMPs) has been observed in a variety of diseases, such as emphysema, atherosclerosis, and cancer metastasis. Destruction of connective tissue ensues and elastin is often a key target. Three of the main elastolytic MMPs are the gelatinases MMP-2 and MMP-9 and the metalloelastase MMP-12. To investigate the possibility of using peptides to inhibit the elastolytic activity of these enzymes, we mapped the sites within tropoelastin recognized by MMP-9 and MMP-12. Peptides that correspond to regions overlapping these sites were then tested for their ability to inhibit these MMPs. These included an unmodified peptide directed against MMP-9 (peptide PP), cysteine-containing peptides that mimicked either the MMP-9 (peptide NCP) or the MMP-12 (peptide lin24) cleavage sites in tropoelastin and their cyclized forms (CP and cyc24, respectively), and a peptide containing a zinc-chelating hydroxamate group directed against MMP-9 (HP). The presence of a free sulfhydryl or hydroxamate group capable of chelating the zinc ion in the active site of the MMPs was generally found to increase the inhibitory activity of the peptides. The specificity of the inhibitors varied, with some of the inhibitors showing activity against all of the MMPs examined. None of the inhibitors had any significant effect on the activity of the unrelated serine protease, plasmin. K(i) values for the inhibitors were in the micromolar range. Our results suggest ways of developing other MMP inhibitors based on substrate recognition sites that may provide greater levels of inhibition.