Microfibril-associated glycoprotein 4 forms octamers that mediate interactions with elastogenic proteins and cells.

Microfibril-associated glycoprotein 4 (MFAP4) is a 36-kDa extracellular matrix glycoprotein with critical roles in organ fibrosis, chronic obstructive pulmonary disease, and cardiovascular disorders, including aortic aneurysms. MFAP4 multimerises and interacts with elastogenic proteins, including fibrillin-1 and tropoelastin, and with cells via integrins. Structural details of MFAP4 and its potential interfaces for these interactions are unknown. Here, we present a cryo-electron microscopy structure of human MFAP4. In the presence of calcium, MFAP4 assembles as an octamer, where two sets of homodimers constitute the top and bottom halves of each octamer. Each homodimer is linked together by an intermolecular disulphide bond. A C34S missense mutation prevents disulphide-bond formation between monomers but does not prevent octamer assembly. The atomic model, built into the 3.55 Å cryo-EM map, suggests that salt-bridge interactions mediate homodimer assembly, while non-polar residues form the interface between octamer halves. In the absence of calcium, an MFAP4 octamer dissociates into two tetramers. Binding studies with fibrillin-1, tropoelastin, LTBP4, and small fibulins show that MFAP4 has multiple surfaces for protein-protein interactions, most of which depend upon MFAP4 octamer assembly. The C34S mutation does not affect these protein interactions or cell interactions. MFAP4 assemblies with fibrillin-1 abrogate MFAP4 interactions with cells.

Structural and physical basis for the elasticity of elastin.

Elastin function is to endow vertebrate tissues with elasticity so that they can adapt to local mechanical constraints. The hydrophobicity and insolubility of the mature elastin polymer have hampered studies of its molecular organisation and structure-elasticity relationships. Nevertheless, a growing number of studies from a broad range of disciplines have provided invaluable insights, and several structural models of elastin have been proposed. However, many questions remain regarding how the primary sequence of elastin (and the soluble precursor tropoelastin) governs the molecular structure, its organisation into a polymeric network, and the mechanical properties of the resulting material. The elasticity of elastin is known to be largely entropic in origin, a property that is understood to arise from both its disordered molecular structure and its hydrophobic character. Despite a high degree of hydrophobicity, elastin does not form compact, water-excluding domains and remains highly disordered. However, elastin contains both stable and labile secondary structure elements. Current models of elastin structure and function are drawn from data collected on tropoelastin and on elastin-like peptides (ELPs) but at the tissue level, elasticity is only achieved after polymerisation of the mature elastin. In tissues, the reticulation of tropoelastin chains in water defines the polymer elastin that bears elasticity. Similarly, ELPs require polymerisation to become elastic. There is considerable interest in elastin especially in the biomaterials and cosmetic fields where ELPs are widely used. This review aims to provide an up-to-date survey of/perspective on current knowledge about the interplay between elastin structure, solvation, and entropic elasticity.

Determining the Sequence Dependency of Self-Assembly of Elastin-Like Peptides Using Short Peptide Analogues with Shuffled Repetitive Sequences.

Synthetic elastin-like peptides (ELPs) that possess characteristic tropoelastin-derived hydrophobic repetitive sequences, such as (VPGVG)n, exhibit thermoresponsive reversible self-assembly. Although their thermoresponsive properties have been well-studied, the sequence-dependent and structural requirements for self-assembly remain ambiguous. In particular, it is still unclear whether the amino acid sequences derived from tropoelastin are necessary for self-assembly. In this study, 11 sequence-shuffled ELP analogues based on (FPGVG)5, which is a previously developed short ELP (sELP), were designed to elucidate the sequence-dependent and structural requirements for their self-assembly. Among them, eight shuffled peptides exhibited self-assembling properties, whereas the other three peptides were difficult to dissolve in water. Structural analyses revealed that the structural characteristics of the three insoluble peptides were different from those of their thermoresponsive analogues. Furthermore, the secondary structures of the peptide analogues possessing the self-assembly abilities were different from each other. These results suggest that the potential for self-assembly and water solubility of sELPs depend on the primary structure in each repeated unit. Moreover, several shuffled analogues exhibited more potent self-assembling properties than the original (FPGVG)5, indicating that shorter ELPs can be obtained using their novel motifs as repetitive units. We also observed that the presence of Pro-Gly sequence in the repeating units was advantageous in terms of peptide solubility. Although further analysis will be necessary to elucidate the molecular mechanism underlying the self-assembly of these sELPs, this study provides insights into the relationship between the amino acid sequence and the self-assembling ability of the peptides for developing new sELPs for various applications.

Fabrication of Insoluble Elastin by Enzyme-Free Cross-Linking.

Elastin is an essential extracellular matrix protein that enables tissues and organs such as arteries, lungs, and skin, which undergo continuous deformation, to stretch and recoil. Here, an approach to fabricating artificial elastin with close-to-native molecular and mechanical characteristics is described. Recombinantly produced tropoelastin are polymerized through coacervation and allysine-mediated cross-linking induced by pyrroloquinoline quinone (PQQ). A technique that allows the recovery and repeated use of PQQ for protein cross-linking by covalent attachment to magnetic Sepharose beads is developed. The produced material closely resembles natural elastin in its molecular, biochemical, and mechanical properties, enabled by the occurrence of the cross-linking amino acids desmosine, isodesmosine, and merodesmosine. It possesses elevated resistance against tryptic proteolysis, and its Young's modulus ranging between 1 and 2 MPa is similar to that of natural elastin. The approach described herein enables the engineering of mechanically resilient, elastin-like materials for biomedical applications.

Changes in elastin structure and extensibility induced by hypercalcemia and hyperglycemia.

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.

Globule and fiber formation with elastin-like polypeptides: a balance of coacervation and crosslinking.

Elastic fiber assembly is a complex process that requires the coacervation and cross-linking of the protein building block tropoelastin. To date, the order, timing, and interplay of coacervation and crosslinking is not completely understood, despite a great number of advances into understanding the molecular structure and functions of the many proteins involved in elastic fiber assembly. With a simple in vitro model using elastin-like polypeptides and the natural chemical crosslinker genipin, we demonstrate the strong influence of the timing and kinetics of crosslinking reaction on the coacervation, crosslinking extent, and resulting morphology of elastin. We also outline a method for analyzing elastin droplet network formation as a heuristic for measuring the propensity for elastic fiber formation. From this we show that adding crosslinker during peak coacervation dramatically increases the propensity for droplet network formation.

Domains 12 to 16 of tropoelastin promote cell attachment and spreading through interactions with glycosaminoglycan and integrins alphaV and alpha5beta1.

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 α5ß1 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 α5ß1 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.

The evolutionary background and functional consequences of the rs2071307 polymorphism in human tropoelastin.

Elastin is a major polymeric protein of the extracellular matrix, providing critical properties of extensibility and elastic recoil. The rs2071307 genomic polymorphism, resulting in the substitution of a serine for a glycine residue in a VPG motif in tropoelastin, has an unusually high minor allele frequency in humans. A consequence of such allelic heterozygosity would be the presence of a heterogeneous elastin polymer in up to 50% of the population, a situation which appears to be unique to Homo sapiens. VPG motifs are extremely common in hydrophobic domains of tropoelastins and are the sites of transient ß-turns that are essential for maintaining the conformational flexibility required for its function as an entropic elastomer. Earlier data demonstrated that single amino acid substitutions in tropoelastin can have functional consequences for polymeric elastin, particularly when present in mixed polymers. Here, using NMR and molecular dynamics approaches, we show the rs2071307 polymorphism reduces local propensity for ß-turn formation, with a consequent increase in polypeptide hydration and an expansion of the conformational ensemble manifested as an increased hydrodynamic radius, radius of gyration and asphericity. Furthermore, this substitution affects functional properties of polymeric elastin, particularly in heterogeneous polymers mimicking allelic heterozygosity. We discuss whether such effects, together with the unusually high minor allele frequency of the polymorphism, could imply some some evolutionary advantage for the heterozygous state.

Insights into the biophysical forces between proteins involved in elastic fiber assembly.

Elastogenesis is a complex process beginning with transcription, translation, and extracellular release of precursor proteins leading to crosslinking, deposition, and assembly of ubiquitous elastic fibers. While the biochemical pathways by which elastic fibers are assembled are known, the biophysical forces mediating the interactions between the constituent proteins are unknown. Using atomic force microscopy, we quantified the adhesive forces among the elastic fiber components, primarily between tropoelastin, elastin binding protein (EBP), fibrillin-1, fibulin-5, and lysyl oxidase-like 2 (LOXL2). The adhesive forces between tropoelastin and other tissue-derived proteins such as insoluble elastin, laminin, and type I collagens were also assessed. The adhesive forces between tropoelastin and laminin were strong (1767 ± 126 pN; p < 10-5vs. all others), followed by forces (≥200 pN) between tropoelastin and human collagen, mature elastin, or tropoelastin. The adhesive forces between tropoelastin and rat collagen, EBP, fibrillin-1, fibulin-5, and LOXL2 coated on fibrillin-1 were in the range of 100-200 pN. The forces between tropoelastin and LOXL2, LOXL2 and fibrillin-1, LOXL2 and fibulin-5, and fibrillin-1 and fibulin-5 were less than 100 pN. Introducing LOXL2 decreased the adhesive forces between the tropoelastin monomers by ∼100 pN. The retraction phase of force-deflection curves was fitted to the worm-like chain model to calculate the rigidity and flexibility of these proteins as they unfolded. The results provided insights into how each constituent's stretching under deformation contributes to structural and mechanical characteristics of these fibers and to elastic fiber assembly.

Transglutaminase-Mediated Cross-Linking of Tropoelastin to Fibrillin Stabilises the Elastin Precursor Prior to Elastic Fibre Assembly.

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.

Elastases and elastokines: elastin degradation and its significance in health and disease.

Elastin is an important protein of the extracellular matrix of higher vertebrates, which confers elasticity and resilience to various tissues and organs including lungs, skin, large blood vessels and ligaments. Owing to its unique structure, extensive cross-linking and durability, it does not undergo significant turnover in healthy tissues and has a half-life of more than 70 years. Elastin is not only a structural protein, influencing the architecture and biomechanical properties of the extracellular matrix, but also plays a vital role in various physiological processes. Bioactive elastin peptides termed elastokines - in particular those of the GXXPG motif - occur as a result of proteolytic degradation of elastin and its non-cross-linked precursor tropoelastin and display several biological activities. For instance, they promote angiogenesis or stimulate cell adhesion, chemotaxis, proliferation, protease activation and apoptosis. Elastin-degrading enzymes such as matrix metalloproteinases, serine proteases and cysteine proteases slowly damage elastin over the lifetime of an organism. The destruction of elastin and the biological processes triggered by elastokines favor the development and progression of various pathological conditions including emphysema, chronic obstructive pulmonary disease, atherosclerosis, metabolic syndrome and cancer. This review gives an overview on types of human elastases and their action on human elastin, including the formation, structure and biological activities of elastokines and their role in common biological processes and severe pathological conditions.

Covalent Biofunctionalization of the Inner Surfaces of a Hollow-Fiber Capillary Bundle Using Packed-Bed Plasma Ion Implantation.

Hollow-fiber capillary bundles are widely used in the production of medical devices for blood oxygenation and purification purposes such as in cardiopulmonary bypass, hemodialysis, and hemofiltration, but the blood interfacing inner surfaces of these capillaries provide poor hemocompatibility. Here, we present a novel method of packed-bed plasma ion implantation (PBPII) for the modification of the inner surfaces of polymeric hollow-fiber bundles enclosed in a cassette. The method is simple and can be performed on an intact hollow-fiber bundle cassette by the placement of a hollow cylindrical electrode, connected to a negative high-voltage pulse generator, around the cassette. The method does not require the insertion of electrodes inside the capillaries or the cassette. Nitrogen gas is fed into the capillaries inside the cassette by connecting the inlet of the cassette to a gas source. Upon the application of negative high-voltage bias pulses to the electrode, plasma is ignited inside the cassette, achieving the surface modification of both the internal and external surfaces of the capillaries. Fourier transform infrared-attenuated total reflectance spectroscopy of the PBPII-treated capillaries revealed the formation of aromatic C═C bonds, indicating the progressive carbonization of the capillary surfaces. The PBPII treatment was found to be uniform along the capillaries and independent of the radial position in the cassette. Atomic force microscopy of cross sections through the capillaries revealed that the increased stiffness associated with the carbonized layer on the inner surface of the PBPII-treated capillary has a depth (∼40 nm) consistent with that expected for ions accelerated by the applied bias voltage. The modified internal surfaces of the capillary bundle showed a greatly increased wettability and could be biofunctionalized by covalently immobilizing protein directly from the buffer solution. The direct, reagent-free protein immobilization was demonstrated using tropoelastin as an example protein. Covalent binding of the protein was confirmed by its resistance to removal by hot sodium dodecyl sulfate detergent washing, which is known to disrupt physical binding.

Single-step, acid-based fabrication of homogeneous gelatin-polycaprolactone fibrillar scaffolds intended for skin tissue engineering.

Blends of natural and synthetic polymers have recently attracted great attention as scaffolds for tissue engineering applications due to their favorable biological and mechanical properties. Nevertheless, phase-separation of blend components is an important challenge facing the development of electrospun homogeneous fibrillar natural-synthetic polymers scaffolds; phase-separation can produce significant detrimental effects for scaffolds fabricated by electrospinning. In the present study, blends of gelatin (Gel; natural polymer) and polycaprolactone (PCL; synthetic polymer), containing 30 and 45 wt% Gel, were prepared using acetic acid as a 'green' sole solvent to straightforwardly produce appropriate single-step Gel-PCL solutions for electrospinning. Miscibility of Gel and PCL in the scaffolds was assessed and the morphology, chemical composition and structural and solid-state properties of the scaffolds were thoroughly investigated. Results showed that the two polymers proved miscible under the single-step solution process used and that the electrospun scaffolds presented suitable properties for potential skin tissue engineering applications. Viability, metabolic activity and protein expression of human fibroblasts cultured on the Gel-PCL scaffolds were evaluated using LIVE/DEAD (calcein/ethidium homodimer), MTT-Formazan and immunocytochemistry assays, respectively. In vitro results showed that the electrospun Gel-PCL scaffolds enhanced cell viability and proliferation in comparison to PCL scaffolds. Furthermore, scaffolds allowed fibroblasts expression of extracellular matrix proteins, tropoelastin and collagen Type I, in a similar way to positive controls. Results indicated the feasibility of the single-step solution process used herein to obtain homogeneous electrospun Gel-PCL scaffolds with Gel content ≥30 wt% and potential properties to be used as scaffolds for skin tissue engineering applications for wound healing.

Elastic fiber ultrastructure and assembly.

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.

A tribute to Ladislas Robert.

This Thematic Minireview Series of Matrix Biology focused on elastin, from structure to disease celebrates the memory of Ladislas Robert, a pioneer in Matrix Biology in France and Europe. Since his first publication on elastin and elastases in 1957, the huge development in matrix biology led to major findings on elastic fibers and their component proteins including elastin architecture, the role of fibrillins and microfibril-binding proteins on elastin assembly, the effects of sequence variants of human tropoelastin on its assembly, structure and functions, the role of elastin peptides in health and diseases, the identification of neuraminidase-1 as a member of the elastin receptor complex, and the fate of elastic fibers upon aging, which are reviewed in this series. Two other reviews, focused on the design and use of elastin-like recombinamers as biomaterials, and on the circadian rhythms in skin and other elastic tissues, complete this series.

Avidity and Cell Uptake of Integrin-Targeting Polypeptide Micelles is Strongly Shape-Dependent.

We describe a genetically encoded micelle for targeted delivery consisting of a diblock polypeptide with segments derived from repetitive protein motifs inspired by Drosophila melanogaster Rec-1 resilin and human tropoelastin with a C-terminal fusion of an integrin-targeting fibronectin type III domain. By systematically varying the weight fraction of the hydrophilic elastin-like polypeptide (ELP) block and molecular weight of the diblock polypeptide, we designed micelles of different morphologies that modulate the binding avidity of the human wild-type 10th fibronectin domain (Fn3) as a function of shape. We show that wormlike micelles that present the Fn3 domain have a 1000-fold greater avidity for the αvß3 receptor compared to the monomer ligand and an avidity that is greater than a clinically relevant antibody that is driven by their multivalency. The amplified avidity of these micelles leads to significantly increased cellular internalization, a feature that may have utility for the intracellular delivery of drugs that are loaded into the core of these micelles.

Elastin architecture.

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.

Sequence variants of human tropoelastin affecting assembly, structural characteristics and functional properties of polymeric elastin in health and disease.

Elastin is the polymeric protein responsible for the physiologically important properties of extensibility and elastic recoil of cardiovascular, pulmonary and many other tissues. In spite of significant advances in the understanding how monomeric tropoelastin is assembled into the polymeric elastic matrix, details of this assembly process are still lacking. In particular it is not clear how the various architectures and more subtle elastic properties required by diverse elastic tissues can arise from the protein product of a single gene. While monomeric tropoelastin has the intrinsic ability to self-assemble into fibrillar structures, it is clear that in vivo assembly is guided by interactions with cells and other matrix-associated components. In addition, the multiplicity of reported mRNA isoforms of human tropoelastin, if translated into protein variants, could modulate not only interactions with these matrix-associated components but also self-assembly and functional properties. Critical information identifying such protein isoforms of human tropoelastin is only now emerging from mass spectrometric studies. Increased levels of complexity of the assembly process provide additional opportunities for production of polymeric elastins with aberrant architectures and sub-optimal functional properties that could affect the longer-term structural integrity of elastic matrices. Biophysical techniques, such as SAXS, NMR and molecular dynamics, have provided a means to discern details of the effects of sequence variants, including both alternate splicing isoforms and genetic polymorphisms, on the dynamic flexibility of elastin required for its elastomeric properties. Such approaches promise to provide important new insights into the relationship between sequence, structural characteristics, assembly and functional properties of elastin in both health and disease.

Tropoelastin-Coated Tendon Biomimetic Scaffolds Promote Stem Cell Tenogenic Commitment and Deposition of Elastin-Rich Matrix.

Tendon tissue engineering strategies that recreate the biophysical and biochemical native microenvironment have a greater potential to achieve regeneration. Here, we developed tendon biomimetic scaffolds using mechanically competent yarns of poly-ε-caprolactone, chitosan, and cellulose nanocrystals to recreate the inherent tendon hierarchy from a nano-to-macro scale. These were then coated with tropoelastin (TROPO) through polydopamine (PDA) linking, to mimic the native extracellular matrix (ECM) composition and elasticity. Both PDA and TROPO coatings decreased surface stiffness without masking the underlying substrate. We found that human adipose-derived stem cells (hASCs) seeded onto these TROPO biomimetic scaffolds more rapidly acquired their spindle-shape morphology and high aspect ratio characteristic of tenocytes. Immunocytochemistry shows that the PDA and TROPO-coated surfaces boosted differentiation of hASCs toward the tenogenic lineage, with sustained expression of the tendon-related markers scleraxis and tenomodulin up to 21 days of culture. Furthermore, these surfaces enabled the deposition of a tendon-like ECM, supported by the expression of collagens type I and III, tenascin, and decorin. Gene expression analysis revealed a downregulation of osteogenic and fibrosis markers in the presence of TROPO when compared with the control groups, suggesting proper ECM deposition. Remarkably, differentiated cells exposed to TROPO acquired an elastogenic profile due to the evident elastin synthesis and deposition, contributing to the formation of a more mimetic matrix in comparison with the PDA-coated and uncoated conditions. In summary, our biomimetic substrates combining biophysical and biological cues modulate stem cell behavior potentiating their long-term tenogenic commitment and the production of an elastin-rich ECM.