Elastin-like systems for tissue engineering

14 Elastin-like systems for tissue engineering J. R O D R I G U E Z - C A B E L L O, A. R I B E I R O, J. R E G U E R A, A. G I R O T T I and A. T E S T E R A, Universidad de Valladolid, Spain

14.1

Introduction

Modern biomaterials science is characterized by a growing emphasis on identification of specific design parameters that are critical to performance, and by a growing appreciation of the need to integrate biomaterials design with new insights emerging from studies of cell-matrix interactions, cellular signalling processes, and developmental and systems biology.1 This leads us to a new concept, with an extraordinary use: ‘Nanobiotechnology’. The initial impetus for the development of technology at the nano-scale came from its relevance to electronics. However, interest has quickly been focused on biological systems. The search for common goals between this focus and biology arises from the idea that biology offers the most complex and sophisticated collection of functional nanostructures that exist. Understanding biological nanostructures will be enormously stimulating for nanotechnology; designers of nanomachines and biomolecular motors have much to learn from biological systems. Nanotechnology offers to biology new tools and biology offers nanotechnology access to new types of functional nanosystems (components of the cell) that are unquestionably interesting and possibly quite useful in the near future. Combined together, both could provide infinite possibilities for new designs in materials science.2 One of the most promising approaches in the fabrication of biomaterials is the bottom-up strategy, where materials are self-assembled, molecule by molecule, in order to perform new and hierarchically ordered complexes. This could be considered as a part of nanomaterials building, and a deep understanding of individual molecular behaviour and properties is required. For instance, the knowledge of the self-assembly of biological molecules provides an outstanding tool to obtain desired and functional supramolecular structures. Today, several self-assembled polymeric materials are well known and used as biomaterials. Natural biopolymers are an impressive example of advanced materials. Besides their unmatched biocompatibility and biodegradability, their bioactivity is one central feature of these biomaterials. 374

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Bioactive materials interact with biological systems and can interact with both the soft and hard tissues, promoting the healing and regeneration processes of surrounding tissues.

14.2

Genetic engineering of protein-based polymers

For many years, materials scientists have been investigating the possibilities of obtaining higher levels of control in polymer synthesis. Although important progress has been made, especially in recent years, the level of control of the characteristics of biomacromolecules was not reached using chemical synthesis. The development of molecular biology and genetic engineering techniques nowadays allow the design and bioproduction of polymers with absolute control of molecular architecture and the absence of randomness in the primary structure. Using these new molecular biology techniques, we are now able to obtain materials that mimic natural biomaterials, once we can create almost any DNA duplex coding in any amino-acid sequence. Many advantages can come from this approach. First, genetically-engineered protein-based polymers (GEPBPs)3 will, in principle, be able to show any simple or complex properties present in natural proteins. In this sense, this method offers an opportunity to exploit the huge resources, in terms of functionality, hoarded and refined to the extreme by biology during the long process of natural selection. GEPBPs can easily make use of the vast amount of functionality present in hundreds of thousands of different proteins existing in living organisms, from the smallest prions to viruses, for example. On the other hand, as we can construct coding gene, base by base, by following our own original designs and without being restricted to gene fragments found in living organisms, we can design and produce GEPBPs to obtain materials, systems and devices exhibiting functions of particular technological interest that are not displayed in living organisms. Moreover, from the point of view of a polymer chemist, the degree of control and complexity attained by genetic engineering is clearly superior to that achieved by even the most sophisticated polymer synthesis technologies. GEPBPs are characterized as being strictly monodisperse and can be obtained from a few hundred Daltons to more than 200 kDa; and this upper limit is continuously increasing.4 The number of different combinations attainable by combining the 20 natural amino acids is practically infinite. In a simple calculation, if we consider how many different combinations are possible to obtain small proteins consisting of, for example, 100 amino acids (their modest molecular mass would range in between 5.7 and 18.6 kDa), the figure is as high as 1.3 × 10130 possibilities, and the matter necessary to produce just a single copy of them would be 55 orders of magnitude higher that the whole known dark and luminous matter of the universe.3

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14.3

Genetic strategies for synthesis of proteinbased polymers

In synthesizing genes encoding repetitive protein based polymers, the techniques of molecular biology are typically employed to self-ligate monomer DNA fragments in a process of oligomerization. The monomer fragments must be oligomerized in a ‘head-to-tail’ orientation, and can be seamless in sequence or can contain intervening linkers between the desired repeats. Approaches to oligomerization can be broadly classified as either iterative, random or recursive, although these modes can be sequentially combined within the same implementation. Each of these methods is illustrated in Fig. 14.1. For iterative techniques, a DNA segment is oligomerized in a series of single, uniform steps; each step grows the oligomer by one unit length of the monomer gene. In random methods, an uncontrolled number of monomer DNA segments are oligomerized in a single step, creating a population of oligomerized clones of different lengths. This random approach of selfligation is referred to as ‘concatenation’ because the DNA segment is concatenated in a reaction that is analogous to the propagation step in chemical polymerization. Finally, in recursive approaches, DNA segments are joined E

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14.1 Schematics of three approaches to DNA oligomerization, (a) iterative, (b) random, (c) recursive.

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in sequential steps, with the length of the ligated segments growing geometrically in each step.5

14.4

State-of-the-art in genetically-engineered protein-based polymers (GEBPs)

Nowadays, genetic engineering of PBPs is still in its early infancy. The radically different approach in the methodology used to produce these polymers has resulted in the fact that, even now, a limited number of research groups and companies have made the effort to make this transition. Among these pioneer groups, the main interest has been mainly concentrated in two major polymer families, spider-silk like polymers6 and elastin-like polymers (ELPs), although some other interesting protein polymers have also been studied. Those include coiled-coil motifs and their related leucine zippers,7–9 β-sheetforming polymers, poly(allylglycine) and homopolypeptides such as poly(glutamic acid). Several families of polymers based on other elastic proteins such as resilin,10 abductin11 or gluten12 have attracted attention in recent years.2

14.5

Elastin-like polymers

ELPs are non-natural polypeptides composed of repeating sequences. They have their origin in the repeating sequences found in the mammalian elastic protein elastin that confers elasticity to structures such as skin and blood vessels. The most striking and longest sequence between cross-links in pig and cow is the undecapentapeptide (VPGVG)11.13,14 Along with this repeating sequence, others can be pointed out such as (VPGG) n, 15 (APGVGV)n.16 The importance of these polymers reside in the fact that they show a versatile and ample range of interesting properties that are difficult to find together in other materials, and that goes beyond their simple mechanical performance. Certainly, ELPs show a set of properties that places them in an excellent position towards designing advanced polymers for many different applications, including the most cutting edge biomedical and nanobiotechnological uses. Regarding their properties, some of their main characteristics are derived from the natural protein on which they are based. For example, the cross-linked matrices of these polymers retain most of the striking mechanical properties of elastin,17 i.e an almost ideal elasticity with Young’s modulus, elongation at break, etc. in the range of natural elastin and an outstanding resistance to fatigue.18,19 In addition, this mechanical performance is accompanied by an extraordinary biocompatibility.20 The polymer poly(VPGVG) is considered as a model for the ELPs. Most of the ELPs are based on the pentapeptide VPGVG (or its permutations),

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with amino-acid side-chains, excluding glycine, comprising simple aliphatic and mainly hydrophobic chains, without further functionalization. A wide variety of ELPs have been (bio)synthesized, based on the model ELP with the general formula VPGXG, where X represents any natural or modified amino acid except proline.21–23 All functional ELPs exhibit a reversible phase transition in response to changes in temperature,23 i.e. they show an acute thermo-responsive behaviour, associated with the existence of an inverse temperature transition (ITT). In aqueous solution, below the transition temperature Tt, the free polymer chains remain disordered, random coils in solution24 that are fully hydrated, mainly by hydrophobic hydration. This hydration is characterized by ordered clathrate-like water structures surrounding the apolar moieties of the polymer;25,26 structures somewhat similar to those described for crystalline gas hydrates,27 although more heterogeneous, and of varying perfection and stability. 26 However, above T t , the chain hydrophobically folds and assembles to form a phase-separated state of 63% water and 37% polymer in weight.28 In the folded state, the polymer chains adopt a dynamic, regular, non-random structure, called a β-spiral, involving type II β-turns as the main secondary feature, and stabilized by intra-spiral, inter-turn and inter-spiral hydrophobic contacts23 (see Fig. 14.2). This behaviour is the result of the ITT. In its folded and associated state, the chain loses essentially all of the ordered water structures of hydrophobic hydration.25 During the initial stages of polymer dehydration, hydrophobic association of the β-spirals takes on fibrillar form. This process starts from the formation of filaments composed of three-stranded dynamic polypeptide β-spirals that grow to several hundred nm before settling into a visible phase-separated state.23,29 The process of the ITT is completely reversible, and it goes back to the first state when the temperature is lowered below Tt.23 We have to keep in mind that, with this kind of polymer, and with the huge amounts of water playing an active role in increasing the dynamics of the

β-spiral formation

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14.2 Mechanism for the inverse temperature transition of elastin-like polymers. From left to right: β-spiral formation of ELP molecules with three pentapeptides per turn, formation of twisted filaments or supercoil of β-spirals and their aggregation into microaggregates. Adapted with permission from J. Biomater. Sci. Polymer Edn.2

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polymer chain, regular polymer conformations cannot be understood as the fixed structures can be found, for example, in crystalline polymers. The folded state of the ELPs is characterized by a high chain mobility in which the β-spiral could be not the actual fixed structure but rather the ideal conformation around which the polymer chain oscillates.30 The transition can be easily followed either by turbidity measurements or by calorimetric methods, measuring the heat flow during the transition. The first method is characterized by a turbidity profile showing a sharp step, this increase in turbidity being caused by the formation of aggregates. Tt can be taken as the temperature at 10% or 50% change in the relative turbidity (Figure 14.3). In contrast, the differential scanning calorimetric (DSC) measurements are always characterized by a broad peak, expanding 20°C or more. In this case, Tt can be considered either as the onset of peak temperature; furthermore with this technique it is possible to obtain the enthalpy of the process as the area of the peak (Figure 14.3).30

14.6

Self-assembly behaviour of peptides and proteins

The key challenge in nanotechnology is to produce systems with desired functionalities at the nanometer scale. Over recent years, many synthetic strategies have been developed to obtain advanced nanodevices in an attempt to mimic the behaviour of biology in nature. Understanding the forces governing thermodynamic stability and specificity of the self-assembly events have become the central goal for biomimicking processes. Fabricating proteinbased devices through bottom-up approaches have been widely investigated since the preliminary studies of Drexler in 1981.31 Peptides and proteins are useful building blocks to obtain ordered nanostructures via self-assembly due to their well-stabilized folding, stability and protein–protein interactions.32 The attractive benefit of peptide self-assembly comes from the precise knowledge of their structural and functional information, allowing the construction of novel materials with tailored morphologies dictated by the individual building blocks.2

14.7

Self-assembly of elastin-like polymers (ELPs)

The self assembly of polymers is an emerging new field within material sciences, offering many potential applications in nanotechnological and nanobiotechnological fields. In relation to self-assembly, elastin undergoes a self-aggregation process in its natural environment. It is produced from a water soluble precursor, tropoelastin, which spontaneously aggregates into a covalently cross-linked fibrillar polymeric structure.33 The self-assembling ability of elastin resides in certain relatively short amino-acid sequences, as

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14.3 Thermal responsiveness of poly(VPGVG). (a) Turbidity profile as a function of temperature for a poly(VPGVG) 5 mg/L sample dissolved in water and DSC thermogram of a 20 µl (50 mg/L) aqueous solution of the same polymer (heating rate 5°C/min). (b) Photographs of aqueous solution (5 mg/mL) of this poly(VPGVG) below (5°C) and above (40°C) its Tt. Adapted with permission from Nanomedicine.30

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has been proposed by Yang et al.34 working on recombinant ELPs. The tendency to self-assemble in nanofiber conformation of ELPs can be extended to other topologies and nanostructural features.35–37 With the potential afforded by genetic engineering in designing new polymers, the growing understanding in the molecular behaviour of ELPs, and the enormous wealth of experimental and theoretical background gained during the past decade on the self-assembling characteristics of different types of block co-polymers, novel self-assembly properties are being unveiled within the ELP family. Reguera et al. showed that the ELP [(VPGVG)2-(VPGEG)-(VPGVG)2]15, previously found to be pH responsive, is able to form polymer sheets with self-assembled nanopores.37 AFM study of the topology of this ELP, containing equally spaced glutamicacid residues along the polymer chain, deposited by spin coating on a Si hydrophobic substrate at temperatures below Tt showed that in acid conditions, the deposited polymer presents a flat surface without any particular topological features. However, from neutral-basic solutions, the polymer monolayer clearly has an aperiodic pattern of nanopores (ca. 70 nm wide and separated by ca. 150 nm). This different behaviour as a function of pH has been explained in terms of the polarity of the free γ-carboxyl group of the glutamic acid. In the carboxylate form, this moiety shows a markedly higher polarity than the rest of the polymer domains and the substrate itself. Under this condition, the charged carboxylates impede any hydrophobic contact in their surroundings, which is the predominant mode of assembly for this kind of polymer. The charged domains, along with their hydration sphere, are then segregated from the hydrophobic surrounding giving rise to nanopore formation.2

14.8

Biocompatibility of ELPs

ELPs show an additional property which is highly relevant for the use of those polymers in advanced biomedical applications such as tissue engineering and controlled drug release. This is their tremendous biocompatibility. The complete series of the American Society for Testing Materials (ASTM) generic biological tests for materials and devices in contact with tissues, tissue fluids and blood demonstrate unmatched biocompatibility.20 Apparently the immune system just ignores these polymers because it cannot distinguish them from the natural elastin. Furthermore, monoclonal antibodies to the (GVGVP)n have not yet been produced, despite intensive efforts to do so.38 Incidentally, nowadays, it is believed that the high segmental mobility shown by the β-spiral, the common structural feature of ELPs, greatly helps in preventing the identification of these foreign proteins by the immune system.39 In addition, the secondary products of their bioabsorption are just simple and natural amino acids.

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14.9

Natural-based polymers for biomedical applications

Biomedical applications

Due to the new biomolecular techniques we are now capable of designing materials that will perform the same role in an organism as the natural ones. Here we can find the ELPs that, presenting a nice set of properties like smart-behaviour, self-assembly and extreme biocompatibility, are excellent for biomedical applications. This is applicable not only for ELPs that show a tailored bioactivity but also for the most basic ELPs; in this scenario we can find the polymers resulting from the repetition of the pentapeptides (VPGVG) and (VPGAG). Two different fields of interest have been targeted with ELPs in biomedical applications. The first application is for drug delivery systems, in which a polymer that can be in a matrix aggregate or device, releases drug gradually or when a certain stimulus triggers it. The second major application is the use of ELPs for tissue engineering in which the polymer, normally in the form of a matrix, works as a temporal scaffold that is gradually substituted by endogenous growing tissue. In many modern designs both functions, bioactive scaffolding and controlled release, come together to create a new generation of bioactive supports for cells.

14.10 ELPs for drug delivery ELP drug carriers can be divided into three different classes based on their intended end-use as carriers: (1) ELP homopolymers and pseudorandom copolymers for the systemic delivery of chemically conjugated radionuclides and chemotherapeutics;40 (2) block copolymers that thermally assemble into micelles or vesicles, designed for the encapsulation of hydrophobic drugs;41,42 and fusion proteins for the delivery of protein therapeutics.5 The first ELP-based drug delivery systems were reported by Urry. They were quite simple devices in which γ-radiated cross-linked poly(VPGVG) hydrogels of different shapes were loaded with a model water-soluble drug (Biebrich Scarlet).43 This drug was then released by diffusion. In this simple design, the extraordinary biocompatibility and the lack of pernicious compounds during the reabsorption of the device were exploited. The designs then became more complex. The basic VPGVG pentapeptide was functionalized by including some glutamic acids whose free carboxyl groups were used for cross-linking purposes. The cross-linker was of the type that forms carboxyamides, which were selected because of their ability to hydrolize at a given and controlled rate, releasing the polymer chains and, concurrently, any drug entrapped within the cross-linked slabs.44 Another example is the case of poly(VPAVG), were the self-association properties of the polymer were employed by our group in the development of various applications as controlled drug release devices. For example, Molina et al. 46 tested self-assembled nano- and micro-particles of

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poly(VPAVG), another version of ELP, as carriers of the model drug dexamethasone phosphate, in order to develop injectable systems for controlled drug release. In these particles, the drug is entrapped while the particles selfassemble as the temperature is raised above Tt. The slow diffusion of the drug has been considered as the main mechanism of drug delivery for this simple model, although more complex polymer designs based on this are possible. In a different approach, Betre et al. evaluated ELPs that aggregate below body temperature as a potential injectable depot for intra-articular drug delivery.46 Biodistribution studies revealed that the aggregating ELP has a 25-fold longer half-life in the injected joint than an equivalent molecular weight ELP that remains soluble and does not aggregate. These results suggest that the intra-articular delivery of ELP fusion proteins may be a viable strategy for the prolonged release of protein drugs for osteoarthritis.47 In an alternative approach, involving not only ELPs, injectable depots of silk-ELP hybrids were formed in situ for local delivery of DNA.48 This approach demonstrated the potential for sustained release of DNA from ELP depots, and may also be applicable for the release of other high molecular weight species such as proteins.47 The growing complexity of these systems is reaching nowadays levels that will lead us to almost perfect systems for controlled drug release. For example, Chilkoti’s group has produced nice examples of ELPs specially design for targeting and intracellular delivery, mainly in solid tumours. Taking advantage of the tunable Tt of ELPs they developed a non-invasive thermal targeting to solid tumours. This Tt when combined with hyperthermia treatment – the application of mild heat to the site of the tumour to promote the uptake of ELP – allows the drug to conjugate within the tumours.40,49,50 Actually, they showed, with in vivo studies of ELP delivery to two different types of implanted tumours in nude mice, that thermal targeting provided a nearly 23 fold increase in tumour localization when compared with non-heated controls.40,49 In summary they showed that, taking advantage of the Tt of the ELPs and local hyperthermia of tumours, it is possible to target the drugs, usually harmful for healthy cells, in a more precise and effective way.

14.11 Tissue engineering A tissue is composed of two major components: the cells and the extracellular matrix that the cells construct. Each tissue has a particular set of physical and chemical functions in fulfilling its role of sustaining the organ/or organism of which it is a part.51 The design of functional biomaterials that elicit cellular behaviour constitutes a major challenge for the fields of tissue engineering and materials science. Efforts to develop such materials have principally involved the design of scaffolds and hydrogels to mimic the dynamic interactions between cells and the extracellular matrix in vivo,52–54

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and the incorporation of extracelllar adhesion ligands and growth factors into engineered materials has proven effective in directing cellular responses in many applications.55–57 Knowing the central issues in tissue engineering, we can ask why sometimes materials that are used fail. Looking to all the processes that a mature or stem cell passes when it divides and spreads in growing tissue we clearly see that the cell is passing through the most vulnerable stage of its life cycle. And this is the main reason why materials that work in biomedical uses may fail when used for tissue engineering (the failure can be caused both by the material itself and by its biodegradation products). Additionally, we have to keep in mind that when we design a matrix for tissue engineering, we are trying to substitute for the natural extracellular matrix (ECM), at least transiently. Therefore, many aspects have to be taken into consideration in designing an adequate artificial ECM. Initially, the material developer must have a clear concern about the mechanical properties of the artificial scaffold. It is well know that, when properly attached to the ECM, cells sense the forces to which they are subjected via integrins. In this way, cells continuously sense their mechanical environment and respond by producing an ECM that adequately withstands the forces. In this sense, cells are very efficient force transducers. Therefore, any artificial ECM has to properly transmit forces from the environment to the growing tissue. Only in this way, can the new tissue build an adequate natural ECM that will eventually replace the artificial ECM. However, a stronger or too weak artificial ECM will cause its substitution by a too weak or too dense natural ECM, which can seriously compromise the success of tissue regeneration. In addition, growth tissues seem to need the input of mechanical deformation to create better structured tissues.38 The proteins of the natural ECM (fibronectin, collagen, elastin, etc.) contain in their sequences a huge number of bioactive peptides that are of crucial importance in the natural processes of wound healing. Those sequences include, of course, not only the well-known cell attachment sequences. In the natural ECM we find target domains for specific protease activity. Proteases, such as the metalloproteinases of the ECM, are only expressed and secreted to the extracellular medium when the tissue needs to remodel its ECM.58 They act on specific sequences that are present only in the proteins of the ECM, so they cannot cause damage to other proteins in their vicinity. It is also known that some fragments of these hydrolyzed ECM proteins, once released, show strong bioactivity, which includes the promotion of cell differentiation, spreading and angiogenesis, among other activities. Finally, growing tissue is delicately controlled by a well performed symphony of growth factors and other bioactive substances that are secreted by the cells. This is the scenario that tissue meets when passing through the difficult processes of growing and regenerating. One can say now, and after

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understanding how cells work when they are attaching and spreading in a scaffold, that the general aim of tissue engineering is to design temporary functional scaffolds that mimic essential elements of the structural and functional state of a tissue. Lots of materials have been applied in the field of tissue engineering, like the petroleum-based polymers and lately natural based polymers. The results are being improved with the passing of time but until today, and due to a lack of total understanding how cells work, the ideal material has not been found. This material has to be highly compatible; present excellent mechanical properties and include bioactive peptide sequences. A good candidate arises from this need, the ELPs. These polymers, besides presenting these requirements, are also smart materials that respond to changes in their environment. As a pioneer group in the developing of ELPs, Urry’s group was the first to develop systems based on these polymers for tissue engineering. Soon after they discovered the extraordinary biocompatibility of the (VPGVG)based ELPs,20 they tested the capability of these materials as raw materials for designing new scaffolds. The first candidates were simple VPGVG polymers and their cross-linked matrices. Surprisingly, tests on the cross-linked poly(VPGVG) matrices showed that cells did not adhere at all to this matrix and no fibrous capsules forms around it when it was implanted.59 Accordingly, this matrix and other states of the material have potential use in prevention of post-operative, post-trauma adhesions.59 Restoring an injured tissue to its normal state in many cases demands a means to prevent adhesion; that is, preventing abnormal bands of connective tissue binding tissues together inappropriately.51 In one of their works, Urry and his co-workers showed that when placed between an injured abdominal wall and an injured loop of bowel, a sheet of the cross-linked poly(GVGVP) prevented significant adhesion formation in 80% of the cases.60 Other applications of this basic polymer are in strabismus surgery, in preventing adhesion between rectus muscle and sclera of the eye;61 and in spinal surgery to correct herniated intervertebral discs.62 The absolute lack of cell adherence of the poly(GVGVP) could be seen as a drawback in the intended use of this kind of material for tissue engineering, but it is not. This polymer is ideal as a starting material since it provides adequate mechanical properties and biocompatibility and lacks unspecific bioactivity. Very soon these simple molecules were enriched with short peptides having specific bioactivities. Due to the polypeptide nature of the ELPs, those active short peptides were easily inserted within the polymer sequence. The first active peptides inserted in the polymer chain were the well-known general-purpose cell adhesion peptide RGD (R = L-arginine, G = glycine, D = L-aspartic acid)63 and REDV (E = L-glutamic acid, V = L-valine), which is specific for endothelial cells.64,65 Using ELPs with the RGD sequence, Urry et al. developed an excellent

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material for urological prostheses and intervertebral-disc restoration. In the first case they found that when using a matrix of the bioactive ELP and stimulating the matrix by filling and emptying, a greater density of bladder cells and extracellular matrix was obtained.38,66 In the second case, the selected bioelastic material was used to restore the proper dimensions and swelling pressure by injection into a disc previously depleted of its pulpous nucleus. The bioactive sequence was included to induce tissue regeneration while the disc is in an improved structural-functional state.51 Another interesting application of bioactive ELPs with cell adhesion sequences is the developing of vascular grafts. Severe atherosclerosis often requires surgical removal of the affected tissue and implantation of an autologous or synthetic vascular graft.67 The most widely used materials in synthetic vascular grafts, when used in small-diameter grafts, are characterized by high failure rates due to thrombosis and intimal hyperplasia.68–70 Tirrel’s group designed new vascular grafts attending two criteria, (a) enhancement of endothelial cell adhesion and (b) tuning the elastic modulus of the material to match that of the affected artery.71 For this, they developed two new ELPs, one containing the CS5 domain from the fibronectin and the other containing the well known RGD sequence. In both cases they substituted some of the valines by lysines for cross-linking purposes. Comparative studies of these two polymers showed that the grafts with the RGD cell-binding domained bound endothelial cells more strongly and elicited a faster cell spreading than the CS5 cell-binding domain.67 Using these ELPs they overwhelmed the two main problems in the vascular graft failure; the difference between veins’ tensile modulus and synthetic grafts and also the absence of a confluent endothelial monolayer.67 We can find in the literature other interesting work with ELPs as a raw material in tissue engineering applications. For example, one with a big potential regarding future applications is the work developed by Chilkoti’s group with genetically engineered ELPs for cartilaginous tissue repair. Cartilaginous tissues, such as articular cartilage, the meniscus, and intervertebral disc, contribute to mechanical load support, load distribution, and flexibility in joints of the body.72 These kinds of tissues virtually enable self-repair because they are avascular with a very low cell density. Taking advantage of the unique and favourable feature of the ELPs to form mechanical functional constructs in situ and their liquid-like behaviour below the temperature transition, Chilkoti’s group developed a method where they mixed chondrocyte cells with the ELPs in a way to create an injectable gel.72 In their studies they also demonstrated the ability of elastin-like polymers polypeptide gels to induce and support the chondrocytic differentiation of human adipose tissuederived adult stems cells in vitro in the absence of exogenous chondrogenic supplements, by promoting the expression of cartilage-specific genes and the accumulation of cartilage-specific extracellular matrix.46 This way

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they showed that ELPs have unique advantages as scaffolds for cartilage repair. There are currently examples based on more complex designs that include various bioactivities and other functionalities in an effort to mimic the complex composition and function of the natural extracellular matrix (ECM). Girotti et al. have bioproduced the ELP polymer depicted in Fig. 14.4.65 This ELP is made from a monomer 87 amino acids in length and has been produced with n = 10 (TE20109) (molecular mass 80.695 kDa). The monomer contains four different functional domains in order to achieve an adequate balance of mechanical and bioactive responses. First, the final matrix is designed to show a mechanical response comparable to the natural ECM, so that the matrix is produced over a base of an ELP of the type (VPGIG)n. This basic sequence assures the desired mechanical behaviour and outstanding biocompatibility. In addition, this basic composition endows the final polymer with smart and self-assembling capabilities, which are of high interest in the most advanced tissue engineering developments. The second building block is a variation of the first. It has a lysine substituting the isoleucine, so the lysine γ-amino group can be used for cross-linking purposes while retaining the properties of elastin-like polymers. The third domain is the CS5 human fibronectin domain. This contains the well-known endothelial cell attachment sequence, REDV, immersed in its natural sequence to potentiate its efficiency. Finally, the polymer also contains elastase target sequences to favour its bioprocessability by natural routes. The chosen elastase target sequence is the hexapeptide VGVAPG, which is found in natural elastin. The presence of this specific sequence in the artificial polymer guarantees that the polymer will be bioprocessed only when the growing tissue decides that it is time to substitute it by a natural ECM, while, in practice, it remains fully functional until that time. In addition, the activity of this domain is not restricted to being an inert target of protease activity. It is well known that these hexapeptides, as they are released by the protease action, have strong cell proliferation activity and other bioactivities related to tissue repair and healing.

n [(VPGIG)2–(VPGKG)–(VPGIG)2–(EEIQIGHIPREDVDYHLYP)–(VPGIG)2(VPGKG)–(VPGIG)2(VGVAPG)3]N

14.4 A schematic composition of the monomer of the ELP design described. The scheme shows the different functional domains of the monomer, which can be easily identified with their corresponding peptide sequences.

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14.12 Self-assembling properties of ELPs for tissue engineering One crucial issue in the interaction between cells and materials is the nanometric level. This interaction is essentially an interfacial interaction, in which the properties of the material surface play the key role. It is well known that certain topological features at the nanometric level can affect the performance of a given material; therefore, this phenomenon must be addressed and controlled. The surface self-assembly displayed by simple ELPs can also be present in more complex polymers such as those designed for tissue engineering. For example, this is the case for the ELP TE20109 shown previously. In this polymer, there are both predominantly apolar domains, such as those containing peptides (VPGIG) and (VGVAPG), and other domains, which can change their polarity by changing the pH, VPGKG and EEIQIGHIPREDVDYHLYP. Deposition of this polymer on an adequate substrate and at a suitable pH leads to a spontaneous nanostructuration of the polymer surface. This can be seen in Figure 14.5a-c.

14.13 Processability of ELPs for tissue engineering Of course, those protein-based materials can also be processed as conventional tissue engineering materials. Several examples can be found in the literature. The way in which matrices and hydrogels from these polymers can be prepared is diverse; for example, by γ-irradiation4 or by placing specific amino acids with functional groups for easy further cross-linking.4,19, 73–75 For instance, by using the free γ-amino group of lysine and hexamethylene diisocyanate as a cross-linker, the polymer His-Tag-{[(VPGIG)2-VPGKG-(VPGIG)2AVTGRGDSPASS-(VPGIG)2-VPGKG-(VPGIG)2]6} (TE20211), forms stable hydrogels (Figure 14.6).76

730 nm (a)

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14.5 Atomic force microscope images of the deposition of a polymer for tissue engineering with different conditions. (a) Polymer solution at pH 7 deposited on hydrophobic silicon substrate. (b) Polymer solution at pH 12 deposited on hydrophobic silicone substrate. (c) Polymer solution at pH 12 deposited on hydrophilic mica.

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14.6 Cross-linked matrix of the RGD-containing polymer TE20211. Reproduced with permission from Nanomedicine.30

Additionally, in this cross-linked state, the polymer still retains its smart nature. In this sense, the matrix tends to hydrate by hydrophobic hydration below its Tt while it shrinks and de-swells above Tt . Interestingly, in the swollen state (below Tt), the weight of the hydrogel shown in Figure 14.6 is approximately 2 g, and only 20 mg of polymer have been used to form the matrix. Therefore, the ELPs can be considered as exceptional superabsorbents. Also, the ITT can still be observed by DSC. Of course, the microstructures and properties are different in both states. Below Tt, the matrix shows a honeycomb porous structure that is lost at temperatures above Tt (Figure 14.7). That greatly affects, among many other properties, its mechanical and diffusion properties. Thinking in terms of 3D tissue engineering, the porous structure found at temperatures below Tt is not wide enough to permit the cells to colonize the interior of the matrix. However, many well established techniques for the formation of pores can be used to achieve a more convenient porous scaffold. For example, by using dimethylformamide (DMF) as the solvent during cross-linking, a salt (NaCl) leaching method can be used to obtain adequate porous scaffolds (Figure 14.8).30

14.14 Future trends The conjugation of nanotechnological concepts and the know-how of biological processes leads us to a step where we are able to understand the bases of the

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10 µm

20 µm (a)

(b)

14.7 Scanning electron microscope micrographs of a TE20211 crosslinked hydrogel (a) above 37°C and (b) below 15°C, its Tt. Reproduced with permission from Nanomedicine.30

20 µm

14.8 Scanning electron micrograph of a TE20211 cross-linked porous hydrogel obtained by SALT leaching as explained in the text. Reproduced with permission from Nanomedicine.30

design of new materials. Although nanobiotechnology is in its childhood, the current work is laying the foundations for the future on materials science in tissue engineering and other areas. Since the potential of genetic-engineering techniques has not been fully exploited we can look at this tool as unlimited. The design of new materials is getting closer and closer to the performance

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of materials and molecular machines found in nature. Due to the close collaboration between biology, medicine and nanotechnology, we can now assume that in the near future we will be witnesses to the appearance of a new generation of biomaterials with astonishing properties, as they will progressively integrate the new concepts that come out every day in the most diverse areas. Taking into account all the recent applications described previously and their interesting and unconventional properties, the elastin-like polymers will play a key role in this new generation of materials.

14.15 References 1 Langer R and Tirrel D A (2004), Designing materials for biology and medicine, Nature, 428, 487–492. 2 Rodriguez-Cabello J C, Prieto S, Reguera J, Arias F J and Ribeiro A (2007), Biofunctional design of elastin-like polymers for advanced applications in nanotechnology, J Biomater Sci Polymer Edn, 18(3), 269–286. 3 Rodríguez-Cabello J C, Reguera J, Girotti A, Arias F J and Alonso M (2006), Genetic Engineering of Protein-Based Polymers: The Example of Elastin-like Polymers, Adv Polym Sci, 200, 119–167. 4 Lee J, Macosko C W and Urry D W (2001), Elastomeric polypentapeptides crosslinked into matrixes and fibers, Biomacromolecules, 2, 170–179. 5 Chilkoti A, Dreher M R and Meyer D E (2002), Design of thermally responsive, recombinant polypeptide carriers for targeted drug delivery, Advanced Drug Delivery Reviews, 54, 1093–1111. 6 Gosline J M, Guerette P A, Ortlepp C S and Savage K N (1999), The mechanical design of spider silks: from fibroin sequence to mechanical function, J Exp Biol, 202, 3295–3303. 7 Yu Y B (2002), Coiled-coils: stability, specificity, and drug delivery potential, Adv Drug Deliv Rev, 54, 1113–1129. 8 Bilgicer B, Fichera A and Kumar K (2001), A coiled coil with a fluorous core, J Am Chem Soc, 123, 4393–4399. 9 Potekhin S A, Medvedkin V N, Kashparov I A and Venyaminov S Y (1994), Synthesis and properties of the peptide corresponding to the mutant form of the leucine zipper of the transcriptional activator GCN4 from yeast, Protein Eng, 7, 1097–1101. 10 Elvin C M, Carr A G, Huson M G, Maxwell J M, Pearson R D, Vuocolo T, Liyou N E, Wong D C C, Merritt D J and Dixon N E (2005), Synthesis and properties of crosslinked recombinant pro-resilin, Nature, 437, 999–1113. 11 Bochicchio B, Jimenez-Oronoz F, Pepe A, Blanco M, Sandberg L B and Tamburro A M (2005), Synthesis of and structural studies on repeating sequences of abductin, Macromolecular Bioscience, 5, 502–511. 12 Shewry P R, Halford N G, Belton P S and Tatham A S (2002), The structure and properties of gluten: an elastic protein from wheat grain, Philos. Trans R Soc Lond Ser B: Biol Sci, 357, 133–142. 13 Sandberg L B, Leslie J G, Leach C T, Torres V L, Smith A R and Smith D W (1985), Elastin covalent structure as determined by solid-phase amino-acid sequencing, Pathol, Biol, 33, 266–274.

392

Natural-based polymers for biomedical applications

14 Yeh H, Ornstein-Goldstein N, Indik Z, Sheppard P, Anderson N, Rosenbloom J C, Cicilia G C, Yoon K and Rosenbloom J (1987), Sequence variation of bovine elastin mRNA due to alternative splicing, Collagen Rel Res, 7(4), 235–247. 15 Sandberg L B, Soskel N T and Leslie J G (1981), Elastin structure, biosynthesis, and relation to disease states, New Engl J Med, 304(10), 566–579. 16 Indik Z, Yeh H, Ornstein-Goldstein N, Sheppard P, Anderson N, Peltonen L, Rosenbloom J C and Rosenbloom J (1987), Alternative splicing of human elastin mRNA indicated by sequence analysis of cloned genomic and complementary DNA, Proc Natl Acad Sci, 84, 5680–5684. 17 Ayad S, Humphries M, Boot-Handford R, Kadler K and Shuttleworth A (1994), The Extracellular Matrix Facts Book, San Diego, CA, Academic Press. 18 Urry D W, Luan C H, Harris C M and Parker T (1997), ‘Protein-based materials with a profound range of properties and applications: the elastin Tt hydrophobic paradigm’, in McGrath K, Kaplan D, Protein Based Materials, Boston, MA, Birkhauser, 133– 177. 19 Di Zio K and Tirrell D A (2003), Mechanical properties of artificial protein matrices engineered for control of cell and tissue behavior, Macromolecules, 36, 1553–1558. 20 Urry D W, Parker T M, Reid M C and Gowda D C (1991), Biocompatibility of the Bioelastic Materials, Poly(GVGVP) and Its Gamma-Irradiation Cross-Linked Matrix – Summary of Generic Biological Test-Results, J Biact Compat Polym, 6, 263–282. 21 Gowda D C, Parker T M, Harris R D and Urry D W (1994), in Peptides: Design, Synthesis and Biological Activity, Basava C and Anantharamaiah G M (Eds), Boston M A, Birkhäuser, p 81. 22 Martino M, Perri T and Tamburro A M (2002), Biopolymers and biomaterials based on elastomeric proteins, Macromol Biosci, 2, 319–328. 23 Urry D W (1993), Molecular machines: how motion and other functions of living organisms can result from reversible chemical changes, Angew Chem Int Edn, 32, 819–841. 24 San Biagio P L, Madonia F, Trapane T L and Urry D W (1988), Overlap of elastomeric polypeptide coils in solution required for single phase initiation of elastogenesis, Chem Phys Lett, 145, 571–574. 25 Urry D W (1997), Physical chemistry of biological free energy transduction as demonstrated by elastic protein-based polymers, J Phys Chem B, 101, 11007–11028. 26 Rodríguez-Cabello J C, Alonso M, Perez T and Herguedas M M (2000), Differential scanning calorimetry study of the hydrophobic hydration of the elastin-based polypentapeptide, poly(VPGVG), from deficiency to excess of water, Biopolymers, 54(4), 282–288. 27 Pauling L and Marsh R E (1952), The structure of chlorine hydrate, Proc Natl Acad Sci USA, 38, 112–118. 28 Urry D W, Trapane T L and Prasad K U (1985), Phase-structure transitions of the elastin polypentapeptide-water system within the framework of compositiontemperature studies, Biopolymers, 24, 2345–2356. 29 Manno M, Emanuele A, Martorana V, San Biagio P L, Bulone D, Palma-Vittorelli M B, McPherson D T, Xu J, Parker T M and Urry D W (2001), Interaction of processes on different length scales in a bioelastomer capable of performing energy conversion, Biopolymers, 59, 51–64. 30 Rodriguez-Cabello J C, Prieto S, Arias F J, Reguera J and Ribeiro A (2006), Nanobiotechnological approach to engineered biomaterial design: the example of elastin-like polymers, Nanomedicine, 1(3), 267–280.

Elastin-like systems for tissue engineering

393

31 Drexler K E (1981), Molecular engineering: An approach to the development of general capabilities for molecular manipulation, Proc Natl Acad Sci USA, 78, 5275– 5278. 32 Rajagopal K and Schneider J P (2004), Self-assembling peptides and proteins for nanotechnological applications, Curr Opin Struct Biol, 14, 480–486. 33 Miao M, Cirulis J T, Lee S and Keeley F W (2005), Structural Determinants of Cross-linking and Hydrophobic Domains for Self-Assembly of Elastin-like Polypeptides, Biochemistry, 44, 14367–14375. 34 Yang G C, Woodhouse K A and Yip C M (2002), Substrate-Facilitated Assembly of Elastin-Like Peptides: Studies by Variable-Temperature in Situ Atomic Force Microscopy, J Am Chem. Soc, 124, 10648–10649. 35 Wright E R and Conticello V P (2002), Self-assembly of block copolymers derived from elastin-mimetic polypeptide sequences, Adv Drug Deliv Rev, 54, 1057–1073. 36 Lee T A T, Cooper A, Apkarian R P and Conticello V P (2000), Thermo-reversible self-assembly of nanoparticles derived from elastin-mimetic Polypeptides, Adv Mater, 12, 1105–1110. 37 Reguera J, Fahmi A, Moriarty P, Girotti A and Rodríguez-Cabello J C (2004), Nanopore formation by self-assembly of the model genetically engineered elastin-like polymer [(VPGVG)2(VPGEG)(VPGVG)2]15, J Am Chem Soc, 126, 13212– 13213. 38 Urry D W et al. (1997), in Domb A, Kost J and Wiseman D, Handbook of Biodegradable Polymers, Chur, Harwood Academic Publishers, 367–386. 39 Urry D W (2005), What Sustains Life? Consilient Mechanism for Protein-based Machines and Materials, NY, Springer-Verlag. 40 Meyer D E, Kong G A, Dewhirst M W, Zalutsky M R and Chilkoti A (2001), Targeting a genetically engineered elastin-like polypeptide to solid tumors by local hyperthermia. Cancer Res, 61(4), 1548–1554. 41 Chung J E, Yokoyama M, Yamato M, Aoyagi T, Sakurai Y and Okano T (1999), Thermo-responsive drug delivery from polymeric micelles constructed using block copolymers of poly(n-isopropylacrylamide) and poly(butylmethacrylate), J Controlled Release, 62(1-2), 115–127. 42 Chung J E, Yokoyama M, Aoyagi T, Sakurai Y and Okano T (1998), Effect of molecular architecture of hydrophobically modified poly(n-isopropylacrylamide) on the formation of thermoresponsive core-shell micellar drug carriers, J Controlled Release, 53, 119–130. 43 Urry D W, Gowda D C, Harris C M and Harris R D (1994), ‘Bioelastic materials and the ∆Tt-Mechanism in drug delivery’, in: Ottenbrite R M, Polymeric Drugs and Drug Administration, Washington DC, CRC Press, 15. 44 Urry D W (1990), Preprogrammed drug delivery systems using chemical triggers for drug release by mechanochemical coupling, Polym Mater Sci Eng, 63, 329–336. 45 Herrero-Vanrell R, Rincón A C, Alonso M, Reboto V, Molina-Martinez I T and Rodriguez-Cabello J C (2005), Self-assembled particles of an elastin-like polymer as vehicles for controlled drug release, J Control Rel, 102(1), 113–122. 46 Betre H, Ong S R, Guilak F, Chilkoti A, Fermor B and Setton L A, (2006), Chondrocytic differentiation of human adipose-derived adult stem cells in elastin-like polypeptide, Biomaterials, 27, 91–99. 47 Chilkoti A, Christensen T and MacKay J A (2006), Stimulus responsive elastin biopolymers: applications in medicine and biotechnology, Current Opinion in Chemical Biology, 10, 1–6.

394

Natural-based polymers for biomedical applications

48 Megeed Z, Haider M, Li D, O’Malley J B W, Cappello J and Ghandehari H (2001), In vitro and in vivo evaluation of recombinant silk-elastinlike hydrogels for cancer gene therapy, J Control Release, 94, 433–445. 49 Chilkoti A, Dreher M R, Meyer D E and Raucher D (2002), Targeted drug delivery by thermally responsive polymers, Advanced Drug Delivery Reviews, 54, 613–630. 50 Meyer D E, Shin B C, Kong G A, Dewhirst M W and Chilkoti A (2001), Drug targeting using thermally responsive polymers and local hyperthermia, Journal of Controlled Release, 74, 213–224. 51 Urry D W (1999), Elastic molecular machines in metabolism and soft tissue restoration, TIBTECH, 17, 249–257. 52 Kleinman H K, Philp D and Hoffman M (2003), Role of the extracellular matrix in morphogenesis, Curr Opin Biotechnol, 14, 526–532. 53 Hubbell J A (2003), Materials as morphogenetic guides in tissue engineering, Curr Opin Biotechnol, 14, 551–558. 54 Maskarinec S A and Tirrell D A (2005), Protein engineering approaches to biomaterials design, Curr Opin in Biotechnol, 16, 1–5. 55 Griffith L G and Naughton G (2002), Tissue engineering – current challenges and expanding opportunities, Science, 295, 1009–1014. 56 Hirano Y and Mooney D J (2004), Peptide and protein presenting materials for tissue engineering, Adv Mater, 16(1), 17–25. 57 Sakiyama-Elbert S E and Hubbell J A (2001), Functional biomaterials: design of novel biomaterials, Annu Rev Mater Res, 31, 183–201. 58 Sternlicht M D and Werb Z (2001), How matrix metalloproteinases regulate cell behavior, Annu Rev Cell Dev Biol, 17, 463–516. 59 Urry D W, Nicol A, Gowda D C, Hoban L D, McKee A and Williams T (1993), ‘Medical applications of bioelastic materials’ in: Gebelein C G, Biotechnological Polymers: Medical, Pharmaceutical and Industrial Applications, Atlanta, GA: Technomic Publishing Co. Inc., 82–103. 60 Hoban L D, Pierce M, Quance J, Hayward McKee A and Gowda D C (1994), The use of polypenta-peptides of elastin in the prevention of postoperative adhesions, J Surg Res, 56, 179–183 61 Elsas F J, Gowda D C and Urry D W (1992), Synthetic polypeptide sleeve for strabismus surgery, J Pediatr Ophthalmol Strabismus, 29, 284–286. 62 Alkalay R N, Kim D H, Urry D W, Xu J, Parker T M and Glazer P A (2003), Prevention of post-laminectomy epidural fibrosis using bioelastic materials, Spine, 28, 1659–1665. 63 Urry D W, Pattanaik A, Xu J, Woods T C, McPherson D T and Parker T M (1998), Elastic protein-based polymers in soft tissue augumentation and generation, Journal of Biomaterials Science-Polymer Edition, 9(10), 1015–1048. 64 Panitch A, Yamaoka T, Fournier M J, Mason T L, Tirrell D A (1999), Design and biosynthesis of elastin-like artificial extracellular matrix proteins containing periodically spaced fibronectin CS5 domains, Macromolecules, 32, 1701–1703. 65 Girotti A, Reguera J, Rodríguez-Cabello J C, Arias F J, Alonso M and Testera A M (2004), Design and bioproduction of a recombinant multi(bio)functional elastin-like polymer containing cell adhesion sequences for tissue engineering purposes, J Mater Sci- Mater Med, 15, 479–484. 66 Urry D W and Pattanaik A (1997), Elastic protein-based materials in tissue reconstruction, Ann New York Acad Sci, 831, 32–46. 67 Liu J C, Heilshorn S C and Tirrell D A (2004), Comparative cell response to artificial

Elastin-like systems for tissue engineering

68 69

70 71 72

73

74

75

76

395

extracellular matrix proteins containing the RGD and CS5 cell-binding domains, Biomacromolecules, 5, 497–504. Nugent H M and Edelman E R (2003), Tissue engineering therapy for cardiovascular disease, Circ Res, 92, 1068–1078. Salacinski H J, Tiwari A, Hamilton G and Seifalian A M (2001), Cellular engineering of vascular bypass grafts: role of chemical coatings for enhancing endothelial cell attachment, Med Biol Eng Comput, 39, 609–618. Bos G W, Poot A A, Beugeling T, van Aken W G and Feijen J (1998), Small-diameter vascular graft prostheses: current status, Arch Physiol Biochem, 106, 100–115. Heilshorn S C, Liu J C and Tirrell D A (2005), Cell-binding domain context affects cell behavior on engineered proteins, Biomacromolecules, 6, 318–323. Betrea H, Setton L A, Meyer D E and Chilkoti A (2002), Characterization of a genetically engineered elastin-like polypeptide for cartilaginous tissue repair, Biomacromolecules, 3, 910–916. Welsh E R and Tirrel D A (2000), Engineering the extracellular matrix: a novel approach to polymeic biomaterials.I. Control of the physical properties of artificial protein matrices designed to support adhesion of vascular endothelial cells, Biomacromolecules, 1, 23–30. Trabbi-Carlson K, Setton L A and Chilkoti A (2003), Swelling and mechanical behaviors of chemically cross-linked hydrogels of elastin-like polypeptides, Biomacromolecules, 4, 572–580. McMillan R A, Caran K L, Apkarian R P and Conticello V P (1999) High-resolution topographic imaging of environmentally responsive elastin-mimetic hydrogels. Macromolecules, 32, 9067–9070. Prieto S, Espirito-Santo V, Alonso M, Testera A M, Mano J and Rodriguez-Cabello J C, Physical properties of artificial extracellular matrix of a cross-linked elastin-like polymer designed for tissue engineering purposes. (in preparation).