Acta Biomaterialia 10 (2014) 1751–1760

Contents lists available at ScienceDirect

Acta Biomaterialia

journal homepage: www.elsevier .com/locate /actabiomat

Review

Designing ECM-mimetic materials using protein engineering q

1742-7061/$ - see front matter � 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.actbio.2013.12.028

q Part of the Special Issue on Biological Materials, edited by Professors Thomas H.Barker and Sarah C. Heilshorn.⇑ Corresponding author. Mailing address: 476 Lomita Mall, McCullough Room

246, Stanford University, Stanford, CA 94305-4045, USA. Tel.: +1 650 723 3763;fax: +1 650 498 5596.

E-mail address: heilshorn@stanford.edu (S.C. Heilshorn).

Lei Cai, Sarah C. Heilshorn ⇑Department of Materials Science and Engineering, Stanford University, Stanford, CA 94305, USA

a r t i c l e i n f o a b s t r a c t

Article history:Available online 21 December 2013

Keywords:Protein engineeringExtracellular matrixTissue engineeringRecombinant protein synthesisThree-dimensional hydrogels

The natural extracellular matrix (ECM), with its multitude of evolved cell-instructive and cell-responsiveproperties, provides inspiration and guidelines for the design of engineered biomaterials. One strategy tocreate ECM-mimetic materials is the modular design of protein-based engineered ECM (eECM) scaffolds.This modular design strategy involves combining multiple protein domains with different functionalitiesinto a single, modular polymer sequence, resulting in a multifunctional matrix with independent tunabil-ity of the individual domain functions. These eECMs often enable decoupled control over multiple mate-rial properties for fundamental studies of cell–matrix interactions. In addition, since the eECMs arefrequently composed entirely of bioresorbable amino acids, these matrices have immense clinical poten-tial for a variety of regenerative medicine applications. This brief review demonstrates how fundamentalknowledge gained from structure–function studies of native proteins can be exploited in the design ofnovel protein-engineered biomaterials. While the field of protein-engineered biomaterials has existedfor over 20 years, the community is only now beginning to fully explore the diversity of functional pep-tide modules that can be incorporated into these materials. We have chosen to highlight recent examplesthat either (i) demonstrate exemplary use as matrices with cell-instructive and cell-responsive propertiesor (ii) demonstrate outstanding creativity in terms of novel molecular-level design and macro-levelfunctionality.

� 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction: the rationale for creating engineeredextracellular matrices

The native extracellular matrix (ECM) is a complex and hetero-geneous material containing numerous multifunctional proteinsthat provide cells with structural support and biochemical signalsto facilitate a series of important cellular processes [1]. First, theECM presents integrin-specific ligands and binding affinity to othercell-surface receptors that combine to initiate cell adhesion. Sec-ond, through complex and dynamic interactions with cells, the ma-trix provides a three-dimensional (3D) mechanical framework.These biochemical and biomechanical cues activate highly regu-lated signaling pathways that allow for ensuing cellular responsessuch as spreading, migration, proliferation, and differentiation.

In order to mimic these important functions of native ECM inthe design of biomaterial scaffolds, the strategy of using isolatedECM components (collagen, fibronectin, etc.) or their mixtures har-vested from tissues has been widely adopted [2]. Although oftenhighly cell adhesive, these biomaterial scaffolds have proved

challenging to standardize and use for in vivo applications due topoorly defined chemical structure, inconsistent batch-to-batchreproducibility and risk of immunogenicity. In addition, it is extre-mely difficult to manipulate and customize the ECM scaffolds for aspecific cellular microenvironment or to study fundamental as-pects of cell–material interactions, because all material factorsare intertwined and coupled together, resulting in largely observa-tion-based outcomes.

Motivated to design tunable biomaterials that emulate the na-tive ECM, researchers have been developing engineered ECM(eECM) that combines multiple structural and biofunctional fea-tures [3,4]. Using recombinant protein technologies, eECM offersenormous possibilities in the design of reproducible, highly tunable,and modular protein scaffolds [5–9]. The four major advantages ofcreating eECM using protein engineering are: (i) to gain better con-trol over decoupled material variables for mechanistic studies ofcell–matrix interactions; (ii) to achieve more physiologically rele-vant in vitro cultures; (iii) to create more reproducible materialsfor clinical therapies; and (iv) to create more complex and dynamicmaterials with multifunctionality, responsiveness and bioactivity.These four advantages are discussed in more detail below.

Towards goal 1, eECM can be customized to have consistentmaterial properties with only one variable factor of interest, suchas cell-adhesive ligand density, matrix compliance, structural for-mation and cell-instructive biochemical signals. For example,

1752 L. Cai, S.C. Heilshorn / Acta Biomaterialia 10 (2014) 1751–1760

elastin-like protein (ELP) hydrogels have been designed with eithera cell-adhesive arginine–glycine–aspartic acid (RGD) ligand ornon-adhesive, sequence-scrambled RDG in their otherwise identi-cal primary amino acid sequences [10]. Thus, blending these twoengineered proteins together prior to crosslinking into a bulkhydrogel affords direct control over the bioactive ligand density.Independently, the matrix stiffness of these hydrogels can be tunedby altering the density of crosslinks [11]. This system has beenused to evaluate the independent effects of RGD ligand densityand matrix stiffness on neurite outgrowth from 3D cultures of dor-sal root ganglia [12].

Towards goal 2, once synthesized, eECM proteins can be fabri-cated through a variety of techniques to create matrices that mimiccertain structural features of the native ECM. These material struc-tures include 2D surface patterning at the micro- and nanoscale[13], 3D hydrogels [12,14], porous scaffolds [15] and fibrous struc-tures [16]. The eECM can then be seeded with cells to create either2D or 3D cultures that recapitulate aspects of the cell niche andproduce cell responses distinct from standard 2D tissue culturepolystyrene with ECM coatings. These in vitro cultures may resultin cell morphologies and levels of gene expression that are morereminiscent of in vivo tissue.

Towards the creation of consistent materials for clinical thera-pies, protein engineering offers a highly reproducible syntheticstrategy. Because of the high fidelity of protein translation, recom-binant proteins present precisely controlled, monodispersed se-quences and biochemical compositions at the molecular level, afeature that is normally improbable in natural or synthetic materi-als [17]. In addition to reproducibility and customizability, eECM isalso biodegradable and yields non-toxic degradation products,which is desirable for clinical usage.

Towards goal 4, the modular design strategy of eECM enablesdirect incorporation of diverse peptide building blocks into thebackbone of a single protein sequence. This modular approach re-sults in the synthesis of multifunctional materials that combine thefunctionality of each individual peptide domain. For example, no-vel, protein-engineered, cell-delivery vehicles have been developedusing several peptide-based gelation mechanisms, includingleucine-zipper self-assembly [18–20], enzyme-triggered self-assembly [21,22], chemical crosslinking [11,12,14,23] andheteroassembly of molecular recognition peptides [24–27]. Inaddition to these domains that enable structural gelation, ECM-mimetic domains that are either cell-instructive or cell-responsivecan be included. Examples include cell-adhesive [28–32],growth factor mimetic [33–38] or enzyme-degradable domains[11,39–41]. Finally, more complex designs can be achieved byadding functional domains that interact with inorganic materials[42–45] or respond to dynamic environmental stimuli [46–48].

In this review, we describe the toolbox that is currently used togenerate protein-engineered eECM biomaterials in Section 2. Wefocus our attention on eECM fabricated purely from protein-engi-neered materials using canonical amino acids. In Section 3, we de-scribe the wide selection of peptide building blocks and domainsavailable for the modular design of eECM, with an emphasis oninteractions with mammalian cells. Finally, we discuss emergingnew functionalities and peptide modules for eECM design in Sec-tion 4, including binding domains with inorganic materials, anti-microbial peptides, immunomodulatory peptides and dynamicpeptides triggered by environmental stimuli.

2. Toolbox to design and synthesize eECM

2.1. Direct peptide synthesis versus recombinant protein synthesis

As protein technologies continue to evolve, there are now sev-eral toolkits available to achieve reproducible and tunable eECM

with virtually limitless possibilities. To design a multifunctionaleECM, the first step is the selection of primary amino acid se-quences with desirable structural, gelation, degradation or bioac-tive roles. Once the desired amino acid sequence has beendesigned, two common techniques can be used to synthesize theeECM: direct chemical synthesis of small peptide building blocksor recombinant biochemical synthesis of large proteins (Fig. 1). Di-rect peptide synthesis is usually realized through solid-phase pep-tide synthesis (SPPS) using a peptide synthesizer. The advent ofpeptide synthesizers allows for automatic and efficient productionof up to 30–50 amino acid residues in sub-gram quantities at rea-sonable cost [49]. These small peptides can be ligated together tocreate much longer sequences of up to 150 amino acids [50]. SPPSensures precise chemical structures and enables the addition ofmodifications to the functional groups of individual amino acidsduring synthesis. Liquid-phase peptide synthesis (LPPS) is a classi-cal method that requires sequential incorporation of amino acidsfollowed by removal of protecting groups [51]. LPPS is still com-monly used for large-scale production of gram-scale quantities ofa given peptide, although this synthetic route is much slowerand more labor intensive than SPPS.

Recombinant protein synthesis can produce much larger pro-teins with more complex structural features, resulting in morecomplex functionalities. This biochemical synthesis strategy re-quires several genetic engineering steps, each of which may re-quire optimization to achieve efficient yields of functionalprotein. First, a DNA template that encodes the target amino acidsequence is designed and chemically synthesized. This engineeredgene is then inserted into a plasmid vector that enables gene rep-lication and transcription. The vector is transformed into a hostorganism that expresses the target protein. The target protein isthen harvested and purified from other endogenous proteins andcontaminants. This templated synthesis offers precise control oflong protein sequences with multiple functional modules each pre-sented at a specific location, facilitating independent tuning ofmechanical properties and bioactivity within the final biomaterial.

2.2. Protein engineering strategies for designing eECM

The most common strategy for designing engineered proteinbiomaterials with multifunctionality is to first design individualpeptide domains and then to mix and match these peptide do-mains to create a versatile family of ECM-mimetic materials. Thismodular design strategy can use peptide domains derived from na-tive sequences, predicted through computational modeling oridentified by high-throughput combinatorial screening. Commonpeptide modules that are rationally derived from native sequencesinclude silk-like [52], elastin-like [53] and resilin-like [54] do-mains. Protein sequences based on these peptide modules oftendisplay properties reminiscent of their native proteins. Rationalmodification of the native sequences is often performed usingsite-specific amino acid mutation, which requires knowledge ofthe underlying protein structure–function relationship and identi-fication of appropriate target sites for mutation [55].

To better choose sites for mutation and to predict the resultantproperties, many computational algorithms have been developedthat employ physics-based modeling to estimate energetic proteininteractions. As an example, the WW domain, a small protein inter-action module that undergoes heteroassembly with a proline-richpeptide sequence, has been engineered using a computer-basedmodel to construct an artificial sequence that functions like its nat-ural counterpart [56,57]. This molecular recognition has been fur-ther utilized to create physical hydrogels that encapsulate cellswithout environmental triggers [24]. A new computational methodhas recently been used to design self-assembling proteins withhigh accuracy. This method includes a symmetrical docking of

Fig. 1. Schematic of peptide and recombinant protein synthesis to construct eECM biomaterials. Peptide synthesis is conducted by either solid- or liquid-phase synthesis thatresults in short peptide chains for self-assembly or conjugation into eECMs. Recombinant protein synthesis involves the modular design of peptide modules derived fromnative sequences, predicted through computational modeling or identified by high-throughput combinatorial screening. These modules are concatenated and translated intoa DNA template, which is then cloned into a genetic vector and transformed into the host cell of choice, where production of the engineered protein occurs. After purification,the target protein is further processed via self-assembly, crosslinking or electrospining into 3D eECM biomaterials.

L. Cai, S.C. Heilshorn / Acta Biomaterialia 10 (2014) 1751–1760 1753

peptides in a target with symmetric architecture and a subsequentdesign of low-energy, protein–protein interfaces to drive self-assembly [58]. Besides rational or computational engineering ofnative sequences, combinatorial engineering using high-through-put screening methods provides a powerful alternative to generat-ing proteins with complex properties. Also known as directedevolution, this technology involves first creating a diverse geneand corresponding protein library, then screening and identifyingmutants with the desired properties [11,42,59–61]. For example,combinatorial engineering has been used to identify clones withhigh affinity to specific integrin receptors [60]. It is also a widelyused method to identify peptides or proteins capable of bindingto target inorganic material surfaces, such as hydroxyapatite [42].

3. Protein modules to create eECMs

3.1. Structural domains for cell-compatible encapsulation

The formation of 3D ECM-mimetic matrices entrapping viablecells is a crucial step for functional tissue engineering [62]. To con-struct a 3D structure for cell encapsulation, a crosslinking mecha-nism is often needed for eECM materials. Current state-of-the-arttechniques can be classified into two main categories: physicalcrosslinking, which includes self-assembly and molecular recogni-tion; and chemical crosslinking, which typically utilizes primaryamine and thiol groups to form covalent bonds between proteinpolymer chains. A combination of both crosslinking mechanismshas also been used to design hydrogels for potential cell encapsu-lation [63]. Physical hydrogels are generally endowed with shear-thinning and self-healing properties via reversible sol–gel transi-tions, but they are subject to quick erosion and low mechanicalmoduli. In comparison, chemical hydrogels are typically muchmore stable in situ and possess higher mechanical rigidity,although special attention must be made to developing a cell-com-patible crosslinking chemistry.

One classic example of a self-assembly module is the leucinezipper domain, which enables reversible self-assembly throughcoiled-coil associations [64]. This motif contains hydrophobic leu-cine residues and charged residues that form amphiphilic a-helicalstructures that multimerize into coiled-coils as junction points inthe engineered hydrogel network. The gelation process can bereversibly triggered by external stimuli such as temperature, pHand ionic strength [19]. These injectable hydrogels display rapidrecovery after injection, and more than 95% of seeded cells survivethe injection process [65]. These cytocompatible leucine zipper

hydrogels were modified with RGD cell-binding ligands to promoteadhesion, spreading and polarization of human fibroblast cells,which remained rounded on unmodified hydrogels [20]. Whenpresented as 2D substrates, these materials promote the viabilityand proliferation of human fibroblasts, human umbilical veinendothelial cells and rat neural stem cells [66]. One potentialdrawback to the use of leucine zipper hydrogels is their stabilityin biological environments, as they erode when placed in contactwith cell culture medium. However, leucine zipper domains canbe stabilized to inhibit dissolution by subsequent chemical cross-linking using end-linked cysteine residues that form disulfidecovalent bonds [18].

Molecular recognition is another important motif for creatingphysical hydrogels. In one example, a mixing-induced two-compo-nent hydrogel (MITCH) system, where one component is a blockcopolymer containing multiple repeats of the WW domain thatspecifically recognize proline-rich peptide domains encoded in asecond component, the two components undergo a sol–gel transi-tion by forming physical associations upon mixing without theneed for any external environmental triggers [24,67]. The resultinghydrogel is shear-thinning, injectable and able to self-heal, facili-tating the encapsulation of neural stem cells and supporting theirgrowth and differentiation [24]. In addition, MITCH has been usedas a stem cell delivery vehicle for adipose-derived stem cellsin vivo in a subcutaneous mouse model. MITCH delivery resultedin higher levels of cell survival and retention compared to othercommon hydrogels, such as collagen and alginate [68] (Fig. 2).

The design of reversible hydrogels was also demonstrated usingmolecular recognition between a protein and a peptide conjugatedto a multi-arm polyethylene glycol (PEG) spacer. For example, a re-combinant tax-interactive protein-1 (TIP1) with a PDZ domain wasfused to each end of the triangular trimeric CutA protein. Uponmixing with PDZ-binding peptides conjugated to PEG, a 3D physi-cal gel was formed at physiological pH and temperature, entrap-ping chondrocytes with high viability [69]. TIP1 was furthermodified into a fusion protein with four binding sites and mixedwith self-assembled nanofibers formed by TIP1 binding polypep-tides, leading to the formation of molecular hydrogels [27]. Simi-larly, a physical hydrogel design was created based on astructural tetratricopeptide repeat (TPR) that recognizes TPR-bind-ing peptide modules conjugated to PEG. These two componentsform self-supporting hydrogels upon mixing at room temperature[70]. As a further example, a self-assembling dock-and-lock systemwas used to fabricate hydrogels through molecular recognition be-tween an A-kinase anchoring protein domain and a docking anddimerization domain [71]. This specific, dynamic and rapidly

Fig. 2. A physically crosslinked hydrogel formed upon mixing of two modularly designed polypeptides for the encapsulation of mouse adipose-derived stem cells (mASCs).(a) Schematic of the MITCH network (left) formed after mixing of individual components (right). Protein sequences are shown using single-letter amino acid abbreviations.(b) Images of mASC (Fluc+) transplant sites in nude mice; sites are demarcated with dotted lines (day 0, left) and with bioluminescence (BLI) total flux overlay (day 3, right).(c) BLI measurements of cell retention. Data normalized to day 1 and reported as mean ± SEM; n = 5 or 6; ⁄p < 0.0001. Adapted from Ref. [100], copyright 2012, reprinted withpermission from Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

1754 L. Cai, S.C. Heilshorn / Acta Biomaterialia 10 (2014) 1751–1760

associating protein–ligand interaction enabled two components toform shear-thinning and rapidly self-healing hydrogels for cyto-compatible encapsulation of human mesenchymal stem cells(hMSCs) [71].

As an alternative to the above-described physical hydrogels thatrely on transient crosslinking, permanent chemical crosslinkingmechanisms have been widely used to create 3D protein hydrogelsfor cell encapsulation. In one strategy, enzymatic reactions enablemild and biocompatible crosslinking reactions to occur betweenprotein polymers that act as enzyme substrates. For example, tis-sue transglutaminase (tTG), a multifunctional enzymatic cross-linker that stabilizes tissues [72], has been used to catalyzecovalent bonding between lysine and glutamine amino acids. Inone demonstration, 3D hydrogels were created using tTG to formcrosslinks between two classes of protein polymers containingeither lysine or glutamine reactive sites that were evenly spacedalong the protein backbone. Under physiological conditions, com-plete crosslinking through tTG occurred within 2 min. Mouse 3T3and primary human fibroblasts encapsulated in this 3D hydrogelshowed high cell viability and displayed spreading on 2D gel sur-faces [21]. In another study, tTG was used to crosslink elastin-likepolypeptides (ELPs) designed for potential cartilage repair thera-pies. Cells maintained their chondrocytic phenotype in the ELPhydrogels in vitro and restructured the ELP matrix to deposit car-tilage ECM components [73].

In addition to enzymatic crosslinking, chemical hydrogels canalso be formed using small molecule crosslinkers. As an example,ELP materials are frequently modified to include lysine residues,which enable primary amine reactive crosslinking. These ELPhydrogels have been created using various chemical crosslinkers,including tris-succinimidyl aminotriacetate [74], disuccinimidyl

suberate [11] and phosphine-based catalysts [12,14,23]. Similarly,resilin-like polypeptides (RLPs), which are based on the elasto-meric structural protein resilin, have been crosslinked by phos-phine-based catalysts to form ECM-mimetic hydrogels [39,75,76].Recently, another novel family of recombinant elastomeric pro-teins has been reported based on abductin, a protein found in theinner hinge of bivalves [82]. Numerous types of cells have beenencapsulated in these eECM hydrogels to probe cell–material inter-actions, as well as to build up tissue-engineered constructs. Forexample, by encapsulating dorsal root ganglia within a tunablefamily of ELP matrices, the effects of integrin ligand density andmatrix stiffness on neurite outgrowth were analyzed in a decou-pled manner, showing longer neurite outgrowth in gels with high-er ligand density and lower stiffness [12] (Fig. 3). As a furtherexample, an integrin-binding RLP hydrogel with stiffness close tohuman cartilage tissue supported the adhesion, spreading and 3Dencapsulation of hMSCs [39,75].

3.2. Cell-instructive domains

While the structural and crosslinking domains described in theprevious section provide scaffolding interactions to build up 3Dmatrices, a host of other peptide modules can be introduced intothe eECMs to impart additional biofunctionality. To promote celladhesion and spreading, well-known cell-adhesive domains, suchas the RGD, IKVAV and YIGSR amino acid sequences, naturallyfound in various ECM proteins, are frequently integrated into thematerial [29–32]. In addition to the naturally evolved cell-adhesivesequences, domains identified through protein engineering strate-gies may be particularly useful in eECM development. For example,the RGD sequence has been engineered into a cystine-knot peptide

Fig. 3. A chemically crosslinked ELP hydrogel for the encapsulation of dorsal root ganglia (DRG). (a) ELPs were designed with modular repeats of bioactive (grey) and elastin-like (green) sequences. The bioactive domains were either an extended RGD sequence or a non-adhesive, scrambled RDG sequence. (b) The elastin-like structural domainsincluded lysines (purple) for site-specific crosslinking with amine-reactive crosslinkers yielding a 3-D hydrogel network. (c) Photograph (left) and schematic (right) of ELPhydrogel within a 5-mm silicone mold (orange) to encapsulate a single DRG cluster for culture within a 24-well plate. (d) Neurites extended from the initial DRG explant inhydrogels with 0 and 1.9 � 107 cell-adhesive RGD ligands lm�3 over the course of 7 days in culture. (e) Neurites extended by day 1 in 0.5 kPa hydrogels, by day 3 in 1.5 kPahydrogels but not until day 7 in 2.1 kPa hydrogels. Adapted from Ref. [12], copyright 2012, reprinted with permission from Elsevier.

L. Cai, S.C. Heilshorn / Acta Biomaterialia 10 (2014) 1751–1760 1755

motif to modulate its integrin-binding affinity and specificity[60,77].

RGD ligands have been encoded into ELP, RLP and silk-like pro-teins to promote interactions with a variety of cell types [12,14,78].In a particularly elegant example, RGD accessibility was tuned byfusion to a coiled-coil domain, which was functionalized on goldnanoparticles and subsequently immobilized on substrates. RGDaccessibility and thereby cell adhesion were reversibly controlledvia co-immobilization and removal of leucine-zipper coiled coilsthrough heterodimerization [79]. An alternative cell-binding do-main, CS5, has been added into ELPs to illustrate the effects of dis-tal amino acid selection on the resultant accessibility and affinityof the cell-binding domain [28]. In another study, N-cadherin, akey cell–cell adhesion protein in neural development, was fusedto the Fc region of the IgG antibody to create an artificial ECM sur-face that enables neural stem cells to maintain their undifferenti-ated state and preserve their differentiation potential [80]. Inanother novel design, a fibronectin type III domain from humantenascin-C was used in the creation of ECM-mimetic hydrogels thatencourage the spreading of human lung fibroblast cells [81].

In addition to cell-adhesion domains, other bioinstructive moi-eties, such as growth factors and cell signaling components, havebeen added to the protein-engineered biomaterials toolbox. Forexample, fibroblast growth factor-2 (FGF-2) fused to a fibronectinfragment presented a synergistic effect on osteoblast cell adhesionand proliferation [33]. However, the direct encoding of longer bio-active sequences into the protein backbone can lead to decreasedfunctionality, since conjugation at the N- and/or C-termini mayhinder the complex folding that is typical of these factors. There-fore, as an alternative strategy, fully folded soluble factors havebeen immobilized to the scaffold, either by covalent conjugationor affinity binding. As an example, vascular endothelial growth fac-tor (VEGF), a heparin-binding growth factor, served as elasticcrosslinks in a noncovalently assembled hydrogel network. In thepresence of VEGF receptors, the hydrogel is selectively eroded,and the VEGF released from the hydrogel increases the prolifera-tion of VEGF-responsive cell lines [34].

An alternative method for growth factor immobilization withineECMs is to conjugate the bioactive signals to an ECM-binding

domain, which binds to ECM matrices via affinity-based interac-tions. For example, transforming growth factor-beta has beenrecombinantly engineered to contain a collagen-binding domain[35]. Similarly, genetically engineered human bone morphogeneticprotein-2 (BMP-2) was fused to a collagen-binding domain for po-tential bone repair. A collagen matrix loaded with the engineeredBMP-2 induced better bone formation in a rabbit mandible defectmodel [36,37]. Additionally, platelet-derived growth factor BB(PDGF-BB) has been immobilized to a collagen-binding domain.The engineered PDGF-loaded collagen scaffolds encouraged cellproliferation in vitro and were uniformly cellularized and vascular-ized in vivo [38].

3.3. Cell-responsive domains

The native ECM is able to respond to changes in the local micro-environment, as cells survive, migrate and secrete their own ECM.To mimic this functionality, amino acid sequences that can becleaved at specific sites by cell-secreted proteases have been intro-duced into eECM biomaterials. One common motif is domainsdegradable by matrix metalloproteinases (MMPs). As an exampleof eECM multifunctionality, a single RLP has been engineered witha cell-adhesive RGD sequence, a heparin-binding domain for non-covalent immobilization and release of growth factors, and anMMP-cleavable site to enable proteolytic degradation [83]. TheMMP-sensitive domains are susceptible to MMP-1 enzymatic deg-radation within 2 days. hMSCs encapsulated in these RLP hydrogelswith various degradation rates displayed high viability [39]. Simi-larly, silk-elastin-like protein polymers have been modified to in-clude a sequence that is sensitive to MMP, which yieldedcomplete cleavage of all full-length polymers in 2 days [41].

As an alternative approach, peptide sequences that are sensitiveto degradation by the proteases tissue plasminogen activator (tPA)and urokinase plasminogen activator (uPA) have been encodedinto ELP hydrogels. These domains were designed to be responsiveto neuronal growth cones, which locally secrete tPA and uPA, in or-der to clear a path for elongating neurites. Minor amino acid se-quence mutations yielded hydrogel variants with identical initialmechanical properties but with degradation kinetics that spanned

1756 L. Cai, S.C. Heilshorn / Acta Biomaterialia 10 (2014) 1751–1760

two orders of magnitude [40]. Neuronal cell adhesion, neuronaldifferentiation and neurite outgrowth were all supported by thesematrices [11].

4. Emerging functionalities for eECMs

While the majority of domains used in eECMs to date have beeninspired by commonly occurring motifs in the native ECM, func-tional domains from non-matrix proteins can be repurposed to cre-ate more complex eECM designs. These emerging functionalitiesinclude linkage with inorganic materials, antimicrobial activities,immune modulators and dynamically responsive modules. Thesenew functionalities represent the versatility of protein sequencesand expand the potential of eECM biomaterials for a plethora of tis-sue engineering and biotechnology applications.

4.1. Linkage with inorganic materials

Many peptides are endowed with the capability to specificallybind to or control the synthesis of diverse inorganic materials[84]. These inorganic-binding peptide modules offer new possibil-ities to create composite eECMs that include both organic and inor-ganic components. The biogenesis of hybrid inorganic/organiccomposites, such as bone, often includes the process of templatedmineralization [42]. Thus, biomimetic synthesis of bone-like struc-tures requires novel organic scaffolds that mediate the mineraliza-tion of hydroxyapatite (HAP), the major inorganic component ofbone. Using phage display, a 12-residue peptide has been identifiedthat binds to single-crystal HAP. This peptide, mimicking the tri-peptide repeat of type I collagen, was found to template the nucle-ation and growth of crystalline HAP [42] (Fig. 4). Similarly, a silk/silica fusion protein was designed by fusing the self-assemblingdomains of spider dragline silk with the silaffin-derived R5 peptidethat initiates silica mineralization [43–45]. This engineered silkprotein served as the template for the formation of 3D, composite,porous networks with tunable silica morphologies and distribu-tions. hMSCs were found to attach, proliferate and differentiate to-ward osteogenic lineages with early bone formation on thesecomposite structures [44]. Besides bone-related applications,cell–material interactions have been probed using a series of pep-tides bound to gold, platinum, glass and titanium with RGD se-quences. These inorganic-binding peptides control the adhesionand spreading of fibroblast cells through the immobilization ofRGD ligands on solid surfaces [85]. In a highly creative application,the rational design of thermostable vaccines was achieved byengineering biomimetic nucleating peptides to induce virus

Fig. 4. Design of short HAP-binding peptides. (a) Schematic diagram of the phage displayof the collagen-like peptide CLP12 with the single-crystalline HAP surface, showing closelAdapted from Ref. [42], copyright 2011, reprinted with permission from American Chem

self-biomineralization under physiological conditions. The engi-neered, self-biomineralized vaccine was found to have better ther-mostability for long-term storage at ambient temperature [86].

4.2. Antimicrobial activity

Multidrug-resistant bacteria are a severe threat to public health.Conventional antibiotics are becoming increasingly ineffective as aresult of evolving drug resistance, and therefore it is imperative tofind new antibacterial strategies. Natural antimicrobial peptides(AMPs) are innate immune system effectors to defend host organ-isms against microbes, but most of them have relatively modestantibiotic activity. Enhanced variants have been developed usingrational design, optimization strategies and computer-assisted de-sign strategies [87]. Induced amphipathic a-helical conformationswere found to play an important role in the antimicrobial activityof these peptides [88]. Additionally, most AMPs tend to form amy-loid-like structures to destabilize phospholipid bilayers, suggestingthat these aggregation-prone structures may have served as tem-plates from which AMPs were evolutionarily derived [89]. Basedon these structural guidelines and computer-assisted technologiesthat relate primary sequence to peptide structure, more potent,cost-effective, broad-spectrum peptides are being identified as po-tential next-generation anti-infective peptides [87]. Some AMPshave already been included as eECM modules to create multifunc-tional biomaterials for medical use. In one study, three new fusionproteins were designed, cloned and evaluated for function by fus-ing the sequence of dragline spider silk with three different antimi-crobial peptides. These engineered proteins demonstratedcompatibility with mammalian cells while offering resistance toGram-negative Escherichia coli and Gram-positive Staphylococcusaureus [90].

4.3. Immune modulatory peptides

Understanding the immune responses against eECMs is of sig-nificant importance in their ultimate clinical use. Peptides are gen-erally poor immunogens and typically require coadministrationwith adjuvants to elicit antibody responses; however, their immu-nogenicity can increase significantly upon assembly and multimer-ization of supramolecular stuctures [91]. Self-assembling peptideshave shown significant variability in immunogenicity, with manypeptides inducing minimal antibody responses, but others induc-ing strong responses in the absence of any supplemental adjuvants[91–94]. Rudra et al. exploited these interactions by designingself-assembling peptides for use as chemically defined immune

process for single-crystalline HAP whiskers. (b) A model of the proposed interactiony matched distances between adjacent hydroxyl residues and the HAP crystal lattice.

ical Society.

Fig. 5. Schematic of a self-assembling peptide system that can be modulated to either raise a strong antibody response or to avoid such a response. The immune system (left)consists of antigen-presenting cells (APC), T cells and B cells. Peptide assemblies containing the OVA323–339 antigen (top middle) elicit strong antibody responses, whileassemblies without the antigen (bottom middle) elicit no significant responses. The engineered peptide system underwent self-assembly to form nanofibrils (right). Adaptedfrom Ref. [94], copyright 2012, reprinted with permission from American Chemical Society.

L. Cai, S.C. Heilshorn / Acta Biomaterialia 10 (2014) 1751–1760 1757

adjuvants [92]. Peptide epitopes, assembled into nanofibers via ashort synthetic fibrillization domain, elicited high antibody titerswithout any adjuvant. This strategy represents a simple, chemi-cally defined method to dramatically elevate antibody responsesto peptide epitopes [93]. These same ideas can also be used to cre-ate eECMs with minimal immunogenicity. For example, the self-assembling peptide, OVA-Q11, which elicits a strong, T-cell-depen-dent antibody response in mice, was modified by deleting the ami-no acid regions in the peptide that are recognized by T cells,thereby greatly diminishing immune responses [94] (Fig. 5). Takentogether, this work demonstrates that eECMs can be modulatedeither to raise a strong antibody response, and hence find potential

Fig. 6. A dynamic, protein-based hydrogel using a hinge-motion protein, calmodulin (Cextended (left) and collapsed (right) conformations. Cysteine residues engineered intovolume changes as a result of trifluoperazine (TFP) ligand binding. Photomicrographs shoconformation (middle). The volume decrease was recovered when gels were returned to[47], copyright 2007, reprinted with permission from WILEY-VCH Verlag GmbH & Co. K

use in immunotherapies, or to avoid such a response, and hencefind use as scaffolds for regenerative medicine applications.

4.4. Dynamically responsive peptides

Hydrogels that harness protein motion to achieve dynamicresponsiveness have great potential in biotechnology applications.Through careful design, protein motion at the molecular level canbe translated into macroscopic changes in hydrogel properties.For example, calmodulin, a hinge motion protein, has been de-signed into hydrogels that collapse upon binding of calcium ionsor other specific biochemical ligands, leading to significant

aM). (a) Schematic representation of hydrogel network structure with CaM in thethe protein are delineated by boxes. (b) CaM-based hydrogels undergo substantialwing CaM-based hydrogels with CaM in extended conformation (left) and collapsedan environment favoring the extended CaM conformation (right). Adapted from Ref.GaA, Weinheim.

1758 L. Cai, S.C. Heilshorn / Acta Biomaterialia 10 (2014) 1751–1760

decreases in hydrogel volume [95]. In one example, a mutant ver-sion of calmodulin was used as a crosslinker to form a PEG-basedhydrogel network [46]. In another design, PEG–calmodulin conju-gates were photocrosslinked to form dynamic hydrogels that couldundergo tunable volume change (up to 80%) based on the gelationconditions [47] (Fig. 6).

In addition to calmodulin, catalytic enzymes have been used asmodules in the design of dynamic biomaterials. One study em-ployed a protein that underwent a nanoscale conformationalchange upon binding to a substrate, which was translated intomacroscale mechanical motion and control of hydrogel swelling[48]. As another example, a glucose-responsive hydrogel was fab-ricated by conjugating a glucose/galactose-binding protein withinan acrylamide hydrogel network, resulting in a quantitative, accor-dion-like dynamic response upon addition of glucose [26]. Dy-namic hydrogels can also be generated by metallothioneinconformational changes that respond to heavy metal ions. Uponbinding of various toxic heavy metal ions, such as mercury andcadmium, metallothionein undergoes collapse from an extendedcoil to a more compact, globular morphology. When incorporatedinto poly(acrylamide) hydrogels, metallothionein’s conformationalshift was translated into an up to 80% decrease in hydrogel swell-ing [96].

Photoswitchable self-assembly is another emerging strategy inthe fabrication of smart, functional eECMs. Reversible optical con-trol of protein structure and function offers the possibility of prob-ing and manipulating complex cell microenvironments [97].Photocontrollable peptides that are reversible are often based ona helix–coil transitions. For example, a simple light-activated sys-tem employed a peptide designed to self-assemble into hydrogelsdepending on its intramolecular folded conformational state [98].This system utilized a photocaged peptide that remains unfoldedin aqueous medium and a freely soluble unfolded peptide that isstable to ambient light. Irradiation of the solution released thephotocage and triggered peptide folding to produce amphiphilica-hairpins that self-assembled into viscoelastic hydrogels. Fibro-blasts seeding indicated that the gel surface supported cell adhe-sion, proliferation and migration [98]. Azobenzene is anotherwidely used, light-responsive molecule. In one example, an azo-benzene-linked, symmetrical gemini a-helical peptide was de-signed to enable light-switched, self-assembly due to reversiblemolecular transitions between Z- and U-structures. The self-assembled morphology was observed to reversibly change be-tween nanofibers and nanospheres in acidic medium, and betweennanospheres and vesicles in basic medium [99].

5. Conclusions

In summary, protein eECMs have been designed with molecularprecision to fabricate 3D biomaterial scaffolds that mimic many ofthe features of native ECMs. With the advancement of recombinantprotein engineering technology and various crosslinking mecha-nisms, eECMs have been designed with independently controlledfunctional modules that encompass fibril structural domains,highly tunable mechanical compliance, cell-instructive biochemi-cal functionalities and tailored cell-responsive degradation pro-files. The repertoire of peptide modules that can be successfullydesigned into protein-based eECMs is fast developing, which en-ables the virtually limitless design of multifunctional materials.Furthermore, additional functionality using synthetic organicmaterials or inorganic components can be combined and seques-tered in the eECM matrix with ease. This emerging class of bioma-terials offers a versatile platform with decoupled materialparameters for the development of physiologically relevantin vitro cultures. These scaffolds are well suited for fundamental

biological studies of cell–matrix interactions, as well as for transla-tional applications in clinical therapies.

Acknowledgements

The authors acknowledge support from NSF (DMR-0846363),NIH (R01-DK085720, DP2-OD006477, R21-AR062359) and the Cal-ifornia Institute for Regenerative Medicine (RT2-01938).

Appendix A. Figures with essential colour discrimination

Certain figures in this article, particularly Figs. 1–5, are difficultto interpret in black and white. The full colour images can be foundin the on-line version, at http://dx.doi.org/10.1016/j.actbio.2013.12.028.

References

[1] Lutolf MP, Hubbell JA. Synthetic biomaterials as instructive extracellularmicroenvironments for morphogenesis in tissue engineering. Nat Biotechnol2005;23:47–55.

[2] Badylak SF, Freytes DO, Gilbert TW. Extracellular matrix as a biologicalscaffold material: structure and function. Acta Biomater 2009;5:1–13.

[3] Barker TH. The role of ECM proteins and protein fragments in guiding cellbehavior in regenerative medicine. Biomaterials 2011;32:4211–4.

[4] Collier JH, Segura T. Evolving the use of peptides as components ofbiomaterials. Biomaterials 2011;32:4198–204.

[5] Maskarinec SA, Tirrell DA. Protein engineering approaches to biomaterialsdesign. Curr Opin Biotechnol 2005;16:422–6.

[6] Romano NH, Sengupta D, Chung C, Heilshorn SC. Protein-engineeredbiomaterials: nanoscale mimics of the extracellular matrix. BiochemBiophys Acta Gen Subj 2011;1810:339–49.

[7] Sengupta D, Heilshorn SC. Protein-engineered biomaterials: highly tunabletissue engineering scaffolds. Tissue Eng Part B Rev 2010;16:285–93.

[8] Kiick KL. Biosynthetic methods for the production of advanced protein-basedmaterials. Polym Rev 2007;47:1–7.

[9] DiMarco RL, Heilshorn SC. Multifunctional materials through modular proteinengineering. Adv Mater 2012;24:3923–40.

[10] Liu JC, Tirrell DA. Cell Response to RGD density in cross-linked artificialextracellular matrix protein films. Biomacromolecules 2008;9:2984–8.

[11] Straley KS, Heilshorn SC. Independent tuning of multiple biomaterialproperties using protein engineering. Soft Matter 2009;5:114–24.

[12] Lampe KJ, Antaris AL, Heilshorn SC. Design of three-dimensional engineeredprotein hydrogels for tailored control of neurite growth. Acta Biomater2013;9:5590–9.

[13] Sengupta D, Gilbert PM, Johnson KJ, Blau HM, Heilshorn SC. Protein-engineered biomaterials to generate human skeletal muscle mimics. AdvHealthc Mater 2012;1:785–9.

[14] Chung C, Lampe KJ, Heilshorn SC. Tetrakis(hydroxymethyl) phosphoniumchloride as a covalent cross-linking agent for cell encapsulation withinprotein-based hydrogels. Biomacromolecules 2012;13:3912–6.

[15] Werkmeister JA, Ramshaw JAM. Recombinant protein scaffolds for tissueengineering. Biomed Mater 2012;7 (Article No. 012002).

[16] Benitez PL, Sweet JA, Fink H, Chennazhi KP, Nair SV, Enejder A, et al.Sequence-specific crosslinking of electrospun, elastin-like protein preservesbioactivity and native-like mechanics. Adv Healthc Mater 2013;2:114–8.

[17] Gomes S, Leonor IB, Mano JF, Reis RL, Kaplan DL. Natural and geneticallyengineered proteins for tissue engineering. Prog Polym Sci 2012;37:1–17.

[18] Shen W, Lammertink RGH, Sakata JK, Kornfield JA, Tirrell DA. Assembly of anartificial protein hydrogel through leucine zipper aggregation and disulfidebond formation. Macromolecules 2005;38:3909–16.

[19] Petka WA, Harden JL, McGrath KP, Wirtz D, Tirrell DA. Reversible hydrogelsfrom self-assembling artificial proteins. Science 1998;281:389–92.

[20] Mi L, Fischer S, Chung B, Sundelacruz S, Harden JL. Self-assembling proteinhydrogels with modular integrin binding domains. Biomacromolecules2005;7:38–47.

[21] Davis NE, Ding S, Forster RE, Pinkas DM, Barron AE. Modular enzymaticallycrosslinked protein polymer hydrogels for in situ gelation. Biomaterials2010;31:7288–97.

[22] Toledano S, Williams RJ, Jayawarna V, Ulijn RV. Enzyme-triggered self-assembly of peptide hydrogels via reversed hydrolysis. J Am Chem Soc2006;128:1070–1.

[23] Nettles DL, Kitaoka K, Hanson NA, Flahiff CM, Mata BA, Hsu EW, et al. In situcrosslinking elastin-like polypeptide gels for application to articular cartilagerepair in a goat osteochondral defect model. Tissue Eng Part A2008;14:1133–40.

[24] Foo CTSWP, Lee JS, Mulyasasmita W, Parisi-Amon A, Heilshorn SC. Two-component protein-engineered physical hydrogels for cell encapsulation.Proc Natl Acad Sci USA 2009;106:22067–72.

L. Cai, S.C. Heilshorn / Acta Biomaterialia 10 (2014) 1751–1760 1759

[25] Yuan W, Yang J, Kopeckova P, Kopecek J. Smart hydrogels containingadenylate kinase: translating substrate recognition into macroscopicmotion. J Am Chem Soc 2008;130:15760.

[26] Ehrick JD, Luckett MR, Khatwani S, Wei Y, Deo SK, Bachas LG, et al. Glucoseresponsive hydrogel networks based on protein recognition. Macromol Biosci2009;9:864–8.

[27] Zhang X, Chu X, Wang L, Wang H, Liang G, Zhang J, et al. Rational design of atetrameric protein to enhance interactions between self-assembled fibersgives molecular hydrogels. Angew Chem Int Ed 2012;51:4388–92.

[28] Heilshorn SC, Liu JC, Tirrell DA. Cell-binding domain context affects cellbehavior on engineered proteins. Biomacromolecules 2005;6:318–23.

[29] Liu JC, Heilshorn SC, Tirrell DA. Comparative cell response to artificialextracellular matrix proteins containing the RGD and CS5 cell-bindingdomains. Biomacromolecules 2004;5:497–504.

[30] Nakamura M, Mie M, Mihara H, Nakamura M, Kobatake E. Construction of amulti-functional extracellular matrix protein that increases number of N1E–115 neuroblast cells having neurites. J Biomed Mater Res B Appl Biomater2009;91:425–32.

[31] Elloumi I, Kobayashi R, Funabashi H, Mie M, Kobatake E. Construction ofepidermal growth factor fusion protein with cell adhesive activity.Biomaterials 2006;27:3451–8.

[32] Jung JP, Moyano JV, Collier JH. Multifactorial optimization of endothelial cellgrowth using modular synthetic extracellular matrices. Integr Biol QuantBiosci Nano Macro 2011;3:185–96.

[33] Jang J-H, Chung C-P. Engineering and expression of a recombinant fusionprotein possessing fibroblast growth factor-2 and fibronectin fragment.Biotechnol Lett 2004;26:1837–40.

[34] Yamaguchi N, Zhang L, Chae BS, Palla CS, Furst EM, Kiick KL. Growth factormediated assembly of cell receptor-responsive hydrogels. J Am Chem Soc2007;129. 3040–+.

[35] Andrades JA, Wu LT, Hall FL, Nimni ME, Becerra J. Engineering, expression,and renaturation of a collagen-targeted human bFGF fusion protein. GrowthFactors 2001;18:261–75.

[36] Chen B, Lin H, Zhao Y, Wang B, Zhao Y, Liu Y, et al. Activation of demineralizedbone matrix by genetically engineered human bone morphogenetic protein-2with a collagen binding domain derived from von Willebrand factorpropolypeptide. J Biomed Mater Res Part A 2007;80:428–34.

[37] Chen B, Lin H, Wang J, Zhao Y, Wang B, Zhao W, et al. Homogeneousosteogenesis and bone regeneration by demineralized bone matrix loadingwith collagen-targeting bone morphogenetic protein-2. Biomaterials2007;28:1027–35.

[38] Lin H, Chen B, Sun W, Zhao W, Zhao Y, Dai J. The effect of collagen-targetingplatelet-derived growth factor on cellularization and vascularization ofcollagen scaffolds. Biomaterials 2006;27:5708–14.

[39] Li L, Tong Z, Jia X, Kiick KL. Resilin-like polypeptide hydrogels engineered forversatile biological function. Soft Matter 2013;9:665–73.

[40] Straley KS, Heilshorn SC. Dynamic, 3-D-Pattern formation within enzyme-responsive hydrogels. Adv Mater 2009;21. 4148–+.

[41] Gustafson JA, Price RA, Frandsen J, Henak CR, Cappello J, Ghandehari H.Synthesis and characterization of a matrix-metalloproteinase responsive silk-elastin-like protein polymer. Biomacromolecules 2013;14:618–25.

[42] Chung WJ, Kwon KY, Song J, Lee SW. Evolutionary screening of collagen-likepeptides that nucleate hydroxyapatite crystals. Langmuir 2011;27:7620–8.

[43] Foo CWP, Patwardhan SV, Belton DJ, Kitchel B, Anastasiades D, Huang J, et al.Novel nanocomposites from spider silk–silica fusion (chimeric) proteins. ProcNatl Acad Sci USA 2006;103:9428–33.

[44] Mieszawska AJ, Nadkarni LD, Perry CC, Kaplan DL. Nanoscale control of silicaparticle formation via silk–silica fusion proteins for bone regeneration. ChemMater 2010;22:5780–5.

[45] Huang J, Wong C, George A, Kaplan DL. The effect of genetically engineeredspider silk–dentin matrix protein 1 chimeric protein on hydroxyapatitenucleation. Biomaterials 2007;28:2358–67.

[46] Murphy WL, Dillmore WS, Modica J, Mrksich M. Dynamic hydrogels:translating a protein conformational change into macroscopic motion.Angew Chem Int Ed Engl 2007;46:3066–9.

[47] Sui Z, King WJ, Murphy WL. Dynamic materials based on a proteinconformational change. Adv Mater 2007;19. 3377–+.

[48] Yuan W, Yang J, Kopeckova P, Kopecek J. Smart hydrogels containingadenylate kinase: translating substrate recognition into macroscopicmotion. J Am Chem Soc 2008;130:15760–1.

[49] Amblard M, Fehrentz JA, Martinez J, Subra G. Methods and protocols ofmodern solid phase peptide synthesis. Mol Biotechnol 2006;33:239–54.

[50] Raibaut L, Ollivier N, Melnyk O. Sequential native peptide ligation strategiesfor total chemical protein synthesis. Chem Soc Rev 2012;41:7001–15.

[51] Bayer E, Mutter M. Liquid-phase synthesis of peptides. Nature 1972;237:512.[52] Megeed Z, Cappello J, Ghandehari H. Genetically engineered silk-elastin-like

protein polymers for controlled drug delivery. Adv Drug Deliv Rev2002;54:1075–91.

[53] Nettles DL, Chilkoti A, Setton LA. Applications of elastin-like polypeptides intissue engineering. Adv Drug Deliv Rev 2010;62:1479–85.

[54] Elvin CM, Carr AG, Huson MG, Maxwell JM, Pearson RD, Vuocolo T, et al.Synthesis and properties of crosslinked recombinant pro-resilin. Nature2005;437:999–1002.

[55] Urry DW, Luan CH, Parker TM, Gowda DC, Prasad KU, Reid MC, et al.Temperature of polypeptide inverse temperature transition depends on meanresidue hydrophobicity. J Am Chem Soc 1991;113:4346–8.

[56] Macias MJ, Gervais V, Civera C, Oschkinat H. Structural analysis of WWdomains and design of a WW prototype. Nat Struct Biol 2000;7:375–9.

[57] Russ WP, Lowery DM, Mishra P, Yaffe MB, Ranganathan R. Natural-likefunction in artificial WW domains. Nature 2005;437:579–83.

[58] King NP, Sheffler W, Sawaya MR, Vollmar BS, Sumida JP, Andre I, et al.Computational design of self-assembling protein nanomaterials with atomiclevel accuracy. Science 2012;336:1171–4.

[59] Harris JL, Backes BJ, Leonetti F, Mahrus S, Ellman JA, Craik CS. Rapid andgeneral profiling of protease specificity by using combinatorial fluorogenicsubstrate libraries. Proc Natl Acad Sci USA 2000;97:7754–9.

[60] Silverman AP, Levin AM, Lahti JL, Cochran JR. Engineered cystine-knotpeptides that bind avb3 integrin with antibody-like affinities. J Mol Biol2009;385:1064–75.

[61] Sidhu SS, Bader GD, Boone C. Functional genomics of intracellular peptiderecognition domains with combinatorial biology methods. Curr Opin ChemBiol 2003;7:97–102.

[62] Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920–6.[63] Sallach RE, Cui W, Wen J, Martinez A, Conticello VP, Chaikof EL. Elastin-

mimetic protein polymers capable of physical and chemical crosslinking.Biomaterials 2009;30:409–22.

[64] Landschulz WH, Johnson PF, McKnight SL. The leucine zipper: a hypotheticalstructure common to a new class of DNA binding proteins. Science1988;240:1759–64.

[65] Olsen BD, Kornfield JA, Tirrell DA. Yielding behavior in injectable hydrogelsfrom telechelic proteins. Macromolecules 2010;43:9094–9.

[66] Fischer SE, Liu X, Mao H-Q, Harden JL. Controlling cell adhesion to surfaces viaassociating bioactive triblock proteins. Biomaterials 2007;28:3325–37.

[67] Mulyasasmita W, Lee JS, Heilshorn SC. Molecular-level engineering of proteinphysical hydrogels for predictive sol–gel phase behavior. Biomacromolecules2011;12:3406–11.

[68] Parisi-Amon A, Mulyasasmita W, Chung C, Heilshorn SC. Protein-engineeredinjectable hydrogel to improve retention of transplanted adipose-derivedstem cells. Adv Healthc Mater 2013;2:428–32.

[69] Ito F, Usui K, Kawahara D, Suenaga A, Maki T, Kidoaki S, et al. Reversiblehydrogel formation driven by protein–peptide-specific interaction andchondrocyte entrapment. Biomaterials 2010;31:58–66.

[70] Grove TZ, Osuji CO, Forster JD, Dufresne ER, Regan L. Stimuli-responsive smartgels realized via modular protein design. J Am Chem Soc 2010;132:14024–6.

[71] Lu HD, Soranno DE, Rodell CB, Kim IL, Burdick JA. Secondaryphotocrosslinking of injectable shear-thinning dock-and-lock hydrogels.Adv Healthc Mater 2013;2:1028–36.

[72] Greenberg CS, Birckbichler PJ, Rice RH. Transglutaminases: multifunctionalcross-linking enzymes that stabilize tissues. FASEB J 1991;5:3071–7.

[73] McHale MK, Setton LA, Chilkoti A. Synthesis and in vitro evaluation ofenzymatically cross-linked elastin-like polypeptide gels for cartilaginoustissue repair. Tissue Eng 2005;11:1768–79.

[74] Trabbic-Carlson K, Setton LA, Chilkoti A. Swelling and mechanical behaviorsof chemically cross-linked hydrogels of elastin-like polypeptides.Biomacromolecules 2003;4:572–80.

[75] Renner JN, Cherry KM, Su RSC, Liu JC. Characterization of resilin-basedmaterials for tissue engineering applications. Biomacromolecules2012;13:3678–85.

[76] Li L, Teller S, Clifton RJ, Jia X, Kiick KL. Tunable mechanical stability anddeformation response of a resilin-based elastomer. Biomacromolecules2011;12:2302–10.

[77] Moore SJ, Hayden Gephart MG, Bergen JM, Su YS, Rayburn H, Scott MP, et al.Engineered knottin peptide enables noninvasive optical imaging ofintracranial medulloblastoma. Proc Natl Acad Sci USA 2013;110:14598–603.

[78] Kambe Y, Yamamoto K, Kojima K, Tamada Y, Tomita N. Effects of RGDSsequence genetically interfused in the silk fibroin light chain protein onchondrocyte adhesion and cartilage synthesis. Biomaterials2010;31:7503–11.

[79] Wang X, Riesberg JJ, Shen W. Reversible regulation of bioactive ligandspresented on immobilized gold nanoparticles. Soft Matter 2012;8:2812–5.

[80] Yue XS, Murakami Y, Tamai T, Nagaoka M, Cho CS, Ito Y, et al. A fusion proteinN-cadherin–Fc as an artificial extracellular matrix surface for maintenance ofstem cell features. Biomaterials 2010;31:5287–96.

[81] Lv S, Bu T, Kayser J, Bausch A, Li H. Towards constructing extracellular matrix-mimetic hydrogels: an elastic hydrogel constructed from tandem modularproteins containing tenascin FnIII domains. Acta Biomater 2013;9:6481–91.

[82] Su RSC, Renner JN, Liu JC. Synthesis and characterization of recombinantabductin-based proteins. Biomacromolecules 2013;14:4301–8.

[83] Charati MB, Ifkovits JL, Burdick JA, Linhardt JG, Kiick KL. Hydrophilicelastomeric biomaterials based on resilin-like polypeptides. Soft Matter2009;5:3412–6.

[84] Dickerson MB, Sandhage KH, Naik RR. Protein- and peptide-directedsyntheses of inorganic materials. Chem Rev 2008;108:4935–78.

[85] Khatayevich D, Gungormus M, Yazici H, So C, Cetinel S, Ma H, et al.Biofunctionalization of materials for implants using engineered peptides.Acta Biomater 2010;6:4634–41.

[86] Wang GC, Cao RY, Chen R, Mo LJ, Han JF, Wang XY, et al. Rational design ofthermostable vaccines by engineered peptide-induced virus self-biomineralization under physiological conditions. Proc Natl Acad Sci USA2013;110:7619–24.

[87] Fjell CD, Hiss JA, Hancock REW, Schneider G. Designing antimicrobialpeptides: form follows function. Nat Rev Drug Discov 2012;11:37–51.

1760 L. Cai, S.C. Heilshorn / Acta Biomaterialia 10 (2014) 1751–1760

[88] Blondelle SE, Houghten RA. Design of model amphipathic peptides havingpotent antimicrobial activities. Biochemistry 1992;31:12688–94.

[89] Torrent M, Valle J, Nogués MV, Boix E, Andreu D. The generation ofantimicrobial peptide activity: a trade-off between charge and aggregation?Angew Chem Int Ed 2011;50:10686–9.

[90] Gomes SC, Leonor IB, Mano JF, Reis RL, Kaplan DL. Antimicrobialfunctionalized genetically engineered spider silk. Biomaterials2011;32:4255–66.

[91] Collier JH, Rudra JS, Gasiorowski JZ, Jung JP. Multi-component extracellularmatrices based on peptide self-assembly. Chem Soc Rev 2010;39:3413–24.

[92] Rudra JS, Tripathi PK, Hildeman DA, Jung JP, Collier JH. Immune responses tocoiled coil supramolecular biomaterials. Biomaterials 2010;31:8475–83.

[93] Rudra JS, Tian YF, Jung JP, Collier JH. A self-assembling peptide acting as animmune adjuvant. Proc Natl Acad Sci USA 2010;107:622–7.

[94] Rudra JS, Sun T, Bird KC, Daniels MD, Gasiorowski JZ, Chong AS, et al.Modulating adaptive immune responses to peptide self-assemblies. ACS Nano2012;6:1557–64.

[95] Topp S, Prasad V, Cianci GC, Weeks ER, Gallivan JP. A genetic toolbox forcreating reversible Ca2+-sensitive materials. J Am Chem Soc2006;128:13994–5.

[96] Esser-Kahn AP, Iavarone AT, Francis MB. Metallothionein-cross-linkedhydrogels for the selective removal of heavy metals from water. J Am ChemSoc 2008;130. 15820–+.

[97] Woolley GA. Photocontrolling peptide a helices. Acc Chem Res2005;38:486–93.

[98] Haines LA, Rajagopal K, Ozbas B, Salick DA, Pochan DJ, Schneider JP. Light-activated hydrogel formation via the triggered folding and self-assembly of adesigned peptide. J Am Chem Soc 2005;127:17025–9.

[99] Chen C-S, Xu X-D, Li S-Y, Zhuo R-X, Zhang X-Z. Photo-switched self-assemblyof a gemini a-helical peptide into supramolecular architectures. Nanoscale2013;5:6270–4.

[100] Parisi-Amon A, Mulyasasmita W, Chung C, Heilshorn SC. Protein-engineeredinjectable hydrogel to improve retention of transplanted adipose-derivedstem cells. Adv Healthc Mater 2012;2:428–32.