REVIEW SUMMARY◥

MATERIALS SCIENCE

Advances in engineering hydrogelsYu Shrike Zhang and Ali Khademhosseini*

BACKGROUND:Hydrogels are formed throughthe cross-linking of hydrophilic polymer chainswithin an aqueous microenvironment. The ge-lation canbe achieved througha variety ofmech-anisms, spanning physical entanglement ofpolymer chains, electrostatic interactions, andcovalent chemical cross-linking. The water-richnature of hydrogels makes them broadly appli-cable tomanyareas, including tissueengineering,drugdelivery, soft electronics, andactuators. Con-ventional hydrogels usually possess limitedme-chanical strength and are prone to permanentbreakage. The lack of desired dynamic cues andstructural complexity within the hydrogels hasfurther limited their functions. Broadened appli-cations of hydrogels, however, require advancedengineering of parameters such as mechanicsand spatiotemporal presentation of active or bio-activemoieties, as well asmanipulation ofmulti-scale shape, structure, and architecture.

ADVANCES:Hydrogels with substantially im-proved physicochemical properties have beenenabled by rational design at the molecular

level and control overmultiscale architecture.For example, formulations that combine per-manentpolymernetworkswith reversibly bond-ing chains for energy dissipation show strongtoughness and stretchability. Similar strategiesmay also substantially enhance the bondingaffinity of hydrogels at interfaces with solidsby covalently anchoring the polymer networksof tough hydrogels onto solid surfaces. Shear-thinning hydrogels that feature reversible bondsimpart a fluidic nature upon application ofshear forces and return back to their gel statesonce the forces are released. Self-healing hy-drogels based on nanomaterial hybridization,electrostatic interactions, and slide-ring con-figurations exhibit excellent abilities in spon-taneously healing themselves after damages.Additionally, harnessing techniques that candynamically and precisely configure hydrogelshave resulted in flexibility to regulate theirarchitecture, activity, and functionality. Dy-namic modulations of polymer chain physicsand chemistry can lead to temporal altera-tion of hydrogel structures in a programmed

manner. Three-dimensional printing enablesarchitectural control of hydrogels at high pre-cision, with a potential to further integrateelements that enable change of hydrogel con-figurations along prescribed paths.

OUTLOOK:We envision the continuation ofinnovation in new bioorthogonal chemistriesfor making hydrogels, enabling their fabri-

cation in the presence ofbiological species with-out impairing cellular orbiomolecule functions.Wealso foresee opportunitiesin the furtherdevelopmentofmore sophisticated fab-

rication methods that allow better-controlledhydrogel architecture across multiple lengthscales. In addition, technologies that preciselyregulate the physicochemical properties ofhydrogels in spatiotemporally controlledmannersare crucial in controlling their dynamics, suchas degradation and dynamic presentation ofbiomolecules. We believe that the fabricationof hydrogels should be coupled with end ap-plications in a feedback loop in order to achieveoptimal designs through iterations. In the end,it is the combination ofmultiscale constituentsand complementary strategies that will enablenew applications of this important class ofmaterials.▪

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The list of author affiliations is available in the full article online.*Corresponding author. Email: alik@bwh.harvard.eduCite this article as Y. S. Zhang, A. Khademhosseini, Science356, eaaf3627 (2017). DOI: 10.1126/science.aaf3627

Engineering functional hydrogels with enhanced physicochemical properties. Advances have been made to improve the mechanicalproperties of hydrogels as well as to make them shear-thinning, self-healing, and responsive. In addition, technologies have been developed tomanipulate the shape, structure, and architecture of hydrogels with enhanced control and spatial precision.

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REVIEW◥

MATERIALS SCIENCE

Advances in engineering hydrogelsYu Shrike Zhang1,2,3 and Ali Khademhosseini1,2,3,4,5*

Hydrogels are formed from hydrophilic polymer chains surrounded by a water-richenvironment.They have widespread applications in various fields such as biomedicine, softelectronics, sensors, and actuators. Conventional hydrogels usually possess limitedmechanical strength and are prone to permanent breakage. Further, the lack of dynamiccues and structural complexity within the hydrogels has limited their functions. Recentdevelopments include engineering hydrogels that possess improved physicochemicalproperties, ranging from designs of innovative chemistries and compositions to integrationof dynamic modulation and sophisticated architectures. We review major advances indesigning and engineering hydrogels and strategies targeting precise manipulation of theirproperties across multiple scales.

Hydrogels, a class of three-dimensional (3D)networks formed by hydrophilic polymerchains embedded in a water-rich environ-ment, possess broadly tunable physical andchemical properties (1–3). A variety of nat-

urally derived and synthetic polymers can be pro-cessed into hydrogels, from those formed throughphysical entanglement to ones stabilized via co-valent cross-linking. Hydrogels may be furthertuned toward the integration of chemically andbiologically active recognition moieties such asstimuli-responsivemolecules and growth factorsthat enhance their functionality. The versatilityof the hydrogel systemhas endowed it withwide-spread applications in various fields, includingbiomedicine (1–3), soft electronics (4, 5), sensors(6–8), and actuators (9–14). As an example, whena hydrogel is created with proper stiffness andbioactive moieties, it modulates the behavior ofthe embedded cells (15, 16). In addition, chemicallyactivemoieties and light-guiding properties allowhydrogels to sense substances of interest and per-form on-demand actuation (7, 17).Advances in chemical methods—such as click

chemistry, combination of gelation mechanisms,and doping with nanomaterials—have producedhydrogels with more controlled physicochemicalproperties. Furthermore, the static and uniformmicroenvironments characteristic of traditionalhydrogel matrices do not necessarily replicatethehierarchical complexity of the biological tissues.Innovative fabrication strategies have been de-

veloped not only to achieve dynamic modula-tion of the hydrogels that can evolve their shapesalong predefined paths, but also to control thespatial heterogeneity that will determine local-ized cellular behaviors, tissue integration, and de-vice functions.

Hydrogel formation

Hydrogels are formed by cross-linking polymerchains dispersed in an aqueousmedium througha myriad of mechanisms, including physical en-tanglement, ionic interactions, and chemical cross-linking (Fig. 1). Majority of the physical gelationmethods depend on the intrinsic properties of thepolymers. This dependence limits the ability tofine-tune the attributes of hydrogels, but gela-tion is easy to achieve without the need formodi-fying the polymer chains and is usually easy toreverse when necessary. Conversely, chemical ap-proaches can be used to allow for more control-lable, precise management of the cross-linkingprocedure, potentially in a spatially and dynam-ically defined manner.Manynatural polymers such as seaweed-derived

polysaccharides and proteins from animal originform thermally driven hydrogels. During the gela-tion process, physical entanglement of the poly-mer chains occurs in response to a temperaturechange. This change is typically caused by an al-teration in their solubility and the formation ofpackedpolymer backbones that are physically rigid(Fig. 1A) (18, 19). An increase or decrease in tem-peraturemay result in thermal gelation, inwhichthe transition temperatures are defined as lowercritical solution temperature (LCST) and uppercritical solution temperature (UCST), respectively(20, 21). The gelation mechanism, however, mayvary with specific types of polymers. Macromole-cules exhibitingUCST includenatural polymerssuchas gelatin aswell as synthetic polymers such as poly-acrylic acid (PAA) that gel as the temperature dropsto below their respectiveUCSTs. In contrast, someother macromolecules show LCST behavior, suchas synthetic polymer poly(N-isopropylacrylamide)

(PNIPAAM), that gels above its LCST. The LCST/UCST of the thermo-responsive polymersmay betuned by their molecular weight, ratio of the co-polymers, and/or the balance of the hydrophobic/hydrophilic segments (22, 23).Noncovalentmolecular self-assembly is adop-

ted as a common strategy, especially for protein-based hydrogels (24).Weak noncovalent bondingmechanisms—including hydrogen bonds, van derWaals forces, andhydrophobic interactions—causemacromolecules to fold into scaffolds possessingwell-defined structures and functionality. A nota-ble example is the hierarchical self-assembly ofcollagen, themost abundant protein in thehumanbody (Fig. 1B). The assembly procedure relies onthe regular arrangement of the amino acids incollagen molecules that are rich in proline or hy-droxyproline (25). These molecules facilitate theformationof the triple helix termed tropocollagen.Subsequent stabilization upon further packing ofthe tropocollagen subunits into fibrils/fibers even-tually forms a collagen hydrogel (24–26). Inspiredby this mechanism, biomimetic supramolecularformulations have been designed that can followsimilar hierarchical self-assembly processes, suchas collagen-mimetic peptides (26) and those basedonpeptide-amphiphiles andhydrogelators (27, 28).Spontaneousphysical gelationmay alternatively

depend on chelation or electrostatic interactions.Alginate, apolysaccharidecomposedofa-L-guluronicacid (G) and b-D-mannuronic acid (M) residuesderivedmainly frombrown algae, is a prominentexample of hydrogel formation based on chelation(29). The G-blocks in alginate rapidly gel in pres-ence of certain species of divalent cations such asCa2+ orBa2+ in an “egg-box” form, inwhich pairs ofhelical chains pack and surround ions that arelocked in between (Fig. 1C) (30). The sophisticatedstructures of natural macromolecules often ren-der them varying degrees of electrostatic chargesalong the backbone. Althoughmany natural poly-mers are negatively charged at neutral pH be-cause of the presence of carboxyl groups (such ashyaluronic acid and alginate), somemay also pre-sent positive chargeswhenaminegroupsdominate(such as gelatin and chitosan). In contrast, synthet-ic polyelectrolytes offer much better control overthe electrostatic properties. A common exampleis the poly(L-lysine) (PLL)/PAApair (31,32).Whenthe solutions containing polyelectrolytes of oppo-site charges aremixed, thepolymer chains entangleto formcomplexes that become insoluble becauseof mutual shielding (Fig. 1D) (33).Chemical cross-linking is better at stabilizing a

hydrogel matrix because it allows substantiallyimproved flexibility and spatiotemporal precisionduring the gelation process as compared withphysicalmethods. Chemically activemoieties pen-dant to the backbones or side chains of macro-molecules in anaqueous solution can formcovalentbondsunderproper circumstances toobtainhydro-gels (Fig. 1E). Conventional mechanisms includecondensation reactions (for example, carbodiimidechemistry between hydroxyl groups/amines andcarboxylic acids), radical polymerization, aldehydecomplementation, high-energy irradiation, andenzyme-enabled biochemistry, among others (34).

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1Biomaterials Innovation Research Center, Division ofBiomedical Engineering, Department of Medicine, Brighamand Women’s Hospital, Harvard Medical School, Cambridge,MA 02139, USA. 2Harvard-MIT Division of Health Sciencesand Technology, Massachusetts Institute of Technology,Cambridge, MA 02139, USA. 3Wyss Institute for BiologicallyInspired Engineering, Harvard University, Boston, MA 02115,USA. 4Department of Bioindustrial Technologies, College ofAnimal Bioscience and Technology, Konkuk University,Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic ofKorea. 5Department of Physics, King Abdulaziz University,Jeddah 21569, Saudi Arabia.*Corresponding author. Email: alik@bwh.harvard.edu

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Over the past decade, click chemistry—proposedto be fast, orthogonal, high-yield, and typicallyamenable to cells and bioactive agents—has under-gone tremendousdevelopment. This class of precisechemical reactions aims to address the challengesassociatedwith traditional chemical reactions, suchas lowyield, prolonged reaction times, and extremereaction conditions (35–37). A rich variety of bio-compatible click reactions have been devised foruse in direct bioconjugation and hydrogel forma-tion in the presence of cells within the matrices,such as thiol-vinyl sulfone and thiol-maleimideMichael addition reactions, azide-alkyne andazide-alkyne cycloaddition reactions, and thiolenephoto-coupling reaction (37).Although each of these gelation methods has

its advantages and limitations, the field has beenmarching toward combiningmultiple componentsand/ormechanisms to achieve improvedhydrogelformulations (38–42). These hydrogels typically ex-hibit excellent physicochemical properties, suchas substantially enhancedmechanics, injectability,self-healing, and possibility to undergo dynamicmodulation, as further discussed below.

Tuning the strength

Conventional hydrogels typically possess a lowto intermediate stretchability within a few timestheir original length, and fracture energies of<100 Jm−2. Applications such as load-bearing bio-materials (39, 43), soft robotics (44), andwearabledevices (4–6) have spurred interest in developingtough and highly stretchable hydrogels as a classof soft and hydrated substrates.

One strategy lies in the use of hydrogels derivedfrom natural proteins that may present excep-tional elasticity (45). For example, elastin is awidelydistributed structural protein required tomain-tain tissue integrity and confer elasticity and is pres-ent in tissues such as blood vessels, heart, bladder,and skin. To this end, recombinant tropoelastin,and its enzymatically cross-linked form termedelastin-like polypeptides (ELPs), have been engi-neered (46, 47). These engineered elastin-basedhydrogels typically possess strong elasticity byusing a pentapeptide repeat, VPGXG, where Xis any amino acid except proline (46). They canachieve stretchability of up to ~400% their orig-inal lengths (47), which is much higher thanmostexisting naturally derived hydrogels.Nevertheless,the high cost of these protein-based materialslimits their use for applications requiring largehydrogel amounts.Alternatively, hydrogels formed throughhybrid-

izationwith nanomaterials (39, 48), via crystallitecross-linking (49), or bymixingmultiple compo-nents (38, 41, 50), may possess substantially im-proved mechanical properties (51). For all thesemechanisms, the key lies in combination of care-fully selected components that formulate thehydrogels.Nanomaterials (such as inorganicnano-particles, carbon nanotubes, and graphene), be-cause of their highly tunable surface propertiesand usually strong mechanics, contribute to en-hancedmechanical performances of bulk hydro-gels through interacting with polymer chainsforming the hydrogels (39, 42, 48). The use of nano-materials as cross-linkers would reduce the re-

strictions exerted by dense cross-links that areotherwise present in pure polymernetworks. Forexample, by use of inorganic clay nanoplateletsas anchors for grafting the polymer chains uponcross-linking, the resulting PNIPAAM hydrogelnetwork could be stretched to up to 1400% itsoriginal length (48). The stable grafting pointsof these clay-PNIPAAM networks was attributedto the tunable surface properties of the clay nano-platelets (48). The filler materials may provideadditional functionality, such as enhancement ofelectrical conductivity (39), or promotion of tissueregeneration (52).Multiple cross-linking mechanisms, often in-

volvingphysical entanglement and chemical bond-ing of two types of polymer chains, have beencombined to achieve hydrogels with superiortoughness and high fracture energy (41, 53–56).A highly stretchable and tough hydrogel was syn-thesized bymixing alginate, ionically cross-linkedby Ca2+, and covalently cross-linked long-chainpolyacrylamide (PAAm). The two components,alginate and PAAm, were further intertwinedthrough covalent bonding between their respec-tive carboxyl andaminegroups (Fig. 2A) (41).Duringthe stretching process, the PAAm network remainsintact while the alginate network unzips progres-sively to efficiently dissipate energy, which canreform upon removal of the stress, thus exhibit-ing excellent hysteresis and negligible gross defor-mation. As such, this class of hybrid hydrogelscould achieve >20 times the elongation of theiroriginal lengths with fracture energies of up to9000 Jm−2. Alginatemolecules of different chainlengthsmay further be coembedded in the alginate-PAAm system in order to synthesize hydrogelscontaining improved density of chelation, thusachieving extraordinarily high fracture energiesof up to 16,000 J m−1 without compromising thetoughness (38). Introduction of crystallizationwithin an entangled polymer network representsanother approach to achieve tough and stretch-able hydrogels (49, 57). As an example, ratherthan the alginate component, polyvinyl alcohol(PVA) molecules could be incorporated, whichupon annealing formed crystallites within theintertwined network of PVA and PAAm (49, 57).The dried network was then rehydrated to formthe tough and stretchable hydrogel, in which thecrystallites of PVA functioned as the zippable,energy-dissipating segments.In another strategy, molecular sliding was de-

vised to synthesize extremely stretchable hydro-gels with good toughness (50). Hydrogels werederived from anetwork of structures composed ofa copolymer, ofN-isopropylacrylamide (NIPAAM)andsodiumacrylic acid (AAcNa), anda-cyclodextrinconjugated with poly(ethylene glycol) (PEG)(Fig. 2B). The free movement and sliding of thea-cyclodextrin rings along the polymer chains, en-abledby the inclusionof the ionicmonomerAAcNatomodulate ionization, rendered the hydrogelshighly stretchable to up to 400 to 800% their orig-inal lengths. The degree of stretching, however,was also dependent on the type of cross-linkerused, either hydroxypropylatedpolyrotaxane cross-linker (HPR-C)orN,N′-methylenebisacrylamide (BIS).

Zhang and Khademhosseini, Science 356, eaaf3627 (2017) 5 May 2017 2 of 10

Fig. 1. Cross-linking of hydrogels. (A to D) Physical cross-linking. (A) Thermally induced entanglementof polymer chains. (B) Molecular self-assembly. (C) Ionic gelation. (D) Electrostatic interaction. (E) Chemicalcross-linking.

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Strong bonding between hydrogels and solidmaterials as diverse as metals, glass, ceramics,and silicone is required for a wide range of ap-plications in areas spanning from biomedicineto soft electronics (5, 58). Nevertheless, rationaldesign of a robust interaction between thesetwo mechanically distinctive classes of materialshas remained historically challenging. Followingan approach used for making a tough compositehydrogel from intertwined polymer networks, thelong-chain polymer molecules were further cova-lently attached to the surface of a solid throughanchored chemical bonds (Fig. 2C) (40, 59). Theresistance to scission of the anchored polymerchains during peeling generates the adhesion, andthe energy is dissipated by the reversible physicalchelation of the ionically cross-linked alginate. Thistype of bonding may reach interfacial toughnessvalues of over 1000 J m−2, which is comparablewith the toughest bonding found between atendon and a bone in humans (800 J m−2). Inaddition to potential applications in biomedicine,optimization of hydrogel bondingwith other typesof materials, such as elastomers, may further ex-pedite the development of the field of soft elec-tronics. For example, stretchable soft electronicshave been designed by coating an electrolyte-containing hydrogel layer onto a substrate of elas-tomeric tape without strong bonding between thetwo surfaces (4).

Break and heal

Hydrogels capable of regaining their initial me-chanical properties and undergoing autonomoushealing upon damage can be useful in variousapplications such as in biomedicine, externalcoatings, and flexible electronics. Such propertiestypically rely on reestablishing molecular inter-actions in the water-rich microenvironment afterthe hydrogel is subjected to external forces or dam-age. This ability is achieved by the incorporationof moieties that present reversible but typicallystrong physical interactions in the hydrogel’spolymer network. These mechanisms can rangefrom electrostatic interactions (55, 60, 61) andhydrogen bonding (62, 63) to hydrophobic inter-actions (64, 65) and caged guest-host interac-tions (66, 67). The resulting hydrogelsmay furtherbe stimuli-dependent that are modulated by, forexample, pH (62) or oxidative state (66).Besides functioning as chemical cross-linkers,

clay nanoplatelets may also be physically mixedwithpolymer chains to formshear-thinninghydro-gels. Synthetic clay nanoplatelets that have ananisotropic charge distribution, positive alongthe edges and negative on the two surfaces, mayresult in a net negative charge in an aqueousmedium (60). Therefore, when complexed withpositively charged supramolecules such as gela-tin type A at neutral pH, a shear-thinning hydro-gel is formed because of the strong electrostaticinteractions between the clay nanoplatelets andthe polymer chains (Fig. 3A). The resulting hydro-gel temporarily reduces its viscosity upon applica-tion of shear stress to endow injectability througha plug flow process, while returning to its originalviscosity at low shear owing to electrostatic inter-

actions (68). The system is simple and may beextended to many charged species of nanopar-ticles and polymer chains to produce injectableformulations (61). These shear-thinning hydro-gels have found utility in hemostatic dressing(60), endovascular embolization (68), tissue engi-neering (69), and drug and gene delivery (61).Systemswith electrostatic interactions between

polymer chains containing oppositely chargedside groups have also been developed (Fig. 3B)(55). Polyampholytes, synthesized through ran-

dom copolymerization of oppositely chargedmono-mers, were induced to spontaneously formphysical hydrogels containing electrostatic bondswith a wide distribution of strengths. In this sce-nario, the strong ionic bonds serve as semiper-manent cross-links to maintain the shape of thehydrogel,whereas theweakbondsdissipate energythrough reversible bond breakages and forma-tions, enabling improved toughness as well asshear-thinning properties. The physical cross-linking imparted by the electrostatic interactions,

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Fig. 2. Tuning the mechanics of hydrogels. (A) Stretchable hydrogel made from long-chain polymersand reversible physically cross-linked polymers. (Right) The hydrogel could be stretched to 21 times itsinitial length, where the stretch l is the final length of the unclamped region divided by the original length;stress-stretch curves of the alginate, PAAm, and alginate-PAAm hydrogels, each stretched to rupture,where the nominal stress s is defined as the force applied on the deformed hydrogel, divided by the cross-sectional area of the undeformed hydrogel. [Adapted with permission from (41), copyright 2012 NaturePublishing Group] (B) Stretchable hydrogel based on a sliding ring mechanism. (Right) Photo of anelongated NIPAAM-AAcNa-HPR-C hydrogel, and stress-strain curves of different hydrogels: (i) NIPAAM-AAcNa-BIS [0.65 weight % (wt %)], (ii) NIPAAM-AAcNa-BIS (0.065 wt %), (iii) NIPAAM-AAcNa-HPR-C(2.00 wt %), (iv) NIPAAM-AAcNa-HPR-C (1.21 wt %), and (v) NIPAAM-AAcNa-HPR-C (0.65 wt %). Thepercentages denote those of the cross-linkers. [Adapted with permission from (50), copyright 2014 NaturePublishing Group] (C) Tough bonding of hydrogels with smooth surfaces. (Right) The peeling process of atough hydrogel chemically anchored on a glass substrate, and curves of the peeling force per width ofhydrogel sheet versus displacement for various types of hydrogel-solid bonding. [Adaptedwith permissionfrom (59), copyright 2016 Nature Publishing Group]

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however, further enable these types of hydrogelsto self-heal even upon complete damage betweentwo freshly cut or aged surfaces (55).Self-healing through hydrogen bonding can

be attained by incorporating side groups suchas amine or carboxyl into the polymer backbone

of the hydrogel network (Fig. 3C) (62). It furtherallows a pH-dependent healing capacity of thedamaged hydrogels. In the case of carboxyl pendantgroups, at pH lower than the pKa values of themolecules (where Ka is the acid dissociationconstant), the functional groups become proton-

ated and can form hydrogen bonds, whereas intheir deprotonated state at pH values above pKa,the charged groups exhibit strong electrostaticrepulsion. A host-guest system forms another basisof generating self-healing hydrogels. The classicalmolecule cyclodextrin, when conjugated to the poly-mer backbone, functions as the host to engulf hy-drophobic moieties dangling as side chains ofanother polymer backbone to achieve self-healing.If an environment-responsive moiety—such asPAAm-ferrocene (PAAm-Fc), which undergoes a re-versible hydrophobic-charge transition at reduced/oxidized states—is included in a hydrogel, self-healing could further be selectively promoted uponintroduction of favorable stimuli (Fig. 3D) (66).Whereas current strategies in synthesizing self-healing hydrogels heavily rely on physical andchemical methods, we envision the emergence ofbioinspired approaches in which bioactive speciesmay as well facilitate the healing process of thehydrogels upon damage. In nature, self-healingof biological tissues occurs frequently through anorchestrated cascade of cellular events, in whichcells residing in the local microenvironment activelyrespond to the wound by selectively clearing thedebris, secreting bioactive cues, and depositing newextracellular matrix (ECM) molecules to achievehealing. Therefore, rational combination of thephysicochemical methods and biologically activespecies (such as enzymes, microorganisms, or evenmammalian cells) may represent a new self-healingstrategy.

Dynamic modulation

No single existing system,whether it is biologicalor synthetic, is purely static. Rather, dynamic evo-lutions are universally present, in which the capa-bility tomodulatehydrogel behavior in a temporallydependent manner becomes particularly attractive.When engineering biologically relevant systems,it is strongly desirable that the hydrogel micro-environment evolves with time through intrin-sically embeddedmoieties that respond to externalstimuli, either in an explicitly user-defined fashionor imposed by coexisting cells. Various approacheshave been devised in the past decade toward in-corporationofdynamicphysiological signalswithinhydrogel platforms to precisely modulate cell-matrix interactions (1, 70). Dynamic modulationof hydrogels further opens up opportunities forconstruction of actuators and robotics (9–12).Photopatterning is a long-established approach

for introducing heterogeneity in a hydrogel vol-ume by means of controlled spatial immobiliza-tion of bioactive molecules (71–73). The additionof specific biochemical cues into thematrix throughphotopatterning allows localized presentation ofsignals for cells to respond. These signals mayinclude growth factors, proteins, hormones, cell-adhesion peptides, and cell-repellingmoiety (73).Notably, the reverse process—on-demand releaseof certainmoieties from a hydrogelmatrix—is alsosometimes desirable to trigger temporal changeswithin the hydrogel. In realizing this capability,photodegradation (asopposed tophotocross linking),has been developed on the basis of the inclusionof photolabile moieties that undergo photolysis

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Fig. 3. Shear-thinning and self-healing hydrogels. (A) Shear-thinning hydrogel through nanocomposit-ing. (Bottom) Recovery of the nanocomposites was observed by subjecting the hydrogel to alternatinghigh and low strain conditions (100% strain and 1% strain) while monitoring the moduli of the composite.[Adapted with permission from (60), copyright 2014 American Chemical Society] (B to D) Self-healinghydrogels based on ionic interactions, hydrogen bonds, and host-guest coupling. (B) Ionic interactions.(Bottom left) Recovery of the sample for different waiting times. (Bottom right) Self-healing betweeneither two freshly cut surfaces (red and blue) or a fresh and an aged surface (white) of samples; [Adaptedwith permission from (55), copyright 2013 Nature Publishing Group] (C) Hydrogen bonds. (Bottom) Thehealed hydrogels at low pH separate after exposure to a high-pH solution (with pH > 9), and the separatedhydrogels could reheal upon exposure to acidic solution (pH < 3). [Adapted with permission from (62),copyright 2012 the National Academy of Sciences] (D) Host-guest coupling. (Bottom) The cut hydrogelspread with NaClO aqueous solution did not heal after 24 hours, but readhesion was observed 24 hoursafter spreading reuced glutathione aqueous solution onto the oxidized cut surface. [Adapted withpermission from (66), copyright 2011 Nature Publishing Group]

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upon two-photon illumination (Fig. 4A) (74). Thesemoieties can be designed to be biocompatible,enabling photodegradation of the hydrogels in thepresence of bioactive molecules or cells, as wellas time-dependent degradation. Photopatterningandphotodegradationmaybecombined toachievefull control over thedynamics of the spatial patternsin a hydrogel system (36, 75). For example, a photo-reversible patterning strategy can be used to firstdefine spatial distributionof biomolecules throughphotomediated ligation, followed by subsequentremoval upon further exposure to a different light(Fig. 4B) (75). More recently, attention has beenfocused ondynamicmodulation of biophysical cuesinside the hydrogel such as matrix mechanics,which can be used to tune the behavior of encap-sulated cells (76, 77).Cellsmay be another source of external stimuli

to trigger a dynamic alteration of the hydrogelmatrix inwhich they reside (78–80). In the humanbody, a critical characteristic of the ECM lies in itssusceptibility to proteolysis triggered by the re-siding cells to facilitate their mobility and sub-sequent remodeling of the microenvironment.To achieve this functionality within a hydrogel,a specially designed linker moiety typically sen-sitive to an enzyme can be copolymerized into thehydrogel so as to allow for localized protease-mediateddegradationof thepolymericnetwork (79).In combinationwith a growth factor-sequesteringcomponent, these cell-instructive hydrogelmatricesare able to reversibly immobilize growth factorsondemand (81). The concept of protease-mediateddegradation has been recently taken a step for-ward by implementing a negative feedback loopthrough a combinatory effect of an enzyme-labilemoiety and an enzyme inhibitor coembedded inthehydrogelmatrix (80). In this scenario, anenzyme-sensitive peptide [for example, to matrix metal-loproteinase (MMP)] is used as the linker of thehydrogel network, whereas the enzyme-inhibitor[for example, tissue inhibitor ofMMPs (TIMP)] issimultaneously liberatedby the samecleavingevent.Concurrent inclusion of the enzyme-inhibitor pairinduces balanced local activation and inhibitionof the enzyme activity, therefore modulating thehydrogel degradation (Fig. 4C). Compared withconventional unconfined stimuli-responsiveness,the introduction of the feedback system clearlydemonstrates its advantage in providing moreprecise manipulation of the matrix properties.Thismechanism, based on cell-responsiveMMPs,has also been adapted for other types of hydro-gels [such as PEG-heparin (82)]. Alternatively,instead of introducing stimuli-responsive moi-eties, selective modification of the macromole-cules before formation of hydrogelsmay allow formanipulation of hydrogel properties. For exam-ple, when heparin was selectively desulfated, theresulting hydrogels demonstrated altered releaseprofiles of affinity-immobilizedgrowth factors com-pared with the release in the case of pristine hep-arin (83). This strategy canbe extended to chemicalmodifications so as to further enable dynamic con-trol of biomolecule sequestration and release.Morphogenesis is an important feature of living

organisms. As an organism develops, it altersmor-

phologies along a certain path, to produce the finalshape and architecture working in a coordinatedmanner that renders the entire organism func-tional. Throughmeticulous selection of polymersthat make up the hydrogels, they can also be pro-cessed to ensue bioinspired shape-morphing capa-bilityupondesired stimulation (12) suchashumidity(10, 84), temperature (85), pH (86, 87), ionic strength(87, 88), magnetism (89, 90), or light (11), amongother stimulants. A heterogeneous or multilayerconfiguration containing materials with varyingdegree of responsiveness is typically required toenable a dynamic shape change. For example, adual-layer hydrogel formed by two polymerswithdistinctive swelling capacities (for example, PEGand alginate) presents directional bending uponabsorption of water (84). Another example is thecombination of thermo-sensitive (such as PNI-PAAM) and thermo-inert hydrogelmaterials thatmodulate bidirectional bendingof fabricated struc-ture, at temperatures above or below which thethermo-sensitive hydrogel component is shaped(Fig. 4D) (85). Also, the inclusion of pH-sensitivemoieties into a hydrogel leads to pH-mediatedswellingor deswelling (86). Although thesemodelsrely on the intrinsic properties of the polymersthat constitute hydrogels, functional materialssuch asmagnetic nanoparticles (90) and carbonnanomaterials (11) may be added to the hydro-gel matrices in order to achieve shape actuationupon external stimuli ofmagnetic field and light-induced local heating, respectively.Cell-mediated traction force can also contribute

to shape-morphing of hydrogels (91). Carefullydesigned microstructures seeded with cells areable to self-fold into predefined architecturesthrough origami (92). Alternatively, cells thatspontaneously contract enable mechanical move-ment of the substrate onwhich they reside, leadingto generation of soft bioactuators (93, 94).Whereasearlier bioactuators were mainly based on elasto-meric materials, hydrogel materials have recentlyattracted increasing attention because of theirbetter biocompatibility. To this end, a combina-tion ofmyocytes (skeletalmuscle cells or cardiomyo-cytes) and hydrogels that enable communicationsbetween these cells is usually adopted. For example,carbon nanotube–composited gelatin methacryloylhydrogels have been processed into flexible sub-strates as large as centimeter scales, and cardio-myocytes spontaneously and synchronously beatingon the surfaces allow these bioactuators to ex-hibit rhythmic contraction or extension and swim-mingbehaviors (95,96). Engineeredskeletalmuscles,when bound to bioprinted hydrogel frameworks,could also actuate andmove the devices in definedpatterns (13, 14). These bioactuators relying on celltraction forces may be remotely controlled by avariety of external stimuli such as electrical signals(95–97) and light (14, 94). To be suited for buildingrobust bioactuators, hydrogels must be both bio-compatible and mechanically stable.

Shaping the hydrogels

Biological tissues are exceedingly complex andmay possess a hierarchically assembled architec-ture featuring a variety of nano- or microscale

bioactivemolecules in conjunctionwithmultiplecell populations functioning in synergy. For ex-ample, almost all organs in the human body con-tain repeating building units that assemble intodistinctive compartments and interfaces [for ex-ample, the myocardium, endocardium, and peri-cardium in the heart (98, 99) and the lobulesconstituting the liver (99, 100)]. Most organs areembeddedwith perfusable vasculature of sophis-ticated tortuosity that supplies oxygen and nu-trients and transports bioactive molecules (suchas growth factors and hormones). Such complex-ity further extends to the architecture of the bloodvessels, themselves defined by layered structuresof tunica intima, tunicamedia, and tunica externa(99). Consequently, engineering a biomimeticorgan requires the capacity to reconstitute itsnative characteristics and physiology throughrational design that ensures compositional, ar-chitectural, mechanical, and functional accura-cies. Soft actuators and electronic devices madefrom hydrogels may similarly demand spatiotem-poral control to render heterogeneity required forfunctionality.Microengineering provides a versatile approach

to engineer well-definedhydrogels fromminiatur-ized building blocks through programmed spon-taneous assembly.Multiple types of hydrogel units,individually fabricated to contain desired cell typesandphysicochemical cues, canbeused tomimic themicrotissue units in the human organs. Earlier ver-sions of packing strategies rely on shape comple-mentarity in which these different blocks can beprocessed by using, for example, lithographic orphotopatterning techniques. These engineeredbuilding blocks of microgels with complemen-tary geometrical configurations subsequently un-dergo directed assembly into the bulk form undera combinatory influenceof external energy, surfacetension, and microgel dimensions at liquid-liquidinterfaces (Fig. 5A) (101). Assembled microgelsmay be further cross-linked to stabilize the bulkarchitecture. Using this strategy, arrays of ring-shapedmicrogels were assembled into a 3D tubu-lar construct with interconnected lumens in abiphasic system via stimulation applied by fluidshear (102). Microgels could also be assembledinto large-scale structures on a surface patternedwith hydrophilic and hydrophobic regions at highfidelity (103). A class of hydrophobic hydrogels hasbeen proposed by stabilizing a layer of hydropho-bic microparticles on the surface of conventionalhydrogels to allow them to float on aqueousmedia(104). These hydrophobic hydrogels with comple-mentary shapes could self-assemble because of hy-drophobic interactions directly on the surface ofwaterwhenbrought into proximitywith each other.Although this type of self-assembly is convenientand scalable, it does not confer strong control overthe assembly process because it is limited by thesole reliance on theminiaturization of interfacialsurface free energy (101).Alternatively, the self-assembly properties of

nucleic acids provide a powerful approach for thefabricationof sophisticatedpatterns, structures, anddevices. Sequence complementarity inDNA/RNAstrands can be encoded to induce self-organization

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Fig. 4. Dynamic modulation of hydrogel microenvironment. (A) Photo-degradation through photolabile moieties. (Bottom left) Hydrogels demon-strated surface erosion upon irradiation. (Bottom right) Hydrogel was erodedspatially through masked flood irradiation, where feature dimensions werequantified with profilometry after different periods of irradiation. Scale bars,100 mm. [Adapted with permission from (74), copyright 2009 AmericanAssociation for the Advancement of Science] (B) Dynamic photopatterningand photorelease. (Bottom) Patterned primary antibodies are visualized witha fluorescent secondary antibody,which were subsequently photoreleased toform a secondary pattern. Scale bar, 3 mm. [Adapted with permission from(75), copyright 2015 Nature Publishing Group] (C) Cell-responsive cleavageand anticleavage negative feedback system. (Bottom) recombinant TIMP-3

(rTIMP-3) activity was measured by its ability to inhibit a recombinant MMP-2(rMMP-2) solution, in which (left) bound rTIMP-3 did not substantially reducerMMP-2. (Bottom)Hydrogels with (solid symbols) andwithout (open symbols)encapsulated rTIMP-3 were incubated with (squares) or without (triangles)rMMP-2, in which encapsulated rTIMP-3 attenuated rMMP-2-mediated hydro-gel degradation. [Adapted with permission from (80), copyright 2014 NaturePublishing Group] (D) Thermo-responsive shapemorphing hydrogel. (Bottom)Serial images of a closed gripper with poly(propylene fumarate) (PPF) seg-ments on the outside opening as the temperature was decreased to below36°C and then folding back on itself to become a closed gripper, but with thePPFsegments on the inside. Scale bar, 2mm. [Adaptedwith permission from(85), copyright 2015 American Chemical Society]

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into a predefined structure, when the comple-mentary segments pair up under proper physicalconditions (105). On the basis of this principle, a

plethora of molecular assemblies from syntheticnucleic acids have been generated such as tubes(106–108), ribbons (108), lattices (107, 109), and

other complex shapes (106, 110). This concept hasbeen adapted to assemble microgel units, sim-ilar to the abovementioned approach but in amuch more precisely controlled manner. In thissense, the “giant DNA glue” is decorated onto theprescribed surfaces of a nonspherical hydrogelunit to produce asymmetric glue patterns on het-erogeneous surfaces. These microscale buildingunits, through combination of themolecular pro-grammability of the DNA glue and the shapecontrollability of themicrogels, can achievemulti-plexed assembly of complex structures of hydrogelsacross multiple scales (Fig. 5B) (111). The assemblyof diverse hydrogel structures—including dimers,linear chains, and large-scale networks—couldpotentially be used to build hierarchical tissuearchitectures and biomedical devices. Nucleicacid–directed self-assemblymay further be adaptedfor programmed synthesis of 3D biological tis-sues (112). Instead of hydrogels, dissociated singlecells may be chemically functionalized with oli-gonucleotides, which are complementary to DNAsequences coated on desired surfaces to allow forcellular assembly into the third dimension via alayer-by-layer approach. The oligonucleotideswere designed to be degradable, thus achievingon-demand reversible binding once the micro-tissues formed (112).Three-dimensional printing represents another

facile technique for constructing volumetric objects(113, 114). Three-dimensional printing empowersextremelyhighprecision through the roboticmanip-ulation of the prepolymer (the ink), thus allowingfor reproducible manufacturing of objects withminimal aberration in their shape, architecture,and functionality. The 3Dprinting technologywasinitially introduced in the form of stereolithog-raphy, in which defined objects aremanufacturedthrough a pull-out procedure from a liquid bathof prepolymer via layer-by-layer photopatternedcross-linking (115). Precise control of locally avail-able oxygen concentrations during the stepwiseradical polymerization further enables continuousmanufacture of large-sized 3D shapes at substan-tially improved speed (116). Using such a manu-facturing principle, hydrogel objects with complex3D architecture andmultiplexedmaterials may begenerated with high spatial resolution (100, 117).Complementary to stereolithography, nozzle-

based 3D printing strategies typically providehigher degrees of flexibility in the production ofhydrogel objects with defined architecture. Theink, deposited through positioned ejection froman orthogonally movable printhead, builds 3Dstructures in a layer-by-layer manner. Whereasstereolithography solely relies on photopolymer-ization, a rich variety of cross-linkingmechanismsmay be used for nozzle-based printing throughmeticulous selection of the inks combined withrational design of the printhead. For example,physical cross-linking of alginate by Ca2+ can beachieved with a core-sheath nozzle design to co-deliver the two components during the extrusionprocedure, whereas photocrosslinking may stillbe applied to the methacryloyl-modified hydro-gels after printing (118, 119). Alternatively, shear-thinning inks may be directly printed, followed

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Fig. 5. Shaping macroscale hydrogels. (A) Self-assembly through shape complementarity. (Bottom)(Top row) Hydrogels assembled by capillary force. Scale bar, 200 mm. (Bottom row) Hydrophobic hy-drogels assembled through hydrophobic interactions on the surface of water. Scale bars, 5 cm. [Adaptedwith permission from (101), copyright 2008 the National Academy of Sciences, and (104), copyright 2016American Chemical Society] (B) Self-assembly using sequence-complementing DNA glues. (Bottom)(Top row) DNA-assisted hydrogel self-assembly across multiple length scales. Scale bars, 1 mm. (Bottomrow) Directed formation regular dimers and T-junction based on their surface DNA glue patterns. Scalebars, 1 mm. [Adapted with permission from (111), copyright 2013 Nature Publishing Group] (C) Embeddednozzle-based bioprinting. (Bottom left) A continuous hollow knot written with fluorescent microspheres ina granular hydrogel. Scale bar, 3 mm. (Bottom right) A freely floating hydrogel jellyfish model retrievedafter printing and dissolution of the granular hydrogel. (Inset) The printed structure before the removal ofthe supporting hydrogel matrix. Scale bars, 5 and (inset) 10 mm. [Adapted with permission from (125),copyright 2015 American Association for the Advancement of Science] (D) 4D biomimetic printingthrough a printed dual-layer structure with unbalanced swelling. (Bottom) Time-lapse photographs show-ing bioprinted simple flower-like structures undergoing shape-morphing during the swelling process, forthe double layers of different orientations. Scale bars, 5 mm. [Adapted with permission from (140),copyright 2016 Nature Publishing Group]

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by subsequent chemical cross-linking (120). Also,theymay functionas sacrificial templates in certain3D printing applications so as to obtain hollowhydrogel structures after selective removal (121–123).Tough and highly stretchable hydrogels can alsobe printed into complex architecture (124). Thecapacity of nozzle-based 3D printing has beensubstantially boosted with recent developmentof the technique based on embedded printing(125–127). Conventionally, extruded hydrogelma-terials would not stand their own weights whencomplex structures containing large cavities, thin-walled tubes, and suspended elements were cre-ated. The ability to resist gravitational force hasthus been proposed by the use of a supportinghydrogel bath, which is both shear-thinning,allowing for convenient deposition of the ink, andself-healing, ensuring shape-maintenance of theprinted fine structures within the volume (Fig. 5C).Often, the ability to combine multiple types of

hydrogels is desired over a singlematerial to fabri-cate compositionally complex patterns. Printerscarrying several printheads have thus been devel-oped to deposit selected hydrogel inks, or inkswith polymer scaffolding in a prescribedmanner(120, 128, 129). The emergence of microfluidicprintheads capable of codelivering dual materialtypes further enhanced the speed of multimate-rial printing via improved switching between thedifferent materials (118, 119, 130, 131). Instead, byusing a microscale fluid mixing device inside theprinthead, the two materials may be homo-genized to achieve direct printing of gradientstructures through precise control over the ratiobetween the inks infused into the printhead (132).

Outlook

Hydrogels represent an important class of mate-rials possessing awatery environment andbroadlytunablephysicochemical properties. Efforts devotedto engineering hydrogels with enhanced propertiesin the past decade have expanded their opportu-nities in numerous applications, including bio-medicine, soft electronics, sensors, and actuators.For instance, the mechanics of hydrogels havebeen engineered to become stiff and tough whilemaintaining a high water content. Self-healingmechanisms and dynamicmodulation have beenincorporated within hydrogel systems to achievecontrol over their behaviors over time. Advancedbiofabrication techniques have further improvedour capability to construct sophisticated hydrogelarchitectures that feature hierarchically assembledstructures across multiple length scales.However, several key challenges persist. Clinical

translation of hydrogels requires rigorous testing(133), and their approval by the U.S. Food andDrug Administration for clinical applications hasbeen limited to a few types of hydrogel materials(134).Maintaining or improving themechanics ofhydrogels in a wide range of media has remainedanother challenge. Swelling is a commonphenome-non of hydrogels owing to water uptake causedby unequal osmotic pressure when immersed inan aqueousmediumof lower osmolarity. Swellingoften causes the weakening of the hydrogel net-works upon long exposures in aqueous environ-

ments. In tackling themechanical instability causedby swelling of the hydrogel network, an “anti-swelling” (or “nonswellable”) hydrogel consistingof a network integrating hydrophilic and thermo-responsive polymer units was proposed (135). Itresisted swelling at elevated temperature whenthe thermo-responsive polymer chains condensed,thus preserving the mechanics of the pristinehydrogel. Although this strategy seems universal,continuation in the innovation of new chemistriesand compositions to further optimize the pro-cesses becomes critical.The potential to integrate hydrogel formula-

tions with advanced biofabrication techniquesis exciting aswell, despite that strict optimizationprocesses should be carried out to meet properfabrication requirements. For example,whenhighlystretchable hydrogels are combined with bio-printing, elastic substrates of any arbitrary shape,pattern, and architecture may be obtained (124).As analternative strategy todirect hydrogel printing,the mechanical properties of hydrogels can bereinforcedby integrating themwithprintedmicro-fibrous scaffolds (136). When encapsulation ofcells is desired during the fabrication processes,the need for hydrogels with suitable mechanicalproperties and biocompatibility becomes critical(114). In addition, printing should be carried outunder physiologically relevant conditions in orderto ensure cell viability and phenotype mainte-nance (137).Furthermore, printed hydrogel structuresmay

be dynamically modulated. Throughmaterial de-signs, it is feasible to add an extra dimension—time—into the 3D architecture. In 4D printing(138–143), compositionally heterogeneous inks aredeposited to form the initial layout, which is capa-ble of shape transformation in a preprogrammedmanner over time under stimuli. This techniqueallows for performance-driven functionality to bedesigned into the printed materials. For example,a hydrophilic polymer printed in between rigid,nonswelling segments swells after encounteringwater andexpands intohydrogel structures, forcingthe rigid materials to bend until they stop whenhitting neighboring elements (138, 139). Thefolding pattern of printed structures can be ac-curately predicted throughmathematical mod-eling, which serves as a guideline for the design ofthe printing path to achieve prescribed temporaltransformation (140).We envision that with increasing adoption of

interdisciplinary approaches, it is possible to ratio-nally combine multiple strategies, leading to crea-tion of hydrogels that possess enhancedproperties.The design ofmaterials inwhichmultiple constit-uents or phases are assembled across multiplelength scales may harness synergistic effects andfacilitate novel functionalities. The combinationsof properties can emerge as a result of the par-ticular arrangement or interactions between themultiscale constituents, providing opportunitiesfor further innovations in hydrogels. Ultimately,the advancements in engineeringhydrogels shouldbe coupled with their end applications in a feed-back loop to achieve optimal designs through iter-ative optimization cycles.

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ACKNOWLEDGMENTS

The authors acknowledge funding from the National Institutes ofHealth (grants AR057837, DE021468, D005865, AR068258,AR066193, EB022403, and EB021148) and the Office of NavalResearch Presidential Early Career Award for Scientists andEngineers (PECASE). Y.S.Z. acknowledges the National CancerInstitute of the National Institutes of Health Pathway toIndependence Award (K99CA201603). We greatly appreciateS. Masoud Moosavi-Basri for assistance with figure illustrations.We also thank S. Hassan, J. Seo, and W. Wang for their valuablediscussions.

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Advances in engineering hydrogelsYu Shrike Zhang and Ali Khademhosseini

DOI: 10.1126/science.aaf3627 (6337), eaaf3627.356Science 

, this issue p. eaaf3627Scienceapplications.advances in making hydrogels with improved mechanical strength and greater flexibility for use in a wide range ofused as dynamic, tunable, degradable materials for growing cells and tissues. Zhang and Khademhosseini review the

Hydrogels are highly cross-linked polymer networks that are heavily swollen with water. Hydrogels have beenWet, soft, squishy, and tunable

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