RESEARCH

◥ manner. Three-dimensional printing enables REVIEW SUMMARY architectural control of hydrogels at high pre- cision, with a potential to further integrate elements that enable change of hydrogel con- MATERIALS SCIENCE figurations along prescribed paths.

OUTLOOK: We envision the continuation of Advances in engineering hydrogels innovation in new bioorthogonal chemistries for making hydrogels, enabling their fabri- Yu Shrike Zhang and Ali Khademhosseini* cation in the presence of ◥ ON OUR WEBSITE biological species with- out impairing cellular or Read the full article BACKGROUND: Hydrogels are formed through level and control over multiscale architecture. at http://dx.doi. biomolecule functions. We the cross-linking of hydrophilic polymer chains For example, formulations that combine per- org/10.1126/ also foresee opportunities within an aqueous microenvironment. The ge- manent polymer networks with reversibly bond- science.aaf3627 in the further development ...... lation can be achieved through a variety of mech- ing chains for energy dissipation show strong of more sophisticatedfab- anisms, spanning physical entanglement of toughness and stretchability. Similar strategies rication methods that allow better-controlled polymer chains, electrostatic interactions, and may also substantially enhance the bonding hydrogel architecture across multiple length covalent chemical cross-linking. The water-rich affinity of hydrogels at interfaces with solids scales. In addition, technologies that precisely nature of hydrogels makes them broadly appli- by covalently anchoring the polymer networks regulate the physicochemical properties of Downloaded from cable to many areas, including tissue engineering, of tough hydrogels onto solid surfaces. Shear- hydrogels in spatiotemporally controlled manners drug delivery, soft electronics, and actuators. Con- thinning hydrogels that feature reversible bonds are crucial in controlling their dynamics, such ventional hydrogels usually possess limited me- impart a fluidic nature upon application of as degradation and dynamic presentation of chanical strength and are prone to permanent shear forces and return back to their gel states biomolecules. We believe that the fabrication breakage. The lack of desired dynamic cues and once the forces are released. Self-healing hy- of hydrogels should be coupled with end ap-

structural complexity within the hydrogels has drogels based on nanomaterial hybridization, plications in a feedback loop in order to achieve http://science.sciencemag.org/ further limited their functions. Broadened appli- electrostatic interactions, and slide-ring con- optimal designs through iterations. In the end, cations of hydrogels, however, require advanced figurations exhibit excellent abilities in spon- it is the combination of multiscale constituents engineering of parameters such as mechanics taneously healing themselves after damages. and complementary strategies that will enable and spatiotemporal presentation of active or bio- Additionally, harnessing techniques that can new applications of this important class of active moieties, as well as manipulation of multi- dynamically and precisely configure hydrogels materials.▪ scale shape, structure, and architecture. have resulted in flexibility to regulate their architecture, activity, and functionality. Dy- The list of author affiliations is available in the full article online. ADVANCES: Hydrogels with substantially im- namic modulations of polymer chain physics *Corresponding author. Email: [email protected] proved physicochemical properties have been and chemistry can lead to temporal altera- Cite this article as Y. S. Zhang, A. Khademhosseini, Science enabled by rational design at the molecular tion of hydrogel structures in a programmed 356, eaaf3627 (2017). DOI: 10.1126/science.aaf3627

on February 25, 2018

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

Zhang and Khademhosseini, Science 356, 500 (2017) 5 May 2017 1of1 RESEARCH

◥ (PNIPAAM), that gels above its LCST. The LCST/ REVIEW UCST of the thermo-responsive polymers may be tuned by their molecular weight, ratio of the co- polymers, and/or the balance of the hydrophobic/ MATERIALS SCIENCE hydrophilic segments (22, 23). Noncovalent molecular self-assembly is adop- ted as a common strategy, especially for protein- Advances in engineering hydrogels based hydrogels (24). Weak noncovalent bonding mechanisms—including hydrogen bonds, van der Yu Shrike Zhang1,2,3 and Ali Khademhosseini1,2,3,4,5* Waals forces, and hydrophobic interactions—cause macromolecules to fold into scaffolds possessing Hydrogels are formed from hydrophilic polymer chains surrounded by a water-rich well-defined structures and functionality. A nota- environment. They have widespread applications in various fields such as biomedicine, soft ble example is the hierarchical self-assembly of electronics, sensors, and actuators. Conventional hydrogels usually possess limited collagen, the most abundant protein in the human mechanical strength and are prone to permanent breakage. Further, the lack of dynamic body (Fig. 1B). The assembly procedure relies on cues and structural complexity within the hydrogels has limited their functions. Recent the regular arrangement of the amino acids in developments include engineering hydrogels that possess improved physicochemical collagen molecules that are rich in proline or hy- properties, ranging from designs of innovative chemistries and compositions to integration droxyproline (25). These molecules facilitate the of dynamic modulation and sophisticated architectures. We review major advances in formation of the triple helix termed tropocollagen. designing and engineering hydrogels and strategies targeting precise manipulation of their Subsequent stabilization upon further packing of properties across multiple scales. the tropocollagen subunits into fibrils/fibers even- Downloaded from tually forms a collagen hydrogel (24–26). Inspired by this mechanism, biomimetic supramolecular ydrogels, a class of three-dimensional (3D) veloped not only to achieve dynamic modula- formulations have been designed that can follow networks formed by hydrophilic polymer tion of the hydrogels that can evolve their shapes similar hierarchical self-assembly processes, such chains embedded in a water-rich environ- along predefined paths, but also to control the as collagen-mimetic peptides (26)andthosebased ment, possess broadly tunable physical and spatial heterogeneity that will determine local- on peptide-amphiphiles and hydrogelators (27, 28). H http://science.sciencemag.org/ chemical properties (1–3). A variety of nat- ized cellular behaviors, tissue integration, and de- Spontaneous physical gelation may alternatively urally derived and synthetic polymers can be pro- vice functions. depend on chelation or electrostatic interactions. cessed into hydrogels, from those formed through Alginate, a polysaccharide composed of a-L-guluronic physical entanglement to ones stabilized via co- Hydrogel formation acid (G) and b-D-mannuronic acid (M) residues valent cross-linking. Hydrogels may be further Hydrogels are formed by cross-linking polymer derived mainly from brown algae, is a prominent tuned toward the integration of chemically and chains dispersed in an aqueous medium through example of hydrogel formation based on chelation biologically active recognition moieties such as a myriad of mechanisms, including physical en- (29). The G-blocks in alginate rapidly gel in pres- stimuli-responsive molecules and growth factors tanglement, ionic interactions, and chemical cross- ence of certain species of divalent cations such as that enhance their functionality. The versatility linking (Fig. 1). Majority of the physical gelation Ca2+ or Ba2+ in an “egg-box” form, in which pairs of of the hydrogel system has endowed it with wide- methods depend on the intrinsic properties of the helical chains pack and surround ions that are 30 spread applications in various fields, including polymers. This dependence limits the ability to locked in between (Fig. 1C) ( ). The sophisticated on February 25, 2018 biomedicine (1–3), soft electronics (4, 5), sensors fine-tune the attributes of hydrogels, but gela- structures of natural macromolecules often ren- (6–8), and actuators (9–14). As an example, when tion is easy to achieve without the need for modi- der them varying degrees of electrostatic charges a hydrogel is created with proper stiffness and fying the polymer chains and is usually easy to along the backbone. Although many natural poly- bioactive moieties, it modulates the behavior of reverse when necessary. Conversely, chemical ap- mers are negatively charged at neutral pH be- the embedded cells (15, 16). In addition, chemically proaches can be used to allow for more control- cause of the presence of carboxyl groups (such as active moieties and light-guiding properties allow lable, precise management of the cross-linking hyaluronic acid and alginate), some may also pre- hydrogels to sense substances of interest and per- procedure, potentially in a spatially and dynam- sent positive charges when amine groups dominate form on-demand actuation (7, 17). ically defined manner. (such as gelatin and chitosan). In contrast, synthet- Advances in chemical methods—such as click Many natural polymers such as seaweed-derived ic polyelectrolytes offer much better control over chemistry, combination of gelation mechanisms, polysaccharides and proteins from animal origin the electrostatic properties. A common example anddopingwithnanomaterials—have produced form thermally driven hydrogels. During the gela- is the poly(L-lysine) (PLL)/PAA pair (31, 32). When hydrogels with more controlled physicochemical tion process, physical entanglement of the poly- the solutions containing polyelectrolytes of oppo- properties. Furthermore, the static and uniform mer chains occurs in response to a temperature site charges are mixed, the polymer chains entangle microenvironments characteristic of traditional change. This change is typically caused by an al- to form complexes that become insoluble because hydrogel matrices do not necessarily replicate teration in their solubility and the formation of of mutual shielding (Fig. 1D) (33). the hierarchical complexity of the biological tissues. packed polymer backbones that are physically rigid Chemical cross-linking is better at stabilizing a Innovative fabrication strategies have been de- (Fig. 1A) (18, 19). An increase or decrease in tem- hydrogel matrix because it allows substantially perature may result in thermal gelation, in which improved flexibility and spatiotemporal precision the transition temperatures are defined as lower during the gelation process as compared with 1Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham critical solution temperature (LCST) and upper physical methods. Chemically active moieties pen- and Women’s Hospital, Harvard Medical School, Cambridge, critical solution temperature (UCST), respectively dant to the backbones or side chains of macro- MA 02139, USA. 2Harvard-MIT Division of Health Sciences (20, 21). The gelation mechanism, however, may moleculesinanaqueoussolutioncanformcovalent and Technology, Massachusetts Institute of Technology, vary with specific types of polymers. Macromole- bonds under proper circumstances to obtain hydro- Cambridge, MA 02139, USA. 3Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, cules exhibiting UCST include natural polymers such gels (Fig. 1E). Conventional mechanisms include USA. 4Department of Bioindustrial Technologies, College of as gelatin as well as synthetic polymers such as poly- condensation reactions (for example, carbodiimide Animal Bioscience and Technology, Konkuk University, acrylic acid (PAA) that gel as the temperature drops chemistry between hydroxyl groups/amines and Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of to below their respective UCSTs. In contrast, some carboxylic acids), radical polymerization, aldehyde Korea. 5Department of Physics, King Abdulaziz University, Jeddah 21569, Saudi Arabia. other macromolecules show LCST behavior, such complementation, high-energy irradiation, and *Corresponding author. Email: [email protected] as synthetic polymer poly(N-isopropylacrylamide) enzyme-enabled biochemistry, among others (34).

Zhang and Khademhosseini, Science 356, eaaf3627 (2017) 5 May 2017 1of10 RESEARCH | REVIEW

strictions exerted by dense cross-links that are otherwise present in pure polymer networks. For example, by use of inorganic clay nanoplatelets as anchors for grafting the polymer chains upon cross-linking, the resulting PNIPAAM hydrogel network could be stretched to up to 1400% its original length (48). The stable grafting points of these clay-PNIPAAM networks was attributed to the tunable surface properties of the clay nano- platelets (48). The filler materials may provide additional functionality, such as enhancement of electrical conductivity (39), or promotion of tissue regeneration (52). Multiple cross-linking mechanisms, often in- volving physical entanglement and chemical bond- ingoftwotypesofpolymerchains,havebeen combined to achieve hydrogels with superior toughness and high fracture energy (41, 53–56). A highly stretchable and tough hydrogel was syn- thesized by mixing alginate, ionically cross-linked

by Ca2+, and covalently cross-linked long-chain Downloaded from polyacrylamide (PAAm). The two components, alginate and PAAm, were further intertwined through covalent bonding between their respec- tive carboxyl and amine groups (Fig. 2A) (41). During the stretching process, the PAAm network remains intact while the alginate network unzips progres- http://science.sciencemag.org/ sively to efficiently dissipate energy, which can Fig. 1. Cross-linking of hydrogels. (A to D) Physical cross-linking. (A) Thermally induced entanglement reform upon removal of the stress, thus exhibit- of polymer chains. (B) Molecular self-assembly. (C) Ionic gelation. (D) Electrostatic interaction. (E)Chemical ing excellent hysteresis and negligible gross defor- cross-linking. mation. As such, this class of hybrid hydrogels could achieve >20 times the elongation of their original lengths with fracture energies of up to Over the past decade, click chemistry—proposed One strategy lies in the use of hydrogels derived 9000 J m−2. Alginate molecules of different chain to be fast, orthogonal, high-yield, and typically from natural proteins that may present excep- lengths may further be coembedded in the alginate- amenable to cells and bioactive agents—has under- tional elasticity (45). For example, elastin is a widely PAAm system in order to synthesize hydrogels gone tremendous development. This class of precise distributed structural protein required to main- containing improved density of chelation, thus

chemicalreactionsaimstoaddressthechallenges tain tissue integrity and confer elasticity and is pres- achieving extraordinarily high fracture energies on February 25, 2018 associated with traditional chemical reactions, such ent in tissues such as blood vessels, heart, bladder, of up to 16,000 J m−1 without compromising the as low yield, prolonged reaction times, and extreme and skin. To this end, recombinant tropoelastin, toughness (38). Introduction of crystallization reaction conditions (35–37). A rich variety of bio- and its enzymatically cross-linked form termed within an entangled polymer network represents compatible click reactions have been devised for elastin-like polypeptides (ELPs), have been engi- another approach to achieve tough and stretch- use in direct bioconjugation and hydrogel forma- neered (46, 47). These engineered elastin-based able hydrogels (49, 57). As an example, rather tion in the presence of cells within the matrices, hydrogels typically possess strong elasticity by than the alginate component, polyvinyl alcohol such as thiol-vinyl sulfone and thiol-maleimide using a pentapeptide repeat, VPGXG, where X (PVA) molecules could be incorporated, which Michael addition reactions, azide-alkyne and azide- is any amino acid except proline (46). They can upon annealing formed crystallites within the alkyne cycloaddition reactions, and thiolene photo- achieve stretchability of up to ~400% their orig- intertwined network of PVA and PAAm (49, 57). coupling reaction (37). inal lengths (47), which is much higher than most The dried network was then rehydrated to form Although each of these gelation methods has existing naturally derived hydrogels. Nevertheless, the tough and stretchable hydrogel, in which the its advantages and limitations, the field has been the high cost of these protein-based materials crystallites of PVA functioned as the zippable, marching toward combining multiple components limits their use for applications requiring large energy-dissipating segments. and/or mechanisms to achieve improved hydrogel hydrogel amounts. In another strategy, molecular sliding was de- formulations (38–42). These hydrogels typically ex- Alternatively, hydrogelsformedthroughhybrid- vised to synthesize extremely stretchable hydro- hibit excellent physicochemical properties, such ization with nanomaterials (39, 48), via crystallite gels with good toughness (50). Hydrogels were as substantially enhanced mechanics, injectability, cross-linking (49), or by mixing multiple compo- derived from a network of structures composed of self-healing, and possibility to undergo dynamic nents (38, 41, 50), may possess substantially im- a copolymer, of N-isopropylacrylamide (NIPAAM) modulation, as further discussed below. proved mechanical properties (51). For all these and sodium acrylic acid (AAcNa), and a-cyclodextrin mechanisms, the key lies in combination of care- conjugated with poly(ethylene glycol) (PEG) Tuning the strength fully selected components that formulate the (Fig. 2B). The free movement and sliding of the Conventional hydrogels typically possess a low hydrogels. Nanomaterials (such as inorganic nano- a-cyclodextrin rings along the polymer chains, en- to intermediate stretchability within a few times particles, carbon nanotubes, and graphene), be- abled by the inclusion of the ionic monomer AAcNa their original length, and fracture energies of cause of their highly tunable surface properties to modulate ionization, rendered the hydrogels <100 J m−2. Applications such as load-bearing bio- and usually strong mechanics, contribute to en- highly stretchable to up to 400 to 800% their orig- materials (39, 43), soft robotics (44), and wearable hanced mechanical performances of bulk hydro- inal lengths. The degree of stretching, however, devices (4–6) have spurred interest in developing gels through interacting with polymer chains was also dependent on the type of cross-linker tough and highly stretchable hydrogels as a class forming the hydrogels (39, 42, 48). The use of nano- used, either hydroxypropylated polyrotaxane cross- of soft and hydrated substrates. materials as cross-linkers would reduce the re- linker (HPR-C) or N,N′-methylenebisacrylamide (BIS).

Zhang and Khademhosseini, Science 356, eaaf3627 (2017) 5 May 2017 2of10 RESEARCH | REVIEW

Strong bonding between hydrogels and solid actions (68). The system is simple and may be dom copolymerization of oppositely charged mono- materials as diverse as metals, glass, ceramics, extended to many charged species of nanopar- mers, were induced to spontaneously form and silicone is required for a wide range of ap- ticles and polymer chains to produce injectable physical hydrogels containing electrostatic bonds plications in areas spanning from biomedicine formulations (61). These shear-thinning hydro- with a wide distribution of strengths. In this sce- to soft electronics (5, 58). Nevertheless, rational gels have found utility in hemostatic dressing nario, the strong ionic bonds serve as semiper- design of a robust interaction between these (60), endovascular embolization (68), tissue engi- manent cross-links to maintain the shape of the two mechanically distinctive classes of materials neering (69),anddrugandgenedelivery(61). hydrogel, whereas the weak bonds dissipate energy has remained historically challenging. Following Systems with electrostatic interactions between through reversible bond breakages and forma- an approach used for making a tough composite polymer chains containing oppositely charged tions, enabling improved toughness as well as hydrogel from intertwined polymer networks, the side groups have also been developed (Fig. 3B) shear-thinning properties. The physical cross- long-chain polymer molecules were further cova- (55). Polyampholytes, synthesized through ran- linking imparted by the electrostatic interactions, lently attached to the surface of a solid through anchored chemical bonds (Fig. 2C) (40, 59). The resistance to scission of the anchored polymer chains during peeling generates the adhesion, and the energy is dissipated by the reversible physical chelation of the ionically cross-linked alginate. This type of bonding may reach interfacial toughness values of over 1000 J m−2, which is comparable with the toughest bonding found between a − tendon and a bone in humans (800 J m 2). In Downloaded from addition to potential applications in biomedicine, optimization of hydrogel bonding with other types of materials, such as elastomers, may further ex- pedite the development of the field of soft elec- tronics. For example, stretchable soft electronics have been designed by coating an electrolyte- http://science.sciencemag.org/ containing hydrogel layer onto a substrate of elas- tomeric tape without strong bonding between the two surfaces (4).

Break and heal Hydrogels capable of regaining their initial me- chanical properties and undergoing autonomous healing upon damage can be useful in various applications such as in biomedicine, external coatings, and flexible electronics. Such properties

typically rely on reestablishing molecular inter- on February 25, 2018 actions in the water-rich microenvironment after the hydrogel is subjected to external forces or dam- age. This ability is achieved by the incorporation of moieties that present reversible but typically strong physical interactions in the hydrogel’s polymer network. These mechanisms can range from electrostatic interactions (55, 60, 61) and hydrogen bonding (62, 63) to hydrophobic inter- actions (64, 65) and caged guest-host interac- tions (66, 67). The resulting hydrogels may further be stimuli-dependent that are modulated by, for example, pH (62) or oxidative state (66). Besides functioning as chemical cross-linkers, clay nanoplatelets may also be physically mixed with polymer chains to form shear-thinning hydro- Fig. 2. Tuning the mechanics of hydrogels. (A) Stretchable hydrogel made from long-chain polymers gels. Synthetic clay nanoplatelets that have an and reversible physically cross-linked polymers. (Right) The hydrogel could be stretched to 21 times its anisotropic charge distribution, positive along initial length, where the stretch l is the final length of the unclamped region divided by the original length; the edges and negative on the two surfaces, may stress-stretch curves of the alginate, PAAm, and alginate-PAAm hydrogels, each stretched to rupture, result in a net negative charge in an aqueous where the nominal stress s is defined as the force applied on the deformed hydrogel, divided by the cross- medium (60). Therefore, when complexed with sectional area of the undeformed hydrogel. [Adapted with permission from (41), copyright 2012 Nature positively charged supramolecules such as gela- Publishing Group] (B) Stretchable hydrogel based on a sliding ring mechanism. (Right) Photo of an tin type A at neutral pH, a shear-thinning hydro- elongated NIPAAM-AAcNa-HPR-C hydrogel, and stress-strain curves of different hydrogels: (i) NIPAAM- gel is formed because of the strong electrostatic AAcNa-BIS [0.65 weight % (wt %)], (ii) NIPAAM-AAcNa-BIS (0.065 wt %), (iii) NIPAAM-AAcNa-HPR-C interactions between the clay nanoplatelets and (2.00 wt %), (iv) NIPAAM-AAcNa-HPR-C (1.21 wt %), and (v) NIPAAM-AAcNa-HPR-C (0.65 wt %). The thepolymerchains(Fig.3A).Theresultinghydro- percentages denote those of the cross-linkers. [Adapted with permission from (50), copyright 2014 Nature gel temporarily reduces its viscosity upon applica- Publishing Group] (C) Tough bonding of hydrogels with smooth surfaces. (Right) The peeling process of a tion of shear stress to endow injectability through tough hydrogel chemically anchored on a glass substrate, and curves of the peeling force per width of a plug flow process, while returning to its original hydrogel sheet versus displacement for various types of hydrogel-solid bonding. [Adapted with permission viscosity at low shear owing to electrostatic inter- from (59), copyright 2016 Nature Publishing Group]

Zhang and Khademhosseini, Science 356, eaaf3627 (2017) 5 May 2017 3of10 RESEARCH | REVIEW however, further enable these types of hydrogels of the hydrogel network (Fig. 3C) (62). It further ated and can form hydrogen bonds, whereas in to self-heal even upon complete damage between allows a pH-dependent healing capacity of the their deprotonated state at pH values above pKa, two freshly cut or aged surfaces (55). damaged hydrogels. In the case of carboxyl pendant the charged groups exhibit strong electrostatic Self-healing through hydrogen bonding can groups, at pH lower than the pKa values of the repulsion. A host-guest system forms another basis be attained by incorporating side groups such molecules (where Ka is the acid dissociation of generating self-healing hydrogels. The classical as amine or carboxyl into the polymer backbone constant), the functional groups become proton- molecule cyclodextrin, when conjugated to the poly- mer backbone, functions as the host to engulf hy- drophobic moieties dangling as side chains of another polymer backbone to achieve self-healing. If an environment-responsive moiety—such as PAAm-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 upon introduction of favorable stimuli (Fig. 3D) (66). Whereas current strategies in synthesizing self- healing hydrogels heavily rely on physical and chemical methods, we envision the emergence of bioinspired approaches in which bioactive species may as well facilitate the healing process of the

hydrogels upon damage. In nature, self-healing Downloaded from of biological tissues occurs frequently through an orchestrated cascade of cellular events, in which cells residing in the local microenvironment actively respond to the wound by selectively clearing the debris, secreting bioactive cues, and depositing new extracellular matrix (ECM) molecules to achieve http://science.sciencemag.org/ healing. Therefore, rational combination of the physicochemical methods and biologically active species (such as enzymes, microorganisms, or even mammalian cells) may represent a new self-healing strategy.

Dynamic modulation No single existing system, whether it is biological or synthetic, is purely static. Rather, dynamic evo- lutions are universally present, in which the capa-

bility to modulate hydrogel behavior in a temporally on February 25, 2018 dependent manner becomes particularly attractive. When engineering biologically relevant systems, it is strongly desirable that the hydrogel micro- environment evolves with time through intrin- sically embedded moieties that respond to external stimuli, either in an explicitly user-defined fashion or imposed by coexisting cells. Various approaches have been devised in the past decade toward in- corporation of dynamic physiological signals within hydrogel platforms to precisely modulate cell- matrix interactions (1, 70). Dynamic modulation of hydrogels further opens up opportunities for construction of actuators and robotics (9–12). Photopatterning is a long-established approach for introducing heterogeneity in a hydrogel vol- Fig. 3. Shear-thinning and self-healing hydrogels. (A) Shear-thinning hydrogel through nanocomposit- ume by means of controlled spatial immobiliza- ing. (Bottom) Recovery of the nanocomposites was observed by subjecting the hydrogel to alternating tion of bioactive molecules (71–73). The addition high and low strain conditions (100% strain and 1% strain) while monitoring the moduli of the composite. of specific biochemical cues into the matrix through [Adapted with permission from (60), copyright 2014 American Chemical Society] (B to D) Self-healing photopatterning allows localized presentation of hydrogels based on ionic interactions, hydrogen bonds, and host-guest coupling. (B) Ionic interactions. signals for cells to respond. These signals may (Bottom left) Recovery of the sample for different waiting times. (Bottom right) Self-healing between include growth factors, proteins, hormones, cell- either two freshly cut surfaces (red and blue) or a fresh and an aged surface (white) of samples; [Adapted adhesion peptides, and cell-repelling moiety (73). with permission from (55), copyright 2013 Nature Publishing Group] (C) Hydrogen bonds. (Bottom) The Notably, the reverse process—on-demand release healed hydrogels at low pH separate after exposure to a high-pH solution (with pH > 9), and the separated of certain moieties from a hydrogel matrix—is also hydrogels could reheal upon exposure to acidic solution (pH < 3). [Adapted with permission from (62), sometimes desirable to trigger temporal changes copyright 2012 the National Academy of Sciences] (D) Host-guest coupling. (Bottom) The cut hydrogel within the hydrogel. In realizing this capability, spread with NaClO aqueous solution did not heal after 24 hours, but readhesion was observed 24 hours photodegradation (as opposed to photocross linking), after spreading reuced glutathione aqueous solution onto the oxidized cut surface. [Adapted with has been developed on the basis of the inclusion permission from (66), copyright 2011 Nature Publishing Group] of photolabile moieties that undergo photolysis

Zhang and Khademhosseini, Science 356, eaaf3627 (2017) 5 May 2017 4of10 RESEARCH | REVIEW upon two-photon illumination (Fig. 4A) (74). These phologies along a certain path, to produce the final bioactive molecules in conjunction with multiple moieties can be designed to be biocompatible, shape and architecture working in a coordinated cell populations functioning in synergy. For ex- enabling photodegradation of the hydrogels in the manner that renders the entire organism func- ample, almost all organs in the human body con- presence of bioactive molecules or cells, as well tional. Through meticulous selection of polymers tain repeating building units that assemble into as time-dependent degradation. Photopatterning thatmakeupthehydrogels,theycanalsobepro- distinctive compartments and interfaces [for ex- and photodegradation may be combined to achieve cessed to ensue bioinspired shape-morphing capa- ample, the myocardium, endocardium, and peri- full control over the dynamics of the spatial patterns bility upon desired stimulation (12) such as humidity cardium in the heart (98, 99) and the lobules in a hydrogel system (36, 75). For example, a photo- (10, 84), temperature (85), pH (86, 87), ionic strength constituting the liver (99, 100)]. Most organs are reversible patterning strategy can be used to first (87, 88), magnetism (89, 90), or light (11), among embedded with perfusable vasculature of sophis- define spatial distribution of biomolecules through other stimulants. A heterogeneous or multilayer ticated tortuosity that supplies oxygen and nu- photomediated ligation, followed by subsequent configuration containing materials with varying trients and transports bioactive molecules (such removal upon further exposure to a different light degree of responsiveness is typically required to as growth factors and hormones). Such complex- (Fig. 4B) (75). More recently, attention has been enable a dynamic shape change. For example, a ity further extends to the architecture of the blood focused on dynamic modulation of biophysical cues dual-layer hydrogel formed by two polymers with vessels, themselves defined by layered structures inside the hydrogel such as matrix mechanics, distinctive swelling capacities (for example, PEG of tunica intima, tunica media, and tunica externa which can be used to tune the behavior of encap- and alginate) presents directional bending upon (99). Consequently, engineering a biomimetic sulated cells (76, 77). absorption of water (84). Another example is the organ requires the capacity to reconstitute its Cells may be another source of external stimuli combination of thermo-sensitive (such as PNI- native characteristics and physiology through to trigger a dynamic alteration of the hydrogel PAAM) and thermo-inert hydrogel materials that rational design that ensures compositional, ar- matrix in which they reside (78–80). In the human modulate bidirectional bending of fabricated struc- chitectural, mechanical, and functional accura- body, a critical characteristic of the ECM lies in its ture, at temperatures above or below which the cies. Soft actuators and electronic devices made Downloaded from susceptibility to proteolysis triggered by the re- thermo-sensitive hydrogel component is shaped from hydrogels may similarly demand spatiotem- siding cells to facilitate their mobility and sub- (Fig. 4D) (85). Also, the inclusion of pH-sensitive poral control to render heterogeneity required for sequent remodeling of the microenvironment. moieties into a hydrogel leads to pH-mediated functionality. To achieve this functionality within a hydrogel, swelling or deswelling (86). Although these models Microengineering provides a versatile approach a specially designed linker moiety typically sen- rely on the intrinsic properties of the polymers to engineer well-defined hydrogels from miniatur- sitive to an enzyme can be copolymerized into the that constitute hydrogels, functional materials ized building blocks through programmed spon- http://science.sciencemag.org/ hydrogel so as to allow for localized protease- such as magnetic nanoparticles (90) and carbon taneous assembly. Multiple types of hydrogel units, mediated degradation of the polymeric network (79). nanomaterials (11) may be added to the hydro- individually fabricated to contain desired cell types In combination with a growth factor-sequestering gel matrices in order to achieve shape actuation and physicochemical cues, can be used to mimic the component, these cell-instructive hydrogel matrices upon external stimuli of magnetic field and light- microtissue units in the human organs. Earlier ver- are able to reversibly immobilize growth factors induced local heating, respectively. sions of packing strategies rely on shape comple- on demand (81). The concept of protease-mediated Cell-mediated traction force can also contribute mentarity in which these different blocks can be degradation has been recently taken a step for- to shape-morphing of hydrogels (91). Carefully processed by using, for example, lithographic or ward by implementing a negative feedback loop designed microstructures seeded with cells are photopatterning techniques. These engineered through a combinatory effect of an enzyme-labile able to self-fold into predefined architectures building blocks of microgels with complemen- moiety and an enzyme inhibitor coembedded in through origami (92). Alternatively, cells that tary geometrical configurations subsequently un- 80 the hydrogel matrix ( ). In this scenario, an enzyme- spontaneously contract enable mechanical move- dergo directed assembly into the bulk form under on February 25, 2018 sensitive peptide [for example, to matrix metal- ment of the substrate on which they reside, leading a combinatory influence of external energy, surface loproteinase (MMP)] is used as the linker of the to generation of soft bioactuators (93, 94). Whereas tension, and microgel dimensions at liquid-liquid hydrogel network, whereas the enzyme-inhibitor earlier bioactuators were mainly based on elasto- interfaces (Fig. 5A) (101). Assembled microgels [for example, tissue inhibitor of MMPs (TIMP)] is meric materials, hydrogel materials have recently maybefurthercross-linkedtostabilizethebulk simultaneously liberated by the same cleaving event. attracted increasing attention because of their architecture. Using this strategy, arrays of ring- Concurrent inclusion of the enzyme-inhibitor pair better biocompatibility. To this end, a combina- shaped microgels were assembled into a 3D tubu- induces balanced local activation and inhibition tion of myocytes (skeletal muscle cells or cardiomyo- lar construct with interconnected lumens in a of the enzyme activity, therefore modulating the cytes) and hydrogels that enable communications biphasic system via stimulation applied by fluid hydrogel degradation (Fig. 4C). Compared with between these cells is usually adopted. For example, shear (102). Microgels could also be assembled conventional unconfined stimuli-responsiveness, carbon nanotube–composited gelatin methacryloyl into large-scale structures on a surface patterned the introduction of the feedback system clearly hydrogels have been processed into flexible sub- with hydrophilic and hydrophobic regions at high demonstrates its advantage in providing more strates as large as centimeter scales, and cardio- fidelity (103). A class of hydrophobic hydrogels has precise manipulation of the matrix properties. myocytes spontaneously and synchronously beating been proposed by stabilizing a layer of hydropho- This mechanism, based on cell-responsive MMPs, on the surfaces allow these bioactuators to ex- bic microparticles on the surface of conventional has also been adapted for other types of hydro- hibit rhythmic contraction or extension and swim- hydrogels to allow them to float on aqueous media gels [such as PEG-heparin (82)]. Alternatively, ming behaviors (95, 96). Engineered skeletal muscles, (104). These hydrophobic hydrogels with comple- instead of introducing stimuli-responsive moi- when bound to bioprinted hydrogel frameworks, mentary shapes could self-assemble because of hy- eties, selective modification of the macromole- could also actuate and move the devices in defined drophobic interactions directly on the surface of cules before formation of hydrogels may allow for patterns (13, 14). These bioactuators relying on cell water when brought into proximity with each other. manipulation of hydrogel properties. For exam- traction forces may be remotely controlled by a Although this type of self-assembly is convenient ple, when heparin was selectively desulfated, the variety of external stimuli such as electrical signals and scalable, it does not confer strong control over resulting hydrogels demonstrated altered release (95–97)andlight(14, 94). To be suited for building the assembly process because it is limited by the profiles of affinity-immobilized growth factors com- robust bioactuators, hydrogels must be both bio- sole reliance on the miniaturization of interfacial pared with the release in the case of pristine hep- compatible and mechanically stable. surfacefreeenergy(101). arin (83). This strategy can be extended to chemical Alternatively, the self-assembly properties of modificationssoastofurtherenabledynamiccon- Shaping the hydrogels nucleic acids provide a powerful approach for the trol of biomolecule sequestration and release. Biological tissues are exceedingly complex and fabrication of sophisticated patterns, structures, and Morphogenesisisanimportantfeatureofliving may possess a hierarchically assembled architec- devices. Sequence complementarity in DNA/RNA organisms. As an organism develops, it alters mor- ture featuring a variety of nano- or microscale strands can be encoded to induce self-organization

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on February 25, 2018

Fig. 4. Dynamic modulation of hydrogel microenvironment. (A) Photo- (rTIMP-3) activity was measured by its ability to inhibit a recombinant MMP-2 degradation through photolabile moieties. (Bottom left) Hydrogels demon- (rMMP-2) solution, in which (left) bound rTIMP-3 did not substantially reduce strated surface erosion upon irradiation. (Bottom right) Hydrogel was eroded rMMP-2. (Bottom) Hydrogels with (solid symbols) and without (open symbols) spatially through masked flood irradiation, where feature dimensions were encapsulated rTIMP-3 were incubated with (squares) or without (triangles) quantified with profilometry after different periods of irradiation. Scale bars, rMMP-2, in which encapsulated rTIMP-3 attenuated rMMP-2-mediated hydro- 100 mm. [Adapted with permission from (74), copyright 2009 American gel degradation. [Adapted with permission from (80), copyright 2014 Nature Association for the Advancement of Science] (B) Dynamic photopatterning Publishing Group] (D) Thermo-responsive shape morphing hydrogel. (Bottom) and photorelease. (Bottom) Patterned primary antibodies are visualized with Serial images of a closed gripper with poly(propylene fumarate) (PPF) seg- a fluorescent secondary antibody, which were subsequently photoreleased to ments on the outside opening as the temperature was decreased to below form a secondary pattern. Scale bar, 3 mm. [Adapted with permission from 36°C and then folding back on itself to become a closed gripper, but with the (75), copyright 2015 Nature Publishing Group] (C) Cell-responsive cleavage PPFsegments on the inside. Scale bar, 2 mm. [Adapted with permission from and anticleavage negative feedback system. (Bottom) recombinant TIMP-3 (85), copyright 2015 American Chemical Society]

Zhang and Khademhosseini, Science 356, eaaf3627 (2017) 5 May 2017 6of10 RESEARCH | REVIEW into a predefined structure, when the comple- plethora of molecular assemblies from synthetic other complex shapes (106, 110). This concept has mentary segments pair up under proper physical nucleic acids have been generated such as tubes been adapted to assemble microgel units, sim- conditions (105). On the basis of this principle, a (106–108), ribbons (108), lattices (107, 109), and ilar to the abovementioned approach but in a much more precisely controlled manner. In this sense, the “giant DNA glue” is decorated onto the prescribed surfaces of a nonspherical hydrogel unit to produce asymmetric glue patterns on het- erogeneous surfaces. These microscale building units, through combination of the molecular pro- grammability of the DNA glue and the shape controllability of the microgels, can achieve multi- plexed assembly of complex structures of hydrogels across multiple scales (Fig. 5B) (111). The assembly of diverse hydrogel structures—including dimers, linear chains, and large-scale networks—could potentially be used to build hierarchical tissue architectures and biomedical devices. Nucleic acid–directed self-assembly may further be adapted for programmed synthesis of 3D biological tis- sues (112). Instead of hydrogels, dissociated single

cells may be chemically functionalized with oli- Downloaded from gonucleotides, which are complementary to DNA sequences coated on desired surfaces to allow for cellular assembly into the third dimension via a layer-by-layer approach. The oligonucleotides were designed to be degradable, thus achieving on-demand reversible binding once the micro- http://science.sciencemag.org/ tissues formed (112). Three-dimensional printing represents another facile technique for constructing volumetric objects (113, 114). Three-dimensional printing empowers extremely high precision through the robotic manip- ulation of the prepolymer (the ink), thus allowing for reproducible manufacturing of objects with minimal aberration in their shape, architecture, and functionality. The 3D printing technology was initially introduced in the form of stereolithog-

raphy, in which defined objects are manufactured on February 25, 2018 through a pull-out procedure from a liquid bath of prepolymer via layer-by-layer photopatterned cross-linking (115). Precise control of locally avail- able oxygen concentrations during the stepwise radical polymerization further enables continuous manufacture of large-sized 3D shapes at substan- tially improved speed (116). Using such a manu- facturing principle, hydrogel objects with complex 3D architecture and multiplexed materials may be generated with high spatial resolution (100, 117). Complementary to stereolithography, nozzle- based 3D printing strategies typically provide Fig. 5. Shaping macroscale hydrogels. (A) Self-assembly through shape complementarity. (Bottom) higher degrees of flexibility in the production of (Top row) Hydrogels assembled by capillary force. Scale bar, 200 mm. (Bottom row) Hydrophobic hy- hydrogel objects with defined architecture. The drogels assembled through hydrophobic interactions on the surface of water. Scale bars, 5 cm. [Adapted ink, deposited through positioned ejection from with permission from (101), copyright 2008 the National Academy of Sciences, and (104), copyright 2016 an orthogonally movable printhead, builds 3D American Chemical Society] (B) Self-assembly using sequence-complementing DNA glues. (Bottom) structures in a layer-by-layer manner. Whereas (Top row) DNA-assisted hydrogel self-assembly across multiple length scales. Scale bars, 1 mm. (Bottom stereolithography solely relies on photopolymer- row) Directed formation regular dimers and T-junction based on their surface DNA glue patterns. Scale ization, a rich variety of cross-linking mechanisms bars, 1 mm. [Adapted with permission from (111), copyright 2013 Nature Publishing Group] (C)Embedded may be used for nozzle-based printing through nozzle-based bioprinting. (Bottom left) A continuous hollow knot written with fluorescent microspheres in meticulous selection of the inks combined with a granular hydrogel. Scale bar, 3 mm. (Bottom right) A freely floating hydrogel jellyfish model retrieved rational design of the printhead. For example, after printing and dissolution of the granular hydrogel. (Inset) The printed structure before the removal of physical cross-linking of alginate by Ca2+ can be the supporting hydrogel matrix. Scale bars, 5 and (inset) 10 mm. [Adapted with permission from (125), achieved with a core-sheath nozzle design to co- copyright 2015 American Association for the Advancement of Science] (D) 4D biomimetic printing deliver the two components during the extrusion through a printed dual-layer structure with unbalanced swelling. (Bottom) Time-lapse photographs show- procedure, whereas photocrosslinking may still ing bioprinted simple flower-like structures undergoing shape-morphing during the swelling process, for be applied to the methacryloyl-modified hydro- the double layers of different orientations. Scale bars, 5 mm. [Adapted with permission from (140), gels after printing (118, 119). Alternatively, shear- copyright 2016 Nature Publishing Group] thinning inks may be directly printed, followed

Zhang and Khademhosseini, Science 356, eaaf3627 (2017) 5 May 2017 7of10 RESEARCH | REVIEW

by subsequent chemical cross-linking (120). Also, ments. In tackling the mechanical instability caused REFERENCES AND NOTES they may function as sacrificial templates in certain by swelling of the hydrogel network, an “anti- 1. J. A. Burdick, W. L. Murphy, Moving from static to dynamic 3D printing applications so as to obtain hollow swelling” (or “nonswellable”)hydrogelconsisting complexity in hydrogel design. Nat. Commun. 3, 1269 (2012). hydrogel structures after selective removal (121–123). of a network integrating hydrophilic and thermo- doi: 10.1038/ncomms2271; pmid: 23232399 135 2. D. Seliktar, Designing cell-compatible hydrogels for Tough and highly stretchable hydrogels can also responsive polymer units was proposed ( ). It biomedical applications. Science 336, 1124–1128 (2012). be printed into complex architecture (124). 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Zhang and Khademhosseini, Science 356, eaaf3627 (2017) 5 May 2017 10 of 10 Advances in engineering hydrogels Yu Shrike Zhang and Ali Khademhosseini

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

Wet, soft, squishy, and tunable Hydrogels are highly cross-linked polymer networks that are heavily swollen with water. Hydrogels have been used as dynamic, tunable, degradable materials for growing cells and tissues. Zhang and Khademhosseini review the advances in making hydrogels with improved mechanical strength and greater flexibility for use in a wide range of applications. Downloaded from Science, this issue p. eaaf3627 http://science.sciencemag.org/

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