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4D bioprinting: the next-generation technology for biofabrication enabled by stimuli-responsive

materials

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2017 Biofabrication 9 012001

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Biofabrication 9 (2017) 012001 doi:10.1088/1758-5090/9/1/012001

TOPICAL REVIEW

4D bioprinting: the next-generation technology for biofabricationenabled by stimuli-responsive materials

Yi-Chen Li1,2,3,7, Yu Shrike Zhang1,2,3,7, Ali Akpek1,2,4, SuRyon Shin1,2,3,8 andAli Khademhosseini1,2,3,5,6,8

1 Biomaterials InnovationResearchCenter, Division of Engineering inMedicine, Department ofMedicine, BrighamandWomen’sHospital, HarvardMedical School, Cambridge,MA02139,USA

2 Harvard-MITDivision ofHealth Sciences andTechnology,Massachusetts Institute of Technology, Cambridge,MA02139,USA3 Wyss Institute for Biologically Inspired Engineering, HarvardUniversity, Boston,MA02115,USA4 Department of Biomedical Engineering, Istanbul Yeni Yuzyil University, Istanbul 34010, Turkey5 Department of Physics, King AbdulazizUniversity, Jeddah 21569, Saudi Arabia6 College of Animal Bioscience andTechnology, Department of Bioindustrial Technologies, KonkukUniversity, Hwayang-dong,

Kwangjin-gu, Seoul 143-701, Korea7 YCLi andY SZhang contributed equally to this work.8 S R Shin andAKhademhosseini contributed equally as corresponding authors to this work.

E-mail: sshin4@partners.org and alik@bwh.harvard.edu

Keywords: 4Dbioprinting, 3D bioprinting, shape-changing, smart biomaterials

AbstractFour-dimensional (4D) bioprinting, encompassing awide range of disciplines including bioengineer-ing,materials science, chemistry, and computer sciences, is emerging as the next-generationbiofabrication technology. By utilizing stimuli-responsivematerials and advanced three-dimensional(3D) bioprinting strategies, 4Dbioprinting aims to create dynamic 3Dpatterned biological structuresthat can transform their shapes or behavior under various stimuli. In this review, we highlight thepotential use of various stimuli-responsivematerials for 4Dprinting and their extension intobiofabrication.We first discuss the state of the art and limitations associatedwith current 3Dprintingmodalities and their transition into the inclusion of the additional time dimension.We then suggestthe potential use of different stimuli-responsive biomaterials as the bioink thatmay achieve 4Dbioprintingwhere transformation of fabricated biological constructs can be realized.Wefinallyconcludewith future perspectives.

1. Introduction

Engineered artificial tissues that biologically andphysiologically resemble their counterparts in vivo canbe used to repair damaged or diseased tissues [1–4].Alternatively, in vitro tissue models have also recentlyattracted increasing attention with an aim of improv-ing current platforms to more accurately predictresponses of pharmaceutical compounds in thehuman body, thus expediting the drug developmentprocess [5–10]. To this end, three-dimensional (3D)bioprinting has emerged as a versatile technique tocreate biomimetic tissue constructs at unprecedentedspatial precision and freedom [11–13].

In 1986, the first 3D printing technology wasreported by Hull and co-workers [14]. They descri-bed a process termed stereolithography (SLA), where3D constructs could be fabricated via layer-by-layer

crosslinking of the resin through synchronized UVexposure and upward movement of the stage. Thislayer-by-layer technique enabled direct generationof complex objects at very high precision not achiev-able with the traditional manufacturing andmachin-ing procedures. A series of other 3D printingmethods have also been developed over the yearssuch as fused deposition modeling [15], selectivelaser melting [16], and selective laser sintering tech-niques [17–19]. While the conventional 3D printingstrategies such as inkjet, microextrusion, and laser-assisted printers were used for generation of non-cellular structures, more recently these techniqueshave been further developed to enable 3D fabricationof biological structures directly encapsulating cellsand bioactive agents, including various biomimetictissues such as the blood vessels, liver, heart, andtumors [20–22].

RECEIVED

4 July 2016

REVISED

12 September 2016

ACCEPTED FOR PUBLICATION

7October 2016

PUBLISHED

1December 2016

© 2016 IOPPublishing Ltd

Most native tissues exhibit complex 3D shapes,structures, microarchitectures, and extracellularmatrix compositions, as well as possessing uniquefunctions that are achieved through dynamic changesin tissue conformations. For example, the heart has afour-chambered configuration, which along withrhythmic contraction governed by the intrinsic elec-trical system, exhibits a strong and regular pumpingbehavior to achieve blood circulation through thebody [23]. In addition, similar electrical signals trans-mitted from the brain can induce themovement of themusculoskeletal system, such as the peristaltic movesof esophagus or gut that consist of sequential, alternat-ing waves of contraction and relaxation of alimentarywall smooth muscles, which act to propel food along[24–26]. The movements can also be directed by otherbiochemical cues such as nitric oxide that leads tovasodilation and caffeine that causes vasoconstriction.Most these dynamic conformational changes of tissuesthat render them functional are caused by built-inmechanisms that respond to intrinsic and/or externalstimuli, adding a fourth dimension to the biologicalsystem.

Therefore, the conventional 3D structures featur-ing static properties and behaviors obtained from 3Dbioprinting may not suffice the need for use in biome-dicine. The recently developed four-dimensional (4D)bioprinting technology based on 3D bioprinting butwith the embedded ability of shape transformationmay solve this challenge and more accurately mimicthe dynamics of the native tissues. In this strategy, sti-muli-responsive biomaterials are integrated with the3D bioprinting technology to fabricate biologicallyactive constructs that can alter their shapes upondesired stimulation to achieve prescribedfunctionality.

In this review, we highlight recent advances in thedevelopment of 4D bioprinting techniques and theirpotential applications in biomedicines. We discuss thestate of the art 3D printing and bioprinting modalitiesalong with their associated challenges. In addition, thepotential use of different stimuli-responsive biomater-ials, including thermal-, humidity-, electrical-, magn-etic-, and photo-sensitive materials as programmablebioinks, is discussed for their capability to achieve 4Dbioprinting. We finally propose future directions andperspectives to further develop novel 4D bioprintingplatforms in mimicking architectures and function ofnative biological tissues.

2. Current 3Dbioprinting techniques andtheir limitations

2.1. 3Dprinting andbioprintingmodalitiesA variety of techniques have been developed for 3Dprinting [21, 27–29]. Fused deposition modeling is themost common modality due to its low cost andsimplicity [30]. In fused deposition modeling, the

melted material is extruded out through a nozzle in alayer-by-layer manner until a desired 3D model iscreated.Materials suitable for fused depositionmodel-ing include thermoplastics such as poly(lactic acid)(PLA). However, fused deposition modeling generallypossesses relatively low resolution and long operationtimes. Laminated object manufacturing is anothertechnique used for 3D printing. During a typicalprocedure, a belt of target material is cut out by thelaser in the desired shape and repeated in a layer-by-layermanner, where all the layers are adhered togetherby the assistance of heating [31]. Laminated objectmanufacturing is a fast fabrication methodology butthe substantial amount of waste material during theprinting process has increased its cost. In selective lasermelting, the printing material in the powder form fillsthe reservoir, which is subsequently exposed to a high-energy laser thatmelts the powders into desired shapes[32]. Repeated melting and solidification cycles willeventually lead to the fabrication of 3D constructs.Similar to selective laser melting, selective laser sinter-ing also shapes models by laser [33], while the mostcommonmaterials that are employed in selective lasersintering are metals and ceramics [34]. Similar toselective laser melting, the granulated material fillingthe reservoir is exposed by laser. Unlike selective lasermelting, the major difference of selective laser sinter-ing lies in that the granules of the material are sinteredinstead of melting, leaving larger grains and theporosity of the fabricated objects [35]. Additionally,SLAhas also beenwidely used as a versatile 3Dprintingtechnology [27]. It is an additive manufacturingmethod, during the process of which a photosensitivepre-polymer is crosslinked layer-by-layer using pat-terned light, synergized with upward pulling of theprinted object from the reservoir [28]. Recently thisbioprinting technology has been adapted for applica-tions in biofabrication. For example, Shaochen andcolleagues developed a digital light processing-basedstereolithographic process to create multi-componentconstructs with human induced pluripotent stemcells-derived hepatocyte-like cells in a biomimeticliver lobule pattern [36]. SLA printing enables highexquisiteness and resolution in the range of0.05–0.15 mm.

Although these methodologies allow for con-venient 3D printing of thermoplastics, metals, andceramics, they are not necessarily compatible with bio-materials embedded with cellular components. Dur-ing the past decade tremendous progress has beenmade in the development of 3D bioprinting, whereboth cells and bioactive molecules especially those inthe form of hydrogels are integrated with 3D printers[11–13, 37–39]. Three common techniques of 3D bio-printing including inkjet, microextrusion, and laser-assisted printing, are illustrated in figure 1 [11]. Theinkjet printing relies on ejection of a liquid bioink inthe droplet form forced to build in the 3D volume on asubstrate, through either piezoeletric [40] or thermal

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[41] actuation (figure 1(a)). This type of drop-on-demand bioprinter typically presents high printingspeed at low cost. Another common modality for 3Dbioprinting is microextrusion where the bioinks arephysically extruded by amechanical [42] or pneumatic[43] dispensing system (figure 1(b)). Themajor advan-tages of microextrusion printers include the con-tinuity of the extruded bioinks rather than thediscontinuous liquid droplet for the inkjet printers,thewide viscosity range of the bioinks that can be used,and high cell densities that may be achieved. Anotherwidely used modality for bioprinting is termed laser-induced forward transfer [44–46]. This system con-sists of a laser-energy absorbing layer and bioink layer.When the focused laser pulses on the absorbing layersuch as gold or titanium, a droplet containing thebioink and encapsulated cells is forced to deposittowards the collecting substrate to form the desiredpattern (figure 1(c)). Since laser-induced forwardtransfer does not need a nozzle, the clogging problemwith nozzle-based bioprinters can be avoided [47–49].

2.2. Challenges associatedwith 3DbioprintingDue to the versatility of the technique, 3D printinghas been extensively used inmany areas such as thosein electrical, mechanical, and biomedical engineer-ing. However, there are several challenges associatedwith 3D printing especially when it comes tobiomedicine. For example, the choice over thematerials (i.e. the bioinks) has to be accurate toensure robust transformation into 3D structureswhile maintaining sufficient viability and function-ality of the embedded cells coherently [13]. On onehand, bioprinted structures may exhibit inhomoge-neous cell densities in case the bioinks have overlylow viscosities [50] and on the other hand, high-viscosity bioinks may cause increased shear stressesduring the extrusion process that may affect cellviability and functions [51]. To this end, a micro-fluidic technique has been recently proposed forconvenient deposition of low-viscosity bioinks,

where a carrier sheath flow was used to assist theextrusion of the bioink delivered through the core ofthe printhead [52]. Moreover, a new techniquetermed embedded bioprinting further allowed directanti-gravity writing of 3D freeform structures in asupporting matrix, which could be selectivelyremoved to retrieve the bioprinted volumetricobjects [53–55].

The inability of most current 3D bioprinting plat-forms to fabricate biological structures with high com-positional complexity creates another major obstacle,since most systems can only achieve bioprinting of asingle bioink during each deposition process. Thus,efforts have been presented during the last decade forthe development of multi-material bioprinting mod-alities [52, 56–59]. For example, the Sun, Lewis, andAtala Groups separately developed multi-nozzle bio-printers capable of sequentially extruding several typesof bioinks as well as polymeric scaffolding materialsfor the generation of complex tissue architectures[56, 59, 60]. Alternatively, a single microfluidic print-head design devised by the Khademhosseini and LewisGroups enabled continuous and/or simultaneousdeposition of two types of bioinks for production ofheterogeneous or gradient structures [52, 58, 61].

While these two challenges require further techno-logical developments in materials and instrumenta-tion, the third obstacle described below might bereadily overcome by rational integration of existingtechnologies. The conventional 3D bioprinted micro-environment may not be able to elicit proper biologi-cal responses since the structures cannot activelytransform once printed, in contrast to the highlydynamic and constantly changing morphologies ofnative tissues against surrounding stimulants [62, 63].Therefore, it is crucial to develop new methodologiesfor bioprinting objects that can undergo prescribedtransformation as needed, by elegantly combiningcurrent 3D bioprinting platforms with a rich variety ofalready known stimuli-responsive biomaterials, thusleading to the emergence of 4Dbioprinting.

Figure 1.Threemost common 3Dbioprinting techniques: (a) inkjet bioprinters, (b)microextrusion bioprinters and (c) laser-assistedbioprinter. (Reprintedwith permission from [12]Copyright (2013)Wiley.)

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3. Transition to 4Dprinting andbioprinting

Shape transformation is regarded as the fourth dimen-sion added onto printed 3D structures.Mechanisms ascomplex as those in facilitating protein folding andorigami that employ directed transformation areexciting and expected to evolve into 4D bioprinting inthe years to come [64–69]. One of the simplestmechanisms that can be adopted for 4D printing andbioprinting however, lies in the deposition of hetero-geneous materials that possess differential properties(e.g. swelling) to achieve folding and unfolding underproper conditions. Indeed, two pioneering examplesusing such strategy have opened new avenues forimproved designs of engineered tissues, biomedicaldevices, and soft robotics, among others.

Tibbits and colleagues envisioned a combinationof insights on both design and engineering aspect, andindicated that material programmability, multi-mat-erial printing techniques, and meticulous designs foraccurate transformations are vital requirements for4D printing [70]. In an early demonstration on 4Dprinting by the team, the transformation of the printedgrid structures was reported. Materials capable ofexpanding to different degrees were used as the jointsbetween two adjacent rigid segments, where the fold-ing direction and angle of the joints could be con-trolled by regulating the material properties andtuning the position of the angle limiters (figure 2(a)).Consequently, under suitable stimulus the printedgrid could transform into the desired sinusoidal waveor hyperbolic surface (figures 2(b) and (c)). Moresophisticated transformation could be designed usingthis approach, such as the gradual conversion of theshape resembling ‘MIT’ into a pre-defined form ‘SAL’(figure 2(d)).

Recently, a 4D biomimetic printing techniquebased on hydrogel materials was further reported byLewis and co-workers [71]. They developed a printableink consisting of synthetic hectorite clay, nanofi-brillated cellulose, and the N, N-dimethylacrylamideor N-isopropylacrylamide (NIPAM) monomers, forfabrication of shape-morphing natural analogues suchas the flowers, bracts, and leaves that could transformunder stimulus. The technology relied on the aniso-tropic swelling properties controlled by aligning theorientations of cellulose fibrils within the hydrogel.During the process of printing, the extruded fiberspossessed anisotropic swelling behaviors along thelongitudinal direction since the cellulose componentunderwent a shear-induced alignment [72] as the inkwas squeezed through the nozzle [73]. They harnessedthe anisotropic swelling property of this bioink to pre-cisely control their relative orientations and locationsin bilayer structures to achieve different folding uponswelling (figure 2(e)). Figure 2(f) clearly demonstratesthe folding of printed bilayer patterns into 3D flower-like structures using this biomimetic 4D printing

technique. In particular, when the petals were printedin a bilayer structure with a 90°/0° angle arrangement,the resulting structure could mimic the closure of theflower (figure 2(f) (i)); on the contrary, if the fibers inthe petals were printed at a 45°/45° configuration, aconvoluted folding path was obtained (figure 2(f) (ii)).Combination of these folding mechanisms furtherenabled multiple shape-transformation events for thegeneration of a structure mimicking the orchid Den-drobium helix, by discrete dispositions of each petalwhen swollen inwater (figures 2((f) (iii))–((f) (vi))).

Besides swelling, Studart and colleagues proposedanother interesting idea of 4D printing based onmagnetic materials. They developed an ink consistingof poly(urethane acrylate) (PUA) doped withmodifiedaluminum platelets that could respond to an exter-nally applied low magnetic field [74]. The aluminumplatelets (shown in brown color) dispersed in the prin-ted architecture underwent directed pattern transfor-mation upon the application of amagnetic field duringthe printing and subsequent curing processes(figure 2(g)). Microscopic images further confirmedthat the local orientations and alignments of the plate-lets well matched the patterns as programmed(figure 2(g) (iii) (vi)).

These few pioneering examples clearly demon-strated the possibility to incorporate the additionaltime scale into 3D printing to achieve transformationof fabricated objects along prescribed paths. However,these pioneering 4D printing techniques still requireovercoming major challenges in mimicking the com-plex dynamic deformation of the native tissues such asthe pumping behavior of the heart and the peristalsismoves of the esophagus/gut, among others. Combin-ing bioactive agents and specific cell types with the 4Dprinting strategies provides a potential method tosolve these challenges. To this end, it is necessary todevelop a library of biocompatible stimuli-responsivematerials that possess good cytocompatibility, providemechanical support, and enable proper biophysical/biochemical signaling for the cells to induce pro-grammed deformation under physiological stimuli,therefore achieving 4D bioprinting. In the next sectionwe will introduce several categories of stimuli-respon-sive biomaterials that can be potentially used for 4Dbioprinting to generate biomimetic tissue constructswith extended applications in biomedicine.

4. Stimuli-responsivemechanisms for 4Dbioprinting

As the field of 4D bioprinting is just starting to emerge,we expect its rapid expansion in the years to come. Thesignificantly enhanced usability and functionality ofthe bioprinted objects due to their capability to trans-formwith timewill likely findwidespread applicationsin areas including but not limited to tissue engineer-ing, regenerative medicine, bioelectronics, robotics,

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actuators, and even medical devices. Since there islimited number of demonstrations currently availablein this new field, here we will review a series oftransformation mechanisms based on either cells orstimuli-responsive biomaterials and discuss theirpotential integration with the 4D bioprintingtechnology.

4.1. Cell traction force (CTF) for 4DbioprintingIn addition to using material properties to providetransformation ability to the structures, anotherpossibility lies in the utilization of cells as the activefolding element to move 3D microstructures into

pre-defined shapes. Cell origami is a technique thatharnesses living cells as the driving force to imple-ment the transformation of cell-laden patterns[75, 76]. CTF is considered as the major mechanismof cell origami due to the contractile force exerted bythe cells originating from actin polymerization andactomyosin interactions [75]. CTF plays a criticalrole in many biological processes to regulate thecellular behaviors such as wound healing [77],angiogenesis [78], metastasis [79], and inflammation[80]. Also, CTF regulates several fold-forming pro-cesses of tissues during the development of anorganism. For instance, the tension plays an

Figure 2. (a)The transformation of 4Dprinted objects. The transformation of the printed grid into (b) a sinusoidal wave and (c) ahyperbolic surface. (d)A time-varying curve showed the change of shape fromMIT to SAL. (Reprintedwith permission from [70]Copyright (2014)Nature PublishingGroup.) (e)The schematic of programming the anisotropy of ink. (f)Themorphologies of flowerfabricated by biomimetic 4Dprinting. (i) 90°/0° bilayer. (ii)−45°/45° bilayer. The (iii) print bah, (iv) printed structure, and (v)swollen structure of a flower inspired by (vi)Dendrobiumhelix. (Reprintedwith permission from [71]Copyright (2016)NaturePublishingGroup.) (g) Internal helicoidally staircase was fabricated by 3Dprinted object containingmagnetic nanoplates. (g) (i), (ii)Opticalmicrographs of the top layer of the structure show the local different alignment of aluminumplateletsmatch the programmedarchitecture. (g) (iii), (vi)Opticalmicrographs of the bottom layer of the structure, showing the in-plane oriented platelets (III) andout-of-plane oriented platelets aligningwith programmed directions (IV–VI). (Reprintedwith permission from [74]Copyright(2015)Nature PublishingGroup).

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important role in gastrulation and neurulation dur-ing the body’s development [81, 82].

Recently, Takeuchi and colleagues have appliedbasic principles of cellular origami to fabricate a 3Dmicrostructure induced by CTF [83]. They developeda model where cells were seeded across two micro-plates attached onto a piece of glass substrate(figure 3(a)). Upon detaching from the glass direc-tional folding of these microplates could be initiated(figure 3(b)). Subsequently, 3D cell-laden micro-structures were fabricated using an assembly of multi-ple two-dimensional (2D)microplates (figure 3(c)). Itwas further possible to create various 3D cell-ladenmicrostructures by properly designing the geometricalpatterns of the assembled 2D microplates. In part-icular, with fibroblasts the group was able to success-fully generate various hollowmicrostructures such as acube, a dodecahedron, and a cylindrical helical tube,without using flexible joints or supporting materials(figures 3(d) and (f)). As indicated in figure 3(f), thecylindrical tube was fabricated by rolling up micro-plates along the diagonal lines, whose diameters couldbe controlled by modifying the angles of these lines.Using such a method, vessel-like structures were alsofabricated by culturing vascular cells including humanumbilical vein endothelial cells and human arterialendothelial cells. In addition, it was also demonstratedthat this cell-enabled folding technique had the cap-ability to produce 3D microstructures such as thedodecahedron in a high-throughput manner(figure 3(g)). Confocal images confirmed that hollowstructures were present within these cell-laden dode-cahedrons (figure 3(g)). These features have indicatedthe feasibility to fold 2Dmicroplates into 3D cell-ladenmicrostructures by taking advantages of CTF. Cell ori-gami is a simple technique that potentially enables 4Dbioprinting, and when multiple cell types are com-bined complex cell-laden structures mimicking theirnative counterparts may be spontaneously formed in apost-printing procedure.

4.2. Smart biomaterials for 4Dbioprinting4.2.1. Humidity-responsive materialsHumidity-responsiveness is widely present in nature.For example, when the humidity is low the flake ofpinecone will spontaneously open to assist the seeddispersion and the wheat awns will twist due to theheterogeneity present in these structures [84–86]. Asthe humidity shifts in the surrounding environment,some of the biological systems release or absorbmoistures. These phenomena thus inspired the devel-opment of humidity-responsive materials [87, 88].Naumov and co-workers developed a new materialwhosemotion could be quickly actuated upon humid-ity change [89]. They combined the poly(ethyleneglycol) (PEG)-conjugated azobenzene derivative(PCAD) and agarose (AG) (PCAD@AG) to fabricate acomposite substrate. AG is a natural material capable

of rapid absorption of water but low desorption. Onthe contrary, PCAD was used to decrease the rate ofabsorption and enhance desorption to establish ahumidity-responsive system. This design was criticalfor the rapid locomotion of PCAD@AG film, whichdeflected upon exposure to the environmental humid-ity (figure 4(a)).

Thiele and co-workers further formulated ahumidity-responsive cellulose-based material [90].They fabricated a bilayer film consisting of a cellulosestearoyl ester with a low substitution degree of 0.3(CSE0.3) and a high substitution degree of 3 (CSE3).When CSE0.3 was exposed to humidity on one side, itssurface absorbed water molecules and generated hor-izontal and vertical swelling forces ( Fs) (figure 4(b)).On the contrary, CSE3 was hydrophobic and hadtemperature-responsive properties. Therefore, thebilayer film possessed both hydrophilic and hydro-phobic surfaces each exposed to one side. As shown infigure 4(b), the CSE0.3/CSE3 bilayer film was initiallymaintained at 55 °C and 5.9% relative humidity toachieve steady states of both layers and stabilize thefilm in a flat configuration. Subsequently, as the temp-erature was cooled down to 22 °Cwith relative humid-ity increased to 35% the bilayer film started to bendfrom the CSE3 side, which slightly contracted; theCSE0.3 layer followed to roll up due to swelling toeventually form a tight roll of the film. This processcould be readily reverted when the bilayer film wasagain exposed to its original environment at 55 °C and5.9% relative humidity. Additionally, the authors indi-cated that making the CSE0.3 film with a gradientthickness could result in a non-symmetric bendingmovement for the cellulose-based materials. Theseexamples recall the earliest versions of 4D printingtechnology and the concept of a multi-layer structurecan be easily adapted to many future designs of 4Dbioprinting, in conjunction with biocompatiblehumidity-responsive materials. However, the cell cul-ture environment with a constantly high humidity (i.e.within themedium)will limit the transformation abil-ity of each material to only a single time. In addition,the cells require a specific osmotic pressure. Therefore,the extent of shape-changing for humidity-responsivematerials may be limited due to the limitation in thehumidity/osmotic pressure that can be applied to theconstructs. These challengesmight be resolved by tun-ing the sensitivity of the humidity-responsive materi-als that can readily transform within the range of cellendurance.

4.2.2. Temperature-responsivematerialsThermo-responsive materials can employ the exogen-ous temperature changes as stimuli to achieve shapetransformation [91]. In particular, the transformationprinciple of thermo-responsive hydrogels is based ontheir wettability and solubility alteration following thechange of temperature [92, 93]. Temperature respon-siveness is probably one of the most commonly

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investigated mechanisms to achieve user-definedfunctionality [94, 95]. Hu and colleagues provided abasic idea to fabricate a double-network hydrogel that

combined two components with drastically differentthermal properties induced by crystallization of onecomponent [93, 96–98]. Based on this concept, a

Figure 3.The 3D cell-laden structure formed by cellular origami. (a)Cells adhered on the twomicroplates. (b)The illustration ofmicroplate was folded byCTF. (c)Three different types of 3D structures were formed byCTF. (d)–(f)Three transformed geometricstructures: regular tetragon, regular dodecahedron, and cylindrical tube. (g) Fluorescent image ofmultiple dodecahedrons. (h)Confocal cross-sectional images of the hollow dodecahedron structure. Green: actin, blue: nucleus. Scale bars = 50 μm. (Reprintedwith permission from [83]Copyright (2012)Public Library of Science.)

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hydrogel was fabricated with poly(vinyl alcohol)(PVA) capable of crystallization interpenetrated with achemically crosslinked PEG (figure 5(a)) [98]. Whenthe hydrogel containing 70% PVA and 30% PEG(PEG-30) went through three cycles of freezing/thawing (PEG-30-3), the morphology of the hydrogelcould be stabilized as a helix. The hydrogel was able totransform back to a straight line after 15 s of immer-sion in hot water (90 °C), at which temperature thecrystalline domain of PVA melted. Additionally,deformed angle and x-ray diffraction analyses indi-cated that the crystallinity would increase when thefreezing/thawing cycle was increased [99]; thereforePEG-30 with only one freezing/thawing cycle (PEG-30-1) showed fastest response compared to othersamples that underwent more cycles. These resultsrevealed that the degree of thermo-responsivenesscould be regulated by the crystallinity of the polymersforming the hydrogel.

Xu and co-workers combined dynamic ionicbonds and covalent bonds to develop a new thermal-responsive thiol-ene-functionalized polybutadiene(PB) rubber [100]. As shown in figure 5(b), the ionicbonds were formed by the functionalized PB-COOHand PB-NH2 and then crosslinked by different con-centrations of trithiol crosslinker (i.e. synthesized PB(SPB)-0%, SPB-2%, and SPB-6%). The SPB-6%hydrogel was processed into a permanent spiral-likestructure by photocrosslinking the SPB film rolled ona rod. When the spiral hydrogel was heated to 60 °C,the structure transformed into a temporary flat shape,which could be further fixed upon cooling.

Interestingly, it was observed that the spiral-like struc-ture spontaneously recovered after being reheated to60 °C. The mechanism of transformation might becaused by the thermo-reversibility of the ionic bonds.During the heating process, the broken ionic bondsresulted in the transformation of the film, which werethen reconstructed after cooling to lock the shape;during the reheating stage, the bonds became brokenagain and the stored strain was released, and conse-quently the recovery stress drove the film to return toits original spiral structure.

Poly(N-isopropylacrylamide) (pNIPAM) isknown as one of the most used thermo-responsivematerials [101]. In pNIPAM hydrogel the polymerchains become hydrophilic when the environmentaltemperature is decreased to lower than the low criticalsolution temperature [102]. Breger and colleaguesphotocrosslinked acrylic acid-functionalized pNI-PAM (pNIPAM-AAc) to polypropylene fumarate(PPF) [103]. As the temperature was raised to above36 °C, the pNIPAM-AAc component transformed to ahydrophobic state to expose the PPF segments andthus achieve shape transformation (figure 5(c)). At thesame time water was also expelled as the temperaturewas elevated resulting in increased ratio of dry weight-to-swollenweight of the hydrogel.

Moreover, Chen and colleagues utilized4-hydroxybutyl acrylate (4HBA) as a crosslinker togenerate a pNIPAM-OHhydrogel with a gradient por-ous morphology (figure 5(d)) [104]. By increasing thetemperature to 40 °C, the sliced hydrogel disk foldedupwards to assume a tubular shape within 35 s,

Figure 4. (a)The shape change of PCAD@AG film before (a) (i) and after (a) (ii) exposure of humidity. Humidity-induced bending ofPCAD@AG film and pure AGwith sizes of (a) (iii) 2 cm×0.5 cm×100 μmand (a) (iv) 2 cm×0.5 cm×200 μmloadedwith acargo of 370 mg. (a) (v)The doped versus undoped PCAD@AG film in humidity-induced bending. (a) (vi)Effect of cargoweight onPCAD@AG film. (Reprintedwith permission from [89]Copyright (2015)Nature PublishingGroup.) (b)The illustration and shapechange of the humidity-responsive CES film is triggered by the absorption and desorption ofwater. (Reprintedwith permission from[90]Copyright (2015)Nature PublishingGroup.)

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Figure 5. (a) (i)Photographs of thermal transformation of PEG30-3. (a) (ii)Plots of deformed angle versus temperature and (a) (iii)x-ray diffraction patterns of PEG-30 hydrogel underwent different freezing/thawing cycle. (Reprintedwith permission from [98]Copyright (2015)AmericanChemical Society.) (b)The image andmechanismof shapememory behaviors of SPB-6%at a cooling(20 °C) and heat/reheat (60 °C) temperature. (Reprintedwith permission from [100]Copyright (2015)Royal Society of Chemistry.)(c) Schematics of the transformion behaviors of the thermal-responsivemicrogripper. (Reprintedwith permission from [103]Copyright (2015)AmericanChemical Society.) (d) (i)Themorphology change of a gradient porous hydrogel at 20 °Cand 40 °C. (d)(ii)The thermal responsive time of the hydrogel stripwas placed at 40 °Cwater. (d) (iii)The reversible size change and responsive timeof the hydrogel after thermal stimulation. (d) (iv)The end-to-end distance of the hydrogel strip after the alternative heating/coolingstimulation. (d) (v)The folding behaviors of sliced hydrogels with random thickness after thermal stimulation. (Reprintedwithpermission from [104]Copyright (2015) JohnWiley & Sons).

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through anisotropic motion caused by the gradient inpore sizes. The hydrogel also presented a strong rever-sibility, where no significant differences after 10 cyclesof shape changes were observed. Also, Spinks and co-workers used a thermal-responsive alginate/pNIPAMink to print a smart valve to control the flow of waterby the hydrogel valve at 20 °C (swollen, closed state)and 60 °C (shrunken, open state) [105].

These different types of thermo-responsive mate-rials have been widely used in various applicationsincluding tissue engineering, drug delivery, and sen-sing, and are thoroughly characterized for their prop-erties including for example, biocompatibility.Therefore, they potentially offer a great value for 4Dbioprinting where shape transformation can beachieved by temperature alteration within a reason-able range. However, these pNIPAM-based thermal-responsive materials lack bioactivity and generallyrequire relatively high transformation temperaturesleading to insufficient cell viability and functions. Thischallenge might be solved by using copolymers withpNIPAM to reduce the transformation temperature toaround 37 °C to maintain high cell viability, and bycombining bioactive peptides (e.g. arginine–glycineas-partic acid (RGD)) andmolecules (e.g. growth factors)to improve the cell adhesion and growth.

4.2.3. Electrical andmagnetic-responsivematerialsMost electrical-responsive smart materials usually relyon their electrically conductive characteristic. Theproperties of such materials can be regulated by theintensity or the direction of the externally appliedelectrical field [106]. This feature provides new appli-cations for biomimetic or bioinspired systems sincethe materials can change their size and/or shape inresponse to the electric stimulation [107, 108]. Forexample, dimensional changes in some electricallyconductive materials such as polyaniline [109] andpolypyrrole (PPy) [110] have been confirmed due tothe electrochemical doping that transports ionsbetween the materials and the surrounding electrolyteunder applied electrical potential, thus facilitatingshape transformation using these materials. Saido andcolleagues presented a method where PPy film wasdopedwith tetrafluoroborate, and then connected by acopper wire and a silver paste on the two ends,respectively (figure 6(a)) [111, 112]. Figure 6(b) showsa folded accordion-like construct, which was com-pressed and annealed to fix its shape. Upon stimula-tion by the electric potential, the PPy constructsexpanded rapidly due to the desorption of water vaporby Joule heating, and achieved a maximum strain of147% [103]. The elongation of the construct underelectrical stimulation could be further controlled bythe balance of the forces. A biomorphic robot wasaccordingly created by connecting two accordion-likeconstructs in series. Plastic plates were assigned aspawls for this biomorphic robot. When a voltage of3 V was applied for 5 s, the front pawl was pushed to

move forward through expansion of the first accor-dion while the rear hook was fixed by the ratchet teethon the substrate to prevent it from sliding backwards;when the electric field was turned off for 10 s, thecontraction force pulled the rear hook forward.

Carbon nanotubes (CNTs) are a class of bio-compatible one-dimensional materials possessingexcellent conductivity [113, 114]. Du and colleaguescoated a sheet of CNTs with a shape-memory polymerVeriflex® [115]. Figure 6(c) shows the recovery of theshape-memory composite blended with 1.8 g CNTnanopaper under the application of an electric currentof 0.09 A. It was observed that the flat shape-memoryspecimen was bended to a ‘U’-like temporary shape,but restored to its original shape within 120 s uponrelease of the current. Therefore, it was confirmed thatthe electrical conductivity and the speed of electro-active response of the shape-memory composite couldbe significantly improved by CNTs. This informationindicated that electrical-responsive materials mightimprove the electrical property of other materials andbe useful for the applications in shape transformationormemory.

Besides, magnetic field is broadly used in the bio-medical applications [116, 117]. For example, magn-etic-responsive ferrogels have been used to control therelease of a number of drugs [118, 119]. Mooney andcolleagues recently developed an alginate-based scaf-fold with magnetic-sensitive capability and 3D inter-connected macropores for drug and cell delivery[120]. To increase the adhesive and release abilities,they conjugated a cell-binding domain, RGD withalginate and iron oxide particles were embeddedwithin the RGD-modified alginate. Under the magn-etic field, strong and rapid compression could be acti-vated for this macroporous ferrogel to drive theoutward movement of water from the internal pores,triggering the release of cells or biological agent. Asshown in figure 6(d), comparing the alginate-basedferrogels with the two pore sizes, the nanoporous fer-rogels reduced only 5% in height at 38 Am−2, whilethe macroporous ferrogels was compressed nearly70% when subjected to the same magnetic field. Fur-thermore, the release of the cells from these ferrogelscould also be controlled by the adhesion properties ofthe alginate matrix. Upon stimulation of a cyclicmagnetic field, more cells could be released from thescaffold when the RGD modification degree wasreduced for alginate in the ferrogels.

Furthermore, composite biocompatible inksbased on amixture of CNTs ormagnetic particles withhydrogels have been recently developed for bioprint-ing of both 2D patterns and 3D structures [114, 120].The bioprinted constructs were able to support theadhesion and growth of cells while showing high elec-trical conductivity. It is expected that, these bioinkswhen programmed to deposit into desired structurescombined with proper electrical or magnetic stimuliwill make 4D bioprinting possible. Although the

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Figure 6. (a)The fabrication process of electrical-responsive construct (a) (i) before and after annealing at (a) (ii) 0 V and (a)-(iii) 2 V.(b) (i)–(v)The serial action images of the biomorphic robot. (Reprintedwith permission from [112]Copyright (2013) JohnWiley&Sons.) (c)Photographs of the shape-memory effect of shape-memory polymer (SMP)material with 1.8 g carbon nanofiber (CNF)nanopaper. (Reprintedwith permission from [115]Copyright (2013) IOPPublishing.) (d) (i)Ananoporous ferrogel decreased itsheight of 5%under a verticalmagneticfield. (d) (ii)Themacroporous ferrogel reduced its height of nearly 70%under the samemagnetic field. (e) SEM images of a freeze-driedmacroporous ferrogel at undeformed/deformed states. Scale bar=500 μm. (f)Mechanism ofmarcoporous gel deformation and release of cells carried by outflowingmedium. (g)Proliferation and spreading of thereleased cells from the ferrogels replanted on the culture dish. (Reprintedwith permission from [120]Copyright (2010)NationalAcademy of Sciences.)

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electrical-/magnetic-sensitive materials have thepotential to be applied to 4D bioprinting, local chan-ges of pH value and temperature of themediummightoccur during the electrical or magnetic stimulations.To prevent the changes that may cause adverse effectson cells, it is necessary to optimize the voltage of theelectrical field and intensity of themagnetic field whentriggering the transformation of the 4D bioprintingstructures.

4.2.4. Light-responsivematerialsLight-sensitive materials may convert externallyapplied optical stimulant intomechanical responses inamore precise manner than the previously mentionedstimuli since light can be conveniently pinpointed tothe location of interest [121, 122]. Optical stimuli maybe applied to a localized region at extremely highspatial resolution, where the light intensity can directlymodulate the behaviors of these materials. Photo-isomerization and photodegradation in the polymerchains are the most important mechanisms for light-responsive materials [123–125], which have beenwidely applied in switches, artificial muscles, androbotics [126]. Recently, CNT and graphene-basedmaterials are considered as good candidates for theirusage as light-sensitive elements due to the largearomatic (π-configuration) interface that endowsthemwith excellent optical responsiveness (i.e. photo-thermal effect) in addition to the electrical properties[114, 127, 128]. Chen and colleagues combinedreduced graphene oxide (rGO), CNT, and poly(dimethylsiloxane) (PDMS) to fabricate a crawler-typerobot [129]. When one side of this robot was facedwith a light source it started to expand due to heating,while the transformation on the shaded side wasmuchsmaller due to the lack of irradiation, thus inducting

the robot to continuously roll forward (figure 7(a)).This observation revealed that the rGO/CNT-basedrobot had the capability to rapidly and continuouslyrespond to optical stimulation.

Moreover, Zhu and co-workers presented otherapplications of light-responsive graphene-basedmate-rials [130]. They used a polydopamine-functionalizedGO nanosheet (GO-PDA nanosheet) to fabricate a flatpattern. The GO-PDA paper implemented a directedtransformation to form a box after 2.6 s upon expo-sure of a near-infrared (NIR) light, which couldreverse back to the original shape after release of thelight (figure 7(b)). Based on the same concept, an arti-ficial hand and a microbot were further assembled. Asshown in figures 7(c) and (d), the artificial hand couldfold, grasp, and even hold an object when stimulatedwith NIR light, whereas the microbot crawled forwardupon the same stimulation. The major advantages ofthis type of light-triggered transformation lies in itsprecision and the ability for remote control. Addition-ally, Tirrell, Anseth, and their colleagues utilized PEG-based hydrogels to create another category of light-responsive materials that possessed photodegradationability. Tirrelland co-workers synthesized a light-responsive hydrogel based on PEG doped with aphoto-labile peptide (N3–DGPQGIWGQGDK(N3)–NH2) through a strain-promoted azide-alkynecycloaddition reaction [109]. They could regulate theimmobilization and release of proteins in desired pat-terns within the 3D hydrogel by the oxime-ligationand the ortho-nitroenzyl ester photocleavage reac-tions, respectively, upon exposure of light at selectedwavelengths. On the other hand, Anseth and co-work-ers developed a photodegradable hydrogel by the useof a photodegradable crosslinker, arginine-glycine-aspartic acid-serine (RGDS) tether polymerized with

Figure 7. (a) (i)Photographs of themotion of a crawler-type robot fabricated by rGO/CNT-coated PDMSdriven by light to passacross an obstacle. (Reprintedwith permission from [129]Copyright (2015) JohnWiley& Sons.) (b)Transformation of a box and arobotic hand, and themovement of amicrorobot, all based onGO-PDAnanopaper triggered by light irradiation. (Reprintedwithpermission from [130]Copyright (2015)AmericanAssociation for theAdvancement of Science.)

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PEGmonomarylate (PEGA) [124]. Under proper lightstimulation, 3D features in the hydrogel could be gen-erated due to localized degradation. Cells were alsoencapsulated in this material where they could bedirected to migrate along the degraded patterns [131].Moreover, they also confirmed that the chondrogenicdifferentiation of human mesenchymal stem cells andglycosaminoglycans production could be enhancedwhen the RGDS tether was cleaved. These results indi-cated that the cells could sense and actively respond tothe changes of the dynamicmicroenvironments.

Light-responsiveness has so far been widelyapplied in a wide range of biomedical applicationssuch as controlled drug delivery or cancer therapy[132]. To extend its applications to 4D bioprinting, itis envisioned that they can be rationally combinedwith existing bioinks to allow for direct bioprintingfollowed by optical stimulation, to achieve desireddeformation or degradation of hydrogels and thus fab-rication of biological constructs and biomimeticrobotics. The strong attenuation of light by biologicaltissues may post a challenge to this strategy causingnon-uniform illumination and transformation. Alter-natively, NIR light sources can be used to improve thepenetration of light in bioprinted photo-sensitive tis-sue constructs.

5. Conclusions and perspective

Overall, significant advancements have been achievedin the evolution of 3D printing technologies duringthe past few decades. In particular, 3D bioprinting thatutilizes 3Dprinters to fabricate objects frombiomater-ials and cellular species have seen tremendous progressin the last 5–10 years.Merging smart biomaterials withinnovative instrumentation, 4D bioprinting is nowexpected to become the next-generation technology tocreate transformable objects for applications in bio-medicine. Essentially, 4D bioprinting endows pat-terned structures with the unique ability to transformby exploiting the shape-changing features of thevarious types of stimuli-responsive biomaterials. Theinnovative technology has paved new avenues tofabricate sophisticated structures with on-demanddynamically controllable shapes by integrating anadditional dimension of time.

However, several challenges remain that need to beaddressed tomake a great leap to this exciting new field.First and foremost, the bioinks have to be optimized toachieve successful bioprinting. The myriad types of sti-muli-responsive biomaterials discussed above, althoughhave been thoroughly investigated for the properties andused to generate constructs via conventional biofabrica-tion approaches, their direct translation for usage as thebioinks may not be straightforward. Ideally a bioinkshould possess a few basic properties [13, 133, 134]: (i)strong biocompatibility to ensure proper cellular

behaviors and functionality for the bioprinted objects;(ii) appropriate rheological parameters to ensure properprintability [11, 135–137]; (iii) suitable stabilizationmechanisms, either physical (e.g. ion-induced, thermal)gelation or chemical crosslinking to ensure the archi-tectural integrity of the bioprinted structures [60, 135];and (iv) the interplay between the cellular viability/func-tion and the dynamic modulation of the bioprintedobjects, where it has to be ensured that the cells can sur-vive the stimuli necessary to endow these 4D bioprintedmaterialswith shape-changing capacitywhile such capa-city should not be diminished by the inclusion of cells.Therefore, engineering the properties of existing sti-muli-responsive biomaterials has become extremelyimportant to render them compatible with the require-ments for use as bioinks, thus achieving 4Dbioprinting.

Furthermore, ways to obtain robust shape-chan-ging capability of the 4D bioprinted constructs posesanother challenge. For example, Tibbits and collea-gues proved that the mechanical properties of printedobjects degraded severely after repeatedly execution offolding/unfolding procedures, where the scaffoldscould only fully recover to its original shape for a smallnumber of repeated processes [70]. To this end, it isimperative to design the mechanics of the stimuli-responsive bioinks and thus printed constructs espe-cially when repeated responsiveness is desired.

Moreover, complexity in the shape transformationalso needs improvement to suit for increasing demandin 4D bioprinting to construct objects of a wide spec-trum of applications. For example, biological tissuestypically require multiple folding processes to achievefull functionality especially for those undergoingdevelopment, and biorobotics may need on-demand,repeated but distinctive folding events to render themdesired utilities. To advance the innovation in 4D bio-printing, it is consequently crucial to incorporaterational computer designs of sophisticated multiplestimuli-responsive procedures in order to furtherboost its dissemination in building complex self-morphing objects for widespread applications in bio-medicine such as tissue engineering, soft robotics, andbiomedical devices.

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