4D printed shape-shifting biomaterials for tissue engineering and regenerative medicine applications

The existing 3D printing methods exhibit certain fabrication-dependent limitations for printing curved constructs that are relevant for many tissues. Four-dimensional (4D) printing is an emerging technology that is expected to revolutionize the field of tissue engineering and regenerative medicine (TERM). 4D printing is based on 3D printing, featuring the introduction of time as the fourth dimension, in which there is a transition from a 3D printed scaffold to a new, distinct, and stable state, upon the application of one or more stimuli. Here, we present an overview of the current developments of the 4D printing technology for TERM, with a focus on approaches to achieve temporal changes of the shape of the printed constructs that would enable biofabrication of highly complex structures. To this aim, the printing methods, types of stimuli, shape-shifting mechanisms, and cell-incorporation strategies are critically reviewed. Furthermore, the challenges of this very recent biofabrication technology as well as the future research directions are discussed. Our findings show that the most common printing methods so far are stereolithography (SLA) and extrusion bioprinting, followed by fused deposition modelling, while the shape-shifting mechanisms used for TERM applications are shape-memory and differential swelling for 4D printing and 4D bioprinting, respectively. For shape-memory mechanism, there is a high prevalence of synthetic materials, such as polylactic acid (PLA), poly(glycerol dodecanoate) acrylate (PGDA), or polyurethanes. On the other hand, different acrylate combinations of alginate, hyaluronan, or gelatin have been used for differential swelling-based 4D transformations. TERM applications include bone, vascular, and cardiac tissues as the main target of the 4D (bio)printing technology. The field has great potential for further development by considering the combination of multiple stimuli, the use of a wider range of 4D techniques, and the implementation of computational-assisted strategies.


Introduction
There is a rising interest in engineering tissues to closely mimic the physiological structure and the composition of their native counterparts, so that they can be used for the repair or regeneration of damaged tissues as well as for the development of highly reliable in vitro disease models.Most tissue engineering approaches use scaffolds, which are matrices supporting cell attachment, proliferation, and extracellular matrix deposition [1,2].Most common conventional techniques for scaffold fabrication include solvent-casting particulate leaching [3], gas foaming [4], fiber bonding [5], melt molding [6], freeze-drying [7,8], and solution casting [9].However, these techniques lack sufficient control over the internal geometry of the scaffold, as well as the tailored distribution and interconnectivity of pores in the scaffolds.
These challenges have been partially addressed by the introduction of three-dimensional (3D) printing, which enables the fabrication of sophisticated, patient-specific, and biomimetic constructs with precise spatial control over the material-forming scaffold in a layer-by-layer and reproducible manner.Common 3D printing methods for tissue engineering and regenerative medicine (TERM) applications include material extrusion (ME), material jetting (MJ), powder bed fusion (PBF) and vat photopolymerization (VP).A detailed overview of the working principles as well as the applications of the biofabrication techniques can be found elsewhere [10][11][12].Printing using cell-laden biomaterials, also known as bioinks, is collectively referred to as (3D) bioprinting.Bioprinting has had some level of success in the fabrication of several tissue types in vitro, including muscle [13], cartilage [14], bone [15], skin [16], vasculature [17], and neuronal tissue [18].
While 3D printing is a promising biofabrication method, it has significant limitations in the engineering of complex out-of-plane features and shapes, and in achieving temporal variations of the properties of multi-material constructs [19].These limitations are particularly evident in the fabrication of tubular or curved living structures [20], which usually requires the use of sacrificial materials and supports [21], introducing additional post-printing processing steps and, thus, increasing the total fabrication time [19,22].For instance, using one of the available 3D printing approaches, such as ME, to fabricate multimaterial, curved and porous scaffolds would require a significant number of print-head changes, for sacrificial material, as well as complex design and coding.Moreover, optimal tissue formation may require temporal changes in the geometry and other properties of the scaffold.These spatial and temporal complexities can be best addressed with four-dimensional (4D = 3D + time) printing as the next generation of biofabrication technologies.
Four-dimensional printing combines the same fabrication principles as 3D printing with the use of stimuli-responsive biomaterials while introducing a post-printing phase.During this phase, the application of one or more stimuli triggers a transformation of the printed structure, inducing either a structural or a functional change in the construct.The term 4D printing was coined in 2013 by Skylar Tibbits, the director and founder of the Self-Assembly Lab (MIT), who demonstrated the humidity-induced self-assembly of 3D printed structures into complex shapes [23].In another early study, Sydney Gladman et al developed a biomimetic hydrogel composite ink from cellulose fibrils embedded in an acrylamide matrix, and used it to 3D print complex, plant-inspired structures that could self-transform into 'flowers' upon immersion in water [24].In this way, 4D printing simplifies the fabrication of curved or tubular structures by using a rectangular bilayer which, upon proper stimulation, self-rolls into a tube or a dome-like structure [25][26][27][28].Furthermore, the combination of 4D printing and shape-memory polymers enables the fabrication of constructs that can be compressed to smaller sizes, to allow their implantation through minimally invasive procedures, and which could regain their original or predesigned size and shape upon reaching the defect site [29].
As 4D printing is gaining momentum, different definitions have arisen.For instance, gradual degradation of 3D printed scaffolds [30] as well as maturation of 3D printed tissues [31] have been considered a type of 4D printing, due to their time-dependency.Furthermore, a functional change of the scaffold, that is not accompanied by a shape-change, has been suggested as an additional dimension to 3D printing, such as controlled enzyme-mediated processes or supramolecular tuning of cell-hydrogel interactions [32,33].In this review, we adopt the definition of Tibbits and regard the fourth-dimension as 'the transformation over time, emphasizing that printed structures are no longer simply static, dead objects; rather, they are programmably active and can transform independently.' [23] Earlier attempts to summarize the advances of 4D printing have been made [31,34,35].However, the swift development of the field requires an equally updated overview.This review aims to highlight the developments of the technology for achieving controlled shape-changing of constructs for TERM applications.Toward this aim, the printing methods, the stimuli, the shape-changing mechanisms as well as the cell incorporation strategies are reviewed and discussed.Finally, future research directions and perspectives of 4D (bio)printing for TERM applications are proposed.

Search method and general output
Four databases were used to search for 4D printing articles: Pubmed, Scopus, Research Gate and Google Scholar.The following keywords were used: '4d bioprinting' OR '4d printing' AND 'shape memory' OR 'shape changing' OR 'shape shifting' OR 'shape morphing' OR 'self folding' OR 'stimuli responsive' OR 'self assembling' AND 'tissue' , and the title of the papers was selected as the search field.Of the functional rather than a spatial change of the printed structure.Furthermore, reference tracking of the included studies lead to the inclusion of four more articles.Finally, 28 papers were considered suitable for the present review.As 4D printing is a relatively new technology, all the included studies were published between 2015 and 2023 and, thus, no time filtering was necessary.The selection procedure as well as the distribution of the papers per publication year are summarized in figure 1(a).The first 4D study in which cells were bioprinted was in 2017, and the first in vivo study was in 2020 in a rabbit model (figure 1(b)).The main target journals are mainly focused on the biofabrication and materials fields.
4D printing-based shape-shifting scaffolds are fabricated with 3D printing techniques and are, subsequently, exposed to one or more external stimuli that trigger their shape transformation.The techniques used in 4D studies include extrusion-based approaches (e.g.fused deposition modelling (FDM)), cryogenic 3D printing (cryo-3DP), direct ink writing (DIW)), stereolithography (SLA), digital light processing (DLP), melt-electrowritting (MEW), and inkjet printing (figures 1(c) and (d)).Unlike conventional 3D printing techniques, bioprinting methods use a modified technical approach that is compatible with the deposition of living cells.The distribution of the studies per year based on the fabrication technique used demonstrates that 4D bioprinting has recently become more relevant (figure 1(c)).From the previously mentioned techniques, only extrusionbased techniques and DLP have been used in studies implementing a shape-shifting 4D bioprinting approach for TERM applications (figure 1(d)).
Shape transformation in 4D (bio)printed scaffolds is triggered by the application of one or more stimuli, post-fabrication.The reported stimuli include hydration, temperature, and light, as well as multiple stimuli with the most studied being temperature and hydration (figures 1(e) and (f)).In particular, hydration is noted to be almost exclusively used as stimulus for 4D bioprinting studies in the last years (figure 1(g)).

Printing methods for shape-shifting scaffolds
The following sections describe each of the techniques used in the reviewed article (table 1).

FDM
FDM has become the most widespread 3D printing technique after it was first introduced in the late 1980s [36].During the printing process, a thermoplastic polymer in powder or filament form is introduced to the heating chamber of the device and is melted (figure 2(a)).Subsequently, the molten polymer is extruded from the nozzle and deposited layer-by-layer on the printing surface to create a scaffold [37].Hendrikson et al used FDM to fabricate scaffolds using shape-memory polyurethane [38].Interestingly, the authors observed that the recovery of the shape of the compressed scaffolds back to its original state resulted in the elongation of mesenchymal stem cells (MSCs) seeded on the structure post-printing, thus demonstrating that even a single mechanical stimulus is sufficient to initiate morphological changes to the adherent cells.In a different approach, shape-changing scaffolds were FDMprinted with graded, interconnected pores, from a castor oil/polycaprolactone (PCL) triol mixture [39].
The graded porosity mimicked the nonuniform distribution of the pores present in natural tissues and promoted the adhesion, proliferation, and differentiation of human MSC (hMSC).Self-fitting porous scaffolds for bone defects have also been printed with FDM using polylactide (PLA) and hydroxyapatite as materials [40,41].

Cryo-3DP
Cryo-3DP, another extrusion-based technique, utilizes the liquid to solid phase transition of a hydrogel ink [42].The extruded material is printed in a liquid coolant, such as liquid nitrogen [43] or an isopropanol bath with solid carbon dioxide (CO 2 ) [42], where it is cooled below its freezing point (figure 2(b)).This method permits the fabrication of stable, complex structures from hydrogels with low viscosity values.Cryogenic 3D printing has been used for the fabrication of shape-changing scaffolds from β-tricalcium phosphate and poly(lactic acidco-trimethylene carbonate) nanocomposite solutions for the treatment of irregular bone defects [29].In that study, the scaffolds were printed at −10 • C and were compressed to a very small volume.They then regained their shape upon exposure to near-infrared irradiation.The implantation of the compressed constructs in rat cranial defects resulted in improved formation of new bone.The majority of the reviewed studies used an extrusion-based fabrication method, whether it was for conventional printing or bioprinting.This trend may be justified by the simplicity of the extrusion printing technologies as well as the option they offer to use hydrogels with or without cells directly as inks (e.g. in DIW, cryogenic 3D printing).

DIW
DIW is a printing technique that utilizes liquid or paste-like materials as inks, which exhibit viscoelastic behaviour in heat-free environments [44,45].This enables the use of a broader range of ink materials, as compared to FDM, that can be applicationspecific.However, DIW requires post-processing steps for solidification that can be time-consuming.Materials that have been used in DIW applications include nanocomposite polymeric solutions [46], hydrogels [47], resins embedded in a supporting matrix [48], metal/bioceramic-based inks [49] and ceramic slurries [50].The inks are placed in a reservoir or cartridge and extruded as continuous filaments using a pneumatic pump or a mechanical piston or screw (figure 2(c)).The printed constructs solidify through either a temperature phase-change or a crosslinking/gelation mechanism.DIW has been implemented for the fabrication of highly stretchable scaffolds with shape memory and self-healing properties, using an ink containing urethane diacrylate and PCL, which can be used as 'patches' for vascular repair [51].In addition, Wang et al [52] used DIW for the fabrication of shape-shifting scaffolds from alginic GelMA and PEGDM [82] acid or alginate (AA)/pluronic F127 diacrylate hydrogels, illustrating their potential application in TERM.

Extrusion-based bioprinting
In extrusion-based bioprinting, the bioink is extruded through one or more nozzles as a continuous filament based on either a pneumatic or mechanical extrusion system, as described for DIW (figure 2(c)).A pneumatic system increases the structural integrity of the printed scaffold [53], while its mechanical counterpart can allow for a more direct control over the bioink flow than the pneumatic system which can suffer from a delay of the compressed gas used.Although this printing method is a subcategory of DIW technologies, the use of inks combined with living cells is a unique feature which has resulted in the term extrusion-based bioprinting to prevail in the TERM field.A wide range of bioinks can be printed using this technology, including hydrogel precursor solutions [54] as well as cell pellets or aggregates [55].The viscosities of the bioinks used to date range between 30 mPa•s to ⩾6 × 10 7 mPa•s [56].However, the mechanical stress developed between the inner walls of the nozzle and the bioink can become very high and have a negative impact on cell viability [20,57].To alleviate this issue, materials with shear thinning behavior (i.e. a decrease in viscosity upon the increase of the applied shear rate) are often preferred for extrusion applications.For such materials, the high shear rate value at the nozzle-tip acts as a printing optimization parameter.As the viscosity decreases under applied shear stress, the bioink flows more smoothly through the nozzle and, upon deposition on the printing platform and in the absence of applied shear stresses, the viscosity increases again resulting in the shape-retention of the deposited filament.Nevertheless, maintaining a high cell viability remains a challenge and requires careful tailoring of certain printing parameters, such as the inner nozzle diameter and geometry, the applied pressure, and the printing speed, depending on the viscosity of the bioink used in this process.A detailed overview on bioink printability and the different parameters that affect it can be found elsewhere [58,59].
As an example of 4D bioprinting, an extrusionbased system developed by Kirillova et al [60] have been used for the fabrication of self-folding tubes composed of two types of cell-laden hydrogels based on AA and hyaluronic acid (HA).When submerged in aqueous media, printed mats with thickness values varying between 2 µm and 16 µm folded (in the order of seconds) into hollow, tubular structures with diameters comparable to the diameters of the smallest blood vessels and could support cell survival for at least 7 days [60].In a different approach, a gelatin methacryloyl bioink was simultaneously extruded through a nozzle to produce thin hydrogel fibers and was exposed to electrical stimulation before crosslinking, which induced gelatin and cell alignment.The fibers were deposited on a self-folding gelatin film to mimic the formation of myotube bundles [28].

SLA
SLA uses a single-beam ultraviolet (UV) laser to crosslink or polymerize a photopolymer resin in a layer-by-layer manner [37].The UV laser is directed to the desired coordinates across the xy plane, tracing the geometry of each layer and curing the resin in a point-by-point manner (figure 2(d)).Using soybean oil, epoxidized acrylate as resin, Miao et al fabricated temperature-activated complex scaffolds using SLA [61] and demonstrated their successful applications as tubular nerve guidance conduits in a later study [25].Moreover, by combining SLA with photolithography, the authors reported the fabrication of selffoldable scaffolds capable of supporting cardiomyogenic differentiation, thus showing the potential of these 4D constructs for cardiac TERM applications [62].

DLP
DLP is a printing method similar to SLA.The primary difference between the two techniques is the light source: DLP uses light from a projector (UV or white light) instead of a laser (figure 2(e)).Additionally, in DLP, the light source remains stationary, curing a complete layer of resin at a time [63].Weems et al used DLP for the fabrication of shape-memory scaffolds from aliphatic polycarbonate urethanes for the induction of adipose tissue repair [64].

SLA-and DLP-based bioprinting
SLA and DLP have also been modified for bioprinting.The working principles of such systems are similar to the ones described before: a photocrosslinkable bioink is solidified layer-by-layer by a light source.High resolution in combination with rapid fabrication make SLA and DLP attractive bioprinting methods.However, certain drawbacks of these methods, such as the continuous photocrosslinking of already printed layers should be considered, as they can affect the functionality of the structure by introducing a stiffness gradient, which can then be used as 4D strategy.For instance, a novel 4D bioprinting system based on DLP and a UV-curable, cell-laden silk fibroin bioink was employed by Kim et al [65] for the fabrication of trachea-mimetic tissue scaffolds that can swell to a desired curved shape.Patterned sheets with various designs, including flowers, Dionaea, clams, and stars were also printed achieving a layer thickness of 40 µm [65].

MEW
MEW is a 3D printing technique based on melt electrospinning principles that combines some elements of electro-hydrodynamic fiber attraction and melts extrusion [66] (figure 2(f)).MEW allows for the consistent production of fibers in the submicron range with a relatively high surface area and can be used to fabricate 3D patterned scaffolds with increased dimensions along the extrusion axis.MEW has been used for the fabrication of methacrylated AA and PCL bilayers capable of self-folding when immersed in aqueous solutions.The tubes were found to support high myoblast viability and proliferation as well as an increased degree of cell orientation along the direction of the PCL electrowritten fibers post-4D transformation [67].
Although not considered as 3D printing, electrospinning has also been used in 4D approaches to create shape-changing structures.For instance, uniaxially aligned PCL-poly(glycerol sebacate) (PCL-PGS) and randomly aligned methacrylated HA fibers were combined in bilayers and self-folded into tubular structures upon immersion in an aqueous buffer [68].In a similar approach, a layer of aligned PCL fibers was electrospun on top of a layer of anisotropic methacrylated alginate (AA-MA) fibers.The scroll-like selffolded mats directed the differentiation of myoblasts into aligned myotubes that contracted upon electrical stimulation [69].

Inkjet-based
Inkjet printing is the most commonly used type of printing for both non-biological and biological applications [20].In inkjet printing, the ink is loaded in a cartridge connected to a printing head, which ejects the material in drops, following the object profile.There are two kinds of drop-on-demand heads: piezoelectric and thermal (figure 2(g)).In thermal systems, a heating element (e.g.thin-film resistor) is attached to the inner walls of the head.When an electrical pulse is applied, a high current flow through the resistor, causing the ink in contact with it to evaporate and form a bubble.The expansion of this bubble increases the pressure in the ink, leading to the ejection of a droplet from the nozzle.In piezoelectric inkjet printing, a piezoelectric actuator is attached to the printing head and, upon the application of a voltage pulse, induces a volumetric change to the ink reservoir.This change leads to a pressure increase which subsequently results in droplet ejection [70].Thermal inkjet printers have a higher printing speed and lower fabrication cost as compared with their piezoelectric counterparts.However, piezoelectric systems possess a high control on the size and directionality of the generated droplets without increasing the ink temperature [20].The selection of the inkjet system depends on the desired properties of the scaffold and the shape transformation strategy.
An inkjet platform for 4D printing was developed by Cui et al to fabricate bilayers synthesized from gelatin-based materials [71].Immersion in aqueous media resulted in the self-rolling of the scaffolds into microtubes that could successfully mimic microvessels and support the encapsulation and proliferation of endothelial cells.Droplet-based printing has also been used for producing synthetic cells that could self-assemble into tissue-like structures capable of folding in a controlled manner after light stimulation, thus showing potential for 4D bioprinting [72].

Stimuli used for shape transformation
Shape transformation in 4D (bio)printed scaffolds is triggered by the application of one or more stimuli, post-fabrication.In this section, we review the different stimuli that have been used for TERM applications.

Hydration
Hydration was identified as one of the most popular stimuli investigated in current 4D (bio)printing approaches for shape-shifting.Many native tissues are responsive to changes in the humidity of their environment.The responses can range in complexity from the curling of drying leaf to self-burial of the Erodium cicutarium seeds [83].Another common example of tissue response to environmental humidity is that of the pine cones.Pine cones, which are natural hygromorphs, are closed in the presence of a humid environment and 'open' when they dry [84].Such hygroscopic behaviors inspired the study and development of humidity-responsive materials as 4D printing inks for the fabrication of shape-changing structures using hydrogels.
Hydrogels are hydrophilic, three-dimensional networks of physically or chemically crosslinked polymers that can absorb and retain a large fraction of water while remaining insoluble.They are composed of an aqueous matrix and, in addition to humidity, may respond to other external stimuli, such as temperature [85] or light [86], as well as demonstrate shape-memory behavior [52].Hydrogels that have been used for the preparation of inks for 4D printing of shape-shifting structures include HA [27,60], AA [27,60,67,80], gelatin [28,71,80], and silk fibroin [65].Stimulation of shape transformation with humidity is achieved by submerging the printed hydrogel scaffolds in a swelling medium (e.g.water, PBS, or cell culture medium).Humidity is a stimulus suitable for both cell-free and cell-laden constructs.For the latter, the swelling medium is a cell culture medium, and the swelling temperature is 37 • C. Apsite et al fabricated bilayer scaffolds consisting of a layer of aligned PCL fibers and a layer of anisotropic AA-MA fibers [69].When submerged in water, the bilayers rolled and formed tubular scrolllike structures, due to the swelling of the AA layer.The diameters of these microtubes and the folding side of the scaffolds could be tuned by changing the orientation of the PCL fibers as well as the thickness of the AA layer.In a different 4D bioprinting approach, cell-laden bars consisting of a layer of oxidized AA-MA and a layer of GelMA exhibited controlled bending upon immersion in growth medium (figure 3(a)) [80].Incorporation of hMSCs in the hydrogel formulations as well as the use of osteogenic medium resulted in bone-like tissue formation.Multiple synthetic polymers can be modified to acquire shape-shifting properties induced by humidity.For instance, Naficy et al investigated the reversibility of shape-change in scaffolds consisting of a bottom part made from a poly(HEMA)-based hydrogel and a top part made from a PNIPAAm-based hydrogel [26].In a recent study, a low-swelling bioink made of AA and HA and a high-swelling HA ink were used to fabricate bilayers with self-bending behaviors [27].The final aim was to 4D bioprint a scaffold that mimicked the curved multilayered cellular structure and organization of the native articular cartilage which is a curved structure that needs to have a very smooth surface and is, thus, extremely difficult to fabricate using a layer-by-layer, extrusion 3D bioprinting system.

Temperature
Temperature is a widely investigated stimulus for shape-shifting in smart materials [31].This trend was also apparent in 4D printing for TERM applications (figure 1(e) and (f)).The ease of application and the ability to tune the triggering temperature by changing the material composition are the main advantages that have made temperature-responsive materials attractive for TERM applications, with many of the reviewed studies selecting temperature as the trigger of the shape-transformation phase, either as a single stimulus [38,40,41,47,51,61,73,76,77,87,88] or in combination with other types of stimuli [25,29,62,89].
The materials primarily used for the fabrication of temperature-responsive scaffolds in TE are shape memory polymers (SMPs) (figure 3(b)).For a thorough overview of the applications of thermoresponsive polymers in the biomedical field, the reader is referred to [90].Different 4D applications have been described for such SMPs.For instance, scaffolds made of shape-memory polyurethane were 3D printed, compressed to a temporary shape at 65 • C, and cooled at 4 • C to fix the temporary shape.The samples were heated to 30 • C, below the glass transition temperature of the polymer (T g = 32 • C) before cell seeding, and subsequently recovered their initial shape upon heating above their T g [38].This allowed the researchers to create a biomimetic environment in which to study the effects of dynamic mechanical properties, typical in an active body, on tissue regeneration.Such approaches have the potential to direct cell activity through mechanotransductive cues.A similar approach was adopted by Miao et al [73] using 3D printed shape-memory scaffolds made of PCL.The potential for implanting such scaffolds by using minimally invasive procedures for treating bone defects was demonstrated by uniaxially compressing the samples by almost 30% and allowing them to fully recover to their initial shape when the temperature increased up to 37 • C.

Light
Apart from hydration and temperature, light has also been used to trigger shape changes in 4D printing.The advantages of light as a stimulus include its local nature that allows for accurate focusing as well as rapid switching.The potential of using light as a stimulus for shape transformation in TERM was demonstrated by Luo et al who fabricated 3D printed, biphasic scaffolds, consisting of an AA/polydopamine (PDA) phase and a cell-laden AA/gelatin methacryloyl phase [79].The incorporation of PDA, which has a near infrared radiation (NIR)-responsive behavior and has been used in various biomedical applications to trigger drug release and hyperthermia [91], resulted in NIR-triggered dehydration of the rectangular scaffolds, transforming them to saddle-like structures.The dehydrated scaffolds could maintain their shape in culture media and the incorporated cells showed high viability for up to 14 days [79].Myocardium-mimetic smart scaffolds consisting of an SMP and graphene (figure 3(c)) have also been fabricated using NIR laser stimulation for triggering the shape-shifting effect.The smart scaffolds were found to support uniform cell alignment as well as tissue maturation [78].

Multiple stimuli
A combination of two or more of the stimuli presented above can be used to trigger shape transformation multiple times.Miao et al developed scaffolds from soybean oil, epoxidized acrylate which could selftransform upon immersion in ethanol (figure 3(d)) and also possessed a shape-memory behavior that was triggered by temperature increase [25,62].A photothermal-responsive ink was developed for 4D printing scaffolds to treat critical-size bone defects by incorporating black phosphorus nanosheets in β-tricalcium phosphate/shape memory poly(lactic acid-co-trimethylene carbonate) (TCP/P(DLLA-TMC)).NIR-induced hyperthermia of the fabricated structures triggered their shape shifting from a temporary compressed configuration to a permanent one to facilitate minimally invasive delivery to the damaged tissue [29].

Shape-shifting mechanisms
To transform the shape of 3D printed scaffolds, materials are used that undergo large changes, either physical or chemical, as a consequence of exposure to various stimuli.Based on the application, the engineered scaffolds could be implanted after the 4D shape transformation has occurred, for instance in scaffolds intended to guide maturation in vitro and consider implantation at a later stage [28,60,65].However, most of the studies reviewed here considered the case of triggering the shape-change after the scaffold was implanted at the site of interest [51,61,73,77].Two shape transformation mechanisms have been mostly explored for 4D printed scaffolds intended for TERM applications, namely, shape-memory and differential swelling.

Shape-memory effect
Shape-memory materials can memorize permanent shapes, be brought to a temporary shape, and recover to their original conformation upon exposure to a stimulus, such as temperature [92], moisture [93], light [94], or a magnetic field [95].A full cycle of a shape-memory material includes two phases, namely the programming and the recovery steps.During the programming step, the material is deformed to a temporary shape, while during the recovery step it selftransforms back to its original, permanent shape, following stimulus application.Here, we will focus on thermoresponsive SMPs (figure 4(a)), as they have attracted significant interest for shape-memorybased 4D printing applications (table 2).
Polymers exist in a glassy state at low temperatures and a rubbery state at elevated temperatures.In the rubbery state, polymers can withstand large deformations.If the temperature is lowered from the rubbery state, while the deformation of the polymer is kept constant, a temporary shape can be fixed in the glassy state.As the glassy modulus is at least two orders of magnitude higher than the rubbery modulus, the elastic stress stored in the polymer is not large enough to drive shape recovery after the load is removed in the glassy state.Upon reheating to the rubbery state, ordinary polymers are incapable of completely restoring the residual inelastic deformation.On the contrary, thermoresponsive SMPs can recover almost the entire residual deformation [96].The driving force for the shape recovery is a change in the mobility of the polymer chains and a transition from an ordered, temporary configuration to a thermodynamically favored configuration of higher entropy [40].The threshold T f, high : the heating temperature for the beginning of the programming step.T f, low : the cooling temperature for fixing the temporary shape.Tr: the shape-recovery temperature.RT: Room temperature.
To assess the efficiency of shape recovery in SMPs, two parameters are usually determined: the shape recovery rate (R r ) and the shape fixity rate (R f ).R r quantifies the extent of shape recovery whereas R f measures the ability of the SMP to maintain its deformed shape in the glassy state after the external load is removed [98].R r and R f are determined by the following formulas: where ε m is the maximum strain of the SMP that is kept constant during the cooling phase of the programming step, ε p is the residual strain that is left at the end of the recovery step, ε u is the recovered strain after removing the load from the structure during the programming step, and N is the loading cycle.In applications where the fabricated objects are deformed by bending or rolling during the programming step, R r and R f can be determined using the deformation angle [52,61,73] or cylinder diameter [88], respectively.In 4D printing, the permanent shape of SMPbased inks is defined by the 3D printed structure.This structure is subsequently deformed and cooled for fixing the temporary shape.Upon increasing the temperature to reach the rubbery state of the applied SMPs, the deformed structure recovers its original, 3D printed shape.In biomedical applications and especially for TE and RM, the threshold temperatures for the shape-memory cycle must be tailored wisely to be compatible with the tolerance of the incorporated functional molecules, cells, and/or surrounding tissues.So far, a wide temperature range has been used with fixing temperatures varying between −18 • C [25, 61, 62, 73] and 25 • C [76] and recovery temperatures ranging from 37 • C [38,73,76] to 70 • C [40].
The main focus of 4D printed, shape-memory scaffolds demonstrated to date for TERM applications appears to be the development of constructs that can be implanted via minimally invasive strategies at a defect site.More precisely, by reducing the size of the scaffold during shape fixation, its delivery and fitting to the site of interest can be optimized.Decreasing the diameter of cylindrical structures in the programming step allows for the in vitro introduction of endoluminal [77] and vascular [51,75] stents that could securely anchor to the native tissue (trachea and blood vessels, respectively), upon recovering to their original shape.Furthermore, compressed scaffolds capable of recovering to a larger geometry have been used for filling bone cavities (figure 4(b)) [29,40,41,73].In addition, more advanced 4D printing approaches using shape-memory materials have been proposed.For instance, a multiwell array insert printed by projection micro-SLA was recently reported for culturing patient-derived glioblastoma organoids [76].The array could be stretched to a 96-well plate conformation (temporary shape) and, upon heating to >T trans , recover to its original shape which was programmed to precisely fit in a histology cassette, thus facilitating post-processing tissue analysis (figure 4(c)).
In addition to thermo-responsive SMPs, there are other shape-memory mechanisms, such as ionicresponsive SMPs, that could be of interest in 4D printing applications.For instance, Lu et al used a combination of Ca 2+ or Fe 3+ ions and high temperature solvents to trigger reversible molecular switches to coordinate metal-ligand and host-guest interactions [99].Despite the novel aspects of such mechanisms, the concentration of ions and the high temperature used needs to be revised to ensure cell viability and its applicability in TERM applications.

Differential swelling
Differential swelling is another mechanism that has been widely employed in 4D printing to fabricate shape-shifting scaffolds, using hydrogel-based inks for TERM applications.Differential swelling has been used for producing mainly tubular or curved structures [28,60,65,71,74].One of the approaches using this mechanism is based on the use of a bilayer scaffold containing two materials with distinct swelling profiles [100].The presence of a hydrophilic layer and a hydrophobic or less hydrophilic layer enables the controlled bending of the bilayer in its thickness direction upon the immersion of the specimen into aqueous solutions (figure 5(a)).Díaz-Payno, Kalogeropoulou et al 4D bioprinted a flat bilayered scaffold that was able to curve into a cartilage-mimicking structure using a high swelling HA-tyramine (HAT) and a low swelling HA-tyramine containing AA bioink (AHAT) (figure 5(b)) [27].A second approach is the control of the photocrosslinking degree throughout the thickness of a scaffold.This can establish a swelling gradient within a single-material to achieve a bending effect [60,80].For instance, Kirillova et al presented a 4D bioprinting approach for the fabrication of cell-laden hydrogel scaffolds from AA or HA crosslinked from the top capable of self-rolling into hollow tubes when immersed in aqueous media [60].The tubes retained their shape without showing any signs of degradation even 6 months after their fabrication (figure 5(c)).A third approach for enhancing the swelling-induced shape-shifting is the patterning of one of the two layers (figure 5(d)) [28,65].In a different study, the differential swelling of a hydrogel bilayers was used as a hinge-like mechanism for the fabrication of selfassembling cubes (figure 5(e)) [26].
To quantify the degree of shape-change as well as the time needed to reach a stable state, certain material properties and parameters can be determined.A commonly used indicator of the amount of liquid a hydrogel can absorb is the (mass) swelling ratio (Q m ) which is defined as: where m s is the mass of the swollen hydrogel polymer and m d is the mass of the dry hydrogel.The swelling ratio and, by extension, the degree of shapechange of 4D printed scaffolds can be controlled by tailoring several parameters.In terms of ink-related parameters, the composition of the hydrogel [80] as well as the crosslinking degree [28,60,67] have been used to control how far the scaffolds self-bend.For instance, photocrosslinking Gel-MA films for 10, 30, and 60 min resulted in structures with swelling ratios of ∼150%, ∼300%, and ∼350%, respectively [28].
The scaffold design has also been reported to affect the final shape.By tuning the thickness ratio of the two layers [67,71], the thickness of the whole scaffold [28,60,71,80] as well as the pattern design or orientation [65,67], the shape-shifting can be further controlled.Finally, a swelling-dependent system can be affected by the composition of the swelling medium [60,65,67,80] as well as the concentration of certain ions in the medium [68,69], which can modulate the degree of shape-change.More specifically, the reported swelling ratios of HA-based mats varied between 2000%, 3000%, and 4000% after 24 h in water, PBS, and, culture medium, respectively.An overview of the characteristic parameters of the reviewed studies is presented in table 3.
In several studies, efforts have been made to predict the swelling-induced bending and curvature values [12,24,71].Those predictive models are inspired by the Timoshenko equation, which was originally developed to predict the bending of bi-metal beams subjected to uniform heating [101].In differential swelling, however, the driving force of bending is the difference between the swelling ratios of both layers, instead of their coefficients of thermal expansion.Therefore, certain assumptions and modifications of the classic model have been proposed.Firstly, as the bending is not triggered by temperature variations, the temperature difference can be ignored to a certain extent.Secondly, the 3D printed layers of the structure are considered uniform and isotropic, but this is not always the case.Thus, the introduction of correction coefficients to compensate structural and material heterogenicity as well as the addition of the swelling ratios to the original equation have also been suggested [71].

Other mechanisms
Apart from thermoresponsive shape-memory effect and differential swelling, other shape-changing mechanisms have also been used for 4D printing.The coil-globule transition of PNIPAAm hydrogels is a mechanism underlying large, reversible volume changes at a critical temperature (≈32 • C − 35 • C) and results in a dramatic decrease in the water content when the temperature is increased above that critical value.By combining PNIPAAm with AA, interpenetrating polymer network hydrogels compatible with extrusion (bio) printing were prepared and, subsequently, used for the fabrication of thermally actuating scaffolds that swell and shrink reversibly [47].In a different approach, electrospun PCL-PNIPAAm-PCL trilayers capable of folding into tubular structures were fabricated [89].The thermo-sensitivity of PNIPAAm defined the folding degree and side while the fiber orientation of the PCL layers defined the folding direction [89].Furthermore, the drying of AA has been used as a mechanism for inducing shape-shifting of 3D printed scaffolds [79].The combination of more than one shapechanging mechanisms in the same scaffold has also been reported.For instance, laser energy attenuation during the SLA fabrication process has been identified as the source of a crosslinking gradient that resulted in the accumulation of internal stresses in soybean oil-based scaffolds [25,62].Immersion in ethanol resulted in stress release and scaffold deformation.A subsequent immersion of the structure in ethanol and water triggered the scaffold to recover and then re-deform, respectively.In addition, the printed scaffolds exhibited shape-memory and could, thus, recover their initial shape after deformation and heating above their T trans .

Strategy 1: Cell incorporation post-fabrication
This strategy includes the fabrication of scaffolds with a conventional 3D printing technique and their subsequent stimuli-induced shape-shifting.Most of the printing methods following this strategy are well characterized methods but most often not cell friendly.As cell encapsulation is not considered in this process, these scaffolds can be used as drug carriers, biomolecules [52,88], biomedical devices [51,75,77], or as off-the-shelf tissue-conductive scaffolds that can be colonized by infiltrating cells post-4D fabrication [25,41,61,64,73].This approach allows for greater versatility in the printing method and scaffold design.Additionally, a wide spectrum of different materials can be used, from shape-memory alloys [102] to elastomers [103].These materials can be processed at elevated temperatures or be crosslinked with potentially cytotoxic radiation wavelengths (UV 365-405 nm) [104] into scaffolds of complex geometries.Unlike the other cell-incorporation approaches, in Strategy 1, the selected stimulus for the 4D transformation may divert from the strictly cell-friendly triggers, without any adverse effects on the biocompatibility of the final scaffold.The mechanical properties of the resulting constructs can also vary greatly, while not being limited to the materials suitable for cell-encapsulation, such as hydrogels.For instance, Wang et al fabricated photothermal-responsive shape memory scaffolds for precise fitting in irregular bone Figure 6.A schematic illustration of the three cell-seeding strategies in 4D printing.From top to bottom: In strategy 1 (S1), no cells are involved in the fabrication process, but after the shape-change has taken place (either added for in vitro test or migrated from host upon implantation).In strategy 2 (S2), a cell-free scaffold is printed and is seeded with cells before the stimulation.In strategy 3 (S3), the cells are involved from the beginning of the fabrication process.The scaffold is, thus, bioprinted, using a cell-laden material, and is then exposed to stimulation to change its shape.defects capable of releasing peptides for enhancing osteogenesis [29,31].Nonetheless, it is worth noting that this strategy could require prolonged cell migration times, as the cells are not incorporated in the construct itself, potentially leading to non-uniform tissue growth.To address this challenge, a careful selection of tissue-instructive materials [105], either as the printing material, or as a subsequent coating, may compensate for any fabrication-related issues.

Strategy 2: Cell seeding post-printing but pre-4D stimulus
In this strategy, the cell seeding follows the scaffold fabrication but precedes the application of the stimuli.Therefore, the structure undergoes shape-shifting after cells have adhered to its surface.This strategy allows for decoupling of the printing process of the two-dimensional unfolded patterns from the incorporation of the cells before the scaffold can transform to a more complex 3D structure [38,64,67,89].This is particularly useful if the printing technology or crosslinking mechanism utilized is not cell-friendly or leads to a significant decrease in cell viability.For instance, Senatov and colleagues investigated the selffitting efficiency of shape-memory 3D printed scaffolds that were fabricated using extrusion printing at 180 • C-220 • C. Upon cooling, the scaffolds were used for in vitro studies with hMSCs, and demonstrated good biocompatibility and adhesion capacity [41].
Similar to Strategy 1, a broader palette of materials and printing methods can be considered in Strategy 2 as compared to the use of bioinks (Strategy 3).Additionally, a finer degree of control over the initial cell distribution could be achieved with this approach, as different cell types at various densities can be precisely seeded on specific sites of the 3D printed scaffolds.By decoupling the cell seeding and shape-shifting processes, uniform tissue formation is promoted.Following this strategy, Wang et al have fabricated patterned cardiac patches that were used for the triculture of hMSCs, hECs, and hIPSC-CMs, using different cell seeding densities for each cell population [78].In the same study, a comparison of the cell seeding efficiency before and after the application of the 4D stimulus revealed that precurved patches were unable to form uniform cardiac tissue, since the cells settled and aggregated at the bottom of the scaffolds due to gravity effects.
Regarding the selection of the proper 4D approach in Strategy 2, it becomes clear that the selected stimulus will be limited by their degree of biocompatibility.In other words, since the shapeshifting occurs after cell seeding, only cell-friendly stimuli can be used to ensure high viability.Moreover, if the selected stimulus is either physiological temperature or/and humidity, the cell seeding step could inadvertently trigger the shape-shifting of the scaffold.This limitation could be potentially addressed through sacrificial materials that delay the programmed shape transformation until the desired moment.For example, Cui et al fabricated gelatinbased bilayers, which were kept flat using a sacrificial AA/gelatin layer.After the adhesion, spreading, and proliferation of HUVEC cells, the sacrificial layer was dissolved, allowing the bilayered mat to fold into a tube, due to the difference in the swelling ratios of Mouse fibroblasts Differential swelling GelMA and PEGDM [82] both layers [71].While the limitations of Strategy 2 may be mitigated through meticulous selection of materials, stimuli, and seeding techniques, it is worth acknowledging that the complexity of this approach may inevitably extend the fabrication timeline.

Strategy 3: Cell encapsulation pre-printing
The third strategy refers to the 4D bioprinting processes when the cells are incorporated in the ink prior to the printing and shape-transformation processes [27,28,60,65,79,80].The advantage of this strategy is the ability to place cells in the desired location during fabrication, while the disadvantage is that the process relies on cell friendly bioinks, which still need further development in terms of printing resolution and fidelity.
In this single-step approach, cells are integrated directly into the structural matrix of the scaffold during the fabrication process, thus reducing the fabrication time and complexity.A good homogeneity of the printed scaffolds can also be achieved, because cells are incorporated inside the ink, instead of adhering to its surface.Nonetheless, it is essential to acknowledge the constraints imposed by the limited range of materials available for this bioprinting strategy in comparison to its counterparts.This limitation arises from the fact that bioinks should be suitable for cell encapsulation and provide suitable biochemical cues for the proliferation, growth, and/or differentiation of the cells.To date, the majority of the bioinks presented in the literature are hydrogel precursors, which often need to be cross-linked either during or after the scaffold fabrication [58].The selection of the cross-linking method calls for careful experimental design to minimize potential cytotoxic or genotoxic effects.Furthermore, the bioprinting method itself could possess inherent features that could negatively affect the incorporated cells.For instance, a high shear stress in extrusion bioprinting could result in a significant decrease in cell viability, especially for bioinks with high cell densities and/or high viscosity values [106].Nonetheless, recent studies have proposed strategies to minimize the printing-induced cell damage.For example, Pan and colleagues have proposed a nature-inspired strategy for cell protection during bioprinting using a pyrogallol-alginate cell encapsulation system, which mimics the dormant state of plant seeds when they are exposed to adverse environmental conditions [107].
Apart from the selected bioink, crosslinking mechanism, and printing approach, the selected shape-shifting stimulus should also be compatible with living cells in Strategy 3. In a recent study, Luo et al printed scaffolds from AA and GelMA loaded with human embryonic kidney cells, which selftransformed into saddle-like structures upon NIR irradiation [79].The scaffolds were able to maintain their deformed structures for at least 14 d, while the printed cells retained significant viability.Overall, this approach requires a delicate parameter optimization and selection of cross-linking method and 4D stimulus.In certain cases, the complexity of the system could be reduced by opting for a stimulus inherent to bioprinting, such as physiological temperature or humidity.For instance, Ding and colleagues developed a jammed micro-flake hydrogel from ionically crosslinked OMA as a bioink for 4D bioprinting [81].The hydrogels were crosslinked using UV light and a crosslinking gradient along the thickness direction of the scaffolds was established.Immersion of these constructs in the culture medium resulted in self-bending.Three different cell types were investigated (i.e.NIH3T3, HeLa and hMSCs) and cartilage-like tissue formation was demonstrated in bent hydrogel bars.In another study, Díaz-Payno, Kalogeropoulou et al 4D bioprinted a scaffold that recapitulated some aspects of the complex multilayered curved structure of articular cartilage based on differential swelling of the layers using a tyramine-HA containing AA bioink embedded with hMSCs [27].

Discussion and future directions
4D printing is an emerging technology that is expected to revolutionize the TERM field.4D printing consists of 3D printing a scaffold that transforms into a distinct stable state upon the application of one or more stimuli, featuring the introduction of time as the fourth dimension (4D).In this review, we presented an overview of the recent developments in this exciting area of research that aims to enable the biofabrication of highly complex structures through the shape-change of (bio)printed constructs for TERM applications.Our findings show that the two most common mechanisms used in TERM applications are shape-memory (e.g. with such materials as PLA, PGDA, and polyurethanes) and differential swelling with different combinations of AA, HA, or gelatin (figure 7(a)).The selection of the shape transformation mechanism is closely correlated to the cell seeding strategy.For instance, in most shape-memory studies in which cells are used, they are seeded posttransformation.On the other hand, most swellingbased studies explore the addition of the cells to the material from the very beginning of the fabrication setup (figure 7(b)).Several biomedical applications have been described, but the most widely studied target tissues to date are bone, vascular, and cardiac tissues (figure 7(c)).Despite the remarkable 4D printing applications already reported, there are several challenges that are yet to overcome.
Firstly, there is a limited number of suitable inks, most of which are responsive to a single type of stimulus.However, in an environment as complex as the human body, tissues are exposed to multiple stimuli and regulatory processes.Therefore, a possible future research direction could be the use of the complexity of the human body as a mechanism to increase the capabilities of 4D printed structures.For instance, the use of multi-responsive smart materials or a combination of multiple single-responsive materials in a single structure could allow for the fabrication of scaffolds with more than two stable states, capable of performing complex functions without the need for any external stimuli.Secondly, the effects of the stimuli on the cells need to be better characterized.For instance, although light is a promising stimulus for 4D printing applications in TE, more in-depth research is required to address concerns regarding potential toxic or damaging effects of light and the photo-initiators used on the cells, as well as the risk of tissue damage caused by heat generation during photo-thermal conversion [84].
Other stimuli, already used in 4D printing, such as magnetic and electric fields, could be useful for TERM applications.The main advantages of magnetic field include the remote stimulus application as well as the ability to easily and harmlessly penetrate most materials [109].Zakharenko et al developed self-rolling and unrolling tubes by combining thermo-(PCL) and magneto-responsive materials (PNIPAAm) [110] that could be used for controlled cell encapsulation and release.In another innovative 3D bioprinting approach proposed by Betsch et al, a conventional extrusion printer was modified by adding a magnet close to the printing bed [111], which allowed for the application of a magnetic field to the iron-containing agarose/collagen bioink during extrusion.In this way, it was possible to achieve collagen fiber alignment parallel to the direction of the applied field, producing chondrocyte-laden scaffolds that mimicked the native collagen architecture of the human articular cartilage.
The use of electric fields is particularly attractive for the fabrication of scaffolds that promote the regeneration of electrically active tissues.To date, very few attempts have been made at electrically assisted 4D printing.For instance, Cvetkovic et al used SLA to print hydrogel poly(ethylene glycol) diacrylate (PEGDA) 'bio-bots' , powered by the contraction of engineered skeletal muscle [112].Electric fields have also been used to direct the orientation of muscle cells.In a recent study by Yang et al, an electric field-assisted 3D bioprinting system was presented for the fabrication of scaffolds with a cell-alignment cue [28].Gelatin-based bioinks were extruded through a modified nozzle while simultaneously exposed to an external electric field to induce the alignment of the embedded myoblasts.The printed fibers were deposited on top of a patterned gelatin film that underwent self-rolling upon immersion in water, resulting in the formation of cell-laden fibrous bundles.
Thirdly, 4D bioprinting seems to be limited to extrusion and DLP-based bioprinting, but there are other fabrication techniques, such as inkjet-based bioprinting and laser-assisted bioprinting with high potential for TERM 4D applications.Inkjet bioprinting was the first printing method that was combined with cell-laden materials [113] and is based on the same working principles as conventional inkjet printing.A cell-containing hydrogel precursor is loaded in the ink cartridge, which is connected to the printing head and is ejected in droplets.The main advantages of inkjet bioprinting include high printing speed, relatively high cell viability, low cost due to the similarity of the devices to the commercially available printers as well as a relatively high printing resolution, with droplets containing one or two cells printed in ∼ 50 µm wide patterns [114].However, there are several points to take into consideration when using bioinks.For example, in thermal inkjet bioprinting, the localized temperature increase, ranging from 200 • C to 300 • C, may be detrimental to cell viability and the stability of biological molecules if applied for more than ∼ 2 µs [20].In addition, the bioink must be liquid to be effectively ejected from the printing head.Thus, a common issue of this technology is headclogging due to high bioink viscosity, material drying within the printing head, or cell settling in the cartridge.
Laser-assisted bioprinting (LAB) originates from the laser-induced forward transfer technology [115].The system consists of a focused laser pulse that causes volatilization of a liquid bioink at the donor/bioink interface, generating a high-pressure bubble that ejects the cell-containing material toward the collector substrate.Since LAB is a non-contact printing method, both clogging and shear-induced cell damage are avoided.It is a bioprinting technology that is compatible with a moderately wide range of viscosities (1 mPa•s-300 mPa•s) and is capable of depositing high cell densities with resolutions down to a single cell per drop [116].However, the side effects that laser exposure may have on cells are not yet fully comprehended.Additionally, LAB systems are expensive in comparison to inkjet or extrusion bioprinting and the printing procedure is complex and often requires a pre-printing preparation step which significantly increases the fabrication time [20].
Despite the several challenges of the 4D (bio)printing process, current shape-changes relying on bilayers are relatively simple.To increase the complexity of such changes and mimic natural processes of high complexity, there is a need to include dual or even multiple shape-changing mechanisms.Previously, researchers have combined 3D printing with self-folding origami to program shape-shifting of flat soft matter with several patterned surfaces [117].Such strategies could contribute to the future developments of 4D (bio)printing.
Finally, the development of material-specific models for reliable prediction of the final shape of the scaffolds is needed.Although individual studies have tried to create databases for modeling the shape-shifting behavior of 4D printed scaffolds [65], these are based on equations that have been optimized for each particular application and conditions.Therefore, developing computational models that can be easily modified for a wide spectrum of 4D printing applications could provide a valuable tool for designing complex shape-shifting patterns, optimizing experiments as well as comparing results among different studies.

Conclusions
4D printing has emerged as a cutting-edge technology, enabling the fabrication of highly sophisticated scaffolds with controllable, dynamic shapes by introducing the additional dimension of time.This has marked the beginning of a new era in the field of biofabrication.This review focused on the shapeshifting aspect of 4D printing and its application in the field of TERM.We observed that the most common 4D mechanisms are based on shape-memory materials when printing without cells and is mainly limited to swelling-based differential growth when working with embedded cells.This field is still in its infancy and there is a need to develop smart biocompatible strategies that can use a wider range of 4D mechanisms, stimuli, and printing techniques.

Figure 1 .
Figure 1.A schematic representation of the search strategy and results.(a) A flowchart depicting the filtering procedure for acquiring the reviewed papers focused on shape-change 4D printing for TERM applications.(b) The distribution of the included research papers per year of publication highlighting the first 4D bioprinting study in 2017 and the first in vivo study using a rabbit model for trachea regeneration.(c) The distribution of the reviewed studies according to their fabrication method and year (with part of 2023).(d) The distribution of the reviewed studies according to the applied 4D printing (pie chart) or 4D bioprinting technique (rectangular chart).(e) The distribution of the articles per stimulus and per year.(f) A pie chart showing the distribution of papers for each of the stimuli.(g) The distribution of 4D bioprinting studies according to their stimulus type and publication year.n: number of articles included in each category.

Figure 3 .
Figure 3. Application of various stimuli in 4D printing.(a) Biofabricated grippers with multiple arms before (top left inset) and after deformation.Hydrogel deformation was performed in a growth medium at 37 • C. Reprinted from [80], Copyright (2021), with permission from © 2021 The Authors.Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd.(b) A compressed structure recovers to its original shape of a standing hollow vase after being heated.Scale bar: 6 mm.Reprinted with permission from [51].Copyright (2018) American Chemical Society.(c) The images of a U-shaped structure recovered by NIR laser stimulation.Reprinted with permission from [78].Copyright (2021) American Chemical Society.(d) 4D printing using stress-induced shape transformation based on multiple stimuli: hydration (water) and dehydration (ethanol).Scale bar: 2 mm.[25] John Wiley & Sons.© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figure 4 .
Figure 4. Shape memory-based 4D printing.(a) A schematic illustration of the programming and recovery steps of a thermoresponsive shape-memory scaffold.Created with BioRender.(b) In vitro void filling of a DLP printed polyurethane-based implant, demonstrating the shape-memory behavior of the porous scaffold.(i) The temporary shape after programming, (ii) shape recovery in the void, without visible deformation of the void-bearing structure and (iii) removal of the scaffold for further inspection of the recovery process.Scale bars: 1 cm.Reproduced from [64].CC BY 4.0.(c) (i) Stretched 4D culture array to a 96-well plate configuration (temporary shape) and its recovery to a histology megacassette configuration.(ii) The megacassette containing patient-derived glioblastoma organoids cultured in a 6-well plate.Reprinted from [76], Copyright (2020), with permission from Elsevier.

Figure 5 .
Figure 5. Differential swelling-based 4D printing.(a) An illustration of the bending of a bilayer scaffold due to the distinct swelling ratios (Qm) of both layers, upon stimulation with an aqueous solvent.Created with BioRender.(b) A proof-of-concept of the fabrication of a curved multilayered cellular construct made of AA with HA-tyramine (AHAT) and HA-tyramine alone (HAT) where bioprinted human mesenchymal stromal cells (hMSCs) remained in their corresponding layers after the 4D transformation.Reproduced from [27].CC BY 4.0.© 2022 The Authors.Advanced Healthcare Materials published by Wiley-VCH GmbH.(c) (left) Bending of a scaffold made from oxidized and AA-MA (OMA) with a continuous crosslinking gradient across its thickness.Scale bar: 2 mm.(right) A magnified image clearly showing the gradient.Reprinted from [80], Copyright (2021), with permission from © 2021 The Authors.Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd.(d) The printing pattern of a cubic box and its hinges (actuating parts) and some images of the box in its as-printed (top) and swollen (bottom) states.The base of the box was printed using a NIPAM-based ink while the hinges were printed from a poly(HEMA) based ink.[26] John Wiley & Sons.© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.(e) Finite element analysis (FEA) simulation (top) and experimental results (bottom) of a bi-layered scaffold made from silk fibroin with a patterned layer.Reprinted from [65], Copyright (2020), with permission from Elsevier.

Figure 7 .
Figure 7. Biomaterials, mechanisms, and the targeted tissue of shape-based transformation 4D (bio)printing for TERM applications.(a) A list of the materials used in 4D (bio)printing research articles organized by the number of articles.(b) The percentage of the articles in which the different shape-changing mechanisms are used in each of the different seeding strategies (S1, S2, S3).(c) The target tissues in 4D (bio)printing applications.

Takatani S 2005
Biocompatible inkjet printing technique for designed seeding of individual living cells Tissue Eng.11 1658 [115] Barron J A, Ringeisen B R, Kim H, Spargo B J and Chrisey D B 2004 Application of laser printing to mammalian cells Thin Solid Films 453-454 383 [116] Guillotin B et al 2010 Laser assisted bioprinting of engineered tissue with high cell density and microscale organization Biomaterials 31 7250 [117] van Manen T, Janbaz S and Zadpoor A A 2018 Programming the shape-shifting of flat soft matter Mater.Today 21 144

Table 1 .
Classification of the reviewed studies on the applications of 4D printing in TERM, based on the (bio) printing method, the printing resolution, and the material used as ink.

Table 2 .
The shape-memory parameters used in the reviewed 4D printing applications.

Table 3 .
The differential swelling parameters used in 4D printing applications showing different layer composition, pattern design (or orientation), swelling strategy and bending control.

Table 4 .
The cell-seeding strategies and their parameters.