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Emerging 4D Printing Strategies for Next-Generation

Tissue Regeneration and Medical Devices
Yue Wang, Haitao Cui, Timothy Esworthy, Deqing Mei, Yancheng Wang,*
and Lijie Grace Zhang*
printing. First introduced by Massachu-

The rapid development of 3D printing has led to considerable progress in setts Institute of Technology researcher
the field of biomedical engineering. Notably, 4D printing provides a potential Skylar Tibbits during a Technology, Enter-
strategy to achieve a time-dependent physical change within tissue scaffolds tainment, Design (TED) talk in 2013, and
or replicate the dynamic biological behaviors of native tissues for smart tissue then subsequently expanded upon in pub-
lished research,[2] 4D printing has gained
regeneration and the fabrication of medical devices. The fabricated stimulus-
substantial research attention in recent
responsive structures can offer dynamic, reprogrammable deformation or years and has become the subject matter
actuation to mimic complex physical, biochemical, and mechanical processes of a substantial number of recent publica-
of native tissues. Although there is notable progress made in the develop- tions (Figure 1).
ment of the 4D printing approach for various biomedical applications, its The additional dimension over 3D
printing, namely, the 4th dimension, is a
more broad-scale adoption for clinical use and tissue engineering purposes is
time component.[3] Therein, 4D printing
complicated by a notable limitation of printable smart materials and the sim- can be defined as a 3D printed structure
plistic nature of achievable responses possible with current sources of stimu- that can self-transform in shape, proper-
lation. In this review, the recent progress made in the field of 4D printing by ties, and/or functionality when exposed
discussing the various printing mechanisms that are achieved with great to predetermined stimuli, such as light,[4]
emphasis on smart ink mechanisms of 4D actuation, construct structural heat, applied magnetic field (MF),[5] or
electrical field (EF),[6] changes in pH,[7]
design, and printing technologies, is highlighted. Recent 4D printing studies
and/or various combinations thereof post-
which focus on the applications of tissue/organ regeneration and medical printing.[8] Since its invention, the 4D
devices are then summarized. Finally, the current challenges and future per- printing approach has been utilized for
spectives of 4D printing are also discussed. multitudinous applications, such as the
fabrication of sensors,[9] actuators,[10] soft
robotics,[11] biomedical scaffolds/devices,
1. Introduction drug delivery systems,[12] and others.[13] Presently, 4D printing,
which is still in its infancy, has become an exciting branch of
3D printing, also known as additive manufacturing (AM)[1] or AM and has spurred great interest from both academia and
rapid prototyping, has been utilized for a multitude of novel industry alike.[14]
applications and has spurred the creation of new materials and In this review article, we present a general overview of
nuanced manufacturing methods alike. One such recent manu- the 4D printing field and recent achievements. Our review
facturing method which traces its lineage to 3D printing is 4D includes the discussion on the structural design of 4D printing,
Y. Wang, D. Mei, Y. Wang H. Cui, T. Esworthy, L. G. Zhang

State Key Laboratory of Fluid Power and Mechatronics Systems Department of Mechanical and Aerospace Engineering
Zhejiang University The George Washington University
Hangzhou 310027, China Washington, DC 20052, USA
E-mail: yanchwang@zju.edu.cn E-mail: lgzhang@gwu.edu
Y. Wang, D. Mei, Y. Wang L. G. Zhang
Key Laboratory of Advanced Manufacturing Technology Department of Electrical and Computer Engineering
of Zhejiang Province The George Washington University
School of Mechanical Engineering Washington, DC 20052, USA
Zhejiang University L. G. Zhang
Hangzhou 310027, China Department of Biomedical Engineering
The George Washington University
Washington, DC 20052, USA
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adma.202109198. L. G. Zhang
Department of Medicine
The George Washington University
DOI: 10.1002/adma.202109198 Washington, DC 20052, USA
Adv. Mater. 2022, 34, 2109198 2109198 (1 of 42) © 2022 Wiley-VCH GmbH
15214095, 2022, 20, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/adma.202109198 by National Research Tomsk Polytechnic University, Wiley Online Library on [09/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
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Figure 1. Statistics of publications on 4D printing from Web of Science. A) The number of publications every year and B) the number of citations every
year. C) The leading countries in 4D printing publication. Statistics are provided by the Web of Science and collected by November 2021.
4D smart polymeric materials (i.e., shape memory polymers 2. Printing Technologies for 4D Manufacturing
(SMPs), liquid crystal (LC) polymers (LCPs), stimuli-responsive
hydrogels, and others), the stimuli-responsive mechanisms of As a relatively new additive manufacturing technology, 4D
the shape transformation (or function shift), and recent studies printing platforms are primarily based on a small, core set of
on 4D printed scaffolds/constructs and devices (e.g., vascular 3D printing approaches. Since its inception in the mid-1980s,
tissues and stents, cardiac patches and valves, brain constructs, 3D printing has evolved into a multitude of diverse fabrication
neural scaffolds and conduits, bone scaffolds, muscle scaffolds, technologies, including extrusion-based methods (fused depo-
and tracheal implants). Finally, crucial regulatory and com- sition modeling (FDM),[15] liquid extrusion printing, direct ink
mercialization details pertaining to 4D printing are discussed, writing (DIW),[16] and laminated object manufacturing, light-
and the current challenges and foreseeable progress are also assisted types (stereolithography (SLA or SL),[17] selective laser
summarized. It is envisioned that upon the assurance of key sintering (SLS)),[18] selective laser melting (SLM),[19] two-photon
regulatory demands, such technology will become translatable polymerization (TPP),[20] digital light processing (DLP),[21] and
for numerous biomedical applications, which require the fab- inkjet printing.[22] Currently, the most commonly applied 4D
rication of constructs with dynamic and functionality complex printing technologies are extrusion-based (DIW and FDM) and
structures. light-assisted (SLA and DLP) printing.
2.1. Extrusion-Based Printing Technology (laser) to photocrosslinking liquid polymers and resins into
solid or gel-like constructs.[29] The principal advantages of
2.1.1. FDM Printing SLA are its high printing resolution and excellent achievable
surface finish of the fabricated constructs compared to the
FDM, also deemed fused filament fabrication by some literature, extrusion-based printing technologies.[30] SLA has been widely
is by far the most common and best-characterized form of extru- utilized to print a multitude of polymeric materials such as
sion-type 3D printing and has been widely used in both industry photocrosslinkable hydrogels and photocurable resins (SMPs
and laboratory settings, as this printing methodology offers rela- or LCPs) in order to achieve the fabrication of constructs that
tively fast printing speed.[10,23] FDM printing relies on a heated can actuate 4D shape changes. Laser curing is a complex and
nozzle-based extruder with a drive gear to melt and deposit ther- dynamic process which is affected by many factors such as
moplastic filaments in a line-by-line fashion to form stacked curing speed, curing depth, laser energy, and polymerization
layers in the production of a 3D object.[10] The extruder apparatus shrinkage.[31]
in most FDM printers are primarily driven by a mechanical-
(piston or screw)-based extrusion and the method in which plastic
filaments are deposited in an ordered fashion typically relies on 2.2.2. DLP Printing
the application of high temperatures, but can also involve the
application of electric fields, magnetic fields, and light).[23] Which DLP printing has become a promising tool to rapidly fabricate
method is used to deposit polymer fibers is largely dependent on complex 3- and 4D structures with micro- to nanoscale struc-
the stimuli-responsive properties of 4D filament materials used tural features using light as a crosslinking source. DLP-based
or the addition of functional fillers. However, a major concern is printing can achieve a considerable degree of printing resolu-
the waste material during the preparation of printing filaments, tion, high printed shape-to-design fidelity, and offers a selection
as well as the relatively low printing resolution of FDM printing of printing materials.[32] Unlike the SLA method, DLP replaces
technologies compared to the other printing methods. the scanning laser beam with a digital light pattern to cure the
liquid resin in a reservoir in a timescale of seconds per layer.[33]
Owing to its surface-projection-based fabrication method,
2.1.2. DIW Printing DLP can reach high printing speeds, on the order of multiple
times faster than what is achievable with SLA printing. Mean-
As another widely employed extrusion-based 3D printing tech- while, commercially available projectors used to construct DLP
nology, DIW printing technology refers to a printing method printers are considerably economical, and due to the simplistic
that utilizes a computer-controlled print head to extrude shear- design of most DLP orienting systems, they can be readily con-
thinning liquids through the nozzle(s) or needle(s) under structed by novice engineers, as they primarily involve only
pressure to construct complex geometries in a line-by-line two functional components 1) a projector and 2) a Z-direction
manner.[24] Three types of DIW extruders have been commonly motor.[34] Similar to SLA printing, the degree of crosslinking of
used in current research, including pneumatic-,[25] mechanical- DLP printing systems is a collaborative parameter for the struc-
(piston or screw), or solenoid-based extrusion systems.[26] For tural design of tunable transformation,[35] which may result in
a more comprehensive review of DIW-based 4D printing tech- different swelling ratios, inner stresses, and mechanical proper-
nologies and their various applications, the reader is referred ties such as compressive modulus of the printed constructs.[36]
to the recent review produced by Prof. Leng from the Harbin Among these printing methods, both SLA and DLP have the
Institute of Technology.[27] The use of micronozzles in DIW greatest potential to spur the development of commercial and
enables high printing resolution. The DIW technique displays lab-made materials such as photocrosslinkable 4D SMPs or
marked advantages over other printing methodologies, in that LCPs, which can be crosslinked with fast printing speed and
this approach offers the greater choice of 4D printable materials excellent printing resolution.
(e.g., hydrogels, SMPs, and LCPs (or LC elastomers (LCEs))), a
small number of raw materials, and notable feasibility for mul-
timaterial printing. The DIW method also has the advantages of 2.2.3. TPP Printing
excellent interface bonding between printing ink filaments and
a wide adjustment of mechanical properties after curing, which Through scanning a tightly focused femtosecond laser beam in
can be applied to a variety of high-viscosity resins.[28] However, a liquid photopolymer, TPP, also known as direct laser writing,
the ink preparation needs to be carefully regulated because the can directly produce 3D nanostructures with a spatial resolu-
rheological performance must be tightly controlled. Addition- tion far beyond the diffraction limit of material as a function of
ally, the printing speed of DIW-type printers is considerably the nonlinear threshold effect.[37] Presently, TPP-based printing
slower than face-by-face printing methodologies such as DLP. can achieve a notably high resolution (down to 10 nm features),
which is the highest precision among current 3D printing tech-
nologies. Companies such as Nanoscribe GmbH offer commer-
2.2. Light-Assisted Printing Technology cialized TPP equipment and resins, which have been used in a
variety of research institutions. Compared with DLP printing,
2.2.1. SLA Printing the need for supporting materials, high surface tension, oxygen
inhibition, and a rough surface can largely be avoided.[38] How-
SLA is another well-characterized 3D printing process that ever, the limitation of TPP 4D printing is the product size
utilizes the emission of various frequencies of beams of light (height) which is typically on a millimeter-scale due to the
Table 1. Comparison of typical 4D printing technologies and adapting materials.
Printing methods
Spraying Extrusion-based Light-polymerization Powder bed fusion
Inkjet FDM DIW SLA DLP TPP SLS/SLM
Primary features Drop-by- Line-by-line; Line-by-line; Dot-by-dot; Face-by-face; Dot-by-dot; the Dot-by-dot; the laser
drop; liquid thermoplastic polymer viscous fluid the laser beam projection onto femtosecond laser beam focuses onto
binder jetting; through a heated extruded through focuses onto the photocuring beam focuses onto a powder bed
drop-on-powder nozzle the nozzle the photocuring polymer solution the photocuring
polymer solution polymer
Support structure Required Required Required Not required Not required Not required Not required
Printing speed Slow Medium-fast Medium-fast Medium-fast Fast Fast Fast
Resolution X–Y plane 1 µm position 0.1–0.4 mm ≈0.1 mm 25–300 µm 0.6–200 µm 80–160 nm 1–150 µm
repeatability
Z Direction ≈2 µm 0.5 to 0.127 mm <100 µm 0.05 to 0.015 mm 0.1 mm 100 nm 0.05 to 0.01 mm
Typical materials in Hydrogels, Thermoplastic polymers Metal particles,[54] Photocuring Photocuring Photocuring Metal powders,
4D printing e.g., gelatin and composites, ceramics,[24] resins, hydrogels, resins,[36,59] LCEs, resins, hydro- ceramic powders,[64]
methacryloyl e.g., polylactide hydrogels,[55] LCEs, multi- hydrogels,[60] gels[62] LCEs[63] polymer powders,[65]
(GelMA) and (PLA)/poly(ε-caprolactone) LCEs, SMPs and materials, etc. multimaterials[61] e.g., thermoplastic
Gel-COOH-MA[50] (PCL),[51] carbon black multimaterial[56] polyurethane (TPU)
and PLA,[52] PLA, e.g., LCEs,[57] and neodymium–
highimpact-polystyrene, hydrogel[55,58] iron–boron
and acrylonitrile-buta- (Nd2Fe14B)[64,65]
diene-styrene,[10] LCE[53]
Stimuli resources Ions,[50] etc. Temperature,[51,66] Temperature,[56] EF,[68] etc. [36] Tempera-
solvent,[62] light,[63] MF,[65] etc.
humidity,[23] etc. mechanical,[67] etc. ture,[69] light,[70] pH or ions,[71] etc.
humidity,[61] etc.
short working distance of the high-numerical-aperture focal materials simultaneously, and ease of scalability.[45] The mate-
lens,[39] which is used in TPP devices. Additionally, the cost of rial throughput is largely dependent on the size of the printer
the equipment can be prohibitory considering the femtosecond and the number of operational jets. Interestingly, the US Food
laser generator, the optical system, and the positioning system and Drug Administration (FDA) first approved 3D printed med-
used in the devices. icine was fabricated employing inkjet printing.[46] As a tradi-
tional 3D printing technology, inkjet printing has advantages in
manipulating single cells or cell mass in a thin hydrogel layer
2.2.4. SLS/SLM Printing for 4D swelling with different (e.g., thermosensitive, ionic, and
pH) responsiveness. However, the resolution of inkjet printing
The SLS (or SLM) technology used a CO2 or neodymium-doped is limited by the printhead nozzle diameter, the rheology of
yttrium aluminum garnet (Nd:YAG; Nd:Y3Al5O12) laser beam droplet formation, and ink spreading on the substrate.[47] Mean-
for scanning continuous layers of powdered materials to create while, the inkjet-printed structure lacks structural integrity and
a 3D object.[40] Sintering can be divided into three categories: hardness, which is essential for tissue scaffolds.[48] Also, another
solid-state sintering, where materials always remain solid; limitation of the inkjet printing method is that the drop-by-drop
liquid-phase assisted sintering, commonly used for materials deposition of ink used in this method of printing method leads
difficult to sinter; and complete melting,[41] which is used for to poor cytocompatibility through the printing process and a
metallic and ceramic materials. Generally, both shape memory high fatality rate of encapsulating cells (Table 1).[49]
ceramics and alloys are suitable for the SLS printing to fabri-
cate the 4D constructs.
3. Design of 4D Transforming
2.3. Inkjet/PolyJet Printing To date, there are five basic types of shape deformation pro-
cesses which can be actuated by 4D printed structures: 1)
Inkjet printing, also known as the drop-on-demand printing,[42] rolling, 2) compression, 3) torsion, 4) stretching, and 5) folding
is one of the first widely used 3D printing technologies and was (or bending) (Figure 2). The complex design of programmed
inspired by the use of common commercial inkjet printers,[43] patterns or material compositions, such as those which com-
which recreate digital images in 2D by propelling droplets of bine two or more types of the basic deformation structures,
liquid ink onto printer paper.[44] Inkjet printing has been high- allows for the more complex shape deformations to be achieved
lighted as a promising additive manufacturing method due by a given 4D printed construct. The wide variety of struc-
to its precision, accuracy, low cost, ability to deposit multiple tural and compositional designs available offers boundless
Figure 2. Five basic types of shape deformation are achievable by 4D printed constructs, including A) rolling. Reproduced with permission.[75] Copyright
2021, American Chemical Society. B) Torsion. Reproduced with permission.[76] Copyright 2021, American Chemical Society. C) Folding. Reproduced with
permission under the terms of the Creative Commons Attribution-Non Commercial license.[72a] Copyright 2017, The Authors, published by American
Association for the Advancement of Science. D) Compression. Reproduced with permission.[77] Copyright 2020, Elsevier. E) Stretching. Reproduced
under the terms of the Creative Commons CC BY license.[78] Copyright 2016, The Authors, Springer Nature. F) Folding. Reproduced with permission
under the terms of the Creative Commons Attribution-Non Commercial license.[72a] Copyright 2017, The Authors, published by American Association
for the Advancement of Science.
applications for 4D printing. In this section, we will discuss the gami structures[24,72] can be achieved through these relevant
mechanisms of shape transformation in regard to the struc- structural design concepts,[73] and are inspired by the Japanese
tural design and material composition design together with art of paper folding enables the production of various 3D sculp-
the generally applied mathematical methods and simulation tures simply by folding 2D sheets. This is of great interest in
approaches for guiding 4D transformation. microelectronics, soft sensors, actuators, mechanical metama-
terials, and biomedical devices.[74]
3.1. Structural Design for 4D Printing

3.1.1. Bi-/Multilayer Structural Design
There are a variety of structural changes which can be achieved
by 4D printed constructs to recapitulate dynamic biological pro- Using bi-/multilayer composites, which combine materials with
cesses and functionalities. We will primarily discuss the struc- different properties to fabricate reversibly deformable struc-
tural designs associated with these construct conformational tures, have been demonstrated to be effective in fabricating 4D
changes from two aspects: 1) bi-/multilayer structure design enabled structures in previous research, which works to expand
and 2) programmed patterns. For example, Origami and Kiri- the range of available materials.[79] As one of the most common
Figure 3. Typical examples of bi-/multilayer structures triggered by the different mechanical properties between two layers. A) Mathematical modeling
of multilayered PLA/TPU (left), and its conceptual illustration (right) of the statistic status, and the Euler–Bernoulli bending caused by internal stress.
Reproduced with permission.[79] Copyright 2021, Elsevier. B) Photographs of the different self-buckling or folding behaviors of the laterally adhered
or pattern-programmed composite films with different external strains. Reproduced with permission.[80] Copyright 2021, American Chemical Society.
C) Schematic illustration of manufacturing and shape-shifting of 4D printed polyurethane (PU)–shape memory polymer polyvinyl chloride (SMPVC)
bilayer structures stimulated by water and heat. Reproduced under the terms of the Creative Commons Attribution License (CC BY).[81] Copyright 2016,
Springer Nature.
types of 4D printed constructs, bi-/multilayer structures are other solvents, over a short period.[82b] In the future, bi-/mul-
readily employed for a wide range of structural designs and tilayer constructs will be able to be fabricated by multimaterial
have notable utility in dynamically controlled systems such printers. Moreover, TPP or multiphoton lithography technolo-
as actuators.[82] In the bi-/multilayered design, there are two gies will continue to develop and will have the ability to rap-
primary components: 1) the active component (e.g., higher idly fabricate soft actuators with higher resolution and shorter
swellability, higher elasticity, higher stress[80,82a]) and 2) a pas- response times (e.g., 500 ms).[82b] In addition, some of these
sive component (sometimes also referred to as the static layer). designed structures exhibit reversible structural changes due to
In particular, sophisticated multimaterial printing technologies the dynamically changeable mechanics or swell/de-swell behav-
allow the spatial arrangement of active (and passive) elements iors, which allow for a wide array of 4D motions such as[81]
to be configured with ease.[69,83] Meanwhile, force analysis and bending, torsion, and others[84] (Figure 3C). The combination
finite element simulations are also beneficial in predicting of the water-swellable elastomer PU and the heat-shrinkage
the shape change of the printed structures (Figure 3A), which SMPVC can yield a bilayer structure which can produce a pro-
promotes the ability to effectively predict the influence of each grammable deformation in response to both temperature and
parameter on the reversible deformation of the composite lami- water exposure. As such, the reversible transformation has
nate structure in accordance to mathematical models based on been applied for use in sensors,[85] actuators,[80] biomedical
the principle of deformation.[79] devices,[86] and soft robotics.[86] Bi-/multilayer constructs are
Moreover, a bi-/multilayer structure can be pattern-pro- arguably the simplest structures which can achieve the most
grammed, as shown in Figure 3B, wherein the multistimuli complex structural conformation changes. Through the com-
response can drive the dynamic shape transformation bination of both substrate and functional materials, it enables
(Figure 3C). It would be expected that further geometrical the actuation of dynamic 4D shape changes.[87] However, the
control in 4D constructs, such as those fabricated from hydro- realization of complex structural design requires sophisticated
gels[81] and SMPs, can provide rapid actuation through uptake and often customized printers to achieve robust spatial distri-
and release of internal or external stimuli, such as water or butions of different materials.[82a]
3.1.2. Programmed Pattern Design the optimal printing parameters to achieve the most desirable
4D actuation.[56,92]
In addition to the above-mentioned bi-/multilayer struc-
tural design, anisotropic properties, as is seen in pattern-pro-
grammed structures, can be included as another structural 3.2. Composition Design for 4D Printing
design method to achieve dynamic 4D shape-changing pro-
cesses within printed constructs.[88] With the assistance of 3D In addition to the structure design, the material composition is
printing technologies (such as FDM, SLA, DLP, DIW), com- also crucial for the achievement of 4D shape change. To better
plex curved constructs are readily obtained after induced 4D characterize the relevant mechanisms of deformation under-
bending.[89] Notably, complex programmed pattern design lying many 4D processes in terms of printing materials (or
could not correspond to the simple deformation types dis- material composition), 4D printing can also be roughly divided
cussed above. Pattern programming of printing structure into two general categories: 1) constructs fabricate using single
requires essential support of 3D printing technology, which print material,[78] and 2) constructs fabricate using multiple
would determine the concrete paths (magnitude and direc- materials[93] (Figure 5). And more details about polymeric mate-
tion angle) of 4D shape change. Additionally, the changes in rials employed in 4D printing will be discussed in the next
the physicochemical properties of the 4D material before and section.
after the stimulation also affect the patterning programming
of printed objects. Here, we will introduce two types of pro-
grammed pattern design: 1) microscale patterned compos- 3.2.1. Single-Material-Based 4D-Constructs
ites,[90] and 2) macroscale patterned[91] structures, of which both
reflect the advantages of complex structure manufacturing An individual or single material can readily respond to exog-
technologies. enous sources of stimulation. Therein, a 4D printed construct
An illustrative example of a micro-scale patterned com- composed of a single material can complete an array of “pre-
posite is displayed in Figure 4A. Kim et al. recently developed programmed” movements such as construct bending, when
a composite ink for 4D printing applications which consisted subjected to an appropriate degree of environmental stimula-
of magnetic Nd2Fe14B alloy microparticles and fumed silica tion. Kuang et al. developed a single-vat grayscale DLP printing
nanoparticles embedded within a silicone rubber matrix.[90] method to fabricate a complex helical structure and artificial
Therein, the ferromagnetic particles embedded in the com- arm using a two-stage curing ink which could obtain function-
posite ink were redirected by an external MF placed around the ally graded layers with mechanical gradients up to three orders
dispensing nozzle.[90] A mathematical model effectively pre- of magnitude and high resolution. The advantage of generating
dicted the transformation of the complex 3D printed structures 4D printed constructs utilizing only a single printing material
concerning the parameters: domain pattern, MF strength, and is the ease at which a fabricated 4D structure can be immedi-
actuation fields, and the torques on the embedded ferromag- ately reprogrammed to actuate a specific movement after the
netic particles were evaluated, resulting in a joint material printing process, as outlined in Figure 5A.[3]
macroscopic response.[90]
This microstructural design can be reliably extended to
macroscale patterned structures. For instance, researchers 3.2.2. Multiple-Material-Based 4D Constructs
proposed a preprogrammed shape change of a printed planar
construct triggered by high temperatures, as indicated in Multimaterial-based 4D structures often require more pro-
Figure 4B.[88] The proposed technique has led to a variety of cessing steps during the manufacturing workflow before the
design strategies that can be used alone, or in combination construct can be programmed to undergo a dynamic shape
to achieve unprecedented levels of complexity in the final 3D change. The additional manufacturing steps involving multi-
constructs.[88] Similarly, our research group also developed a material 4D printing often include changing of printable fila-
programmed 4D constructs methodology, which utilizes dif- ments or inks, as well as sequential pre-curing processes. A
ferent printed line orientations and line combinations between notable advantage of multimaterial 4D printing over single
layers to reach different self-bending and self-folding ele- material-based constructs is that the fabricated multimaterial
ments (Figure 4C[91]). Therein, the anisotropy of printed layers structures often do not have to be manually reprogrammed, as
caused the differences in transverse and longitudinal defor- the complex internal stresses conferred by the different mate-
mation as well as other directionally complex deformation.[91] rials and the print infill of the printed construct contain all
Moreover, the ultimate structures were predicted by the inten- of the necessary structural information needed to actuate the
tionally bidirectional anisotropy, which was controlled by the dynamic shape-changing process (Figure 5B,C). As is shown
crosslinking degree, the printed layer thickness, and the direc- in Figure 5B,[69] composite material printing applies the same
tion of lining patterns.[92] Generally, these structural design materials in different ratios, which has great significance in
methods to fabricate complex 3D constructs are one of the the development of 4D printing research.[69] Moreover, when
most convenient means to obtain dynamic 4D transformation. more composites are introduced, the printed structures can
However, the complex structural design necessary to achieve display the ability to increase the number of deformation types
the 4D effect relies on the experience of researchers and often and stimuli-response methods. Figure 5C[94] shows that the
requires a large number of repeated experiments to determine active hinges of the construct were printed with oligomeric
Figure 4. Typical examples of programmed patterned structures. A) Schematics of the printing process and the material composition. The ferromag-
netic particles embedded in the composite ink were reoriented by the applied MF, which was generated by a permanent magnet or an electromagnet
placed around the dispensing nozzle. Schematic designs, finite-element simulations, and experimental results for an annulus encoded with alternating
domains that are equidistant (a–c); an annulus encoded with alternating domains that vary in size. Reproduced with permission.[90] Copyright 2018,
Springer Nature. B) The initially flat dual strands together with semipassive connectors resulted in a DNA-inspired self-twisting shape after twisting.
Other samples of the monolayer panels with small grooves were used to determine the folding lines and create the desired self-bending, self-rolling,
and self-twisting 4D structures. Reproduced under the terms of the Creative Commons Attribution 3.0 Unported Licence.[88] Copyright 2017, The Royal
Society of Chemistry. C) Illustration of an SLA 4D printing process. A cross-architecture was printed by the SLA printer, and the claw architecture was
formed after stress relaxation. Various 4D transformations were performed by different pattern designs. Reproduced with permission.[91] Copyright
2018, Wiley-VCH.
LCE inks, whose rigid mesogens aligned along the print path 125 °C (right). The LCE hinges that form the central square and
during the HOT-DIW (top) process. After printing, the LCE the four LCE hinges that point toward the center of the struc-
ink cross-linking was initiated to lock the desired guide align- ture (left) are mountain folds, whereas the other LCE hinges
ment. Structural tiles were then printed with ink composed of are valley folds. When considering external multiplex signals,
acrylate resin which chemically bonded to the LCE hinges when multimaterial 4D printed constructs may have more potential
photoinitiated crosslinking (below). A more complex, square- to perform effectively concerning deformation or function com-
twist reconfigurable structure was printed (left) and actuated at pared to those fabricated from a single material.[95]
Figure 5. Based on the number of printed materials, 4D printing can be fabricated using: (I) single material. A) Grayscale-based DLP printed SMP con-
structs and their sequential shape recovery process in hot water, programmed by the grayscale pattern. Reproduced with permission under the terms
of the Creative Commons Attribution-NonCommercial license 4.0.[3] Copyright 2019, The Authors, some rights reserved; exclusive licensee American
Association for the Advancement of Science. (II) Multimaterials. B) Lattice structure. Reproduced with permission under the terms of the Creative
Commons Attribution-NonCommercial license.[69] Copyright 2017, The Authors, published by American Association for the Advancement of Science.
C) Mountain fold and valley fold are printed by two different inks and programmed to reach the 4D printed soft robotic matter. Scale bar = 1 cm.
Reproduced under the terms of the Science Journals Default License.[94] Copyright 2019, The Authors, some rights reserved; exclusive licensee American
Association for the Advancement of Science.
3.3. Mathematical Modeling and Simulation a programmed and predictable shape-morphing construct with
for Programming Structure the assistance of FEM.[96] The obtained experimental shapes
closely matched the finite element simulations and geometric
Within the field of 4D printing, researchers often prefer to con- prediction of the system with unidirectional deformation mag-
struct a simulation model to evaluate the 4D shape-shifting nitude (Figure 6B). Additionally, Zhou et al. performed an FEM
process. Primarily, these simulations are based on a finite ele- simulation on their designed 4D four-finger structure, which
ment method (FEM). The simulation process can help guide was triggered by electric voltage, of which the relationship
the programming process of the 4D printed construct that is between the actuator bending angle and the voltage increment
initially designed digitally. FEM focuses on multiphysics cou- was investigated.[98] Additionally, they studied the movement of
pling, which is typically suitable for the 4D printed structure the actuator under a voltage induction and compared it with the
deformation process as a function of time. The numerical anal- simulation results with consideration to the mechanical and
ysis method provides a tool for precisely controlling the shape viscoelastic relaxation model of the dielectric elastomer. More-
deformation process. With the assistance of simulations, highly over, Lei et al. utilized FEM to generate materials with negative
reprogrammable 4D designs can be rendered, which can offer Poisson’s ratios termed as auxetic metamaterials, and further
greater possibilities and control for fabricating sophisticated generate a viscoelastic model (Figure 6C).[97] The thermome-
structures. chanical behavior of the material and the boundary conditions
Recently, Boley et al. have made remarkable progress in pro- were set periodically for higher computational efficiency, and
ducing structurally programmed lattices with multimaterials. both the Maxwell and theoretical models were built for the
Specifically, they have effectively 4D printed multiple materials deformation in 2D and 3D.
into heterogeneous lattice designs (Figure 6A).[56] The thin Generally, mathematical models and simulations enrich
printed shape-shifting structures were limited in morphing the quality of materials research. The FEM simulation results
into complex and doubly curved shapes, where the transfor- and corresponding experimental results can be mutually veri-
mation required not only a large in-plane expansion and con- fied, which by extension is quite promising for the design of
traction gradient but also control of the external curvature. In advanced pattern-programmed structures. Therefore, the
addition, it was demonstrated that the approach could be used mathematical model is a sufficient method for researchers to
to fabricate a hemispherical antenna that could shift its reso- design the pattern structure, and even the transformation of an
nance frequency as it changed shape, as well as a flat lattice that irregular shape can be easily obtained compared to the shape
transformed into a 4D human face. Kuenstler et al. developed change of more simplistic configurations.
Figure 6. Numerical analysis samples applied in 4D constructed models. A) A lattice multimaterial plane and its simulation results in shapeshifting
to a human face. Reproduced with permission.[56] Copyright 2019, National Academy of Sciences of the United States of America. B) Shape
transformation via localized photothermal inclusions, which were buckled into different shapes in both experimental demonstration and FEM
predictions. Reproduced with permission.[96] Copyright 2020, Wiley-VCH. C) A cylindrical shell with negative Poisson’s ratio and its football shell
deformation under stretching and its saddle shell deformation under compression. Reproduced with permission.[97] Copyright 2019, American
Chemical Society.
4. Polymeric Materials Employed in 4D Printing shape upon exposure to an external stimulus; during the pro-
and Applicable Printing Technologies cess, the external stimuli energy, such as heat and light, is
converted into a change in strain. SMAs have thoroughly dem-
Presently, 4D printing studies rely on the combination of onstrated their unique advantages over both irreversible and
3D printing and active materials, herein referred to as shape super mechanical properties, making them a superior class of
responsive materials (SRMs). These SRMs are primarily capable actuation materials.[104b] The SMCrs with oligocrystalline struc-
of actuating four broad types of 4D behaviors, including 1) self- tures possess a set of unique properties, including high energy
assembly,[74,99] 2) self-actuation,[100] 3) self-sensing,[52] and 4) self- output and high-temperature usage.[64] Generally, the SMAs
healing.[101] Among SRMs, shape-memory materials (SMMs) and SMCrs have potential clinical applications for smart bone
often involve metal alloys.[102] The term of shape memory effect implants due to their high mechanical modulus.[109] However,
(SME) refers to the ability of the shape memory alloys (SMAs) the flexibility of metal or ceramics implants is limited, and the
to achieve the transformation between two or several crystallo- resulting compliance mismatch affects their application in soft
graphic phases through diffusion-less transformations (the so- tissues implantation.[110]
called “reversible martensitic transformations”[103]). Presently, Polymeric SRMs such as SMPs, LCPs, and stimuli-respon-
the smart SRMs include SMAs,[104] shape memory ceramics sive hydrogels are a class of intelligent or adaptive materials
(SMCrs),[64,105] SMPs,[28,106] LCPs,[107] stimuli-responsive hydro- with broad prospects for a multitude of applications.[111] Com-
gels,[86,108] and composites containing shape-changing mate- pared to SMAs and SMCrs, polymeric SRMs are lightweight,
rials. The SME can be extended to the ability of all SMMs that low-cost, and electrical/thermal insulators, which can be easily
memorize temporary shape(s) and revert to their permanent manufactured using standard polymer processing t echnologies.
Their main advantages are 1) their ease of operation, synthesis, to trace patterns at various print speeds, and the resultant
and functionalization. Unlike the metallic materials that are printed scaffolds were used to culture and promote the car-
processed under high pressure and higher than 1000 °C, the diomyogenesis of human marrow mesenchymal stem cells
processing methods typically used for polymeric materials (hMSCs).[117] In addition, epoxy-based SMPs are another type
are generally milder (mostly lower than 200 °C). Polymeric of 4D printing material that is highly desirable, as they dis-
materials have been widely synthesized and functionalized in play high mechanical strength, chemical resistance for prac-
organic solvents using a wide variety of chemical reactions. 2) tical applications, and excellent thermal stability.[28] Using
Their excellent printability and elasticity. Compared to non- a DIW printing approach along with a two-step assisted UV
polymeric materials (e.g., the pressure to extrude most SMAs cured postprocessing method, 4D printed high-toughness
is between 50 and 500 MPa), polymeric materials are easily epoxy composites were fabricated and showed favorable shape
extruded. For example, the required operating pressures for memory properties, which can work to enhance the tensile
the deformation of SMPs are between 1 and 3 MPa. Moreover, toughness and tunable properties for high-performance and
the stretchability of polymers (up to 800%) is much higher functional applications of thermoset 4D constructs.[118] How-
than that of SMAs (less than 8%).[112] 3) Their biodegradability ever, the limitation of thermoset polymers such as (meth)
and biocompatibility (for the biopolymers). Therefore, consid- acrylate materials and epoxy polymeric material is that their
ering their universality and benefits in biomedical applications, permanent shape cannot be changed to be further modified
in this review paper, we will majorly discuss the deformation after synthesis due to the high degree of covalent linkages
behavior of polymeric SRM in various manners applied in 4D within the polymer networks. As such, the transformation
printing. of geometric structures is primarily from a temporary shape
For most polymeric materials, the applied 3D printing tech- to the original shape, which further limits their usage for 4D
nologies are extrusion-based and light-assisted printing. DIW printing applications.
(or FDM) printing is used to extrude liquid (or fused) polymeric In contrast to thermosets, 4D printed thermoplastic SMPs
materials with specific viscosities, while DLP (or SLA) printing are inherently reprogrammable due to the ephemeral nature
is suitable for all photocrosslinkable materials. of their crosslinked networks, and as such, they can be readily
used as extrusion-based printing inks. Some thermoplastic
SMPs have one broad reversible phase transition which
4.1. Shape Memory Polymers exhibits dual-, triple-, and even quadruple-SME, which are
highly tunable without the need to alter the material composi-
SMPs are a group of smart polymers that can maintain a tem- tion. FDM printing is the most common fabrication method
porary shape and recover their initial shape in the presence for generating constructs composed of thermoplastic mate-
of an external stimulus, such as exposure to heat, light, and rials, as their filaments are easily obtained by the extrusion
chemicals.[14] SMPs have been explored for various structure- of bulk thermoplastic materials. Recently, Xie synthesized
morphing applications,[113] especially for biomedical applica- a perfluorosulphonic acid ionomer (PFSA), which is a com-
tions, such as biological sutures, tissue engineering scaffolds, mercial thermoplastic polymer with a polytetrafluoroethylene
stents, and bladder sensors. Owing to their notable SMEs, backbone and perfluoroether sulfonic acid side-chains.[106] The
medical devices made using SMPs can be implanted into the results of their study demonstrated that the dynamic shape-
human body in a contracted or folded state through minimally changing behavior of the thermoplastic PFSA can be readily
invasive surgery and be restored to the desired original shape utilized to fabricate geometrically complex multifunctional
at the target location.[113b] Among the SMPs, heat-activated devices.[72b]
SMPs are one of the most commonly used classes of SMPs, Interestingly, some thermoplastic and thermoset SMPs dem-
as they demonstrate a broad range of tunable mechanical, onstrate a notable self-healing capability, which can be adapted
thermal, and optical properties.[70] They exhibit a significant for potential applications in tissue regeneration and other
change in elastic modulus when they shift between the glass biomedical devices such as vascular repair constructs.[101a,119]
state and the rubber state around their glass transition tem- Kuang et al. proposed a novel 4D printed, highly stretchable
perature (Tg).[114] Here, Tg is also referred to as the transforma- semi-interpenetrating polymer network elastomer in which
tion temperature(Ttrans) of a 4D printed structure. Thus, the a semicrystalline thermoplastic polymer was embedded and
mechanical properties of the heat-active (or thermoresponsive) printed using a DIW printer.[120] This 4D printed construct
SMPs can be dramatically different below or above their Tg,[76] could change its shape with time and displayed a notable self-
as well as their electrical resistance properties if they have healing functionality. Recently, Huang et al. synthesized ther-
any.[115] More details of thermoresponsive SMPs will be dis- moplastic vulcanizates (TPVs), which displayed tri-triggered
cussed in Section 5. SMEs with thermal, magnetic, and light induction. The damage
Recent studies of SMP-based 4D printing materials utilized of polylactide (PLA)/epoxidized natural rubber (ENR)/Fe3O4
photocrosslinkable (meth)acrylate, which is a commonly used TPVs could be healed synergistically through three events: 1)
thermoset polymer.[8] Recently, our group utilized a renew- the SME of TPVs led to the physical contact of damaged sur-
able plant-based soybean oil epoxidized acrylate (SOEA) ink faces, 2) the ENR/Fe3O4-bound rubber desorption–absorp-
and a custom-built SLA bioprinter to fabricate simple tissue tion promoted interdiffusion of ENR chains, resulting in the
scaffolds. The ink was developed using SOEA with bis(2,4,6- self-healing of ENR phase, and 3) ENR was grafted onto PLA
trimethyl benzoyl)-phenyl phosphine oxide as a photopolym- segments to assist PLA rearrangement and entanglement recy-
erization agent.[116] The ink was cured by activating a UV laser cling to achieve the repair of the TPVs.[121]
In the future, it is expected that finely controlled complex range[129] from tissue engineering to drug delivery,[130] and have
structures will be precisely fabricated using 4D printed SMPs. many functional advantages due to their high water content
Moreover, the transformation temperature of these SMPs is and solid-like mechanical properties.[131] Generally, hydrogels
expected to be controlled and tuned to be around that of physi- are prepared by covalent or physical crosslinking of hydrophilic
ological temperature (37 °C) for potential clinical applications polymers to form an insoluble network.[132] Owing to their high
such as smart punctal plugs.[122] elasticity and softness, biocompatibility, and multifunctionality,
hydrogels have been widely utilized across various fields of bio-
medical research, such as bioengineering, smart devices, soft
4.2. Liquid Crystal Polymers robotics,[86] and actuators.[133] Stimulus-responsive hydrogels
primarily achieve dynamic shape changes as a function of their
LCPs are polymers whose monomeric liquid-crystalline subunits swelling properties (including the effects of pH, biochemical
have a high degree of order and[107b] usually contain aromatic signals, and ion concentration) as well as their temperature-
rings as mitogens. LCPs are typically used for reversible shape- sensitive properties. Hydrogels are one of the most important
morphing inks for 4D printing due to their reversible, large, classes of smart materials, as they can utilize various external
and rapid mechanical actuation under the external stimulus.[123] sources of stimulation such as heat, pH,[130] light, chemicals,[71]
The most common application of LCPs are actuators, which can and electricity to actuate controllable and reversible shape trans-
show large and reversible shape deformations in response to a formations. With the exception of 4D shape changes induced
variety of applied stimuli such as heat, light,[107b] and a change by the gradient density of crosslinking, hydrogel-based shape
in the chemical environment. When heated above their nematic- morphing is primarily realized through the complementa-
to-isotropic transition temperature (TNI), the programmed LCPs tion of bilayer structuring. Many review articles focus on the
shrink along the alignment direction of their directors and polymeric hydrogel theory[134] and the applications of hydrogel-
expand in the orthogonal direction. Once cooled below TNI, based actuators along with other specific examples.[133] Com-
this process reverses.[124] LCPs also include LCEs[53] and liquid pared to the SMPs and LCPs, hydrogels are water-based
crystal networks.[107b] A recent review paper on the 4D printing materials and are far more similar to living tissues concerning
of LCEs[14] has outlined their broad potential applications in soft molecular structure and bulk material characteristics. The
robotics,[57] actuators,[125] and artificial muscles.[107a] Orientational highly biomimetic nature of engineered hydrogels enables
order in LCEs is obtained when the precursors are processed in them to have the most diverse set of biomaterial applica-
an LC phase (i.e., nematic phase).[107a] Saed et al. formulated tions, such as a distinct utility for use in tissue scaffolds, arti-
nematic inks via the self-limiting “thiol-acrylate” Michael’s addi- ficial muscles and valves, wound dressings, and many other
tion reaction between a nematic diacrylate and an isotropic clinical applications.[135] DIW is the most common technique
dithiol, and using a DIW technique, the inks were patterned in applied for the fabrication of both physically and chemically
molecular order within 3D LCEs geometries.[107a] Barnes et al. crosslinked smart hydrogels.[136] Moreover, DLP and SLA are
proposed a catalyst bath-assisted printing method utilizing an also widely used in the manufacturing of 4D photocrosslink-
LCE precursor solution to produce complex architectures via able hydrogels.[130]
direct printing.[126] LCEs with tunable actuation temperatures Among these hydrogels, thermoresponsive poly(N-isopropy-
and actuation strains can be utilized for the fabrication of intel- lacrylamide) (PNIPAm) is a typical shape memory hydrogel that
ligent constructs such as soft robotics and implantable medical has been used for a wide range of biomedical applications,[100]
devices. such as cell sheeting technologies.[137] PNIPAm becomes hydro-
These mechanically active LCPs provide critical benefits phobic and insoluble in water above a lower critical solution
and may replace traditional actuators in applications with low temperature (LCST), whereas it is soluble below this value.[137]
density, large shape change, and autonomous activation.[107a] The great interest for PNIPAm resides in the fact that its LCST
Additionally, recyclability and self-repair have also been dem- value ranges between 32 to 35 °C, which is similar to human
onstrated in the development of LCPs.[127] As a typical example physiological temperature. This characteristic of PNIPAm has
of biomedical applications, Babakhanova et al. demonstrated been recently exploited by Sanzari et al. as they developed cell-
notable cell alignment on polymerized smectic A LCE coatings sheeting tissues generated from patients’ autologous cells for
with nanogrooves, which was synthesized by mixing the typical cardiac tissue engineering.[137] At 37 °C, the cells easily grew
smectic mesogen, 4′-(octyloxy)-4-biphenylcarbonitrile, with a on hydrophobic PNIPAm surfaces. When the temperature
reactive monomer 2-methyl-1,4-phenylene bis(4-((6-(acryloyloxy) was reduced, PNIPAm became hydrophilic and promoted the
hexyl)oxy)benzoate) (RM82).[128] This study notably extended detachment of cell sheets. This thermosensitive behavior of
the methods of controlling cells behaviors through the topog- 4D hydrogels enables the fabrication of constructs with great
raphy of substrates, which effectively illustrates this method’s potential applications in the fields of bioengineering and bio-
potential utility in tissue engineering. technology.[138] However, there are also some notable drawbacks
to using 4D hydrogels, such as low mechanical strength, low
biodegradability, and limited loading or release capacity of
4.3. Hydrogels drugs/cells.[139] Therefore, more 4D hydrogels with high perfor-
mance are expected to be synthesized and utilized for future
Hydrogels usually consist of a large amount of water and 3D biomedical studies.
cross-linked polymer networks. Hydrogels are an attractive class Similar to the other polymeric materials applied in 4D
of materials suitable for many biomedical applications, which printing, polymeric hydrogels also possess the ability to self-heal
after physical/chemical/mechanical damage in order to fully or to be developed via the combination of multiresponsive mate-
partially restore their original properties, which enhance their rials that have complementary advantages. For instance,
prospects for application in various therapeutic and clinical taking advantage of the stretchability/elasticity of hydrogels,
settings.[101b] Moreover, multifunctional hydrogels with shape DEA-based carbon nanotube/polyvinyl alcohol (PVA) hydrogel
memory and self-healing capabilities are expected to be fur- electrodes were demonstrated to have an enhanced degree of
ther developed shortly in order to be adapted for the repair and stretchability and self-healing capabilities.[144] In another study,
regeneration of functional tissues. This is largely due to their the assembly of agarose hydrogels, SMPs, and enhancing com-
capability for notable geometric change, which can notably ponents, yielded 4D constructs which could be reversibly sof-
facilitate the implantation of different tissue geometries and tened by heating and hardened by cooling, largely as a function
the spatiotemporal distribution of the multiple cell types. of the sol–gel transformation behavior of agarose.[145]
4.4. Others 5. Stimuli and Mechanism of 4D Printing

Whereas there is a multitude of functional materials which Here, different stimuli-responsive mechanisms are discussed
can directly respond to external stimuli in order to actuate in each section based on the various internal and external
dynamic 4D shape changes, there is also a variety of important stimuli, which have been used to enact the 4D effect, including
polymeric materials which are non-responsive themselves but temperature, chemical induction, light, magnetism, and mul-
can nonetheless contribute to the generation of multimaterial- tistimulation sources. The classification of basic stimuli used
based 4D constructs. For example, the low mechanical modulus to trigger 4D printed architectures is illustrated in Figure 7,
of SMPs is a notable drawback for various biomedical appli- where the type of stimuli can be divided into internal (inner)
cations; therefore, material improvements are being investi- and external stimuli.
gated employing the incorporation of reinforcing fibers within Generally, the most common stimuli we have included are
printed constructs. Whereas the printing parameters need to external, considering that internal stimuli typically only refer to
be adjusted due to the introduction of new components in this internal (inner) force-triggered shape deformation (or mechan-
regard, typical printing methods such as DIW and FDM can ical-triggered shape transformation). Currently, the external
still be readily utilized toward this aim.[85] stimuli used to trigger the 4D effect of a printed construct can
Moreover, the introduction of nanomaterials into 4D com- be summarized into four types: 1) chemical, 2) thermal, 3) light,
posite materials has been investigated to enhance their 4D and 4) magnetism. The chemically based trigger mechanisms
functionalities. One of the most commonly 4D materials is can be further subdivided into pH, ion, solvent, humidity, and
graphene (or graphene oxide), which is readily incorporated biochemical signal-induced types. Meanwhile, the thermally
into SMP composites and allows 4D constructs to achieve based stimuli can be enacted by direct heating (temperature),
near-infrared (NIR)-triggered 4D shape change.[119] Specifically,
graphene-based composites can absorb the photons from NIR
light and then transfer the light energy into heat, in order to
induce a rise in local temperature within a given SMP, which
ultimately leads to a thermoresponsive shape transforma-
tion.[76] In addition to composites functionalized by nanopar-
ticles which make them sensitive to the photothermal effect,
magnetic nanoparticle compounds such as carbon porous
nanocookies coupled with high-frequency MF enable SMPs to
respond to the MF stimulus.[140] In recent years, the applica-
tion of magnetic compounds and externally applied MF in the
fabrication and modulation of soft robotics and actuators has
gained substantial research attention. Owing to their excellent
mechanical response to exogenously applied MF, including
induced magnetic torque and magnetic force, and the fast
response to the external stimuli compared to optical or acoustic
stimuli, magnetic nanoparticle compounds exhibit great poten-
tial for remote control of dynamic biomaterial processes for
various biomedical applications.[141]
Furthermore, dielectric elastomers are widely used to
develop artificial muscles because of their excellent properties
under applied EF.[142] The typical application of dielectric elas-
tomers is in the production of dielectric elastomer actuators
Figure 7. The manufacturing process of 4D printing is primarily based
(DEAs), which function as promising soft transducers. Funda-
on existing 3D printing technologies. Classification of 4D printing can
mentally, DEAs operate as compliant capacitors, in which the be sorted by stimulation sources, including two categories: inner stimuli
dielectric elastomer deforms in response to an applied EF.[143] and external stimuli, and subtypes under five subcategories: inner-, mag-
In the future, more functional smart materials are expected netism-, light-, heat-, and chemicals-stimuli.
electrothermal heating, and magnetothermal heating. More- z ( t ) z ( t )
over, the light-triggered stimuli usually can be subdivided into ∫ σ dz = ∫ E ( ε − ε s ) dz = 0 (1)

0 0
NIR light, UV light, and visible light sources. This section will
review the recent developments pertaining to the mechanisms, Here z (t ) is the solidified thickness, σ is the stress, and
materials, and applications of stimuli-responsive 4D printing. σ = E(ε − εs) = Eεe, which is determined by Young’s modulus E,
and ε, εs, and εe are total strain, the shrinkage strain, and elastic
strain, respectively. Here, the elastic strain is obtained by the
5.1. Internal (Inner) Stress-Triggered superposition of bending and stretching
z
Unlike the shape changes which are triggered by external ε e = ε − ε S + ε b + κ  z −  (2)
 2
stimuli, internal stress-triggered shape change is driven by the
incorporation of the “stress-relaxation” phenomenon during the where εb is the bending strain of the midplane at half of z (t ),
material manufacturing process. As indicated in Figure 8A,[72a] and κ is the bending curvature, which can be calculated by the
Zhao et al. proposed an internal stress-induced method to force equilibrium and moment equilibrium.
actuate a dynamic 4D transformation. When the polymeric Our research team also proposed an inner stress-triggered
film generated in their study was gradually photopolymerized multiresponsive 4D bioprinted architecture which has notable
from one side, the initially cured material was allowed to shrink utility for neural engineering applications, as indicated in
to release internal stress. By contrast, the newly cured mate- Figure 8B.[91] An autonomous, reversible, and programmed
rials shrank under the confinement of the previously printed shape configuration was achieved, which was triggered by the
layers of material. After the structures were removed from the light-induced graded internal stress within the printed con-
printing platform, the structures assumed a curved configura- struct, and subsequent solvent-induced relaxation.[91] The 4D
tion by releasing the internal stress, enabling them to reach a SLA printing technique used to generate the 4D neural scaffolds
static balance. The sequential shrinkage-induced bending cur- relied on a laser head to directly draw the desired structural
vature was affected by both irradiation time and illumination patterns on the photocrosslinkable SOEA resin. Interestingly,
light intensity. They also established a theoretical model based the resultant pattern design was able to achieve more complex
on the beam theory. The mechanical behavior of films under shape-changing such as torsion. After immersing the sample in
illumination was controlled by force equilibrium along the ethanol, the uncrosslinked SOEA dissolved, which triggered the
thickness direction, where release of internal stresses and drove the dynamic shape change
Figure 8. 4D printed structures and their shape change modalities achieved by internal stress. A) By changing the exposure time in DLP printing,
different composites led to dynamic shape changes, though the structure was fabricated using a single material. Reproduced with permission under
the terms of the Creative Commons Attribution-NonCommercial license.[72a] Copyright 2017, The Authors, published by American Association for the
Advancement of Science. B) The internal stress-induced deformation was controlled by varying the crosslinking density. Reproduced with permission.[91]
Copyright 2018, Wiley-VCH. C) The SLA printed cardiac tissue structure with tunable programmed curvature. Reproduced with permission under the
terms of the Creative Commons Attribution-NonCommercial license 4.0.[31] Copyright 2020, The Authors, some rights reserved; exclusive licensee
American Association for the Advancement of Science.
process. In this study, the mechanical information was neces- of printed constructs. For example, NIR light, MF, and EF are
sary to drive the 4D effect was directly incorporated within the also able to induce a localized change in temperature, which
fabrication process as a function of the different crosslinking have been demonstrated to effectively drive the shape recovery
densities of the upper- and lower-layers of the construct. process of SMPs. Figure 9A shows the molecular mechanism
Moreover, our research group applied a similar technology to of thermoresponsive SMPs, illustrating their thermal dis-
fabricate a 4D printed cardiac patch, as illustrated in Figure 8C. tinction between the glass and rubber states. Generally, ther-
The hydrogel-based cardiac patch was fabricated using an SLA momechanical characterization tests are conducted with a
printer, and the printing speed and laser intensity were varied dynamic mechanical analysis machine to obtain consecutive
as required, which enabled the ability to tailor the degree of elasticity (shape memory) cycles (Figure 9B).[72b] A typical pro-
crosslinking of the inks and, thereby had a profound effect cess of programming 4D constructs is indicated in Figure 9C,
on the physicochemical properties of the resultant hydrogels. when the external temperature surrounding an SMP is raised
Additionally, we also established a mathematical model to cal- to high levels (above its Tg or Ttrans), the bonds of the polymer
culate the 4D curvature of the hydrogel structure. The relation- molecules become weaker, which allows for the constructs to
ship between the redundant length of the stretchable structure be more easily deformed into temporary configurations under
ΔL and the ventricular curvature κ in the systole state is applied external force (such as bending or twisting). Once the
temperature of the SMP decreases below its Tg, the tempo-
cos−1 (1 − D 2κ 2 /2) rary shape can be affixed without the application of additional
∆L = −D (3)
κ external force. When the temperature of the SMP has increased
The redundant length was designed to mimic the differences again (approaching its Tg), it will recover to its original shape.
between the systole and diastole phases of the beating heart. As Here, a parameter Rret is defined as the shape retention ratio
such, the 4D printed hydrogel patch enabled a highly stretch- ε
R ret = × 100% , with εload and ε representing the strains
able structure with very limited deformability. It is believed that ε load
this increase in material elasticity and decrease in bulk deform- at the Tg or Ttrans before and after load removal, respectively.
ability contributed to a distinct reduction in both e adverse The shape fixity ratio (Rf ) for a typical elastic shape memory
effects on the resident cardiomyocytes and the material fatigue. ε
cycle is defined by a similar equation R f = × 100% . The
A major benefit of the internal stress-triggered shapeshifting ε load
effect is the ease at which the 4D structure is able to be pro- critical difference lies in that ε in Rf refers to the temporarily
grammed.[69] The direction of the shapeshifting is determined by fixed strain, whereas ε in Rret is the permanent strain. These
the heterogeneous crosslinking densities of the fabricated struc- 4D printed thermoresponsive structures are primarily fabri-
tures. These methods provide a novel and simple means to create cated using DLP,[60,147] SLA,[116] and FDM[80,148] technologies and
metamaterials and 4D constructs with utility for a wide range of the Tg of these SMPs typically varies from room temperature
applications. By adjusting the irradiation time and crosslinking to hundreds of degrees Celsius.[147,149] Due to the wide range of
degree, a complex, two-side folded origami structure can also responsive temperatures, thermoresponsive SMPs have been
be achieved,[72a] which can potentially be adapted for biomed- utilized as sensors, soft robotics,[150] actuators,[147] and other
ical devices and drug delivery applications. Among the SRMs, various applications.
photocrosslinkable materials are primarily utilized for internal In terms of the fabrication of intricate architectures, ther-
stress-triggered 4D printing since the modulation of the degree moresponsive SMPs display temperature-dependent plasticity.
of crosslinking of the material is relatively simple to implement. Yang et al.[151] created a 4D printed, reconfigurable, deployable,
and mechanically tunable metamaterial. Due to the consider-
able differences in the mechanical properties of the compo-
5.2. Thermoinduced nents of the constructs, such as the tensile, compressional, and
flexural moduli under different temperatures, the 4D structures
A typical 4D structural programming process such as rolling were utilized as tunable springs, which were found to be as stiff
and folding of thermally sensitive constructs, including hydro- as wood at 30 °C and as soft as a sponge at 90 °C (Figure 9D).
gels, LCPs, and SMMs, usually results from the change of Additionally, the thermo-induced shape recovering behavior can
molecular structure, anisotropic nature, or different swelling also be utilized for complex structure design, such as origami-
behaviors with the variation of temperature. Although ther- like artifacts (Figure 9E)[24] and bionic structures (Figure 9F).[69]
moresponsive hydrogels and LCPs have been applied in 4D Thermoresponsive SMPs have also been utilized for the
printing, few studies focus on their biomedical applications. fabrication of superhydrophobicity-memory surfaces. Recently,
Additionally, thermoresponsive SMPs (also known as thermoa- Bai et al. fabricated a construct with a superhydrophobic sur-
dapt SMPs)[146] have recently gained considerable attention and face using a femtosecond laser SLA approach. It was found
have been the central biomaterial used in several 4D printing- that the surface morphology and wettability of the printed con-
based recent biomedical studies. Thermoresponsive SMPs struct could be completely recovered before and after heating
exhibit excellent biocompatibility and phase/mechanical trans- (Figure 9G).[149] Due to the superhydrophobicity-memory
formation properties at body temperature, as discussed above microstructures on the surface, the printed material was able
(Section 4). As such, we will majorly discuss the mechanism to perform a thermoresponsive self-cleaning process. Moreover,
and applications of thermoresponsive SMPs here. Shao et al. developed a femtosecond-laser-assisted SLA printed
In addition to direct temperature-triggered stimuli, indi- surface with a micropillar array that could switch transfer
rect heating has also been utilized to drive the 4D actuation between superhydrophobic surface and hydrophilicity.[152] The
Figure 9. A) A schematic illustration of thermoresponsive SMPs whose network is thermally distinct. B) The consecutive elasticity cycles of a typ-
ical shape memory polymer. Reproduced with permission under the terms of the Creative Commons Attribution-NonCommercial license 4.0.[72b]
Copyright 2016, The Authors, published by American Association for the Advancement of Science. C) Conventional schematic diagram of the defor-
mation and recovery process in the SMPs fold-deploy tests. Reproduced with permission.[60] Copyright 2019, Elsevier. D) The time-lapsed images of
an SMP tetrakaidekahedron Kelvin foam loading under 30 and 90 °C. It shows a shape memory circle of an SMP microlattice whose shape is pro-
grammed through three steps: heating, deformation, and cooling. Reproduced under the terms of the Creative Commons Attribution 3.0 Unported
Licence.[151] Copyright 2019, The Royal Society of Chemistry. E) 4D printed Origami structure via DIW printing. The 3D-printed elastomeric lattices can
be transformed into a curved configuration with a honeycomb-like internal structure. Reproduced under the terms of the Creative Commons Attribu-
tion license.[24] Copyright 2018, American Association for the Advancement of Science. F) A thermoresponsive 4D printed structure with multimaterial
composites fabricated by a direct 4D printing approach to achieve a programmed shape. The printed flower consists of multiple petals on multiple
layers where the petals at multiple layers bloom into different configurations with different curvatures. The pictures with a yellow background demon-
strate different configurations upon heating can be programmed and can also recover their permanent shapes. Reproduced with permission under
the terms of the Creative Commons Attribution-NonCommercial license 4.0.[69] Copyright 2017, The Authors, published by American Association for
the Advancement of Science. G) A 4D printed superhydrophobicity-memory surface assisted with a femtosecond laser, whose surface can be shifted
both at macro- and microscales. The reversible morphology transformation of the 4D printed surface between the original micropillar array and the
deformed micropillar array determines the construct’s wettability. The image illustrates the liquid transportation on a curved track of the SMP surface.
Reproduced with permission.[149] Copyright 2020, Elsevier.
pattered microarrays of the construct surface could be triggered (pH[155]), cations[156] such as Ca2+ and Fe3+, solvents, humidity,
to raise or lower by increasing or decreasing the temperature. or water,[84] have important applications in the fields of tissue
These results illustrate that the morphology and wettability of engineering and biomedicine. The most common chemical-
the 4D printed thermoresponsive surface can be transferred responsive materials are hydrogel, which has received growing
through a heating process, implying their potential in the regu- attention due to its similarity to soft tissues, as we discussed
lation of cell behaviors and mass transportation. In the future, above in Section 4. Generally, the primary mechanism of chem-
it is expected that 4D printed microarrays or micropatterns can ical triggered 4D shape change is the swelling behaviors of
function as rewritable platforms to achieve directional transpor- hydrogels.
tation of cell-containing liquid droplets or droplet-based micro-
reactors. Furthermore, 4D printed microneedle arrays with
dynamic backward-facing barbs have been fabricated toward 5.3.1. Solvent
the aim of enhancing tissue adhesion.[153]
A critical factor for 4D printed tissue constructs is the The swelling behavior of hydrogels in aqueous environments
tuning of the shape-recovery temperature of SMPs, which (or humid environments) is the most prominent driving force
must operate around physiological temperature in order to be for their shape deformations. To evaluate the swellability of
employed for cell culture or in vivo implantation purposes. hydrogels such as poly(ethylene glycol) diacrylate (PEGDA),
Therein, thermosensitive SMPs with high Ttrans may lead to the gelatin, gelatin methacroyloyl (GelMA), and other hydro-
death of resident cells, whereas SMPs with considerably low gels, the equilibrium swelling ratio Qs is usually estimated by
Ttrans may cause a premature change of shape during culturing. immersing the dried hydrogel samples into PBS or deionized
As numerous choices of thermoresponsive SMPs or compos- water at 37 °C for a certain period (up to 7 days,[157] or shortly
ites with different Ttrans (Tg) are currently available, the Ttrans of 6 h[158]) in order to promote water ingress into the hydrogel
4D printed constructs can be optimized to be suitable for bio- bulk (swelling). The Qs definition is according to the following
medical applications.[148] Recently, our research group has syn- equation
thesized a biocompatible SMP whose Tg is around physiological
Ws − Wd
temperature.[154] The thermoresponsive constructs we devel- Qs = (4)
oped can be deformed at a specific temperature above Tg using Wd
a conventional thermomechanical reprogramming technique. where Ws and Wd are the weights of the swollen samples and
After the temporary shape of the printed constructs was affixed the dried samples, respectively.
at a lower temperature (below Tg), cells were seeded onto the Taking advantage of the swellability of hydrogels, the
flat temporary shape’s surface. After the cells were evenly dis- swelling degree of hydrogels relies on their internal structures,
persed and attached to the culturing surface, the temporary including microstructural anisotropy, crosslinking density, and
shape reverted to the original 3D structure, forming a perma- hydrophilicity. While a homogeneously crosslinked gel displays
nent, complex 3D tissue structure with a uniform cell distribu- isotropic volume change, an anisotropic bilayer structure pos-
tion. The biomedical applications of temperature-responsive 4D sessing different internal structures causes stress and results
printing will be discussed in more detail in Section 6. in large amplitude folding and buckling. Hao et al. proposed
Generally, as one of the most widely used 4D materials, tem- a novel method for achieving a Kirigami-design 4D shape-
perature-responsive SMPs exhibit several advantages for the bio- changing effect for hydrogels by exploiting the discrepancy
medical applications: 1) a benign and straightforward nature of between active and passive elements in hydrogel structures.[159]
the thermal stimulation source, as opposed to the harsh chemicals The red, blue, and gray portions of the graphical depiction of the
and applied electrical/magnetic fields, 2) a 4D transformation trig- 4D hydrogel in Figure 10A represent the high-swelling soft poly
gered at body temperature, and 3) a greater diversity of achievable (acrylamide-co-2-acrylamido-2-methylpropanesulfonic acid) gel,
shape transformations compared to internal stress induced 4D the soft nonswelling polyacrylamide gel, and the nonswelling
shape change. We expect that future development of thermore- stiff poly(methacrylamide-co-methacrylic acid) gel, respec-
sponsive SMPs will yield more biodegradable and biocompatible tively. This design led to the active portion of the hydrogel con-
SMPs formulations which can actuate 4D transformation around structs becoming more deformed, whereas the rigid portions
physiological temperatures, which will effectively broaden their remained largely unchanged. Therein, the determining factor
application in tissue engineering and other biomedical applica- for which portion of the construct underwent swelling or not
tions. It should be noted, however, that thermally responsive SMPs was the exposure time to the solvent. This controlled swelling
require a “thermomechanical reprogramming” process to achieve of hydrogel constructs may further inspire future applications
a repeatable 4D transformation, which limits their utility to func- in biomedical devices to accompany the multistable morphing
tion as actuators of autonomously cyclic deformations, such as configurations achieved by these hydrogels.
with biorobots. In this case, LCPs have greater utility toward this Additionally, Schwartz and Boydston[160] utilized a highly
application, and as such, the development of biocompatible LCPs novel multimaterial actinic spatial control method to fabricate
with a biocompatible Ttrans will be necessary for future studies. a 4D structure with two components in the same resin tank.
Under long-wavelength irradiation, the curing of the acrylate
components of the ink formulation was achieved within the
5.3. Chemical-Triggered mixed resin. Meanwhile, under short-wavelength irradiation,
the combined ingredients of the composition (acrylates and
Chemical-responsive materials, whose physical properties epoxides) were cured together. Thus, the different swelling
respond to chemicals in the surroundings, such as H+/OH− ratios between the two composites led to the deformation of the
Figure 10. A) The 4D out-of-plane deformation of preswollen composite hydrogel in water after patterning, which can control the buckling direction
of each strip. Scale bar = 1 cm. Reproduced with permission.[159] Copyright 2020, Wiley-VCH. B) pH-responsive shape deformation of the M1S0.5A0.5
hydrogel, which is spontaneously twisted in 0.1 m HCl solution after the first shape recovery, and the original shape was recovered in 0.1 m NaOH solu-
tion for 10 min. The picture of the 5th and 10th spontaneous twisting and recovery demonstrate the repeatability of shape deformation. Reproduced
with permission.[161] Copyright 2018, Wiley-VCH. C) Solvent (hydration)-induced shape memory performance of a “D” shaped construct. Reproduced
with permission.[158] Copyright 2020, Elsevier. D) Illustration of the working mechanism and its repeatability of the pH-responsive hydrogel (top). The
schematic diagram and experimental diagram (bottom) show that a well-designed microcage structure is used to capture a microsphere. The SEM
image in the bottom right corner is an image of the captured microsphere in the microcage. Scale bars: 10 µm. Reproduced with permission.[130] Copy-
right 2020, Wiley-VCH. E) Photographs of the reversible folding properties of PCL/AA-MA bilayer self-folded tube. Actin staining images of cells on the
AA/MA layer and PCL/AA-MA bilayer. Actin (green) and nuclei (blue). Reproduced with permission.[75] Copyright 2021, The American Chemical Society.
4D printed structure. Shape transformation occurs as an out-of- crosslinked by glutaraldehyde to form permanent net-points.
plane motion when nonuniform in-plane stresses induced by The coordination interaction between the molecular chain of
swelling are released. The solvent-induced stimulus is relatively sodium alginate and calcium ion was used to create tempo-
easy because a 4D construct can achieve the shape transfor- rary crosslinks. With a change in the concentration of calcium
mation or functional transformation only through immersion ions, the hydrogel exhibited a favorable ion-responsive SME.
into a solvent such as water as indicated in Figure 10C.[158] It is Constante et al. developed a bilayer hydrogel which was com-
ultimately preferable for water to serve as the primary trigger posed of a 3D extruded methacrylated alginate (AA-MA) layer
mechanism for the dynamic 4D effect of the 4D tissue scaf- and a separate layer of melt-electro-written PCL. The diameter
fold, as water is the primary-most component of the natural of the resultant tube construct could be precisely controlled by
microenvironment of the human body, which is composed of varying concentrations of Ca2+ ions in the culturing media and
more than 70% water by volume. If the 4D constructs can be by varying the PCL/AA-MA thickness ratio. In addition, seeded
efficiently used to mimic the native dynamic culture matrix, it myoblasts demonstrated favorable proliferation on the AA-MA
is expected that solvent-induced 4D constructs may be readily film and the bilayer film, and displayed a high degree of cel-
combined with lab-on-chip technologies, which show notable lular orientation on the PCL: fibers (Figure 10E).[75] Addition-
promise to become fully dynamic experimental platforms ally, Han et al. prepared a novel zinc-ion-inspired hydrogel with
which can replicate the functionalities of multiorgan systems a triple SME.[163] Meanwhile, Wang et al.[156] also developed a
within a fluid environment. polyacrylamide/sodium alginate (PAM/SA) hydrogel and used
six divalent cations, including Fe3+, Cu2+, Ag+, Ca2+, Ni2+, and
Co2+, to characterize the hydrogel’s 4D shifting behavior. They
5.3.2. pH found that the metal ions, such as Ag+, Zn2+, and Cu2+, dis-
played notable antibacterial and antitumor properties, and
pH-responsive hydrogels are commonly used to actuate shape could be used in topical dressings or other biomedical appli-
change of 4D structures as a function of the significant shift cations. Meanwhile, catalytically active Fe3+, Ni2+, and Co2+
in their swelling degree when the ratio of H+/OH− ions in an could pass through water for redox degradation of harmful
aqueous solution are varied. In a recent study, a spontaneous substances.
shape change was achieved by a 4D printed construct after it
was manually twisted into a temporary helical shape and fixed
by immersion in 0.1 m HCl solution. The shape change achieved 5.3.4. Biochemicals
by the construct was induced by continuously varying the pH
by immersing it in NaOH and HCl solutions alternately.[161] The Biochemicals, such as glucose, are highly desirable for devel-
recovery time of the construct was controlled and modulated to oping an artificial physiological system for 4D printing appli-
be around 90 s, with the recovery process also being demon- cations. In addition to modulating the concentrations of pH
strated to be repeatable (Figure 10B).[161] Moreover, Hu et al.[130] and ions a given printed construct is exposed to, biochemical
developed a novel botanically inspired 4D printed hydrogel con- signals have great significance for in vivo dynamic environ-
struct using a femtosecond laser direct writing approach. The ments. For instance, a closed-loop system capable of inducing
deformation mechanism of the resultant construct was largely an insulin release in response to a glucose concentration under
attributed to the electrostatic repulsion between the carboxy- physiological conditions was developed.[164] Additionally, Zhao
late ions, leading to a significant increase in the size of the et al. synthesized a pH and glucose dual-responsive hydrogel
polymer mesh. Conversely, when the pH is lower than 9, the through the covalent cross-linking of imine bond and phenyl
carboxyl group accepts protons and is therefore deionized. The boronate esters using phenylboronic-modified chitosan and
ionization effect of carboxyl groups is much more forceful than oxidized dextran.[155a] During the in situ cross-linking process,
their deionization processes. As such, the hydrogel exhibits proteins, drug compounds, and living cells were encapsulated
stronger or weaker swelling in alkaline or acidic solutions, within the developed hydrogel. The sustained pH/glucose-trig-
respectively (Figure 10D).[130] The drawback of currently avail- gered drug release from the hydrogel was observed to enhance
able pH-induced 4D constructs is that the pH variation is much wound healing, which allowed the hydrogel to be used as a bio-
larger than the pH range observed in the human body. Novel active dressing for diabetic wound repair.[155a] This work dem-
pH-responsive hydrogels should be synthesized to achieve 4D onstrated that the pH/glucose dual-stimuli hydrogels could play
transformation with high pH sensitivity, whose value is close to a role in a delivery platform for various therapeutic proteins/
that of the human body. live cells for wound-healing applications.
Moreover, Song et al. fabricated a novel glucose-responsive
sensor using an inkjet printing method. The glucose oxidase
5.3.3. Ions and platinum nanoparticles were combined into the fabri-
cated chemoresistance sensor to produce a two-step catalytic
In addition to the H+/OH− ions, some divalent ions, such as process. The first step involved the oxidase-driven catalysis
zinc (Zn2+), calcium (Ca2+), and iron (Fe2+), have wide applica- of glucose to yield hydrogen peroxide. The second step of the
tions in driving the shape memory or shape change behavior process involved the catalysis of hydrogen peroxide to produce
of hydrogels through the adjustment of the degree of swelling hydroxide ions utilizing the incorporated platinum nanoparti-
degree. Recently, Xu et al.[162] developed a hydrogel with a cles to cause local pH changes near the reaction site.[165] This
gelatin–alginate interpenetrating network, where gelatin was work proposed a simple printing method for fabricating a rapid
and disposable glucose-sensing device that can be employed in Additionally, thermomagnetic and electromagnetic-induced
an on-demand printable point-of-care diagnostic kit for blood shape changes have been extensively investigated in recent
glucose measurement and may have the potential for broad- studies.[171,177] The mechanism of the thermomagnetic SME of
scale clinical commercialization. magnetic SMPs (M-SMPs) can be described by the Néel–Brown
Generally, the chemical-responsive materials primarily model.[5] In an alternating MF with a relatively high frequency,
involve three major shape change conditions: 1) swelling in a the magnetic vector cannot follow the change of the MF and
given solvent solution, 2) ionization/deionization of polyelec- is out of phase with the MF, which results in the magnetic
trolytes, and 3) the exchange of ions. The advantages of chem- energy being dissipated and converted into heat.[5] The relaxa-
ical-responsive materials are primarily related to their capacity tion time depends on the particle size and the materials used:
for reversible shape transformation, considerable biocompat- for magnetite particles below 10 nm, fast Néel model relaxa-
ibility, and wide selection of ionic triggers. However, there are a tion dominates; by contrast, larger particles tend to follow the
few noteworthy drawbacks that should be addressed for future Brown model. Therefore, obtaining a magnetic composite with
applications of chemical-responsive 4D printing through the excellent shape memory properties requires strict control of the
swelling of hydrogels: 1) the response speed tends to be very size of the magnetic particles, and also requires them to be uni-
low, especially for large objects, because the expansion mecha- formly dispersed in the polymer matrix.[178] Zhang et al. utilized
nism is fundamentally limited by mass diffusion, 2) the actua- a PLA/Fe3O4 4D structure to achieve the shape recovery of a 4D
tion shape is often unstable due to the volatility of water, and 3) printed structure in an applied MF. An aschematic example
most hydrogels are relatively brittle. Loss of shape fidelity and (Figure 11B) showed the application of the 4D scaffolds for
material integrity may occur during the process of hydration/ bone repair, which may have great potential in the engineering
dehydration.[166] Additionally, the low mechanical properties of of other tissues.[173] Additionally, Wei et al. utilized a 30 kHz
hydrogel-based materials are a significant concern. alternating MF to magnetically guide and remotely actuate the
self-expanding behavior of an artificial vascular stent with Fe3O4
nanoparticles.[179]
5.4. Magnetic Field-Triggered Mohr et al. developed a noncontact triggering method of
shape-change via inductive heating of magnetic nanoparticles
Magnetism-driven 4D printing has attracted tremendous atten- embedded within thermoplastic SMPs.[180] The shape change
tion in addressing problems of the requisite contact between process of the TFX100 composite demonstrated that a strip with
the printed constructs and the stimulation source (e.g., heat a temporary spiral shape could recover its permanent flat shape
transfer media or liquid) to achieve or maintain the 4D shape due to the thermomagnetic effect. Moreover, the magnetic
change, as is seen with thermoresponsive and chemical-induced response effect has been applied for embossing technology.
trigger regimes. Magnetism-responsive materials deform under Testa et al. developed a novel M-SMP, which could undergo
magnetic forces exerted by an applied MF, in which the forces an exceptional stiffening transition, whose shear modulus can
still exist and operate at a critical distance between the printed increase almost 30 times with the influence of the MF.[172]
constructs and the magnets used to drive the transformation An advanced method of combining direct magnetism-
process. This unique feature facilitates the remote control of 4D induced and thermomagnetic SME has attracted much
shape change. Magnetism-responsive materials have been widely research interest in the past few years. Prof. Qi from the
used to design and fabricate various actuation devices due to Georgia Institute of Technology and Prof. Zhao from the Ohio
their excellent mechanical response to an external MF, including State University have performed relevant research on the actua-
magnetic force and torque, for diverse applications such as tion of MF and heating MF in manipulating thermoresponsive
actuators,[167] soft robotics,[168] biomedical devices,[169] tissue M-SMPs.[175,176] Additionally, Ze et al. produced a multicom-
engineering scaffolds,[170] and drug delivery systems.[171] Current posite construct consisting of two types of magnetic particles in
research in magnetism-driven 4D printing largely focuses on an amorphous M-SMP matrix.[175] As illustrated in Figure 11C,
the deformation of magnetism-responsive polymer composites the working mechanism of this design is that the deformation
achieved by SLA, SLS, and DIW printing. Through the addition of stiff M-SMP could not be driven by the actuation MF (Ba)
of magnetic metal particles and their oxides, there are three major at low temperatures, while M-SMP became soft and could be
methods by which to induce the shape change effects using mag- deformed under a heating MF (Bh). After closing the Bh, the
netism-responsive composites including: 1) direct magnetism,[172] M-SMP was cooled to harden it and to affix the temporary
2) thermomagnetism,[173] and 3) electromagnetism.[174] shape. With the programming of MF, the M-SMP could be
Wu et al. adopted a popular SLS method to fabricate a direct applied as a four-armed soft robotic grippers construct, deploy-
magnetism-driven 4D printed architecture consisting of mag- able/reconfigurable antennas, sequential actuation devices,
netism-responsive composites (thermoplastic polyurethane and and digital logic circuits. As depicted in Figure 11D,[176] the
magnetic particle (Nd2Fe14B)), whose programmed shape can DIW-based 4D printed magnetic multimaterial construct was
be changed through the application of an external MF.[65] As fabricated by integrating M-SMPs and magnetic soft materials,
illustrated in Figure 11A,[65] magnetic particles were arranged and the resultant enhanced multimodal shape transformation
synchronously along the direction of the external MF during the with tunable properties and shiftable mechanical behaviors
magnetization processes. Therein, the magnetization direction was investigated. The study results showed that by combining
of the grippers could be controlled by changing the direction of MF and the heating MF, different deformation modes could be
the applied MF, resulting in the controllable deformation of the achieved when these different responsive materials were ade-
grippers and the subsequent 4D effect printing. quately distributed into the predesigned structure.
Figure 11. A) Schematic diagram of the SLS-based 4D printed magnetism-responsive gripper. Reproduced with permission.[65] Copyright 2021, The
American Chemical Society. B) Shape recovery behavior of a 4D printed composite structure in an applied MF with an application illustration of bone
repair. Reproduced with permission.[173] Copyright 2019, Elsevier. C) The working mechanism of M-SMP and its application in the mechanical claw lifting
of a lead ball. Reproduced with permission.[175] Copyright 2016, Wiley-VCH. D) DIW-based 4D printing of the magnetic multimaterial system and its
working mechanism. Four different modes of deformation are achieved by cooperatively controlling the temperature and magnetic field. Reproduced
with permission.[176] Copyright 2021, The American Chemical Society.
Generally, magnetism-responsive materials applied in 4D 5.5. Light-Triggered

printing have excellent capabilities for contactless stimulation
for remote manipulation, diverse achievable deformation, and Light-induced 4D shape-changing materials can be divided into
reversible/reproducible shape transformation. M-SMPs also three types based on the type of light sources used, including
demonstrate superior shape manipulation performance by easily 1) UV light, 2) NIR light, and 3) visible light. The application of
realizing reprogrammable, untethered, fast, and reversible shape light-responsive SMP materials can be characterized into two
transformation and shape locking in one material system, which models: one is the direct light-induced SME, and the other is the
can serve as a universal platform for a wide range of biomedical indirect effect, of which the principal mechanism of action is the
applications for minimally invasive surgery, biorobots, and tar- photothermal SME. Typically, due to the photothermal properties
geted drug delivery. However, the M-SMPs still exhibit several of NIR light, the NIR-responsive materials tend to have secondary
key disadvantages. For example, the M-SMPs mostly rely on the thermal properties. Additionally, UV and visible light-responsive
thermomagnetic effect of thermal responsive SMP to fix their 4D actuators have also been utilized to manipulate small amounts of
shapes, and thus necessitate strict control over temperature flux liquids and provide contactless spatial and temperature control.
to have utility in biomedical and clinical applications. Moreover, Recently, we reported an application study of an NIR-photo-
the biosafety of magnetic nanoparticles is another concern for thermal responsive biocompatible SMP for the 4D printing of
the in vivo use of magnetism-responsive 4D materials.[169,171] We complex geometric structures (such as flowers, hand gestures,
expect that more studies on the remote actuation of such unteth- and biomedical models), as illustrated in Figure 12A.[154] The
ered, complex, and fast shape-shifting soft materials based on MF novel SMP composite also displayed a conductive feature due
stimulation will be further explored in the future, and will create to the addition of graphene to the ink formulation and could be
new possibilities for biomedical applications in implantable flex- utilized as a conductive material to construct a dynamic circuit
ible electronics, soft biorobots, and other biomedical devices. switch. We used this novel NIR-sensitive material to generate a
Figure 12. A) Dynamic and controllable conversion of 4D printed constructs. NIR responsive transformation behavior of flower, hand, controllable circuit
switch, folded brain, and dilated heart. Reproduced with permission.[154] Copyright 2019, Springer Nature. B) Multiwavelength photoresponsive behaviors
of an SMP film under irradiation of UV light (365 nm) and NIR light (808 and 980 nm).181 Reproduced with permission.[181] Copyright 2019, Wiley-VCH.
C) Molecular mechanism of UV light-responsive SMP and its different shapes. Reproduced with permission.[182] Copyright 2005, Springer Nature.
D) Series of photographs showing time profiles of the flexible robotic arm motion of the CLCP laminated film induced by irradiation with UV and visible light
at room temperature. Arrows indicate the direction of light irradiation. Reproduced with permission.[183] Copyright 2009, The Royal Society of Chemistry.
4D bioprinted cardiac tissue. Additionally, Zhou et al. introduced under NIR light irradiation.[184] Recently, Yamada et al. prepared
a rapid light-responsive SMP based on photothermal W18O49 a crosslinked LCP, which had both LC and elastomer proper-
nanowires, which could respond to both UV and NIR light, as ties.[183] When heated above the LC-isotropic phase transition
shown in Figure 12B.[181] Furthermore, Lendlein et al. proposed temperature, the polymer would shrink along the alignment
a typical direct light-induced SME,[182] in which the material direction of the LC. By incorporating the azobenzene moiety, the
chromophore was covalently grafted to a permanent polymer photochemical reaction of these azobenzene chromophores was
network to form a photoreversible crosslinked temporary shape. able to cause a greater deformation. A “hinge joint” transition
The fixation and restoration of the shape were achieved by irra- between a curved shape and a flat shape at laminated parts of the
diating UV rays with appropriate wavelengths. Its irreversible actuators was controlled by the exposure of UV light (366 nm,
UV light (λ > 260 nm or λ < 260 nm) response at room tempera- 240 mW cm−2) and visible light (>540 nm, 120 mW cm−2)
ture under stress-controlled and/or strain-controlled conditions at room temperature as indicated in Figure 12D.[183] Generally,
is illustrated in Figure 12C.[182] Bai et al. synthesized an NIR NIR responsive light actuators are more suitable for clinical
responsive photo-thermoset SMP with a significantly reconfig- applications, as NIR light is more biocompatible than UV light,
urable and self-healing polymer which was sensitive to a photo and has a more pronounced tissue penetrating capability.
thermal triggered exchange reaction.[119] After being reconfig-
ured seven times, the composite material still showed excellent
light-induced shape memory performance, with the shape fixity 5.6. Other Stimuli-Induced
ratio and shape recovery ratio exceeding 95%. Similarly, Li et al.
also developed self-healing NIR-responsive SMPs which contain Among other stimuli-responsive materials, EF-responsive mate-
poly(acrylic acid)-grafted graphene oxide matrix with PVA. The rials have many advantages due to their ability to enable the
fabricated SMP-based flower structure was capable of blooming highly sensitive triggering of reactions,[185] the programmability of
Figure 13. A) An instance of a 3D printed flexible dielectric elastomer soft actuator and its shape morphing under different voltage applied. Repro-
duced with permission.[98] Copyright 2019, Elsevier B.V. All rights reserved. B) A flow diagram of the NIR-thermal dual-stimulus responsive SME where
the transformation could be reached both under the irradiation of NIR light and external temperature manipulation. Reproduced with permission.[154]
Copyright 2019, Springer Nature. C) A schematic of pH-thermal dual-stimulus responsive molecular illustration and the self-healing process. Repro-
duced with permission.[131] Copyright 2019, The Royal Society of Chemistry. D) A novel hydrogel designed for a light and MF dual stimuli-response.
The walking of the fabricated actuator was triggered by light under a rotating magnetic field. E) Light triggered walking under rotating magnetic fields.
D,E) Reproduced with permission.[188] Copyright 2019, American Association for the Advancement of Science.
fabricated structures, and the reversibility of 4D transformation. pronounced degree of motion. The primary operational mecha-
Recently, Zhou et al.[98] developed a novel approach to fabricate nism for this type of material relies on the electrothermal trig-
3D printed flexible structures by utilizing a dielectric elastomer gering of SMPs, which included carbon nanotubes to provide an
actuator. The FDM printed multimaterial soft gripper could reach, increase in the local material temperature. In their recent study,
grasp, and hold motion through the modulation of externally Ling et al. further introduced a multitude of electrothermally
applied 5 kV voltage, as displayed in Figure 13A. The maximum controlled 2D/3D/4D structures.[186] Currently, the EF-responsive
bending angle was ≈128°, and the maximum blocked force was materials remain limited both in the number of available mate-
around 24 mN. Sachyani Keneth et al. prepared a trilayer actuator rials and their applications, e.g., soft robotics and actuators.[187]
that contained an (electro)thermal-responsive SMP to achieve The use of EF-responsive materials in tissue regeneration is lim-
reversible continuous movements.[6] Compared to bilayer actua- ited by the complexity and availability of power devices and the
tors, the trilayer actuator could achieve a more complicated and potentially harmful effects of the applied voltages on cells.
Additionally, there are several kinds of multistimulus Generally, multistimuli-responsive materials exhibit the sig-
responses used in 4D printing, such as pH/temperature, light/ nificant advantage of being able to enhance the responsiveness
temperature, magnetism/temperature, etc. The magnetic- and and complexity of 4D printed structures, which by extension,
electric-thermal dual-responsive SMPs have been discussed can ultimately work to augment their applications in multistep
above; therefore, we will introduce other types of multistimulus change or complex environment situations. By introducing the
responsive materials here. Recently, Zou et al. synthesized a aforementioned stimuli-responsive materials which are suitable
light-programmable thermotriggered SMP, which could be pro- for 4D printing, it is expected to yield progress toward the full
grammed by a topographical patterning mechanism, so that the realization of the capabilities and functionalities of 4D pro-
patterned SMP had thermomechanical properties.[4] This photo- grammed matter. Moreover, this progress may in turn poten-
and thermo-dual responsive material could be likely be utilized tially revolutionize various biomedical industries shortly.[29]
for artificial muscle and flexible electronics applications. Our
research group also recently developed photo- and thermo-dual
responsive SMP with a Tg around 40 °C, which can be regarded 6. Biomedical Applications of 4D Printing
as being cell-benign for 4D printed tissue regeneration pur-
poses.[31,76] An illustration of the dual response mechanism is The gap between the increasing demand for personalized
shown in Figure 13B.[154] medicine and the shortage of effective therapeutics (e.g., organ
Yan et al. proposed a straightforward fabrication approach donors or advanced biomedical devices) is a significant chal-
to generate a biodegradable pH/thermo dual-responsive inter- lenge toward improving human health and quality of life.
penetrating polymer network hydrogel with multifunction- Regenerative medicine has the potential to heal or replace
ality (Figure 10C). Its shape memory property was erasable damaged tissues or organs,[189] and has the potential to revolu-
and pH-dependent, allowing it to achieve an excellent shape tionize conventional therapeutic strategies. Presently, tremen-
memory property at a high pH range and could be erased in dous effort has been made to expand and scale-up regenerative
acidic conditions. An in vitro dissolution test indicated the technologies for tissue/organ regeneration applications. Of
great potential for programming the multistaged degradation which, 3D/4D printing has one of the most popular methods
of such composites in response to sequential pH or thermal to fabricate 3D artificial tissue constructs with highly complex
stimuli.[158] Recently, Huang et al. proposed a synergistic self- structures and functions.[190] Moreover, 3D/4D printing has also
healing mechanism, wherein the shape recovery of TPVs led to been widely applied to develop various advanced biomedical
the physical contact of damaged surfaces.[121] The desorption/ devices, such as bionic eyes/ears, biosensors, and biorobots,
absorption of epoxidized natural rubber (ENR)/Fe3O4 promoted which have had a profound effect on the development of future
the self-healing of the ENR phase. With the assistance of the biomedicine.[191]
grafting reaction between PLA and ENR, PLA segments were Beyond conventional 3D printing, 4D printing is currently
rearranged and entangled again to achieve a self-healing pro- a promising and innovative biomanufacturing method that
cess of the TPVs. This multistimulus responsive shape mem- can be used to mimic various dynamic processes of living
ory-assisted self-healing TPV was first reported in the field of tissues and can facilitate the fabrication of complex tissue/
dynamic vulcanization and may have considerable potential medical products, which can respond to stimulation by com-
application in the fabrication of smart medical devices. plex biological signals, such as bioelectrical or biochemical sig-
Furthermore, Lv et al. prepared a double network hydrogel nals (Figure 14).[192] Owing to its ability to generate constructs
based on pH-sensitive bonds.[131] The smart hydrogel had excel- with distinct self-morphing capacities, 4D printing may offer
lent thermoprocessability properties, a pH-driven SME, and a more favorable fabrication approach over 3D printing, as 4D
displayed a notable self-healing behavior. The material was com- constructs can respond to internal and/or external stimuli, as
plexed with borax in an aqueous solution to form borate ester we discussed in Section 4. The unique advantages 4D printing
bonds, which could impart excellent self-healing properties to offers for the fabrication of biomedical devices include but are
the hydrogel, as shown in Figure 13C. Additionally, a hydrogen not limited to 1) ease of obtaining uniform cell distribution
bond-based double-helix structure was achieved through the on irregular 3D microstructures due to its ability to transform
modulation of local temperatures, which exhibited the ability from a temporary 2D flat structure to a complex and irregulated
to be thermoprocessed. Recently, Wang et al. proposed a gen- 3D structure (original shape) under a certain stimulus, 2) facili-
eral strategy for fabricating a double layer self-deformed shape tation of minimally invasive surgery and seamless integration
memory hydrogel with a pH-responsive memorizing layer, and within tissue defects, 3) the replication of dynamic biological
a thermoresponsive actuating layer.[100] The fabricated actuator behaviors (e.g., tissue growth, joint bending, and muscle con-
had complex structures and could respond to both tempera- traction/relaxation) of native tissues, 4) robust biocompatible
ture and changes in OH−/ H+ ions. Additionally, Li et al. pre- stimuli-responsiveness, and 5) self-driven transformation and
pared a fast and programmable locomotion SMP that contained shape/functional changeability.
hydrogel–metal hybrids that could respond to light and applied Recently, several review papers have been published which
magnetic fields.[188] The working mechanism for the program- discuss 3D printing for biomedical applications,[193] 4D inks,
mable SMP is illustrated in Figure 13D. Light illumination and 4D printed structures.[190,192,194] In this review article,
could influence and change the water content of the hydrogel instead of summarizing the general concepts of 4D printing
such that the magnetic particle content concentration could and various applications, we majorly focus on discussion of
be increased. As such, the trajectory of the 4D structure was recent studies of 4D printed tissues and biomedical devices
guided under the application of an MF, as shown in Figure 13E. regarding in vitro tissue maturation and in vivo implantation to
Figure 14. Schematic illustration of 4D printing for tissue regeneration. A) Most commonly used 4D printing technologies: FDM or DIW, SLA, and
DLP. B) Biomaterial and bio-ink strategy applied in tissues/organs regeneration. C) 4D transformation process of the printed scaffolds triggered by
external stimuli.
elaborate on their potential clinical use. We also include some materials include gelatin,[198] collagen,[199] GelMA,[200] algi-
exciting examples of 4D dynamic constructs fabricated by non- nate,[201] hyaluronic acid (HA),[104a,202] decellularized extracel-
printing methods[74,195] . The recent studies on 4D printed tissue lular matrices (dECMs),[203] and their (methyl)acrylate derives.
constructs are briefly summarized in Table 2. Cell-seeded scaffolds are easy to produce, as the printed con-
structs are simply printed, washed, and then topically adminis-
tered cell sources and/or growth factors. The advantage of this
6.1. Employed Materials of 4D Printing in Tissue Engineering methodology is the broad choice of printable materials, such as
synthetic plastic polymers.[196] Here, these 4D materials used
The fabrication process of dynamic tissue scaffolds involves the for tissue engineering function as an artificial ECM, which
utilization and placement of a 4D ink solution, the filament of provides biophysical and biochemical cues for cells to mimic
a (bio)material, or a mixture of several (bio)materials,[196] which the intrinsic cues of the native ECM, which leads to improved
either directly encapsulates desired cells together with growth cellular proliferation and differentiation.[197] Compared to tra-
factors[197] (or loading of cells postprinting).[190] Generally, there ditional 4D printing materials we have discussed above, 4D
are two types of printed tissue scaffolds, i.e., cell-laden scaffolds materials applied in biomedical applications require 1) a proper
and cell-seeded scaffolds. The cell-laden scaffolds are fabricated print temperature and 4D Ttrans, which do not exceed physio-
by the simultaneous deposition of bioinks that consist of biological temperature if the cells are encapsulated or if they are
compatible material and cells. The most commonly used bioink to be utilized for in vivo use; 2) mild cross-linking or gelation
Table 2. Typical 4D printing examples for biomedical applications.
No. Applications Materials Cells Printing methods Stimuli Ref.

1 Vascular tissue AA-MA/methacrylated hyaluronic Mouse bone marrow DIW Solvent [211]
acid (AA-HA) stromal cells
2 Vascular tissue Gel-MA/Gel-COOH-MA bilayer Human umbilical vein Inkjet Solvent [50]
endothelial cells
3 Vascular stent Thermoplastic copolyester elastomer – FDM Temperature [212]
4 Vascular stent Fe3O4/PLA/dichloromethane/ – DIW MF (thermomagnetism) [179]
benzophenone C
5 Vascular stent PLA – FDM Temperature [213,214]
6 Vascular occluder PLA-based magnetic nanocomposites In vivo implantation FDM MF (thermomagnetism) [215]
7 Cardiac patch GelMA/PEGDA Human-induced pluripotent stem SLA Internal stress [31]
cell-derived cardiomyocytes
(hiPSC-CMs)/human endothelial
cells (hECs)/hMSCs
8 Cardiac patch Bisphenol A diglycidyl ether, hiPSC-CMs/hECs/hMSCs DLP and DIW NIR light and [76]
poly(propylene glycol) bis(2-aminopropyl) temperature
ether, and decylamine
9 Cardiac tissue SOEA hMSCs cardiomyogenic behavior SLA Temperature [117]
10 Neural tissue Poly(methyl methacrylate) Neural stem cells (NSCs) SLA Temperature [148]
11 Neural conduit SOEA/graphene hMSCs SLA Internal stress [91]
12 Bone tissue PCL/irondoped hydroxyapatite hMSCs FDM MF [216,217]
13 Bone repair Fe3O4/PLA Osteoblast FDM MF (thermomagnetism) [77]
14 Bone repair Black phosphorus nanosheet/osteogenic Rat mesenchymal stem cells DIW NIR light and [218]
peptide/β-tricalcium phosphate/ temperature
poly(lactic acid-co-trimethylene carbonate)
15 Cartilage tissue Agarose/collagen type I Human knee articular DIW and inkjet MF [219]
chondrocytes/hMSCs printing
16 Muscle tissue PLA/PCL/ SOEA hMSCs FDM Temperature [15]
17 Muscle tissue AA-MA/PCL C2C12 mouse muscle cells FDM Solvent [195b]
18 Muscle tissue SOEA hMSCs FDM Temperature [116]
19 Trachea repair Silk fibroin methacrylate Chondrocytes/turbinate-derived DLP Solvent [220]
(Sil-MA) hydrogel mesenchymal stem cells (TBSCs)
20 Tracheal stent PCL/ isocyanato ethyl methacrylate – SLA Temperature [221]
21 Tissue scaffolds Alginate/polydopamine Human embryonic kidney DIW Solvent and NIR light [89]
cells, 293T and temperature
22 Tissue scaffolds Polyurethane/PCL hMSCs Extrusion-based Temperature [222]
Commercial polymers printing
23 Tissue scaffolds PEG(700)DA Mouse embryo NIH 3T3 eGFP cells DLP Solvent [223]
24 Medical protective Shape memory – UV-assisted FDM Temperature [51]
devices aliphatic copolyester
conditions if the cells are encapsulated; 3) their components tubular structures can be found within the human body, and
and degradation products must be nontoxic and biocompatible, include tissues such as[205] muscle fibers, nerve bundles,[195a]
and 4) a favorable environment for cell adhesion and growth in and tendons.[206] In a recent review article, we have summarized
tissue regeneration application. pertinent studies on 3D printing for cardiovascular regenera-
tion, in which the basic knowledge of the vascular system, e.g.,
vessel types, cell composition, and function, was discussed at
6.2. Vascular Grafts and Stents length.[207] Within native vessels, ≈79% of the vessel wall com-
position is water, with the remaining biological components
Vascular regeneration and vascular stents represent one of the consisting of collagen, elastin, proteoglycans, and vascular
first biologically relevant applications for 4D printed constructs, cells.[208] The most widely utilized cell types in vascular regener-
as their tubular structure is simple to obtain by a 4D rolling ation are endothelial cells (ECs)[209] and vascular smooth muscle
or stretching process. In addition to blood vessels, various[204] cells.[210] Camasão and Mantovani published a critical review
Figure 15. 4D vascular graft and stent devices. A) Schematic of the DIW printed AA-MA or HA-MA polymer membrane, which can self-fold into tubes
in water, PBS, or cell culturing medium. The representative fluorescent images of the cell-laden self-rolling tubes in which live cells (green) and dead
cells (red) are stained after 1, 2, and 7 days of culture and exhibited favorable tube formation. Reproduced with permission.[211] Copyright 2017, Wiley-
VCH. B) A thermoresponsive vascular stent was applied for vascular regeneration showing that the nascent fiber-based vessel scaffold could mimic the
microstructure of native arteries. Reproduced with permission.[179] Copyright 2017, American Chemical Society. C) Crimping and delivery of 4D printed
vascular stent prototype. The printed stent was placed inside the crimpling device and then transferred into the transapical delivery device. After being
pushed out of the delivery system, it could self-expand. Reproduced with permission.[212] Copyright 2017, Mary Ann Liebert, Inc.
paper in which they discuss the mechanical characterization of utilized DIW-based printing to create a bilayer membrane
blood vessels and their substitutes.[208] Blood vessels are perfus- with AA-MA and HA-MA, which can self-fold into a tube after
able and elastic to transport blood and withstand high blood being immersed into water, PBS, or culture media due to the
pressures, which typically range between 90 and 110 mmHg different crosslinking degrees in the bilayer structures, as indi-
in the arteries and 5–15 mmHg in the veins.[208] Thus, there is cated in Figure 15A. The visible green light was applied in the
a higher mechanical requirement for the mechanical proper- crosslinking process to replace traditional UV light emission as
ties of the printed vessels and stents, in which some unique it is more cell benign than UV light. The study results showed
mechanical features in response to the physiological forces that this cell-laden tube could achieve internal diameters as low
such as i) nonlinearity, ii) anisotropy, iii) viscoelasticity, and iv) as 20 µm, with unprecedented control over architectural features
compliance, should be considered. with high-resolution, and as such, could effectively mimic small
3D printing has shown its capacity in generating complex in vivo vessels. Additionally, self-rolling tubes could support cell
network structures[224] and high-resolution micromanufac- survival for at least seven days, suggesting that there were no
turing[204] for vascularized tissue regeneration.[221] With regards adverse effects on the cell viability after 4D printing.[211] More-
to 4D printing, the dynamic shape transformation enables over, many nonprinting 4D technologies have also been used
the facile implantation of 4D printed vascular tissues/vessels to fabricate vessels. For example, a trilayered bacterial cellulose
to promote the development of minimally invasive medical membrane was fabricated with three types of cells, i.e., human
devices. As one of the earliest examples of a 4D bioprinting umbilical vein endothelial cells, human aortic smooth muscle
vessel, Prof. Ionov’s lab from the University of Bayreuth has cells, and human skin fibroblasts, which exhibited self-rolling
and tunable shape memory properties.[225] We expect that these transformed into a preprogrammed coil shape in the renal
exciting ideas can help to inspire researchers toward the further artery of a pig model system.
development of increasingly more complex 4D printed vessel The key advantage of 4D printing utilized for vascular regen-
technologies. eration and medical devices is its ability to achieve minimally
Moreover, another important application of 4D printing is to invasive surgical implantation through shape-memory effects,
produce minimally invasive stents. 4D printed stents can easily such as the rolling of vascular grafts or the stretching of vas-
be implanted within native vascularized tissues and recover cular stents. Therein, the initially compressed structures would
their original conformations to help open blocked vessels and expand and have functional application in the prevention of
improve blood flow. Generally, polymeric stents for minimally vascular blockage. It is expected that more stimulus-responsive
invasive applications need to meet a set of essential criteria, materials, including but not limited to, thermoresponsive bio-
such as sufficient radial force to maintain their position after degradable SMPs, magnetic- and light-responsive SMPs, can
deployment, and limited plastic deformation to ensure initial also be utilized and developed in future studies. With future
valve functioning until host body integration.[213] Boire et al. advancements in 4D printing, 4D vascular grafts can be fab-
synthesized a new class of thermoresponsive SMPs with pen- ricated to have more advanced geometries than simple linear
dant photocrosslinkable allyl groups, which could be utilized tubes. In addition to the mechanical parameters of printed
as a biocompatible stent, as it exhibited adequate mechanical constructs (tensile stress and shear stress) we discussed above,
strength and a shape recovery near body temperature.[226] 4D other crucial features of the vessel must be emphasized for 4D
printed stents were further studied for vascular compatibility printing vessel regeneration, including perfusability, perme-
using a murine model of hindlimb ischemia. The in vivo results ability, vasculoactivity, and durability over time.
after two weeks of culture showed that the stained tubular con-
structs revealed successful cellularization without significant
inflammatory response and did not show the common forma- 6.3. Cardiac Patches and Valves
tion of foreign body giant cells, nor observable fibrotic tissue
formation. These results suggested that SMP constructs had In contrast to vascular grafts or stents which are mostly tubular
mild host immune response and favorable biocompatibility, in structure, cardiac scaffolds/patches must achieve curved
which would be a promising material for the treatment of vas- and aligned architectures to effectively attach to the surface
cular thrombus. Recently, Wu et al. also developed 4D struc- of the heart to repair injured myocardial tissue. Compared to
tures with a negative Poisson’s ratio to fabricate self-expanding 3D printing for cardiovascular regeneration as discussed in a
vascular stents.[227] In addition to the thermoresponsive mate- previous review,[230] 4D printing can enable the fabrication of
rials, there have been other stimuli-responsive 4D stents that a physiologically relevant curved surface with incorporated
have been developed. For example, a recent study presented dynamic mechanical stimulation. For example, an SLA printed
an MF-triggered 4D stent that could be manipulated remotely 4D structure was fabricated into a curved cardiac patch by our
(Figure 15B[179]). This remote manipulation of the stent system research group, in which the cardiomyogenic differentiation
offers an effective strategy to design 4D active shape-changing of hMSCs was modulated on the 4D thermoresponsive pat-
vascular stents with multifunctional properties. Addition- terned patches.[117] An SOEA ink was cross-linked using an SLA
ally, Cabrera et al. proposed a similar proof-of-concept study printer to generate a microchannel patterned surface, which
to implant 4D printed self-expandable biocompatible polymer could guide the alignment of hMSCs.[117] The structure could
stents for minimally invasive heart valve regeneration through be fixed at −18 °C and recover its original shape at 37 °C over
the use of FDM technology.[212] The authors utilized a flexible 60 s. The 4D fabricated cardiac patches enabled significant car-
thermoplastic copolyester elastomer for their stent, as polyes- diomyogenesis of seeded cardiomyocytes, as was confirmed by
ters are notably bioabsorbable and biodegradable polymers, immunofluorescence staining and gene expression analysis.
largely as a function of their easily hydrolyzed ester bonds (as Additionally, a hydrogel-based 4D cardiac patch with physi-
is shown in Figure 15C).[212] This work demonstrated that FDM ological adaptability was also fabricated in our lab.[31] The
printed thermoresponsive polymeric stents can be promising shapeshift of the printed pattern was triggered by a crosslinking
for future clinical use owing to their ease of degradation and density-induced internal stress. As illustrated in Figure 16A, by
distinct 4D shape transformation. Additionally, Shin et al. fab- combining the unique 4D self-deformability with the expand-
ricated a shape memory tube using an optimized microfluidic able microstructure, our design not only improved the biome-
validation and computational fluid dynamics modeling regime chanical properties of the patch itself but also improved the
in order to prevent vascular stenosis, which ultimately resulted dynamic combination of the patch and the beating heart.[31] The
in a successfully implanted in vivo stent, which was placed in vitro dual mechanical stimulation yielded substantial car-
within a vessel with a diameter smaller than 3 mm.[228] diac maturation after two weeks of culture, and the long-term
Similar to the designs for vascular stents, a permanent 4D development of the implantable cardiac constructs in a murine
embolization construct designed to fit arteries of various diam- chronic MI model demonstrated the potential therapeutic effect
eters has been created to treat aneurysms.[229] A notably stiff of the 4D constructs in vivo.
and tough radiopaque hydrogel was fabricated, which exhibited Moreover, our recent study demonstrated a new design for
a body-temperature-triggered SME and demonstrated sponta- manufacturing a cardiac patch with an adjustable curvature and
neous transformation from a coil to a rod conformation. The aligned microgroove patterns, in which hiPSC-CMs, hMSCs,
hard radiopaque hydrogel strip could be passed through a cath- and hECs were tricultured to mimic the cellular composition
eter under the protection of cold saline and could be quickly of native myocardial tissue.[76] The patches were fabricated
Figure 16. 4D cardiac tissue scaffolds and devices. A) Illustrations of the working principle of a cardiac patch. The cardiac cell-laden hydrogel patch
can match the change of heart surface curvature induced by cardiac muscle contraction, which exhibited an excellent therapeutic efficacy via an in vivo
MI model surgery. Reproduced with permission under the terms of the Creative Commons Attribution-NonCommercial license.[31] Copyright 2020, The
Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. B) An illustration of an NIR-light-responsive
cardiac patch and immunofluorescent image of the hiPSC-CMs cells laden on the 4D printed cardiac patch. Reproduced with permission.[76] Copyright
2021, American Chemical Society. C) Schematic illustration of the prototype of the 4D occluder. The programmed shape recovery process of a magnetic
PLA 4-arm occluder was performed under an MF, and an in vitro study of an atrial septal defect occlude demonstrated its feasibility. Reproduced with
permission.[231] Copyright 2019, Wiley-VCH.
with the assistance of DLP printing, and exhibited controllable cerebrovascular 3D printed models in the clinical setting.[234]
deformation under the stimuli of temperature and NIR light, as Superior to 3D printing, 4D printing has great potential for
indicated in Figure 16B. When transitioning to a permanently replicating the development of native complex tissue in a
curved surface, the cardiac patch was entirely covered with a manner of spatiotemporal change. For example, the cerebral
uniform layer of aligned myofibers, and thus prevented cell cortex folding process of the brain can be mimicked via 4D
aggregation. The uniform distribution of tricultured cells and bioprinting, which has been discussed in our previous review
the excellent myocardial maturation indicated that this meth- paper (Figure 17A).[235] Currently, a substantial amount of
odology provided an effective means to achieve complex curved research is being committed to studying the outer surface of the
tissue structures.[76] brain, which is known as the cortex or cortical tissue. Specifi-
Furthermore, nonprinting 4D fabrication has been applied in cally, there is interest in exploring how the cortical tissues of the
minimally invasive surgery for scaffold delivery. For example, brain enfold to generate its convoluted surface. Therein, what
a flexible shape-morphing scaffold was fabricated using a we learn about the expansion and folding of the cerebral (and
molding technique for minimally invasive delivery in cardiac cerebellar) cortices can shed light on the etiologies of various
tissue.[232] This UV-crosslinked elastomeric poly(octamethylene neurodevelopmental and cognitive diseases (Figure 17B).[236]
maleate (anhydride) citrate) exhibited hydrolytic biodegrada- Recent work on the folding mechanism of the brain focuses
bility, adjustable mechanical properties, nontoxicity, and min- primarily on the human cerebral cortex.[237] It largely involves
imal inflammatory properties. The in vivo implantation data directional growth, axon tension, or differential expansion
illustrated that the scaffold yielded an excellent capacity to spur models of elastic materials, which generate contractile forces
tissue repair. It is expected that this minimally invasive scaf- to drive mechanical instabilities that cause folding.[238] Hence,
fold type could be adapted to the 4D printing approach to repair the notion of employing the 4D printing approach to mimic the
skin, liver, and other tissues in the future. cerebral cortex folding process has been raised. Moreover, the
In terms of cyclical formation and deformation, the utility of combination of 4D printing and simulation of cerebral cortex
4D printing is expounded by its excellent mechanical proper- folding can significantly aid our understanding of how mechan-
ties and repeatability for the fabrication of biomedical devices. ical forces and cell processes affect cerebral folding.
Most commercial occlusion devices for clinical use are made of Taking advantage of the differences in the swelling ratio of
nondegradable metals, which tend to yield adverse effects such hydrogels, Tallinen et al. fabricated a physical model of the
as perforation, allergic/inflammatory response, and erosion. brain (Figure 17C).[239] Their 4D material model of cortical
To address these challenges, Lin et al. developed a 4D biopol- folding was created using hemispherical polydimethylsiloxane
ymer-based occlusion device that incorporated Fe3O4 mag- (PDMS) gel, coated with a top layer of PDMS that could swell
netic particles into a shape memory PLA matrix, which could by absorbing different solvents, such as hexane. By changing
be applied to facilitate interventional therapy for atrial septal the crosslinking density, the samples with varying ratios of two-
defects. The magnetism-driven approach matched the com- layer modulus were fabricated to capture different wrinkling or
mand of the actuators in enclosed and restricted spaces in the folding regimes. Additionally, a theoretical numerical model of
left and right atrium, as indicated in Figure 16C.[231] The occlu- gyrification with the corresponding simulation calculations was
sion device could respond to the magnetic stimulus rapidly, also proposed.[240] They developed a quantitative approach to
all while holding its temporary shape for an extended period mimic spherical brain configuration and its increasingly folded
during cell culturing without applying the MF. The in vivo and surface with brain size growth.
in vitro results demonstrated favorable biocompatibility and Another application for 4D printing of brain tissue is to
biodegradation, cytocompatibility, histocompatibility of the achieve a uniform cell distribution on the wrinkled surface of
M-SMPs. The results illustrated the advantage of M-SMPs, in the brain construct. Recently, our research group has devel-
terms of their capacity for remote control and biocompatibility, oped a 4D NIR-responsive brain construct for brain tissue
for replacing the currently used metal occlusions. regeneration.[154] Due to the photothermal effect of NIR light,
Presently, the development of 4D printing has shown its when exposed to the NIR light, the 4D brain constructs can
potential in cardiac tissue engineering and cardiac devices with controllably transform for structural conformation in the cul-
regard to the specific requirement of shape changeability and ture medium. Figure 17D shows the distribution of green flu-
dynamic controllability. In the future, it is expected that more orescent protein-modified NSCs on the 4D brain constructs,
in-depth research will be focused on various cardiac-related where the planar shape of the construct was converted to a
applications. For example, 4D-induced mechanical forces can wrinkled shape 37 °C. The study results demonstrated that the
be explored to improve cardiac function in future research. brain tissue layer could be formed on the surface of brain-like
scaffolds without cell aggregation. Therefore, the 4D printing
strategy can not only be used to create complex structures but
6.4. Bionic Brain Constructs can also provide a potential method for building cell-laden bio-
logical tissues with the uniform cellular distribution.
The brain is considered to be the most complex and myste- Based on current research, using hydrogel swellability to
rious organ of the human body.[233] Through the conversion of mimic cerebral cortex folding is feasible and easy to implement
patient-specific geometries rendered from diagnostic imaging for 4D printing compared to the traditional molding method.
studies, such as a computed tomography (CT) and magnetic It is expected that biocompatible hydrogels can be utilized to
resonance imaging (MRI), into STL files using specific software 4D print a cell-laden model by SLA or DLP printing through the
and hardware, 3D printing has helped to develop personalized solvent-triggered swelling.[84] To date, there is a dearth of current
Figure 17. 4D brain tissues and models. A) A schematic of a flow diagram of the 4D bioprinting in the brain model. Reproduced under the terms of the
Creative Commons CC BY license.[235] Copyright 2019, Taylor & Francis Group. B) With the assistance of a 3D printed brain model, a gel-brain coated
with a thin layer was demolded. As a function of time, a brain-like gel model was formed. Reproduced with permission.[236] Copyright 2016, Springer
Nature. C) Utilizing the different swelling ratios of the hydrogels at the different layers induces the various folds on the hemisphere model, which may
have notable utility in the future of 4D direct bioprinting. Reproduced under the terms of the Creative Commons CC BY license.[239] Copyright 2014,
the National Academy of Sciences of the United States of America. D) A side view of the brain-like tissue which transforms from a flat surface to a
curved one under the stimulation of NIR light and its corresponding photothermal image (left). GFP-NSCs distribution when 4D brain constructs
transformed from a flat shape to a folded shape, suggesting no significant effect on the cell attachment after the 4D change (right). Reproduced with
permission.[154] Copyright 2014, Springer Nature.
research on brain tissue regeneration and devices, largely owing has become one of the most promising strategies for repairing
to the complexities and poorly understood circuitry of the brain’s peripheral nerve injury or spinal cord injury. 3D printing of
cortical tissues. We anticipate the development of future neuro- neural conduits,[243] nerve chips,[244] and nerve patches have
science could accelerate more related research on 4D printing of been extensively studied to provide new treatments for nerve
brain scaffolds to regenerate the tissues of the brain. Moreover, injuries, which has been discussed in some recent review arti-
4D printing of implantable brain devices could be used to detect cles.[91,245] Here, we will focus on the recent development of 4D
and simulate bioelectric signals in the future, such as applying printed neural scaffolds to illustrate the unique features of 4D
brain–computer interfaces[241] to realize brain-to-morphing and printing for neural tissue engineerings, such as dynamic self-
shape-to-function based on brain-to-text technology. enturbulation and seamless integration.
Our research group recently fabricated a novel repro-
grammed nerve guidance conduit using an SLA technology
6.5. Neural Scaffolds and Conduits with the photocrosslinkable SOEA ink, which is responsive
to changes in temperature.[91] The 4D plus reprogramming
Nerve regeneration is a complex, and poorly understood biolog- of constructs could be triggered by both the laser-induced
ical phenomenon. Since nerve injuries with a large defect gap graded internal stress and SME. The fabricated nerve conduct
have drastically limited regeneration capabilities, nerve graft demonstrated a notable capacity for 4D shape transformation
surgery is usually required.[242] Recently, the artificial nerve graft to achieve a minimally invasive, surgical delivery with in situ
Figure 18. 4D neural conduits and constructs. A) A serial image of bird flying structures fabricated with different graphene contents (0–0.8%). The 4D
nerve guidance conduit was manufactured without graphene (white) and with 0.8% graphene. Schematic illustration shows the full enturbulation of the
4D nerve conduit via thermomechanical programmed shape transformation. Immunofluorescent images of neurogenic differentiation of hMSCs. The
cell alignment originated from the microstrip orientation of the printed architectures, in contrast to UV-cured conduits.[ Reproduced with permission.[91]
Copyright 2018, Wiley-VCH. B) The fabrication procedure of a novel 4D neural scaffold with a time-dependent topographic transformation. It was used
to provide a new platform for modulating desired extracellular microenvironments from NSC aggregation at an early stage to highly aligned axons for
NSCs differentiation. Reproduced under the terms of the Creative Commons CC BY license.[148] Copyright 2020, The Authors, published by Wiley-VCH.
shape activation, as shown in Figure 18A. In addition, neural was completed for setting the shape. This device could then be
differentiation of hMSCs growing on the 4D printed constructs deformed away from the final shape to enable implantation and
suggested that the 4D printed constructs exhibited favorable would recover its shape. In vivo experiments demonstrated a
potential for neural tissue regeneration purposes due to their nerve cuff electrode wrapped around the vagus nerve of the rat
ability to dynamically and seamlessly integrate into the stumps to drive the nerve activity. Though this study is an example of a
of a damaged nerve. nonprinting fabrication approach, the material could be applied
Additionally, a 4D programmable culture substrate with a as 4D inks in a DIW printer to fabricate the electrodes.[16]
self-morphing capability was fabricated to enhance dynamic Generally, the 4D scaffolds with dynamically topographic
cell growth and induce differentiation of stem cells by our change we discussed above are used for the first time to manip-
research group.[91,148] A PVA sacrificial mold was fabricated by ulate stem cell functions, which is promising for other cell
FDM printing to create a microgroove array of scaffolds. With types and provides a platform for the next generation of cul-
the assistance of SLA printing, a mold of micropillars was fab- ture substrate. Due to the particularity of nerve grafts, 4D nerve
ricated to create the microwell substrates on the microgroove grafts do not require large-scale or complex shape transforma-
arrays, as indicated in Figure 18B. The study results illustrated tion but change from plane to conduit one-time, compared with
that the 4D culture substrate exhibited a time-dependent self- other tissue scaffolds or biomedical devices. Therefore, it has
morphing process, which could regulate NSC behaviors in a a higher commercial availability for clinical use in the future.
spatiotemporal manner for enhancing neural differentiation of
NSCs along with significant axonal alignment.[148] This work is
the first study to employ an intelligent 4D culture substrate to 6.6. Bone Regeneration
replicate the dynamic process of neurodevelopment with time-
dependent complex topographic transformation. In addition to the tremendous advances achieved in the appli-
Moreover, a 4D nerve cuff electrode with SME was developed cations above, many other impressive 4D printing results have
by Ware et al.[195a] The thiol–ene/acrylate SMP was utilized to demonstrated their advantages for regenerating other tissues,
create softening substrates for stimulation electrodes. The cuff such as bone.[205,246] Bone repair is one of the earliest applica-
substrate system was softened in vivo from more than 600 to tions of 3D printing in tissue engineering.[247] 4D printing of
6 MPa. Metal electrodes were deposited on a sacrificial glass bone scaffolds has significant advantages in their reconfigurable
substrate through a shadow mask. These electrodes were trans- capability for easy implantation with minimally invasive surgery
ferred to a partially polymerized substrate, and an insulating and perfect shape fitting in irregularly shaped bone defects.[218]
layer was cast on the electrodes. The electrodes were then You et al. synthesized a dual stimulus-responsive SMP that
deformed into the desired final shape, and the polymerization can achieve the swelling-induced macroscopic morphing and
Figure 19. 4D scaffolds for bone tissue engineering. A) The schematic illustration of the 4D printed bilayer membrane for bone repair. 4D printing of
bilayer membrane. The macroscopic curvature was programmed by controlling the light exposure. The application examples of 4D printed complex
macroscopic geometries and the custom fitting of 4D printed bilayer membranes for bone repair. Reproduced with permission.[248] Copyright 2021,
Wiley-VCH. B) Thermoresponsive shape memory cycle of fabricated 4D scaffolds, changing from the original shape to the deformed shape, and finally
recovering to the original shape. Schematic illustration of in vivo scaffold implantation in irregular rat cranial defects with the assistance of NIR irradia-
tion; typical µ-CT images of regenerated cranial bone defects from coronal view and top view, new bone tissue was labeled with pink color. Reproduced
with permission.[218] Copyright 2020, IOP Publishing.
heat-induced microscopic morphing.[248] The dynamic cell cul- DIW printing to facilitate the implantation of irregular bone
turing surface, the static micropillar surface, and the static flat defects. They incorporated black phosphorus nanosheets and
surface were printed using a DLP printer (Figure 19A). The osteogenic peptides into β-tricalcium phosphate/poly(lactic
study results showed the continuous spreading of the cytoskel- acid-co-trimethylene carbonate) nanocomposite scaffolds.
eton on the dynamic surface. By contrast, the nuclear shape on When NIR irradiation was applied on-demand, the temperature
the dynamic surface was slightly elliptical at an initial stage and of bone scaffolds rapidly increased to 45 °C, which enabled the
was changed to anisotropy after the micropattern transforma- scaffold shape reconfiguration for easy implantation and precise
tion. The images also reflected the effect of the dynamic surface fitting in irregular bone defects (Figure 19B). This is an enlight-
on the mechanical cue provided additional evidence to support ening and inspiring work of the next-generation 4D printed
its favorable function in controlling cell fate. By combining the scaffolds for bone repair, suggesting their potential applications
SMP and hydrogel layers, this 4D printed membrane could in regenerating other tissues, such as heart and cartilage, when
realize the time control of multiscale structural deformation for their mechanical properties match the demand.[218]
bone repair in vivo. The photo-programmable hydrogel layer Moreover, there exist some nonprinting 4D bone constructs;
realized the macroscopic fitting of the 4D membrane into geo- for example, Xie et al. seeded mouse myoblast C2C12 cells on
metrically complex bone defects, and the SMP layer provided an electroactive SMP-based scaffold, and the enhanced osteo-
on-demand switching of microscale topography. The dynamic genic differentiation of C2C12 cells showed potential for bone
topography acted as a cell fate switch to promote bone forma- regeneration.[246] Bao et al. also fabricated fibrous poly(d,l-
tion through rapid cell proliferation and effective osteogenic lactide-co-trimethylene carbonate) scaffolds with an SME by
differentiation, potentially offering a versatile platform for pre- electrospinning to assess the cellular responsive behavior of
cise and personalized bone therapy. In addition, Wang and co- osteoblasts. These biomimetic 4D SMP scaffolds ultimately
workers fabricated a thermoresponsive 4D bone scaffold using enhanced the bone regeneration efficacy upon transplantation,
where the ECM architecture provided a favorable milieu to nat- were fabricated for cell alignment and enhancement of myo-
urally regulate cell adhesion, proliferation, and differentiation. genic differentiation (Figure 20B).[251] Bioelectric cues (electric
The study results also demonstrated it also offered favorable stimulation) were used as a significant regulator of muscle cell
biomechanics for tissue remodeling upon the activation of the responses, including cell proliferation, migration, orientation,
SME in a well-controlled manner. With a similar concept, this and differentiation. Similar to the mechanism of muscle con-
SMP material could be fabricated into a more complex struc- traction in vivo, this in vitro process was activated through the
ture for regulating the bone remodeling by the 4D printing voltage-gated calcium channels in the cell membrane. Results
method such as DIW. indicated that when treated with an EF (>0.4 kV cm−1), cells at
Current studies have demonstrated their ability in shape-mor- medium density (15 × 106 cells mL−1) show great alignment.
phing with time for dynamic culture, which can encourage more Generally, the alignment methods of 4D muscle scaffolds
studies of the 4D printed bone tissues and implants.[220,248,249] discussed above could broaden their applications in many other
In addition to the biodegradation and biocompatibility of the tissues with high organization and anisotropy. Especially, the
4D bone scaffolds, more deep studies in terms of in vitro dif- use of EF for initiating cellular activities, such as migration,
ferentiation and in vivo implantation should be performed in alignment, and differentiation, has been widely applied in
the future. Considering the in vivo implantation, mechanical other nonprinting methods. For example, some electroactive
properties such as compression strength and fatigue strength cells, such as neurons and skeletal (or cardiac) muscle cells,
are another consideration for the choice of 4D bone materials. could react to EF stimulation for mediating their physiological
activities, including synapse formation and muscle contraction.
Therefore, we expect that EF responsive 4D materials could be
6.7. Muscle Tissue Regeneration applied to incorporate the electrical cues into the 4D printed
scaffolds for inducing cell-cell interactions or providing a physi-
Skeletal muscle is one of the most abundant tissues in the ologically relevant environment.
human body, comprising around 45% of the body mass.[250] The
muscle tissue is dynamic and involved in the voluntary control
and movement of the body, which is composed of muscles fiber 6.8. Trachea Repair
bundles. The myogenic differentiation of myoblasts requires
a high degree of myotube formation and spatial alignment of The trachea, also known as the windpipe, is a cartilaginous tube
myotubes.[251] 3D printing has emerged as a promising tech- that connects the larynx to the bronchi of the lungs, allowing the
nique that enables the fabrication of engineered muscle tis- passage of air.[253] The trachea is surrounded by 16–20 C-shaped
sues with heterogeneous structures and mechanical properties. rings of hyaline cartilage, which is connected by the tracheal
However, it lacks dynamic mechanical cues such as stretching muscle. The generation of a[254] 4D printed trachea is another
or folding to generate myogenic alignment and functional mat- important application due to its ability to fold into C-shaped
uration. As such, 4D printing has become a promising method rings. Kim and co-workers prepared a photocurable Sil-MA
to solve this problem. hydrogel using DLP printing, and its 4D shape transforma-
Currently, one of the major applications of 4D printing is tion was driven by moisture-induced swelling. The fabricated
the mechanical actuators, such as artificial muscle, which are bilayer hydrogel sheet could change the shape when immersed
usually fabricated by LCEs.[252] Unlike the robotic muscles into a cell culturing medium, as illustrated in Figure 21.
without the requirement of biocompatibility, we focus on the The bilayer structure consisting of a concave–convex patterned
4D biologically active muscle tissues or devices for biomed- layer and a base layer, to induce the 4D shape transformation,
ical applications in this section. Our group has developed 4D was designed correctly through simulation modeling results of
printed skeletal muscle tissue constructs with anisotropy. We the shape-morphing. As such, the cell-laden hydrogel sheet con-
took advantage of the specific features of FDM printing, where structs with a base layer (Cell1: TBSCs for respiratory mucosa)
the contours exhibited directionally topographical structure. It and a pattern layer (Cell2: chondrocytes for tracheal cartilage)
could serve as an integrated approach to creating anisotropic were printed and then incubated in a culture medium to induce
or aligned scaffolds using different biomaterials. The FDM the 4D transformation for generating the desired trachea shape
printed alignment on the plane scaffolds could efficiently after a predetermined time. The chondrocytes and TBSCs
induce the alignment and skeletal muscle differentiation of laden C-shaped bilayer constructs were implanted into a dam-
hMSCs, as indicated in Figure 20A.[117] The thermoinduced aged trachea of rabbits for eight weeks. Results showed that the
shape-changing process led to the rolling of the plane scaffolds implants were integrated with the host trachea naturally, and
for warping the muscle defect area. Additionally, a bioreactor both epithelium and cartilage were formed at the defect sites.
with a rotor was also employed for uniformly culturing 3D cell- This work suggested the excellent therapeutic effect of 4D
laden constructs. Immunofluorescence staining and real-time printing in trachea regeneration, thereby raising our optimism
quantitative polymerase chain reaction (RT-PCR) analysis sug- for the future clinical application of 4D printing.
gest that the topographical alignment created by the staircase Besides, Zarek et al. fabricated a 4D printed tracheal stent with
effect strategy significantly enhanced the expression of myo- a thermoresponsive resin by the DLP printing. This personal-
genic proteins and genes, including myoblast differentiation ized digital model of a tracheobronchial tree was obtained from
protein-1, desmin, and myosin heavy chain-2 (MHC). a middle-aged male through an MRI scanning. The 4D printed
Moreover, Yang et al. proposed an EF-assisted extrusion-based stent could transfer its temporary shape to the permanent shape
4D printing system in which cell-laden GelMA microfibers with the stimulus of heat. The tunable shape transformation
Figure 20. 4D scaffolds for muscle tissue engineering. A) Illustration of the fabrication process for the development of 4D aligned scaffolds using
the FDM and surface coating technique. Effect of topographical alignment on myogenic differentiation of hMSCs. Fabrication of patient-specific 4D
scaffolds, and immunofluorescence staining of 4D scaffolds and controls (flat samples) on 7 and 14 days, respectively. Desmin (green), and DAPI
(blue). Myogenic gene expression on 14 days was determined by RT-PCR analysis. Reproduced with permission.[117] Copyright 2019, IOP Publishing.
B) Schematic of electrically assisted cell-printing process and one-way shape morphing process and the folding illustration of the printed gelatin film.
The cell-laden GelMA fibers and the stained DAPI/MHC images of bundled GelMA fibers after 21 days of culture. Reproduced under the terms of the
Creative Commons Attribution License.[251] Copyright 2021, Ivyspring International Publisher.
ensured the stable fixation of the 4D printed stent to match the irregular-shaped defect of the damaged tissue, the per-
the shape of the native trachea, which could not be achieved fect implantation is still a challenge during surgical operation
by 3D printing technologies. However, its shape transforma- despite 3D printing can theoretically achieve a patient-specific
tion required an outside thermal chamber which is difficult for fabrication of implants. As such, the 4D printing scaffolds facil-
in vivo applications. It is expected that other stimuli-responsive itate the seamless integration between the scaffold and defect
materials can be introduced into the 4D printing of tracheal tissue through the inherent shape-changing effect.[91] Addition-
implants or stents to achieve a facile and remote control. Com- ally, due to its shape-changing capability, 4D printing also offers
pared to other applications, 4D printing of trachea tissue or stent promising possibilities in fabricating next-generation biomed-
exhibited great clinical translation potential due to its relatively ical devices without power supply, including self-tightening
low biocomplexity and high regeneration efficiency. sutures,[195a] vascular stents,[179,212,213,256] neural stent,[91] drug
As discussed above, 3D printed scaffolds seeded with cell delivery carriers,[153] biosensors,[227,257] and bioactuators.[6,187]
populations postprinting often cause heterogeneous cell dis- Currently, commercial culturing platforms utilize a 2D or
tributions on their irregular surface due to gravity.[255] Due 3D microenvironment, e.g., well plate, petri dish, Matrigel, and
to its ability to change the shape upon stimulation, 4D bio- scaffolds, only to support the growth and proliferation of cells.
printing offers a potential means to circumnavigate the issue of By applying a unique self-morphing mechanism, novel 4D
uneven cell settling on the scaffold surface. Another advantage culture substrates would be fabricated for modulating cellular
of 4D printed scaffolds is that they enable a facile and mini- behavior in terms of their ability to contain dynamic biophysical
mally invasive shape activation, reducing trauma in situ and cues such as topographies, surface properties, and biomechan-
significantly improving patient comfort.[8] Considering that ical properties.[148] They may revolutionize current cell culture
Figure 21. 4D scaffolds for tracheal repair. Schematic of the DLP 4D bioprinting process for tracheal repair in vitro and in vivo study. Shape morphing
was accomplished during in vitro culture. Scale bars: 1 cm. The transplantation of the 4D printed trachea. The engineered trachea was trimmed and
implanted into a damaged rabbit trachea. It was sutured to the surrounding trachea. Scale bars: 1 cm. Bronchoscopy images of the native trachea and
the 4D-bioprinted tissue-engineered trachea 1, 2, 4, 6, and 8 weeks after the surgery. Follow-up bronchoscopy showed the full recovery of the tracheal
lumen and the epithelial mucosa at six weeks. Arrows indicate implanted engineered trachea. Reproduced with permission.[220] Copyright 2019, IOP
Publishing.
technologies and have a breakthrough impact on enhancing designed using Aprecia’s proprietary ZipDose formulation
tissue regeneration and mimicking disease progression. technology, incorporating powder liquid 3D printing as the
Furthermore, beyond the cellular level characteristics such as centerpiece of the manufacturing process. There are still no
complex 3D macro-/microstructures and ECM compositions, available FDA-approved 4D products; therefore, we can refer to
natural tissues/organs have unique characteristics in terms the policy of FDA-approved 3D products written by Di Prima.
of their dynamic changes in structures and functions, which It presents an overview of the additive manufactured medical
involve the dynamic environment of the ECM. There are a lot products published by the FDA agencies, including the Center
of examples, including the electrical signal transmission from for Devices and Radiological Health, the Center for Drug Evalu-
the nerves,[258] the vasculoactivity of the blood vessel (e.g., nitric ation and Research, and the Center for Biologics Evaluation and
oxide causes vasodilation[259] and caffeine leads to vasoconstric- Research.[263] As we concluded in Table 2, these examples of 4D
tion[260]), embryonal development (e.g., the structure is always printing remain in the Research & Development phase, and
changing), tissue growth (e.g., the size is growing from child no clinical trials are proposed.[264] Most of them are proof-of-
to adult), and some specific dynamic tissues (e.g., the heart is a concept studies; thus, more in vitro and in vivo experiments are
dynamic beating organ). Inspired by these native phenomena, ready to be evaluated. Regarding the future commercialization
the self-motivated 4D printed structures are expected to respond of 4D printing, according to a new market research report pub-
to the biostimuli of in vivo microenvironment without offering lished by Global Market Estimates, the 4D Bioprinting Market is
any exogenous energy or power for biomedical applications in required to develop at a compound annual growth rate of 28.5%
the future. during the forecast period (2020–2026) reported from Mordor
Intelligence Inc. Although this technology is still in its infancy
due to its complexity and challenges, it has shown great pros-
7. Conclusions and Future Perspectives pects for realizing tissue/organ manufacturing and implantable
devices, ultimately addressing the problem of organ shortages,
7.1. FDA Regulation and Commercialization saving lives, and extending healthspan.[265]
The bioproducts and medical devices manufactured by 4D

printing or other technologies are subject to regulation by the 7.2. Conclusion and Challenges
FDA. With the development of 3D printing technology for bio-
medical applications,[204,261] the regulatory requirements have 4D printing has become increasingly popular in academia and
prompted the FDA to create a working group to assess tech- industry alliances since it was first proposed in 2012. In addi-
nical and regulatory considerations regarding the 3D printing tion to the original simple construct’s deformations without
of medical devices. The book “3D and 4D Printing in Biomed- functions, 4D printing has currently been utilized for a multi-
ical Applications: Process Engineering and Additive Manufac- tude of different applications such as soft robotics, biomedical
turing”,[262] described the first FDA-approved pharmaceutical devices, tissue engineering, etc. In this review, we have given
product, SPRITAM (levetiracetam), which was the first product an overview of the divisions and classifications of 4D inks,
assistant technologies, the associated simulation methodolo- Heart Association Transformative Project Award for financial support.
gies, and their recent biomedical applications. However, there Y.W. also thanks the support of Zhejiang University for Striving for
remain some limitations to this novel technology. Outstanding Doctoral Dissertations. Many thanks to Han Shuo for his
contribution to the literature collation.
First, there is a stark limitation on the number of available
materials suitable for 4D printing, as most materials are not sen-
sitive to exogenous stimulation, especially for biocompatible and
biodegradable materials. The thermoresponsive SMPs account Conflict of Interest
for a large part of the existing SMPs used in 4D printing. The
The authors declare no conflict of interest.
other modalities of SMPs or other SRMs are largely uninvesti-
gated. Although there is much interest in contactless manipu-
lation (stimulation) sources, many of these stimuli or related
materials are unsuitable for manipulating cell-laden 4D con- Keywords
structs. However, it is expected that contactless manipulation
will remain an emerging research area for biomedical applica- 4D printing, additive manufacturing, biofabrication, medical devices,
tissue regeneration
tions and will see the expansion of more cell-friendly variations
of remote stimulation sources such as applied MF, EF, and NIR Received: November 13, 2021
light.[266] The external stimulation such as MF and NIR light Revised: December 17, 2021
could penetrate the skin to allow a contactless or remote stim- Published online: March 28, 2022
ulus-response, which might be utilized in noninvasive interven-
tional surgery in vivo and biomanufacturing in situ.
Furthermore, the shortage of reversible transformation is
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Yue Wang is a Ph.D. student in the School of Mechanical Engineering at Zhejiang University. She
received her B.S. degree at Zhejiang University (2017). She was a visiting student in Dr. Zhang’s
lab at GW (2019-2020). Her research interests include 3D printing and 4D printing and their appli-
cations in biomedical engineering.
Yancheng Wang is a professor in the School of Mechanical Engineering at Zhejiang University. He

received his Ph.D. in mechanical engineering from Zhejiang University. Before joining ZJU, he was
a postdoctoral research fellow in mechanical engineering at the University of Michigan. Now he is
the deputy director of Zhejiang Provincial Key Laboratory of Advanced Manufacturing Technology
and deputy director of National Experimental Teaching Demonstration Center of Mechanical
Engineering. His major research interests include 3D printing and manufacturing, advanced
manufacturing and biomechatronics, tactile sensing, and wearable sensors.
Lijie Grace Zhang is a professor and the associate dean for research in the School of
Engineering and Applied Science at George Washington University. She is also the director of the
Bioengineering Laboratory for Nanomedicine and Tissue Engineering at GW. She received her
Ph.D. in biomedical engineering at Brown University in 2009 and did her postdoctoral training at
Rice University and Harvard Medical School. Her major research interests include 3D/4D printing,
complex tissue engineering, stem cell engineering, biorobots, and cancer metastasis model.

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Emerging 4D Printing Strategies for Next-Generation

printing. First introduced by Massachu-

Y. Wang, D. Mei, Y. Wang H. Cui, T. Esworthy, L. G. Zhang

Table 1. Comparison of typical 4D printing technologies and adapting materials.

3.1. Structural Design for 4D Printing

4.4. Others 5. Stimuli and Mechanism of 4D Printing

electrothermal heating, and magnetothermal heating. More- z ( t ) z ( t )

over, the light-triggered stimuli usually can be subdivided into ∫ σ dz = ∫ E ( ε − ε s ) dz = 0 (1)

Generally, magnetism-responsive materials applied in 4D 5.5. Light-Triggered

Table 2. Typical 4D printing examples for biomedical applications.

No. Applications Materials Cells Printing methods Stimuli Ref.

The bioproducts and medical devices manufactured by 4D

Yancheng Wang is a professor in the School of Mechanical Engineering at Zhejiang University. He

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Best Review

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Review

Emerging 4D Printing Strategies for Next-Generation

printing. First introduced by Massachu-

Y. Wang, D. Mei, Y. Wang H. Cui, T. Esworthy, L. G. Zhang

Table 1. Comparison of typical 4D printing technologies and adapting materials.

3.1. Structural Design for 4D Printing

4.4. Others 5. Stimuli and Mechanism of 4D Printing

electrothermal heating, and magnetothermal heating. More- z ( t ) z ( t )

over, the light-triggered stimuli usually can be subdivided into ∫ σ dz = ∫ E ( ε − ε s ) dz = 0  (1)

Generally, magnetism-responsive materials applied in 4D 5.5. Light-Triggered

Table 2. Typical 4D printing examples for biomedical applications.

No. Applications Materials Cells Printing methods Stimuli Ref.

The bioproducts and medical devices manufactured by 4D

Yancheng Wang is a professor in the School of Mechanical Engineering at Zhejiang University. He

You might also like

over, the light-triggered stimuli usually can be subdivided into ∫ σ dz = ∫ E ( ε − ε s ) dz = 0 (1)