4D Bioprinting Tissue Like Constructs

Review
www.advmattechnol.de
Natural Origin Biomaterials for 4D Bioprinting

Tissue-Like Constructs
Patrícia D. C. Costa, Dora C. S. Costa,* Tiago R. Correia, Vítor M. Gaspar,
and João F. Mano*
biomorphic features.[4] Particularly, 3D

Leveraging 4D biofabrication for engineering biomimetic living constructs bioprinting has been rapidly emerging as
is rapidly emerging as a valuable strategy for recapitulating native tissue a key AM technique for biomedical appli-
dynamics, via on-demand stimuli, or in a naturally evolving mode. Carefully cations, due to its versatility, ability to pro-
duce evermore complex 3D architectures,
selecting smart materials with suitable responsiveness and cell-supporting
and the possibility of fabricating cell-
functionalities is crucial to take full operational advantage of this next-genera- embedded structures on the fly, by mixing
tion technology. Recent endeavors combining naturally available polymers or living cells with biomaterial inks.[5]
hybrid smart materials improve the potential to manufacture volumetrically The concept of 3D printing was ini-
defined, cell-rich constructs that may display stimuli–responsive properties, tially introduced in the 1980s,[6] and its
shape memory/shape morphing features, and/or dynamic motion in time. In underlying mechanism is based on the
controlled and consecutive deposition of
this review, natural origin biomaterials and the stimuli that can be exploited
several layers of a given material to obtain
for granting dynamic morphological features and functionalities post-printing a 3D object with a well-defined structure.
are highlighted. A broad overview of recent reports focusing on 4D-bioprinted Within the scope of tissue engineering
constructs for tissue engineering and regenerative medicine is also provided (TE), this AM approach gained visibility
and critically discussed in light of current challenges, as well as foreseeable when biocompatible materials in combi-
nation with cells and bioactive molecules
advances. It is envisioned that upon assurance of key regulatory demands,
started to be employed as bioinks, to
such technology will become translatable to numerous biomedical applica- produce 3D constructs with an accurate
tions that require fabrication of constructs with dynamic functionality. control over their architecture, thus giving
origin to the term 3D bioprinting.[7] How-
ever, there has been some ambiguity in
1. Introduction the literature when it comes to the terms related to 3D printing
applied to the biomedical sciences,[1] most likely due to the
Up-to-date, extensive research has been conducted within the rapid emerging of this technology and the fast-growing number
fields of tissue engineering and regenerative medicine (TERM), of studies being published in recent years.[8] In fact, while
with the intent of developing artificial biological elements some authors recognize the term “bioprinting” to encompass
capable of replacing, restoring, maintaining, or improving bio- any printed construct that is either i) suitable for biomedical
logical functions of damaged tissues or organs.[1] Even though applications, ii) biocompatible and viable for human trans-
there have been many advancements in this field, researchers plant, or iii) loaded with living cells within its structure,[9] it is
are still striving to fully replicate the natural cellular heteroge- our understanding that a more accurate definition of the term
neity and dynamic biofunctionality of living tissues in biomate- “bioprinting” is the one where it refers only to constructs that
rial-based platforms.[2,3] Among the several techniques available have been printed from bioinks containing both the biomaterial
for tackling these challenges, additive manufacturing (AM), precursor (usually a polymer or polymer mixture) and living
allied with the use of computer-aided design (CAD) specialized cells. Additionally, these bioinks may also include other biomol-
software, provides a user-defined, reliable, and reproducible ecules, such as growth factors.[7] From this perspective, another
methodology to fabricate complex 3D-printed structures with distinction can be made between the term bioink, defined pre-
viously, and biomaterial ink, which consists of a biomaterial
used for the controlled deposition of 3D constructs with precise
P. D. C. Costa, Dr. D. C. S. Costa, Dr. T. R. Correia, Dr. V. M. Gaspar,
Prof. J. F. Mano spatial arrangements that do not include embedded cells in its
Department of Chemistry composition.[10,11] 3D constructs printed from biomaterial inks
CICECO—Aveiro Institute of Materials can then be seeded with cells’ postprinting and used for several
University of Aveiro biomedical applications.[11]
Campus Universitário de Santiago, Aveiro 3810-193, Portugal
3D bioprinting may also enable the manufacture of individu-
E-mail: doracosta@ua.pt; jmano@ua.pt
ally designed constructs customized for each patient, providing
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/admt.202100168.
a new ground for advancing personalized medicine.[12] This
is generally materialized, by combining imaging techniques,
DOI: 10.1002/admt.202100168 such as magnetic resonance imaging (MRI) and computed
Adv. Mater. Technol. 2021, 2100168 2100168 (1 of 21) © 2021 Wiley-VCH GmbH
www.advancedsciencenews.com www.advmattechnol.de
tomography (CT), to generate patient-specific data that are Within the AM spectrum, 4D printing and 4D bioprinting,
converted to digital CAD models and then 3D-bioprinted as i.e., manufacturing approaches based on the 3D bioprinting of
personalized living constructs.[13] Building on this, the possi- stimuli–responsive constructs, are building momentum owing
bility of combining patient’s own cells with bioactive bioma- to their potential for providing smart materials to operate as
terial inks further expands the potential of this customized autonomous soft robotics/actuators,[22–24] for designing intri-
approach, while preventing or mitigating constructs rejection cate drug delivery systems,[25,26] for improving adhesion upon
upon implantation.[14] Adding to this versatility, recent advances implantation,[27] and/or for generating 4D cell-laden constructs
in 3D bioprinting techniques exploiting suspension bioprinting mimicking living tissues dynamics. In 4D bioprinting, manufac-
have also enabled the fabrication of truly freeform constructs tured constructs are built from functional biomaterial inks that
with more detailed biomorphic features that can be highly valu- undergo a shape or functionality change over time, upon expo-
able for specific biomedical applications.[15] sure to certain stimuli, or derived from the progressive tissue
Despite the fact that highly complex geometries can be maturation due to naturally occurring cellular processes (e.g.,
generated by employing 3D bioprinting methods, such tech- cell organization, intra-/intercommunication, and/or de novo
nique still lacks the ability to mimic the active modifications matrix deposition) that take place along time—fourth dimen-
that native tissues experience as a result of their interactions sion.[28] Postprinting modifications can be programmed before-
with their naturally dynamic surrounding environment.[16] In hand, so that when constructs are exposed to certain stimuli,
the human body, natural tissues’ microenvironment not only they evolve, in a predictable manner, toward a specific goal.[16]
provides support for their development, but also entangles a One important aspect to point out is that the controlled degra-
series of different biochemical and biophysical signals that dation of 3D-printed biomaterials should not be mistaken by
regulate their biological functions. Considerations regarding 4D functionality, and therefore should not be considered within
the resemblance of artificial bioconstructs with living tissues, the scope of 4D bioprinting.[29] This means that a more accurate
where they will be implanted, are becoming a primary concern reproduction of the native target tissues and its dynamic inter-
in AM, alongside with the biocompatibility, biodegradability, actions with cells may be partially achieved with 4D bioprinting.
and mechanical properties of the developed biomaterials.[2] This plays a key role in achieving promising results regarding
With a view to better recapitulate the features of each living the control over cellular processes and over the final therapeutic
tissue, it is important to proceed carefully within the selec- outcome.[13] For instance, the control over hydrogels’ stiffness,
tion of the biomaterials that will be used for producing 3D by employing hydrogels with stimuli-dependent viscoelastic
constructs, acknowledging that underlying bioactive proper- behavior, allows us to guide mesenchymal stem cells (MSCs)
ties and interactions with cells/host tissues will strongly affect differentiation toward specific phenotypes.[30,31] This feature
the overall biological performance. This selection is highly can, ultimately, translate in an enhancement of both in vitro
dependent on the nature of the targeted tissue and on the and in vivo performances of the produced constructs.[32]
specific physicochemical, biomolecular, topographical, and To date, the dynamic features of 4D bioprinting have granted
mechanical properties that are required to restore full func- exceptional progress in several areas within the tissue engi-
tionality.[17] For an optimal outcome of the engineered bioma- neering domain, namely tissue vascularization,[33] cardiac,[34]
terial ink, the choice of embedded cells is also a fundamental bone,[35] muscle,[36] and neural[22] tissue engineering and
aspect to be considered, as natural tissues are highly hetero- stents[37] production. While there are still many challenges
geneous, increasing the degree of complexity for their in vitro to be overcome, the 4D bioprinting technique has the poten-
replication via AM techniques. The possibility of seamlessly tial to change the paradigm of tissue engineering, by being
combining different biomaterials and cell types in a 3D-bio- able to input a level of dynamism and response to stimuli
printed construct allows researchers to closely resemble some currently unattainable by more conventional biofabrication
of the anisotropic features of native tissues.[2] Moreover, cel- techniques.[9,29]
lular spatial distribution, either in single or multicellular 3D To materialize such dynamic constructs with programmable
aggregates, has shown to considerably influence numerous and controlled shape or functionality changes, smart polymeric
intra-/intercellular processes occurring in constructs during materials of natural, synthetic, or hybrid origin, processed
in vitro maturation or upon in vivo implantation, and must into stimuli–responsive and shape memory hydrogels, have
also be considered in the design stages.[2,18] Fortunately, both become the main source of biocompatible bioinks for 4D bio-
3D and 4D bioprinting techniques allow a suitable control printing.[2,38] Such kinds of materials are able to change their
over this parameter, thus granting the possibility of producing properties upon the variation of external variables in a highly
heterogeneous scaffolds, comprised of several biomaterials nonlinear manner.[39] Here we present a brief overview of the
and cell types, with a more controlled spatial arrangement 4D bioprinting technology and its operational features, its use-
for each final application when compared to other available fulness to move toward the biofabrication of biomimetic tis-
technologies[19] (e.g., electrospinning, solution casting, particu- sues, as well as provide a broad overview on the myriad of bio-
late leaching, and micromolding, in which cell distribution is materials and stimuli that can be exploited. A particular focus
generally random[20,21]). Additionally, it is extremely important is given to seminal studies that explore the potential applica-
for future engineered biomimetic scaffolds to be able to grasp tions of 4D bioprinting within the scope of tissue engineering.
the intrinsic dynamism of the supporting extracellular matrix Systems containing natural-based polymers will be focused as
(ECM), moving away from the traditional 3D paradigm of bio- they are often assumed as materials with adequate bioinstruc-
printed constructs as inanimate structures, and toward a time- tive and structural properties for a variety of biomedical applica-
spanned 4D approach. tions.[40] Finally, the most pressing challenges to be overcome,
as well as future perspectives for this emerging technology, are in a single material[45]) are frequently employed to convey shape
outlined and critically discussed in light of envisioned future change over time to 3D-bioprinted constructs.[7]
advances. In recent years, both natural and synthetic stimuli–responsive
polymers have been explored as potential bioinks for numerous
4D bioprinting applications, such as skeletal muscle[46] or bone
2. Biomaterials for 4D Bioprinting tissue engineering,[29] among several others. In this regard,
proteinaceous materials and polymers derived from natural
The development of 4D bioconstructs implies the use of sources, which are generally regarded as biocompatible, have
stimuli–responsive materials as a design rule, for enabling the advantage of possessing bioactive properties when com-
researchers to preprogram a particular shape or enable a func- pared to their more inert synthetic counterparts;[7,47–49] pro-
tionality change, depending on the envisioned final biomedical teins obtained from human plasma, for example, provide a
application.[7] Besides stimuli responsiveness, for bioprinting very interesting source of biomaterials in a personalized per-
purposes it is also crucial to take into consideration the phys- spective.[50] Yet such materials generally present considerable
icochemical and rheological properties of the bioink formula- batch-to-batch variability and less tailorable mechanical prop-
tion, to assure printability without compromising cell viability erties when in a pristine state, two parameters that must be
during the printing process,[13] as well as guarantee the main- taken into consideration during the design of bioinks for 4D
tenance of bioprinted 3D constructs structural stability in the bioprinting. Conversely, synthetic polymers provide a higher
desired time frame postprinting.[2] When working within the control over batch-to-batch variability, molecular weight dis-
scope of biomedical-related applications, these materials must persity, and a higher versatility over crosslinking mechanisms
meet important additional requirements, including i) biocom- and mechanical properties.[51] However, these polymers are
patibility, ii) nonimmunogenicity, iii) mechanical robustness, generally highly bioinert, lacking the presence of intrinsic cues
and, in some cases, iv) biodegradability,[16] as well as being able to induce biological activity (e.g., cell adhesion molecules and
to be processed and perform shape change under cell compat- morphogens binding).[7] Several bioinks developed for 3D/4D
ible conditions.[41] bioprinting comprise natural polymers or a combination of nat-
The realm of biomaterials that are currently being explored ural and synthetic polymers. Nevertheless, there have also been
as precursors for fabricating 4D-bioprinted structures is mostly reports where synthetic polymers alone have been used with
based on natural, synthetic, or hybrid smart polymers,[7,42] with interesting results but mainly in 3D printing applications that
an emphasis on the use of stimuli–responsive polymeric hydro- do not include cells during the printing process; instead, cells
gels,[38] which can also possess shape memory[4] and inject- are generally seeded in scaffolds post printing—top-down engi-
ability[43] properties. Besides single-component materials, the neering. For instance, Hendrikson et al. employed a synthetic
use of multicomponent polymeric bioinks and other additives thermoresponsive shape memory polymer, polyurethane, to
(e.g., biomolecules and nanomaterials) has become a valu- fabricate 4D-printed scaffolds to assess cellular behavior upon
able approach to further extent the library of biomimetic 4D modification of the mechanical properties.[52] The researchers
constructs that can be materialized.[2,30,43] These topics will be performed cell seeding after fixing the temporary shape of
addressed in further detail throughout this section, and a par- the scaffold and observed that, after recovery to its perma-
ticular focus will be given to natural origin materials owing to nent shape, cells experienced morphological changes, while
their wide availability, versatile chemical processability, bioac- maintaining a high viability. These approaches hold particular
tivity, general biocompatibility, and biodegradability. potential for supporting the advancement of cutting-edge tissue
regeneration applications; however, the efficacy and yield of cell
seeding in such top-down approaches remain to be optimal
2.1. Smart Polymeric Materials when compared to bioink formulations laden with cells on the
fly.
The use of polymeric materials for biomedical applications has In terms of natural origin smart polymers, the reports from
been well established over the years, not only regarding their Miao et al.[35,53] are particularly interesting owing to its use of a
biocompatible properties, but also owing to their similarities relatively unexplored and renewable polymer—namely soybean-
to some components of natural tissues or their surrounding oil-epoxidized acrylate (SOEA)—to produce 4D smart scaffolds
environment, and to their ability to be processed under rela- with shape memory features. This plant-derived oil polymer is
tively mild conditions.[7] In the context of 4D bioprinting, a responsive to temperature changes, enabling temporary shape
group of smart polymers frequently used are shape memory fixation at very low temperatures (−18 °C) and permanent shape
polymers, which can retain memory of a deformed temporary recovery at 37 °C (i.e., physiological temperature). Biocompati-
shape, and then recover to their initial permanent shape when bility assessment of this material revealed a suitable attachment
an external stimulus has been particularly explored.[44] Besides and proliferation of human bone marrow mesenchymal stem
the conventional shape memory process, 4D-printed smart cells (hMSCs).[35]
polymers can also undergo other types of transformations, Due to the beneficial properties of biopolymers, these mate-
namely self-deformation, self-assembly, and/or self-healing.[41] rials are being extensively explored to formulate bioinks for
For example, shape morphing anisotropic polymeric materials both 3D/4D bioprinting. To build 4D bioconstructs, the pres-
exhibiting bending deformation (which can result from either ence of stimuli–responsive groups is generally required. In the
the combination of two materials with different properties or event that polymer precursors do not exhibit responsive proper-
the gradient/pattern-driven/nematic arrangements distributed ties to external stimuli, or in case their responsive properties are
not suitable for certain biomedical applications, it is often pos- possess an exceptional trait for ECM-biomimetic hydrogel
sible to use precision chemical tools for imprinting functional bioinks’ formulation, since, in essence, they are natural-ECM-
groups in the polymeric backbone and that confer stimuli– derived biomaterials,[70] i.e., collagen proteins make up a sig-
responsive functionality in a user-programmed and application- nificant portion of the natural ECM in mammalian tissues,
oriented mode.[16] This strategy can also be applied to achieve and gelatin corresponds to the denatured form of collagen,
other desirable properties,[16] such as solubility in water,[54] which significantly improves cell attachment, proliferation, and
control over the degradation rate,[55] and enhanced mechanical activity, making them particularly suitable for bioprinting appli-
properties.[56] Gathering on this, some of the most explored cations and TERM.[7] This feature, allied with the inherent tem-
natural polymers for 4D bioprinting including alginate,[33,57] perature[61] and pH[32] responsiveness of gelatin, makes it a par-
collagen,[58] gelatin,[34,59,60] hyaluronic acid,[18,33] and chitosan[61] ticularly suitable precursor for 4D biomaterial inks’ formulation
are depicted in Figure 1. Within 3D bioprinting applications, and dynamic constructs’ manufacture. Moreover, gelatin modi-
alginate hydrogel bioinks are undoubtedly one of the most fied with methacrylic groups (methacrylated gelatin, GelMA)
broadly researched natural biomaterials. The extremely rapid has been frequently employed as a bioink.[12,43,57] The use of
crosslinking when in contact with divalent ions (e.g., calcium, GelMA for bioprinting provides efficient means to perform
barium, and magnesium), suitable biocompatibility, and rheo- covalent crosslinking of the bioconstruct generally initiated
logical properties render it an excellent choice for assuring by UV light exposure. The photo-crosslinking process can be
good printability without significantly compromising cell sur- controlled to endow GelMA-based bioconstructs with 4D func-
vival during and after the bioprinting process, making it a tionality, by controlling the crosslinking degree throughout the
reference in this field.[62,63] To be employed in 4D bioprinting, constructs, thus generating a differential crosslinking, making
alginate can be further chemically modified using precision the resulting bioconstructs responsive to humidity/moisture
chemistry tools (e.g., zero length coupling,[64] grafting of click- due to the differential swelling degree, when in contact with a
chemistry moieties,[65] and/or caged cell adhesion motifs[66]) to solution.[46]
induce stimuli responsiveness onto the bioprinted material.[33] Similarly, hyaluronic acid biopolymers are also an asset for
Moreover, the possibility of using alginate in combination with the fabrication of bioconstructs with close resemblance to the
a variety of stimuli–responsive biomaterials has the potential natural ECM, as they can also be found in several native tis-
to generate bioinks with pH,[67] temperature,[68] and acoustic sues (in particular, in some connective tissues and cartilage),
responsiveness,[69] thus attesting its enormous versatility for and are known to contribute for cell viability and to activate
being used in 4D bioprinting applications. Collagen and gelatin key cellular signaling pathways both in healthy and diseased
Figure 1. Chemical structures of natural and bio-based polymers which can be used in 4D bioprinting.
tissues.[7,71] While chitosan has been recognized as a natural presents an overview of several polymers and additive mate-
polymer with very interesting properties for several biomedical rials that have been reported to produce bioconstructs with
applications,[72] being easily modified using well-established 4D functionality. Here, the biofabrication technique, type of
chemical routes,[73] its use for cell-laden bioinks formulation stimuli employed, and envisioned final applications are also
has not been extensively explored, mainly due to the fact that presented. For the most part, the works presented in Table 1 are
chitosan is insoluble at physiological pH (≈7.4)—instead, a solu- described in more detail throughout this review. An analysis
tion of weak acids (e.g., acetic acid) is required to dissolve this on Table 1 shows that while several 4D bioconstructs include
biopolymer. At this pH, cellular viability is significantly affected cells within the bioprinted material, and therefore in the bioink
(depending on the time of incubation in these conditions), formulation, in some cases, cell embedding is not required
making chitosan hydrogels unsuitable for long-term cell encap- for the intended final application. In those instances, there is
sulation. However, this limitation might be overcome by using a higher degree of freedom in the bioink formulation, as the
multicomponent material bioinks or by chemically modifying concern for cell viability only poses after the bioprinting pro-
chitosan polymeric backbone with certain functional groups to cess takes place. The production of composites, including the
improve its properties including solubility.[13,74] combination of hydrogels with additive materials to improve
As favorable as natural polymer-based materials are for bio- their performance, has also been the subject of several research
medical applications, they also encompass some disadvantages. works.[84] For example, nanomaterials, such as graphene[22] and
Even though hydrogels derived from synthetic polymers may iron nanoparticles,[58] represent a class of materials that have
mitigate some of these shortcomings, they are also more likely been added to bioinks to enhance electric and mechanical prop-
to contain cytotoxic components, such as unreacted mono- erties of bioconstructs intended to be explored for neural and
mers, initiators, and crosslinkers, as well as generate toxic muscle tissue engineering.
reaction byproducts, which means that this type of polymers It has become increasingly important to investigate the pos-
must be carefully selected in order not to compromise bio- sibilities of combining several materials for multicomponent
compatibility.[13] Poly(N-isopropylacrylamide) (PNIPAAm) and bioink formulations, considering that in general the use of
poly(ethylene glycol) (PEG) are examples of synthetic polymers different elements results in more versatile biomaterials, with
that have been frequently used in combination with natural improved biomimetic features. Besides combining different
polymers for biofabrication purposes, including bioprinting. materials in the bioink formulation, different bioinks can also
PNIPAAm bioinks are often formulated together with biopoly- be alternately used to print different layers or different seg-
mers such as sodium alginate[75] or hyaluronic acid[76] to convey ments of materials that will make up the final construct. The
temperature responsiveness onto them. On the other hand, recent development of multinozzle 3D printers facilitates the
the use of PEG derivative forms (namely poly(ethylene glycol) buildup of these heterogeneous scaffolds with distinct bioinks.[2]
diacrylate (PEGDA) and poly(ethylene glycol) methacrylate
(PEGMA)) has been known to improve mechanical proper-
ties of natural-based hydrogels.[2] Other polymers derived from 3. Stimuli in 4D Bioprinting
renewable sources,[77] including polylactic acid (PLA),[37,78] and
other biodegradable polymers, such as polycaprolactone,[79,80] In general, any material that comprises stimuli–responsive
have been widely used for 4D bioprinting. properties has the ability to bear certain changes when a given
stimulus is applied.[13] These changes can occur in terms of
alteration of biomaterials chemical, physical, mechanical, or
2.2. Combinatorial and Hybrid Smart Materials electromagnetic properties and changes in constructs shape,
size, or even ability to perform dynamic movement, depending
Analogously to what has been previously described for the on the type of stimuli–responsive properties exhibited by
smart polymeric materials, 4D bioinks can also be formulated the materials/constructs as a whole and the type of stimulus
by the combination of different natural biopolymers (i.e., poly- applied. Often the combination of several polymers, respon-
saccharides or proteinaceous materials[81,82])—combinatorial sive to different stimuli, can also yield multistimuli–respon-
materials—or by the combination of natural and synthetic poly- sive constructs, which allows us to produce extremely versatile
mers to attain complementary properties[2]—hybrid materials— biomaterials. Interestingly, from an applicability point of view,
to overcome some limitations for more demanding biomedical their ability to undergo transformation of their physicochemical
applications (for instance, improved mechanical properties properties (e.g., viscoelastic behavior and mechanical proper-
for bone tissue engineering[83]). Such combinations are also ties) or shape (e.g., self-folding, self-assembly, and morphing) in
highly valuable to better mimic the complexity, heterogeneity, response to a particular stimulus (e.g., temperature, humidity,
or topography of natural ECM.[30] A relatively straightforward light, and enzymes) renders them suitable for simulating the
practice used to tune construct properties is to formulate multi- dynamic and anisotropic nature of native tissues and organs in
component bioinks, which can comprise two or more different engineered 4D bioconstructs in the time dimension.[2,29]
polymers, two or more different cell types, and a wide variety of
biomolecules or other additive materials.[2]
So far, there are already a significant number of combina- 3.1. Stimuli Classification
torial and hybrid polymer mixtures (including biopolymers
only, synthetic polymers only, or a mixture of both), which have The time-dependent change characteristics of 4D bioprinting
been employed to develop 4D-bioprinted constructs. Table 1 can be induced through a variety of different stimuli, which,
Table 1. Examples of natural-based polymeric materials and/or combined with other materials/additives reported for 4D bioconstructs fabrication.
Polymers/ Type of Biofabrication technique Stimuli to induce 4D behavior Cell type Cell culture Applications Reference
Additives biomaterial 4D functionality
Alginate/PDOPAa) Combinatorial 3D bioprinting: extrusion Near-infrared Shape morphing 293 T cells Cell-laden material Various TE applica- [57]
+ (NIR) light behavior tions (including skin,
Alginate/GelMAb) cartilage, and cardiac
tissue)
Agarose/type I Combinatorial 3D bioprinting: drop-on- Magnetic field Stimuli–response hKACsc) Cell-laden material Cartilage TE [58]
collagen/iron demand (DoD) (alignment)
nanoparticles
SOEAd) Natural based 3D printing: Temperature Shape memory hMSCse) Cell seeding post Various TE [35]
stereolithography behavior fabrication applications
SOEA Natural based 3D printing: photolitho- Moisture/humidity Shape morphing hMSCs Cell seeding post Cardiac TE [53]
graphic–stereolitho- and behavior fabrication
graphic–tandem strategy temperature and
(PSTS) shape memory
behavior
HA–MAf) Natural based 3D bioprinting: extrusion Moisture/humidity Shape morphing mBMSCsh) Cell-laden material Tissue [33]
AA–MAg) and behavior vascularization
CaCl2/EDTA and
Shape memory
behavior
Gelatin + GelMA Natural based 3D bioprinting: extrusion Moisture/humidity Shape morphing C2C12 cells Cell-laden material Muscle tissue [46]
fibers behavior engineering
Alginate/ Hybrid 3D printing: extrusion Temperature Shape memory – – Smart valve [68]
PNIPAAmi) behavior
Gelatin/chitosan Combinatorial 3D printing: extrusion Temperature Stimuli–response mBMSCs Cell seeding post Tissue [61]
(patterning) fabrication vascularization
SOEA/graphene Natural based 3D printing: Temperature Shape memory hMSCs Cell seeding post Nerve regeneration [22]
stereolithography behavior fabrication
GelMA/iron oxide Natural based 3D bioprinting: extrusion Magnetic field Stimuli–response C2C12 cells Cell seeding post Muscle tissue [59]
nanoparticles (alignment) fabrication and engineering
cell-laden material Soft robotics
PLAj) Natural-based 3D printing: extrusion Temperature Shape memory – – Vascular stents [37]
behavior
GelMA/PEGDAk) Hybrid 3D printing: Mechanical stimuli Shape morphing hiPSC-derived Cell seeding post Cardiac TE [34]
beam-scanning behavior CMsl) fabrication
stereolithography
hECsm)
hMSCs
GelMA/ Natural based 3D printing: Moisture/humidity Shape morphing HUVECsn) Cell seeding post Various TE applica- [60]
GelCOOHMA inkjet behavior fabrication tions (including
intestinal, lung fat,
and linear tissues)
a)Polydopamine; b)Methacrylated gelatin; c)Human primary knee articular chondrocytes; d)Soybean-oil-epoxidized acrylate; e)Human bone marrow mesenchymal stem cells;
f)Methacrylated hyaluronic acid; g)Methacrylated alginate; h)Mouse bone marrow stromal cells; i)Poly(N-isopropylacrylamide); j)Polylactic acid; k)Poly(ethylene glycol) dia-
crylate; l)Human induced pluripotent stem-cell-derived cardiomyocytes; m)Human endothelial cells; n)Human umbilical vein endothelial cells.
in the context of biomedical applications, require special Cell-responsive systems, usually based on the concept of “cell
attention, since they must not be harmful for the cells or origami,” have also recently emerged to induce shape change
tissues. There are two possible approaches to induce pro- in cell-laden micro-/macrostructures by taking advantage of the
grammed responses in 4D bioconstructs: i) the use of physical contractile forces that occur naturally, referred to as cell traction
or chemical stimuli or ii) cell-responsive systems. The most forces (CTFs). Here, CTFs is exploited as a biological stimulus
common one is the bioprinting of materials that are intrin- to provoke folding of a cell-laden 2D structure into a 3D struc-
sically responsive to physical or chemical stimuli (e.g., tem- ture, according to specific predesigned patterns, to achieve dif-
perature, pH, water, light, magnetic field, and electric field)[13] ferent geometries. Figure 3 exemplifies the application of this
(Figure 2). strategy in a work developed by Kuribayashi-Shigetomi et al.[85]
Figure 2. Representation of the 4D bioprinting process: bioink formulation and time/stimuli-dependent 4D behavior (top); examples of possible
changes that can occur in 4D-bioprinted constructs (bottom).
Although in this case the authors explored CTFs to promote bear in mind that a certain degree of freedom within the matrix
bending of microplates with flexible joints, it is expected that is necessary to allow this cell-induced shape change process to
the same principle can be employed in cell-rich 3D-bioprinted occur. Additionally, the mechanical properties of the biocon-
structures/constructs as the substrates. The presence of hinges struct, particularly porosity,[86] viscoelasticity,[87] and stiffness,[88]
in these bioconstructs can also be explored to facilitate the are other important aspects to be considered. For example, the
shape change into the intended geometries.[29] When applying material cannot be too soft that it cannot withstand the envi-
this technique onto bioprinted constructs, it is important to ronmental conditions in which it will be applied, and at the
Figure 3. a) Folding process by cell traction forces (cell origami): a) after adhering and stretching across the microplates in which cells are seeded, cell
traction forces generated toward the center of the cell causes the folding of these structures. b) Schematic of self-folding into a dodecahedron structure.
c) Schematic of self-folding into a cylindrical tube structure. Adapted with permission.[85] Copyright 2012, Plos One.
same time cannot be too stiff; otherwise, the cells might not be (at temperatures above 30 °C, gelatin is in a soluble state,
able to reshape it. Besides being able to promote shape change, while at temperatures below 25 °C gelatin is in a gel state).[61]
the cellular process taking place after bioprinting also leads to Importantly, these temperature ranges are highly dependent
the progressive maturation of the tissue construct, which may on gelatin origin, with fish skin gelatin presenting an entirely
endow the biomaterial with certain functionalities over time.[63] different temperature profile (for example, 4–8 °C gelling and
Interestingly, shape changes such as self-folding can also be 16–18 °C melting[90]), which is also dependent on the type of
obtained by using multiple components with different volume fish living areas (i.e., cold or warm water).
expansion properties (e.g., swelling ratio or thermal expansion)
or by using single-component-based hydrogels with a differen-
tial gradient in their intrinsic properties.[7] 3.1.2. pH
Both physicochemical stimuli-induced changes and cellular-
induced shape change or tissue maturation play a key role in pH is another parameter that can be altered to trigger modi-
achieving 4D biomaterials, which mirror native tissues more fications (including, but not limited to, shape memory effect)
accurately. However, within the scope of 4D bioprinting, the of some polymers or hydrogels. Biopolymers containing ion-
cellular-induced shape change approach is much less fre- izable chemical groups in their structure are susceptible to
quently employed than the use of smart materials sensitive to pH changes in their environment.[91] For instance, the con-
physicochemical stimuli.[29] figuration of natural proteins like collagen, gelatin, and ker-
atin undergoes globule-to-coil transition upon pH alteration.
Macroscopically, these changes can translate into behaviors like
3.1.1. Temperature swelling, shrinking, or bending.[32]
Among the different stimuli, temperature has been one of the

most studied over the years, particularly for exploring shape 3.1.3. Moisture or Humidity
memory.[32] In fact, there are several polymers known to pos-
sess temperature-sensitive properties. However, considering Liquid-responsive materials, which alter their shape or prop-
that most of them are of synthetic origin, only a few of them erties when in the presence, for example, of water or cell cul-
hold the intrinsic properties required to produce bioactive bio- ture medium, are very useful for applications in the sphere
material inks/4D bioinks. In this context, the use of tempera- of actuator materials and soft robotics, due to their swelling/
ture as the shape or functionality change inducing stimuli is deswelling behavior that can be used to impart motion through
only viable when this transition occurs at temperatures close reversible bending.[9] In general, this feature is accomplished by
to the physiological one, which limits the number of thermore- employing a single anisotropic material, with a differential gra-
sponsive polymers that could potentially be employed for 4D dient in their swelling ratio or multiple materials with different
bioprinting applications, since those which require tempera- volume expansion properties.[7] For example, chitosan under-
tures considered to be too extreme for cell survival are not suit- goes a reversible glass transition with the presence of mois-
able for most biomedical applications.[29] The combination of ture[92] that was used to produce scaffolds with shape memory
thermoresponsive macromolecules with biopolymers, such induced by hydration.[93] We believe that this characteristic
as polysaccharides, is a strategy to induce temperature-sensi- could be found in other biopolymers and further explored in
tive properties in natural systems.[89] PNIPAAm is a common the context of 4D bioprinting.
example of a synthetic thermoresponsive polymer widely
used for tissue engineering and drug delivery applications.[28]
Recently, Miao et al. reported on the use of a naturally derived 3.1.4. Light
material, SOEA, to print biocompatible constructs with tem-
perature-responsive shape memory effect, where, after fixation The use of photoresponsive materials provides an opportunity
of a temporary shape, the recovery of the permanent shape to exploit light as an on-demand, user programmed stimuli to
of the material takes place at the physiological temperature generate modifications on constructs shape or size, including
(37 °C).[35] Mammalian-derived gelatin is another example of contraction, bending, or volume changes.[32,94] Light can also
a thermoresponsive naturally derived polymer that can experi- be used as a heat source, producing thermal energy that may
ence reversible sol–gel transitions upon temperature variations trigger a localized change on photothermal-responsive groups
or molecules. This mechanism has been applied to trigger When trying to achieve biomimetic artificial tissues, either
shape memory effect on some polymers and hydrogels and will the use of multiple stimuli–responsive materials or the com-
be further discussed in the following sections.[7] bination of several materials that respond to different stimuli
(for example, gelatin, which exhibits both temperature and
pH responsiveness) is most often a more interesting option to
3.1.5. Magnetic and Electrical Fields better recapitulate the complex interactions and transforma-
tions that occur in native ECM.
Several studies have also focused on the prospect of employing
magnetic and electrical fields as a means to drive certain altera-
tions within biomaterials. In the case of magnetic stimulus, 3.2. Shape Memory and Shape Morphing
the use of composites is generally required to impart the mag-
netic responsiveness onto the biomaterials.[95] Iron and iron Within the group of stimuli–responsive materials, two dif-
oxide nanoparticles (IONPs) are common examples of additives ferent types of behavior regarding shape modifications can be
used to prepare materials sensitive to magnetic fields.[58,59,96,97] distinguished, specifically shape memory behavior and shape
Besides being used to promote alterations after printing, the morphing behavior (Figure 2). In terms of 4D bioprinting appli-
application of a magnetic field during the bioprinting process cations, the use of stimuli–responsive hydrogels either with
of magnetized bioinks can be harnessed to control the ori- shape memory or shape morphing abilities has emerged as a
entation of the magnetic-responsive particles, thus attaining viable option.
bioconstructs with anisotropic properties that can be directed The shape memory ability involves the transition
according to the envisioned final application.[58] In the case of between a temporary and a permanent shape, in a prepro-
electrical stimulus, the electric responsiveness may derive from grammed manner, upon exposure to a specific stimulus.[107]
the use of electrically conductive polymers or additives with elec- Shape memory hydrogels typically comprise two types of
trical conductive properties (e.g., graphene, carbon nanotubes, crosslinking networks—a covalently crosslinked network,
and other metal nanoparticles).[16] These types of biomaterials which is responsible for fixing structures’ permanent shape,
are currently being explored for the development of smart nerve as these are irreversible bonds, and a supramolecular or
guidance conduits.[22] reversible crosslinked network, which will, at a first instance,
fix structures as a temporary shape. Then upon exposure
to a determined stimulus (e.g., temperature, pH, or light),
3.1.6. Biomolecules the material will return to its original permanent shape, as
these are reversible bonds.[108] Some shape memory materials
Biological stimulus, namely the presence of certain biomole can sustain several cycles of temporary shape deformation/
cules, represents an additional possibility of inducing pro- fixation and permanent shape recovery, showing reversibility
gressive changes to the bioprinted constructs. For instance, and possibility of repeating the stimuli–responsive behavior
Devillard et al. developed a 4D-printed hydrogel loading two several times, while others can even be preprogrammed
different enzymes, alkaline phosphatase and thrombin, which to enable more than one shape change—multiple shape
promoted calcification and fiber formation over time, respec- memory effect.[109]
tively, thus imparting calcification and vascularization function- On the other hand, the shape morphing ability comprises an
alities to the bioconstruct postprinting.[98] irreversible change of the material’s properties or morphology
in response to a particular stimulus.[110] An example of such
structures is provided in the study conducted by Luo et al.[57]
3.1.7. Mechanical Forces The key difference between shape memory behavior and shape
morphing behavior is that the first is reversible and the con-
Finally, the use of mechanical stimuli (i.e., pressure, deforma- struct can therefore return to a previous form, while the latter is
tion, and load) also poses an interesting alternative to promote irreversible. Biomaterials capable of undergoing shape memory
changes within mechanoresponsive materials. Hydrogel systems or shape morphing effects have gathered considerable attention
have been commonly studied for this purpose, and they have for biomedical applications, mainly due to their ability to adapt
been known to be able to change some of their physicochemical to specific defect sites and potential for implantation through
properties such as strength,[99,102] viscosity,[100,102] color,[101,102] and minimally invasive methods.[16] The shape morphing and shape
topography[103] upon mechanical stimulation. memory processes in 4D-bioprinted constructs are depicted
Given that, in general, biological systems are constantly sub- in Figure 4a,b, respectively, where the reversible nature of the
jected to mechanical stimuli, and that mechanical cues from shape memory behavior and irreversible nature of shape mor-
the surrounding environment are recognized by cells and phing behavior are evidenced.
trigger a specific cellular response (i.e., the so-termed mecha- The anisotropic and reversible shape memory behavior of
notransduction), this stimulus has been broadly reported in the stimuli–responsive hydrogels is also the basis for most studies
literature.[104–106] Exploiting the mechanical responsiveness of conducted on hydrogel-based soft actuator materials.[111] In
artificial systems can be a great asset for the development of this context, and inspired by skeletal muscle movement,
smart biomimetic materials, particularly for wound repair scaf- Bakarich et al. developed an alginate/PNIPAAm hydrogel,
folds, drug delivery systems, and fabrication of artificial tissues where the reversible temperature-responsive volume transi-
or biosensors.[102] tions of the PNIPAAm network endowed this material with
Figure 4. a) Illustration of the shape morphing process in a methacrylated alginate hydrogel: i) printing/bioprinting step; ii) photo-crosslinking
with green light, where a differential crosslinking degree is created (higher at the top, since it absorbs more light); iii) folding into tubes induced by
differential swelling degree when in contact with a solution, due to the differential crosslinking degree. b) Illustration of the shape memory process
in a methacrylated alginate hydrogel: i) permanent shape, fixed by an irreversible covalent crosslinking network—photoinitiated covalent bonds
due to the presence of methacrylic groups; ii) temporary shape, fixed by a secondary reversible crosslinking network—alginate crosslinking with
calcium ions; iii) return to the permanent shape, after the removal of calcium ions by an EDTA solution. Adapted with permission.[33] Copyright 2017,
Wiley-VCH.
thermoresponsive actuation behavior. Through the 4D printing 4.1. Tissue Vascularization

of supporting materials, a prototype for a smart valve was pro-
duced for controlling water flow, triggered by changes in the The in vivo performance of engineered tissues depends signifi-
water temperature.[68] cantly on the ability of seeded or encapsulated cells to survive,
Hydrogel-based soft robot actuators with dynamic move- proliferate, integrate into host tissues, and eventually carry out
ment triggered by exposure to temperature variations,[112] elec- their natural functions. To assure full functionality, an essential
tric fields,[113] and magnetic fields[114] have also been reported. aspect that must be considered is the vascularization of artificial
Studies employing these unique concepts and fully natural tissues, in order to allow gas (e.g., O2 and CO2), nutrients, pro-
origin biopolymers are still scarce but if materialized they may teins, and waste products’ exchange.[13] From this perspective,
enable a unique set of opportunities and open new avenues for it is understandable that many research efforts are focusing on
advanced biomedical applications that can take advantage of exploring bioprinting for developing new ways to produce vas-
such dynamics. cularized tissues or to induce the production of blood-vessel-
like tubular structures.[116]
Extrusion 3D bioprinting-based methods have been inves-
4. Biomedical Applications tigated to fabricate hollow tubular constructs; however, the
high shear forces that are employed in these approaches to
Although 4D bioprinting is a relatively recent technology, its produce tubes of smaller diameter can be deleterious for cell
unique features contribute for its ever growing arrays of appli- viability. The use of 4D-bioprinted materials, where the tubular
cations in many fields including TERM, biomedical devices, structure is formed after printing (i.e., by self-folding), has
and soft robotics/actuators.[115] Some of the most relevant bio- been explored as a viable option to circumvent this problem.
medical-related applications, in which 4D printing/bioprinting Several studies showed promising results, namely the hollow
is currently being employed, are indicated in Figure 5 and will tubular cell-laden structures developed by Kirillova et al.[33] In
be outlined hereafter. this report, the authors developed self-folding hollow tubular
Figure 5. Scope of the possible biomedical applications for 4D bioprinting.
structures from bioprinted planar hydrogel sheets, based on consequently disrupted the Ca2+–alginate crosslinking network.
the disparity of the crosslinking degree observed between the This approach shows that besides self-folding, the AA–MA hydrogel
top and bottom layers of bioprinted materials. Two modified was capable of undergoing a reversible shape modification.
biopolymers, specifically methacrylated alginate (AA–MA) and Using another strategy, Luo et al. produced a near-infrared
methacrylated hyaluronic acid (HA–MA) were tested to produce (NIR)-responsive shape-changing hydrogel that transformed
bioprinted hydrogel films, in which the photo-crosslinking from planar into a tubular structure.[57] Alginate and polydo-
reactions were initiated using visible green light. Since the top pamine (PDOPA) were the constituents of the bioinks used to
layer of these hydrogels absorbs a larger amount of light than produce these hydrogel constructs, and the self-deformation
the bottom layer, the crosslinking degree was higher at the behavior into tubular structures was driven by NIR-induced
top, which caused these structures to bend into hollow tubes dehydration, upon laser irradiation. The switch was induced
when immersed in water, phosphate-buffered saline (PBS), by the photothermal effect of NIR, which led to a tempera-
and cell culture media. The mouse bone marrow stromal cells ture increase with the printed hydrogel and consequent loss of
(mBMSCs) encapsulated within these tubes showed homoge- water, resulting in the shrinkage and folding of the alginate/
neous distribution and good viability after 7 days. Self-folding polydopamine scaffold in specific directions. Furthermore,
tubular constructs with inner diameters ranging from 20 to researchers were able to control the bending angle of the struc-
150 µm, which are quite similar to the size of small blood ves- ture by adjusting the laser power, irradiation time, and designed
sels, were obtained (Figure 4). patterns of the printed construct, resulting in different shape
Interestingly, the authors also found that when the folded AA– changes (i.e., tubular and saddle-like forms). The combination
MA hydrogel was placed in a CaCl2 solution, the crosslinking of stimuli–responsive alginate/polydopamine biomaterial inks
interactions between the alginate chains and Ca2+ ions led to and cell-laden alginate/GelMA bioinks allowed the production
its unfolding, as a result of the deswelling induced by this addi- of biphasic scaffolds, capable of sustaining shape morphing
tional crosslinking mechanism. Refolding could then be restored while also supporting cell survival.
by immersing the unfolded hydrogel in an ethylenediaminetet- PEG bilayered hydrogels, consisting of two different mole-
raacetic acid (EDTA) solution, which captured the Ca2+ ions, and cular weight PEG polymers, have also been described to be
able to self-fold into hollow tubes, promoted by the differential the previously used standard bone grafts, as it prevents disease
swelling of the hydrogel bilayers when in contact with an transmission and is not dependent on donor availability.[118,119]
aqueous solution. These cell-laden structures showed long-term Therefore, this has been a widely researched area in terms of
cell viability.[117] potential biomaterials and strategies used for bone defect repair.
In a slightly different approach, Wen et al. took advantage Taking into account that bone defects are usually irregular and
of the temperature–responsive character of gelatin and pH- can vary in size, 4D bioprinting presents several beneficial
responsive character of chitosan to fabricate 4D dynamic features for this application, such as the opportunity to tailor
tubular constructs through the printing of stimuli–respon- the biomaterial to the specificities of tissues injuries, to enable
sive hydrogels.[61] Here, an initial solid tubular structure was progressive tissue maturation, to impart functionality onto the
obtained via 3D printing of gelatin and chitosan (at pH < 5). engineered constructs, and to attain complex construct archi-
Afterward, this material was immersed in a sodium citrate tectures, with further similarities to the native bone tissue. The
solution, leading to the formation of a second electrostatic incorporation of different inorganic composites within bioinks,
crosslinking network due to the interaction of chitosan and including silicates, hydroxyapatite, tricalcium phosphate (TCP),
citrate ions at low pH. By controlling the diffusion time of and bioactive glass, among others is also an asset to promote
citrate ions into the structure, the researchers managed to stem cells’ osteogenic differentiation and to mimic natural
limit this secondary crosslinking network to a certain thick- bone-building blocks. Moreover, as previously mentioned, the
ness of the cylindrical object, thus generating a dual network 4D bioprinting technique is also being exploited to create artifi-
shell surrounding the single network core. By cutting off cial tissues with vascular and nervous networks, which will sub-
the tips of the cylinder structure and immersing it in warm stantially enhance its regenerative action upon implantation in
water, the core will act as a sacrificial material and will be the bone tissue.[2,29]
removed, with only the hollow tubular structures remaining In the interest of developing a biomaterial resembling the
in the end. structure and functionality of vascularized alveolar bone, Dev-
Considering the current efforts that are being made into illard et al. fabricated a 4D-printed PEGDA hydrogel.[98] Alka-
researching this topic, it is feasible to assume that, in the line phosphatase and thrombin were mixed with the PEGDA
future, 4D bioprinting of vascularized architectures will be a polymer precursor to be entrapped within the printed struc-
widespread process, allowing us to obtain vascularized multi- ture (Figure 6a,b), respectively. The authors found that over
material constructs with multiple cell types, thus getting one time, these enzymes could promote both calcification and fiber
step closer to the ultimate goal of creating tissue mimicking formation (comparable to blood vessels) on the printed con-
bioactive constructs. Once the vascular network engineering is struct (Figure 6c).
refined, it may also be possible to engineer nervous and lym- Besides bone, 4D bioprinting also finds application for carti-
phatic components onto these scaffolds.[2] lage tissue engineering. For instance, Betsch et al. took advan-
tage of the magnetic responsiveness of a bioink composed of
agarose, type I collagen, iron nanoparticles, and human pri-
4.2. Bone and Cartilage Tissue Engineering mary knee articular chondrocytes (hKACs), to force the align-
ment of collagen fibers (due to unidirectional motion provoked
The repair of bone defects through bone-tissue-engineered in the iron nanoparticles by the application of a magnetic field)
structures has been established as a better approach than using at the moment of bioprinting. Since the native cartilage tissue
Figure 6. Schematic of 4D activity of a) calcification promoted by alkaline phosphatase and b) fibrin formation promoted by thrombin. c) 4D-printed
bioconstruct with calcification and fibrin formation activity, promoted by alkaline phosphatase and thrombin enzymes, respectively. Adapted with
permission.[98] Copyright 2018, Wiley-VCH.
Figure 7. SOEA/graphene 4D construct for nerve regeneration: a) implantation process of the SOEA/graphene nanohybrid through shape memory
process—a temporary planar shape recovers its permanent tubular shape at physiological temperature, allowing the 4D construct to wrap itself around
the two stumps of a severed nerve. b) 4D smart nerve conduit integrated in a severed nerve. Adapted with permission.[22] Copyright 2018, Wiley-VCH.
is composed of several layers of collagen fibers aligned in dis- scaffolds built from a top layer of uniaxially aligned polycaprol-
tinct orientations, a heterogeneous construct composed of two actone–poly(glycerol sebacate) (PCL–PGS) and a bottom layer of
distinct layers of horizontally aligned and randomly distrib- randomly aligned HA–MA. This material proved to be capable
uted collagen was produced. The study revealed that bilayered of shape transformation from a flat to a tubular configuration
constructs exhibited higher potential for cartilage tissue repair upon immersion in an aqueous solution, and was suitable to
than constructs built with only one hydrogel layer. In this study, sustain neural cells cultured on its top layer with high adhe-
the time dimension is not employed exactly in the same way sion, viability, and proliferation being obtained.[121]
as it is viewed in other studies concerning 4D bioprinting—
i.e., the hydrogel morphology and functionality change occur
during bioprinting, in response to its exposure to a magnetic 4.4. Muscle Tissue Engineering
field.[58] In the future, developments in this field are envisioned
to include more complex structures for cartilage regeneration, Skeletal muscles are a substantial component of the human
including constructs for osteochondral repair. body, and therefore, the accurate and viable reproduction of
muscle tissue is a subject of much interest among the tissue
engineering field.[36] Within this scope, Tognato et al.[59]
4.3. Neural Tissue Engineering reported the biofabrication of a multiple-stimuli–responsive
nanocomposite hydrogel, comprised of GelMA and IONPs
Tissue-engineered nerve grafts have become an increasingly in the context of the magnetic-force-based tissue engineering
viable option to treat peripheral nerve injury, by integrating the concept. In this work, the magnetic responsiveness of the
use of biomaterials with biological, physical, and chemical cues, bioink due to the presence of IONPs was harnessed to pro-
to promote nerve regeneration.[120] In this domain, Miao et al. mote the aligned organization of the IOPs into filaments in
reported the 4D bioprinting of a smart nerve guidance conduit the constructs. This anisotropic arrangement of the IONPs
from a bioink composed of SOEA, graphene, and hMSCs. The was stabilized by decreasing the temperature prior to hydrogel
4D functionality was achieved as a result of the light-induced photo-crosslinking. It was observed that the C2C12 skeletal
graded internal stress, during the crosslinking step at the bio- myoblasts within these scaffolds aligned to the same axes of the
printing stage, which later on caused the bioprinted structure to IONP filaments, and differentiated into myotubes, proving that
bend, when in contact with a solution. Graphene was used both this kind of structure possesses the necessary cues to guide cell
as a promoting agent for hMSCs’ differentiation into neural cell behavior in the direction of muscle tissue formation.
types and as an approach to enhance biomaterial conductivity. More recently, Yang et al. also sought to replicate skeletal
Since SOEA possesses shape memory property, with permanent muscle tissue by using a combination of 3D and 4D bioprinting
shape recovery triggered by physiological temperature, this fea- techniques. In this study, the researchers produced cell-laden
ture was further explored to perform dynamic self-entubulation GelMA fibers through a modified 3D printing process, which
and seamless integration of the tissue-engineered nerve graft included an applied electrical field to stimulate cell alignment
with the damaged nerve. Succinctly, the bioprinted planar sheet and myogenic differentiation. Then, a 4D-printed gelatin film
is sequentially exposed to ethanol and water to induce the with shape morphing behavior was used to hold these fibers
shape change into a tubular structure, which corresponds to together in bundles (Figure 8a). Here, the self-folding behavior
the permanent shape of the biomaterial. Then, this structure of gelatin resulted from the grooved pattern applied during
is opened and flattened, fixing this as the temporary shape, to the 3D printing process of the gelatin film, which caused it
facilitate its implementation in vivo, and recovering its perma- to experience different swelling degrees throughout its struc-
nent shape at body temperature on the defect site, thus wrap- ture when placed in a liquid environment. By placing the cell-
ping itself around both stumps of the damaged nerve.[22] The in laden GelMA fibers on top of the 4D-printed gelatin film and
vivo implant processes aided by the shape memory process and then exposing it to culture medium, the gelatin film folded,
its result are illustrated in Figure 7. wrapping around the cell-laden GelMA fibers, thus creating
Recently, the 4D functionality was also employed in scaffolds a biomimetic skeletal muscle-like structure. The diamidino-
produced by the electrospinning technique for the biofabrica- 2-phenylindole/myosin heavy chain (DAPI/MHC) images
tion of artificial nerve grafts. In this case, Apsite et al. produced obtained for these fibers after 21 days of culture evidence the
Figure 8. a) Schematic representation of the electrically assisted bioprinting and 4D process employed. b) DAPI/MHC images of cell-laden GelMA
fibers after 21 days of culture. c) Cross-sectional scanning electron microscopy (SEM) image of the cell-laden GelMA fibers enwrapped in the gelatin
outer structure. Adapted with permission.[46] Copyright 2021, Ivyspring International Publisher.
alignment and myotube differentiation of the C2C12 cells cardiac cycle. The shape-morphing ability of the 4D-bioprinted
within the fibers (Figure 8b).[46] GelMA and PEGDA construct was achieved due to the uneven
crosslinking of the bioprinted structure during the bioprinting
process—the higher crosslinking density on the bottom layer
4.5. Cardiac Patches allows the structure to bend into the desired curved conformation.
Furthermore, the 4D ability of this anisotropic patch to transition
Seeing that cardiac problems are to this day one of the major between flat and curved architectures promoted an excellent inte-
health concerns worldwide, there has been a growing necessity gration with the dynamic process of the beating heart.[34]
to develop increasingly functional and improved means to treat Following up on this work, Wang et al. developed a 4D car-
damages in cardiac tissues.[122] diac patch with NIR-light-induced shape memory behavior.[123]
In a recent study, Cui et al. focused on the development of To this end, researchers prepared a bioink composed of temper-
cardiac patches produced from cell-laden smart hydrogels via ature-responsive shape memory polymers, namely bisphenol A
4D bioprinting, in order to obtain a biomaterial capable of fully diglycidyl ether (monomers), poly(propylene glycol) bis(2-amino-
adapting to the native physiology and function of the heart. Cui’s propyl) ether (crosslinker), and decylamine (crosslinking modu-
team aimed at obtaining a cardiac patch with a curvature similar lator), along with graphene as an additive nanomaterial. Due to
to the heart curvature, with the possibility of reversibly stretching the photothermal effect triggered upon NIR light exposure, this
and shrinking, according to the movement of the heart in its construct was able to change its shape from a flat to a curved
Figure 9. 4D bioprinting of dynamic constructs for cardiac tissue engineering. a) Representation of the developed 4D cardiac construct to promote
myocardial repair. The use of a construct capable of shape changing into a curved form as an alternative to a precurved construct prevents cell aggre-
gation and stimulates uniform cell distribution and alignment, as well as organ-specific shape fitting. b) Schematic representation of the 4D cardiac
construct production process and shape memory behavior. Adapted with permission.[123] Copyright 2021, ACS Publications.
configuration (Figure 9b), in order to adjust to the natural cardiac In a more recent study, Cui et al. developed a new approach
tissue morphology. The addition of graphene onto this construct to produce shape morphing 4D-printed hydrogels from gel-
promotes heat absorbance, thus facilitating the photothermal- atin-based bioinks (GelMA and GelCOOHMA).[60] In this
induced shape change process. The 4D shape changing feature approach, the bioprinting of the shape morphing hydrogel is
allows the cardiac patch to adjust its curvature according to the performed on a glass slide coated with a sacrificial layer of algi-
region of the heart where it will be applied, thus ensuring seam- nate/gelatin, to allow detachment and folding of this structure
less and organ personalized integration and increased perfor- in due time, after umbilical vein endothelial cells are properly
mance for myocardial tissue regeneration. The shape memory seeded and cultured on its surface. After detaching, the dif-
effect also enabled us to perform uniform cell seeding onto the ferent swelling rate of GelMA and GelCOOHMA in aqueous
flat temporary shape of the bioconstruct, and the microgroove solutions causes the hydrogels to self-fold. By modifying a
pattern printed onto this 4D biomaterial promoted cell align- few parameters during the bioprinting process (which bioink
ment and mechanical support (Figure 9a). When transitioning is used for the top and bottom layers, and if there is rotation
into the permanent curved shape, this cardiac patch is already of bioinks, for example), it was possible to create a variety of
laden with a uniform layer of aligned myofibers throughout its conformations with these hydrogels that could be employed in
entire surface, thus preventing cell aggregation, which so far has various applications. Constructs with grooves and ridges resem-
been one of the biggest challenges to overcome when producing bling the topographical traits of intestinal villi were obtained,
curved bioconstructs for cardiac tissue repair. which could possibly be used to engineer intestinal tissues.
Hollow spherical structures were also obtained by promoting
self-folding into polyhedron structures, which could be useful
4.6. Additional Biomedical Applications to mimic tissues such as lung alveoli or as lipid compartments
characteristic of fat tissues. Tubular structures with potential to
Due to its versatility and advantageous features, the 4D bio- be used in linear tissues were also achieved.
printing technique has been branching out in many biomedical With the increasing amount of research on this subject, cer-
applications, and several studies have reported the development of tainly many more applications for 4D bioprinting will arise in
4D bioconstructs that hold the potential to be applied in different a near future. Besides bioprinting, the 4D functionality is also
tissues. For example, Luo et al. reported the production of a mul- finding applications in other biofabrication techniques, namely
ticomponent shape morphing scaffold, by alternatively employing electrospinning,[121,124] which indicates that this is becoming a
an AA/GelMA/human embryonic kidney cells bioink and an AA/ transversal functionality to other methods.
PDOPA ink during the bioprinting process, thus obtaining a
heterogeneous scaffold.[57] The photothermal responsiveness of
PDOPA allowed inducing of shape morphing behavior (bending) 5. Limitations and Future Perspectives
onto the AA/PDOPA segment of the scaffold, through localized
NIR-light-induced heating, generating a curved structure, which 4D bioprinting technology is driven by the ever-growing need
could potentially be employed for cartilage and skin tissue repair, to develop artificial biomaterials with closer resemblance to
as well as cardiac patches for myocardial repair. native tissues’ and organs’ dynamics, in an effort to enhance
its potential as a solution for translational TERM applica- evaluate the mechanical properties of 4D-bioprinted materials,
tions. However, due to the incredible complexity and dynamics considering the requirements for the different stages of the
inherent to the natural extracellular environment, the construc- biomaterial’s practical applications. For instance, in the case
tion of biomaterials capable of accurately mimicking living of shape memory hydrogels, the mechanical properties may
tissues in their whole intricacy is a challenging endeavor, vary significantly from the permanent to the temporary shape
even with advanced fabrication techniques available at the and may not recover completely after the permanent shape
moment.[17] Furthermore, considering that the biological envi- recovery. Typically, the mechanical performance, namely the
ronment may be different for each individual, one must con- elastic moduli, is lower in the permanent state as compared
sider this added layer of complexity prior to translation into a with the temporary state, which can limit some load-bearing
clinical setting.[16] applications of the construct. Moreover, in the cases of mul-
The 4D bioprinting technology presents many beneficial tiple shape memory effect, in which the materials can undergo
aspects including the fact that cells can be distributed in a shape deformation and recovery several times, their mechan-
spatially controlled manner throughout the bioconstructs, the ical performance tends to decrease with the number of cycles
possibility of attaining complex tissue alike structures (such as carried out.[126] For the case of natural-based polymers, macro-
vascularized tissues, nerve graft conduits, and tracheal stents) molecular design strategies may be used to produce hydrogels
and the ability to mimic the dynamic changes that occur in with improved mechanical properties that could be integrated
the natural tissues over time.[9] However, considering that the in 4D-bioprinted principles.[127] For that, more work will be
4D functionality is still a rather recent technology, there are needed to build larger libraries of chemically modified nat-
still many challenges and limitations to be overcome in this ural polymers, including polysaccharides,[128] proteins,[129] and
domain. One limitation of the bioprinting techniques in gen- human plasma derivatives.[130]
eral, whether it is 3D or 4D, is that the physical entrapment of Furthermore, the 4D functionality requires the precursor
cells within the bioprinted constructs may hinder some cellular material or a mixture to contain stimuli–responsive compo-
process, namely spreading, migration, and organization, which nents, which further limits the number of candidates suitable
may compromise the overall therapeutic performance of the to be explored for this new technology. Besides this, the stim-
constructs.[60] Controlling cell distribution within the bioprinted ulus needed to induce a specific change upon 4D-bioprinted
construct in the long term is also a very challenging aspect of constructs must also be cell friendly, which means that mate-
3D/4D bioprinting, since the cellular processes taking place rials responsive to any stimuli considered too extreme for cells
post printing may alter the cell distribution within the material, (for instance, temperature and pH outside the physiological
which may result in inhomogeneous cell distribution and even range) will not be adequate for the vast majority of applications
the formation of cell clusters within the bioprinted material.[125] related to TERM.[14,41]
Many of the studies carried out on 4D-bioprinted constructs Although natural polymeric materials present several
focus a lot more on the shape or functionality change process intrinsic advantageous properties for use in biomaterials fab-
of the final material and end up lacking more comprehensive rication, their use also entails some difficulties. For instance,
studies on how these changes affect complex cellular processes, their mechanical properties are often not suitable for highly
oftentimes only performing biological assays based on staining demanding biomedical applications, and in some cases their
techniques (e.g., live/dead viability assay). To safely move fast biodegradation rate may pose as a disadvantage, as it
toward clinical applications, more advanced biological tests reduces biostability of the construct. Common strategies to
will most likely be required.[16] Besides cell viability and pro- overcome such drawbacks include the combination of natural
liferation, in vitro studies should also comprise the evaluation and synthetic materials, as well as chemical modifications
of cell morphology, adhesion, differentiation, and activity, and using precision chemistry tools (i.e., covalent, dynamic cova-
then a transition into in vivo implementation will be necessary lent, and guest–host moieties) to tune their physicochemical
to better understand how the fabricated biomaterial responds properties.[131]
when included in a living host system—analysis of inflamma- Additionally, the currently developed bioprinters do not pos-
tory responses, assessment of biofunctionality, biodegradation, sess the necessary resolution to fabricate certain structures with
and overall effectiveness are examples of crucial data that need high precision[16] (for instance, the diameter of the smallest
to be acquired by in vivo studies before moving toward human blood capillaries ranges from around 5 to 10 µm, which, in
clinical trials. terms of 3D bioprinting resolution, are values remarkably dif-
There are also a number of limitations concerning the ficult to attain[132]). On the other hand, the bioprinting of very
materials available to employ for 4D bioprinting. For one, it large constructs (such as bone tissue) in a high-throughput
is essential that they comprise all the necessary characteris- manner is also an issue that this technology is not yet prepared
tics to be used in biomedical applications, which have been to solve.[2] Technological solutions, involving the combina-
discussed previously. Among these, the mechanical properties tion of different materials (including the inclusion of distinct
assume a significant role, and should be in good agreement stimuli–responsive components in different regions of the con-
with the final applications for which the 4D bioconstructs are struct), will continue to be developed in the coming years, as
intended, in order to guarantee an adequate performance. well as the integration of different techniques to process multi-
However, taking into account that the 4D functionality pre- scale and complex structures.[133]
supposes changes in the constructs over time, it is reason- Besides the technical challenges and safety assurance
able to assume that the mechanical properties may also be regarding 4D-bioprinted constructs, there are other critical
altered. Therefore, it is crucial for researchers to critically issues that need to be addressed to be able to fully implement
4D bioprinting as a standard clinical practice. For instance, the national funds through the FCT/MEC and when appropriate co-financed
evaluation of the cost effectiveness of this technique, the need by FEDER under the PT2020 Partnership Agreement. This work
for personnel training, and the ability to comply with current was also supported by the Programa Operacional Competitividade
e Internacionalização (POCI), in the component FEDER, and by
legal and ethical requirements are all factors that need to be national funds (OE) through FCT/MCTES, in the scope of the projects
considered of and settled before moving forward to a well- MARGEL (PTDC/BTM-MAT/31498/2017), MIMETic (FCT–POCI-01-
established clinical implementation.[134] 0145-FEDER-031210), and PANGEIA (PTDC/BTM-SAL/30503/2017).
Despite the above-mentioned challenges, 4D bioprinting is The MARGEL project was acknowledged for the individual Junior
still a very promising technology to achieve numerous break- Researcher contract of D.C.S.C. and M.Sc. Scholarship of P.D.C.C. The
throughs within the biomedical field, being highly expected MIMETic project was acknowledged for the individual Junior Researcher
contract of T.R.C. FCT was also acknowledged by financial support
that the scientific community will continue to pursue new
through an individual contract as Junior researcher attributed to V.M.G.
endeavors in this topic. Currently, the most pressing matters (CEECIND/01410/2018).
requiring progress in this field are i) the development of new
materials (or upgrade the functionalization of existing ones);
ii) the development of more precise bioprinting methods; iii)
the improvement of the biological assay component in these Conflict of Interest
research studies, to eventually translate to clinical trials and The authors declare no conflict of interest.
ultimately, clinical applications.
Keywords
6. Conclusion 4D bioprinting, biofabrication, natural origin polymers, stimuli–
The development of 4D bioprinting has led to incredible responsive biomaterials, tissue engineering
progress in several areas of tissue engineering, allowing the
Received: February 11, 2021
attainment of more complex and dynamic structures, with Revised: April 5, 2021
a better resemblance of the native tissues. Biocompatible Published online:
stimuli–responsive shape memory and shape morphing hydro-
gels have been established as promising systems to apply with
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Patrícia D. C. Costa concluded her Master’s degree in Chemical Engineering in 2016, with a
specialization in Biosystems at the University of Coimbra. Her Master’s thesis research was
conducted in the area of nanomaterials, focusing on the stabilization of nanotube suspen-
sions for further applications in chemical stabilization of soft soils. Currently, she is working at
Aveiro University, as a member of the COMPASS group at CICECO. Her research work is mainly
focused on the chemical functionalization of polymers of natural origin, for tissue engineering
applications.
Dora C. S. Costa is a junior researcher of the COMPASS Group at CICECO—Aveiro Institute

of Materials, Portugal. She got her Ph.D. in Chemistry in 2019 from Universidade Federal
Fluminense (Niterói, RJ, Brazil), her M.Sc. in organic chemistry and natural products in 2011, and
her B.Sc. Biochemistry in 2009 from University of Aveiro (Portugal). She has a strong background
in organic synthesis, working with several families of compounds. Currently she is working in
biopolymers chemical functionalization for tissue engineering.
Tiago R. Correia is a junior researcher of the COMPASS Group at CICECO—Aveiro Institute of

Materials, University of Aveiro, Portugal. He obtained his Ph.D. in Biochemistry from University
of Beira Interior, Portugal, in 2017. His current research interests include the design of biomate-
rials for 3D printing and the microfabrication of innovative microplatforms for bottom-up tissue
engineering applications.
Vítor M. Gaspar is a junior research fellow in the COMPASS research group at the University of
Aveiro. His main research interests include the precision chemical modification of natural origin
biomaterials to formulate nanoscale-to-macroscale materials that aim to be applied for tissue
engineering and in vitro 3D disease modeling. His recent research is also focused on devel-
oping new 3D bioprinting techniques and bioactive, multicomponent, bioinks for biomedical
applications.
João F. Mano is a full professor at the Chemistry Department of University of Aveiro, Portugal.
He is the director of the COMPASS Research Group, from the research unit CICECO—Aveiro
Institute of Materials, focusing on the use of advanced biomaterials and cells toward the progress
of transdisciplinary concepts to be employed in regenerative and personalized medicine. He is
the Editor-in-Chief of Materials Today Bio (Elsevier). He has been coordinating several national
and European research projects, including Advanced and Proof-of-Concept Grants from the
European Research Council. He is an elected fellow of the European Academy of Sciences.

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Review

Natural Origin Biomaterials for 4D Bioprinting

biomorphic features.[4] Particularly, 3D

Among the different stimuli, temperature has been one of the

thermoresponsive actuation behavior. Through the 4D printing 4.1. Tissue Vascularization

Figure 5. Scope of the possible biomedical applications for 4D bioprinting.

Dora C. S. Costa is a junior researcher of the COMPASS Group at CICECO—Aveiro Institute

Tiago R. Correia is a junior researcher of the COMPASS Group at CICECO—Aveiro Institute of

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