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3D Bioprinting for Organ Regeneration

REVIEW
Haitao Cui, Margaret Nowicki, John P. Fisher, and Lijie Grace Zhang*

Presently, tissue engineering approaches

Regenerative medicine holds the promise of engineering functional tissues have been widely studied in cartilage, bone,
or organs to heal or replace abnormal and necrotic tissues/organs, offering skin, vascular tissue and nerve regenera-
hope for filling the gap between organ shortage and transplantation needs. tion, among others.[4–7] When designing
a tissue engineered scaffold, the combi-
Three-dimensional (3D) bioprinting is evolving into an unparalleled bio-
nation of material, biological and engi-
manufacturing technology due to its high-integration potential for patient- neering requirements must be considered
specific designs, precise and rapid manufacturing capabilities with high in an application-specific manner.[8–11] Bio-
resolution, and unprecedented versatility. It enables precise control over mimetic design of the scaffolds, including
multiple compositions, spatial distributions, and architectural accuracy/ 3D structural characteristics and physical
properties, can substantially enhance
complexity, therefore achieving effective recapitulation of microstructure,
the physiological performance through
architecture, mechanical properties, and biological functions of target appropriate cell–cell and cell–matrix
tissues and organs. Here we provide an overview of recent advances in interactions, further enhancing biolog-
3D bioprinting technology, as well as design concepts of bioinks suitable ical functions.[10,12,13] However, most 3D
for the bioprinting process. We focus on the applications of this technology scaffolds currently fabricated with tradi-
for engineering living organs, focusing more specifically on vasculature, tional techniques lack these qualities.[14]
Although significant successes have been
neural networks, the heart and liver. We conclude with current challenges
achieved in engineered tissues, both in
and the technical perspective for further development of 3D organ research and clinical applications, it is
bioprinting. obvious that complex 3D organs require
more precise multicellular structures
with vascular and neural network integra-
1. Introduction tion. These requirements cannot be fulfilled using traditional
methods.[15–17]
Human organs are highly complex structures formed by the 3D printing is a rapid prototyping and additive manufac-
combined, functional organization of multiple tissue types. The turing technique used to fabricate complex architecture with
cells in these organs are highly specialized and group together high precision through a layer-by-layer building process.[18]
to perform distinctive functions.[1] Organ dysfunction or failure This automated, additive process facilitates the manufac-
is drastically increasing due to traumatic injury and disease.[2] turing of 3D products having precisely controlled architecture
Often, clinical treatments are limited by a paucity of avail- (external shape, internal pore geometry, and interconnectivity)
able donors and immune rejection of donated tissue.[3] In the with highly reproducibility and repeatability.[9,19] Therefore, in
search for alternatives to conventional treatment strategies for the regeneration field, it can provide an excellent alternative
the repair or replacement of missing or malfunctioning human for biomimetic scaffold fabrication by accurately positioning
tissues and organs, tissue engineering approaches are being multiple cell types and biofactors simultaneously into complex
explored as a promising solution.[2–4] multi-scale architectures that better represent the structural
and biochemical complexity of living tissues or organs.[18,20,21]
In the past three decades, 3D bioprinting has been widely
Dr. H. Cui, M. Nowicki, Prof. L. G. Zhang
developed to directly or indirectly fabricate 3D cell scaffolds
Department of Mechanical and Aerospace Engineering or medical implants for the field of regenerative medicine.
Department of Biomedical Engineering It offers very precise spatiotemporal control on placement of
Department of Medicine cells, proteins, DNA, drugs, growth factors, and other bioactive
The George Washington University substances to better guide tissue formation for patient-specific
3590 Science and Engineering Hall
800 22nd Street NW, Washington, DC 20052, USA therapy.[20,22,23] 3D printing of bioactive scaffolds contains
E-mail: [email protected] two types of scaffold fabrication: acellular functional scaf-
Prof. J. P. Fisher folds which incorporate biological components, and cell-laden
Fischell Department of Bioengineering constructs aiming to replicate native analogues.[21,24] Both of
University of Maryland them aim to produce biocompatible, implantable constructs
3238 Jeong H. Kim Engineering Building
College Park, MD 20742, USA
for tissue/organ regeneration, thus we refer to 3D printing in
the context of bioactive scaffold fabrication as “bioprinting”.
DOI: 10.1002/adhm.201601118 In this regard, the term bioprinting does not indicate whether

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cells are directly printed or involved at any stage of the fabrica-

REVIEW

Haitao Cui received his

tion process.
Ph.D. in chemistry and physics
An essential requirement for reproducing the complex,
of polymers in 2015 from
heterogeneous architecture of functional tissues or organs is a
Changchun Institute of Applied
comprehensive understanding of the composition and organi-
Chemistry, Chinese Academy of
zation of their components.[10,11] Therefore, medical imaging
Sciences. He joined the Prof.
technology is an indispensable tool to provide information on
Zhang’s group in March 2015.
3D structure and function at the cellular, tissue, organ and
His research currently focuses
organism levels, aiding the design of a patient-specific con-
on the development of novel
struct.[20,25] It commonly offers noninvasive imaging modality,
biomaterials, 3D bioprinting,
including computed tomography (CT) and magnetic resonance
and complex tissue/organ
imaging (MRI). Computer-aided design (CAD) and computer-
regeneration.
aided manufacturing (CAM) tools and mathematical modeling
are also used to collect and digitize the complex tomographic
and architectural information for tissues.[25] The 3D imaged
tissue or organ model is divided into 2D horizontal slices Margaret Nowicki is currently
that are imported into a 3D bioprinter system for the layer- completing her PhD in
by-layer deposition. Considering the available 3D bioprinting Mechanical Engineering at The
techniques, the cell types (differentiated or undifferentiated), George Washington University
biomaterials (synthetic or natural), and supporting biochem- School of Engineering and
ical factors are then selected, and the configuration of these Applied Sciences under the
printing components drives the construction of the 3D tissues supervision of Dr. Lijie Grace
and organs. This integrated technique (imaging-design-fabri- Zhang. Her research interests
cation) can recreate more complex 3D organ level structures include 3D/4D bioprinting,
and incorporate mechanical as well as biochemical cues that complex tissue engineering,
are crucial elements of the whole organ architecture.[20,26] In and nanobiomaterials.
addition, this technique has the capacity to build a 3D tissue-
or organ-specific microenvironment by mimicking the natural,
highly dynamic yet variable 3D structures, mechanical proper-
ties, and biochemical microenvironments.[27] In this manner,
Lijie Grace Zhang is an
3D bioprinting for organ regeneration involves additional strat-
associate professor in the
egies for printing multiple living cells, including vasculature
Department of Mechanical and
and neural network integration, and eventually developing the
Aerospace Engineering at the
specific functions of 3D bioprinted organ analogues.
George Washington University.
Charles W. Hull, in 1986 received a patent for the liquid,
She obtained her Ph.D. in
photopolymer-based manufacturing technology of stereolithog-
Biomedical Engineering at
raphy; this proved to be the pioneering work for future 3D
Brown University in 2009
printing techniques.[28] In 2003, a cellular bioprinting technique
and did her postdoctoral
based on traditional 2D inkjet technology was proposed.[29] In
training at Rice University
2009, Organovo and Invetech created one of the first commer-
and Harvard Medical School.
cial 3D bioprinters.[30,31] Finally in 2016, the Food and Drug
She is the director of the
Administration (FDA) issued draft guidance, titled “Technical
Bioengineering Laboratory for Nanomedicine and Tissue
Considerations for Additive Manufactured Devices”, which
Engineering at GW. Her research interests include 3D/4D
provided guidance for 3D printing techniques and products.[32]
bioprinting, complex tissue engineering, nanobiomaterials,
Currently, with the increasing global interest and need, more
stem cell engineering, drug delivery and breast cancer bone
and more businesses have been established in the expanding
metastasis.
bioprinting market, such as 3D Systems (Rock Hill, SC, USA),
Hewlett-Packard (Palo Alto, CA, USA), Novogen MMX Bio-
printer (Organovo, Inc., San Diego, CA, USA), 3D Bioplotter
(EnvisionTEC, Gladbeck, Germany), Oxford Performance
Materials (South Windsor, CT, USA), and Commercial Blood In this review, we focus on general principles, techniques,
Vessel Bioprinter (Revotek, Sichuan, China) among others.[31] and other essential elements pertaining to the application of
By 2022, the global 3D bioprinting market is expected to reach 3D bioprinting technologies for generating 3D tissues and
$1.82 billion and will include products and materials for dental, organs. We propose a stepwise process of regenerating a com-
medical, analytical, and food applications.[31] Although still in plex tissue/organ, and also present recent advances in 3D bio-
its infancy considering the complexity and functionality, this printing for organ regeneration. Furthermore, we discuss cur-
technology appears to show great promise for advancing tissue rent challenges and exciting opportunities of 3D bioprinting
engineering toward organ fabrication, ultimately mitigating technologies toward creating realistic organs that further funda-
organ shortage and saving lives. mental research and translational medicine (Figure 1).

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REVIEW
Figure 1. Schematic diagram outlining information covered in this review. Reproduced with permission.[139] Copyright 2015, the American Association
for the Advancement of Science.

2. 3D Tissue/Organ Bioprinting and Related further modification before being imported into the printer.
Manufacturing Strategies The slicing program can parse the solid object into a stack of
thin, axial cross sections; each respective 2D cross section is
2.1. Fundamental Principles reproduced integrating various infill patterns, as programmed.
In this step, the printer reads the stl file and deposits succes-
3D bioprinting is basically a rapid prototyping and additive sive layers of liquid, powder, or several other materials to build
manufacturing technique used to fabricate artificial implants the 3D model from a series of 2D cross-sections. Several 3D
or complex tissue constructs through a layer-by-layer building printing techniques are capable of using multiple nozzles (mul-
process for patient-specific therapy. 3D bioprinting shares three tiple materials), adjustable angles, and even multiple printing
basic concepts with ordinary 2D printing – desktop printer combinations.
(3D printer), print file (3D model file), ink (bioink consisting 3D bioprinting for tissue engineering applications can be
of biomaterials, bioactive components and cells), and paper divided into two forms, with and without incorporated living
(print platform). Unlike 2D printing, 3D bioprinting is a com- cells printed directly into the constructs. Cellular bioprinting
prehensive process requiring various design considerations, techniques can directly deposit bioinks with viable cells to form
including imaging, modeling, printer choice, bioink selec- a 3D living structure. Based on the working strategies, they
tion, culture condition, and 3D construct development among can primarily be classified into three categories, droplet-based,
others. Generally, the manufacturing activities can be divided extrusion-based and laser-assisted bioprinting.[20,33] Variations
into three steps: pre-bioprinting (modeling), bioprinting, and in the available bioprinting technologies also affect the charac-
post-bioprinitng. teristics of living tissue/organ constructs. Comparatively, acel-
Pre-bioprinting, also known as modeling, mainly includes lular bioprinting techniques provide more extensive choices for
3D imaging acquisition, digital 3D design and bioink/bioma- tissue regeneration applications.[18] Without the consideration
terial selection based on the type of 3D bioprinting model.[20] of cell viability or bioactive components, several 3D printing
Several imaging technologies, such as 3D scanner, CT, MRI techniques with higher temperatures, chemicals and other
and others, are applied to collect and digitize the complex tomo- harsh environments can be utilized to manufacture implants.[34]
graphic and architectural information of tissues. Giesel et al. Considering the specific requirements of the targeted tissues/
described and discussed the various methods of 3D imaging organs properties, the design must take into account the capa-
technology for 3D printing applications in detail.[25] The desired bilities and properties of the bioprinting systems (both bioinks
structure of digital 3D models is precisely created using CAD and bioprinters), which we discuss next in detail.
software and stored as a stereolithography (stl) files. Bioink or Finally, post-bioprinting, which involves the development
biomaterial selection depends on the specific bioprinter type of biomimetic structures, mechanical supports and biological
and the product properties required. functionality, is an essential step to develop mature tissues/
The 3D structure with patient-specific design is then printed organs for living applications.[35,36] Several additional manu-
in layer-by-layer deposition modeling process in the bio- facturing techniques, including substrate supports and sacrifi-
printing phase. According to the program design of different cial templates, among others, are potentially required to create
printers, the 3D design files can be directly loaded into the higher mechanical elasticity/strengths, more precise structures,
printer, or must first be passed through a slicing program for more complex structures or multiple biological functions due

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REVIEW

Figure 2. (a) A tree-diagram of the various 3D bioprinting techniques and (b) Simplified illustrations of typical 3D bioprinting techniques for tissue/
organ regeneration.

to current printing technique limitation.[16] More importantly, depositing cells onto the constructs after acellular bioprinting.
in vitro culture (preference in a bioreactor), in vivo implanta- In contrast, the direct cellular bioprinting is a one-step process
tion, or even in situ bioprinting will be performed to induce of generating a rapid prototyped tissue by accomplishing both
and enhance construct maturation thereby transforming con- the construct fabrication and cellularization jointly.
structs into functional tissues/organs.[26] Figure 2 shows a
tree-diagram of the various 3D bioprinting techniques with
simplified illustrations of typical 3D bioprinting techniques. 2.2.1. Cellular Bioprinting

Cellular 3D bioprinting directly employs living cells in the con-

2.2. Accustomed Bioprinting Techniques struct fabrication process together with the inherent advantages
of 3D printing-based rapid prototyping. Diverse techniques
Typically, ASTM (F2792) standard terminology for 3D printing have been developed to create 3D living tissue/organ ana-
technologies consists of several parts including vat photopo- logues, and each of them has different features (strengths and
lymerization, material jetting, material extrusion, powder bed limitations) in terms of the available conditions such as bio-
fusion, binder jetting, sheet lamination, and directed energy dep- logical materials, resolution, printing speed and cell viability.
osition.[37] To some extent, the terminology of bioprinting tech- Depending on the printing modality (bioink deposition mecha-
niques more specifically refers to both the bioink formulations nism), the representative techniques of cellular bioprinting can
and printing modality. Acellular bioprinting is divided into two be categorized into three types: droplet-based, extrusion-based,
forms, direct implantation and cell post-seeding. The acellular and stereolithography.[14,20,22,38]
implant serves as a nonliving implant device or artificial graft Droplet-based bioprinting relies on various energy sources
substitute, while the cellular implant often requires an extra step (thermal-, electric-, laser beam-, acoustic- or pneumatic- mechanisms)

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to pattern the bioink micro-droplets of living cells and other printer.[44] Ultrasound parameters, including pulse, duration

REVIEW
biologicals in a high-throughput manner. It offers greater and amplitude, can be adjusted to control the size of droplets
advantages due to its simplicity and agility with precise control and the rate of ejection. The acoustic radiation is capable of
on deposition of biologicals including cells, growth factors, and generating and controlling uniform droplet size and ejection
genes for tissue/organ regeneration. It has also been the most directionality. However, the acoustic frequencies used in these
common for pharmaceutical use due to its simplicity, versatility, printers have the potential to induce damage of the cell mem-
and high-through put capability. brane and cell lysis. Additionally, several modified inkjet tech-
Extrusion-based (dispensing, or direct writing) bioprinting niques with multi-jets have been developed to build complex
which originates from fused deposition modeling (FDM) tissue and organ prototypes by arranging multiple cell types
printing uses pneumatic-, mechanical- or electromagnetic- and other tissue components.[45] Overall, the inkjet bioprinting
driven systems to deposit cells based on a “needle-syringe” technique ensures rapid fabrication with highly repeatable pat-
type. During bioprinting, bioink dispensed by a deposition terns; additionally, small volume droplets enable high printing
system precisely prints cell-laden filaments forming desired 3D resolution (lower to 50 µm). Moreover, inkjet bioprinting typi-
structures. cally exhibits over 80% cell viability after cellular bioprinting.[20]
Stereolithography-based (vat-photopolymrization) bioprinting Inkjet bioprinters do, however, still have limitations on material
mainly utilizes laser energy to deposit cell-laden bioink in a viscosity, cell density and mechanical strength. Devices are typi-
reservoir via beam scanning or image projection modeling, cally compatible with low solution viscosities (below 0.01 Pa•s)
allowing the molding of the high-precision patterns. It offers and low cell concentrations (fewer than 10 million cells/mL),
greater advantages due to the precise control on deposition of avoiding high shear stress and nozzle clogging.[20]
biologicals and high resolution. The electrohydrodynamic jetting (electrospraying or elec-
trospinning) applies an electric potential difference between
2.2.1.1. Droplet-Based Cellular Bioprinting (DCB): The key fea- a positively charged needle and a grounded electrode to gen-
ture of droplet-based bioprinting is that the droplets of cell- erate repulsive Coulombic force. Droplet ejection occurs, in
laden bioink (hydrogels or slurries) are generated and deposited the micrometer to nanometer size range, when the charged
to pre-defined locations on the substrate. As a noncontact bio- medium exiting the needle enters the high-intensity electric
printing technique, it provides a high-throughput method for field.[39,46,47] The size and distribution of these droplets can be
depositing multiple cells or biologicals in small droplets onto controlled through the applied potential difference, the flow
a targeted spatial position. Droplet techniques can be clas- rate to the needle, the distance of electrodes and the liquid solu-
sified into four categories: inkjet, electrohydrodynamic jet- tion properties.[48] Inkjet bioprinting dimensions are currently
ting, pneumatic pressure assisted-, and laser assisted- droplet limited by the diameter of the jetting needle. Typically, droplet
bioprinting.[39–42] diameter is approximately two times the size of the needle
The inkjet bioprinting, which is granted with the earliest cel- diameter; as such, this technology has limitations in the size
lular printing patent, is originated from commercial 2D inkjet range of tens of nanometers. In contrast to inkjet technology,
printing.[29] The necessary equipment is easily remolded from electrohydrodynamic jetting does not suffer from these limi-
2D inkjet desktop printers making this technology widely avail- tations and can be used to process concentrated suspensions
able and relatively inexpensive. In this technique, the bioink from needles that are a few hundred micrometers in size yet
solution including biomaterial, bioactive factors, and cells is are capable of generating droplet deposits a few micrometers
stored in a cartridge or reservoir, and then transferred to the ink in size and smaller. Furthermore, no adverse effects on cell via-
chamber for droplet ejection. The droplets can be generated by bility have been observed when jetting the cell-laden bioinks.[49]
two mechanisms, thermal or piezoelectric actuation, which can The pneumatic pressure technique uses a set of electro-
be ejected from the inkjet-head nozzle to the print surface.[43] mechanical micro-valves where the droplets are produced by
They operate similar to the traditional “drop-on-demand” 2D opening the micro-valve under constant pneumatic pressure.[42]
inkjet printers. The thermal actuation is based on a heating This technique uses various types of liquid biomaterials with
element, which can superheat the bioink to create vapor bub- viscosities of up to 200 Pa•s, and controls the droplet volume by
bles for ejecting the droplets. Although the temperature reaches adjusting the pressure to the fluidic pathway and valve gating
200∼300 °C, the process only persists for a few microseconds time. Although a higher liquid viscosity can be applied, there
(∼2 µs) resulting in an overall temperature rise of ∼10 °C in the remain several concerns regarding droplet controls and cell via-
printer head. Many results have demonstrated that this increase bility. In order to obtain a favorable printing structure, the effect
in temperature causes minimal damage to the viability of both of the printing conditions must be fully considered to include
printed cells and other integrated biologicals. The piezoelectric substrate stiffness, material preparation, droplet size, printing
technique employs a voltage to induce a rapid shape change speed, surfactant usage, and agitation among others.[22]
of the piezoelectric material, which generates a pressure pulse However, for the inkjet and pneumatic pressure assisted
in the fluid forcing a droplet of ink from the nozzle. Droplet bioprinting, the viscosity of the available bioinks is too low to
shape and size can be adjusted by tuning the applied voltage to facilitate the rapid generation and sustainment of 3D struc-
the piezoelectric material. It allows a wider variety of inks than tures. Therefore, additional cross-linking methods are applied
thermal inkjets as there is no requirement for a volatile com- to address this limitation, including UV/Vis light, pH, tem-
ponent, and no issue with coagulation. The acoustic radiation perature or chemical reagents. The photocurable materials
force associated with the ultrasound field is also utilized to eject can be independently used, associated as the droplet bioinks,
the droplets from an air-liquid interface on the piezoelectric or deposited as supporting materials to assist in 3D molding.

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Additionally, multi-jet bioprinting strategies can be used to co- (or direct writing) provides a simpler and more direct method
REVIEW

print the bioinks and the cross-linkers in turn, allowing the of controlling the bioink printing.[51–53] The piston system
generation of a solid, stable 3D structure. The crosslinking commonly composed of syringes and needles is suitable to a
procedure may slow down the printing process, and several fluid with low viscosity, whereas the screw system is capable
crosslinking methods may result in excessive damage of cell of generating a larger pressure for dispensing the bioinks with
viability and biological functionality; these risks must be con- higher viscosities.[50] However, a large shearing force along the
sidered and managed carefully during fabrication. nozzle in mechanical micro-extrusion can potentially harm the
In addition to the high resolution, simple processing, and laden cells. Solenoid (or electromagnetic driven) microextru-
low cost benefits of these bioprinting techniques, another sion applies electrical pulses to open a valve by canceling the
advantage is the potential to introduce concentration gradients magnetic pull force generated between a floating ferro-mag-
of cells, materials or growth factors throughout the 3D struc- netic plunger and a ferro-magnetic ring magnet.[42] Mechanical
ture by altering droplet densities or sizes. Recent developments dispensing systems might provide more direct control over
of this technique have reported controlling droplet sizes and the material flow, because of the delay of the compressed gas
deposition rates ranging from 1 pL to 300 pL in volume with volume in pneumatic systems and the high complexity of elec-
up to 10 000 droplets/s. Droplet bioprinting also shows great tromagnetic driven systems. Materials with viscosities ranging
promise for “scaffold-free” bioprinting by depositing layers of from 30 to >6 × 107 mPa•s have been shown to be compatible
cells into a sacrificial mold. with microextrusion bioprinters, with higher-viscosity materials
Besides the three aforementioned droplet techniques, laser- often providing structural support for the printed construct and
assisted droplet systems have also been developed.[40] Differing lower-viscosity materials providing a suitable environment for
from several other reviews that listed it as a laser-assisted bio- maintaining cell viability and function.[52]
printing technique individually, we combine it into droplet In addition to dispensing systems, the extrusion printers
bioprinting because of some similarities. Laser-assisted droplet include a stage and one or more cartridges (i.e., syringes or
bioprinting (also known as laser-induced forward transfer or pens) that can be loaded with cell-laden bioinks or other bio-
LIFT), consists of a pulsed laser source, a donor layer (this logicals for printing. The materials inside the cartridges may be
includes a laser-energy-absorbing layer, such as gold or tita- dispensed using a microextrusion system. The printing process
nium, and a bioink layer) and a receiving substrate. It utilizes a can be controlled by the dispensing procedure, speed, nozzle
focused laser to pulse on the absorbing layer generating a high- size, the displacement of the cartridge, and/or the stage motion
pressure bubble that propels cell-laden droplets onto the sub- in x, y, and z axes. Moreover, several advanced techniques
strate. The absorbing layer is used to transfer heat for bioink have been developed for the cartridges and the stages such
droplet production; this prevents from direct laser. The droplet as: temperature-controlled cartridge (nozzle) or stage systems,
volume can be controlled from 10 to 7000 pL by adjusting the multiple independently controlled nozzles or chambers,
viscosity and thickness of the bioink layer contributing to a multiple direction-controlled nozzle or stage systems, and
higher printing resolution. The resolution is also influenced by coaxial nozzle systems, among others.[20,50,54]
many additional factors, including the laser fluence, the surface Compared to droplet-based bioprinting, extrusion-based bio-
tension, the wettability of the substrate, the air gap between printing enables rapid printing, easy operation and a wide selec-
the donor layer and the substrate, and the thickness and vis- tion of bioinks, including cell aggregates, cell-laden hydrogels,
cosity of the bioink layer. Moreover, this technique is capable of micro-carriers, decellularized matrices and synthetic polymer
employing a high cell densities (up to 108 cells/mL) and as well fibers. Synthetic polymers that have relatively high mechanical
as high bioink viscosity (1∼300 mPa•s) because of its nozzle- strength are often employed to reinforce printed 3D tissue/
free droplet model. Despite these advantages, a relatively low organ analogues. There are two main types of bioinks used in
efficiency, high cost and limited availability of bioinks for this microextusion systems.[50] The first is high-viscosity, cell-laden
technique are still major concerns. solutions or low-modulus cell-laden hydrogels, which need be
rapidly solidified into a 3D construct after extrusion. However,
2.2.1.2. Extrusion-Based Cellular Bioprinting (ECB): Extrusion- the printing conditions of the cell-laden hydrogel are somewhat
based (or dispensing, direct writing) bioprinting is an integrated limited by high shear force management. Another type involves
technique consisting of a fluid-dispensing system for extrusion using spherical and cylindrical multicellular systems with or
control and an automated robotic system for bioprinting.[20,22] without supportive biomaterials as a bioink; cell spheroids and
The bioink is extruded into the manner of cell-laden cylindrical cell-laden microcarriers are two examples of this type of bioink.
filaments or discrete volumes of bioinks that can be precisely After printing, the multicellular systems fuse together to repli-
deposited into the desired 3D structures. Continuous deposi- cate the 3D tissue structure. This technique directly prints solid
tion provides better structural integrity during rapid fabrica- cellular units enabling scaffold-free bioprinting, or printing
tion. Dispensing systems can be classified into three types: free of exogenous biomaterials. In order to obtain appropriate
pneumatic-, mechanical- (piston or screw), and solenoid-based mechanical integrity of a 3D configuration, the molding process
microextrusion.[50] and the properties of bioinks, as well as their interactions, are
Pneumatic-based systems utilize pressurized air to extrude very important considerations for extrusion printing and must
filaments using a valve-free or a valve-based configuration. be addressed in the experimental design.[50] The typical molding
Compared to the valve-free configuration, the valve-based processes include: (1) self-assembly (i.e., shear-thinning mate-
configuration possesses a higher precision due to a controlled rials, self-healing materials), (2) crosslinking agent integration
pressure and pulse frequency.[42] Mechanical micro-extrusion (i.e., pre-crosslinked bioink, bioplotting, coaxial crosslinking,

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aerosol crosslinking or spraying crosslinking system), (3) UV/ face of the photocurable bioinks using a digital light procession

REVIEW
Vis photocuring and (4) environmentally sensitive deposition technique (DLP), which can solidify an entire 2D patterned
(pH, temperature, and others). In extrusion based bioprinting, layer simultaneously.[22,37,57,58] The DLP system uses a digital
bioplotting refers to syringe dispensing system requiring a micromirror device (DMD) to project a set of 2D images from
curing process involving additional solidification over time.[50,55] the horizontally sliced 3D structure. Compared to the beam-
Therefore, we do not separately introduce it as a bioprinting scanning technique, mask-image-projection printing can be
technique. In the bioplotting approach, cell-laden bioinks are much faster due to its ability to simultaneously form the shape
directly extruded into a plotting medium (crosslinking pool) of an entire layer.
to complete the curing process. It requires the use of relatively There is a limited choice of photopolymerizable bioinks,
viscous bioinks printed into plotting medium that can sup- however polymer modification can technically enable more
port the extruded structures temporarily until crosslinking is options.[58] The commonly photocurable bioinks include poly-
complete.[56] ethylene glycol acrylate/methacrylate and its derivatives, meth-
Overall, extrusion-based techniques are capable of greater acrylated/acrylated natural biomaterials (gelatin, hyaluronic
deposition and printing speed and have more tolerance for het- acid, dextran, and others), and methacrylated/acrylated capped
erogeneous formulations, allowing physiologically relevant cell among other synthetic polymers. Overall, the main advantages
densities, which facilitate scalability in a relatively short period of sterelithography-based bioprinting techniques are their
of time. Despite its versatility and great benefits, extrusion-based ability to simply fabricate complex designs with high resolu-
bioprinting still has several challenges mainly involving lower tion and rapidly print constructs without support material.
resolutions, higher shear stresses, and limited material selection Most commercial systems prepare structures with low to 50 µm
among others. The minimum feature size of the technology is features; µSLA systems are capable of preparing structures
generally over 100 µm; nonbiological microextrusion printers with <5 µm features.[57] However, the photopolymerization is
are capable of 5 µm resolution.[20,50] Bioinks should possess driven by a radically induced chemical reaction, and the free
shear thinning ability to overcome surface tension to extrude radicals can damage the cell membrane, proteins, and nucleic
in filament form. The resulting high shear stress at the nozzle acids. This technique can achieve up to 40∼80% cell viability
may decrease the cell viability. Cell viability after microextrusion depending on the laser wavelength, power, exposure time and
bioprinting is typically lower than that with inkjet-based bio- toxicity of photo-intiator.[22,57] Therefore, it is important to apply
printing; cell survival rates are in the range of 40∼86%, control- a cytocompatible photo-initiator. Additionally, the limited avail-
lable by changing extrusion speed and nozzle gauge.[50] ability of photocurable biomaterials and high equipment costs
are major concerns with this technology.
2.2.1.3. Sterelithography-Based Cellular Bioprinting (SCB): Stere-
lithography appearance (SLA) offers an additive manufacturing
technique with very high resolution and accuracy.[20,22,57] The 2.2.2. Acellular Bioprinting
Sterelithography-based bioprinting technique (vat photopoly-
merization) utilizes the spatially controlled irradiation of light Compared to cellular bioprinting techniques, acellular 3D bio-
or laser to solidify a geometrically 2D pattern layered through printing provides more extensive choices for material selection
selective photopolymerization in the bioink reservoir. The 3D and manufacturing method. The aforementioned cellular bio-
structure can be consecutively built on 2D patterned layers in printing techniques can also employ acellular bioinks to fabricate
a “layer-by-layer” fashion, and the uncured bioink can be easily tissue engineered scaffolds. An additional cell seeding technique
removed from the final product. The photo-polymerization of can be employed to create artificial 3D cell-laden scaffolds for
2D patterned layers is the most crucial step in SLA-based bio- tissue/organ regeneration after printing. Here, a universal cell
printing. Traditional SLA-based bioprinting techniques have two seeding procedure can be used in a post-seeding process, or per-
types: beaming-scanning and mask-image-projection.[22,24,58] fusable cell seeding can be obtained using a bioreactor device.
The beam-scanning technique, or laser direct writing (LDW), Also 3D printed grafts without cells can be directly implanted
uses a laser beam to scan photocurable bioinks for solidifica- into injured patients for functional replacement or structural
tion of a 2D patterned layer.[59] The resolution is dependent support during healing. The representative techniques of acel-
on irradiant exposure conditions (laser spot size, wavelength, lular bioprinting fall into two categories: extrusions-based acel-
power, exposure time/velocity and the occurrence of absorp- lular bioprinting (EAB) or laser-based acellular bioprinting (LAB).
tion or scattering of the laser beam), and the selection of photo-
initiator or any UV absorbers.[60,61] The types and concentra- 2.2.2.1. Extrusion-Based Acellular Bioprinting (EAB): Unlike the
tion of bioinks, scanning speed and laser power contribute to previously presented extrusion systems focusing on cellular
the overall mechanical properties of the bioprinted structure. bioprinting, acellular extrusion (or acellular direct writing) can
Additionally, when printing multiple layers, early layers may utilize volatile or easily displaced organic solvents to dissolve
be repeatedly exposed to the laser, causing uneven mechanical polymers, followed by conversion from a highly viscous solu-
strength or undesired 3D structures/patterns. With the develop- tion to solid 3D structures.[51,64] After removing the organic
ment of micro-stereolithography (µSLA) techniques, a resolu- solvent thoroughly, the cells can be seeded and grown on the
tion of about 5 µm in the x/y plane and 10 µm in the z axis can scaffold’s surface for tissue/organ regeneration.
be achieved.[62,63] Fused deposition modeling (FDM) or fused filament fabri-
The mask-image-projection printing system dynamically cation (FFF) was developed in the early 1990s and is a major
generates a defined mask image that is projected onto the sur- acellular, extrusion-based system.[18,21,34,37] It is the most widely

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used and generally well-explored 3D printing strategy because may be printed using several material types such as ceramics,
REVIEW

it is low-cost and relatively fast. This technique employs ther- metals, polymers and their composites.[68] Printing parameters,
moplastic filaments that are heated to their melting point or to such as energy source, particle size, particle shape, free packing
a semimolten state, passed through an extrusion nozzle and density, and thermodynamic variations of materials play crit-
allowed it to solidify on the printing stage without any addi- ical roles in the fabrication process. The resolution of the dif-
tional crosslinking requirment.[18,65] This method is analogous ferent SLS machines usually ranges from 20 to 100 µm; this is
to conventional extrusion or injection molding except molds achieved and manipulated through a careful balance between
are not used. Multiple print heads can be accommodated to therefore we need to consider a balance between obtaining fine
permit co-printing of temporary support material for complex resolution and allowing for adequate powder dispensability.[68]
overhanging structures or multiple material integration with The un-sintered powder serves as the physical support during
different properties within a single structure for complicated 3D manufacturing, and unused powders may be removed or
construct fabrication. The printer is composed of heating blocks recycled after bioprinting. For polymer powder sintering, the
with temperature controllers, an extrusion block and motors.[58] laser parameters of power, beam size, scanning speed and
The extrusion force is driven pneumatically or mechanically spacing needs to be carefully controlled to avoid polymer degra-
with a lead screw. These models result in an overall resolution dation by overheating.[68]
of >50 µm in layer height and an accuracy of >100 µm.[18] The Selective laser sintering is applied to rapid scaffold proto-
main advantages of the FDM method in tissue engineering typing in much the same fashion as in the industrial fabrication
applications are its simple employment, rapid printing capa- of metal or plastic components. The main advantages of this
bility, diverse synthetic biomaterial availability, and favorable process for tissue engineering applications are the wide range
mechanical properties make it suitable for hard tissue regenera- of available biomaterials. Specifically, ceramics and metals are
tion applications. This technique also eliminates the need for suitable to the fabrication of hard bone replacements or struc-
solvent submersion and has the ability to fabricate large-format tural-supporting materials.[68] Moreover, the powders used in
objects positively impacting scalability. Several synthetic bioma- this technique are more readily available than FDM materials,
terials such as poly(caprolactone) (PCL), poly(lactic acid) (PLA), which are limited by filament prefabrication. Compared to
polyurethane, and their derivatives have demonstrated adequate other 3D printing techniques, the SLS is more expensive, cum-
thermoplastic performance and biocompatibility.[58] Any bioma- bersome, and provides low resolutions for tough, stiff grafts.
terials that can be melted and then re-solidified or thermally Additionally, material oxidation, thermal degradation, material
cross-linked are suitable for FDM printing.[66] Exploiting low- shrinkage, and crystallinity change are concerns about material
temperature thermoplastic biomaterials is preferable in that properties affected by the heating process that must be consid-
biologicals can be added though more mild processes after bio- ered in fabrication.[69] Additionally, a range of fillers can also
printing, preserving their functionality. Moreover, in order to be incorporated into the powder to further modify the appear-
offer a higher and more uniform strength between each layer, ance and properties of the printed parts. Being analogous to the
a conversion from thermoplastic material to thermoset material SLS approach, a technology known as selective heat sintering
can be conducted via an additional crosslinking reaction using (SHS) utilizes a thermal print head rather than a laser to fuse
ionizing radiation or novel material design.[67] The disadvan- the surface of powdered thermoplastic materials into patterned,
tages are the limited material selection related to thermoplastic layered structures.[31]
polymers and it is not suitable for printing with cells due to
the high manufacturing temperature. Therefore, an extra step
is required after the FDM printing to seed cells on constructs 2.2.3. Recent Developments in Bioprinting Techniques
for tissue/organ regeneration.
A recent development in SLA-based 3D printing involves con-
2.2.2.2. Laser-Based Acellular Bioprinting (LAB): Sterelithog- tinuous liquid interface production (CLIP), which is facilitated
raphy techniques can also be applied for fabrication of acellular through a well-controlled oxygen inhibited dead-zone (per-
scaffolds. In these cases, however, more photocuring resins and sistent liquid interface) preventing the resin from attaching
crosslinking conditions are available since acellular constructs to the UV window.[21,58,70] Traditional SLA techniques use the
eliminate the concern of cell damage during the printing pro- bottom-up building approach and therefore require slow solidi-
cess. The increase in material selection is beneficial in that fication to inhibit the adhesion process. CLIP, on the other
it allows for more diverse scaffold properties. Selective laser hand, employs an oxygen-permeable curing window (a thin,
sintering (SLS) is another laser based printing technique that amorphous Teflon film) below the UV image projection plane
uses a high power laser for powder sintering, forming solid 3D to create an oxygen-containing zone between the solid part and
structures on the surface of a powder bed.[21,31,68] The technique the liquid precursor where solidification cannot occur.[58] The
relies on two energy sources, a bed-heater and a high-power rate of resin replenishment in this dead-zone, the initiation
laser. First, the particles are preheated between their melting efficiency, and the resin reactivity all combine to determine the
transition and the temperature necessary for recrystallization rate at which the part can be formed in a continuous, rather
during the cooling cycle. Localized thermal sintering of the par- than layer-by-layer, fashion. This approach allows 3D constructs
ticles is achieved by the controlled additional energy input of production in minutes instead of the hours required with tra-
the a high-power laser, which traces the 2D layer design fusing ditional SLA, and generates structures tens of centimeters in
exposed particles together within the layer as well as connecting size that could contain features with resolutions below 100 µm.
it to the previously scanned underlying layer.[37,68] This process The choice of photocuring resins is fairly broad in CLIP; the

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viscosity and reactivity of the monomers, however, are more tion mechanism and printing modality as discussed above.[77]

REVIEW
critical since they affect the oxygen diffusion within the resin The bioink material is crucial because it should provide the
affecting the permeable curing window.[70] spectrum of biochemical (i.e., chemokines, growth factors,
3D powder printing is a powder-based 3D bioprinting tech- adhesion factors, or signaling proteins) and physical (i.e., inter-
nique that has been developed based on the principles of stitial flow, mechanical and structural properties of extracellular
SLS.[31,71–73] This technology uses a binder solution, such as matrix) cues which promote a favorable environment for cell
water, citric acid or phosphoric acid, among others, to selectively survival, motility, and differentiation.[10] Strategies for bioink
bind the loose powdered biomaterial together in the designed selection can be divided into two categories: functional scaf-
geometry. Available biomaterials for binder integration include fold bioprinting (biomaterials with/without cells as the printing
starch, dextran, gelatin, calcium phosphates, and hydroxyapatite ink) or scaffold-free bioprinting (only use cells as the printing
among others.[31,71,74] This process is reasonably inexpensive ink).[20] In this section, we focus on the design strategies of bio-
compared with other modalities, and provides more options materials for functional scaffold bioprinting; scaffold-free bio-
for tissue engineering and drug-delivery because it avoids the printing will be introduced in detail in section 3.5.
damage of incorporated bioactive components. A major limi- Generally, biomaterials range from cell supportive soft
tation of this system is the difficulty in removing unbound hydrogels, to stiff metal or ceramic implants and from nano-
powder from desired hollow spaces. In addition, the usage of particles and quantum dots for drug delivery and imaging, to
aqueous binding agents exhibits limited mechanical strength complex functioning medical devices.[16,50,78–80] In tissue engi-
and resolution, and requires further post-processing. This tech- neering, the scaffolds fabricated by biomaterials serve as extra-
nique is also difficult for direct depositing or patterning living cellular matrix (ECM) biomimetic structures that organize the
cells. Several researchers are also using the inkjet printer to tissue regeneration, temporary substitutes for tissue functions,
eject the binder droplet. This concept is closer to the traditional and guides for regenerating tissue ingrowth or integration
2D inkjet technique where the powder bed acts like the paper.[31] within a host tissue. Some basic elements including porosity,
A nano-stereolithography technique, also called two-photon interconnectivity, pore dimensions, internal geometry, biodeg-
polymerization printing (TPP), is used to photocure the liquid radation kinetics, mechanical properties, and biocompatibility
polymers by simultaneous two-photon absorption.[75,76] Unlike are also taken into account in the scaffold manufacturing pro-
the single photon polymerization process in SLA, two-photon cess. Therefore, material science and/or engineering play cru-
polymerization allows electron transitions over excited energy cial roles in programming an active and effective building block
levels; the polymerization process occurs when an atom for tissue formation.
absorbs two photons simultaneously. More specifically, a spe-
cific photoinitiator that reacts at low wavelengths simultane-
ously absorbs two photons with high wavelengths, their ener- 3.1.1. Design Principles
gies combine to achieve the energy of one photon with low
wavelength and thus initiate the polymerization process.[76] The The design principles can be combined into four major con-
photopolymerization that is triggered by nonlinear excitation siderations for selection (Figure 3):[22,38,50,52,75,81] (1) Biomate-
happens at the focal point, but other regions are not affected rials must have suitable properties to meet specific bioprinter
by the laser energy. So it has the potential to print precise 3D deposition requirements (printability). Printability refers to
structure with very high resolution, and even enable 3D con- the capability of the material to support manufacturing and
struct printing inside the photocuring material solution without rapid solidification, the printability and interrelation of bioinks
affecting other regions. This technique can achieve spatial in various bioprinters and on associated substrates must be
solidification with a resolution of up to 100 nm.[22] By exploiting evaluated carefully to produce accurate, high-quality patterns. (2)
the high resolution of this technique, many researchers have Biomaterials must possess suitable physicochemical properties,
focused on the realization of 3D environments for cell adhesion including wetting/swelling, internal and external structure char-
and proliferation. Two-photon polymerization printing is an acteristics range from nano- to macro-scale, degradation kinetics,
improvement, but the process and cost of materials often limits mechanical strength and structural stability. (3) Biocompatibility
products to a small scale. and biological activity are necessary for tissue development and
remodeling over long-term in vivo implantation. The bioink
material should facilitate engraftment with the endogenous
3. Material/Cells in 3D Bioprinting: From Bioink to tissue without generating an immune response and provide a
spectrum of biochemical cues (i.e., chemokines, growth factors,
Modular Building Blocks
adhesion factors, or signaling proteins) that promote an environ-
3.1. Design and Selection Principles of Bioink ment for cell survival, motility, and differentiation. (4) The mate-
rials should be affordable, abundant, and commercially available
In addition to the bioprinting techniques chosen for the tar- with appropriate regulations for clinical use.
geted tissue requirements, appropriate bioink selection,
including cells, biomaterials and biochemical signals, is neces-
sary for the successful construct fabrication. As printing and 3.1.2. Bioink Formulations
fabrication depend on the solidifying kinetics of the biomate-
rials and the native, chemically, or environmentally induced Acellular 3D bioprinting technologies, including the deposition
material properties, specific concerns arise based on the deposi- of metals, ceramics and thermoplastic polymers, generally

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REVIEW

Figure 3. Design of bioinks for 3D bioprinting, including design principles, formulations, solidification mechanisms and bio-functionalization.

involve the use of organic solvents, high temperatures, seeded onto the printed constructs after fabrication, avoiding
crosslinking agents or other severe process conditions. As such, conditions harmful to the cells. A dynamic cell seeding method
an extra post-process step such as purification, sterilization, or is often utilized to improve scaffold coverage. The intercon-
other modification is necessary for further biomedical applica- nectivity of pores allows for uniform cell distribution. (2) Soft
tion.[21,78] In contrast, cellular bioprinting requires biocompat- biomaterials such as hydrogels, comprised of synthetic or nat-
ible fabrication processes and biomaterials during printing, ural polymers, possess biomimetic characteristics, providing
ensuring cell viability and development throughout. There- a favorable environment for cells. The cellular bioprinting
fore, Food and Drug Administration (FDA)-approved bioma- technologies currently available are only capable of dispensing
terials are preferred in these applications; the biocompatibility liquid materials or hydrogels (they should be in liquid or paste-
evaluation for newly developed materials and their degradation like form during printing). In order to better mimic the prop-
byproducts needs to be performed in vitro or/and in vivo before erties of natural ECMs, many biomaterial combinations have
gaining approval. been designed for cell printing mimicking the mechanical
In general, the printable biomaterials are divided into two properties and bioactivity of native tissue. Moreover, the more
categories:[22,38,50] (1) Hard biomaterials, such as metals, specific and complex printable materials are steadily being
ceramics, and curing (thermoplastic) polymers. They can developed to match desirable traits for a variety of biomedical
fabricate mechanically robust and durable constructs. These application.
materials typically require high temperatures or toxic sol- In addition to the components of bioink, the resultant forma-
vents to facilitate printing, so that they are not appropriate tion is also an important factor. For example, extrusion-based
for printing together with cells. Therefore, cells are usually and SLA-based bioprinting are very versatile in depositing a

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wide array of bioink types, including hydrogels, microcarriers, to be considered so that deformation of the final construct can be

REVIEW
tissue spheroids, cell pellet, tissue strands and decellularized prevented via the proper selection of bio-ink type.
matrix components. Extrusion-based bioprinting also is capable
of depositing small building blocks in a fugitive liquid delivery
medium, remains flexible in nozzle tip design, and has the 3.1.4. Bio-Functionalization
ability to extrude bioink in near solid state, due to larger nozzle
diameter ranges.[50] An ideal scaffold should possess excellent bioactivity for
regulating cell events. Directly encapsulating growth factors
or cytokines into bioinks is a simple way of regulating cel-
3.1.3. Solidification Mechanisms lular behaviors through diffuse release after bioprinting.[82]
Several conventional approaches can also be used to modify
Some general types of curing approaches are described in the printable biomaterials such as incorporation with bioac-
Figure 2.[38,50,52,81] The curing methods of hard biomaterials can tive factors, enzymatic recognition sites, and adhesion factors
be easily understood. (1) The melt-deposition is based on phase among others.[12,83] The bulk modification before or during the
transition of associated materials around their melting point. (2) printing process may affect the physicochemical properties of
The solution-deposition is mainly used in curable polymers. The the resultant scaffolds, while post-processing surface modifica-
proper organic solvents (volatile or exchangeable) need be chosen tion on printed scaffolds only changes the interactions between
to dissolve the different polymers. For soft biomaterials, cell- cell/tissue and material surface.[66,84–86] Based on the 3D printed
laden hydrogel solutions are the most typical or universal bioinks. biomimetic spatial structure, incorporating bioactive compo-
Various key properties such as concentration, molecular weight, nents into constructs provides the proper spatial distribution
viscosity, gelation kinetics, and stiffness are important determi- of biochemical cues for guiding tissue formation and remod-
nants. The solidification (or gelation) mechanisms include phys- eling.[82,87] Herein, the surface modification involving physical
ical crosslinking and covalent crosslinking. As one of the typical adsorption or chemical conjunction has been widely utilized to
physical crosslinking methods, a phase transition from sol to gel increase cell attachment, proliferation and regulate cell differ-
state can be controlled by the printing environment change (var- entiation by means of interacting with cellular surface ligands
ious external stimuli) such as temperatures, pH, or others. The and/or modulating the signaling pathways.[6,86,88] Moreover,
ionically cross-linked network is formed via multivalent counte- the presence of nanoscale features also affects cell adhesion,
rions, however, these ions could be leached out or exchanged by cell orientation, cell motility, and cytoskeletal assembly.[8,11]
other ionic molecules in long term culture, compromising the Other techniques such as microspheres or hydrogels can be
control over the construct properties. Other physical interactions combined into 3D bioprinted scaffolds to achieve efficiently
such as hydrophobic, electrostatic, hydrogen bond, or inclusion sustained release of various bioactive factors.[89] The strategies
complex can employ the solidification of 3D structures, how- to engineer biomaterials with specific physiological functions
ever, their weak mechanical strength limits their application. requires a comprehensive understanding of the complex bio-
Therefore, covalent network formation is preferred in order to logical mechanisms of the regeneration process, involving the
enhance the mechanical stability. In general, the radical based natural tissue-specific composition, the localization of bioac-
cross-linking can be induced by light, redox, and temperature; the tive components in ECM, and the complex cascade of signaling
non-radical crosslinking methods involve Michael addition, enzy- pathways in normal physiological events.
matic, glutaraldehyde, carbodiimide, and genipin among others, To date, the exploration of new biomaterials for tissue/organ
so the toxicity of the crosslinking agents should be considered. bioprinting is still underway. Emphasis should be given to
In the light-induced deposition or solidification strategies, the those printed scaffolds that play a significant role in cell sur-
photoinitiators or photosensitizers are commonly applied to ini- vival, proliferation, migration and differentiation during and
tiate the radical crosslinking reaction of monomers and/or pre- after bioprinting processes. They also must possess the prin-
polymer solutions, such as D-p-chromophore (known for its high cipal means of mechanical support and biochemical signals
sensitivity in 2PP processes), Irgacure 2959 (I2959; high biocom- for the long term tissue regeneration. Due to the limitations of
patible initiator and UV working range), lithium acylphosphi- material properties relative to specific printing techniques, 3D
nate salt (LAP, biocompatible initiator and visible light working constructs with complex structures and characteristics were dif-
range), VA-086 (high biocompatible initiator and visible light ficult to realize.[7,20,65] Therefore, multiple printing techniques
working range), and camphorquinone (CQ; an initiator with or material systems can be integrated into a 3D construct by
many dental applications and visible light working range) among choosing the proper printer and materials with appropriate
others. Some chemical crosslinking reactions are too slow to printability. Combining 3D bioprinting platforms and tech-
support 3D structures during rapid printing, thus multiple-step niques has been proven to be an effective alternative, especially
crosslinking offers a better choice. Normally, the printing tem- for complex organ manufacturing.[87]
peratures of cells encapsulated in the hydrogels should be around The inherent characteristics of these different printing mate-
the physiological temperature in order to avoid ice nucleation or rials, including solubility, viscosities, melting points, mechan-
overheating, which are harmful to cells. In vivo stabilities, perme- ical properties, and available chemistries for crosslinking and
ability, compatibilities, and degradation rates of polymer hydro- functionalization are responsible for the overall success of the
gels should be seriously considered before the 3D constructs can design. More importantly, the customized approaches described
be implanted, particularly for soft tissues or organs. In addition, above will provide potential strategies for creating versatile
the swelling and contraction characteristics of the bio-inks have materials to support successful bioprinting.

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biocompatible and biodegradable polymers used with FDM

REVIEW

3.2. Common Bioinks and Recent Developments

because of their proper melting temperatures and good solu-
Currently, most research focuses on the development of new bility allowing easy printing and processing.[78,79] In addition
printing techniques, the update of printing parameters (reso- to being used for fabricating tissue engineering scaffolds, they
lution, speed or others) or the bio-application of printed con- can also be used as a 3D structural support for cell-laden soft
structs instead of exploring new printable materials and their materials in the printed constructs. PCL, however, exhibits
functionalization. Therefore, the lack of variety in ideal, print- very slow degradation, due to its semicrystalline structure,
able biomaterials remains a major challenge. The different hydrophobicity, and low water absorption capacity. Poly(lactic-
printing techniques have specific requirements for the proper- co-glycolide) (PLGA) is another material that is ideal for extru-
ties of bioinks as we discussed above. In this section, we will sion bioprinting; degradation can be controlled by adjusting the
briefly cover the traditional and universal biomaterials used in polymerization ratio between the lactide and glycolide groups,
bioprinting, and will further discuss the development of new another favorable feature of this material. Poly(3-hydroxybu-
printable biomaterials. tyrate) (PHB) is a natural thermoplastic polyester produced by
microorganisms that has also attracted attention for 3D bio-
printing applications. Acrylonitrile-Butadiene-Styrene copol-
3.2.1. Hard Biomaterials (Metals, Ceramics, and ymer (ABS) is not widely used in medical devices in compar-
Thermoplastic Polymers) ison to biomaterials such as PCL and PLA which offer greater
native biocompatibility. Although surface modification has been
In the field of tissue engineering, hard biomaterials are derived applied to engineer hydrophilicity and enable biocompatibility,
either from natural or synthesized materials, containing in it shows limited promise for biomedical application. Variations
metallic components, polymers, ceramics or composites.[21,31,78] in the copolymer structure and polymer concentration enable
Metals have been used clinically for bone replacement or the tailoring of mechanical properties for the scaffolds.
repair in the biomedical community because of their high
mechanical strength and in vivo safety.[21] Common metals
such as stainless steel, titanium, and certain alloys have been 3.2.2. Soft Biomaterials (Hydrogels)
studied in 3D printing as well, and some have progressed to
clinical trials.[68,69] However, limited 3D metal printing tech- With the exception of the stiffest tissue types such as bone and
niques, metal corrosion and ageing, and potential toxicity of teeth, hydrogels can recapitulate a range of elastic modulus
metal ions are serious considerations being further evaluated values through manipulation of chemistry, crosslinking density,
for long term implantation. Biodegradable implants are pre- and polymer concentration, thus mimicking the elastic moduli
ferred for tissue regeneration, thus a “biodegradable metal” of most soft tissues in the body.[14,50,94] Soft biomaterials mainly
concept has been proposed.[90] Some magnesium-based, iron- used in the cellular bioprinting techniques are predominantly
based, zinc-based, or other biodegradable metal-based com- based on either naturally occurring polymers and their deriva-
posites, which consist of the pure metals themselves, alloys, or tives (including alginate, gelatin, collagen, chitosan, fibrin and
metal matrix composites have been reported.[90] The favorable hyaluronic acid, often isolated from animal or human tissue) or
biocompatibility, suitable degradation rates, and completed synthetic materials (polyethylene glycol (PEG), Pluronic F127
metabolism can be observed in studies either in vitro, or in and their derivate copolymers, includes polyesters, polypeptides
vivo. As the availability of biodegradable metals increase, more or others).[21,65,79,81] The advantages of natural polymers are
comprehensive research is needed before further clinical appli- their similarity to human ECM, and their inherent bioactivity,
cations can progress. however, they are limited by immunogenicity, weak mechan-
Ceramics and glasses are widely used as biocompatible ical strengths and a lack of control in composition/molecular
materials for dental, joint and bone implantation due to their weight. The advantage of synthetic polymers is that they can
mineralization abilities.[72,91] Therefore, bioceramics containing be tailored with specific physical properties in terms of tissue
both metallic and nonmetallic elements are applied in the 3D response such as specific molecular weight, chemical structure,
bioprinting field ranging from ceramic oxides (inert in the composition and functional group chemistry as well as bioac-
body) to resorbable materials (eventually replaced by regener- tive anchored sites (i.e., adhesion motif or enzyme degradation
ated tissue).[92] Hydroxyapatite (HA) is a primary component sites) to suit particular applications.[50]
in human teeth and bones. It, along with its analogues tri- As the synthetic polymers, both polyethylene glycol PEG and
calcium phosphate (TCP) and calcium phosphate (CaP), has Pluronic F127, are water-soluble polymers, they are intensively
been printed into bone scaffolds with biomimetic structures, used as a representative sacrificial material for fabricating com-
adequate mechanical strength, and the ability to promote plex 3D constructs.[50] Pluronic F127 is of particular interest
osteogenesis.[93] because it possesses the characteristic of thermo-reversible
The physicochemical and mechanical properties of syn- gelation dependent on the solution concentration. Pluronic
thetic polymers can be easily modified for enhancing tissue F127 can transform from a liquid under 4 °C to a gel at over
engineering outcomes, and these materials can be produced 16 °C when above 20% w/w concentration. Both PEG and Plu-
at low cost without immunogenicity. Some FDA-approved ronic F127 should be chemically modified prior to forming
degradable polymers and their copolymers are extensively physical or chemical networks when using as tissue engineered
used in FDM and SLS bioprinting.[22,31,81] Polycaprolac- scaffolds.[95] The typical method for achieving gel formation is
tone (PCL) and poly­ lactide (PLA) are the most widely used acrylation or methacrylation, where the chemically modified

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PEG or Pluronic F127 is generally crosslinked under UV expo- in high concentration, or cell scaffolds in low concentration,

REVIEW
sure. Additionally, their derivative copolymers including poly- due to its rapid gelation property. However, the fibrin hydrogel
esters and polypeptides, among others, have been widely syn- is too soft and fragile to maintain a 3D shape.[104] Decellularized
thesized as physical or chemical hydrogels and used for cell extracellular matrices (dECM) from different tissues contain a
encapsulation, which can also be applied in 3D cellular printing variety of proteins, proteoglycans and glycoproteins of native
and manufacturing. tissue ECM components, so it has been used as bioink capable
Natural polymers and their chemical modifications are the of recapitulating a tissue-specific microenvironment in printed
most widely used as printable biomaterials and encapsulating 3D tissue/organ analogues.[100,105,106] Challenges in tissue decel-
living cells due to the similarity of their components to the lularization are with ensuring the complete removal of cel-
native tissue microenvironment.[20,50,81] They can also pro- lular components while maintaining of the fine vascularture
vide tissue-specific biochemical and physical stimuli to guide and other tissue structures.[107] Additionally, some toxicity has
cellular behaviors including migration, proliferation, differen- been observed when cells are grown on decellularized tissue
tiation, and maturation. For use in bioprinting, natural poly- scaffolds, potentially due to the retention of the decellulariza-
mers have been employed in several ways, to include the use of tion detergent.[105] Other natural polymers such as starch, cel-
the temperature sensitivity or ionic interaction characteristics lulose, and dextran, among others, have been developed for 3D
to facilitate extrusion, and the use of covalent addition of func- printing scaffolds with potential use in tissue engineering or
tional groups to induce chemical crosslinking approaches. other biomedical applications.[22,38,50,65,79,80]
Alginate (Alg) is an anionic polysaccharide derived from
algae or seaweed.[96] This material is composed of two repeating
monosaccharides (i.e., L-guluronic and D-mannuronic acids), 3.2.3. Latest Development of Bioinks
therefore, the typical ionic hydrogel can be formed using multi-
valent cations (i.e., Ca2+, Zn2+) instantaneously, making it attrac- The chemical modification of biomaterials may provide a prom-
tive for 3D tissue/organ printing.[96,97] The crosslinking pro- ising approach for extending 3D printable bioinks.[65,77,94] For
cesses are reversible, however, so the printed structures cannot example, the photocrosslinkable macromers or prepolymers
be maintained for longterm culture applications.[96] Hyaluronic can easily be prepared by acrylated/methacrylated multi-armed
acid (HA), or hyaluronan is a linear polysaccharide compo- oligomers/polymers or branched polymers. Photocrosslinked
nent of the ECM (non-sulfated glycosaminoglycans), which has networks have a high gel content, which indicates a high degree
been used clinically for several decades for treatments such as of crosslinking. However, due to the solvents used in these sys-
therapy for damaged joints and arthritis.[98] Acrylate or meth- tems, shrinkage or swelling of scaffolds may occur after drying
acrylate modified HA can be crosslinked to form a hydrogel or soaking, resulting in changes in structural and mechanical
via light based 3D bioprinting. Thiol-modification of HA can properties. Some macromers can be heated above the melting
form a hydrogel through Michael-type addition reactions with temperature to obtain the suitable viscosity; in such cases,
active vinyl based crosslinkers.[38] Limitations of HA as a bio- no solvent is needed for bioprinting and no obvious material
material for bioprinting are that HA hydrogels are typically too shrinkage is observed after cooling. Additionally, other covalent
soft to form robust structures, and show significant swelling crosslinking systems have been developed in the 3D printable
behavior. Collagen (Col) is a main structural protein of ECMs; inks. A thermally reversible dynamic covalent Diels−Alder reac-
it responds to simple crosslinking via thermosensitive gelation tion was used to synthesize a printable PLA blend for dramati-
under physiological conditions, which can be a major advan- cally improving both strength and toughness of the scaffolds.[67]
tage in 3D printing.[99,100] However, the high cost and weak Currently, composites of polymers and bioactive materials
mechanical strength limit its application. Gelatin (Gel) derived are being developed with the aim of increasing the mechanical
from partially hydrolyzed Col is inexpensive, and also possesses scaffold stability or improving tissue interaction.[21] The com-
thermosensitive properties.[101] Both Col and Gel have abun- posite scaffolds combining a variety of biodegradable polymers
dant proteins including fibronectin, vimentin, vitronectin, and and bioactive ceramics are fabricated by 3D printing and are
arginineglycine-aspartic acid (RGD) peptides, which promote capable of achieving high mechanical strength and good bio-
cell adhesion. The transition temperature of Gel lies around logical activity. Generally, the stiffness of the cured composites
30 °C limiting its direct application as the cell scaffold at physi- will increase with raising the concentration of nanoparticles or
ological temperature, thus it commonly serves as the sacrificial other materials. Synthetic polymers can often be combined with
material in 3D printed structures.[50] The reversible gelation bioactive materials, naturally derived materials or other func-
mechanism in aqueous conditions is based on the formation of tionalized materials to create more complex hybrid structures.
an alpha helix structure below 30 °C and a random coil struc- Moreover, the strongly desired characteristics of advanced
ture above 40 °C. Gelatin methacrylate (or gelatin methacryla- tissue scaffolds involve both biomimetic properties in struc-
mide, GelMA) has been widely used to fabricate scaffolds via ture and the ability to regulate cell behavior.[7,94] Engineering
the various light-based printing platforms.[102] Additionally, the techniques that mimic the critical aspects of natural healing
aforementioned thiol-ene crosslinking method has also applied and growth cascade are preferred to augment the proliferation
to the GelMA system when adding thiol-based materials.[94] and differentiation of the recruited or implanted cells; this is
Fibrin is comprised of fibrinogen monomers that are cleaved often achieved through the integration of growth factors and
with thrombin by a blood coagulation crosslinking mechanism, cytokines that provide suitable biochemical and physicochem-
thus fibrin plays an important role in the blood clotting and ical factors for tissue regeneration. Engineering these dynamic
wound healing processes.[103] It is widely used as surgical glue ECM mechanisms into biomaterials offers further control

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over cell behavior. One challenge is in developing methods challenges are: technique restrictions on the isolation and in
REVIEW

to incorporate these biologically inspired materials into con- vitro culture of cells, finite expansion or regeneration capacity
structs using bioprinting technology. Material printability and of many primary cell types, and the effectiveness of patient-
degradation characteristics such as time and byproduct emis- sourced cells. Pluripotent stem cells including embryonic stem
sion must be better understood for progression toward clinical cells (ES) and induced pluripotent stem cells (iPS) are prom-
applications. Also, it is essential that these materials have well- ising cell types due to their ability to proliferate in an undif-
understood and controllable structural and functional biological ferentiated but multipotent state (self-renewal) and their capa-
effects before advancing to in vivo testing and application. bility to generate multiple functional tissue-specific cell phe-
notypes.[20,38] Especially, iPS derived directly from adult tissues
requires reprogramming the cell type thus overcoming the dif-
3.3. Cell Sources and Selection ficulty and limitations associated with the current cell sources.
Adult mesenchymal stem cells from bone marrow, fat, umbil-
The choice of cells for tissue or organ printing is crucial for ical or other sources can differentiate into osteoblasts, chondro-
functionality of the fabricated construct, especially for future cytes, adipocytes, cardiac cells, endothelial cells, smooth muscle
clinical application.[108] Tissues and organs are comprised of cells, hepatocytes, and neural cells and can be used in many
multiple cell types with specific biological functions that must biomedical applications. Although they have a more limited
be recapitulated in the regenerated tissue. In addition to the multipotent differentiation potential, they are considered safer
primary functional cell types, most tissues contain various cell for clinical uses and show great promise for bioprinting appli-
types that provide supportive, structural or other functions, cations. According to previous studies, high cell viability can be
or are involved in vascularization or provide an essential sur- obtained through optimizing the printing parameters; overall
rounding for functional maintenance and development of the the printing processes have no adverse effects on the stem cell
primary cells.[20] Therefore, the options for printing cells not proliferation and differentiation abilities.[50]
only involve the arrangement of primary cell types in the 3D The 3D printed constructs for the complex tissue or organ
printed construct, but also have a close relationship with other regeneration need to be fabricated with either functional pri-
cells for contributing to complete functionality of complex mary cells with supporting cells or progenitors/stem cells for
tissues/organs. further differentiation.[16,23,26] In cellular printing, multiple
bioinks with different cells need to be prepared to print in par-
allel, requiring complicated and precise control of the printing
3.3.1. Principles of Cell Selection step. Additionally, acellular printing is difficult to post-seed
specific cells on the desired regions of complex 3D structure.
Cells used for 3D printing should take into account several ele- Printing stem cells with the regional bioactive factors, or post-
ments.[16,20,81] (1) Sufficient numbers of cells can be expanded seeding stem cells on the construct with the regional bioactive
in vitro culture for bioprinting; (2) Cells must be robust enough factors, may reduce the complication of the fabrication process
to survive during or after the bioprinting process; (3) Appro- for complex tissue/organ regeneration. Stem cells can be dif-
priate cell proliferation and controllable differentiation in the ferentiated into target cell types by bioactive factors in the com-
3D printed scaffolds are required for either in vitro culture or bination of location or spatial arrangement. Therefore, 3D stem
in vivo implantation; (4) Cellular functions can be maintained cell printing can provide a simple and effective approach for
in vitro to closely mimic the true physiological state, and devel- regenerating complex tissue/organ.
oped after implantation by stimulation with the in vivo envi-
ronment, including physical forces and biological stressors;
(5) Physiological specificity both structural and functional on 3.4. Modular Fabrication of Mini-tissue
different cell types; (6) Interaction of multiple cells for tissue
development involves biological signal paths. Regeneration of a tissue or organ including cellular and extra-
cellular components, needs to reproduce specific cellular
functions, thus a complete understanding of the tissue micro-
3.3.2. Cell Sources environment, such as specific organization and hierarchy of
various cell types, gradients and arrangement of biologicals,
In order to maintain long-term function after implantation, composition of the ECM as well as the native biomechanical
the bioprinted construct must be able to maintain cellular stimulation in vivo.[17] Tissues or organs can be considered as
homeostasis, self-renew, respond to tissue damage or injury, an aggregate structure consisting of small structural and func-
and integrate with host tissue or organ.[16,20] tional components, which can be defined as functional building
Host immune response may be triggered by the implanta- blocks.[23,36,50] Modular fabrication of these building blocks can
tion of exogenous cells. Therefore, the autologous source of easily be assembled to complete tissues. 3D bioprinting tech-
cells is the preference, autologous cells may be obtained from niques can be used to print these building blocks and guide
the patients themselves through the generation and differen- them to assemble into 3D living structures. The process of
tiation of autologous stem cells or through reprogramming tissue/organ development relies on cellular self-organization
approaches, to avoid negative immune responses. However, through direct assembly in scaffold-free conditions or aggre-
some limitations make it difficult to apply autologous cells in gation along with the 3D printed scaffold degradation.[20] This
bioprinted constructs for tissue regeneration. Examples of these spontaneous self-organization happens during development

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in vivo, but has also been recapitulated in numerous in vitro structures compared to all traditional tissue engineering tech-

REVIEW
applications. niques. Although avascular or thin tissue bioprinting has shown
In addition to the traditional “cell-scaffold” approach, cell great promise in current research, complex tissue or organ bio-
aggregation is a typical technique of tissue fabrication, in printing for implantation remains a challenge.[16] Prior to reca-
which solid cellular units are directly used as building blocks pitulating organ-level complexity, creating functional tissue is
to engineer the tissues.[23] This technique enables scaffold-free an essential stage that contains a hierarchical arrangement of
bioprinting, even free of exogenous biomaterials. The con- multiple cell types in a 3D microenvironment. This includes
cept originates from the knowledge of developmental biology the primary functional cells depending on tissue type, along
and the fact is that, tissues and organs are formed without any with a multi-scale network of vasculature in stroma and paren-
scaffolds during embryonic development. Generally, cellular chyma, as well as lymphatic vessels and neural networks.[4,17,26]
aggregation techniques have some typical procedures: (1) Cell The requirements for 3D printing biological tissues and
expansion; (2) Initiation of cell aggregation; (3) Cellular pellet organs can be summarized in three elements: (1) Biomimetic
collection; (4) Geometric molding, such as cylinder or sphe- structure (modeling and resolution); (2) Biocompatible and bio-
roid.[20,23] Moreover, the multiple cell types can also be organ- active components; (3) Bio-microenvironment, either in vitro or
ized during cell aggregation, especially endothelia cells can be in vivo, including biomechanics and biochemistry. The first two
co-cultured to form vascularized cellular aggregates.[109] After have been discussed in the technique and bioink parts of this
obtaining sufficient mechanical integrity, aggregates with diam- manuscript. The various bioprinting techniques and bioinks
eters ranging from 200 to 500 µm can be printed as building can facilitate the hierarchical fabrication of multiple cell types
blocks to form 3D structures. However, the directly printed con- and direct them to differentiate into desirable tissue types.
structs are fragile and lack cohesive tensile strength. Therefore, The post-bioprinting process is crucial to provide an adequate
successful fusion is a very important process for the formation bio-microenvironment including mechanical, chemical or bio-
of 3D structures that rely on the cohesive ability of multicellular logical signals to regulate tissue remodeling and growth.[65]
aggregates and additive properties. The accumulation of ECM, The development of new bioreactor technologies enables the
associated restriction of cell motility and enhancing tissue rapid maturation of tissues, multiscale vascularization for sur-
cohesion in tissue spheroids can change kinetics or impede the vivability of tissues, and mechanical integrity and innervation
tissue spheroids’ fusion process.[23] for implantation.[20,38] In complex tissue/organ bioprinting, the
One advantage of this technique is potentially accelerated post-bioprinting process or the tissues/organ regenerated pro-
tissue organization and the ability to direct the formation of cess involves three phases:
complex structures.[23] Tissue spheroids are thought to possess
material properties that can replicate the mechanical and func- (1) Cell viability. For cellular bioprinting, the time of printing
tional properties of the tissue ECM. Moreover, by manipulating procedure and the sensitivity of cells may impact the cell
the host bioactive composition, self-organization can be con- viability. If the time is long and the cells are fragile, then with-
trolled. Unlike the critical role of bioinks in assisting the ECM out any supplement of extra nutrition, the cell viability will
production in traditional 3D bioprinting, bioprinted self-assem- decrease severely. Therefore, the bioprinting time, including
bling cellular spheroids may produce a suitable ECM environ- preparation time, should be shortened; the incorporation of
ment by themselves.[17,23] culture media during printing can also significantly improve
It is noteworthy that in either biomaterial based bioprinting cell viability.
or scaffold-free bioprinting, cell-laden bioprinting techniques (2) Mass transport and mechanical stimulation. The cells are
require suitable nutrient and oxygen transport for regenerated directly encapsulated in the bioinks in the printing process
tissues/organs through proper pore architectures or vasculature or are post-seeded onto the printed constructs; it is essential
analogues. to supplement them with nutrients and oxygen during the
culture periods. After fabrication, the constructs with viable
cells must not only remain viable for the long culture period,
4. 3D Bioprinting of Organs but also must be able to function as intended. Bioreactors,
typically employing perfusion, tensile or compressive load-
4.1. Definition, Elements and Procedure ing, rotation or other conditioning, may provide a dynamic
surrounding and mechanical stimulation for cell culture in
3D bioprinting of organs is a comprehensive or integrative vitro, mimicking the native fluid environment and physical
approach that offers a pathway for scalable and reproducible forces in vivo.[38,65]
mass production of engineered living organs.[35] It allows the Shear forces created by medium flow through the 3D
precise simultaneous 3D positioning of multiple cell types printed constructs facilitates efficient transfer of nutrients
with high density to mimic their natural counterparts.[110] and oxygen. This perfusion system can be used to mimic
More importantly, it enables the creation of functional com- haemodynamic forces and pressures that occur naturally
plex tissues with vasculature and neural networks.[20] The in the human body thus improving ECM production and
ultimate goal is industrial, scalable, biofabrication of patient- mechanical properties of the artificial tissues/organs.[87]
specific functional 3D living human organs suitable for clinical Compared with slow permeation in static culture, the use of a
implantation.[15] bioreactor, or direct implantation in vivo, could be more ben-
As previous mentioned, bioprinting techniques have the eficial for ensuring homogenous and efficient mass trans-
most tremendous potential on manufacturing the complex port.

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Furthermore, the mechanical cues, present at the on- organs is crucial for transporting oxygen and nutrients and
REVIEW

set of several signaling pathways in normal physiologi- maintaining tissue functions.[113] Cells existing more than
cal conditions, have been known to positively influence 200 µm away from the nearest capillaries will undergo hypoxia,
tissue formation and further integration.[111] In addition apoptosis and ultimately cell death, due to limited diffusion
to the intrinsic mechanical stimulation from biomaterial ability.[112] Therefore, the most critical challenge for complex
composition, periodic stretching, pulsing, or compression tissue and organ regeneration is the integration of a vascular
that mimics the physical forces its corresponding tissues or network, which is also a major problem in 3D bioprinting of
organs experience in vivo can increase strength and flexibil- complex tissues and organs.[26,109,110,115]
ity, as well as increase matrix reorganization and maturation Native blood vessels have complex unique structures in
of the construct.[38] multi-scale and multilayer arrangements. The inner diam-
(3) Construct maturation. In this stage, the cells must proliferate eter of blood vessels ranges from microscopic size, 5∼10 µm
to form the appropriate cell-cell connections for communicat- for the smallest capillaries, to 30 mm, for the largest artery
ing with each other; they must be able to secrete their own (aorta).[116] Walls of the large vessels, namely elastic arteries,
matrix components and perform natural biological functions, muscular arteries and veins have three distinct layers starting
further integrating into the host tissue. The phenomenon from the vessel lumen: intima, tunica media and tunica adven-
involved in maturation that was discussed above can be ac- titia respectively.[117,118] Intima, the innermost layer is a throm-
celerated using techniques such as mechanical conditioning. boresistent confluent monolayer of ECs and is attached to a
The constructs may provide an appropriate environment for basement membrane (40∼120 nm). Media, the middle layer,
cell development, both biomechanically and biologically, and is comprised of a dense population of concentrically organized
can also be replaced by deposited native ECM with increasing SMCs with bands or fibers of elastic tissues, and adventitia, the
time. Over time, cells reach equilibrium states between cell– outermost layer, is a collagenous ECM containing mainly fibro-
matrix adhesions, such as integrins, and cell–cell adhesions, blasts and perivascular nerves.[119] In contrast, the capillaries
such as tight junctions and adherens junctions. These inter- only consist of EC layer. Although grafts can spontaneously vas-
actions between cells and cell–matrix offer the ability of cell cularize after implantation (involving an inflammatory wound-
populations to spontaneously reorganize into 3D tissue.[38] healing response and hypoxia-induced endogenous release of
angiogenic growth factors), the process of angiogenesis and
inosculation with microcirculation in vivo is too slow to provide
After in vitro maturation of 3D printed constructs, the engi- sufficient nutrients and oxygen to the cells inside of the tissue
neered tissue needs to be implanted into the patients’ body for construct.[120] Obtaining a functional vasculature, consisting of
in vivo integration. This phase will involve the issue of bio- adequate vessel geometries and dimensions through 3D bio-
manufactured construct immune acceptance, in vivo safety and printing strategies, is absolutely essential.[112,118,121]
efficacy, and monitoring of construct integrity and function Encapsulating ECs or SMCs into bioprinted constructs
post-implantation.[16,20,50] without prefusable channels has developed for self-assembly
Therefore, as mentioned above, several challenges must of interconnected vasculature.[122] This method of generating
be progressively addressed to make organ printing become a blood vessels in artificial tissue relies on the ability of ECs to
reality. The most critical challenge is the integration of a vas- organize into blood vessels autonomously (angiogenesis), thus
cular network and a neural network in the 3D printed con- only capillaries can be created. Although such designs lead
structs, which is also a problem the majority of tissue engi- to the formation of vascularized tissue, capillaries are too far
neering technologies are facing. In the following section, we from the arteriovenous (AV) loop and the tissue, in vivo, was
will present recent developments on bioprinting scale-ups prone to necrosis after longer implantation times. Addition-
of complex tissue and organ constructs for implantation, ally, from a tissue engineering standpoint, it is not necessary
including vasculature/vascularized tissue, neural regeneration, to consider the fabrication of the capillaries, because they can
and organ constructs. We will also discuss major roadblocks sprout from the large blood vessels based on the native angi-
toward clinical translation and provide potential solutions and ogenesis process (a complex cascade of events including ECs
future perspectives. activation, migration, and proliferation as well as arrangement).
The rate of sprouting angiogenesis is around 1.0 mm per week
in vivo. Compared to the commercial and clinical successes in
4.2. 3D Bioprinting Applications in Organ Regeneration large-diameter vascular grafts, manufacturing small-diameter
(<6 mm) vasculature currently remains a formidable task.[123]
4.2.1. Vasculature 3D bioprinting technology is currently unsuccessful in
fabricating hierarchical and complex structural vasculature,
To maintain metabolic functions, the native tissues or organs where vascular trees spanning arteries and veins down to capil-
require the supplementation of adequate nutrients, gas laries are required to be manufactured to mimic natural vas-
exchange, and metabolic waste removal, all of which are also cular anatomy.[112,114] More importantly, successful maturation
necessary for engineered tissue maturation.[112–114] Without toward functional, mechanically integrated vasculature is still
these, low cell viability and malfunction of artificial tissues/ a challenge. The main unit of the vascular tree is a ‘Y’ shape
organs may result, especially when scaling-up tissues with a branching unit (a bifurcated pattern); the repeat of multi-scale
high volumetric oxygen-consumption rate, such as cardiac, units will form a hierarchical structure with different branching
pancreatic, or liver tissue. Vasculature within the tissues or orders.[109,115] The constructs at submicrometer scale are difficult

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to be printed using the current techniques. Therefore, instead significantly increased cell viability near the channel regions

REVIEW
of fabricating a biomimetic vascular tree, some researchers inside the construct compared with other deeper regions. In an
alternatively print perfusable (bifurcated or branched) chan- earlier study, carbohydrate glass was used as a cytocompatible
nels at micrometer or higher scale to mimic a vascular network, sacrificial template to print rigid 3D filament lattices.[125] After
facilitating medium flow and oxygen supplementation for cell crosslinking the ECM gels, the glass filaments were dissolved
viability, tissue maturation and formation.[87,95,124] to form vessels with intervessel junctions. In co-cultures with
Here, we focus on the manufacturing of interconnected chan- 10T1/2 cells in the interstitial space, endothelial cells lining the
nels or free-standing tubular structures, allowing native blood vascular lumen became surrounded by the 10T1/2 cells and
vessel ingrowth and anastomosis. Over all, three approaches formed single and multicellular sprouts extending from the
have been developed: (1) indirect bioprinting through utilizing patterned vasculature into the bulk hydrogel (Figure 4a).[125]
sacrificial templates (a fugitive ink) that is removed to create Thermosensitive fugitive ink, Pluronic F127 was also explored
hollow channels in a bulk construct; (2) directly bioprinting to print microvascular networks (Figure 4b).[95,124,126] After a
interconnected channels in a construct; (3) direct bioprinting of cooling process, the perfusable channels possessed final diam-
a vasculature network or blood vessel in a tubular shape. eters ranging from ca. 100 µm∼1 mm in the GelMA hydrogel
(Figure 4c).[124] In a thick osteogenic tissue model (>1cm thick
4.2.1.1. Sacrificial Templates: In indirect bioprinting, cell-laden and 10 cm3 in volume), the embedded vascular network ensured
hydrogels serve as the bulk matrix to fabricate vascularized uniform and long-term perfusion culture throughout the con-
tissue constructs; here the vascular networks are printed with struct, promoting the osteogenic differentiation of encapsulated
fugitive inks. The endothelium lumen is generated inside human mesenchymal stem cells (hMSCs).[126] In other studies,
tubular channels after the perfusion culture of endothelial a straight EC-laden gelatin line was printed inside of the col-
cells (ECs). The integration of the vascular network shows lagen layers.[127] The 3D printed constructs were incubated at

Figure 4. (a) 3D printed perfusable vascularized tissue constructs. A confocal z-stack montage demonstrating HUVECs (expressing mCherry, red) were
residing in the vascular space with 10T1/2 cells (expressing EGFP, green) uniformly distributed throughout a bulk fibrin gel, after one day in culture.
Scale bar, 1 mm. A partial z-stack of two intersecting channels demonstrated endothelialization of channel walls and across the intervessel junction,
while in the surrounding bulk gel 10T1/2 cells are seen beginning to spread out in three dimensions. Reproduced with permission.[125] Copyright 2012,
Nature Publishing Group. (b) Fluorescent image of a 3D microvascular network fabricated via omnidirectional printing of a fugitive ink (dyed red)
within a photopolymerized Pluronic F127-diacrylate matrix. (Scale bar = 10 mm) Reproduced with permission.[95] Copyright 2011, Wiley-VCH. (c) Confocal
image of live HUVEC cells lining the microchannel walls using the same fugitive ink method. Reproduced with permission.[124] Copyright 2014, Wiley-
VCH. (d) Confocal fluorescence images of hMSCs and HUVECs co-cultured on various scaffolds in a static culture condition for 5 days. hMSCs
were labeled with cell tracker green, and HUVECs were stained with cell tracker red. The scale bars indicate 200 µm. Reproduced with permission.[85]
Copyright 2016, Wiley-VCH. (e) 3D-printed PPF scaffolds as venous interposition grafts at the time of in vivo implantation. Reproduced with permis-
sion.[130] Copyright 2016, Wiley-VCH. (f) Confocal fluorescence images of hMSCs and HUVECs co-cultured in designed vascular channel regions for
1 week. HUVECs encapsulated in the hydrogel were inclined to aggregate and migrate to form annular ring patterns along the channel. The scale bars
indicate 200 µm. Reproduced with permission.[87] Copyright 2016, Wiley-VCH. (g) Confocal microscopy images show interconnected structures of the
encapsulated HUVECs after migrating to outer regions of the bioprinted fibers at day10. Reproduced with permission.[136] Copyright 2015, Wiley-VCH.

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37 °C for 30 min to complete the collagen gelation and the A biomimetic vascular lumen with capillaries was successfully
REVIEW

gelatin liquefaction. The functional vascular channels and the generated in our engineered construct after the encapsulation
sprouts were generated with fully covered ECs.[127] Although of endothelial cells (Figure 4f).[87]
the bioprinted sacrificial template method for vascular fabrica- In one paradigm for direct extrusion of tubular structures,
tion has exhibited feasibility, flexibility and angiogenic ability, coaxial extrusion nozzles are employed to produce free-standing
this technique still faces some challenges. First, the fabricated fluidic channels. The outer nozzle contains an uncrosslinked
vasculature only possesses endothelium and is unable to repeat biopolymer, while the inner nozzle contains the corresponding
the multilayer cell structure of native blood vessels. Addition- crosslinker. The freestanding tubular constructs are generated
ally, the current studies only suggest the 3D printed channel along with the crosslinking process. Typically, the combina-
could provide sufficient nutrients for cell viability inside of tion of alginate and calcium chloride (CaCl2) are widely used
constructs, which is similar with the interconnected pores in in 3D printed blood vessels.[131,132] The stacks of self-standing
the traditional scaffold design. The functionality and inoscu- fibers are printed using a shell/core nozzle through the con-
lation with microcirculation in vivo has yet to be systemically tinuous extrusion of highly concentrated alginate (16.7% w/w)
explored. Finally, the structure of vascular networks totally rely with PVA (6% w/v) solution.[133] After printing, constructs are
on the bulk hydrogel constructs, thus structural fabrication of transferred to a CaCl2 solution and the hollow fibers are gen-
external constructs is limited, such as the hierarchical struc- erated by the dissolution of the PVA.[133] Furthermore, the low
tural design and the biomimetic distribution of multiple cells. concentrated, cell-laden sodium alginate (2∼6%) has been uti-
lized to fabricate hollow tubes without any supports.[131,134] In
4.2.1.2. Direct Printing of Interconnected Channels: Direct bio- this design, the sodium alginate solution flowing through the
printing of a construct with interconnected channels is another outer tube of a coaxial nozzle is immediately crosslinked by a
approach to fabrication of vascularized tissue. For example, the CaCl2 solution through the inner tube.[134] Although, the coaxial
millimeter-sized branched channels were designed and printed nozzle system offers a very straightforward approach for direct
in poly(ethylene glycol) dimethacrylate (PEGDMA) hydrogels extrusion of tubular structures, this printing method cannot
by SLA.[128] In addition, a high-resolution µSLA technique was currently achieve branched structures.
utilized to print 3D cell/biomaterial patterns with <5 µm reso- The alginate/CaCl2 system has also been applied to inkjet
lution, as well as open fluidic channels with 100 µm diameter printing.[45] Microgel beads were formed by diffusion of Ca2+
for angiogenic cell-encapsulating patches.[63] Although laser into alginate ink droplets by laminating printing to fabricate
based methods are capable of producing extremely high-reso- tubular structures.[47] The wall thickness and the inner diam-
lution features and fluidic channels, architectural complexity eters of the tubular structures could be adjusted respectively
is still largely restricted to uniaxial channels. The open chan- from 35 to 40 µm and from 30 to 200 µm through varying the
nels would be crosslinked by light exposure after printing the diameter of the microgel beads.[47] In addition, a 3D zigzag
subsequent layers. Therefore, other 3D bioprinting techniques tube structure was printed via an inkjet-printing process.[135]
have also been used to produce the multidirectional and inter- By refining the operating conditions, 210 layers of Ca-alginate
connected channels. In a recent study, we developed a 3D bio- droplets were deposited to form a freestanding tube with a
printed vascularized tissue construct with a unique integration height of 10 mm, an overhang angle of 63° and an overhang
of fully interconnected microvascular networks using a FDM height of 5 mm.[135]
printer (Figure 4d).[85,129] The microvascular design of the con- Alginate has shown promising advantages for fabrication
structs can provide similar flow characteristics to native blood of blood vessels, however, it is not an ideal material for living
vessels under pulsatile arterial flow. tissue construction.[50,96,97,136] First, it does not promote cell
adhesion due to its strongly hydrophilic nature, thus ECs cannot
4.2.1.3. Direct Printing of Tubular Constructs: The third approach grow around the tubular construct to form the endothelium
is direct bioprinting of blood vessels or vascular networks in layer. Second, the encapsulated cells have limited proliferation
a tubular shape via: (1) bioprinting of tubular grafts or vascu- due to a lack of natural ECM receptors. Finally, the alginate/
lature; and (2) bioprinting of scaffold-free branched vascular CaCl2 hydrogel is unstable for a long culture time because of
tubes that are printed inside a mold pattern. Compared to its ionic interaction, owing to the exchange reaction with other
other fabrication methods, direct printing of the self-supporting ions. Therefore, the use of more biomimetic materials instead
tubular constructs allows them to be integrated within a mul- of, or along with, alginate gel is required. In a different design,
tiple bioprinting platform and also facilitates patterning them coaxial extrusion was used to create a grid of solid alginate/
into complex spatial structures. GelMA fibers by extruding an alginate/GelMA mixture through
Recently, a biodegradable, 3D-printed, acellular vascular an inner nozzle and calcium chloride through an outer nozzle
graft was fabricated with poly(propylene fumarate) (PPF) (Figure 4g).[136] After a temporary solidification by physical
using a DLP based SLA technique (Figure 4e).[130] Both in vitro crosslinking, GelMA was covalently photocrosslinked to fur-
and in vivo studies showed that this graft-fabrication strategy ther reinforce the fibers. Over the culture period, the ionically
enabled the printing of scaffolds with inner diameters of 1 mm crosslinked alginate disintegrated leaving hollow, intercon-
and wall thicknesses of 150 µm, which sustained patency and nected fluidic channels in the constructs. The results showed
functionality for 6 months after implantation in the venous ECs encapsulated in the fibers could migrate to the edges of the
system of mice.[130] Moreover, our group also developed a com- fibers for endothelium formation. This technique addressed the
plex vascularized tissue construct using a dual 3D bioprinting questions of the instability and weak bioactivity of alginate gels,
technique based on the FDM and SLA bioprinter systems.[87] but it did not provide a biomimetic, hollow, vascular structure.

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Subsequently, they modified the design to fabricate perfus- effect on angiogenesis in our works.[85,87] On the other hand,

REVIEW
able hollow tubes using a blended bioink and alginate removal the design and bioprinting of vascular networks should be
process.[137] easily connected to native blood vessels, thus it should possess
Combining direct extrusion and a supporting slurry of certain properties, such as proper mechanical properties (elas-
hydrogel microparticles was also developed. This approach can ticity and tensile strength) to satisfy retention and burst pres-
print hierarchically branching tubular networks. Photoreactive sure, sufficient interconnectedness of endothelium to prevent
poly(vinyl alcohol) (PVA) was crosslinked after printing and thrombosis, and a high patency rate to support occlusion-free
fully crosslinked structures were recovered from the slurry of circulation. Compared with indirect bioprinting of a vascular
Carbopol particles by immersion in stirred water.[138] Concentri- network, the direct bioprinting of tubular construct can be
cally nested objects were also printed, highlighting the poten- more convenient for anastomosis to the host.
tial for this technique to produce biological structures with
heterogeneous internal structure.[138] In another study, gelatin
hydrogel microparticles were used with freeform reversible 4.2.2. Neural Networks
embedding of suspended hydrogels (FRESH) as a support bath
to print embedded branching arterial tree.[139] The human nervous system is widely distributed throughout
Combination of high-resolution TPP with SLA enables the the body, which is similar with the aforementioned vasculature.
fabrication of refined and complex geometries for printing It is responsible for controlling all the biological processes that
tubular blood vessels. The tubular or bifurcated structures coordinate voluntary and involuntary actions as well as transmit
were created using photo-crosslinkable synthetic polymers and signals among the different parts of the body.[145] It consists of
biopolymers. The high resolution of TPP enabled the fabrica- the central nervous system (CNS) and the peripheral nervous
tion of branched tube structures with 18 µm luminal diameter system (PNS).[145] The CNS consists of the brain and spinal
and wall thicknesses <5 µm, using a polytetrahydrofuranet- cord and is responsible for processing the information from
herdiacrylate (PTHF-diacrylate).[140] The presented artificial the PNS.[146–148] The PNS consists of nerve fiber (Schwann cells
vascular constructs possessed 3D microstructured wall architec- (SCs) wrap around the axons) bundles and connective tissues
tures, including high porosity and high interconnectivity.[140] In that detect and transmit signals between the CNS and limbs/
order to obtain a proper mechanical strength, dithiol-mediated peripheral organs through motor (or efferent) and sensory
chain transfer was also applied to photo-crosslinkable mate- (or afferent) nerves.[149–151] At the cellular level, neurons are the
rials.[141] The printed networks showed the reduced cross- core components of both the CNS and the PNS, which connect
linking density and high contents of reversible H-bonds, gen- to each other to form neural circuits or neural networks.[145]
erating the biomimetic mechanical properties in the range of Based on biological viewpoints, the primary function of the
native porcine carotid arteries.[141] nervous system is to control the whole body via connecting the
Scaffold-free bioprinting provides an alternative method brain to all tissues/organs. Therefore, the fabrication of neural
based on cells and the genuine matrix they secrete while networks is an essential process for complex tissue and organ
avoiding the complexity of biomaterials such as biocompat- regeneration.
ibility, degradation behavior or potential immunogenicity.[135,142] Prior to creating neural networks, the repair of nervous
The extrusion of cell aggregates consisting of either one cell system injuries caused by disease, trauma and disorders
type or several cell types was developed for the fabrication of remains a formidable task.[146,150] In our previous review and
tubular structures. A fully biological self-assembly approach book, we have discussed the self-repair process of the nervous
was proposed to fabricate the scaffold-free, small diameter, vas- system in detail.[152,153] Following the PNS injuries, Wallerian
cular reconstructions.[143] Various vascular cell types, including degeneration commonly occurs, and SCs are activated to con-
SMCs and fibroblasts, were aggregated into discrete units of tribute to forming the bands of Büngner, enabling guided
either multicellular spheroids or cylinders of controllable diam- axonal regeneration. However, the reconstruction process
eter. Then they were printed by molding a sacrificial template cannot take place in CNS injuries because the CNS lacks SCs.
of collagen or agarose.[143,144] After removing the template, Instead, glial scar tissue forms, impeding both axon growth
the fusion of the discrete units resulted in single- and double- and myelination.[152] Therefore, tissue engineering techniques
layered hollow vascular tubes. Unique advantages of this combining cells and scaffolds is the most feasible approach for
method are speed and scalability; it is capable of engineering CNS regeneration.[147,152,154] Although the PNS has a greater
blood vessels with distinct shapes/diameters and hierarchical capacity for axonal regeneration after injury, spontaneous
structure.[143,144] peripheral nerve repair is nearly always incomplete with poor
Although numerous 3D bioprinting approaches have functional recovery.[149,150] Misdirection towards the wrong
emerged to fabricate the vasculature, generating vasculature target reduces functional outcome, thus grafts between the
with multi-scale, multilayer structures that replicate the geom- nerve stumps are required to bridge the gap and support axonal
etry, complexity, and longevity of human vascularized tissues regrowth. Additionally, the PNS shows poor self-repair ability in
remains a challenge. In addition to fabricating the vasculature large defects. Utilization of an autograft in clinical treatments
within the construct, the functionality and anastomosis of in inevitably creates additional nerve injury and loss of function
vivo microcirculation should be taken into account for implan- near the donor site; allografts techniques often induce immu-
tation. On one hand, a strategy combining 3D printing and site- nological rejection. To overcome these disadvantages, current
specific delivery of angiogenic factors should be developed to research is focused on the development of novel tissue engi-
promote vascularization. Several studies have shown a positive neering alternatives to repair peripheral nerve gaps.[150,151]

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Overall, nervous system regeneration involves the surgical growth and experimental differentiation in vitro. The conduits
REVIEW

implantation of a neural scaffold or conduit fabricated in vitro had acceptable handling properties and performed compara-
at the targeted site. The scaffold or conduit bridges the lesion, tively with an autograft control in a thy-1-YFP-H mouse (the
provides a direct framework for neurons to proliferate and pro- YFP+ transgenic mouse strain possesses a population of fluo-
motes cell secretion of inductive factors for axonal elongation rescently labelled peripheral axons) 3 mm gap injury model
and for minimal scar formation.[145,152] In the human body, after 21 days, with the number of unique axons at the distal end
neural cells reside within a 3D ECM with micro/nano architec- in each repair group being similar (Figure 5a).[162]
ture and spatiotemporal chemical and physical cues. Conven- The stem cell replacement has attracted much attention as
tional scaffold or conduit fabrication techniques offer limited a promising therapeutic option for neural tissue regeneration,
control over geometry and internal microstructure, especially especially for CNS regeneration.[163] To support the physiolog-
for the oriented feature. 3D printing techniques offer great pre- ical function of stem cells in the implanted tissue site, the use of
cision and control of the internal architecture and outer shape 3D printed scaffolds that mimic the biologically functional and
of the conduits and even have great potential for replicating the organizational complexity of the tissue has been regarded as an
complicated nervous network.[34,152,153,155] important approach.[152,163] In addition, stem cells can secrete
various cytokines and growth factors that generate a variety of
4.2.2.1. 3D Printing for PNS Regeneration: Inkjet bioprinting has beneficial effects such as anti-inflammation, neural cell protec-
shown great potential for fabricating conduits for engineered tion, and induction of the endogenic recovery systems.[153,163]
neural tissue. In an earlier study, the controlled patterns and The micro-well/channel arrays as topographic network patterns
structures of primary embryonic hippocampal and cortical were made by SLA to guide the growth and differentiation of
neurons were first fabricated using a thermal inkjet printer.[156] embryonic stem cells (ESCs) and MSCs towards a neurogenic
The results showed cellular properties and functional fidelity lineage.[164] Extrusion-based bioprinting was employed to fab-
of neurons, including neuronal phenotypes, and electrophysi- ricate scaffold-free conduits using MSC and ESC cylinders.[165]
ology could be retained after bioprinting.[156] Furthermore, a The multicellular cylindrical units of MSCs and ESCs were pre-
novel bioink based on a microgel suspension (endotoxin-free pared and extruded into molded wells made by agarose rods.
low-acyl gellan gum) in a surfactant-containing tissue culture After the removal of the agarose rods, the fused construct
medium was used to print neuron-like PC-12 cells using two resulted in three hollow channels forming a fully cellular con-
different commercially available inkjet bioprinting systems.[157] duit graft.[165] Compared with autologous graft and commercial
The bioink performed very well in preventing cell aggregation collagen conduit graft, the bioprinted graft performed at a com-
and promoting cellular differentiation which was confirmed by parable level on the sciatic nerve defect model. Both motor and
immunostaining studies.[157] The LIFT technique has also been sensory functions exhibited recovery even though limited axon
utilized to print constructs for neural tissue engineering.[158] growth was observed.[165] The indirect 3D printing technique
Schwann cells and astroglial cells can survive, proliferate and has been developed to fabricate customized conduit molds.
differentiate well after printing.[158] In 3D printing, SLA is the A bio-conduit consisting of adipose-derived stem cell (ASC)
most typical technique to create geometrically patterned con- laden cryopolymerized gelatin methacryloyl (cryoGelMA) gel
structs with high resolution, creating highly aligned nano/ was prepared for PNS regeneration 3D printing (Figure 5b).[166]
micro structures that mimic natural ECM and facilitate the The conduits were fabricated with different geometries using
outgrowth of neurites. Glycidyl methacrylate modified hya- 3D-printed “lock and key” molds, such as the designed multi­
luronic acid (GMHA) was printed as scaffolds with different channel or bifurcating models and personalized structures.
geometries, including hexagonal and circular patterns with The in vitro result showed cryoGelMA scaffolds supported the
different numbers of channels.[159] The authors proposed that attachment, proliferation and survival of the seeded ASCs, and
successful fabrication of conduits with multiple channels par- up-regulated the expression of their neurotrophic factors. After
allel to the long axis of the lumen could mimic nerve fascicles implantation in a rat model, the bio-conduit was capable of sup-
and the branched scaffolds can mimic a nerve plexus.[159] Gradi- porting re-innervation across a 10 mm sciatic nerve gap, with
ents of molecules were patterned along the length of channels results close to that of autografts in terms of functional and his-
during printing and it was hypothesized that such gradients tological assessment.[166]
could be useful in printed nerve guidance conduits.[159] More- Conductive nanobiomaterials have been shown to improve
over, a dual hydrogel approach was developed for a patterned axon outgrowth and enhance connection between artificial sub-
conduit, where PEG hydrogel served as a cell-restrictive region stitutes and target injured nerve tissue.[152,153] They can assist to
supplying structure and a cell-permissive, self-assembling gel stimulate and control neuron activities under electrical stimu-
(Puramatrix or agarose) was made to encapsulate the embry- lation and more effectively guide neural tissue repair. A 3D
onic dorsal root ganglia (DRG).[160] A multilayered, on-demand printable graphene (3DG) composite liquid ink consisting most
3D collagen construction with astrocytes and neurons was of graphene with small amounts of polylactide-co-glycolide,
printed into single-layer and multilayer constructs using a bio- was utilized to create neural constructs via extrusion-based 3D
printer with 4-channel dispenser.[161] The immunostaining sug- printing (Figure 5c).[167] The resulting 3DG material is mechani-
gested the patterned neurons showed neurite outgrowth and cally robust and flexible with electrical conductivities greater
neural connectivity in three dimensions.[161] Nerve guidance than 800 S/m. In vitro experiments, in the absence of neuro-
conduits (NGCs) with ∼50 mm resolution from photocurable genic stimuli, reveal that 3DG supports MSC proliferation and
poly(ethylene glycol) resin was printed using µSL technique.[162] neurogenic differentiation. This coincides with hMSCs adopting
The photocurable form of PEG was permissive for neuronal highly elongated morphologies with features similar to axons

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REVIEW
Figure 5. (a) A PEG nerve guide made with a wall thickness of 50 mm by µSLA. The PEG nerve guide was implanted in to a Thy-1-YFP-H common
fibular mouse, small gap, 3 mm injury model. The nerve graft repair image illustrated intervals marked with sample axon tracing from 4.0 mm interval
position back to 0.0 mm (start) interval. The number of axons at each interval was counted to obtain a sprouting index value; axons were traced from
distal intervals back to 0.0 mm, or a branch point with a previously traced axon (as highlighted in expanded sections with green circles), to calculate
percentage of unique start axons represented at each interval. Reproduced with permission.[162] Copyright 2015, Elsevier. (b) A patient’s sciatic nerve
was reconstructed based on MR neurography, and then a personalized nerve guidance conduit (NGC) was fabricated. The images show an intraop-
erative photograph of the NGCs for nerve regeneration in a rat sciatic nerve transection model with 10 mm gap, and the general observations of the
regenerated sciatic nerve at 16 weeks post-surgery. Reproduced with permission.[166] Copyright 2016, Nature Publishing Group. (c) 3D printed graphene
nerve graft conduit at various sizes. Photograph of tubular nerve conduit that was implanted into a human cadaver via longitudinal transection and
wrapped around the ulnar nerve (white arrows). The nerve conduit was then sutured closed along the previously described longitudinal transection
(white dotted line) as well as to the surrounding epinerium and nerve tissue (inset, yellow circle). Excess 3DG nerve conduit length was then cut with
surgical shears to expose additional nerve tissue. Reproduced with permission.[167] Copyright 2015, American Chemical Society. (d) SEM image of
a 3D printed hollow nerve pathway displaying an axially oriented physical cue on the luminal surface. Photograph of an implanted 3D printed nerve
guide prior to suturing. Cultured primary embryonic neurons on the 3D printed, horizontally oriented physical cue (90° reference angle) stained for
tau (green), while cultured Schwann cells on the horizontally oriented physical cue (90° reference angle) stained for GFAP (green) and laminin (red).
Reproduced with permission.[169] Copyright 2015, Wiley-VCH. (e) Printed gel scaffold comprising optimal 5% w/v alginate, 5% w/v carboxymethyl
chitosan, and 1.5% w/v agarose. NSCs (31 d post-printing, including 21 d differentiation) stained with DAPI (blue) and expressed TUJ1 (red), with cell
clusters interconnected by neurites. The lower right panel shows depth coding of cells along the Z-axis (0–59 µm). Reproduced with permission.[174]
Copyright 2016, Wiley-VCH.

and presynaptic terminals. In vivo experiments using a human growth factor (VEGF)-releasing fibrin gel were printed to con-
cadaver nerve model illustrate that 3DG has exceptional han- struct an artificial neural tissue.[168] Compared to the control
dling characteristics and can be intraoperatively manipulated.[167] samples (fibrin without the VEGF or VEGF printed directly
Neurotrophic factors are endogenous molecules critical to in collagen), the printed C17.2 cells in the collagen hydrogel
the maintenance, survival, proliferation and differentiation of showed high viability, and migrated toward the fibrin gel with
various neuronal populations.[151–153] They have been used in a total distance of 102.4 ± 76.1 µm over 3 days due to the
neural tissue engineering to promote axonal regeneration, neu- VEGF induction.[168] Recently, an imaging-coupled 3D printing
ronal plasticity and neurogenesis. In a study, murine neural approach was developed, facilitating customized neuroregener-
stem cells (C17.2), collagen hydrogel, and vascular endothelial ation in previously inaccessible ways.[169] The custom scaffolds

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were fabricated via a microextrusion printing system. The bifur- the purpose of repairing the nervous system, we believe its suc-
REVIEW

cating pathways were augmented with 3D printed biomimetic cess would also pave the way for regenerating neural networks
physical cues and path-specific biochemical cues (nerve growth for engineered complex tissues/organs.
factor, NGF and glial cell line-derived neurotrophic factor,
GDNF).[169] In vitro studies revealed that 3D printed physical
and biochemical cues provide axonal guidance and chemo- 4.2.3. Organs
tractant/chemokinetic functionality. In vivo studies examining
the regeneration of bifurcated injuries across a 10 mm complex Due to the shortage of organs suitable for transplantation,
nerve gap in rats showed that the 3D printed scaffolds achieved researchers are now exploring development of functional,
successful regeneration of complex nerve injuries, resulting full-sized organs using tissue engineering technology. 3D
in enhanced functional return of the regenerated nerve 3D bioprinting holds great promise for achieving all goals, but
printing (Figure 5d).[169] Our group also developed a novel 3D whole-organ bioprinting, incorporating all of these compo-
patterned scaffold, which has tunable porous structure and nents has remained elusive largely due to organ-level com-
embedded core-shell nanoparticles with sustained neurogenic plexities.[20,23,26,35,110,175] Therefore, current research in the 3D
factor (NGF) delivery, using SLA printing and co-axial electro- printed organ field focuses on the biomimetic fabrication of
spraying techniques.[170] The printed scaffold with nerve growth vascularized tissue and their functionalization. In biology, an
factor (NGF) nanoparticles greatly increased the length of neu- organ is a collection of tissues joined in a structural unit to
rites and directed neurite extension of PC-12 cells along the serve a common function, including sensory organ, visceral
fiber. In addition, the 3D printed nanocomposite scaffolds also organ, and others.[1–3] Herein, we mainly focus on visceral
improved the average neurite length of primary cortical neu- organs (internal organs), such as the heart and liver, due to the
rons 3D printing.[170] unique challenges in replicating their highly complex structures
and functions.
4.2.2.2. 3D Printing for CNS Regeneration: In a study, piezoelec-
tric inkjet printing was used to print two types of adult rat CNS 4.2.3.1. Heart: The heart is a muscular organ in humans,
cells, retinal ganglion cell (RGC) neurons and retinal glia.[171] which pumps blood throughout the body in the circulatory
The printing process did not effect RGC/glial survival and RGC system.[110,176] During embryonic development, the heart is
neurite outgrowth itself. Moreover, the printed glial cells could the first functional organ to be developed from the splanch-
retain their growth promoting properties.[171] In another study, nopleuric mesenchyme cell layer. In anatomy, the heart has
3D brain-like structures were printed using a bio-ink consisting four chambers, four valves and a heart wall. The heart valves
of a novel peptide-modified biopolymer, (arginine-glycine- ensure unidirectional flow of blood: the atrioventricular/inflow
aspartic acid)-gellan gum (RGD-GG), combined with primary valves (mitral and tricuspid) and the semilunar/outflow valves
cortical neurons3D printing.[172] The results demonstrated suc- (aortic and pulmonary).[176] Each valve is composed of leaflets
cessful encapsulation, survival and networking of primary cor- and a fibrous annulus wall (root wall). Leaflets and root walls
tical neurons and glial cells in 3D printed RGD-GG modified mainly contain valve interstitial cells (VIC) and smooth muscle
hydrogels, indicating that cortical neurons responded better to cells (SMC) respectively, with valvular endothelial cells (VEC)
the RGD peptide in RGD-coupled GG than to purified GG.[172] covering on the surface.[176] The heart wall is made up of three
In a recent study, two thermoresponsive water-based biode- layers: the inner endocardium, middle myocardium and outer
gradable polyurethane dispersions were synthesized for use epicardium/pericardium.[176] The endocardium is primarily
on self-developed FDM equipment.[173] The 3D printed neural made up of endothelial cells, which act as a blood–heart bar-
stem cell (NSC)-laden construct was implanted into a brain rier to protect the valves and heart chambers. The myocar-
injury model of adult zebrafish. The results showed the 3D dium consisting of cardiomyocytes is the thick muscular layer
construct promoted the repair of damaged CNS and rescued responsible for contraction and relaxation of the heart. The
the function of impaired nervous systems.[173] Most recently, a pericardium is a double-wall fibroserous sac that acts to pro-
novel 3D neural mini-tissue construct (nMTC) was fabricated tect the heart, anchoring it to the surrounding walls, and pre-
by microextrusion bioprinting.[174] Frontal cortical human NSCs venting it from overfilling with blood.[176] Overall, the heart
were used for in situ differentiation toward functional neurons includes three main cellular components, cardiomyocytes,
and supporting neuroglia. The bioink was comprised of algi- fibroblasts, and endothelial cells. In the treatment of serious
nate, carboxymethylchitosan (CMC), and agarose, which form cardiovascular disease (CVD), traditional approaches, including
a gel by chemical cross-linking following extrusion with hNSC autografts, allografts, xenografts, and artificial prostheses, have
encapsulation.[174] The results showed that the differentiation several disadvantages, such as donor tissue shortage, immune
of hNSCs resulted in GABAergic neurons, together with glial rejection, anticoagulation therapy, and limited durability. Tissue
cells expressing astrocyte and oligodendrocyte lineage markers. engineering techniques have shown a promising approach for
Moreover, the neurons are spontaneously active and show a creating engineered tissues to repair congenital defects and/
bicuculline-induced increased calcium response.[174] or diseased cardiovascular tissues. 3D bioprinting has recently
As we discussed above, 3D printing has the capacity to rap- been an efficient approach to reproduce the complexity of struc-
idly fabricate subtle exterior geometries as well as complex tural and functional cardiac tissues.[110]
interior microarchitectures. Therefore, 3D printing has shown The most fatal CVD is myocardial infarction (MI), caused by
its huge potential for neural tissue regeneration. Although the blockage of the coronary arteries. Myocardial ischemia sets
most research currently focuses on neural regeneration with off a series of complicated and irreversible processes, involving

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cell death, scar formation, and ventricular dysfunction.[177] 4.2.3.2. Liver: In the human body, the liver is composed of

REVIEW
Therefore, engineered myocardial tissue has been explored to highly specialized tissue consisting of mostly hepatocytes. It
restore cardiac functions using 3D printing techniques. LIFT- plays a major role in metabolism with numerous functions,
based cell printing techniques were first applied to prepare a including regulation of glycogen storage, decomposition of
polyester urethane urea (PEUU) cardiac patch seeded with red blood cells, plasma protein synthesis, hormone produc-
HUVECs and MSCs in a defined pattern for cardiac regen- tion, and detoxification.[1,176] In anatomy, the liver is divided
eration.[178] Compared with the random patch, the patterned into four lobes, each of which is microscopically made up of
patch showed increased vessel formation and found significant hepatic lobules. The lobules are roughly hexagonal, and consist
functional improvement of infarcted hearts following implan- of hepatocyte plates radiating from a central vein.[176] Between
tation.[178] Human cardiac derived cardiomyocyte progenitor the hepatocyte plates are liver sinusoids, which connect the
cell (hCMPC)-laden alginate hydrogel was used to print the central vein with the portal triads for mixing of the oxygen-rich
3D cardiac construct.[179] The printed construct retained the blood from the hepatic artery and the nutrient-rich blood from
cardiac phenotype with high cell viability. Moreover, the 3D cul- the portal vein.[176] Histology shows two major types of liver
ture enhanced gene expression of the early cardiac transcrip- cell: parenchymal cells and non-parenchymal cells. The paren-
tion factors Nkx2.5, Gata-4 and Mef-2c as well as the sarcomeric chymal cells are hepatocytes that constitute 70∼85% of the liver
protein TroponinT.[179] volume. The non-parenchymal cells are sinusoidal endothelial
Dysfunctional valves caused by stenosis or regurgitation cells (SECs), phagocytic Kupffer cell (KCs), and hepatic stel-
impair the proper opening and closing of the valves, affecting late cells (HSCs), among others. Owing to its location and
efficient heart performance.[110] Compared with traditional multidimensional functions, the liver is also prone to many
approaches, tissue engineering has great potential to address diseases.[176]
current limitations of non-living prosthetics by providing living The liver has extensive regeneration capacity due to high pro-
constructs that can grow, remodel and integrate in patients. For liferation ability of hepatocytes, even if it is subjected to vast
the engineered heart valve, 3D bioprinting can create anatomi- damages. Therefore, various tissue engineering techniques
cally accurate, living, engineered valves with heterogeneous have been developed to fabricate biomimetic liver tissues. How-
mechanical properties and well -distributed multiple cells. ever, the traditional methods have a limited achievement on the
In an earlier study, native anatomic and axisymmetric aortic volumetric liver tissues with highly intercellular adhesion.[183]
valve geometries (root wall and trileaflets) with 12–22 mm 3D bioprinting facilitates the fabrication of complex liver struc-
inner diameters were 3D printed with a dual-nozzle printer.[180] tures with higher cell densities.[110]
PEG-DA hydrogels and alginate hydrogels were utilized to fab- In an earlier work, a 3D hepatocyte/gelatin construct was
ricate the heterogeneous aortic valve constructs with different printed from a 38 layer assembly.[184] The laminated hepato-
mechanical properties. VIC seeded scaffolds maintained near cytes remained viable and performed biological functions in
100% viability over 21 days.[180] In addition, a 3D simplified the construct for more than 2 months. This technique showed
heart valve construct with root and trileaflets was printed from a promising and stepwise approach to reconstitute the struc-
a HAMA/GelMA based hybrid hydrogel.[181] Human aortic ture of the in vivo microenvironment of human livers.[184]
VICs were encapsulated into the bioprinted hydrogel, which Some researchers also focus on fabricating micro-organs
maintained high viability, and remodeled the initial matrix by or organs-on-a-chip using 3D printing for drug metabolic
depositing collagen and glyosaminoglycans.[181] They also used studies.[110] A multi-nozzle extrusion system was applied to
alginate/gelatin hydrogel to bioprint a living 3D heart valve with fabricate human liver hepatocellular carcinoma cell (HepG2)
anatomical architecture (Figure 6a).[182] Direct encapsulation of laden alginate hydrogel in an organized 3D architecture.[185]
aortic root sinus SMCs in the valve root and aortic VICs in the The biofabricated micro-organ device was performed as an
leaflet were viable (81.4 ± 3.4% for SMCs and 83.2 ± 4.0% for in vitro drug metabolism model. The results showed a bio-
VICs) over 7 days in culture. Moreover, the encapsulated SMCs mimetic drug metabolic process in the micro-organ under
expressed higher alpha-smooth muscle actin (α-SMA) in the continuous perfusion flow.[185] In another study, a liver-on-a-
printed stiff matrix, while the soft matrix elevated vimentin chip platform of 3D human HepG2/C3A spheroids was also
expression of the VICs.[182] developed for drug toxicity assessment.[186] The engineered
Some researchers have developed bioprinted cardiac and bioreactor could be interfaced with a bioprinter to fabricate
valve constructs for cardiac and valve tissue engineering, but 3D hepatic spheroids encapsulated within GelMA hydrogel.
a functional 3D heart construct has yet to be explored. The The engineered hepatic constructs remained functional for 30
whole heart organ not only has complicated structure, but is days while monitoring the secretion rates of albumin, alpha-1
also comprised of multiple cell types with spatial distribution, antitrypsin, transferrin, and ceruloplasmin, as well as immu-
associating with specific and integrated functions comparable nostaining for the hepatocyte markers, cytokeratin 18, MRP2
to native tissue. Recently, the FRESH technique has also been bile canalicular protein and tight junction protein ZO-1.[186] In
developed to create a 3D heart construct of a 5-day-old chick addition, an acetaminophen-induced toxic response test in this
embryo, demonstrating the capability of recapitulating the com- platform was performed to verify the effectiveness similar to
plex trabecular structures of a whole heart through CAD mod- that of animal studies.[186]
eling (Figure 6b).[139] Although it is still not a functional heart Recently, metabolically active, anatomical, 3D hepatic
with a vascular network, it offers a potential approach to gen- tissues have also been developed. For example, a double-
erate a large-scale, complex 3D internal and external anatomical nozzle bioprinting technique was used to fabricate an
architecture. anatomical liver structure with a vascular-like network.[187]

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REVIEW

Figure 6. (a) 3D printed aortic valve conduit. Fluorescent image of first two layers of a printed aortic valve conduit; SMC for valve root were labeled by
cell tracker green and VIC for valve leaflet were labeled by cell tracker red. Live/dead assay for encapsulated VIC (i) in the leaflet and SMC (iii) in valve
root after 7 day culture. Representative image of immunohistochemical staining for aSMA (green) and vimentin (red), and Draq 5 counterstaining for
cell nuclei (blue); Staining for VIC (ii) in the leaflet, and staining for SMC (iv) in the root. Reproduced with permission.[182] Copyright 2012, Wiley-VCH.
(b) A dark field image of an explanted embryonic chick heart. A 3D image of the 5-day-old embryonic chick heart stained for fibronectin (green), nuclei
(blue), and F-actin (red) and imaged with a confocal microscope. A cross section of the 3D CAD model of the embryonic heart with complex internal
trabeculation based on the confocal imaging data. A cross section of the 3D printed heart in fluorescent alginate (green) showing recreation of the
internal trabecular structure from the CAD model. A dark field image of the 3D printed heart with internal structure visible through the translucent heart
wall via FRESH technique. Reproduced with permission.[139] Copyright 2015, the American Association for the Advancement of Science. (c) Images (5×)
taken under fluorescent and bright field channels showing patterns of fluorescently labeled hiPSC-HPCs (green) in 5% GelMA and supporting cells
(red) in 2.5% GelMA with 1% GMHA on day 0. Scale bars, 500 µm. Grayscale images (5×) and confocal immunofluorescence images (40×) showing
albumin (Alb), E-cadherin (E-Cad), and nucleus (Dapi) staining of hiPSC-HPCs in 3D triculture constructs. Scale bars, 500 µm in bright field and 100 µm
in fluorescent images. Reproduced with permission.[190] Copyright 2016, National Academy of Sciences.

Adipose-derived stromal cells (ADSCs) were encapsulated throughout the 3D tissue. Moreover, the 3D printed liver tis-
within a gelatin/alginate/fibrinogen hydrogel to form a sues also underwent other biochemical studies for six weeks.
vascular-like network, and hepatocytes laden gelatin/algi- In addition to the liver-specific functions, it also exhibited a
nate/chitosan hydrogel was placed around it. The ADSCs clinically relevant injury response. These results demonstrate
were induced to differentiate into endothelial-like cells with the potential utility of human 3D bioprinted liver tissues
endothelial growth factor. The albumin secretion level of in drug discovery and development.[189] Most recently, a 3D
the embedded hepatocytes increased during the 2 week cul- hydrogel-based triculture model that embeds hiPSC-derived
ture, while the levels of urea and alanine transaminase were hepatic progenitor cells (hiPSC-HPCs) with human umbil-
decreased after an increasing profile. These results indicate ical vein endothelial cells and adipose-derived stem cells in a
that this double-nozzle assembly technique could be a pow- microscale hexagonal architecture was developed using a DLP
erful tool for fabricating complex liver constructs with special technique (Figure 6c).[190] In comparison with 2D monolayer
intrinsic/extrinsic structures.[187] Using NovoGenTM bio- culture and a 3D HPC-only model, the 3D triculture model
printing technology (Organovo Holdings, Inc., San Diego, showed both phenotypic and functional enhancements in
CA, USA), 3D liver constructs were fabricated containing the hiPSC-HPCs over weeks of in vitro culture, especially for
architecturally and physiologically relevant features for two improved morphological organization, higher liver-specific
hepatic cell lines and primary hepatocytes.[188,189] Bioprinted gene expression levels, increased metabolic product secretion,
3D hepatic neotissues were further enhanced in complexity and enhanced cytochrome P450 induction.[190]
with the addition of ECs and HSCs. Biochemical studies Overall, although 3D printing of complex and large 3D
demonstrated that several critical liver functions were pre- organs currently remains an arduous challenge, it has shown
sent, and tight junction protein expression was observed promising results toward the generation of ‘mini-organs’ that

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contain the same functional components of large organs.[191] technology is the rapid or accelerated tissue maturation pro-

REVIEW
Mini-organs can be considered a future trend in organ cess, where printed organ constructs should undergo rapid
printing and might be a gateway to fully functional organs. matrix deposition, remodeling, and maturation toward a solid
They can be built in smaller scale than their natural coun- living tissue with bioink degradation, ensuring structural integ-
terparts while closely performing the most vital function of rity, mechanical rigidity and biological functionality for implan-
the associated organ. From the current studies, the simulta- tation. As an alternative, in situ bioprinting is an advanced
neous printing of multiple materials could allow fabrication of trend in organ regeneration, where living cells can be printed
engineered tissues with heterogeneous internal organization in the human body during an operation instead of the post-
of cells and ECM, which could more accurately model native bioprinting process in vitro.[26,50] It can enable growth of thick
tissue organization. Additionally, multiple organ-specific cell tissues in critical defects with the help of vascularization driven
types are required to be spatially organized to form functional by natural processes in the body. Although in situ bioprinted
organs, especially for the integration of vasculature within the skin and bone have been tested, the manufacturing of complex
constructs. organs is still uncertain in view of the limited success of their
fabrication in vitro.[26]
Finally, in order to ensure effective industrial translation and
5. Current Challenges and Future Perspectives commercialization of organ printing technology, the key issue
is quality assurance and regulation of bioinks, bioprinters and
Organ printing is a multidisciplinary technology including bioprinted products. This customizable 3D product requires
biology, engineering, materialogy, computer science, and medi- a comprehensive regulation to assure quality control in every
cine, which requires a multidisciplinary team and long-term sus- step of the process: production of printing equipment and raw
tainable financial support. Over the past few years, researchers materials (bioprinter, biomaterials, biological factors and cells),
not only have demonstrated proof-of-concept examples of artifi- design control of the 3D printed model, validation of the manu-
cial tissue fabrication using different bioprinting technologies, facturing along with its governing software, product testing and
but also have shown possibilities that 3D bioprinting offers finally the implantation process. The FDA issued draft guidance
an effective method of recapitulating structural and functional on May 10, 2016, titled “Technical Considerations for Additive
complexity for engineered tissue fabrication, especially for func- Manufactured Devices”, which provides guidance for manufac-
tional organ fabrication. Many of the challenges facing the 3D turers who are producing devices through 3D printing tech-
bioprinting field relate to specific technical, material, and cel- niques. In March, the FDA approved Aprecia Pharmaceuticals’
lular aspects. SPRITAM® (levetiracetam), which was the first FDA-approved
Considering the pathway from 3D organ printing to implanta- drug manufactured using 3D printing. In addition to regula-
tion into a human in a reasonable amount of time, standardized tion, ethical concerns will be considered for future attempts.
protocols involving patient-specific design, fabrication tech- Although the majority of the trials have been made on animals,
niques, maturation processes, surgical operations and post- ethical concerns will be raised when printing tissues or organs
operative care are essential for customized functional organ for implantation in humans.
fabrication. First, considering printing techniques, current New niches for technological advancements on instrumen-
3D tissue/organ printing has to address many technical chal- tation, with improved spatial and temporal resolutions as well
lenges to increase the resolution, printing speed and flexibility as optimized bioinks and cell sources for specific organs,
with relevant biomaterials for creating more complex and com- provide promise that 3D bioprinting will eventually become
posite tissue/organ structures at clinically relevant sizes. Sec- one of the most efficient, reliable, and convenient methods
ondly, bioinks and cells must be considered. The 3D construct to biofabricate tissue constructs in the near future. The high
generated by bioprinting serves as a biomimetic construct with flexibility and controllability of 3D bioprinting enables com-
desired composition and cellular contribution to support func- plex and tailored release profiles of multiple active pharma-
tionality. Moreover, it is unlikely that any single material and ceuticals with spatiotemporal gradients for regulating cellular
single cell possess all the properties required to recapitulate functions during tissue/organ regeneration.[82,192] A unique
tissue function. Therefore, developing appropriate bioink formu- aspect of this technology is its ability to achieve a personalized
lations associating with cell sources with sufficient supply are therapeutic schedule to address individual patient needs.[193]
very important. The third consideration is fabrication strategy. Moreover, advanced materials engineering approaches fea-
Organs have highly complex architectures and properties; as turing biologically dynamic variations will further allow tem-
such they may require a combination of several bioprinting poral evolution of bioprinted tissue constructs that potentially
techniques along with specifically designed bioinks to introduce meet the requirements of dynamic tissue remodeling during
structural heterogeneity and functionality. Highly repeatable and developmental processes. For instance, 4D bioprinting tech-
straightforward technologies and protocols should be developed niques have been proposed,[27] and our lab has developed
to print the organs in logical steps, from simple to complex. The some 4D bioprinted constructs for regulating cell/scaffold
fourth consideration is the manufacturing process. Considering behavior.[61,194] Furthermore, 3D bioprinting techniques have
the micro-scale resolution in cellular bioprinting, the process shown the potential to facilitate the development of real-
requires enough nutrients and oxygen supplementation for istic tissue/organ models, therefore this technology is also
sustaining scalable living constructs in a stable, sterile printing expected to translate advancing the needs of other specific
system over long printing times. The post-bioprinting process applications such as models for pharmaceutical/toxicological
is the fifth consideration. Another challenge for organ printing screening.[36,195]

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