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Title
Bioinks for 3D bioprinting: an overview.
Permalink
https://escholarship.org/uc/item/4520s2n6
Journal
Biomaterials science, 6(5)
ISSN
2047-4830
Authors
Gungor-Ozkerim, P Selcan
Inci, Ilyas
Zhang, Yu Shrike
et al.
Publication Date
2018-05-01
DOI
10.1039/c7bm00765e
Peer reviewed
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University of California
Biomaterials
Science
REVIEW
Cite this: DOI: 10.1039/c7bm00765e
Received 22nd August 2017,
Accepted 12th January 2018
DOI: 10.1039/c7bm00765e
rsc.li/biomaterials-science
Bioinks for 3D bioprinting: an overview
P. Selcan Gungor-Ozkerim,†
a,b,c
Ilyas Inci, †
a,b
Yu Shrike Zhang, †
a,b,d
Ali Khademhosseini
a,b,d,e,f,g,h,i,j
and Mehmet Remzi Dokmeci *
a,b,d,e,f
Bioprinting is an emerging technology with various applications in making functional tissue constructs to
replace injured or diseased tissues. It is a relatively new approach that provides high reproducibility and
precise control over the fabricated constructs in an automated manner, potentially enabling high-
throughput production. During the bioprinting process, a solution of a biomaterial or a mixture of several
biomaterials in the hydrogel form, usually encapsulating the desired cell types, termed the bioink, is used
for creating tissue constructs. This bioink can be cross-linked or stabilized during or immediately after
bioprinting to generate the final shape, structure, and architecture of the designed construct. Bioinks may
be made from natural or synthetic biomaterials alone, or a combination of the two as hybrid materials. In
certain cases, cell aggregates without any additional biomaterials can also be adopted for use as a bioink
for bioprinting processes. An ideal bioink should possess proper mechanical, rheological, and biological
properties of the target tissues, which are essential to ensure correct functionality of the bioprinted
tissues and organs. In this review, we provide an in-depth discussion of the different bioinks currently
employed for bioprinting, and outline some future perspectives in their further development.
1. Introduction
Biofabrication is an emerging research area and includes the
creation of tissue constructs with a hierarchical architecture.
Conventional biofabrication techniques include, for example,
particulate leaching, freeze-drying, electrospinning, and micro-
engineering.
1
Although these techniques can all generate
three-dimensional (3D) structures with a wide range of bioma-
terials, they typically possess limited reproducibility and versa-
tility in their fabrication procedures. The most recent defi-
nition of biofabrication is the generation of biologically func-
tional products in an automated manner with structural
organization by using bioactive molecules, living cells, and cell
aggregates, such as micro-tissues, biomaterials, or hybrid cell-
material constructs via bioassembly or bioprinting, and sub-
sequent tissue maturation processes.
2
More recently, 3D bio-
printing has emerged as a novel biofabrication method,
offering significantly improved control over the architecture of
the fabricated tissue constructs with high reproducibility
endowed by the automated deposition process.
3–5
Essentially,
bioprinting allows for the fabrication of 3D tissue constructs
with pre-programmed structures and geometries containing
biomaterials and/or living cells (together termed the bioink)
by synchronizing the bioink deposition/cross-linking with the
motorized stage movement. Although bioprinting is still in its
early developmental stages, its versatility has continued to
accelerate the applications in tissue engineering.
6
The main 3D bioprinting modalities (Fig. 1),
3
in general
can be classified as: laser-assisted bioprinting (LaBP), inkjet
bioprinting/droplet bioprinting, and extrusion-based
bioprinting.
7–10
In addition, the use of multi-head deposition
systems (MHDSs) allows the simultaneous or subsequent
printing of multiple materials. Besides, there are several
custom-made bioprinting systems developed with various
application-specific functions.
7–10
In these methods, 3D con-
† These authors contributed equally to this work.
a
Biomaterials Innovation Research Center, Division of Engineering in Medicine,
Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School,
Cambridge, MA 02139, USA
b
Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute
of Technology, Cambridge, MA 02139, USA.
E-mail: mdokmeci@rics.bwh.harvard.edu
c
Department of Biomedical Engineering, Faculty of Engineering and Natural Sciences,
Biruni University, Istanbul 34010, Turkey
d
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston,
MA 02115, USA
e
Department of Radiology, David Geffen School of Medicine, University of California-
Los Angeles, Los Angeles, CA 90095, USA
f
Center for Minimally Invasive Therapeutics (C-MIT), University of California-Los
Angeles, Los Angeles, CA 90095, USA
g
Department of Bioindustrial Technologies, College of Animal Bioscience and
Technology, Konkuk University, Seoul 143-701, Republic of Korea
h
Center for Nanotechnolog y, King Abdulaziz University, Jeddah 21569, Saudi Arabia
i
Department of Bioengineering, Department of Chemical and Biomolecular
Engineering, Henry Samueli School of Engineering and Applied Sciences, University
of California-Los Angeles, Los Angeles, CA 90095, USA
j
California NanoSystems Institute (CNSI), University of California-Los Angeles,
Los Angeles, CA 90095, USA
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structs are programmed in a computer-aided design/computer-
aided manufacturing (CAD/CAM) system. In all of these
different bioprinting strategies, however, the bioinks are an
essential component, and are cross-linked or stabilized during
or immediately after the bioprinting to create the final shapes
of the intended tissue constructs. The selection of the bioink
depends on the specific application (e.g., target tissue) and the
type of cells as well as the bioprinter to be used. While the
field has seen significant development in the bioprinting mod-
alities (reviewed elsewhere),
7–10
their applications have been
limited by the lack of appropriate bioinks, which both need to
meet the requirements for bioprinting and to have the proper
bioactivity of the different cell types.
An ideal bioink should possess the desired physicochemical
properties, such as proper mechanical, rheological, chemical,
and biological characteristics.
11
These properties should lead
to: (i) the generation of tissue constructs with adequate
mechanical strength and robustness, while retaining the
tissue-matching mechanics, preferably in a tunable manner;
(ii) adjustable gelation and stabilization to aid the bioprinting
of structures with high shape fidelity; (iii) biocompatibility
and, if necessary, biodegradability mimicking the natural
microenvironment of the tissues; (iv) suitability for chemical
modifications to meet tissue-specific needs; and (v) the poten-
tial for large-scale production with minimum batch-to-batch
variations.
12
Since determining the optimal cell-laden bioink
formulation is the vital step toward successful bioprinting, to
date, various natural and synthetic biomaterials with specific
features have been utilized as bioinks.
13
Moreover, standar-
dized bioink formulations are urgently needed that allow for
their use in different bioprinting applications.
The goal of this review is therefore to present an in-depth
overview of the state of the art in existing bioinks. The review
includes both natural and synthetic biomaterials used either
alone or in combination, and in certain cases, multicellular
spheroids used as bioinks (Table 1). Those studies in which
biopolymers are printed without embedded cells and those
where the cells are seeded post-printing are excluded from the
review, and only bioinks that contain cells are discussed. We
first describe natural bioinks and then move on to synthetic
bioinks and multicellular spheroids, followed by several com-
mercial bioinks, where the main advantages and drawbacks of
P. Selcan Gungor-Ozkerim
Selcan Gungor Ozkerim is a
bioengineer and holds an MSc in
Biomedical Engineering. Her
PhD was based on designing
complex 3D tissue scaffolds for
tissue engineering purposes. She
developed a novel multilayered
nanofibrous scaffold system by
using various polymeric bioma-
terials in combination with con-
trolled release systems. She
worked at Harvard –
Massachusetts Institute of
Technology (MIT) in the Health
Sciences & Technology (HST) division as a postdoctoral research
fellow and studied the 3D bioprinting of cell-laden tissue con-
structs. She generated viable and perfusable functional vascular
constructs that could be utilized for the vascularization of various
tissue scaffolds.
Ilyas Inci
Ilyas Inci received his Ph.D in
Bioengineering at Hacettepe
University in Turkey. His Ph.D
study was on the effect of cryogel
scaffolds for calvarial defect
repair. After his Ph.D, he worked
as a post-doctoral research
fellow in Sabanci University in
Turkey and then he attended
Harvard-Massachusetts Institute
of Technology (MIT), Health
Sciences and Technology division
to do research on tissue engin-
eering as a post-doctoral
research fellow. His research interests mainly focus on using bio-
compatible scaffolds to develop therapeutic methods in tissue
engineering and analyzing the effects of applied tissue engineering
methods on molecular signaling during tissue regeneration.
Fig. 1 Schematic representation of the main 3D bioprinting technologies. (a) Inkjet/droplet bioprinting. (b) Extrusion-based bioprinting. (c) Laser-
assisted bioprinting.
3
Reproduced with permission Copyright Nature Publishing Group, 2014.
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each category are discussed. We subsequently provide a short
section to elaborate on some of the new bioink formulation
strategies that are not mentioned in the preceding sections.
We finally conclude with future perspectives on the remaining
challenges associated with the current bioinks and the further
need for standardization.
2. Hydrogel-based bioinks
Hydrogels have numerous attractive features for use as tissue
scaffolds. For example, they are biocompatible and typically
biodegradable, and a majority of them possess specific cell-
binding sites that are desirable for cell attachment, spreading,
growth, and differentiation. In addition, some of these bio-
materials in their modified forms can be readily photocross-
linked.
14
Hydrogel biomaterials, including alginate, gelatin,
collagen, fibrin/fibrinogen, gellan gum, hyaluronic acid (HA),
agarose, chitosan, silk, decellularized extracellular matrix
(dECM), poly(ethylene glycol) (PEG), and Pluronic, and their
use as bioinks will be discussed in the following section.
2.1. Protein-based bioinks
Collagen is the main structural protein in the extracellular
matrix (ECM) of mammalian cells. Collagen thus possesses
tissue-matching physicochemical properties, together with
superior in vitro/in vivo biocompatibility, and has been widely
used in biomedical applications.
15
Koch et al. used collagen as
a bioink formulation with encapsulated keratinocytes and
fibroblasts and bioprinted multilayer 3D skin tissue constructs
via LaBP.
16
They investigated the viability and morphological
functions of both cell types following bioprinting. Their find-
ings demonstrated the presence of intercellular communi-
cations between different cell types and suggested that the fab-
ricated skin grafts had tissue-specific functions, which are
promising for fabricating complex multicellular tissue con-
structs. The same group further evaluated the in vivo appli-
cations of these constructs and demonstrated the formation of
3D skin-like tissue via proliferation and proper differentiation
of the cells.
17
Another study also utilized a collagen bioink for
skin tissue engineering and created mature tissues with dis-
tinct cell layers.
18
Moon et al. developed a valve-based droplet
ejector system to bioprint smooth muscle cells (SMCs)-laden
collagen droplets, which formed line patterns with layer-by-
layer deposition and demonstrated uniform cell seeding with
controlled resolution.
19
In another study, the differentiation
potential of bioprinted mesenchymal stem cells (MSCs) was
investigated.
20
Here, MSCs were encapsulated in collagen
alone or collagen–agarose blend bioink and were bioprinted
using an extrusion-based platform. The collagen-only matrix
supported the spreading of the cells following printing, in con-
trast to the behavior of the cells in the hybrid matrix made
with agarose, which kept its structural integrity but did not
allow cell spreading. The results revealed that the anisotropic
soft collagen-rich matrices were better suited for osteogenesis,
whereas the isotropic stiff agarose-rich matrices supported adi-
pogenesis of the bioprinted 3D constructs. Collagen may also
be combined with alginate for use as a composite bioink. Also,
preosteoblasts and human adipose tissue-derived stem cells
(ASCs) were encapsulated in this bioink to bioprint 3D porous
cellular blocks.
11
Here, the cells were first cultured on a col-
lagen gel and then the cell-laden collagen gel was combined
with alginate. The results showed that the osteogenic potential
of the collagen-alginate bioink was higher than the one with
alginate alone. Moreover, hepatic lineage differentiation of the
ASCs was also achieved in the bioprinted blocks, implying that
Yu Shrike Zhang
Yu Shrike Zhang received his Ph.
D. from Georgia Institute of
Technology in the Wallace
H. Coulter Department of
Biomedical Engineering in 2013.
He is currently a Faculty and
Associate Bioengineer in the
Division of Engineering in
Medicine at the Brigham and
Women’s Hospital, Harvard
Medical School. Dr Zhang’s
research is focused on innovating
medical engineering technologies
to recreate functional biomimetic
tissues and tissue models, including biomaterials, bioprinting,
organs-on-chips, medical devices, biomedical imaging, and biosen-
sing. He is actively collaborating with a multidisciplinary team
encompassing biomedical, mechanical, electrical, and computer
engineers as well as biologists and clinicians to ultimately trans-
late these cutting-edge technologies into clinics.
Ali Khademhosseini
Ali Khademhosseini is the Levi
Knight Professor of
Bioengineering, Chemical
Engineering and Radiology at
the University of California-Los
Angeles (UCLA). He is the
Founding Director of the Center
for Minimally Invasive
Therapeutics (C-MIT) at UCLA
and the Associate Director of
the California NanoSystems
Institute. Previously, he was a
Professor of Medicine at Harvard
Medical School. He is recognized
as a leader in combining micro- and nano-engineering approaches
with advanced biomaterials for regenerative medicine applications
and has authored ∼500 journal papers (H-index >98 & >35 000
citations). He is a fellow of AIMBE, BMES, RSC, FBSE, and AAAS.
Read more at: http://www.tissueeng.net/.
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this new bioink could be used in various tissue engineering
applications. Collagen is also widely used as a biopaper in bio-
printing applications. Biopaper is the substrate used in bio-
printing and is the analog of the media used in standard print-
ing processes and it is commonly referred to the hydrogel
surface on which the cell-laden bioink or cell spheroids can be
bioprinted.
21–23
Gelatin is produced by the denaturation of collagen.
24
It
can be derived from bones, tendons, or skins of animals via
acidic or basic hydrolysis. Its solution is thermosensitive and
can form a hydrogel at lower temperatures in a concentration-
dependent manner, and consequently it is one of the most
widely used natural polymers for many biomedical appli-
cations. Some of the superior advantages of gelatin include
biocompatibility, biodegradability, low antigenicity, inclusion
of intrinsic Arg-Gly-Asp (RGD) motifs, accessible active groups,
absence of harmful byproducts, ease of processing, and low
cost.
25–27
All of these properties, and especially its cellular
affinity, make it a versatile material for applications in tissue
engineering and bioprinting. For bioprinting applications,
gelatin with a wide range of concentrations has been used as a
bioink material and/or as a composite with other polymers. In
addition, its modified forms, which can be chemically cross-
linked, have also been adapted for bioprinting, such as gelatin
methacryloyl (GelMA).
28
Zhang et al. used gelatin–alginate composite bioinks to
encapsulate myoblasts and investigated the mechanical pro-
perties of the bioprinted soft tissue constructs.
29
By using a
dual-nozzle system, they bioprinted 3D filaments with
different structural configurations, such as layers with specific
angles. The process consisted of a two-step cross-linking pro-
cedure; the physical cross-linking of gelatin at a low tempera-
ture during bioprinting and ionic cross-linking of the alginate
with Ca
2+
ions following the bioprinting step. They observed
that the mechanical strength of the cell-laden constructs
decreased during the culture period but the low-porosity and
the angled geometry of the structures supported their mechan-
ical durability. Although bioprinting at low temperatures
resulted in a drastic decrease in cell viability during the
first few days, cell proliferation increased over culture. In
another study, a gelatin–alginate composite bioink was used
for bioprinting hard tissue constructs.
30
The authors reported
that human osteosarcoma cells remained in a non-proliferat-
ing state within this composite bioink matrix, so they pro-
posed filling the construct with an agarose overlay following
bioprinting. Moreover, they added [polyphosphate (polyp).
Ca
2+
-complex] into the overlay for better mineral deposition.
This combined system significantly improved both the cellular
proliferation and mechanical properties of the cell-laden con-
structs, indicating that this system could be extended for other
tissue engineering applications by adapting it for specific
tissue needs. Another study utilized gelatin–alginate hydrogels
for bioprinting cell-laden aortic valve conduits designed from
micro-computed tomography images.
31
They simultaneously
bioprinted encapsulated SMCs in the valve root part and valve
leaflet interstitial cells in the leaflet part of the constructs in
line with their anatomical regions by using an extrusion-based
bioprinter. The bioprinted constructs were close to clinical
dimensions and were cross-linked in a CaCl
2
solution post-
printing. It was noted that the optimal ratio of the gelatin and
alginate combination in the blend was crucial for the printing
quality for enabling proper cell growth, spreading, and pheno-
typic maturation. Using the same bioprinting system, Wüst
et al. fabricated 3D tubular constructs using a composite
hydrogel with tunable properties.
32
Additionally, they used
syringe tip heaters to control the temperature of the bioink,
which prevented clogging and hence improved the bioprinting
process. They supplemented the gelatin/alginate bioink with
hydroxyapatite (HAp), which is an osteoinductive agent with
numerous applications in bone tissue engineering.
Due to its thermoresponsive property, gelatin can be tuned
and physically cross-linked during bioprinting by thermal gela-
tion, which helps to maintain the shapes of the bioprinted
structures. However, temperature-induced gelation is typically
slow and unstable. To address this problem, gelatin has been
further modified with photopolymerizable methacryloyl
groups, enabling covalent cross-linking by UV light under mild
conditions following the bioprinting process.
33–37
This function-
alized form of gelatin, namely GelMA, is a promising bioink
material because its cross-linking density can be easily con-
trolled during methacryloyl group activation or during photo-
polymerization, which determines the physicochemical pro-
perties of the final construct. In bioprinting, since preservation
of the integrity and mechanical strength of the bioprinted
constructs are two of the most important criteria, GelMA is
a suitable material for meeting these requirements. Our
group reported a strategy for bioprinting cell-laden GelMA
bioinks by using an extrusion-based bioprinter.
38
Various
GelMA and cell concentrations and different UV exposure
times were explored to evaluate the printability of cell-laden
Mehmet Remzi Dokmeci
Mehmet Remzi Dokmeci is an
Associate Adjunct Professor of
Radiology at the University of
California-Los Angeles (UCLA).
Previously, he was an Instructor
of Medicine at Brigham and
Women’s Hospital, Harvard
Medical School. He has also
worked at Corning-Intellisense, a
MEMS foundry and was a faculty
member in the Department of
Electrical and Computer
Engineering at Northeastern
University. Dr Dokmeci has long
standing expertise in micro- and nanoscale sensors and devices
and related applications to biomedical devices, organs on a chip,
regenerative medicine and implantable biosensors. He has written
over 116 journal articles, 110 conference publications, 4 book
chapters, and has 4 patents.
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