Recent Advances in Bioink Design for 3D Bioprinting of Tissues and Organs

Abstract

There is a growing demand for alternative fabrication approaches to develop tissues and organs as conventional techniques are not capable of fabricating constructs with required structural, mechanical, and biological complexity. 3D bioprinting offers great potential to fabricate highly complex constructs with precise control of structure, mechanics, and biological matter [i.e., cells and extracellular matrix (ECM) components]. 3D bioprinting is an additive manufacturing approach that utilizes a “bioink” to fabricate devices and scaffolds in a layer-by-layer manner. 3D bioprinting allows printing of a cell suspension into a tissue construct with or without a scaffold support. The most common bioinks are cell-laden hydrogels, decellulerized ECM-based solutions, and cell suspensions. In this mini review, a brief description and comparison of the bioprinting methods, including extrusion-based, droplet-based, and laser-based bioprinting, with particular focus on bioink design requirements are presented. We also present the current state of the art in bioink design including the challenges and future directions.

Full text

April 2017 | Volume 5 | Article 231
MINI REVIEW
published: 05 April 2017
doi: 10.3389/fbioe.2017.00023
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org
Edited by:
Giovanni Vozzi,
University of Pisa, Italy
Reviewed by:
Piergiorgio Gentile,
University of Shefeld, UK
Arti Ahluwalia,
University of Pisa, Italy
*Correspondence:
Murat Guvendiren
muratg@njit.edu
Specialty section:
This article was submitted to
Bionics and Biomimetics,
a section of the journal
Frontiers in Bioengineering and
Biotechnology
Received: 25January2017
Accepted: 21March2017
Published: 05April2017
Citation:
JiS and GuvendirenM (2017) Recent
Advances in Bioink Design for 3D
Bioprinting of Tissues and Organs.
Front. Bioeng. Biotechnol. 5:23.
doi: 10.3389/fbioe.2017.00023
Recent Advances in Bioink Design
for 3D Bioprinting of Tissues and
Organs
Shen Ji and Murat Guvendiren*
Instructive Biomaterials and Additive Manufacturing (IBAM) Laboratory, Otto H. York Department of Chemical Biological and
Pharmaceutical Engineering, New Jersey Institute of Technology, Newark, NJ, USA
There is a growing demand for alternative fabrication approaches to develop tissues and
organs as conventional techniques are not capable of fabricating constructs with required
structural, mechanical, and biological complexity. 3D bioprinting offers great potential to
fabricate highly complex constructs with precise control of structure, mechanics, and
biological matter [i.e., cells and extracellular matrix (ECM) components]. 3D bioprinting
is an additive manufacturing approach that utilizes a “bioink” to fabricate devices and
scaffolds in a layer-by-layer manner. 3D bioprinting allows printing of a cell suspension
into a tissue construct with or without a scaffold support. The most common bioinks are
cell-laden hydrogels, decellulerized ECM-based solutions, and cell suspensions. In this
mini review, a brief description and comparison of the bioprinting methods, including
extrusion-based, droplet-based, and laser-based bioprinting, with particular focus on
bioink design requirements are presented. We also present the current state of the art in
bioink design including the challenges and future directions.
Keywords: additive manufacturing, biofabrication, tissue engineering, regenerative medicine, hydrogel, cell
printing, extracellular matrix
INTRODUCTION
Tissue engineering is a multidisciplinary eld currently focused on two major areas: (i) developing
new methods to repair, regenerate, and replace damaged tissues and organs and (ii) creating invitro
tissue models to better understand tissue development, disease development, and progression and
to develop and screen drugs (Langer and Vacanti, 1993; Grith and Naughton, 2002; Benam etal.,
2015; Tibbitt etal., 2015; Nguyen etal., 2016; Zhang etal., 2016). Despite recent advances in tissue
engineering, there is a continuous lack of tissues and organs for transplantation and a shortage
for tissue models for drug discovery and testing (Bajaj et al., 2014). Conventional techniques,
such as porogen-leaching, injection molding, and electrospinning, are generally recognized as the
bottleneck due to limited control over scaold architecture, composition, pore shape, size, and
distribution (Murphy and Atala, 2014; Groen etal., 2016; Shaee and Atala, 2016). 3D bioprinting
enables fabrication of scaolds, devices, and tissue models with high complexity (Murphy and
Atala, 2014; Mandrycky etal., 2016; Ozbolat etal., 2016, 2017; Shaee and Atala, 2016). 3D print-
ing allows construction of tissues from commonly used medical images (such as X-ray, magnetic
resonance imaging, and computerized tomography scan) using computer-aided design. Custom
and patient-specic design, on-demand fabrication, high structural complexity, low-cost, and

FIGURE 1 | 3D bioprinting techniques for bioprinting of tissues and organs. Figure reproduced with permission from Miller and Burdick (2016). Copyright
2016, American Chemical Society.
2
Ji and Guvendiren Bioinks for Tissue and Organ Printing
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org April 2017 | Volume 5 | Article 23
high-eciency are some of the major advantages of 3D printing
making it very attractive for medicine (Guillemot etal., 2010;
Guvendiren etal., 2016).
3D bioprinting is a technology to fabricate constructs from
living cells with or without a carrier material in a layer-by-layer
manner (Dababneh and Ozbolat, 2014; Murphy and Atala, 2014;
Mandrycky etal., 2016; Shaee and Atala, 2016; Cui etal., 2017).
e material that is printed is referred to as a “bioink,” which can
be dened as an ink formulation that allows printing of living
cells. Here, we would like to note that many of the biomaterial
ink formulations are not suitable for cell printing. For instance,
polycaprolactone (PCL) and poly(lactic acid) (PLA) are the most
widely used biomaterials in 3D printing. However, they could
only be printed at elevated temperatures in the form of a polymer
melt or when dissolved in organic solvents as a polymer solution.
erefore, they are not considered as bioinks in this review, as
both approaches are not suitable for live cell printing (Jose etal.,
2016; Munaz etal., 2016). In this paper, we discuss the most com-
monly used bioinks, including cell-laden hydrogels, extracellular
matrix (ECM)-based solutions, and cell suspensions (Levato
etal., 2014; Adam etal., 2016; Guvendiren etal., 2016; Panwar
and Tan, 2016), and give the current state of the art in bioink
design with challenges and future directions. A brief description
and comparison of the bioprinting methods with particular focus
on bioink design requirements are also given.
3D BIOPRINTING TECHNOLOGIES
3D bioprinting process should be relatively mild and cell friendly
as it is required to allow cell printing (Ozbolat etal., 2016, 2017).
is requirement limits the number of 3D printing techniques
that are suitable for bioprinting (Figure 1). It is important to
note that the 3D printing technology determines the require-
ments for printability of a material, and not all of the 3D printing
technologies are suitable for bioprinting. Currently available
3D printing technologies allow a wide range of materials to be
printed using diverse ink formulations (Guvendiren etal., 2016).
Fused deposition modeling (FDM) is an extrusion-based print-
ing and utilizes synthetic thermoplastics and their composites
with ceramics and metals (Turner et al., 2014). For FDM, the
form of ink material is a lament, and it is extruded at elevated
temperatures (140–250°C) in melt state, which eliminates FDM
as an option for bioprinting. Direct ink writing (DIW) is also an
extrusion-based printing and allows extrusion of high viscosity
solutions, hydrogels, and colloidal suspensions (Ozbolat and
Hospodiuk, 2016). DIW allows printing of cell suspensions
and/or aggregates with or without a carrier. Inkjet printing is
another technology for cell printing. e processing principle
is deposition of polymeric solutions, colloidal suspensions,
and cell suspensions, with relatively low viscosities [<10 cP
(mPa⋅s)] at relatively high shear rates (105–106s−1) in the form
droplets (~50μm in diameter) (Mironov etal., 2003; Wilson and
Boland, 2003a,b; Nakamura etal., 2005; Gudapati etal., 2016).
As compared to extrusion-based bioprinters, inkjet bioprinters
are not readily available, yet there are commercially available
inkjet print heads that are suitable for bioprinting (Nishiyama
etal., 2008; Choi et al., 2011). Selective laser sintering utilizes
metals, ceramics, polymers, and composites in powder form
(10–150µm in diameter) and is not suitable for bioprinting. In
this technique, a directed laser beam locally melts either directly
the powder or a polymeric binder onto the bed surface (Shirazi
etal., 2015). Layers of fresh powder are continuously supplied
aer each layer is created. Stereolithography (SLA) requires a
viscous photocurable polymer solution or a prepolymer, which
is exposed to a directed light (such as UV or laser) to spatially
cross-link the solution (Skoog etal., 2014). SLA could potentially
be considered for printing live cells as long as a cell-laden pre-
polymer formulation is used and the photocuring takes place in
a mild, cell friendly condition, which are the two major issues for
SLA in bioprinting (Elomaa etal., 2015; Wang etal., 2015; Morris
etal., 2017). When 3D printing technologies are considered for
bioprinting, the most commonly used technologies are DIW and
inkjet printing (Ozbolat etal., 2016, 2017). In addition to these
technologies laser-induced forward transfer (LIFT) is also shown
to be suitable for bioprinting (Barron etal., 2004a,b; Ringeisen
etal., 2004; Hopp etal., 2005; Doraiswamy etal., 2006; Koch etal.,
2010). In this technique, ink solution is coated onto a glass slide
and coated with a laser absorption layer (metal or a metal oxide).
Laser is directed to the laser absorption layer with an ablation
spot size between 40 and 100 µm in diameter (Barron et al.,
2004a,b; Koch etal., 2010) creating a local pressure to eject the
ink layer to the substrate.

3
Ji and Guvendiren Bioinks for Tissue and Organ Printing
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org April 2017 | Volume 5 | Article 23
BIOINK DESIGN
The ideal bioink formulation should satisfy certain material
and biological requirements. Material properties are print-
ability, mechanics, degradation, and functionalizability.
Biological requirements mainly include biocompatibility,
cytocompatilibilty, and bioactivity. When material properties
are considered, printability is the most important parameter.
Printability comprises two parts: (i) the processability of
the bioink formulation and (ii) the print fidelity associated
with the mechanical strength of the printed construct to
self-sustain a 3D structure post-printing. Depending on the
printing process, printability could potentially involve solu-
tion viscosity, surface tension, and cross-linking properties.
Viscosity is a crucial parameter for a bioink formulation as it
affects both the print fidelity and cell encapsulation efficiency.
High viscosity polymer solutions are less likely to flow easily
so that the printed structure could hold its shape at longer
times post-printing. However, they require higher pressures
to flow, limiting the gage size and smallest achievable print
size (mainly for DIW). In this regard, Tirella et al. (2009)
investigated the processing window for alginate hydrogels
using pressure-assisted microfabrication (DIW technique).
They successfully developed a 3D phase diagram showing the
interplay between bioink viscosity, print velocity, and applied
pressure to obtain high print fidelity (Tirella etal., 2009). The
bioink formulation is preferred to have a tunable viscosity to
be compatible with different bioprinters. For instance, bioinks
for inkjet or droplet-based bioprinters have viscosity values
close to 10 mPa⋅s (Gudapati et al., 2016); the viscosity of
bioinks for extrusion-based DIW bioprinting ranges from 30
to 6×107mPa⋅s (Hölzl etal., 2016; Ozbolat etal., 2016, 2017);
for laser-assisted bioprinting, the bioink viscosity is in the
range of 1–300mPa⋅s (Guillotin etal., 2010; Hölzl etal., 2016).
For high viscosity bioinks used in extrusion and droplet-based
print, the shear-thinning characteristic is desired to compen-
sate for the high shear stress associated with high viscosity. The
overall mechanics, i.e., achievable stiffness, is important not
only to create self-supporting constructs but also to control
and direct cellular behavior. Degradation is important for
the functional integration of the printed construct invivo by
enabling cells to gradually replace the construct with their
ECM. Both the bioink and the degradation products should
not contain materials that induce inflammatory host response
when implanted. Functionalizability is required to incorporate
biochemical cues, i.e., bioactivity, to direct cellular behavior,
such as adhesion, migration, and differentiation. In addition
to biocompatibility and cytocompatibility, high cell viability,
both prior- and post-printing, is crucial for the ink formula-
tion. In addition to bioink design, a recent study showed the
importance of the print substrate for live cell inkjet printing. In
this work, computational and experimental studies confirmed
that the stiffness of the print substrate directly influences the
impact forces acting on the droplet, which affects the overall
cell survival (Tirella etal., 2011). Below we will discuss the
commonly used bioinks including current state of the art in
ink design.
CURRENTLY AVAILABLE BIOINKS
e most commonly used bioinks for tissue and organ printing
are cell-laden hydrogels, decellularized extracellular matrix
(dECM)-based solutions, and cell suspensions (Figure 2).
Cell-laden hydrogels are particularly attractive due to their
tunable properties and their ability to recapitulate the cellular
microenvironment (Fedorovich etal., 2007). ECM-based bioink
formulations or decellulerized tissue inks are an emerging eld
due to their inherent bioactivity and ease of formulation into a
printable bioink (Pati etal., 2014). Cell suspension inks based on
cell aggregates are a viable option to create scaold-free biological
constructs (Forgacs and Foty, 2004; Marga etal., 2007).
Cell-Laden Hydrogels
Cell-laden hydrogels are the most commonly used bioinks as they
can be easily formulated for extrusion-based (DIW), droplet-
based (inkjet), and laser-based (SLA and LIFT) bioprinting tech-
nologies. Cell-laden hydrogel bioink formulations utilize natural
hydrogels such as agarose, alginate, chitosan, collagen, gelatin,
brin, and hyaluronic acid (HA), as well as synthetic hydrogels
such as pluronic (poloxamer) and poly(ethylene glycol) (PEG), or
blends of both. Natural hydrogels oer inherent bioactivity except
for agarose and alginate and display a structural resemblance to
ECM. For instance, brin and collagen hydrogels with inherent
lamentous structure display strain-stiening property, mimick-
ing the non-linear elastic behavior of the so tissues in our body
(Gardel eta l., 2004; Storm etal., 2005). Synthetic hydrogels permit
but do not promote cellular function, yet there are many ways to
tether bioactive cues into synthetic hydrogels (Guvendiren and
Burdick, 2013). When compared to natural hydrogels, synthetic
hydrogels generally oer tunable mechanical properties. Many
natural polymers (such as gelatin and HA) have functionaliz-
able backbone side chains enabling them to be functionalized
with chemical moieties to induce cross-linking (chemical- and/
or photo-cross-linking) or additional bioactivity (Burdick and
Prestwich, 2011). Blends of synthetic and natural polymers
have been used to develop mechanically tunable hydrogels with
user-dened bioactivity. Finally, the mechanical properties and/
or bioactivity can also be tuned by incorporating small amounts
of nanoparticles into bioink formulation (Ribeiro etal., 2015).
Usually, all hydrogel bioink formulations require printing of
a polymer solution followed by subsequent cross-linking. is
requires a highly viscous polymer solution (polymer wt% >3%)
and rapid cross-linking to develop self-supporting structures.
ere are two forms of cross-linking: physical and chemical
cross-linking. Physical cross-linking is a non-chemical approach
that utilizes hydrophobic interactions, ionic interactions, and
hydrogen bonding. Chemical cross-linking relies on the forma-
tion of covalent bonds, which could be a radical polymerization
(such as photo-cross-linking) or Michael-type addition reaction.
e chemically cross-linked hydrogels form a mechanically robust
network as compared to the physically cross-linked hydrogels,
which is particularly important for the stem cell behavior includ-
ing dierentiation (Huebsch etal., 2010; Khetan etal., 2013).
Pluronic and PEG are the most common synthetic polymers
for bioprinting. Pluronic, a poloxamer-based triblock copolymer

FIGURE 2 | (i) 3D printed constructs in various forms (a,b) using poly(ethylene glycol)–alginate–nanoclay hydrogels. Red food dye was incorporated into some of the
bioink formulations for visibility. Live/dead assay of cells (c) in a collagen infused mesh from (b). Reprinted with permission from Hong etal. (2015). Copyright 2015,
John Wiley and Sons. (ii) Tissue construct printed from decellularized extracellular matrix (dECM) (a), SEM images of hybrid constructs from dECM supported with
polycaprolactone framework (b,c), and uorescent images of cells (d). Scale bars are 5mm for (a), 400µm for (b,c), and 100µm for (d). Adapted with permission
from Pati etal. (2014). Copyright 2014, Nature Publishing Group. (iii) Cell aggregate (500-µm average diameter) congurations in simulations (A,B,K,L) and
experiments. C–J correspond to cell aggregates embedded in a neurogel with RGD fragments (C,D) and collagen gels of concentration 1.0mg/ml (E,F), 1.2mg/ml
(G,H), and 1.7mg/ml (I,J). Figure adapted with permission from Jakab etal. (2004). Copyright 2004, National Academy of Sciences.
4
Ji and Guvendiren Bioinks for Tissue and Organ Printing
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org April 2017 | Volume 5 | Article 23
composed of two hydrophobic groups between a water-soluble
group, has been widely used in extrusion-based bioprinting as
it gels at room temperature but ows at temperatures below
10°C. However, it is not very stable and erodes within hours.
us, it is generally used as a supporting material (Kang etal.,
2016). Lewis Lab took an advantage of this property and printed
pluronic within a photopolymerizable hydrogel to create micro
channels (Wu et al., 2011). Müller et al. (2015) developed an
acrylated pluronic to create UV cross-linked stable gels post-
printing. e most common forms of PEG for bioinks are
PEG-diacrylate (PEG-DA) and PEG-methacrylate, which are
suitable for extrusion-based, droplet-based, and laser-based
printing technologies (Cui etal., 2012; Hribar etal., 2014; Wüst
etal., 2015). PEG is hydrophilic and not adhesive to proteins and
cells; therefore, it requires blending with other natural polymers
or functionalization with biochemical cues. It is possible to form
strong robust hydrogels using PEG-based polymers. For instance,
Hockaday et al. (2012) printed aortic valve geometries using
PEG-DA hydrogels blended with alginate and achieved 10-fold
range in elastic modulus from ~5 to ~75kPa. Hong etal. (2015)
reported 3D printing of tough and biocompatible, cell-laden
PEG–alginate–nanoclay hydrogels infused with collagen. Rutz
etal. (2015) developed partially cross-linked PEG-based multi-
material bioink formulations with tunable viscosity to enhance
print delity and secondary cross-linking ability to stabilize the
constructs.
Alginate is one of the most commonly used natural polymers
to formulate bioinks for inkjet and DIW printing. For inkjet
printing, calcium chloride is jetted onto alginic acid solution
(Boland et al., 2007). For extrusion-based printing, alginate is
printed as a viscous solution, and the constructs are exposed to
CaCl2 solution to induce post-printing cross-linking. Alginate is
not cell adhesive, thus it is generally blended with other natural
polymers (e.g., gelatin and brinogen) to induce cell adhesion
and biological activity (Xu etal., 2009; Jia etal., 2014; Yu et al.,
2014; Lim et al., 2016; Pan etal., 2016). Note that, the major-
ity of the natural polymers are used as a component of bioink
formulation. HA and gelatin that have been utilized extensively
in the form of functionalized polymers thus fall into the synthetic
polymer category, which is discussed below.
Gelatin is commonly used in the form of gelatin methacry-
loyl (GelMA)-based hydrogel for DIW (Bertassoni etal., 2014;
Loessner etal., 2016). Lim etal. (2016) recently reported a visible
light photo-cross-linking system to minimize the oxygen inhi-
bition in photopolymerized GelMA hydrogels. ey reported
higher print delity and cell viability for ruthenium/sodium
persulfate visible photo-initiator as compared to UV photo-
initiator Igracure 2959. Similar to gelatin, HA has been modied
in many ways to create cell-laden bioinks (Highley etal., 2015;
Rodell etal., 2015; Ouyang etal., 2016). For instance, Burdick
lab reported HA-based supramolecular hydrogels cross-linked
by cyclodextrin–adamantane host–guest interactions, which

5
Ji and Guvendiren Bioinks for Tissue and Organ Printing
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org April 2017 | Volume 5 | Article 23
are capable of shear-thinning and self-healing (Highley etal.,
2015). e non-covalent bonds allow direct writing of inks
into support gels. HA hydrogels were developed to display
both shear-thinning behavior due to guest–host bonding and
stabilization post-printing via UV-induced covalent cross-
linking (Ouyang et al., 2016). Supramolecular hydrogels are
particularly attractive for extrusion-based printing as they
could ow under shear and self-heal immediately aer print-
ing, leading to high print delity. In addition to guest–host
bonding, self-assembling peptides (Raphael etal., 2017) and
polypeptide–DNA hydrogels (Li etal., 2015) are other emerg-
ing candidates for bioink design.
Cell Suspension Bioinks
Modied inkjet printers have long been used to print cells into
cellular assemblies. For instance, endothelial cells were printed
from cell suspension (1×105cells/ml) in growth media (Wilson
and Boland, 2003a,b). Bioprinting of scaold-free constructs
utilizes cell aggregates in the form of mono- or multicellular
spheroids as a bioink (Mironov etal., 2003; Norotte etal., 2009;
Jakab etal., 2010; Christensen etal., 2015). e bioink formula-
tion undergoes a fully biological self-assembly without or in the
presence of a temporary support layer (Norotte etal., 2009). is
technique relies on tissue liquidity and fusion, which allow cells
to self-assemble and fuse due to cell–cell interactions (Forgacs
etal., 1998; Jakab etal., 2004; Fleming etal., 2010). For instance,
Norotte etal. developed spheroids and cylinders of multicellular
aggregates with controlled diameter in the range of 300–500µm
and showed that post-printing fusion led to single- and double-
layered vascular tubes. Organovo is the rst medical research
company that uses a similar approach to create functional human
tissues toward invitro disease models. e company has devel-
oped liver models using high density bioinks from parenchymal
cells or non-parenchymal cells that are printed via extrusion-
based printing (Nguyen etal., 2016). Tissues were allowed to
mature in a bioreactor for at least 3days to form scaold-free
tissues. Levato et al. (2014) developed an alternative approach
by combining bioprinting with microcarrier technology, which
allowed extensive expansion of cells on cell-laden PLA-based
microcarriers. Tan etal. (2016) used poly(,-lactic-co-glycolic
acid) porous microspheres enabling cells to adhere and prolifer-
ate before printing.
dECM-Based Bioinks
Decellularized extracellular matrix-based bioinks involve decel-
lularization of a tissue of interest by removing the cells while pre-
serving the ECM. e ECM is then crushed into a powder form
and dissolved in a cell friendly buer solution to formulate the
bioink. A carrier polymer could be used to increase the solubility,
to tune the viscosity, or to induce/enhance post-cross-linking of
the bioink. In this regard, Pati etal. (2014) printed 3D constructs
using dECM-based bioinks supported by a PCL framework.
For this purpose, dECM was obtained from fat, cartilage, and
heart, using a combination of physical, enzymatic, and chemical
processes. ese ink materials were initially solubilized in an
acidic buer, and pH was adjusted to accommodate cells. is
formulation was soluble at 10°C and gelled at 37°C. Following
this study, the same group showed that the dECM bioink can
be pre-gelled using vitamin B2-induced covalent cross-linking
(Jang etal., 2016a,b,c). Using this approach, a 3D printed cardiac
patch composed of multiple-cell lines including human cardiac
progenitor cells and mesenchymal stem cells was developed
(Jang etal., 2016a,b,c). Although dECM bioinks provide novel
opportunities to fabricate tissue specic constructs, the decel-
lularization process requires multiple steps including precise
quantication of the DNA and the ECM components, making it
a costly approach.
SUMMARY AND FUTURE PERSPECTIVES
3D printing has a strong potential to become a common fabrica-
tion technique in medicine as it enables fabrication of modular
and patient-specic scaolds and devices, and tissue models,
with high structural complexity and design exibility (Murphy
and Atala, 2014; Jang et al., 2016a,b,c; Kang etal., 2016; Kuo
etal., 2016; Zhang etal., 2016). ere is a signicant interest in
designing novel bioink formulations toward the goal of achiev-
ing the “ideal” bioink for each bioprinting technology (Hölzl
etal., 2016). Cell-laden hydrogels are the most common bioinks,
oering novel strategies including multi-material printing,
shear-thinning capability, and sequential cross-linking toward
self-supporting constructs. dECM-based bioinks provide an
alternative approach utilizing decellulerized tissues, yet the pro-
cessing of decellulerized tissue increases the cost of the bioinks.
Cell aggregate printing enables direct printing of cells into tissue
constructs, but the size of these constructs is currently limited
as the process requires large quantities of cells. In addition to
bioink development, there is also need for bioprinters with high
resolution, which is particularly important to develop vascular-
ized constructs. Considering future perspectives, supramolecular
hydrogels with reversible cross-linking mechanism (Rodell
etal., 2015) and stimuli responsive materials for biomimetic 4D
printing (Sydney Gladman etal., 2016) are potentially the most
interesting candidates for bioink design. Finally, there are still
many regulatory challenges to move the 3D bioprinted constructs
into clinic.
AUTHOR CONTRIBUTIONS
SJ and MG wrote the manuscript, and MG edited the manuscript.
ACKNOWLEDGMENTS
Authors would like to thank Dr. Chya-Yan Liaw for her fruit-
ful comments. Authors are very grateful to National Science
Foundation (DMR-1714882) (MG) and New Jersey Institute of
Technology (MG and SJ) for the funding.
FUNDING
is work is funded by National Science Foundation (DMR-
1714882) and New Jersey Institute of Technology (NJIT) through
Faculty Seed Grant and new faculty startup funds.

6
Ji and Guvendiren Bioinks for Tissue and Organ Printing
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org April 2017 | Volume 5 | Article 23
REFERENCES
Adam, E. J., Alexandra, L. R., and Ramille, N. S. (2016). Advancing the eld of 3D
biomaterial printing. Biomed. Mater. 11, 014102. doi:10.1088/1748-6041/11/1/
014102
Bajaj, P., Schweller, R. M., Khademhosseini, A., West, J. L., and Bashir, R. (2014).
3D biofabrication strategies for tissue engineering and regenerative medicine.
Annu. Rev. Biomed. Eng. 16, 247–276. doi:10.1146/annurev-bioeng-071813-
105155
Barron, J. A., Spargo, B. J., and Ringeisen, B. R. (2004a). Biological laser printing
of three dimensional cellular structures. Appl. Phys. A Mater. Sci. Process. 79,
1027–1030. doi:10.1007/s00339-004-2620-3
Barron, J. A., Wu, P., Ladouceur, H. D., and Ringeisen, B. R. (2004b). Biological
laser printing: a novel technique for creating heterogeneous 3-dimensional cell
patterns. Biomed. Microdevices 6, 139–147. doi:10.1023/B:BMMD.0000031751.
67267.9f
Benam, K. H., Dauth, S., Hassel, B., Herland, A., Jain, A., Jang, K. J., etal. (2015).
Engineered invitro disease models. Annu. Rev. Pathol. 10, 195–265. doi:10.1146/
annurev-pathol-012414-040418
Bertassoni, L. E., Cardoso, J. C., Manoharan, V., Cristino, A. L., Bhise, N. S., Araujo,
W. A., etal. (2014). Direct-write bioprinting of cell-laden methacrylated gelatin
hydrogels. Biofabrication 6, 024105. doi:10.1088/1758-5082/6/2/024105
Boland, T., Tao, X., Damon, B. J., Manley, B., Kesari, P., Jalota, S., et al. (2007).
Drop-on-demand printing of cells and materials for designer tissue constructs.
Mater. Sci. Eng. C 27, 372–376. doi:10.1016/j.msec.2006.05.047
Burdick, J. A., and Prestwich, G. D. (2011). Hyaluronic acid hydrogels for
biomedical applications. Adv. Mater. Weinheim 23, H41–H56. doi:10.1002/
adma.201003963
Choi, W. S., Ha, D., Park, S., and Kim, T. (2011). Synthetic multicellular cell-to-
cell communication in inkjet printed bacterial cell systems. Biomaterials 32,
2500–2507. doi:10.1016/j.biomaterials.2010.12.014
Christensen, K., Xu, C., Chai, W., Zhang, Z., Fu, J., and Huang, Y. (2015). Freeform
inkjet printing of cellular structures with bifurcations. Biotechnol. Bioeng. 112,
1047–1055. doi:10.1002/bit.25501
Cui, H., Nowicki, M., Fisher, J. P., and Zhang, L. G. (2017). 3D bioprinting for organ
regeneration. Adv. Healthc. Mater. 6, 1601118. doi:10.1002/adhm.201601118
Cui, X., Breitenkamp, K., Finn, M. G., Lotz, M., and D’Lima, D. D. (2012). Direct
human cartilage repair using three-dimensional bioprinting technology. Tissue
Eng. Part A 18, 1304–1312. doi:10.1089/ten.tea.2011.0543
Dababneh, A. B., and Ozbolat, I. T. (2014). Bioprinting technology: a current
state-of-the-art review. J. Manuf. Sci. Eng. 136, 061016. doi:10.1115/1.4028512
Doraiswamy, A., Narayan, R. J., Lippert, T., Urech, L., Wokaun, A., Nagel, M.,
et al. (2006). Excimer laser forward transfer of mammalian cells using a
novel triazene absorbing layer. Appl. Surf. Sci. 252, 4743–4747. doi:10.1016/j.
apsusc.2005.07.166
Elomaa, L., Pan, C.-C., Shanjani, Y., Malkovskiy, A., Seppälä, J. V., and Yang, Y.
(2015). ree-dimensional fabrication of cell-laden biodegradable poly(eth-
ylene glycol-co-depsipeptide) hydrogels by visible light stereolithography.
J. Mater. Chem. B 3, 8348–8358. doi:10.1039/c5tb01468a
Fedorovich, N. E., Alblas, J., De Wijn, J. R., Hennink, W. E., Verbout, A. B. J., and
Dhert, W. J. A. (2007). Hydrogels as extracellular matrices for skeletal tissue
engineering: state-of-the-art and novel application in organ printing. Tissue
Eng. 13, 1905–1925. doi:10.1089/ten.2006.0175
Fleming, P. A., Argraves, W. S., Gentile, C., Neagu, A., Forgacs, G., and Drake,
C. J. (2010). Fusion of uniluminal vascular spheroids: a model for assembly of
blood vessels. Dev. Dyn. 239, 398–406. doi:10.1002/dvdy.22161
Forgacs, G., and Foty, R. A. (2004). “Biological relevance of tissue liquidity and
viscoelasticity,” in Function and Regulation of Cellular Systems, eds A. Deutsch,
J. Howard, M. Falcke, and W. Zimmermann (Basel: Birkhäuser Basel), 269–277.
Forgacs, G., Foty, R . A., Shafrir, Y., and Steinberg, M. S. (1998). Viscoelastic proper-
ties of living embryonic tissues: a quantitative study. Biophys. J. 74, 2227–2234.
doi:10.1016/S0006-3495(98)77932-9
Gardel, M. L., Shin, J. H., MacKintosh, F. C., Mahadevan, L., Matsudaira, P.,
and Weitz, D. A. (2004). Elastic behavior of cross-linked and bundled actin
networks. Science 304, 1301. doi:10.1126/science.1095087
Grith, L. G., and Naughton, G. (2002). Tissue engineering – current challenges and
expanding opportunities. Science 295, 1009–1014. doi:10.1126/science.1069210
Groen, N., Guvendiren, M., Rabitz, H., Welsh, W. J., Kohn, J., and de Boer,
J. (2016). Stepping into the omics era: opportunities and challenges for bio-
materials science and engineering. Acta Biomater. 34, 133–142. doi:10.1016/j.
actbio.2016.02.015
Gudapati, H., Dey, M., and Ozbolat, I. (2016). A comprehensive review on
droplet-based bioprinting: past, present and future. Biomaterials 102, 20–42.
doi:10.1016/j.biomaterials.2016.06.012
Guillemot, F., Mironov, V., and Nakamura, M. (2010). Bioprinting is com-
ing of age: report from the International Conference on Bioprinting
and Biofabrication in Bordeaux (3B’09). Biofabrication 2, 010201.
doi:10.1088/1758-5082/2/1/010201
Guillotin, B., Souquet, A., Catros, S., Duocastella, M., Pippenger, B., Bellance, S.,
etal. (2010). Laser assisted bioprinting of engineered tissue with high cell den-
sity and microscale organization. Biomaterials 31, 7250–7256. doi:10.1016/j.
biomaterials.2010.05.055
Guvendiren, M., and Burdick, J. A. (2013). Engineering synthetic hydrogel
microenvironments to instruct stem cells. Curr. Opin. Biotechnol. 24, 841–846.
doi:10.1016/j.copbio.2013.03.009
Guvendiren, M., Molde, J., Soares, R. M. D., and Kohn, J. (2016). Designing bio-
materials for 3D printing. ACS Biomater. Sci. Eng. 2, 1679–1693. doi:10.1021/
acsbiomaterials.6b00121
Highley, C. B., Rodell, C. B., and Burdick, J. A. (2015). Direct 3D printing of
shear-thinning hydrogels into self-healing hydrogels. Adv. Mater. 27, 5075–5079.
doi:10.1002/adma.201501234
Hockaday, L. A., Kang, K. H., Colangelo, N. W., Cheung, P. Y. C., Duan, B., Malone,
E., etal. (2012). Rapid 3D printing of anatomically accurate and mechanically
heterogeneous aortic valve hydrogel scaolds. Biofabrication 4, 035005.
doi:10.1088/1758-5082/4/3/035005
Hölzl, K., Lin, S., Tytgat, L., Van Vlierberghe, S., Gu, L., and Ovsianikov, A. (2016).
Bioink properties before, during and aer 3D bioprinting. Biofabrication 8,
032002. doi:10.1088/1758-5090/8/3/032002
Hong, S., Sycks, D., Chan, H. F., Lin, S., Lopez, G. P., Guilak, F., etal. (2015). 3D
printing of highly stretchable and tough hydrogels into complex, cellularized
structures. Adv. Mater. Weinheim 27, 4035–4040. doi:10.1002/adma.201501099
Hopp, B., Smausz, T., Kresz, N., Barna, N., Bor, Z., Kolozsvári, L., etal. (2005).
Survival and proliferative ability of various living cell types aer laser-induced
forward transfer. Tissue Eng. 11, 1817–1823. doi:10.1089/ten.2005.11.1817
Hribar, K. C., Soman, P., Warner, J., Chung, P., and Chen, S. (2014). Light-assisted
direct-write of 3D functional biomaterials. Lab. Chip 14, 268–275. doi:10.1039/
C3LC50634G
Huebsch, N., Arany, P. R., Mao, A. S., Shvartsman, D., Ali, O. A., Bencherif, S.
A., et al. (2010). Harnessing traction-mediated manipulation of the cell/
matrix interface to control stem-cell fate. Nat. Mater. 9, 518–526. doi:10.1038/
nmat2732
Jakab, K., Marga, F., Norotte, C., Murphy, K., Vunjak-Novakovic, G., and Forgacs,
G. (2010). Tissue engineering by self-assembly and bio-printing of living cells.
Biofabrication 2, 022001–022001. doi:10.1088/1758-5082/2/2/022001
Jakab, K., Neagu, A., Mironov, V., Markwald, R. R., and Forgacs, G. (2004).
Engineering biological structures of prescribed shape using self-assembling
multicellular systems. Proc. Natl. Acad. Sci. U.S.A 101, 2864–2869. doi:10.1073/
pnas.0400164101
Jang, J., Kim, T. G., Kim, B. S., Kim, S. W., Kwon, S. M., and Cho, D. W. (2016a).
Tailoring mechanical properties of decellularized extracellular matrix
bioink by vitamin B2-induced photo-crosslinking. Acta Biomater. 33, 88–95.
doi:10.1016/j.actbio.2016.01.013
Jang, J., Park, H. J., Kim, S. W., Kim, H., Park, J. Y., Na, S. J., etal. (2016b). 3D printed
complex tissue construct using stem cell-laden decellularized extracellular
matrix bioinks for cardiac repair. Biomaterials 112, 264–274. doi:10.1016/j.
biomaterials.2016.10.026
Jang, J., Yi, H. G., and Cho, D. W. (2016c). 3D printed tissue models: present and future.
ACS Biomater. Sci. Eng. 2, 1722–1731. doi:10.1021/acsbiomaterials.6b00129
Jia, J., Richards, D. J., Pollard, S., Tan, Y., Rodriguez, J., Visconti, R. P., etal. (2014).
Engineering alginate as bioink for bioprinting. Acta Biomater. 10, 4323–4331.
doi:10.1016/j.actbio.2014.06.034
Jose, R. R., Rodriguez, M. J., Dixon, T. A., Omenetto, F., and Kaplan, D. L. (2016).
Evolution of bioinks and additive manufacturing technologies for 3D bioprint-
ing. ACS Biomater. Sci. Eng. 2, 1662–1678. doi:10.1021/acsbiomaterials.6b00088

7
Ji and Guvendiren Bioinks for Tissue and Organ Printing
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org April 2017 | Volume 5 | Article 23
Kang, H. W., Lee, S. J., Ko, K. I., Kengla, C., Yoo, J. J., and Atala, A. (2016). A 3D
bioprinting system to produce human-scale tissue constructs with structural
integrity. Nat. Biotechnol. 34, 312–319. doi:10.1038/nbt.3413
Khetan, S., Guvendiren, M., Legant, W. R., Cohen, D. M., Chen, C. S., and Burdick,
J. A. (2013). Degradation-mediated cellular traction directs stem cell fate in
covalently crosslinked three-dimensional hydrogels. Nat. Mater. 12, 458–465.
doi:10.1038/nmat3586
Koch, L., Kuhn, S., Sorg, H., Gruene, M., Schlie, S., Gaebel, R., etal. (2010). Laser
printing of skin cells and human stem cells. Tissue Eng. Part C Methods 16, 5.
doi:10.1089/ten.tec.2009.0397
Kuo, C.-Y., Eranki, A., Placone, J. K., Rhodes, K. R., Aranda-Espinoza,
H., Fernandes, R., etal. (2016). Development of a 3D printed, bioengi-
neered placenta model to evaluate the role of trophoblast migration
in preeclampsia. ACS Biomater. Sci. Eng. 2, 1817–1826. doi:10.1021/
acsbiomaterials.6b00031
Langer, R., and Vacanti, J. P. (1993). Tissue engineering. Am. Assoc. Adv. Sci. 260,
920–926. doi:10.1126/science.8493529
Levato, R., Visser, J., Planell, J. A., Engel, E., Malda, J., and Mateos-Timoneda, M. A.
(2014). Biofabrication of tissue constructs by 3D bioprinting of cell-laden
microcarriers. Biofabrication 6, 035020. doi:10.1088/1758-5082/6/3/035020
Li, C., Faulkner-Jones, A., Dun, A. R., Jin, J., Chen, P., Xing, Y., etal. (2015). Rapid
formation of a supramolecular polypeptide–DNA hydrogel for insitu three-di-
mensional multilayer bioprinting. Angew. Chem. Int. Ed. 54, 3957–3961.
doi:10.1002/anie.201411383
Lim, K. S., Schon, B. S., Mekhileri, N. V., Brown, G. C. J., Chia, C. M., Prabakar, S.,
etal. (2016). New visible-light photoinitiating system for improved print delity
in gelatin-based bioinks. ACS Biomater. Sci. Eng. 2, 1752–1762. doi:10.1021/
acsbiomaterials.6b00149
Loessner, D., Meinert, C., Kaemmerer, E., Martine, L. C., Yue, K., Levett, P. A.,
etal. (2016). Functionalization, preparation and use of cell-laden gelatin meth-
acryloyl-based hydrogels as modular tissue culture platforms. Nat. Protoc. 11,
727–746. doi:10.1038/nprot.2016.037
Mandrycky, C., Wang, Z., Kim, K., and Kim, D. H. (2016). 3D bioprinting for
engineering complex tissues. Biotechnol. Adv. 34, 422–434. doi:10.1016/j.
biotechadv.2015.12.011
Marga, F., Neagu, A., Kosztin, I., and Forgacs, G. (2007). Developmental biology
and tissue engineering. Birth Defects Res. C Embryo Today 81, 320–328.
doi:10.1002/bdrc.20109
Miller, J. S., and Burdick, J. A. (2016). Editorial: special issue on 3D print-
ing of biomaterials. ACS Biomater. Sci. Eng. 2, 1658–1661. doi:10.1021/
acsbiomaterials.6b00566
Mironov, V., Boland, T., Trusk, T., Forgacs, G., and Markwald, R. R. (2003). Organ
printing: computer-aided jet-based 3D tissue engineering. Trends Biotechnol.
21, 157–161. doi:10.1016/S0167-7799(03)00033-7
Morris, V. B., Nimbalkar, S., Younesi, M., McClellan, P., and Akkus, O. (2017).
Mechanical properties, cytocompatibility and manufacturability of chi-
tosan:PEGDA hybrid-gel scaolds by stereolithography. Ann. Biomed. Eng. 45,
286–296. doi:10.1007/s10439-016-1643-1
Müller, M., Becher, J., Schnabelrauch, M., and Zenobi-Wong, M. (2015).
Nanostructured pluronic hydrogels as bioinks for 3D bioprinting. Biofabrication
7, 035006. doi:10.1088/1758-5090/7/3/035006
Munaz, A., Vadivelu, R. K., St John, J., Barton, M., Kamble, H., and Nguyen, N.-T.
(2016). ree-dimensional printing of biological matters. J. Sci. Adv. Mater.
Devices 1, 1–17. doi:10.1016/j.jsamd.2016.04.001
Murphy, S. V., and Atala, A. (2014). 3D bioprinting of tissues and organs. Nat.
Biotechnol. 32, 773–785. doi:10.1038/nbt.2958
Nakamura, M., Kobayashi, A., Takagi, F., Watanabe, A., Hiruma, Y.,
Ohuchi, K., etal. (2005). Biocompatible inkjet printing technique for designed
seeding of individual living cells. Tissue Eng. 11, 1658–1666. doi:10.1089/
ten.2005.11.1658
Nguyen, D. G., Funk, J., Robbins, J. B., Crogan-Grundy, C., Presnell, S. C., Singer,
T., etal. (2016). Bioprinted 3D primary liver tissues allow assessment of organ-
level response to clinical drug induced toxicity invitro. PLoS ONE 11:e0158674.
doi:10.1371/journal.pone.0158674
Nishiyama, Y., Nakamura, M., Henmi, C., Yamaguchi, K., Mochizuki, S., Nakagawa,
H., etal. (2008). Development of a three-dimensional bioprinter: construction
of cell supporting structures using hydrogel and state-of-the-art inkjet technol-
ogy. J. Biomech. Eng. 131, 035001. doi:10.1115/1.3002759
Norotte, C., Marga, F. S., Niklason, L. E., and Forgacs, G. (2009). Scaold-free
vascular tissue engineering using bioprinting. Biomaterials 30, 5910–5917.
doi:10.1016/j.biomaterials.2009.06.034
Ouyang, L., Highley, C. B., Rodell, C. B., Sun, W., and Burdick, J. A. (2016). 3D print-
ing of shear-thinning hyaluronic acid hydrogels with secondary cross-linking.
ACS Biomater. Sci. Eng. 2, 1743–1751. doi:10.1021/acsbiomaterials.6b00158
Ozbolat, I. T., and Hospodiuk, M. (2016). Current advances and future perspec-
tives in extrusion-based bioprinting. Biomaterials 76, 321–343. doi:10.1016/j.
biomaterials.2015.10.076
Ozbolat, I. T., Moncal, K. K., and Gudapati, H. (2017). Evaluation of bioprinter
technologies. Addit. Manuf. 13, 179–200. doi:10.1016/j.addma.2016.10.003
Ozbolat, I. T., Peng, W., and Ozbolat, V. (2016). Application areas of 3D bioprinting.
Drug Discov. Today 21, 1257–1271. doi:10.1016/j.drudis.2016.04.006
Pan, T., Song, W., Cao, X., and Wang, Y. (2016). 3D bioplotting of gelatin/alginate
scaolds for tissue engineering: inuence of crosslinking degree and pore
architecture on physicochemical properties. J. Mater. Sci. Technol. 32, 889–900.
doi:10.1016/j.jmst.2016.01.007
Panwar, A., and Tan, L. P. (2016). Current status of bioinks for micro-extru-
sion-based 3D bioprinting. Molecules 21, 6. doi:10.3390/molecules21060685
Pati, F., Jang, J., Ha, D. H., Won Kim, S., Rhie, J. W., Shim, J. H., et al. (2014).
Printing three-dimensional tissue analogues with decellularized extracellular
matrix bioink. Nat. Commun. 5, 3935. doi:10.1038/ncomms4935
Raphael, B., Khalil, T., Workman, V. L., Smith, A., Brown, C. P., Streuli, C., etal.
(2017). 3D cell bioprinting of self-assembling peptide-based hydrogels. Mater.
Lett. 190, 103–106. doi:10.1016/j.matlet.2016.12.127
Ribeiro, M., de Moraes, M. A., Beppu, M. M., Garcia, M. P., Fernandes, M. H.,
Monteiro, F. J., etal. (2015). Development of silk broin/nanohydroxyapatite
composite hydrogels for bone tissue engineering. Eur. Polymer J. 67, 66–77.
doi:10.1016/j.eurpolymj.2015.03.056
Ringeisen, B. R., Kim, H., Barron, J. A., Krizman, D. B., Chrisey, D. B., Jackman,
S., etal. (2004). Laser printing of pluripotent embryonal carcinoma cells. Tissue
Eng. 10, 483–491. doi:10.1089/107632704323061843
Rodell, C. B., MacArthur, J. W., Dorsey, S. M., Wade, R. J., Wang, L. L., Woo, Y.
J., et al. (2015). Shear-thinning supramolecular hydrogels with secondary
autonomous covalent crosslinking to modulate viscoelastic properties invivo.
Adv. Funct. Mater. 25, 636–644. doi:10.1002/adfm.201403550
Rutz, A. L., Hyland, K. E., Jakus, A. E., Burghardt, W. R., and Shah, R. N. (2015).
A multimaterial bioink method for 3D printing tunable, cell-compatible hydro-
gels. Adv. Mater. 27, 1607–1614. doi:10.1002/adma.201405076
Shaee, A., and Atala, A. (2016). Printing technologies for medical applications.
Trends Mol. Med. 22, 254–265. doi:10.1016/j.molmed.2016.01.003
Shirazi, S. F. S., Gharehkhani, S., Mehrali, M., Yarmand, H., Metselaar, H. S. C.,
Kadri, N. A., etal. (2015). A review on powder-based additive manufacturing
for tissue engineering: selective laser sintering and inkjet 3D printing. Sci. Tech.
Adv. Mat. 16, 033502. doi:10.1088/1468-6996/16/3/033502
Skoog, S. A., Goering, P. L., and Narayan, R. J. (2014). Stereolithography in
tissue engineering. J. Mater. Sci. Mater. Med. 25, 845–856. doi:10.1007/
s10856-013-5107-y
Storm, C., Pastore, J. J., MacKintosh, F. C., Lubensky, T. C., and Janmey, P. A. (2005).
Nonlinear elasticity in biological gels. Nature 435, 191–194. doi:10.1038/
nature03521
Sydney Gladman, A., Matsumoto, E. A., Nuzzo, R. G., Mahadevan, L., and Lewis,
J. A. (2016). Biomimetic 4D printing. Nat. Mater. 15, 413–418. doi:10.1038/
nmat4544
Tan, Y. J., Tan, X., Yeong, W. Y., and Tor, S. B. (2016). Hybrid microscaf-
fold-based 3D bioprinting of multi-cellular constructs with high compres-
sive strength: a new biofabrication strategy. Sci. Rep. 6, 39140. doi:10.1038/
srep39140
Tibbitt, M. W., Rodell, C. B., Burdick, J. A., and Anseth, K. S. (2015). Progress in
material design for biomedical applications. Proc. Natl. Acad. Sci. U.S.A. 112,
14444–14451. doi:10.1073/pnas.1516247112
Tirella, A., Orsini, A., Vozzi, G., and Ahluwalia, A. (2009). A phase diagram for
microfabrication of geometrically controlled hydrogel scaolds. Biofabrication
1, 4. doi:10.1088/1758-5082/1/4/045002
Tirella, A., Vozzi, F., De Maria, C., Vozzi, G., Sandri, T., Sassano, D., etal.
(2011). Substrate stiffness influences high resolution printing of living
cells with an ink-jet system. J. Biosci. Bioeng. 112, 79–85. doi:10.1016/j.
jbiosc.2011.03.019

8
Ji and Guvendiren Bioinks for Tissue and Organ Printing
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org April 2017 | Volume 5 | Article 23
Turner, B. N., Strong, R., and Gold, S. A. (2014). A review of melt extrusion additive
manufacturing processes: I. Process design and modeling. Rapid Prototyp. J. 20,
192–204. doi:10.1108/rpj-01-2013-0012
Wang, Z., Abdulla, R., Parker, B., Samanipour, R., Ghosh, S., and Kim, K. (2015).
A simple and high-resolution stereolithography-based 3D bioprinting
system using visible light crosslinkable bioinks. Biofabrication 7, 045009.
doi:10.1088/1758-5090/7/4/045009
Wilson, W. C. Jr., and Boland, T. (2003a). Cell and organ printing 1: protein and
cell printers. Anat. Rec. A Discov. Mol. Cell Evol. Biol. 272, 491–496. doi:10.1002/
ar.a.10057
Wilson, W. C. Jr., and Boland, T. (2003b). Cell and organ printing 1: protein and cell
printers. Anat. Rec. A Discov. Mol. Cell. Evol. Biol. 272, 491–496. doi:10.1002/
ar.a.10057
Wu, W., DeConinck, A., and Lewis, J. A. (2011). Omnidirectional printing of 3D
microvascular networks. Adv. Mater. Weinheim 23, H178–H183. doi:10.1002/
adma.201004625
Wüst, S., Müller, R., and Hofmann, S. (2015). 3D Bioprinting of complex channels –
eects of material, orientation, geometry, and cell embedding. J. Biomed. Mater.
Res. A. 103, 2558–2570. doi:10.1002/jbm.a.35393
Xu, T., Baicu, C., Aho, M., Zile, M., and Boland, T. (2009). Fabrication and
characterization of bio-engineered cardiac pseudo tissues. Biofabrication 1,
035001–035001. doi:10.1088/1758-5082/1/3/035001
Yu, Z., Rui, Y., Liliang, O., Hongxu, D., Ting, Z., Kaitai, Z., et al. (2014).
ree-dimensional printing of Hela cells for cervical tumor model in vitro.
Biofabrication 6, 035001. doi:10.1088/1758-5082/6/3/035001
Zhang, Y. S., Duchamp, M., Oklu, R., Ellisen, L. W., Langer, R., and
Khademhosseini, A. (2016). Bioprinting the cancer microenvironment. ACS
Biomater. Sci. Eng. 2, 1710–1721. doi:10.1021/acsbiomaterials.6b00246
Conict of Interest Statement: e authors declare that the research was con-
ducted in the absence of any commercial or nancial relationships that could be
construed as a potential conict of interest.
e reviewer, AA, and handling Editor declared their shared aliation, and the
handling Editor states that the process nevertheless met the standards of a fair and
objective review.
Copyright © 2017 Ji and Guvendiren. is is an open-access article distributed under
the terms of the Creative Commons Attribution License (CC BY). e use, distribu-
tion or reproduction in other forums is permitted, provided the original author(s)
or licensor are credited and that the original publication in this journal is cited, in
accordance with accepted academic practice. No use, distribution or reproduction is
permitted which does not comply with these terms.