REVIEW

Biofabrication www.advhealthmat.de

‘Printability’ of Candidate Biomaterials for Extrusion Based

3D Printing: State-of-the-Art
Stuart Kyle, Zita M. Jessop, Ayesha Al-Sabah, and Iain S. Whitaker*

biological and physical sciences, including

Regenerative medicine has been highlighted as one of the UK’s 8 ‘Great Tech- cell biology, molecular biology, mate-
nologies’ with the potential to revolutionize patient care in the 21st Century. rials science, engineering and clinical
Over the last decade, the concept of ‘3D bioprinting’ has emerged, which medicine.[2]
In recent years, the ability to print
allows the precise deposition of cell laden bioinks with the aim of engineering
biological ‘inks’, rather than plastic and
complex, functional tissues. For 3D printing to be used clinically, there is metal inks of traditional 3D printing,
the need to produce advanced functional biomaterials, a new generation of has resulted in the birth of the new bio-
bioinks with suitable cell culture and high shape/print fidelity, to match or printing research field.[3] The global 3D
exceed the physical, chemical and biological properties of human tissue. With bioprinting market was estimated to be
$487 million in 2014 and is predicted
the rapid increase in knowledge associated with biomaterials, cell-scaffold
to reach $1.82 billion by 2022 (Grand
interactions and the ability to biofunctionalize/decorate bioinks with cell rec- View, 2015). 3D bioprinting allows the
ognition sequences, it is important to keep in mind the ‘printability’ of these potential to replicate complex native-like
novel materials. In this illustrated review, we define and refine the concept tissue architecture in the laboratory with
of ‘printability’ and review seminal and contemporary studies to highlight the potential to biomanufacture physio­
the current ‘state of play’ in the field with a focus on bioink composition and logically relevant multicellular tissue con-
structs on demand. The ultimate success
concentration, manipulation of nozzle parameters and rheological properties.
of this platform technology depends on
the process itself, plus answers the funda-
mental scientific questions regarding the
1. Introduction most appropriate blend of tissue-specific cell source, suitable
scaffold and ideal microenvironment.[4]
Congenital or acquired tissue loss and dysfunction is an enor- The ultimate goal of 3D bioprinting is to produce functional
mous socioeconomic burden for healthcare systems globally. biomimetic composite tissues. A key tenet of this paradigm
In the Western World, hundreds of thousands of people are is that cell biology in 2D and 3D culture are significantly dif-
on transplant waiting lists, and although hundreds of millions ferent and in order to fabricate a successful tissue engineering
are registered as organ donors there is simply not an adequate substrate, 3D constructs must best match native tissue in vitro.
supply (www.unos.org; www.organdonation.nhs.uk). Globally, it Contemporary high resolution 3D printers are able to do this by
is impossible to estimate the number of individuals who would controlling the nano-, micro- and macrostructure.[5,6] Current
benefit from functional tissue engineering to replace and bioprinting technology enables cells and scaffolds to be printed
restore function. simultaneously, allowing accurate control of cell distribution,
Regenerative medicine has the potential to revolutionize high resolution, scalability and cost-effectiveness.[7]
patient care in the 21st Century.[1] One of its major branches, Several bioprinting strategies have been explored with
tissue engineering, holds promise to repair or replace human the ultimate goal of fabricating functional tissue constructs
tissues and organs in order to restore normal function. Tissue (Table 1). As extrusion based bioprinting is the most widely
engineering is a multidisciplinary field that involves aspects of used in current research and the commercial sphere, this article
focuses on the applicability of contemporary biomaterials with
this strategy in mind.
Dr. S. Kyle, Ms. Z. M. Jessop, Dr. A. Al-Sabah, Prof. I. S. Whitaker One of the current key research questions is what the
Reconstructive Surgery & Regenerative Medicine Group (ReconRegen)
ideal properties of a ‘bioink’ actually are. The characteristics
Institute of Life Sciences
Swansea University Medical School of an ideal bioink can be seen in Table 2. It is essential that
Swansea SA2 8PP, UK suitable bioinks have the desired functional and mechanical
E-mail: iainwhitaker@fastmail.fm properties that closely match the tissue it is to replace. A suc-
Dr. S. Kyle, Ms. Z. M. Jessop, Prof. I. S. Whitaker cessful, printable bioink must have suitable properties that
The Welsh Centre for Burns and Plastic Surgery maintain the bioink and cell integrity post-printing. It is well
Morriston Hospital
Swansea SA6 6NL, UK documented that various cell phenotypes are exquisitely sensi-
tive to subtle variations in mechanical properties of the micro-
DOI: 10.1002/adhm.201700264 environment. Hence many bioinks have been developed and

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engineered for specific cell types. Moreover, printing parame-

ters must be clearly defined for different bioinks and cell types. Stuart Kyle is currently a
The term ‘bioink’ refers to (1) the ‘biopaper’ which generally Clinical Lecturer in Plastic
is hydrogel-based and provides structural and mechanical sup- Surgery at Swansea University
port, and (2) specialized cells, such as stem cells. In order to Medical School, UK.
create a functional 3D construct, the ‘bioink’ must be carefully Following his Wellcome Trust-
selected in addition to an appropriate bioprinter (Figure 1). funded PhD (Biomaterials)
The biofabrication window paradigm (Figure 2) defines the in 2010 from the University
compromise between suitability for fabrication (printability), of Leeds, he completed his
and the ability to encapsulate cells and maintain cell via- medical degree (MBChB
bility.[10,15] Traditionally there has been a compromise between with Honors) in 2014. He
the ideal bioink for 3D printing and cell culture. Many lacked was awarded the prestig-
shape fidelity and printability, had inadequate mechanical ious Foulkes Foundation
strength post-printing and had poor bioactivity and biodeg- Fellowship (one of only 275 in the past 40 years). He has
radability profiles. Higher concentrations of some polymeric published widely in the field of biomaterials and nano-
bioinks introduces more cross-linking and stiffness. However technology and his current research interests are in the
these networks can often hinder cell migration, proliferation development of novel biomaterials for 3D bioprinting for
and matrix deposition. Conversely, less dense networks can lead use in reconstructive surgery.
to poor shape fidelity as it can be difficult to maintain shape
post-printing. Therefore more traditional bioinks have com- Ayesha Al-Sabah is a Post-
promised with a moderate degree of crosslinking, but with far Doctoral Research Fellow in
from ideal cell culture environments. More advanced bioinks the Reconstructive Surgery
are now being designed to significantly improve printability and Regenerative Medicine
and biocompatibility. These can be achieved through careful Research Group, Swansea
control of various physical, chemical and biological proper- University Medical School,
ties, such as rheology (viscosity, shear-thinning, viscoelasticity), UK where she supports
biofunctionalization, biodegradation, gelation kinetics and cell a large team of Clinical
function (cytocompatibility, cell adhesion, migration, prolifera- Researchers. Following the
tion, differentiation). award of her PhD from Cardiff
There is a wealth of current literature reviewing bioprinting University (Cartilage Tissue
techniques, candidate biomaterials for use as bioinks, and the Engineering), she was a Post-
applications of bioprinting for tissue engineering and transla- Doctoral Researcher in Cardiff before joining the team in
tional purposes. There is a paucity of articles concentrating on Swansea. Her research interests are currently in the field of
the ‘printability’ of bioinks, which is especially important when mesenchymal tissue engineering with a focus on stem cell
complex 3-dimensional structures are required, to our knowl- biology, bioenergetics and 3D bioprinting.
edge there are no comprehensive reviews in the literature.
Iain Whitaker is Professor of
Plastic Surgery and Director
of the Reconstructive
2. Concept of ‘Printability’ Surgery and Regenerative
Medicine Research Group
The core processes of 3D bioprinting can be seen in
(ReconRegen) based at
Figure 3. The product (printed biological constructs) must be
Swansea University Medical
precise (with high resolution, shape fidelity) and reproducible.
School. After reading medi-
Yet how are printability, fidelity and reproducibility measured,
cine at Cambridge University
and is it adequate? Key studies that have identified parameters
he completed Plastic Surgery
that potentially help control and measure ‘printability’ of var-
training in the UK, Sweden,
ious biomaterials are highlighted in Table 3. The desired struc-
USA, Australia & France and
ture of the finished construct is highly precise and, in order to
was awarded a PhD from the University of Utrecht. He
mimic these structures, it is necessary for the printing process
was the first plastic surgeon to receive the Rowan Nick’s
to be equally precise and for the construct to maintain this
Award to work In Melbourne, followed by the European
shape over a period of cell culture.
Association of Plastic Surgeons Young Plastic Surgeon
Printability is an important concept as the product of the bio-
Scholarship to work in Paris. His current research interests
printing process must mimic both the cellular architecture and
involve 3D biomanufacture and tissue engineering.
shape. This is particularly critical when tissues of an intricate
nature are required, such as a heart valve or ear. Geometry is
as important in tissue structure as it is function. Heart valves
for example have to be durable whilst allowing appropriate novo valve scaffolds have been engineered over the years which
blood flow at varying valvular pressures. For this reason, spa- involved manually shaping leaflet geometries using various
tial and mechanical heterogeneity is needed. A number of de polymers, mostly based on polylactide,[45] polyglycolide,[46]

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Table 1.  Comparison of four types of bioprinting modalities.
Bioprinting modality Advantages
■ Fast printing speeds (1–10,000
droplets s−1)
■ Low cost
■ Small volume droplets (picoliters)
■ Intricate structures with high
resolution (10–50 µm)
■ Readily adapted with multiple
nozzles for multimaterial printing
■ Fast gelation to allow for rapid
fabrication
■ High resolutions (≈1 µm) and
cell viabilities (>85%)
■ Increased fabrication speed
■ Reduced fabrication time
■ No limitation of viscosities
■ Low cost
■ High resolutions
(pico- to microscale)
■ Small printing volumes
(pico-to-nanoliter)
■ No dispensing nozzle so no
clogging issues
■ Reduces issues of shear stress
which can affect cell viability
■ Multiple cell/material delivery
■ Higher viscosity biomaterials
can be printed (30–6 × 107 mPa.s)
■ Room temperature processing
■ Direct incorporation of cells and
homogenous distribution of cells
■ Large and intricate structures
with moderate resolutions
(200–1000 µm)
■ Shape fidelity is high post-printing
Adv. Healthcare Mater. 2017, 1700264
Disadvantages
■ Require low viscosity (3–12 mPa.s)
environments to prevent clogging
■ Low thermal conductivity
■ Small volumes droplets may restrict
scaling up to fabricate larger organ
structures
■ Difficult printing vertical 3D structures
■ Limited to photosensitive materials
■ Possibility of DNA damage by UV
light, but as this method typically
uses UV-visible light (anywhere from
365–530 nm), many studies have
shown no/little damage to cells
or DNA for the brief duration
of exposure
■ Fast gelation kinetics and fast
moving stage for fabrication
can be troublesome
■ High cost
■ Complex design
■ Heat from laser may damage cells
■ Slow print speed (µm s−1)
■ Critical timing of gelation time
■ Specific matching of material
densities to preserve intricate shapes
■ Small diameter nozzles (150 µm)
can lower cell viability (68.6%)
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Applications Reference
■ Fabrication of 3D cell-laden [8–10]
constructs in a drop by drop
manner
■ Tissue engineering of blood
vessels, cartilage, bones and
neurons
■ Uses lasers/projectors that [11]
polymerize light sensitive polymer
materials which enable 3D
structures to be created in
bottom-up assembly
■ Tissue engineering of blood
vessels, liver, bone and cartilage
■ Organ on a chip
■ Uses a laser as the energy [9,12]
source to propel and deposit
bioinks onto a collecting
substrate in a precise manner
■ Tissue engineering of bone,
adipose tissue, skin and blood
vessels
■ Produces a continuous bioink [13,14]
filament onto a stationary sub-
strate from a microscale syringe
tip driven by either pneumatic
pressures or mechanical force
■ Tissue engineering of blood
vessels, muscle, bone, cartilage,
heart valves, liver and neurons
■ Organ on a chip
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Table 2.  Ideal properties of bioinks.

Property Examples Reference

Form scaffolds with adequate mechanical strength, maintaining shape ■ Ionic crosslinking [14,16,17]
fidelity at a high resolution ■ Photochemical crosslinking
■ Sacrificial scaffolds
■ Quantifying shear stress during bioprinting
■ Concomitantly printed scaffolds
Exhibit tunable gelation to facilitate extrusion ■ Co-axial nozzle [17–20]
■ Inks loaded with
○ nanoparticles

○ spheroids

○ micro-carriers

■ Blending

Form scaffolds that resemble the native cellular microenvironment ■ Natural [17,21,22]
○ Alginate

○ Collagen

○ Gelatin

○ Agarose

○ Chitosan

○ Cellulose

○ Hyaluronic acid
■ Synthetic

○ Poly(lactic acid)
○ Poly(lactic acid-co-glycolic acid)
○ Polyethylene glycol

○ Polycaprolactone

○ Polyurethane

○ de novo designed peptides

Exhibit biocompatibility ■ Improved cell adhesion and viability [5,17,19,23–25]

■ Improved cell binding
■ Support differentiation and matrix deposition

■ Matrix remodeling

Be amenable to chemical modifications (functionalization/decoration) ■ RGD modification [17,25,26]

■ Cyclic RGDS
■ PEGylation

■ MMP-sensitive sequences

Capable of large scale synthesis with minimal batch-to-batch variation ■ Fabrication of large-volume constructs using 3D printing [17,27]
system capable of providing enhanced printability

polyhydroxybutyrates[47] and polyhydroxyalkanoates.[48] Hence,
a more automated manufacturing technology that could fabri-
cate tissue engineered scaffolds more rapidly with higher
anato­mical precisions was required. One group implemented
an on-board photocrosslinking system that enabled simulta-
neous 3D extrusion printing and curing of hydrogels in com-
plex aortic geometries.[38] Native anatomic and axisymmetric
aortic valve geometries with 12–22 mm inner diameters were
3D printed with poly-ethyleneglycol-diacrylate (PEG-DA) hydro-
gels supplemented with alginate (Figure 4A–F). 3D printing
geometric accuracy/shape fidelity was quantified and com-
pared using micro computerized tomography (micro-CT)
(Figure 4G–H). It was found that the 3D printing strategy
generated scaffolds with high geometric precision, but accu-
racy decreased somewhat with reduced size. Higher shape
fidelity was seen with larger valves (22 mm), compared to
smaller valves but more overprint error (regions printed out-
side the target print area) was seen for 12 mm valves, which
suggested that print paths were too wide. Print paths are soft-
ware generated dimensions of path height and width defined
after importing Standard Tessellated Language file (STL) geom-
etries acquired using micro-CT. As print paths were too wide
for filling in smaller regions, careful control and manipulation
of print paths (height and/or width) is critical for improving
print resolution and shape fidelity. Hence in order to optimize
future studies, smaller nozzle diameters to increase print reso-
lution, and a lower extrusion rate may be required. In analysing
CT slices to assess internal geometric fidelity, they found that
overlapping area percentage decreased as the size of the image-
derived valve scaffolds decreased. They also reported on the
importance of swelling in printed constructs. The scaffolds in
this study were found to expand outward but not inward, pos-
sibly suggesting surface tension on the inner walls prevented
this. In fact, surface tension is an important factor when con-
sidering printability, where contact angles between two media
can be measured and manipulated.[7] Surface tension has been
shown to be important in droplet formation in inkjet-printing
and this could be extended to extrusion-based systems, since
the affinity of the nanofluid for the substrate can be formulated
from the solid surface tensions.[49] Surface tension of the bioink

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Figure 1.  Functional 3D constructs can be fabricated through careful control of the (1) ‘bioink’
(specialized cells + biopaper) and (2) the bioprinter.
can also influence printing quality, resolution and width of
printed lines.[50] Hockaday et al (2012) therefore proposed that
hydrogel scaffolds should therefore be printed thinner than the
target size so that the expanded final state matches the native
model.[38] This study demonstrated that 3D printing of ana-
tomical scaffolds with high shape fidelity was possible but por-
trayed the complexities associated with these techniques.
2.1. Pore and Filament Dimensions
Whilst is it clear that the properties of bioinks critically influ-
ence their delivery and the integrity of the structure formed,
the concept of ‘printability’ has only been investigated in a
handful of papers. One early study investigated the printability
of alginate and gelatin with calcium chloride as a cross linking
agent in an extrusion-based printing system.[41] They found
that the properties were changed favourably when gelatin was
included at 2 and 4% Alg-Gel. In addition to other methods of
testing printability such as rheology and ink consistency (dis-
cussed later), they compared sample dimensions inputted into
the software and those obtained after fabrication. Although
they had obviously tried to make some headway in measuring
pore diameters and filament widths, it simply was not robust
enough as a means to correlating intended dimension parame-
ters to those that were actually measured. The study highlighted
that alginate-gelatin showed better resolution with respect to
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pore diameter and filament width at higher
percentage compositions. However, quanti-
tatively there were large variations in dimen-
sions compared to the intended dimensions.
Visually on the other hand, better resolution
of pores and widths was evident (Figure 5).
2.2. Computer Simulation and Modeling
There is no question that quantitative anal-
ysis of post-printing structures is difficult,
and it has often been trial and error. In order
to achieve a successful 3D bioprinting proce-
dure however, parameters must be carefully
tuned. Many reports have focussed on illus-
trating how bioink properties and printing
parameters can either affect cell viability or
bioink printability following printing. Yet it is
not until the last couple of years that studies
have tried to highlight the importance of
the two combined. Good progress has been
made in trying to semi-quantify printability
using mathematical models or more quanti-
tative methods using computational analysis.
Extrusion-based bioprinting systems use
mini-tissues as bioinks.[51,52] Mini-tissues
combine biomimicry with complex, self-
assembly processes to produce large-scale
functional tissue. Biological structure forma-
tion can be characterized by the coalescence
of discrete mini-tissues in the form of multi-
cellular spheroids or cylinders.[52] Shape evolution during tissue
fusion[53] has been shown to be analogous to merging of liquid
drops.[51,54] This liquid analogy has been exploited to develop
an experimental-theoretical-computational formalism.[55,56]
One group[37] have made great progress in this area using a
computer simulation method known as the cellular particle
dynamics (CPDs). This method can predict the shape evolution
of multicellular systems that undergo shape-changing biome-
chanical relaxation (Mathematic details of the CPD formalism
can be found in[55]). Furthermore, it has been developed with
the intention to make extrusion bioprinting more predictive,
thus reproducible and more time efficient.[37] They found that
complex, mathematical modeling via CPD simulations can be
used to predict post-printing structure formation even in the
case of volume changing bioink units. They went on to highlight
that tubes printed with cylindrical bioink formed nearly twice
as fast as one printed with spheroidal bioink. Figure 6 shows
snapshots from the start and end of CPD simulations through
the fusion of stacked rings formed by spherical and cylindrical
cellular aggregates. CPD simulation was then used to predict
the tube formation for tubes built with spherical (21.7 hours)
and cylindrical (11.8 hours) bioink units. Albeit, this would
appear to be more appropriate for tubular organ structures such
as fabricating vascular grafts. It certainly appears to be a sig-
nificant step in the right direction by being the conduit between
needing a 3D tissue engineering construct and potentially pro-
ducing an accurate, reproducible structure with high fidelity.
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Figure 2. The biofabrication window. Advanced bioinks are being rationally designed to have excellent biocompatibility with high shape fidelity con-
structs. There has been a paradigm shift from the more traditional bioinks which have seen a stand-off between shape fidelity and biocompatibility
(top left and bottom right). Stiffer, more dense polymeric networks result in better printable bioinks but lead to a poor cell culture microenvironment.
Adapted with permission.[10,15] Copyright 2016, Springer.

More recently, Ouyang et al. (2016) have systematically
studied the influence of bioink properties and printing para­
meters on bioink printability and embryonic stem cells viability
using extrusion-based cell printing.[24] The study used three
extrusion states: under gelation, proper gelation and over gela-
tion (Figure 7A). In this study, gelation is defined as hydrogel
fixation using post-processing chemical crosslinking (calcium
chloride) over a range of temperatures. In ideal gelation con-
ditions, the extruded filament would demonstrate clear mor-
phology with smooth surface and constant width in three
dimensions resulting in regular grids and square holes in the
fabricated construct. In under gelation conditions, a more
liquid-like state was created where upper and lower layers
would fuse and create holes that are more circular. They then

Figure 3.  Core fabrication process for 3D bioprinting technology.

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Table 3.  Key parameters for measuring printability.

Parameter(s) Conclusions Reference

Rheology • Printing accuracy determined by objectively quantifying printability over a 1 cm2 area and evaluated in terms of adhering [28]
to the programmed 1 cm2 dimensions
• Printing accuracy was calculated by dividing 1 cm2 by the actual printed area and multiplying by 100%
• More accurate (>95%) printing was found in higher viscosity bioinks (chitosan, chitosan-collagen,
methylcellulose-hyaluronan) which prevented them from spreading out over the printing area
• More viscous bioinks could only be printed via syringe deposition, compared to less viscous bioinks which were
printed using pressure-driven printers
• Oxidized alginate biofunctionalized with cell recognition motif, RGD resulted in a practical method of controlling viscosity [29]
and biodegradation
• Optimal range of viscosity suitable for high fidelity printing was found to be between 400 mm2 s−1 and 3000 mm2 s−1
• Hydroxyapatite made the bioink more viscous and difficult to print [30]
• Lower viscosity hydrogels led to watery, soft structures that could not hold their shape post-printing [31]
• Highly viscous materials were difficult to print and deposit smoothly
• Better rheological properties achieved through structural support of nanocellulose and cross-linking ability of alginate [32]
• Viscoelasticity was found to be the decisive factor for printability in microextrusion-based 3D printing technology [33]
• Incorporating collagen into the bioink enhances extrudability, and alters rheological properties to allow better control over [34]
shape fidelity
• Gelation viscosity remained on the same level when bioink concentration was constant, suggesting that gelation viscosity [24]
could act as an intrinsic property parameter representing bioink rheology
• Longer gelation times resulted in poor printability
• High resolution bioprinting of viscous materials was possible at lower shear stress [35]
• All samples of alginate with calcium chloride showed shear thinning properties [36]
• Incorporating graphene oxide increased viscosity and led to different thixotropic properties with a better quality of the
printed construct
Bioink composition • Stable constructs created when gelatin added to alginate [30]
and concentration
• Incorporating nanocellulose into alginate improved shape fidelity [32]
• Ideal printing conditions that gave best resolution were with 7.5% gelatin with 1% alginate and a printing temperature [24]
of 27.5 and 30 °C
Computer simulation/ • Predicts shape evolution of multicellular systems that undergo shape-changing relaxation [37]
modeling • Printing using cylindrical bioinks form twice as fast as using spheroidal bioinks
• Fluid-dynamic modeling used to control shear stress at the nozzle site, which could be adjusted by varying printing [35]
pressure, hydrogel viscosity and nozzle diameter
Nozzle variables • Smaller nozzle diameters can lead to better print resolution [38]
• Print scaffolds thinner than target size as post-printing expansion occurs
• Smaller diameter nozzles do not necessarily enhance printing resolution [39]
• Higher pressures may be necessary if printing with fine nozzles and highly viscous materials to overcome shear resistance
at the cost of resolution loss
• Addition of nozzle tip heaters allowed homogenous deposition of hydrogel and eliminated clogging at tip [30]
• Control of nozzle parameters and diffusion rate at lattice intersections is important for printing intricate structures with [40]
high shape fidelity
• Air pressure was found to be the most important parameter as it determined extrusion output
Pore and/or filament • Correlated intended and measured dimensions [41]
dimensions • Quantitatively, large variations between intended and measured dimensions
• Better resolution of filament widths and pore size with alginate-gelatin bioinks by controlling temperature of formulations
Grid geometry • Solidified filament morphology demonstrated with sufficient mechanical strength to support the deposition of upper layers [42]
• Semi-quantifiable method developed to assess printability based on fibre/grid morphology [24]
Effect of printing angles • Shorter distances between nozzle tips and cross-linking agents such as calcium chloride led to better mechanical [43]
on shape fidelity properties, support and printing quality when printed at varying angles for printing complex, 3D structures
• Printing quality was worse with acute angles compared to obtuse and right angles [40]
Resolution determined • Incorporation of hydroxyapatite led to enhanced visibility of hydrogel and enhanced resolution of intricate microstructures [30]
by micro CT

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Table 3. Continued.

Parameter(s) Conclusions Reference

• Used to quantify scaffold structural properties, such as fiber diameter, which was used as a metric to assess printing quality [44]
• Linear regression models used to determine the impact of material and printing parameters on printing accuracy
• Using a direct melt extrusion system and comparing needle size on printing quality,
○ material properties were found to play a more important role in printing quality with smaller diameter needles

○ printing parameters, such as pressure and speed became more significant in determing fiber quality when using large

diameter needles
• 3D printing geometry accuracy was quantified and compared using micro-CT (slice-by-slice comparison) [38]
• Micro-CT generated slices were compared to the corresponding image-derived layers of the original STL files in the XY-plane
• Slices were overlapped and compared using Boolean operations
Oxygen concentration in • Oxygen inhibition had a detrimental effect on shape fidelity which was overcome by increasing UV or visible light [34]
printing environment photoinitiator concentration, or light irradiation intensity

Figure 4.  Printing heterogeneous valve and scaled valve constructs. A) Porcine aortic valve model was B) printed with PEG-DA hydrogels. C) Scaffolds
were printed with inner diameters of 22, 17 and 12 mm. D) Axisymmetric valve model was E) printed with two blends of hydrogels F) and at 22, 17
and 12 mm inner diameter. Fidelity analysis of fully hydrated printed valve scaffold geometry compared to model geometry using microCT-derived STL
files. G) Heat maps and average % frequency histograms representing surface deviations between printed porcine scaffold versus model geometries
and H) printed simplified scaffolds versus model geometries. Warmer colours indicate positive deviation while cooler colours indicate negative devia-
tion in millimetres. % frequency histograms show the deviations that lie within ± 10% tolerance (green), and outside the tolerance (blue and pink).
Reproduced with permission.[38] Copyright 2012, IOP Publishing.

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Figure 5.  Macrostructure of alginate-gelatin scaffolds with concentrations of (A) 1%, (B) 2% and (C) 4%. Extrusion printing of alginate-gelatin scaffold
is seen in (D). A mechanically robust 2% alginate-gelatin scaffold after crosslinking with calcium chloride is seen in (E). Dimensions of alginate-gelatin
scaffolds +/− calcium chloride is highlighted in (F). Reproduced with permission.[41] Copyright 2013, The Royal Society of Chemistry.

applied mathematical formulae as seen in Equations (1) and (2) π 1 L2

to define bioink printability (Pr).
4π A (1)
C=
L2 larity (C) so C is equal to 1, and the closer values are to 1, the
Figure 6.  Tube formation from CPD simulations at the start (t = 0) and
end (t = 620). Time (t) units are CPD related and when mathemati-
cally processed, correlate with tube formation times of 21.7 hours and
11.8 hours for spherical and cylindrical bioink units, respectively. Different
colours are used to emphasize the absence of mixing during the fusion
process. Reproduced with permission.[37] Copyright 2015, IOP Publishing.
Pr = . = (2)
4 C 16 A
They use the principles that circles have the highest circu-
more circular they are (Equation (1)). They then define printa-
bility (Pr) based on a square using Equation (2). They state that
for an ideal gelation condition or perfect printability status, the
interconnected channels of the constructs would demonstrate
a square shape, with a Pr value of 1. Hence, larger Pr values
would correlate with a greater degree of gelation of the bioink
and vice versa. The study used varying compositions of gelatin
and alginate at different nozzle temperatures. It highlighted
very nicely how important controlling design parameters are
when trying to printing materials with defined, intricate struc-
ture and high fidelity. Under proper gelation conditions, smooth
and uniform filaments were continuously extruded, which
resulted in grid constructs with distinguished layers (Figure 7B).
When they analysed printability, Pr over time, they found
that the 7.5% gelatin with 1% alginate at 27.5 °C and 30 °C,
were the proper printing temperatures and 25 °C resulted in
over gelation after 10 minutes (Figure 7C). They then went on
to demonstrate that cell-laden constructs showed high shape

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Figure 7.  Bioink printability assessment under different printing parameter combinations. A) Evaluation of printability (Pr) under different gelation
states – under-, proper- and over-gelation. B) Optical microscope images at 30 mins. C) Semi-quantified Pr value construct for gelatin/alginate
bioink with different concentrations under different printing temperatures. Scale bars are 1 mm. Reproduced with permission.[24] Copyright 2016, IOP
Publishing.

fidelity and clear contours, and that embryonic stem cells were
uniformly distributed in the hydrogel filaments. In terms of
assessing printability based on the degree of gelation, this study
has made excellent progress in trying to help define printability,
or at least provide some quantification so that a trial and error
approach is minimized. It further demonstrates how careful
control of design and process parameters can lead to constructs
with precise and accurate uniformity.
2.3. Printer/Nozzle Parameters and Printing Angles
A more recent study has tried to address the apparent lack of
systematically investigating and controlling printing para­meters
as a means of printing structures with high fidelity, reso­lution
and accuracy.[40] They highlighted important factors that must
be considered in order to produce accurate structures, from
printing lines in 1D to printing lattices in 2D to ultimately
printing 3D structures. Hydrogels were created using alginate
and gelatin, with calcium chloride as the crosslinking
agent. They demonstrated that line printing was affected by
(1) air pressure (the most important parameter as it determines
extrusion output), (2) nozzle feed rate and (3) distance between
nozzle and substrate. Printing quality was also found to be
worse with acute angles compared with obtuse and right angles,
but could be improved when the extrusion rate is controlled by
the motor speed (Figure 8A–D; Table 3). They found that in
sharp angle printing, there was an overlap problem in which
extrusion of the hydrogel was doubled. To overcome this, the
extrusion rate was reduced. The double extrusion at the overlap
(Figure 8C) could therefore be released by doubling the nozzle
feedrate and creating a more uniform extrusion (Figure 8D).
In order to produce cell-laden scaffolds, lattice structures
have been frequently used and so precise control of printing
quality is critical. They found that line distance and the inter-
section area of the lines affected lattice quality, in relation to

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Figure 8.  High fidelity 3D printing of alginate/gelatin hydrogels. Overlap in sharp corner printing. Line shape of (A) obtuse and (B) right angle printing.
C) Double extrusion at overlap area. D) Uniform extrusion by doubling the nozzle federate in the overlap area. Hydrogel diffusion at the intersections
of lines. The model and shape of lattice intersections when line distance was (E) 5 mm and (F) 2 mm. Hydrogel diffusion and fusion when lattice
scaffolds had line distances of (G) 4 mm, (H) 3 mm, (I) 2 mm and (J) 1 mm. Printability of 3D hydrogel scaffolds. K) Digital model of 3D scaffold.
L) An outline of the first layer of the scaffold with line width 2 mm. M) First layer of the scaffold shape and N) three views of hydrogel scaffolds formed
at different angles (30 layers printed with a height of 15 mm, fidelity of the top surface is approximately 80%). Reproduced with permission.[40] Copy-
right 2016, Nature Publishing Group.

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diffusion rates. Hence larger line distances led to a drop in dif-
fusion rate. Diffusion rates appeared to have a direct affect on
shape fidelity as when reticular shapes were printed one on top
of another, the pore would radially narrow along the second
hydrogel layer line and did not change axially. Overlapped
hydrogels diffused due to gravity which resulted in diffusion
between lattice intersections (Figure 8E–F). It was also apparent
that for larger structures with larger line distances within lat-
tices, diffusion and fusion would be mitigated (Figure 8G–J),
yet for smaller, more intricate structures this may prove more
problematic. Thus translating this in to 3D printing to produce
high shape fidelity objects was possible (Figure 8K–N).
2.4. Oxygen Concentration in the Printing Environment
A novel method in trying to improve print fidelity has recently
been reported.[34] This study was one of the first to highlight
the importance of oxygen inhibition on print fidelity in photo-
polymerized gelatin-methacryloyl hydrogel constructs. Oxygen
can act as a radical scavenger. In certain applications this has
many benefits, however in 3D bioprinting it can often lead to
incomplete or insufficient crosslinking which may affect the
resulting shape fidelity.[18,57] It has been shown that free radi-
cals formed from photolysis of photoinitiators are converted
to peroxyl radicals that do not react with ester bonds.[57] These
peroxyl radicals can form hydroxyperoxides or alcohols which
prevent covalent crosslinking. Inert atmospheres involving
nitrogen purging has been used widely by many to prevent
entry of oxygen, yet this would be unsuitable in 3D bioprinting
involving cell-laden constructs. Lim et al. (2016) utilized light-
activated polymerization systems consisting of water-soluble
photoinitiators ruthenium (Ru) and sodium persulfate (SPS)
compounds which can absorb photons in the visible light
range.[34] They compared this system to the conventional UV/
Irgacure 2959 system and found that oxygen inhibition had
a detrimental effect on shape fidelity which was overcome by
increasing UV or visible light photoinitiator concentration, or
Figure 9.  3D plotting of gelatin+methacryloyl+collagen constructs that have been photopolymerized in UV+l2959 and Vis+Ru/SPS systems. Different
shape fidelity can be seen post-irradiation and post-swelling with the different systems. Reproduced with permission.[34] Copyright 2016, American
Chemical Society.
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light irradiation intensity. They also incorporated a small con-
centration of collagen I (0.6 wt %) which was found to enhance
extrudability of the bioink, and alter rheological properties to
allow for better control over shape fidelity. In the Vis+Ru+SPS
system, cell cytocompatibility was found to be significantly
improved and the resulting porous biofabricated constructs
were found to be less susceptible to the effects of oxygen inhibi-
tion and incomplete crosslinking. This was evident by the lower
percentage change in fiber diameter compared to the UV+l2959
system post-swelling (Figure 9).
2.5. Viscosity and Rheology
Viscosity is another key parameter to consider for successful
3D bioprinting. Many groups report on viscosity and rheo­
logy studies in conjunction with printability assessment for
bioprinting materials. Hydrogels have received much more
attention than other biomaterials as viscosity can be carefully
controlled.[22,58,59] Hydrogels must be sufficiently viscous to
retain their shape during the printing process and they should
have crosslinking capabilities so that the 3D structure is not
altered after printing. Low viscosity materials have been shown
to be more attractive for bioprinting as cells can grow well in
the low pressure environment.[60] However, it is well known
that 3D printed structures tend to collapse due to low viscosity.
Although many research groups have focussed on adjusting
the concentration and molecular weight of various hydrogels
in order to change viscosity, shape fidelity has often not been
achieved after printing. In this respect, hydrogels have often
been printed simultaneously with other materials in order to
increase structural fidelity. Fugitive materials have also been
employed in order to maintain or improve shape fidelity with
low viscosity bioinks.[58] Recently, nanocellulose has been
combined with alginate to prepare sponges used for adipose
tissue[61] and for cell encapsulation.[62] Markstedt et al. (2015)
found that in order to improve shape fidelity of alginate, nano-
cellulose can be incorporated into the bioink formulation.[32]
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They found that the low zero-shear viscosity of alginates gave
poor shape fidelity when printing. Nanocellulose alone had a
higher viscosity at low shear rates, however when printed the
shape was destroyed when mechanical force applied. The com-
bination of better rheological properties using nanocellulose
with the cross-linking ability of alginate yielded the best results.
Photocrosslinking has emerged as a promising strategy
to address several challenges associated with lower viscosity
bioinks, cell viability and printability. Since light is minimally
invasive, spatiotemporally controlling the application of light in
order to crosslink photosensitive cell-laden bioinks with high
shape fidelity and without significantly reducing cell viability
has been reported.[63] In one study, photocrosslinkable hydro-
gels were generated using methyacrylated hyaluronic acid and
methyacrylated gelatin, and human aortic interstitial cells were
encapsulated within the hydrogels for 3D bioprinting.[31] They
found that systematically varying hydrogel concentration had
a significant impact on viscosity and material stiffness. When
viscosity values were lower than 100 Pa⋅s, the material was too
fluidic to hold the printed shape, and when values were higher
than 104 Pa⋅s, materials were too viscous and difficult to deposit
smoothly.
The combination of alginate and gelatin with varying con-
centrations of hydroxyapatite have been 3D bioprinted to form
hydrogels for use in bone tissue engineering. One group sug-
gested that combining gelatin and alginate improves instanta-
neous stability by maintaining initial bonding between single
layers due to the gelation of gelatin as well as long-term stability
of the whole construct by chemically crosslinking alginate.[30]
The addition of hydroxyapatite led to enhanced visibility of the
hydrogel when seen with micro CT and led to higher resolution
of intricate microstructures. Introducing hydroxyapatite into
the alginate-gelatin composite rendered solutions more viscous
and more difficult to handle and print. They emphasized that
viscosity affects the printing process primarily at the dispense
tip which is the most critical location during extrusion. They
also highlighted that control of the tip temperature affected vis-
cosity of the bioink, insomuch that the dual action of a heatable
Figure 10.  A) 3D bioprinting with bi-phasic support liquid for the manufacturing of thin-walled, multi-layered hydrogel structures. B) Schematic illus-
tration of the velocity and shear stress distribution as well as the stress impinged on cells inside a bioprinter nozzle. Reproduced with permission.[35]
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print-head and additional tip heaters allowed homogenous
deposition of hydrogels. Thus eliminating clogging issues at
dispense tips, and also maintaining liquidity of the bioink so
that the printing substrate could be cooled down sufficiently to
guarantee instantaneous gelation of gelatin. This then created
stable 3D constructs that maintained the shape of the prede-
fined geometry.
An understanding of shear stress is another important factor
to consider in bioprinting applications. Studies have demon-
strated that shear stress can be influenced by varying nozzle
diameters, printing pressures and viscosity of the material to be
printed.[64] It has also been well documented that shear stress
plays a pivotal role in cell biology, particularly in relation to cell
signaling[65] and differentiation.[66] Recent research has shown
that hydrogel viscosity and nozzle size directly affect shear
stress.[35] They presented a microvalve-based bioprinting system
for the manufacturing of high resolution, multi-material 3D
structures (Figure 10A). They then used fluid-dynamic mode­
ling to precisely control shear stress at the nozzle site, which
could be adjusted by varying the printing pressure, hydrogel
viscosity and nozzle diameter. Subsequently, it was highlighted
how cell viability and proliferation potential were affected by
different levels of shear stress (Figure 10B). At lower rates of
shear stress (<5 kPa), L929 mouse fibroblasts remained viable
at 96%, however this viability decreased to 91% and drasti-
cally to 76% at shear stress ranges from 5–10 kPa and >10 kPa
respectively. They further demonstrated that high-resolution
bioprinting of viscous materials is possible at a low shear stress
level, whereby computer aided design software was trans-
formed into multi-layered drop models using custom designed
algorithms. Although this method was specific to microvalve-
based bioprinting, and was not extrusion-based, it certainly is
pioneering in using fluid-dynamics and mathematical analysis
to understand the importance of shear stress on high resolu-
tion, drop-on-demand bioprinted 3D constructs.
It is evident that very few reports in the literature have dis-
cussed the relationship between rheological properties of
bioinks and 3D printability. We have seen that alginates have
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
been a mainstay as scaffolds for tissue engineering. Yet native
alginate is bioinert with a poor degradation profile. This has led
many groups to functionalize alginate to make it more bioac-
tive, and chemically modify it in order to improve biodegra-
dability. Oxidized alginates have been shown to have a better
degradation process with promising results for tissue engi-
neering applications.[67] Further studies have shown that these
biodegradable alginates have potential in 3D bioprinting.[29]
Oxidized alginate conjugated with the cell recognition motif
RGD, and human adipose-derived stem cells were printed in
a point-to-point method. They found that altering oxidation
and concentration resulted in a practical method of controlling
the viscosity of degradable alginates. In order to asses printing
resolution, dot arrays were printed using different biodegrad-
able alginates to examine the effect of viscosity on dot fidelity
and size. Hence an optimal range of viscosity suitable for high
fidelity printing was found to be between 400 mm2 s−1 and
3000 mm2 s−1. Hence rheological properties are key players in
controlling printability and fidelity of 3D constructs.
In order for a bioink to be printable, it should ideally be
thixotropic i.e. exhibit non-Newtonian shear thinning behav-
iour whereby the higher the stress they experience, the lower
their viscosity, and have a reasonable recovery time. Thixotropy
provides information on how quickly and how much viscosity
bioinks recover post-printing, whilst the recovery time is the
time given to the bioink in order for viscosity to be recovered
during bioprinting. Since not all bioinks are thixotropic, it is
essential that bioinks must be rheologically evaluated to deter-
mine the effects of 3D printing on print quality. Studies have
shown that materials that exhibit a linear relationship between
viscosity and shear rate improves print quality.[5] Hence, finding
relationships between piston speeds of bioprinters, viscosity,
shear rates and thixotropy are fundamentally important. This
was highlighted by one group who performed rheological
studies on 3D printability of alginate-based hydrogels and inves-
tigated the effects of introducing graphene oxide into these
hydrogels.[36] Graphene oxide is known to have excellent bio-
compatibility, mechanical stiffness and is ultra-strong so may
be able to modify scaffold properties. This study demonstrated
that measurable parameters can be defined for quantifying the
quality of 3D printing. For instance, they found that all sam-
ples of alginate with calcium chloride showed shear thinning
properties. Recovery time decreased as alginate concentration
increased, but most samples could not recover after a few sec-
onds, and they needed more than 30 seconds to recover their
viscosities to 83% of the initial values. They found that incor-
porating graphene oxide increased viscosity and led to dif-
ferent thixotropic properties with a better quality of the printed
construct. This is another study that has defined measureable
parameters in order to fabricate high fidelity and accurate 3D
printed constructs.
3. Conclusions
Research in the field of 3D bioprinting is very topical, and
research efforts have accelerated worldwide over the last few
years. 3D printing was highlighted in The Economist as having
enormous potential.[68] The inception of new journals focusing
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entirely on the field (The International Journal of Bioprinting,
Bioprinting, Journal of 3D Printing in Medicine and 3D
Printing in Medicine) attest to the emerging volume of litera-
ture. Early studies focussed on the assessment of ‘printability’ of
bioinks whilst investigating different biofabrication platforms.
Basic correlations of printability was compared to mechanical
properties and biocompatibiity of the bioink. More recently,
more precise control of process parameters during the printing
process have been interrogated and correlated with fidelity and
reproducibility of 3D constructs. It is clear that bioink compo-
sition and concentration, rheology and manipulation of nozzle
parameters are key factors in order to evaluate and achieve
good printability with high shape fidelity and reproducibility.
The potential to biofunctionalize and decorate bioinks with cell
recognition sequences may enhance physical, chemical and bio-
logical properties of next generation, advanced bioinks.
Advanced research methodology and technical advantages
pave the way for emerging 4D printing methods, adding in
temporal dimensions. The success of such strategies will allow
biofabricated constructs to adapt and transform to the new
micro-environment and a range of physico-chemical cues over
time, resulting in accurate biomimicry and smart biomaterials.
This advanced and innovative technology has great potential in
the field of biomedicine, particularly in areas such as regenera-
tive medicine and biomedical devices.
Despite increased research infrastructures, man power,
funding and technological advances, significant challenges
remain. The design and manufacture of specialized bioprinters
is still niche, and high cost, meaning a small number of research
groups do not have ready access to them. However, costs have
reduced considerably in the past few years with commercial
bioprinters being sold for ≈$5000, and systems being built with
open-source designs for <$1000. Development of mechanically
appropriate, biomimetic, durable bioinks that encourage cell
proliferation and survival whilst maintaining shape fidelity still
remains the holy grail. 4D bioprinting, improvements in print
resolution and modification at the molecular scale are key areas
of research to watch closely in the future.
It is in the interests of patients, scientists, clinicians and
industry to see translation of 3D bioprinting to routine clinical
application, and this will depend on the aformentioned devel-
opments in research, reducing the cost and improving repro-
ducibility of the bioprinting process and overcoming significant
regulatory/legal hurdles. To date, the FDA has not imposed
specific restrictions on bioprinting technology which will facili-
tate future developments. Bioprinting potentially has to face a
dilemma for mass market application. Whilst the technology
and products have to be scalable in order to be economically
viable, the advance of personalized medicine means that a
number of products will be made to order rather than available
‘off the shelf’.
3D bioprinting as a field may be considered analogous to the
introduction of tissue engineering over 30 years ago—where
interdisciplinary researchers including materials scientists,
cell biologists, engineers, computational modelers, biophysi-
cists and clinicians came together to try and help the problems
with tissue/organ dysfunction and failure. Although the field
has progressed slower than most hoped, modern technological
and scientific advances coupled with advances in the internet
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
and advances in international collaboration have brought us
closer to creating artificial tissues/organs that closely mimic
native structures. Bioprinting offers the opportunities to fine
tune such processes to produce a paradigm shift in medical
practice.
Acknowledgements
Mr Steve Atherton RMIP MIMI, Medical Illustrator, ABMU Health Board
for Figures in Table 1, and Figures 1– 3.
Conflict of Interest
The authors declare no conflict of interest.
Keywords
biofabrication, bioprinting, extrusion-based 3D printing, printability,
printing parameters
Received: February 28, 2017
Revised: May 2, 2017
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