RESEARCH ARTICLE

Concentric bioprinting of alginate-based tubular

constructs using multi-nozzle extrusion-based
technique
Edgar Y. S. Tan 1,2 and Wai Yee Yeong1,2*
1
2

School of Mechanical and Aerospace Engineering, Nanyang Technological University, Singapore 637372, Singapore
Singapore Centre for 3D Printing, Singapore 6397798, Singapore

Abstract: Bioprinting is a layer-by-layer additive fabrication technique for making three-dimensional (3D) tissue and
organ constructs using biological products. The capability to fabricate 3D tubular structure in free-form or vertical configuration is the first step towards the possibility of organ printing in three dimensions. In this study, alginate-based tubular structures of varying viscosity were printed vertically using multi-nozzle extrusion-based technique. Manufacturing challenges associated with the vertical printing configurations are also discussed here. We have also proposed
measurable parameters to quantify the quality of printing for systematic investigation in bioprinting. This study lays a
foundation for the successful fabrication of viable 3D tubular constructs.
Keywords: 3D printing, alginate, viscosity, rapid prototyping, additive manufacturing, extrusion
*Correspondence to: Wai Yee Yeong, Singapore Centre of 3D Printing, Singapore 6397798, Singapore; Email: wyyeong@ntu.edu.sg

Received: May 7, 2015; Accepted: June 17, 2015; Published Online: July 2, 2015
Citation: Tan E Y S and Yeong W Y, 2015, Concentric bioprinting of alginate-based tubular constructs using multi-nozzle extrusion-based technique. International Journal of Bioprinting, vol.1(1): 4956. http://dx.doi.org/10.18063/IJB.2015.01.003.

1. Introduction

ubular structures play an important role in multicellular organisms by improving the efficiency of transportation of nutrients, growth factors
and specific signals into and out of different tissue
regions. This vast improvement in transporting materials within the organism has enabled multicellular
cells to develop into larger organisms. As such, the
need for tubular structures in tissue engineering (TE)
has been growing in importance as demands for replacement of these structures grow. Tubular structures
in TE have served a multitude of applications ranging
from micro-scaled products such as nerve guides to
larger products such as vascular channels[13] and gastrointestinal substitutes[4, 5].
In recent years, various emerging medical products

have emerged from the use of synthetic tubular structures. These products have been developed using
technologies such as electrospinning[2, 6], melt spinning[79], dip coating[1012] and extrusion[13, 14]. These
methods usually use poly(-caprolactone) (PCL), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA),
polyglycolic acid (PGA) and poly(ethylene-co-vinyl
acetate) (PEVA) to fabricate products; The product fabricated can reach a resolution of approximately 7 m[15].
Although these methods have proven to be effective in
creating tubular structures, these synthetic polymeric
materials lack adhesion factors such as arginine-glycine-aspartic acid (RGD) which are important for cell
adhesion, proliferation and matrix production[16, 17].
Moreover, these materials usually degradealthough
only after long periods of timeand would induce
foreign body response by the host[18] which could be

Concentric bioprinting of alginate-based tubular constructs using multi-nozzle extrusion-based technique. 2015 Edgar Y. S. Tan and Wai Yee Yeong.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License
(http://creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.

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Concentric bioprinting of alginate-based tubular constructs using multi-nozzle extrusion-based technique

lethal to the patient in the long run[19]. Furthermore,

the degradation residues of these materials could potentially cause substantial damage to the tissue around
it[20].
In light of such issues, researchers have begun to
develop hydrogel tubular structures for TE purposes.
These tubular structures are usually cast using a
mold[21, 22], centrifuged in tubes[23, 24] or co-axially
extruded[25, 26]. Although the strength of such gels are
much weaker than polymers in terms of tensile
strength and ductility, the use of some of these gels
improves cell compatibility and have been shown to
reduce autoimmune responses[27].
Recently, bioprinting has emerged as a potential
method for fabricating cell-encapsulated hydrogel
tubular construct[28, 29]. This method of fabrication
increases the flexibility and versatility in the printing
process, allowing various forms and sizes of tubular
structures to be manufactured with design-driven repeatability[30].
Tubular structures fabricated using bioprinting have
been demonstrated with multiple materials in several
reports. Examples of some of these materials used in
printing include hyaluronan[31], alginate[32] and gelatin-derived products[33]. They are usually built horizontally as the weight from the structure itself would
not allow for sufficient structural integrity of the base
if it was to be built vertically. However, such method
of producing tubular structure would cause irregularities in the diameter if the diameter is too large. Fabricating a tubular structure in the horizontal configuration also restricts the potential in constructing tubular
branches on a 3D scale.
The main objective of this study is to investigate
the feasibility of fabricating vertical tubular structure
using a multi-nozzle extrusion-based technique. This
method was derived based on concurrent deposition of
cross-linking agent into the concentric tubular wall
during each layer of deposition. Alginate was selected
as the model material to demonstrate the feasibility of
this versatile and simple method. This method could
be extended for different hydrogels and their crosslinking agents.

2. Materials and Methods

2.1 Hydrogel
Sodium alginate powder (Sigma-Aldrich) was dissolved at 0.06 g/mL in phosphate-buffered saline solution
under constant stirring. Next, 0.01 g/mL to 0.03 g/mL
50

of xanthan gum (XG) from Xanthomonas campestris

(Sigma-Aldrich) was dissolved into the alginate solution. Thereafter, 0.022 g/mL of calcium chloride
(CaCl2) (Sigma-Aldrich) was added dropwise to the
mixture. Additionally, 500 mmol/L of CaCl2 was prepared as an additional cross-linking solution.
2.2 Hydrogel Characterisation
Rheology of the hydrogel was characterized using a
Discovery Hybrid Rheometer 2 (TA Instruments) using a 40 mm parallel plate geometry with a measurement gap of 0.5 mm and Peltier plate thermal control.
After loading, the samples were conditioned by subjecting to 30 s pre-shear at 500 s1 followed by one
minute equilibrium before measurements were taken.
Shear-dependent viscosity was evaluated using a
stepped ramp of shear rate from 11000 s1 and the
process was done at 25C. In this method, the hydrogel was used as it is with the exception of the addition
of excess CaCl2. Measurements were taken at 10
points per decade.
2.3 Printing Process
Tubular structure design and process was input into
the bioprinter using BioCad (RegenHu). First, a circular structure with radius of 6 mm was defined in the
system as the extrusion route for the hydrogel. Next, a
secondary loop of radius of 4 mm which is concentric
with the hydrogel path was made for the dispensing
route for CaCl2. The hydrogel was placed into a timebased extruder while CaCl2 was placed in a microvalved controlled dispenser. The printing process was
done at room temperature. The printing process is
shown in Figure 1.
The pressure of the bioprinter was set at 1.5 bar for
the hydrogel and 0.5 bar for CaCl2. Tubular constructs
were printed using RegenHus Biofactory. Hydrogel
was printed through a 0.25 mm syringe needle
(Needle DD-135N-N4) while the CaCl2 solution was
dispensed through a 0.3 mm needle tip. The path
speed of the hydrogel was 500 mm/min while the path
speed of the CaCl2 was 100 mm/min. CaCl2 solutions
path speed was much lower than the hydrogel to allow
it to sufficiently fill up the tube during dispensing. The
layer thickness of the hydrogel was set at 0.2 mm. To
allow sufficient time for the layers to fuse before the
gel cross-linked, the printer was programmed to dispense CaCl2 only after 3 layers have been built. Subsequent layers were added in the vertical axis after
gelling interaction was achieved in the first 3 layers,

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Edgar Y. S. Tan and Wai Yee Yeong

Figure 1. Fabricating vertical tubular structure using multi-nozzle extrusion-based technique.

providing it with sufficient structural integrity to support the next few layers of hydrogel. To show the feasibility of this printing strategy, the structure was
printed to a height of 4.8 mm.
2.4 Image Analysis for Measurement of Printing
Quality
Images obtained were processed using ImageJ (National Institiute of Health, USA). The images were
calibrated to correlate dimension of the physical objects to the pixel size. The images were then converted
into an 8-bit image before undergoing image segmentation. The tubular structure was measured in terms of
size, area and perimeter with the image processing
methods. Circularity results were calculated by comparing the difference in the ratio between the radius
obtained from the perimeter and the area. When the
ratio is approximated to 1, there is no distortion in the
circle.

3. Results and Discussion

3.1 Challenges in Bioprinting Tubular Structures
Printing tubular structures in the vertical configuration
is very challenging. The strength of the base material
must be robust enough to withstand the weight of the
entire structure. This is especially difficult as hydrogel
is a soft material with high water content. Insufficient
structural strength of the hydrogel base will result in

the collapse of the tubular structure in vertical configuration. Therefore, the viscosity of the material must
be relatively high in order to sustain the compressive
pressure induced by the upper layers of the tubular
structure. However, high shear force will be needed
for extrusion of viscous materials, which might impact
the cells negatively and reduce cell viability. Moreover, from the Stokes-Einstein equation (see equation
(1)), it can be predicted that the increase in viscosity
would have an inverse effect on the diffusion rate of
molecules[34]. Low diffusion rate in the hydrogel will
inhibit nutrient exchange into the hydrogel, which is
critical for cellular growth and survival. Thus, there is
a need to optimize the viscosity of the printable hydrogel for printability and diffusion rate.
(1)
Where, D represents the diffusion constant of the hydrogel, k is the Boltzmanns constant, T is the absolute
temperature, is the dynamic viscosity and Rs is the
radius of the particle.
3.2 Optimizing the Viscosity of Printable Hydrogel
The printable hydrogel needs to be optimized to have
low viscosity during printing, yet have sufficient mechanical strength upon being printed, with good diffusion rate for nutrient exchange. One of the ways to
overcome such conflicting requirements would be to

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Concentric bioprinting of alginate-based tubular constructs using multi-nozzle extrusion-based technique

complete gelation after printing. Although some research has been done on cross-linking alginate gels
simultaneously during printing, the process was
shown to be relatively slow as they were done in the
cross-linking solution or on the surface of the alginate
solution. Thus, the resolution of the fabricated structure is influenced by parameters such as surface tension and the speed of the extrusion, rendering a
non-direct way to control resolution.
In this study, XG was selected to formulate an optimal viscosity for alginate to retain its shape fidelity
after printing. XG is an anionic polysaccharide produced by the bacterium Xanthomonas capestris. XG
has been used in multiple applications ranging from
food[35], agriculture[36], petroleum[37] and in the pharmaceutical industry[38]. It is used mainly as a viscosity
enhancer and stabilizer in blends and has been reported to contain bio-adhesive properties[39]. Since XG
has only weak interactions with CaCl2, it serves as a
pure filler and will have minimum effect in the
cross-linking process. To understand the viscosity of
the hydrogel and its shear thinning properties, the rheology behaviour of the hydrogel was characterized
(Figure 2). With increasing amounts of XG, the solution tends toward a more viscous behavior as expected.
This viscosity will affect the volume of hydrogel extruded and the overall printing fidelity of the construct.
3.3 Quality of Printing
Tubular constructs were bioprinted successfully in the
vertical configuration using the concentric printing method developed in this study. In general, the designed

Figure 2. Viscosity as a function of shear rate for alginatexanthan gum gel. Xanthan gum concentrations varied from 1.0%
(blue diamond), 1.5% (orange square), 2.0% (grey triangle), 2.5%
(yellow cross) and 3.0% (blue asterisk).

computer-aided design profile was replicated successfully in the bioprinted hydrogel. The printing
quality was represented using the following measurements, namely tubular length (t), wall thickness (w),
and roundness (R). For calculation of roundness in the
equation 2, we require the perimeter (P) and the area
(A) of the inner cavity of the tube. Printing effects
in Figure 3 such as Spreading (e1 and e2) and opaque
layer thickness (OW) were also proposed and discussed.
Tubular length (t) helps to quantify if the layer
thickness was calibrated correctly such that sufficient
hydrogel was deposited to form layers additively to
eventually achieve the desired tubular height in vertical configuration. To optimize the printing, a group
of printing parameters such as extrusion speed, nozzle
diameter and pressure of the extrusion were synchronized to enable the calibrated system to deposit material
accurately on top of the previously printed layers[40].
With proper calibration, defects and delamination

Figure 3. Parameters for measurements recorded to quantify the printing quality of a bioprinted tubular construct.
52

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Edgar Y. S. Tan and Wai Yee Yeong

between the layers should not occur. The wall thickness, w, determines the minimum resolution of material deposited onto the platform. The spreading effect, e, was calculated as an angle from the platform.
Spreading displayed the effect of viscosity on the
physical dimensions of the construct. The opaque
layer thickness (OW) shows the degree of calcium
diffusion into the material. As the calcium diffuses
into the material, the ions cross-link within the alginate-XG solution. This reaction also caused the structure to turn white, allowing it to be quantifiable
through the thickness of the diffusion zone.
3.4 Wall Thickness
The wall thickness was measured to develop a presentation of how the viscosity affects the resolution.
From the results in Figure 4, it can be seen that at 2%
XG concentration, the width variation is minimal; indicating that printing these material is most stable at
that viscosity range. It can also be established that at
the concentration of 2%, less material has spread as
compared to at the 1% XG concentration. In this study,
as pressure was fixed at a specific value for the print,
the thickness of the wall was allowed to be a variable
factor. Thus, the optimal surface area can be determined to be sufficient to support the weight of the gel
deposited on it.

rence pixel for the perimeter of the cavity, P.

R=

( P )2

(2)
4 . A
The ideal roundness of a tubular construct is 1.0,
which implies that the area and perimeter are equal. In
this experiment, we showed that the pre-designed
roundness was achieved with 2% XG (see Figure 5),
indicating that an apparent optimal viscosity threshold
exist for a desired roundness value. The printed tubular structure became increasingly out-of-roundness
when the viscosity was too low at 1% XG or too high
at 3% XG. It was estimated that the movement of the
printing platform may have affected the shape fidelity
as the extrusion pressure and speed were kept constant
for all viscosity materials. When the viscosity is too
high, the volume of hydrogel that was dispensed may
have been insufficient, causing the material to be unstable and become out-of-round shape.

Figure 5. Circularity at different concentrations of xanthan gum.

3.6 Spreading

Figure 4. Wall thickness versus concentrations of xanthan gum.

Better shape fidelity can be derived from the spreading angle of the construct. Mirroring the concept of
contact angle for droplet spreadability, the right and
left angles of the base layer were measured and averaged. The optimal spread angle would be 90, indicating no spreading has occurred and thus representing

3.5 Roundness of Tubular Construct

Generally, there are multiple methods for measuring
the roundness of an object. In this case, roundness of
the construct was determined from the ratio between
the square of the perimeter and the area of the cavity
(see equation (2)). To calculate A and P in equation (2),
ImageJ was utilized to analyzed the cavity. Segmentation was performed on the image and the result was
calculated based on the number of pixels for area of
the cavity, A, and the length of the overall circumfe-

Figure 6. Spread angle at the different concentrations of xanthan gum.

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Concentric bioprinting of alginate-based tubular constructs using multi-nozzle extrusion-based technique

the best printing quality with the material as seen

in Figure 6. best printing quality was achieved for
formulation of 1.5% to 2.5%. Spreading of the material increased at low and high viscosity indicating an
optimal threshold of spreading could be related to the
viscosity of the material. The decrease in the spreading angle at 3% could indicate that the initial layer did
not have sufficient surface area to provide sufficient
compressive strength to withstand the weight of the
hydrogel that was layered on it.
3.7 Opaque Layer Thickness
The clear variation between the opaque area and the
transparent area indicates that the hydrogel is relatively viscous with the diffusion coefficient of the gel
reaching close to 1[41].The constructs develop an opaque interior due to the cross-linking effect of calcium
ion on the alginate-XG gel. The opaque wall is related
to the diffusion rate of the cross-linking agent in the
hydrogel. Based on estimation from equation (1), the
thickness of the opaque area should be inversely proportional to viscosity. The increase in concentration of
XG increases the number of molecular chains blocking the path of the ions as it diffuses into the gel. Thus,
the viscosity increases, reducing the diffusion rate as
shown in Figure 7.

The photos of the printed construct are shown in

Figure 8. When the concentration of XG used is too
low (1%), we can see that there is an overall spreading
of the hydrogel as compared to the rest of the construct. Additionally, in Figure 8(E), the hydrogel
printed showed substantial shrinkage after cross-linking. This further strengthens the need of an optimal
material viscosity as cross-linking diffusion was too
slow at high viscosity, thus affecting the overall shape.
This can be seen from the defects and cave-in shown
in Figure 8(J).
The constructs developed an opaque interior due to
the cross-linking effect of CaCl2 on the alginate-XG
gel, creating a gradient of mechanical properties radially towards the outer wall of the gel wall. From the
results, it could be concluded that using 2% XG produced the best printing quality with optimal roundness,
minimum spreading and optimal diffusion rate.
3.8 Extending Process Capability
Demonstrating the process capability of this novel
approach, a tubular construct of 15 mm length was
fabricated in the vertical configuration using the 2%
XG-alginate gel as shown in Figure 9.

Figure 9. A tubular construct of 15 mm in the vertical configuration.

Figure 7. Opaque layer thickness of tubular construct at different concentrations of xanthan gum.

Compared to printing of the 4.8 mm tubular structure, there seems to be a shrinkage effect caused by
the CaCl2 gelation interaction at higher tube height.
This shrinkage could potentially be resolved by using
a lower concentration of CaCl2. In summary, printing
at 2% XG is effective despite an increase in printed
length of tubular structure with minimal deviation
from its designated shape and minimal spreading.

4. Conclusion
Figure 8. Printed alginate-xanthan gum tubular structure at
different concentrations of xanthan gum.
54

This paper has shown that tubular constructs can be

successfully bioprinted in vertical configuration by
controlling the viscosity of the hydrogel and through

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Edgar Y. S. Tan and Wai Yee Yeong

the use of a new multi-nozzle printing approach to

achieve in-process cross-linking. Hydrogel tubular
structures of large diameter with good shape fidelity
and integrity were demonstrated. Limitation of this
method includes shrinkage induced during the crosslinking process, which require further optimization of
the process. We also demonstrated the capability of
this fabrication technique with a tall hydrogel tubular
structure of at least 15 mm in length in the vertical
configuration. There is potential to further develop
this concentric printing method to create a branching
tubular tree structure with further fine-tuning of the
tool path design.
This paper also proposed quantifiable parameters
for printing quality which could help in characterizing
new materials for bioprinting in terms of shape fidelity.
We proposed that shape fidelity can be quantified as
tubular length, wall thickness and roundness. Printing
effects such as spreading and opaque layer thickness
(cross-linking layer thickness) were also proposed.
The definition of these parameters would be crucial in
developing systematic studies in research as we move
closer towards developing printing of new organs and
tissues.

6.

7.

8.

9.

10.

11.

Conflict of Interest and Funding

No conflict of interest was reported by the authors.
This work has been funded by NTU Start-Up Grant
(SUG).

12.

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International Journal of Bioprinting (2015)Volume 1, Issue 1