Original Micle

3D Bioprinting of Cellulose Structures

from an Ionic Liquid
Kajsa Markstedt,' Johan Sundberg,1 and Paul Gatenholm1

Abstract

This article reports on 3D bioprinting of dissolved cellulose to produce small feature structures with a tailored design of
regenerated cellulose. The process consists ofdissolving cellulose with different origins and molecular weight in an ionic liquid
(l -ethyl-3 -methylimidazolium acetate), c ntrolled multilayered dispensing, and coagulation. The printability was examined by
studying the viscosity of cellulose solutio ns and by varying the settings of the printer setup regarding flow rate and needle
dimensions. Water was added as a nonso Ivent, enabling a coagulation process to form a gel structure of the printed solutions.
By printing on a coagulating gel, the printed solutions were regenerated within afew seconds. Rheology analysis showed that
higher concentrations of cellulose and cellulose ofa high molecular weight were shear thinning; providingfavorable printing
properties. Printing3D structures ofcellulose dissolved in an ionic liquidfollowed by coagulation by a nonsolvent was possible.
Both complex patterns of2D structures as well as multilayered prints were created to obtain 3D structures. This novel method
allows for theproduction ofspatially tailored 3D gels or membrane structures madefrom cellulose.

Introduction
THE USE 0F 3D PRINTING as a bottom -up
fabrication method is increasing and is
becoming more user-friendly and
affordable. A wide range of materials can
be printed using multiple different
methods.' The common 3D printing
technologies such as ink-jet printing,2
fused deposition modeling,3 and selective
laser sintering4 are based on fusing
powdered materials or extrusion of
thermoplastic materials. There are also
some methods to solidify curable
polymers with laser or UV, called
stereolithography.5 3D printing has been
applications. Henke et al. have shown the
feasibility to use wood -based bulk
materials in a 3D printing process to
generate macroscaled 3D
bioprinting is an emerging technology in
which suspensions, hydrogels, or
solutions are dispensed by a syringe -
needle system or an ink-jet nozzle. The
print is solidified by physical or chemical
crosslinking, or phase transition
processes such as coagulation or
precipitation. 3D bioprinting technology
can be used for fabrication of acellular
scaffolds for tissue engineering or when
used with cells to fabricate living tissues
and organs. The applications go far
Cellulose (1,4 -glucan) is apolysaccharide
that shows several desirable properties
such as renewability biodegradabfflty
and high mechanical performance.
Cellulose and cellulose -based materials
have a very broad field of applications,
for example, papers, tissues, hygiene
products, packages, and food and drug
compounds.I The supermolecular
structure of cellulose'2"3 makes it
insoluble in most solvents at moderate
conditions, and the processing of
cellulose is therefore restricted to the use
of fibers, fibrils, or microcrystals. In
recent years, research has shown that
ionic liquids (ILs), for example, 1 -ethyl -

used to produce relative complex bilayer beyond the biomedical field since 3D 3 -methylimidazolium acetate (EminiAc),
tablets,6 force sensors,7 soft dielectric bioprinting enables the fabrication of 3D can be used to dissolve cellulose'4 in a
actuators,8 and soft scaffolds for tissue objects with high resolution and more environmentally friendly way
engineering,9 just to mention a few multiflinctionality compared with other solvents, which

1Blopolymer Technc1og Chalmers Unersdy of Technology, Gothenburg Sweden.

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 Markstedt et al.
often are volatile, toxic, costly, or difilcult
to recover.
Previously, cellulose dissolved in ILs has
mainly been studied to produce thin
hirns and fibers ofrecovered cellulose)'7
If cellulose is to be successful as an
alternative to polymers based on fossil
fuels,'8 it is important to develop ways to
control the structure and properties of
the materials made from cellulose. This
would enable applications where a
defined structure ofthe cellulose material
is necessary; for example, in packaging,
medicine tablets, scaffolds for tissue
engineering, and sensors.°
The aim of the study was to control the
structure and properties of cellulose via
bottom -up fabrication. Because cellulose
cannot be melted, extruded 3D
bioprinting technology was found
suitable to use when investigating the
feasibility of printing 3D structures of
cellulose. The printability was examined
by studying the viscosity of the cellulose
solutions and by varying the settings of
the printer setup regarding pump rate,
printing speed, and needle dimensions.
To retain the 3D structure of the
dispensed solutions, water was added,
enabling a coagulation process turning
the print into a geL
Materials and Methods
Bacterial Nanocellulose
Biosynthesis
Production and cleaning of the bacterial
nanocellulose (BNC) was done according
to previously described protocols.21a2
The clean BNC was homogenized (IKA
T25; Fisher Scientific) for 20mm at
25,000 rounds per minute, and the
suspension was then frozen at -80°C. The
BNC was freeze-dried (Heto PowerDry
PL3000). The dry BNC sponges were kept
at 0% relative humidity (RH) until use.
The BNC was torn into small flakes
before being added to the IL.
Dissolution of Cellulose in EmimAc
Cellulose with three different degrees of
polymerization (DP) -for example,
molecular weight (Avicel PH -101; Fluka
Sigma Aldrich; DP 150-30(Y1), dissolving
116 30 PRINTING
Kopie von subito e.V., geliefert für Bibliothèque nationale du Luxembourg (SLl03X00241E)
pulp (Domsjö AB; DP 750), and BNC
(DP 2000 -8000") --was dissolved in the
IL EmimAc (BASF ~90%; Sigma
Aldrich). Three different concentrations
(1%, 2%, and 4% cellulose [w/wl) ofeach
type of cellulose were prepared. The
cellulose was slowly added to a 50 ml
bottle containing Emin,Ac kept at 85°C,
in order to minimize the risk of thermal
degradation of the cellulose.24 The
samples were stirred until all cellulose
was completely dissolved and a clear
homogenously flowing solution was
attained (-'3 h for Avicel). The dissolving
pulp and BNC samples were stirred at
50°C overnight. The solutions used for
printing were dyed dark blue by adding
blue mineral pigment Ultramarin
(Villafärg) to the EmimAc before
addition of cellulose in order to better
visualize the prints.
Rheological Measurements
For evaluation of the viscoelastic
properties of the IL solution, rheology
studies were performed on a Bohlin
Rheometer CS 30 (Malvern Instruments).
The measurements were taken using a
cone -and-plate geometry with a diameter
of 25 mm and a cone angle of 5.4°.
Steady-state sheer viscosity was
measured at shear stresses in the range of
0.24-370 Pa. IL solutions prepared from
Avicel, dissolving pulp, and BNC with
cellulose concentrations of 1%, 2%, and
4% (w/w) were measured at room
temperature (25°C). The solutions were
kept in dosed containers until analysis to
minimize potential ambient moisture
absorption. Measurements at 50°C and
80°C were conducted for solutions of 4%
dissolving pulp, 2% BNC, and 4% BNC
(w/w). Both oscifiatory measurements
and stress viscosity measurements were
taken for each solution.
Flow Rate, Pressure, and Needle
Size Simulation
MatLab (R2013b; MathWorks) was used
to simulate shear rates and pressures for
different needle sLzes based on results
from the rheological measurements to
ensure an optimized setup when
printing. The theoretical shear rate and
the pressure in needles with different
limer radius were calculated by assuming
-
capillary flow. For Newtonian liquids,
the shear rate, t is given by Equation 1.25
MARY ANN LIEBERT, INC. ¯
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2014
The pressure drop in the needle, AP, the
length of the needle, L, the radius of the
needle, R, and the viscosity; j, are all
constants.
¯ dv 4PR
(1)
For non -Newtonian liquids the power -
law viscosity model is used (Equation 2),
which describes the dependence of
viscosity on shear rate,26 where k and n
are two fitting parameters.
n-1
(2)
The fitting parameters were determined
by linear regression of the graphs from
the rheological measurements. To decide
for which region of the graph to perform
the linear regression, an apparent shear
rate at the wail, 7app,25 was calculated,
defined as in Equation 3, where Q is the
flow rate and R is the radius of the needle.
7app (3)
2rR3
Printing
Printing was done by a customized
commercial available printer (MakerBot
ReplicatorlM2; MakerBot) where the
heating element and the filament head
were removed and a syringe holder was
fitted. The parts needed for the syringe
holder were designed in Pro/Engineer
(Wildfire 4.0; PTC) and produced in a
MakerBot ReplicatorTM2 (MakerBot).
The syringe holder held both the 5m1
syringe containing the IL cellulose
solution and a vertical syringe actuator
controlled by a syringe pump
(Alladin -1000; World Precision
Instruments) (Figure 3B). Attached to
the syringe was a dispensing needle with
a flat tip (Drifton AIS). Three sizes of
dispensing needles were tested (Table I).
To ease the filling of the syringe, the
solutions were heated to 50°C to reduce
the viscosity. During printing, the flow
rate of the syringe pump was set to
10 il/min and printing was controlled by
MakerWare software (Version 2.2.2.89;
MakerBot) (Figure 3A). The printing
speed was set to 10mm/s.
Coagulation
Water was used as a nonsolvent for
coagulation of dispensed solutions.
¯
DCI: 10.1089/3dp.2014.0004

lii!
I ¯I
Color Gauge
Blue 22
Red 23
Clear 25
The needles are color coded according to The manufacturer.
Different methods of coagulation were
evaluated: (1) Spraying water manually
using an atomizing nozzle (LEE
Company). (2) Coagulation bath: water
was sprayed closely around the print
using a syringe enabling contact between
the extruded solution and the nonsolvent.
(3) Coagulating gel: the IL solution was
printed onto an agar gel, and the agar gel
was prepared by mixing 0.75 g agar and
50 ml water; the agar solution was
autoclaved (121°C, 20mm), poured
warm into Petri dishes with a diameter of
13.5 cm, and set to cool until a gel was
formed; for prints exceeding 8mm in
height, vertical agar supports were used;
the agar was poured warm into a negative
mold of the desired print structure, and
the printing was then done into the agar
mold.
Scanning Electron Microscopy
Samples for scanning electron
microscopy (SEM) were quenched in
liquid nitrogen and freeze-dried (Heto
PowerDry P13000) until completely dry
Samples were cut and mounted vertically
onto stubs in order to visualize the cross
section. The samples were sputter coated
for 60s with gold (Edwards Sputter
Coater SI5OB). The samples were
analyzed using a Leo Ultra 55 FEG
scanning electron microscope.
Results and Discussion
Dissolving Cellulose
Cellulose is a homopolymer that gives a
direct correlation between the molecular
weight and the DP. Depending on the
molecular weight, different amounts of
cellulose can be dissolved in EmimAc.
Cellulose of higher molecular weight is
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Kopie von subito e.V., geliefert für Bibliothèque nationale du Luxembourg (SLIO3XOO24IE)
3D Bioprinting of Cellulose Structures
-II1(*IltI:I.lr:l.1u1!:Ii
Inner Length
dIameter (mm) (mm)
0.41 12.7
0.25 12.7
0,2 12,7
more difficult to dissolve because of the
high viscosity of the resulting solution,
BNC being the hardest to dissolve and
Avicel the easiest. Up to 10% (wlw)
Avicel can successfully be dissolved in
EmimAc, whereas only 5% (w/w) BNC
can be dissolved at the same conditions.
Finer flakes and powders of cellulose
dissolve faster. At higher concentrations
than 10% Avicel and 5% BNC, it takes
longer time and higher temperature to
dissolve the cellulose, which potentially
can cause the cellulose to degrade.24 The
dissolution of cellulose in EmimAc at the
condition used in this study results in a
molecular solution where all the macro -
morphological structures and crystal
structures are removed. The regenerated
structure is therefore completely
anisotropic and amorphous.'6
Rheology
The measured viscosities are visualized
as a function of shear rate in Figure 1.
The graph shows that at lower molecular
weights and lower concentrations of
cellulose, the viscosity is independent of
shear rate and therefore is Newtonian.
These results correlate well to previous
findings by Gericke et aL27 and Sescousse
et aL28 At higher molecular weights and
higher concentrations, the curve is
sloped, showing a non-Newtonian liquid
with shear thinning similar to the
findings of Kuang et ai.29 Solutions with
large shear thinning (2% and 4% BNC,
and 4% dissolving pulp), seen in Figure
1, are the most interesting for printing
since they have properties resembling
Bingham plastics. The high shear rates
during dispensing will decrease the
viscosity, making the solution flow. Once
the solution is dispensed and the
shearing is stopped, the viscosity will
quickly increase, making the print almost
¯
DCI: 10108913dp.2014.0004
gel -like again despite that it has not been
coagulated. This is crucial for the
dispensed pattern to keep its shape and
structure.
The viscosity's temperature dependence
at different shear rates was also analyzed.
As seen in Figure lB, for low shear rates
the viscosity of a cellulose solution
at certain concentrations decreased
drastically at higher temperatures. At
high shear rates, on the other hand, the
viscosity did not change much when the
temperature was increased. This implies
that if the solution is dispensed at high
shear rates, a change in temperature will
not affect the viscosity greatly. For
printing optin-iization, a cold solution is
favorable since the difference in viscosity
at different shear rates is larger, enabling
a more gel -like solution once it has been
dispensed.
The viscosity as a function of the shear
rate ofthe solutions was measuredbefore
and after the dispensing process to
ensure that no degradation of the
cellulose polymer chains because of the
high shear rate occurred (Supplementary
Figures S1 -S6; Supplementary Data are
available online at www.liebertpub
.com/3dp). The viscosity of the solutions
was maintained after dispensing,
consequently proving that the cellulose
polymer chains did not degrade.
Flow rate, Pressure,
and Needle Size
The results from the rheological
measurements show that a high shear
rate is required in order to get good
printing properties where a high shear
thinning during printing is followed by
an increased viscosity at no sheai The
shear rate depends on the flow rate, Q,
and the inner diameter of the needle, R,
and to achieve high shear rates a high
pressure is needed.
The apparent shear rate calculated from
Equation 3 was 62.87 s1 for Q=lo il/min
and R=0.3 mm. The value for Q was
chosen since it proved experimentally to
give a stable flow, and the value for R was
an average size of the studied needles.
Based on the slope of 4% dissolving pulp
at 25°C in Figure lB, a linear regression
30 PRINTING 117
 Markstedt et al.

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Shear rate (.1) Shear rate (s.4)
Figure'l. (A) Shear viscosity graph of BNC, dlssoMng pulp, and Avicel dissolved In Emlrn,c at concentrations of 1%, 2%, and 4% (wtw). .AJl
measurements were taken at 25'C. (B) Shear viscosity graph showing temperature dependence for 4% BNC, 4% dissoMng pulp, and 2% BNC
IwIw) at three difterent temperatures: 25cC, 505C, and 805C. BNC, bactefal nonoceilulose; Emirr,c, 1 -efhy1 -3-meth1lmidazollum acetate, Color
Images available onine at ww.lIebertpub.corrV3dp -

was made for shear rates between 0.1
and 100 s. The linear regression follows
the equation
J1=93.991y
-0.428
where the constants correspond to
the fitting parameters in Equation 2;
k = 93.991 and n = 0.572 for (n 1) =
-0.428.
The fitting parameters were used to
calculate the shear rate and pressure ofthe
non -Newtonian liquid during capifiary
flow. By using Rabonowitsch correction
in Equation 3, the true shear rate as a
function of the flow rate, Q, is given by
¯ (3n+1 . (3n+1) Q
i, 4n )
(4) By combining Equations 1 and 2 and
using the expression for shear rate from
Equation 5, the expression for the pressure
as a function of the flow rate is derived.
-
=1Q(3n+1)1 2kL
L nirR3
From Equation 6 it is seen that shorter
needle lengths, L, reduce the pressure
needed to achieve the desired flow. The
shear rate (Figure 2A) and the pressure
(Figure 2B) are plotted as a function of
the flow rate for needles ofthree different
sizes. Figure 2A and B shows that the
(5) shear rate and pressure required for the
0.2 -mm-diameter needle is almost 6 bar
for aflow rate of 10 pl/min. The dispensing
needles should not be used at pressures
above 6 bar and therefore printing at
lower pressures is preferred. The
0.25 -mm -diameter needle shows a much
lower pressure at the same flow rate, and
(6) at the same time it still has a shear rate
] above 100s', which reduces the viscosity
Dispensing
Experimentally it was found that 4%
dissolving pulp worked best for printing.
It had a viscosity high enough to keep its
shape after dispensing while the pressure

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Flow rate, Q (t hmm) Flow rate, Q (& hmm)
Figure 2. Graphs of shear rate and pressure as a funcflon of flow rate of 4% (wfw) dlssoMng pulp dissolved in EmirnAc. The three different curves
represent needles of different sizes. The needle length Is 12.7mm and the Inner diameters are 0.2, 0.25, and 0.41 mm. (A) Shear rate as a funcflon
of flow rate. (B) Pressure as a function of flow rate. Color images available online at www.liebertpub.com/3dp

118 3D PRINTING MARYANIN IJEBERT, INC. ¯

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 3D B)oprinhing of Celulose Structures

ri Coagulation baths where the print was

A dispensed into a shallow pool of the
nonsolvent were affècted by the
L. .
movement of the printer, resulting in
poor layer-layer adhesion.

Coagulating Gel
By printing the IL solution onto an agar
gel, a good interchange with the
nonsolvent was achieved. The solution
coagulated within seconds after it was
printed onto the gel (Figure 3C and E,
and Supplementary Videos Vi and V2),
ensuring high resolution as seen in
Figure 4A, where a print on glass is
compared with a print on gel. The
nonsolvent diffuses through the print
from the bottom up, so when another
layer of solution is added, the top of the
first layer is not completely gelled or
covered in nonsolvent, making it possible
for the next layer to adhere to the first
one. While printing, water keeps on
Figure 3. OvervIew of the printing process. (A) Printing controlled by MakerWare software utiulng diffusing from the gel and through the
stereolithography file format (Sm) flies. (B) Customized MakerBot 3D ptinter, set up to print ionic print so that every added layer comes in
liquid solutions on agar gel. (C) Printing of 4% (w/w) dissolving pulp dissolved in EmimAc on an contact with the nonsolvent.
agar plate to obtain instant coagulation. (D) A close-up showing how the 'Ascous liquid is
dispensed layer upon loyer, (E) Schematic showing the diffusion of water from the agar plate to
the ptinted structure. Color images available online at www.liebertpub,conV3dp At approximately six deposited layers,
the print reached a critical height of
about 8mm, where the passive diffusion
needed for dispensing was low enough to multilayer interconnected prints. of nonsolvent through the print did not
be produced by the syringe pump. The Applying the nonsolvent by spraying coagulate at a sufficient speed, causing
customized printer ensured continuous using an atomizing nozzle ensured good the top of the print to slide and
flow, constant speed, and an even resolution retainment but caused subsequently collapse. The print height
movement enabling printing of complex complications when multiple layers were could be extended by progressively
shapes. To achieve the flow rates needed extruded on top of each other because decreasing the print speed, but eventually
for high -quality prints, needles with a of poor adhesion between layers. the print came to an almost halt. In order
minimum inner diameter of 0.41mm
were used. Figure 3 shows a schematic
image of the printing process.
Stereolithography file format (.STL) files
loaded to MakerWare software (Version
2.2.2.89; MakerBot) (Figure 3A) were
used to control the movement of the
customized printer (Figure 3B). The
patterns printed had complex geometries,
and Figure 3C shows an example of a
pattern extruded and coagulated on an
agar gel. The alignment of sequential
layers was also good, enabling multilayer
prints as seen in Figure 3D.

Coagulation 20 mm

Water was used as a nonsolvent for
coagulation of dispensed solutions. The
nonsolvent application method turned
out to be critical in order to achieve
Figure 4. (A) Ptinted tree of 4% (w/w) dissoMng pulp In EmimAc. The left halt shows ptinted
solution without coagulation on a glass plate. In compaison, the right hait has been Instantly
coagulated by the agar gel. (B) Cylinders ptinted using vertical agar supports where the highest
structure is about 25mm. (C) Top view of the cytnders in Figure 4B. Color images available
online at ww,lieberipub.corrV3dp

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Kopie von subito e.V., geliefert für Bibliothèque nationale du Luxembourg (SL103X00241 E)
 Markstedt et ai,
FIgure 5. Scanning electron microscopy Images of a regenerated and freeze-dried cellulose
structure printed by an EmimAc solution of 4% (w/w) dlssoMng pulp. (A) 10.000 x magnification.
(B) 25,000x magnification.
to print taller structures, vertical agar
supports were used to ensure a constant
and continuous coagulation of the print.
In Figure 4B and C, several cylinders made
by this technique are depicted. The slight
slanting of the structures in Figure 4 was
because of imperfections in the vertical
agar supports used to produce them and
not because of settling effect after
removal of the print from the support.
Structure of Prints
After complete removal of the IL, the
printed structures are made up by
amorphous cellulose gels.'6 The porosity
of the gel structures is depicted in Figure
5, showing SEM micrographs of freeze-
dried gels. The gels have an
interconnected porous structure.
Conclusions
The results of the rheology analysis
showed that higher concentrations of
cellulose and cellulose of higher
molecular weights gave more viscous
solutions. These solutions were also
shear thinning, providing favorable
printing properties: allowing for low
viscosity during dispensing and high
viscosity after dispensing, preserving the
structure of the dispensed liquid. The
solution's temperature proved to be less
important at the pressures used while
dispensing. The inner diameter of the
needles used for dispensing was
simulated and optimized.
Complex patterns of 2D structures as
well as multilayered prints were created
to obtain 3D structures. The printing
1 20 30 PRINTING
Kopie von subito e.V., geliefert für Bibliothèque nationale du Luxembourg (SLIO3XOO24IE)
optimization depends strongly on the
needle size, the viscosity of the solution,
the pressure during dispensing, and the
coagulation method. A solution of 4%
dissolving pulp dispensed with a needle
with an inner diameter of 0.41mm
enabled prints with a high spatial
resolution, and the coagulating gel
ensured fast progressive coagulation of
multilayer constructs. Vertical coagulation
gel supports were used to produce
constructs past six deposited layers.
This work has shown that printing 3D
structures of cellulose dissolved in IL
followed by coagulation of a nonsolvent
is possible. This novel method allows for
the production of spatially tailored gel
structures made from cellulose with
many potential industrial applications.
Acknowledgments
The Knut and Alice Wallenberg
Foundation is gratefully acknowledged
for funding the Wallenberg Wood
Science Center. Tekn. Lic Linda Härdelin
and Prof. Bengt Hagström at Swerea IVF
are acknowledged for their help during
the rheology study.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Paul Gatenholm
Arvid Waligrens Backe 20, lvi 4
SE-41346 Göteborg
Sweden
E-mail: paul.gatenholm@chalmers.se
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