Rheological and Printability Assessments on Biomaterial Inks of Nanocellulose/Photo-Crosslinkable Biopolymer in Light-Aided 3D Printing

Abstract

Biomaterial inks based on cellulose nanofibers (CNFs) and photo-crosslinkable biopolymers have great potential as a high-performance ink system in light-aided, hydrogel extrusion-based 3D bioprinting. However, the colloidal stability of surface charged nanofibrils is susceptible to mono-cations in physiological buffers, which complexes the application scenarios of these systems in formulating cell-laden bioinks. In this study, biomaterial inks formulated by neutral and negatively surface charged CNFs (GrowInk-N and GrowInk-T) and photo-crosslinkable biopolymers (gelatin methacryloyl (GelMA) and methacrylated galactoglucomannan (GGMMA)) were prepared with Milli-Q water or PBS buffer. Quantitative rheological measurements were performed on the ink formulations to characterize their shear flow recovery behavior and to understand the intermolecular interactions between the CNFs of different kinds with GGMMA or GelMA. Meanwhile, printability assessments, including filament extrudability and shape fidelity of the printed scaffold under varying printing conditions, were carried out to optimize the printing process. Our study provides extensive supporting information for further developing these nanocellulose-based systems into photo-crosslinkable bioinks in the service of cell-laden 3D bioprinting.

Full text

Rheological and Printability
Assessments on Biomaterial Inks of
Nanocellulose/Photo-Crosslinkable
Biopolymer in Light-Aided 3D Printing
Qingbo Wang
1
, Oskar Backman
1
, Markus Nuopponen
2
, Chunlin Xu
1
and Xiaoju Wang
1
,
3
*
1
Laboratory of Natural Materials Technology, Faculty of Science and Engineering, Åbo Akadem i University, Turku, Finland,
2
UPM-
Kymmene Comporation, Biomedicals, Helsinki, Finland,
3
Pharmaceutical Sciences Laboratory, Faculty of Science and
Engineering, Åbo Akademi University, Turku, Finland
Biomaterial inks based on cellulose nanofibers (CNFs) and photo-crosslinkable
biopolymers have great potential as a high-performance ink system in light-aided,
hydrogel extrusion-based 3D bioprinting. However, the colloidal stability of surface
charged nanofibrils is susceptible to mono-cations in physiological buffers, which
complexes the application scenarios of these systems in formulating cell-laden bioinks.
In this study, biomaterial inks formulated by neutral and negatively surface charged CNFs
(GrowInk-N and GrowInk-T) and photo-crosslinkable biopolymers (gelatin methacryloyl
(GelMA) and methacrylated galactoglucomannan (GGMMA)) were prepared with Milli-Q
water or PBS buffer. Quantitative rheological measurements were performed on the ink
formulations to characterize their shear flow recovery behavior and to understand the
intermolecular interactions between the CNFs of different kinds with GGMMA or GelMA.
Meanwhile, printability assessments, including filament extrudability and shape fidelity of
the printed scaffold under varying printing conditions, were carried out to optimize the
printing process. Our study provides extensive supporting information for further
developing these nanocellulose-based systems into photo-crosslinkable bioinks in the
service of cell-laden 3D bioprinting.
Keywords: cellulose nanofibrils, photo-crosslinkable biopolymer, light-aided extrusion-based 3D printing,
printability assessment, rheology
INTRODUCTION
In the research context of regenerative medicine and tissue engineering, three-dimensional (3D)
printing techniques are advantageous to fabricate the biomimetic hydrogels of site-specific geometry
and with a complex structure (Hinton et al., 2015). From the hydrogel fabrication point of view, these
additive manufacturing methods highlight the convenient workability of automation,
reproducibility, and, in particular, a personalized design aided by a digital model (Gillispie et al.,
2020). An extrusion-based 3D printing system with a piston-derived syringe and microscale needle is
mostly adopted among the 3D printing methods that are suitable for hydrogel printing (Kang et al.,
2016;Gillispie et al., 2020). Furthermore, a variety of ink formulations consisting of high
concentration water-soluble polymers such as alginate, gelatin, and collagen have been
developed for the printing process (Rhee et al., 2016;Lewis et al., 2018;Yang et al., 2018).
Recently, attributed to structural similarity to extracellular matrix, low cytotoxicity, and desirable
Edited by:
Per Stenius,
Aalto University, Finland
Reviewed by:
Liangliang Lin,
Jiangnan University, China
Arne Reinsdorf,
Evonik Industries, Germany
*Correspondence:
Xiaoju Wang
xiaoju.wang@abo.fi
Specialty section:
This article was submitted to
Chemical Reaction Engineering,
a section of the journal
Frontiers in Chemical Engineering
Received: 10 June 2021
Accepted: 29 July 2021
Published: 21 September 2021
Citation:
Wang Q, Backman O, Nuopponen M,
Xu C and Wang X (2021) Rheological
and Printability Assessments on
Biomaterial Inks of Nanocellulose/
Photo-Crosslinkable Biopolymer in
Light-Aided 3D Printing.
Front. Chem. Eng. 3:723429.
doi: 10.3389/fceng.2021.723429
Frontiers in Chemical Engineering | www.frontiersin.org September 2021 | Volume 3 | Article 7234291
ORIGINAL RESEARCH
published: 21 September 2021
doi: 10.3389/fceng.2021.723429

rheological properties, the gel-like cellulose nanofibrils (CNFs)
have attracted increasing attention as an ingredient when
formulating the bio(material) inks for hydrogel extrusion-
based 3D bioprinting (Shin et al., 2017;Heggset et al., 2019).
To accurately reproduce the structure of the digital model and to
achieve adequate shape fidelity are challenging factors in the
scenarios of extrusion-based 3D printing because of the soft
nature of the CNFs-based hydrogels, which typically have a
water content greater than 95%. CNFs can be either printed as
a monocomponent hydrogel as a platform biomaterial (Ajdary
et al., 2019) or more often in binary ink formulations with other
biopolymers, such as gelatin and alginate (Markstedt et al., 2015;
Ojansivu et al., 2019), where CNFs are more often seen as a
rheological modifier to facilitate the extrudability/printability and
to promote the shape fidelity performance of the formulated
bioink. In order to improve the ink fidelity, different crosslinking
strategies such as ionic crosslinking (Rees et al., 2015), thermal
crosslinking (Xu et al., 2018b), enzymatic crosslinking (Huang
et al., 2020), and photo-crosslinking (Ma et al., 2020) were
implemented in previous studies. In an up-to-date research
context, light-aided 3D bioprinting is a prevailing approach
for biofabrication. Here, the hydrogel bioink that contains the
photo-crosslinkable biopolymer such as gelatin methacryloyl
(GelMA) or hyaluronic acid methacrylate (HAMA) can be
rapidly cross-linked into a covalent network via free-radical
chain polymerization upon UV irradiation. This process can
take place in situ either right after the ink being extruded
through the nozzle on the collecting board or while the ink is
still being run through the nozzle of a special type that allows UV
penetration (Lim et al., 2020). Attributed to beneficial biological
properties, GelMA, a derivative of gelatin that still preserves the
residual cell attachment motif from native ECM, has been widely
used in such types of photo-crosslinkable bioink formulations. Xu
et al. (2019a) reported 3D printing of a binary biomaterial ink of
TEMPO-mediated oxidized CNFs and low-concentration
GelMA. The printed hydrogels showed mechanical strength in
the range of 2.5–5 kPa and promoted the proliferation of
fibroblasts. Adjusting the printed hydrogels’mechanical
properties enables them to mimic biomechanical properties of
natural biological tissues that could regulate the fate of the
attached cells (Engler et al., 2006;O’Brien, 2011). Xu et al.
(2019b) developed a wood-derived biomaterial ink composed
of TEMPO-mediated oxidized CNFs and galactoglucomannan
methacrylates (GGMMAs) with a different substitution degree of
methacrylates. The results showed that the fabricated hydrogels
displayed a broad and tunable mechanical strength ranging from
2.5 to 22.5 kPa. Fan et al. (2020) incorporated 10 wt% of cellulose
nanocrystals into GelMA/HAMA to reinforce the printed
hydrogels’mechanical property, which serves as a structure-
supporting material in a hybrid printing strategy (Fan et al., 2020).
In hydrogel extrusion-based 3D printing, the ink fidelity of the
CNFs/photo-crosslinkable biopolymer inks is largely determined
by the rheological properties of the hydrogel ink, which are
intrinsically dictated not only by the physiochemical properties
of CNFs, such as nanodimension and surface charge density, but
also by interactions between CNFs and photo-crosslinkable
polymers (Hubbe et al., 2017;Xu et al., 2019b). CNFs
prepared solely by mechanical defibrillation have a relatively
larger dimension, and the colloidal stability of the dispersion
is mainly attributed to the physical entanglement between CNFs
with a high aspect ratio (Nechyporchuk et al., 2016). As there is
no oxidation involved in the preparation process, the
mechanically defibrillated CNFs typically have a rather low
negative surface charge density. This makes this type of CNFs
insensitive to the ionic strength of metal ions in terms of the
microstructure stability within a hydrogel, which is a preferred
scenario in cell-laden 3D bioprinting when applied to
formulating bioinks upon mixing with cell-containing
physiological buffers, e.g., phosphate buffered saline (PBS).
CNFs can also be prepared by mechanical defibrillation in
combination with such a pretreatment as TEMPO-mediated
oxidation. Owing to the small nanodimension of fibrils and
the negative surface charge induced by the oxidation reaction,
the TEMPO-oxidized CNFs possess a higher water retention
ability and are more transparent when forming hydrogels
(Benhamou et al., 2014). Hence, they are accepted as a generic
type of biomaterial in supporting the suspended 3D cell culture as
well as in constructing biomedical hydrogels (Lou et al., 2014).
Furthermore, quite a few studies have reported the development
of biomaterial inks with the TEMPO-oxidized CNFs for 3D
printing (Xu et al., 2018a;Li et al., 2018). It is worth noting
that the colloidal stability of TEMPO-oxidized CNFs dispersion
dispersion is sensitive to monovalent cations as the nanofibrils are
stabilized by the electronic repulsion between the negatively
charged nanofibrils (Fukuzumi et al., 2014;Levaničet al.,
2020). Eventually, this aspect challenges its application in cell-
laden 3D bioprinting as the monovalent cations when introduced
with the PBS buffer in cell mixing can cause flocculation of
TEMPO-oxidized CNFs, and it further hinders its printability.
In the current study, biomaterial inks engaging mechanically
defibrillated CNFs or TEMPO-oxidized CNFs with GelMA and
GGMMA (with or without PBS buffer) were formulated, and the
ink printability in light-aided extrusion-based 3D printing was
investigated. The influence of PBS buffer on the formulated inks
was analyzed by quantitatively characterizing the rheological
properties and photo-crosslinking kinetics. In addition, the
printability of the formulated inks was evaluated by a series of
tests, including single filament extrusion uniformity and the
filament fusion test in multi-layer cross-hatch grids under
different printing conditions. We have aimed to optimize and
validate the printability of these UV-curable and CNFs-based
biomaterial inks and to provide principal information for future
use of these inks in cell-laden 3D bioprinting aided by photo-
curing.
MATERIALS AND METHODS
Materials
Mechanically defibrillated CNFs (GrowInk-N, 2.5 wt%) and
TEMPO-oxidized CNFs (GrowInk-T, 2 wt%) were donated by
UPM Biomedicals. Phosphate buffered saline tablets (PBS,
Biotechnology grade) were purchased from VWR Life Science.
Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and
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Wang et al. Nanocellulose-Based Light-Aided 3D Biomaterial Printing

deuterium oxide (D
2
O) containing 0.75 wt% 3-(trimethylsilyl)
propionic-2,2,3,3-d
4
acid (TMSP) were purchased from Sigma-
Aldrich. Dulbecco’s modified eagle’s medium (DMEM, high
glucose) was purchased from Biowest. GelMA was purchased
from ALLEVI (Philadelphia, United States). GGMMA was in-
house synthesized following a reported method (Xu et al., 2019b).
Method
Characterization of the Ink Ingredients
The nanomorphologies of CNFs in GrowInk-N and GrowInk-T
were obtained via a transmission electron microscope (TEM,
JEM-1400 PLUS). The charge density of GrowInk-N and
GrowInk-T was determined by the potentiometric titration
method reported by Chinga-Carrasco et al. (2014). The degree
of methacryloylation (DM) of GGMMA (0.862 mmol/ g) and
GelMA (0.463 mmol/ g) was determined by
1
H NMR
spectroscopy using a method reported by Claaßen et al.
(2018). The results were displayed in Figure 1.
Ink Formulation
The ink formulations in this study are listed in Supplementary
Table S1. Briefly, the inks were prepared by diluting GrowInk-N
(2.5 wt%) and GrowInk-T (2 wt%) with PBS buffer or Milli-Q
water. Then, the lyophilized powder of GGMMA or GelMA was
mixed with diluted GrowInk-N or GrowInk-T at 50°C using a
vortex mixer. LAP was selected as the photoinitiator and added to
each ink to make a final concentration of 0.1 wt%. The inks were
stored in a cold and dark place for 24 h before measurement.
Rheological Measurements
Rheological properties of the inks were measured by using an
MCR 702 MultiDrive rheometer (Anton Paar GmbH) with a
PP25 parallel-plate at 25°C (diameter: 25 mm and gap: 0.5 mm).
Viscosity curves were obtained through shear flow measurement
with a shear rate ramp-up and ramp-down of 0.01–1000 s
−1
in
logarithmic scale with 1 s per data point. Amplitude sweep was
performed with a strain range from 0.01 to 500% at a constant
frequency of 1 Hz at 25°C. The data acquisition time was 10 s per
data point. The thixotropic behavior of the inks was analyzed by
shearing the sample at 0.1 s
−1
for 60 s, followed by shearing at
700 s
−1
for 10 s and then at 0.1 s
−1
for 60 s. The samples were pre-
sheared at 1 s
−1
for 20 s and equilibrated for 60 s before all
measurements were taken. Photo-crosslinking kinetics of the
inks were measured at a constant strain and frequency of 0.1%
and 1 Hz, respectively. The samples were irradiated with UV
(bluepoint 4 ecocure UV lamp, The Hönle Group, Germany) after
1 min, and the storage modulus was registered.
Printability Assessment
Printability assessment of the inks was performed at room
temperature with a 3D bioprinter from ROKIT INVIVO
(ROKIT, South Korea), equipped with a piston-driven
extrusion nozzle. The inks were loaded into a 3 ml syringe
with a steel needle gauge of 25 GA (inner diameter: 0.25 mm)
for printability assessment. A UV-LED spotlight (wavelength:
365 nm and energy output: 10 mW/ cm
2
; bluepoint LED eco, The
Hönle Group, Germany) was applied for photo-crosslinking
during the printing process. The schematic image of the 3D
bioprinter and the hydrogel extrusion-based printing process is
displayed in Supplementary Figure S1.
Uniformity Ratio Measurement
The uniformity analysis of the printed filaments was followed by a
reported method with modifications (Gao et al., 2018). Briefly,
three filaments were printed on a microscopic slide under a
controlled input flow rate and printing speed for each ink.
The input flow rate was set at 100, 110, or 120%, and the
printing speed was set at 4, 8, 12, 16, or 20 mm/ s. The shape
of each filament was recorded by a digital microscope (Nikon
ECLIPSE E200 with a Nikon DIGITAL SIGHT DS-US camera).
The filaments’outline length on both sides and diameter were
manually measured by ImageJ software. The uniformity ratio was
determined by the ratio between the length of the filament outline
and the filament itself.
Semi-Quantification of Printability
10-layer cross-hatch scaffolds of each ink with five vertical and
five horizontal lines with a span width of 1.25 mm were printed to
evaluate the printability. The layer height was set at 0.07, 0.08, or
0.09 mm, and the input flow rate was set at 100, 110, or 120%. The
printed scaffold was washed and soaked in PBS buffer for 24 h
after printing, and then the photos of the printed scaffold were
taken by using a microscope. The printability value (Pr) and
diffusion rate value (Dr) were adopted for printability evaluation
(Ouyang et al., 2016;Habib et al., 2018). The Pr value is defined as
in Eq. 1 by comparing the circularity of a square (π/4) with the
outcome pores,
Pr
L2
16A(1)
where L and A are the perimeter and area of the outcome pores,
respectively.
The Dr value is defined as in Eq. 2 by comparing the difference
between the actual area of outcome pores and the theoretical area
defined from the line spacing and nozzle size,
Dr
At−Aa
At
(2)
where A
t
and A
a
mean the theoretical area and actual area,
respectively. The perimeter and area of the pores were
manually measured by ImageJ software.
Scaffold Stability
36-layer cross-hatch scaffolds with a layer height of 0.09 mm of
each ink were printed for scaffold stability analysis. The printed
scaffolds were incubated in PBS buffer and DMEM for 7 days.
Mechanical Test
The mechanical property of the cast hydrogel discs (diameter:
8 mm and height: 4.5 mm) was measured with an MCR 702
MultiDrive rheometer under the compression mode. The
compression speed and displacement were set at 0.1 mm/ s
and 3.5 mm, respectively. The compressive Young’s modulus
was calculated according to a reported method (Xu et al., 2019b).
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Wang et al. Nanocellulose-Based Light-Aided 3D Biomaterial Printing

RESULTS AND DISCUSSION
Rheological Property
Rheological studies were carried out to understand the flow
behavior and viscoelastic properties of the CNFs-based inks.
The viscosity of each formulated ink as a function of the shear
rate was recorded in ramp-up and ramp-down experiments, as
displayed in Figure 2. The shear-thinning response upon
shearing, a prerequisite ink property for extrusion-based 3D
printing, could be observed in all formulated inks. For inks
with the same composition, their viscosity increases with the
increase in CNF content. As shown in Figures 2A and B,
GrowInk-N-PBS displayed a higher viscosity and hysteresis
behavior than GrowInk-N-water. This is mainly due to the
moderate electrostatic screening effect caused by monocations
in PBS buffer, which reduces the distance between CNFs
(Saarikoski et al., 2012). The CNFs are more likely to
aggregate into flocs, which, thus, increases the ink’s viscosity
at rest or at a low shear rate. However, the flocs are likely to be
dissociated into nanofibrils at a high shear rate and difficult to
recover immediately, leading to hysteresis behavior (Oh et al.,
2020). Meanwhile, the flocs will also form an uneven
microstructure of the ink, which is not conducive to its
printability. However, the hysteresis behavior of GrowInk-N-
PBS disappeared after the incorporation of GGMMA, as shown in
Figure 2F. This might be attributed to the steric stabilization by
the GGMMA, which is a high-molecular-weight and water-
soluble heteropolysaccharide that has an intrinsic affinity/
adsorption to the nanocellulose surface via hydrogen bonding
(Xu et al., 2019b). It is inferred that the adsorbed GGMMA tends
to prevent the closely approaching CNFs from aggregating into
flocs, thus consequently preventing the hysteresis behavior and
uneven microstructure of the GrowInk-N-PBS inks (Winter et al.,
2010;Hubbe et al., 2017). On the other hand, the electrostatic
screening effect in GrowInk-N-GGMMA-PBS inks still exists,
leading to a higher viscosity than the GrowInk-N-GGMMA-
water inks, as shown in Figure 2E. A significant increase in
viscosity was observed in Figures 2I and J by introducing 5% of
GelMA to GrowInk-N-GelMA–based inks. Moreover, GelMA
also showed the ability to reduce the hysteresis behavior in
GrowInk-N-GelMA-PBS inks, as shown in Figure 2J.
Incorporation of GGMMA or GelMA with GrowInk-N would
FIGURE 1 | (A) TEM images of GrowInk-N and GrowInk-T and (B)
1
H NMR spectra of GGMMA and GelMA.
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Wang et al. Nanocellulose-Based Light-Aided 3D Biomaterial Printing

increase the inks’viscosity, owing to increased total mass content
in the ink, as shown in Figures 2E and I.
In GrowInk-N, the entanglement of large-dimension
nanofibrils dominates its gel-like structure. Whereas, the gel-like
structure of GrowInk-T composed of relatively small-dimension
nanofibrils is mainly governed by the strong fibril–fibril repulsion
induced by its negatively charged surface groups (Benhamou et al.,
2014). Therefore, the microstructure stability of GrowInk-T is
more sensitive to the variation of ionic strength. As shown in
Figures 2C and D, the electrostatic repulsion between nanofibrils
in GrowInk-T was disordered by cations, causing the flocculation
of CNFs. Therefore, GrowInk-T-PBS displayed a higher zero shear
viscosity than GrowInk-T-water (Sim et al., 2015). As shown in
Figures 2G and H, a similar trend in viscosity was observed in
GrowInk-T-GGMMA–based inks. Similar to GrowInk-N-
GelMA–based inks, the incorporation of GelMA greatly
increased the viscosity of GrowInk-T-GelMA–based inks, as
shown in Figures 2K and L. Moreover, the viscosity of
GrowInk-T-GelMA–based inks was much higher than that of
GrowInk-N-GelMA–based inks at the same concentration as
shown in Figures 2I and K. This is mainly attributed to the
ionic interaction between the positively charged GelMA and
negatively charged GrowInk-T under neutral pH (Xu et al., 2019a).
Amplitude sweep is used to determine the linear viscoelastic
region (LVER), which indicates the viscoelastic character of the
test samples. The LVER is necessary to be registered prior to
frequency sweep and photorheology. In addition, amplitude
sweep could provide information such as yield stress (τ
y
, the
value of the shear stress at the limit of the LVER) and flow stress
(τ
f
, the value of the shear stress at the crossover point where G’
equals G”).
As shown in Figures 3A and B,G’,τ
y
, and τ
f
of GrowInk-N-
PBS increased drastically compared to the values of GrowInk-N-
water. This is mainly due to the aggregation of CNFs flocs by the
electrostatic screening effect as mentioned above. The τ
y
and τ
f
of
1.5% GrowInk-N-PBS were higher than that of 2% GrowInk-N-
PBS. This might be due to higher ionic strength, leading to severe
CNFs entanglement in 1.5% GrowInk-N-PBS, as a larger volume
of PBS buffer was used in the preparation of 1.5% GrowInk-N-
PBS than that used in 2% GrowInk-N-PBS. As shown in Figures
3A and C,G’of GrowInk-N-GGMMA inks was increased after
incorporating GGMMA, whereas the τ
f
was slightly increased.
This indicates that the GGMMA could enhance the elasticity of
the GrowInk-N–based ink without increasing its flow resistance.
Meanwhile, G’and τ
f
of GrowInk-N-GGMMA-PBS inks were
decreased after incorporating GGMMA as shown in Figures 3B
and F, except for 2% GrowInk-N + 2% GGMMA-PBS. This is
because the steric stabilization effect of GGMMA could reduce
the agglomeration effect and frictional interaction and thus leads
to a lower τ
f
(Araki et al., 2001). The steric stabilization effect of
GGMMA could help prevent the aggregation of GrowInk-N
CNFs flocs caused by cations, as shown in Figures 3E and F.
FIGURE 2 | Flow curves of formulated inks as a function of the shear rate [(A–D) GrowInk-N and GrowInk-T, (E–H) GGMMA incorporated GrowInk-N–and
GrowInk-T–based inks, and (I–L) GelMA incorporated GrowInk-N–and GrowInk-T–based inks diluted with PBS buffer and water, respecti vely. Closed symbol: ramp-up
and open symbol: ramp-down].
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As shown in Figures 3I and J, incorporating 5% of GelMA in
GrowInk-N significantly increases both G’and τ
f
. Besides, the
effect of PBS to GrowInk-N-GelMA–based inks was not
significant on τ
f
and G’. Except for the 1% GrowInk-N + 5%
GelMA-PBS ink, τ
f
was lower than that of the 1% GrowInk-N +
5% GelMA-water ink. It might be because the interactions
between GelMA and GrowInk-N were disturbed under
relatively higher ionic strength, and this resulted in a weaker
microstructure maintenance ability with the imposed stress and
strain. In addition, it is also indicated as shown in Supplementary
Figures S2I and J, where the G”of 1% GrowInk-N + 5% GelMA-
PBS ink displayed an apparent overshot at the end of the LVER
compared to 1% GrowInk-N + 5% GelMA-water ink (Hyun et al.,
2002).
As shown in Figures 3A and C,G’and τ
f
of GrowInk-T-water
were higher than those of GrowInk-N-water at the corresponding
solid content level due to the strong electrostatic repulsion
between nanofibrils. As shown in Figures 3C and D,G’and
τ
f
of GrowInk-T-PBS were increased in the absence of PBS buffer,
which is similar to those of GrowInk-N. However, different from
GrowInk-N-GGMMA inks, incorporation of GGMMA in
GrowInk-T and GrowInk-T-PBS showed a limited effect on
their G’and τ
f
, as shown in Figures 3G and H.Tobe
noticed, the flow transition index (τ
f
/τ
y
) of GrowInk-N and
GrowInk-T was low, where the G’displayed a sudden drop at
the end of the LVER, as shown in Figures 3A–D. However, the
flow transition index was much higher after the incorporation of
GGMMA, as shown in Figures 3E–H. This illustrates the
structure transition of inks from the “brittle”to the “soft”
material and demonstrates the effect of GGMMA on the
viscoelastic property of CNFs-based inks (Corker et al., 2019).
A similar phenomenon on the flow transition index was also
observed in GrowInk-GelMA inks, as shown in Figures 3I–L.As
shown in Figures 3K and L, the τ
f
of the GrowInk-T-GelMA inks
increased drastically, which is mainly attributed to the strong
electrostatic interaction between GrowInk-T and GelMA. In
addition, the τ
f
of GrowInk-T-GelMA-PBS inks also displayed
a decreased value when diluted with PBS buffer.
In light-aided, hydrogel extrusion–based 3D printing, the
printing resolution is dictated by the shape maintenance
ability of ink materials in between being extruded out of the
needle and photo-curing. A viscosity recovery test is performed to
monitor the viscosity change of the formulated inks post
extrusion. A 3-stage shear test was adopted to mimic the
applied shear rate on the inks and time scale during printing.
In stage 1, a low shear rate of 0.1 s
−1
was applied for 60 s to
simulate at the rest condition. Following this, a high shear rate of
700 s
−1
was applied immediately for 10 s to simulate the extrusion
condition of the inks in the needle during printing in stage 2. The
shear rate is calculated based on the printing condition of a
printing speed of 12 mm/ s and an input flow rate of 100%. Then,
a low shear rate of 0.1 s
−1
was applied again to monitor the
FIGURE 3 | Amplitude sweep of formulated inks plotted against the shear stress [(A–D) GrowInk-N and GrowInk-T, (E–H) GGMMA incorporated GrowInk-N and
GrowInk-T, and (I–L) GelMA incorporated GrowInk-N–and GrowInk-T–based inks diluted with PBS buffer and water, respectively. Closed symbol: G’and open
symbol: G”].
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Wang et al. Nanocellulose-Based Light-Aided 3D Biomaterial Printing

FIGURE 4 | Thixotropic behavior of the inks (A–D) GrowInk-N-GGMMA–and GrowInk-T-GGMMA–based inks, (E–H) GelMA incorporated GrowInk-N–and
GrowInk-T–based inks diluted with PBS buffer and water, respectively, and (I) schematic illustration of the microst ructure of the inksduring the extrusion process. CNFs
showed a stable microstructural network in water. The nanofibrils were orderly arranged during the extrusion condition and slowly recovered to a network structure after
extrusion, whereas the CNFs in PBS buffer were aggregated into flocs at the rest condition and broke into individual nanofibrils during the extrusion process. The
nanofibrils were not able to form into flocs immediately after extrusion. In contrast, the photo-crosslinkable polymer of GGMMA or GelMA absorbed on the surface of
CNFs could significantly prevent floc formation, thus increasing the microstructure network stability during the extrusion process.
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Wang et al. Nanocellulose-Based Light-Aided 3D Biomaterial Printing

viscosity change of the inks after being extruded out of the needle.
The viscosity recovery test was performed at 25°C to mimic the
printability test performed in this study. As shown in Figure 4, all
inks exhibited a fast recovery to stabilize viscosity with no lag
observed within 10 s after a high shear rate. This suggests that the
ink could maintain shape fidelity after extrusion in the process
(Paxton et al., 2017). Meanwhile, the viscosity of all inks in stage 3
did not recover to the initial value in 60 s, indicating that their
microstructure was changed. This is associated with the
phenomenon in which randomly entangled CNFs at stage 1
were orderly arranged during high shear in stage 2, and the
change would take longer to recover to the original state. As
shown in Figure 4, the viscosity recovery ability of GrowInk-
GelMA inks was more vulnerable to PBS than GrowInk-
GGMMA inks. This might be owing to the monovalent cation
in PBS that disordered the electrostatic repulsion between CNFs
and GelMA, thus leading to difficult recovery phase-separated at
a high shear rate.
For hydrogel extrusion–based 3D printing, efficient
crosslinking of ink materials is the key to achieve good shape
fidelity. Rapid and robust crosslinking of the extruded filament is
demanded to support the layer-by-layer fabrication of printed 3D
objects in complex structures and to prevent distortion against
gravity. In our study, photo-crosslinkable GGMMA or GelMA
was formulated together with GrowInk to facilitate the covalent
crosslinking of the interpenetrating polymer networks with UV
irradiation. Photo-rheology was applied to monitor the photo-
crosslinking kinetics of the inks, and the G’was recorded upon
kicking off the UV irradiation. Theoretically, a minimum G’of
2.15 kPa to support an extruded filament span over a distance
5 times larger than the nozzle diameter can be established using a
simple beam theory with the following equation:
G’≥1.4cs4D(3)
where γis the specificweightoftheink,sisthespandistance(L/
D), L is the span width, and D is the nozzle diameter. The acceptable
deflection is 0.05D (Smay et al., 2002;Schaffner et al., 2017).
As shown in Figure 5, maximum G’(G’
max
) increased with the
increase in CNFs content within inks with the same content of
GGMMA or GelMA. The G’of all inks was increased
dramatically upon UV irradiation. The G’
max
of all inks was
higher than 2.15 kPa, and the required time of G’to exceed
2.15 kPa is summarized in Supplementary Table S2. As shown in
Figures 5A and B, the G’
max
of most GrowInk-N-PBS-based inks
was higher than that of GrowInk-N-water–based inks, except 2%
GrowInk-N + 5% GelMA, whereas the G’
max
of GrowInk-N-
PBS–based inks was lower than that of GrowInk-T-water–based
inks, as shown in Figures 5C and D.
FIGURE 5 | Photorheology profiles of the formulated inks: (A) GrowInk-N-GGMMA–based inks, (B) GrowInk-N-GelMA–based inks, (C) GrowInk-T-
GGMMA–based inks, and (D) GrowInk-T-GelMA–based inks diluted with PBS or water, respectively.
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Wang et al. Nanocellulose-Based Light-Aided 3D Biomaterial Printing

Printability Assessment
Rheological measurements are performed with the given
measuring parameters to mimic the ink flow conditions of the
printing process. These measurements could provide quantitative
information on the viscosity, viscoelastic properties, recovery
behavior, and photo-crosslinking kinetics of the inks.
However, the rheological simulation could not perfectly
reproduce the printing process, and the rheological evaluation
could not define the absolute criteria for printability (Paxton
et al., 2017). Therefore, comprehensive printability assessments
are required to provide essential information for adapting new
ink formulations in hydrogel extrusion–based 3D printing.
Considering the practical scenario of saline introduced by cell-
mixing in 3D bioprinting, the printability was mainly evaluated
for the GrowInk-based inks diluted with PBS. However, as we
often experienced clogging in the needle when printing GrowInk-
T-PBS–based inks, the printability was evaluated for the
GrowInk-T–based inks diluted with water. Electrostatic
repulsion between GrowInk-T CNFs contributes to their gel-
like structure and desired viscoelasticity, as revealed in
rheological studies. Meanwhile, it also determines that the
colloidal stability of GrowInk-T is sensitive to ionic strength
when diluted with PBS.
Extrudability is the ability to produce uniform and continuous
filaments under given printing conditions, which is the
fundamental requirement in extrusion-based 3D printing. In
the extrudability analysis, the printing speed and the input
flow rate were controlled, and the uniformity ratio and width
of the printed filaments were evaluated. As shown in
Supplementary Figure S3A, the filaments of GrowInk-N-
GGMMA-PBS inks displayed a smooth outline and the
uniformity ratio was not dependent on the GrowInk-N
loading, printing speed, and input flow rate. In contrast, the
uniformity ratio of GrowInk-T-GGMMA-water inks increased
with the increase in printing speed and decreased with the
increase of GrowInk-T loading. As shown in Supplementary
Figure S3C, both GrowInk-N-GelMA-PBS and GrowInk-T-
GelMA-water inks displayed quite good uniformity regardless
of the printing speed, input flow ratio, and CNF loading.
The width of the filament indicates its ability to hold a
cylindrical shape and also against spreading. As shown in
Supplementary Figures S3B and D, the overall filament width
decreased with the increase of the GrowInk-N and GrowInk-T
loading, attributed to the increasing G’and crosslinking kinetics.
Due to the difference in the physicochemical properties of the
photo-crosslinkable polymer, GrowInk-N–based inks
incorporated with GGMMA or GelMA behaved differently
under the same extrusion condition. The filament width of
GrowInk-N-GGMMA-PBS inks decreased with the increase in
printing speed under the same input flow, but the filament width
of GrowInk-N-GelMA-PBS inks increased with the increase in
the input flow rate, as shown in Supplementary Figure S3D.
Meanwhile, the GrowInk-T-GelMA-water inks displayed an
outstanding performance against spreading. This might be
attributed to the strong interaction between GrowInk-T and
GelMA, besides the relatively higher G’and faster crosslinking
kinetics. Speaking of the printing resolution as defined by both
the filament width and uniformity, 1.5 and 2% GrowInk-N + 2%
GGMMA-PBS inks and 1% GrowInk-T+5% GelMA-water ink
allowed a decent printing resolution of uniform filaments less
than 500 µm. Based on the extrudability evaluation and aiming to
decrease shear stress at the needle, a printing speed of 12 m/ s was
applied in the following printability assessment by printing layer-
by-layer cross-hatch scaffolds.
When printing a cross-hatch scaffold with an ideally gelated
ink, the outcome pores would display a square shape (Ouyang
et al., 2016). A Pr value was developed to quantify filament
uniformity and gelation condition by comparing the circularity of
the outcome pore to a perfectly square pore (Ouyang et al., 2016).
However, in practice, the gel-like behaviour of the printed
filament would lead to fusion between the stacking filaments,
especially for inks with an under-gelation status. Thus, the
outcome square pores would display varying degrees of
circularity. As observed in Figure 6, all inks displayed an
under-gelation status under different printing conditions, in
which the Pr values were in the range from 0.8 to 1.0. In this
case, the diffusion rate (Dr) value should also be considered to
evaluate their printability because filament spreading would also
lead to printing failure by losing printing accuracy. As observed in
Figures 6D and E, the Pr values did not display a noticeable
difference between the two scaffolds of 1.5% GrowInk-N + 5%
GelMA-PBS that were printed with a constant layer height of
0.07 mm but at different input flow rates; however, the filament
spreading resulted in almost closure of pores in the grid as the
input flow increased. Hence, the Dr value was carried out to
evaluate the pore closure effect caused by filament spreading
during the printing by comparing the outcome pore area with its
theoretical area (Habib et al., 2018). These two factors could
quantitatively evaluate the printability of the inks. In this study,
10-layer cross-hatch scaffolds were printed under the given
printing conditions for the ink formulations that showed
promising extrudability. As shown in Figure 6, Dr values
increased with the increase in the input flow rate for the same
formulation, which indicated a more severe spreading of the
filaments and closure of the pore. Meanwhile, Dr values decreased
with an increase in the layer height. The filaments of under-
gelation inks are not likely to hold an ideal cylindrical structure.
Thus, the layer height should be considered and adjusted to reach
an acceptable resolution.
To further analyze the printability of inks and evaluate the
structural stability of printed objects, 36-layer scaffolds were
printed under the printing parameter set at a layer height of
0.09 mm and input flow rate of 100% and were further incubated
in PBS buffer and DMEM for 7 days, respectively. As shown in
Figure 7, all scaffolds displayed a good height maintenance
ability, and no apparent collapse was observed. Subsequently,
the printed scaffolds were incubated in PBS and DMEM to
evaluate their structural stability in the cell culture medium.
All scaffolds displayed structural integrity, where no apparent
contraction and swelling was observed after 7 days.
Mechanical Property
The mechanical property of hydrogel scaffolds is one of the vital
aspects considering its application in the biomaterials field. As
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Wang et al. Nanocellulose-Based Light-Aided 3D Biomaterial Printing

FIGURE 6 | (A) Pr and Dr values and (B–I) optical microscope images of the cross-hatch scaffold printed with formulated inks under different printing parameters:
(B and C) printed with 1.5% GrowInk-N + 2% GGMMA-PBS with a layer height of 0.07 mm and an input flow rate of 100 and 110%, respectively, (D and E) printed with
1.5% GrowInk-N+5% GelMA-PBS with a layer height of 0.07 mm and an input flow rate of 100 and 110%, respectively, (F and G) printed with 1.5% GrowInk-T + 2%
GGMMA-PBS with a layer height of 0.07 mm and an input flow rate of 100 and 110%, respectively, (H and I) printed with 1.5% GrowInk-T + 5% GelMA-PBS with a
layer height of 0.07 mm and an input flow rate of 110 and 120%, respectively. Scale bar: 1 mm)
FIGURE 7 | Side and top views of the printed hydrogel scaffolds incubated in PBS buffer and DMEM for 7 days.
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Wang et al. Nanocellulose-Based Light-Aided 3D Biomaterial Printing

shown in Figures 8A and C, Young’s modulus and the yield
strain of the GrowInk-GGMMA–based hydrogels increased with
the increase of the CNF loading. In addition, Young’s modulus of
the GrowInk-T-GGMMA–based hydrogels was higher than that
of the GrowInk-N-GGMMA–based hydrogels for the same CNF
loading, whereas the fracture strain of the GrowInk-T-
GGMMA–based hydrogels was slightly smaller than that of
the GrowInk-N-GGMMA–based hydrogels. This indicated a
stiffer and more brittle mechanical property of the GrowInk-
T-GGMMA–based hydrogels. More likely, the small and surface-
charged GrowInk-T-CNFs could evenly disperse in GGMMA
and form a comparatively homogeneous hydrogel than the
GrowInk-N-GGMMA–based ink (Takeno et al., 2020). A
similar trend in Young’s modulus could also be observed in
the GrowInk-GelMA–based hydrogels, as shown in Figures 8B
and D. Unexpectedly, the hydrogel of 2% GrowInk-N + 5%
GelMA-PBS displayed a drop in Young’s modulus compared to
the hydrogel of 1.5% GrowInk-N + 5% GelMA-PBS. This might
be because the high solid loading of GrowInk-N inhibited the
crosslinking of GelMA, leading to a lower crosslinking density.
Meanwhile, the yield strain of GrowInk-N–based hydrogels was
higher than that of the GrowInk-T–based hydrogels, as shown in
Figures 8A and B. It is inferred that the CNFs in the GrowInk-N
hydrogel are entangled to a more significant extent, which
facilitates energy dissipation. Besides, Young’s modulus of
the GelMA-based hydrogels is much lower than that of the
GGMMA-based hydrogels. One aspect to account for is the
relatively lower DM of GelMA than GGMMA, which leads to
a lower crosslinking density and softer structure. The result of
Young’s modulus is in line with the G’in photo-rheology. Overall,
Young’s modulus of the hydrogel disks is tunable in a wide range
from 9.35 to 68.32 kPa.
CONCLUSION
When CNFs are used as a major constituent in formulating
biomaterial inks with a photo-crosslinkable biopolymer, the
nanodimensions of and the surface charge on CNFs together
dominate the rheological properties of the as-prepared
nanocellulose-based inks and, consequently, dictate the ink
printability in light-aided, hydrogel-extrusion–based 3D
printing. On using PBS buffer as the formulation medium, the
gel structure of TEMPO-oxidized CNFs-based inks (GrowInk-
T–based ink) is sensitive to the variation of ionic strength, which
would potentially make complex its application in cell-laden 3D
bioprinting. Owing to the specific absorption of GGMMA onto
CNFs via hydrogen bonding, the addition of 2% GGMMA in
GrowInk-N diminished the hysteresis behavior of the GrowInk-
N-PBS ink by preventing the formation of uneven
FIGURE 8 | Compressive stress–strain curves and compressive Young’s modulus of hydrogels fabricated by GrowInk-N and T with (A and C) GGMMA and (B
and D) GelMA.
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Wang et al. Nanocellulose-Based Light-Aided 3D Biomaterial Printing

microstructures of flocs upon variation of ionic strength.
Furthermore, the addition of 2% GGMMA significantly
decreased the G’value and the flow stress. In comparison, the
addition of 5% GelMA in GrowInk-T largely increased the flow
stress because of the electrostatic interaction between GelMA and
TEMPO-oxidized CNFs. With the same content of CNFs, 2%
GGMMA-containing inks exhibited faster crosslinking kinetics
and resulted in stiffer hydrogels than 5% GelMA-containing inks
because of their high DM and intrinsic absorption onto
nanofibrils. Meanwhile, the GelMA-containing hydrogels were
soft but more elastic, which showed an extended strain at break.
Compared to GrowInk-T–based inks, the GrowInk-N–based inks
with the addition of either GGMMA or GelMA in PBS buffer
showed quite good printability on optimizing the printing
parameters. Upon UV-crosslinking, the printed scaffolds
displayed an under-gelation status and the Dr value was more
significant in the printability assessment than the Pr value. The
shape fidelity of the formulated inks is mainly determined by the
input flow rate, layer height, and CNFs concentration.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in
the article/Supplementary Material; further inquiries can be
directed to the corresponding author.
AUTHOR CONTRIBUTIONS
All authors contributed to the experimental design, planning,
execution, and data analysis. QW and OB carried out the main
experimental work. QW and XW drafted the original draft
manuscript. MN and CX provided critical revision to the
manuscript. XW is the main scientist who supervised the
work. The manuscript has been approved by all the co-authors
for submission.
FUNDING
QW was supported by the research grants from the China
Scholarship Council (Student ID 201907960002) and KAUTE
Foundation (Project number 20190031) at Abo Akademi
University (AAU), Finland. The major sponsor of research
resources used in the current research is the Academy of
Finland (AoF) (333158). The AoF project has received funds
for its open access publication fee.
ACKNOWLEDGMENTS
QW would like to acknowledge the financial support from the
China Scholarship Council (Student ID 201907960002) and
KAUTE Foundation (Project number 20190031) to his
doctoral study at Abo Akademi University (ÅAU), Finland.
XW would like to thank the Academy of Finland (333158) as
well as Jane and Aatos Erkko Foundation for their funds to her
research at AAU. This work is also part of activities within the
Johan Gadolin Process Chemistry Centre (PCC) at AAU. Luyao
Wang is gratefully acknowledged for assisting the TEM analysis.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at:
https://www.frontiersin.org/articles/10.3389/fceng.2021.723429/
full#supplementary-material
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Conflict of Interest: Author MN was employed by the company UPM-Kymmene
Comporation, Biomedicals.
The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential
conflict of interest.
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Frontiers in Chemical Engineering | www.frontiersin.org September 2021 | Volume 3 | Article 72342913
Wang et al. Nanocellulose-Based Light-Aided 3D Biomaterial Printing