Carbohydrate Polymers 220 (2019) 12–21

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Bioinspired 3D printable pectin-nanocellulose ink formulations T

a,1 a,1 a,b a
Alexandra I. Cernencu , Adriana Lungu , Izabela-Cristina Stancu , Andrada Serafim ,
⁎
Ellinor Heggsetc, Kristin Syverudc, Horia Iovua,d,
a
Advanced Polymer Materials Group, University Politehnica of Bucharest, 1-7 Gh. Polizu Street, 011061, Bucharest, Romania
b
Faculty of Medical Engineering, University Politehnica of Bucharest, 1-7 Gh. Polizu Street, 011061, Bucharest, Romania
c
RISE PFI, Høgskoleringen 6B, NO-7491 Trondheim, Norway
d
Academy of Romanian Scientists, 54 Splaiul Independentei, 050094, Bucharest, Romania

A R T I C LE I N FO A B S T R A C T

Keywords: The assessment of several ink formulations for 3D printing based on two natural macromolecular compounds is
3D printing presented. In the current research we have exploited the fast crosslinking potential of pectin and the remarkable
Polysaccharide shear-thinning properties of carboxylated cellulose nanofibrils, which is known to induce a desired viscoelastic
Hydrogels behavior. Prior to 3D printing, the viscoelastic properties of the polysaccharide inks were evaluated by rheo-
Pectin
logical measurements and injectability tests. The reliance of the printing parameters on the ink composition was
Cellulose nanofibrils
established through one-dimensional lines printing, the base units of 3D-structures. The performance of the 3D-
printed structures after ionic cross-linking was evaluated in terms of mechanical properties and rehydration
behavior. MicroCT was also used to evaluate the morphology of the 3D-printed objects regarding the effect of
pectin/nanocellulose ratio on the geometrical features of scaffolds. The proportionality between the two poly-
mers proved to be the determining factor for the firmness and strength of the printed objects.

1. Introduction toxic biopolymers is pivotal. Although there are numerous biocompa-

Three-dimensional (3D) printing stands out as a breakthrough
technology in the fabrication field due to the capability of providing
specific structural features in terms of shape and porosity. The 3D
printing technology subsumes a plethora of deposition techniques in-
cluding extrusion (direct ink writing), inkjet and drop-on-demand sys-
tems, and one must consider the printing setup as the main determining
factor of the printability of an ink. 3D bioprinting has been recently
recognized as one of the most promising technologies to fabricate
scaffolds appropriate as in vitro models and constructs for tissue en-
gineering and regenerative medicine (Moroni et al., 2018). This tech-
nology ensures the spatial and temporal control of acellular and cellular
components, thus recapitulating in 3D the complexity of targeted tis-
sues. The development of such printable formulations combining cells,
macromolecular compounds and bioactive clues, recognized under the
term bioink, becomes critical for the success of functional tissue re-
placements (Moroni et al., 2018).
Biomimetic 3D engineered materials able to enact specific physio-
logical responses have gained attention in both research and develop-
ment. In this respect, the use of sustainable, biodegradable, and non-
tible polymers reported in literature, few are fulfilling the considerable
requirements for ink formulations and subsequent production of 3D
scaffolds (Donderwinkel, van Hest, & Cameron, 2017; Liu et al., 2019).
Among them, hydrogel-based inks are appealing to fabricate extra-
cellular matrix bio-inspired analogues, considering their elastic hy-
drated intrinsic network. Engineered inks based on natural polymers
such as proteins (gelatin, collagen, silk fibroin) and polysaccharides
(alginate, chitosan, agarose) are currently under development as
highlighted by recent reviews (Guvendiren, Molde, Soares, & Kohn,
2016; Hospodiuk, Dey, Sosnoski, & Ozbolat, 2017; Moroni et al., 2018).
The stability of the printed polymeric constructs is crucial and cross-
linking by suitable physical and chemical methods is therefore applied.
For instance, alginate gelation can be induced through ionic cross-
linking upon addition of divalent cations (such as Ca2+), gelatin forms
physical gels upon lowering the temperature, while methacrylamide
modified gelatin undergoes photo-crosslinking to obtain permanent gels
(Ojansivu et al., 2019; Park, Lee, Lee, Park, & Lee, 2018; Pepelanova,
Kruppa, Scheper, & Lavrentieva, 2018).
More recently, the nanoscale versions of cellulose in the form of
cellulose nanofibrils (CNFs), proved to be versatile and sustainable bio-

⁎
Corresponding author at: Advanced Polymer Materials Group, University Politehnica of Bucharest, 1-7 Gh. Polizu Street, 011061, Bucharest, Romania
E-mail address: horia.iovu@upb.ro (H. Iovu).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.carbpol.2019.05.026
Received 21 January 2019; Received in revised form 7 May 2019; Accepted 7 May 2019
Available online 08 May 2019
0144-8617/ © 2019 Published by Elsevier Ltd.
A.I. Cernencu, et al.
nanomaterials that holds a great potential for fabrication of 3D con-
structs. The various types of nanocellulose with specific morphology
and surface chemistry bring benefits to rheological and mechanical
behavior and can be used either as a main component of 3D-printable
inks or as reinforcement for hybrid inks in various polymer-based
composite inks. Several works describe the 3D printing of nanocellulose
in combination with polymers such as alginate or gelatin (Ávila,
Schwarz, Rotter, & Gatenholm, 2016; Heggset et al., 2019; Leppiniemi
et al., 2017; Müller, Öztürk, Arlov, Gatenholm, & Zenobi-Wong, 2017;
Ojansivu et al., 2019; Xu et al., 2018).
Among the pretreatment methods of cellulose pulp, chemically
modified CNFs could be obtained as dense and transparent gel-like
solutions through different oxidative reactions (Hoeng, Denneulin, &
Bras, 2016; Torstensen et al., 2018). The use of 2,2,6,6-Tetra-
methylpiperidine 1-oxyl (TEMPO) as a mediator in the cellulose oxi-
dation has become increasingly popular since a controlled oxidation
process for cellulose pulp is developed. The production of chemically
modified CNFs is achieved nowadays by several methods, e.g. TEMPO
mediated oxidation or carboxymethylation and periodate oxidation
(Grossman & Nwabunma, 2013; Rees et al., 2015). Chemically modified
CNFs can be employed to overcome some typical challenges in 3D
printing providing a shear-thinning behavior to the inks that subse-
quently enhances both the printability and the shape fidelity after
printing (Chinga-Carrasco, 2018). For tissue engineering applications,
CNFs are used as rheological modifier ensuring a cytoprotective role
when cells are loaded into the gels prior printing.
A review of literature on the 3D (bio)printing pointed out numerous
papers focused on describing alginate-nanocellulose ink formulations
for biomedical applications (Chung et al., 2013; Fedorovich, De Wijn,
Verbout, Alblas, & Dhert, 2008; Paxton et al., 2017). As both polymers
contain carboxyl groups, an ionically crosslinking process in the pre-
sence of Ca2+ ions is considered. An alginate and nanocellulose con-
sisting bio-ink, commercialized under the trademark CELLINK, was
designed to mimic the cells natural environment combining the shear
thinning behavior of CNFs and the versatility of alginate (Ávila et al.,
2016).
Lately, pectin, another heterogeneous polysaccharide extracted
from biomass, was reported as a suitable hydrocolloid used to develop
edible model systems for 3D food printing (Vancauwenberghe,
Katalagarianakis et al., 2017; Vancauwenberghe, Mbong et al., 2017). It
is widely known that pectin, along with cellulose, are the main struc-
tural polysaccharides that compose the plant cells, pectin being also the
determining factor of flexibility in cell walls since it could be highly
hydrated (Boyer, 2016; Shtein et al., 2017). In addition to biodegrad-
ability and cytocompatibility, pectin has also a facile gelling ability that
depends on the methoxylation degree. Low methoxylated pectin
(< 50%) has a gelation mechanism similar to alginate, forming ionic
hydrogels in the presence of divalent or multivalent cations with ap-
plications in tissue engineering (Banks, Guo, Chen, Kumpaty, & Zhang,
2017; Radhakrishnan, Subramanian, Krishnan, & Sethuraman, 2016;
Willats, Knox, & Mikkelsen, 2006). A recent paper describes the func-
tionalization of pectin that allows hydrogels formation by ionic cross-
linking and UV photopolymerization to design new types of bioinks
(Pereira, Sousa, Barrias, Bártolo, & Granja, 2018).
The focus of this research is to develop new bio-based ink for-
mulations suitable for 3D printing using pectin and TEMPO-oxidized
CNFs. To the best of our knowledge, the employment of this combi-
nation of biological materials to produce 3D printable composite inks is
not yet reported and holds tremendous potential. Moreover, the out-
standing property of pectin to modulate the elasticity in plant cell walls
was exploited as a bio-inspired design strategy to fabricate 3D printed
objects.
Herein extrusion-based printing was used to build up self-sup-
porting polysaccharide constructs that were easily and rapidly cross-
linked in cell-friendly conditions immediately after dispensing. A series
of preliminary printability experiments were performed to understand
13
Carbohydrate Polymers 220 (2019) 12–21
the relationship between the composition of the ink formulations and
the printing parameters (deposition speed, extrusion pressure) and to
ensure an accurate printing. Also, a correlation between the material’s
rheological properties and printing behavior has been established. The
newly developed aqueous printing inks based on these polysaccharides
are appealing for further development as platforms for tissue en-
gineering since the obtained formulations could be loaded with pro-
teinaceous materials that natively exhibit cell-interactive clues (for in-
stance RGD-rich sequences).
2. Materials and methods
2.1. Materials and ink preparation
High-methoxylated pectin (HM-pectin) from apple (70–75% ester-
ification, Sigma-Aldrich) was chemically demethylesterified to obtain
low-methoxylated pectin (LM-pectin) using an alkaline saponification
method described in literature (Fraeye et al., 2009; Vancauwenberghe,
Katalagarianakis et al., 2017). Briefly, an aqueous solution of 2.5% HM-
pectin was set at pH 11 using NaOH [2 M]; this pH value was kept
constant for 1 h (carboxyl groups formed during the saponification of
methyl esters lead to a drop in pH). The reaction was stopped by re-
ducing the pH to a value of 4.5 using HCl [0.5 M]. The solution was
then dialyzed for 2 days, followed by adjusting the pH to 6 and freeze
dried at −80 °C. The degree of methylesterification (DM) of LM-pectin
was then evaluated by infrared spectrometry following the protocol of
Kyomugasho, Christiaens, Shpigelman, Van Loey, and Hendrickx
(2015). LM-pectin with a DM varying from 12 to 25% was produced
and used within ink’s compositions.
Carboxylated-cellulose nanofibrils (CNFs) as a viscous and trans-
parent gel suspension was provided by RISE PFI from Norway with a
solid content of 1.19%. CNF gel was obtained by TEMPO-mediated
oxidation from bleached sulfite pulp.
Sodium hydroxide 98% (pellets), hydrochloric acid ACS reagent
37%, calcium chloride ≥93.0% (anhydrous, granular, ≤7.0 mm) and
ethanol (absolute puriss) were also purchased from Sigma-Aldrich.
Phosphate buffered saline (PBS) powder from Sigma-Aldrich was used
to prepare PBS [0.01 M], containing NaCl [0.138 M] and KCl
[0.0027 M], with a pH of 7.4.
Three composite inks for 3D printing were prepared by mixing CNF
suspension with LM-pectin gel; three formulations with mass ratio of
CNF/LM-pectin of 3:1, 1:1 and 1:3 were obtained and further referred
as CNFP31, CNFP11 and CNFP13 respectively. To prepare the printing
mixtures, an appropriate mass of LM-pectin was gradually added into
the nanocellulose gel and intensively stirred at room temperature until
complete dissolution. Pure CNFs were used as a reference gel.
The total solid content (T%) of the studied water-based inks was
also considered as it represents a key parameter to prevent typical
collapsing during printing experiments. T (%) was calculated as total
solid weight per volume and it gradually increases by the addition of
pectin from 1.59% (CNFP31), 2.38% (CNFP11) to 4.76% (CNFP13).
Moreover, considering the significant contribution of aqueous media to
the precursor material properties, the initial water content (IWC%) was
calculated and subsequently correlated with specific features of the
printed products.
2.2. Determination of ink injectability
To correlate the ink composition with their printability, preliminary
measurements of the formulations’ injectability were performed. The
injection force required to extrude the precursor suspensions through a
nozzle was evaluated using a Brookfield CT3 Texture Analyzer
equipped with a 4500 g cell load in compression mode. A 5 mL Luer-
Lock syringe filled with the polymeric formulations was fixed in a
holder and various needles with different gauges were attached: 21 G
(Ø 0.51 mm), 23 G (Ø 0.33 mm), 25 G (Ø 0.25 mm) and 27 G (Ø
A.I. Cernencu, et al.
0.20 mm). The length of the needles was kept constant (6.35 mm).
During the preparation of the precursors, the homogeneity and absence
of air bubbles is very important for the reproducibility of the experi-
ments. The plunger end of the syringe was placed in contact with the
load cell and was gradually pressed with an increased load at a constant
testing speed (0.5 mm/s). The applied force for injecting the gels
through the needles was measured. All measurements were performed
in triplicate for each sample, at room temperature (Chen, Wang et al.,
2017).
2.3. Rheological evaluations
The rheology data were obtained using a Kinexus Pro rheometer
(Malvern Instruments) equipped with a Peltier element for temperature
control. In order to measure the shear viscosity of the precursor sus-
pensions a cone-plate geometry was used. A cone angle of 4° and a
diameter of 40 mm was employed. A water lock was used to protect the
samples from drying at the edges during measurements. Shear viscosity
and shear stress as a function of shear rate were recorded at 25 °C by
steady shear measurements, with shear rate ranging from 0.01–1000
s−1.
To evaluate the mechanical behavior of the resulting composite
hydrogels, dynamic oscillatory measurements were also performed at
25 °C. To this end, hydrogel samples were synthesized in cylindrical
molds and subsequently crosslinked in CaCl2 solution (3%, 10 min). The
samples were placed in distilled water for 1 h to swell into equilibrium
and measured with a digital caliper before testing. The average di-
mensions of the swollen disks were 20 mm diameter and a thickness of
1.2 mm. The samples were placed on the bottom plate of the rheometer
and a parallel-plate geometry was used. Dehydration of the hydrogels
was prevented using a water lock. The storage modulus G’ and loss
modulus G” were evaluated within the linear viscoelastic region,
through oscillatory shear tests with a frequency sweep between
0.10–10 Hz. All the experiments were repeated three times and the
average data were used to plot the curves.
2.3.1. 3D printing
The CNFP ink formulations were tested and printed using a multi-
headed 3D Discovery bioprinter (RegenHU, Switzerland). The inks were
extruded using the PH2 print-head (pneumatic driven print-head,
DD135 N). To avoid the formation of air bubbles, the printing materials
were loaded using a spatula into a 3 mL syringe having attached a cy-
lindrical nozzle of 27-gauge (0.20 mm inner diameter, 6.35 mm in
length). The nozzle height was set at 0 mm. All the samples were
printed at room temperature onto a glass microscope slide in a layer-by-
layer approach.
Prior to 3D printing, the printability of the gels was firstly evaluated
through qualitative ways to ensure successful printing. Multiple ex-
perimentally trials were carried out to print one dimensional line
having 20 mm length by adjusting two essential printing parameters:
printing pressure and material feed rate. To find the optimal processing
parameters, a range of pneumatic pressure from 50 to 100 kPa in 10 kPa
increments and a range of feed rate from 5 to 40 mm/s in 5 mm/s in-
crements were studied for each sample. Therefore, we tested 48 com-
binations of printing parameters. Right after the fabrication, the lines
were photographed with a scale bar and the width of the printed lines
was measured at three random locations using ImageJ software.
Subsequently, optimal processing parameters were used for each ink
formulation for printing 3D constructs using a print pressure in the
range 50–80 kPa and a print speed between 20–35 mm/s. The 3D model
was designed with the BioCAD software as square grids of 20 mm x 20
mm with a crosshatch infill of 2.5 mm spacing between lines in 10
layers with a 0°-90° deposition direction. After printing, the 3D printed
constructs were crosslinked in a bath of 3% CaCl2 aqueous solution for
10 min at room temperature to allow the Ca2+ ions to diffuse through
the objects.
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Carbohydrate Polymers 220 (2019) 12–21
2.4. Morphology
Micro computed tomography (CT) was used to visualize the 3D
structure of the printed gels using a Skyscan 1272 equipment (Bruker
microCT, Belgium). 3D printed gels cut as rectangular samples (5 × 5
x15 mm) were placed in sealed 2 ml polypropylene test tubes and fixed
on the sample holder using modeling clay. All samples were scanned
without filter at a voltage of 40 kV and a source current of 200 μA. The
images were registered at a resolution of 2452 × 1640 pixels and a
pixel size of 7 μm, with a rotation step of 0.3°. All images were pro-
cessed using CT NRecon software and reconstructed as colorful scien-
tific illustrations of 3D object using CTVox.
2.5. Rehydration of the 3D objects in PBS
Rehydration of the 3D objects in PBS was evaluated as swelling
behavior. Briefly, the crosslinked 3D printed grids were washed three
times in freshly prepared PBS. Progressive dehydration was performed
at room temperature, through sample incubation for 15 min in aqueous
ethanolic solutions (with increasing concentration 20%, 40%, 60%,
80%, 100%; the last incubation in pure ethanol was repeated three
times). The samples were left overnight to complete desiccation by air
drying. The sample number for each material combination was n = 3.
The obtained 3D dried scaffolds were weight to determine the dry
weight (Wd) and then immersed in PBS pH 7.4 at 37 °C. The wet weight
(Ww) was measured after 24 h post immersion. The measured values of
Wd and Ww were used to estimate both the maximum swelling degree
(%MSD) and the PBS equilibrium content (%ECPBS) after rehydration by
applying Eq (1) and Eq (2), respectively.
Ww − Wd
% MSD= ∙ 100
Wd (1)
Ww − Wd
% ECPBS = ∙ 100
Ww (2)
3. Results and discussion
Best-known for their structural function in vegetal tissues, superior
mechanical properties and high-water affinity, plant-derived poly-
saccharides became increasingly popular since these properties could
be exploited to develop a new class of bio-inspired inks for 3D printing
(Guvendiren et al., 2016; Liu et al., 2019; Radhakrishnan et al., 2016).
3.1. Injectability tests
The injection force revealed by the texture analyzer was further
employed to predict the ease of injectability of the formulation. The
injection force needed to extrude the polymeric formulations from a
syringe with different needles gauges was evaluated. The applied force
increases at the beginning of the experiment then it remains constant
until the end of the test. Fig. 1 illustrates the values of maximum force
applied to the piston for the extrusion of each gel formulations.
All the CNF-based inks are injectable due to the presence of a con-
siderable amount of water within the polysaccharide matrices. As ex-
pected, increasing needle gauge, and thereby reducing needle diameter,
the injection force increases (Chen, Wang et al., 2017). Also we ob-
served that the force applied in order to inject CNFP31 and CNFP11 gels
through all the selected needles decreases (50–120 Pa depending on the
needle gauge) in comparison with nanocellulose reference gel
(100–180 Pa). On the other hand, the injection force required for
CNFP13 with the highest pectin content was close to the reference
nanocellulose. This result indicates that the addition of pectin influ-
ences ease of injection, improving the extrusion of the formulations.
Balancing the CNF/pectin ratio in favor of pectin, the CNFP13 requires
a higher injection force just for the 27 G nozzle gauge with the smallest
A.I. Cernencu, et al.
Fig. 1. Maximum injection force for the CNFP composite inks injected through
different needle gauges.
nozzle diameter as a consequence of very high T% (T% is 4.76 for
CNFP13 in comparison with T% of 2.38 for CNFP11 and of 1.59 for
CNFP31).
3.2. Rheological evaluation
The injectability assessment of the formulated inks is a key starting
point to establish the extrusion pressure during the printing process.
However, when evaluating gels as precursor materials for 3D printing,
there are many factors to consider besides the needed stress to be ap-
plied for a proper injection such as viscosity, shear-thinning properties
and yield stress. Therefore, in addition to injectability tests, rheological
analysis of shear response was performed for all the ink formulations to
predict the printing parameters and shape retention after printing. To
evaluate the gels’ printability and the influence of pectin amount over
the nanocellulose, the shear stress and the viscosity were investigated at
25 0C and the results are shown in Fig. 2.
The bicomponent combinations forming the printing inks provide
specific rheological features. The plot of shear stress over shear rate
exhibit a gradual increase of the shear stress with the increase of shear
rate. The rheological response of CNF-based formulations is dependent
on the nanocellulose properties which combine both colloidal gels and
nanofibers suspensions properties (Martoïa, Dumont, Orgéas,
Belgacem, & Putaux, 2016). In this context a significant influence over
the non-Newtonian behavior of CNF-based precursors in both low and
high shear rate regime is expected, where the systems exhibit solid- and
liquid-like properties, respectively. The data show linearity in the low
shear rate regime up to 1 s−1, while above it the formulations exhibit
distinctive rheological behaviors in comparison with the starting con-
stituent nanocellulose. The assessment of the rheological features at
higher shear rates is of great interest in describing printable materials as
the typical shear rates of extrusion process is 100-1000 s−1 (Hubbe
et al., 2017).
Thus, a slight negative curvature was observed above 1 s−1 for CNF
Fig. 2. Shear stress (a) and apparent viscosity (b) as a log-log function of shear rates for the formulated inks.
15
Carbohydrate Polymers 220 (2019) 12–21
reference probably due to the weak assembling of the suspension that is
easily interrupted by increasing shear rates. In contrast, CNF/pectin
blends with 3/1 and 1/1 ratio maintain a constant increasing trend in
the high shear rate regime and no obvious disruption of the network
can be observed as a consequence of pectin loading. Moreover, the
pectin-rich formulation presents a noticeable increase of shear stress
within high shear rate regime (from 1 to 1000 s−1) indicating a more
stable gel structure formed through (physical) interactions among the
polysaccharides (Fu & Rao, 2001).
A well-known desirable feature that improves the gels’ printability
providing a high printing fidelity is the shear-thinning phenomenon
meaning that the viscosity progressively decreases as the shear rate
increases (Murphy & Atala, 2014). The viscosity experimental data
show that all the formulations have a shear-thinning behavior conferred
by CNFs, this being the main reason for engaging CNFs in printing
precursors. The viscosity of the gels is clearly dominated by the nano-
cellulose and the addition of pectin does not alter the nanocellulose
shear-thinning behavior, but rather intensifies this phenomenon by
increasing the viscosity drop. As it can be observed from Fig. 2(b), the
viscosities of the studied mixtures exhibit a slight increase with in-
creasing the pectin content, especially at shear rates above 1 s−1 as the
viscosity is highly reliant on the total solid content. Addition of pectin
up to equivalence leads to a modification of viscosity noticed in the
shear rate interval from 1 to 100 s−1 while at higher shear rates the
modification is insignificant. Further increasing the pectin content, a
viscosity increase is better emphasized for the shear rates regime from 1
to 1000 s−1, while the shear thinning behavior is also enhanced
(viscosity drops within the typical shear rates of extrusion process from
1.5 Pa s at 100 s−1 to 0.4 Pa•s at 1000 s-1 for CNFP13).
The experimental data obtained from rheological characterization
were used to quantify the flow behaviors of the formulated nano-
cellulose/pectin mixtures. The Herschel-Bulkley method, a common
rheological mathematical model used to aid the data analysis for non-
Newtonian fluids, has been applied to fit the flow curves as follows:
τ= τ0 + K γ̇ n (3)
where τ is the shear stress (Pa), τ0 yield shear stress (Pa), K the con-
sistency index (Pa•sn), γ̇ the shear rate (s−1) and n the flow behavior
index. The regression analysis was conducted to calculate the τ0, K and
n of each mixture sample.
The values of the Hershel-Bulkley fitting parameters (Table 1) de-
scribe the yielding of shear-thinning gels (n < 1) and reveal that the
amount of pectin influences the rheological behavior of the CNF-based
formulation.
The dynamic yield stress, τ0 is a significant parameter that implies
the necessary force for a material to be extruded and reflects mainly the
flow resistance of the fluid (Mohtaschemi et al., 2014). Thus, τ0 is an
inherent material property directly related to the gel strength to support
subsequent 3D printed layers. The CNF gel with 0% pectin has a low
yield stress of about 13 Pa while the formulations containing pectin
afford a higher yield stress relative to reference at about 20 ± 1 Pa.
A.I. Cernencu, et al.
Table 1
Rheological parameters of the studied formulations from Hershel-Bulkley
model.
Sample code τ0 (Pa) K (Pa•sn) n R2
CNF 13 5.47 0.458 0.94
CNFP31 21 6.39 0.482 0.99
CNFP11 21 5.47 0.468 0.97
CNFP13 19 17.41 0.457 0.98
where R2 is the regression coefficient.
This fact indicates that the yield stress is conditioned by the physico-
chemical characteristics of the fluid and the pectin loading plays an
important role in enhancing the printability of ink formulations. 3D
printed objects using materials with a low yield stress is expected to
exhibit a loss of shape fidelity by addition of sequential layers, while a
high yield stress will induce a better extrusion and implicitly a better
printing resolution and structural integrity.
The values of the flow curve parameters show that the flow index,
n < 1 corresponding to shear-thinning behavior, is approximately 0.45
for all formulations and is nearly independent on the solid content. This
pronounced shear-thinning phenomenon could be a consequence of the
formation of water-rich boundary layers that will induce an apparent
slippage within the network (Kumar, Ottesen, Syverud, Gregersen, &
Toivakka, 2017; Martoïa et al., 2016). Yet all the formulations exhibit a
similar flow behavior index (n), the values of the consistency index
parameter (K) for the tested fluids can be evaluated in contrast to
comprehend the fluid’s viscosities. The value of K is around 5 (Pa•sn) for
the CNF, CNFP31 and CNFP11 gels while the pectin-rich sample
(CNFP13) shows a three times higher K index that could indicate
stronger interactions induced by the preponderance of the pectin
chains. These fundamental rheological concepts are quantitative in-
dicators of a gel extrudability and shape retention after printing. The
correlation coefficient value (R2) of the sample is higher than 0.94,
showing a proper fitting of the dataset.
The rheological measurements are frequently correlated with the
injectability however, in 3D printing of shear-thinning gels, the mate-
rial extrusion is affected by a combination of shear and extensional
flow. Thus the injectability through a syringe needle is directly de-
pendent by the yield stress in flow regime.
3.3. Ink printability assessment
Multiple printability experiments were conducted to define the
optimum parameters that affect the printing resolution of 3D con-
structs. The influence of most important parameters, printing pressure
and printing speed was firstly evaluated with a simple printing design
such as lines.
Line printing tests were performed at 6 different extrusion pressures
from 50 to100 kPa (with 10 kPa increments and 8 different nozzle feed
rates between 5–40 mm/s (in 5 mm/s increments).
In Fig. 3 a quantitative comparison of extruded filaments width
obtained is presented, based on several trials and error experiments for
each of the materials composition. For all these tests, a 0.20 mm dis-
pensing nozzle was selected. The extruded lines collected as rod-shaped
strands were evaluated mainly in terms of continuous deposition and
thickness.
A continuous ink filament with smooth shape and uniform thickness
is considered as a proper gelation and is more likely to maintain the
shape fidelity of the 3D fabricated scaffold. Some of the inks were non-
uniformly dispensed, leading to successive droplets instead of con-
tinuous filaments; this is a consequence of a too high printing speed that
caused material under extrusion (CNFP13 over 25 mm/s printing speed
as visible in Figs. 3 and 4). Regardless the operation pressures and feed
rate, the lines thickness for all the studied compositions is much larger
with respect to the nozzle diameter, because of the material’s post-
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Carbohydrate Polymers 220 (2019) 12–21
extrusion swelling (also known as the Barus effect). This expansion
appears to be reduced with pectin loading, so that the filament width
progressively decreases at various nozzle speeds.
The composition of the tested inks critically affects their delivery
and the integrity of the structure formed, the filament resolution being
strongly dependent both on the printing speed and the extrusion feed
rate. These two fabrication parameters exhibit an inversely propor-
tional effect over the thickness of the lines: at low printing speeds
(5–10 mm/s) and high pressures (80–100 kPa) high diameters are re-
corded. When CNFs were printed slowly, the measured thickness of the
extruded lines was around 1–2.5 mm. The pectin addition expresses a
different influence depending on the nanocellulose / pectin ratio. For
the nanocellulose-rich (CNFP31) and balanced (CNFP11) formulations,
higher filament widths are generated, whereas the composition with
1:3 nanocellulose: pectin ratio exhibits minimum filament width. For
example, at the same operating parameters such as 70 kPa and 20 mm/
s, a diameter of 1.2 mm for CNFP31 and of 1.4 mm for CNFP11 have
been measured while a three times lower value (0.40 mm) was recorded
for the CNFP13. Hence, the ink with pectin preponderance seems to
exhibit a less pronounced Barus effect that leads to a good printability
being able to preserve the 3D printed object resolution. Increasing the
printing speed over 20 mm/s, higher pressures are required, out of the
studied range, for uniform deposition first and foremost due to higher
total solid content of the CNFP13 sample.
Following these results, a printability region was defined for each of
the tested formulations as a parameter reliance range that provides
uniform and defect-free filament deposition and it was depicted as a
shadowed area in their corresponding graph in Fig. 3. The acceptable
resolutions interval was chosen in a certain feed rate/pressure range
comprising the optimum processing parameters.
Afterwards, as a direct result of the line deposition, the optimum
printing set-up was selected for each formulated ink based on the
printing behavior which relies on the nanocellulose/pectin ratio, as
outlined above. Fig. 4 depicts representative photographs of filaments
extruded at a constant pressure in the range of feed rate from 5 to
40 mm/s in 5 mm/s increments. The operating pressures were chosen so
that the filaments deposition is continuous and constant in width cor-
related with the total solid content of the inks. In order to ensure a
successful 3D printing and to fabricate 3D scaffold structures, we
decided to choose a feed rate that will provide a line width around
0.6 ± 0. 05 mm (outlined in red in each image).
3.4. 3D scaffold fabrication
Through harmonizing all the parameters mentioned above, 3D
printing of CNF-based formulations was further performed. The pho-
tographs of the fabricated 3D scaffolds are shown in Fig. 5. Prior to the
printing process various water-soluble dyes were employed within the
inks precursors to emphasize the differences between the formulations
and to facilitate the visualization of the 3D objects.
A good printability of the designed 3D pattern for all the formulated
inks was observed. A visual examination of the infill shapes within the
printed grids was carried out after fabrication. In comparison with the
reference (CNFs), the 3D scaffolds including small amounts of pectin
(CNFP31, CNFP11) are losing their shape fidelity during the printing
process. These scaffolds show an obvious collapse in the filaments
overhang region and consequently the cavities area appears to be di-
minished and in some cases the strands are completely merged.
However, the printed 3D objects based on pectin-rich composition
(CNFP13) exhibit a higher printing resolution, maintaining the shape
fidelity after fabrication in a more precise manner. The scaffold printed
using the CNFP13 in. presents homogeneous filaments and pores.
Therefore, the ratio of 1:3 nanocellulose to pectin proved to be the most
promising combination for 3D printing, in terms of shape fidelity and
post-printing stability. The square-grids display accurately nice lines
that illustrates how the viscoelastic properties improve printing
A.I. Cernencu, et al.
Fig. 3. Line thickness of extruded filaments under different printing parameter combinations.
resolution.
After printing, all the 3D scaffolds were crosslinked through im-
mersion in CaCl2 bath. As both nanocellulose and pectin include car-
boxyl groups (−COO–) the two polysaccharides were conjointly
crosslinked in the presence of Ca2+ ions to generate ionic inter-chain
links. After crosslinking, the constructs withstand their shape and can
be easily manipulated, as presented in Supplementary data S1 (Fig. 1S).
3.5. Morphology - MicroCT
MicroCT was used to further assess the morphology of the 3D
printed objects in terms of influence of the ink composition on the
geometrical features of the printed objects such as homogeneity of the
porous scaffolds and shape fidelity. The micro-architecture was eval-
uated from the point of view of the homogeneity, pores size and shape
(Fig. 6). The results are in good agreement with the data from Fig. 5.
It can be noticed that the addition of pectin into the ink formulation
modifies the morphological features of the 3D printed objects. The 3D
printed scaffolds based on pure CNF hydrogels present homogeneous
filaments and circular pores, such features stating for a partial in-
stability of the pure CNF filaments. Adding pectin modifies the mor-
phology of the bicomponent 3D printed porous scaffolds. The printed
objects using CNFP11 and CNFP31 show non-homogeneous pores, with
circular shape and partly closed or filled with a continuous film formed
Fig. 4. Representative photographs for filaments of each composition printed at constant pressure in the range of feed rate from 5 to 40 mm/s.
17
Carbohydrate Polymers 220 (2019) 12–21
due to the lack of stability of the strands. When printing with CNFP13,
the post-printing stability of the ink is significantly improved, the fab-
ricated porous object is homogeneous, it presents square-like pores
homogeneous in both shape and size. The addition of pectin in a ratio of
3:1 with respect to nanocellulose, in CNFP13 in., is sufficient to keep
the 3D printed filaments from collapsing during printing, post printing
and after cross-linking with calcium ions. Adding pectin modifies the
pores morphology, the most homogeneous and post-printing stable
scaffold being obtained with CNFP13 in.
The investigation of the morphology and shape stability as depicted
in this work allows a correlation between the composite ink formula-
tion and the architecture of the 3D printed hydrogel scaffolds. It also
confirms the potential of pectin to increase the post-printing stability of
nanocellulose-based composite inks. Using only CNF with the same
solid content, the stability could have been good too. However, it is
difficult to produce TEMPO oxidized CNF with such high solid content.
3.6. Rehydration of the 3D objects in PBS
The 3D printed structures were completely dried and soaked in PBS
buffer (pH 7.4, 37 °C) that simulates the physiological environment to
assess both their shape stability and the swelling behavior. The shape of
the objects was macroscopically monitored, while the PBS uptake was
evaluated after 24 h of incubation as the maximum swelling degree (%
A.I. Cernencu, et al.
Fig. 5. Top-down view of 3D printed objects with CNF (a); CNFP31 (b); CNFP11 (c); CNFP13 (d) ink formulations, before crosslinking.
MSD) in Fig. 7a and the PBS equilibrium content (%ECPBS) in Fig. 7b
(described in Materials and Methods section).
All the 3D printed hydrogel scaffolds show the ability to rehydrate
after dehydration. At the end of the incubation in PBS, all the samples
maintained their stability, no fragmentation was noticed while the
porosity is preserved, proving that the obtained polysaccharide-based
constructs are stable networks and the cross-linking process was ef-
fective.
CNF control sample exhibited the lowest PBS rehydration capacity
(MSD value of about 720% - Fig. 7a), as cellulosic chains naturally
stiffen upon drying (Diniz, Gil, & Castro, 2004; Torstensen et al., 2018).
Pectin addition to CNF gels was expected to generate bicomponent
systems with enhanced hydrophilic character considering the re-
cognized affinity for water of pectin (Boyer, 2016). While the smallest
pectin amount (CNFP31) does not significantly enhance the rehydration
in PBS (MSD value of about 785%), increasing its content up to weight
equivalence (CNFP11) leads to a nearly double MSD value (about
1370%). Further addition of pectin in CNFP13 leads to a stronger in-
crease of the PBS rehydration ability, the MSD becomes three times
higher (about 1860%) than the reference.
The rehydration potential of the 3D printed samples is illustrated in
Fig. 7b as ECPBS relative to the initial water content (IWC) of the inks.
Comparing the mass gain during rehydration it was observed that the
chemical composition of the material has a strong impact over the
mechanism of aqueous media imbibing. The addition of pectin sig-
nificantly improves the PBS-uptake of the bicomponent systems. It is
18
Carbohydrate Polymers 220 (2019) 12–21
important to notice that the PBS content reaches the initial water
content of the ink only when the highest amount of pectin is used,
CNFP13 exhibiting the best rehydration properties. Such behavior was
expected considering the total solid content (T%) and the interactions
between the two polymers.
3.7. Rheological characterization of the hydrogels
The influence of the pectin component on the rheological properties
of the bicomponent hydrogels was determined. The hydrogels obtained
using a cross-linking protocol similar to that used for the 3D printed
samples, were subjected to oscillatory shear stress applied by a rhe-
ometer. The log-log trend of G’ and G” vs. frequency for these poly-
saccharide hydrogels is plotted in Fig. 8.
All the studied hydrogel formulations revealed a higher storage
modulus (G′) compared to the viscous one (G′′), indicating the gel-like
nature of the materials. In addition, it can be observed that within the
studied oscillation range (0.1–10 Hz), the samples show high stability,
no breakage of cross-link bonds appears, since G’ values are nearly
independent of frequency. Considering that the mechanical stability of
the CNF-based gels is not influenced by the external forces, their dis-
tinct rheological behavior could be exclusively attributed to the nano-
cellulose/pectin ratio. The differences in the dynamic moduli are not
surprising considering that the addition of pectin will increase the
number of rheologically-effective network crosslinks. As shown in
Fig. 8, the reference hydrogel exhibited the lowest elastic modulus (G’)
A.I. Cernencu, et al.
Fig. 6. Morphology of the porous scaffolds as revealed through microCT images in front view of CNF (a), CNFP31 (b), CNFP11 (c), CNFP13 (d) printed scaffolds
(scale bar 500 μm).
Fig. 7. Effect of pectin content on the rehydration in PBS of the printed polysaccharide-based scaffolds a – as MSD after immersion in PBS for 24 h, b – as ECPBS after
immersion for 24 h, relative to the IWC of inks. The error bars depict the standard deviations.
Fig. 8. Elastic and viscous moduli (G′, G′′) versus frequency for polysaccharide-
based hydrogels.
at about 13 kPa as a consequence of a low number of carboxylic groups’
content, the CNF specimens proving a high elasticity. Meanwhile, for
CNF/pectin formulations the value of G’ was about 20 kPa for CNFP31
and increased three times for CNFP11 and CNFP13 when compared to
19
Carbohydrate Polymers 220 (2019) 12–21
monocomponent CNF control, proving a less elastic behavior of the
resulting bicomponent hydrogels. Furthermore, within the latter for-
mulations, the effect of pectin amount was apparent since G’ profiles
show a similar rheological behavior. Specifically, they have comparable
G’ values because stronger hybrid hydrogels are obtained with the ad-
dition of pectin since it provides additional sites for crosslinking. Hence,
the increase of pectin percentage leads to an upward trend in stiffness
as the rheological response is directly correlated with the material
structure. As an outcome the moduli of the studied hydrogels closely
match the stiffness of various types of tissue, including liver (1–20 kPa),
breast (4–60 kPa), or arterial wall tissues (10 kPa-10 MPa) (Nemir &
West, 2010).
The interest for the development of 3D printable bioinks based on
CNFs increased in the last five years due to their suitability to support
cell viability and proliferation (Sultan, Siqueira, Zimmermann, &
Mathew, 2017). Most of the reported formulations contain alginate as a
key component involved in the stability of the 3D printed constructs
due to its fast-ionic crosslinking (Ávila et al., 2016; Heggset et al., 2019;
Müller et al., 2017; Ojansivu et al., 2019; Rees et al., 2015).
Pectin, a plant-derived polysaccharide with applicability in tissue
engineering (Chen, Ni et al., 2017; Ninan et al., 2013) and typically
used as excipient for pharmaceutical dosage forms (Minzanova et al.,
A.I. Cernencu, et al.
2018), has been recently investigated for 3D food printing
(Vancauwenberghe, Katalagarianakis et al., 2017, Vancauwenberghe,
Mbong et al., 2017). Considering its native structural function in ve-
getal tissues, conferring specific mechanical properties and high-water
affinity, this polymer has been selected for combination with CNFs to
obtain new bio-inspired inks with appealing features for 3D bioprinting.
To the best of our knowledge, this is the first study reporting the de-
velopment of CNF - pectin formulations for 3D printing and the as-
sessment of printability and post-printing stability becomes mandatory.
Such CNF/pectin formulations present the potential to be employed in
the obtaining of hydrogels using 3D bioprinting concepts. Since CNFs
resemble the collagen fibrils found in extracellular matrix of several
tissues, the obtained self-supporting CNF/pectin constructs could en-
sure a cell-friendly micro-environment. Considering cell adhesion as a
crucial feature for the successful application in tissue engineering, ef-
ficient strategies to introduce specific bioactive molecules (peptides,
enzymes) onto cellulose substrates could be also employed (Weishaupt
et al., 2015).
4. Conclusions
In this research, novel bio-based ink formulations were designed as
suitable for 3D printing by using pectin and TEMPO-oxidized CNFs.
Their printing properties were considered within a comprehensive
study on the influence of ink properties and printing parameters. The
ease of injection of gels through various syringe needles, revealed a
good correlation with the precursors’ composition and gave a sig-
nificant insight on their printability.
The rheological investigation revealed that the amount of pectin
influences the rheological behavior of the suspension, preserving the
shear-thinning property while increasing the yield stress, though im-
proving the printability of ink formulations. The specific rheological
response of the nanocellulose based-matrices enabled printing, and the
process set-up was established considering the filaments resolution. The
low speed printing might be regarded as a drawback as extruded fila-
ment is thick and the finer details of the design would be lost after
fabrication. In optimized processing conditions for all the formulated
inks, a good printability of the designed 3D pattern was observed.
Nonetheless, CNFP13 can be extruded under low-expansion there-
fore exhibiting the best shape fidelity and post-printing stability, also
remarked after crosslinking by means of MicroCT. The investigation of
the morphology highlights the correlation between the composite ink
formulation and the architecture of the 3D printed hydrogel scaffolds:
pure CNF filaments present a partial instability of the filaments that
generate circular pores while with increasing the amount of pectin and
consequently the T% the printed scaffold present square-like pores.
Moreover, the evaluation of the ability to rehydrate in PBS indicated
a pectin dependent behavior of the cross-linked hydrogels, CNFP13
exhibiting enhanced rehydration properties. Further on the dynamic
rheological measurements of the crosslinked hydrogels reveal that
pectin contributed to the viscoelastic properties of the hydrogel. The
rheological response is directly correlated with the material structure,
thus an increase in pectin concentration resulted in a corresponding
increase in stiffness.
The rheological and printing behavior of the bio-based cellulose-
pectin formulations along with fast gelation properties suggest that
these inks are candidate biomaterials for extrusion based 3D printing.
Author contributions
All authors have given approval to the final version of the manu-
script.
Acknowledgement
The 3D printing experiments and MicroCT analysis were possible
20
Carbohydrate Polymers 220 (2019) 12–21
due to European Regional Development Fund through Competitiveness
Operational Program 2014-2020, Priority axis 1, ID P_36_611, MySMIS
code 107066, INOVABIOMED. A. Lungu would like to thank for the
financial support provided by a grant of the Romanian Ministery of
Research and Innovation, CCCDI – UEFISCDI, project number PN-III-P1-
1.2-PCCDI-2017-0782 /REGMED – project 4 TUMOR, within PNCDI III.
The authors would like to acknowledge the helpful discussions with
Claudiu Patrascu on matters regarding rheology. Parts of this work has
also been funded by the Research Council of Norway through the
NORCEL project (Grant no. 228147).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.carbpol.2019.05.026.
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