Development of phenol-grafted polyglucuronic acid and its application to extrusion-based bioprinting inks

Carbohydrate Polymers 277 (2022) 118820

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

Development of phenol-grafted polyglucuronic acid and its application to
extrusion-based bioprinting inks
Shinji Sakai a, *, Takashi Kotani a, Ryohei Harada a, Ryota Goto a, Takahiro Morita a,
Soukaina Bouissil b, Pascal Dubessay b, Guillaume Pierre b, Philippe Michaud b,
Redouan El Boutachfaiti c, Masaki Nakahata a, Masaru Kojima a, Emmanuel Petit c,
C´edric Delattre b, d
a

Division of Chemical Engineering, Department of Materials Engineering Science, Graduate School of Engineering Science, Osaka University 1-3 Machikaneyama-Cho,
Toyonaka, Osaka 560-8531, Japan
b
Universit´e Clermont Auvergne, Clermont Auvergne INP, CNRS, Institut Pascal, F-63000 Clermont-Ferrand, France
c
UMRT INRAE 1158 BioEcoAgro – BIOPI Biologie des Plantes et Innovation, SFR Condorcet FR CNRS 3417, Universit´e de Picardie Jules Verne, Amiens, France
d
Institut Universitaire de France (IUF), 1 rue Descartes 75005, Paris, France

A R T I C L E I N F O

A B S T R A C T

Keywords:
Polyglucuronic acid
Bioprinting

3D-printing
Horseradish peroxidase
Tissue engineering

In this present work, we developed a phenol grafted polyglucuronic acid (PGU) and investigated the usefulness in
tissue engineering field by using this derivative as a bioink component allowing gelation in extrusion-based 3D
bioprinting. The PGU derivative was obtained by conjugating with tyramine, and the aqueous solution of the
derivative was curable through a horseradish peroxidase (HRP)-catalyzed reaction. From 2.0 w/v% solution of
the derivative containing 5 U/mL HRP, hydrogel constructs were successfully obtained with a good shape fidelity
to blueprints. Mouse fibroblasts and human hepatoma cells enclosed in the printed constructs showed about 95%
viability the day after printing and survived for 11 days of study without a remarkable decrease in viability.
These results demonstrate the great potential of the PGU derivative in tissue engineering field especially as an ink
component of extrusion-based 3D bioprinting.

1. Introduction
Polyglucuronic acid (PGU) also called glucuronan is a high molecular
weight homopolymer of glucuronic acid composed of [→4)-β-D-GlcpA(1→] residues partially acetylated at the C-3 and/or the C-2 position
produced by the strain Sinorhizobium meliloti M5N1CS (Heyraud, Cour­
tois, Dantas, Colin-Morel, & Courtois, 1993). First described in cell walls
of Mucor rouxii (Deruiter, Josso, Colquhoun, Voragen, & Rombouts,
1992), these polyuronides have since been isolated from other sources
such as in the cell walls of green algae (Redouan et al., 2009) but the
most described polysaccharide was obtained from the Rhizobia strains.
However, recent progress in the oxidation of primary hydroxyl groups
by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) reagents per­
mits obtaining PGU mimick derivatives from cellulose and xanthan on a

large scale-up and concomitantly new polysaccharide lyase family able
to degrade these PGU have been identified (Delattre et al., 2015;
Elboutachfaiti, Delattre, Petit, & Michaud, 2011).

Different applications of poly- and oligo-glucuronic acids have been
published as scientific articles or patents. Courtois-Sambourg et al.
patent described the biocompatibility of PGU and its use in food prod­
ucts, farming, pharmaceutics, cosmetics, or water purification, partic­
ularly as a gelling, thickening, hydrating, stabilizing, chelating, or
flocculating agent (Courtois-Sambourg, Courtois, Heyraud, Colin-Morel,
& Rinaudo-Duhem, 1993). Another application concerned the immu­
nostimulating effects on human blood monocytes, low molecular weight
PGU enhanced the production of cytokines IL-1, IL-6, and TNF-α
(Courtois-Sambourg & Courtois, 1998). Cosmetic applications of PGU
have been claimed by Lintner in association with an algal

* Corresponding author.
E-mail addresses: (S. Sakai), (T. Kotani), (R. Harada),
(R. Goto), (T. Morita), (S. Bouissil),
(P. Dubessay), (G. Pierre), (P. Michaud), (R. El Boutachfaiti), nakahata@
cheng.es.osaka-u.ac.jp (M. Nakahata), (M. Kojima), (E. Petit), (C. Delattre).
/>Received 8 July 2021; Received in revised form 22 October 2021; Accepted 25 October 2021
Available online 28 October 2021
0144-8617/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license ( />

S. Sakai et al.

Carbohydrate Polymers 277 (2022) 118820

polysaccharides extracted from Haematococcus pluvialis (Lintner, 1999),
or by Fournial et al. for oligo-PGU stimulating of elasticity of the dermis
and epidermis (Fournial, Grizaud, LeMoigne, & Mondon, 2010). Bio­
logical activities of these low molecular weight glucuronans modified by
sulphonation were also investigated on a model of injured extensor

digitorum longus muscles on rats and demonstrated that the regenera­
tion activity is not induced only by the presence of sulfate groups, but
also by acetyl groups (Petit et al., 2004). The renewal process of cells is
regulated by specific signals (or communication peptides such as growth
factors) of the extracellular matrix. These signals are stored, protected,
and positioned on a family of large polysaccharides called heparan
sulfates. In cases of injury, specific enzymes destroy heparan sulfates,
which no longer protect the specific signals. Other enzymes called pro­
teases then destroy specific signals along with other structural proteins
of the extracellular matrix. Due to their resistance to natural enzymes
from the extracellular matrix, the biological effect of these modified
bacterial polysaccharides could be explained (Petit et al., 2004).
Here, we synthesize PGU derivatives possessing phenolic hydroxyl
moieties (PGU-Ph) and demonsrate the potency for use as a component
of hydrogels in tissue engineering applications. Especially, we investi­
gate the potency by using PGU-Ph as a component of bioink for threedimensional (3D) bioprinting. Phenolic hydroxyl (Ph) moieties were
introduced to PGU for a rapid formation of stable hydrogels through
horseradish peroxidase (HRP)-catalyzed cross-linking (Fig. 1a–c). The
gelation mediated by HRP has been revealed as an effective route for
obtaining cell-laden hydrogels from various derivatives of natural and
synthetic polymers such as alginate (Sakai & Kawakami, 2007), hyal­
uronic acid (Kurisawa, Chung, Yang, Gao, & Uyama, 2005), gelatin
(Sakai, Hirose, Taguchi, Ogushi, & Kawakami, 2009), dextran (Jin,
Hiemstra, Zhong, & Feijen, 2007), and poly(vinyl alcohol) (Sakai et al.,
2013). Recently, HRP-mediated gelation was applied to 3D bioprinting
(Sakai et al., 2018; Sakai, Ueda, Gantumur, Taya, & Nakamura, 2018),
in which rapid curation of inks ejected from needles is required for

fabricating 3D constructs with higher fidelity to blueprints. 3D bio­
printing is a technique of fabrication of cell-laden constructs based on

digital blueprints. The resultant cell-laden constructs are fabricated for
the sake of wound dressing and tissue engineering for drug screening
and regenerative medicine (Gungor-Ozkerim, Inci, Zhang, Kha­
demhosseini, & Dokmeci, 2018; Murphy & Atala, 2014). The biological
properties required for the components of inks are different in each
application. Therefore, the development of novel components for inks,
which have unique biological properties, is believed to extend the
application of the bioprinted hydrogel constructs for tissue engineering
(Gungor-Ozkerim et al., 2018). Due to the unique biological features of
PGU described above (Courtois-Sambourg et al., 1993; Courtois-Sam­
bourg & Courtois, 2000; Elboutachfaiti et al., 2011; Petit et al., 2004; Tai
et al., 2019), the PGU-based inks will be an attractive choice for 3D
bioprinting inks. One of the most advantages of PGU as new
polysaccharides-based bioink is its microbial origin. Extracellular
polysaccharides (EPS) including PGU are the most studied microbial
polysaccharides to date and the easiest to be purified because they are
directly excreted in the culture medium without covalent bonding to the
bacterial envelopes (Delattre, Laroche, & Michaud, 2008). They are
found in many species of microorganisms isolated from marine and
terrestrial ecosystems (Delattre, Pierre, Laroche, & Michaud, 2016). In
addition, bacterial polysaccharides are considered to be very advanta­
geous compared to the most common polysaccharides extracted from
natural resources such as alginate, carrageenan, and chitosan, because
fermentation parameters and conditions such as carbon source, tem­
perature, pH, aeration, and agitation can be controlled in terms of
optimizing production. PGU produced in bioreactors is easily extracted
and purified with the eco-friendly process without the use of drastic
conditions such as acidic/basic extraction process in the case of alginate,
carrageenan, or chitosan for example which may in some cases lead to
their partial depolymerization and increase the cost of production

(Elboutachfaiti et al., 2011). More, EPS such as polyglucuronic acid

Fig. 1. (a) Synthetic scheme of PGU-Ph, (b) cross-linking scheme of PGU-Ph through HRP-mediated reaction, and (c) photo of PGU-Ph hydrogel obtained through
HRP-mediated reaction.
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Carbohydrate Polymers 277 (2022) 118820

(PGU) is a bio-polymer whose recovery has many advantages such as the
absence of dependence on political, climatic and ecological hazards that
can sometimes affect the supply, quality and cost of polysaccharides
extracted from algae (in case of alginate, carrageenan…), plants (pec­
tins, starch…) or animals (hyaluronic acid, chitosan) (Delattre et al.,
2008).
In this study, we synthesized for the first time PGU-Ph and investi­
gated the gelation property of its aqueous solution, cytocompatibility,
and cell adhesiveness of the resultant hydrogels. Then, we investigated
the possibility of using PGU-Ph as a component of inks gellable through
HRP-catalyzed cross-linking for 3D bioprinting.

H2O2 solutions were sequentially added to the well with stirring the
PGU-Ph solution using a magnetic stirrer bar (10 mm long) at 60 rpm.
The gelation was signaled when magnetic stirring was hindered and the
surface of the solution swelled.
2.6. Mechanical property measurement
Mechanical properties of hydrogels (disc: 8-mm diameter and 3-mm
height) were determined by measuring the repulsion forces toward

compression (10 mm/min) using a Table-Top Materials Tester (EZ-test,
Shimadzu, Kyoto, Japan). The hydrogels were obtained by pouring 0.15
mL PGU-Ph solution containing HRP and H2O2 into wells of 8-mm in
diameter and 3-mm depth and then stand at 25 ◦ C for 12 h. Young's
moduli were calculated from the data of 1–5% strain.

2. Materials and methods
2.1. Materials
Tyramine hydrochloride and water-soluble carbodiimide (WSCD)
were obtained from Combi-Blocks (San Diego, CA) and Peptide Institute
(Osaka, Japan), respectively. N-Hydroxysuccinimide (NHS), HRP (180
U/mg), and H2O2 aqueous solution (31 w/w%) were purchased from
Fujifilm Wako Pure Chemical Industries (Osaka, Japan). Mouse fibro­
blast 10T1/2 cells and human hepatoma HepG2 cells obtained from the
Riken Cell Bank (Ibaraki, Japan) were grown in Dulbecco's modified
Eagle's medium (DMEM, Nissui, Tokyo, Japan) supplemented with 10 v/
v% fetal bovine serum in a 5% CO2 incubator.

2.7. Cytocompatibility
10T1/2 cells were seeded in the wells of 96-well cell culture plate at
4 × 103 cells/well and incubated in a medium for 20 h in a humidified
5% CO2 incubator at 37 ◦ C. Subsequently, the medium was changed to
the medium (0.2 mL) containing PGU or PGU-Ph at 0.5 w/v% and
incubated for an additional 24 h. Then, the medium containing the
polymers was changed to the medium (0.2 mL) containing 1/20 vol of
the reagent from a colorimetric mitochondrial activity assay kit (Cell
Counting Kit-8, Dojindo, Kumamoto, Japan). After 2 h of incubation, the
absorbance at 450 nm was measured using a spectrophotometer. So­
dium alginate (Alg) and alginate possessing Ph moieties (Alg-Ph) were
used as controls.

Cytocompatibility of PGU-Ph was also evaluated using hydrogels.
The solution containing 1.0 w/v% PGU-Ph, or 1.0 w/v% PGU-Ph + 1.0
w/v% Gelatin-Ph, and 5 U/mL HRP was poured into the wells of 12-well
cell culture dish at 0.5 mL/well. Subsequently, the dish was put in a
plastic container. The air containing 8 ppm H2O2 obtained by bubbling
air in 0.5 M H2O2 aqueous solution flowed into the plastic container at
10 L/min. After 15 min of exposure to the air containing H2O2, the wells
coated with hydrogels were rinsed sequentially with PBS and medium.
10T1/2 cells were suspended in a medium containing 0.3 mg/mL
catalase and poured into each well at 6 × 104 cells/well.

2.2. PGU production and extraction
The Sinorhizobium meliloti M5N1CS mutant strain was grown at 30 ◦ C
in a 20 L bioreactor (SGI) with 15 L of Rhizobium complete medium,
supplemented with sucrose 1 w/v% (RCS medium). The inoculum was a
1.5 L of RCS medium inoculated with S. meliloti M5N1CS and was
incubated for 20 h at 30 ◦ C on a rotary shaker (120 rpm). After 72 h of
incubation, the broth was centrifuged at 33,900 ×g for 40 min at 20 ◦ C.
The supernatant was purified by tangential ultrafiltration on a 100,000
normal-molecular-weight cutoff (NMWCO) polyethersulfone membrane
from Sartorius (Goettingen, Germany) against distilled water. Finally,
the retentate solution was freeze-dried to obtain the PGU.
2.3. PGU-Ph synthesis
PGU was dissolved in 2-(N-morpholino)ethanesulfonic acid (MES)
buffered solution (pH 6.0) at 1 w/v%. Tyramine hydrochloride, NHS,
and WSCD were sequentially added at 45 mM, 10 mM, and 20 mM,
respectively, and stirred for 20 h at room temperature. The resultant
polymer was precipitated in an excess amount of acetone and then
washed with 90% ethanol +10% water until the absorbance at 275 nm
attributed to the existence of free tyramine became undetectable in the

supernatant. Successful synthesis was evaluated by measuring 1H NMR
and UV–Vis spectra using an NMR spectrometer (JNM-ECS400, JEOL,
Tokyo, Japan) and a UV–Vis spectrometer (UV-2600, Shimadzu, Kyoto,
Japan), respectively.

2.8. 3D bioprinting
An extrusion-based 3D printing system developed by modifying a
commercial 3D bioprinting system (Bio X, Cellink, Gothenburg, Sweden)
was used for 3D bioprinting. The system consisted of a syringe pump for
flowing ink, a 27-gauge needle (0.2 mm inner diameter, 0.4 mm outer
diameter) for extruding the ink, a bubbling system for supplying air
containing 8 ppm H2O2, and a stage for layering the extruded ink. Inks
containing 2.0 w/v% PGU-Ph and 5 U/mL HRP were used. The effect of
the extrusion with the inks on cells was evaluated by measuring the
viabilities of 10T1/2 and HepG2 cells suspended in the inks at 3 × 105
cells/mL. The inks containing cells were collected at the tip of the needle
and the cells were stained with trypan blue dye for the measurement
using a hemocytometer. The viabilities of the cells enclosed in the
hydrogels obtained through the printing process were determined by
staining the cells with fluorescent dyes, Calcein-AM, and propidium
iodide (PI). Mechanical property of hydrogel discs (8-mm diameter, 3mm height) prepared by extruding the ink non-containing cells in the
air containing 8 ppm H2O2 was measured as mentioned in 2.6.

2.4. Shear rate-viscosity profile
Shear rate-viscosity profiles of solutions were measured using a
rheometer (HAAKE MARS III, Thermo Fisher Scientific, Waltham, MA)
equipped with a parallel plate of a 20-mm radius with a 0.5-mm gap at
20 ◦ C.
2.5. Gelation time

2.9. Statistical analysis

The gelation time was measured for phosphate-buffered saline (PBS,
pH 7.4) containing PGU-Ph at 20 ◦ C based on our previously reported
method (Sakai & Kawakami, 2007). The PGU-Ph solution was poured
into a 48-well plate at 0.2 mL/well. Then, 0.01 mL HRP and 0.01 mL

Comparisons between groups were made using student's t-test.
Values of p < 0.05 were considered to indicate significance.
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3. Results
3.1. PGU-Ph synthesis and PGU-Ph solution property
Synthesis of PGU-Ph was confirmed by 1H NMR and UV–Vis spectra
analysis. From the 1H NMR spectra of PGU and PGU-Ph, the peaks
attributed to the existence of phenol groups (6.7–7.2 ppm) were found
only for PGU-Ph (Fig. 2a). In addition, compared to PGU solution, PGUPh solution showed a UV absorbance peak around 275 nm corre­
sponding to the absorbance of phenol group (Fig. 2b). These results
demonstrate the successful synthesis of PGU-Ph. The content of Ph
groups in PGU-Ph calculated from the standard curve obtained from a
known percentage of tyramine solution was 3.7 × 10− 4 mol-Ph/g of
PGU-Ph.
Fig. 3 shows the shear rate-viscosity profiles of 1 and 2 w/v% PGUPh solutions. The viscosity of 2 w/v% PGU-Ph solution was larger than
that of 1 w/v% PGU-Ph solution. For example, at a shear rate of 1 s− 1,
the viscosity of 2 w/v% PGU-Ph solution was about 8-times larger than

that of 1 w/v%. The viscosity of 2 w/v% PGU-Ph solution decreased
significantly with increasing shear rate.

Fig. 3. Shear rate-viscosity profiles of 1 and 2 w/v% PGU-Ph solution at 20 ◦ C.

3.2. Gelation of PGU-Ph solutions
PGU-Ph solutions were gellable through HRP-catalyzed reaction in
the presence of H2O2 (Fig. 1c). Fig. 4a shows the effect of PGU-Ph
concentration on gelation time at 5 U/mL HRP and 1 mM H2O2. The
gelation time decreased with increasing PGU-Ph concentration: The
values at 0.5, 1.0, and 2.0 w/v% were 9.8, 7.3, and 3.5 s, respectively.
Fig. 4b and c show the effects of HRP and H2O2 concentrations on
gelation time measured for 2.0 w/v% PGU-Ph solutions. Gelation time
decreased as HRP concentration increased from 76 s at 0.1 U/mL to 2.5 s
at 10 U/mL (Fig. 4b). Gelation time increased as H2O2 concentration
increased from 1 mM to 50 mM from 3.5 to 25 s (Fig. 4c).
3.3. Mechanical properties of PGU-Ph hydrogels
As shown in Fig. 5a, the stiffness of hydrogels increased with
increasing PGU-Ph concentration at 5 U/mL HRP and 1 mM H2O2. The
Young's modulus at 2.0 w/v% (2.0 kPa) was twice larger than that at 0.5
w/v% (1.0 kPa, p = 0.003). The concentrations of HRP and H2O2 also
affected the stiffness of PGU-Ph hydrogels. The Young's modulus
increased with increasing HRP concentration from 0.1 (1.2 kPa) to 5 U/
mL (2.0 kPa, p = 0.006, Fig. 5b). When the HRP concentration further
increased to 10 U/mL, the value decreased to about 40% (0.85 kPa) of
that at 5 U/mL (p = 0.002). The Young's modulus of PGU-Ph hydrogels
increased 5-fold when the concentration of H2O2 increased from 1 to 10
mM (Fig. 5c). However, the value decreased when H2O2 concentration
was further increased to 30, and 50 mM.
3.4. Cytocompatibility of PGU-Ph

For evaluating the cytocompatibility of PGU-Ph, 10T1/2 cells were
incubated in a solution containing the polymer. The solutions containing
PGU, Alg, or Alg-Ph were used as controls. Fig. 6a and b show the
morphologies of cells at 20 h of culture in the mixture solutions of me­
dium (50 vol%) and PBS (50 vol%) containing PGU or PGU-Ph at 0.5 w/
v%. There were no remarkable differences in cell morphology specific to
the exposure to PGU-Ph. In addition, there was no significant decrease in
the mitochondrial activity of cells incubated in the mixture solutions
caused by Ph moieties introduced in PGU (p = 0.45), as the same with
the cells incubated in the mixture solutions containing 0.5 w/v% Alg and
Alg-Ph (p = 0.28, Fig. 6c). The mitochondrial activities of the cells

Fig. 2. (a) 1H NMR spectra of PGU and PGU-Ph in D2O. (b) UV–Vis spectra of
0.1 w/w% PGU and PGU-Ph in PBS (pH 7.4).
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Fig. 4. Dependence of gelation time for concentrations of (a) PGU-Ph at 5 U/mL HRP and 1 mM H2O2, (b) HRP at 2.0 w/v% PGU-Ph and 1 mM H2O2, and (c) H2O2 at
2.0 w/v% PGU-Ph and 5 U/mL HRP. Data: mean ± standard deviations (n = 4).

bioprinting, PGU-Ph solutions containing 5 U/mL HRP were extruded
onto a substrate based on a blueprint for printing a hexagonal cell with 2
mm height (Fig. 8a). Hydrogel was not obtained when 2.0 w/v% PGU-Ph
ink was extruded in the air free of H2O2 (Fig. 8b). Hydrogel construct
with a better shape fidelity was obtained from 2.0 w/v% PGU-Ph ink
(Fig. 8d) than that obtained from 1.0 w/v% PGU-Ph ink (Fig. 8c) by

extruding these inks in air containing H2O2. By using 2.0 w/v% PGU-Ph
ink, varieties of hexagonal cell constructs were obtained, including the
constructs with 10 mm height (Fig. 8e–j). The Young's modulus of the
hydrogel obtained through the printing process was 1.5 ± 0.2 kPa
(mean ± S.D., n = 6).
The effects of the 3D printing process and embedding in PGU-Ph
hydrogels on cells were evaluated by printing 2.0 w/v% PGU-Ph
hydrogel constructs enclosing 10T1/2 and HepG2 cells. The viabilities
of 10T1/2 and HepG2 cells the day after bioprinting determined through
staining with Calcein-AM and PI were 95% and 94%, respectively. This
result demonstrates the printing process using PGU-Ph solution as the
ink was not harmful to these cells. Regarding the morphologies of the
enclosed cells, 10T1/2 cells kept a round shape during 11 days of study
without the formation of cell aggregates (Fig. 9a, c, e). In contrast,
HepG2 cells formed aggregates in the hydrogel constructs. The size of
the aggregates increased with increasing culture period (Fig. 9b, d, e).
There was no obvious increase in dead cells for both the cells during 11
days of study. The behaviors of 10T1/2 and HepG2 cells in PGU-Ph
hydrogels were almost the same as the cells enclosed in 2 w/v% AlgPh hydrogels (Fig. S1).

Fig. 5. Dependence of Young's modulus of hydrogels for concentrations of (a)
PGU-Ph at 5 U/mL HRP and 1 mM H2O2, (b) HRP at 2.0 w/v% PGU-Ph and 1
mM H2O2, and (c) H2O2 at 2.0 w/v% PGU-Ph and 5 U/mL HRP. Data: mean ±
standard deviations (n = 4).

incubated in the solutions containing PGU and PGU-Ph were about 20%
higher than those incubated in the solutions containing Alg and Alg-Ph
(p < 0.03).

4. Discussion

Here, we present a functionalization of PGU for use as a component
of hydrogels for tissue engineering applications, and to demonstrate this,
we applied this derivative to a bioink for 3D bioprinting. To accomplish
our objective, we conjugated PGU with tyramine for introducing Ph
groups, which enabled us to induce gelation of its aqueous solution
through HRP-catalyzed cross-linking of the Ph groups. Our results
confirmed the good cytocompatibility of PGU-Ph, and low cell adhe­
siveness of the hydrogels obtained from PGU-Ph alone. Furthermore, our
results confirmed the printing PGU-Ph hydrogel constructs with a good
shape fidelity and without giving severe damage to cells under appro­
priate printing conditions. Our motivation for developing PGU-Ph was
that the excellent biocompatibility (Courtois-Sambourg et al., 1993) and
specific biological activity inducing the production of cytokines (Cour­
tois-Sambourg & Courtois, 1998) would be useful in the future for the
biofabrication of functional tissues.

3.5. Cell behavior on PGU-Ph hydrogels
The hydrogels containing PGU-Ph alone and both PGU-Ph and
Gelatin-Ph were used for evaluating the cytocompatibility and cell
adhesiveness of hydrogels containing PGU-Ph. The day after seeding, the
majority of 10T1/2 cells were floating on PGU-Ph hydrogels (Fig. 7c).
During the subsequent incubation period, the cells continued to float on
PGU-Ph hydrogels, and some cells formed small aggregates (Fig. 7d). A
small number of cells adhered to the hydrogels but did not elongate. In
contrast, the 10T1/2 cells seeded on PGU-Ph + Gelatin-Ph hydrogels
adhered, elongated, and proliferated as the same as those on a cell
culture dish (Fig. 7a, b, e, f). No remarkable morphological difference
was found between the 10T1/2 cells on the PGU-Ph + Gelatin-Ph
hydrogels and those on the cell culture dish.
3.6. 3D printing

For evaluating the feasibility of PGU-Ph solution as inks of
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Fig. 6. Microphotos of 10T1/2 cells incubated for 20 h in mixture of medium (50 vol%) and 1 w/v% (a) PGU or (b) PGU-Ph (50 v/v%, i.e., final concentration 0.5 w/
v%) at 37 ◦ C. Bars: 100 μm. (c) Mitochondrial activity of 10T1/2 cells expressed as absorbance of Wst-8 reagent at 450 nm after 2 h of incubation in medium at 37 ◦ C.
Data: mean ± standard deviations (n = 4).

4.1. PGU-Ph synthesis, hydrogel mechanical properties, and the factors
affecting printability

extrusion due to a rise in viscosity. The mechanism of shear-thinning of
polymer solutions is explained by the disentanglement and alignment of
polymer chains, which are randomly oriented at rest, as the shear rate
increases, and the return of the polymer chains to the random orienta­
tion as the shear rate decreases (Schwab et al., 2020). The greater
change in viscosity observed for 2.0 w/v% PGU-Ph solution with
increasing shear rate than 1.0 w/v% PGU-Ph solution (Fig. 3) can be
explained by the increase in the anionic polymer chains.
The shape fidelity of extruded inks is also influenced by the time
necessary for gelation. Therefore, next, we investigated the factors
affecting the gelation time and found that the gelation time of PGU-Ph
solution is controllable by changing the concentrations of PGU-Ph,
HRP, and H2O2 as the same with other solutions of polymer-Phs (Kur­
isawa et al., 2005; Ogushi et al., 2007; Sakai & Kawakami, 2007; Sakai,
Liu, Matsuyama, Kawakami, & Taya, 2012). The decrease in gelation

time with increasing PGU-Ph concentration at fixed concentrations of
HRP and H2O2 (Fig. 4a) can be explained from a stoichiometric view­
point. The decrease in gelation time with increasing HRP (Fig. 4b) is
intuitively understandable because HRP catalyzes the cross-linking re­
action of Ph groups. The increase in gelation time with increasing H2O2
concentration is explained by the inactivation of HRP by H2O2

Firstly, we conjugated tyramine and PGU through a carbodiimide/
active ester-mediated coupling reaction. The successful synthesis of
PGU-Ph confirmed by 1H NMR and UV–Vis measurements (Fig. 2) is
consistent with the preceding literature for the conjugation of tyramine
and acidic polysaccharides such as alginate (Sakai & Kawakami, 2007),
hyaluronic acid (Kurisawa et al., 2005), and carboxymethylcellulose
(Ogushi, Sakai, & Kawakami, 2007).
Then, we studied about shear-rate viscosity profile of PGU-Ph solu­
tions and confirmed that PGU-Ph solution has attractive rheological
properties as bioinks for extrusion-based bioprinting. The trend we
observed for PGU-Ph solution was that viscosities of PGU-Ph solutions
decreased with an increase in shear rate (Fig. 3). Therefore, we found
that PGU-Ph solution is a shear-thinning fluid. Shear-thinning is typi­
cally exhibited by inks often used in extrusion-based bioprinting because
the property greatly influences printability (Schwab et al., 2020; Wilson,
Cross, Peak, & Gaharwar, 2017). The property is related to the ease of
extrusion with a decrease in viscosity during the extrusion phase where
the shear forces increase, and the preservation of the printed shape after
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Carbohydrate Polymers 277 (2022) 118820

Fig. 7. Microphotos of 10T1/2 cells at (a, c, e) 1, and (b, d, f) 4 days of seeding on (a, b) cell culture dish (Dish), (c, d) PGU-Ph hydrogel, and (e, f) PGU-Ph + GelatinPh hydrogel. Bars: 100 μm.

(Bayntona, Bewtrab, Biswasb, & Taylor, 1994). A limitation of the re­
sults for the studies of gelation time (Fig. 4) is that the values obtained
under mixing of PGU-Ph solution in the presence of HRP and H2O2 are
not the same as the time necessary for gelation of the extruded PGU-Ph
inks, where gelation progresses at rest. However, the findings can be
used as an indicator for setting the conditions of printing based on the
correlation with the results for printability.
Regarding the mechanical properties of hydrogels obtained by mix­
ing of PGU-Ph, HRP, and H2O2 in a solution, the increase in Young's
modulus with increasing PGU-Ph concentration (Fig. 5a) can be
explained by the increase in polymer volume fraction in the hydrogels.
The Young's modulus of PGU-Ph hydrogels decreased with increasing
HRP concentration from 5 to 10 U/mL (Fig. 5b) and increased with
increasing H2O2 concentration from 1 to 10 mM (Fig. 5c). These results
indicate that stiffer hydrogels are not necessarily obtained from the
condition giving faster gelation. Similar results that the mechanical
properties are independent of the gelation rate have been reported for

Alg-Ph (Sakai, Hirose, Moriyama, & Kawakami, 2010) and hyaluronic
acid possessing phenolic hydroxyl moieties (HA-Ph) (F. Lee, Chung, &
Kurisawa, 2008). A possible explanation for the decrease in Young's
modulus when HRP concentration increased from 5 to 10 U/mL is the
decrease of a homogeneous microscopic structure of hydrogels due to
faster gelation. The formation of the stiffer hydrogel at 10 mM H2O2
than that at 1 mM H2O2 would be due to the increase in crosslinking
density between Ph moieties due to the abundance in H2O2 as a substrate

of HRP-catalyzed crosslinking. Lee et al. reported that stiffer HA-Ph
hydrogels were obtained with increasing H2O2 concentration from
0.15 to 1.25 mM at 0.062 U/mL HRP but the stiffness decreased with
further increase in H2O2 concentration (Lee et al., 2008). They explained
that the decrease in the stiffness of the HA-Ph hydrogel was caused by
the increase in the effect of HRP inactivation by H2O2. The Young's
modulus of the hydrogel obtained from the ink containing 2.0 w/v%
PGU-Ph and 5 U/mL HRP through the printing process in air containing
H2O2 (1.5 ± 0.2 kPa) was smaller than those obtained by mixing 2.0 w/v
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Carbohydrate Polymers 277 (2022) 118820

Fig. 8. (a) Blueprint of a hexagonal cell with 2 mm height, and photos of (c) 1.0 w/v% and (b, d) 2.0 w/v% PGU-Ph inks extruded onto substrates based on the
blueprint in air (b) non-containing and (c, d) containing H2O2. Photos of printed 2.0 w/v% PGU-Ph constructs with (e) triple hexagonal cells and (f) picked double
hexagonal cells put on skin. (g) Blueprint of a hexagonal cell with 10 mm height, and (h, i) photos of 2.0 w/v% PGU-Ph hydrogel constructs taken from different
viewpoints and (j) the hydrogel construct threaded with glass tube. The content of HRP in inks was 5 U/mL.

% PGU-Ph, 5 U/mL HRP, and H2O2 in solution (Fig. 5c). In the printing
process, H2O2 is supplied from air to the non-stirred solution containing
PGU-Ph and HRP. Therefore, it is difficult to predict the mechanical
properties of the printed hydrogels from the data of the hydrogels ob­
tained by mixing all the components in a solution, at least at present.
However, the prediction may become possible with the accumulation of
data in future. The findings obtained for the hydrogels prepared by
mixing all the components in a solution would be useful for applications
of PGU-Ph hydrogels other than 3D bioprinting.

2.0 w/v% PGU-Ph ink containing HRP extruded in air free of H2O2
(Fig. 8b) indicates the necessity of HRP-catalyzed cross-linking of Ph
groups for the bioprinting. This result is consistent with the results re­
ported for inks containing polymer-Phs (Sakai, Mochizuki, et al., 2018;
Sakai, Yoshii, Sakurai, Horii, & Nagasuna, 2020). The better shape fi­
delity of the printed hexagonal cell with 2 mm height obtained from 2.0
w/v% PGU-Ph ink than that obtained from 1.0 w/v% ink is attributed to
the higher viscosity and shorter gelation time at higher PGU-Ph con­
centrations (Figs. 3, 4a). The successful printing of the construct with 10
mm height also demonstrates the feasibility of 2.0 w/v% PGU-Ph solu­
tion containing 5 U/mL HRP for 3D bioprinting (Fig. 8g–j). As described
above, it is known that the shape fidelity of extruded inks is governed by
the time necessary for gelation and shear-thinning properties (Schwab
et al., 2020; Wilson et al., 2017). Therefore, it may be possible to
fabricate the hydrogel constructs with good shape fidelity even from 1.0
w/v% PGU-Ph solution by altering the concentrations of HRP and H2O2
for shortening the gelation time. In addition, increasing the content of
Ph groups in PGU-Ph would also be effective. It has been reported that
the gelation time of polymer-Phs decreased with increasing the content
of Ph groups (Sakai et al., 2012; Sakai & Kawakami, 2007). Even the
hydrogels obtained from 2.0 w/v% PGU-Ph ink containing HRP
(Fig. 8d–f) had slightly rounded corners, even though the width of the
side (average of 6 sides: 1.95 mm) was almost the same as that of the
blueprint (2.0 mm). The printing conditions giving shorter gelation time
would sharpen the corners. In addition, we used a 27-gauge needle (0.2
mm inner diameter, 0.4 mm outer diameter) to extrude the ink. The use
of finer needles would also improve the printing resolution. We aimed to
demonstrate the feasibility of PGU-Ph solution as inks for 3D bio­
printing, thus, the optimization of conditions for each PGU-Ph solution

is out of the scope of this study. The important finding of this study is
that it is possible to fabricate PGU-Ph hydrogel constructs with good
shape fidelity by setting the appropriate conditions.
We also confirmed the effectiveness of PGU-Ph solution as an ink for
bioprinting based on the >90% viabilities of 10T1/2 and HepG2 cells on
the day after printing and the behaviors of these cells during the sub­
sequent culture period (Fig. 9). The round shape of individual 10T1/2
cells without increasing each size indicates that PGU-Ph hydrogel is
unsuitable for their growth due to the poor cell adhesiveness as indi­
cated from the result of the cells seeded on PGU-Ph hydrogel (Fig. 7c).
On the other hand, the growth of HepG2 cells, confirmed by the increase
in the size of cell aggregates (Fig. 9b, d, f), indicates that PGU-Ph
hydrogels are not necessarily unsuitable for cell growth. These results

4.2. Cytocompatibility of PGU-Ph
Before the studies of bioprinting, we investigated the cytocompati­
bility of PGU-Ph by contacting cells with PGU-Ph as a solute in solution
or as a hydrogel. The no significant differences in shape and mito­
chondrial activity (p = 0.45) of the mouse fibroblast 10T1/2 cells after
20 h of incubation in a medium containing PGU-Ph with those in a
medium containing PGU (Fig. 6) indicate that the introduction of Ph to
PGU does not induce adverse effects on cells. In addition, the mito­
chondrial activity not lower than that obtained for the cells incubated in
media containing Alg with excellent cytocompatibility (Augst, Kong, &
Mooney, 2006; Lee & Mooney, 2012) also supports the finding. The
exact reason is unclear, but the higher mitochondrial activity of cells
incubated in the medium containing PGU-Ph than those in media con­
taining Alg and Alg-Ph may attribute to the intrinsic biological activity
of PGU. It was reported that the metabolic activity of human fibroblasts
was highly increased by the stimulation with PGU (Delattre, Michaud,

Chaisemartin, Berthon, & Rios, 2012). We also confirmed the good
cytocompatibility of PGU-Ph from the no different morphology and
growth of 10T1/2 cells seeded on PGU-Ph + Gelatin-Ph hydrogels with
those on the cell culture dish (Fig. 7a, b, e, f). This result suggests that
the floating of the majority of 10T1/2 cells on PGU-Ph hydrogel (Fig. 7c,
d) was not due to the cytotoxicity of PGU-Ph hydrogel but the poor cell
adhesiveness of the hydrogel. PGU is a hydrophilic polymer and does not
contain a cell-adhesive ligand such as the arginine–glycine–aspartic acid
sequence.
4.3. Bioprinting
In the final stage of our investigation, we evaluated the printability of
PGU-Ph solution and the behaviors of 10T1/2 and HepG2 cells
embedded in the printed hydrogels. The no formation of hydrogels from
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Carbohydrate Polymers 277 (2022) 118820

Fig. 9. Merged microphotos of (a, c, e) 10T1/2 cells and (b, d, f) HepG2 cells enclosed in 2.0 w/v% PGU-Ph hydrogels through bioprinting at (a, b) 1, (c, d) 4 and (e,
f) 11 days of printing. The cells were stained using Calcein-AM (green) and PI (red). Bars: 200 μm. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)

are consistent with the behaviors of murine fibroblasts (Hunt, Smith,
Gbureck, Shelton, & Grover, 2010) and HepG2 cells (Coward et al.,
2009) encapsulated in alginate hydrogels known as poor cell adhesive­
ness. We also confirmed that the results were not specific to PGU-Ph
hydrogels from the similar behavior of 10T1/2 and HepG2 cells
enclosed in Alg-Ph hydrogels (Fig. S1). For cell-laden hydrogels, the

properties that promote cell adhesion and elongation are often desired.
One approach to provide good cell adhesiveness is the use of PGU-Ph
with Gelatin-Ph as indicated by the adhesion and elongation observed
for 10T1/2 cells on PGU-Ph + Gelatin-Ph hydrogel (Fig. 7e, f). Another
approach is the incorporation of cell adhesion ligands to PGU-Ph as the
same methodology which has been applied for promoting cell attach­
ment to the native polysaccharides which do not promote significant
adhesion (Lei, Gojgini, Lam, & Segura, 2011; Rowley, Madlambayan, &
Mooney, 1999). The use of PGU-Ph with other polymer-Ph and the

modification of PGU-Ph should change the gelation profiles and the
viscoelastic properties of solutions. These points are needed to be
investigated in the applications in which cell adhesiveness of PGU-Phbased hydrogels is required.
5. Conclusion
In this study, we investigated for the first time the modification of
PGU for use as a component of hydrogels for tissue engineering appli­
cations, and also investigated as an ink component allowing gelation in
3D bioprinting. The aqueous solution of PGU-Ph obtained by incorpo­
rating Ph groups to PGU was efficiently gellable through HRP-mediated
cross-linking of Ph groups in the presence of H2O2. The shortest time
necessary for gelation of 2.0 w/v% PGU-Ph solution containing 5 U/mL
HRP was 3.5 s. The superior cytocompatibility was confirmed from the
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S. Sakai et al.

behaviors of 10T1/2 cells exposed to the medium dissolving PGU-Ph and

seeded on PGU-Ph-based hydrogels. The hydrogel obtained from PGUPh alone showed low cell adhesiveness. The 3D printed PGU-Ph
hydrogel constructs using 2.0 w/v% PGU-Ph solution containing 5 U/
mL by extruding in air containing 8 ppm H2O2 had a good shape fidelity
to blueprints. The viabilities of 10T1/2 and HepG2 cells enclosed in the
constructs through bioprinting showed about 95%. In addition, the cells
survived for 11 days of study without a remarkable increase in dead
cells. The HepG2 cells grew in the printed hydrogel. From these results,
we conclude that PGU-Ph is a promising material in tissue engineering
applications, especially as a component of inks for extrusion-based
bioprinting.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.carbpol.2021.118820.

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Data availability
All experimental data within the article and its Supplementary in­
formation are available from the corresponding author upon reasonable
request.
CRediT authorship contribution statement
Shinji Sakai: Conceptualization, Methodology, Writing - Original
Draft, Writing - Review & Editing, Visualization, Supervision.
Takashi Kotani: Validation, Development or design of methodol­
ogy; creation of models, Investigation, Writing - Review & Editing.
Ryohei Harada: Development or design of methodology; creation of
models, Investigation, Writing - Review & Editing.
Ryota Goto: Development or design of methodology; creation of
models, Investigation, Writing - Review & Editing.
Takahiro Morita: Validation, Development or design of methodol­
ogy; creation of models, Investigation, Writing - Review & Editing.
Soukaina Bouissil: Methodology, investigation.
Pascal Dubessay: Writing - Review & Editing.
Guillaume Pierre: Writing - Review & Editing.
Philippe Michaud: Writing - Review & Editing.
Redouan El Boutachfaiti: Methodology, Writing - Original Draft,
Writing - Review & Editing.
Masaki Nakahata: Writing - Review & Editing.
Masaru Kojima: Writing - Review & Editing.

Emmanuel Petit: Methodology, Writing - Original Draft, Writing Review & Editing.
´dric Delattre: Conceptualization, Methodology, Writing - Orig­
Ce
inal Draft, Writing - Review & Editing, Visualization, Supervision.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This work was supported by the PHC SAKURA 2019 program; JSPS
Bilateral Joint Research Projects, Grant number 43019NM; and JSPS
Fostering Joint International Research (B), Grant number 20KK0112.
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