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

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.

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 (http://creativecommons.org/licenses/by/4.0/)

Development of phenol-grafted polyglucuronic acid and its application to

extrusion-based bioprinting inks

Shinji Sakaia,*, Takashi Kotania, Ryohei Haradaa, Ryota Gotoa, Takahiro Moritaa,

Soukaina Bouissilb, Pascal Dubessayb, Guillaume Pierreb, Philippe Michaudb,

Redouan El Boutachfaitic, Masaki Nakahataa, Masaru Kojimaa, Emmanuel Petitc,

C´edric Delattreb,d

aDivision of Chemical Engineering, Department of Materials Engineering Science, Graduate School of Engineering Science, Osaka University 1-3 Machikaneyama-Cho,

Toyonaka, Osaka 560-8531, Japan

bUniversit´e Clermont Auvergne, Clermont Auvergne INP, CNRS, Institut Pascal, F-63000 Clermont-Ferrand, France

cUMRT INRAE 1158 BioEcoAgro – BIOPI Biologie des Plantes et Innovation, SFR Condorcet FR CNRS 3417, Universit´e de Picardie Jules Verne, Amiens, France

dInstitut Universitaire de France (IUF), 1 rue Descartes 75005, Paris, France

A R T I C L E I N F O

Keywords:

Polyglucuronic acid

Bioprinting

3D-printing

Horseradish peroxidase

Tissue engineering

A B S T R A C T

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, & CourCour-tois, 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: sakai@cheng.es.osaka-u.ac.jp (S Sakai), t.kotani@cheng.es.osaka-u.ac.jp (T Kotani), harada.vn@cheng.es.osaka-u.ac.jp (R Harada),

gotoryota@cheng.es.osaka-u.ac.jp (R Goto), morita-t@cheng.es.osaka-u.ac.jp (T Morita), soukaina.bouissil@etu.uca.fr (S Bouissil), pascal.dubessay@uca.fr

(P Dubessay), guillaume.pierre@uca.fr (G Pierre), philippe.michaud@uca.fr (P Michaud), redouan.elboutachfaiti@u-picardie.fr (R El Boutachfaiti), nakahata@ cheng.es.osaka-u.ac.jp (M Nakahata), kojima@cheng.es.osaka-u.ac.jp (M Kojima), emmanuel.petit@u-picardie.fr (E Petit), cedric.delattre@uca.fr (C Delattre)

Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

https://doi.org/10.1016/j.carbpol.2021.118820

Received 8 July 2021; Received in revised form 22 October 2021; Accepted 25 October 2021

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 three-

dimensional (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

(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

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

2.2 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

2.4 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

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

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

2.7 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

2.8 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, 3-

mm height) prepared by extruding the ink non-containing cells in the air containing 8 ppm H2O2 was measured as mentioned in 2.6

2.9 Statistical analysis

Comparisons between groups were made using student's t-test Values of p < 0.05 were considered to indicate significance

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, PGU-

Ph 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% PGU-

Ph 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

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)

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

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)

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

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% Alg-

Ph hydrogels (Fig S1)

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

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)

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)

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

affecting printability

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

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

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)

(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

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 + Gelatin-

Ph hydrogel Bars: 100 μm

% 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

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

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

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

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-Ph- based 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

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.)

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

seeded on PGU-Ph-based hydrogels The hydrogel obtained from PGU-

Ph 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

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

C´edric Delattre: Conceptualization, Methodology, Writing -

Orig-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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