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.
Carbohydrate Polymers 277 (2022) 118820 Contents lists available at ScienceDirect 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), 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 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 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: 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) https://doi.org/10.1016/j.carbpol.2021.118820 Received 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 (http://creativecommons.org/licenses/by/4.0/) 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 (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 S Sakai et al 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 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 × 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 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 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 ppm H2O2 obtained by bubbling air in 0.5 M H2O2 aqueous solution flowed into the plastic container at 10 L/min After 15 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 × 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 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 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 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 ppm H2O2, and a stage for layering the extruded ink Inks containing 2.0 w/v% PGU-Ph and 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 × 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 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 S Sakai et al Carbohydrate Polymers 277 (2022) 118820 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− mol-Ph/g of PGU-Ph Fig shows the shear rate-viscosity profiles of and w/v% PGUPh solutions The viscosity of w/v% PGU-Ph solution was larger than that of w/v% PGU-Ph solution For example, at a shear rate of s− 1, the viscosity of w/v% PGU-Ph solution was about 8-times larger than that of w/v% The viscosity of w/v% PGU-Ph solution decreased significantly with increasing shear rate Fig Shear rate-viscosity profiles of and 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 U/mL HRP and 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 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 U/mL HRP and 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 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 U/mL (p = 0.002) The Young's modulus of PGU-Ph hydrogels increased 5-fold when the concentration of H2O2 increased from 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 (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) S Sakai et al Carbohydrate Polymers 277 (2022) 118820 Fig Dependence of gelation time for concentrations of (a) PGU-Ph at U/mL HRP and mM H2O2, (b) HRP at 2.0 w/v% PGU-Ph and mM H2O2, and (c) H2O2 at 2.0 w/v% PGU-Ph and U/mL HRP Data: mean ± standard deviations (n = 4) bioprinting, PGU-Ph solutions containing U/mL HRP were extruded onto a substrate based on a blueprint for printing a hexagonal cell with 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 w/v% AlgPh hydrogels (Fig S1) Fig Dependence of Young's modulus of hydrogels for concentrations of (a) PGU-Ph at U/mL HRP and mM H2O2, (b) HRP at 2.0 w/v% PGU-Ph and mM H2O2, and (c) H2O2 at 2.0 w/v% PGU-Ph and 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) 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 S Sakai et al Carbohydrate Polymers 277 (2022) 118820 Fig Microphotos of 10T1/2 cells incubated for 20 h in mixture of medium (50 vol%) and 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 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 S Sakai et al Carbohydrate Polymers 277 (2022) 118820 Fig Microphotos of 10T1/2 cells at (a, c, e) 1, and (b, d, f) 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 to 10 U/mL (Fig 5b) and increased with increasing H2O2 concentration from 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 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 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 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 S Sakai et al Carbohydrate Polymers 277 (2022) 118820 Fig (a) Blueprint of a hexagonal cell with 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 U/mL % PGU-Ph, 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 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 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 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 S Sakai et al Carbohydrate Polymers 277 (2022) 118820 Fig 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) 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 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 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 U/mL HRP was 3.5 s The superior cytocompatibility was confirmed from the Carbohydrate Polymers 277 (2022) 118820 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 U/ mL by extruding in air containing 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 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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 References Augst, A D., Kong, H J., & Mooney, D J (2006) Alginate hydrogels as biomaterials Macromolecular Bioscience, 6, 623–633 Bayntona, K J., Bewtrab, J K., Biswasb, N., & Taylor, K E (1994) Inactivation of horseradish peroxidase by phenol and hydrogen peroxide: A 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S., Gantumur, E., Carranza, M S., Yoshii, A., Sakai, S., & Delattre, C (2019) Use of anionic polysaccharides in the development of 3D bioprinting technology Applied Sciences, 9(13), 2596 Wilson, S A., Cross, L M., Peak, C W., & Gaharwar, A K (2017) Shear-thinning and thermo-reversible nanoengineered inks for 3D bioprinting ACS Applied Materials & Interfaces, 9(50), 43449–43458 11 ... 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... 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 Materials and methods 2.1... of Young''s modulus of hydrogels for concentrations of (a) PGU-Ph at U/mL HRP and mM H2O2, (b) HRP at 2.0 w/v% PGU-Ph and mM H2O2, and (c) H2O2 at 2.0 w/v% PGU-Ph and U/mL HRP Data: mean ± standard