Lénaïc Soullard

Pierre-Alain Bayle

Christine Lancelon-Pin

Sébastien Rolere

Isabelle Texier

Bruno Jean

Guillaume Nonglaton
email: guillaume.nonglaton@cea.fr

Optimization of the methacrylation of carboxymethylcellulose and use for the design of hydrogels and cryogels with controlled structure and properties

Keywords: Carboxymethylcellulose, methacrylation, photo-crosslinking, hydrogel, cryogel

Carboxymethylcellulose (CMC) was functionalized using methacrylic anhydride (MA) to produce photo-crosslinkable methacrylated carboxymethylcellulose (mCMC). Optimization of the synthesis led to a wide range of mCMC samples with well-controlled degrees of methacrylation (DM) up to 76 ± 6 %, surpassing the literature data. Aqueous mCMC formulations with different DM and concentrations of mCMC were UV-cured to obtain hydrogels, which were transformed into cryogels after freeze-drying. An innovative 13 C CP-MAS solid-state NMR methodology was used to calculate the crosslinked methacrylate density in cryogels and thus in hydrogels. These values (from 4.3×10 -3 to 1.3×10 -2 mmol/cm 3 ) were correlated with both the DM and the mCMC concentration in formulations. These parameters were used to control the material microstructure and rheological properties. As a result, the swelling ratio of hydrated cryogels could be modulated for applications requiring high or low water absorption. Moreover, it was shown that the elastic moduli G' of the hydrated cryogels were superior to those of the original hydrogels, over the entire frequency range (0.1-10 Hz).

Introduction

Polysaccharides such as hyaluronic acid, dextran, or cellulose, have been used for many years in biomedical applications due to their interesting features such as biocompatibility, biodegradability or biological properties (mucoadhesion, antimicrobial properties, etc.) [START_REF] Rebelo | Biopolymers in Medical Implants: A Brief Review[END_REF]. These raw materials can eventually be chemically modified to improve their mechanical properties, to confer biological properties (specific cell targeting, biodegradability, etc.) [START_REF] Luo | Modifications of Polysaccharide-Based Biomaterials under Structure-Property Relationship for Biomedical Applications[END_REF] or defined shapes and porosities [START_REF] Muir | Chemically Modified Biopolymers for the Formation of Biomedical Hydrogels[END_REF]. For example, the methacrylation of these natural polymers can be used to form hydrogels by photo-crosslinking. The resulting materials are threedimensional polymer networks with entrapped water, with potential applications in bone reconstruction [START_REF] Poldervaart | 3D Bioprinting of Methacrylated Hyaluronic Acid (MeHA) Hydrogel with Intrinsic Osteogenicity ». Édité par Masaya Yamamoto[END_REF], tissue engineering [START_REF] Chen | Functional Human Vascular Network Generated in Photocrosslinkable Gelatin Methacrylate Hydrogels[END_REF], wound healing (Baier [START_REF] Leach | Photocrosslinked Hyaluronic Acid Hydrogels: Natural, Biodegradable Tissue Engineering Scaffolds[END_REF], drug delivery [START_REF] Matricardi | In Situ Cross-Linkable Novel Alginate-Dextran Methacrylate IPN Hydrogels for Biomedical Applications: Mechanical Characterization and Drug Delivery Properties[END_REF] or 3D cell culture [START_REF] García-Lizarribar | Composite Biomaterials as Long-Lasting Scaffolds for 3D Bioprinting of Highly Aligned Muscle Tissue[END_REF]. It is also possible to remove the entrapped water by freezedrying to obtain cryogels [START_REF] Darpentigny | Ice-Templated Freeze-Dried Cryogels from Tunicate Cellulose Nanocrystals with High Specific Surface Area and Anisotropic Morphological and Mechanical Properties[END_REF][START_REF] Hossen | Wet Stable and Mechanically Robust Cellulose Nanofibrils (CNF) Based Hydrogel[END_REF], which are more stable and robust than hydrogels [START_REF] Memic | Effect of Polymer Concentration on Autoclaved Cryogel Properties[END_REF]). These spongy materials can absorb a large amount of solvent thanks to their high porosity [START_REF] Pacelli | Dextran-Polyethylene Glycol Cryogels as Spongy Scaffolds for Drug Delivery[END_REF], which is interesting for the transport of nutrients or cells through scaffolds [START_REF] Razavi | Three-dimensional Cryogels for Biomedical Applications[END_REF]. Their robustness is also useful for bone tissue regeneration [START_REF] Gu | Comparative Study of Gelatin Cryogels Reinforced with Hydroxyapatites with Different Morphologies and Interfacial Bonding[END_REF].

Although dextran and hyaluronic acid are the most studied polysaccharides for such applications [START_REF] Dovedytis | Hyaluronic Acid and Its Biomedical Applications: A Review[END_REF], carboxymethylcellulose (CMC) is also of great interest for the elaboration of hydrogels by photo-crosslinking [START_REF] Reeves | Synthesis and Characterization of Carboxymethylcellulose-Methacrylate Hydrogel Cell Scaffolds[END_REF]. This cellulose derivative is biocompatible, water-soluble, and displays carboxylate and hydroxyl groups amenable to chemical functionalization [START_REF] Rahman | Recent Developments of Carboxymethyl Cellulose[END_REF]. One of the most studied reaction for the grafting of photo-crosslinkable methacrylate groups onto CMC consists of the condensation of the hydroxyl groups with methacrylic anhydride (MA) to yield methacrylated carboxymethylcellulose (mCMC) [START_REF] García-Lizarribar | Composite Biomaterials as Long-Lasting Scaffolds for 3D Bioprinting of Highly Aligned Muscle Tissue[END_REF][START_REF] Hossen | Wet Stable and Mechanically Robust Cellulose Nanofibrils (CNF) Based Hydrogel[END_REF][START_REF] Dutta | Temperature, PH and Redox Responsive Cellulose Based Hydrogels for Protein Delivery[END_REF]. After photo-crosslinking, CMC hydrogels have been considered for biomedical applications such as cell encapsulation [START_REF] Reza | Characterization of Novel Photocrosslinked Carboxymethylcellulose Hydrogels for Encapsulation of Nucleus Pulposus Cells[END_REF], tissue engineering [START_REF] Lin | Lower Crosslinking Density Enhances Functional Nucleus Pulposus-like Matrix Elaboration by Human Mesenchymal Stem Cells in Carboxymethylcellulose Hydrogels: Crosslinking Density Impacts HMSC Chondrogenesis[END_REF] or drug delivery [START_REF] Dutta | Temperature, PH and Redox Responsive Cellulose Based Hydrogels for Protein Delivery[END_REF]. mCMC cryogels have also been studied for their water absorption capacity [START_REF] Hossen | Wet Stable and Mechanically Robust Cellulose Nanofibrils (CNF) Based Hydrogel[END_REF].

In order to benefit from tunable structures and properties (e.g. mechanical) of the CMC hydrogels and cryogels, which will allow targeting one or the other biomedical application, it is firstly of crucial importance to be able to control the degree of methacrylation (DM) of the mCMC, as already highlighted for other polymers such as methacrylated dextran (van [START_REF] Dijk-Wolthuis | Degradation and Release Behavior of Dextran-Based Hydrogels[END_REF]) and gelatin [START_REF] Chen | Functional Human Vascular Network Generated in Photocrosslinkable Gelatin Methacrylate Hydrogels[END_REF]. However, in all previously reported studies, the DM of the mCMC never exceeded 60 % [START_REF] Melilli | DLP 3D Printing Meets Lignocellulosic Biopolymers: Carboxymethyl Cellulose Inks for 3D Biocompatible Hydrogels[END_REF], which limits the possible uses of such products. The influence of parameters such as the extent of methacrylate conversion and polymer concentration, which can also play a major role in the structural and mechanical properties of hydrogels and cryogels based on mCMC, have also been poorly investigated yet.

Herein, we report successful efforts to control the degree of methacrylation of mCMC samples and extend its range to values never achieved before. Beyond this optimization of the DM, we also investigated for the first time to the best of our knowledge the influence of the DM and concentration on the structural and physical properties of photocrosslinked mCMC materials, either in the form of hydrogels or cryogels, and studied their swelling behavior. Useful information were also obtained from the evaluation by solidstate NMR of the conversion ratio in photo-crosslinked mCMC samples (cryogels).

Materials and Methods

Materials

Sodium salt of CMC (NaCMC, reference BLANOSE 7LP EP, Ashland, France) with a weightaverage molar mass (Mw) of 90.5 kg/mol and a degree of substitution (DS) of 0.7, was used as received. Methacrylic anhydride (MA) containing 2,000 ppm topanol A as inhibitor, 2hydroxy-4ʹ-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), anhydrous sodium hydroxide (NaOH) pellets and deuterium oxide (D2O) were purchased from Sigma Aldrich (France).

Methacrylation of carboxymethylcellulose

The protocol was adapted from [START_REF] Melilli | DLP 3D Printing Meets Lignocellulosic Biopolymers: Carboxymethyl Cellulose Inks for 3D Biocompatible Hydrogels[END_REF]. NaCMC was solubilized at 2 wt% in deionized water at room temperature under stirring. MA was then added dropwise to the CMC solution. Different molar ratios between MA and CMC hydroxyl groups were used (namely 1.24, 2.48, 3.72 and 4.96) to study the effect of this parameter on the degree of methacrylation of the resulting mCMC samples. Homogenization during the reaction was performed by magnetic stirring at 700 rpm or by mechanical stirring at 2,000 rpm with a glass shaft anchor stirrer, to investigate the influence of the stirring method on the reaction. The reaction was continued for 24 h at room temperature. The pH of the medium was constantly adjusted to 8 with 10 M NaOH during the reaction. The resulting mixture was purified for a week by dialysis against deionized water using a cellulose mixed ester membrane (MWCO = 12-14 kg/mol; Roth, Germany). Finally, the mCMC product was freeze-dried for five days with a CHRIST Beta 2-8 LS plus lyophilizer (Martin Christ Gefriertrocknungsanlagen, Germany).

Hydrogel and cryogel preparations

The freeze-dried synthetised mCMC samples were dissolved at 2, 3 or 4 wt% in deionized water at 50 °C during 12h under magnetic stirring. The photoinitiator Irgacure 2959 was then added at room temperature with a constant molar ratio compared to the number of moles of methacrylate (MA) in solution (nPI/nMA = 0.55). Irgacure 2959 was chosen because it is water-soluble (0.5 wt% maximum), non-toxic, and absorbs at 365 nm [START_REF] Tomal | Water-Soluble Photoinitiators in Biomedical Applications[END_REF]. The formulations were treated for 15 min in an ultrasonic bath at 50 °C and then agitated with a multi-rotator PTR-35 (Grant, UK) at 6 rpm for 12 h at room temperature. 650 µL of each formulation was then placed in a PTFE circular mold of 15 mm diameter and 1 cm height and then insolated for 5 min under a UV-light source with a wavelength of 365 nm and an irradiance of 82 mW/cm² (Promaker V6000, Prodways, France). All resulting hydrogels had a thickness of 3.6 mm and a diameter of 15 mm. They were placed in an aluminum basket, filled with liquid nitrogen and freeze-dried overnight with the same lyophilizer to yield cryogels.

Characterization techniques

Nuclear magnetic resonance (NMR)

1 H NMR spectroscopy 1 H NMR spectroscopy was used to quantify the degree of methacrylation (DM) of the mCMC samples. 4.5 mg of mCMC was dissolved in 650 µL of D2O at 50 °C for 12 h using a ThermoMixer C (Eppendorf, Germany). All 1 H NMR spectra were recorded on a Spectrospin 300 MHz spectrometer (Bruker, Germany) at 25 °C from 64 successive scans with a relaxation time D1 of 3 s.

13 C Non-Quaternary Suppression (NQS) and Cross-Polarization Magic-Angle Spinning (CP-MAS) solid state NMR spectroscopy 13 C NQS and CP-MAS solid-state NMR experiments were carried out to identify and quantify carbons before and after crosslinking of mCMC. 13 C CP-MAS solid-state NMR spectroscopy was also used to measure the crosslinking ratio of UV-cured cryogels. 80 mg of crosslinked mCMC cryogel was crushed and placed in a 3 mm diameter rotor. All 13 C NQS and CP-MAS solid-state NMR spectra were recorded on a Neo 500 MHz spectrometer (Bruker, Germany) operating at 125.8 MHz. The acquisition time was 28 ms and the sweep width was 37 kHz and at least 2,048 successive scans were recorded at 25 °C.

Fourier-transformed infrared (FTIR) spectroscopy

FTIR by attenuated total reflectance (ATR) was performed with a MIRacle 10 instrument (Shimadzu, Japon) to analyze the characteristic chemical bonds of CMC and mCMC samples. Absorbance spectra were acquired within the 4,000 -550 cm -1 wavenumber range (64 scans, 4 cm -1 resolution).

Scanning electron microscopy (SEM)

SEM was used to investigate the cryogel morphology and measure the pore size distribution. Samples were fractured horizontally and pasted on metallic stubs with carbon cement, fractured and sputter-coated with Au/Pd. Secondary electron images were recorded with a FEI Quanta 250 (Thermo Fisher Scientific, US) scanning electron microscope equipped with a field emission gun operating at 2.5 kV. All pores were observed on the cutted surface. The pore size was determined by image treatment with ImageJ. Horizontal or vertical lines were drawn on each pore giving the average pore size. This procedure was performed on 25 pores per image and 4 images per sample. Pores whose walls were broken by the cutting method were not measured.

Swelling ratio and hydrogel mesh size calculation

Swelling tests were performed on cryogels. Measurements were made on 3 cryogels per condition. The samples were immersed in deionized water at room temperature and the cryogels transformed this way into hydrogels (or re-hydrated cryogels) were regularly weighed. These steps were repeated until mass equilibrium was reached to determine the swelling ratios (SR) according to Eq. ( 1).

𝑆𝑅 = 𝑤

! /𝑤 " (1)
where w0 is the cryogel weight and wt is the weight at measurement time t. Swelling ratios were averaged over measurements carried out on three different samples.

The hydrogel mesh size, ξ, was estimated from swelling tests, based on the previously described Flory-Rehner theory used by Baier [START_REF] Leach | Photocrosslinked Hyaluronic Acid Hydrogels: Natural, Biodegradable Tissue Engineering Scaffolds[END_REF] and [START_REF] Lin | Lower Crosslinking Density Enhances Functional Nucleus Pulposus-like Matrix Elaboration by Human Mesenchymal Stem Cells in Carboxymethylcellulose Hydrogels: Crosslinking Density Impacts HMSC Chondrogenesis[END_REF]. First, the mass ratio between the hydrogel and cryogel, Qw, was calculated using Eq. ( 2):

𝑄 # = 𝑤 $ /w " ( 2
)
where wh is the hydrogel weight and w0 is the cryogel weight. The volumetric ratio, Qv, between the hydrogel and the polymer volumes, was determined from Qw using Eq. ( 3):

𝑄 % = 1 + * & !"#" & $%&'( (𝑄 # -1). (3)
where 𝜌 '()( is the density of the considered dried mCMC measured by pycnometry, by analyzing pellets of each mCMC using an AccuPyc II 1340 pycnometer (Micromeritics, US) (Supplementary data -Table S1); 𝜌 #*!+, is the solvent density (𝜌 #*!+, = 1 g. cm -. ). The average molecular weight between two crosslinking points (M / ) was calculated using a simplified version of the Flory-Rehner equation ( 4) demonstrated by [START_REF] Hoti | Effect of the Cross-Linking Density on the Swelling and Rheological Behavior of Ester-Bridged β-Cyclodextrin Nanosponges[END_REF]:

𝑀 0 = 2 $%&'( 34
$ ) $ *" 5 + , -4 - . $ ) $ *"

56

-7894:-

$ ) $ *" 5; $ ) $ *" ;<( $ ) $ *"
)²@

(4) where 𝑉 #*!+, is the molar volume of the solvent at room temperature (Vwater = 18 mol.cm -3 ), 𝑤 A is the hydrogel weight, 𝑤 $( is the weight of the hydrated cryogel at the maximum of swelling, χ is the Flory polymer-solvent interaction parameter and f is the functionality of the crosslinking, i.e. the maximum number of chemically linked polymer chains at a crosslinking point (f = 2.3 for CMC samples with DS = 0.7). Considering that CMC and mCMC have a similar structure, a value of 0.45 was used for χ, which corresponds to the CMC-water interaction parameter determined by [START_REF] Xia | Determination of Crosslinking Density of Hydrogels Prepared from Microcrystalline Cellulose[END_REF]. The differences between CMC, mCMC and crosslinked mCMC were assumed to be negligible based on the work of Baier [START_REF] Leach | Photocrosslinked Hyaluronic Acid Hydrogels: Natural, Biodegradable Tissue Engineering Scaffolds[END_REF] on hyaluronic acid (HA), methacrylate HA (mHA) and the crosslinked mHA.

The hydrogel mesh size, 𝜉 was determined by Torres-Lugo and Peppas (1999) using Eq. ( 5):

ξ = Q B + , = CD / EEEE CD 0 > ? r " F ( 5
)
where Mw is the molar mass of the mCMC, calculated depending on the DM, and 𝑟 " F is the root-mean-square distance between two crosslinking points that can be linked to the molar mass Mn of the mCMC by [START_REF] Cleland | Ionic Polysaccharides. IV. Free-Rotation Dimensions for Disaccharide Polymers. Comparison with Experiment for Hyaluronic Acid[END_REF] using Eq. ( 6):

? r " F = ?

FG 1 H² D 2 BM I (6)
where 𝑀 , is the molecular weight of the mCMC unit. 𝐶 9 is the Flory characteristic ratio of CMC (11.5) determined by [START_REF] Picout | On the Chain FLexibility of Arabinoxylans and Other B-(1 4) Polysaccharides[END_REF] and 𝑙 is the lengh of a virtual bond spanning a monosaccharide unit (0.545 nm) determined by [START_REF] Holtzer | The Molecular Configuration and Hydrodynamic Behavior of Cellulose Trinitrate[END_REF].

Finally, the hydrogel mesh size was calculated using Eq. ( 7):

ξ = ? FG 1 H² D 2 ? M / Q B + , (7)

Rheology measurements

The rheological properties of each hydrogel were measured at least on three different samples per hydrogel composition using a Netzsch Kinexus Pro+ parallel plates rheometer (Germany). Frequency sweep measurements were performed at a constant temperature (25 °C) from 0.1 to 10 Hz. According to preliminary amplitude sweep measurements, all tests were carried out in the linear viscoelastic region at 1 % strain. The gap between the plate and the mobile plane was set to 0.4 mm. Rheology measurements were also performed on hydrated cryogels at equilibrium swelling using the same procedure.

Results and discussion

Control and optimization of the methacrylation of CMC

Photo-crosslinkable methacrylate groups were successfully grafted onto CMC using the reaction between methacrylic anhydride (MA) and the polymer hydroxyl groups (Fig. 1a).

In the literature, the reaction was only performed at low temperature (0 or 4 °C) [START_REF] Reza | Characterization of Novel Photocrosslinked Carboxymethylcellulose Hydrogels for Encapsulation of Nucleus Pulposus Cells[END_REF][START_REF] Qian | Delivery of Doxorubicin in Vitro and in Vivo Using Bio-Reductive Cellulose Nanogels[END_REF][START_REF] Dutta | Temperature, PH and Redox Responsive Cellulose Based Hydrogels for Protein Delivery[END_REF][START_REF] Melilli | DLP 3D Printing Meets Lignocellulosic Biopolymers: Carboxymethyl Cellulose Inks for 3D Biocompatible Hydrogels[END_REF]). However, no influence of temperature was observed in our study when comparing reactions performed at 0 °C or at room temperature (Supporting Information Fig. S1). Therefore, the reactions described below were more conveniently performed at room temperature and were fully optimized by varying protocol parameters.

All mCMC samples were characterized by FTIR spectroscopy, and the appearance of a carbonyl band at 1,705 cm -1 on the spectra of methacrylated samples when compared to the CMC spectrum (Supporting Information Fig. S2) qualitatively proved the success of the methacrylation (Supporting Information Fig. S2). Then, the samples were characterized by liquid 1 H NMR and compared to raw CMC (Fig. 1b) to determine the degree of methacrylation. According to the literature, protons of the CMC unit (HCMC) are located in the bulk between 2.8 and 4.7 ppm [START_REF] Kono | NMR Characterization of Sodium Carboxymethyl Cellulose: Substituent Distribution and Mole Fraction of Monomers in the Polymer Chains[END_REF], and account for 8.4 protons considering a DS of 0.7. For mCMC samples, three additional peaks corresponding to the vinyl (Ha: 6.0-6.4 ppm, Hb: 5.65-6.0 ppm) and methyl (Hc: 1.7 -2.2 ppm) protons of the methacrylate groups were also found after purification, indicating that methacrylation occurred under our experimental conditions (Van Hove, Wilson & Benoit 2013). The deprotonation of hydroxyl groups is achieved by adding NaOH. The resulting hydroxide ion reacts with one of the carbons in the carbonyl group of MA, leading to the liberation of methacrylic acid. CMC methacrylation should first preferentially occur on the remaining 30 % of C6 primary hydroxyl groups, which are more reactive [START_REF] Fedorova | A Study of the Relative Reactivity of the Hydroxyl Groups of Cellulose in Esterification in an Acidic Medium[END_REF]. Once all C6 hydroxyl groups are consumed, methacrylation will then occur on the C2 and C3 secondary hydroxyl groups. The reaction between the carboxylate group of CMC and MA is unlikely, as the carboxylate group is stabilized by mesomeric effects. Furthermore, mCMC remains water-soluble, proving the conservation of carboxylates. It is however difficult to verify these hypotheses by conventional characterizations. Nevertheless, recent advances in dynamic nuclear polarization NMR (DNP NMR) may allow determining the regioselectivity of the reaction as [START_REF] Berruyer | Advanced Characterization of Regioselectively Substituted Methylcellulose Model Compounds by DNP Enhanced Solid-State NMR Spectroscopy[END_REF] did with a substituted methylcellulose.

The degree of methacrylation of mCMC samples was calculated from the respective integrals of vinyl (Ha + Hb) and CMC (HCMC) protons, according to Eq. ( 8).

DM (%) = J 3 5 J 6 J 787 × K.M F×F.. × 100 (8)
The influence on the final DM of both the molar ratio between MA and CMC hydroxyl groups (MA/OHCMC) and the agitation method was investigated. First, by increasing the MA/OHCMC ratio from 0 to 4.96, a continuous increase in the integrals of all methacrylate peaks (Ha -Hc) was noticed (Fig. 1b), leading to a linear increase in the calculated DM (Fig. 1c; R² > 0.94). A maximum DM value of 76 ± 6 % was obtained for the mCMC prepared using a MA/OHCMC ratio of 4.96 under mechanical stirring at 2,000 rpm, indicating that methacrylate groups successfully substituted 76 of the initial hydroxyl groups over 100 units. Such results therefore show a very easy and high degree of tunability of the DM by simply playing with the MA/OH ratio.

As for reactions performed using magnetic stirring at 700 rpm, a linear increase with the number of equivalents was observed. However, lower DM values were systematically obtained for the same molar ratio, which was attributed to a lower diffusion of MA in the solution. For example, for a MA/OHCMC ratio of 4.96 under magnetic stirring, a DM of only 47 ± 4 % was obtained. The reaction was therefore found to be more efficient with mechanical stirring at 2,000 rpm.

It is worth noticing that the maximum DM obtained in this work is 76 ± 6 %, which is 16 percentage points higher than the maximum DM reported in the literature (DM = 60 %) by [START_REF] Melilli | DLP 3D Printing Meets Lignocellulosic Biopolymers: Carboxymethyl Cellulose Inks for 3D Biocompatible Hydrogels[END_REF]. According to the present results and since no plateau value was reached, it is quite likely that even higher DM values could be obtained by using a higher initial MA/OHCMC ratio, but the influence of molar ratios larger than 4.96 was not studied here.

This methacrylation could be considered for other cellulosic derivatives. Methylcellulose has already been functionalized by MA and the development of the synthesis could be the same as the one presented here [START_REF] Stalling | Development of Photocrosslinked Methylcellulose Hydrogels for Soft Tissue Reconstruction[END_REF][START_REF] Gold | Development of Crosslinked Methylcellulose Hydrogels for Soft Tissue Augmentation Using an Ammonium Persulfate-Ascorbic Acid Redox System[END_REF]. Similarly, hydroxypropyl cellulose has been functionalized by MA in N,Ndicyclohexylcardodiimide with pyridine instead of NaOH (A. [START_REF] Qi | Hydroxypropyl Cellulose Methacrylate as a Photo-Patternable and Biodegradable Hybrid Paper Substrate for Cell Culture and Other Bioapplications[END_REF]). The synthesis conditions described in this work could be adapted to this cellulose derivative. However, these conditions could not be suitable for cellulose acetate which is not water-soluble.

The mCMC samples discussed below were all synthesized using mechanical stirring, with initial MA/OHCMC ratio of 1.24, 2.48 and 4.96 and had final DM values of 34 ± 4 % (sample A), 50 ± 5 % (sample B) and 76 ± 6 % (sample C), respectively.

Hydrogels and cryogels characterization

The three mCMC samples (A-C) were then used to prepare eight photo-crosslinkable liquid formulations with mCMC concentrations between 2 and 4 wt% (Table 1). Due to its lower solubility in water, sample C (DM = 76 ± 6 %) was only used at 2 and 3 wt%. The influence of the UV insolation duration (30, 60 and 300 s) was then investigated by characterizing photo-crosslinked cryogel samples C3 using 13 C CP-MAS solid-state NMR (Supporting Information Fig. S2). The methacrylate conversion ratio calculated for these cryogels were 45 %, 54 % and 62 %, respectively, and rapidly reached a plateau value around 60 % (Supporting Information Fig. S4). Based on these results, the photocrosslinking reaction was considered completed after 5 min, and this time was selected for the subsequently-described experiments.

Determination of the conversion ratio in photo-crosslinked mCMC

The methacrylate conversion ratio (CR) of all 8 cryogels was investigated by NMR spectroscopy. First, the 13 C CP-MAS spectra of the mCMC samples before crosslinking were used to attribute the signals of the different carbons, as already detailed (H. [START_REF] Qi | Homogenous Carboxymethylation of Cellulose in the NaOH/Urea Aqueous Solution[END_REF]). The heterocyclic carbons (C2-5) were found between 55 and 90 ppm, while the anomeric carbon was found at 104 ppm (Fig. 2a). The signals at 168 and 176 ppm were attributed to the carbonyl of the methacrylate (C9) and carboxylate (C8) groups, respectively. [START_REF] Capitani | 13 C Solid-State NMR Determination of Cross-Linking Degree in Superabsorbing Cellulose-Based Networks[END_REF] showed that the raw unmethacrylated CMC only has a carbonyl signal at 176 ppm, in good accordance with this attribution. Finally, the signals at 18, 123 and 133 ppm were respectively attributed to the methyl carbon (C12) and to both vinyl carbons (C10-11) of the methacrylate group (Fig. 2a). A few differences were observed in the 13 C CP-MAS and NQS spectra of photo-crosslinked samples and attributed using sample C3 spectra (Fig. 2b and Supporting Information Fig. S3). During the UV insolation, the methacrylates of the C3 sample were progressively converted by vinyl polymerization, leading to a progressive decrease of carbons C9-12 signal intensities. Simultaneously, the apparition of new carbon signals was observed and attributed to the crosslinking points formed by vinyl conversion: C9' (163 ppm), C10' (46 ppm), C11' (115 ppm) and C12' (28 ppm). The conversion ratio of methacrylates in the cryogel samples was then calculated by integrating the area of the C12' carbon peak from the C12 of the crosslinked mCMC, normalized by the anomeric carbon 1, which remained constant during crosslinking. Two carbons of Irgacure 2959 were also integrated at the same chemical shift (24 ppm). However, considering a constant molar ratio between the photoinitiator and the methacrylate groups (0.55), the Irgacure contribution does not modifiy the calculation of CR. The CR was therefore calculated with Eq. ( 9):

𝐶𝑅 = QR( +-> S Q(( +-) ; QR( +-> S (9)
where A(C12') and A(C12) correspond to the integrals of the C12' (28 ppm) and C12 (18 ppm) carbon signals of crosslinked mCMC, respectively.

Finally, the CR of methacrylates was calculated for all cryogels to determine its dependence with the initial degree of methacrylation (DM) and concentration of the mCMC in the formulations. Table 1 summarizes the extracted values that lie between 50 ± 2 % and 62 ± 2 %. It is very interesting to note that the mCMC concentration negatively impacted the final CR while the DM had little influence (Table 1). Indeed, the CR did not change significantly with increasing DM for the same initial concentration, while, on the other hand, for the same DM, the higher the concentration, the lower the CR. Considering that the volume of all cryogels was constant and equal to 0.64 cm 3 , it was possible to calculate the density of crosslinked methacrylates in mmol/cm 3 from the DM and CR values (Table 1). Interestingly, the crosslinked methacrylate density increased linearly with the DM at a constant concentration (R² > 0.99; Fig. 3a), as well as with the concentration for a constant DM (R² > 0.98; Fig. 3b). The hydrogel crosslink density can thus be controlled over a significant range (3-fold increase between the lowest and highest densities) by modulating the initial DM of the considered mCMC, but also the mCMC concentration in the photo-crosslinkable formulation.

Relationship between swelling and hydrogel and cryogel structure

Water swelling ratios (SR) reflecting the capacity of cryogels to absorb and retain water without breaking were measured. A swelling equilibrium was achieved in less than 10 hours for all cryogels (Supporting Information Fig. S5). Interestingly, the final swelling ratio decreased with increasing concentration, with A2, A3 and A4 cryogels absorbing 81, 66 and 45 times more water than their initial mass after 48 h at room temperature, respectively. Similarly, the swelling ratio decreased with increasing DM. For example, swelling ratios of 81, 62 and 37 were obtained for the cryogels A2 (DM = 34 %), B2 (DM = 50 %) and C2 (DM = 76 %), respectively. The same trends were observed between the swelling ratio and the crosslinked methacrylate density (Fig. 4a). When the crosslinked methacrylate density in the cryogel increased, the network became denser and less stretchable, and thus absorbed less water. The hydrated cryogel swelling ratios were then compared to the water content of initial hydrogels (Fig. 4b). All cryogels absorbed more water than that contained in the hydrogels, as the swelling was conducted to its maximum capacity for the hydrated cryogels, whereas the equilibrium was probably not reached for the initial hydrogels. Interestingly, differences in the swelling ratio between the two materials were observed depending on the sample. For example, hydrated cryogels A2 with the lowest crosslink density was twice as heavy as the initial hydrogel A2, whereas the most crosslinked cryogel B4 absorbed only 7 % more water than the initial hydrogel B4 had. This could be explained by the freezing, which causes the polymer chains to aggregate [START_REF] Cassanelli | Role of Gellan Gum Microstructure in Freeze Drying and Rehydration Mechanisms[END_REF]. The swelling ratio at equilibrium strongly depends on the intrinsic capacity of the polymer chains to absorb water [START_REF] Ganji | Theoretical Description of Hydrogel Swelling: A Review[END_REF]. In the case of cryogels, the collapsed polymer chains were held together by hydrogen bonds, making it more difficult for water to hydrate them. This phenomenon became even more significant when mCMC concentration increased, which could involve enlargement of the size of the pore walls as observed by [START_REF] Cassanelli | Role of Gellan Gum Microstructure in Freeze Drying and Rehydration Mechanisms[END_REF] with gellan gum cryogels. Finally, the water absorption capacity of cryogels also appeared to depend on the free methacrylate density (Supporting Information Fig. S6), since methacrylates are hydrophobic. The possibility to finely tune the swelling capacity of hydrated cryogels makes it possible to consider several types of medical applications. Indeed, it has been reported that the higher the water content of hydrogels, the more they provide a functional environment for cells. Such a property would be beneficial for applications such as drug delivery [START_REF] Chang | Superabsorbent Hydrogels Based on Cellulose for Smart Swelling and Controllable Delivery[END_REF] or wound healing [START_REF] Kanikireddy | Carboxymethyl Cellulose-Based Materials for Infection Control and Wound Healing: A Review[END_REF][START_REF] Uzun | A Review of Wound Management Materials[END_REF]. Moreover, higher water content allows for better biodegradability [START_REF] Capanema | Superabsorbent Crosslinked Carboxymethyl Cellulose-PEG Hydrogels for Potential Wound Dressing Applications[END_REF][START_REF] Chang | Superabsorbent Hydrogels Based on Cellulose for Smart Swelling and Controllable Delivery[END_REF]). However, for other applications such as bone repair, a lower swelling ratio is recommended [START_REF] Bai | Bioactive Hydrogels for Bone Regeneration[END_REF]. The density of crosslinked methacrylates also had an influence on the material structure.

The hydrogel mesh size, representing the distance between two crosslinks, was evaluated using the Flory-Rehner theory for each formulation (Eq. 7). As expected, the mesh size decreased as the concentration and DM of mCMC increased (Table 1). The more methacrylate groups in the formulation, the more crosslinks and the smaller the mesh size. However, these results did not lead to significant variations in the cryogel pore sizes determined by SEM (Fig. 5, Supporting Information Fig. S7) as the most crosslinked sample (B4) had an average pore size of 13.9 ± 5.3 µm and the less crosslinked sample (A2) displayed an average pore size of 18.9 ± 6.5 µm. The variations would rather be reflected in the thickness of the walls constituting the pores [START_REF] Plieva | Pore Structure in Supermacroporous Polyacrylamide Based Cryogels[END_REF]. In fact, the pore size and direction in the cryogels happen to be predominantly controlled by the freezedrying process, and not directly by the material crosslink density. The influence of the freeze-drying process is currently under investigation. Regarding the size of the pores, they are too small for applications such as bone repair, for which pores around 200 µm are required to allow osteoblasts to penetrate the network [START_REF] Abbasi | Porous Scaffolds for Bone Regeneration[END_REF][START_REF] Sindhu | Medical Applications of Cellulose and Its Derivatives: Present and Future[END_REF]. To overcome this issue, it could be possible to shape grids by 3D printing with an adequate porosity. However, the range of pore size we could reach would be able to allow neovascularization or fibroblast growth [START_REF] Annabi | Controlling the Porosity and Microarchitecture of Hydrogels for Tissue Engineering[END_REF].

Rheological behavior

The impact of the crosslink density on the hydrogel mechanical properties was investigated by rheology. The evolution of the elastic modulus, G', viscous modulus, G'' and tan(𝛿) =

T"

T > were first compared for A2 and B4, the hydrogels bounding the range of rheological properties (Supporting Information Fig. S8). Over the entire frequency range (0.1 to 10 Hz) at 1 % strain and at 25 °C, G' is always superior to G'', which evidenced the solid behavior of the gels. Moreover, the same behavior was observed for all the hydrogels, irrespective of the formulation. Finally, the elastic and viscous moduli of hydrogels and hydrated cryogels maintained the same order as a function of DM and mCMC concentration in the formulations (Supporting Information Fig. S9). Consequently, the comparison of the solid rheological properties of the materials was performed by considering G' at 1 Hz. The same study could be done with G'' for the viscous behavior.

For hydrogels, G'(f) of the initial hydrogels increased with mCMC concentration (Fig. 6a). At 1 Hz, hydrogel G'(1 Hz) values of 1.8 ± 0.50, 3.5 ± 0.90 and 8.0 ± 0.40 kPa were measured for B2, B3 and B4, respectively. However, there was no significant trend observed with the DM. The same trend was observed with mCMC concentration for the hydrated cryogels (Fig. 6b). Hydrated cryogel G'(1 Hz) values of 8.6 ± 2.2, 13.1 ± 0.8 and 16.7 ± 1.7 kPa were measured for B2, B3 and B4, respectively. At 1 Hz, the ratio of elastic moduli (G'(hydrated cryogel)/G'(initial hydrogel) was superior to 1 for each formulation. Noticeably, even if they contain more water, the elastic moduli of the hydrated cryogels were significantly higher than those of the initial hydrogels. This ratio also decreased as the mCMC concentration increased. While at 2 wt% mCMC, the G' ratio increased with decreasing DM, no significant G' ratio evolution with DM was observed at 3 wt% and 4 wt% (Fig. 6c).

Such a difference in the rheological properties could be due to the liquid nitrogen-freezing step before freeze-drying. As observed in the SEM images along the cross-section (Supporting Information Fig. S10), cryogel pores were oriented along the freezing axis (from the bottom to the top) after freeze-drying, whereas they could be randomly oriented in the hydrogels. It is conceivable that this orientation implies a stronger network organization in the cryogel than in the initial hydrogel. Indeed, cryogel swelling did not affect the variation of the elastic modulus G' of hydrated cryogels as a function of DM and mCMC concentration compared to hydrogels (Fig. 6a,b). The elastic moduli of both materials increased with mCMC concentration and did not show significant variation with DM. However, the less crosslinked methacrylate density in the formulation, the more water the initial hydrogel contained (Fig. 4b). It is possible that upon freezing the pore orientation and network organization is partly governed by the quantity of water in the initial hydrogel. In other words, the formed crystals could be larger in the presence of larger amounts of water. The microstructure could have deformed more easily and become more robust upon freeze-drying when more water was entrapped in the initial hydrogel. Moreover, the freezing process collapses the chains and can strengthen the structure of the 3D network. Indeed, the G' ratio decreased with the hydrogel water content (Fig. 6d). For B2, B3 and B4, G' ratios were 4.7 ± 0.10, 3.8 ± 0.61 and 2.1 ± 0.10 for a hydrogel water content of 38 ± 1.2, 28 ± 0.43 and 24 ± 0.010, respectively (Fig. 6d).

Finally, our results evidence that the elastic modulus G' of both initial hydrogels and hydrated cryogels can be tuned by varying the mCMC concentration, rather than the DM. Depending on the material targeted application, specific DM and concentration can be selected to achieve suitable microstructure and mechanical properties. As an example, hydrogels are generally mechanically brittle for bone repair applications [START_REF] Bai | Bioactive Hydrogels for Bone Regeneration[END_REF][START_REF] Sindhu | Medical Applications of Cellulose and Its Derivatives: Present and Future[END_REF]. It is shown here that it is possible to increase the viscoelasticity of hydrogels without increasing the amount of water in the hydrogel, as in the case of the B4 sample. However, the obtained elastic moduli are too low for skeletal tissues. On the other hand, they could be considered for use as extracellular matrix (ECM) or for soft tissue repair, such as lungs or brain [START_REF] Chaudhuri | Effects of Extracellular Matrix Viscoelasticity on Cellular Behaviour[END_REF].

Conclusion

In this study, the reaction between carboxymethylcellulose and methacrylic anhydride was first investigated to produce photo-crosslinkable methacrylated carboxymethylcellulose. By controlling the synthesis parameters, a wide range of mCMC samples with degrees of methacrylation ranging between 0 and 76 ± 6 % were obtained. Eight aqueous formulations with different DM and concentrations of mCMC were used to prepare UV-cured hydrogels, which were subsequently transformed into cryogels by freeze-drying. A new 13 C solid-state NMR method was used to measure the methacrylate conversion ratio (CR, from 50 to 62 %) and deduce the density of crosslinked methacrylates (from 4.3×10 -3 to 1.3×10 -2 mmol/cm 3 ) of the final cryogels, which was found to be correlated with both DM and initial concentration of the mCMC formulation. Furthermore, correlations between the density of crosslinked methacrylate and the swelling ratio of cryogels on the one hand, and the microstructure on the other hand, were demonstrated. In addition, these results help to explain the evolution of the elastic moduli of hydrogels and hydrated cryogels, which depended mainly on the initial concentration of mCMC in the formulation. Furthermore, the difference between the two elastic moduli depended on the quantity of water in the initial hydrogel and the crosslinked methacrylate density. In conclusion, we highlighted the possibility of finetuning the microstructure (crosslinked methacrylate density; mesh size between 0.56 and 5.4 nm) as well as the cryogel swelling ratio (25-80) and rheological properties (2-8 kPa for hydrogels, 4.5 -17 kPa for swollen cryogels) of UV-cured mCMC hydrogels and cryogels, by controlling the methacrylation conditions of a commercial CMC. According to the selected properties, these crosslinked mCMC hydrogels and cryogels could serve different purposes in biomedicine, such as 3D scaffolds for soft tissue regeneration, or cell encapsulation. Additionally, photo-crosslinkable mCMC could be easily shaped by 3D printing technics to obtain complex 3D-printed artefacts. Finally, these materials could also be considered in fields such as agriculture, food and textile where porous materials are generally used to absorb solvents, dyes, water or liquids harmful to the analyzed environment [START_REF] Rahman | Recent Developments of Carboxymethyl Cellulose[END_REF]).

Fig. 1

 1 Fig. 1 (a) Scheme of the methacrylation of CMC with MA in water at 25 °C to yield mCMC. (b) 1 H NMR spectra for CMC in black and mCMC in red (2.48 eq, mechanical stirring, sample B). (c) Evolution of the degree of methacrylation with the molar ratio between MA and CMC hydroxyl groups for syntheses performed with mechanical and magnetic stirring. Error bars depend on the repetition of the synthesis (n= [1-3]) and the protons integration processing of the NMR spectra.

Fig. 2

 2 Fig. 2 13 C CPMAS solid-state NMR spectra of (a) C mCMC sample (DM = 76 %) and (b) C3 cryogel.

Fig. 3

 3 Fig. 3 Density of crosslinked methacrylates according to (a) mCMC DM for a given concentration, and (b) mCMC concentration for a given DM.

Fig

  Fig 4 (a) Influence of the density of crosslinked methacrylates in cryogel samples on the swelling ratio of the hydrated cryogel after 48h. (b) Comparison of hydrogel water content and hydrated cryogel swelling ratio at equilibrium. Error bars are standard deviations for 3 different samples per formulation.

Fig. 5

 5 Fig. 5 SEM images displaying the cryogel morphologies and corresponding pore size distributions for (a) A2, (b) B2 and (c) C2. The average pore size was determined after around 25 measurements on 4 images per sample. Pores whose walls were broken by the cutting section method were not taken into account.

Fig

  Fig 6 (a) Evolution of the elastic moduli G' of the initial hydrogels (at 1 Hz, 1% strain and 25 °C) versus the mCMC concentration in each formulation. (b) Evolution of the elastic moduli G' of the hydrated cryogels (at 1 Hz, 1% strain and 25 °C) versus the mCMC concentration in each formulation. (c) Ratios of the elastic moduli of hydrated cryogels over that of the corresponding initial hydrogels versus mCMC concentration. (d) Evolution of the ratios of the elastic moduli of hydrated cryogels over that of the corresponding hydrogels versus hydrogel swelling ratio. Error bars come from the standard deviation of three independent measurements.

Table 1

 1 Composition of the photo-crosslinkable mCMC formulations, and structural properties of the corresponding hydrogels and cryogels.

			mCMC	Cryogel	Cryogel
					Conversion	Crosslinked	Hydrogel
	Formulation Sample DM (%)	Concentration (wt%)	ratio of methacrylates	methacrylate density	Mesh size 𝜉 (nm)
					(CR) (%)	(10 -3 mmol/cm 3 ) a
	A2			2	53 ± 2.0	4.3 ± 0.29	5.4 ± 1.6
	A3	A	34 ± 4	3	59 ± 2.0	6.4 ± 0.35	3.9 ± 1.2
	A4			4	50 ± 2.0	8.1 ± 0.41	2.8 ± 0.96
	B2			2	58 ± 2.0	6.6 ± 0.39	4.2 ± 1.0
	B3	B	50 ± 5	3	54 ± 2.0	9.1 ± 0.50	2.8 ± 0.50
	B4			4	53 ± 2.0	12 ± 0.65	0.79 ± 0.27
	C2	C	76 ± 6	2	62 ± 2.0	9.9 ± 0.62	1.3 ± 0.38
	C3			3	54 ± 2.0	13 ± 0.95	0.56 ± 0.14

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ACKNOWLEDGEMENTS

The authors acknowledge the NanoBio chemistry platform (ICMG UAR 2607) for granting access to the electron microscopy facilities. The authors also acknowledge Ashland for kindly offering carboxymethylcellulose.

Availability of data and material Additional information regarding this manuscript can be found free of charge in the supporting information.

Author contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Lénaïc Soullard, Sébastien Rolere, Bruno Jean, Isabelle Texier, Guillaume Nonglaton, Pierre-Alain Bayle and Christine Lancelon-Pin.

The first draft of the manuscript was written by Lénaïc Soullard and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding