# Thermoresponsive and cytocompatible infernan-based hydrogels exhibiting poroelastic properties

Arnaud Fillaudeau, Marion Desdouits, Corinne Sinquin, Sylvia

Colliec-Jouault, Agata Zykwinska, Stéphane Cuenot

► To cite this version:

Arnaud Fillaudeau, Marion Desdouits, Corinne Sinquin, Sylvia Colliec-Jouault, Agata Zykwinska, et al. Thermoresponsive and cytocompatible infernan-based hydrogels exhibiting poroelastic properties. Applied Materials Today, 2025, 42, pp.102544. 10.1016/j.apmt.2024.102544. hal-04861386

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Submitted on 7 Jan 2025

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Distributed under a Creative Commons Attribution 4.0 International License
Thermoresponsive and cytocompatible infernan-based

hydrogels exhibiting poroelastic properties

Arnaud Fillaudea¹, Marion Desdouitsa¹, Corinne Sinquin a, Sylvia Colliec-Jouault a, Agata

Zykwinska a,*, Stéphane Cuenot b,*.

<footnote>ᵃ Ifremer, MASAE Microbiologie Aliment Santé Environnement, F-44000 Nantes, France.</footnote>

<footnote>b Nantes Université, CNRS, Institut des Matériaux de Nantes Jean Rouxel, IMN, F-44000 Nantes,
France.</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail addresses: Stephane.Cuenot@cnrs-imn.fr (S. Cuenot), Agata.Zykwinska@ifremer.fr (A. Zykwinska).</footnote>
**Abstract**

Thermoresponsive polymers sensitive to temperature changes are particularly well adapted for applications in tissue engineering due to their *in situ* sol-gel transition at physiological temperature, enabling a rapid gel formation after injection. Infernan, a bacterial exopolysaccharide (EPS) of marine origin endowed with glycosaminoglycan-mimetic properties was grafted with a thermoresponsive polymer, poly(N-isopropylacrylamide) (pNIPAM) using carbodiimide chemistry. The resulting infernan-pNIPAM polymers were shown to form thermoresponsive hydrogels at physiological temperature. Their mechanical properties determined in fully hydrated state at 37 °C by Atomic Force Microscopy (AFM) through force-indentation and force-relaxation experiments revealed their poroelastic behavior. Based on AFM measurements, a set of mechanical parameters was extracted, including the elastic modulus, the solvent diffusion coefficient and the pore size. Cytocompatibility of hydrogels evaluated in 3D culture using HEK293 cells was impacted by polysaccharide amount within the hydrogel and the cross-linking density of the network. Elucidation of the structure-function relationship of infernan-pNIPAM hydrogels was essential for their further optimal use in cartilage engineering.

*Keywords: exopolysaccharide; pNIPAM; AFM; cell viability; glycosaminoglycan-mimetic.*
# 1. Introduction

Tissue engineering, an interdisciplinary field involving cellular biology, materials sciences and engineering, aims to develop biomaterials able to restore damaged tissues, such as articular cartilage, bone or skin, by combining cells and growth factors with three dimensional (3D) matrices. Such 3D scaffolds are designed to support cell growth and formation of the neo-tissue [1-4]. Articular cartilage is a specialized connective tissue produced by a unique cell type, the chondrocyte, able to synthesize an extracellular matrix (ECM) composed of collagen fibrils and proteoglycans, mainly aggrecans, constituted of sulfated glycosaminoglycans (GAGs) [5,6]. The resulting aggrecan/collagen network provides the tissue its resistance to tensile and compressive strengths. In some cases, e.g. accidental trauma, osteochondritis dissecans or excessive articular stresses, the integrity of this hydrated pressure-resistant network can be altered. However, due to its low cellularity and absence of vasculature, cartilage displays only limited ability to repair. To promote cartilage regeneration, designing of deformable biomaterials, such as hydrogels, i.e. highly hydrated polymeric matrices, was shown promising [7-9]. An appropriate hydrogel matrix should constitute a microenvironment providing biological and mechanical cues to support cell migration, proliferation, differentiation and secretory activities [10,11]. Amongst all hydrogels already developed for tissue engineering applications, thermoresponsive matrices seem particularly interesting for cartilage regeneration due to their unique characteristics [12-14]. Indeed, polymeric solution can easily be injected in a minimally invasive manner into the injured area, thus allowing to precisely match both shape and size of the lesion before *in situ* sol-gel transition [15]. To optimize biological properties of hydrogels in order to promote tissue repair, their composition should closely mimic that of the ECM. In particular, presence of anionic polymers that could regulate cellular behaviors in a similar manner to sulfated GAGs would be highly beneficial [16,17]. Mechanical properties of hydrogels, modulated by the nature of the
crosslinking involved in the matrix formation as well as its density, also strongly contribute to the observed cellular responses. Indeed, recent insights into cell mechanobiology have revealed that the whole mechanical forces, both exerted by cells and subjected to cells, regulate their behaviors (cell migration, proliferation, secretion and differentiation) [18–23]. Chemically crosslinked hydrogels exhibit elastic behaviors that decrease cell proliferation and may lead to cell death due to the restriction of cell volume expansion by elastic stresses. In contrast, physical hydrogels are stabilized by noncovalent bonds, including hydrogen bonds, hydrophobic and ionic interactions that can easily be disrupted by cells and reformed immediately after. These responses to applied load result from viscoelastic properties of physical hydrogels, that ensure network deformation, thus enabling cell migration, proliferation and production of the appropriate ECM.

Infernan exopolysaccharide (EPS), secreted by a deep-sea hydrothermal vent bacterium *Alteromonas infernus* [24,25], is a promising candidate for the formation of hydrogels, mimicking, through the presence of uronic acids and sulfate groups, the composition of sulfated GAGs and their biological properties. Indeed, this high-molecular weight anionic polysaccharide has a natural affinity to growth factors, including TGF-β1 and BMP-2, involved in cartilage and bone regeneration [26–28]. It was previously shown that addition of infernan into silanized hydroxypropyl methylcellulose (Si-HPMC) hydrogel supported the 3D culture of functionally competent chondrocytes able to produce type II collagen and sulfated GAGs. In addition, this infernan-enriched hydrogel implanted in the subcutis of nude mice for three weeks allowed the synthesis of a cartilage-like ECM by implanted chondrocytes without any detectable inflammation [28]. For its further use in cartilage engineering such as a thermoresponsive hydrogel, this biologically relevant polysaccharide was recently grafted with a thermosensitive water-soluble polymer, poly(N-isopropylacrylamide) (pNIPAM) [29]. Due to its rapid coil-to-globule phase
transition at lower critical solution temperature (LCST ~32°C) below physiological temperature, pNIPAM became one of the most widely studied smart polymer for tissue engineering applications [30,31]. Above LCST, the rearrangement of water molecules near hydrophobic groups of the polymer leads to clustering of exposed hydrophobic surface and polymer collapse [32,33]. The physicochemical and thermoresponsive properties of infernan grafted with pNIPAM were previously characterized at the molecular scale [29]. It was shown that both their composition and their grafting densities, modulated by the EPS:pNIPAM molar ratio applied during synthesis, highly impacted their functional properties in highly or semi-diluted solutions.

In this context, the aim of the present study was to evaluate the potential of infernan-
pNIPAM polymers to form thermoresponsive hydrogels with mechanical and biological properties
suitable for tissue engineering applications. Atomic Force Microscopy (AFM) was applied to
determine the mechanical properties of EPS-pNIPAM hydrogels in fully hydrated state at
physiological temperature through force indentation and force relaxation experiments. The origin
of energy dissipation involved in the time-dependent mechanical properties of hydrogels was
firstly determined from the force relaxation curves allowing to apply the appropriate mechanics
models. Then, a set of mechanical parameters characterizing the hydrogels was extracted from
these curves, such as the solvent diffusion coefficient, the Poisson's ratio and the pore size. In
addition, the elastic component of the hydrogels was determined from both the approach force-
indentation curves and force relaxation curves. Cytocompatibility of infernan-pNIPAM hydrogels
evaluated in 3D culture using HEK293 cells was impacted by the polysaccharide amount and the
cross-linking density of the network. A better understanding of the relationship between the
physicochemical characteristics (composition and grafting density) of infernan-pNIPAM polymers
and functional (biological and mechanical) properties of resulting hydrogels remains crucial for their further optimal use in cartilage engineering.

## 2. Material and methods

### 2.1. Materials

Amine-terminated pNIPAM (Mn 5,500 g/mol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and *N*-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich and were used as received without any further purification. Phosphate Buffered Saline (PBS) 1X, pH 7.4 and Dulbecco's Modified Eagle medium (DMEM) were purchased from Gibco. AlamarBlue® solution was purchased from G-Biosciences. Cultrex™, Cultrex RGF BME was purchased from Bio-Techne.

### 2.2. *Production and molecular composition of native infernan EPS*

Infernan was produced by *A. infernus* fermentation (25 °C, pH 7,4) in a 30 L bioreactor (Techfors, INFORS, Switzerland), as previously reported [24]. The monosaccharide composition and sulfur content of EPS were determined according to the methods previously described by Gélébart et al [26].

### 2.3. *Production and molecular characterization of infernan-pNIPAM polymers*

Infernan-pNIPAM polymers were prepared by carbodiimide chemistry with EDC/NHS as coupling agent, as previously described [29]. The amine-terminated (*R*'-NH₂) pNIPAM (Mn 5,500 g/mol) (p5) was grafted on the carboxyl (R-COOH) groups of uronic acids of infernan (Mn 900,000 g/mol). Three EPS-pNIPAM polymers were prepared by varying the COOH:NH₂ molar ratio (0.65, 1.0, 2.1), while maintaining COOH:EDC:NHS molar ratio constant at 1:1.6:1.6. EPS (10, 20, 40 mg) and pNIPAM-NH₂ (160, 200, 200 mg) were separately dissolved in 1 mL of MES
buffer (50 mM, pH 4.8) at room temperature. After overnight solubilization, both solutions were mixed, and EDC (3.0, 5.9, 11.8 mg) and NHS (1.8, 3.6, 7.1 mg) were added. The reaction mixture was stirred at 250 rpm for 6 h at 20 °C before a new EDC/NHS addition to potentialize the pNIPAM-NH₂ graft. After overnight incubation, the products were dialyzed (MWCO 50,000 g/mol) against 0.3 M NaCl (24 h) and Milli-Q water (48 h) and freeze-dried [29].

EPS-pNIPAM polymers were characterized in term of their grafting density (i.e. the percentage of uronic acids of EPS grafted by pNIPAM), their molecular weight and conformation in solution by Frit Inlet-Asymmetrical Flow Field-Flow Fractionation coupled with Multi-Angle Light Scattering and Refractive Index detectors (FI-AF4-MALS-dRI). Three grafted EPS were obtained: EPS-p5-3%, EPS-p5-8% and EPS-p5-16%, where % indicates their grafting density [29].

## 2.4. UV-visible spectroscopy

The thermosensitive properties of EPS-pNIPAM solutions were evaluated by UV-visible spectroscopy (Varioskan LUX, ThermoFisher). EPS-pNIPAM polymers were solubilized overnight at room temperature in PBS buffer (1X, pH 7.4, Gibco) at 10 % w/v for EPS-p5-16% and EPS-p5-8% or 7 % w/v for EPS-p5-3%. Solutions (100 µL) were then deposited in a 96-well plate. The thermal phase transition was followed using a microplate reader (preheated at 37 °C for 10 min) by measuring the absorbance at 550 nm at 37 °C for 10 minutes.

## 2.5. Swelling of infernan-pNIPAM hydrogels

Due to reversible gelation, a specific procedure was developed to determine the swelling ratio of infernan-pNIPAM hydrogels. For this purpose, EPS-pNIPAM polymers were solubilized in PBS buffer (1X, pH 7.4, Gibco) at 10 % w/v for EPS-p5-16% and EPS-p5-8% or 7 % w/v for EPS-p5-3%. After overnight dissolution at room temperature, 100 µL of each solution were deposited in a Transwell insert (Greiner Bio-one, ThinCert™ 24 wells, 0.4 µm, polyethylene
terephthalate) and placed in a 24-well plate (Greiner Bio-one). Solutions were firstly preincubated at 37 °C for 1 h (5% CO₂, controlled humidity), allowing for gel setting. The initial mass of the hydrogel thus obtained was then determined (mass (initial hydrogel)). Then, 1 mL of DMEM (DMEM 1X, 0.45 % w/v D-glucose, 0.058 % w/v L-glutamine, 0.011 % w/v sodium pyruvate, from Gibco) at 37 °C was added to the well and 200 µL on top of the hydrogel (t = 0 h). At each time point (1 h, 2 h, 4 h, 6 h, 22 h, 30 h, 47 h and 55 h), DMEM in the well and on the top of the hydrogel was removed and weighed. It was then replaced by the fresh thermostated DMEM and incubated at 37 °C. The mass of DMEM absorbed by the hydrogel (mass (absorbed DMEM)) was then deduced from the difference between the mass of DMEM initially added and the mass of DMEM weighed at each time point. The swelling ratio of EPS-pNIPAM hydrogels was then determined from the equation (1):

$$ \text{Swelling ratio} = \frac{\text{mass (absorbed DMEM)} + \text{mass (initial hydrogel)}}{\text{mass (initial hydrogel)}} \quad (1) $$

Measurements were carried out in triplicate.

## 2.6. Imaging and mechanical properties of infernan-pNIPAM hydrogels by Atomic Force Microscopy (AFM)

Prior to AFM experiments, infernan-pNIPAM solutions were prepared in PBS buffer (1X, pH 7.4, Gibco) at 10 % w/v for EPS-p5-16% and EPS-p5-8% or 7 % w/v for EPS-p5-3%. After overnight dissolution at room temperature, 30 µL of each solution were deposited on a Petri dish heated at 37 °C for 30 min (Scienceware CultureTemp™), allowing for gel setting. DMEM preheated at 37 °C was then added. All AFM experiments were carried out using NanoWizard II® Atomic Force Microscope (JPK, Germany).

The surface of infernan-pNIPAM hydrogels was firstly imaged using the intermittent contact mode to limit the tip-surface shear component. A cantilever (MSCT, Bruker) with a free
resonance frequency of 20 kHz, a spring constant of 0.07 N/m and a tip radius of ~ 10 nm was used. High-resolution images (10 µm x 10 µm) were recorded in DMEM at 37 °C before to be processed using JPK Data Processing software (JPK, Germany).

Mechanical properties of EPS-pNIPAM hydrogels were performed between 30 min and 2 h after the preparation of the hydrogels in DMEM at 37 °C, imposed by a temperature controller with an accuracy of 0.1 °C. Force-distance curves and force relaxation curves were measured on several surface points on two hydrogels per synthesis condition (EPS-p5-3%, EPS-p5-8%, EPS-p5-16%). Cantilevers with a 5 µm diameter glass sphere (SQube) were used for these experiments. Their normal spring constant, systematically calibrated by the thermal noise method (implemented in the JPK software) before experiments, was comprised between 0.1 and 0.2 N/m. Force-distance curves were recorded with a constant speed of 1 µm/s to neglect the hydrodynamic drag forces exerted on the cantilever by cell culture medium (proportional to the cantilever velocity). These curves were then converted into force-indentation curves, and the approach part was fitted by the Hertz model (equation 2), using the least-squares method to determine the elastic modulus of hydrogels. Equation (2) describes this model relating the force F applied by a spherical AFM tip of radius R to generate indentation δ by considering the sample as a semi-infinite material characterized by its elastic modulus E and Poisson coefficient ν as [34]:

$$F = \frac{4\sqrt{R}}{3(1-\nu^2)} E \delta^{3/2} \qquad (2)$$

As the maximum indentation depth (~ 1 µm) was lower than the hydrogel thickness (~ 3 mm), the hypothesis of semi-infinite thickness of material was appropriate. The other mechanical experiments conducted by AFM on these hydrogels consisted in adding a holding phase of few seconds (between 2 s and 5 s) between the approach and retraction parts of force-distance curves.
These modified force curves, commonly called force relaxation curves, were obtained after rapid indentation of the AFM tip (at high speed of 20 µm/s) into the hydrogel until to reach a predefined indentation depth, by recording the force decay while the indentation depth was kept constant. To avoid overestimation of mechanical parameters, force relaxation curves were systematically corrected by the hydrodynamic drag force, which is proportional to the cantilever velocity. Only the initial applied force measured at high speed of 20 µm/s (i.e. when the predefined indentation depth was reached) was corrected following the procedure previously described, while the velocity was zero in the holding phase [35].

## 2.7. Cytocompatibility of the infernan-pNIPAM hydrogels

Cytocompatibility of infernan-pNIPAM hydrogels was assessed on human embryonic kidney cells line, HEK293, in complete DMEM (DMEM, fetal bovine serum (VWR) (10 % v/v), penicillin/streptomycin (Gibco) (1 % v/v) and GlutaMAX 100X (Gibco) (1 % v/v)). Cells were passaged once a week using Trypsin 0.05 % - EDTA (Gibco). For experiments with hydrogels, HEK293 cultures were washed once with PBS, detached using pre-warmed Trypsin 0.05 % - EDTA, resuspended in complete DMEM and counted using KOVA Glassic Slides (Kova international). EPS-pNIPAM polymers were solubilized overnight at room temperature in PBS buffer (1X, pH 7.4, Gibco) at 10 % w/v for EPS-p5-16% and EPS-p5-8% or 7 % w/v for EPS-p5-3%. Solutions were then heated for 30 min at 80 °C to sterilize the samples. Absence of bacterial contamination was ensured by incubating each solution on plate count agar (PCA) medium for 72 h at 30 °C. HEK293 cells ($10^6$ cells/mL) were gently mixed with EPS-pNIPAM solutions and 30 µL of each solution were deposited in triplicate in a 24-well plate (Nunclon Delta, Thermo
Scientific), and incubated at 37 °C for 1 h (5 % CO₂, controlled humidity), allowing for gel setting.
Complete culture medium (1 mL) at 37 °C was then added to each well.

Cell viability at days 3 and 7 after cell seeding was evaluated using AlamarBlue® staining. Cells recovered from hydrogels after temperature decrease were incubated at 37 °C for 5 h with AlamarBlue® solution (G-Biosciences) diluted at 20 % v/v in complete DMEM. Fluorescence in the supernatants was measured using Varioskan LUX (ThermoFisher) (excitation: 560 nm; emission: 590 nm). Cell viability in EPS-pNIPAM hydrogels was normalized by comparing to positive control (cells seeded inside Cultrex™, Cultrex RGF BME, Bio-Techne). Cell viability was evaluated in triplicate in three independent experiments.

## 2.8. Statistical analysis.

All experimental results were expressed as a mean value ± standard deviation (SD). Cell viability data were analyzed by Student *t* test to determine statistical differences between different hydrogels. Levels of significance were set at **p** < 0.05.

## 3. Results and Discussion

Covalent grafting of the amine extremity (R'-NH₂) of a commercial pNIPAM (Mn 5,500 g/mol, p5) onto uronic acids (R-COOH) of the native high-molecular weight infernan EPS (Mw 1,600,000 g/mol) *via* carbodiimide chemistry led to EPS-pNIPAM polymers (EPS-p5), as recently described (Fig. 1) [29]. By varying COOH:NH₂ molar ratio, three polymers, EPS-p5-3%, EPS-p5-8% and EPS-p5-16%, showing different densities of uronic acids grafted by pNIPAM (3 %, 8 % and 16 %) as well as decreasing polysaccharide content (24 %, 14 % and 9 %) were obtained. In semi-diluted solutions (0.1 % w/v) at room temperature, grafted polysaccharide chains were shown to form aggregates of very high molar weight (> 10⁹ g/mol) and dense spherical conformation,
resulting from inter-chain interactions between pNIPAM chains grafted on the polysaccharide
backbone. Due to the ability of pNIPAM to adopt a temperature-dependent structural change above
its LCST of ~ 32 °C [30], a decrease in the hydrodynamic diameter of EPS-pNIPAM polymers in
semi-diluted solutions was observed at 37°C confirming thus the collapsed state of polymers [29].
Indeed, inter-chain interactions between pNIPAM chains led to folding of the polysaccharide
chains, allowing a more compact structure to be adopted.

<figure><img src="image_2.png" /><figcaption>Fig. 1. Infernan grafting with pNIPAM using carbodiimide chemistry.</figcaption></figure>

3.1. *Swelling properties of infernan-pNIPAM hydrogels*

Thermosensitive properties of infernan-pNIPAM polymers previously observed at 37 °C in semi-diluted solutions led to gel formation at 37 °C, at high polymer concentrations: 10 % w/v for
EPS-p5-16% and EPS-p5-8%, and 7% w/v for EPS-p5-3%. At 37 °C, a rapid sol-gel transition of EPS-pNIPAM solutions of ~4 min was determined by measuring their absorbance at 550 nm (Fig. S1). Since sol-gel transition of EPS-pNIPAM polymers is reversible, a specific protocol was developed to measure the swelling ratio of the hydrogels obtained. The evolution of the swelling ratio over time of the EPS-pNIPAM hydrogels was followed in cell culture medium DMEM at 37 °C (Fig. 2A, Fig. S2). During the first hour of incubation, all three hydrogels presented similar initial state of hydration, which evolved differently depending on the hydrogel composition. EPS-p5-16% hydrogel, displaying the highest grafting density and the lowest EPS amount in the hydrogel (9%), presented the lowest swelling ratio with the equilibrium reached at 6 h. A three-fold increase in the swelling ratio was noticed for less grafted EPS-p5-8% hydrogel (14% EPS amount), which reached its swelling equilibrium at 24 h. In contrast, slightly grafted EPS-p5-3% (24% EPS amount) swelled over time with a six-fold ratio observed at 47 h. These results indicated that the hydrogel swelling was proportional to the EPS content inside hydrogels and inversely proportional to the EPS grafting density. The degree of crosslinking of the hydrogel network plays a crucial role in the integrity of the polymeric network formed and impacts its swelling properties [36,37]. The most grafted EPS-p5-16%, having the lowest polysaccharide amount, formed dense, highly crosslinked network mediated by inter-chain pNIPAM-pNIPAM interactions with low ability to swell (Fig. 2B). Less grafted EPS-p5-8% and EPS-p5-3% with higher EPS amounts were able to swell to form looser networks. Indeed, even if their initial states after gel setting were
similar to a dense network, their lower crosslinking densities favored their swelling over time (Fig. 2B).

<figure><img src="image_3.png" /><figcaption>Fig. 2. (A) Evolution of the swelling ratio in time of infernan-pNIPAM hydrogels at 10 % w/v for EPS-p5-16% and EPS-p5-8%, and 7 % w/v for EPS-p5-3% in DMEM at 37 °C. (B) Schematic representation of EPS-pNIPAM hydrogel network (with respect to the degree of grafting and EPS amount in the grafted samples) during swelling: initial state after gel setting and final swollen state. EPS chains are presented in blue and pNIPAM chains in red.</figcaption></figure>

## 3.2. Investigation of infernan-pNIPAM hydrogels by Atomic Force Microscopy (AFM)

For future tissue engineering applications, mechanical properties of the thermoresponsive infernan-pNIPAM hydrogels were determined in fully hydrated state (DMEM) at physiological temperature using AFM. Indeed, contrary to other techniques, AFM offers the ability to measure the mechanical properties and to image the sample surface of soft materials under liquid environment, while controlling the temperature. In addition, spherical AFM tips with 5 µm in diameter, comparable to the cell size, can be used to probe the mechanical responses of hydrogels as perceived by cells. Firstly, AFM was used to image the surface of EPS-pNIPAM hydrogels immersed in DMEM at 37 °C (Fig. 3A). Highly dense network was observed for all three hydrogels
independently from grafting density and EPS amount.

<figure><img src="image_4.png" /><figcaption>Fig. 3. AFM experiments performed in fully hydrated state (DMEM) at 37 °C to image the surface topography of EPS-pNIPAM hydrogels and to measure their mechanical properties. (A) Representative height image (10 µm x 10 µm) of EPS-pNIPAM hydrogel surface (EPS-p5-3% hydrogel, 7 % w/v) obtained 1 h after DMEM hydration. (B) Experimental force indentation and force relaxation curves measured on EPS-pNIPAM hydrogels (EPS-p5-16% hydrogel, 10 % w/v).</figcaption></figure>

Mechanical properties of these hydrogels were then probed (Fig. 3B). Amongst various AFM-based methods developed to investigate the mechanical properties of soft materials, the most common consists in recording the force when the tip approaches the sample surface (approach curve) and during its withdrawal (retraction curve). These combined approach and retraction curves are usually called force-distance curves. Such curves were performed on several locations on EPS-pNIPAM hydrogels, as shown in Fig. 4A. For similar indentation depths of about 400 nm, the elastic modulus *E* of the investigated hydrogels was extracted by adjusting this single parameter in the Hertz model (equation (2)) using the least-squares method. A good agreement between the experimental curves and the theoretical ones resulting from the Hertz model, was observed. All values of the elastic modulus measured at different locations on hydrogels were
collected in the boxplot representation (Fig. 4B). The highest *E* values comprised between 3.6 kPa and 6.0 kPa with a mean value of 4.9 ± 0.9 kPa were measured for the slightly grafted EPS-p5-3% hydrogel, while the lowest ones between 2.2 kPa and 2.9 kPa with a mean value of 2.5 ± 0.3 kPa were obtained for the highly grafted EPS-p5-16% hydrogel. Intermediate *E* values between 3.2 kPa and 3.8 kPa (mean value of 3.5 ± 0.2 kPa) were determined for EPS-p5-8% hydrogel. It appeared that the elastic modulus of hydrogels was proportional to the EPS amount in each hydrogel (24 %, 14 %, 9 % for EPS-p5-3%, EPS-p5-8% and EPS-p5-16%, respectively) and inversely proportional to the degree of grafting of pNIPAM chains onto the polysaccharide backbone. Even if less pNIPAM-pNIPAM interactions can be established at low grafting density (EPS-p5-3% and EPS-p5-8%), high EPS content in these hydrogels led to a dense physical network reinforced by hydrophilic interactions between polysaccharide chains. In contrast, less hydrophilic interactions between polysaccharide chains can be formed in EPS-p5-16% hydrogel of low EPS content, even if more pNIPAM-pNIPAM cross-links can be set. This observation put forward high impact of the polysaccharide chains on the network structuring even at low grafting densities.

<figure><img src="image_5.png" /><figcaption>Fig. 4. (A) Experimental force-indentation curves measured on three EPS-pNIPAM hydrogels displaying three grafting densities (EPS-p5-3%, EPS-p5-8% and EPS-p5-16%) in DMEM at 37 °C. The approach curves were fitted</figcaption></figure>
by the Hertz model using the least-squares method with a single parameter, the elastic modulus $E$. (B) Boxplot representation of all elastic modulus values measured on the three hydrogels.

Since time-dependent mechanical properties of hydrogels are characterized by both elastic and dissipative components, the elastic component determined for EPS-pNIPAM hydrogels need to be completed with the dissipative one. This latter component was revealed by the hysteresis occurring between the approach and retraction curves for all hydrogels (Fig. 4A). In contrast, for a purely elastic material these curves are superimposed without hysteresis. Indeed, hydrogels with their high-water content possess a dissipative character resulting either from poroelastic or viscoelastic properties [35]. During the hydrogel compression by the AFM tip, mechanical responses of hydrogels are due either to solvent migration and its redistribution through the network structure (i.e. poroelasticity), or arise from unbinding of weak noncovalent crosslinks and their subsequent re-establishment (i.e. viscoelasticity) [38]. These two dissipation mechanisms, poroelasticity or viscoelasticity, can be distinguished by exploiting, respectively, the dependence or independence of the mechanical responses with the tip-surface contact size. By far, the common mean to modify the tip-surface contact size in AFM experiments is to vary the indentation depth. Indeed, the tip-surface contact radius $a$ is directly related to the indentation depth $\delta$ as described by the Hertz contact theory for a spherical indenter of radius $R$ by [34]:

$$a = \sqrt{R\delta} \qquad (3)$$

Here, force relaxation curves were measured at different indentation depths (200 nm, 650 nm, 800 nm) to probe the mechanical responses of the EPS-pNIPAM hydrogels, while varying the tip-surface contact size. Such curves were obtained by adding a holding phase between the approach and retraction curves of conventional force-distance curves, and the force was recorded as a function of time while the indentation depth was kept constant at a predefined value. Fig. 5A shows three force relaxation curves measured for the EPS-p5-16% hydrogel for indentation depths
comprised between 200 nm and 800 nm. For such indentation depths, the AFM tip-surface contact size varied by a factor two, between 1.4 µm and 2.8 µm. To analyze the dependence of these relaxation curves with the experimental contact size, raw curves were normalized in force using $(F - F_\infty)/(F_0 - F_\infty)$, where $F_0$ corresponds to the instantaneous force response measured when the indentation depth reached the predefined value (which determines the initial time), and $F_\infty$ is the constant force reached at equilibrium [39]. As these normalized curves were not superimposed into a single one, the dissipative character of the hydrogels was dependent on the tip-surface contact size, emphasizing poroelastic behavior of EPS-pNIPAM hydrogels (Fig. 5B). In addition, normalized curves revealed that relaxations were slower for larger contact sizes, due to longer migration distance of the solvent through the hydrogel network deformed by the AFM tip. To confirm the poroelastic origin of dissipative character of hydrogels, normalized curves were further normalized by dividing the experimental time by the poroelastic relaxation time $\tau$, which is defined by the ratio between the tip-surface contact area and the diffusion coefficient $D$ of the solvent within the hydrogel [40]:

$$ \tau = \frac{a^2}{D} \qquad (4) $$

The normalized experimental relaxation curves were then superimposed into a master curve indicating that experimental relaxations were proportional to the size of deformed network (Fig. 5C). An analytical expression describing the force response of a poroelastic material indented by a spherical indenter has been proposed by Hu et al. from finite-element simulations [39]:

$$ \frac{F(t)-F_{\infty}}{F_0-F_{\infty}} = 0.491 \exp^{-0.908\sqrt{\theta}} + 0.509 \exp^{-1.679\theta} \qquad (5) $$

where $\theta$ corresponds to the measured time normalized by the poroelastic time $\tau$. The good agreement obtained between normalized experimental relaxation curves and the universal curve (i.e. the equation 5 without any adjustable parameter) provided additional confirmation of the
poroelastic behaviour of thermoresponsive EPS-pNIPAM hydrogels (Fig. 5C). The same poroelastic origin of relaxation mechanisms was also revealed for EPS-p5-3% and EPS-p5-8% hydrogels (Fig. S3 and S4).

The poroelastic character of other pNIPAM-based hydrogels and more generally for polyacrylamide hydrogels was previously evidenced [41–43]. In contrast, it has recently been shown that hydrogels formed by infernan physically crosslinked by calcium, showed viscoelastic properties [35,44,45]. Thus, distinct dissipative mechanisms may occur for physically crosslinked infernan hydrogels, depending directly on the crosslink nature in the network. Indeed, weak ionic crosslinks in infernan/calcium hydrogels can easily be disrupted under the applied AFM force and reformed after, conducting to viscoelastic responses [35]. In the case of thermoresponsive infernan-pNIPAM hydrogels, hydrophobic interactions between pNIPAM chains grafted on the polysaccharide backbone were strong enough, compared to ionic crosslinks, to generate a poroelastic behavior, characterized by the solvent movement and its redistribution inside the network. Such a poroelastic behavior is usually observed for chemically crosslinked hydrogels with strong covalent bonds [38].
<figure><img src="image_6.png" /><figcaption>Fig. 5. (A) Experimental force relaxation curves measured on EPS-p5-16% hydrogels in solvent at 37 °C for three indentation depths of ~200 nm, ~650 nm and ~800 nm. (B) Non-superimposition of force relaxation curves after force normalization, where $F_0$ is the instantaneous force response and $F_\infty$ is the force reached at the equilibrium state. (C) Superimposition of force relaxation curves after both force and time normalizations, where $\tau$ is the poroelastic relaxation time, with the universal curve given by the Hu's equation. (D-F) Boxplot representation of poroelastic parameters determined on the EPS-pNIPAM hydrogels, (D) the diffusion coefficient $D$, (E) the elastic modulus $E$, (F) the Poisson's ratio $\nu$.</figcaption></figure>

In the following, poroelastic theory developed by Tanaka *et al.*, based on the theory originally formulated by Biot, was applied to determine the mechanical parameters characterizing poroelastic EPS-pNIPAM hydrogels [39,46,47]. The diffusion coefficient $D$ was obtained by incorporating the equation (4) into the equation (5) to fit the experimental relaxation curves with $D$ as the single adjustable parameter. In this way, the three experimental relaxation curves presented in Fig. 5A were simultaneously fitted using the least-squares method, with a best result obtained for a diffusion coefficient of $4.5 \cdot 10^{-12} \text{ m}^2/\text{s}$. All diffusion coefficient values obtained on
different hydrogels were reported on the boxplot representation in Fig. 5D. The mean *D* value in EPS-p5-3% hydrogels was 3.3 ± 0.9 µm²/s, 3.3 ± 1.1 µm²/s for EPS-p5-8% and 3.5 ± 1.0 µm²/s for EPS-p5-16%. These values, very close for the three hydrogels, were slightly lower than values reported in the literature for water diffusion in polyacrylamide and pNIPAM hydrogels in the range 10-30 µm²/s [43]. This difference by a factor three could be attributed to higher viscosity of DMEM containing 4.5 g/L of glucose compared to pure water [48]. Poroelastic relaxation times for these hydrogels were then comprised between ~550 ms and ~1.15 s, by considering an indentation depth of 1 µm and by combining equations (3) and (4).

At short times, the poroelastic hydrogel behaves like an incompressible elastic material because the fluid flow is negligible and the elastic modulus of the hydrogel can be determined from the instantaneous force [39]:

$$E = \frac{9}{16} \frac{F_0}{\delta^{3/2} R^{1/2}} \qquad (6)$$

The elastic modulus determined from the force relaxation curves are presented in Fig. 5E with a mean value of 4.6 ± 0.4 kPa, 3.5 ± 0.2 kPa and 2.0 ± 0.4 kPa for EPS-p5-3%, EPS-p5-8% and EPS-p5-16% hydrogels, respectively. All these values were in very good agreement with those previously determined by fitting the Hertz model on the approach force curves (Fig. 4A). The elastic modulus values obtained for thermoresponsive EPS-pNIPAM hydrogels corresponded well to elastic modulus obtained for physically crosslinked infernan/calcium hydrogels, varying from ~1 kPa to ~8 kPa depending on the polysaccharide and calcium concentrations [35,44].

After the solvent redistribution through the pores of the hydrogel network, the Poisson's ratio *v* of the new equilibrium state reached by the hydrogels can be determined from the ratio between the instantaneous force and the equilibrium force as [39]:

$$\frac{F_0}{F_\infty} = 2(1-\nu) \qquad (7)$$
Poisson's ratio with a mean value of 0.33 ± 0.02 kPa, 0.38 ± 0.02 kPa and 0.34 ± 0.02 kPa were obtained for EPS-p5-3%, EPS-p5-8% and EPS-p5-16% hydrogels, respectively (Fig. 5F). These values are in good agreement with those reported for others polyacrylamide-based hydrogels in the range 0.3-0.4 kPa [41,43]. From all these extracted mechanical parameters, an estimation of the pore size φ of the hydrogel network can then be obtained [49]:

$$ \phi = 2 \sqrt{\frac{D\eta(1+\nu)(1-2\nu)}{E(1-\nu)}} \qquad (8) $$

where η is the solvent viscosity. Using the determined mean values of D, E, ν and using the DMEM viscosity at 37 °C of 0.73 mPa.s [48], the pore size was comprised between 1.2 nm and 1.8 nm for the three EPS-pNIPAM hydrogels. The porosity of these hydrogels was shown nanometric, in agreement with the AFM image of the hydrogel surface (Fig. 3A). Similar pore sizes comprised between 1 nm and 10 nm were reported in the literature for polyacrylamide hydrogels, with a dispersity coming from the synthesis conditions [41,43]. Nanoporous hydrogels based on calcium crosslinked alginate were also previously reported [23,50].

### 3.3. Cytocompatibility of infernan-pNIPAM hydrogels

Evaluation of the cytocompatibility of infernan-pNIPAM hydrogels constitutes a key-step for their further preclinical use. HEK293 cells are commonly used for viability assessment in hydrogels, including pNIPAM-based hydrogels [51–54]. Viability of HEK293 cells seeded inside EPS-pNIPAM hydrogels was evaluated using AlamarBlue staining. Fluorescence measured at day 3 (D3) and day 7 (D7) were normalized with those obtained for cells seeded inside Cultrex™, used as positive control (Fig. 6A). At D3, the lowest cell viability was measured for the highly grafted EPS-p5-16% (20 %) hydrogel, compared to less grafted EPS-p5-8% (77 %) and EPS-p5-3% (62 %) hydrogels. With increasing incubation time, at D7, cell viability increased up to 50 % for EPS-
p5-16% and around 85 % for both EPS-p5-8% and EPS-p5-3% hydrogels. In these less grafted hydrogels, cells were organized in large spheroids whose size has increased compared to D3 (~ 30 µm vs ~ 150 µm) (Fig. 6B), as observed for Cultrex™ (Fig. S5).

<figure><img src="image_7.png" /><figcaption>Fig. 6. (A) Viability of HEK293 cells seeded (at 10⁶ cell/mL D0) in infernan-pNIPAM hydrogels at 10 % w/v for EPS-p5-16% and EPS-p5-8% and 7 % w/v for EPS-p5-3%. (B) Optical microscopic images of HEK293 cells in infernan-pNIPAM hydrogels (Gx10). Cultrex™ was used as a positive control (Fig. S5).</figcaption></figure>

It appeared that both the EPS content within infernan-pNIPAM hydrogels and the number of pNIPAM chains grafted on the polysaccharide backbone were determinant for cell viability. Indeed, less grafted hydrogels rich in EPS (EPS-p5-8% and EPS-p5-3%), showed important swelling (Fig. 2), leading to loose networks, that sustained cell growth. In contrast, dense network formed by highly grafted EPS-p5-16% hydrogel appeared less favorable for cell growth most likely by preventing cell volume expansion, thus limiting cell proliferation and/or cell metabolism.

Lee et al. (2017) reported that mechanical confinement regulates cartilage matrix formation by chondrocytes in nanoporous not degradable viscoelastic alginate/calcium hydrogels. In slow relaxing hydrogels, spacial confinement of cell volume activated IL-1β signaling, resulting in
increased cell death and catabolic gene expression. Moreover, restriction of cartilage matrix
deposition by chondrocytes in these hydrogels contributed to cell death. In contrast, in fast relaxing
hydrogels, elastic stresses resisting expansion of the chondrocyte volume were relaxed, allowing
the cells to expand [22]. Recently, viscoelastic fast relaxing alginate/calcium hydrogels were
shown to be more permissive to cell migration and fusion of encapsulated mesenchymal stem cell
spheroids, compared to slow relaxing hydrogels [55]. Indeed, spheroids have gained particular
attention in the field of tissue engineering due to their enhanced regenerative properties [56].

In the case of infernan-pNIPAM hydrogels with fast poroelastic relaxations lower than 1.2 s, HEK293 cells were able to grow and associate in large spheroids (Fig. 6B), suggesting that cells were able to deform the network, which then dissipated through the solvent movement inside. In particular, the solvent redistribution was facilitated in highly swelled less grafted EPS-p5-8% and EPS-p5-3% hydrogels, which likely positively contributed to nutrient and waste exchanges, thus favoring cell development. However, high density of crosslinks through pNIPAM hydrophobic interactions in EPS-p5-16% hydrogel led to hardly deformable hydrogel unable to swell, considerably restricting cell volume expansion. Similarly, viability of nucleus pulposus cells of the intervertebral disc was shown strongly affected by the network density in hyaluronic acid (HA)-pNIPAM hydrogels [57,58]. Cell viability in HA-pNIPAM hydrogel displaying loose network, formed by high-molecular weight HA (1,700,000 g/mol) and pNIPAM of 20,000 g/mol, was greater (90 %) to that measured in a dense network, obtained with pNIPAM of higher molecular weight (35,000 g/mol) (69 % at D3 and 50 % at D7) [58]. Lower cell viability was correlated with a decrease in hydrogel volume (by 30 %) due to chain collapse, more important for pNIPAM with longer chains. Cytocompatible HA-pNIPAM hydrogel was then shown
biocompatible, causing no interferences with the intrinsic healing response, when injected into a
rabbit osteochondral defect [59].

4. Conclusion

Infernan, a marine bacterial EPS grafted with pNIPAM was shown to form
thermoresponsive hydrogels through hydrophobic interactions established between pNIPAM
chains grafted on the polysaccharide backbone. Due to the synthesis conditions applied, hydrogels
with different polysaccharide rate and grafting densities were obtained. Both parameters were
shown crucial for the network structuring and resulting mechanical properties. Determination of
the mechanical properties of fully hydrated hydrogels maintained at physiological temperature by
AFM revealed that infernan grafted with pNIPAM formed poroelastic hydrogels. This implies that
the energy accumulated during deformation is dissipated through the solvent movement within the
network. It was further shown that the elastic modulus of the hydrogels was impacted by both the
polysaccharide grafting density and the polysaccharide amount. It was observed that the elastic
modulus *E* of hydrogels decreased from 4.9 kPa, 3.5 kPa to 2.5 kPa with decreasing polysaccharide
content (from 24 %, 14 % to 9 %) and increasing amount of grafted pNIPAM (i.e. by increasing
grafting densities from 3 %, 8 % to 16 %). Within the first hour of incubation in cell culture
medium after gel setting at 37 °C, all hydrogels formed similar compact structures through the
collapse of pNIPAM chains. However, higher amount of high molecular weight infernan within
the hydrogels led to stronger network reinforced by hydrophilic polysaccharide-polysaccharide
interactions. During incubation of these thermoresponsive hydrogels in cell culture medium at
37 °C, distinct swelling behaviors were observed. Indeed, dense network with only low ability to
swell was evidenced for the highly grafted infernan (EPS-p5-16%). On the opposite, for slightly
grafted infernan (EPS-p5-3% and EPS-p5-8%) with lower density of pNIPAM-pNIPAM
crosslinks, hydrogels were able to swell over time, leading to loose networks. Such loose networks were further shown to be more favorable for cell growth, enabling HEK293 cells to form large spheroids. Expansion of cell volume in these deformable networks seemed crucial for their development. The present study enabled to elucidate the relationship between the composition of grafted infernan and the biological and mechanical properties of the resulting thermoresponsive infernan-pNIPAM hydrogels. Based on these findings, slightly grafted infernan hydrogels will further be investigated for their use in cartilage engineering.
**Declarations of competing interest:** The authors declare no conflict of interest.

**Acknowledgments**

Financial support was provided by the French National Research Agency within the framework of the SmartIEs project (ANR-22-CE52-0005-01).

A CC-BY public copyright license has been applied by the authors to the present document and will be applied to all subsequent versions up to the Author Accepted Manuscript arising from this submission, in accordance with the grant's open access conditions.

<figure><img src="image_8.png" /></figure>

**Author contribution**

**Arnaud Fillaudeau:** Conceptualization, Methodology, Investigation, Formal analysis,

Validation, Writing – Original Draft, Writing – Review & Editing; **Marion Desdouits:** Conceptualization, Methodology, Validation, Writing – Review & Editing; **Sylvia Colliec-Jouault:** Conceptualization, Funding acquisition, Writing – Review & Editing; **Corinne Sinquin:** Writing – Review & Editing; **Agata Zykwinska:** Conceptualization, Methodology, Investigation,

Formal analysis, Validation, Funding acquisition, Writing – Original Draft, Writing – Review & Editing; **Stéphane Cuenot:** Conceptualization, Methodology, Investigation, Formal analysis,

Validation, Funding acquisition, Writing – Original Draft, Writing – Review & Editing.

**Data availability**

Data will be made available on request.
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