Dynamic Structuration of Physical Chitosan Hydrogels - Langmuir

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DYNAMIC STRUCTURATION OF PHYSICAL CHITOSAN HYDROGELS Nicolas Sereni, Alin Alexandru Enache, Guillaume Sudre, Alexandra Montembault, Cyrille Rochas, Philippe Durand, Marie-Hélène PerrardDurand, Grigore Bozga, Jean-Pierre Puaux, Thierry Delair, and Laurent David Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02997 • Publication Date (Web): 11 Oct 2017 Downloaded from http://pubs.acs.org on October 12, 2017

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DYNAMIC STRUCTURATION OF PHYSICAL CHITOSAN HYDROGELS

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Nicolas Sereni1, Alin Enache2, Guillaume Sudre1, Alexandra Montembault1, Cyrille Rochas3,

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Philippe Durand4, Marie-Hélène Perrard4, Grigore Bozga2, Jean-Pierre Puaux1, Thierry

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Delair1, Laurent David1*

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1

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Matériaux Polymères IMP@Lyon1, 15 bd Latarjet, 69622 Villeurbanne Cedex France

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2

Université de Lyon, Université Claude Bernard Lyon 1, CNRS UMR 5223 Ingénierie des

Centre for Technology Transfer in the Process Industries, Department of Chemical

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Engineering, University POLITEHNICA of Bucharest, 1 Polizu Street, RO-011061

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Bucharest, Romania

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3

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Recherches sur les Macromolécules Végétales, boîte postale 53, F-38041 Grenoble Cedex

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France

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Université de Grenoble, Université Joseph Fourier, CERMAV-CNRS UPR5301 Centre de

Kallistem, Ecole Normale Supérieure de Lyon, 46 allée d'Italie 69364 Lyon Cedex 07

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*corresponding author. Email : [email protected]., Laboratoire Ingénierie des Matériaux Polymères IMP@Lyon1, 15 bd Latarjet, 69622 Villeurbanne Cedex France. Téléphone : +33 (0)4 72 43 16 05.

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ABSTRACT

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We studied the microstructure of physical chitosan hydrogels formed by neutralization of

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chitosan aqueous solutions highlighting the structural gradients within thick gels (up to

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thicknesses of 16 mm). We explored a high polymer concentrations range (Cp ≥ 1.0 % w/w)

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with different molar mass of chitosan and different concentrations of the coagulation agent.

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The effect of these processing parameters on the morphology was evaluated mainly through

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small angle light scattering (SALS) measurements and confocal laser scanning microscopy

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(CLSM) observations. As a result, we reported that the microstructure is continuously

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evolving from the surface to the bulk, with mainly two structural transitions zones separating

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3 hydrogel types. The first zone (zone I) is located close to the surface of hydrogel and

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constitutes a hard (entangled) layer formed in fast neutralization conditions. It is followed by

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a second zone (zone II) larger in thickness (≈ 3-4 mm), where in some cases, large pores or

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capillaries (diameter~10µm), oriented parallel to the direction of gel front are present. Deeper

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in the hydrogel (zone III), a finer oriented microstructure, with characteristic sizes lower than

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2-3 µm, gradually replace the capillary morphology. However, this last bulk morphology

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cannot be regarded as structurally uniform, since the size of small micron-range oriented

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pores continuously increase as the distance to the surface of hydrogel increases. These results

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could be rationalized through the effect of coagulation kinetics impacting the morphology

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obtained during neutralization.

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II

II-III

III

tubular Micro-range porous capillaries microstructure

I

primary membrane

top of hydrogel

direction of the gel front

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INTRODUCTION

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Hydrogel material engineering is a emergent field of research, with numerous applications.1–5

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Hydrogels are cross-linked networks of hydrophilic polymers capable of retaining large

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amounts of water yet remaining insoluble.6 Due to their hydrophilicity combined with water

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and because of their soft and rubbery consistence, biocompatible hydrogels closely resemble

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living tissues and have emerged as promising materials in biomedical science.7–13 Hydrogels

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are usually characterized by their swelling, transport, and mechanical properties. These

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properties are in turn influenced by the structural parameters of the network.3 In the case of

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natural polymers, this approach often requires the understanding of the material morphology

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at macroscopic, micrometric, sub-micrometric to nanometric scales (i.e. a ‘multiscale

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approach’) in order to understand and interpret the physical and biological properties of

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hydrogels. Hydrogels materials are extremely diverse and can be classified in a number of

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ways, according to their origin (natural and synthetic polymer), cross-linking method

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(chemically and physically), structure (according to the composition of the network and

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additional morphology details at higher scales: inter-penetrating networks, homopolymer

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networks, double networks, semi-crystalline systems, porous hydrogels), charge (anionic,

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cationic, amphoteric, and non-ionic) and specific properties such as degradability and bio-

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degradability. Physically cross-linked hydrogels represent a hydrogel class that imply physical

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intermolecular forces such as hydrogen bonds, hydrophobic interactions, electrostatic ionic

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interactions or, at a higher scale, intermolecular assemblies such as guest–host inclusions,

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stereo-complexation and complementary binding.14,15 These interactions can be triggered by

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external stimuli such as temperature, pH, ionic strength, electric fields, light, static pressure,

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sound waves or the presence of specific molecules or ions. Physical cross-linking methods

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provide simple and safe approaches to prepare hydrogels for biomedical applications since

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they potentially offer gelation routes in water, avoiding the addition of possibly toxic

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crosslinkers or catalysts. Naturally-derived hydrogels, such as collagen, elastin, alginate,

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chondroitin sulfate, heparin, hyaluronic acid, and chitosan display multiple advantages over

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synthetic polymer gels for biomedical applications with respect to their inherent

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biocompatibility, biodegradability, and cytocompatibility properties.16–19

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Chitosan in particular is a polysaccharide derived from chitin consisting in N-acetyl-D-

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glucosamine (GlcNAc) and D-glucosamine (GlcN) residues linked by ß(1→4) glycosidic

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linkages. Such a family of polysaccharides can be considered, from structural arguments, as 3 ACS Paragon Plus Environment

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biosynthetic glycosaminoglycans. A specific difficulty in chitosan physico-chemistry is the

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correct determination of the degree of acetylation (DA i.e. molar fraction of GlcNAc), the

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molecular mass distributions, and the intra-molecular repartition of the acetylated and non-

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acetylated residues.20,21 Chitosans are generally known to exhibit exceptional biological

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properties as biocompatibility, biodegradability and bioactivity, and chitosan based materials

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are envisioned in many applications.22–32 However, these properties are related to the

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structural parameters of chitosan and also its diverse physical forms (solutions, nanoparticles,

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films, hydrogels, etc.). Chitosan is soluble in aqueous acidic solutions thanks to the

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protonation of the amine groups of GlcN residues that limits the establishment of interchain

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interactions (hydrophobic junctions and H-bonding). In solution, the behavior and the

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organization of chitosan chains are influenced by the combination of two groups of factors: (i)

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structural parameters of the polymer and (ii) environmental parameters (pH, ionic strength,

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quality of the solvent and temperature).33–35 Several works studying the behavior of chitosan

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chains in a dilute and semidilute regime evidenced the presence of supramolecular structures

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which vary from aggregates to nanoparticules, depending on the physico-chemical context,

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polymer concentration and DA.36–38 It has been additionally reported that the morphologies of

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chitosan in the solid and gel states are inherited by the structural organization of the initial

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chitosan solution through a ‘continuum’ of structural analogies.38

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Physical chitosan hydrogels can be prepared with a variety of ways. Ionotropic gels can be

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formed by complexation with different metallic cations 39,40 or anions (sulfate ions, phosphate

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ions)41–43. Polyelectrolyte complexes are readily formed by cooperative ionic interactions

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between chitosan in the protonated state and negatively charged polyanions.14,44,45 Chitosan is

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also capable of forming hydrogels by itself without the use of any polyion or complexation

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agent. Starting from an aqueous acidic chitosan solution, an increase of the pH in the proper

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physico-chemical conditions leads to aqueous physical chitosan hydrogels. During

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neutralization, as the pH increases close to the apparent pKa of the amine groups, the apparent

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charge density of the chitosan chains decreases, and the chains become more flexible (the

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electrostatic contribution of the persistence length vanishes); the balance of hydrophobic and

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hydrophilic interactions is changed up to a critical point that induces chitosan gelation.46 Such

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gelation in contact with a base (e.g. sodium hydroxide, potassium hydroxide or ammonia)

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needs further washing steps to eliminate salts and excess of base. No organic solvent or toxic

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crosslinker are necessary for this ‘hydrophobic’ gelation process. Polyols such as propanediol

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or glycerol can be added to the initial chitosan aqueous solutions to obtain hydro-alcoholic 4 ACS Paragon Plus Environment

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solutions. The drying of such solutions yields alcogels.47–49 The neutralization of alcogels

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yields in turn hydrogels with different physical and biological properties than those obtained

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by the direct aqueous neutralization route.50

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Rivas et al. studied the morphology of hydrogels formed by neutralization of chitosan

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solutions (aqueous or hydro-alcoholics) and alcolgels by gaseous ammonia or sodium

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hydroxide solution.51 In the case of the neutralization of chitosan solutions and under specific

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conditions, they observed the formation of capillary structures parallel to the direction of gel

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front in chitosan hydrogels. The preparation of chitosan hydrogels by neutralization of

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solutions may induce localized flow occurring in the vicinity of the gel front. This

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phenomenon is known to induce the formation of ‘capillaries’ under specific conditions and

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was first evidenced and modeled in the case of alginate gels.52–56 Gelation was obtained by

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diffusion of copper ions (Cu2+) within an aqueous solution of sodium alginate. The periodic

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capillary organization resulted from the formation of local vortexes (at the gel front) induced

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by friction between the contracting alginate chains and surrounding solution. In case of

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alginate gels, formation and size of capillary depend on a number of parameters such as

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viscosity of the initial alginate solution, chain density, thickness of the contraction zone,

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friction coefficient between contracting chains and surrounding solution, and the diffusion

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coefficient of the coagulation agent. The presence of a concentration gradient at the gel front

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resulting in the collapse of chitosan chains from the solution to the gel via convective flow is

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responsible to the formation of capillaries. In this context, Rivas et al. studied the morphology

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of chitosan hydrogels formed by neutralization of chitosan solution with a polymer

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concentration ranging from 0.01 to 1.0 % (w/w). They observed the formation of a periodic

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capillary structure only at high concentrations. In fact, they showed the chitosan solution

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viscosity plays a key role to control the morphology at micron scale since the transition

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between isotropic structural regime to capillary formation occurs for a critical viscosity value.

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In addition, the nature and the concentration of the coagulating agent also determine the

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kinetics of gelation, influencing the resulting gel microstructure. However, other authors

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described microstructure gradients from the surface of the gels.57,58

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In this work, we aimed to study the microstructural organization of physical chitosan

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hydrogels formed by neutralization of chitosan aqueous solutions, systematically taking into

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account the structural gradients within thick gels (up to thicknesses of 16 mm). We explore a

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high polymer concentration range (Cp ≥ 1.0 % w/w) and obtain complex multilayered

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hydrogels with oriented structures at different concentrations of the coagulation agent and 5 ACS Paragon Plus Environment

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molar mass of chitosan. These results could be rationalized through the effect of coagulation

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kinetics impacting the morphology obtained during neutralization. The microstructure of

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hydrogels was studied convergently using optical microscopy in combination with small angle

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light scattering (SALS) and confocal laser scanning microscopy (CLSM).

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EXPERIMENTAL SECTION

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Materials. The initial highly deacetylated chitosans, produced from squid pens or shrimp

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shells, was supplied by Mahtani Chitosan Pvt.Ltd. (India, Mahtani batch indexes 114 and

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243). Sodium hydroxide pellets, ammonium hydroxide solution at 28–30 % (w/w) and glacial

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acetic acid were purchased from Sigma Aldrich.

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Chitosan Purification. In order to obtain a high-purity material, chitosan was dissolved at

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0.5% (w/v) in an aqueous acetic acid solution, by the addition of the necessary amount of acid

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to achieve the stoichiometric protonation of the -NH2 sites. After complete dissolution, the

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chitosan solution was consecutively filtered through Millipore membranes with pore sizes of

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3, 0.8 and 0.45 μm. Then, dilute ammonia was added to the filtered chitosan solution to fully

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precipitate the polymer. Finally, the precipitate was repeatedly rinsed with distilled deionized

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water until a neutral pH was achieved. Then it was centrifuged and lyophilized.

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1

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was calculated from 1H nuclear magnetic resonance spectroscopy.59 10 mg of purified

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chitosan was dissolved in 1 mL of D2O containing 0.06 mM of HCl. Spectra were recorded on

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a Bruker Avance III 400 US+ spectrometer (400 MHz) at 25°C. The DA was deduced from

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the ratio of the area of the peaks of the methyl protons of the N-acetyl glucosamine residues to

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that of all of the H2 to H6’ protons of both glucosamine and N-acetyl glucosamine residues.59

H Nuclear Magnetic Resonance Spectroscopy. The degree of acetylation (DA) of chitosan

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Size Exclusion Chromatography Coupled with Multiangle Laser Light Scattering. The

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mass average molar mass of chitosan and its dispersity were determined by size exclusion

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chromatography (SEC) coupled on line with a differential refractometer (Waters R410, from

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Waters-Millipore) and a multiangle laser-light scattering detector operating at 632.8 nm

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(Wyatt Dawn DSP). The refraction index increment dn/dc depends on the DA as determined 6 ACS Paragon Plus Environment

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in previous studies.60 For the chitosan samples used in this study, dn/dc is close to 0.198

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mL/mg. A 0.15 M ammonium acetate / 0.2M acetic acid buffer (pH = 4.5) was used as eluent

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at a flow rate of 0.5 mL/min on Tosoh TSK PW 2500 and TSK PW 6000 columns. The

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polymer solutions were prepared by dissolving 1 mg of polymer in 1 mL of buffer and then

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refiltered through a Millipore membrane with a pore size of 0.45 µm before an injection of

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100 μL.

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Thermogravimetric Analysis. The water content of freeze-dried chitosan samples was

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evaluated on 10 mg with a TA Instrument Q500 thermogravimetric analyser (TGA) operating

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at a ramp of temperature of 5°C/min under helium flow. The full characterization of the

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different chitosan samples used in this work are given in Table 1.

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Table 1. Characteristics of the freeze-dried Chitosan Samples Used in This Work: mass

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average molar mass (Mw), Dispersity (Đ), Degree of Acetylation (DA), and Water

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Content (W). Batch index

Source

Mw (kg.mol-1)

Đ

DA (%)

W (%)

114

Squid pens

570 ± 10

1.5 ± 0.3

4.0 ± 0.1

3.2 ± 0.3

243

Shrimp shells

170 ± 2

1.7 ± 0.2

1.1 ± 0.2

3.4 ± 0.1

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Preparation of Chitosan Hydrogels. Physical chitosan hydrogels were prepared by gelation

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of chitosan acetate aqueous solutions containing different chitosan concentrations ranging

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from 0.75 to 4.00 % (w/w).61 The solutions were first obtained by dispersing purified and

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neutralized chitosan lyophilizates into water, and then adding acetic acid was added in

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stoichiometric amount to achieve the protonation of -NH2 sites. In order to limit the

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evaporation of water, the solutions were mechanically stirred in a closed reactor. After

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polymer dissolution (almost 16 hours), the resulting solutions were placed in syringes and

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centrifuged for 10 minutes at 5000 rpm using ProcessMate 5000 centrifuge (Nordson EFD) to

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remove air bubbles. In order to study the gel formation, the solutions were then extruded (with

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Performus I, Nordson EFD) through a needle tip of 1.54 mm diameter into a transparent

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“Special Optical Glass” cell (volume 700 μL) manufactured by Hellma Analytics (Hellma ref

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100-OS, height = 45 mm, width = 12.5 mm, and light path = 2 mm). The coagulation of the 7 ACS Paragon Plus Environment

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chitosan solutions was performed by placing the glass cells in a bath containing sodium

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hydroxide agitated magnetically at room temperature. After a coagulation time of 24 hours,

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the optical cell now filled with physical chitosan hydrogel was placed in deionized water bath

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and was washed until neutral pH was reached and complete elimination of salts. Washing did

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not induce any significant change in the structure of hydrogels.

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Rheological Measurements. Rheological measurements were carried out with a stress-

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controlled rheometer AR 2000 (TA Instruments), in Couette or cone-plate geometries.

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Measurements were carried out at 25°C with shear rates ranging from 10-3 to 10-2 s-1. The

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Cross model was used to fit experimental results and determine the zero-shear viscosity value

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( ):

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(Equation 1)

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where

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characterizing the shear-thinning behavior.62

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Table 2. Parameters of the Cross equation (see Equation 1) for chitosan acetate

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solutions.

is the steady shear rate,

is a characteristic rheological time and

Batch index 114 Cp (% w/w)

(Pa.s)

is an exponent

Batch index 243

(s)

(Pa.s)

(s)

1.00

3.3

0.0414

0.57

/

/

/

1.50

22

0.193

0.59

/

/

/

2.00

95

0.823

0.61

/

/

/

2.50

285

2.14

0.55

/

/

/

3.00

912

7.17

0.46

4.8

0.00730

0.51

4.00

/

/

16

0.0290

0.58

5.00

/

/

55

0.105

0.56

6.00

/

/

173

0.397

0.53

7.00

/

/

582

1.64

0.51

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Small Angle Laser Light Scattering. SALS measurements were performed by using an

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experimental setup equipped with a helium-neon laser (Spectra-Physic, USA, P=1mW) 8 ACS Paragon Plus Environment

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working at a wavelength of 633 nm with a beam cross-section close to 1 mm2. The setup

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included a Fresnel lens with a 13 cm focal length and a beam stop magnetically fixed onto the

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Fresnel length. The resulting signal scattered by the sample was captured with a two-

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dimensional detector (CCD camera Micam VHR 1000). One-dimensional profiles are

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obtained after averaging intensities from only thin rectangular horizontal selections centered

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on the beam stop (conventional radial averages were not performed with anisotropic samples).

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For these measurements, the cell was positioned (within the focal plane) to locate the laser

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beam at different distances below the surface of the hydrogel.

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Kinetics of coagulation. The kinetics of the coagulation was investigated by measuring the

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propagation of the gel front into a transparent “Special Optical Glass” cell (volume 350 μL)

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manufactured by Hellma Analytics (Hellma ref 100-OS, height = 45 mm, width = 12.5 mm,

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and light path = 1 mm). In this aim, we were captured images of the formed gel layer, at

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different coagulation times, using an Olympus BX41 (4x objective) optical microscope,

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coupled with an Olympus DP26 camera, connected on-line to a PC. All the experiments were

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conducted at room temperature. In each experiment, the cell filled with chitosan solution was

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immobilized (by a double sided tape) on the bottom of a Petri dish (100 mm in diameter)

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containing the coagulation base solution, and the camera was focused and positioned

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adequately to follow the variation of gel thickness inside the cell. Thereafter, a volume of

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approximately 80 mL of aqueous sodium hydroxide solution was poured into the Petri dish

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when the chronometer was started. The aqueous NaOH solution was not mechanically

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agitated, due to technical difficulties (lack of space, due to the volume occupied by the

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microscope).

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Confocal Laser Scanning Microscopy. Hydrogels were placed directly on a 150 µm thick

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glass slide with an excess of water to maintain the hydrated state of the samples during their

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observation. They were observed using an inverted confocal laser-scanning microscope (Zeiss

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LSM 510) powered by an argon laser, and available at the Centre Technologique des

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Microstructures at Université Claude Bernard Lyon 1. Samples were excited at a wavelength

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of 488 nm. The samples were viewed using an oil immersion 40x lens (1.3 numerical

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aperture). The signal is given by the reflective or fluorescent properties of chitosan hydrogels

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without any specific probe. 9 ACS Paragon Plus Environment

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RESULTS AND DISCUSSION

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In order to avoid confusions with the term “thickness”, we define the parameter d (for depth),

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which represents the distance from the observation point to the top (or ‘first surface’) of the

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gel.

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Structural organization of a physical chitosan hydrogel as a function of d

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Initially we chose to study the structural organization of a physical chitosan hydrogel prepared

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by neutralization of an aqueous chitosan acetate solution (Mw = 570 kg.mol-1; DA = 4.0 %)

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concentrated to 1.5 % (w/w) with a 1 M sodium hydroxide solution. According to the works

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of Rivas et al., these conditions of gelation are ideal for the formation of capillaries in the

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chitosan hydrogel (accounting for the structural parameters of chitosan, viscosity of the

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solution, concentration of the base). The observations under the optical microscope of a

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physical chitosan hydrogel indeed confirmed the presence of oriented structures, parallel to

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the direction of propagation of the gel front i.e. growth direction of the gel layer (Figure 1). base solution

top of hydrogel

primary membrane

=0 oriented structures

base flux OH-

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structural gradient

bottom of hydrogel

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Figure 1. Optical microscopy observation of a physical chitosan hydrogel showing oriented

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microstructures. These microstructures show an orientation parallel to the alkaline flux. The

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hydrogel was prepared from an aqueous chitosan solution (Mw = 570 kg.mol-1, DA = 4.0 %,

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and Cp = 1.5 % w/w) neutralized in a 1 M sodium hydroxide solution. 10 ACS Paragon Plus Environment

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These oriented structures are similar to those described in the work of Rivas et al.51 However,

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they are not present in all the hydrogel (Figure 1). The zone close to the top of the hydrogel,

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which corresponds to the zone of initial contact with the alkaline solution, does not contain

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oriented structures. This zone is about 200 micrometers thick, it is compact and homogeneous

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with high mechanical properties as shown by Fiamingo et al.57 In the case of physical alginate

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hydrogels, which comprise capillaries in their structure, the presence of a zone without

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capillary called “primary membrane” close to the top of hydrogel was also observed.56 In the

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following, we will show that the size of this zone lies between 100 micrometers and several

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millimeters depending on the conditions of gelation. Besides this difference of structural

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organization of hydrogels between the first membrane and the deeper “oriented structures

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zone”, there is a strong structural gradient within the zone where the oriented structures are

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present (Figure 2). A

B

C

D

E

F

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Figure 2. Two-dimensional SALS images of a physical chitosan hydrogel obtained at

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different distances from the top of the gel. All images were obtained with the same detector

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gain. The scattering vector q ranges from 0 up to 4 x 10-3 nm-1. The hydrogel was prepared

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from an aqueous chitosan solution (Mw = 570 kg.mol-1, DA = 4.0 %, and Cp = 1.5 % (w/w))

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neutralized by a 1 M sodium hydroxide solution. The base flux is vertical. 11 ACS Paragon Plus Environment

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At all investigated distances d, the two dimensional SALS images of Figure 2 are

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characteristic of the presence of anisotropic structures in hydrogels. SALS analyses confirm

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the formation of objects elongated in the direction of the gel front. However, SALS

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observations of hydrogel for d < 6 mm are quite different from those obtained with larger

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values of d, where the kinetics of gelation is slower. Figure 2A and 2B display sharp scattered

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streaks in the equatorial (horizontal) direction close to the beamstop, whereas the SALS

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patterns of figure 2C-F display a correlation peak at larger angles. Thus, the morphology of

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physical chitosan hydrogels is characterized by different length scales: close to the surface of

300

the gel, (d~2 to 4 mm) large oriented structures are present and can be evidenced by confocal

301

microscopy (see below). In the depth of the hydrogel at d > 6 mm, smaller and distinct

302

oriented structures are present and give rise to the correlation spots observed in figure 2C-F.

303

When the distance d increases, the scattering vector

304

peak maximum (

of the correlation

) is shifted to lower values (Figure 3).

300 ξ (µm)

3.5

d = 4 mm d = 6 mm d = 8 mm d = 10 mm d = 12 mm d = 14 mm d = 16 mm

3.0 2.5 2.0 1.5 1.0

200

0.5 0

0

2

4

6

8 10 12 14 16 18

d(mm)

I (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 35

100

0 0

1.10

-3

2.10

-3

3.10

-3

4.10

-3

-1 q (nm )

305 306

Figure 3. Equatorial SALS plots for the physical chitosan hydrogel measured at different

307

distances from the top of the gel. The hydrogel was prepared from an aqueous chitosan


Page 13 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

308

solution (Mw = 570 kg.mol-1, DA = 4.0 %, and Cp = 1.5 % (w/w)) neutralized by a 1 M

309

NaOH solution. “a.u.”: arbitrary units.

310 311

The Bragg correlation length (

) defining the periodicity of small oriented

312

structures, thus increases when the distance d increases. The periodicity of small oriented

313

structures is ranging from 2 µm to 3 µm at distance d of 8 mm and 16 mm respectively. In

314

contrast, closer to the surface at 0.2 < < 6 mm, the size of oriented structures is much larger

315

with a different morphology, as shown by confocal laser scanning microscopy (CLSM)

316

evidencing the presence of larger oriented capillary microstructure (Figure 4).

317 318

Figure 4. CLSM micrographs of a physical chitosan hydrogel measured at 1 mm from the top

319

of the gel. The hydrogel was prepared from an aqueous chitosan solution (Mw = 570 kg.mol-1,

320

DA = 4.0 %, and Cp = 1.5 % (w/w)) neutralized by a 1 M NaOH solution.

321 322

As expected,51 these large oriented capillaries are parallel to the direction of the gel front.

323

However, they appear as non-continuous voids with cigar shapes of about 50 µm long,

324

possibly because of the misalignment between the observation plane and the local axis of the

325

capillary structures. Moreover, their lateral size (~10 μm at the center) is well above that of

326

small oriented microstructures described previously (appearing at d > 6 mm). The CLSM

327

observations of a physical chitosan hydrogel at various distances d reveal that such capillary

328

microstructures in the 10-50 µm size range are present at depth

329

“surface zone” or “primary membrane” (d < 200 μm, see Figure 1) does not contain oriented

330

microstructures. The CLSM observations thus also confirm the previous optical microscopy

331

observations which show a compact zone close to the surface of hydrogels (figure 1). From a

332

distance d higher than 4 mm, the large oriented “tubular pores or capillaries” gradually


between 0.2 to 6 mm. The

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 35

333

disappear and the structure of hydrogel is dominated with small (micron-range) oriented

334

microstructures.

335

The reflexion at the surface of large pores in the hydrogel is responsible of the sharp streaks

336

observed in the equatorial direction on SALS images (see Figure 2). This sharp streaks

337

appears only at distances d (2 and 4 mm) where the tubular pores/capillaries were observed in

338

CLSM. Moreover, this signal is more pronounced closer to the surface (at depth distance

339

2 mm) in comparison with

340

fewer.

341

Microstructures similar to tubular pores described here were also observed in physical

342

collagen hydrogels. Furusawa et al. showed that macroscopic (mm size) tubular pores are

343

formed in physical collagen hydrogels prepared by gelation of an aqueous collagen solution

344

with a phosphate buffer solution.63 The CLSM observations of collagen hydrogels showed

345

that these tubular pores are parallel to the direction of the gel front and that they do not

346

constitute continuous structures throughout the gels. Such structures were studied by SALS,

347

and images also revealed the presence of sharp streaks perpendicular to the gel front direction,

348

as it is observed here in figure 2A and 2B. Thus, according to the system under investigation

349

and the gelation conditions, large tubular capillary pores may be much larger than the ones

350

observed in this work.

351

As a first conclusion, a convergent analysis combining optical microscopy, SALS, and CLSM

352

showed that the microstructure is continuously evolving from the surface to the bulk, with

353

mainly two structural transitions zones separating 3 hydrogel types (physical chitosan

354

hydrogel prepared by neutralization of an aqueous acetate chitosan solution MW =

355

570 kg/mol; DA = 4.0%; polymer concentration: 1.5 % w/w). The first zone (zone I) is

356

located close to the surface of hydrogel and constitutes a hard (entangled) layer formed in fast

357

neutralization conditions. It is followed by a second thicker zone (zone II) (t ≈3-4 mm), where

358

large oriented pores or capillaries, parallel to the direction of gel front are present. Deeper in

359

the hydrogel (zone III), smaller oriented objects with characteristic sizes lower than 2-3 µm

360

gradually replace the capillary morphology. However, this last bulk morphology cannot be

361

regarded as structurally uniform, since the size of small micron-range oriented pores

362

continuously increase as the distance to the surface of hydrogel increases. (Figure 5).

of

= 4 mm (see Figure 2A and 2B) where tubular pores become


Page 15 of 35

I

II

top of hydrogel

direction of the gel front

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

dI = 0 mm dII = 0,1-0,2 mm tubular capillaries

zone I zone II II-III

dIII = 2-3 mm

zone III III

Micro-range porous microstructures

363 364

Figure 5. Structural scheme and corresponding CLSM micrographs of a physical chitosan

365

hydrogel obtained at different distances from the top of the gel. The hydrogel was prepared

366

from an aqueous chitosan solution (Mw = 570 kg.mol-1, DA = 4.0 %, and Cp = 1.5 % w/w)

367

neutralized by a 1 M NaOH solution.

368 369

Our analysis yields a microstructural panorama of a specific physical chitosan hydrogel as a

370

function of the depth d, as shown in figure 5. However, this description reflects the particular

371

evolution of the neutralization kinetics, from instantaneous gelation conditions (in zone I) to

372

much slower neutralization deeper in the bulk of the hydrogel. In addition, it was previously

373

shown that other parameters of the gelation conditions, in particular the chitosan solution

374

viscosity and the nature of the coagulation bath strongly influence the formation and resulting

375

size of oriented microstructures at a fixed depth of 10 mm.51 Thus, to obtain a meaningful

376

microstructural description of chitosan physical hydrogels in general, it is necessary to study

377

the impact of the gelation parameters on the structural organization of chitosan physical

378

hydrogels accounting for the structural gradients and transitions, thus spatially resolved.

379


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 35

380

Effect of the polymer concentration

381

The key factor in the formation of capillary structures is the viscosity of the solution from

382

which hydrogel is formed.51,53 When the viscosity of the chitosan solution was increased from

383

1 to 4 Pa.s (varying the chitosan concentration from 0.01 to 1% (w/w)), the formation of

384

microcapillaries was promoted, hence defining an lower concentration threshold for their

385

formation.51 Here, we determined globally the influence of the polymer concentration on the

386

hydrogel structure in the chitosan concentration range from 0.75 to 4.0 % (w/w). The

387

observations performed under the optical microscope showed that the dense membrane

388

thickness (in zone I) increases when the polymer concentration increases (Supplementary

389

Figure S1). The thickness of the first zone is close to 100 micrometers when the chitosan

390

concentration used is 1.0 % (w/w). In contrast, this zone is about 0.5mm thick when the

391

polymer concentration used is higher than 3.0 % (w/w) (Table 3).

392

Table 3. Influence of the polymer concentration on the thickness of zone I. Cp (% w/w)

Thickness zone I (µm)

1.0

90 ± 10

1.5

190 ± 20

2.0

190 ± 20

2.5

310 ± 30

3.0

510 ± 20

3.5

620 ± 40

4.0

760 ± 60

393 394

The shift of the first microstructural transition (from external membrane to capillary zone) to

395

higher depth values is also accompanied with a shift of the micron-range oriented

396

microstructures, since for a given distance d, the inter-distance separating micron-range

397

oriented microstructures decreases with the chitosan concentration of the parent solution.

398

Accordingly, the SALS analyses show that the scattering maximum shifts to higher q values

399

when the polymer concentration increases (Figure 6).


Page 17 of 35

300

Cp = 0.75 % Cp = 1.00 % Cp = 1.50 % Cp = 1.75 % Cp =2.00 % Cp =2.50 %

200

I (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

100

0 0

1.10

-3

2.10

-3

3.10

-3

4.10

-3

-1

q (nm ) 400 401

Figure 6. Plots of scattered intensity I vs scattering vector q for physical chitosan hydrogels

402

prepared from aqueous chitosan solutions with polymer concentrations ranging from 0.75 to

403

2.50 % (w/w). Hydrogels were processed from aqueous chitosan solutions (Mw = 570 kg/mol,

404

DA = 4.0 %) neutralized by a 1 M sodium hydroxide solution. SALS profiles were obtained at

405

d = 15 mm from the top of the gels.

406 407

Quantitatively, at d = 15 mm, the periodicity distance between small oriented microstructures

408

decreases from 4.9 μm to 1.7 μm when the polymer concentration ranges from 1.0 % (w/w) to

409

2.0 % (w/w) respectively. No scattering maximum could be observed on the 2D SALS images

410

of hydrogels prepared with polymer concentrations above 2.5 % (w/w). In these gelation

411

conditions, micron-range oriented structures probably exhibit a size smaller than a 1 µm,

412

scattering at angles that are out of the assessable range. As a result of combined impact of the

413

analysis depth d (i.e. neutralization kinetics) and chitosan concentration (i.e. solution viscosity

414

and dynamics) on the characteristic size of the morphology, there seem to be a compensation

415

law: lower characteristic sizes of oriented structures occurring for both at higher concentration

416

or low analysis depth d when the relative neutralization time vs chain relaxation time is low.

417

Dynamic effects on the capillary morphology were described previously. For example, Treml 17 ACS Paragon Plus Environment

Langmuir

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Page 18 of 35

418

et al., in the case of physical alginate hydrogels, showed that the value of coagulant diffusion

419

coefficient impacts the formation and size of oriented microstructures.53

420

Accordingly, from a qualitative standpoint, the characteristic size of oriented microstructure

421

morphology ( ) should be mainly determined by the kinetic ratio ( ):

422

(Equation 2)

423

Where

424

width of the sol-gel transition layer.

425

For a given polymer concentration, when the depth analysis of hydrogels

426

decreasing values from 15 mm to 10 mm (see figure 5 and Supplementary Figure S3),

427

is the front gel speed and

an apparent chain disentanglement time and

should decrease, resulting in a smaller characteristic distance

is the

is carried out at and

. This situation is

428

analogous at lower molecular mobility, i.e. for higher viscosities and polymer concentrations

429

resulting in a higher relaxation time

430

range morphology, disentanglement of chains would result in micron-range elongated pores.

431

Such morphology was evidenced by CLSM in the deepest zones (see figure5 and

432

Supplementary Figure S2)

433

In fact, the neutralization kinetics is also expected to be impacted by the polymer

434

concentration because the number of protonated amine functions to be neutralized is

435

proportional to (1-DA)*Cp. Hence, we quantitatively determined the influence of the polymer

436

concentration on the kinetics of gelation (Figure 7).

. This yields physical interpretation of the micron-


Page 19 of 35

(A) 8000 7000

Thickness (µm)

6000 5000 4000 Cp = 1.0 % (w/w) Cp = 1.5 % (w/w) Cp = 2.0 % (w/w) Cp = 2.5 % (w/w) Cp = 3.0 % (w/w) Cp = 3.5 % (w/w) Cp = 4.0 % (w/w)

3000 2000 1000 0 0

437

500

1000 1500 2000 2500 3000 3500 4000

Gelation Time (s) (B) 8000 7000 6000

Thickness (µm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

5000 4000 Cp = 1.0 % (w/w) Cp = 1.5 % (w/w) Cp = 2.0 % (w/w) Cp = 2.5 % (w/w) Cp = 3.0 % (w/w) Cp = 3.5 % (w/w) Cp = 4.0 % (w/w)

3000 2000 1000 0 0

438

20

40

60

80

Square root of Time, t1/2 (s1/2)

439

Figure 7. Measured gel thickness as a function of gelation time (A) and square root of

440

gelation time (B), for different chitosan concentrations. Hydrogels were processed from

441

aqueous chitosan acetate solutions (Mw = 570 kg.mol-1, DA = 4.0 %) neutralized by a 1 M

442

sodium hydroxide solution. 19 ACS Paragon Plus Environment

Langmuir

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Page 20 of 35

443

The propagation velocity V of gel front (determined by optical microscopy) is indeed slower

444

when the polymer concentration increases. Surprisingly, a 4 fold variation of the polymer

445

concentration only induces a 2 fold variation (or less) in the neutralization time. This could be

446

due to a change of critical polymer neutralization ratio [NH3+]/[NH2] necessary to reach the

447

gel point (the gel could form at a higher charge density at higher polymer concentration, when

448

the availability of neutralized chain segments is sufficient). Nevertheless, other physico-

449

chemical effects may act to shorten the gelation time at high concentrations, such as the pre-

450

entangled state of the solution and the presence of aggregates (pre-gelated domains) within

451

the solution.38 Indeed, based on a rheological gelation criterion,46 Montembault et al. showed

452

that the gel time effectively decreases with solution polymer concentration at stoichiometric

453

protonation.

454

At polymer concentrations above 2.5 % (for Mw = 570 kg.mol-1), the size of the fine micron-

455

range oriented microstructures exited the accessible size window of SALS and the scattered

456

intensity strongly decreased. At a shorter size scale, as explored by SAXS, we did not detect

457

anisotropic scattered patterns, an indication that the anisotropic patterns could vanish at high

458

concentrations or at high viscosities, i.e. for highly entangled systems.

459

At a larger scale, the formation of capillary microstructures was basically related to the

460

creation of convective flows in the vicinity of the gel front.53 Thus, if viscosity is high the size

461

of convective vortexes is firstly reduced and finally their absence prevents the formation of

462

capillary microstructures. As expected, we observed that the solution viscosity and polymer

463

concentration has also an effect on the formation of capillaries. The CLSM observations

464

showed that their formation is prevented when the polymer concentration exceeds 2.0 %

465

(Figure 8).

466 467

Figure 8. CLSM micrographs of physical chitosan hydrogels prepared from aqueous chitosan

468

solutions with polymer concentrations at (A) 1.0 % (w/w), (B) 1.5 % (w/w), and (C) 2.0 % 20 ACS Paragon Plus Environment

Page 21 of 35

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Langmuir

469

(w/w). Hydrogels were processed from aqueous chitosan solutions (Mw = 570 kg.mol-1, DA =

470

4.0 %) neutralized by a 1 M sodium hydroxide solution. CLSM micrographs were made at d

471

= 1 mm from the top of the gels.

472 473

The polymer concentration weakly affects the capillary diameter (approximately 10 μm at the

474

center of the capillary. The spatial distribution of the capillary is rather homogeneous for a

475

concentration of 1.0 % (w/w), whereas the capillaries were localized in domains with

476

concentrations of 1.5 and 2.0 % (w/w). At concentrations higher than 2.5 % (w/w), no

477

capillaries could be observed. Thus, hydrogels prepared in the interval of concentration

478

ranging from 2.5 to 4.0 % (w/w) only two distinct structural zones (i.e. primary membrane +

479

underlying hydrogel with fine micron-range oriented microstructure) are present in the

480

hydrogels. Optical microscopy investigations of physical chitosan hydrogels in the

481

concentration range from 2.5 % to 4.0 % (w/w) showed the presence of oriented structures in

482

the second zone (see Supplementary Figure S1). Such large scale morphology could be

483

representative of an additional structural organization level with polymer concentration

484

fluctuations at a higher scale, and probably needs further investigations.

485

If this large-scale level is again connected to microfluidic motions in the sol-gel transition

486

layer, then several vortex length scales should be invoked to explain their resulting “in-

487

printing” in physical chitosan hydrogels in the form of large zones of low or high polymer

488

concentration, but with absence of well-defined capillaries. The possible evolution of the size

489

of such vortexes is not in line with the features of the micron-range oriented structures that

490

exhibit a coarser morphology in the depth of the hydrogel, whereas the capillaries appear only

491

in the first millimeters from the surface. In addition, in figure 8C, capillaries can appear

492

isolated at high concentrations, thus their mechanism is not related to the establishment of a

493

periodic array of vortexes in the sol-gel transition layer yielding a periodic capillary

494

structure.53 In this regime, it thus appears that the formation of the capillaries should be due to

495

another structuration mechanism. Nie et al. studied the morphology of hydrogels formed by

496

neutralization of chitosan aqueous solutions by sodium hydroxide solutions.58 Under specific

497

conditions, they observed by confocal microscopy the formation of oriented structures parallel

498

to the direction of gel front in chitosan hydrogels. To explain their formation mechanism, they

499

considered the gel in formation consisting in layer units stacked along the base diffusion

500

direction. The originally homogeneous chitosan solution turned to hydrogel containing

501

chitosan-rich domains and water- rich domains based on phase equilibrium of polymer 21 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 35

502

solution. The propagation of such fluctuations from one layer to the next layer during gelation

503

is due to the entanglements causing mobility restrictions in the gel-sol interface. As a result,

504

the structuration in a given layer units were imposed by the previous gelated unit and the

505

persistence of chitosan-rich and water-rich zones will form oriented microstructures.

506 507

Effect of molar mass

508

The influence of the polymer concentration on the formation of micron-range pores and

509

capillaries shows that the microstructure of a physical chitosan hydrogel depends on the

510

gelation conditions. The microstructure of the gels also depend on the macromolecular

511

parameters of chitosan. For example, Rivas et al. compared the structure of physical chitosan

512

hydrogels obtained with chitosans of two different molar masses (Mw = 515 and 200 kg/mol).

513

They prepared hydrogels with the same polymer concentration (Cp = 1.0 % w/w). They

514

observed that the structure of the hydrogel prepared with chitosan of smaller molar mass

515

(solution viscosity = 0.7 Pa.s) was less anisotropic than the hydrogel prepared with chitosan

516

of higher molar mass (solution viscosity = 4.1 Pa.s). It was concluded that the viscosity of the

517

solution was a key parameter in the formation of oriented microstructures.

518

In this context, we compared the hydrogel microstructures prepared with solutions having

519

similar viscosities but by using two chitosans with different molar masses (Mw = 575 and 170

520

kg.mol-1). Indeed, the SALS analyses show that the micron-range structure of hydrogels

521

obtained with the chitosan of smaller molar mass are less anisotropic (Figure 9).


Page 23 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Mw = 170 kg.mol-1

Mw = 570 kg.mol-1

A

B

C

D

522 523

Figure 9. Two-dimensional SALS images of physical chitosans hydrogels obtained by

524

neutralization of aqueous chitosan solutions with viscosity : (A) 3.3 Pa.s (Mw = 570 kg.mol-1,

525

DA = 4.0 %, Cp = 1.0 % (w/w)), (B) 4.8 Pa.s (Mw = 170 kg.mol-1, DA = 1.0 %, Cp = 3.0 %

526

(w/w)), (C) 22 Pa.s (Mw = 570 kg.mol-1, DA = 4.0 %, Cp = 1.5 % (w/w)), and (D) 16 Pa.s

527

(Mw = 170 kg.mol-1, DA = 1.0 %, Cp = 4.0 % (w/w)). Solutions were neutralized by a 1 M

528

sodium hydroxide solution. SALS images were made at 10 mm from the top of the gels.

529

Images (C) and (D) were recorded with a detector gain four times higher than images (A) and

530

(B).

531 532

A similar result was obtained at a distance d of 15 mm (Supplementary Figure S4). As a

533

result, the viscosity of the chitosan solution cannot be regarded as the only key parameter in

534

the formation of small oriented microstructures. The chitosan molar mass, playing on the

535

chain mobility and disentanglement ability, is also a significant intrinsic parameter to be taken

536

into account in the structural development of the gels.

537

Surprisingly, at a larger scale, no capillary tubular pores could be observed in hydrogels

538

prepared with the chitosan of smaller molar mass (MW = 170 kg.mol-1), the polymer 23 ACS Paragon Plus Environment

Langmuir

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Page 24 of 35

539

concentration ranging from 3.0 to 7.0 % (w/w), whereas the high molecular mass hydrogels,

540

prepared from solutions of similar viscosities but at lower concentrations, exhibited the

541

capillary morphology (see Figure 8).

542

Empirically, we noticed that the formation of tubular pores can be connected to the

543

conformation of chitosan chains in solution and the resulting entanglement density. Several

544

works related to the structural organization of chitosan solutions showed that chitosan was an

545

hydrophobic polyelectrolyte, and described according to two conformational regimes.36–38,64.

546

When the physicochemical context is favorable to a strong polyelectrolyte in solution (high

547

density of charge, low ionic strength, hydrophilicity), the chains are extended in the form of

548

polyelectrolyte strings with high entanglement density. When hydrophobicity is favored, then

549

the conformation of the chains is more compact and is dominated by the presence of

550

aggregates (pearls) with a lower density of inter-chain entanglements. Boucard et al. showed

551

the existence of a critical polymer concentration Cb where the structural organization of the

552

chitosan solution passes from a “strings” (highly entangled) dominated regime to a (partly

553

disentangled) “pearls” dominated regime.36 Several other works devoted to physical chitosan

554

hydrogels showed the existence of a critical concentration,46–49 above which the chitosan

555

solution is structured into nano-aggregates, which are precursors of the hydrogel

556

microstructure.38 A careful examination of the conditions for the formation capillaries can be

557

related to a strong polyelectrolyte regime with extended chain conformation. Such regime is

558

met when chitosan concentration is lower than Cb ~ 2 % (w/w) at low ionic strength and low

559

DA. In other physico-chemical conditions, nano-aggregates are present in the solution, the

560

final microstructure of hydrogel could correspond to the association of these nano-aggregates

561

resulting in a globular morphology observed by cryoSEM.65 Such ‘string’-dominated

562

conformation in solution, retaining a high entanglement density, could result in strong

563

intermolecular associations after fast gelation (such as in crystallites of hydrated allomorph of

564

chitosan), and local syneresis yielding the formation and propagation of solvent-rich and

565

solvent-poor domains at various scales. In brief, this observation well explains the impact of

566

chitosan concentration (and the threshold value of 2.0 % w/w) and of the DA.36,37 Indeed, we

567

previously showed that the critical concentration limiting the “strings” entangled regime and

568

the “pearls” disentangled regime is close to 2.0 % (w/w) for a chitosan with a DA of 4.0 %.37

569

This impact of chain conformation is also consistent with the absence of capillaries in the

570

interval of polymer concentration ranging from 2.0 to 7.0 % (w/w). Thus, our observations

571

underline the role of entanglements at the sol-gel interface in the multi-scale structuration of 24 ACS Paragon Plus Environment

Page 25 of 35

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Langmuir

572

chitosan physical hydrogels. They are fairly in line with the structural propagation model of

573

Nie et al, since entanglements should play a major role in the constraints of a gelated layed

574

onto the next liquid layer. They could be related with the vortex-induced morphology

575

model51- 53 if the role of entanglements on the formation of the vortexes within the sol-gel

576

interface could be clarified.

577 578

Effect of the base concentration

579

We proposed above that the kinetics of neutralization, due to the base diffusion through the

580

gel, impacts the forming hydrogel microstructure through the establishment of different

581

gelation regimes, resulting in different gel morphologies. Accordingly, the concentration of

582

the alkaline solution should be an important parameter to modulate the structure of hydrogels.

583

First, optical microscopy observations consistently showed that when the base concentration

584

increases, the thickness of the first zone increases (Table 4).

585

Table 4. Influence of the NaOH concentration on the thickness of zone I. [NaOH] = 1 M

[NaOH] = 7 M



1.00

90 ± 10

150 ± 20

1.50

190 ± 20

360 ± 20

2.00

190 ± 20

490 ± 10

.50

310 ± 30

720 ± 20

3.00

480 ± 20

1000 ± 10

3.50

620 ± 40

1200 ± 20

4.00

760 ± 60

2640 ± 40

Cp (% w/w)

586 587

The shift of the morphology towards higher depths with the concentration of the coagulation

588

bath should imply that at a given depth d, in Zone III, the size of the micron-range

589

morphology also varies according to the base concentration. Again, the SALS analyses show

590

that the scattering maximum shifts to greater values of q when the NaOH concentration

591

increases (Figure 10).


Langmuir

300

NaOH 1 M NaOH 2 M NaOH 3 M NaOH 4 M NaOH 5 M NaOH 6 M

250 200

I (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 35

150 100 50 0 0

1.10 -3

2.10

-3

3.10

-3

4.10

-3

-1

q (nm ) 592 593

Figure 10. Plots of scattered intensity I as a function of the scattering vector q for physical

594

chitosan hydrogels obtained by neutralization with sodium hydroxide concentrations ranging

595

from 1 to 6 M. Hydrogels were prepared from aqueous chitosan solutions (Mw = 570 kg.mol-

596

1

, DA = 4.0 %, Cp = 1.5 % (w/w)). SALS profiles were made at 15 mm from the top of the

597

gels.

598

As an example, at d = 15 mm, the periodicity of the micron-range morphology, decreases

599

from 3.5 µm to 1.9 µm when the hydrogel was neutralized with a 1 M or 3 M sodium

600

hydroxide solution respectively. No correlation halo could be completely observed on the 2D

601

SALS images of hydrogel neutralized with a 6 M sodium hydroxide solution. Oriented

602

microstructures in the hydrogels prepared with this base concentration (at depth d = 15 mm)

603

thus exhibit a characteristic distance lower than 1 µm. Similar SALS observations were

604

performed at a distance d of 10 mm (Supplementary Figure S5). When the base

605

concentration increases, the propagation velocity of gel front increases (see figure 7). The

606

resulting effect is to shift the morphology to larger depths, thus confirming that the different

607

stages of the morphology are dynamically controlled by the gel front velocity, also evidencing

608

that the mobility of chitosan chains plays a central role in the development of the morphology,

609

as described in the dynamic ratio r. As expected, the base concentration used to neutralize the 26 ACS Paragon Plus Environment

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610

hydrogel exhibit a dynamic effect on the formation of small oriented microstructures, and

611

determine the evolution of the characteristic periodicity of small oriented microstructures ξ

612

with analysis depth d.

613

In contrast, the concentration of the base weakly impacts the formation of capillaries. The

614

CLSM observations show that the diameter and number of capillaries formed is similar

615

whatever the concentration of the base used to form the hydrogel (Figure 11).

616 617

Figure 11. CLSM micrographs of physical chitosan hydrogels obtained by neutralization

618

with : (A) NaOH 1 M, (B) NaOH 4 M and (C) NaOH 7 M. Hydrogels were prepared from

619

aqueous chitosan solution (Mw = 570 kg.mol-1, DA = 4.0 %, Cp = 1.0 % (w/w)). CLSM

620

micrographs were recorded at 1 mm from the top of the gels.

621 622

This result may be an indication that the mechanisms at work for the formation of capillaries

623

and the micron-range pores should be widely different.

624

In addition, purely geometrical factors of the diffusion/gelation process may also impact the

625

capillary structure. When the surface to thickness ratio was much larger than in the optical

626

cells, i.e. when neutralization occurred in a Petri dish, we observed (see SI figure S7) the

627

coexistence of capillaries with small (~5µm) and large (≥20µm) diameters, possibly

628

originating from a transition layer (between chitosan solution and chitosan gel) presenting

629

vortexes of different scales. The establishment of the precise origin of this vortexes network at

630

two well defined scales needs further study.

631 632

SUMMARY AND CONCLUSION


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Page 28 of 35

633

The morphology of physical hydrogels is complex and multiscale. In this work, we describe 3

634

gelation regimes, yielding a 3-layered structure with different microstructures:

635

- gels formed at low values of

636

time

637

high entanglement density in the hydrogel.

638

- gels formed at high values of

639

of micro-range pores in the depth of the hydrogel and inducing static light scattering and also

640

confocal microscopy contrast due to chitosan density fluctuation.

641

Increasing acetic acid concentration well above the stoichiometric conditions is likely to

642

decrease the neutralization kinetics due to the direct consumption of OH- groups for the

643

neutralization of the acid; this should in turn impact the microstructure of the gels since the

644

dynamic ratio r could be decreased at high acetic acid concentrations.

645

In addition, a capillary microstructure will develop in conditions that are not governed by the

646

dynamic ratio . Such morphology will impact the transport and mechanical properties of

647

hydrogels. Such properties are important for their applications as bioreactors where the

648

diffusion of oxygen and nutrients is essential in the survival and maturation of cells within

649

hydrogel compartments.66 The presence of capillaries is also essential in the colonization of

650

hydrogels by macrophages and other cells when hydrogels are implanted in vivo.57,61 The

651

mechanical properties of the gels may also impacted by the disentanglement of chains

652

resulting from neutralization, and the presence of capillaries could also contribute to decrease

653

the tenacity of hydrogels and solids formed by drying of such hydrogels, such as in wet fiber

654

spinning processes.67 The interplay between the gelation dynamics and the disentanglement

655

dynamics is expected to play a role in the gelation of other types of chitosan hydrogels

656

obtained in different physico-chemical contexts. 68,69

657

ACKNOWLEDGEMENTS

658

The authors would like to acknowledge the Centre Technologique des Microstructures at

659

Université Claude Bernard Lyon 1 for their expertise and assistance in Confocal Laser

660

Scanning Microscopy analyzes.

in the dynamic conditions where the gelation

is short vs molecular relaxation time

, yielding a dense surface membrane with

where disentanglement is favored, resulting in the formation

661


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Dynamic Structuration of Physical Chitosan Hydrogels - Langmuir

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