Rheometric Study of the Gelation of Chitosan in Aqueous Solution

Biomacromolecules 2005, 6, 653-662

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Rheometric Study of the Gelation of Chitosan in Aqueous Solution without Cross-Linking Agent Alexandra Montembault,†,‡ Christophe Viton,‡ and Alain Domard*,‡ Laboratoires Genevrier, 280 rue de Goa, Z.I. Les Trois Moulins, 06901 Sophia-Antipolis, France, and Laboratoire des Mate´ riaux Polyme` res et des Biomate´ riauxsUMR CNRS 5627, Domaine Scientifique de La Doua, 15 Bd. A. Latarjet, Bat. ISTIL, 69622 Villeurbanne Cedex, France Received July 16, 2004; Revised Manuscript Received November 10, 2004

A new process of formation of chitosan physical hydrogels in aqueous solution, without any organic solvent or cross-linking additive, was studied. The three conditions required for the physical gelation were an initial polymer concentration over C*, a critical value of the balance between hydrophilic and hydrophobic interactions, and a physicochemical perturbation responsible for a bidimensional percolating mechanism. The time necessary to reach the gel point was determined by rheometry, and gelations were compared according to different initial conditions. Thus, we investigated the influence of the polymer concentration and the degree of acetylation (DA) of chitosan on gelation. The number of junctions per unit volume at the gel point varied with the initial polymer concentration, i.e., the initial number of chain entanglements per unit volume or the number of gel precursors. The time to reach the gel point decreased with both higher DAs and concentrations. For a chitosan of DA ) 36.7%, a second critical initial concentration close to 1.8% (w/w) was observed. Above this concentration, the decrease of the time to reach the gel point was higher and fewer additional junctions had to be formed to induce gelation. To optimize these physical hydrogels, to be used for cartilage regeneration, their final rheological properties were studied as a function of their degree of acetylation and their polymer concentration. Our results allowed us to define the most appropriate gel for the targeted application corresponding to a final concentration of chitosan in the gel of near 1.5% (w/w) and a DA close to 40%. Introduction Biopolymer-based physical hydrogels have been reported to be particularly interesting for their biological properties and, consequently, their potential use in biomedical and pharmaceutical fields. Among polysaccharides, glycosaminoglycans, especially chitosan, constitute a very interesting family showing the rare property of bioactivity.1 Chitosan is produced from an N-deacetylation of chitin under alkaline conditions.2 Chitin occurs mainly in the cuticles of arthropods, the endoskeletons of cephalopods, and fungi. Previous studies reported that chitosan is biodegradable and has a good biocompatibility.1 Moreover, it plays an important role in cell regulation and tissue regeneration.3-6 Chitosan, as chitin, belongs to the family of linear copolymers of (1f4)-2amino-2-deoxy-β-D-glucan (GlcN) and (1f4)-2-acetamido2-deoxy-β-D-glucan (GlcNAc). DA, the degree of acetylation, corresponding to the molar fraction of acetyl units constituting the polymer chains, is a very important parameter. It is considered that chitosan refers to polymers soluble in dilute acidic solutions and then to DAs below 60%. The physical gelation of chitosan was already reported in the literature.7-9 Vachoud and Domard extensively studied the formation of gels during the N-acetylation of chitosan * Corresponding author. Phone: + 33 (0) 4 72 44 85 87. Fax: + 33 (0) 4 72 43 12 49. E-mail: [email protected]. † Laboratoires Genevrier. ‡ Domaine Scientifique de La Doua.

with acetic anhydride in a solution containing an equivalent amount of water and 1,2-propanediol.10 They showed that gelation was achieved for a critical value of the balance between hydrophilic and hydrophobic interactions depending on various external parameters. In our two previous papers we described, for the first time, the formation of a true chitosan physical hydrogel with no external cross-linking agent.11,12 For that, the initial polymer concentration had to be over C*. Indeed, the entanglements could be used as physical junctions of a three-dimensional network of polymer chains via possible nanometric precursors. The balance between hydrophilic and hydrophobic interactions had also an important role in the gel formation and had to reach a critical value to allow the gelation. In the case of chitosan, we decided to modify this value thanks to a homogeneous decrease of the polymer ionization and then a decrease of its apparent charge density. These conditions were put together when a solution of chitosan in a hydroalcoholic medium was evaporated. During this process, a percolating gelation occurred thanks to the progressive displacement of an interphase of a sol-gel transition from the top to the ground of the solution. Thus, chitosan was first dissolved in an acetic acid aqueous solution in order to achieve the stoechiometric protonation of the -NH2 sites. After complete dissolution, 1,2-propanediol was added. The mixture was stirred and allowed to evaporate up to gelation. The role of the alcohol was to

10.1021/bm049593m CCC: $30.25 © 2005 American Chemical Society Published on Web 01/21/2005

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reduce the dielectric constant of the medium and possibly to participate in the formation of hydrophobic junctions between polymer chain segments. Under these typical conditions, the chitosan acetate salt was less formed. Moreover, since acetic acid was not highly soluble in such a solvent, the evaporation contributed to eliminate both water and acetic acid. The consequence was a decrease of the apparent charge density of chitosan chains up to a critical value of gelation. Our new objective was to form physical hydrogels without any organic solvent. Then, new hydrogels were directly formed from an aqueous chitosan solution. In fact, the solution was simply put in contact with gaseous ammonia to induce gelation. From this new method, we confirmed that the following three conditions had to be observed to induce chitosan gelation: (1) the initial concentration of the solutions had to be over C*, the critical concentration of chain entanglements; (2) the hydrophobic/hydrophilic balance had to achieve a critical value; and (3) an interphase, corresponding to a bidimensional sol-gel transition, had to be created uniformly. The aim of this work was first to describe and explain the formation of these new gels as a function of various parameters. The time to reach the gel point was determined by rheometry. Once damaged, the articular cartilage has very little capacity for spontaneous healing; because of the avascular nature of the tissue,13 only a fibrous material forms with poor mechanical properties.14 Homogenic and allogenic tissue transplantation procedures are then currently performed, followed by an autologous chondrocytes transplantation processing.15 Tissue engineering is tentatively used with only cells and matrixes, but signaling substances or genetic measures are also involved for the re-establishment of a structurally and functionally competent cartilage.15 Actually, numerous studies are processed to design a suitable replacement material of damaged cartilage. The matrixes can be broadly categorized into matrixes based on proteins, carbohydrate polymers, and synthetic polymers.15 But, to date, specific biological and technical problems are commonly encountered. Moreover, tissue engineering is still only at an experimental stage of development and a limited number of patients have been treated with these new biomaterials.15 Cartilage tissue is constituted of a complex physical hydrogel ensuring viscoelastic properties. This is at the origin of the idea to use a bioinspired structure to repair cartilage diseases. The study of the rheological properties of our hydrogels seemed important, considering the application of these materials, and then the study expanded to define the optimal material for the targeted use. Experimental Section Purification and Acetylation of Chitosan. The initial chitosan, a highly deacetylated sample, produced from squid pens, was purchased by France Chitin (batch number 114). This chitosan was purified as follows. The polymer was dissolved at 0.5% (w/v) in a stoechiometric amount of aqueous acetic acid. After complete dissolution, it was

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filtered successively on 3, 0.8, and 0.45 µm pore size membranes (Millipore). Then, the solution was precipitated with dilute ammonia up to a constant pH close to 9 and centrifuged. The precipitate was repeatedly rinsed with distilled deionized water, centrifuged until a neutral pH was achieved, dispersed in water, and freeze-dried. Samples of different DAs were prepared by reacetylating the initial chitosan with acetic anhydride as reactant, in a water/alcohol solution.10 Thus, an aqueous acetic acid solution of this chitosan was prepared at a concentration of about 1% (w/w), and acetic acid was added to achieve the stoechiometric protonation of the -NH2 sites. This solution was mixed with 1,2-propanediol to achieve a final polymer concentration of 0.5% (w/v). The acetylating reactant was constituted of a freshly prepared solution of acetic anhydride in a small amount of 1,2-propanediol.10 This was slowly added under stirring to the chitosan solution and the medium was allowed to stand for 3 h. The amount of acetic anhydride corresponded to the stoechiometric amount necessary to achieve a given degree of acetylation. The polymer was then fully precipitated by addition of aqueous ammonia and repeatedly washed and centrifuged with distilled deionized water. The product was finally freeze-dried. Characterization of Chitosans. After lyophilization, DAs of the different samples were characterized from 1H NMR spectra recorded on a Brucker 250 spectrometer (250 MHz) at 25 °C. Thus, 10 mg of chitosan was solubilized in 1 g of D2O containing 0.21 wt % HCl. The DA was then evaluated as proposed by Hirai et al. from the ratio of the area of the peaks of the methyl protons of the N-acetylglucosamine residues to those of all the H2 to H6′ protons of both glucosamine and N-acetylglucosamine residues.16 The DA of the initial chitosan was found to be close to 5.2%. 1H NMR also allowed us to check for the absence of propanediol in the reacetylated chitosans. The weight-average molecular weight (Mw) and the root-mean-square of the z-average value of the gyration radius (RG,z) of the samples were determined by size exclusion chromatography (SEC) coupled online with a multiangle laser light scattering (MALLS) detector.17 SEC was perfomed by means of an IsoChrom LC pump (Spectra Physics) connected to Protein Pack glass 200 SW and TSK gel 6000 PW columns. A Waters R 410 (Waters-Millipore) differential refractometer and a multiangle laser-light scattering detector, operating at 632.8 nm (Wyatt Dawn DSP), were connected online. A 0.15 M ammonium acetate/0.2 M acetic acid buffer (pH 4.5) was used as eluent. The flow rate was 0.5 mL/min. The polymer solutions were prepared by dissolving 1 mg of polymer in 1 mL of buffer solution and then filtered after complete dissolution on a 0.45 µm pore size membrane (Millipore) before injection of 100 µL. The water content of freeze-dried chitosan samples was evaluated on 10 mg with a DuPont Instrument 2950 thermogravimetric analyzer (TGA) operating at a temperature ramp of 2°C/min under a flow of helium. Preparation of Solutions, Gelation, and Washing of Gels. The critical concentration of chain entanglement C* was evaluated from the reverse of the intrinsic viscosity and found to be close to 0.1% (w/w) (measurements of the intrinsic viscosity performed at 22°C, acetic acid/ammonium

Rheometric Study of the Gelation of Chitosan

acetate buffer used as solvent, pH 4.5, ionic strength of 0.15 M). We approximated that this kind of evaluation could be applied to the case of a chitosan chain conformation different from true random coils. For all the experiments described hereafter, the initial polymer concentration was above or equal to 0.5% (w/w). Thus, chitosan was dispersed in water and acetic acid was added to achieve the stoechiometric protonation of the -NH2 sites. After complete dissolution (almost 12 h of stirring), the solution was put in contact with gaseous ammonia. Practically, a Petri dish (diameter ) 35 mm) containing nearly 3 g of the chitosan solution was placed in a glass reactor (Figure 1). This Petri dish was displayed over 40 mL of an aqueous solution of ammonia. The concentration of the ammonia bath was 1 mol/L. The sample stood for 48 h in the reactor. When the hydrogel was taken off the reactor, it was then washed thoroughly with distilled deionized water to eliminate ammonium acetate and the excess ammonia, until the pH of the distilled water used for washing was achieved. For experiments of gelation, the concentration of the ammonia bath was lower, to slow the processing and thus to allow the measurement of rheological moduli throughout the gelation; 400 mL of ammonia solution at 0.02 mol/L was used. Rheometry. A dynamic mode was used for rheology measurements: it consisted of applying an oscillatory strain to the sample and then measuring the resulting stress calculated from the torque. The rheological studies were performed thanks to a “Rheometrics” rheometer fitted with a plate-plate tool. The diameter of the plates was 25 mm. The gap varied with the sample from 1 to 1.5 mm. The normal force sensor of the rheometer was used to measure the first contact between the hydrogel slab and the upper plate. For experiments on gelation, to allow us to determine the pH at the gel point, several identical samples containing 1.2 g of chitosan solution were prepared. They were then individually loaded on the rheometer platform, at different times during gelation. The temperature was 22 °C for all rheology measurements. pH Measurements. The pH of the samples throughout gelation was measured directly with a contact electrode, model IQ 240 from IQ Scientific Instruments. Evaluation of the Polymer Concentration. Samples of hydrogels were freeze-dried after use to evaluate their polymer concentration. The water content of freeze-dried hydrogels was determined by thermogravimetric analysis. Thus, the weight of polymer in the samples was known precisely. From the initial weight of the samples of hydrogels and the water content in the lyophilized samples, we deduced the weight concentrations in polymer of the gels. Results and Discussion Starting Materials. To work with a homogeneous series of chitosans with the same distribution of degrees of polymerization but different DAs, a starting sample of chitosan of DA 5.2% was reacetylated. Acetylation was performed with acetic anhydride in a hydroalcoholic medium. The conditions led to a random distribution of acetyl groups

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Figure 1. Experimental setup for the gelation of a chitosan solution in a gaseous ammonia atmosphere.

along the polymer chains and they were sufficiently soft to avoid any chain degradation.18-20 Characteristics of the various prepared chitosan samples are given in Table 1. Formation of Physical Hydrogels of Chitosan. First, the initial polymer concentration had to be over C*. Indeed, under these conditions, the chain entanglements could be used to give rise to physical junctions responsible for the formation of precursors of a three-dimensional network of polymer chains. Then, the balance between hydrophilic and hydrophobic interactions had to reach a critical value to allow the gelation. This value could be achieved thanks to a decrease of the polymer ionization and thus a decrease of its apparent charge density. Moreover, this perturbation had to occur simultaneously within a layer constituting the solgel transition interface. In a first set of experiments, an aqueous ammonia solution was put in contact with a chitosan solution to generate a gelling surface. For that, a chitosan solution was poured inside a dialysis tube and this reactor was placed in an ammonia bath. Inside the tube, a thick film of gel appeared at the interface between the dialysis bag and the polymer solution, but in the bulk a precipitate rather than a gel appeared. In this case, the gelling surface was not uniform, and the process of gelation occurred too rapidly and only locally. A similar trial was tested by Rodriguez-Sanchez and Rha, but with the use of an additive.21 Thus, a chitosan solution in a mixture of acetic acid, glycerol, and water was dialyzed against sodium hydroxide for 24 h. The resulting gel showed a local precipitation of chitosan.22 In fact, the third condition required for gelation, consisting of the formation of a uniform gelling bidimensional interface, was not really achieved. This last condition was fulfilled in the case of the gelation in a hydroalcoholic medium studied in our previous works.11,12 In the latter case, when the sample evaporated, a percolating gelation occurred with the progressive displacement of an interphase of a sol-gel transition from the surface of the sample to the ground of the reactor. We decided to use an alkaline gas instead of an alkaline solution. The fact that the polymer solution was directly in contact with a gas made it possible to give rise to a uniform sol-gel transition layer, thus allowing the gelation. Therefore, the three conditions necessary to observe a percolating gelation were put together when a solution of chitosan was subjected to gaseous ammonia. Indeed, under these conditions, gelation could occur, corresponding to the progressive displacement of an interphase of a sol-gel transition from the surface of the sample to the bottom of the reactor. Ammonia was chosen for its high volatility at both ambient pressure and temperature. Thus, the ammonia concentration could rapidly equilibrate in the atmosphere of a closed reactor containing both aqueous solutions of ammonia and polymer. Therefore, gaseous ammonia could easily diffuse in the acidic chitosan solution for neutralization. Typically, chitosan was

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Table 1. Characteristics of the Different Chitosan Samples Used in This Work: Degree of Acetylation (DA), Water Content, Weight-Average Molecular Weight (Mw), Weight-Average Degree of Polymerization (DPw), Polydispersity Index (Ip), and Root Mean Square of the z-Average Value of the Gyration Radius (RG,z) DA (%)

water content [% (w/w)]

Mw (g/mol)

DPw

Ip

RG,z (nm)

5.2 ( 0.3 20.8 ( 1.0 31.6 ( 1.6 36.7 ( 1.8 40.4 ( 2.0 52.1 ( 2.6

8.1 ( 0.8 11.1 ( 1.1 9.6 ( 1.0 9.6 ( 1.0 12.8 ( 1.3 9.4 ( 0.9

534 000 ( 27 000 477 000 ( 24 000 511 000 ( 26 000 466 000 ( 23 000 526 000 ( 26 000 549 000 ( 27 000

3 300 ( 300 2 800 ( 300 2 900 ( 300 2 600 ( 300 3 000 ( 300 3 000 ( 300

2.1 ( 0.2 2.0 ( 0.2 1.8 ( 0.2 1.8 ( 0.2 2.1 ( 0.2 2.0 ( 0.2

130 ( 10 130 ( 10 120 ( 10 110 ( 10 130 ( 10 120 ( 10

Figure 2. Example of the final hydrogel: diameter ) 33 mm and height ) 4 mm [DA of chitosan ) 36.7%, final polymer concentration (Cpolymer) ) 1.5% (w/w)].

dissolved in an acetic acid aqueous solution to achieve the stoichiometric protonation of the -NH2 sites. The solution was then allowed to stand for degassing and put in contact with gaseous ammonia for gelation. The ammonia gas dissolved into the chitosan solution and contributed to the neutralization of amine functions. The consequence was a decrease of the apparent charge density of chitosan chains. Initially, chitosan molecules had a high charge density and behaved more like semirigid chains due to high intramolecular electrostatic repulsions. When the pH became higher, the charge density decreased and the chains were more flexible.23 The molecular mobility was also favored by the local increase of temperature due to the high exothermicity of the reaction of deprotonation of the ammonium sites of chitosan (to be published). Under these conditions, the formation of hydrophobic interactions and hydrogen bonding was favored. At the end, the hydrogel was washed thoroughly with water in order to eliminate ammonium acetate and the excess ammonia. Thus, the final gel only contained water and chitosan (photograph in Figure 2). This gelation process had the advantage of occurring only from a simple aqueous solution of chitosan. We observed that gels did not “disappear” on heating, under stirring, in water, showing the existence of interactions other than hydrogen bonding, that should be necessarily of hydrophobic interaction type. Conditions for Rheological Measurements. The values of the strain amplitude were verified to ensure that all measurements were performed within the linear viscoelastic region, so that the storage (or elastic) modulus G′ and the loss (or viscous) modulus G′′ were independent of the strain. Thus, a strain sweep test at a frequency of 10 rad/s was performed to define this region. As the strain increased, the moduli first remained constant and then decreased after a critical value of the strain. Then, from the linear region, an appropriate strain was selected to be as high as possible to

Figure 3. Variation of G′, G′′, and the modulus of η* versus the frequency for a chitosan solution [DA ) 5.2%, initial Cpolymer ) 1.5% (w/w)].

avoid a too low torque. For the study of gelation, the applied strain varied from 200 to 10% between the beginning of the experiment and the gel point. For the gels, it was near 5%. Using this critical strain, the storage and loss moduli were measured from a constant-strain frequency sweep within frequency ranges of 100-0.05 rad/s for gels and 100-0.2 rad/s for gelation experiments. The system evolved with time, partly due to the solvent evaporation; thus, at very low frequencies, the evolution of the system with time became non-negligible compared to the measurement time. Gelation Experiments. Behavior of Chitosan Solutions. The rheology curves in Figure 3 show the typical shear thinning behavior of a chitosan solution for an initial concentration over C*, with a dynamic viscosity η* that decreases when the frequency increases. This phenomenon corresponds to the disentanglement of the polymer chains, in relation with the breakdown of the low-energy intermolecular interactions.14 This shear thinning became more pronounced for solutions of higher concentration. This behavior was already observed, for example, by Merkovich et al. for solutions containing 2.4% (w/w) of chitosan with two different molecular weights equal to 330 000 and 550 000 g/mol.24 For the initial solutions, the values of the zero shear rate viscosity (η0) were evaluated for low frequencies from the extrapolated Newtonian plateau on the curves of the variation of the dynamic viscosity (Figure 3). η0 showed a typical variation with DA illustrated in Figure 4 (solutions concentrated at 1.5% (w/w)). This behavior was in good agreement with the general law of behavior previously described.12,19,20 Then, when DA decreased below 25%, chitosan behaved as a polyelectrolyte with a high charge density responsible for


Figure 4. Influence of DA on η0, the zero shear rate viscosity of the initial solutions [initial Cpolymer ) 1.5% (w/w)].

Figure 5. Influence of the polymer concentration on η0, the zero shear rate viscosity of the initial solutions of DA ) 36.7%.

strong electrostatic repulsions, inducing the chain backbone expansion and then a zero shear rate viscosity increase. Due to the disappearance of the behavior of a strong polyelectrolyte, this parameter became constant between 25 and 50%. The higher values of η0 for higher DAs were attributed to the presence of aggregates.19,20 On the other hand, for solutions of DA ) 36.7%, η0 strongly increased with the concentration of chitosan (Figure 5). This result was mainly related to the role of the degree of entanglement of the polymer chains on η0. Therefore, the influence of the polymer concentration on η0 appeared as much higher than the influence of DA. It was interesting to notice two different ranges with a frontier located near 1.8%. In the first one, the viscosity increase was much higher than in the second, thus revealing a difference of molecular organization in the two ranges.


Evolution of the Moduli G′ and G′′ during Gelation and Determination of the Gel Point. A continuous measurement of G′(t) and G′′(t) was done to investigate the evolution of the moduli when a chitosan solution was put in contact with gaseous ammonia produced from an aqueous solution (Figure 6). The conditions of this experiment were the following: DA ) 5.2%, Cpolymer ) 0.5% (w/w), CNH3 bath ) 1.4 mol/L. A 90 mL portion of an ammonia solution was shared equally among three small dishes, displayed around the chitosan solution already placed between the plates of the rheometer. Initially, G′(t) and G′′(t) remained stable and small. Then, ammonia baths were placed around the chitosan solution and both moduli were increasing. G′(t) rapidly rose over G′′(t), in relation with the increasing number of physical junctions responsible for the formation of the polymer network. The crossover of the curves of the variation of the two moduli, chosen to define the gel point, for one frequency, is a rough approximation.25 A much better representation of G′(t) and G′′(t) curves was achieved by means of a description based on the scaling concept and percolation theories.26,27 In fact, universal-scaling laws predicted equations for the zero shear rate viscosity η0 and the storage modulus G0 at very low frequencies near the gel point. The detection of η0 and G0 seemed to depend on very precise criteria we could not experimentally validate. Indeed, it is rash to apply zero shear predictions to measurements performed at nonzero shear rates. Moreover, the determination of η0 near the gel point became tricky as the terminal relaxation domain was shifted toward lower frequencies.27 As a consequence, these models could not be applied in our case. To solve the problem of unreliable measurements, Winter and Chambon characterized the system by dynamical mechanical analysis and measured the complex modulus G* at varying frequencies.28 The evolution of the gelling process was monitored by the behavior of the curves G′(ω) and G′′(ω) (Figure 7). During gelation, the behavior of the sample changed from that of a liquid to that of a solid. The frequency-dependent components of G*(ω), i.e., the storage modulus G′(ω) and the loss modulus G′′(ω), were measured. When G′ < G′′, the behavior of the sample was liquidlike, whereas when G′ > G′′, elastic properties dominated.29 Both G′(ω) and G′′(ω) of the initial solutions showed a slight increase with ω (Figure 7). The gel point was determined as the time where G′(ω) and G′′(ω) followed a power law:

Figure 6. Plots of G′ and G′′ versus time at a frequency of 10 rad/s, during the gelation of a chitosan solution.

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Figure 8. Influence of DA on t0, the time to reach the gel point [initial Cpolymer ) 1.5% (w/w)]. Figure 7. Plots of variation of G′ and G′′ versus frequency during the gelation of a chitosan solution.

G′ ) k′ωn and G′′ ) k′′ωn, with the same relaxation exponent n.30 In our case, the proportionality constants k′ and k′′ were almost equal. The value of n was found to be near 0.5, for all gelation experiments, in good agreement with the proposition of Chambon and Winter.28 This value has already been found in other works, for example, for physical gels combining chitosan and glycerol phosphate31 or in the system gelatinwater during the thermoreversible gelation process.30 The value of n was also found to be near 0.5 in the gelation of a solution of chitosan in a hydroalcoholic medium we previously described.11 The values of the viscous and elastic moduli at the gel point were found to be close to 10 Pa at the frequency of 2.5 rad/s for all the systems with different DAs, at a constant initial polymer concentration. This had to be related to the number of junctions per unit volume, globally the same, whatever the DA. As a consequence, the balance between hydrogen bonding and hydrophobic interactions responsible for the formation of junctions, which depends on DA, did not play a major role in this parameter. Concerning the influence of the initial polymer concentration, the values of the moduli increased with this concentration. Therefore, the gel point was not achieved for an absolute critical number of junctions, per unit volume, but was particularly influenced by the initial conditions of chain entanglement and more generally by the molecular organization in the solution. Then, the values of the moduli at the gel point were only depending on the polymer concentration. Influence of the Degree of Acetylation on the Gelation Kinetics. The influence of DA on the gelation mechanism was investigated at first. Figure 8 shows the variation of t0, the time necessary to reach the gel point, for a series of chitosan solutions with different DAs and at an initial concentration of 1.5%. t0 continuously decreased on increasing DA. Figure 8 confirms that acetyl groups are strongly involved in the gelation mechanism, in relation with the major role they play in the formation of hydrophobic interactions32 and then in the formation of physical junctions responsible for gelation. The curve also illustrates the classical three ranges of DA: below 25%, between 25% and 50%, and above 50%. These

three parts agree with the typical law of behavior already illustrated in other studies.12,19,20 In the first range, chitosan is a polyelectrolyte which apparent charge density increases on decreasing DA. Over 50%, it behaves as a polymer bearing isolated charges which hydrophobic environment increases with DA, leading to polymer chain self-associations. Between these two domains, we notice a transition range where the curve makes a plateau and where the polyelectrolyte behavior is progressively counterbalanced by hydrophobic interactions and hydrogen bonding, but also where the value of the intrinsic pK of the amine functions increases with DA.20 Thus, for a low DA, the high charge density responsible for strong electrostatic repulsions disfavors the formation of physical junctions between chain segments. On the contrary, for high DAs, the low charge density, in connection with the presence of numerous hydrophobic groups, favors the formation of nanoaggregates constituting precursors for gelation. This has also to be related to a greater ease to form hydrophobic junctions than hydrogen bonds, the latter requiring a preliminary deprotonation of the amine sites. The plateau found between 25% and 50% indicated that the kinetics of gelation was almost constant in this range. The curve t0 ) f(DA) for the physical gelation of chitosan in a hydroalcoholic medium studied in our previous works showed the same law of behavior with the three ranges of DA.11,12 This underlines the importance of acetyl units in the chitosan physical gelation and the importance of this typical law of behavior of chitosan properties as a function of DA. Influence of the Polymer Concentration on the Gelation Kinetics. Figure 9 shows the influence of the polymer concentration in the initial solution on t0, the time to reach the gel point. The solutions were prepared with a chitosan of DA equal to 36.7%. The first result was a continuous decrease of this time on increasing the concentration. This is in agreement with more entangled polymer chains as the initial concentration increased and then, initially, with a more favorable situation for the formation of physical junctions. As a consequence, the formation of additional junctions became less and less necessary. The curve shows a law of behavior with two linear domains separated by a frontier corresponding to a critical concentration located at near 1.8%. Thus, the decrease of t0


Figure 9. Influence of the polymer concentration in the initial solution on t0, the time to reach the gel point (DA ) 36.7%).

was faster above this concentration and the formation of new junctions was much less necessary than in the first range. This phenomenon must be related to the behavior already mentioned in Figure 5. We may propose the existence of a C** as a second critical concentration, where the medium undergoes a drastic change of molecular organization to decrease the energy of the system. Then, from this value, the mechanism of gelation becomes easier, and over C**, the solution would initially contain precursors of gelation constituted of polymer chain self-associations responsible for the formation of aggregates of nanometric size. These particles should correspond to true initiators of gelation, previously described in the literature.29 Their number and size would increase with the initial polymer concentration, thus explaining the fast decrease of t0. This typical state should not exist at C < C**, and then, in this range, solutions would need more time to sufficiently modify the physicochemical parameters allowing their formation. Whatever the initial concentration, the number of junctions was not sufficient to directly induce a gel state, except, possibly, for a critical concentration close to or over 3.5% (extrapolation of t0 at 0 time), difficult to achieve experimentally. But, the behavior could also change and then never reach t0 ) 0. The value of C** should depend on DA, but this had to be verified (see below). Finally, the sol-gel transition below and over C** could be schematized in Figure 10. pH at the Gel Point. The initial pH was the same for all the solutions and was found as close to 5. As shown on Figure 11, the pH at the gel point decreased continuously with DA. For low DAs, the polymer behaved as a polyelectrolyte and a higher pH was necessary to reach the gel point and then to sufficiently reduce the extent of electrostatic repulsions and allow more molecular mobility. The curve clearly demonstrates this behavior, especially the fact that the ionized form of amine groups disfavored both hydrophobic interactions and hydrogen bonding. On increasing DA, the presence of acetyl units, although an increase of the intrinsic pK was observed,20 required a lesser deprotonation of amine groups to reach the gel point. Finally, this curve confirmed that the charge density, in association with hydrophobic interactions brought about by acetyl residues, was an important parameter in the mechanism of this typical gelation.


The evolution of pH at the gel point, as a function of the polymer concentration in the initial solution, can be observed in Figure 12. To have better information on the law of behavior, the curve was represented considering the variation of pH as a function of the logarithm of the concentration. This pH decreased continuously with the concentration. The law of behavior illustrated by the curve agrees quite well with that of Figure 9. Thus, the higher the concentration, the more the chains were initially entangled, and then, gelation occurred more easily, even if the medium was slightly acidic. It is also noteworthy to find again the behavior illustrating a critical concentration, we termed C**, near 1.8%, over which the pH at the gel point became more and more close to the initial pH of the solution as C increased. This justifies that below C**, the solution needs a sufficient physicochemical modification, illustrated by a pH increase, to form precursors of gelation. Over C**, these precursors are initially present and the change in pH only contributes to increase their size up to their collapse. We may also consider that over C**, the formation of these precursors should be favored by a strong ionic condensation, thus favoring an easier formation of physical junctions due to hydrophobic interactions and hydrogen bonding. Figure 12 also confirms that another critical concentration close to 3.5%, for the studied DA, could be considered as the concentration from which gelation spontaneously occurs, since the extrapolated pH on this point is that of the initial solution. In fact, this condition could not be achieved experimentally and, as above, we may also imagine a change of behavior in the curve, just over the pH of the initial solutions. Characterization of the Gels. Behaviors of Gels during Washing. These new kinds of gels were necessarily fully neutralized during gelation. We could notice that during gelation, contrary to the case of numerous polysaccharide physical hydrogels, no syneresis was observed. The gels were then washed to eliminate both the salt formed during gelation (ammonium acetate) and the excess ammonia. It was important to know the behavior of these gels during washing. Indeed, they could either swell or exclude their solvent. Thus, depending on their possible depletion or swelling, the gels should not have the same final concentration in polymer. This is particularly important for their use as cell culture media, especially for tissue engineering with chondrocytes or other cells. The polymer concentration in hydrogels at the end of their processing was measured and compared to the concentration of the corresponding initial solutions, which, in fact, was close to the concentration of the unwashed hydrogel (Table 2). The difference between the two concentrations tended to increase when DA decreased. Thus, gels of low DA (especially below 31.6%) showed a depletion during their washing. The presence of salts before washing certainly favored water retention and consequently tended to disfavor hydrogen bonding. In these gels of low DA, the physical junctions were mainly composed of hydrogen bonding counterbalancing the low amount of acetyl groups in favor of hydrophobic interactions. During washing, the salts were

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Montembault et al.

Figure 10. Scheme of the gelation of chitosan as a function of the polymer concentration (DA ) 36.7%). In fact, the most important events arise in the transition range. Nethertheless, the results obtained in the present work do not allow us to propose a more precise representation of this interphase.

Figure 11. Influence of DA on pH at the gel point [initial Cpolymer ) 1.5% (w/w)].

Figure 12. Influence of the polymer concentration on pH at the gel point (DA ) 36.7%).

eliminated and a shrinking occurred due to formation of additional hydrogen bonds. On the contrary, for gels with DA over 40%, the final concentration was very close to that of the initial solution and washing had no significant influence on these gels. Indeed, they were constituted with more hydrophobic interactions than hydrogen bonds. Moreover, for these DAs, when pH decreased, the pKa was higher than for low Das,20 favoring the reprotonation of some amine groups and maintaining the hydration of the chains. Thus, both phenomena of shrinking and hydration were compensated. In this case, the elimination of the salt, although favoring hydrogen bonding, disfavored hydrophobic interactions. The variation of the final polymer concentration in the washed hydrogel, as a function of the polymer concentration in the initial solution, was observed for two DAs: 5.2% and

Table 2. Difference of Polymer Concentration between the Initial Solution and the Final Gel for Different DAsa degree of acetylation (%) 5.2 20.8 31.6 36.7 40.4 52.1 concn of the initial soln 1.20 1.30 1.50 1.50 1.60 1.70 (% (w/w)) concn of the 1.82 1.42 1.65 1.67 1.62 1.75 corresponding final gel (% (w/w)) a

The precision of the concentrations was (0.02% (w/w).

40.4%. In the range of concentration studied (0.5-3% (w/ w)), the variation of the final concentration as a function of the initial concentration was quite linear (not shown). The slope of 2.32 for DA ) 5.2% was higher than that of 1.25 found for DA ) 40.4%, in agreement with the phenomenon of shrinking described above for low DAs. For the two DAs,


Figure 13. Influence of DA on Ge, the equilibrium storage modulus, for different gels. Final concentration of the gels after washing, Cpolymer ) 1.8% (w/w).

in the range of concentration studied here, the result showed that the shrinking appeared whatever the initial polymer concentration in the solution. Equilibrium Storage Modulus Ge. In the second part of this work, the final gels were characterized (after washing). We decided to investigate the influence of DA and polymer concentration of the gel on the equilibrium storage modulus (Ge), corresponding to the value of G′ at the plateau for low frequencies. Thus, the final gels with different DAs and polymer concentrations could be compared, and the best hydrogel could be chosen for biomedical uses. In the case of a physical gel, a very slight decline in G′ and the very small extent of G′′ are mentioned at low frequencies. Nethertheless, these moduli are nearly frequency independent.33,34 These variations were also observed for our gels. Influence of DA. Figure 13 shows the variation of Ge, the equilibrium storage modulus, for different DAs and a concentration of 1.8% (w/w) in the gel. This modulus depended on the number of junctions per unit volume. We observed that Ge increased with DA. The curve shows again the importance of the N-acetylglucosamine residues contributing to the formation of hydrophobic interactions responsible for the increase of Ge. As described previously, for high DAs, polymer chain self-associations occur. The increase of intermolecular cross-linking should increase the fraction of stable hydrophobic interactions. This explains why Ge has the highest value for a DA above 40%. Three parts can be shown: 0-25%, 25-40%, and above 40%. In the first range, hydrogels are mainly constituted of hydrogen bonding, whereas in the third one, hydrophobic interactions predominate. This curve illustrates again the general law of behavior of chitosan properties as a function of DA. It shows also the very important difference of viscoelastic behavior of gels whether hydrogen bonding or hydrophobic interactions predominate. We may consider that the gels formed in the first and last range of DA must be regarded as different in behavior but also in molecular organization. The number of junctions per unit volume is higher in the case of high DAs, partly in relation with a greater ease to form hydrophobic junctions at high DA than hydrogen bonding at low DA, requiring first a deprotonation of the amine sites.


Figure 14. Influence of the final polymer concentration of the gel on Ge, the equilibrium storage modulus, for different gels (DA ) 36.7%).

Influence of the Polymer Concentration. The evolution of Ge as a function of the final polymer concentration within the gel is reported in Figure 14, for a DA of 36.7%. Ge increased with the concentration. The presence of more polymer chains was necessarily responsible for more entangled junctions (see above). The influence of the polymer concentration on Ge was then more important than that of DA. This seems to agree with the presence of more entangled polymer chains having more influence on the number of junctions per unit volume than the proportion of acetyl units in the sample. Above a concentration of 1.5%, the modulus of amylose gels showed a seven-power dependence concentration.35 This high dependence is quite different from that of other biopolymer gels, such as gelatin,36 agarose, or carrageenans,37 showing more a square dependence. In the concentration range studied, we found that Ge was approximately proportional to C2.3. Moreover, the curve Ge ) f(C) could be separated in two parts, revealing again a critical concentration near 1.8% (w/w). Below this concentration, hydrogels had a relatively constant storage modulus at equilibrium, and above, the increase of this modulus with the polymer concentration was much higher. Since for this DA (see Table 2) the concentration in the gel was not too different from the concentration in the initial solution, we found again the critical value of C** already discussed. Loss Tangent: tan δ. In addition to the elastic modulus, the viscoelastic nature of the gels was further evaluated from tan δ, the loss tangent. As a measure of the ratio of the energy loss to the energy stored, tan δ reflects the overall viscoelasticity of the sample.38 Thus, as tan δ becomes smaller, the elastic modulus of the material increases, while the viscous behavior is reduced. tan δ decreased with the increase of DA or the polymer concentration. Thus, the elastic contribution became higher with the polymer concentration or DA. These hydrogels are then not homogeneous in term of viscoelastic behavior. Conclusion The gelation mechanism of chitosan solutions studied in this paper is particularly interesting, since the initial solution only contained water and chitosan. Thus, no organic solvent

662


or other additive was necessary for the gelation. The evolution of the zero shear rate viscosity of solutions enabled us to better understand the organization of the chains just before the beginning of gelation. Then, rheological experiments allowed us to study the influence of both DA and the polymer concentration on gelation. The role of acetyl groups in physical gelation of chitosan was again underlined. They played an important kinetics role in the formation of hydrophobic interactions, mainly responsible for gelation. In this study, we again observed our general law of behavior. Initially, when C > C*, the number of chain entanglements is always insufficient as well as the number of physical junctions of hydrophobic nature. It is then necessary to increase this number. Then, DA and the initial concentration play a conjugate role on both the formation of these junctions and their number. The number of junctions per unit volume at the gel point varied only with the initial polymer concentration, i.e., the initial number of chain entanglements per unit volume. Above C close to 1.8% (w/w), the time to reach the gel point was much reduced, suggesting a high junction density and also, probably, the presence of gel precursors due to a reorganization of the solutions to lower the energy, inducing a faster construction of a physical network. Therefore, it would be interesting to consider a second critical concentration C** corresponding to the presence of nanoobjects in the initial solution which increase in size with time up to their collapse. This behavior also depends on DA. Thus, the influence of the polymer concentration and DA of chitosan on this typical gelation and, consequently, the mechanism of gelation are now relatively well understood. The second aim of this work was to characterize the gels by rheology measurements in order to choose a gel with optimal characteristics for our biomedical applications. A DA near 40% seemed to be optimal if we considered the value of Ge, since gels made from a chitosan of higher DA could encounter problems of redissolution at pH of a biological medium. Consequently, the following characteristics for the final gel were chosen: a DA near 40% and a final polymer concentration close to 1.5% (w/w). Acknowledgment. This work was financially supported by the Laboratories Gene´vrier, the ANRT, and the center of medical technology of Saint-Etienne (France). We also are indebt for the help of Professor Ph. Cassagnau. We thank Aure´lien Guicherd and also Mahtani Chitosan for the gift of chitosan samples. References and Notes (1) Domard, A.; Domard, M. In Polymeric Biomaterials, Dumitriu, S., Ed; Basel: New York, 2002; Chapter 9, p 187-212.

Montembault et al. (2) Roberts, G. A. F. Chitin chemistry; Macmillan Press Ltd: London, 1992. (3) Klokkevold, P. R.; Vandemark, L.;.Kenney, E. B.; Bernard, G. W. J. Periodontol. 1996, 67, 1170. (4) Muzzarelli, R. A. A.; Biagini, G.; Pugnaloni, A.; Filippini, O.; Baldassarre, V.; Castaldini, C.; Rizzo, C. Biomaterials 1989, 10, 598. (5) Muzzarelli, R. A. A.; Zucchini, C.; Ilari, P.; Pugnaloni, A.; Mattioli Belmonte, M.; Biagini, G.; Castaldini, C. Biomaterials 1993, 14, 925. (6) Muzzarelli, R. A. A.; Biagini, G.; Bellardini, M.; Simonelli, L.; Castaldini, C.; Fratto, G. Biomaterials 1993, 14, 39. (7) Shumilina, E. V.; Shchipunov, Y. A. Colloid J. 2002, 64, 372. (8) Draget, K. I.; Vaarum, K. M.; Moen, E.; Gynnild, H.; Smidsroed, O. Biomaterials 1992, 13, 635. (9) Chenite, A.; Chaput, C.; Combes, C.; Selmani, A.; Jalal, F.; Bio Syntech, Ed.; Canada US 6344488, 2002. (10) Vachoud, L.; Zydowick, N.; Domard, A. Carbohydr. Res. 1997, 302, 169. (11) Montembault, A.; Viton, C.; Domard, A. Biomaterials 2004, in Press. (12) Montembault, A.; Viton, C.; Domard, A. Biomaterials 2005, 26, 933. (13) Buckwalter, J. A.; Mankin, H. J. J. Bone J. Surg. 1997, 79-A, 600. (14) Pelletier, S.; Hubert, P.; Payan, E.; Marchal, P.; Choplin, L.; Dellacherie, E. J. Biomed. Mater. Res. 2001, 54, 102. (15) Hunziker, E. B. Osteoarthritis Cartilage 2001, 10, 432. (16) Hirai, A.; Odani, H.; Nakajima, A. Polym. Bull. 1991, 26, 87. (17) Domard, A. In Chitin Enzymology; Muzzarelli, R. A. A., Ed.; European Chitin Society Publisher: Ancona, Italy, 1993; pp 441446. (18) Lamarque, G.; Lucas, J.-M.; Viton, C.; Domard, A. Biomacromolecules 2004, in press. (19) Schatz, C.; Viton, C.; Delair, T.; Pichot, C.; Domard, A. Biomacromolecules 2003, 4, 641. (20) Sorlier, P.; Denuzie`re, A.; Viton, C.; Domard, A. Biomacromolecules 2001, 2, 765. (21) Rodriguez-Sanchez, D.; Rha, C. J. Food Technol. 1981, 16, 469. (22) Cairns, P.; Miles, M. J.; Morris, V. J.; Ridout, M. J.; Brownsey, G. J.; Winter, W. T. Carbohydr. Res. 1992, 235, 23. (23) Schatz, C.; Pichot, C.; Delair, T.; Viton, C.; Domard, A. Langmuir 2003, 19, 9896. (24) Merkovich, E. A.; Carruette, M.-L.; Babak, V. G.; Vikhoreva, G. A.; Gal’braikh, L. S.; Kim, V. E. Colloid J. 2001, 63, 350. (25) Kavanagh, G. M.; Ross-Murphy, S. B. Prog. Polym. Sci. 1998, 23, 533. (26) De Gennes, P. G. Scaling concepts in polymer physics; Cornwell University Press: Ithaca; 1979. (27) Stauffer, D.; Coniglio, A.; Adam, M. AdV. Polymer Sci. 1982, 44 (Polym. Networks), 103. (28) Winter, H. H.; Chambon, F. J. Rheology 1986, 30, 367. (29) Fuchs, T.; Richtering, W.; Burchard, W.; Kajiwara, K.; Kitamura, S. Polym. Gels Networks 1997, 5, 541. (30) Borchard, W.; Burg, B. In Molecular basis of polymer networks, 42nd ed.; Baumga¨rtner, A.; Picot, C. E., Eds.; Springer-Verlag: Berlin, 1989; pp 162-168. (31) Chenite, A.; Chaput, C.; Wang, D.; Combes, C.; Buschmann, M. D.; Hoemann, C. D.; Leroux, J. C.; Atkinson, B. L.; Binette, F.; Selmani, A. Biomaterials 2000, 21, 2155. (32) Amiji, M. M. Carbohydr. Polym. 1995, 26, 211. (33) Ross-Murphy, S. B.; McEvoy, H. Br. Polym. J. 1986, 18, 2. (34) Clark, A. H.; Ross-Murphy, S. B. AdV. Polym. Sci. 1987, 83, 57. (35) Ellis, H. S.; Ring, S. G. Carbohydr. Polym. 1985, 5, 201. (36) Ferry, J. D. AdV. Protein Chem. 1948, 4, 1. (37) Guenet, J.-M. ThermoreVersible Gelation of Polymers and Biopolymers; Academic Press: London, 1992. (38) Ferry, J. D. Viscoelastic properties of polymers; Wiley: New York, 1980.

BM049593M

Rheometric Study of the Gelation of Chitosan in Aqueous Solution

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