Morphological and Rheological Characterization of Chitosan Liquid-Crystalline Solutions and Gels

Hugo M. Lisboa1,2, João P. Borges2, Marcus V. L. Fook1, Ana M. Ramos3, Maria

T. Cidade2

1 CertBio, Unidade Académica de Engenharia dos Materiais, Universidade Federal Campina Grande, Campina Grande,

PB, Brasil2 Departamento de Ciência dos Materiais and Cenimat/I3N, Faculdade de Ciências e Tecnologia, FCT, Universidade

Nova de Lisboa, UNL, 2829-516, Caparica, Portugal3Departamento de Química and REQUIMTE, Faculdade de Ciências e Tecnologia, FCT, Universidade Nova de Lisboa,

UNL, 2829-516, Caparica, Portugal

AbstractChitosan is a biopolymer used in biomedical applications, which has the ability to form lyotropic

mesophases (chiral nematic) in several solvents. However, chitosan chiral nematic phase is

only observed at high polymer concentrations where the system is in a non-homogeneous gel-

like state which has limited or no practical interest. In this work the formation of chitosan liquid-

crystalline solutions and gels was investigated. For the first time, a chiral nematic mesophase

was observed in low viscosity solutions of chitosan, the novelty of this study. Malic and

hydrochloric aqueous solutions with different concentrations of chitosan were prepared and the

gel formation was followed by rheological measurements (crossover between the values of G’

and G’’ and evolution of tan vs c for different ). The concentration, CGel, at which the gel is

formed, was found to be dependent on the acid used, the malic aqueous solutions presenting

higher CGel. For the chitosan/malic acid system the chiral nematic mesophase appeared in

solution and was preserved in the gel (for C>CGel) while for the chitosan/hydrochloric acid

system the mesophase was only observed in the gel. This behavior was correlated with the type

of interaction polymer/solvent. Swirl-like fingerprint textures typical of cholesteric mesophases

were observed by Polarized Optical Microscopy (POM) and the helical pitch was determined

from POM images. The existence of mesophases in the gel, in both chitosan aqueous solutions,

was also confirmed by rheometry. The results here obtained can be of key importance in the

field of Tissue Engineering. The biomedical interest in liquid crystalline systems of chitosan lies

in the possibility of mimicking the structure of the main component of the extra-cellular matrix in

connective tissues, collagen type I, which has analogous liquid crystalline ordering in acidic

solutions.

Keywords: chitosan, morphology, rheology, cholesteric, liquid-crystals, gels

Introduction

Chitin and chitosan are glycosaminoglycans with the rare property of bioactivity. These

polysaccharides are in fact copolymers of -(1→4)-linked 2-acetamido-2-deoxy-D-

glucopyranose and 2-amino-2-deoxy-D-glucopyranose. The difference between the two is

determined by the DA, the degree of acetylation, corresponding to the molar fraction of acetyl

units constituting the polymer chains. It is considered that chitosan refers to polymers with DAs

below 50 % (more commonly 10-30 %) (Khor, 2001). Chitin is considered the second most

abundant polymer in nature but due to its low solubility has limited applications and therefore is

mostly used in the deacetylated form, as chitosan. Chitosan is the product of the alkali

hydrolysis of chitin and is soluble in dilute acidic solutions (Tolamaite et al., 2003). The reason

why chitosan presents a higher solubility than chitin is the presence of protonated free amine

groups below pH 6.2 [Park et al., 1983] and this polycation material has several applications in

different fields, from agriculture, waste and water treatment, food and beverages, cosmetic and

toiletries, biopharmaceuticals to biomedical applications [Rinaudo, 2006 ,Majeti et al., 2000]. In

biomedical and pharmaceutical applications chitosan is used to produce hydrogels, due to its

gelation properties. Several reports on literature can be found concerning the production of

chemical [Guo et al., 1998, Roberts et al., 1989] and physical (Shumilina et al., 2002, Chenite

et al., 2002,] hydrogels from chitosan. Hamdine (Hamdine et al., 2005, Hamdine et al., 2006)

reported an extensive work regarding chitosan gelation in different acids and concluded that

chitosan has different behaviours when dissolved in acid malic and in hydrochloric acid. In the

first case, due to the considerable volume of the acid molecule, that hinders the interactions

between chitosan chains, gelation is inhibited. However, when chitosan is dissolved in HCl, a

short chain solvent, gelation occurs due to neutralization and limited solubility of chitosan. The

present work focus on the formation of lyotropic liquid crystalline phases (chiral nematic) both

on sol and gel phases of chitosan dissolved in hydrochloric and malic acid aqueous solutions.

Liquid crystalline structures are common in nature, with many fluid and solid biological tissues

revealing liquid crystalline architectures [Giraud-Guille et al., 2008, Rey, 2010). This situation

encompasses fluid cell membranes bearing orientational order to other more solid but clear

templates which possess a liquid crystal arrangement [Mosser et al., 2006]. Human bones and

extracellular cuticles of arthropods are examples of that. It is assumed that cells produce highly

concentrated liquid crystalline polymers that later become the support matrix for minerals in

order to reinforce the neo structure. This type of reinforcement “freezes” the liquid crystal

arrangement by a chemical cross-link. Several rigid or semi-rigid natural polymers have been

found to form a lyotropic mesophases. Examples include polypeptides (Uematsu et al., 1894),

proteins (Bernal et al., 1941), poly(nucleic acids) (Yevdokimv et al, 1988, Robinson, 1961,

Spencer et al., 1962, Iizuka et al., 1977) and cellulosics (Gray, 1983, Godinho et al., 2002).

Chitosan belongs to another family of liquid-crystalline biopolymers, which was first reported in

1982 by Ogura et al. (Ogura et al., 1983) and since then, quite a number of lyotropic solutions of

chitosan derivatives have been found (Terbojevich et al., 1991, Rout et al., 1993a, Rout et al.,

1993b, Rout et al., 1994, Dong et al., 2001, Chang et al., 2007). Few of these studies

concerned the effects of structural factors on the critical concentration (C*) for forming the

lyotropic mesophases (Wu et al., 2002, Dong et al., 2004, Dong et al., 2000). M. Prabaharan

(Prabaharan et al., 2006) have performed a systematic monitoring of the liquid crystalline

behaviour of chitosan in different acidic solutions, especially at high concentration of polymer,

when gel-like material can be produced. Chitosan was found to form lyotropic mesophases in

concentrated acidic solutions of formic, acetic and monocholoroacetic acids. The influence of

acid concentrations on the C* and on the helical pitch of the cholesteric (P) was investigated.

The C* values increased with increasing the concentration of acids in the solvent, reaching the

maximum of 35 wt% for 30 % (v/v) formic, 50 % (v/v) acetic and 30 % (wt/v) monochloroacetic

acids. The maximum value of P was found to be 12.5 μm for the C* of 30 wt% at 30 % (v/v)

acetic acid. As expected, P was found to decrease with increasing concentration of chitosan in a

given solvent. The results suggested that 30 % (v/v) acetic acid would be a good solvent for the

preparation of chitosan gels exhibiting liquid crystalline properties.

Biopolymers, like collagen, cellulose, and chitin extracted from biological tissues, had already

revealed the same type of liquid crystal arrangement when in concentrated solutions. It seems

clear that, when dealing with a material for human tissue reparation and regeneration, the

design of the original tissue must be considered. Proper cell signalling and cell proliferation

must then be obtained in the synthetic material so that new tissue starts to form. In the

biomedical field, chitosan lyotropic mesophases can be very interesting since a very similar

cholesteric ordering was observed in solutions of collagen type I at high concentrations (>20

mg/mL) in acetic acid (Hulmes et al., 2002); this could be an indication that chitosan could

mimic such kind of structures of the main component of the extracellular matrix in connective

tissues. However, all reports in literature show that the chitosan mesophases are obtained at

high polymer concentrations, ie, in gels, which are heterogeneous due to the difficulty in

dissolving chitosan and, thus have limited or no practical interest.

In the present work we study the formation of chitosan lyotropic solutions and gels by

rheological measurements, supported by POM characterization and for the first time it was

observed the presence of a chiral nematic phase in low viscosity solutions of chitosan.The

formation of the mesophase in a low viscosity solution was dependent on the type of acid used

and was correlated with the polymer/solvent interaction.

Experimental Section

Chitosan Characterization

Chitosan from Sigma Aldrich (High Molecular Weight grade), was characterized by Size

Exclusion Chromatography (SEC) and Fourier Transform InfraRed (FTIR) in order to determine

the average molecular weight and degree of deacetylation of purified chitosan.

The FTIR samples were prepared as described by Brugnerotto et al, 2001. Brieflly, pellets with

1mg of chitosan, mixed with 100mg of KBr, were analyzed using a Nicolet 6700 FT-IR

spectrometer. The IR spectra were acquired by the accumulation of 64 scans with a 4 cm -1

resolution. For SEC characterization a series of packed columns (Waters Hydrogel) were used

to characterize chitosan diluted solutions using 0.3M Acetic Acid/0.2 M Sodium Acetate as

eluent. 100µL of 0.1% chitosan solution was injected and a flow of 1mL/min was used. It was

found that chitosan has an average molecular weight, Mw, of 417 000 and a deacetylation

degree of 88%.

Preparation of chitosan solutions

Solutions of different chitosan concentrations with increments of 0.4% (wt/wt) (0.2 wt% in some

critical range of concentrations), were prepared separately in small glass vials. For

solubilization, two aqueous solutions were used, malic acid (ACROS, reagent grade) 0.15 M

and hydrochoric acid (Sigma Aldrich, reagent grade) 0.1M.

After preparation, all samples were left to dissolve and rest for 3 days, prior to use.

pH and Ionic StrengthWhile dissolved in acidic solutions, chitosan acts like a weak base (Roberts et al., 1992) so it’s

expected that with increasing concentration, pH values also increase until a certain value that

reveals the limit of solubilization and the buffering capacity of glucosamine units. The solution’s

ionic strength was determined by the following equation (Segel et al., 1976) :

(Eq.1)

where c and Z are the concentration and the charge number of ion i, and the degree of

dissociation of the acid. The ionic strength is expressed by molarity (M).

Polarised Optical Microscopy (POM)

Chitosan solutions of different concentrations were observed with an Olympus BHA optical

microscope, under polarized light, at room temperature, which was controlled by a Mettler

FP82HT hot stage.

Rheological Characterization

The rheological behavior of the chitosan solutions was studied with a Bohlin Gemini HRnano

rotational rheometer, using a cone and plate (cone angle: 2º; diameter: 40 mm) at 25ºC.

Oscillatory and steady state shear measurements were performed, which allowed for the

determination of the elastic and viscous modulus and complex viscosity in function of the

angular frequency, and the steady state viscosity as function of the shear rate. Both

measurements were performed for 0-6.8 wt% chitosan solutions allowing for the determination

of the sol-gel transition. In the case of the steady state measurements, after temperature

equilibration, and before starting the measurements, the samples were submitted to a pre-

shearing stage, with a pre-shear of 1 s-1 applied for 60 s, followed by an equilibration time of 180

s. All measurements were performed with a pristine sample.

Results and Discussion

Polarised Optical Microscopy (POM)

Typical swirl-like fingerprint patterns indicative of the existence of cholesteric ordered states

were observed either for chitosan/malic acid or chitosan/hydrochloric acid systems. These

ordered states appeared in chitosan/malic acid solutions and in gels obtained from the

chitosan/hydrochloric acid system. Figure 1a shows, as an example, the POM image of the gel

obtained for 4.4 wt% chitosan in diluted hydrochloric acid solution. Similar textures were

observed for cholesteric lyotropic phases of chitosan in diluted malic acid solution. Below the

critical concentration for the mesophase formation, C*, a biphasic region was observed for the

chitosan/malic acid system. The biphase observed by POM is presented in Figure 1b.

(a)

(b)

Figure 1 – POM observations of chitosan textures: a) fingerprint lines for 4.4 wt% of chitosan in

hydrochloric acid; b) biphase observed for a solution of

3.6 wt% of chitosan in malic acid.

In the case of the chitosan/hydrochloric acid system no biphase was observed which is probably

due to the fact that a gel is formed before the appearance of the cholesteric phase. The POM

images were also used to determine the chiral nematic pitch (P) of the mesophases. Figure 2

shows the variation in pitch with concentration for chitosan in malic, (a) and hydrochloric, (b),

acids. Chitosan volume fractions (%) were calculated from weight fractions considering for all

solvents the density equal to 1.0 g.cm-3 and for chitosan the density 1.42 g.cm-3, obtained from

chitosan membranes (Silva et al., 2004). Chitosan shows long-pitch cholesteric structures in

diluted solutions of malic and hydrochloric acids. The pitch decreases with increasing polymer

concentration and it was verified that

(Eq.2)

where is the volume fraction of the polymer. Similar results have been reported for

(hydroxypropyl)cellulose in water (Onogi et al., 1980) and (acetoxypropyl)cellulose in acetone

(Bhadani et al., 1983). Experimental results for the variation of the pitch with concentration for

polypeptides can also be fitted by a general power law

(Eq.3)

where x has a value between 1 and 2 (Toriumi et al., 1981). The magnitude of x depends on the

solvent, temperature and molecular weight.

(a)

(b)

Figure 2 – Variation in helicoidal pitch with volume fraction of chitosan in diluted malic acid

solution (a) and hydrochloric acid solution (b). Results obtained from POM images Please insert

Rheological Characterization

Figures 3 and 4 show the effect of shear rate on the measured viscosity for the chitosan in malic

acid and hydrochloric acid aqueous solutions at different concentrations. By increasing the

chitosan concentration in both systems we observe an increase in the viscosity, which is

explained by the number of interactions and entanglements that increases with concentration

and restricts the motion of individual chains. For low concentrations the solutions exhibit a

Newtonian behavior for lower shear rates and a shear-thinning behavior for higher shear rates,

but increasing the polymer concentration, only a non-Newtonian shear-thinning behavior is

observed in the shear rate range studied. The shear-thinning effect is strengthened with

increasing polymer concentration. The observation that the shear-thinning effect becomes

stronger as the concentration increases has already been reported for different chitosan

systems (Desbrières, 2002, Mucha, 1997). The decrease in the viscosity with increasing shear

rate can be attributed to the disruption of the molecular interactions and entanglements; that is,

the rate of junction disruption exceeds the rate at which new entanglement associations can be

re-formed, therefore increasing the mobility of the polymer chains and decreasing the solution

viscosity.

Figure 3– The effect of the shear rate and the chitosan concentration on the rheological curves

of malic acid solutions.

Figure 4– The effect of the shear rate and the chitosan concentration on the rheological curves

of hydrochloric acid solutions.

Small amplitude oscillatory shear tests were performed to investigate the effect of chitosan

concentration on the dynamic rheological properties of concentrated chitosan acid aqueous

solutions. Figures 5 and 6 show, the storage (G’) and loss (G’’) modulus as function of angular

frequency, in terms of chitosan concentration for malic acid and hydrochloric acid solutions,

respectively. Both G’ and G’’ increase with increasing chitosan concentration, for both systems.

For the less concentrated solutions, at low frequencies, a predominant viscous character (G’’>

G’) is evident but, increasing the angular frequency, the elastic modulus grows faster than the

viscous one and G’ curve crosses the G’’ curve at a frequency called cross-over frequency (ωc);

at frequency higher than ωc, the elastic modulus prevails on the viscous one and the system

shows a predominant elastic character.

(a)

Figure 5 –Effect of the chitosan concentration on the storage (G’) and loss (G’’) modulus as a

function of angular frequency, for malic acid solutions

(a)

Figure 6 –Effect of the chitosan concentration on the storage (G’) and loss (G’’) modulus as a

function of angular frequency, for hydrochloric acid solutions.

The features of entangled system are also visible in the loss tangent (tan δ = G’’/G’, where δ is

the phase angle) curve, Figure 7. At the cross-over frequency the loss tangent is equal to one

while it is greater than one at lower frequencies and lower than the unit at higher frequencies. In

Figure 7, we can observe, for both systems, the concentrations for which the solutions become

predominantly elastic, being this value 6.0 wt% for malic acid solutions and 2.6 wt% for

hydrochloric acid solutions. Similar values have been obtained for other aqueous solutions of

chitosan. Desbrières (Desbrières, 2002) found a sol-gel transition for chitosan in 0.3 M acetic

acid /0.05 M sodium acetate of 43 g/l L (circa 4.3 wt %) and Iversen (Iversen et al., 1997) found,

for chitosan in 1% acetic acid, a CGel of 3.9 wt %.

(a)

(b)

Figure 7 –The effect of chitosan concentration on the loss tangent as function of angular

frequency, for the (a) malic acid solutions and the (b) hydrochloric acid solutions.

Looking at Table 1, in which the cross-over frequencies and the relaxation times for both

chitosan systems are reported, it can be noticed that for the malic acid solutions the cross-over

frequency was first verified for a solution concentration of 2.8 wt%. Corresponding to the highest

cross-over frequency value, the relaxation time is the lowest for the concentration of

2.8 wt% while it increased with the polymer concentration, varying from 0.083 to 0.226 s for a

concentration of 5.2 wt%. For the hydrochloric acid solutions, the cross-over frequency was

observed for solution concentrations between 1.6 and 2.4 wt%. As shown in Table 1, the cross-

over frequencies decreased with the increase in polymer concentration, from 94.6 to 89.9 rad/s,

with the consequent increase in the relaxation time from 0.066 to 0.070.

Table 1 – The concentration, the cross-over frequency and the relaxation time for both

chitosan systems.

Malic acid solutions Hydrochloric acid solutions

Concentration (wt%)

Cross-over frequency

(rad/s)

Relaxation time (s)

Concentration (wt%)

Cross-over frequency

(rad/s)

Relaxation time (s)

2.8 75.5 0.083 1.6 94.6 0.0663.2 51.4 0.122 2.0 93.8 0.0673.6 30.4 0.207 2.4 89.9 0.0704.0 29.3 0.214 2.6 - -4.4 31.6 0.199 3.2 - -4.8 36.7 0.1715.2 27.8 0.2266.0 - -6.8 - -

At the gel point, G’ and G’’ curves become more or less parallel to each other and the power law

behavior (G’ G’’ ~ ωm) in frequency is observed. Above this point G’ becomes higher than G’’.

This sol-gel transition is observed for a polymer concentration of about 6.0 wt% for the malic

acid solutions and for concentrations of about 2.6 wt% for the hydrochloric acid solutions, as

can be seen in Figures 5 and 6. To determine more accurately this value, the evolution of the

loss tangent as a function of polymer concentration (at several frequencies) is presented in

Figure 8. These curves converge to a sole one for concentrations equal or higher than the

gelation concentration, CGel, when the G’(ω) and G’’(ω) become parallel and the loss tangent

values are independent of the frequency. The found gelation concentrations, CGel, are equal to

2.6 wt% for the hydrochloric acid solutions, and very close to 6.0 wt% for the malic acid

solutions.

(a)

(b)

Figure 8 – Influence of chitosan concentration on the variation of tan δ at different frequencies,

for both (a) malic and (b) hydrochloric acid solutions.

The viscosity curves, along with the curves obtained in oscillatory measurements, Figures 3 and

6, give, also, an indication of the concentration for which a transition from an isotropic to an

anisotropic solution or gel occurs. In fact, a careful observation of the curves in Figures 3 and 5,

(b), shows that, for the malic acid, a biphase appears for concentrations between 3.6 and 4.0 wt

%, with a critical concentration (appearance of the anisotropic phase, C*) between 4.0 and 4.4

wt%, followed by a decrease of the viscosity for 4.8 wt% solutions and, again, an increase of the

viscosity for higher concentrations. This same result may be seen in Figure 9 (a) where the

dependence of the viscosity with the concentration is presented; this transition is perfectly clear,

even though the curve is not the usual one. In fact this tiny decrease could be thought as an

experimental error, however, it is perfectly reproducible. This observation is in accordance with

the well known concentration dependence of the viscosity in the isotropic-nematic transition

found in lyotropic liquid crystalline polymers (Doi, 1981, Larson, 1999). This situation is

explained by the decrease of the viscosity at the transition due to the fact that the molecules can

slide past each other more readily in the nematic state than in the isotropic one, due to their

orientation. It is important to note that our lyotropic system presents, at rest, cholesteric

mesophase, however it is known that the helix unwound under shear forces, giving rise to a

nematic structure, at shear rates below the lowest values considered in our experiments

(Cidade et al., 1995). The same phenomena occurs for the hydrochloric acid solutions, Figures

4 and 6 (b), with C* between 2.6 and 2.8 wt%. The viscosity as function of the concentration

curve is not so clear in this case (see figure 9 (b)), probably due to the coexistence of the

nematic-isotropic transition and the sol-gel transition. In fact, even present, the nematic phase

behavior is masked by the physical cross-linking occurring in the gel phase. However, in the

absence of the nematic phase a continuous increase of the viscosity could be anticipated, which

is not the case, until concentrations equal or higher than about 4%, meaning that, at these

higher concentrations the nematic arrangement is no longer possible. The relaxation times

presented in Table 1, in the case of malic acid solutions are in accordance, as expected, with

these values of C*; in fact the relaxation time increases with concentration until a value of 4.0 wt

%, then decreases until around 4.8 wt% to increase again for higher concentrations. In the case

of hydrochloric acid since the anisotropic phase appears already in the gel phase, an indication

of C* cannot be given by this method.

(a)

(b)

Figure 9 – Influence of the chitosan concentration on the viscosity of malic (a) and hydrochloric

(b) acid solutions.

These critical concentrations, confirmed by the ones found using optical microscopy, clearly

show that a transition from an isotropic to an anisotropic phase occurs in these systems. A point

of interest is the fact that while in the malic acid system the isotropic-nematic transition appears

before the sol-gel transition (in the solution state), in the case of hydrochloric acid C* is higher

than CGel, which means that the anisotropic phase appears after the gel is formed. Montembault

and coworkers (Montembault et al., 2005a, Montembault et al., 2005b) have shown that

gelation’s mechanism of chitosan in acidic solutions is attributed to the formation of physical

junctions due both to hydrophobic interactions involving N-acetyl groups and hydrogen bonding.

The balance between these hydrophobic interactions and H-bonds depend on the DA. For low

DA (< 25 %), as in the case of the present work, the initial charge density of the polymer is high,

the content of hydrophobic functions is low and hydrogen bonding plays a more important role in

the gelation mechanism (Montembault et al., 2005b). Malic acid is an organic carboxylic diacid

with three functionalities (two carboxyl and one hydroxyl groups) capable of establishing H-

bonds with either amine or hydroxyl groups of chitosan chain which is not the case of the

mineral hydrochloric acid. Therefore, as it is a weaker acid, and its molecule has a higher

volume, it acts like a spacer, reducing the possibility of proximity of the chains and then a lower

number of intermolecular H-bonds between acetylated groups of chitosan chains are

established when this biopolymer is dissolved in malic acid, resulting in a higher CGel. The low

concentration of hydrochloric acid may also be the reason for lower Cgel. As it can observed at

table 2, the solution’s pH increase sharply between concentrations of 2.0 and 2.8 wt%, and that

this is the limit concentration for chitosan solubilization in hydrochloric acid, resulting the

formation of the gel.

Table 2 – Concentration, pH and ionic strength for both chitosan systems

If the protonation ability of chitosan’s amino groups by the two acids is considered, the values of

the acid dissociation constant, pKa is - 6.2 for hydrochloric acid (Robinson et al., 1971) and 3.4

and 5.2 for the first and the second carboxylic group of malic acid to be dissociated (Dawson et

al., 1959). These values mean that hydrochloric acid has a higher capacity to protonate

chitosan, which introduce a large amount of positive charges in a smaller number of polymer

chains, originating the consequent repulsion phenomena between them. Probably, these might

be the explanation for the observed higher C* in the chitosan/malic acid system. In this system,

for this frame of time, the possibility of malic acid to behave as a crosslinker for chitosan, as

reported in literature (Chang et al., 2007) does not seem to be very significant, according to the

results obtained, which must be due to the low concentrations of the acids used. Currently there

is still no agreement on the conformation of chitosan chains. While some authors [Cölfen et al.,

2001, Fee et al., 2003, Kasaai et al., 2006, Terbojevich et al., 1991) point to a rigid-

rod conformation others [Berth et al., 1998, Brugnerotto et al., 2001, Lamarque et al., 2005,

Mazeau et al., 2004, Rinaudo et al., 1993, Shatz et al., 2003, Velásquez et al., 2008, Vold,

2004, Morris et al., 2009) adopt a  semi-flexible coil model. Through Flory’s model (Flory,

1956)  it is possible to estimate the theoretical value of the critical concentration, with this value

being 17.2 wt%, in both acids. The difference between our experimental values (C* ~ 2.6-2.8 wt

% for hydrochloric acid; C* ~ 4.0 – 4.4 wt% for malic acid) and this may be related to the fact

that this model assumes that the chitosan chains are in a rigid rod conformation, so this result

indicates that the chitosan in the present system is in semi-flexible coil conformation. For the

range of pH and ionic strength used in the present work (see table 2), and accordingly to the

work of Chen and Tsaih [Chen et al., 1998], chitosan chains with a molecular weight higher than

223 000 assume a less stretched conformation, indicating the adoption of a semi-flexible coil

conformation instead of a rigid-rod.

CONCLUSIONS

In this work, hydrochloric and malic acid solutions of chitosan were microscopically and

rheologically characterized, which allowed for the determination of the concentration at which a

gel is formed (CGel) and also the concentration at which a chiral nematic phase appears (C*) in

the solution or gel. CGel was determined from the crossover between the G’ and G’’ as a function

of the angular frequency, as well as by the evolution of the tan as a function of the

concentration for different angular ferquencies, while C* was determined by the evolution of the

viscosity curve and relaxation times, obtained, once again, from the crossover mentioned

before. CGel obtained are equal to 2.6 wt% for the hydrochloric acid solutions, and very close to

6.0 wt% for the malic acidsolutions. Concerning C* the values obtained were between 4.0 and

4.4 wt% for the malic acid solutions and between 2.6 and 2.8 for the hydrochloric acid solutions.

A point of interest is the fact that while in the malic acid system the isotropic-nematic transition

appears before the sol-gel transition (in the solution state), in the case of hydrochloric acid C* is

higher than CGel, which means that the anisotropic phase appears after the gel is formed. The

explanation for this fact lies in the different acid strength of the two acids and their capability in

forming hydrogen bonds with chitosan. Typical swirl-like fingerprint patterns, observed under

polarized optical microscopy, showed that the mesophases presented by the chitosan aqueous

solutions were of the chiral nematic type. The pitch of the mesophase has shown to decrease

with increasing polymer concentration with a power law dependence of 3. The possibility of

obtaining liquid crystalline phases of chitosan from low viscosity acidic solutions opens new

perspectives in materials science, and especially in the Tissue Engineering field. These

mesophases can be “freezed” in a gel, for example, by a pH-triggered sol-gel transition. The

obtained gels can mimic the organization of collagen I and are good analogues of the

extracellular matrix, with a structure close to that of biological tissues. These materials can be

used either in tissue repair or as models for the culture of cells in 3D, to study their migration

and signaling activities, in a manner close to physiological conditions.

Acknowledgements Hugo Lisboa acknowledges Fundação para a Ciência e Tecnologia for grant SFRH/ BDE/

15557/2005

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