development and rheological investigation of novel alginate/n-succinylchitosan hydrogels

Development and Rheological Investigation of NovelAlginate/N-Succinylchitosan Hydrogels

M. R. NOBILE,1 V. PIROZZI,1 E. SOMMA,1 G. GOMEZ D’AYALA,2 P. LAURIENZO2

1Department of Chemical and Food Engineering, University of Salerno,Via Ponte Don Melillo 84084 Fisciano, Salerno, Italy

2Institute of Polymer Chemistry and Technology (ICTP) - CNR, Via Campi Flegrei 34, 80078 Pozzuoli, NA, Italy

Received 30 November 2007; revised 13 March 2008; accepted 17 March 2008

DOI: 10.1002/polb.21450

Published online in Wiley InterScience (www.interscience.wiley.com).

ABSTRACT: In the present article alginate hydrogels and novel hydrogels based on

blends of alginate/N-succinylchitosan have been realized in water solution at neutral

conditions. The gels have been obtained by crosslinking via the internal setting

method using calcium carbonate (CaCO3) as calcium ions source. A rheological inves-

tigation of both the plain alginate and the alginate/N-succinylchitosan blend hydro-

gels has been performed by means of oscillatory dynamic measurements. The effect of

the inclusion of different amounts of CaCO3 on the critical deformation (cc) character-

izing the limit of the linear viscoelastic regime has been studied for the plain alginate

gels. The frequency response in small amplitude oscillatory experiments of the plain

alginate gels has been investigated in terms of the storage (G0) and loss (G00) modulus

behavior. The dynamic data have been interpreted in terms of the Friedrich and Hey-

mann model. The inclusion of the N-succinylchitosan, in the range 10–50% w/w, had

no effect on the cc values. On the contrary, when the 10% w/w of the N-succinylchito-

san is added to the plain alginate gels, a significant increase in the storage modulus

values is recorded for all the systems analyzed. The gelation kinetics has been inves-

tigated and the results indicate that the kinetics process can be accelerated increas-

ing the percentage of Ca12 ions and/or including the N-succinylchitosan in the plain

alginate systems. Finally, the morphological analysis of scaffolds obtained from the

hydrogels through freeze-drying revealed an interconnected porous structure. VVC 2008

Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 46: 1167–1182, 2008

Keywords: alginate gels; biomaterials; gels; rheology

INTRODUCTION

Hydrogels are a class of materials of growing in-

terest in view of their numerous biomedical

applications. They are applied as space-filling

agents, as drug delivery vehicles and as three-

dimensional structures that organize cells to

direct the formation of a damaged tissue.1–3

Naturally derived polysaccharide-based hydro-

gels are appealing scaffold materials because

they are either components of or structurally

similar to the natural extracellular matrix of

many tissues, can often be processed under rela-

tively mild conditions and are able to absorb body

fluid for transfer of cell nutrients and metabolites

through the material. Such hydrogels play an

important role also in the field of drug release

and cosmetics, particularly for their high bio-

compatibility and low cost. Among them, algi-

nate-based hydrogels are ones of the commonly

investigated.4–8

Correspondence to: M. R. Nobile (E-mail: [email protected])

Journal of Polymer Science: Part B: Polymer Physics, Vol. 46, 1167–1182 (2008)

VVC 2008 Wiley Periodicals, Inc.

1167

Briefly, alginate is a safe biocompatible poly-

saccharide obtained from marine algae. It is a

linear polysaccharide copolymer of (1-4)-linked b-

mannuronic acid (M) and a-guluronic acid (G)

monomers. Within the alginate polymer, the M

and G monomers are sequentially assembled in

either repeating (MM or GG) or alternating (MG)

blocks. Alginate hydrogels are formed when diva-

lent cations, such as Ca21, cooperatively interact

with blocks of G monomers to form ionic bridges

between different polymer chains.9 Hydrogel sys-

tems based on alginate are also intended for

injectable scaffolds,10 that means that they can

be easily injected to the wanted sites and solidi-

fied in situ. Alginate crosslinking via calcium

ions is viewed as a mild process involving non-

toxic components and pH, osmolarity, and tem-

perature suitable for preserving mammalian

cells survive. On the other hand, it is important

to note that ionically crosslinked alginates show

low mechanical stability.11,12 To overcome single

polymer-based hydrogel drawbacks as weak me-

chanical properties, in the recent years heteroge-

neous hydrogels derived from polymer blends,

block copolymers, or interpenetrating polymer

networks, have been widely investigated. Blend-

ing is a simple method to combine the ad-

vantages of different polymers. The resulting

polymer blends may show synergistic proper-

ties,13,14 and are expected to have improved me-

chanical characteristics.

Because of its intrinsic antibacterial activity

and biocompatibility,15 chitosan is a good part-

ner for alginate in hydrogel development.16,17

Chitosan is a bioresorbable natural polysac-

charide, obtained by partial de-acetylation of

chitin.18–20 Structurally, chitosan is a linear

polysaccharide consisting of b(1-4) linked D-glu-

cosamine residues with a variable number of

randomly located N-acetylglucosamine groups.

Chitosan-based hydrogels can be formed by

change in pH value, ionic21 or covalent cross-

linking.22 A combination of chitosan with polyol

anionic salts has been reported to give tempera-

ture-controlled pH-dependent chitosan solu-

tions.23 Nevertheless, the acidic solubility and

gelation methods employed so far will surely

limit the application of chitosan as biopolymer

for in situ forming gels. Chemical derivatization

of chitosan, based on the reactivity of the pri-

mary amino groups, is a reported method to

induce solubility in neutral aqueous solutions,24

but the possibility to crosslink chitosan via ionic

interactions or by further reactions with chemi-

cal crosslinking agents such as glutaraldehyde

or genipin is strongly reduced upon modifica-

tion. The realization of blends with the easily

crosslinkable alginate may also represent a way

to overcome this problem.

In a previous work25 the preparation and

characterization of a novel composite based on

calcium sulfate (CaSO4) embedded in alginate or

in a blend of alginate with a water soluble chito-

san derivative (N-succinylchitosan, sCh) has

been reported. It has been found that the algi-

nate/sCh-based materials showed a considerable

improvement in Young modulus and yield

strength values with respect to the plain algi-

nate-based material, with a maximum corre-

sponding to a given composition of the alginate/

sCh blend. Such results have been tentatively

attributed to a synergistic effect of the N-succi-

nylchitosan in chelating calcium ions during the

alginate gelation process, which occurs as a con-

sequence of the slow calcium ions release from

CaSO4.

On the basis of this result, in the present ar-

ticle a novel hydrogel based on alginate and N-

succinylchitosan has been prepared in water so-

lution at neutral conditions. The gels have been

obtained by crosslinking via the internal setting

method,26,27 using calcium carbonate (CaCO3) as

calcium ions source. This technique allows a

controlled gelation of alginate through a slow

release of calcium ions at neutral pH, thus lead-

ing to the formation of a very regular gel

network. Uniform cell distribution and well-

controlled material properties are, indeed, neces-

sary in tissue engineering applications.28,29 The

plain alginate gels obtained by the CaCO3-GDL

(D-glucone-d-lactone) system are themselves

characterized by satisfactory mechanical pro-

perties for tissue engineering applications.28

Therefore, aim of this article is to show that the

inclusion of the N-succinylchitosan can lead to a

novel biocompatible hydrogel characterized by

good mechanical properties. At this regard, it

will be shown that a rheological study can be

successfully used to investigate the physical

characteristics of both the alginate gels and the

novel hydrogels, as also reported in the litera-

ture for different gel systems.30–47 Indeed, the

viscoelastic properties, the gelation kinetics, as

well as indications of the gels structure, will be

determined by the oscillatory dynamic study of

the gels. First, the effect of the calcium content

on the dynamic mechanical behavior of the plain

alginate will be presented. It is noteworthy that

1168 NOBILE ET AL.

Journal of Polymer Science: Part B: Polymer PhysicsDOI 10.1002/polb

there are no articles in the literature that inves-

tigate the effect of the inclusion of different

amounts of Ca21 ions both on the limit of the

linear viscoelastic regime as well as on the elas-

tic and viscous response of alginate gels on

the frequency. Then, alginate/N-succinylchitosan

blends of different compositions will be analyzed

to evidence the influence of the chitosan succi-

nate on the plain alginate properties, with the

goal to obtain a novel hydrogel with higher or

equivalent mechanical properties and structural

uniformity to satisfy the needs of different bio-

medical applications.

Furthermore, scaffolds obtained from the

hydrogels through freeze-drying have been

physically characterized by means of scanning

electron microscopy.

EXPERIMENTAL

Materials

Medium molecular weight chitosan (Ch) [75–

85% deacetylation degree; viscosity ¼ 200–800

cP; Mw ¼ 1 3 106 Da], succinic anhydride (SA),

calcium carbonate (CaCO3) and D-glucone-d-lac-

tone (GDL) were all purchased from Fluka-

Aldrich and used as received. Pyridine (Fluka)

was distilled under reduced pressure prior to

use. Medium molecular weight sodium alginate

(Alg) was supplied by Seaweed Company

(Shangai, China). The main chemical and physi-

cal characteristics of the alginate are reported

in Table 1. All the solvents were of analytical

grade.

N-Succynilchitosan Preparation Methodology

The succinylation reaction was carried out

according to a previously reported method.24

Briefly, a 2% chitosan solution (w/v) in aqueous

HCl (0.37%) was prepared and a 12.5% (w/v) an-

hydride succinic solution in pyridine was drop-

wise added at room temperature under vigorous

stirring. The reaction pH was maintained at 7.0

by simultaneous addition of a NaOH solution.

After 40 min of reaction, the modified polymer

was precipitated in a large excess of methanol,

washed with acetone and desiccated. To remove

the unreacted SA, the obtained product was dis-

solved in water and dialysed in a NaOH solution

for 48 h using a dialysis membrane bag with a

molecular weight cut-off of 10 kDa. (yield ¼95%)

FTIR: 1720 (C¼¼O acid stretching), 1640

(C¼¼O amide stretching), 1560 (N��H amide

bending) cm�1.1H NMR: d 4,6 (s, 1H), 3.7 (m, 6H), 2,5 (d,

4H).

Hydrogel Preparation Methodology

Alginate and alginate/N-succinylchitosan hydro-

gels were realized by the internal setting

method.27,28 To 10 mL of a 2% w/w polymeric so-

lution at different Alg/sCh ratios (100/0; 90/10,

80/20, 70/30, 60/40) a given amount of CaCO3

was added and the resulting mixture was left

under stirring for 30 min. Then a 9% D-glucone-

d-lactone (GDL) solution was added and the sys-

tem was kept for 45 s under vigorous stirring.

The concentrations of calcium carbonate used

were 0.1, 0.2, 0.5, and 0.7% w/w respect to poly-

meric total fraction. GDL was always used in

stoichiometric ratios respect to CaCO3 to obtain

a neutral gel.

The obtained solution was poured into a Petri

dish with diameter of 8 cm and left at room tem-

perature for 48 h to allow the complete alginate

crosslinking. The viscoelastic behavior of the al-

ginate and alginate/N-succinylchitosan hydrogels

was, then, studied in strain and frequency sweep

rheological tests, as described in details in the

‘‘Rheological measurements’’ section. Instead,

the obtained solution was quickly put on the rhe-

ometer plate to study the alginate and alginate/

N-succinylchitosan gelation kinetics.

It is noteworthy that the amount of Ca21 ions

theoretically necessary to saturate all the car-

boxylic groups in any alginate molecule corre-

sponds to the inclusion of the 0.2% w/w of

CaCO3.

As an example, to obtain 13 g of alginate

gel at 0.2% of CaCO3 concentration, 0.26 g of

Table 1. Main Physical (Weight Average Molecular

Weight, Mw; Number Average Molecular Weight, Mn;

P.I., Polydispersity Index) and Chemical (Percentage

of Overall Guluronic, FG, and Mannuronic, FM, Acidic

Residues; Number–Average of Guluronic Units in GG

Acidic Residues, NG) Characteristics of the Alginate

Used in This Work

Mw (Da) Mn (Da) P.I.

FG

(%)

FM

(%) NG

1.0838�106 1.9791�105 5.5 62 38 7.9 6 10%

RHEOLOGICAL INVESTIGATION OF NOVEL ALGINATE HYDROGELS 1169


alginate are dissolved in 11.3 g of demineralised

water under stirring for 1 h at room tempera-

ture; then, 0.03 g of CaCO3 are dispersed in the

solution by stirring at room temperature for

30 min over. Then 1.2 mL of a 9% GDL solution

is added and the mixture is kept under vigorous

stirring for 45 s.

Spectroscopic Measurements

FTIR characterization was carried out by using

a Perkin–Elmer spectrometer, model Paragon

500 (average of 20 scans at resolution of 4

cm�1). The dry materials were powdered,

ground with exhaustively dried KBr powder,

and the discs were prepared by compression

under vacuum.1H NMR spectra were recorded with a Bruker

avance DPX300 apparatus operating at 300

MHz. The sample was prepared by dissolving

15 mg of polymer in 0.75 mL of deuterated sol-

vents. Particularly, Ch was dissolved in a 20%

CD3COOD/D2O solution, while D2O was used

for sCh.

Rheological Measurements

The rheological investigation was performed on

the alginate and alginate/N-succinylchitosan

hydrogels with a strain controlled rotational

rheometer, ARES TA, equipped with a parallel

plate configuration (gap ¼ 1.5 mm, R ¼ 12.5

mm). Dynamic oscillatory strain sweep experi-

ments were performed on the hydrogels to deter-

mine the limit of the linear viscoelastic region.

Consequently, frequency sweep tests were per-

formed in the linear regime for all the samples.

The test temperature in all the experiments was

25 8C and the samples were tested under a con-

tinuous nitrogen purge.

The isothermal gelation kinetics was studied

at the temperature of 25 8C in quiescent condi-

tions by means of a rotational stress rheometer,

SR5000, Rheometric Inc., in a parallel plate con-

figuration (gap ¼ 1.5 mm, R ¼ 20 mm) at the

constant oscillation frequency of 1 rad/s under

nitrogen atmosphere. The formation of the gel

was analyzed by following the time evolution of

the storage (G0) and the loss (G00) moduli.

Scanning Electron Microscopy

The morphology of materials was investigated

by Scanning Electron Microscopy (SEM). The

hydrogels were prepared as described in Mor-

phological Analysis (left for 48 h at room tem-

perature). Then they were frozen in liquid nitro-

gen and lyophilized. The dried hydrogels were

fractured in liquid nitrogen and the surface of

fracture was observed. Samples were mounted

on a stub and coated with an Au/Pd alloy. Micro-

graphs were obtained using a scanning electron

microscope Philips XL 20.

RESULTS AND DISCUSSION

Synthesis of N-Succinylchitosan

To obtain a water-soluble polymer, the chitosan

was modified by reacting with succinic anhy-

dride. Further details of the synthesis and char-

acterization of the obtained N-succinylchitosan

polymer have been published elsewhere.25 The

succinylation reaction consists of a condensation

between polysaccharide amino groups and car-

bonylic groups of the anhydride, with conse-

quent formation of an amidic bond (Scheme 1).

The resulting product is a water-soluble poly-

mer. FTIR and 1H NMR analyses, performed on

both plain and modified chitosan, confirmed the

occurrence of succinylation.

In fact, the infrared spectrum of modified chi-

tosan (not shown), compared with that of chito-

san, shows the appearance of new absorption

peaks at 1720 cm�1 and 1640–1560 cm�1, which

correspond respectively, to carboxylic acid (C¼¼O

stretching of ��COOH) and amidic groups

(C¼¼O stretching and N��H bending). Moreover,

absorption increase of the peaks at 2930 and

1075 cm�1 (due to ��CH2��CH2�� of succinic

moieties) and broadening of the band at 3400

cm�1 (due to ��OH stretching) were also

detected.

The 1H NMR spectrum of the modified poly-

mer shows a new signal at 2.5 ppm correspond-

ing to the ��CH2�� moieties of succinic acid

(H-7, H-8). Furthermore, the shifting of the

peak relative to H-2 proton from 3 ppm in the

Ch spectrum to higher values in the sCh spec-

trum (where it overlaps the multiplet at 3.7

ppm) is in line with the formation of the

amidic bond. The degree of polymer succinyla-

tion has been estimated through the ratio

between peak areas of the grafted succinic

protons ��CH2�� CH2�� (2,5 ppm) and the

polysaccharide protons at 3,7 ppm, and turned

out to be around 90%. The high degree of

1170 NOBILE ET AL.


succinylation obtained is responsible for the

good water solubility of sCh.

Hydrogel Preparation

Alginate hydrogels have been widely investi-

gated in view of their numerous applications in

biomedical, cosmetic, and food fields. No exam-

ples exist in literature of hydrogels based on al-

ginate and chemically modified chitosan. In this

work, a novel alginate/N-succinylchitosan-based

hydrogel was prepared and investigated, and

compared with plain alginate gel.

The alginate and alginate/N-succinylchitosan

hydrogels were prepared by the internal setting

method.27,28 By this way it is possible to obtain

hydrogels with an uniform concentration of algi-

nate. In general, this method uses an inactive

form of the crosslinking ion, either bound by a

sequestering agent such as phosphate, citrate or

EDTA, or as an insoluble salt, for example

CaSO4 and CaCO3. In association with the diva-

lent ion, usually a solution of a slowly hydrolyz-

ing lactone, like D-glucono-d-lactone, is then

added to the mixture of alginate and crosslinker.

Because of the acidic pH generated by GDL, the

calcium ions are gradually released and cap-

tured by guluronic residues of alginate. In this

work, the CaCO3-GDL system was used to real-

ize the hydrogels. The slow release of calcium

ions allowed obtaining homogenous and trans-

parent hydrogels; the use of stoichiometric

CO2�3 /GDL ratio allowed for neutral pH. An

accurate premixing of alginate solution with

CaCO3 before the addition of GDL is necessary

to obtain a homogeneous dispersion of the

CaCO3 inside the alginate solution, to create a

regular network while the Ca21 ions are pro-

gressively released after the addition of GDL,

according to the following Scheme 2.

Rheological Analysis

The Alginate Hydrogels

The Strain Sweep Behavior. Alginate hydrogels

with a polymer concentration of 2% w/w and dif-

ferent amounts of Ca21 ions were investigated

under oscillatory strain sweep tests to define the

critical deformation (cc) characterizing the limit

of the linear viscoelastic regime as a function of

the calcium carbonate concentration. The values

of (cc) for gels are not often reported in litera-

ture, indeed only recently articles have ap-

peared on this subject.30–33,42,43,46,47 In particu-

lar, there are no articles that investigate the

effect of the inclusion of different amounts of

Ca21 ions on the limit of the linear viscoelastic

regime of alginate gels.

The existence and the extent of a linear visco-

elastic range for the alginate hydrogels studied

in this work has been first investigated at the

constant frequency of 0.5 rad/s. In Figure 1(a,b)

Scheme 2. Schematic representation of alginate gel-

ification process in presence of CaCO3/GDL system.29

Scheme 1. Succinylation reaction of chitosan.



the storage (G0) and loss (G00) moduli are shown

at T ¼ 25 8C as a function of the % of strain am-

plitude for the different alginate hydrogels with

a concentration of CaCO3 ranging between 0.1

and 0.7% w/w.

At the frequency of 0.5 rad/s the rheological

experiments lasted no more than 700 s, after

the loading procedure of about 500 s. The values

of the storage and loss moduli are constant up

to cc [Fig. 1(a,b)], clearly indicating that evapo-

ration of water did not occur during the test

time. Indeed, at the frequency of x ¼ 0.5 rad/s,

the same sample was also subjected to two re-

petitive tests that lasted in total about 2000 s

and a very good reproducibility was obtained,

confirming the absence of water evaporation

during the time of our measurements. The

strain sweep experiments were, then, also per-

formed at the frequencies of 1 and 10 rad/s that

lasted less than 20 min.

In Figure 1(a,b), all alginates show a general

trend not different from that usually expected

for gels: a linear viscoelastic behavior at small

strain amplitudes and a nonlinear behavior beyond

a critical deformation, cc. The low values of cc (0.6–

8%, as discussed later) allow an immediate distinc-

tion to be made between gels30–33,42,43,46,47 and

entanglement networks, for which the linear vis-

coelastic region may extend to large strains which

are orders ofmagnitude higher than gels.

The onset for nonlinearity (cc) was assumed

to be equal to the strain above which both mod-

uli differ more that 3% from the corresponding

linear values.32 As shown in Figure 2(a), the ccvalues significantly decrease as the amount of

Ca21 ions increases. Indeed, a value of cc � 8%

has been evaluated for the lowest CaCO3 con-

tent of 0.1%, then cc significantly decreases to

�2.4% when the amount of 0.2% w/w of CaCO3,

theoretically necessary to saturate all the car-

boxylic groups in any alginate molecule, is

added (see the experimental section), and finally

it reaches a plateau value of �0.6% at the high-

est CaCO3 concentrations.

The value of cc � 2.4%, obtained for our algi-

nate with a polymer concentration of 2% and

the theoretically stochiometric amount of 0.2%

of CaCO3, very well compares with previous

findings shown by Moresi et al.30 The authors,

indeed, report a value of cc of �2% for an algi-

nate with a polymer concentration of 1.75% and

the theoretically stochiometric amount of Ca21

ions, named ‘‘Guluronic type 3’’ in their Table 2.

It is noteworthy that their system is character-

ized by FG ¼ 63% and Mn ¼ 217.7 KDa, very

similar to the values of FG ¼ 62% and Mn ¼ 198

KDa of our alginate gels.

Moreover, Moresi et al. showed that the value

for the onset of nonlinearity is quite independ-

ent of polymer concentration and Mn in the

range investigated (polymer concentration of 1–

1.75%, Mn of 73–217.7 KDa) for high-G algi-

nates (FG ¼ 63%). On the other hand, the

authors found that the value of cc significantly

increases in the case of high-M alginates. The

different response, previously reported,30 of the

guluronic alginate to shear (cc � 2%) and com-

pression tests (cc � 8%),34 is also confirmed by

our results in shear flow (cc � 2.4).

Figure 2(a) clearly evidences that the cc �2.4% value still decreases with increasing the

amount of CaCO3, due to the increased cross-

Figure 1. Alginate hydrogels at different CaCO3

concentrations. T ¼ 25 8C, x ¼ 0.5 rad/s. (a) G0 versus

strain (%), (b) G00 versus strain (%).

1172 NOBILE ET AL.


linking network density, and it reaches the cc �0.6% plateau value at the concentration of 0.5%

CaCO3 w/w. As known from literature,29 indeed,

a stoichiometric amount of calcium ions is not

sufficient to saturate all the carboxylate groups

of alginate, because of linkage conformations of

the guluronate residues in the chain, therefore

an excess of calcium is necessary to reach the

plateau value. Increasing the calcium ion

amount from 0.5 to 0.7% does not increase the

number of crosslinkings, since all the accessible

carboxylate groups have been saturated, conse-

quently the value of cc � 0.6% remains constant.

The G0 and G00 moduli, instead, still increase

going from 0.5 to 0.7% [Fig. 1(a,b)], because the

increase in modulus is generally caused not only

by a higher number of junction zones, but also

by a higher strength of these junction points in

gels, formed by ions of high binding strength.

In Figure 1(a,b), G0 and G00 data in the non-

linear region are also shown. Beyond the linear

viscoelastic limit, cc, the storage modulus, G0,

decreases with increasing the strain amplitude,

Figure 1(a). Looking at the Figure 1(b) a pecu-

liar behavior in the G00 profile is observed

beyond cc: in the nonlinear region, the loss mod-

uli data first increase, reaching a maximum,

and then decrease with increasing strains. A

similar trend in G0 and G00 has been reported for

different alginate30,31,43 and scleroglucan gels32

and it is a peculiar feature of structured sys-

tems. At this regard, it is noteworthy that differ-

ent papers have been recently published that

show how large amplitude oscillatory measure-

ments (LAOS) can be a very important tool to

study and classify complex fluids as biological

gel molecules, polyelectrolytes, surfactants, sus-

pensions.48–52 In particular, the article by Hyun

et al.48 classifies the types of large amplitude os-

cillatory shear (LAOS) behavior into four types:

Type I, strain thinning (G0 and G00 decreasing);

Type II strain hardening (G0 and G00 increasing);

Type III, weak strain overshoot (G0 decreasing,

G00 increasing followed by decreasing); Type IV,

strong strain overshoot (G0 and G00 increasing

followed by decreasing). Based on this classifica-

tion we, therefore, define our G0 and G00 results

as Type III.

The results of G00, reported in Figure 1(b), are

summarized in Figure 2(b,c). In particular, the

ratio between G00 maximum and the linear visco-

elastic loss modulus values (G00max/G

000) de-

creases as the amount of CaCO3 increases [as

shown in Figure 2(b)], reaching a plateau value

Figure 2. Alginate hydrogels x ¼ 0.5 rad/s. T ¼25 8C. (a) Linear viscoelastic limit (cc %) versus

CaCO3 concentration, (b) G00max/G

000 versus CaCO3

concentration, (c) cmax (%) values versus CaCO3 con-

centration.



at the concentration of 0.5% CaCO3 w/w. In Fig-

ure 2(c) it is evident that the strain value (cmax)

corresponding to G00max dramatically decreases

from �62% (for the lowest CaCO3 content) to a

value of �6.5% at 0.2% of CaCO3, finally reach-

ing a plateau value of cmax � 2.5% at the high-

est CaCO3 amounts investigated.

These results confirm that the viscoelastic

properties G0 and G00 are very sensitive to the

gel structure, with a dramatic variation in ccand cmax recorded in correspondence of the Ca21

ions amount theoretically necessary to saturate

all the carboxylic groups in any alginate mole-

cule. Our results also showed that plateau val-

ues in cc and cmax were obtained at CaCO3

amount of 0.5% w/w.

The onset for nonlinearity (cc) has been found

to be independent of frequency for tests per-

formed in the range 0.5–10 rad/s, as shown in

Figure 3(a,b) where the values of G0/G00 versus

strain amplitude is reported for the samples

with CaCO3 0.5 and 0.7% w/w, respectively. The

frequency sweep tests, discussed in next section,

are performed in the linear viscoelastic regime

with strain values lower than cc. The choice of

the frequency and strain range used in this

work has also ensured that no slipping of the

gel disks from the plates occurred during the os-

cillatory shear tests.

The Frequency Behavior. In this section the fre-

quency response of the alginate gels in the range

10�1 to 102 rad/s is analyzed and discussed. All

the frequency sweep tests were performed in the

linear viscoelastic regime at the constant strain of

0.25%, sensibly lower than cc. The measurements

lasted about 800 s after the loading procedure of

about 500 s. The test time is, then, comparable

with the test time of the strain sweep experiments

and therefore, as discussed previously, we can

assume that evaporation of the water did not

occur during our measurements.

Following Almdal et al.39 a gel is defined as a

soft solid or solid-like material which consists of

two or more components one of which is a sub-

stantial quantity of a liquid and shows an approx-

imately flat mechanical spectrum in small ampli-

tude oscillatory experiments. In Figure 4 the

G0(x), G00(x), and g*(x) data for our alginate gel

with CaCO3 amount of 0.2% w/w show profiles

characterized by two straight lines nearly parallel

to each other with a slight increase with fre-

quency. Moreover G0 is about one order of magni-

tude greater than G00. This behavior is shown by

all the alginate gels (Fig. 5) that, therefore, well

correspond to the definition of gels as given by

Figure 3. G0/G00 versus strain (%), at different fre-

quencies. T ¼ 25 8C. (a) Alginate hydrogel with

CaCO3 ¼ 0.5% w/w, (b) Alginate hydrogel with CaCO3

¼ 0.7% w/w.

Figure 4. G0, G00 and g* versus frequency, for the al-

ginate hydrogel with CaCO3 ¼ 0.2% w/w. T ¼ 25 8C.

1174 NOBILE ET AL.


Almdal et al.39 Indeed, the investigated alginate

gels can be classified as strong gels33 that under

small deformation conditions manifest the typical

behavior of viscoelastic solids. The results

reported in Figure 4 very well compare with pre-

vious findings by Segreen et al.35 for a Ca21-algi-

nate gel (1.4% w/w) and by Moresi et al. for a se-

ries of alginates gels.30,31

The effect of different amounts of Ca21 ions

on the G0(x) and G00(x) values has been studied

and the results are shown in Figure 5(a,b),

respectively. In all the cases, the mechanical

spectra are characterized by a quite flat depend-

ence of G0(x) and G00(x) on the frequency with

G0 about one order of magnitude greater than

G00. As expected, the storage and the loss moduli

both increase as the CaCO3 content is increased,

in agreement with previous findings by Kuo and

Ma28 that showed how compressive modulus

and strength increased with calcium content. In

particular, a strong increase in G0 is recorded at

the CaCO3 0.2% w/w concentration theoretically

necessary to saturate all the carboxylic groups

in any alginate molecule, confirming the results

discussed in the strain sweep section.

In the literature it has been proven that the

generalized Maxwell model33 can be successfully

applied to reconstruct the linear viscoelastic re-

gime of alginate gels submitted to compressive

tests.34 Nevertheless, Moresi et al.30 showed

that the evolution of G0 and G00 in small ampli-

tude oscillatory experiments of alginate gels

could be better described by means of the model

proposed by Friedrich and Heymann,36 that can

predict the linear viscoelastic behavior of gels

not only at the sol–gel transition, but also below

and above the gel point.

The proposed Friedrich and Heymann model

is an extension of the constitutive equation

introduced by Chambon and Winter37 to

describe only the gel point. In particular, the

extended model is also able to describe a start-

ing viscosity function for states below the gel

point and the existence of an equilibrium modu-

lus in the state above the gel point.

It is based on an extended relaxation function

depending on four parameters, namely

GðtÞ ¼ G1;a þS�a

Cð1� aÞt�a expð�t=kaÞ ð1Þ

where S�a is the strength, a is the order of the

relaxation function, G?,a and ka are the equilib-

rium modulus and the mean relaxation time per-

taining to a, respectively. In their article, Frie-

drich and Heymann36 clearly showed that the

extended relaxation function is able to describe

the evolution of G0 and G00 in linear viscoelasticity

during crosslinking reactions for all the reaction

stages in the entire frequency range.

In the linear regime, the knowledge of G(t)

allows the calculation of all the others visco-

elastic functions.53 In particular, the storage

and loss moduli can be computed as follows:

Figure 5. Alginate hydrogels at different CaCO3

concentrations. T ¼ 25 8C. (a) G0 versus frequency, (b)

G00 versus frequency.

G0ðxÞ ¼ G1;a þ x

Z

1

0

GðtÞ �G1;a

� �

cosðxtÞdt ð2aÞ

G00ðxÞ¼G1;aþx

Z

1

0

GðtÞ�G1;a

� �

sinðxtÞdt ð2bÞ



According to Friedrich and Heymann,36 eqs 1,

2a, and 2b turn out in

G0ðxÞ ¼ G1;a þ

ffiffiffi

2

p

r

S�aka

�aðxkaÞ

3sinbð1� aÞ arctanðxkaÞc

1þ ðxkaÞ2

h i1�a2

ð3aÞ

G00ðxÞ ¼

ffiffiffi

2

p

r

S�aka

�aðxkaÞcosbð1� aÞ arctanðxkaÞc

1þ ðxkaÞ2

h i1�a2

ð3bÞ

At moderate and high frequencies eqs 3a and

b give the same slope for G0 and G00 (G0 / xa,

G00 / xa), while at low frequencies they may

describe either liquid (G0 / x2, G00 / x) or solid

(G0 / G1;a, G00 / x) behavior.

It is noteworthy that Friedrich and Hey-

mann36 proved that, near the gel point or in the

high frequency range after the sol-gel transition

(xka � 1), the model reduces to:

G0ðxÞ ¼ G1;a þ

ffiffiffi

2

p

r

S�a xa cos

ap

2ð4aÞ

G00ðxÞ ¼

ffiffiffi

2

p

r

S�a xa sin

ap

2ð4bÞ

These equations are, then, independent of the

relaxation time ka. Moreover, the authors

pointed out that the ratio:

tan d ¼ G00=G0 ¼ tanap

2ð5Þ

is exact at the gel point, when the equilibrium

modulus is equal to zero (Chambon and Winter

model) and holds in the gel state at moderately

or high frequencies, when the equilibrium mod-

ulus can be neglected. Therefore, the article by

Friedrich and Heymann showed that eqs 4a and

b can be used in the gel state at high frequen-

cies when G?,a can be neglected.

In the literature, the evolution of the storage

and loss moduli, G0 and G00, in small amplitude os-

cillatory experiments of alginate gels in the high

frequency range has been successfully described

within the frame of the Friedrich and Heymann

model by means of eqs 4 and 5.30–31,43,47

Figure 5(a,b) show that the storage and loss

moduli of our alginate gels follow a power law

and remain parallel to each other in the high

frequency range investigated. Indeed, both mod-

uli are quite flat and behave as xa. Their ratio

is nearly constant and compares well with eq 5

within the experimental error. Following the lit-

erature suggestions we have, then, used eqs 4

and 5 to fit our dynamic moduli data. The agree-

ment is rather good, as it can be seen in Figure

6 for the CaCO3 0.2% w/w alginate gel. The best

values of the parameters S�a and a for the three

samples are reported in Table 2. The results

show a significant increase of the strength. S�a

with increasing the CaCO3 content from the

0.2% w/w concentration (corresponding to the

Ca21 ions amount theoretically necessary to sat-

urate all the carboxylic groups in any alginate

molecule) to the 0.5 and 0.7% w/w concentration.

The values of the order of the relaxation func-

tion a are in the range 0.05–0.1 confirming the

strong gel state of our samples. They well com-

pare with the a data reported in the literature

for alginate gels.30

Based on these results and on the strain

sweep behavior, the alginate with the 0.5% has

been chosen as the reference system to investi-

gate the influence of the chitosan succinate on

the plain alginate dynamic mechanical proper-

Figure 6. G0, G00 and tand versus frequency for the

alginate hydrogel with CaCO3 ¼ 0.2% w/w. T ¼ 25 8C.

Fit with the Friedrich and Heymann model (line).

Table 2. Strength (S�a) and Values of the Order of

the Relaxation Function (a) for Alginate Hydrogels at

Different CaCO3 Concentrations

CaCO3 Content (w/w %) a Sa* (Pa sa)

0.2 0.055 7,000

0.5 0.085 25,000

0.7 0.101 56,000

1176 NOBILE ET AL.


ties. This system, indeed, corresponds to a gel

with the Ca21 ions amount that reached the pla-

teau values in cc and cmax for the plain alginates

[see Fig. 2(a,c)] and shows a significant increase

in the strength values. For comparison the sys-

tems with 0.2 and 0.7% w/w CaCO3 content

have also been studied and the results are

shown in the following sections.

The Alginate/N-Succinylchitosan Hydrogels

With the intent to obtain a novel hydrogel with

improved biocompatibility, the hydrogels based

on the blends of alginate and N-succinylchitosan

with a total polymer concentration of 2% w/w

and different amounts of N-succinylchitosan

were examined under oscillatory tests. Indeed, it

was verified whether the dynamic mechanical

properties of the plain alginate would depend on

the amount of N-succinylchitosan.

The Strain Sweep Behavior. The critical defor-

mation (cc), characterizing the limit of the linear

viscoelastic regime, has been determined for the

different alginate/N-succinylchitosan blends.

The results, in terms of G0 and G00 versus % of

strain amplitude, are reported in Figure 7(a,b)

for the CaCO3 0.5% w/w system. As clearly

shown in the figures, the cc and cmax are equal

to the values previously reported for the plain

alginate gel with 0.5% w/w of CaCO3 [respec-

tively, of �0.6 and 2.5%, Fig. 2(a,c)], independ-

ently of the amount of N-succinylchitosan, in

the range analyzed. To better evidence this

behavior, the normalized G0/G00 and G00/G00

0 are

reported in the inset of Figure 7(a,b), while the

results in terms of cc and cmax as a function of

the amount of N-succinylchitosan are summar-

ized in Figure 8(a,b). Similar results have been

also obtained for the blends at CaCO3 concentra-

tions of 0.7 and 0.2% w/w.

The Frequency Behavior. The effect of the inclu-

sion of the N-succinylchitosan on the frequency

behavior of the plain alginates gels has been,

then, studied in the range 10�1 to 102 rad/s. In

Figure 9(a–c) the results in terms of G0(x) for

the systems with 0.5, 0.7, and 0.2% w/w of

CaCO3, respectively, are reported. In all cases

the mechanical spectra are slightly dependent

on the frequency, with G0 about one order of

magnitude higher then G00 (not reported in the

figures). The hydrogels based on the alginate

and N-succinylchitosan blends can be, therefore,

classified as strong gels, analogously to the plain

alginate gels.

As reported in Figure 9(a), the alginate gel

with 0.5% w/w of CaCO3 shows significantly

increased values of the storage modulus ((40%)

when the amount of 10% w/w of N-succinylchito-

san (Alg/sCh 90/10) is blended to the plain algi-

nate, in all the frequency range tested. On the

other hand, G0 values for the Alg/sCh 70/30

blend are very similar to those for the plain algi-

nate gels, while a further increase in the N-suc-

cinylchitosan, determines a reduction in G0 val-

ues, as indicated for the Alg/sCh 60/40 and 50/

50 blends.

This behavior has been quite confirmed in the

case of the Alg/sCh system with 0.7% w/w of

CaCO3. Nonetheless, the improvement of the

Figure 7. Alg/sCh blends with CaCO3 ¼ 0.5%w/w, x

¼ 0.5 rad/s. T ¼ 25 8C. a) G0 versus strain(%), inset:

G0/G00 versus strain (%), b) G00 versus strain (%),

inset: G00/G000 versus strain (%).



storage modulus is only (10% for the inclusion of

the amount of 10% w/w of N-succinylchitosan

while a decrease in the mechanical properties is

already evident at the 20% amount of N-succi-

nylchitosan (Alg/sCh 80/20), Figure 9(b).

Finally, in Figure 9(c) the Alg/sCh 90/10 blend

for the system with 0.2% w/w of CaCO3 again

shows a quite satisfactory improvement of (20%

in G0 values if compared to the plain alginate

gel. The higher concentrations of N-succinylchi-

tosan were not analyzed since the Alg/sCh

blend 90/10 was considered the most interesting

system.

The values of the Strength and a for the algi-

nate/N-succinylchitosan hydrogels have been

evaluated by the Friedrich and Heymann,36 as

described in the frequency behavior section for

the plain alginates. The results are summarized

Figure 8. Alg/sCh blends with CaCO3 ¼ 0.5%w/w, x

¼ 0.5 rad/s. T ¼ 25 8C. (a) Linear viscoelastic limit (cc%) versus Alg/sCh ratios, (b) cmax (%) values versus

Alg/sCh ratios.

Figure 9. G0 versus frequency for different Alg/sCh

blends at T ¼ 25 8C: (a) CaCO3 ¼ 0.5% w/w; (b)

CaCO3 ¼ 0.7% w/w; c) CaCO3 ¼ 0.2% w/w.

1178 NOBILE ET AL.


in Table 3. In all cases the Alg/sCh 90/10 blend

shows the highest Strength values, suggesting

that a stronger network is obtained for the

inclusion of 10% w/w of N-succinylchitosan in

all the plain alginate gels analyzed. In particu-

lar, the system based on 0.5% w/w of CaCO3

shows the most significant increase of �40% in

the strength values.

The 90/10 composition may represent a favor-

able condition at which most of the chitosan

molecules are effectively interacting with the al-

ginate network and might cocrosslink with algi-

nate through calcium ions; in turn, this gives

rise to a stronger gel, as found. At higher chito-

san concentration, the excess of chitosan is

bound to the alginate gel network through sim-

ple entanglements. Consequently, the final net-

work is partially a ionically crosslinked network

and partially a physically entangled network,

with a consequent decrease of the viscoelastic

parameters respect with the plain alginate gel.

The blends studied, at all the concentrations,

were characterized by quite similar values for a,

thus exhibiting similar rates of relaxation.

Gelation Kinetics of the Alginate andAlginate/N-Succinylchitosan Hydrogels

In this section the effect of different amounts of

CaCO3 as well as the effect of the blending with

N-succinylchitosan on the gelation rate of the al-

ginate gels is analyzed and discussed.

To investigate the formation and time evolu-

tion of gels, rheometrical techniques based on

the measurement of dynamic properties are usu-

ally applied. Indeed, in this work, the time evo-

lution of the storage and the loss moduli has

been monitored at the constant oscillation fre-

quency of 1 rad/s. The freshly prepared alginate/

CaCO3 as well as alginate/CaCO3/ N-succinyl-

chitosan solutions were poured on the lower

plate of the rheometer and the upper plate was

lowered down fast to reach the final gap. The

critical problem of sample edge drying was

solved by leaving an excess of the solution all

around the plates, to avoid water evaporation in

the tested sample. Thanks to this procedure the

evolution of dynamic moduli is reliable during

the time of our experiments.

The constrain of a small deformation has

been chosen to be within the linear viscoelastic

regime and to avoid to modify the continuity of

the growing network.33 In all the cases the

strain was �1% at the beginning of the mea-

surement and decreased at �0.2% when the

crossover between G0 and G00 occurred. In Figure

10 the results in terms of G0(t) and G00 (t) versus

time for the system of plain alginate at the con-

centration of 0.2% w/w of CaCO3 show that the

isothermal gelation kinetics is characterized by

an induction time, in agreement with previous

results on ion-mediated gels of alginate, pectate

and polygalacturonate, prepared in situ by

means of a controlled release of the counter-

Table 3. Strength (S�a) and Values of the Order of

the Relaxation Function (a) for Different Alg/sCh

Blends and the Plain Alginate hydrogel; at

CaCO3 ¼ 0.5% w/w; at CaCO3¼0.7% w/w; and at

CaCO3 ¼ 0.2% w/w.

Alg/sCh w/w Content a Sa* (Pa sa)

CaCO3 ¼ 0.5% w/w

Alg 0.085 25,000

Alg/sCh (90/10) 0.085 35,000

Alg/sCh (70/30) 0.085 27,000

Alg/sCh (60/40) 0.085 20,000

Alg/sCh (50/50) 0.085 15,000

CaCO3 ¼ 0.7% w/w

Alg 0.101 56,000

Alg/sCh (90/10) 0.101 60,000

Alg/sCh (80/20) 0.095 50,000

Alg/sCh (70/30) 0.095 40,000

CaCO3 ¼ 0.2% w/w

Alg 0.055 7,000

Alg/sCh (90/10) 0.06 8,500

Figure 10. G0 and G00 versus time for the alginate

system with CaCO3 ¼ 0.2% w/w, x ¼ 1 rad/s. T ¼25 8C.



ions.38 In particular, as reported in Table 4, the

value of �1800 s for the induction time of the

plain alginate system has been evaluated at

0.2% w/w CaCO3. The sol–gel transition occurs

at the incipient prevalence of the elastic compo-

nent over the viscous one, observed at �3470 s,

corresponding to the G0-G00 crossover time.

The system with a concentration of 0.7% w/w

CaCO3 has also been studied and the corre-

sponding G0 versus time values are compared

with G0 values for the 0.2% w/w system in Fig-

ure 11. The induction time of �450 s and a G0-

G00 crossover time of �620 s are recorded for the

gel with 0.7% w/w CaCO3 (see Table 4). The

results, then, clearly indicate that the kinetics

process can be significantly increased if the per-

centage of the Ca21 ions is increased.

Finally the effect of the inclusion of the N-

succinylchitosan is analyzed for the 0.2% w/w

CaCO3 system. In Figure 12 it is evident that

the inclusion of the 10% w/w of the N-succinyl-

chitosan accelerates the gelation kinetics with

respect to the case of the plain alginate gel. The

induction time of �1450 s and the G0-G00 cross-

over time of �2550 s are measured for the Alg/

Ch (90/10) blend, as reported in Table 4. This

indicates once more that the sCh has a coopera-

tive effect in chelating calcium ions, at least for

the 10% wt amount here investigated.

Morphological Analysis

According to what already reported in litera-

ture,28 alginate hydrogels obtained by the

CaCO3/GDL system are uniform, transparent

and three-dimensionally well defined. Hydrogels

based on alginate/sCh blends retain these char-

acteristics.

One of the alginate most promising feature is

its ability to be processed into porous structures

for use in tissue regeneration. Porous structures

can be obtained by freezing and lyophilizing al-

ginate gels. Hydrogels crosslinked with 0.5%

CaCO3 have so been frozen and lyophilized and

then analyzed through scanning electron micros-

copy to investigate on their morphology. SEM

micrographs of scaffolds of plain alginate, Alg/

sCh 90/10 and Alg/Ch 60/40 are reported as

examples in Figure 13(a–c). The structures

shown are typical of scaffolds obtained by the

freeze-drying technique. It is evident that all

the samples show an open, interconnected pore

structure, with regular and adequate pores

dimension (100–200 lm). The addition of sCh to

Table 4. Onset Time (tonset) and G0-G00 Crossover

Time (tcrossover) for the Plain Alginate System and the

Blend (90/10) Alg/sCh with CaCO3 ¼ 0.2% w/w, and

the Plain Alginate System with CaCO3 ¼ 0.7% w/w

Sample tonset (s) tcross over (s)

Alg CaCO3 ¼ 0.7% w/w 450 620

Alg CaCO3 ¼ 0.2% w/w 1,800 3,470

Alg/sCh (90/10)

CaCO3 ¼ 0.2% w/w

1,450 2,550

Figure 11. G0 versus time. Comparison between the

alginate systems with CaCO3 ¼ 0.7% w/w and 0.2%

w/w, x ¼ 1 rad/s. T ¼ 25 8C.

Figure 12. G0 versus time. Comparison between the

plain alginate system and the Alg/sCh (90/10) blend,

CaCO3 ¼ 0.2% w/w, x ¼ 1 rad/s. T ¼ 25 8C

1180 NOBILE ET AL.


plain alginate does not seem to significantly

influence the cell size; a detailed insight into the

cell wall morphology at higher magnification did

not evidence any phase separation between the

polymers.

CONCLUSIONS

In this work, a novel alginate/N-succinylchito-

san-based hydrogel was prepared by cross-

linking via the internal setting method using

calcium carbonate (CaCO3) as calcium ions source.

It was shown that a rheological study can be

successfully used to investigate the physical

characteristics of both the alginate gels and the

novel hydrogels. Indeed, the viscoelastic proper-

ties G0 and G00 are very sensitive to the gel

structure, with a dramatic variation in cc and

cmax recorded in correspondence of the Ca21 ions

amount theoretically necessary to saturate all

the carboxylic groups in any alginate molecule.

Our results also showed that an excess of cal-

cium was necessary to reach the plateau value

of cc � 0.6%, since the stoichiometric amount of

calcium ions is not sufficient to saturate all the

carboxylate groups of alginate because of link-

age conformations of the guluronate residues in

the chain.

It was shown that blending is a simple

method to combine the advantages of the two

different polymers. The 90/10 alginate/N-succi-

nylchitosan composition may represent a favor-

able condition at which most of the chitosan

molecules are effectively interacting with the al-

ginate network and might cocrosslink with algi-

nate through calcium ions; in turn, this gives

rise to a strong gel with higher storage modulus

values.

The significant acceleration of the gelation ki-

netic observed for the Alg/sCh 90/10 with

respect to the case of the plain alginate gel was

attributed to a synergistic effect of the N-succi-

nylchitosan in chelating calcium ions during the

alginate gelation process. This result is quite

interesting in view of possible applications as

in situ gelling systems.

Finally, porous structures were obtained by

freezing and lyophilizing alginate/N-succinylchi-

tosan gels. SEM micrographs showed that all

the samples retain the pores dimension and

interconnection characteristic of plain alginate,

thus they are potentially useful as scaffolds for

use in tissue engineering.

The authors are very grateful to M. Malinconico for

the many helpful discussions. They thank G. Narciso

from ICTP for technical support in SEM analysis, and

S. Zambardino (NMR Service of Istituto di Chimica

Biomolecolare (ICB) of CNR, Pozzuoli, Italy) for tech-

nical support.

Figure 13. SEM micrographs of the freeze dired

hydrogels with CaCO3 ¼ 0.5% w/w: (a) plain alginate

hydrogel; (b) Alg/sCh (90/10) hydrogel; (c) Alg/sCh

(60/40) hydrogel.



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development and rheological investigation of novel alginate/n-succinylchitosan hydrogels

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