Novel hydrogel obtained by chitosan and dextrin-VA co-polymerization.

Abstract

A novel hydrogel was obtained by reticulation of chitosan with dextrin enzymatically linked to vinyl acrylate (dextrin-VA), without cross-linking agents. The hydrogel had a solid-like behaviour with G' (storage modulus) > G'' (loss modulus). Glucose diffusion coefficients of 3.9 x 10(-6) +/- 1.3 x 10(-6) cm(2)/s and 2.9 x 10(-6) +/- 0.5 x 10(-6) cm(2)/s were obtained for different substitution degrees of the dextrin-VA (20% and 70% respectively). SEM observation revealed a porous structure, with pores ranging from 50 microm to 150 microm.

Full text

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Abstract
reticulation of chitosan with dextrin enzymati-
cally linked to vinyl acrylate (dextrin-VA), with-
out cross-linking agents. The hydrogel had a
solid-like behaviour with G¢ (storage modu-
lus) >> G† (loss modulus). Glucose diffusion
coefficients of 3.9 · 10–6± 1.3 · 10–6cm2/s and
2.9 · 10–6± 0.5 · 10–6cm2/s were obtained for
different substitution degrees of the dextrin-VA
(20% and 70% respectively). SEM observation
revealed a porous structure, with pores ranging
from 50 lm to 150 lm.
A novel hydrogel was obtained by
Keywords
Diffusion coefficient Æ Hydrogel Æ Rheology
Chitosan Æ Dextrin-VA Æ
Introduction
Hydrogels are a class of three-dimensional, highly
hydrated polymeric networks (Peppas et al. 2000).
They are suitable for biomedical applications
because of their high tissue compatibility mainly
caused by the high water content of the gels
(Drury and Mooney 2003). Hydrogels made of
natural polymers are suitable for tissue engi-
neering applications because they are compo-
nents of, or have macromolecular properties
similarto, thenatural
(Montembault et al. 2005).
Chitosan (poly[b-(1-4)-2-amino-2-deoxy-D-glu-
copyranose]) is a non-toxic and biocompatible
co-polymer of N-glucosamine and N-acetyl-glu-
cosamine produced by partial deacetylation of
chitin. Chitosan is susceptible to structural mod-
ifications due to the high number of hydroxyl and
amine reactive groups. It has been extensively
studied for applications in areas as diverse as
wastewater treatment (flocculating properties,
chelation and ion exchange properties), cosmetic
(hair and skin care), paper and textiles (condi-
tioning polymer), biomedicine and biology (drug
delivery, enzyme immobilization, orthopaedic/
periodontal, tissue engineering and wound heal-
ing), and agro-industries (seed treatment) (Jaw-
orska et al. 2003).
Dextrin is a glucose-containing polysaccharide
linked by a-(1 fi 4)
same general formula as starch, although it is
smaller and less complex. This polysaccharide is
produced by partial hydrolysis of starch, which
can be accomplished by the use of acids and/or
enzymes (J.M. Carvalho, C. Gonc ¸alves, A.M. Gil,
F.M Gama, submitted).
In the present work, we describe the develop-
ment of a novel hydrogel obtained by polymeri-
zation of chitosan with dextrin-VA.
extracellular matrix
D-glucose units, with the
R. Ramos Æ V. Carvalho Æ M. Gama (&)
Centro de Engenharia Biolo ´gica, Universidade do
Minho, Campus de Gualtar, 4710-057 Braga, Portugal
e-mail: fmgama@deb.uminho.pt
Biotechnol Lett (2006) 28:1279–1284
DOI 10.1007/s10529-006-9087-6
123
ORIGINAL PAPER
Novel hydrogel obtained by chitosan and dextrin-VA
co-polymerization
Reinaldo Ramos Æ Æ Vera Carvalho Æ Æ Miguel Gama
Received: 22 February 2006/Accepted: 24 April 2006/Published online: 27 June 2006
? Springer Science+Business Media B.V. 2006

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Materials and methods
Materials
D2O, chitosan and chitin from crab shells were
obtained from Sigma. Proleather FG-F, protease
was obtained from Amano Enzyme Co. Dextrin -
Koldex 60 starch was a generous gift from Tate &
Lyle. Vinyl acrylate (VA) was from Aldrich,
dimethylsulfoxide (DMSO) and acetone were
from AppliChem. (Germany).
DMSO was dried with 0.4 nm molecular sieves
overnight before use. Regenerated cellulose
tubular membranes with 3500 MWCO were
obtained from Membrane Filtration Products.
Enzymatic synthesis of dextrin-VA
The enzyme-catalyzed modification of dextrin,
with various degrees of substitution (DS) was as
described elsewhere (J.M. Carvalho, C. Gonc ¸al-
ves, A.M. Gil, F.M Gama, submitted). DS is
defined as the number of acrylate groups per 100
glucopyranose residues. Briefly, dextrin (4 g) and
two volumes of VA (650 ll gave a DS of 20% and
2.6 ml a DS of 70%) were dissolved in anhydrous
DMSO (60 ml).
The reaction was initiated by adding 600 mg of
Proleather FG-F. The reaction mixture was then
incubated at 50?C with magnetic stirring for 72 h.
Figure 1, reaction A, (see below) shows the
chemical reaction of dextrin activation. The mix-
tures were then dialyzed for 5 days against
deionized water, pH 3 (adjusted with 6 M HCl),
at 4?C. The aqueous solutions of dextrin-VA were
evaporated and freeze-dried.
The degree of substitution was determined by
1H NMR according to Ferreira et al. (2002).
Briefly,1H NMR spectra were recorded in D2O
(10 mg in 1 ml) and DS was calculated using Eq.
(1):
DS% ¼x
y?7
3? 100
ð1Þ
where,intheNMRspectra,xisthesumofthepeak
intensities corresponding to the protons from vinyl
group(dintherangeof6.44–6.00 ppm)andyisthe
sumofthepeakintensitiesofalldextranprotons(d
in the range of 5.50–3.10 ppm).
Preparation of chitosan-dextrin-VA hydrogels
Chitosan-dextrin-VA hydrogels were obtained
after reticulation of chitosan with dextrin-VA.
Chitosan was dissolved in 0.1 M acetic acid. The
pH was raised to 6 so that a higher number of
reactive amines (NH2) were available. The
chitosan solution was mixed with various quanti-
ties of solid dextrin-VA and transferred to a
casting mould. The polymerization occurred at
room temperature for 24 h.
Fig. 1 (A) Reaction between dextrin and vinyl acrylate forming dextrin-VA and acetaldehyde. (B) Reaction between
chitosan and dextrin-VA leading to hydrogel formation
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Rheology
Rheological experiments were performed at 25?C
to characterize the mechanical behaviour of the
hydrogels. The intrinsic mechanical properties of
the gel network are determined submitting the
material to compression
cycles. Rheological analyses of the hydrated
hydrogels, with different concentrations of dex-
trin-VA, (25 mm diam. disk, 2 mm thick) were
carried out using a Reologica StressTech HR
reometer in parallel-plate geometry, with a vari-
able gap. Smallamplitude oscillatory shear mea-
surements were recorded with gaps varying from
0.8 mm to 1.8 mm to determine the variation of
storage and loss module (G¢ and G† respectively),
with frequencies ranging from 0.01 Hz to 100 Hz
under various compression levels. The linear vis-
cosity region was determined by stress sweep
measurements to determine at which values the
gel structure remained constant.
and decompression
Diffusivity
Diffusion coefficients of glucose in the chitosan-
dextrin-VA hydrogels were calculated. The dif-
fusion cell is a modification of Teixeira et al.
(1994). It is made of Perspex and consists of two
chambers of 60 ml, divided by a Perspex plate,
and held together with screws. The hydrogel
(diameter 20 mm; thickness of 0.5 mm) was
inserted in the Perspex plate. The Perspex plate
was supported by a squared mesh and sealed with
O-rings. Agitation was obtained using magneti-
cally driven bars in both chambers.
Samples were collected in the upper chamber
through a port, and the soluble sugars were
determined using the dinitrosalicylic acid method.
Concentration of glucose in the lower chamber
was 100 g/l.
The diffusion coefficients were calculated using
lag-time analysis (Teixeira et al. 1994):
Qts¼ADC1
l
ts?l2
6D
??
ð2Þ
where Q is the total amount of solute transferred
throughthemembrane,Athearea,Dthediffusion
coefficient, C the concentration, l the membrane
thickness and t the time.
The intercept of the linear part of the curve
obtained by plotting Q versus time is the lag time.
The diffusion coefficients were calculated
from the lag time and the membrane thickness.
Diffusion coefficients of glucose were deter-
mined for hydrogels with a dextrin-VA concen-
tration of 60 mg/ml.
SEM analysis
The hydrogels were first frozen at –20?C and
lyophilized. The samples were coated with gold
particles in a Fisons Instruments Polaron SC502
Sputter Coater and observed by electron micros-
copy (Leica, Cambridge S360).
Results and discussion
Chitosan-dextrin-VA Hydrogels
The
obtained upon reticulation of chitosan with dex-
trin-VA. The amine group reacts with the acrylate
groupof dextrin-VA
according to a conjugate 1, 4 addition mechanism.
In this reaction, the nucleophile (amine group)
reacts with the C=C double bond of the a,b-
unsaturated acrylate group.
Since only neutral amines of chitosan are reac-
tive, thepHwas carefullyadjusted inorderto have
solublechitosanwithasignificantfractionofamine
groups in the neutral form. The pKaof chitosan is
approximately 6.5, which means that at this pH,
50% of the amine groups are available to react.
Adjusting the pH of the chitosan solutions is rela-
tively slow, due to its high viscosity. It was possible
to increase the pH to 6.3, while keeping the poly-
mer soluble, with chitosan at 1.5% (w/v). After
reaching the desired pH, the dextrin-VA was
addedtothechitosansolution,andthemixturewas
allowed to react for 24 h at room temperature.
The pH of the reaction medium strongly affects
the polymerization kinetics (Fig. 2).
When the chitosan solution had a pH of 4.7,
(flask A, Fig. 2), the mixture did not gel after
chitosan-dextrin-VAhydrogelswere
(Fig. 1,reaction B),
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24 h. This may due to the low number of neutral
amines at this pH. The consistency of the hydro-
gel was very low, as compared to the one obtained
at higher pH (flask C, Fig. 2). However, after
3 days, the hydrogel became more consistent and
similar to the one obtained at pH 6.0 after 24 h.
The reaction was slower due to the lower con-
centration of NH2, but as the reaction proceeded
NH3
react with the dextrin-VA. Nevertheless, the
process was very slow. Figure 2 (B vs. C) showed
also that chitosan reacted with dextrin-VA, but
not with dextrin without linked VA. The results
confirmed the reaction mechanism suggested in
Fig. 1.
+dissociated, and more NH2was available to
Rheology
Figure 3A demonstrates the dynamic moduli G¢
(storage modulus) and G† (loss modulus) of the
different hydrogels. The gels displayed a solid-
like behaviour, G¢ >> G†. Figure 3B shows the
viscoelastic material functions G* and g. Below
10 Hz, g varied linearly with a negative slope
while G* remained constant. No significant dif-
ferences were observed for the different hydro-
gels. In fact, the different concentration of
dextrin-VA did not affect the rheological prop-
erties of the hydrogels. G¢ and G* of ca.
5 · 103Pa were obtained. These values are
relatively high, as compared to those for poly[N-
(2-hydroxypropyl)methacrylamide]
and hyaluronic acid hydrogels (1 · 102
5 · 102Pa, respectively) (Hou et al. 2005; Woerly
et al. 2001 respectively). G* and g functions are
similar to values determined for brain tissue: a
constant G* and g that increased with decreasing
frequency (Woerly et al. 2001).
(PHPMA)
and
Diffusivity
Dextrin-VA with two DS were used. Diffusion
coefficients of 3.9 · 10–6± 1.3 · 10–6cm2/s and
2.9 · 10–6± 0.5 · 10–6cm2/s, for the hydrogels
Fig. 2 Effect of the reaction medium pH (A vs. C) and of
the dextrin substitution with vinyl acrylate (B vs. C) on the
gelification. (A) 3 ml chitosan pH 4.7 + 30 mg dextrin-
VA; (B) 3 ml chitosan pH 6 + 30 mg dextrin; (C) 3 ml
chitosan pH 6 + 30 mg dextrin-VA
Fig. 3 The rheological experiments were performed with
gels obtained using three different concentrations of
dextrin-VA (60, 120 and 180 mg/ml); (A) Dynamic moduli
G¢ and G†, as function of the frequency. (B) Complex
shear modulus G* and viscosity g
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with a DS of 20 and 70% respectively, were
obtained, using the lag-time analysis, as illus-
trated in Fig. 4. The results were calculated from
four independent assays. The diffusion coefficient
was slightly lower when using a higher substitu-
tion degree due to the higher number of acrylate
groups. More reactive groups were available to
react with chitosan and a more stable and retic-
ulated hydrogel was obtained, exhibiting a lower
glucose diffusion coefficient. The results were in
the range of the ones obtained by Teixeira et al.
(1994) for the diffusion of glucose in alginate
membranes.
SEM analysis
Figure 5 provide images of the hydrogel obtained
by electron microscopy at 200· magnification. The
hydrogel presents irregular pores ranging from
50 lm to 150 lm, with the smaller range being in
the majority. This porous structure may be con-
venient for cell culture. In fact, the hydrogel may
be suitable for three-dimensional neural tissue
culture because of its positive charge.
Conclusion
A novel hydrogel was obtained upon polymeri-
zation of chitosan and dextrin-VA without initi-
ators. These hydrogels are simple to produce and
present interconnected micro and macropores. By
varying the proportion of chitosan to dextrin-VA,
and the dextrin-VA degree of substitution,
hydrogels
porosity, strength) can be obtained. Further
studies, such as swelling experiments and bio-
compatibility assays, will be performed to better
characterize this new hydrogel, and assess its
potential for biomedical applications, such as tis-
sue engineering and drug delivery.
withdifferent properties(charge,
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Fig. 5 SEM micrographs
of Dextrin-VA-CS
hydrogels with 200·
magnification. (A) 120 mg
dextrin-VA/ml CS; (B)
180 mg dextrin-VA/ml
CS. Bar = 200 lm
Fig. 4 Amount of glucose transferred through the hydro-
gel in the diffusion cell
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