21º CBECIMAT - Congresso Brasileiro de Engenharia e Ciência dos Materiais
09 a 13 de Novembro de 2014, Cuiabá, MT, Brasil
A STUDY OF SODIUM ALGINATE AND CALCIUM CHLORIDE INTERACTION
THROUGH FILMS FOR INTERVERTEBRAL DISC REGENERATION USES
Andréia Grossi Santos de Laia, Ezequiel de Souza Costa Júnior, Hermes de Souza
Costa
[email protected]
Material Engineering Department – Centro Federal de Educação Tecnológica de
Minas Gerais – Avenida Amazonas, 5253, Nova Suiça, Belo Horizonte – MG – Brazil
ABSTRACT
The injured intervertebral disc (IVD) requires some measures in order to promote its
regeneration. The sodium alginate in conjunction with CaCl2 forms a net, potentiating
its mechanical properties so it may be an alternative for IVD treatment. In this work,
the viability of films of sodium alginate crosslinked with CaCl2 and submitted to
variations in their solutions’ preparations is verified, comparing the effects of the
addition of CaCl2 through their immersions, before and after drying the films. The
films had their physicochemical properties analyzed by FTIR, DSC and XRD. The
results indicated that films with a greater proportion of CaCl2 were more stable in the
DSC analysis when compared to films with smaller proportions of CaCl2. These
results indicate alginate’s modulation capacity which may be useful for IVD
regeneration.
Key words: sodium alginate, calcium chloride, intervertebral disc, films.
INTRODUCTION
The regeneration of the intervertebral disc (IVD) is a challenging pathology which,
due to the inefficiency of current treatments, demands the development of new
approaches and therapeutical techniques(1). Among the most utilized materials for
intervertebral discs regeneration, polymers are the most used ones. Alginate (Alg) is
a natural polymer, soluble in water and composed of the repetition of two different
units, (1.4)-α-L-gulunorate or unit G and (1.4)-β-D-mannuronate or unit M(2), Fig. 1.
The alginate is extracted from algae and is commonly used in pharmaceutical and
food industry(3). It is considered biocompatible and biodegradable and has the
capacity of forming porous gel matrixes with mucoadhesive properties(4,5). An
important property of alginate is its ability to interact and form gel when in contact
with divalent cations, such as Ba2+, Ca2+ and Sr2+. The gel formation is a result of the
crosslinking which occur with divalent cations, when they become lodged among the
polymer chains, forming the net structure (Fig. 2).
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21º CBECIMAT - Congresso Brasileiro de Engenharia e Ciência dos Materiais
09 a 13 de Novembro de 2014, Cuiabá, MT, Brasil
Figure 1 – Representation of the structure of mers and the natural
polymer alginate.
(a) acid β-D-mannuronic acids (M block) mers;
(b) α-L-guluronic (G block);
(c) natural polymer alginate.
(6)
Source: ERTESVÁG; VALLA,1998 .
The quantity of Ca2+ ions present in the system interferes in the stability of these
polymeric nets, forming permanent or temporary inter-chain associations. There are
studies showing that the chemical structure and the size of the alginate molecule, as
well as the gel formation kinetic connected to the type of ion used are determining
factors
on
its
properties,
such
as
swellability,
porosity,
biodegradability,
biocompatibility, gel resistance and its immunological characteristics(7).
Figure 2 – Calcium alginate net formation.
(8)
Source: SACCHETEIN, 2009 .
Once these materials demonstrate importance in aiding recovery from IVD injures,
this work goal was to analyze and characterize the interaction between alginate and
calcium chloride, as well as their physicochemical properties and biocompatibility.
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21º CBECIMAT - Congresso Brasileiro de Engenharia e Ciência dos Materiais
09 a 13 de Novembro de 2014, Cuiabá, MT, Brasil
MATERIALS AND METHODS
Sodium alginate powder from Sigma-Aldrich® (molar weight (MW) = 100.000 g/mol,
with approximately 61% of mannuronic acid and 39% of guluronic acid, with a M/G
relation of 1.56), dihydrated calcium chloride (CaCl2 from Vetec, PM 147.01) and
acetic acid at 1% (CH3COOH - Cat. #49199, Sigma-Aldrich®) were the reagents used
in this work. Salts and reagents in analytical grade were used in all the solutions as
well as Milli-Q water with minimal resistivity (18. 0MΩ.cm) at 25ºC.
Preparation of the solution containing sodium alginate and calcium chloride
(CaCl2). The sodium alginate solution was prepared from 1% (wt/v) and kept for 24
hours under agitation of 100rpm. Later, still under agitation an aqueous solution of
calcium chloride at 2% was added drop by drop. After, the system was kept under
agitation for more 30 minutes. The solution was degassed under vacuum at a
pressure of 350mmHg for 120 minutes. After 24 hours the solution was poured into
polystyrene Petri dishes of 8.5 cm diameter and dried at 50ºC in oven boards for 24
hours. The obtained films were immersed in a calcium chloride solution at 2%
(wt/v) for 1 hour and dried in room temperature for 48 hours.
Preparation of films containing sodium alginate and calcium chloride (CaCl2).
The sodium alginate and CaCl2 films were prepared as described in the previous
item. Next, four mixtures were prepared as described below:
- Film 1: 100ml of sodium alginate at 1% solution (alginate control film – AC).
- Film 2: 100ml of Alg at 1% solution + 10ml of CaCl2 at 2% solution (alginate film
containing CaCl2 only in the solution – ACS).
- Film 3: 100ml of Alg at 1% solution + 10ml of CaCl2 at 2% solution (alginate film
containing CaCl2 at 2% both in the solution and in the immersion – ACSI).
- Film 4: 100ml of Alg at 1% solution (alginate film containing CaCl 2 at 2% only in the
immersion – ACI).
Characterization of the sodium alginate and calcium chloride films.
- Thermal analysis of the films. The different films were submitted to thermal analysis
by differential scanning calorimetry (DSC). In order to obtain the DSC curve, samples
from different films were placed in an aluminum crucible, at a 10ºC/min-1 heating rate,
starting at 30 until 500ºC in a N2 dynamic atmosphere at a flow rate of 90mL.min-1(9).
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21º CBECIMAT - Congresso Brasileiro de Engenharia e Ciência dos Materiais
09 a 13 de Novembro de 2014, Cuiabá, MT, Brasil
- Determination of the crystallinity parameters of the films. The crystallinity
parameters of the films were obtained by the X-ray diffraction (XRD) in SHIMADZU
XRD 7000, with 2θ ranging from 4.00 to 90.00º and step of 0.06º(10). The degree of
crystallinity was calculated based on Eq. (A).
(A)
- Analysis of chemical groups of films. The characterization of chemical groups was
carried out by infrared spectroscopy – FTIR in a SHIMADZU Corporation
spectrophotometer, model IRPrestige-21 equipped with Fourier transform, FTIR8400S and ATR accessory between 4,000-400cm-1.
RESULTS AND DISCUSSION
Differential scanning calorimetry – DSC. The DSC curve of the obtained films is
presented in Fig. 3, which showed endothermic peak for the AC film at 64.8ºC, for the
ACS film at 78.65ºC, for the ACSI film at 73.8ºC and for the ACI film at 86.4ºC. These
endothermic peaks can be associated to the dehydration process of the sample.
Figure 3 – DSC curves to the sodium alginate and calcium chloride
films.
(a) AC film; (b) ACS film; (c) ACSI film; (d) ACI film.
Peaks of greater intensity were observed for the ACS and ACSI films, which may be
associated to the presence of CaCl2 in their solutions. The ACI film presented higher
dehydration temperature and lower heat intensity when compared to the ACS and
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21º CBECIMAT - Congresso Brasileiro de Engenharia e Ciência dos Materiais
09 a 13 de Novembro de 2014, Cuiabá, MT, Brasil
ACSI films. A second exothermic peak presented apex at 209.3ºC for the AC films, at
201.5ºC for the ACS films, at 200.2ºC for the ACSI films and 207.8ºC for the ACI film.
This peak could be associated to the sample degradation process. A higher peak
intensity is observed for the ACS and ACSI films, which may be associated to the
presence of CaCl2 in these films’ solution. The ACSI film has higher intensity when
compared to the ACS film probably due to the presence of a greater proportion of
CaCl2, both in the solution and in its immersion, granting more stability to the sample.
A third exothermic peak was observed for all films. This peak was attributed to the
carbonization temperature of the samples. This phenomenon was visually observed
after their analysis.
Fourier transform infrared spectroscopy – FTIR. The infrared spectrum of the
sodium alginate powder (Fig. 4) showed a broad absorption band between 3,500cm-1
and 3,100cm-1 referring to the stretching of the O-H group. As the quantity of CaCl2
increases there is an enlargement of this region, which may be observed mainly in
the ACSI film which contains the higher proportion of CaCl2. Between 2,965cm-1 and
2,913cm-1 the band related to the stretching of the C-H symmetric and asymmetric
doublet is observed, which is almost nonexistent in the ACSI film, indicating a
probable interaction between sodium alginate and CaCl2.
Figure 4 – FTIR spectrum of sodium alginate and calcium chloride
films.
(a) AC Film; (b) ACS film; (c) ACSI film; (d) ACI film.
The wavenumbers in 1,592-1 and in 1,394cm-1 refer to the COO- asymmetric
stretching, showing an intensity decrease in this region in the films which contain
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21º CBECIMAT - Congresso Brasileiro de Engenharia e Ciência dos Materiais
09 a 13 de Novembro de 2014, Cuiabá, MT, Brasil
CaCl2; in 1,294cm-1 and in 1,073cm-1, to the C-O stretching; in 1,119cm-1 and in
843cm-1, to the C-C stretching; in 1,018cm-1 to the C-O-C stretching; in 965cm-1, to
the C-O stretching; in 912cm-1, to the C-C-H; in 841cm-1, to the C-C; and in 822cm1
, to the C-C-O. It can be observed in the films that by increasing the CaCl2
proportions there is an intensity decrease in these regions. These values are similar
to those found in Nery (2014)(11) studies.
X Ray Diffraction – XRD. The degree of crystallinity of the films was estimated
by using the software Microcal Origin® version 8.0. Fig. 5 presents the diffraction
pattern obtained. The AC film presents a 10.7% crystallinity degree; the ACSI film, a
24%; the ACS film, a 36%; and the ACI film, a 10%. In the diffraction pattern analysis
it is possible to observe that the areas where the sodium alginate peaks took place
there is an increase related to calcium chloride.
Figure 5 – Infrared spectrum of the sodium alginate and calcium
chloride films.
(a) AC Film; (b) ACS film; (c) ACSI film; (d) ACI film.
The peak for diffraction at around 13º for the sodium alginate increases in the films
composed of sodium alginate and calcium chloride, it has great intensity at 2θ=130
(d= 0.657nm) in the ACSI film and it is almost nonexistent in the ACI film. This
suggests that the interaction between the sodium alginate and the calcium chloride
favors the structural organization increasing the sodium alginate crystallinity. This
diffraction reflex intensity proportion changes when the content of Ca + ions increases.
Such results have also been observed in the studies of Fábia, Slusarezyk,
Gawlowski (2005)(12).
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21º CBECIMAT - Congresso Brasileiro de Engenharia e Ciência dos Materiais
09 a 13 de Novembro de 2014, Cuiabá, MT, Brasil
CONCLUSIONS
In the present study sodium alginate films were prepared and crosslinked with
calcium chloride. The results showed that the presence of calcium in different
proportions in the sodium alginate films modulate its physicochemical properties,
making its structure more thermally stable and more crystalline, and may also
contribute to the intervertebral disc regeneration.
ACKNOWLEDGMENTS
The authors would like to thank CAPES for its financial support and the Laboratory of
Biomaterials of CEFET-MG for allowing this work to be carried out.
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09 a 13 de Novembro de 2014, Cuiabá, MT, Brasil
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