Materials Letters 119 (2014) 146149

Contents lists available at ScienceDirect

Materials Letters
journal homepage: www.elsevier.com/locate/matlet

Evaluation of in vitro bioactivity of Chitosan/Mimosa

tenuiora composites
S.A. Martel-Estrada a,n, I. Olivas-Armendriz b, E. Santos-Rodrguez c, C.A. Martnez-Prez b,
P.E. Garca-Casillas b, J. Hernndez-Paz b, C.A. Rodrguez-Gonzlez b, C. Chapa-Gonzlez b
a

Instituto de Arquitectura, Diseo y Arte, Universidad Autnoma de Cd. Jurez, Av. Del Charro 450 Norte, Col. Universidad, C.P. 32310 Cd. Jurez, Chihuahua,
Mexico
b
Instituto de Ingeniera y Tecnologa, Universidad Autnoma de Cd. Jurez, Av. Del Charro 450 Norte, Col. Universidad, C.P. 32310, Cd. Jurez, Chihuahua,
Mexico
c
ICTP Meso-American Centre for Theoretical Physics (ICTP-MCTP)/Universidad Autnoma de Chiapas, Ciudad Universitaria, Carretera Zapata Km. 4, Real del
Bosque (Tern), Tuxtla Gutirrez, Chiapas, C.P. 29040, Mexico

art ic l e i nf o

a b s t r a c t

Article history:
Received 7 August 2013
Accepted 4 January 2014
Available online 10 January 2014

[Porous biocomposite scaffolds of Chitosan/Mimosa tenuiora (Ch/Mim) were fabricated for tissue
engineering applications by thermally induced phase separation and lyophilization techniques. The
in vitro bioactivity evaluation of the scaffolds was carried out by analyzing the apatite layers produced on
them using SBF as the incubation medium. The apatite formation was analyzed using FTIR spectroscopy
and Field Emission Scanning Electron microscopy coupled to Energy-Dispersive Electron X-ray Spectroscopy. The cumulative results obtained from IR spectra and SEM-EDS suggest that the developed
composites might have potential applications in tissue engineering.
& 2014 Elsevier B.V. All rights reserved.

Keywords:
Bioactive materials
Chitosan
Mimosa tenuiora
SBF

1. Introduction
Mimosa tenuiora (Willd.) Poiret is a species of the Mimosoideae, a subfamily of the Fabaceae botanical family that is
distributed from Mexico to Venezuela and Brazil [1]. It is a plant
with a high content in tannins and large amount of starch [2].
M. tenuiora is traditionally used as an aqueous extract [3] for the
treatment in wounds and venous leg ulcers due to its healing and
antiseptic properties [4]. It has been used in the treatment of
burns and infections in both humans and animals because of its
antimicrobial and cicatrizing effects [4,5].
The chemical composition of the M. tenuiora cortex has been
identied as a group of alkaloids, mainly N-N-dimethyltryptamine,
serotonin, triperpenoid glycosides, and steroidal saponins: 3-O-D-glucopiranosil campesterol, 3-O--D-glucopiranosil estigmasterol, and 3-O--D-glucopiranosil -sitosterol [4].
On the other hand, Chitosan is a semi-crystalline polysaccharide
derived from chitin and possesses antimicrobial activity [6]. Due to
its properties, Chitosan is a good candidate for preparation of
biomaterials that could substitute missing or damaged tissue [7].
One of the most important characteristics of a bioactive material
is the possibility that a biologically active carbonate hydroxyapatite

Corresponding author: +52 656 688 48 20.

E-mail address: adriana.martel@uacj.mx (S.A. Martel-Estrada).

0167-577X/$ - see front matter & 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.matlet.2014.01.004

layer can form on their surface [8]. Calcium phosphate is the main
constituent of bone tissue [9]. This substance could be formed on the
material surface by an inorganic chemical reaction similar to that
occurring in bones. This ability has been related to osteoconduction
and bone bonding [10], because the properties of the apatite layer
can affect cell viability and proliferation [8]. In order to predict
bioactivity, a simulated body uid (SBF) has been used [11].
With these characteristics, it could be reasonable to believe in
the bioactivity of a Chitosan/M. tenuiora composite. So, in this
study, a biocomposite of Chitosan/M. tenuiora composite was
manufactured through a thermally induced phase separation
method. The composite was used to investigate the in vitro
bioactivity of scaffolds by analyzing the apatite layers produced
in them using SBF as an incubation medium.
2. Materials and methods
Materials: Chitosan was purchased from Carbomer, Inc (United
States). The bark of M. tenuiora was obtained directly in the
region of Jiquipilas Chiapas. Ethanol was provided by CTR Scientic
(Mexico). Glacial acetic acid (Mallinckrodt, United States) was used
as a solvent. SBF was prepared in our laboratory according to a
previously published method [7].
Preparation of composites: Chitosan/M. tenuiora was prepared
using the thermally induced phase separation technique. Briey,

S.A. Martel-Estrada et al. / Materials Letters 119 (2014) 146149

Chitosan and M. tenuiora were dissolved in 1% (%v/v) aqueous

acetic acid solution. The solutions were mixed together at 80/20
ratio. Finally, the composite was frozen and freeze dried for 2 days.
FTIR spectroscopy: To evaluate the Chitosan/M. tenuiora composites and mineralized scaffold, FTIR spectra were recorded using
a transmission mode in an IR spectrometer (Nicolet 6700, Thermo
Scientic, USA). For each spectrum, 100 scans at16 cm 1 resolution were averaged.
Characterization of morphology and in vitro bioactivity: A previously studied method was used for the in vitro bioactivity study
[7]. Briey, porous samples of approximately 250 mm3 were
soaked in 5 mL of tetraethoxyl orthosilicate for 2 h in a vacuum
oven at 60 1C. The samples were rinsed with ethanol and were
dried in air at room temperature for 1 day. Then, the samples were
soaked in 5 mL of 1.5  SBF, pH 7.4, in an incubator at 37 1C. After
the incubation period, the specimens were washed carefully with
deionized water and nally dried.
Field Emission Scanning Electron Microscopy (FE-SEM, JEOL
JSM-7000 F) was applied to investigate the morphology of the
composites and Field Emission Gun Scanning Electron Microscopy
(Jeol JSM-7000 F) coupled with Energy Dispersive X-ray Spectroscopy (15 kV) to analyze the dispersion and distribution of apatite
particles and to detect the concentration of calcium and phosphorous on the surface after the treatment for the different
experimental conditions. The Ca/P ratio was estimated for the
various conditions. Also, the pore size was measured using the
Scandium Universal SEM Imaging Platform software.

147

3. Results and discussion

FTIR of the samples: Fig. 1 shows that the NH band of Chitosan
in the composite was affected and shifted from 1543 cm 1 to
1553 cm 1. On the other hand, the peak that corresponds to the
primary alcohol groups of Chitosan (CO stretching) was shifted
from 1418 cm 1 to 1459 cm 1 for the composite. Furthermore, it
is possible to observe a reduced intensity on the band at
1021 cm 1 that could imply an interaction of the ether bond of
Chitosan with the M. tenuiora. All these events suggest that there
is an interaction between the amino group of the Chitosan and
carboxyl groups in M. tenuiora or hydroxyl groups in Chitosan
and carboxyl groups of the M. tenuiora in the blend.
Characterization of morphology and in vitro bioactivity: Pores
with sizes around 50100 m are essential for bone ingrowth [12].
As shown in Table 1, the composite Chitosan/M. tenuiora has
more homogenous porosity than that of Chitosan, with an average
pore size of 211.6113.74 m. The composite also showed an openpore structure with high porosity (Fig. 2). The pore size and
porosity obtained is shown in Table 1.
According to SEM images (Fig. 2e and f), the particles formed
in vitro have a spherical shape. Some large particles with a
diameter of about 10 mm and smaller crystals around 3 mm were
observed. With an increase in immersion period a more compact
mineral layer was formed on the surface. The images revealed that
apatite crystals grow in a layer-by-layer manner. The Ca/P peak
intensity ratio for Chitosan at 28 days was 1.13, which might

Fig. 1. FTIR spectra of Mimosa tenuiora, Chitosan and 80/20 Ch/Mimosa tenuiora composite before incubation.

Table 1
Pore size distribution. It shows the average pore size (mm) and the distribution of porosity in Chitosan and 80/20 Chitosan/Mimosa tenuiora.
Composite

Average pore size (mm)

o50 mm (%)

50100 mm (%)

100200 mm (%)

200300 mm (%)

300400 mm (%)

400500 mm (%)

4500 mm (%)

Chitosan
80/20 Ch/Mim

150.56 714.65
211.61 713.74

15
12

30
16

35
24

10
22

4
12

1
12

5
2

148

S.A. Martel-Estrada et al. / Materials Letters 119 (2014) 146149

Fig. 2. SEM micrographs exhibit (a) Chitosan/Mimosa tenuiora composite and (b) Chitosan scaffold without any treatment. Then, the gure shows the Chitosan/Mimosa
tenuiora scaffolds at 21 days of SBF treatment at (c) 600  , (d) Chitosan at 28 days at 1000  and Chitosan/Mimosa tenuiora at 28 days of treatment at (e) 1000  and
(f) 1700  .

Fig. 3. FTIR of the (a) Chitosan/Mimosa tenuiora composite before the treatment and (b) Chitosan and (c) Chitosan/Mimosa tenuiora composite immersed for 28 days in
1.5  SBF. The image shows evidence of carbonate (CO23 ). Also the FTIR spectra revealed the presence of the v3 stretching mode of PO bonds.

S.A. Martel-Estrada et al. / Materials Letters 119 (2014) 146149

indicate the presence of a Ca-decient apatite. After immersion,

nucleation and growth have occurred on the surface, resulting in a
cluster of mineral precipitates. At higher magnication, the
spheres appear as needle-like deposits as shown in Fig. 2f. The
EDS analysis revealed that the Ca/P relation on the samples
Chitosan/M. tenuiora was 1.36 at 21 days and 1.86 at 28 days.
FTIR of composite before and after soaking in SBF showed the
formation of an apatite-like layer (Fig. 3). New bands can be
assigned to the phosphate group at 1044 cm 1 (PO stretching
(3) vibration), 956 cm 1 and 624 cm 1 (PO bending vibration,
4) and to the carbonate group at 1454 cm 1 (CO23 stretching
group) and 848 cm 1 (CO23 ) [9]. It is obvious from the results
described previously that the composites Chitosan/M. tenuilora
could develop a carbonate containing hydroxyapatite layer on the
surface.
4. Conclusions
Biodegradable Chitosan/M. tenuiora composites have been
successfully prepared by using a thermally induced phase separation method. On the Chitosan/M. tenuiora composite scaffold
there are many amine groups which could act as nucleation sites.
As a result, apatite could be formed more efciently on the
composite scaffolds than on the Chitosan scaffolds.
Also, a porous and interconnected morphology with enhanced
bioactivity that could be appropriate for tissue engineering has
been found. The ability of the composites to form on their surface
a mineralized layer was evident through the cumulative results
obtained from IR spectra, SEM and EDS. Future research must be
focused on testing the biocompatibility of this composite.

Acknowledgments
The authors acknowledge the nancial support of the Mexican
Public Education Secretary and the Mexican National Council for

149

Science and Technology (CONACyT) through Projects SEP-CONACyT

CB 2012-01-180909 and PROMEP/103.5/13/2347.

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