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Journal of Alloys and Compounds 643 (2015) S119S123

Contents lists available at ScienceDirect

Journal of Alloys and Compounds


journal homepage: www.elsevier.com/locate/jalcom

Biocompatibility of chitosan/Mimosa tenuiora scaffolds for tissue


engineering
Santos Adriana Martel-Estrada a, Brenda Rodrguez-Espinoza b, El Santos-Rodrguez c,
Florinda Jimnez-Vega b, Perla E. Garca-Casillas d, Carlos A. Martnez-Prez d, Imelda Olivas Armendriz d,
a

Instituto de arquitectura diseo y arte, Universidad Autnoma de Ciudad Jurez, Ave. Del Charro #610 norte, Col. Partido Romero, C.P. 32320 Cd. Jurez, Chihuahua, Mexico
Instituto de Ciencias Biomdicas, Universidad Autnoma de Ciudad Jurez, Anillo envolvente del PRONAF y Estocolmo, C.P. 32320 Cd. Jurez, Chihuahua, Mexico
ICTP Meso-American Centre for Theoretical Physics (ICTP-MCTP)/Universidad Autnoma de Chiapas, Ciudad Universitaria, Carretera Zapata Km. 4, Real del Bosque (Tern), C.P. 29040
Tuxtla Gutirrez, Chiapas, Mexico
d
Instituto de Ingeniera y Tecnologa, Universidad Autnoma de Ciudad Jurez, Ave. Del Charro #610 norte, Col. Partido Romero, C.P. 32320 Cd. Jurez, Chihuahua, Mexico
b
c

a r t i c l e

i n f o

Article history:
Available online 20 January 2015
Keywords:
Composite
Biocompatibility
Mimosa tenuiora
Chitosan

a b s t r a c t
In search of a plant that exhibits osteogenic activity, Mimosa tenuiora (M. tenuiora) cortex represents
the opportunity to create a biomaterial that, together with the chitosan, is osteoconductive and promote
better and rapid regeneration of bone tissue. Thus, the composite of chitosan/M. tenuiora cortex fabricated will have properties of biocompatibility and allow the osteoblast proliferation. Composites were
developed with different concentrations of chitosan/M. tenuiora cortex (w/w) using thermally induced
phase separation technique (TIPS). To analyze the effects of composite on osteoblasts, primary cultures,
each sample was collected on days 1, 3 and 7 after seeding. The evaluation of composites consisted of
viability and proliferation tests in which we observed the metabolic activity of the cells using MTT
reagent and determined the DNA concentration by means of uorescence. The expression of the marker
alkaline phosphatase (ALP) using p-nitrophenyl phosphate was examined, allowing the observation to
the activity of proliferation and differentiation of osteoblastic cells. Moreover, an analysis of biomineralization was performed using scanning electron microscopy (SEM), energy dispersive spectroscopy, infrared spectroscopy and X-ray diffraction. The results showed that 80/20 chitosan/M. tenuiora cortex
biocomposite has the best performance with osteoblasts compared to biomaterials 100/0 and 70/30
chitosan/M. tenuiora composites. Finally, it was determined that the composite of chitosan/M. tenuiora
cortex presents no cytotoxicity and increases the capacity of the osteoblasts to proliferate and
differentiate.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Bone tissue is an important structure for the support and movement of the body. The bones have the ability to be constantly
renewed through a process called remodeling, which is sometimes
not very effective. In recent years the use of chitosan in bone
regeneration has proved to be an effective treatment because it
is osteocompatible, biodegradable, and osteoconductive [1].
The idea of nding a natural substance that allows the enhancement of the properties of chitosan on bone tissue has been focused
towards plants that are osteogenic. Some plants have demonstrated the ability to regenerate bone such as, Elephantopus mollis
and African Spilanthes, plants that are located in Cameroon (Africa).
Corresponding author. Tel.: +52 656 6884887.
E-mail address: iolivas@uacj.mx (I.O. Armendriz).
http://dx.doi.org/10.1016/j.jallcom.2015.01.034
0925-8388/ 2015 Elsevier B.V. All rights reserved.

The results have shown that extracts of the leaves and branches of
E. mollis and S. African whole plant increased mineral deposition in
tissue, causing the growth of the bone where the fracture or lesion
is located. This increase is attributed to the differentiation of progenitor cells of the bone marrow, ontogenetic lineage cells, and
increased recruitment of osteoblasts at the fracture site [2]. There
is also the case of Cissus quadrangularis, a medicinal plant that has
an activity osteogenesis, and that is gaining great interest as a therapeutic agent to improve healing of bones [3].
In Mexico, M. tenuiora is a plant with osteogenic activity, which
can easily be acquired, is accessible and is economically affordable.
M. tenuiora cortex has been used for decades as a remedy in
the treatment of wounds and burns of the skin [4] due to their ability to heal, anti-inammation and antimicrobial activity. The
action of the components of the cortex on the epithelial tissue have
demonstrated their potential to stimulate the proliferation of

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S.A. Martel-Estrada et al. / Journal of Alloys and Compounds 643 (2015) S119S123

broblasts and epithelial cells. Due to the organic compounds it


contains, it is possible that this is the reason for its reaction to
the tissue of the skin. Triterpenes saponins indentied as
Mimonosidas A, B, and C; tannis; steroids as campesterol-3-Ob-D-glucopyranosyl, stigmasterol-3-O-b-D-glucopyranosyl, and
b-sitosterol-3-O-b-D-glucopyranosyl, are some of the components
of the cortex that are believed to be responsible for this activity [5].
Because broblasts and osteoblasts are similar cell lines, M. tenuiora could activate the osteoblast proliferation, as it does with
broblasts. Incorporating M. tenuiora cortex to chitosan would
obtain a composite containing the benecial properties of the
two components, leading to a better bone regeneration. Although
the effects of M. Tenuiora cortex in the bioactivity of the composite chitosan/M. Tenuiora cortex has been evaluated previously in
our laboratory [6] and demonstrated the ability of the composites
to form on their surface a mineralized layer, the interaction of cells
with the material must be tested. So, in this study we investigated
the behavior of primary osteoblast cultured in the presence of
chitosan and Mimosa tenuiora. Then, biomineralization, alkaline
phosphatase (ALP) activity, cell viability and total DNA of osteoblasts in the scaffolds were evaluated.
2. Experimental
For the preparation of 80/20, 70/30, and 100/0 (w/w) chitosan/M. tenuiora
composites were used in a methodology previously reported [7]. Chitosan (Carbomer, United States) and M. tenuiora cortex (Jiquipilas Chiapas, Mexico) were slowly
added to the solution aqueous of 1% (%v/v) acetic acid (Mallinckrodt, United States).
The solution was placed in a freezer at 40 C for three hours and lyophilized for
72 h. Finally, the composites were neutralized.
All composites were seeded with primary osteoblasts calvaria cells obtained
from Wistar rats by collagenase digestion. 50,000 cells per sample were cultured,
at different time periods, in a-MEM supplemented with fetal bovine serum (FBS),
penicillinstreptomicin, b-glycerolphosphate, and ascorbic acid. The cell viability
was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. At each time point, MTT solution (50 lL) and a-MEM (450 lL)
containing FBS and antibiotic were added to each composite. After 3 h incubation,
400 lL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan product,
and the absorbance at 570 nm was measured using a Benchmark Plus, micro-plate
spectrophotometer. The cell number was determined by using a standard curve.
The analysis of cell proliferation by counting total DNA was evaluated in a QuantiT PicoGreen dsDNA kit. The scaffolds were washed with PBS and 1 mL of MilliQ-deionized water was added, followed by three freezethaw cycles. The samples
were centrifuged to remove the debris of the scaffolds and the supernatant was
used in a total DNA quantication. Briey, 287 lL of sample, 713 lL of TE buffer,
and 1000 lL of picogreen working solution were added in a quartz cell. After
8 min of incubation in the dark, the quartz cell was read in a spectrouorimeter
at 480 nm and the uorescence emission intensity at 520 nm was measured. The
amount of DNA was determined based on the standard curve. The ALP activity
was measure with the same sample as used for DNA assay. Subsequently, 100 lL
of each sample was added to 100 lL of p-nitrophenylphosphate in a 96-well plate
and incubated for 30 min. The reaction was stopped by the addition of 50 lL of 2 M
NaOH solution and the plate at 405 nm.
For SEM examination the samples were rinsed with PBS and immersed in 3%
gluteraldehyde solution for 1 h. Samples were dehydrated through a series of ethanol solutions. Once dried, they were mounted on aluminum stubs and a scanning
electron microscopy (JEOL JSM-7000F) coupled with an energy-dispersive system
(EDS 7557 INCA Oxford Instruments) was used to characterize the morphology
and cell proliferation. FTIR was carried out using a Nicolet 6700 FT-IR Thermo Scientic spectrometer. All spectra were recorded in a wavenumber range of 4000
500 cm 1.

3. Results and discussion


SEM was used in order to observe the morphology of each composite. Fig. 1 shows the highly porous composites, whose interconnected pores are observed with a variable size (Table 1). However,
the 80/20 chitosan/M. tenuiora composite has more homogenous
porosity morphology with pores of 100200 lm (26%), >500 lm
(25%) and 200300 lm (21%). This indicates that the obtained
composites are composed of pores, which theoretically enable

the introduction of cells, and can allow the biological processes


necessary for their adaptation on the supports [8,9].
According to ISO 10993-5:2009, MTT is a test method used to
assess the in vitro cytotoxicity which measures the viability of
the cells through their metabolic activity and made it possible to
correlate the number of viable cells with the absorbance of the formazan crystals dissolved in DMSO. The growth of osteoblast cells
increased on all composites over the period of study (Fig. 2). However, it is possible to observe a low proliferation cell in the control
with respect to the composites, and this can be attributed to the
cells were seeded in a 2D environment. This is because the composites are three dimensional scaffolds that work as a template
in which the cells inltrate, thus serving as a guide in the differentiation and proliferation of the cells [8]. On the other hand, some
research shows that the scaffolds of chitosan support the in vitro
differentiation and proliferation of osteoblastic cells [10]. This
can be seen in the number of cells and DNA concentration obtained
on each day of analysis of the biomaterial 100/0 chitosan/M. tenuiora compared with the control (Fig. 3). However, introduction of
the powder of M. tenuiora cortex in composites indicates that the
mixture between the two components involve a synergy that
results in improved viability and proliferation cells, because the
80/20 and 70/30 chitosan/M. tenuiora composites show higher
values in cell viability and DNA amount compared to 100/0 chitosan/M. tenuiora composite. Nevertheless, a pattern was clearly
evident at day 7, 80/20 chitosan/M. tenuiora composite induced
better cell proliferation compared to all other samples (consistent
with the SEM images). Although, the DNA quantication assay on
the same day indicated that there is no difference between the
80/20 and 70/30 chitosan/M. tenuiora composites. However, at 1
and 3 days 80/20 chitosan/M. tenuiora composite showed a higher
concentration of DNA than other composites, which means that
there is a signicant increase growth rate response to the cells.
These results suggest that the relationship between the chitosan
and M. tenuiora cortex concentration play an important role in
the proliferation and viability of osteoblasts cells. These ndings
corroborate those reported by Zippel and coworkers [11], in that
a group of broblasts cells showed a strong improvement in viability and proliferation due to the present of arabinogalactan extract
(present in M. tenuiora cortex), indicating that primary cellular
target of the components of M. tenuiora were broblast within
connective tissue, suggesting that the same is happening with
osteoblasts cells.
The alkaline phosphatase activity is an indicator of osteoblast
activity and an early stage of differentiation of the same cells
[12]. The results show the ALP expression increased over time in
all composites (Fig. 4). On the other hand, at 1 and 3 days does
not show that the ALP activity increased with the presence of M.
tenuiora cortex (on 80/20 and 70/30 chitosan/M. tenuiora composites), there were no signicant differences in comparison with
the 100/0 chitosan/M. tenuiora composite. This suggests that M.
tenuiora had no inuence on the expression of the membrane protein to earlier periods of time, and therefore the osteodifferentiation is occurring at the same time that the osteoblasts present in
the chitosan/M. tenuiora composite. However, at 7 day ALP activity of 80/20 chitosan/M. tenuiora composite signicantly
increased compared to the other composites, indicating that M.
tenuiora cortex has a late positive effect. Chevallay and Herbage
refer to that chitosan composites have a limitation to support the
spread of the cytoplasmic membrane in the surface, thereof,
obstructing osteoblasts differentiation [13]. However, the difference in ALP expression in the 80/20 chitosan/M. tenuiora composite at 7 day may be due to the same composition of chitosan/M.
tenuiora cortex, so that illustrates the potential of M. tenuiora
cortex in inducing differentiation of osteoblasts cells.

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Fig. 1. SEM image of (a) 100/0, (b) 80/20, and (c) 70/30 chitosan/M. tenuiora composites before cultured with osteoblasts.

Table 1
The average pore size (lm) and pore size distribution (%) in 100/0, 80/20, and 70/30 chitosan/M. tenuiora composites.
Composite chitosan/M.
tenuiora

Average pore size


(lm)

<50 lm
(%)

50100 lm
(%)

100200 lm
(%)

200300 lm
(%)

300400 lm
(%)

400500 lm
(%)

>500 lm
(%)

100/0
80/20
70/30

320.70 211.68
351.76 190.92
201.96 173.82

2
2
8

9
2
26

26
26
28

16
21
17

20
15
12

10
9
2

17
25
7

Fig. 2. Number of osteoblast cells measured by MTT assay of 100/0, 80/20, 70/30
chitosan/M. tenuiora composites and cultures without materials (control) for 1, 3,
and 7 days. : signicant difference compared with 100/0 chitosan/M. tenuiora
composite; : signicant difference compared to control; #: signicant difference
compared to 70/30 chitosan/M. tenuiora composite; : signicant difference
compared to 80/20 chitosan/M. tenuiora composite. (P = 0.05, comparison is made
between samples of the same day).

SEM imaging and EDS analysis reveals broblasts cells attached


to the surface of the scaffolds and initiated the process of proliferation and mineralization (Fig. 5a, d, and g). According to the ALP
activity results, the regularization of mineralization and phosphate
transport is being conducted from the 1st day, conrmed by EDS
analysis, in which the presence of phosphorus and calcium were
found in the three types of scaffolds during the study period,
although the Ca/P atomic ratio is different from the hydroxyapatite
(Ca/P = 1.67). On the other hand, on day 3 it was noted the beginning of the secretion of extracellular matrix (Fig. 5b, e and h) and
the elongated structure of the cells. This is a positive sign that
the cells have adherent more intensely to the scaffolds. Furthermore, FTIR spectra (Fig. 6) support the biomineralization process
on materials to nd the characteristic peaks of phosphates and
carbonates. Moreover, these same spectra show that secretion of
extracellular matrix, to nd the characteristic bands of amide
groups (CONH2). This suggests the presence of glycosaminoglycans (GAG), proteoglycans (PG), and collagen, among other

Fig. 3. Determination of DNA (ng/ml) from the osteoblasts present in the


composites 100/0, 80/20, and 70/30 chitosan/M. tenuiora composites and control,
after 1, 3 and 7 days of incubation. : signicant difference compared 100/0
chitosan/M. tenuiora composite; : signicant difference compared to control; #:
signicant difference compared to 70/30 chitosan/M. Tenuiora composite.
(P = 0.05, comparison is made between samples of the same day).

Fig. 4. ALP expression measure in absorbance at 405 nm of 100/0, 80/20, 70/30


chitosan/M. tenuiora composites and control, after 1, 3, and 7 days of incubation. :
signicant difference compared to 100/0 chitosan/M. tenuiora composite; :
signicant difference compared to control; #: signicant difference compared to
70/30 chitosan/M. tenuiora composite. (P = 0.05, comparison is made between
samples of the same day).

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Fig. 5. Representative SEM images of adhered cells on 100/0 (ac), 80/20 (df), and 70/30 (gi) chitosan/M. tenuiora composites, culturing for 1, 3, and 7 days, respectively.

Fig. 6. Subtraction of the infrared spectra of 80/20 chitosan/M. tenuiora composite before and after cultivation: (a) 1 day, (b) 3 days, and (c) 7 days.

proteins. This is important because GAG and PG are associated


with the organization of the MEC and the mineralization of the
same [14,15]. It is believed that the crystals found on the composite (Fig. 5c, f, and i) are amorphous calcium phosphate, according to

the XRD analysis (Fig. 6). These results are supported by Weiner
[16], who referred to the initial solid-phase of calcium phosphate
formed in the bone is amorphous, which is replaced by poorly
crystalline apatite to subsequently transform a mature crystalline

S.A. Martel-Estrada et al. / Journal of Alloys and Compounds 643 (2015) S119S123

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References

Fig. 7. Diffractogram of the composites (a) 100/0, (b) 80/20 and (c) 70/30 chitosan/
M. tenuiora at 7 days after cell seeding and incubation.

apatite. Furthermore, the process of bone desorption exhibit the


amorphous portion of the mineral part of bone more metabolically
active than the crystalline portion [17]. Based on this, the presence
of amorphous calcium phosphate is due to the osteoblastic cells
that were still heading mineralization process of bone formation
(see Fig. 7).
4. Conclusions
This study has demonstrated the possibility of combining the
chitosan and M. tenuiora cortex in scaffolds for bone tissue regeneration. The active constituents of M. tenuiora cortex enhanced
the proliferation and differentiation of osteoblasts cells.
Acknowledgements
The authors gratefully acknowledge the nancial support of the
Mexican Council for Science and Technology and the Mexican
Public Education Secretary (SEP-CONACyT 2012-180909) and the
support of Susana Garcia during language edition of this document.

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