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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
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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
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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
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S.A. Martel-Estrada et al. / Journal of Alloys and Compounds 643 (2015) S119S123
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
<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).
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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.
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
S123
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.