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The treatment of bone loss due to different etiologic factors is difficult and many
techniques aim to improve the repair, including a wide range of biomaterials and recently,
photobioengineering. This work aimed to assess by histological analysis the repair of bone
defects grafted with biphasic synthetic micro-granular HA + β-TCP associated with LED
phototherapy. Forty rats were divided into 4 groups (Clot, LED, Biomaterial and LED +
Biomaterial) each subdivided into 2 subgroups according to the time of animal death
(15 and 30 days). Surgical bone defects were prepared on the femur of each animal with
a trephine drill. In animals of the Clot group the defect was filled only by blood clot, in
the LED group the defect filled with the clot was further irradiated. In the animals of
Biomaterial and LED + Biomaterial groups the defect was filled by biomaterial and the
last one was further irradiated (λ=850±10 nm, 150 mW, Φ ~ 0.5 cm2, 20 J/cm2 - session,
140 J/cm2- treatment) at 48-h intervals for 2 weeks. Following animal death, samples
were taken and analyzed by light microscopy. Using the degree of maturation of the
bone by assessment of the deposition/organization of the basophilic lines in the newly
formed bone tissue, the LED + Biomaterial group was the one in a more advanced stage
of bone repair process at the end of the experiment. It may be concluded that the use
of LED phototherapy was effective in positively modulating the process of bone repair
of bone defects in the femur of rats submitted or not to biomaterial grafting.
Repair of Surgical Bone Defects Grafted
with Hydroxylapatite + β-TCP and
Irradiated with λ=850 nm LED Light
Luiz Guilherme P. Soares1, Aparecida Maria C. Marques1,2, Milena G. Guarda1,
Jouber Mateus S. Aciole1, Antonio Luiz B. Pinheiro1,2,3, Jean Nunes dos Santos4
1Center of Biophotonics, Dental
School, UFBA - Federal University
of Bahia, Salvador, BA, Brazil
2National Institute of Optics and
Photonics, Physics Institute of São
Carlos, USP - University of São
Paulo, São Carlos, SP, Brazil
3Camilo Castelo Branco University,
São José dos Campos, SP, Brazil
4Laboratory of Surgical
Pathology, Dental School,
UFBA - Federal University of
Bahia - Salvador, BA, Brazil
Correspondence: Prof. Dr. Antônio
Luiz Barbosa Pinheiro, Av. Araújo
Pinho, 62, Canela, 40110-150
Salvador, BA, Brasil. Tel.: +55-71-
3283-9010. e-mail: albp@ufba.br
Key Words: biomaterial,
bone repair, hydroxyapatite,
LED phototherapy.
Introduction
Bone loss is a frequent challenge for surgeons, as it may
be caused by several etiologic factors (1) and the defects
may be too large for spontaneous and physiologic repair.
Autologous bone is the most common type of graft used to
help the repair and it may be harvested from several parts
of the skeleton. Biocompatibility and osseointegration,
as well as substantial osteogenic potential characterize
autologous bone grafts (2,3).
Recent studies have shown that LED phototherapy
(LED-PT) induces a quicker repair process, with good
quality newly formed bone. These features were observed
in many studies in which this group used similar parameters
to the ones carried out with Laser Phototherapy (LPT) or
LED-PT (2-8). It seems that the beneficial effects of LED-
PT are similar to those of the laser. It is possible that the
mechanism involved is similar to the light absorption by
the cytochrome-C-oxidase present in the mitochondrial
membrane (9,10). Despite the increase of successful reports
of applications of different phototherapies in many areas,
their use in bone repair associated with bone grafting with
biomaterials needs to be further studied (4-8).
The combination of HA + β-tricalcium phosphate
(β-TCP) graft and phototherapies seems promising due to
the fact that the biomaterial possesses osteoconductive
properties and phototherapy influences function,
proliferation, secretion of growth factors such as bone
morphogenetic proteins (BMPs), platelet-derived growth
factor (PDGF) and transforming growth factor-β (TGF-β)
by different types of cells. This association may modulate
the repair of bone defects in a manner similar to what
is observed following the use of autologous bone graft,
preventing its complications and limitations (2,4,5,8,11,12).
However, the use of autologous grafts remains the gold
standard for the treatment of bone defects (2,3).
The aim of this study was to assess, by light microscopy,
the repair of bone defects grafted or not with biphasic
synthetic micro-granular HA + β-TCP combined or not
with LED phototherapy (λ=850±10 nm).
Material and Methods
Ethics, Animal Model, Housing and Sampling
The Animal Ethics Committee of the Federal University
of Bahia approved this work (Protocol 08.2010). Forty
healthy adult male Wistar rats (~2 months old, average
weight 295±25 g) were kept under natural conditions
of light, humidity and temperature at the Laboratory of
Animal Experimentation of the Dental School of the Federal
University of Bahia during the experimental period. The
animals were fed a standard laboratory diet (Labina®; Purina,
ISSN 0103-6440
Brazilian Dental Journal (2015) 26(1): 19-25
http://dx.doi.org/10.1590/0103-6440201300055
Braz Dent J 26(1) 2015
20
L.G.P. Soares et al.
São Paulo, SP, Brazil) and water ad libitum. The animals
were kept in individual micro-isolators and accommodated
in ventilated shelves (Insight Equipamentos Ltda, Monte
Alegre, Ribeirão Preto SP, Brazil). This system provides a
controlled environment with decreased risk of infection and
good sanitary condition. Controlled day/night light cycle
and temperature was performed during the experimental
period. The animals were randomly distributed into 4
groups and then divided into 2 subgroups according to
the moment of animal death (Table 1).
Surgical Procedure
Prior to intramuscular general anesthesia, the animals
were sedated (0.04 mL/100g of atropine subcutaneously)
and 20 min later general anesthesia with 10% ketamine
(0.1 mL/100g, Cetamin®, Syntec, São Paulo, SP, Brazil) and
2% xylazin (0.1 mL/100g; Xilazin®, Syntec) was carried out.
The animals had their right leg shaved and a 3-cm-long
incision was performed at the right tibia with a #15 scalpel
blade. Skin and subcutaneous tissues were dissected down
to the periosteum, which was gently sectioned exposing
the bone and a standard partial thickness 2-mm round
defect was surgically produced using trephine drill (SIN, São
Paulo, SP, Brazil) mounted on a 16:1 reduction contra-angle
handpiece (NSK; Utsonomya, Japan), maximum resistance
of 35 N with low speed drill, 1.200 rpm, under refrigeration
(Driller 600; SIN) in each animal (Fig. 1A) (4). Defects on
animals in Clot and LED groups were filled only with the
blood clot. Defects in Biomaterial and LED + Biomaterial
groups were filled with biomaterial. The animals in LED
and LED + Biomaterial groups were further irradiated. All
wounds were sutured and the animals received a single dose
of antibiotics (Pentabiotico; 0.2 mL; Fort Dodge Animal
Health, Kansas City, MO, USA). A biphasic synthetic micro-
granular HA + β-TCP (70%/30% respectively) completely
filled the bone defects when indicated, as recommended
by the manufacturer (Fig. 1B) (4).
Phototherapy Protocol
LED phototherapy was carried out using a LED device
(FisioLED; MMOptics , São Carlos, SP, Brazil) λ=850±10
nm, 150 mW, Φ ~ 0.5 cm2, 20 J/cm2 and light was
transcutaneously applied over the defect at 48-h intervals,
the first session carried out immediately after surgery. The
animals were restrained with the use of a plastic apparatus
designed to keep the animal immobile during irradiation,
avoiding the use of sedation. Total energy delivered was
20 J/cm2 per session and 140 J/cm2 per treatment. Energy
densities used were based upon previous studies carried
out by the authors (1-8,11,12). LED output power was
confirmed by using a calibrated power meter (Thorlabs
PM30-121; Thorlabs GmbH, Munich, Germany).
Animal Death and Sample Manipulation
Following animal death, the samples were longitudinally
cut under refrigeration (Buehler Isomet® 1000; Buehler,
Markham, Ontario, Canada) and the specimens kept in 10%
formalin solution for 24 h. The specimens were routinely
processed to paraffin, cut and stained with hematoxylin
and eosin and Sirius red, and underwent histological
analysis (Table 2) at the Laboratory of Surgical Pathology
of the Dental School of the Federal University of Bahia by
an experienced pathologist in a blind manner using a light
microscope (AxioStar®, Zeiss, Jena, Germany).
Results
Clot Group
On the15th day, the defect was partially filled by newly
Table 1. Distribution of study groups
Group (n =
10 animals)
Time of
euthanasia (d) Phototherapy Biomaterial
Clot 15 or 30 - -
Biomaterial 15 or 30 - Yes
LED 15 or 30 Yes -
LED + Biomaterial 15 or 30 Yes Yes
Table 2. Semi quantitative criteria used for light microscopy analysis
Criteria Discrete Moderate Intense
Bone
resorption
Presence of <25% of resorption of
graft remnants and/or surgical bed
Presence of 25-50% of resorption of
graft remnants and/or surgical bed
Presence of >50% of resorption of
graft remnants and/or surgical bed
New bone
formation
Presence of <25% of newly formed bone
similar to adjacent untreated bone tissue
Presence of 25-50% of newly
formed bone similar to adjacent
untreated bone tissue
Presence of >50% of newly formed bone
similar to adjacent untreated bone tissue
Inflammatory
infiltrate
Presence of <25% of inflammatory
cells on the area
Presence of 25-50% of
inflammatory cells on the area
Presence of >50% of inflammatory
cells on the area
Collagen
deposition
Presence of <25% of collagen
deposition in the area
Presence of 25-50% of collagen
deposition in the area
Presence of >50% of collagen
deposition in the area
Braz Dent J 26(1) 2015
21
LED effect on biomaterials
formed bone, which was characterized by the presence
of thin, interconnected or not, trabecular bone showing
osteocytes and irregular basophilic lines within it and
osteoblastic rimming. There was a moderate chronic
inflammation and cartilaginous differentiation. At the end
of the experimental period, the specimens of this group
showed the defect completely filled by newly formed
bone, but unlike the earlier period, the bone trabeculae
were thick and basophilic lines were parallel to each other
(Fig. 2A). Osteocytes within it were also frequent as well as
sometimes a layer of rimming osteoblasts. The inflammation
was chronic and ranged from moderate to severe in both
experimental periods. The collagen deposition was intense
and it was scored mostly as mature (Fig. 2B).
Biomaterial Group
On the 15th day, the defect was completely filled by
newly formed bone characterized by a varied trabecular
pattern, sometimes showing the presence of irregular
osteocytes and rimming of osteoblasts. Often, these
trabeculae were interwoven and surrounding the
amphophilic material entrapped in small or large amounts,
which was interpreted as being remnants of the biomaterial
(Fig. 3A). In the area corresponding to the biomaterial, few
layers of rimming osteoblasts were seen as well as foreign
body reaction. It was characterized by presence of giant
cells around the remnants. Discrete to moderate mixed
inflammation permeated the entire specimen. Mature
collagen was observed in the newly formed bone tissue,
but it was absent in the remnants of the biomaterial. At
the end of the experimental period most of the specimens
of this group showed similar aspect to the previous period.
The defect was completely filled by thick interconnecting
trabecular bone. Sometimes in the form of globules, many
of these trabeculae were seen surrounding the amphophilic
material (Fig. 3B). The collagen deposition was intense and
collagen was graded as mature in most specimens (Fig. 3C).
LED Group
After 15 days, the specimens of this group showed the
defect partially filled by newly formed bone displaying
thin interconnecting bone trabeculae characterized by
osteocytes within and filled with red bone marrow (Fig.
4A). No signs of resorption were present, but a discrete
to moderate inflammation was observed. The collagen
deposition was intense and it was mature in the newly
formed bone. At the 30th day, the specimens showed the
defect either partially or completed filled by newly formed
Figure 1. A: Clinical aspect of the surgical bone defect created on the right tibia of each animal. B: Clinical aspect of the surgical bone defect created
on the right tibia of each animal filled with the biomaterial (4).
Figure 2. A: Photomicrograph of control specimen evidencing the defect completely filled by thick and mature newly formed bone showing osteocytes
and parallel basophilic lines (30 days - HE). B: Photomicrograph of control specimen evidencing newly formed bone with mature collagen throughout
its length (30 days - Picrosirius).
Braz Dent J 26(1) 2015
22
L.G.P. Soares et al.
interconnecting trabecular bone of varied thicknesses,
characterized the presence of osteocytes and nonparallel
basophilic lines. Sometimes the trabeculae showed rimming
osteoblasts (Fig. 4B). There were no areas of bone resorption.
Cartilaginous differentiation was also evident as well as the
presence of chronic inflammation that ranged from discrete
to moderate. Collagen deposition in the bone trabeculae
was intense and mature (Fig. 4C).
LED + Biomaterial Group
At the 15th day, the specimens showed a small surgical
defect filled by newly formed bone, characterized by the
presence of globules or thin interconnecting bone trabeculae
and the presence of osteocytes and parallel basophilic lines
permeated by moderate chronic inflammation (Fig. 5A).
Cartilaginous differentiation was also present, as well as
rare presence of focal areas of an amphophilic material,
interpreted as remnants of the biomaterial, and foreign body
giant cell reaction. The collagen was moderately mature.
At the end of the experimental period, the specimens
of this group showed the defect completely repaired in
which new bone formation was observed with trabecular
bone tissue of variable thickness, sometimes in the form
of globules, showing few osteocytes and basophilic lines
parallel to each other. Many of these trabeculae were seen
surrounding amphophilic material interpreted as remnants
of the biomaterial. Chronic inflammation was observed and
ranged from discrete to moderate (Fig. 5B). Resorption was
observed in only one specimen. The collagen was mature
and intense in all samples (Fig. 5C).
Discussion
The reason for combining HA + β-TCP graft and LED
light in this study was due to the osteoconductive properties
of the biomaterial and previous reported positive effects
of the LED-PT on both function and proliferation of cells.
These effects are observed when the techniques are used
in isolation and have been shown effective in accelerating
the repair process in various experimental models. It
was hypothesized that combination of both techniques
would modulate the repair of bone defects grafted with
a biomaterial and irradiated with LED light to a pattern
Figure 3. A: Photomicrograph of specimen from Biomaterial group showing on the right side the remaining bone from the surgical bed from which
grows thin interconnecting trabecular bone with osteocytes inside and imprisoned remnants of the biomaterial (15 days - HE). B: Photomicrograph
of specimen from Biomaterial group showing new bone formation characterized by predominantly thick trabecular bone with osteocytes inside and
basophilic lines parallel to each other. Note the remaining biomaterial imprisoned by newly formed bone and chronic inflammation (30 days - HE). C:
Photomicrograph of specimen from Biomaterial group showing on the right side bone from the surgical bed from which depart newly formed bone
trabeculae with collagen of similar aspect to the surgical bed, but unevenly distributed in the biomaterial (30 days - HE).
Figure 4. A: Photomicrograph of specimen from LED group showing discrete amount of newly formed bone characterized by thin trabecular bone with
osteocytes and basophilic lines inside (15 days - HE). B: Photomicrograph of specimen from LED group showing discrete amount of newly formed
bone characterized by thin trabecular bone with osteocytes, basophilic lines inside and rimming osteoblasts (30 days - HE). C: Photomicrograph of
specimen from LED group showing new bone formation presenting mature collagen throughout its length (30 days - Picrosirius).
Braz Dent J 26(1) 2015
23
LED effect on biomaterials
similar to what occurs when autologous bone graft is
used as well as on preventing complications and reducing
limitations. However, so far the use of autologous bone
grafting remains the gold standard for the treatment of
bone defects.
Despite being one of the most common synthetic
bone graft and able to successfully regenerate bone in
various osseous defect areas, β-TCP is also known for its
rapid resorption rate which may precede the growth of
new bone. Because of this, its association to HA has been
used, since HA has low osteoconductive activity but a
good space-maintaining capacity, whereas β-TCP is more
bioresorbable and is rapidly replaced by new bone material.
This association makes one part of the graft to be rapidly
resorbed (TCP) and the other (HA) remain in situ for a
longer period. Possibly higher HA/β-TCP ratios yield more
replacement of biomaterial by new bone (13).
There is no full agreement on regards the most effective
ratio of HA/β-TCP. It was suggested that 60% HA and 40%
β-TCP seemed to provide the optimal bone conductive
properties. For periodontal defects, a ratio of 85/15 has been
suggested as optimal for repairing surgically created bone
defects (13). However, a ratio of 60/40 was mentioned most
suitable in a previous animal study (13). In the present study
the HA/β-TCP ratio was 70:30, which could be considered
within the range for optimal tissue response. It has been
suggested that higher ratio of β-TCP could theoretically
increase the degradation of the particles of the biomaterial
and that biphasic HA/β-TCP composite powders exhibit a
solubility between those of HA and β-TCP the dissolution
rate of the Ca-P being strongly dependent on the content
of β-TCP. It was initially thought that different ratios could
induce different outcomes as regards bone formation and
biodegradation. However, previous reports failed to find
different rates of new bone formation when using different
ratios but found as regards resorption of the material (13).
The authors have recently discussed that the utility
of the of residual bone graft materials may vary with the
type of defect where the materials are applied and that the
biomaterials used shall allow osteoblasts to build bridges
between its granules and integrate with other osteoblasts
providing support for both proliferation and differentiation
at earlier phases of the repair. This will then result in intrinsic
stimulation of new bone formation (13).
The biomaterial used on the present study is granular
and porous and besides that, it contains Ca and P that
favor the early phases of repair. The presence of β-TCP in
the graft used on the present investigation has shown to
influence the mechanical stability of the graft and makes
possible its rapid degradation. This aspect results in some
volume instability of the graft that does not allow new
bone formation to keep the original volume. In addition to
the several advantages of using this type of biomaterial, it
also present disadvantages that include poor mechanical
properties, lack of an organic phase, the possibility of the
presence of impurities and non-homogenous particle size
and shape. Another aspect that influences the results of
studies using such type of biomaterials is that the Ca/P
ratio may differ according to the manufacturer (13).
In a previous study it was also mentioned that a usual
concern when using granular biomaterials is granule-
mediated inflammatory reaction (13). In the present study,
inflammatory reaction was well documented in all groups
where the biomaterial was used. In the present study no
sign of fibrosis was seen.
For evaluation of bone repair were chosen two
experimental periods, 15 and 30 days. During the initial
stages of bone repair, the cellular component (mainly
fibroblasts and osteoblasts) is more prominent and more
prone to be affected by light. At 30 days, the repair process
is in an advanced stage and is widely used for evaluation of
bone healing in several published studies (1-3,6,7,14-16).
Figure 5. A: Photomicrograph from LED + Biomaterial group showing new bone formation of globular aspect with biomaterial imprisoned amidst
chronic inflammation (15 days - HE). B: Photomicrograph of specimen from LED + Biomaterial group showing new bone trabeculae and globules
evidencing the osteocytes and parallel basophilic lines inside. Notice presence of the biomaterial in the remaining central region and remaining red
bone marrow (30 days HE). C: Photomicrograph of specimen from LED + Biomaterial group showing mature collagen (intense red color) unlike the
aspect observed in the remnant of the biomaterial surrounded by the newly formed bone (30 days - Picrosirius).
Braz Dent J 26(1) 2015
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L.G.P. Soares et al.
In the present study, it was chosen to conduct a qualitative
histological analysis, to describe the mechanisms involved in
bone repair of defects treated or not with LED phototherapy
associated or not with biphasic HA + β-TCP. The description
followed the guidelines established in the methodology.
The phenomena observed in non-grafted groups
were similar to the previously reported according to the
used methodology. Therefore, no major differences were
observed histologically in the repair process groups, Clot
and LED in both experimental periods. Therefore, it appears
that the irradiated groups showed a more advanced repair
process and quality than observed in the non-irradiated
group (Clot) as may be seen in Figures 2-5 (7,8,17).
The observation of inflammatory response and, in
some cases, the presence of foreign body reaction around
the biomaterial is an expected finding. No matter how
the biomaterial is biocompatible, it will continue to be
a foreign material. It is interesting to observe that in a
previous study using another biomaterial (MTA - mineral
trioxide aggregate), this reaction was detected in a more
striking and intense manner than observed in the present
study. This may be an indication that the material used in
this work is less irritating to the tissue that the MTA (7).
Besides, the fact that inflammation was limited it surely
caused a physiological effect that would in some extent be
deleterious to the repair process resulting in complications
like fibrosis. However, in the present study no sign of fibrosis
was seen. At the end of the experimental period, there was
an intense inflammatory response in the Clot group, where
no additional treatment was used, different from what
was observed in all other groups. This can be explained
by previous studies which indicated that the persistence
of the inflammatory response in the later stages of bone
healing might be the result of phlogistic activity caused
by the remnants of blood clot (8).
For all parameters in this study, the use of the Biomaterial
+ LED showed the best results. This has been described in
previous studies in which the use of LED was associated
with increased proliferation of fibroblasts, chondroblasts
and osteoblasts and hence increased deposition of
collagen, an important precursor of the mineral matrix
deposition (7,8,17). Thus, the increase in bone formation
is probably closely linked with both the increased numbers
of osteoblasts and their secretory activity.
The presence of a cartilaginous precursor was observed
only at 15 days, in Clot and LED groups + Biomaterial, which
denote a reparative process in a more advanced stage than
in other groups. It is interesting to observe that at the end
of the experimental period only the LED group showed this.
The trabecular aspect also varied between groups. In the
initial period of the repair, the trabeculae were thin in Clot,
LED, and LED + Biomaterial groups and in a variable manner
in the other groups. At the end of the experimental period,
the trabeculae were thicker in Clot and Biomaterial groups
and of varied thickness in the other groups. Interesting
was the observation of the remodelling activity, which was
still present at this time, as evidenced by the presence of
basophilic lines. These lines were observed in most groups
at the end of the experimental period and were sometimes
parallel or not. These lines were not observed in Biomaterial
group. In fact, this aspect seems to have influenced mainly
the defects of Clot group in which the presence of cartilage
seems to have influenced the results. It was the group in
which the cartilage apparently progressed to a trabecular
bone which varied from thin to thick along the observation
time. The reason for this needs further clarification and can
be related either to the presence of the biomaterial or to
the light source, which may have accelerated or delayed
the differentiation of cartilage in some way. This could be
demonstrated in a study using intermediate times between
the two this work, as well as use of specific markers for
this tissue.
Regarding the basophilic lines present in bone tissue,
they were observed initially (15 days) in all groups except
in the Biomaterial group. However, only in the LED +
Biomaterial group these lines were deposited in a regular
pattern, parallel to each other, which may be indicative of
a more mature bone already in the initial repair. At the end
of the experimental period the previously observed pattern
was maintained. The fact that the Biomaterial group did
not show these lines may be indicative of a slower or even
delayed remodelling process.
Using the degree of maturation of bone by assessment
of the deposition/organization of the basophilic lines in the
newly formed bone tissue, the LED + Biomaterial group was
the one in a more advanced stage of bone repair process
at the end of the experiment. It may be concluded that
the use of LED phototherapy was effective in positively
modulating the process of bone repair of bone defects in
the femur of rats submitted or not to biomaterial grafting.
Resumo
O tratamento de perdas ósseas devido a diferentes fatores etiológicos é
difícil e muitas técnicas têm por objetivo melhorar o reparo incluindo o uso
de uma ampla gama de biomateriais e, recentemente, a fotobioengenharia.
Este trabalho teve como objetivo avaliar, por meio de análise histológica,
o reparo de defeitos ósseos enxertados com HA bifásica micro-granular
sintética + β-TCP associada à fototerapia LED. Quarenta ratos foram
divididos em quatro grupos (Clot, LED, Biomaterial e LED + Biomaterial),
subdivididos no dois subgrupos de acordo com o momento da morte
(15 e 30 dias). Defeitos ósseos cirúrgicos foram criados em um fêmur
de cada animal com uma broca trefina. Em animais do grupo coágulo, o
defeito foi preenchido apenas pelo coágulo sanguíneo, no grupo de LED
o defeito foi preenchido pelo coágulo e irradiado. Nos animais dos grupos
do biomaterial e LED + biomaterial, os defeitos foram preenchidos com
biomaterial e o último foi adicionalmente irradiado (λ=850±10 nm, 150
mW, Φ ~ 0,5 cm2, 20 J/cm2-sessão, 140 J/cm2-tratamento) a cada 48 h
Braz Dent J 26(1) 2015
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LED effect on biomaterials
por duas semanas. Após a morte dos animais, amostras foram colhidas e
analisadas por microscopia de luz, usando o grau de maturação do osso
como marcador (deposição/organização das linhas basofílicas) no tecido
ósseo neoformado. O grupo de LED + biomaterial apresentou processo de
reparação mais avançado ao fim do experimento. Pode-se concluir que
o uso da fototerapia LED foi eficaz na modulação positiva do processo
de reparo ósseo de defeitos ósseos no fêmur de ratos submetidos ou não
a enxerto com biomaterial.
Acknowledgements
We would like to thank the Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) for providing financial support for this
project.
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Received April 10, 2014
Accepted December 11, 2014
