Biosilicate® and low-level laser therapy improve bone repair in osteoporotic rats.

Abstract

The aim of this study was to investigate the effects of a novel bioactive material (Biosilicate®) and low-level laser therapy (LLLT) on bone fracture consolidation in osteoporotic rats. Forty female Wistar rats were submitted to ovariectomy (OVX) to induce osteopenia. Eight weeks after surgery, the animals were randomly divided into four groups of 10 animals each: a bone defect control group (CG); a bone defect filled with Biosilicate group (BG); a bone defect filled with Biosilicate and irradiated with LLLT at 60 J/cm(2) group (BG60); and a bone defect filled with Biosilicate and irradiated with LLLT at 120 J/cm(2) group (BG120). Bone defects were surgically performed on both tibias. The size of particle used for Biosilicate was 180-212 µm. Histopathological analysis showed that bone defects were predominantly filled with the biomaterial in specimens treated with Biosilicate. LLLT with either 60 or 120 J/cm(2) was able to increase collagen, Cbfa-1, VGEF and COX-2 expression in the circumjacent cells of the biomaterial. A morphometric analysis revealed that the Biosilicate + laser groups showed a higher amount of newly formed bone. Our results indicate that laser therapy improves bone repair process in contact with Biosilicate as a result of increasing bone formation, as well as COX-2 and Cbfa-1 immunoexpression, angiogenesis and collagen deposition in osteoporotic rats.

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Manyauthorssuggest that,in osteoporotic people, there is
a decreased proliferative activity of osteoblast progenitor
cells and gene expression, an impairment in osteoblast
variety of biodegradable polymers, bioactive glasses and
glass–ceramics have been used as grafts in the treatment
of large bone defects, mainly due to their facility to adapt
to defect shape, their potential to stimulate osteogenesis
TERM309
JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE
J Tissue Eng Regen Med 2010; 4: 000–000.
Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/term.309
RESEARCH ARTICLE
Biosilicateand low-level laser therapy improve
bone repair in osteoporotic rats
Paulo S´ ergio Bossini1, Ana Claudia Muniz Renn´ o2*, Daniel Araki Ribeiro2, Renan Fangel1,
Oscar Peitl3, Edgar Dutra Zanotto3and Nivaldo Antonio Parizotto1
1Department of Physiotherapy, Federal University of S˜ ao Carlos (UFSCar), Rodovia Washington Lu´ ıs (SP-310), km 235, S˜ ao Carlos,
SP, Brazil
2Department of Biosciences, Federal University of S˜ ao Paulo (UNIFESP), Avenida Ana Costa 95, Santos, SP, Brazil
3Vitreous Materials Laboratory (LaMaV), Department of Materials Engineering, Federal University of S˜ ao Carlos (UFSCar), Rodovia
Washington Lu´ ıs (SP-310), km 235, S˜ ao Carlos, SP, Brazil
Abstract
The aim of this study was to investigate the effects of a novel bioactive material (Biosilicate) and
low-level laser therapy (LLLT) on bone fracture consolidation in osteoporotic rats. Forty female
Wistar rats were submitted to ovariectomy (OVX) to induce osteopenia. Eight weeks after surgery,
the animals were randomly divided into four groups of 10 animals each: a bone defect control
group (CG); a bone defect filled with Biosilicate group (BG); a bone defect filled with Biosilicate
and irradiated with LLLT at 60 J/cm2group (BG60); and a bone defect filled with Biosilicate and
irradiated with LLLT at 120 J/cm2group (BG120). Bone defects were surgically performed on
both tibias. The size of particle used for Biosilicate was 180–212 µm. Histopathological analysis
showed that bone defects were predominantly filled with the biomaterial in specimens treated with
Biosilicate. LLLT with either 60 or 120 J/cm2was able to increase collagen, Cbfa-1, VGEF and COX-2
expression in the circumjacent cells of the biomaterial. A morphometric analysis revealed that the
Biosilicate + laser groups showed a higher amount of newly formed bone. Our results indicate that
laser therapy improves bone repair process in contact with Biosilicate as a result of increasing bone
formation, as well as COX-2 and Cbfa-1 immunoexpression, angiogenesis and collagen deposition
in osteoporotic rats. Copyright  2010 John Wiley & Sons, Ltd.
Received 17 January 2010; Accepted 14 April 2010
KeywordsBiosilicate; low level laser therapy; bone repair; osteoporotic rats
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1. Introduction
Osteoporosis is defined as a disease characterized by
low bone mass and microarchitectural deterioration of
bone tissue, leading to enhanced bone fragility and a
consequent increase in fracture risk (Anonymous, 1993).
The decreased bone mass and bone mineral density due to
osteoporosis probably lead to a delay in fracture healing
rates and bone repair quality (Hollinger et al., 2008).
*Correspondence to: Ana Claudia Muniz Renn´ o, Avenida Ana
Costa 95, Vila Mathias, Santos, S˜ ao Paulo, 11050-240 Brazil.
E-mail: a.renno@unifesp.br
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function, a diminished osteoblast response to signalling
and animbalance between the couplingof bone formation
and resorption (Kubo et al., 1999; Meyer et al., 2001).
Consequently, bone healing in osteoporotic individuals
may be delayed and new bone quality may be poor
(Hollinger et al., 2008).
In this context, there is a critical need to develop
technologies capable of treating osteoporotic fractures
(Gauthier et al., 2005). One promising treatment is the
use of bioglasses and polymers, which seem to induce
osteogenesis and stimulate fracture healing (Clupper
et al., 2003; Thomas et al., 2005). To date, a wide
Copyright  2010 John Wiley & Sons, Ltd.

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association of bisphosphonate and laser can also increase
the trabecular bone volume in vertebraa in the osteopenic
control group. It would be useful to know whether, and to
what extent, the association between Biosilicate and laser
therapy is able to improve bone repair in osteroporotic
rats, particularly because there are no previous reports. In
tory of the Federal University of S˜ ao Carlos (S˜ ao Carlos,
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and their capability to influence bone bonding (Lu et al.,
2008). However, the success of the biomaterial implant
and the improvement of the fracture consolidation are
dependent on many characteristics of the material, such
as composition, solubility and particle size (Vogell et al.,
2001).
One of the most common and studied bioactive glasses
is Bioglass45S5, which has been reported as the bioac-
tive material with the highest bioactivity index (Clupper
et al., 2003). Recently, our research group has developed
a novel fully-crystallized bioactive glass–ceramic of the
quaternary P2O5–Na2O–CaO–SiO2system (Biosilicate;
Patent Application WO 2004/074199; Funda¸ c˜ ao Uni-
versidade Federal de S˜ ao Carlos, 2004) with improved
properties. Biosilicate has showed a stimulatory effect
on bone cell metabolism (Moura et al., 2007). By com-
paring the growth of osteogenic cells on Biosilicate and
Bioglass45S5 disks for a period of up to 17 days, Moura
et al.(2007)foundthatalthoughnosignificantdifferences
were detected in terms of protein content and alkaline
phosphatase activity at days 11 and 17, Biosilicate sup-
ported significantly larger areas of calcified matrix at day
17. The results indicate that full crystallization of some
bioactive glasses in a range of compositions of the sys-
tem P2O5–Na2O–CaO–SiO2may promote enhancement
of in vitro bone-like tissue formation in an osteogenic cell
culture system.
Similarly, a significant body of evidence has now
accumulated demonstrating that low-level laser therapy
(LLLT) also has a positive effect on bone tissue
metabolismandfractureconsolidation(Lugeret al.,1998;
Ozawa et al., 1998; Renno et al., 2007). When laser is
applied to tissue, the light is absorbed by chromophore
photoreceptors located in the cells. Once absorbed,
the light can modulate cell biochemical reactions and
stimulate mitochondrial respiration, with the production
of molecular oxygen and ATP synthesis (Renno et al.,
2009). These effects are known to increase the synthesis
of DNA, RNA and cell-cycle regulatory proteins, therefore
promoting cell proliferation (Renno et al., 2007). In
vitro studies using osteoblastic cells showed that LLLT is
capable of increasing mitochondrial activity (Renno et al.,
2009), osteoblast DNA and RNA synthesis, bone nodule
formation (Trelles and Mayayo 1987), osteocalcin and
osteopontin gene expression and ALP activity (Oliveira
et al., 2007). Also, the LLLT has demonstrated to be able
of accelerating the process of fracture repair in rabbits
and rats, increasing the callus volume and bone mineral
density (Stein et al., 2005). However, little attention has
beengiventotheeffectofLLLTonbonewithosteopeniaor
osteoporosis. Our group showed that LLLT had a positive
effect on osteogenesis in rats (Matsumoto et al., 2009).
Moreover, Renno et al. (2006) demonstrated that the
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this context, the aim of this study was to investigate the
effects of Biosilicate and laser therapy on bone fracture
consolidation in osteoporotic rats.
2. Material and methods
This study was conducted in accordance with the Guide
for Care and Use of Laboratory Animals and approved by
the Animal Ethics Committee of the Federal University of
S˜ ao Carlos (08/2098). The animals were maintained at
19–23◦C on a 12:12 h light–dark cycle in the Animal
Experimentation Laboratory of the Federal University of
S˜ ao Carlos. They were housed in plastic cages and had
free access to water and standard food.
A total of forty female Wistar rats (age 12 weeks,
weight ±250 g) were submitted to ovariectomy (OVX)
to induce osteopenia. This model is widely used as
an experimental model of animal osteopenia and it
is known to significantly decrease bone mass 8 weeks
post-surgery (Kalu, 1991). Surgery was performed via
bilateral translumbar incisions, under ketamine/xilazine
anaesthesia (80/10 mg/kg). The uterine tubes were
ligated (catgut, 4.0) and after removal of the ovaries
the incisions were closed (catgut, 3.0). Eight weeks after
the OVX, the animals were randomly divided into four
groups of 10 animals each: a control bone defect group
(CG) – the bone defects without any fillers; a Biosilicate
group (BG) – the bone defects filled with Biosilicate;
a Biosilicate group irradiated with LLLT at 60 J/cm2
(BG60); and a Biosilicate group irradiated with LLLT
at 120 J/cm2(BG120).
Bone defects were surgically performed on both tibias.
The defect depth was guided until the rupture of
cortical bone. The animals were anaesthetized with
ketamine/xilazine (80/10 mg/g) and the mid-regions of
the tibias were shaved and disinfected with povidone
iodine. A dermo-periosteal incision was performed to
expose the tibia. A 2 mm diameter cavity defect was
made, using a spherical bur under copious irrigation
with saline solution. In the Biosilicate-treated animals,
the cavities were carefully filled with the corresponding
biomaterial. The cutaneous flap was replaced and sutured
with resorbable polyglactin, and the skin was disinfected
with povidone iodine. The health status of the rats was
monitored daily.
2.1. Biomaterial
High-purity silica and reagent-grade calcium carbonate,
sodium carbonate and sodium phosphate were used to
obtain the Biosilicateparent glass. The chemicals were
weighed and mixed for 30 min in a polyethylene bottle.
Premixed batches were melted in a platinum crucible at
a temperature range of 1250–1380◦C for 3 h in an elec-
tric furnace (Rapid Temp 1710 BL, CM Furnaces Inc.,
Bloomfield, NJ, USA) at the Vitreous Materials Labora-
Copyright  2010 John Wiley & Sons, Ltd.
J Tissue Eng Regen Med 2010; 4: 000–000.
DOI: 10.1002/term

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0.01 M citric acid buffer, pH 6, for three cycles of
5 min each at 850 W for antigen retrieval. The material
was pre-incubated with 0.3% hydrogen peroxide in
phosphate-buffered saline (PBS) solution for 5 min for
inactivation of endogenous peroxidase, and then blocked
with 5% normal goat serum in PBS solution for 10 min.
by a three-point bending test with a 1 kN •load (• • • • •,
Effect of Biosilicateand laser therapy in osteoporotic rats
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SP, Brazil). Samples were cast into a 10 × 30 mm cylin-
drical graphite mould and annealed at 460◦C for 5 h.
To obtain the fully crystallized Biosilicate glass–ceramic,
parent glass cylinders underwent cycles of thermal treat-
ment to promote their crystallization. The first thermal
cycle was performed at a relatively low temperature,
just above the glass transition temperature, to promote
volumetric nucleation of crystals. Afterwards, the nucle-
ated samples were submitted to further treatment at
about 100◦C above the nucleation temperatures. The
detailed compositions and thermal treatment schedules
to obtain the Biosilicate glass–ceramic are described in
the patent WO 2004/074199 (Funda¸ c˜ ao Universidade
Federal de S˜ ao Carlos, 2004). The biomaterial is pre-
sented as a particulate. The size of particles used was
180–212 µm.
2.2. Low level laser therapy
A low-energy GaAlAs (Teralaser, DMCS˜ ao Carlos, SP,
Brazil), 830 nm CW, 0.6 mm beam diameter, 100 W/cm2,
at60and120 J/cm2,withirradiationtimesof17and34 s,
were used in this study. Laser irradiation was initiated
immediately after the osteotomy procedure and it was
performed on days 2, 4, 6, 8, 10 and 12 post-surgery.
On day 14 post-osteotomy, the rats were sacrificed with
an intraperitoneal injection of general anaesthetic. The
tibias were defleshed and the soft tissues were removed
for analysis.
2.3. Histopathological analysis
For the histopathological analysis, the right tibiae were
removed and fixed in 10% buffer formalin (Merck,
Darmstadt, Germany) for 48 h, decalcified in 4% EDTA
(Merck) and embedded in paraffin blocks. 5 µm serial
sections were cut and stained with haematoxylin and
eosin (H&E Merck).
Histopathological evaluation was performed under a
light microscope (Olympus, Optical Co. Ltd, Tokyo,
Japan). Any changes in the bone defects, such as the
presence of woven bone, medullar tissue, inflammatory
process, granulation tissue or even tissues undergoing
hyperplastic, metaplastic and/or dysplastic transforma-
tion, were investigated for each animal.
2.4. Immunohistochemistry
Paraffin was removed with xylene from serial sections
of •4 µm and the sections were rehydrated in a graded
series of ethanols, then pretreated in a microwave with
AQ3
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The specimens were then incubated with anti-Runx2
polyclonal primary antibody (Santa Cruz Biotechnology,
USA) at a concentration of 1:200, anti-COX-2 polyclonal
primary antibody (Santa Cruz Biotechnology) or anti-
VEGF-2 monoclonal primary antibody (Santa Cruz
Biotechnology) at a concentration of 1:200. Incubation
was carried out overnight at 4◦C within the refrigerator.
This was followed by two washes in PBS for 10 min.
The sections were then incubated with biotin-conjugated
secondary antibody anti-rabbit IgG (Vector Laboratories,
Burlingame, CA, USA) at a concentration of 1:200
in PBS for 1 h. The sections were washed twice
with PBS, followed by the application of preformed
avidin–biotin complex conjugated to peroxidase (Vector
Laboratories) for 45 min. The bound complexes were
visualized by the application of a 0.05% solution
of 3,3?-diaminobenzidine solution and counterstained
with Harris haematoxylin. For control studies of the
antibodies, the serial sections were treated with rabbit
IgG (Vector Laboratories) at a concentration of 1:200
in place of the primary antibody. Additionally, internal
positive controls were performed with each staining
batch.
2.5. Morphometric assessment
The morphometry of the area of newly formed bone
in the regions of bone repair previously indentified
in the histopathological observation for each animal
was measured in a blind fashion by an experienced
pathologist, using image analysis system Motican 5.0.
Sections stained with Masson trichrome were observed;
three areas of the cortical region of the defect were
selected and named C1, C2 and C3, corresponding to the
superior, inferior and central cortical areas of the defect.
The neoformed bone tissue presented in these regions
was measured and the area registered at a magnification
of ×10. After the registration, the areas were added,
resulting in the total bone area of the defect. This analysis
was established in a previous study conducted by our
team (Matsumoto et al., 2009).
2.6. Picrosirius polarization method
Histological sections stained by the Picrosirius polar-
ization method were viewed under polarized light
(Garavello-Freitas et al., 2009) to assess the structural
changes inthe neoformingtrabecular matrix. This method
allows an indirect evaluation of the stage of bone matrix
organization, based on the birefringence of the collagen
fibre bundles after staining with Picrosirius.
2.7. Biomechanical analysis
Biomechanical properties ofthe left tibia were determined
AQ4
Copyright  2010 John Wiley & Sons, Ltd.
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USA, 4444 model, 1 KN load cell). Tibiae were placed
on a 3.8 cm metal device, which provided a 1.8 cm
distance between the two supports. The load cell
was perpendicularly positioned in the anterior–posterior
direction at the exact site of the bone defect. A 5 N preload
was applied in order to avoid specimen sliding. Finally,
the bending force was applied at a constant deformation
rate of 0.5 cm/min until fracture occurred. Thus, the
maximum load (N) was obtained.
2.8. Statistical analysis
The normality of all variables’ distribution was verified
using Shapiro–Wilk’s W test. For the variable that exhib-
ited Normal distribution, comparisons among the groups
were made using one-way analysis of variance (ANOVA),
complemented by Tukey HSD post-test analysis. The
Kruskal–Wallis test was performed for morphometric
assessment. STATISTICA version 7.0 (data analysis soft-
ware system, StatSoft Inc.) was used to carry out the
statistical analyses. p < 0.05 was considered statistically
significant.
3. Results
3.1. General findings
Neither postoperative complications nor behavioural
changes were observed in the animals. None of the
animals died during the experiment and no infection
at the surgical site was observed.
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3.2. Histopathological analysis
Regarding the control group, all the defects were
composed by woven bone inside the bone defect after
14 days (Figure 1A). Additionally, the defects were filled
by medullar tissue and some bone fragments, possibly due
to the surgical procedures (Figure 1A). No inflammatory
process was noticed in any of the specimens of this group,
because no acute inflammatory cells were present. In
specimens treated with Biosilicate, the bone defect was
predominantly filled with the biomaterial. No woven
bone was noticed in the majority of specimens for this
group (Figure 1B). In addition, granulation tissue was
present in circumjacent areas to the wall of bone defect.
Regarding the 60 J/cm2laser and Biosilicate group,
we observed the presence of the biomaterial filling all
bone defects, associated with the presence of woven
bone and granulation tissue (Figure 1C). In the group
exposed to laser 120 J/cm2and Biosilicate, a more
pronounced effect was evidenced, in which woven bone
was in apposition to the surface of the biomaterial in
the majority of cases; granulation tissue was present
as well (Figure 1D). Overall, our results indicate, by
means of subjective morphological analysis, that laser
therapy improves the bone repair process in contact with
Biosilicate in osteoporotic rats, particularly exposure to
the 120 J/cm2laser at 14 days after surgery.
3.3. Immunohistochemistry
COX-2 expression was detected predominantly in the
cytoplasm.After14 daysofthesurgery,COX-2immunore-
activity could be seen in medullar tissue for the control
Figure 1. Bone• defects from control group (A) displaying high celullarized woven bone inside the defect (∗) and the medullar
region (M). (B) Biosilicate group, showing biomaterial (#) and granulation tissue (arrow). (C) •G2–Biosilicate + laser 60 J/cm2
containing formed bone (∗), granulation tissue (arrow) and the presence of biomaterial (#). (D) Biosilicate + laser 120 J/cm2
showing woven bone (∗), biomaterial (#) and granulation tissue (arrow). H&E stain; bar = 48 µm
AQ1
AQ2
Copyright  2010 John Wiley & Sons, Ltd.
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Effect of Biosilicateand laser therapy in osteoporotic rats
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Figure 2. Immunohistochemistry for COX-2. (A) immunoexpression predominantly in the medullar tissue in the control group
(arrow). (B) In group treated with Biosilicate, immunopositive cells were detected in contact with biomaterial (arrow); (C and D)
in groups treated with laser, a strong immunoexpression was noticed to circumjacent cells or granulation tissue (arrow) at 14 days
(B) after surgery. Immunohistochemistry stain; bar = 30 µm
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group (Figure 2A). In the group exposed to Biosilicate,
COX-2 immunoexpressivity was seen in cells circumja-
cent to the biomaterial with a weak pattern (Figure 2B).
Interestingly, LLLT, either 60 or 120 J/cm2, was able
to increase COX-2 expression in the circumjacent tis-
sue of the biomaterial as well as granulation tissue
(Figure 2C, D).
Regarding Cbfa-1 immunohistochemistry, this could
be detected in cells from medullar tissue in the control
group (Figure 3A). Biosilicate-treated groups displayed
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expressivity for this immunomarker in areas circumjacent
to the biomaterial (Figure 3B). In the groups treated
with laser, either 60 or 120 J/cm2, a similar pattern
occurred, i.e. Cbfa-1positive cells were noticed near tothe
Biosilicate and in the walls of the bone defect (Figure 3C,
D, respectively).
VEGF expression was predominantly detected in cells
from capillary walls, which are composed of endothelial
cells; 14 days after surgery, VEGF immunoreactivity could
be seen in the capillary walls for the control group
Figure 3. Immunohistochemistry for Cbfa-1. (A) Immunoexpression predominantly in the medullar tissue in the control group
(arrow). (B) In group treated with Biosilicate, immunopositive cells were detected in contact with biomaterial (arrow). (C, D)
The same picture occurred, i.e. positive cells were detected circumjacent to biomaterial (arrow) (C). Immunohistochemistry stain;
bar = 30 µm
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6
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Figure 4. Immunohistochemistry for VEGF after 14 days of surgery. (A) Control group; (B) Biosilicate group; (C) Biosilicate and
laser 120 J/cm2. Arrow indicates VEGF-positive cells. Immunohistochemistry stain; bar = 56 µm
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(Figure 4A). In the group exposed to Biosilicate, VEGF
immunoexpressivity was seen in some capillaries present
in the areas circumjacent to the biomaterial (Figure 4B).
In groups treated with laser + Biosilicate, either 60 or
120 J/cm2, a similar pattern occurred, i.e. VEGF-positive
cells were noticed in the capillary walls of the bone defect
(Figures 4C).
3.4. Morphometry
The area of neoformed bone tissue presented by the
Biosilicate group was significantly smaller than in the
other groups (Figure 5).No difference was foundbetween
the bone defect control group and the Biosilicate group
treated with laser at 60 J/cm2. Interestingly, the animals
treatedwithBiosilicateandexposuretolaserat120 J/cm2
showed a statistically significant increase in the area of
neoformed bone, corroborating the qualitative analysis.
Figure 5. Area of neoformed bone in the defect area (µm2).
Results are expressed as mean ± SD. GC, fracture control;
BG, Biosilicate; BG60, Biosilicate and laser 60 J/cm2; BG120,
Biosilicate and laser 120 J/cm2.∗p < 0,05 vs BC;#p < 0.05 vs
BG120
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3.5. Collagen assessment by Picrosirius
Figure 6 demonstrates the qualitative analysis for the
collagen evaluation. It can be observed that the animals
treated with Biosilicate only showed a smaller amount
of collagen fibre deposition when compared to control
group. Moreover, it seems that the animals exposed to
laser therapy demonstrated a larger amount of collagen
fibres when compared to the control. These findings
corroborate those of the quantitative analysis of the
collagen deposition. Figure 6 shows that the intensity
of pixels demonstrated by the Biosilicate group was
lower when compared to other groups. The groups
treated with Biosilicate and irradiated with laser therapy
showed a higher intensity of pixels, mainly at the
dosage of 120 J/cm2, indicating that these animals
showed a higher amount of collagen fibre deposition
(Figure 6).
Figure 6. Mean and SD of the collagen assessment. CG, control
group; BG, Biosilicate group; BG60, Biosilicate group plus laser
at 60 J/cm2; BG120, Biosilicate group plus laser at 120 J/cm2.
∗vs. control;#vs. BG;@vs. BG60
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described by Hench and Polak (2002), lead to rapid
growth of HCA bone mineral on the glass surface and to
a rapid proliferation of new bone within a bone defect
site, a process termed osteoproduction. It may be that
the short time post-surgery used to perform the analysis
was not enough to produce a proper interaction of the
formation in the cell nucleus and consequently to increase
Effect of Biosilicateand laser therapy in osteoporotic rats
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Figure 7. Biomechanical properties. CG, control group; BG,
Biosilicate group; BG60, Biosilicate group plus laser at 60 J/cm2;
BG120, Biosilicate group plus laser at 120 J/cm2.∗vs. BG120,
p < 0.05
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3.6. Biomechanical analysis
Figure 7 shows the results found in the biomechanical
analysis. The animals treated with Biosilicate and exposed
to laser at 120 J/cm2presented a higher maximal load
compared to the control group and the group treated with
Biosilicate only. No other difference was found.
4. Discussion
Biological supplementation of traditional strategies needs
to be exploited for osteoporotic and geriatric patients with
poor bone density and compromised bone repair potential
(Hollinger et al., 2008). Recently, many treatments have
been proposed, such as ceramics, osteogenic bioglasses
and laser therapy for recalcitrant tibial non-unions (Peter
et al., 2006). In this context, the aim of this study
was to evaluate whether concomitant administration of
Biosilicate and LLLT are able to improve bone repair
in osteoporotic rats. To the best of our knowledge, the
approach has not been addressed previously.
Our results showed that, although the animals treated
with Biosilicate presented smaller amounts of neoformed
tissue, biomechanical properties and a lower amount of
collagen, the qualitative histological analysis showed that,
in most of the specimens of this group, the presence of
woven bone in apposition to the surface of the biomaterial
could be observed. This finding supports the principle of
bioactive glasses or glass–ceramics, which is considered
to be a rapid deposition of a layer of hydroxycarbonate
apatite (HCA) on the surface of the silica gel, as a result of
exchange between calcium and silica ions when bioactive
glass comes into contact with living tissues (Hench and
Polak, 2002). Entirely inorganic reactions, previously
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biomaterial isolated with bone metabolism, which avoids
better responses. Probably if we extended the time of
the experimental design, we could reach more positive
responses. Despite these results, the osteogenic potential
of Biosilicate needs to be highlighted.
This in vivo finding corroborates previous in vitro
results (Moura et al., 2007) which showed that, although
Biosilicate and Bioglass 45S5 discs supported osteoge-
nesis, significantly larger areas of calcified matrix were
detected for the fully-crystallized glass–ceramic (Biosili-
cate) after 17 days in an osteogenic culture system. Also,
in an in vivo study, we showed that the Biosilicate was
efficient to induce bone formation and to increase the
biomechanical properties of the fracture callus, 20 days
after the surgery, to induce tibial bone defects (Granito
et al., 2009). It is worth noting that the special nucle-
ation and growth thermal treatments developed by our
research group to improve the mechanical properties of
glasses were also useful for obtaining a fully crystallized
glass–ceramic (Biosilicate) with the high bioactivity level
and osteoproductive properties exhibited by glasses.
Interestingly, the group exposed to laser therapy
(mainly at 120 J/cm2) and Biosilicate showed a higher
amount of newly formed bone when compared to
the control, in which woven bone was in apposition
to the surface of the biomaterial in the majority
of cases. LLLT is a promising non-invasive method
for stimulating osteogenesis and reducing the time of
fracture consolidation through bioenergetic, bioelectrical,
biochemical and biostimulatory effects on cells (Nicolau
et al., 2003; da Silva et al., 2006; Ribeiro et al., 2008).
Kazem Shakouri et al. (2010) showed that the use of
laser therapy could enhance callus development in the
early stage of the healing process in rabbits, with doubtful
improvement in biomechanical properties of the healing
bone; therefore, laser therapy may be recommended as an
additional treatment in non-union fractures in humans.
Also, LLLT has been demonstrated to stimulate fracture
bone healing in osteoporotic rats (Renno et al., 2006).
Accumulating evidence suggests that inflammation
plays an important role in connective tissue repair (Simon
et al., 2002). Specifically, cyclo-oxygenase (COX) is the
rate-limiting enzyme in the conversion of arachidonic
acid to prostaglandins, of which two isoforms, COX-1
and -2, have been identified during the inflammatory
process. COX-1 is constitutively expressed in many tissues
and mediates the synthesis of prostaglandins required
for normal physiological function. COX-2 is normally
undetectable in most tissues, but is rapidly induced by
proinflammatory or mitogenic stimuli (Kargman et al.,
1996). As demonstrated by Zhang et al. (2002), COX-2
enzymeactsonosteoblastogenesis,regulatingosteoblastic
differentiation genes such as Cbfa1 and osterix. In this
study we were able to evaluate COX-2 expression in
this setting. Our results revealed that laser therapy
promotes an upregulation of COX-2 expression, even
when combined with Biosilicate, in osteoporotic rats.
Since LLLT is able to increase DNA and RNA synthesis
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results are in line with our results, since positive angio-
genesis was detected in early phases of bone repair after
exposure to Biosilicate. Moreover, LLLT, either 60 or
120 J/cm2, induced angiogenesis in a similar manner to
thegroupexposed toBiosilicate only.Theapproachisnew
in the literature, and the results are difficult to discuss.
power laser irradiation improves histomorphometrical parameters
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cell proliferation and differentiation, we believe that the
immunoexpression of COX-2 found in bone tissue could
also be helpful for bone repair. This expectancy was
confirmed in previous studies investigating the role of
COX-2 during bone repair in rats (Ribeiro et al., 2008).
In 1997, Sato et al. (1997) suggested that COX-2 could
be involved in the early stage of osteogenesis, probably
associated with the maturation of osteoblasts. More
recently, it has been postulated that bone cells are able to
produce COX-2 after mechanical trauma (Li et al., 2002),
therefore being important to bone formation (Forwood,
1996). Our results agree with these previous studies.
To further elucidate the putative mechanisms of action
involving Biosilicate and LLLT on bone repair in osteo-
porotic rats, we designed additional immunohistochem-
ical experiments to observe the expression of Cbfa-
1/RUNX2 (core binding factor protein-1), since some
authors have assumed that that COX-2 enzyme acts on
osteoblastogenesis, regulating osteoblastic differentiation
genes such as Cbfa-1 and osterix (Zhang et al., 2002).
Interestingly, our results demonstrated a moderate Cbfa-
1/RUNX2 expression in groups exposed to the biomaterial
in combination or not with laser therapy. Taken as a
whole, it seems that Biosilicate permits osteoblast differ-
entiation during the process of bone repair in osteoporotic
rats.
Accumulating evidence also suggests that neovascular-
ization plays a crucial role in treating many diseases (e.g.
coronary artery disease) and in virtually all approaches
to tissue engineering (Boontheekul and Mooney, 2003).
A lack of vascularization leads to insufficient nutrient
delivery and waste removal, cell death and limited tissue
development and tissue loss. Angiogenesis, new blood
vessel formation by sprouting from the sides and ends of
pre-existing microvascular vessels, has been widely stud-
ied to determine the rules guiding blood vessel formation
inadulttissues (BoontheekulandMooney,2003).Numer-
ousgrowthfactorshavesofarbeenidentifiedasregulators
of this process. For example, vascular endothelial growth
factor (VEGF) is a key mediator of angiogenesis, as it is a
potent mitogen for endothelial cells (ECs) and induces EC
migration by upregulation of several endothelial integrin
receptors. The VEGFs and their corresponding receptors
are key regulators in a cascade of molecular and cellular
events that ultimately lead to the development of the
vascular system by angiogenesis (Ferrara et al., 2003).
In the present study we were able to evaluate the VEGF
expression in this setting. Our results revealed that Biosil-
icate was able to induce angiogenesis, as depicted by
the positive immunoexpression found in this group. By
comparison, Sojo et al. (2005) assumed that angiogenesis
occurs predominantly before the onset of osteogenesis
in bone lengthening in an osteodistraction model. These
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Independent of its mechanism the action, we believe that
the immunoexpression of VEGF found in these groups
could also be helpful to bone repair in osteoporotic rats.
The Picrosirius polarization method showed that the
association of Biosilicate and LLLT, mainly at a higher
dosage, produced a higher concentration of collagen
fibres and a better organization of the fibres, which
can be attributed to the laser stimulation. Garavello-
Freitas et al. (2009) also observed an increase of collagen
deposition in bone defects irradiated with LLLT. The
birefringent shades of yellow to red bands observed by
these authorsinthe irradiated animals isindicative oftype
I collagen-containing collagen fibres areas of trabecular
bone, which are typical of mature bone, and acquired a
mature disposition in newly-formed bone matrix.
Moreover, the most used preclinical animal model
used for osteoporosis research is the ovariectomized rat.
This model mimics bone loss and compromised fracture
repair prevalent in postmenopausal women who are
oestrogen-deficient and prone to osteoporotic fractures;
consequently,ovariectomized ratswere usedtodetermine
the fracture-healing efficacy of the treatments LLLT and
Biosilicate (Hollinger et al., 2003).
5. Conclusion
In summary, our findings indicate that LLLT laser therapy
improves bone healing in tibial defects ofosteoporotic rats
(asaresultofanupregulationofCOX-2expressioninbone
cells), even when associated with Biosilicate. Also, it is
likely that Biosilicate induces osteoblastic differentiation
following bone repair. These findings should be carefully
addressed to elderly people suffering from osteoporosis
and presenting trauma of the tibia, since they represent a
new perspective for osteoporotic patients, although they
do not solve the problem of general osteoporosis. Further
studies would be welcome to elucidate this issue.
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QUERIES TO BE ANSWERED BY AUTHOR
IMPORTANT NOTE: Please mark your corrections and answers to these queries directly onto the proof at the
relevant place. Do NOT mark your corrections on this query sheet.
Queries from the Copyeditor:
AQ1 Figure(s) 1 to 7 are of low clarity. Kindly provide better replacement figure(s) or confirm if we may use the
existing one(s).
AQ2 What does ‘‘G2’’ mean here? Not mentioned anywhere else in paper. Please clarify/amend
AQ3 Section thickness stated here as 4 µm, but in previous paragraph they were stated to be 5 µm. Please amend
AQ4 Is there a supplier’s name missing here? It’s not informative to state ‘‘USA’’ only.
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