471 23-30 sfs Sakallioglul May 5 1132.fm

23 Research report
Fluoride 47(1)23–30
January-March 2014
Effects of excessive fluoride intake on mandibular bone
Sakallioğlu, Muğlalı, Baş, Gulbahar, Lütfioğlu, Aksoy
23
EFFECTS OF EXCESSIVE FLUORIDE INTAKE ON BONE TURNOVER
IN MANDIBLE: AN IMMUNOHISTOCHEMICAL STUDY IN RABBITS
Elif Eser Sakallioğlu,a Mehtap Muğlalı,b Burcu Baş,b M Yavuz Gulbahar,c
Müge Lütfioğlu,d Abdurrahman Aksoye
Samsun, Turkey
SUMMARY: The aim of this report is to analyze the effects of high fluoride (F) intake
on matrix metalloproteinase-2 (MMP-2), tissue inhibitors of metalloproteinase-1
(TIMP-1), and transforming growth factor-β (TGF-β) levels. Twenty-seven rabbits were
divided into control and F groups. The control group comprised 11 rabbits that
received distilled drinking water; the experimental group consisted of 16 rabbits that
received distilled water with excessive F for 70 days. At the end of 70 days, serum F
concentrations were evaluated, and the rabbits were then sacrificed. Bucco-lingual
histological sections were obtained bilaterally from the mandibular bone. MMP-2,
TIMP-1, and TGF-β levels in the bone matrix were determined by
immunohistochemical staining. The difference between the groups was evaluated
with the Mann–Whitney U test, and the intragroup difference in staining intensity was
evaluated with Friedman and Wilcoxon tests. Increased mineralization and high bone
turnover was observed in the fluorosis group. TGF-β immunostaining of the
mandibular bone in the experimental group was significantly more intense compared
with that in the control group (p=0.03). MMP-2 and TIMP-1 staining were not
statistically different between the experimental and the control groups (MMP-2, p =
0.110; TIMP-1, p=0.479). Intra-group comparison revealed significant differences at
MMP-2/TIMP-1 and TIMP-1/TGF-β ratios only in F group (p=0.02 for MMP-2 and p=0.01
for TGF-β). Excessive F uptake may affect bone remodeling process by increasing
TGF-β levels and TIMP/MMP and TIMP/TGF-β ratios. Further studies including other
bone regulating molecules are needed in order to explain bone metabolism in the
presence of high doses of F.
Key Words: Bone turnover with high F; MMP-2; Rabbit; TIMP-1; TGF-β.
INTRODUCTION
1-3
High fluoride (F) levels cause toxicity. At very high doses, skeletal and dental
fluorosis occurs, characterized by debilitating changes in the skeleton and marked
mottling and discoloration of the teeth.4 High F intake increases the accretion,
resorption, and calcium turnover rates of bone tissue, thereby affecting the
homeostasis of bone mineral metabolism.5 F has also been shown to increase bone
metabolic turnover and stimulate bone cell proliferation.6
A number of factors affect bone turnover, including hormones, cytokines, and
mechanical stimuli. All of these factors affect the amount and quality of the tissue
produced.7 Studies have shown that osteoblast-derived transforming growth
factor-β (TGF-β), matrix metalloproteinase (MMP) and tissue inhibitors of
aCorresponding
author: Associate Professor, Department of Periodontology, Faculty of
Dentistry, Ondokuz Mayıs University, 55139-Samsun, Turkey. E-mail: [email protected],
Tel: +903623121919-3780; bAssociate Professor, Department of Oral and Maxillofacial Surgery,
Faculty of Dentistry, Ondokuz Mayıs University, Samsun, Turkey; cProfessor, Ondokuz Mayıs
University, Department of Pathology, Faculty of Veterinary Medicine, Samsun, Turkey;
dAssistant Professor, Department of Periodontology, Faculty of Dentistry, Ondokuz Mayıs
University, Samsun, Turkey; eProfessor, Department of Pharmacology and Toxicology, Faculty
of Veterinary Medicine, Ondokuz Mayıs University, Samsun, Turkey.
24 Research report
Fluoride 47(1)23–30
January-March 2014
Effects of excessive fluoride intake on mandibular bone
Sakallioğlu, Muğlalı, Baş, Gulbahar, Lütfioğlu, Aksoy
24
metalloproteinase (TIMP) play a role in bone metabolism by degrading the bone
matrix.8 TIMP-1 has been used as a marker of inhibition of bone resorption.9
Investigators have found that low concentrations of TIMP-1 stimulate osteoclastic
bone resorption, whereas higher concentrations inhibit the resorption process.10 It
is well known that an imbalance between MMPs and TIMPs leads to the
destruction of connective tissue, which occurs in a number of pathological
events.11 TGF-β has been implicated in regulating a variety of cellular events
involved in bone growth and turnover.12 TGF-β is produced by osteoblasts and
chondrocytes and mainly stored in the mineralized extracellular bone matrix.13
There is no evidence yet about the possible effects of excessive F intake on
osteogenic markers. The aim of this study was to investigate the effects of
excessive F intake on remodelling of mandibular bone via analyzing bone matrix
concentrations of MMP-2, TIMP-1 and TGF-β.
MATERIALS AND METHODS
The animal experiment protocol was approved by Ondokuz Mayıs University
Ethics Committee. Twenty-seven skeletally mature adult male New Zealand white
rabbits (body weight: 2.5–3kg) were divided into a control (n=11) and a F (n=16)
group. The animals were maintained in a dedicated animal holding facility in the
laboratory animal unit of the University. They were housed individually in
stainless steel cages and fed with standard laboratory rabbit chow and water ad
libitum for 70 days. The control group received distilled drinking water. The
experimental group received distilled drinking water with excessive F (40 mg/L).
Following feeding, blood samples were collected from the marginal ear vein and
serum F levels were measured. Animals were sacrificed using a high dose of
pentobarbital at the end of the 70th day. Following sacrifice, bucco-lingual
histological sections were obtained bilaterally from the mandibular bone at the
molar teeth region.
Serum analysis of fluoride: A F-specific ion electrode (ORION 9609BN,
Thermo Electron Corp, MA, US) connected to a digital ion analyzer (ORION EA
940, Orion Research, Inc., US) was used to measure the F ion concentration. The
electrode was previously calibrated with four standard F solutions of 0.19 mg/L,
1.9 mg/L, 19 mg/L, and 190 mg/L, respectively. Before measurement, 1 mL of
each serum sample solution was pipetted into a clean plastic test tube, and 1 mL of
total ionic strength adjustment buffer II (TISAB II, Orion Research, Inc., Beverly,
MA, USA) concentrate with 1,2-cyclohexylenedinitrolotetraacetic acid (Thermo
Orion, MA, USA) was added to each solution. The concentration (millivolt) of
each solution was directly read out on the instrument display. Data concerning F
were recorded in mg/L.
Histochemistry and immunohistochemistry: The mandibles were dissected and
fixed in buffered 10% formalin solution and decalcified in formic acid and sodium
citrate solutions. Sections 5 µm thick were then cut and mounted on organosilane
(3-aminopropyl) triethoxysilane-coated glass slides. The sections stained with
hemotoxylin-eosin (HE) and examined under a light microscope.
25 Research report
Fluoride 47(1)23–30
January-March 2014
Effects of excessive fluoride intake on mandibular bone
Sakallioğlu, Muğlalı, Baş, Gulbahar, Lütfioğlu, Aksoy
25
Commercially available immunohistochemistry kits (Zymed Laboratories, Inc.,
USA) were used to perform the immunohistochemical analysis of the sections
using rabbit-specific polyclonal antibodies against MMP-2 (sc-53630, 4D3 Santa
Cruz Biotechnology, Inc., CA, USA) TIMP-1 (Ab-2 Clone 102-D1 Thermo
Scientific, Fremont, CA, USA), and TGF-β (Santa Cruz Biotechnology, Inc., CA,
USA). All of the sections were preincubated in 10% goat nonimmune serum
(Zymed Laboratories, Inc., USA) at room temperature for 10 min to block
nonspecific binding of second-step antibodies (Zymed Laboratories, Inc., USA).
The tissue sections were then reacted with primary antibodies and then reacted
with biotin-conjugated second-step antibody (Zymed Laboratories, Inc., USA) for
10 min at room temperature and rinsed in phosphate-buffered saline (PBS). To
inactivate endogenous peroxidase, the sections were incubated in 0.3% H2O2/
methanol for 60 min. The sections were again rinsed with PBS and reacted with
the streptavidin-biotin-peroxidase complex (Zymed Laboratories, Inc., USA) for
10 min at room temperature. Following additional rinsing with PBS, the sections
were incubated with 3-amino-9-ethylcarbazole (Zymed Laboratories, Inc., USA)
for 15 min and then counterstained with Mayer’s hematoxylin. Four negative
controls were prepared by omitting the primary antibodies, and then by replacing
them with PBS or unrelated rabbit antibodies. The bone sections were evaluated,
and the immunoreactivity against the antibodies was determined by a score of 0 to
3 (0: no immunoreactivity; 1: light immunoreactivity; 2: moderate
immunoreactivity; 3: strong immunoreactivity). Two separate examiners
performed the scoring immunostaining procedure on both the right and left
mandibular bones of the molar region for each rabbit. They used the same
magnification in all the examinations The mean of the two scores were determined
as the final score.
Statistical analysis: The Mann-Whitney U test was used to determine the
difference between the groups. The intragroup difference in the staining intensity
was evaluated with Friedman and Wilcoxon tests. The statistical significance level
was determined as p<0.05, and the results were expressed as the median (minmax) of the values.
RESULTS
The serum F levels in the experimental group (301.3±52.18 µg/L) were
significantly higher than those in the control group (18.20±1.58 µg/L) (p<0.05).
HE-staining revealed high cellularity in the experimental group, but there was no
evidence of high cellularity in the control group. Increased number of osteoblasts
and osteoclasts were observed in bone formation and resorption areas in the
experimental group (Figure 1A) compared with the control group (Figure 1B).
These features pointed to high bone turnover in the experimental group.
26 Research report
Fluoride 47(1)23–30
January-March 2014
Effects of excessive fluoride intake on mandibular bone
Sakallioğlu, Muğlalı, Baş, Gulbahar, Lütfioğlu, Aksoy
26
Figure 1. Hematoxylin eosin-stained sections [original magnification ×160]. [A] High cellularity
of the bone in the experimental group. The arrows indicate osteoclasts. [B] Low cellularity in
the control group.
TGF-β immunostaining of the mandibular bone in the experimental group was
significantly higher than that in the control group (p=0.03). The osteoblasts in the
experimental group showed high TGF-β expression, whereas the osteoclasts
showed less TGF-β expression (Figure 2A). Both the osteoblasts and the
osteoclasts in the control group showed low levels of TGF-β expression (Figure
2B).
Figure 2. TGF-β stained sections [original magnification ×320]. [A] Strong TGF-β expression in
osteoblasts and low expression in osteoclasts in the experimental group. The arrows indicate
osteoclasts. [B] Low TGF-β expression in the control group.
Although MMP-2 and TIMP-1 immunostaining of the mandibular bone was
slightly more dense in the experimental group, the difference was not statistically
significant (p=0.110 and p=0.479, respectively). The experimental group exhibited
slightly higher MMP-2 expression, especially in the osteoblasts of the bone
resorption areas (Figure 3A-3B).
27 Research report
Fluoride 47(1)23–30
January-March 2014
Effects of excessive fluoride intake on mandibular bone
Sakallioğlu, Muğlalı, Baş, Gulbahar, Lütfioğlu, Aksoy
27
Figure 3. MMP-2 stained sections [original magnification ×640]. [A] slightly higher expression
in the osteoblasts in the experimental group. The arrow indicates osteoclasts. [B] Low MMP-2
expression in the control group.
The osteoblasts and osteoclasts in experimental group also showed slightly
higher TIMP-1 expression (Figure 4A) than those in the control group (Figure 4B).
Figure 4. TIMP-1 stained sections [original magnification ×640]. [A] Area of high turnover in
the experimental group. The arrows indicate osteoclasts. [B] Low TIMP-1 expression in the
control group.
The intra-group comparison of the immunostaining of MMP-2, TIMP-1, and
TGF-β were evaluated and significant differences were found in the experimental
group. MMP-2 and TGF-β showed more dense staining than TIMP-1 (p=0.02 for
MMP-2 and p=0.013 for TGF-β) while TGF-β and MMP-2 staining intensities
were similar (p=0.076) in the experimental group.
DISCUSSION
The action of F on bone has been extensively studied and shown to have an
effect on bone minerals, bone cells, and bone architecture.14 Sodium fluoride
(NaF) promotes the recruitment of active, normally functioning osteoblasts
independent of any coupling between bone resorption and formation.15,16 Several
studies have investigated the pathogenesis16,17 and mechanical properties18 of
bone under excessive F intake. At high concentrations, F may cause the formation
of abnormally mineralized bone and impaired quality of bone.19 It may also result
in hypermineralized bone, as evidenced by a shift toward denser bone and
28 Research report
Fluoride 47(1)23–30
January-March 2014
Effects of excessive fluoride intake on mandibular bone
Sakallioğlu, Muğlalı, Baş, Gulbahar, Lütfioğlu, Aksoy
28
increased hardness.20 Consistent with the literature, our study revealed increased
cellular activity in the fluorosis group.
Although, the dose-dependent effects of F on the mechanical properties of bone
are well known, the underlying mechanisms are still not fully understood. TGF-β
is a very potent stimulator of osteoblastic bone formation, causing chemotaxis,
proliferation, and differentiation in committed osteoblasts.13 The concentration of
TGF-β in the human skeleton appears to be related to bone turnover.21 We
detected increased TGF-β expression in the fluorosis group compared with the
control group. This finding suggests that excessive F intake may induce the
production of TGF-β. We also found increased cellular activity in this group. This
finding could possibly be attributed to the high TGF–β levels.
In an in vitro study, Farley et al have reported that F increases bone turnover
even at normal therapeutic doses.17 On the other hand, Pepene et al. evaluated the
effects of F salts on the concentrations of three growth factors that including TGFβ in patients with osteopetrosis and found no significant relationship.22 Lütfioğlu
et al. suggested that fluorosis altered the homeostasis of periodontal soft tissue by
increasing TGF-β and also TIMP-1 and MMP-2 balance in animal model.16 We
also used an animal model and evaluated the TGF-β levels under excessive F
intake. Consistent with Pepene et al. and Lutfioglu et al., we conclude that it is
likely that F intake influences the composition of the remodeling of matrix,
possibly in relation to TGF-β levels.16,22
Only a few studies have analyzed the possible mechanism by which F acts on
bone turnover.15,16,22,23 Buyukkaplan and Guldag evaluated the amount of
glycosaminoglycan of patients diagnosed with systemic fluorosis and reported
decreased glycosaminoglycan in these patients.23 Farley et al. demonstrated for
the first time in vitro that NaF can increase the proliferation rate of bone cells.15
They also showed that the effect of NaF on [3H] thymidine incorporation can be
modulated by parathyroid hormones and human skeletal growth factors.15
Waddington and Langley investigated the influence of F on MMP and TIMP on
bone matrix remodeling.24 They reported an altered profile of MMP species in the
presence of F.24 They concluded that F is likely to have serious consequences for
the subsequent formation of the extracellular matrix, the prime function of which
is the regulation of mineral deposition and the control of crystal morphology.
Nevertheless, they did not detect immunoreactivity for TIMP-1 and MMP-2.
Consistent with the results of Waddington and Langley, we found no significant
differences in MMP-2 or TIMP-1 levels in the fluorosis and control groups in our
study. However, we found altered TIMP/MMP and TIMP/TGF-β ratios in the
fluorosis group. TIMP-1 staining was more intensive than MMP-2 and TGF- β in
the fluorosis group. This finding could be attributed to an up-regulation effect of
TGF-β on TIMP-1.25 In our opinion, increased TGF-β levels alters the balance
between TIMP-1 and MMP-2 and leads to an inhibition of proteolytic activity.
Although F is known to alter bone metabolism, the mechanism underlying its
action remains unclear. To the best of our knowledge, this is the first in vivo study
to evaluate TGF-β, MMP-2, and TIMP-1 levels in mandibular bone in response to
29 Research report
Fluoride 47(1)23–30
January-March 2014
Effects of excessive fluoride intake on mandibular bone
Sakallioğlu, Muğlalı, Baş, Gulbahar, Lütfioğlu, Aksoy
29
high F concentrations. Increased levels of TGF-β could serve as an important
marker in the evaluation of the pathological action of F in bone tissue. Further
studies that include other bone regulating molecules are required to elucidate the
molecular events controlling the formation of bone matrix under excessive F
intake.
ACKNOWLEDGEMENT
This research was supported by the Ondokuz Mayıs University Scientific
Research Fund [Project number: DHF.058].
The study was presented at the 92nd Annual Meeting of the American
Association of Oral and Maxillofacial Surgeons (AAOMS) in Chicago, Illinois,
(Muğlalı M. Poster board number 64. Effects of excessive fluoride intake on
MMP-2, TIMP-1, and TGF-β expressions of mandibular bone. J Oral Maxillofac
Surg 2010; 68(9 Suppl):e95-6).
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30 Research report
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January-March 2014
Effects of excessive fluoride intake on mandibular bone
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30
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