J Bone Miner Metab (2012) 30:154163

DOI 10.1007/s00774-011-0312-6

ORIGINAL ARTICLE

Assessment of fluoride-induced changes on physicochemical

and structural properties of bone and the impact of calcium
on its control in rabbits
Subarayan Bothi Gopalakrishnan Gopalan Viswanathan

Received: 28 February 2011 / Accepted: 11 August 2011 / Published online: 27 September 2011
The Japanese Society for Bone and Mineral Research and Springer 2011

Abstract Bone deformities caused by the chronic intake

of large quantities of fluoride and the beneficial effect of
calcium on its control have been studied for many years,
but only limited data are available on the quantitative effect
of fluoride intake and the beneficial impact of calcium on
fluoride-induced changes in bone at the molecular level. It
is necessary to determine the degree of fluoride-induced
changes in bone at different levels of fluoride intake to
evaluate the optimum safe intake level of fluoride for
maintaining bone health and quality. The ameliorative
effect of calcium at different dose levels on minimizing
fluoride-induced changes in bone is important to quantify
the amount of calcium intake necessary for reducing fluoride toxicity. Thirty rabbits, 2 months old, were divided
into five groups. Group I animals received 1 mg/l fluoride
and 0.11% calcium diet; groups II and III received 10 mg/l
fluoride and diet with 0.11% or 2.11% calcium, respectively; and groups IV and V received 150 mg/l fluoride and
diet with 2.11% or 0.11% calcium, respectively. Analysis
of bone density, ash content, fluoride, calcium, phosphorus,
and Ca:P molar ratio levels after 6 months of treatment
indicated that animals that received high fluoride with lowcalcium diet showed significant detrimental changes in
physicochemical properties of bone. Animals that received
fluoride with high calcium intake showed notable amelioration of the impact of calcium on fluoride-induced changes in bone. The degree of fluoride-induced characteristic
changes in structural properties such as crystalline size,

S. B. Gopalakrishnan (&)  G. Viswanathan

Department of Pharmaceutical Chemistry,
Manonmaniam Sundaranar University,
Abishekapatti, Tirunelveli 627012, Tamil Nadu, India
e-mail: sgkmsu@yahoo.co.in

123

crystallinity, and crystallographic c-axis length of bone

apatite cells was also assessed by X-ray diffraction and
Fourier transform infrared studies. X-ray images showed
bone deformity changes such as transverse stress growth
lines, soft tissue ossification, and calcification in different
parts of bones as a result of high fluoride accumulation and
the beneficial role of calcium intake on its control.
Keywords Fluoride intake  Crystalline size 
Crystallinity  Hydrogen bonding  Fluorapatite

Introduction
Fluoride is considered an important therapeutic agent for
the treatment of dental caries and some cases of osteoporosis, but the ingestion of excessive quantities causes dental
and skeletal fluorosis in both human beings and animals
[1, 2]. Skeletal fluorosis is characterized by severe pain and
immobilization of joints of the axial skeleton and the major
joints of extremities [35]. Approximately 99% of the
fluoride in the body is associated with skeletal tissues
[6, 7]; nearly 50% of the fluoride absorbed each day
becomes associated with calcified tissues within 24 h [8,
9]. Fluoride is readily incorporated into the apatite lattice
of bones and teeth, where it replaces the hydroxyl ion in the
hydroxyapatite of bone. The mineral exists as extremely
small crystals surrounded by a hydration shell [8, 10],
which leads to the formation of stoichiometric apatite
[1, 11, 12], with intrinsically weaker material properties
than normal bone [12]. The less soluble, hypertrophic,
compact, and coarse bone formation in bones with high
fluoride accumulation may be the result of the incorporation of excess fluoride [13, 14]. The absorbed fluoride is
highly integrated with cancellous bone tissues rather than

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cortical bone [15, 16], as per the following reactions,

Eqs. 1 and 2, as suggested by Giesecke and Rathje [17]:
Ca10 PO4 6 OH2 F ! Ca10 PO4 6 OHF OH
Hydroxyapatite

Hydroxyfluorapatite

[3436]. Even small changes of crystallinity and size and

shape of the bone apatite crystal may greatly affect
physicochemical properties, bonding, and perfection of
bone [35, 3740].

1
Ca10 PO4 6 OHF F ! Ca10 PO4 6 F2 OH

Fluorapatite

Hence, the maximum ill effects are noticed in the neck,

spine, knee, pelvic, and shoulder joints, and it also affects
small joints of the hands and feet and is prone to reduce
bone strength [1820]. Fluoride is known to create calcium
deficiency by its osteogenic activity, which increases the
skeletal requirement for calcium [21, 22]. A high calcium
intake decreases the absorption of fluoride, because
calcium binds with fluoride and forms an insoluble
calcium fluoride in the gastrointestinal tract [23]. From
previous studies it has been reported that the serum fluoride
level was greatly decreased when sodium fluoride was
administered with a high-calcium diet [24, 25]. Even
though the structural changes on bone crystal lattice from
the action of high fluoride content and the beneficial role of
calcium have been studied by different chemical and
physical methods, there was only limited information
available on the quantitative impact of fluoride
incorporation and the quantitative ameliorative influence
of calcium on high fluoride-induced physicochemical and
structural changes in bone. To understand the quantitative
effect of fluoride and fluoride combined with calcium
intake on bones, some of the important physicochemical
and structural properties were examined in bones of rabbits
treated with fluoride combined with low and high calcium.
The characteristic physicochemical properties such as bone
density, ash content, and Ca:P ratio are important for
understanding bone quality. It is generally accepted that the
level of ash content and Ca:P ratio provide a sensitive
measure of bone mineral changes and bone diseases [26,
27]. X-ray diffraction (XRD) is a successful method for
characterizing the crystallographic changes in bone, and
many previous studies examined the fluoride-induced
changes in bone using this tool [28, 29]. Fourier
transform infrared spectroscopy (FTIR) is also a useful
tool to investigate the structural changes and bonding
related to apatite mineral in macroscopic as well
as microscopic samples [3032]. The crystallinity,
crystalline size, and perfection of bone apatite were
assessed using XRD and FTIR as described by Termine
and Posner [33] and later used by Pleshko et al. [34]. The
effect of fluoride on bone is of great biomedical interest,
because many of the in vivo and in vitro studies revealed
that excess fluoride affects crystalline size and shape of the
bone cells, particularly along the crystallographic c-axis

Materials and methods

Experimental design and selection of animals
Thirty 2-month-old male albino rabbits (average weight,
600 g) were randomly distributed into five groups, with six
animals in each group. Animal care and experimental
protocols were in accordance with and approved by the
Institutional Animal Ethical Committee. The description of
experiments on animals of the individual groups is illustrated in Table 1. Animals were allowed to receive fluoridated distilled water (fluoridated using sodium fluoride)
and diet ad libitum from polypropylene bottles. Water and
diet intake were measured for 3 consecutive days in every
month, and the mean level was used to quantify the intake
of fluoride and calcium. After 6 months, the animals were
killed by CO2 asphyxiation. Because of the diverse pattern
of fluoride absorption in different parts of bones, tibia and
patella bones were separately taken for analysis. A high
fluoride dose of 150 mg/l was chosen because in a previous
study rabbits receiving 150 mg/l fluoride for 6 months
show typical osteofluorosis [41]. The high dose of calcium,
2.11% in diet (control diet, 0.11% calcium fortified with
required quantity of calcium gluconate), was fixed for
observing the impact of calcium on fluoride-induced
changes in bone, because the earlier study reported that rats
treated with 2% calcium with fluoride show some beneficial effects against the toxicity of fluoride [24].
Estimation of bone density
Accurately weighed dry tibia and patella bones were used
for the density measurement. Bone density was calculated
by dividing the weight of dry bone by the volume of distilled water occupied by immersion of an accurately
weighed bone sample in the measured amount of doubledistilled water according to the Archimedes principle.
Estimation of ash content
Aliquots of fat-free, cleaned, and dry bones were accurately weighed and ashed for 6 h at 600C in a muffle
furnace. Upon cooling, the weight of bone ash was recorded. Weights obtained from before and after ashing were
used to calculate the percentage of bone ash content as per
the following numerical formula:

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J Bone Miner Metab (2012) 30:154163

Table 1 Experimental design and average of fluoride and calcium intake of various groups of rabbits
Category

Fluoride dose in
distilled water (mg F-/l)

Calcium and fluoride

level in diet

Amount of fluoride
intake (mg/day)

Amount of calcium
intake (g/day)

0.11% Ca and 0.45 mg/kg F2

0.17 0.02

0.18 0.03

Group II

10

0.11% Ca and 0.45 mg/kg F

1.03 0.13

0.18 0.03

Group III

10

2.11% Ca and 0.45 mg/kg F2

1.03 0.13

3.45 0.59

Group IV

150

2.11% Ca and 0.45 mg/kg F2

14.4 1.92

3.45 0.59

Group V

150

0.11% Ca and 0.45 mg/kg F2

14.4 1.92

0.18 0.03

Group I

Fluoride and calcium intake values are in mean SD

% Ash content weight of ash=weight of dry bone  100

3
Preparation of bone powder
The cortical shaft of the tibia and cancellous portion of
patella were crushed separately, Tris-washed to remove
bone marrow, lyophilized, and defatted in 2:1 (v/v) chloroform/methanol. The bone pieces were pulverized and
sieved in a sifter to separate out bone particles less than
100 lm in size.
Estimation of bone fluoride level
Each accurately weighed pulverized bone sample was carefully transferred into a 100-ml standard measuring flask and
dissolved using 3 ml 0.5 M hydrochloric acid; acidity was
neutralized with 0.5 M sodium hydroxide. The volume of the
solution was then made up to 100 ml using double-distilled
water (solution A). About 10 ml of the aliquot from solution
A was mixed with 10 ml of total ionic strength adjustment
buffer (TISAB II) made from cyclohexylene dinitrilotetraacetic acid (CDTA) and was added to the standards as well as
to the samples before the measurement of fluoride. The
concentration of fluoride in the unknown was directly read
from the digital display of the Orion fluoride ion-selective
electrode attached to an ion analyzer. Before measurements,
the instrument was calibrated with two standard fluoride
solutions so chosen that the concentration of one is ten times
the concentration of the other. The concentration of the
unknown falls between those two standards. The slope value
obtained from the calibration was 58.8 mV, which is the
best slope value for fluoride estimation.
Estimation of bone calcium level
Each accurately weighed pulverized bone sample was
carefully transferred into a 100-ml standard measuring
flask and dissolved with 3 ml 0.5 M hydrochloric acid;
acidity was neutralized with 0.5 M sodium hydroxide. The
volume of the solution was then made up to 100 ml using
double-distilled water (solution A). Calcium level in bone

123

was estimated by using 1 ml of the aliquot taken from

solution A, which was transferred into a 25-ml standard
measuring flask and the contents made up to 25 ml using
double-distilled water. The optimal conditions for estimation of calcium in an atomic absorption spectrophotometer
are wavelength, 422.7 nm; slit width, 0.7 nm; and relative
noise, 1.0; the oxidizing lean blue flame obtained from an
airacetylene mixture is used for the aspiration of samples.
Sample solution was then introduced into a Perkin Elmer
flame atomic absorption spectrophotometer calibrated by
using appropriate calcium standards and control. Calcium
level of the unknown sample was read directly from the
digital display of the meter. Calcium level in bone was
calculated from the reading by multiplying with the
appropriate dilution factor [42].
Estimation of bone phosphorus level
About a 5-ml aliquot from solution A treated with
ammonium molybdate reagent gives a blue-colored complex solution in the presence of SnCl2. After appropriate
dilution, light absorption by this blue complex solution was
measured at 690 nm in a Perkin Elmer UVvisible doublebeam spectrophotometer. Phosphorus concentration was
calculated from the standard curve plotted using various
appropriate standards (520 mg/l) [43, 44].
Infrared spectral analysis
Cleaned, dried, and pulverized patella bone samples,
approximately 1 mg, were mixed with 200 mg pre-ground
spectroscopic grade potassium bromide (KBr) powder and
ground for several minutes. The KBrsample mixture was
then placed in a press under high pressure to make a KBr
sample pellet, which was scanned in the spectrophotometer
at 4 cm-1 resolution over the range 4,000400 cm-1. The
FTIR spectrum was recorded using a Jasco FTIR, Japan.
X-ray diffraction analysis
Aliquots of clean, dried, and pulverized patella bone
samples (a single patella bone sample from each group)

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were analyzed with an XPERT-PRO Panalytical diffractrometer using Cu Ka radiation. The crystalline size of each
bone sample was calculated with Scherrers Eq. 4 [45]:
D

57:3Kk
b cos h

parameters were assessed by Fisher ratio. Correlations

between bone fluoride levels and other physicochemical and
structural parameters were performed using SPSS statistical
version 16.0. P\0.05 was considered statistically significant.

where 57.3 is a conversion factor from degree to radians, k

is X-ray wavelength, b is the full width at half maximum of
the 002 peak near 25.8 diffraction angle along the c crystallographic axis, and K is a shape factor constant that
depends on the crystal habit; here, K = 0.9.
The percentage of crystallinity (X) of the bone samples
was evaluated by Eq. 5 [46]:


V112=300
X 1
 300
5
I300
where I300 is the intensity of the 300 diffraction peak and
V112/300 is the intensity ratio of the hollow between the 112
and 300 diffraction peaks of bone apatite.
X-ray analysis
Rabbits were placed on the appropriate window of the Fuji
CR digital X-ray meter (Japan) to scan the total body of
each animal. The fluoride-induced changes on the skeleton
were assessed by viewing the digital image of the X-ray
radiograph.
Statistical analysis
Results represent mean values with standard deviation
between the measured values. Quantitative relationships
between bone fluoride levels with other physicochemical

Results
Changes in physicochemical properties
The observed mean values of physicochemical parameters
with standard deviation of the experimental animal bone
samples are shown in Table 2. Bone density of tibia
(r = 0.666) and patella (r = 0.664) bones slightly
increased with increase of bone fluoride level. Normally,
tibial bone densities are higher than patella bone densities
of all groups of treated rabbits. Bones of rabbits treated
with fluoride and low calcium show higher bone density
(r = 0.484) than bones of rabbits treated with fluoride and
high calcium (r = 0.357). Ash content levels of tibia and
patella are found to increase with increase of fluoride
concentration. The ash content level of bones of rabbits
treated with low calcium and fluoride is higher than the ash
content level of bones of rabbits treated with high calcium
and fluoride. No significant changes were observed in
calcium levels between the bones of rabbits treated with
low calcium with fluoride and high calcium with fluoride.
The obtained F-ratio between fluoride and calcium levels
shows no notable influence of fluoride on bone calcium
level. Phosphorus levels slightly decreased with increase of
fluoride level in tibia and patella bones, but the trend was
insignificant. The Ca:P molar ratio was higher in tibia than
patella normally in all the groups. Significantly higher

Table 2 Physicochemical parameters of bones of control and fluoride with calcium-treated groups of rabbits
Group I

Group 2

Group 3

Group 4

Group 5

Density (g/cm3)

2.07 0.06

2.16 0.06

2.11 0.08

2.24 0.11

2.32 0.18

Ash content (%)

66.3 0.10

66.4 0.14

66.4 0.06

68.2 0.21

71.3 0.24

Tibia (n = 6)

Fluoride (mg/kg)

231 48

676 63

320 46

4526 616

5883 732

Calcium (%)

27.6 0.17

27.2 0.06

27.4 0.08

27.2 0.12

26.8 0.08

Phosphorus (%)

13.4 0.18

12.9 0.21

13.1 0.07

12.8 0.12

13.1 0.06

Ca:P molar ratio

1.65 0.02

1.69 0.03

1.67 0.01

1.70 0.01

1.63 0.01

Density (g/cm3)

1.92 0.04

1.96 0.05

1.96 0.04

2.04 0.11

2.06 0.08

Ash content (%)

68.2 0.15

69.4 0.25

69.1 0.29

72.4 0.37

74.8 0.61

Fluoride (mg/kg)
Calcium (%)

273 51
26.8 0.08

890 77
26.2 0.12

386 65
27.1 0.11

5457 521
26.7 0.16

6371 635
26.2 0.14

Phosphorus (%)

13.2 0.11

13.1 0.09

13.7 0.14

12.7 0.10

13.1 0.06

Ca:P molar ratio

1.62 0.01

1.61 0.02

1.58 0.02

1.68 0.01

1.60 0.01

Patella (n = 6)

Values are means SD

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Table 3 Correlation between bone fluoride levels with other physicochemical parameters
Correlation
coefficient (r)

Significant
level (P)

95% confidence
interval for (r)

Coefficient of
determination (R2)

F-ratio

Tibia fluoride level vs.

Density (g/cm3)

0.6661

0.0001

0.402 to 0.828

0.444

22

Ash content (%)

0.9205

0.0001

0.838 to 0.962

0.845

155
44

Calcium (%)

-0.781

0.0001

-0.890 to -0.585

0.609

Phosphorus (%)

-0.2628

0.1607

-0.5692 to 0.1077

0.0690

Ca:P molar ratio

-0.1651

0.3832

-0.4959 to 0.2075

0.0273

0.664

0.0001

0.3995 to 0.8266

0.4411

22

0.956

0.0001

0.9080 to 0.9789

0.9133

295

Patella fluoride level vs.

Density (g/cm3)
Ash content (%)
Calcium (%)

-0.409

0.025

-0.6706 to -0.0574

0.1674

Phosphorus (%)

-0.585

0.001

-0.7809 to -0.2852

0.3426

15

0.411

0.024

0.0603 to 0.6722

0.1694

Ca:P molar ratio

Low calcium with fluoride-treated groups bone fluoride level vs.

Density (g/cm3)

0.4840

0.003

0.1848 to 0.7010

0.2342

10

Ash content (%)

0.8857

0.0001

0.7859 to 0.9406

0.7845

124

Calcium (%)

-0.4501

0.0059

-0.6784 to -0.1427

0.2026

Phosphorus (%)

-0.0381

0.8256

-0.3621 to 0.2942

0.0014

0.1

Ca:P molar ratio

-0.3786

0.0228

-0.6289 to -0.057

0.1433

High calcium with fluoride-treated groups bone fluoride level vs.

Density (g/cm3)

0.3569

0.0869

-0.0543 to 0.6646

0.1274

Ash content (%)

0.6611

0.0004

0.3515 to 0.8404

0.4371

17

Calcium (%)
Phosphorus (%)

-0.6450
-0.8215

0.0007
0.0001

-0.8320 to -0.3266
-0.9200 to -0.6254

0.4161
0.6749

16
46

0.6721

0.0003

0.3686 to 0.8461

0.4517

18

Ca:P molar ratio

fluoride levels were observed in patella bones than in tibia

bones of the corresponding group of rabbits. Fluoride
levels of bones of rabbits treated with low calcium and
fluoride were significantly higher than in the bones of
rabbits treated with fluoride and high calcium. Quantitative
relationships between bone fluoride levels and other
physicochemical parameters were assessed through Fisher
ratio (Table 3). Fluoride levels of patella bones showed a
higher influence on ash content than tibia bones; also, the
impact of fluoride on ash content is decreased with
increased calcium intake, expressed as F-ratio in Table 3.
Changes in FTIR spectra
Figure 1 shows the combined FTIR spectra of all the groups
of animal bone samples. Sharp and well-resolved free
hydroxyl group stretching frequency bands appeared at
3,570 cm-1 for II, IV, and V animal group bones, but the
bands were less intense for I and III group bone samples. An
intense band for bonded OH group stretching frequency
appeared at around 3,436 cm-1 for I and III group samples,
shifting to 3,420 cm-1 for II, IV, and V group bone samples

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Fig. 1 Combined Fourier transform infrared (FT-IR) spectra bone

samples of control and other groups

(Fig. 1). Reduction of anti-symmetrical stretching of the

phosphate c1 band at 960 cm-1 is observed for II, IV, and V
group bones, and this characteristic frequency band is well
resolved in groups I and III. The characteristic vibrational
spectral region from 900 to 1,200 cm-1 is primarily considered as the symmetrical (c1) and asymmetrical (c3)

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stretching frequencies of phosphate (c1-PO4 and c3-PO4)

[34]. Bone samples of groups I and III animals show bands
for c1-PO4 and c3-PO4 that appeared broad and covered
more curve areas than samples from groups II, IV, and V in
the same region. Well-observed characteristic carbonate
stretching frequency bands ranging from 1,410 to
1,490 cm-1 appeared for group I and group III bone samples, but these respective bands diminished in group II, IV,
and V bone sample spectra. Diminution on bonded carbonyl
stretching intensities at 1,640 cm-1 was observed for group
II, IV, and V bone samples, but this characteristic band was
well resolved in group I and group III bones.
Changes in XRD and X-ray analysis
The X-ray diffraction patterns of bones from groups II, IV,
and V are more highly resolved than those of groups I and
III (Fig. 2). Changes in crystalline size and crystallinity of
the bones have been assessed through XRD analysis using
appropriate equations (Table 4). The observed crystallinity

Fig. 2 X-ray diffraction (XRD) patterns of normal and fluorideintoxicated animal bones

and crystalline size were higher in bones of groups II, IV,

and V than in bones from groups I and II. The XRD
analysis used a single patella bone sample from each
group. Hence, it is not possible to represent statistical
parameters such as mean and standard deviation within the
group in Table 4. Characteristic fluoride-induced changes
such as transverse stress growth lines, soft tissue ossification, and calcification in tibia and patella bones of different
groups of rabbits are marked with arrows in Fig. 3. X-ray
images of forearms and of tibia and pelvis of high fluoridetreated rabbits compared with other groups of animals
(Fig. 3) indicate the appearance of several growth lines
(black arrows) and diffuse calcification (G5) and ossification (G2) in forearms of high fluoride-accumulated rabbit
bones.

Discussion
Fluorosis is irreversible but is preventable by appropriate
and timely intervention. Therefore, a greater understanding
of physicochemical and structural properties at the
molecular levels of disease progression is very important.
Interaction between fluoride, calcium intake, and the
bodys response to fluoride accumulation is important in
understanding the nature of the disease. Bone samples from
low calcium and high fluoride-treated groups (groups II and
V) show higher fluoride level, bone density, and ash content than those of the control (group I), indicating that high
fluoride accumulation affects bone mineral content and
mineralization. It is also evident that high fluoride accumulation in bone causes hypermineralization (overmineralized bone) [39]. Hypermineralization can alter bone
quality and reduce the strength of bone, primarily by
altering the bonding between the bone mineral and collagen matrix [39]. The shift in mineralization toward denser,
more mineralized bone observed in this study is similar to
data on chicks [47] and humans [48]. The increase of bone
density and ash content may result from a greater packing
density of the bone crystals, and accordingly the increase

Table 4 Crystalline size, crystallinity, and X-ray diffraction (XRD) pattern of bones of respective animal groups
Category

Crystalline
size (nm)

Crystallinity
(%)

hkl (002)

hkl (112)

hkl (300)

d002

b002

Intensity (%)

Intensity (%)

Group I

57.66

98.64

3.416

Group II

67.86

98.93

3.420

0.1534

26.0659

70.56

72.00

0.1303

26.0314

39.54

Group III

65.19

98.98

60.81

3.414

0.1357

26.0824

48.04

68.65

Group IV

66.63

Group V

72.44

98.95

3.408

0.1328

26.1293

47.60

67.53

99.23

3.414

0.1221

26.0766

45.52

76.68

hkl (Miller Indices) are the symbolic vector representation for the orientation of an atomic plane in a crystal lattice and are defined as the
reciprocals of the fractional intercepts which the plane makes with the crystallographic axes

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Fig. 3 X-ray images of skeleton of group I(C), group II (G2), group III (G3), group IV (G4), and group V (G5) animals. Arrows indicate
fluoride-related changes (see text)

of crystalline size and crystallinity in high fluoride-accumulated bones. Moreover, higher fluoride and higher ash
content in patella than in tibia (Fig. 4) suggests that cancellous bones tend to accumulate more fluoride and are
prone to hypermineralization, which leads to increased
bone density and the associated risk of fractures. Increases
in the number of microfractures are frequently found in
fluorotic area bones, usually located in highly mineralized
areas of bone with an increased number of dead osteocytes
[40, 49, 50]. Although the rabbits of group III were
exposed to 10 mg/l fluoride with high calcium, fluoride
levels in the bones were found to be much closer to the
fluoride level of bones of animals exposed to 1 mg/l fluoride. Although the rabbits of group IV and V were exposed
to 150 mg/l fluoride with high (2.11%) and low (0.11%)
calcium in the diet, respectively, fluoride levels of bones of
group IV rabbits are significantly lower than in the bones of
group V animals. The results support that intake of a highcalcium diet may have an ameliorative effect on maintaining the composition of bone quality parameters such as

123

bone density, ash content, calcium, phosphorus, and Ca:P

molar ratio levels against high fluoride accumulation
through reducing absorption of fluoride in the gastrointestinal tract by forming insoluble calcium fluoride. Previous
studies reveal that when children with adequate and inadequate calcium nutrition and with comparable intakes of
fluoride were compared, the toxic effect of fluoride was
found to be severe in those with inadequate calcium [51,
52]. Also, previous studies reported that the intake of more
calcium than the normal requirement is required to combat
the toxic effect of fluoride [24, 25]. In this study, group II
and III animals were exposed to tenfold-higher fluoride
than the group I animals for 6 months, but the group III
animals received a high-calcium diet. Hence, the fluoride
accumulation in group III animal bones was nearly two
times lower than in group II animal bones. Similarly, the
fluoride level in the bones of the high calcium-exposed
group animals is nearly 1,300 mg lower than in group V
animals, even though the respective groups received
150 mg/l fluoride. This finding confirms that increment of

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calcium in the diet quantitatively reduces the accumulation

of fluoride in bone. As per infrared spectroscopy, it has
been observed that the intensity area of antisymmetrical
stretching of the phosphate c1 band near 960 cm-1 indicates the crystalline size of apatite. The phosphate
stretching absorbance between 900 and 1,200 cm-1 indicates the crystallinity of bone apatite [34]. The intensity
changes of the component near 1,060 cm-1 is also
important to illustrate the changes on the length of the caxis of hydroxyapatite [34]. The spectral range of carbonate stretching between 860 and 880 cm-1 is also useful
to understand significant changes in the apatite unit cell
[5355]. In most chemical environments, the hydroxyl
group does not exist in isolation, and a high degree of
association is experienced as a result of extensive hydrogen
bonding with other hydroxyl groups [56]. Even though the
structural hydroxyl groups in hydroxyapatite of bone are
arranged in an orderly fashion at the edge of the crystal
lattice of the space group of P63/m, parallel to the c-axis in
the bone cell [5658], no hydrogen bonds were present
between two neighboring structural hydroxyl groups in
Tibia bone
Patella bone

75
74

Y = 68.4 + 0.000895 x, p = 0.005, r = 0.973

Ash content (%)

73
72
71
70
69
68
67

Y = 65.96 + 0.000754 x , p = 0.018, r = 0.937

66
0

1000

2000

3000

4000

5000

6000

7000

Fluoride level in bone (mg/kg)

Fig. 4 Fluoride levels in patella and tibia bones versus ash content of
respective bone samples

hydroxyapatite because the successive oxygen atoms are

too far away (0.344 nm) along the c-axis [57, 58]. However, the incorporation of fluoride on hydroxyapatite in
bone leads to the formation of hydrogen bonds between
fluoride and the adjacent hydroxyl group along the c-axis
because of the perturbation of fluoride on the hydrogen
environment, involving some displacement of both fluoride
and hydroxyl groups from their normal position [59].
Broadening of bonded OH stretching frequency near
3,436 cm-1 in bones of groups I and III indicates the
presence of a large number of hydrogen bonds between the
OH groups with adjacent fluoride in the respective groups
of animal bones. The decreased intensity near 3,420 cm-1
and sharpening of free OH stretching frequencies at
3,570 cm-1 indicates the increasing number of free OH
groups in group II, IV, and V bone samples compared to
those of groups I and III. This finding implies the missing
of bonded OH groups as a consequence of high fluoride
accumulation in bone through high fluoride intake in a lowcalcium environment. The detection of free hydroxyl group
stretching at 3,570 cm-1 is difficult in normal hydroxyapatite, but the appearance of this characteristic band may
be caused by the effect of high fluoride. The characteristic
bonded OH frequency shifts from 3,436 to 3,420 cm-1,
which indicates that the increase of OH bond strength
may be caused by the loss of hydrogen bond between the
fluoride and hydroxyl groups in bone apatite as a result of
high fluoride accumulation. In general, the loss of hydrogen bonds between OH- and F- increase the bond strength
and decrease bond length between the oxygen and hydrogen in the hydroxyl group. The appearance of high-intensity bands for hydrogen-bonded OH out-of-plane bends in
a characteristic band from 850 to 880 cm-1 in group I and
group III bones (see Fig. 1) confirms the presence of the
OHF vibrational band and the decreasing intensities of
this corresponding band in high fluoride-accumulated bone
samples. This finding implies the missing of a few OHF
hydrogen bonds as a result of the exchange of the OH
group by the integration of fluoride (Fig. 5). The loss of
hydrogen bonding may also be caused by the lengthening

Fig. 5 Scheme of mechanism of fluoride-induced changes in bone apatite cells

123

162

of the c-axis because of the increase of distance between

OH and F- (these changes are also shown in Fig. 5).
Decrease of the curve area of phosphate stretching because
of the incorporation of fluoride between 900 and
1,200 cm-1 in high fluoride-accumulated bone samples
indicates the increase in bone crystallinity compared to
high calcium-treated and control bones (Fig. 1). Curve area
reduction near 1,060 cm-1 for high fluoride bones with
low-calcium treatment animal bones confirms the elongation along the c-axis of the bone apatite crystal as reported
earlier [34, 56]. This change may result from the exchange
of hydroxyl ions positioned in the columnar arrangements
parallel to the c-axis of bone apatite. Increase in crystallinity leading to increase of rigidity in bones may the
reason for severe pain in the joints, which is particularly
higher in cancellous bone in people in fluorotic areas. The
lesser curve area of phosphate anti-symmetrical stretching
frequency near 960 cm-1 (c1) and the well-resolved XRD
pattern in bones with high fluoride content elucidate the
increase of crystalline size and crystallinity of bone apatite
compared to bones of low fluoride content. The lowering of
carbonate group intensities and diminishing of carbonyl
stretching frequencies in high fluoride-accumulated bones
indicate missing labile carbonate ions because of the
exchange of fluoride ion. Ingestion of an optimal level of
fluoride stabilizes the crystal structure of bone apatite by
providing additional and strong hydrogen bonds [60], but
the intake of high fluoride eliminates a few hydrogen bonds
from the bone apatite, which may make the bones prone to
becoming weak and easily fragile. The formation of
chunky soft tissue ossification in the pelvis of high fluoride-treated rabbits noted in X-ray analysis confirms the
detrimental skeletal changes resulting from fluoride incorporation and visualizes the formation of hypermineralization and increased bone density. These changes resulting
from the incorporation of fluoride are higher in the patella
type of bones than the tibia, which may cause patellar parts
in the skeletal systems to be highly prone to fluorideinduced fractures. Moreover, the severe pain and restriction
of movement prevalent among people in the fluorotic areas
is also the result of high fluoride with inadequate calcium
intake. As the intake of 2% calcium through the diet shows
a significant ameliorating effect on fluoride-induced toxicity, it has been recommended that people residing in
fluoride endemic areas consume at least 2% calcium in
their diet to combat against fluorosis.In conclusion, the
consumption of more fluoride in drinking water causes
impairment of bone quality and induces negative changes
in physicochemical properties as a result of the high fluoride accumulation. The infrared spectral study confirms the
missing of some bonded OH groups and increase of
nonbonded OH groups because of the incorporation of
fluoride, which provides information about the changes in

123

J Bone Miner Metab (2012) 30:154163

bond strength between the molecules. Excess fluoride

intake may increase crystallinity, crystalline size, and the
length of the c-axis in bones. The observed variations
through XRD and X-ray in bonding and structural variations in high fluoride-treated rabbit bones provoke a negative effect on bone strength and quality. High calcium
intake, 2.11% through the diet, shows significant ameliorative impact against the detrimental effects of high fluoride accumulation in bone.

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