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The effects of fluoride and low calcium on the physical properties of the rat femur.

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The Effects of Fluoride and Low Calcium on the
Physical Properties of the R a t Femur
DEXTER F. BEARY'
Department of Anatomy, Schools of Medicine and Dentistrg,
Lorna Linda University, Lorna Linda, California 92354
ABSTRACT
The effect of fluoride on the physical properties of bone was investigated with the aid of an Instron materials tester. Two groups of weanling female
rats, one on a n adequate (0.6% ) calcium diet and the other on a low (0.1%) diet
were given the following doszge levels of fluoride (as NaF) i n their deionized drinking
water over a fifteen End one-half week period: 3.4 Fpm, 10.0 ppm and 45.0 ppm. In
the adequate calcium group a significant increase i n flexibility in the rat femur was
found only a t the 45.0 ppm dosage level. This was not offset by a significant decrease
in strength. In the low calcium group a similar significant increase in flexibility appeared at the 10.0 ppm dosage level 2s well as the 45.0 ppm, but a significant decrease in strength at the two dosage levels was observed. These were in direct relation to the amount of fluoride given.
This study was designed to determine
the influence of fluoride on the physical
properties of the fresh rat femur, and
indirectly on the common bone disease
osteoporosis. The osteopenia produced by
a calcium deficient diet in the experimental animal shows essentially the same characteristics.
MATERIALS AND METHODS
Eighty weanling, 25 day old, 38-40 gm,
female, Sprague-Dawley albino rats were
employed in the study. They were divided
into two main groups, I and 11, of forty
rats each. Each main group was in turn
divided by random assortment into five
subgroups of eight rats each. Subgroups
of Group I were designated 1-5; those of
Group I1 were designated 6-10.
Group I received 0.6% calcium in their
diet which was considered to be adequate
in other respects. The phosphorus content
was 0.358%. Group I1 received the same
diet except that calcium was reduced to
0.1% of the diet. In both groups a Jones
and Foster ('42) salt mixture without calcium was chosen since it lacks fluoride.
ANAT. REC., 164: 305-316.
The dietary ingredients were as follows:
Diet
ingredients
Casein
Sucrose
Vitamin Mix
Salt Mixture (without
CaC08)
Calcium Carbonate
Corn Oil
Group I
Adequate
Ca diet
Group II
Low in Ca
%
I
24.000
66.250
24.000
65.000
2.000
2.500
1 .SO0
5.000
2.000
2.500
0.250
5.000
It is to be noted that, while the calcium
was reduced in the diet of Group 11, the
phosphorus content was kept the same in
both diets. Thus the calcium to phosphorus
ratio of the adequate diet is about 1.7-1.0
while that of the low calcium diet is approximately 0.3-1.0. The diet was fed to
the rats ad libitum for a period of 106-110
days depending upon the date of sacrifice.
The subgroups consisting of eight rats
each received different dosages of fluoride,
as sodium fluoride, in their deionized
Received Sept. 20, '68; Accepted Feb. 24, '69.
1 Present address: Department of Biology, Southwestern Union College, Keene, Texas 76059.
305
306
DEXTER F. BEARY
drinking water (except for the control subgroups) as follows:
Group I
(Adequate c.cium)
Subgroup
ppm F
Group 11 (LOWcalcium)
Subgroup
ppm F
l(contro1) 0
G(contro1) 0
2
3.4 ( H W ) ' 7
3.4 (HW)1
3
3.4
a
3.4
4
10.0
9
10.0
5
45.0
10
45.0
1 HW, Water from Hereford, Texas, which contained
3.4 ppm F plus other naturally occurring elements.
This was used to see if other naturally occurring elements might have an additional effect.
The rats were weighed once each week,
in order to determine the effect of the low
calcium diet and fluoride on growth (see
table 1). The amount of diet eaten by each
group was weighed and the drinking water
consumed by each subgroup was measured. At the end of the seventh week, the
rats were anesthetized with ether and
x-rayed in order to compare bone den-
sities. The rats were again x-rayed at the
termination of the experiment.
The rats were sacrificed by an overdose
of ether over a period of five days beginning with the one hundred sixth day and
ending with the one hundred tenth day of
the experiment. Their bodies were x-rayed
in groups of four and then both femurs
were extracted. The femurs were cleaned
of muscle tissue, weighed, measured, and
placed in air-tight polyethylene containers
in order to prevent drying before testing.
At the time of sacrifice, the adrenal glands
were removed and weighed in order to determine if the rats had been stressed due
to the added fluoride in their drinking
water.
An Instron Universal Testing Instrument was employed to test the femurs for
strength and flexibility (fig. 1 ) . A load
cell was chosen which measures forces
from 0-200 pounds with chart recording
setting at 0-20 and 0-50 pounds respectively. A high-speed electronic graphic re-
Figure 1
307
FLUORIDE AND PHYSICAL PROPERTIES OF BONE
corder, driven synchronously with the
tester, recorded the forces and deflections
exerted on each femur. The accuracy of
the system is better than 0.25% at all load
ranges.
A special metal holding device for the
femurs was used so that the diaphysis was
loaded anteriorly at the midpoint, after the
method of Bell, Cuthbertson and Om (’41),
and Weir, Bell and Chambers (’49) (see
fig. 2a). The adjustable, metal supports,
A and A’, were so adjusted that points of
contact or distance, between supports was
fixed at 0.75 inch (19.05mm) apart on
the posterior aspect of the bone. The force,
F, was applied midway between these supports on the anterior surface. The metal
supports and metal used for applying the
force were rounded slightly in order to
avoid cutting into the bone as the force
was being applied. Figure 2b is a representation of a photographically enlarged
view of the femur at the fracture site. The
crosshead or screw driven force was applied at a constant rate of 0.1 inch (2.54
mm) per minute. The chart speed was
set at ten inches (254 mm) per minute.
Every inch (25.4 mm) of chart on the y
axis represents 0.01 inch (0.254 mm) deflection (see fig. 3). The Instron machine
allows the testing speed to be precisely
controlled, thus giving a constant rate of
specimen deformation independent of the
load applied. The synchronously driven
chart provided a permanent record of the
change in resistance to the load applied
and the corresponding deflection. The
load at the elastic limit, maximum load,
and load at the breaking point are measured on the x axis, the amounts of deflection at these loads are measured on the y
axis. The amount of energy absorbed is
calculated by dividing the area under the
recorded stress-strain curve by the bone
volume. Based on the preceding data
Young’s modulus of elasticity was applied.
From the latter, stiffness and flexibility
were determined, the one being the reciprocal of the other.
Young’s modulus of elasticity, E, is a
measure of the stiffness of the material
being tested. It is the coefficient of the
elasticity or the ratio of unit stress-unit
deformation. The formula for E, for a
beam loaded at the center, is E =
where “ W is the load at the elastic limit,
“L” is the length between supports A and
A‘, “y” is the amount of strain or deflection
at the elastic limit and “I” is the moment
of inertia (see figs. 2a, 3, table 3 ) . The
latter concerns the bending moment about
the neutral axis and is related to the configuration of the bone at the point of
breaking. Since the cortex is nearly elliptical on cross-section, “I” can be derived
from the formula I = 4$ (BD3-bd3) (see
fig. 2b).
The formula for E assumes that the
configuration of the cortex of the femur
between supports and its cross-sectional
area are constant. Admittedly the femoral
I
Force(W)
I
I
t
t
Figure 2
I*
b
-I
I
I
!
308
DEXTER F. BEARY
DEFLECTION
Y, =elastic limit
rn axi rn u m
Y3=breaking point
Y2
LOAD
Load -deflection
recording
Wl= elastic limit
W2= maximum
W3: breaking point
Figure 3
shaft changes slightly in configuration
along the shaft, being somewhat greater
in diameter towards the ends where the
cortex is proportionately thinner. However, comparison of computer-calculated
and actual volumes of corresponding
lengths of eight femurs, as described below, suggests that one is justified in handling the calculations as though the configuration and surface area of the cortex
remain constant.
The stiffness of the femoral shaft, or its
resistance to bending is proportional to
EI. The flexibility, or ease of bending
would be be proportional to
The
stress, or force per unit area, on the bone
substance is greater on the outer surface
of the cortex where the load is applied.
The amount of stress per unit area of bone
is derived from the formula S = y ( w h e r e
D is the outside diameter of the bone in
the direction of the applied force - see
fig. 2b).
The next step was to derive the bone
volume. For greater accuracy the surface
areas of the bones at the fracture site
were computed by taking graph paper
tracings of the outer and inner circumferences of the cortex of the bone which had
been photographically enlarged. A Photo-
&.
cell Scanner and a Calcomp 685 Plotter
in conjunction with an IBM 1620 computer
were employed for this purpose. The bone
volume was calculated from the area of
each femur and the distance, L, between
supports. This bone volume was utilized
in computing the amount of energy absorbed before breaking.
The reliability of the computer method
of determining the bone volume was confirmed by the water displacement method
which gave figures averaging only 3%
higher than those calculated by the computer. One could never be sure that all the
air bubbles had been cleaned from the
specimens and it is difficult to accurately
read the volumetric scale. The close agreement coupled with difficultly in actually
measuring volumes favored the computer
method. If any error is introduced, it would
be consistent with all bones tested, since
they were compared with those of the control animals.
The bone between supports was cut out
carefully, degreased and weighed. Finally
the fluoride uptake by the femoral shaft
tested between supports A and A' (see fig.
2a) was calculated by dissolving this section of bone in 4 cm3 of a 1 :1 dilution of
concentrated HCl solution. This was then
diluted to 50 cm3 using a 2% NaOH solu-
309
FLUORIDE AND PHYSICAL PROPERTIES OF BONE
tion. The pH was adjusted to 2.07 2 0.03,
since this is considered a desirable range
for reading fluoride ion activity. An Orion
Fluoride Activity Electrode was used in
conjunction with a reference electrode for
the fluoride determination according to
the method devised by Frant and Ross
('66). The accuracy of this method has
been confumed by Lingane ('67). The
calculations were recorded as mg of F
per bone segment and percentage F by
weight (see table 1).
A statistical analysis was done to determine the validity of the conclusions
reached.
perimental period was 2.3 liters per rat
and was essentially the same for the animals in each subgroup. Consequently, the
fluoride assay was directly proportional to
the dosage given. The percentage content
of fluoride by weight was strikingly different in Groups I and I1 at the 10 ppm F and
45 ppm F dosage levels, there being a percentage increase per bone segment in the
low calcium group. There is also a difference between the Hereford water subgroup (3.4 ppm F) and the corresponding
3.4 ppm F in deionized water subgroup,
the former containing less fluoride than the
latter.
O n adrenal weight. Metabolic stress,
RESULTS
due to possible toxic effects of the higher
The principal data is recorded in tables fluoride levels used, was excluded with regard to having an influence on the results
1-5.
since there was no significant difference
Effects of the diet and fluoride
in the adrenal weight between Group I
On body weight. As shown in table 1 and Group I1 or between any of the subthe rats on the low calcium regimen (Group groups (table 1). Because of this nega11) whose diet contained only 0.1% of cal- tive finding a dry weight determination
cium show 7% less weight gain than those on the thymus was not done.
O n linear bone measurements. No apon an adequate calcium diet (Group I).
Group I1 consumed 43.8 kg of diet over the preciable differences in bone lengths were
experimental period; Group I, 47 kg. The seen between Group I and Group I1 or
amount of weight gained was in direct pro- between any of the subgroups. Outside
portion to the amount of diet consumed. diameters of the femoral cortex likewise
It bore no relationship to the amount of were unchanged but the inside diameters
fluoride administered during the experi- showed a marked increase in those rats
maintained on a low calcium diet resulting
mental period.
072 amount of jhoride per bone segment in a marked thinning of the cortex. This
between supports and its percentage value. was reflected in the volume of the cortical
The average water consumed over the ex- bone between supports A and A' (fig. 2a),
TABLE 1
Means of rat femur analysis
ppm
Bqdy
weight
x;tGE
between
supports
I
(Adequate
Ca>
Group
I1
(LowCa)
1 Hereford water.
2
I and I1
z:$z per
"f:l;:
supports
(dry,
fat free)
bone
segment
P~~~~~~ Adrenal
adrenal
Ratio
weight weight wt,body wt
(mg)
(mg/100 pm)
<mm3)
(pm)
<mg)
0
3.4 1
3.4
10.0
45.0
251.250
257.125
248.125
258.625
254.375
102.846
106.310
99.935
102.930
103.775
0.225
0.223
0.218
0.226
0.230
0.014
0.045
0.059
0.148
0.693
0.01
0.02
0.03
0.07
0.30
56.437
52.387
65.362
59.637
55.975
22.520
20.363
26.376
23.151
21.969
0
3.4 1
3.4
10.0
45.0
237.875
239.625
231.428
238.375
230.285
66.981
66.751
65.138
67.211
67.157
0.133
0.144
0.129
0.141
0.130
0.006
0.035
0.041
0.149
0.671
0.02
0.03
0.16
0.76
57.325
59.500
59.042
56.550
60.014
24.041
24.849
25.463
23.891
26.271
(pm)
Group
- groups
Weight 2
Bone weights obtained from corresponding segment of opposite femur.
310
DEXTER F. BEARY
which averaged 35% lower in the low example of the graph recording with the
calcium group (table 1 ) . Since the com- load points at which the load at elastic
puted bone volume in the subgroups of limit, m a x i m u m load, and load at breahGroup I1 is essentially the same in every ing point were taken for each femoral
instance, it is evident that it is the cal- shaft tested. The corresponding defleccium deficiency and not fluoride admin- tions are given for each load. The results,
istration or the lack of it that was re- as seen in the tables, show significant differences when comparing the variables of
sponsible.
O n loads and deflections. The loads corresponding subgroups of Groups I and
and deflections listed in table 2 were cal- I1 and also when comparing certain subculated primarily for use in formulas em- groups within each of these same groups.
ployed in computing the results of the vari- This difference is reflected by the amount
ables listed in table 3. Figure 3 shows an of calcium and/or fluoride given.
TABLE 2
Means o f rat femur analysis - group I and 11
2t$gt
limit
ppm F
(
~
~
Deflection
elastic
limit
~
t
Maximum Deflection
load
maxlmum
~(Newtons)
~
~
)load
(mm)
Group
I
(Adequate
ca>
Group
I1
(Low Ca)
1 Hereford
Load at
bT:
EFg
(Newtons)
Deflection
breaking
point
(mm)
0
3.4'
3.4
10.0
45.0
88.946
85.386
82.744
84.550
82.355
0.255
0.273
0.261
0.263
0.303
108.416
106.106
104.104
107.943
101.100
0.385
0.425
0.401
0.413
0.471
108.416
106.106
104.104
107.943
101.100
0.385
0.425
0.401
0.413
0.471
0
3.4
3.4
10.0
45.0
37.465
35.851
39.129
34.911
23.617
0.276
0.253
0.294
0.274
0.306
57.406
57.802
57.217
53.040
34.432
0.637
0.654
0.704
0.750
1.038
50.815
52.623
48.856
43.889
29.942
0.868
0.854
0.895
1.020
1.591
water.
TABLE 3
Rat femur analysis
group I and I1
-
ppmF
Moment
in2ia
(mm4)
Group
I
(Adequate
Ca 1
Group
I1
(Low Ca)
Stress
elasticat
limit
(N/=S)
Streps at
load
(N/mmz)
¶Young% IFlexImodulus
bility
Stiffnew
1
1Energy
absorbed
(N/mm2) (:l/Nmmz ) (N/mmz) (N-M/mma)
x 10-2
x 105
45.0
3.629
3.648
3.470
3.463
3.671
248.385
236.950
240.453
242.660
226.782
302.887
294.280
302.773
309.919
278.445
142.815
125.327
133.683
134.677
107.986
1.984
2.233
2.193
2.172
2.578
517.308
455.230
460.363
463.099
393.696
2.273
2.350
2.264
2.40
2.59
0
3.4'
3.4
10.0
45.0
2.673
2.876
2.714
3.057
3.019
141.766
129.146
146.796
121.186
81.610
216.900
207.245
211.620
183.190
118.507
73.038
72.187
72.864
61.740
37.546
5.185
4.913
5.225
5.469
9.184
194.633
204.742
195.719
185.712
113.065
5.283
5.287
5.370
5.704
6.409
0
3.4'
3.4
10.0
x10-2
Xlor
Note: 1. The figures have been altered by powers of ten in order to conserve space., e.g.. Young's
modulus mean of subgroup 1 which is actually 14181.500 N/mmZ has been reduced to
142.815 N/mmZ
The loads have all been expressed h terms of Newtons (N), t h e international measuring
standard of force 1 lb. wt. = 4.45 Newtons (N).
3. To convert to kg/cms for E, (Young's modulus) multiply above values by 100/9.8 (approximately 10). e.g.. 14281.5 x 10 = 142815 kg/cmS.
4. The range of standard deviations of data (not shown) from each rat femur, gi*g
v&ables of each group, was not wide enough as to SignMcantly affect the analysls of the
means.
2.
FLUORIDE AND PHYSICAL PROPERTIES OF BONE
On moment of inertia. The moment of
inertia was naturally lower for the calcium
group as the cortex was much thinner.
The fluoride had no effect on this calculation since the thickness of the cortex was
unaffected by the uptake of fluoride. As
previously shown in the section on methods and materials, the moment of inertia
is necessary in applying the formula for
Young's Modulus of Elasticity, E.
On the variables. (Stress at Elastic
Limit, Stress at Maximum Load, Young's
Modulus of Elasticity, Flexibility, and Stiffness)These variables all show highly significant differences between the corresponding subgroups of Group I and Group
11 and therefore for the groups as a whole.
All except flexibility are greater for the
adequate calcium group, flexibility being
greater for the low calcium group.
To determine the effect of fluoride on
the rat femur, Anova (analysis of variance) was run to determine differences
between means of the first five subgroups
and the last five subgroups for each of
the 13 variables listed in tables 1 and 3.
Table 4 gives the variables for which there
were significant differences among means
of the different subgroups 1-5 and 6-10
and their level of significance. For these
variables, t tests were performed between
the control groups and each of the fluoride
treatment groups (see table. 5).
311
tent of bones in fluorotic subjects may
have been due to subnormal living standards and a low nutritional status. McClure
et al. ('58) who examined the bones of a
subject with a history of prolonged exposure to water containing 8 ppm fluoride
found the amount of Ca F in the skeleton
to be quite uniform being within the narrow range of 0.517and 0.653% in the dry
fat-free samples. Soriano ('65) found, in
human skeletal fluorosis, considerable variation in the bone F content, with an observed high of 1.34% in the coccyx and
a low of 0.12% in the tibia.
Experiments with rats have clearly
shown that when fluoride is added to the
drinking water there is an increase in the
size of the apatite crystals and they are
fewer in number (Menczel et al., '63 and
Eanes et al., '65). The fluorapatite crystals in bone while they have a close structural similarity to the bone salt, hydroxyapatite, appear to be less reactive with
body fluids, resulting in decreased resorption (Rich and Ivanovich, '65). Fluoride
in the amount of 50 ppm in the drinking
water of female rats was shown by Gedalia
et al. ('64) to significantly increase the
calcium content of the bone ash without a
corresponding increase in phosphorus. It
is noted, however, that McCann and
Bdloch ('57) found no such increase on a
100 ppm dosage level.
Bell et al. ('41), who used a bending
DISCUSSION
test, found that the midpoint of the rat
There have been numerous reports con- femoral shaft is the weakest. They atcerning the physiology, crystalline stmc- tribute this to the smaller diameter and
ture and ash content of bone on adequate greater bending moment. According to
and low calcium diets, with and without their findings, bone is elastic up to the
fluoride (Boelter and Greenberg, '40; Light moment of breaking. Weir et al. ('49),
and Frey, '41; Harrison and Fraser, '60; after further testing, revised this estimate
El-Maraghi et al., '65). Studies in humans stating that the dry rat femur is elastic
as by Leone et al. ('55) have suggested only up to 79% of its breaking stress. They
that what is generally considered in this calculated the modulus of elasticity (E) to
country to be an excessive fluoride content be 1.6 X lo6 lbs/ing. Evans ('64) finds
in the water ( 8 ppm) and which causes that the method they used for determining
mottling of the teeth, has a beneficial ef- extreme or outer fiber stress is only valid
fect in counteracting osteoporosis. Zipkin at the elastic limit. Evans and Lebow
et al. ('58) found that fluoride in the ('51) using wet human bone samples
drinking water of human subjects up to showed that it is the middle third of the
4 ppm increases their fluoride content femoral shaft that has the highest modulus
proportionally to the amount of exposure of elasticity. The figure they give is 2.11
but does not affect the ash content of the x lo6 lbs/ina. Drying was found to inbones. In their opinion previously re- crease the modulus of elasticity by 17.6%.
ported very high values for fluoride con- Currey ('59) found the value of "E" for
312
DEXTER F. BEARY
TABLE 4
Significant Variables: Means and Levels of Significance
Group I
Subgroups
2
(HW-3.4 pprn F)
Mean (X)
5z
P
4
3
(3.4ppm F)
P
P
(10.0 ppm F)
-X
P
5
(45.0 ppm F)
X
P
Stress at Elas.
Limit (N/mm2) 248.4
237.0 0.058
240.5 0.148
242.7 0.241
226.8 0.000
Stress at Max.
load (N/mm2)
302.9
294.3 0.256
302.8 0.987
309.9 2.76
278.4 0.002
Young’s Mod.
of Elas. (N/
mm2 x
142.8
125.3 0.019
133.7 0.169
134.7 0.238
108.0 0.000
Flexibility (1 /
Nmmt x lo5)
1.98
2.23 0.038
2.19 0.040
2.17 0.050
2.58 0.000
Stiffness (Nmmz
x 10-2)
517.3
455.2 0.028
460.4 0.036
463.1 0.036
393.7 0.000
0.014
0.043 0.000
0.060 0.000
0.145 0.000
0.669 0.000
9
(10.0 ppm F)
10
(45.0 ppm F)
Fluoride Assay
(mg/mm3
x
102)
Group I1
Subgroups
6
(Control)
7
(HW-3.4 pprn F)
Mean (X)
-X
P
8
(3.4 ppm F)
X
P
F
P
-X
P
Load a t Elas.
Limit
(Newtons)
37.47
35.85 0.274
39.12 0.334
34.91 0.141
23.62 0.000
Maximum Load
(Newtons)
57.41
57.80 0.797
57.22 0.699
53.04 0.010
34.43 0.000
Stress a t Elas.
Limit
( N/mm2 )
141.8
129.1 0.080
146.8 0.547
121.2 0.013
81.6 0.000
Stress at Max.
Load
(N/mm2 1
216.9
207.2 0.150
211.6 0.578
183.2 0.000
118.5 0.000
Young’s Mod. of
Elas. (N/mm2
x 10-2)
73.0
72.2 0.788
72.9 0.963
61.7 0.003
37.5 0.000
Flexibility (1/
N m m 2 x 105
5.19
4.91 0.112
5.23 0.875
5.47 0.213
9.18 0.000
Stiffness (Nmm2
x 10-2)
194.6
204.7 0.116
195.7 0.905
185.7 0.246
113.1 0.000
Energy Absorbed
(N-M/rnms
x 104)
5.283
5.287 0.991
5.370 0.836
5.704 0.324
6.409 0.008
0.009
0.054 0.000
0.064 0.000
0.225 0.000
0.991 0.000
Fluoride Assay
(mg/mm3
x
102)
Note:
X. mean of each variable.
p. level of significance of each variable.
313
FLUORIDE AND PHYSICAL PROPERTIES OF BONE
TABLE 5
A list of the correlation coeficients betureen the fluoride assay and
the significant variables of subgroups 1-5 and subgroups 6-10
~-
~~
Group I
Subgroups 1-5
Variable
Assay - Stress at Elas. Limit
Assay - Young’s Mod. of Elas.
Assay - Flexibility
Assay - Stiffness
n
r
P
40
40
40
40
-0.3528
- 0.4827
0.4772
- 0.4256
0.0256
0.0016
0.0019
0.0062
n
r
P
38
38
38
38
38
38
38
38
-0.7702
- 0.8571
-0.7323
- 0.8573
-0.8359
0.8440
-0.8307
0.4532
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0000
0.0043
Group I1
Subgroups 6-10
Variable
Assay - Load at Elas. Limit
Assay - Maximum Load
Assay - Stress at Elas. Limit
Assay - Stress at Max. Load
Assay - Young’s Mod. of Elas.
Assay - Flexibility
Assay - Stiffness
Assay - Energy Absorbed
Note: n number of samples.
r,’correlation coefficient.
p. level of si@cance.
cortical bone from the ox femur to be
about 2.5 X 10‘ lbs/inz or approximately
ten times the value for collagen. Consequently any change in the crystalline structure would significantly alter “E.” Semb
(’66) found no significant difference in
the breaking strength of longitudinal cortical bone sections of equal dimensions
from femurs of normal and osteoporotic
dogs. He states that any weakness of the
osteoporotic bone is due to the thinning
of the cortex. His data is recorded in
Newtons (the metric unit of force).
Ascenzi and Bonucci (’67) studied individual osteons, wet and dry, from both human and ox cortical bone. The tensile
strength and modulus of elasticity were
found to be greater in the dry specimens,
particularly in those in which there was a
marked longitudinal arrangement of collagen fibers in successive lamellae. The
“ E for wet human specimens with longitudinally disposed collagen fibers was
119400 kg/cmz (1.7 X lo6 lbs/in2). For
corresponding ox specimens, the “ E was
149600 kg/cmz (2.12 X lo6 Ibs/ine). Vincentelli (’68) is doing similar work in this
field.
Experimental studies on the effects of
fluoride on breaking strengths and elastic-
ity of bone have been few. Jovanovits
(’44) whose work is summarized by
Uehlinger (’64) found that in both the
very young and the older growing rabbit,
fluorine in the form of various salts in the
amounts of 5mg/kg and 50mg/kg of
body weight per day respectively, over a
150 day period, diminished the modulus
of elasticity proportional to the amount of
fluoride administered. He also noted a significant lowering of the breaking strength.
The effects were more marked in the first
group. Gedalia et al. (’64) also found
that, in young growing rats given 50 ppm
F in their drinking water for four weeks,
the breaking strengths of their dry defatted
femurs were significantly lower than those
of the control animals. It is yet to be confirmed that fluoride administration is
accompanied by the increased calcium content of the bone they have reported. Normally bone strength is directly proportional
to its calcium content (Shah et al., ’67)
and this would not be consistent with
their findings. Where lesser doses of fluoride have been employed no effects on the
physical properties of bone have been
noted. Smith and Keiper ( ’ 6 5 ) , in testing
the viscoelastic properties of the femora
of Beagle dogs, found that 1.0 mg F in
314
DEXTER F. BEARY
their diet over a period of 122 days had
no demonstrable effect on bone elasticity.
Saville ('67) found no significant difference between the breaking strength of the
femur taken from weanling male rats
given 20 ppm M in the drinking water for
123 days and those of the controls.
The results of this study show that,
when the diet of weanling rats is low in
calcium (as seen in the controls), for
fifteen and one-half weeks, there is a
slight loss in body weight (table 1) as also
noted by Boelter and Greenberg ('40).
Fluoride administration at the dosage
levels employed did not appear to be the
cause. On the low calcium diet the outer
dimensions of the femur remain unchanged but the inner diameter is increased, thus producing a thinner cortex
and decrease in bone volume (table 1).
This observation agrees with that found
by Light and Frey ('41) and is not unlike
that seen in the metabolic disease osteoporosis. There is abundant evidence that a
low calcium diet can induce this disease
(McClendon et al., '62; Whedon et al.,
'63).
Each of the subgroups within each
major group drank essentially the same
amount of water (18.6 liters rt 0.4). In
the low calcium group, at the 10 ppm and
45 ppm fluoride administration levels, the
percentage of fluorine in the bone is seen
to be higher than in the adequate calcium
group given the same dosage levels of
fluoride. Comparing the subgroups within
the two major groups given drinking water
from Hereford, Texas, containing 3.4 ppm
F, with those subgroups with the same
amount of fluoride in their deionized
drinking water, it is seen that the fluoride
percentage is less in the Hereford water
subgroup. This could be due either to
the fact that the drinking water supplied
additional calcium, or that other elements
such as magnesium influence fluoride absorption. Such influence finds corroboration in the review article of Marier et al.
('63). Hereford water contains 61 mg/
liter of magnesium in addition to 64mg/
liter of calcium.
With respect to the effects of fluoride
administration on the physical properties
of the femora of subgroups 2-5 of Group
I (adequate calcium diet) it is noted that
there is no significant difference in
strength (maximum load and/or load at
breaking point) at any of three fluoride
dosage levels. A significant increase in
flexibility was apparent only in the 45
ppm F subgroup (tables 3 and 4). Such
an increase in flexibility was also noted
by Jovanovits ('44). While he recorded a
lowering of the breaking strength it was
not proportional to the amount of fluoride
given. Further, the dosages he employed
were much larger than any used in this
study. Gedalia et al. ('64), like the lastmentioned, also record a lowering of
the breaking strengths in femurs of rats
given 50.0 ppm F in their drinking water
and for only four weeks. They give no
indication as to flexibility. The 20.0 ppm
F employed by Saville ('67) over a 14week period had no effect on the breaking
strength of the rat femur.
The effects of fluoride administration in
Group I1 (low calcium diet) are more apparent even at lower dosage levels. A decrease in strength and an increase in
flexibility are significant in both the
10.0 ppm F and 45 ppm F subgroups
which are related proportionately to
the greater percentage of fluoride per
bone segment (tables 1, 4, 5). The significant increase in the amount of energy
absorbed before breaking found only in
the 45 ppm F subgroup indicates that the
bone is able to withstand a greater bending force at this dosage level, even though
it is weaker from the standpoint of load
bearing.
Osteoporosis-like changes (osteopenia)
will develop in the rat if calcium is deficient in spite of fluoride administration in
the drinking water at the relatively high
dosage of 45.0 ppm F. This is demonstrable radiologically as early as the eighth
week. Fluoride in both the adequate and
low calcium subgroups will increase flexibility. However, in the low calcium subgroups the increased flexibility is associated with a significant diminution in bone
strength.
Whether or not extra fluoride prevents
fractures is still a matter of conjecture.
Since fluoride in high doses weakens bone,
it might tend to predispose to compression fractures of vertebrae as is not uncommon in areas where endemic fluorosis
FLUORIDE AND PHYSICAL PROPERTIES OF BONE
is present. Shambaugh and Petrovic ('68)
believe fluoride availability to be a major
contributory factor in the prevention of
osteoporosis as seen in the human since
they believe the fluoride ion slows the resorptive phase of the remodeling process
and promotes calcification. The calcium
levels employed in this experiment were
not such as to test this hypothesis. It
should be noted that Adams and Jowsey
('65) have stressed the importance of
concomitant calcium supplementation in
high fluoride therapy for osteoporosis. The
results of this study, as shown in tables 1,
2 and 3, also clearly indicate that adequate
calcium is needed in the diet in order to
give the bone strength and that fluoride,
at the 45 ppm range, will increase the
flexibility.
Finally, in this study there is some indication that elements other than fluorine,
such as occur naturally in the drinking
water at Hereford, Texas, may influence
the physical properties of bone. One of
these is possibly magnesium as has been
suggestea by Barnetc('54) and Marier et
al. ('63).
ACKNOWLEDGMENTS
Computation for this research was performed by the Lorna Linda University Scientific Computation Facility supported in
part by NIH grant FR 00276-03.
Special thanks is given to Professor
Harold Shryock, Head of the Department
of Anatomy, Lorna Linda University, for
the facilities provided for this project and
for his encouragement. The following individuals, University staff members, were
especially generous of their time: Jerry
Chrispens of the computational facility;
Drs. U. D. Register, J. Riggs, and W. H.
Roberts; and Mrs. Lucille Innes of the
Audiovisual Department did the art work.
Dr. Earl S. Gerard of the Upjohn Company, Kalamazoo, Michigan, suggested the
subject of the investigation and gave many
helpful suggestions.
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