Journal of
Bone and
Mineral
Metabolism
J Bone Miner Metab (1998) 16:88-95
© Springer-Verlag 1998
The effect of calcium supplement given with a mixture of calcium
carbonate and calcium citrate on the mandibular alveolar bone
of pubertal rats
MAN QIN, ZUYAN ZHANG, KENSHI MAKI, MARIKO NAITO, AKIKO MORIMOTO, and MITSUTAKA KIMURA
Department of Pediatric Dentistry, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580, Japan
Abstract: To determine whether the mixture of calcium carbonate and calcium citrate, an oral calcium supplement made
from sea shell that is called UNICAL, had any beneficial
effect on bone debilitation, we examined the mandibular alveolar bone of pubertal Wistar male rats by bone mineral
mass, Ca/P ratio microanalysis, and scanning electron microscopy. (1) Bone mineral mass of UNICAL-fed rats in the lowcalcium experiment group was not significantly different than
the control group, although its value was significantly higher
than in pair-fed standard diet rats. However, although the
bone mineral mass of UNICAL diet rats in the calciumdeficient experiment group was significantly higher than in
pair-fed standard diet rats, it was significantly lower than in
the control group. (2) All Ca:P ratio values in experiment
groups were significantly lower than that in control group.
Ca : P ratio values of UNICAL diet rats were significantly
higher than those of pair-fed standard diet rats in experiment
groups. Separately, Ca:P ratio was not significantly different
between UNICAL diet rats of the calcium-deficient experiment group and standard diet rats of the low-calcium experiment group.
(3) On scanning electron microscope
observation, UNICAL diet animals were observed to have
more sufficient calcareous microdepositions in bone remodeling areas, which was thought to be one of the morphological
indications of bone formation, reflecting active calcium utilization in bone metabolism, than pair-fed standard diet animals. These results suggested that the mixture of calcium
carbonate and calcium citrate had a positive effect on bone
debilitation to a certain extent in growth-period rats.
Key words: calcium carbonate and calcium citrate mixture,
bone debilitation, bone mineral mass, Ca:P ratio microanalysis, scanning electron microscopy
Introduction
Calcium is an essential nutrient for normal growth and
development.
Adequate dietary calcium builds the
skeleton and helps to prevent skeletal disorders during
childhood and adolescence [1-3]. Inadequate dietary
calcium during the critical growth and building period
may result in failure to reach peak bone mass [4]. A
strong bone structure in young adulthood is likely to be
one of the most important factors for preventing
osteoporosis and associated fractures later in life [5,6].
Approximately 20%-30% of peak bone mass can be
influenced by environmental factors; another 60%-80%
is explained by genetic variation [7,8]. Although it is
generally recognized that optimum calcium intake is
best obtained from food sources, calcium supplements
are destined to become an important source of dietary
calcium because the lesser food intake of modern
humans compared with the food intake of humans during evolution has caused difficulty in obtaining an adequate intake of calcium [9-11].
In addition, more and more dentists' attention has
been directed to the influences of systemic bone debili-
tation in the dental field, for instance, on bone repara-
tion after periapical abscess or periapical granuloma
[12,13], reorganization of periodontitis [14,15], or prog-
nosis of an implant [16]. This investigation was undertaken to determine the effect of a calcium supplement,
a mixture of calcium carbonate and calcium citrate
made from sea urchin shell that is called UNICAL, on
the mandibular alveolar bone of pubertal rats.
Materials and methods
Animals and treatments
Offprint requests to: M. Qin
Received: Nov. 25, 1997 / Accepted: Jan. 29, 1998
09
Thirty Wistar male rats 8 weeks old and weighing about
240g,
maintained by Seiwa Experimental Animal
Research Institute, were housed in small cages individu-
OK
M. Qin et al.: Effect of Ca supplement on rat bone
89
ally under similar conditions. All the rats' food was
made by Oriental Yeast (Tokyo, Japan). In the control
group, rats were fed on a standard diet, containing
0.449% calcium, 0.608% phosphorus, and 1000IU vitamin D3 per 100 g, with tap water for 5 weeks. In the lowcalcium experiment group, rats were fed a low-calcium
diet (calcium component is 30% of standard diet) with
tap water for the first 3 weeks; then they were allocated
into two treatment groups and pair-fed on a standard
diet or the UNICAL diet, which is a mixture of calcium
carbonate and calcium citrate, containing 1.697% calcium, 0.612% phosphorus, and 1000IU vitamin D 3 , with
tap water for a further 2 weeks. In the calcium-deficient
experiment group, rats were fed a no-calcium-component diet with distilled water for the first 3 weeks; they
were then treated the same as the low-calcium experiment group for a further 2 weeks. These rats were killed
after being fed for 5 weeks. Their mandibles were removed and divided into two parts symmetrically between the central incisors using a dental diamond disk
(#62; Shfu, Japan) with spraying water, and later treated
for observations of bone mineral mass, Ca/P ratio microanalysis, and scanning electron microscopy.
0.1 mm/s (Fig. 1). The average of peak values of cortex
bone mineral mass on the lingual side and the labial side
was determined for the bone mineral mass of the mandibular alveolar bone and calculated according to thickness of aluminum.
Sample preparation for Ca/P ratio microanalysis
The samples were cut from the incisor alveolar crest to
1 mm in front of the molar area, at thickness of 3 mm,
vertical to the mandibular border, by dental diamond
disk (#62; Shfu with spraying water. After fixation with
10% neutral formalin, these samples were dehydrated
through a graded ethanol series and treated in 2-methyla-propanol, dried in t-butyl alcohol by freeze-drying
(ID-2; Japanese Electric, Japan). Sections were embedded in resin, then abraded, polished, and sputtered with
aurum. Later they were observed under a scanning
electron microscope (JSM T-300; Japanese Electric)
equipped with energy-dispersive X-ray microanalysis
(JED-2000; Japanese Electric) to determine their
calcium-to-phosphorus ratios.
Sample preparation for scanning electron microscopy
Bone mineral mass
The mandibles were kept in 10% neutral formalin until
radiographs were taken. These samples were fixed with
tape on a aluminum plate (length, 15.00 mm and thinned
from 1.50 to 0.01 mm overall. Soft radiographs (ESM-2;
Softex, Japan) were taken at 28 kvp, 6 mA, 60s,
and 70-cm distance. These films were observed by a
Konica Densitometer (PDS-15; Konica, Japan).
The survey light passed through 1 mm in front of the
molar region and vertical to the mandibular border at
The samples were cut as in preparation for Ca: P ratio
microanalysis, and fixed with 2.5% glutaraldehyde for
1 h after being cleaned with 10% sodium hypochlorite
solution by supersonic vibration to eliminate adhesion.
They were rinsed by buffered phosphate acid (pH 7.2)
before postfixation. Postfixation was performed in 1
osmic acid buffer solution (pH 7.2) under 4°C for 2h.
After fixation, these samples were dehydrated through
a graded ethanol series, treated with 2-methyl-apropanol, then dried in t-butyl alcohol by freeze-drying
Fig. 1. Observation of bone mineral mass
in mandibular alveolar bone
survey light pathway
Table 1. Results of bone mineral mass (mmAl)
Low calcium
Group
Mean ± SD
Calcium deficient
Control
UNICAL diet
Standard diet
UNICAL diet
2.09±0.09
1.99±0.11
1.90±0.15
1.85 ± 0.13
Standard diet
1.79±0.17
M. Qin et al.: Effect of Ca supplement on rat bone
90
(ID-2; Japanese Electric). Specimens were sputter-
different than the control group, although its value was
electron microscope (JSM T-300; Japanese Electric).
significantly higher than pair-fed standard diet rats.
coated with gold and later observed under a scanning
a little lower than the control group. Its value was
However, bone mineral mass of UNICAL diet rats in
the calcium-deficient experiment group was signifi-
cantly higher than that of pair-fed standard diet rats, it
Results
was significantly lower than that of the control group
(Tables 1, 2).
Bone mineral mass
Bone mineral mass of UNICAL diet rats in the
low-calcium experiment group was not significantly
Table 2. F-test results of bone mineral mass
A
B
B
C
*
D
E
*
**
*
*
**
**
for calcium (Ca) and phosphorus (P), with a low backD
*
**
**
E
**
**
ratio microanalysis
A typical spectrum of bone demonstrated strong peaks
C
A
Call'
**
**
*
*
*
A, control group; B, low-Ca • UNICAL diet group; C, lowCa • standard diet group; D, Ca-deficient • U NICAL diet group; E,
Ca-deficient -standard diet group
*, P < .05; **, P < .01
ground and no peaks from other elements interfering
with calcium or phosphorus (Fig. 2). All Ca: P ratio
values in experiment groups were significantly lower
than that in the control group. In experiment groups,
Ca : P ratio values of UNICAL diet rats were significantly higher than those of pair-fed standard diet rats.
Separately, the Ca : P ratio was not significantly differ-
ent between UNICAL diet rats of the calcium-deficient
experiment group and standard diet rats of the lowcalcium experiment group (Tables 3, 4).
Sample ID: 8+5-13 standard 72
Energy Range: 0 - 20 keV 10 eV/ch
Preset: Live Time 100
Real Time: 109.79
97. Deadtime
Sec.
Seconds
Live Time: 100.00
Sec.
1320 Counts/Second
Acquisition date: 19-Aug-97
Acquisition time: 10:47:50
Cf s 4Y
N
V
I
1 28
eV
Cursor
9
t
3.689 keV 1
2479
5.930 keV
Fig. 2. The spectrum of normal bone
showing strong, characteristic peaks for
calcium and phosphorus with a low
background, and no peaks from other
elements interfering with calcium or
phosphorus
at
M. Qin et al.: Effect of Ca supplement on rat bone
91
Table 3. Results of calcium-to-phosphorus molar ratio (Ca: P)
Low calcium
Control
Mean ± SD
UNICAL diet
1.963 ± 0.091
standard diet
1.818 ± 0.042
1.715 -} 0.023
Calcium deficient
UNICAL diet
1.713 ± 0.027
standard diet
1.659 ± 0.045
Table 4. F-test results of calcium-to-phosphorus molar ratio
A
A
B
C
D
E
*
**
**
**
B
*
**
*
**
C
**
**
*
D
**
*
E
**
**
*
A, control group; B, low-Ca • UNICAL diet group; C, lowCa-standard diet group; D, Ca-deficient • UNICAL diet group; E,
Ca-deficient -standard diet group
*, P<.05;**,P<.01
Fig. 3. Scanning electron microscopy (SEM) image of bone
surface in control group. Osteocytic lacunae, at the wall of
which bone canalicali are opening, are emerging on the glossy
bone surface. Bar 10Etm
Scanning electron microscopy
In comparison with the control group (Fig. 3), the
experiment groups readily showed large areas of bone
resorption. In calcium-deficient experiment groups,
concave resorption foveas were observed deep with a
Fig. 4a,b. SEM image of calcium-deficient • UNICAL diet
animal. Large area of bone resorption shows deep concave
resorption foveas and clear division with bone matrix background (a). Sufficient calcareous microdepositions appear in
the bone matrix of resorption area while many slender collagen fibers are seen on the upper surface of the bone matrix
(b). Bar 10µm
calcareous microdepositions appearing in the bone ma-
trix of resorption areas and many slender collagen fibers
on the upper surface of bone matrix (Fig. 4). In com-
clear division from the background of bone matrix.
parison with UNICAL diet animals, pair-fed standard
canalicali were opening, for emergence in resorption
foveas. In UNICAL diet animals were found sufficient
ing some calcareous microdepositions. The margins of
There were many lacunae, at the wall of which bone
diet animals showed gross resorption foveas precipitat-
concave resorption fovea were irregular; some areas did
b
92
Fig. 5. SEM image of calcium-deficient • standard diet animal.
The concave resorption foveas are gross with irregular margins; some had no very clear division from the bone matrix
background. Collagen fibers were often observed in the bone
matrix, and in some places were seen crossed into a network
on the upper surface of the bone matrix. Calcareous microdepositions were also found in the resorption area, but less
often than in pair-fed UNICAL diet animals. Bar 10µm
M. Qin et al.: Effect of Ca supplement on rat bone
Fig. 6. SEM image of low-calcium • UNICAL diet animal. The
resorption concave foveas are shallow and flat; their margins
are smooth and clear. Calcareous microdepositions are obviously fewer than in the calcium-deficient group. Bar 10µm
not show a very clear division from the bone matrix
background. Collagen fibers were more often observed
in bone matrix, and in some places they were seen
crossed into networks on the upper surface of the bone
matrix (Fig. 5).
In the low-calcium experiment group, large areas of
bone resorption also were seen. However, the resorp-
tion foveas were shallower and flatter and their margins
were smoother and clearer than in the calcium-deficient
experiment groups. In UNICAL diet animals of the
low-calcium group, fewer calcium microdepositions
were observed than in the calcium-deficient experiment
groups (Fig. 6). Correspondingly, they were rarely seen
in pair-fed standard diet animals (Fig. 7).
Fig. 7. SEM image of low-calcium -standard diet animal. The
resorption concave foveas are shallow and flat; their margins
are smooth and clear. Calcareous microdepositions are rarely
observed in the resorption area. Bar 10 µm
Discussion
Calcium supplements are becoming an important
source of dietary calcium and a basic defense against
prescribed calcium supplements and hundreds of differ-
a cancer-preventive role for calcium [17-19]. Oral cal-
shell and hydroxyatite calcium, and is a preferred
osteopenia [9-11]. Recent studies have also focused on
cium supplement is a very popular method of fortifying
inadequate dietary calcium because of its cheap cost,
convenient way of intake, and the minimal side effects
[9,10], although a controversy still exists as to its effect
in preventing the age-related decrease of bone mass
[20,21]. There are now more than a dozen commonly
ent formulations commercially available [22-25]. Cal-
cium carbonate is the major constituent in sea urchin
source of calcium supplements because of its high
density of elemental calcium, about 40% by weight.
Calcium citrate is another very popular formulation,
and there are now effervescent preparations available.
At 21 % by weight, the citrate preparations have a lower
calcium content but are considered much more soluble
M. Qin et al.: Effect of Ca supplement on rat bone
r
than calcium carbonate, which is especially important
for achlorhydric patients. The citrate ion may help to
modulate the propensity for the development of renal
stones [22].
The mixture of calcium carbonate and calcium citrate
(UNICAL) made from sea urchin shell is a kind of oral
calcium supplement. The results of this study suggested
a positive effect on bone debilitation in animals from
puberty to early adulthood. In low-calcium diet experimental animals, this process was recovered by UNICAL
diet. The bone mass of UNICAL diet experimental animals reached the level of control group; although the
value was a little lower than that of the control group
the difference between them was not significant, while
the value was significantly higher than in pair-fed standard diet animals. The latter had a significantly lower
value than the control group. In calcium-deficient diet
experimental animals, the bone mass of UNICAL diet
animals showed improvement in that the value was
significantly higher than that of pair-fed standard diet
animals, but it had not yet reached the level of the
control group.
Inadequate dietary calcium during the critical growth
and building period may result in failure to reach peak
bone mass, causing osteopenia, osteoporosis, decreased
skeletal integrity, and increased risk of fracture in later
life [4-6]. The importance of ample calcium intake in
early life has been made evident by recent clinical
studies [26-28] and experimental studies [29]. In the
present study, the experimental range was from puberty
to early adulthood of rats, thought to be the critical
period for peak bone mass gain. We gained satisfactory
results of augmented bone mineral mass with the
UNICAL diet in low-calcium experimental animals, but
the result was not completely satisfactory in calciumdeficient experimental animals. The results suggested
that the mixture of calcium carbonate and calcium citrate had a positive effect on bone debilitation to a certain extent, but it had not been observed to help
complete recovery of serious bone debilitation caused
by extremely inadequate dietary calcium in growthperiod rats. In addition, the appearance suggested that
recovery of serious bone debilitation is not easy to
achieve. A longer time of giving the calcium supplement
might be required. Also, it was thought questionable
that complete recovery could be attained, particularly
using only oral calcium supplements.
A threshold nutrient exerts a biological effect at
levels below the threshold intake. The same principle
applies to calcium nutrition [9], and this has been demonstrated convincingly in animals [30]. However, it is
difficult to establish an optimum threshold level of calcium intake for a human population because this value
can be expected to vary both with age and with the
individual. In this study, although the calcium content of
93
the mixture of calcium carbonate and calcium citrate
was nearly 3.8 fold higher than standard diet, it was
because its calcium content was below the threshold
level that the positive effect on bone debilitation occurred. On the other hand, its high calcium content can
be thought to be an important reason why the UNICAL
diet animals could recover better than standard
diet animals in both the low-calcium experiment and
calcium-deficient experiment groups.
Phosphate is another important mineral content of
bone, thought to have a close relation with calcium
metabolism. Phosphate tends to decrease urinary calcium excretion and increases fecal calcium excretion.
However, the net effect of a diet high in phosphate is
probably not to influence the calcium balance significantly [31]. The theories in which the calcium and phosphate moieties initially combine are many and varied,
and much debate continues as to the exact composition
of bone mineral. However, it is generally accepted that
bone is composed of carbonate-apatite (carbanoapatite) with a calcium-to-phosphate molar ratio of 1.667
in humans [32-34]. In the calcium-to-phosphorus ratio
analysis in the present experiment, the typical spectrum
of bone was observed: strong peaks for calcium and
phosphorus with low background, and no peaks from
other elements interfering with calcium and phosphorus. The same appearance was reported by Cassella et
al. [35]. Although Ca: P ratios of UNICAL diet animals
were significantly higher than those of pair-fed standard
diet animals in experiment groups, all values of experiment dietary groups were significantly lower than that
of the control group. Some authors [36,37] have reported similar results. In this study, we noted an interesting phenomenon: the Ca : P ratio of UNICAL diet
animals in the calcium-deficient experiment group was
i mproved upward to the level of standard diet animals
in the low-calcium experiment group. The phenomenon
helped to demonstrate that the mixture of calcium
carbonate and calcium citrate had a beneficial effect
on bone debilitation.
Generally, calcium supplementation is recognized as
accelerating bone formation with less inhibition of
bone resorption [22]. Observation by scanning electron
microscopy was helpful to understand the morphological alteration on the bone surface during the bone remodeling process. Sissons and colleagues [38,39] found
that bone resorption resulting from an inadequate calcium diet was not accompanied by osteocytic resorption
when osteocyte lacunae were observed by scanning
electron microscopy, and reported also that osteocyte
lacunae in bone formed during the period of calcium
deprivation were larger than those in comparable types
of bone in control rats by light microscopy.
In the present study, we caution that the appearance
of calcareous microdepositions in bone remodeling
94
areas, which was thought to be one of the morphological
indications of bone formation, reflected active calcium
utilization in bone metabolism. In UNICAL diet animals, calcareous microdepositions were found more
often than those in pair-fed standard diet animals
in experiment groups. This result suggested that the
mixture of calcium carbonate and calcium citrate had
accelerated action on bone remodeling in animals
with bone debilitation. Meanwhile, by comparison with
the control group and low-calcium experiment group,
the concave resorption foveas in the calcium-deficient
experiment group were deep and rough and their margins were irregular. Many collagen fibers appeared in
the upper surface of the bone matrix of resorption areas
in the calcium-deficient experiment group. It can be
understood nevertheless that there existed some obviously morphological discrepancies among control group
and experimental groups after being reared on calcium
complement diets for 2 weeks.
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