Journal of
Dental Research
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Changes in Acid-phosphate Content in Enamel Mineral during Porcine Amelogenesis
S. Shimoda, T. Aoba and E.C. Moreno
J DENT RES 1991 70: 1516
DOI: 10.1177/00220345910700120801
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Changes in Acid-phosphate Content in Enamel Mineral
during Porcine Amelogenesis
S. SHIMODA, T. AOBA', and E.C. MORENO
Forsyth Dental Center, 140 Fenway, Boston, Massachusetts 02115
The present study was undertaken to investigate changes in the
acid-phosphate content of porcine enamel mineral during its development and to assess separately the HPO42- pools in labile and stable
forms. Enamel samples at the secretary and maturing stages of
amelogenesis were obtained from the permanent incisors of five- to
six-month-old slaughtered piglets. Human enamel from erupted,
extracted teeth, synthetic hydroxyapatite, and carbonatoapatite
containing acid phosphate were included as references. The acidphosphate content of each sample was determined chemically
through its pyrolytic conversion to pyrophosphate. The assessment
of HPO42- in labile forms was made by analysis of samples preequilibrated with solutions containing 3 mmol/L phosphate at pH 11
(to de-protonate the HPO42-species on crystal surfaces). The analytical results of porcine enamel samples showed that: (a) the outermost
secretary (youngest) enamel contained the highest HPO42-, correspondingto about 16% of the total phosphate; (b) the acid-phosphate
content decreased gradually to 10% in the inner (older) secretary and
to 6% in the maturing tissue; (c) a substantial part of the HPO42-in
developing enamel tissue (50-60% of the HPO 2- for the secretary
enamel) was in labile forms; and (d) the pool of the labile HPO42-decreased with the growth ofenamel mineral. In parallel studies with
mature human enamel, it was ascertained that the total acid phosphate was only about 3% of the total phosphate, much lower than in
developingporcine enamel, andthat the labile pool ofHPO42-was also
small, corresponding to about 15% of the total acid phosphate
determined. The overall results indicate that acid phosphate is one of
the major constituents of the early enamel crystals formed during
amelogenesis and that the surfaces of the growing enamel crystallites are rich in HPO42.
J Dent Res (70)12:1516-1523, December, 1991
essential for definition of their thermodynamic solubility or the
drivingforce for precipitation in situ, as well as for prediction oftheir
stability in the challenging environment of cariogenesis.
The present study was designed to fill the gap ofour knowledge
on the state of ionic constituents and the stoichiometry of forming
enamel crystals at various stages of amelogenesis. Previously we
reported (Aoba et al., 1989; Aoba and Moreno, 1990) that significant
parts of the carbonate and fluoride in porcine enamel exist in labile
forms in the early developmental stages. In the present report, our
interest is focused on the acid-phosphate content ofenamel mineral.
Acid phosphate in the enamel ofhuman erupted teeth is a subject of
interest in relation to the understanding of the dynamic processes
of de- and remineralization during cariogenesis. In studies using
infrared spectroscopy (IR), Arends and Davidson (1975) reported
that the acid-phosphate contents of human and bovine enamel in
erupted teeth correspond to about 10% of the total phosphate and
that the acid phosphate of the enamel increases up to 25% in
artificial caries-like lesions. Similarly, attention was given to the
incorporation of acid phosphate in forming enamel mineral in
relation to possible precipitation of acidic precursors, such as
octacalcium phosphate (OCP) (Brown et al., 1984). Previous studies
using Fourier-transform infrared spectroscopy (FTIR) (Rey et al.,
1990; Aoba and Moreno, 1990) showed that both carbonate and acid
phosphate are constituents of enamel mineral formed in the early
stages of porcine amelogenesis. However, no quantitative assessment was reported on the total acid phosphate ofdevelopingenamel
tissues or possible pools of HPO42- in labile and stable forms. The
present work, with the porcine model, was aimed at gaining insight
into changes of the acid-phosphate content of enamel mineral with
advancement ofmineralization and assessing the labile, exchangeable acid phosphate on crystal surfaces.
Introduction.
Materials and methods.
Much work has been conducted on the composition of the inorganic
constituents of enamel over the last three decades. Reports
(Brudevold and Soremark, 1967; Hiller et al., 1975; Robinson et al.,
1982) indicate that the chemical composition of enamel mineral
changes markedly during amelogenesis and after eruption or with
cariogenic attack. However, there remains a paucity of information
about the state or environment of ionic constituents-such as Ca2+
phosphate, carbonate, Mg2+, Na+, OH-, and F-in enamel tissues at
various developmental stages. Specific questions to be answered
are: (a) whether part of each ionic constituent of enamel tissues may
be in labile forms (i.e., organically bound or adsorbed on crystal
surfaces) rather than in a stable form (i.e., incorporated into the
crystal lattice); (b) whether protonated ionic species, e.g., HCO3-and
HPO42-, exist in significant quantities in enamel crystals; and, as a
consequence, (c) what are the nature and lattice stoichiometries of
enamel crystals formed at various stages of enamel mineralization.
A better understanding of the stoichiometry of biominerals is
Porcine enamel.-The mandibles offive- to six-month-oldpiglets
were collected from a local slaughter house, placed on ice, and
brought to the laboratory. Sampling procedures of enamel tissues
were the same as reported previously (Aoba et al., 1987a, b; Aoba
and Moreno, 1990). Briefly, the enamel organs of central incisors
were dissected out of the mandible, the covering soft tissues were
removed, and then the exposed enamel surface was wiped gently
with a paper tissue to remove the cellular debris and traces ofblood.
Dissection ofenamel samples were performed in three ways: (a) the
whole secretary enamel (transparent in appearance and soft in
consistency) and the maturing enamel (a chalk-like appearance
after brief drying and soft but brittle) were dissected with small
spatulas; (b) the outermost (youngest) secretary enamel, approximately 60-100 pm in thickness (adjacent to the ameloblasts in situ),
was scraped with a razor blade, while the underlying secretary
enamel was collected in toto [The amounts of the outermost secretory enamel dissected from single teeth were approximately 1 mg or
less (wet weight). Thus, in order to secure enough (about 100 mg) of
this sample for analyses of its chemical composition, this type of
enamel dissection was performed on 100 teeth]; (c) in order to
investigate changes in the HP042- content taking place during the
secretary stage, the secretary enamel tissue was dissected into five
zones (each about 200 pm in thickness) from the outer to the inner
(older enamel, close to the dentin-enamel junction) zones as re-
Received for publication January 24, 1991
Accepted for publication June 18, 1991
'To whom correspondence and reprint requests should be addressed
This investigation was supported by USPHS Research Grants DE08670
and DE03187 from the National Institute of Dental Research, National
Institutes of Health, Bethesda, MD 20892.
1516
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1517
ACID PHOSPHATE IN PORCINE ENAMEL MINERAL
Vol. 70 No. 12
TABLE 1
EFFECT OF TEMPERATURE ON DETERMINATION OF THE ACID-PHOSPHATE CONTENT IN ENAMEL MINERAL
AND SYNTHETIC APATITES
% of total P as HPO42-
Sample'
Temperature (0C)
Secretory porcine enamel
5.0
Maturing porcine enamel
-
Mature human enamel
0.5
OH-Ap
3.6
10.8
(0.7)
10.4
(0.5)
3.9
(0.2)
-
7.2
(0.4)
8.0
(0.4)
4.0
(0.3)
(0.1)
2.0 (0.1)
3.0
(0.2)
3.0
(0.2)
1.6
(0.1)
(0.3)
7.4 (0.4)
8.6
(0.5)
10.4
(0.6)
11.6
(0.6)
4.0
(0.4)
(0.3)
8.8 (0.5)
4.0 (0.3)
3.8 (0.4)
3.3 (0.3)
1.5 (0.1)
(1) All enamel samples were de-proteinated by plasma-ashing and then ignited for 24 h at specified temperatures.
(2) The results are represented by the average (S.D.) of three determinations.
CO3-AP
ported previously (Aoba et al., 1987b). The enamel samples corresponding to each zone were separated from the paired central
incisors of single animals and pooled. The sample preparation was
performed on the teeth dissected from five animals. All enamel
samples collected according to procedures (a) through (c) were
stored at -30'C until used. Prior to analysis, each pooled enamel
sample was frozen and pulverized by hand-grinding with an agate
mortar and pestle. The powdered samples were dried in vacuum at
room
600
500
400
300
200
temperature.
De-proteination ofporcine enamel samples.-Part of each pooled
enamel sample was ashed in a plasma (oxygen-activated by a radio
frequency) at low temperature (about 600C). Aweighed amount (2030 mg) of the sample was placed in a Pyrex Petri dish inside the
reaction chamber (Plasmod, Tegal Co., Richmond, CA). The oxidation of the organic matter was conducted at a power level of 25 kW
with a constant oxygen pressure (14 kPa). The Petri dish was
weighed at various time intervals, and the treatment was continued
until a constant weight was obtained, which usually occurred within
48 h. The specific surface areas of the de-proteinated secretary and
maturing enamel samples were 79.4 and 63.2 m2/g (by N2 adsorption, Quantachrome), respectively.
Mature human enamel and synthetic calcium phosphates.Human enamel dissected from erupted permanent teeth (extracted
for orthodontic reasons) and synthetic apatites were included in this
study for comparative purposes. The preparation of mature, human
enamel samples was as reported previously (Moreno and Aoba,
1991). Two batches of synthetic apatites were prepared according to
the procedure reported previously (Shimoda et al., 1990). The
chemical compositions of the synthetic samples (in terms of wt%)
were: (1) for carbonatoapatite, CO3-Ap, Ca 37.9, total P 17.2, and
Co3 2.9; and (2) for hydroxyapatite, OH-Ap, Ca 37.5, total P 18.7,
and CO3 0.0. Both samples were composed of large, rod-like crystals,
havingelongated hexagonal cross-sections. FTIR studies ofthe COAp showed that most of the carbonate was substituting both P043
and OH- ions in the apatitic crystal lattice. The specific surface areas
of CO3-Ap and OH-Ap were 6.6 and 26.5 m2/g, respectively (Shimoda
et al., 1990).
Analysis of acid phosphate.-Acid phosphate in the enamel
samples and synthetic apatites was determined indirectly by the
heat-induced pyrophosphate method of Gee and Deitz (1953).
Samples were heated in air at various temperatures (200-600'C) for
periods ranging from one to 144 h. The formed pyrophosphate (PPi)
was hydrolyzed with 1 mol/L HCl04at 100'C for one h. Hydrolyzed
and non-hydrolyzed samples were analyzed for phosphate colorimetrically. The amount of acid phosphate was obtained from the
difference in the phosphate analysis of the two samples. The
estimated error for the HPO4 determination of enamel samples was
± 6% of the analyzed quantity.
Assessment of labile HPO.2-.-Enamel samples (and synthetic
apatites) were equilibrated m a solution containing 3 mmol/L
phosphate at pH 11. The rationale for the use ofthis solution was to
induce de-protonation of HPO42- species exposed on crystal surfaces
and to minimize dissolution of the sample during equilibration.
Parallel studies were conducted using various solutions, namely,
de-ionized water, 3 mmol/L phosphate buffer at pH 7.3, 1 and 10
mmol/L phosphate buffers at pH 11, and 1, 10, and 100 mmol/L
NaOH solutions at pH 11. Prior to the addition of the solid, N2 gas
was bubbled through the experimental solution to expel CO . The
slurry (solid/solution = 200 mg solid/1 mL) was equilibrated withthe
aid of a vortex mixer at room temperature. In preliminary experiments, when equilibration was conducted over various periods (five
to 120 min), it was ascertained that no time-dependent differences
existed in the analytical values ofthe acid-phosphate content. Thus,
in subsequent experiments, the slurry was equilibrated for 15 min
and then separated by centrifugation. The supernatant was removed and its pH value and Ca concentration were determined. A
sample ofthe sedimented solid was taken for the HPO42-determination, and the remaining solid was re-dispersed in fresh solution. The
process was repeated five times. The solid samples recovered after
each of the equilibrations were freeze-dried and then the acidphosphate content was determined according to the procedure
described above. The magnitude of the exchangeable pool was
assessed by the difference in the acid-phosphate content of the
sample before (original solid) and after equilibration.
Calcium, total phosphate, andcarbonate analyses.-The carbonate contentofthe enamel sampleswas determinedby amicrodiffusion
method (Conway, 1950) with CaCO, as standards. The total calcium
and phosphorus contents were determined, respectively, by atomic
absorption spectrophotometry and colorimetry (Vogel, 1961). The
estimated errors for Ca, total P, and CO3 determinations were 4%,
1.5%, and 8% of the analyzed quantities, respectively.
Electron microscopy.-Part ofthe enamel tissue was lyophilized
immediately after dissection and then embedded with POLY/BED
812. Ultra-thin sections were prepared on a Sorvall Porter-Blum
microtome equipped with a diamond knife. The morphologies and
sizes of the unstained crystals were observed at direct magnifications of 50 K to 100 K in a JEM 1200EX electron microscope at 120
kV.
Results.
Chemical analysis of the original (pulverized but not heated)
enamel samples showed that porcine and human enamel contained
no detectable amounts of PPi. The accuracy of the PPi hydrolysis
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1518
J Dent Res December 1991
SHIMODA et al.
TABLE 2
EFFECTS OF HEATING PERIOD ON DETERMINATION OF THE ACID-PHOSPHATE CONTENT
IN ENAMEL MINERAL AND SYNTHETIC APATITES
% of total P as HPO42-
Sample'
Heating Period (h)
3
1
12
6
144
96
72
48
24
Secretory porcine enamel
9.2 (0.5)
(4000C)
(5000C)
9.7 (0.6) 10.4 (0.6) 11.0 (0.5) 10.7 (0.6) 10.6 (0.4) 10.2 (0.5)
9.5 (0.5)
-
6.7 (0.6)
10.0 (0.6) 10.4 (0.6) 10.9 (0.5) 10.6 (0.5) 10.4 (0.6)
9.1 (0.5)
8.1 (0.5)
7.0 (0.4)
-
-
Maturing porcine enamel
-
6.0 (0.7)
-
8.3 (0.6)
8.0 (0.5)
5.8 (0.5)
Mature human enamel
-
-
3.0 (0.2)
2.8 (0.2)
3.0 (0.1)
2.8 (0.2)
Synthetic Apatites
OH-Ap
-
5.5 (0.2)
8.1 (0.5)
8.8 (0.4) 10.4 (0.6) 10.5 (0.4) 10.5 (0.4) 10.4 (0.6)
3.6 (0.2) 3.8 (0.2) 4.2 (0.2) 3.8 (0.3) 3.6 (0.3) 4.0 (0.3) 3.8 (0.4)
.
CO3-Ap
(1) The samples were the same as used for studies on the temperature effect.
(2) Heating of the samples, except for the secretary porcine enamel, was conducted at 500TC for specified periods prior to the determiination of HP042-.
(3) The results are represented by the average (S.D.) of three determinations.
and analysis of phosphorus was verified using Ca2P207, which was
obtained by igniting synthetic CaHPO42H20 at 7000C in air. X-ray
diffraction patterns of the ignited product confirmed the formation
of PPi without a residue of the original dicalcium phosphate. The
analytical values of the PPi yield were in good agreement (with an
experimental error of 2% ofthe total phosphorus content) with the
theoretical contentofHPO42-in the original salt. Tables 1 and2 show
the results of HPO2- determination in the enamel and synthetic
samples, which were ignited at various temperatures and for various heating periods. An unexpected finding was that, whereas the
PPi formation in the synthetic apatites reached a plateau after
±
heating the samples in the range 500-600TC over 24 to 98 h,
maximum yields of PPi for enamel samples (particularly developing
porcine enamel samples) were obtained after heating at lower
temperatures (400TC for the secretary porcine enamel and 500NC for
maturingporcine and mature human enamel) for shorterperiods (612 h). Prolonged ignition, even at 400C, brought about a substantial
decrease of the PPi yield for the immature porcine enamel. Thus, the
assessment ofthe acid-phosphate content was based on the results
obtained at 4000C for 12'h for the secretary porcine enamel (including the outermost tissue), those obtained at 500TC for 12 h for the
maturing porcine and mature human enamel, and the results
TABLE 3
CHEMICAL COMPOSITION OF ENAMEL SAMPLES AT VARIOUS DEVELOPMENTAL STAGES
Sample
Ca wt%
Chemical Composition
CO3wt%
% P as HPO42total P wt%
Molar Ratios
Ca/P
COJP
Porcine enamel
0.168 (0.018)
(0.6)
1.51 (0.02)
1.50 (0.02)
7.0
(0.4)
1.52 (0.02)
0.159 (0.006)
(0.4)
10.8
(0.7)
1.53 (0.02)
0.140 (0.010)
3.8
(0.3)
6.3
(0.5)
1.57 (0.03)
0.129 (0.010)
(0.05)
4.1
(0.3)
8.3
(0.6)
1.57 (0.03)
0.120 (0.015)
(0.16)
3.6
(0.3)
2.8
(0.2)
1.60 (0.02)
0.108 (0.020)
1.60 (0.03)
3.0 (0.2)
17.3 (0.48)
3.6 (0.2)
plasma-ashed 35.7 (0.30)
(1) S1, outer secretary enamel; S2, inner secretary enamel; Ml, maturing enamel; and M2, mature hard enamel.
(2) The results are represented by the average (S.D.) of four determinations.
0.108 (0.010)
S1 original
plasma-ashed
S2 original
plasma-ashed
Ml original
plasma-ashed
Human enamel
M2original
22.8
(0.24)
11.7
(0.12)
3.8
(0.3)
8.0
(0.5)
30.4
(0.45)
15.7
(0.03)
4.2
(0.4)
15.6
25.5
(0.62)
13.0
(0.22)
4.0
(0.2)
31.4
(0.30)
15.9
(0.18)
4.3
30.8
(0.40)
15.2
(0.33)
35.7
(0.85)
17.6
35.6
(0.25)
17.2
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0.138 (0.005)
ACID PHOSPHATE IN PORCINE ENAMEL MINERAL
Vol. 70 No. 12
1519
Fig. 1-Electron photomicrographs of porcine enamel crystals at various developmental stages: (A) the outer secretary enamel (first layer, youngest
mineral); (B) the underlying secretary enamel (second layer); and (C) the innermost secretary enamel (close to the DEJ, corresponding to the fifth layer).
Note the growth of crystallites in thickness with tissue maturation. Horizontal bar = 0.1 pm.
obtained at 5000C for 24 h for the synthetic apatites.
Table 3 shows the chemical compositions of porcine and human
enamel samples. The calcium-to-phosphorus molar ratio of each
enamel sample did not show any significant difference before and
after the plasma-ashing, whereas the CO3per mole of P (or Ca) ofthe
plasma-ashed sample decreased followingthe treatment. This trend
was consistent for all the enamel samples; the most marked reduc-
I
-
%-*,% %.*.
%.Oov
%,w%
-
1.6;
WN
-a
-
1.5
A
1.4v
n
-a
ct
0.10
Z
a -T-
L
.9.
'L
.Z.
r
-
I
0.05
0Ai
L~ayi er
Aninmal
I
2 3 4 5
2 3 4 5
If
I
2 3 4
III
I
5
i
I
1 2 34
IV
I
I
5
1
23 4
5
V
Fig. 2-Chemical composition of secretary enamel samples separated
into five zones from the outer (1) through innermost (5) zones. The sample
corresponding to each zone was obtained from paired central incisors of
single piglets (animals I through V). The values of the total weight loss
(taking place during the plasma-ashing and the following heating at the
specified temperatures, see text) and the Ca/P and COP molar ratios are
represented with solid circles, squares, and hexagons, respectively. Differences in the acid-phosphate content of the dissected enamel tissues are
displayed with vertical bars (mean S.E., n = 4). There were significant
differences (p < 0.05) between the acid phosphate in enamel layer 1 and the
underlying layers.
tion in the CO3/P ratio was attained for the outermost secretary
enamel. Previous studies (Aoba and Moreno, 1990) indicated that
the decrease ofthe COP ratio after the plasma-ashing was caused
by loss of labile carbonate, most probably adsorbed on the crystal
surfaces. With respect to acid phosphate, it was found that, in all
cases, the analytical values of the acid-phosphate content of the
plasma-ashed samples were higher (p < 0.01) than the values
obtained for the corresponding non-treated samples. The differences between the HPO42 values of the original and plasma-ashed
samples were the greatest for the outermost secretary enamel (Si)
and became smaller for the underlying secretary enamel (S2) and
further reduced for the maturing enamel (Ml). Although the analytical values ofthe HPO42-might be underestimated due to possible
ancillary reactions associated with the pyrolytic process, such as
CO32+ 2HPO42-> CO2 + 2PO43+ H20; CO32+ P2074--> C02 + 2PO4&; or
HPO42- +OH--> PO43 + H20 (Quinaux, 1964; Eanes, 1979; Dowker
and Elliott, 1983), we adopted the analytical maximum value
obtained for the plasma-ashed sample as the total acid-phosphate
contentofthe enamel mineral. Theresults shown in Table 3 indicate
that the youngest enamel, S1, contained the highest HPO42-, corresponding to about 16% of the total phosphate. Furthermore, the
acid-phosphate content decreased consistently with advancing development, from 16% of the total phosphate in Si to 11% in S2 and
8% in Mi. Mature human enamel (M2) contained much less HPO42only 3% ofthe total phosphate.
Fig. 1 shows electron micrographs of secretary porcine enamel
dissected into layers from the labial surface of the incisors toward
the enamel-dentin junction (DEJ). Crystallites found in the outermost secretary enamel (Fig. 1A) appeared as thin ribbons, only a few
unit cells of apatite in thickness. The enamel crystallites grew in
thickness duringthe secretary stage, as exemplified in Figs. 1B and
C for older crystals found in the second and fifth zones, respectively.
In Fig. 2 are illustrated the variations in the chemical composition ofthe secretoryporcine enamel separated into layers (1 through
5) from the paired incisors ofindividual animals (animals I through
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SHIMODA et al.
1520
J Dent Res December 1991
TABLE 4
EFFECTS OF REPEATED EQUILIBRATIONS WITH 3 mmolIL PHOSPHATE SOLUTION AT pH VALUES 11 AND 7.3
ON THE ACID-PHOSPHATE ANALYSIS OF ENAMEL MINERAL AND SYNTHETIC APATITE
Sample'
Original
% of total P as HPO42Number of Equilibrations
2
3
1
4
5
Porcine enamel
S1
(pH 11)
15.6
(0.6)
8.0
S2
(pH 11)
10.8
(0.7)
7.6
(pH 7.3)
10.8
(0.7)
9.7
S2
Ml
5.5
(0.4)
(0.5)
(0.6)
(0.3)
2.4
(0.3)
7.9
-
(pH 11)
8.3
(0.6)
5.7
(pH 7.3)
8.3
(0.6)
7.3
Ml,
-
(0.2)
(0.5)
(0.8)
7.2
6.3
9.1
(0.6)
(0.9)
(0.7)
6.7
(0.6)
(0.1)
(0.3)
(0.7)
2.5
(0.2)
8.3
4.5
7.4
6.0
(0.3)
6.2
(0.5)
6.0
(0.3)
(0.6)
5.5
5.7
(0.4)
8.6
(0.3)
8.9
(0.4)
(0.6)
9.3
(0.5)
9.1
8.8
(0.6)
5.3
(0.8)
6.8
(0.3)
6.3
(0.7)
(0.4)
(0.5)
(0.3)
9.5
6.5
(0.5)
(0.3)
(0.5)
(0.4)
7.0
(0.5)
2.6
(0.1)
2.5
(0.3)
2.7
(0.1)
6.4
4.0
6.9
4.8
6.7
Human enamel
M2
(pH 11)
2.8
(0.2)
Synthetic apatites
9.6 (0.3)
9.5 (0.7)
9.4 (0.6)
OH-Ap (pH 11) 10.4 (0.6)
9.2 (0.4)
9.4 (0.5)
4.0 (0.3)
2.8 (0.3)
3.8 (0.2)
3.4 (0.4)
CO,-Ap (pH 11)
3.7 (0.2)
3.5 (0.3)
(1) The samples were the same as used for studies on the temperature effect. S1, outer secretary enamel; S2, inner secretary enamel; Ml,
maturing enamel; and M2, mature hard enamel.
(2) Samples extracted five times with solutions at pH 11 and then with solutions at pH 7.3.
(3) The results are represented by the average (S.D.) of three determinations.
V). All the samples were plasma-ashed prior to analysis. A finding
consistent with the results shown in Table 3, was that the acid
phosphate was the highest in the first, outer zone (shown by the
dotted column) in all the animals. Importantly, the values of the
HPO42-(11 to 14% of the total phosphate) obtained for the first zones
(200 pm in thickness) were always lower than the value (16%, see
Table 3) of the pooled outermost secretary, sample (60-100 PM in
thickness), suggestingthat a gradient ofthe acid-phosphate concentration changes steeply in a narrow region adjacent to the ameloblasts, in which precipitation ofthe thin ribbons (see Fig. 1A) occurs.
Fig. 2 also depicts a steep decline of the HPO42-concentration within
the outer zone, followed by a gradual and marginal decrease of the
HPO42-toward the inner zones. Additionally, it was found that: (a)
the weight losses by the plasma-ashing and ignition were the
greatest for the outer sample and decreased markedly from the
surface to the inner (older) zones; (b) the Ca/P molar ratios were in
the range of 1.50 to 1.57, showing some trend to increase from the
surface to the inner zones; and (c) the CO)P molar ratios were in the
range of 0.13 to 0.20, displaying random variations or differences
among the animals, but without any apparent trend.
Table 4 shows changes in the acid-phosphate contents of porcine
and human enamel minerals (and synthetic apatites) as a function
of the number of successive equilibrations in a phosphate buffer at
initial pH values of 11 and 7.3. Chemical analyses of the supernatants showed that the Ca concentrations were in the range of 0.02
to 0.05 mmolIL and that, when the initial pH value was 11, the pH
values of the solutions were maintained above 10. In each system,
with either enamel (de-proteinated) or synthetic crystals, the acidphosphate content decreased significantly following the first equili-
bration but, importantly, a plateau in the value of the acid-phosphate content was obtained after the third and through the fifth
equilibrations. In studies with solutions containing either 1 mmoll
L total phosphate or 1 to 10 mmol/L NaOH, the pH values decreased
to the range of 8 to 9.5 after equilibration, and no apparent plateau
in the acid-phosphate content was attained within the five equilibrations. The use of either phosphate or NaOH solutions at concentrations higher than 3 mmol/L or 10 mmol/L, respectively, caused
appreciable changes in the x-ray diffraction pattern and FTIR
spectrum of the mineral phase, suggesting the occurrence of reprecipitation (probably a carbonatoapatite) during equilibration.
Equilibrations of the solid samples (including the outer secretary
enamel) in de-ionized water brought about only a marginal, if any,
decrease in the HPO.42% Based on the foregoing results, we adopted
the plateau value (i.e., the average of the analytical HPO42-values
after the third, fourth, and fifth extractions) attained using 3 mmol/
L phosphate buffer at pH 11 as the pool of HPO42- in a stable form
(in the bulk crystal). The HPO42 quantity corresponding to a labile
pool was obtained by subtracting the stable pool from the total acid
phosphate of the original (not equilibrated) sample. From the
results shown in this Table, it is pertinent to note that about 60% of
the total acid phosphate in the -outermost secretary enamel sample
(Si) was lost after equilibration, while the corresponding pools of
HPO42-decreasedto 50% in S2 and to 40% in Mi. Comparison ofthe
results in the first two lines of Table 4 indicate that, although the
original level of acid phosphate was significantly higher (p < 0.01)
in the outer secretary enamel (Si) than in the inner secretary
enamel (S2), the correspondinglevels afterthe third extractionwere
not significantly different. Therefore, these data indicate that there
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Vol. 70 No. 12
ACID PHOSPHATE IN PORCINE ENAMEL MINERAL
1521
then used for determination of the HPO42-. The extent of protein
adsorption was determined in parallel experiments, in which the
pellet in a centrifuge tube was dissolved with 0.5 mol/L acetic acid,
and the proteins extracted in the acid solution were analyzed by a
protein assay (Lowry et al., 1951) and SDS-PAGE (Laemmli, 1970).
The results ofthese experiments are showninTable 5. Followingthe
protein-crystal equilibration, it was verified by the protein assay
that about 85% of the total proteins were sedimented with the
crystals. The SDS-PAGE assay indicated that the proteins coating
the crystals were most of those in the matrix, including the 25-20
kDa amelogenins and other components having higher and lower
4.5 (0.2)
11.0 (0.8)
(B)
molecular weights. A consistent finding obtained for both enamel
and synthetic samples was thatthere were no significant differences
9.3 (0.7)
(C)
in the PPi yield between the original (non-treated) and proteincoated samples. Additionally, it was ascertained that no significant
10.4 (0.9)
OH-Ap
(A)
amount of phosphorus (less than 0.3 wt%) was present with the
this measurementwas conductedfollowingashing
isolatedproteins;
4.6 (0.3)
10.5 (0.7)
(B)
(600'C) of the weighed amount of the isolated proteins and then
hydrolyzing the residues in 1 mol/L HCl. Therefore, it may be
9.6 (0.7)
(C)
* The enamel sample was porcine secretary enamel S2, which was concluded that the concentration of organic phosphorus in the
secretary enamel was far too low to explain the presence of HPO42de-proteinated by the plasma-ashing. (A) Original (non-treated) corresponding to 10% or more of the total phosphate.
samples; (B) the samples were equilibrated with the protein
solution prior to use for the determination ofthe HPO42-; (C) the
samples were treated in the same manner but without the Discussion.
addition of proteins to the solution. The figures are averages
The present results provide further evidence that the composi(S.E.) of samples in triplicate. There were no significant differ- tion and morphology ofenamel crystals change substantially in the
early stage of mineralization occurring in the vicinity of the
ences in these data (p > 0.05).
ameloblast, and that acid phosphate is not a negligible lattice
was no difference in magnitude ofthe stable pool in samples S1 and constituent of enamel crystals at some developmental stages. The
S2, but the labile pool was larger in the youngest enamel sample. maximum value ofthe HPO42- (16% ofthe total phosphate) obtained
ThefiguresinTable4 also showthatthelabilepooloflHPO4H2-inmature so far from the dissected porcine enamel may be underestimated by
human enamel, M2 (and those in synthetic crystals having small the pyrolytic assay because the presence of carbonate in enamel
specific surface areas), was much lower in magnitude (10% or less crystals interferes with the formation of PPi upon heating. Even
ofthe total acid phosphate) than those found for developing porcine with this uncertainty in the analytical results, the finding that
enamel.
younger enamel, containing more carbonate, disclosed a higher
In Table 4 are also shown typical results obtained with the 3 HPO42- content allows us to conclude safely that: (a) The young
mmol/L phosphate buffer at pH 7.3. In this case, the pH values ofthe enamel mineral formed at early stages of mineralization contains
supernatants were in the range 6 to 6.3. Notably, a plateau of the the highest amount of HPO42- and, taken together with the electron
HPO42- was attained following several equilibrations, but the ob- microscopic observation, (b) the total acid phosphate of porcine
tained reduction of the HPO42- was moderate (15-20% of the total enamel mineral decreases concomitantly with the growth of the
acid phosphate) as compared with the reduction attained with the apatitic enamel crystals.
alkaline pH. The data obtained for the extractions of samples (S22
The results in Table 4 show that a substantial part ofthe HPO 2and M12) with solutions having a pH of 7.3 following extractions in developing enamel mineral is in labile forms and that this labile
with the solutions at pH 11 clearly indicate that most of the acid pool of HPO 2- as well as the total acid phosphate (see Table 3),
phosphate removed by the higher pH solution was regained by the decreases substantially with the advancement of enamel minerallabile pool through the treatments with the solutions at pH 7.3.
ization. In this report, the labile pool of HPO42 of enamel mineral
The plasma-ashing removed most of the organic matter, but a was expressed as the decrease in the amount of HPO42- after treatsmall residue remained in the samples. It was then pertinent to ment with 3 mmolfL phosphate buffer at a pH value of 11. This
investigate the possible effect ofthis residual organic matter on the decrease could be explained by: (i) an exchange between HPO42 on
PPi formation upon heating. To this end, part ofthe de-proteinated the solid surface and PO43in solution; (ii) de-protonation of surface
secretary enamel and the OH-Ap were pre-treated with enamel HPO 2-inducedbythelowhydrogen-ion activity (H+) in solution; (iii)
matrix proteins isolated from secretary porcine enamel as reported dissolution of acidic phases, if any, and re-precipitation of a basic
previously (Aoba et al., 1987a, b). The proteins, consisting of mix- apatitic phase on the crystal surfaces; or (iv) additive effects of (i)
tures of amelogenins and minor amounts of non-amelogenins, were through (iii). The marked difference between the plateau values of
dissolved in de-ionized water at 0°C to yield a 0.1% wt/vol protein HPO42-of enamel mineral attained at pH values of 11 and 7.3 (see
solution. Accurately weighed amounts of either the biomineral or Table 4) could indicate that either process (i) or (ii) is favored with
synthetic crystals, to yield 0.6 m2 of total surface areas, were in- increasing pH value and that there is no unequivocal way to
troduced into 5 mL ofthe protein solution. The pH value ofthe slurry distinguish between them. However, since comparable reductions
was adjusted to 7.3 with a minute amount of 0.1 molVL NaOH. The ofthe acid-phosphate content ofenamel mineral were attained with
slurry was equilibrated end-over-end for one h at room temperature. 1 and 10 mmol/L NaOH solutions with and without the addition of
As a control, an aliquot of the solid sample was treated in the same phosphate to the solution, the de-protonation process seems to be
manner but without the addition of proteins to the solution. At the favored by the experimental conditions. Furthermore, it was ascerend, the slurry was separated by centrifugation. The supernatant tained experimentally that a substantial part of the labile acid
was carefully removed and then used for measurements of the phosphate, which was lost after treatment with the phosphate
concentrations of Ca (dissolved from the solid) and proteins (re- buffer at pH 11, was restoredby re-equilibratingthe treated sample
maining unadsorbed). The sedimented solid was freeze-dried and in 3 mmol/L phosphate solution at pH 7.3. The values of the restored
TABLE 5
EFFECT OF PROTEIN COATING OF SECRETARY ENAMEL
MINERAL AND SYNTHETIC APATITE
ON THE YIELD OF PYROPHOSPHATE UPON HEATING
Sample* Amounts of proteins recovered % of total P
as HPO42from crystal surfaces (mg)
10.8 (0.6)
Porcine enamel(A)
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1522
SHIMODA et al.
J Dent Res December 1991
TABLE 6
CHANGES IN CARBONATE AND ACID-PHOSPHATE CONTENTS OF ENAMEL MINERALS TAKING PLACE
DURING THE PLASMA-ASHING AT LOW TEMPERATURE
Molar Ratios
Changes in C03 and HPO4
Sample
CO3/total P
ACO3
AHPO4
AHPO4
HPO4/total P
A
A
B
B
[B - Al
[B - A]
calculated*
Porcine enamel
S1
0.168
0.138
0.080
0.156
-0.030
0.076
0.060
S2
0.159
0.140
0.070
0.108
-0.019
0.038
0.038
Ml
0.129
0.120
0.063
0.083
-0.009
0.020
0.018
Human enamel
0.028
0.030
0.002
0.000
0.000
A and B, Analytical values of mols CO3 and HPO4per mol of total P before and after the plasma-ashing treatment, respectively.
* The theoretical value of AHPO42 calculated from ACO3 and the assumed reaction CO3 + 2HPO4 -> CO2 + 2PO4> + H20.
M2
0.108
0.108
acid-phosphate content for the enamel samples S2 and Ml amounted
to 85% and 80%, respectively, of the amounts originally present in
the samples. Furthermore, the values shown in Table 4 indicatethat
the restored values ofacid phosphate were almost identical to those
obtained after the repeated treatments of the original samples with
the solution at pH 7.3. The apparent "reversibility" of labile acid
phosphate lends further support to the postulated surface reaction.
In relation to possible dissolution/re-precipitation reactions taking
place duringthe treatment ofthe enamel sample, analyses (electron
microscopy, FTIR, and x-ray diffraction) did not give evidence of any
appreciable changes in the nature and structure of the solid residues after the described equilibration, but these analytical techniques may not be sensitive enough to discern very small changes
occurring on solid surfaces.
The present results support the idea that the substantial decrease of acid phosphate (corresponding to up to 60% of the total
HPO 2-) after equilibration with the alkaline phosphate solution
was Jue mainly to de-protonation of acid phosphate exposed to the
solution. It seems reasonable to think that the obtained decrease of
the labile HPO42- of porcine enamel associated with the advancement of mineralization corresponds to the burying of an initially
precipitated domain rich in HPO42- (probably corresponding to the
central dark lines in Fig. 2C) with a subsequent deposition of an
apatitic phase having different stoichiometries from that ofthe thin
ribbons.
The analytical value of the total acid phosphate obtained for
mature human enamel, about 3% of the total phosphate, is about
one-third of the value (10%) obtained spectroscopically by Arends
and Davidson (1975). An advantage ofthe spectroscopic analysis of
acid phosphate, as compared with the pyrolytic assay used in the
present work, is that ignition of the sample is not required and
thereby interference with the pyrolytic process can be ruled out. On
the other hand, the reported complicated procedure of spectral
analysis (i.e., assessment of the CO3-peak intensity in the range of
1400-1420 cm-' and then separation of the overlapping CO32 and
HPO42- bands in the range of 880-870 cmn') easily gives rise to
substantial experimental errors. Notably, Arends et al. (1987)
compared two analytical values of the acid-phosphate content of a
synthetic apatite that were obtained independentlyby the chemical
and spectroscopic procedures. Their results disclosed that the value
of the HPO42- obtained by the Gee and Deitz method was 2.3%,
whereas the value assessed by IR was 6% with a large experimental
error, i.e., + 4.5%.
The plasma-ashing ofenamel samples, prior to pyrolysis at 400-
5000C, yielded a higher PPi content than that obtained in samples
pyrolyzedwithout the plasma-ashingpre-treatment. Two plausible
explanations for such results might be, first, that when the original
enamel sample is heated at 400-500°C, decomposition ofthe organic
matter (with a release of CO and H20) may interfere with the
condensation of HPO42to PPiLIowever, the yields of PPi obtained
with the protein-coated crystals were comparable with those obtained with uncoated crystals, showingthat such an interference of
the organic matter is unlikely. The second explanation is suggested
by an examination ofTable 3. In this Table, it is apparent that the
greatest increase in the determination of acid phosphate brought
about by the plasma-ashing occurred in the samples of secretary
enamel that had the higher content of carbonate; the effect of the
pre-treatment was much less in the maturing and mature enamel
samples. It was reported recently (Aoba and Moreno, 1990) that
adsorbed carbonate on the surfaces of the enamel crystallites is lost
during the plasma-ashing. Consequently, the increase in the PPi
formed mayresultfrom the elimination ofaportion ofthe carbonate,
namely, the fraction on the crystal surfaces. If the reasonable
assumption is made that the phosphate associated with the organic
matter is negligible by comparison with that in the mineral (in fact,
the maximum content of organic phosphorus was determined as
0.3% by weight ofthe matrix proteins in the secretary enamel), it is
possible to explore the interference ofcarbonate on the condensation
of acid phosphate in quantitative terms. To this end, the moles of
CO32. and HPO42-per mole of P are calculated in the original and
plasma-ashed samples from the analytical data shown in Table 3.
The results of such calculations are shown in Table 6. The changes
in C03 andHPO4 arethen compared with those anticipated fromthe
reaction C032-+ 2HPO42-> C02 + 2PO4 + 110. Clearly, as shown in
thelast column ofTable 6, the agreementbetween the experimental
losses and those expected from the reaction above is quite good and
provides a simpleexplanationforthehigheracid-phosphatecontent
in samples ashed in the plasma prior to their pyrolysis. An interesting observation, but not fully understood, is that optimum pyrolytic
conditions (heating temperatures and time) were different for
enamel minerals and synthetic apatites. As shown in Table 2, the
CO3-Ap and the mature enamel samples heated at 500°C, as well as
the secretary enamel heated at 400°C, yielded the maximumformation of PPi in around 12 h. Such a maximum was attained with the
OH-Ap in about 24 h, and it took only six h when the secretary
enamel was heated at 5000C. The observed difference between the
biomineral and synthetic crystals suggests that interference of PPi
formation may not only depend on the total mass of carbonate but
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Vol. 70 No. 12
ACID PHOSPHATE IN PORCINE ENAMEL MINERAL
also on spatial mutual accessibility of the HPO4 and CO32- ions in
the crystal lattice. These accessibilities may differ in the synthetic
and biological crystals. Supporting the importance of carbonate
environment in apatite lattice, Greenfield et al. (1974) demonstrated that, whereas the carbonate in apatite crystals obtained
from an amorphous precursor decreased the PPi yield, the carbonate in amorphous calcium phosphate did not show any significant
effects on the PPi formation upon ignition. At present, no experimental data are available for assessment of the interference of PPi
formation by carbonate ions incorporated in the crystal lattice.
It is likely that the overall changes in the composition of enamel
mineral, particularly the decrease of both the total and labile acid
phosphate with the advancement of mineralization, reflect changes
in the composition ofthe medium in which precipitation and growth
of enamel carbonatoapatite occur. The morphology of thin ribbons
observed in the outermost secretary enamel (see Fig. 2A) has been
advocated as evidence for OCP precipitation (Brown et al., 1981,
1984; Nelson and Barry, 1989). The higher content of HPO42- in early
secretary enamel might be consistent with the formation of such a
precursor, although direct observation of a well-defined OCP phase
has eluded experimental confirmation. Furthermore, formation of
apatitic phases having significant contents ofacid phosphate would
also be consistent with the high content of HPO4' in the mineral
formed closest to the ameloblast. Elucidation ofthis matter requires
further work on the mechanisms involved in the nucleation and
growth of the enamel crystallites.
Acknowledgment.
The authors are indebted to Dr. Y. Make for his valuable help
in electron microscopic work.
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