In vitro study of the mechanism of oriented growth of tooth enamel apatite
Mayumi Iijima and Yutaka Moriwaki
Dental Materials and Technology
Asahi University, School of Dentistry,
1851-1 Hozumi, Hozumi-cho, Motosu-gun, Gifu 501-02, Japan
Introduction
In the early stage of tooth enamel formation, enamel crystals grow as
extremely thin ribbons1-3), and are highly oriented along the c-axis3).
In the mechanism of tooth enamel crystal formation, it is still inconclusive
what regulates the initial lengthwise and oriented growth :Some previous
studies suggested that fibrillar3,4), lamellar5), or tubular6-8) structure
in the enamel matrix assist the orientation of the crystals. The recent
studies9-11) hypothesize that enamel proteins, such as amelogenins, are
related to the oriented growth of enamel crystals. It is also suggested
that streaming of organic molecules12,13), a secretory force produced by
ameloblasts14) , some additional orienting factor15) causes the orientational
enamel growth. These hypotheses, however, do not exclude other possibilities.
We have been studying the mechanism of the lengthwise and oriented growth
of tooth enamel crystals on the basis of a hypothesis : (1) ribbon-like
enamel crystals grow initially as octacalcium phosphate (Ca8H2(PO4)6・5H2O,
OCP)16,17) and (2) ionic diffusion through the layer of ameloblasts promotes
the one directional growth and the orientation of OCP crystals18). This
paper introduces our previous in vitro studies on (1) the mechanism of
the lengthwise and oriented growth of tooth enamel apatite19-22) and (2)
the roles of F- ion on tooth enamel-like apatite formation23-25).
Materials and Methods
In our model system, a synthetic cation selective membrane was used
to control the ionic
diffusion. Several sets of large (500ml) and small (50ml) Pyrex glass bottles
were used. A 50ml glass bottle was used as a Ca2+ ion diffusion vessel
and was put into a 500ml bottle, which contained 300ml of PO4 solution.
A cation selective membrane ( Asahi Glass Co., CMV, about 3cm2) was fixed
to the Ca-bottle by silicon rubber and a plastic screw stopper. The reactions
were carried out using Ca(CH3COO)2・H2O (30mM) and NH4H2PO4+(NH4)2HPO4
(3.6, 7.2, 14.4mM) at various pH levels ranging from 6.0 to 7.4 and at
37 ℃ for 3days. The effect of F- ion was studied in 7.2mM PO4 at pH6.5.
Various amounts of NaF (0.1-2 ppm F ) were added to the PO4 solution.
Details of the experiments are described elsewhere22,23).
After the reaction, the membrane was removed from solution and rinsed
with distilled water for a few seconds, and then dried in the air. The
product was analyzed using a micro-beam X-ray
diffraction camera ( with a collimator of 100μm diameter), an X-ray
diffracto-meter ( XRD ), a scanning electron microscope ( SEM ) and a high
resolution transmission electron microscope (HR TEM).
Results
OCP crystals grew onto the membrane with the c-axis perpendicular to
the membrane on the whole (Fig.1a). Figure 1b represents the XRD patterns
of the powdered and as grown product on the membrane. Since crystals grew
with orientation, as grown product exhibited strong 002 and 004 reflections
of OCP, whereas, powdered product exhibited a strong 100 reflection of OCP
at 1.867nm. In case of 14.4mM and at pH6.0, both OCP and dicalcium phosphate
dihydrate (DCPD) were obtained. In case of 7.2mM and 3.6mM, precipitation
did not take place at pH6.0 and at pH6.5, respectively. The lower the
pH became, the higher PO4 concentration was necessary to yield the precipitation
on the membrane.
Table 1 represents the morphology of OCP crystals grown at various PO4
concentrations and pHs. Figures 2 (a )- (d) are the SEM photographs of
OCP crystals grown in the 7.2mM PO4 solution at various pHs. When the
pH was higher than 7.0, flake-like crystals grew on the membrane (Fig.2a).
The lengthwise growth (R in Table1) took place at pH lower than 6.8.
As the pH decreased, the growth along the c-axis of OCP increased (Fig.2b-2d).
The growth in the c-axis direction of OCP increased with a decrease
in the pH and the PO4 concentration. However, the growth depended more
strongly on the pH than on the concentration of PO4.
Fluoride ions affected the overall property of the crystals grown on
the membrane. Long and thin ribbon-like crystals grew without and in
the presence of 0.1ppm F (Fig.3a, 3b). The XRD photographs of these crystals
showed OCP reflections with orientation (Fig.4a). The crystals obtained
in the presence of 1ppm F had thin and ribbon-like morphology (Fig.3c) but
the XRD photograph was apatite-like (Fig.4b). The morphology and size
of crystals changed drastically between 1 and 2 ppm F. Small needle-like
crystals were obtained in the presence of 2 ppm F (Fig.3d).
In the presence of 0.1 and 1ppm F, epitaxial overgrowth of apatite
on the (100) of OCP took place and apatite/OCP/apatite sandwich structure
was formed (Fig.5). These crystals have long and thin plate-like morphology
and embed an OCP lamellar in the center of the crystals. In the lattice
images of these crystals, the (100) fringes of OCP and the (100) fringes
of apatite were parallel to each other, indicating that apatite grew on
the (100) of thin plate-like OCP crystals epitaxially. Although ribbon-like
crystals grown in the presence of 0.1ppm F appeared superficially homogeneous
(Fig.3b) and they were identified to be OCP by the XRD method, a small
amount of apatite was observed on the marginal part of the plate-like OCP
crystals (Fig.5a). In the presence of 1ppm F, the OCP region was smaller
than that of apatite (Fig.5b). The width of OCP lamellar in the a*-axis
direction was one to several unit cells thick. The main part of the
crystals were apatite, resulting in the XRD pattern of apatite (Fig.4b).
Some crystals embedded a central plane instead of the distinct OCP lamellar.
Figure 6 is a cross-section of the crystal with a central plane. The
thickness of the plane was about 0.8-1.2nm. The central plane in the present
study is probably a variation of the central OCP crystal, as it was formed
in the same reaction chamber. The feature is close to that of tooth enamel
which shows a so-called central dark line. Table 2 summarizes the property
of the crystals obtained under various amounts of F- ions.
Discussion
Concerning to the mechanism of the oriented growth of enamel crystals,
some of the studies on the enamel matrix suggested that the enamel proteins
assist the oriented growth9-11). The present study demonstrated that
the oriented growth took place on the membrane, even though there was no
organic matrix which supports the oriented growth. The ribbon-like crystals
which grew on the membrane were morphologically resemble to the crystals
in the early stage of enamel crystal formation. Morphology of crystal
sometimes can suggest the process or the condition under which crystal
growth took place. In the usual precipitation reaction in solution without
the membrane, OCP grew into rectangular plates even at pH6.5 (unpublished
data, Iijima ). The length in the c-axis direction was much shorter than
that of OCP grown in the membrane system at the same pH. The one-directional
supply of Ca2+ ions through the membrane should cause the oriented and lengthwise
growth of OCP. During the enamel formation, Ca2+ ions are supplied from
the layer of ameloblasts26-30). Such one-directional ionic flow might,
in part, contribute to the oriented and lengthwise growth of tooth enamel
crystals.
Although the F concentration in the enamel fluid is very low (4-7x10-7M31)),
F- ion seems to be included in the process of enamel apatite formation,
because a remarkably small amount of F- ion promotes apatite formation32,33),
increases the crystallinity of apatite34), induces and accelerates the
hydrolysis of OCP to apatite35,36). The OCP/apatite ratio in the crystals
changed between 0.1 and 1ppm F, in other words, F- ions regulated the
predominant growing phase, i.e., OCP or apatite. F- ions might change
the surface energy of the crystals, the driving force of crystallization
and hydrolysis. The thickness of the central OCP lamellar would be small
when the stability of OCP in solution is small.
A cross-sectioned tooth enamel crystal of mammals with 0.01-0.03%wt
F often exhibits a central line. The origin of the central line is still
inconclusive, though it reflects the initial stage of the crystallization
of tooth enamel. Apatite with the central layer has been synthesized in
CO3-containing solution, and moreover, the reaction related to OCP37,38).
The cross-section of such crystals exhibited a central line whose feature
was similar to that of tooth enamel crystal with the " central dark
line". In the present study, it is probable that the central layer
thinner than 1.87nm was a variation of the central OCP lamellar, because
they were formed in the same reaction. Apatite should precipitate on
the OCP crystal immediately after one unit cell thick OCP was formed.
In conclusion, when Ca2+ ions were supplied one directionally through
the membrane, OCP
crystals grew with orientation and attained a ribbon-like morphology in
slightly acidic solution. In the presence of 1ppm F, apatite crystals
embedded a layer in the center, which should relate to OCP, exhibiting a
central line in the cross- section, whose feature was similar to that of
tooth enamel crystal with "the central dark line". The results
strongly support the idea that initially formed thin plate-like OCP crystals
acted as a template for subsequent overgrowth of apatite in the process
of tooth enamel formation37-39).
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Table 1 The morphology of OCP grown on the membrane at various PO4 concentrations
and pHs
at 37℃22).
pH
PO4 (mM) 6.0 6.3 6.5 6.8 7.0 7.4
14.4 Rs R R SP F F
7.2 - Rs Rs LP SP F
3.6 - - - Rs SP F
F : flake-like ; SP : short plate-like ; LP : long plate-like ; R :
ribbon-like crystals ; s : spherical
aggregate of crystals
Table 2 Characterization of the products grown on the membrane in 7.2mM
PO4 solution in the presence of various amount of F at pH6.5 and 37℃25).
F concentration
(ppm) 0 0.1 1 2
Product OCP lamellar mixed lamellar mixed Apatite
crystals of OCP crystals of OCP
and apatite and apatite
SEM Ribbon-like Ribbon-like Ribbon-like Needle-like
XRD OCP OCP Apatite Apatite
HRTEM OCP OCP >> Apatite OCP << Apatite
Apatite
SEM : Scanning electron microscopy ; XRD : Micro-beam X-ray
Diffraction ;
HRTEM : High resolution transmission electron microscopy
Figure captions
{ 現在付図のインストール準備中 }
Fig.1
(a) SEM photograph of the OCP crystals grown on the membrane at pH6.5.
Reactionwas carried out in 14.4mM PO4 solution at 37 ℃ for 3days. (b)
XRD patterns of powdered (lower) and as grown (upper) product on the membrane.
Fig.2
The morphology of OCP grown on the membrane in a 7.2mM PO4 solution at 37℃
for3 days22). The pHs of solutions were (a) pH7.4, (b) pH7.0, (c) pH6.5
and (d) pH6.3. Scale line is 5μm for (a), (b) and (c) and 50μm for
(d).
Fig.3
SEM photographs of the crystals grown (a) without F, and in the presence
of (b)0.1ppm F, (c) 1ppm F and (d) 2ppm F at pH6.5 and 37℃25).
Fig.4
Micro-beam XRD photographs of the products grown (a) without F and (b)
in the resence of 1ppm F.
Fig.5
High resolution TEM photographs of crystals grown in the presence of (a)
0.1ppm and (b) 1ppm F in 7.2mM PO4 solution with the pH of 6.5 and 37 ℃.
Fig.6
A lattice image of a cross-sectioned crystal with a central plane25).
The central plane is parallel to the (100) plane of apatite. Note that
it looks like a centralline of tooth enamel crystal.