ACBTE Vol. 3:57-64, 1993
Histochemical Demonstration of Calcium Phosphate Depositions in the
Enamel Organ of Calcium-loaded Tooth Germs of The Rat
Yoshiro TAKANO and Hayato OHSHIMA
Department of Oral Anatomy II,
Niigata University School of Dentistry,
2-5274 Gakkkocho, Niigata 951, JAPAN
Abstract: In order to test the possible differences in calcium regulatory
mechanisms among the cells of the enamel organ, calcium-binding property
of the plasma membranes of these cells were examined at different stages
of amelogenesis. Thirteen-day-old rats were vascularly perfused with isotonic
sucrose solution containing either physiological (3 mM) or high concentration
of calcium (30 mM) via the aorta and second molar tooth germs were processed
for rapid freeze, freeze substitution. Incisor teeth obtained from vascularly
perfused young rats were similarly processed. Glyoxals bis(2-hydroxyanil)
(GBHA) staining of thick Epon sections of the tooth germs revealed distinct
granular Ca-GBHA deposits in the ameloblast layer at the stage of matrix
formation and also at the region of smooth-ended maturation ameloblasts.
Ca-GBHA deposits were lacking in other types of ameloblasts and in other
enamel organ cells. The data indicate the similarity between the secretory
ameloblasts and smooth-ended maturation ameloblasts of molars and incisors
in terms of calcium-binding properties of plasma membranes.
Introduction
The role of ameloblasts has been implicated in matrix formation and its
subsequent removal as well as regulation of calcium influx in developing
enamel 1, 2). As regards the regulation of calcium, it has been a matter
of controversies whether or not the ameloblast layer functions as an efficient
diffusion barrier to calcium. It appears from previous autoradiographic
observations that the secretory ameloblast layer restricts calcium from
entering in secretory enamel thus making the enamel less mineralizable throughout
the stage of matrix formation (3-5). On the other hand, the same cell layer
seems to allow drastic but intermittent influx of calcium in the enamel
during the stage of maturation (6-8). Data from previous tracer experiments
in that various tracers of different molecular sizes were tested (9-12)
and those from our recent 45Ca autoradiography (13) were in support of
tightness of intercellular junctions of both the secretory and maturation
ameloblasts to paracellular penetration of calcium. In the maturation stage
ameloblast layer the presence of specific regions where calcium moves by
diffusion has also been recognized (11,14,15). Nevertheless, due to lack
of knowledge concerning the actual distribution and movement of intra- as
well as extracellular calcium, the proposed pathways for calcium translocation
across the ameloblast layer and hence the mechanisms whereby calcium influx
is regulated by the ameloblasts have been a matter of extensive debate.
Reith (1983) (16) and Reith and Boyde (1984) (17) proposed a membrane-associated
pathway as a possible candidate for calcium translocation across the ameloblast
layer, based on cytochemical findings of localization of cell calcium using
potassium pyroantimonate as the precipitant for ionic and/or ionizable
calcium. They showed calcium-antimonate precipitates to occur predominantly
in or along the inner aspects of lateral plasma membranes of both the secretory
and maturation ameloblasts. A survey of literature showed minor but significant
differences in results among similar cytological studies regarding the localization
of cell calcium in the ameloblast layer (17-22), implicating the necessity
for further investigations on this matter.
In the present study, the correlation between the physico-chemical properties
of plasma membranes of the ameloblasts and cell calcium was examined using
a histochemical calcium staining method combined with anhydrous tissue processing.
The calcium-related phenomena in the tooth germs of rooted and non-rooted
teeth were compared.
Materials and Methods
The rats of Wistar strain of both sexes (13 day old) were anesthetized
with ether and perfused with isotonic sucrose solution (0.25 M) containing
3 mM or 30 mM CaCl2 via the ascending aorta through the left ventricle.
The rats were perfused for 5 min at a rate of 2 ml/min at room temperature
using a peristaltic pump. Upper second and third molar tooth germs were
carefully excised from the alveolar sockets with the intact enamel organ
attached. These were immediately quenched in liquid propane at liquid nitrogen
temperature and substituted with cold acetone (80 ¡C) for 4 days and subsequently
embedded in Epon 812 as described elsewhere. Some young adult rats of the
same strain (70 g bw) were similarly processed to examine the incisors.
Thick sections were cut dry with glass knives without using trough and
collected on glass slides. Histochemical staining solution for mineralized
as well as unmineralized calcium comprised 75% ethanol containing 3.5%
NaOH and 5% glyoxal bis(2-hydroxyani) (GBHA) (Fluka, Switzerland). GBHA
solution was poured over the sections on the glass slide. After staining
for 5 min, the sections were rinsed in absolute ethanol until the Epon surrounding
the specimen became transparent and then soaked in xylene for 5 to 10 sec,
and mounted with Entellan new (Sigma, Darmstadt, Germany). Some of the
sections were counter-stained with 0.3% methylene blue in absolute ethanol
after ethanol rinse. Observations were made under bright- or darkfield
illumination.
Results
Cell morphology and the organization of tissue architecture of the isolated
tooth germs were sufficiently preserved in our rapidly frozen tissues.
The addition of sucrose (0.25M) in the perfusate improved the quality of
microscopic images of tissues such that drastic damages of cell morphology
due to ice crystal formation could not be recognized at the light microscopic
level even in the pulp cells situated far from the frozen surface. GBHA
staining of anhydrously prepared sections revealed peculiar granular deposits
of Ca-GBHA complex only in specimens pretreated with high calcium-containing
solution.
Molar Tooth Germs
In molar tooth germs perfused with high calcium solution, distinct red
granular deposits appeared in the ameloblast layer at the stage of matrix
formation (Fig.1). In most cases, the granular deposits were associated
exclusively with the ameloblasts and not with other cells of the enamel
organ. In the third molar tooth germs in which whole enamel was at the stage
of matrix formation, the granular deposits in the ameloblast layer became
less distinct near the cusp region (enamel-free area) where the enamel formation
is known to be absent. Due to mechanical damages occurring during extraction
procedures, the observations of presecretory and early secretory stages
could not be made.
In the maturation stage, the ruffle-ended ameloblasts were devoid of granular
GBHA deposits, but exceptionally, a small number of granular GBHA deposits
could be encountered at distal portion of these cells in some regions (Fig.2).
The granular deposits constantly appeared in the specific regions of the
ameloblast layer where the ameloblasts displayed smooth-ended profiles (Fig.3).
The granular deposits were located along lateral cell borders of the smooth-ended
ameloblasts and tended to be confined to the proximal cell ends (Figs.3-5).
In one occasion, a portion of papillary layer and the endothelium of invaginating
capillaries adjacent to the smooth-ended ameloblasts also displayed similar
granular deposits (Fig.5).
Incisors
Compared to molars, the granular deposits of Ca-GBHA complex in the incisor
enamel organ were more distinct than those in molar tooth germs. It was
shown that granular deposits were absent in the ameloblast layer until the
onset of enamel matrix formation. The Ca-GBHA deposits appeared initially
in the proximal cell ends (Fig.6) and gradually extended to the level of
distal intercellular junctions and remained so throughout the stage of matrix
formation (Figs.6,7).
At the transitional stage, the granular deposits in the ameloblast layer
gradually decreased in number as well as diameter and the deposits diminished
before the completion of transformation of transitional ameloblasts to the
ruffle-ended ameloblasts. On the contrary, the granular deposits in the
transitional ameloblasts persisted in such cases that the maturation stage
began with the smooth-ended type ameloblasts. In the maturation stage,
only the smooth-ended ameloblasts displayed intense granular deposits along
lateral cell borders similarly as in the case of molar tooth germs (Figs.8,9).
In most cases, there was an exact correlation between the localization
of GBHA deposits and cyclical changes of ameloblasts' morphology (Fig.10).
Discussion
Cell calcium is thought to be located in the cytoplasm either in the form
of free ions or as bound calcium to certain molecules and play crucial roles
in regulation of various biological cell functions. Recent progress in
methods have made it possible to visualize cell calcium and its real-time
fluctuation in the living cells by introducing certain calcium indicators
such as fluorescent dyes (23) in the cytoplasm. At present, however, the
system is useful only on isolated cells in vitro and hence unavailable
to test the mechanisms of calcium regulation by the organized cell layers
such as the ameloblast layer in situ. The present experiment system allows
the observations of interactions between calcium and organized cell layers
although the condition used is much less biological .
In this experiment, we did not aim at demonstrating the behavior of biological
calcium in the enamel organ but sought possible differences in cell-calcium
interactions among the cells of the enamel organ, particularly in binding
affinity of membranous components of these cells to calcium ions. In this
context, the deliberately non-biological perfusion condition employed in
this system suited the evaluation of calcium-binding property of the material
constituting cell membranes.
GBHA staining is not specific for calcium ions but reacts to a variety
of divalent cations and form precipitates showing various colors (24).
The kind of divalent cations forming granular GBHA deposits thus needed
to be clarified. One of the advantages of our system is that the ultrathin
sections can be made from the same plastic embedded specimen and processed
for electron microscopy and hence X ray microanalysis. The data from previous
analytical studies indicate calcium and phosphate being major constituents
of granular GBHA deposits in the ameloblast layer at both the secretory
and maturation stages (25). It is therefore safe to state that the localization
of granular GBHA reactions coincides with the site of granular calcium phosphate
deposits in the enamel organs occurring after perfusion with high calcium-containing
solution.
The biological significance of the phenomenon is unclear. The granular
deposits of Ca-GBHA occur specifically in the secretory and smooth-ended
maturation ameloblasts and are lacking in the ruffle-ended ameloblasts,
the latter being widely regarded as most actively involved in calcium transport
to the enamel (26). It is highly possible that the lack of Ca-GBHA deposits
implies lack of calcium-binding property in the membrane constituents
of the respective cells. It may also be the case that the lack of precipitates
results from parallel with the lack of the source of sufficient phosphates
available to form calcium phosphate precipitates, since all membrane-associated
precipitates in the ameloblast layer contain high concentrations of phosphorus.
One of the candidates that may cause calcium accumulations to occur along
the biological membrane is cell surface coat which is negatively charged
and attached to the outer surface. This is unlikely to be the case because
the calcium phosphate deposits occur only in association with the cytoplasmic
aspect of plasma membranes as confirmed in our previous study of rat incisors
(25). Another candidate is the phospholipids, major constituent of cell
membranes. It should be noted that phosphatidylserine, one of the phospholipids
constituting cell membranes, has a high affinity for calcium and is predominant
in the inner leaflet of plasma membranes where granular Ca-GBHA deposits
were associated with (27,28). In this context, we are currently investigating
whether or not the relative proportion of phosphatidylserine among the membrane
phospholipids of ameloblasts at different developmental stages fluctuates
in accord with our histochemical findings of depositions of calcium phosphate.
Further efforts are also needed to seek for other possible factors which
may induce calcium depositions along cell membranes.
Acknowledgment:
This study was supported in part by a Grant-in- Aid for Scientific Research
from the Ministry of Education, Science and Culture, Japan (No. 04404069).
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Figure Legends
Plate 1: Molar tooth germs
Fig.1 Granular GBHA reactions associated with secretory ameloblasts (arrows).
Granular deposits are located along lateral cell borders. N nucleus
x620
Fig.2 Ruffle-ended maturation ameloblasts (RA) and adjacent papillary
layer (PL) showing the absence of GBHA reactions. Arrows indicate some
granular deposits near enamel surface. x500
Fig.3 Granular region reactions in the smooth-ended ameloblasts (SA) at
the sulcular region. Asterisk indicate GBHA granules in obliquely cut SA.
PL papillary layer. x380
Fig.4 Enlargement of granular GBHA reactions associated with the smooth-ended
ameloblasts (SA). PL papillary layer x930
Fig.5 Smooth-ended ameloblasts (SA) and adjacent cells of the papillary
layer (PL) showing granular GBHA reactions. Granular deposits are also
seen along the endothelium of the capillaries (C). x850.
Plate 2: Incisors
Fig.6 Darkfield image of GBHA reactions in the longItudinal section of
incisor . Granular deposits first appear at the proximal portion of secretory
ameloblasts (Am) concomitant with onset of enamel formation (arrow). Od
odontoblasts x160
Fig.7 Secretory ameloblasts (Am) at the stage of inner enamel secretion
showing intense granular GBHA deposits along lateral cell borders. Darkfield.
E enamel , x200
Fig.8 Fluctuation of GBHA reactions in the maturation stage. Granular GBHA
reactions are located exclusively in the smooth-ended ameloblasts (SA).
The portion of enamel (E) overlain by SA show intense (red) GBHA reactions
(asterisk). D dentin x65
Fig.9 & Fig.10 Enlargement of GBHA reactions associated with the smooth-ended
ameloblasts (SA). Ruffle-ended ameloblasts with ruffled border (arrowhead)
are lacking GBHA reactions E enamel PL papillary layer x600