Volume 45(2): 213–230, 1997
The Journal of Histochemistry & Cytochemistry

ARTICLE

Pathway and Speed of Calcium Movement from Blood toMineralizing Enamel
Tadafumi Kawamoto and Masaharu Shimizu
Department of Biochemistry, School of Dental Medicine, Tsurumi University, Tsurumi, Yokohama, Japan

We studied by autoradiography the distribution of 45Ca in the enamel organof frozen rats 4.3, 6.1, 7.8, 10.6 and 13.7 sec after an IV injection. The intercellular junctionsof the proximal side of the smooth-ended ameloblast (SA) and the distal side of the ruffleended ameloblast (RA) were closed to calcium. The junctions of the distal side of SA, theproximal side of RA, and both sides of the secretory stage ameloblasts were not. The timerequired for calcium to pass through the ameloblast layer was less than 1.8 sec in the secretory stage and SA region. The time in the RA region was 3.5–6.3 sec. In the transitional region from RA to SA, a band of strong radioactivity appeared from the papillary layer of RA
region towards the enamel of the SA region. The radioactivity in the secretory stage
enamel increased almost linearly with time. The diffusion speed of calcium in the enamel
was more than 50 µ m for 1.8 sec in the maturation stage and less than 15 mm for 9.4 sec inthe secretory stage. These results indicate that in the secretory and SA regions calcium
moves to the enamel surface through the intercellular spaces of ameloblasts and in the RA
region via RA cells. (J Histochem Cytochem 45:213–230, 1997)
SUMMARY

Calcium flux into forming enamel is regulated bychanging the tightness of the intercellular junctions ofameloblasts and the function of cellular calcium transport. The extracellular pathway has been histologically examined by electron microscopy (Sasaki andGarant 1986a,b; Sasaki et al. 1982,1985; Warshawsky 1978; Josephsen and Fejerskov 1977) and tracer
experiments using horseradish peroxidase (HRP), lanthanum [La(NO3)3.6H2O], calcein, EDTA, and various different molecular weight proteins (Kawamotoand Shimizu 1994; Debari et al. 1986; McKee et al.
1986; Sasaki et al. 1983; Kallenbach 1980a,b; Takano
and Crenshaw 1980). Cellular calcium transport has
been discussed from the standpoint of calcium distribution in cells (Takano et al. 1990a; Ashrafi et al.
1989; Chen et al. 1986; Kogaya and Furuhashi 1986;
Lyaruu et al. 1985; Reith and Boyde 1985; Eisenmann
et al. 1982; Engel 1981; Ozawa et al. 1979; Boyde andReith 1978; Deporter 1977; Kashiwa and Sigman,1966) and of enzymatic activity such as Ca-ATPase
Correspondence to: Tadafumi Kawamoto, Dept. of Biochemistry, School of Dental Medicine, Tsurumi Univ., 2-1-3 Tsurmi,
Yokohama, Japan.
Received for publication May 16, 1996; accepted September 13,
1996 (6A3976).
© The Histochemical Society, Inc.

0022-1554/97/$3.30

KEY WORDS
amelogenesis
ameloblast
calcium transport
mineralization
autoradiography
ratactivity (Eisenmann et al. 1990; Sasaki and Garant1986a,1987; Takano and Akai 1987; Inage and Weinstock 1979; Salama et al. 1978). However, a conclusive description of the pathway and the mechanism ofcalcium transport has not yet been achieved, because
the tracers and calcium differ in chemical and physicalproperties, and because the relation of calcium movement to forming enamel and the distribution of calcium and of enzymatic activity are not clear. There
have been attempts to directly follow the pathway ofcalcium movement to forming enamel by 45Ca autoradiography ( Hanawa et al. 1990; Takano et al. 1990b;
Kawamoto and Shimizu 1987; Reith et al. 1984;
Crenshaw and Takano 1982; Reith and Boyde 1981;
Wennberg and Bawden 1978a,b; Nagai and Frank
1975; Munhoze and Leblond 1974; Hammarstrom
1971; Suga et al. 1970; Reith and Cotty 1962). The
distribution patterns of radiolabeled calcium in thecell layer have been inconsistent because calcium inthe cell layer, which is readily soluble, is translocatedand/or lost from the original site during histologicaland autoradiographic procedures. On the other hand,the uptake patterns of radiolabeled calcium intoenamel are established, and the radiolabeled calcium
incorporated into the enamel passes through the cell
213

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layer within 30 sec. Therefore, to determine the pathway and the mechanism of calcium transport in thecell layer, it is necessary to examine the distribution ofradiolabeled calcium in the enamel organ less than 30
sec after the iv injection. However, this distribution
has not yet been demonstrated because of technicaldifficulties such as the need to dissect the specimen
within the time available and the need to prepare thetissue without translocating the tracer.
In this study, we attempted to define the contribution of cells to calcium transport and the primary routeof calcium movement from the blood to the forming
enamel. To accomplish this, the distribution patternsof radiolabeled calcium in the enamel organ within 30
sec after tracer injection were examined by the autoradiographic method (Kawamoto 1990) that is used to
study the distribution of water-soluble tracers in tissues.

Materials and Methods
Preparation of Sections
To examine the distribution of 45Ca in the enamel organ of a
rat lower incisor within 30 sec after the iv injection of tracer,
section preparation and autoradiography were carried out
according to the method described by Kawamoto (1990).
Nine-day-old Wistar rats (weighing about 20 g) wereanesthetized with sodium pentobarbital, and 0.1 ml of calcein (0.04%) in physiological saline (0.1 ml) was injected
into the abdominal cavity to distinguish the developmental
stages of the lower incisor enamel by its distribution. Thirty
min after injection of calcein, 0.05 ml of saline containing
45CaCl (185 KBq/g body weight) (Dupont NEN; Boston,
2MA) was injected into the iliac vein with an automatic syringe driven by an electric motor. The animals were thenrapidly frozen in liquid nitrogen. The time from the beginning of injection of the tracer until dipping the rats in the liquid nitrogen was electrically measured and recorded. The solution in the syringe was injected in approximately 0.1 sec.
Thirty-six rats were used for this study, and they were exposed to radiolabeled calcium for 3–30 sec. Among them,
five rats that were most successfully injected with the tracer
over an optimal period of time and with a desirable time of
exposure to the tracer (4.3, 6.1, 7.8, 10.6, and 13.7 sec)
were selected and examined.
The frozen animals were embedded in a 5% carboxymethyl cellulose solution (CMC) and the CMC was frozen incold hexane (290C). The CMC block was attached to thesample stage of a microtome (LKB PMV 2258; Bromma,
Sweden) in a cryostat kept at 220C and was then trimmed.
The sectioning surface was covered with a sheet of Saran
Wrap coated with a synthetic rubber adhesive (Azia Genshi;
Gifuken, Japan) and frozen sections were cut at 4 and 7 mm
thick. One serially adjacent section was used for light microscopic autoradiography and the other for contact autoradiogaphy.

Contact Autoradiography
After being freeze-dried for 24 hr in the cryostat, 7-mm-thicksections were removed from the cryostat. The boundary be-

Kawamoto, Shimizu
tween the ameloblast layer and the enamel was carefully cut
with a surgical blade under a microscope to separate the cell
layer from the enamel so as to avoid backscatter of radiation
to the enamel from the cell layer. Both the separated cell
layer and the enamel samples were placed on a glass slide
coated with adhesive glue. They were then covered with aplastic film 4 mm thick.
Before the autoradiographic procedures, the distributionof calcein fluorescence in the sections was recorded with aconfocal laser scanning microscope (CLSM) (Olympus LSMGB200; Tokyo, Japan). Then the sample was contacted with
X-ray film (Fuji IX 50; Fuji Photo, Tokyo, Japan) and exposed for 3 weeks. The film was developed with Rendol(Fuji Photo) for 5 min and fixed with Fuji Super Fix (Fuji
Photo) for 5 min. The labeled areas were identified by superimposing the image of the autoradiograph over the distribution patterns of the calcein image on CLSM.

Light Microscopic Autoradiography
After being freeze-dried in the cryostat, the 4-mm-thick sections were covered with an autoradiographic emulsion film
approximately 1 mm thick according to the method described previously (Kawamoto 1990). In brief, 0.4 ml of
0.2% aqueous solution of sodium dioctylsulfosuccinate was
mixed well with 5 g of autoradiographic emulsion (Konika
NR-M2; Konica, Tokyo, Japan) melted at 40C. A small
amount of the emulsion was collected by dipping the tip of a
glass tube into the emulsion and a bubble was blown. The
emulsion film of the bubble was applied to the section when
the film was half dried. The emulsion film was applied twice.
For exposure, the emulsion covered sections were stored for
5 weeks at 280C in a light-tight box containing silica gel.
After exposure, the emulsion on the section was developed
and fixed. The section was then stained with hematoxylin
and eosin. The autoradiographs were examined by light microscopy and CLSM.

Grain Counting
To count all the silver grains in the examined areas, theemulsion layer of the light microscopic autoradiograph was
optically sliced every 1.2 µm at different focal planes bymeans of CLSM. The images of the grain distribution ineach focal plane were recorded in a computer attached to the
CLSM. Then, one in-focus image for all of these focal planes
was made on a monitor. The grain counting was carried out
manually on the monitor while the contrast and the brightness were varied.
In the secretory stage, the location at which the enamel
approximately 50 mm thick was formed was examined. In
this location, 19 areas of 5 3 25 mm2 each [two areas (SR1,
SR2) in the outer enamel epithelium and the stellate reticulum layer, one area (SI1) in the stratum intermedium, six areas (Am1–Am6) in the ameloblast layer, and 10 areas (En1–
En10) in the enamel] were selected for a section, as shown in
Figure 1A. In the maturation stage, the regions of smoothended ameloblasts (SAs) and the apical two thirds of the ruffle-ended ameloblasts (RAs) were examined. In those regions,
16 areas of the same size [two areas (PL1, PL2) in the papillary layer, four areas (Am1, Am2, Am5, Am6) in the ameloblast layer, and 10 areas (En1-En10) in enamel] were se-Calcium Movement in the Ameloblast Layer
lected, as shown in Figure 1B. In the papillary layer region,an area was selected in a region that excluded blood vessels.
Four areas at the same distance from the enamel surface
were counted, and the mean values and SD of each area were
calculated. In addition to those developing stages, the transitional area from RA to SA was examined by the same
method, as shown in Figure 10A. The background counts
were estimated by counting grains in several selected areas
adjacent to the periphery of the sections.

Results
General Morphology and Autoradiography

Although the expanded intercellular spaces and somecracks caused by shrinkage of cells and tissues during
the freeze-drying were observed in almost all sections,the papillary layer cells, stratum intermedium cells andameloblasts were clearly identified (Figures 3, 6, and
8). In contact autoradiographs (CARGs), the localization of radioactivity in the papillary, ameloblast, andenamel layers could be clearly distinguished (Figures 2
and 5). In the light microscopic autoradiographs(LMARGs), chemographies were not detected in thecontrol samples exposed in the same way.
Secretory StageCARG of the lower incisor section of rat frozen at 4.3
sec after iv injection of 45CaCl2 showed high radioac-

Figure 1 Diagrams showing the regions for grain counts in the secretory
stage (A) and the maturation stage(B). Each region represents a unit
area (5 3 25 mm2 ), and the regionsin the enamel are continuously arranged from the greatest enamel surface towards the dentin–enamel junction. SR1, SR2, PL1, and PL2 arearranged in an area excluding theblood vessels, and Am5 and Am6 arein the nuclear portion. SR1, SR2, stellate reticulum SI1, stratum intermedium; PL1, PL2 papillary layer; AM1–
AMb, ameloblast layer; EN1–EN10
enamel; CV, capillary vessels.

215
tivity in a part of the facial artery but not in the veins(Figure 2A2). The LMARG, in addition to these tissues, showed faint radioactivity in the capillary vessels
on the external side of the outer enamel epithelium,
the stellate reticulum, stratum intermedium and in theproximal part of the ameloblast layer (Figures 3A2
and 4; Table 1). However, no radioactivity was located in the distal area of the ameloblast layer and in
the enamel.
At 6.1 sec after injection, intense radioactivity was
detected in most facial arteries and increased significantly. The radioactivity in the capillary vessels also
increased but remained lower than that in the peripheral tissue of the capillary vessels (Figures 3B2 and 4).
In the ameloblast layer, radioactivity was detected
throughout the entire thickness. The radioactivity wasconsiderably lower than that in the stellate reticulum,and it decreased from the proximal side of the ameloblast towards the distal side (Figure 4). In the enamel,
grains showing 45Ca uptake were observed in the surface layer of the enamel (Figures 3B2 and 4; Table 1).
The radioactivity was distributed in the surface layer
10 mm below the enamel surface.
At 7.8 sec, the radioactivity in the stellate reticulum
increased by approximately twofold (Figure 4; Table1). The radioactivity in the ameloblast layer was significantly lower than that in the stellate reticulum(Figure 3C2). The radioactivity decreased from the

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Kawamoto, Shimizu

Figure 2 Contact autoradiographs (right) at the secretory stage of a lower incisor section of 9-day-old rats injected IV with 45CaCl2 and thecalcein distribution (left) in the section used for autoradiography. The ameloblast layer was cut away from the enamel surface. High radioactivity corresponds to the blackened areas. At 4.3 sec, the radioactivity appears in the blood vessels (arrow in A2) in the connective tissues
overlying the enamel organ, but not in the veins (A2). In the enamel, the radioactivity is seen at 7.8, 10.6, and 13.7 sec (C2,D2,E2). Ve, vessel;
Ar, artery; SR1OE, stellate reticulum and outer enamel epithelium; En, enamel; T, transitional area from the secretory to the maturation
stage. (A2) At 4.3 sec; (B2) at 6.1 sec; (C2) at 7.8 sec; (D2) at 10.6 sec; (E2) at 13.7 sec. Bars 5 1 mm.

Calcium Movement in the Ameloblast Layer
Figure 3 Light microscopic autoradiographs at the secretory stage.
The left was focused on the tissues and the right was focused on thesilver grains. Each autoradiograph was prepared with the section adjacent to that used for contact autoradiography. Small silver grains
are seen in the stellate reticulum and the proximal part of theameloblast layer at 4.3 sec after an IV injection of 45CaCl2 (A2), but nograins are seen in the distal part of the ameloblast layer and theenamel. The 45Ca uptake in the enamel is seen at 6.1, 7.8, 10.6 and13.7 sec (B2,C2,D2,E2). SR 1 OE, stellate reticulum and outer enamel
epithelium; SI stratum intermedium; Am, ameloblast layer; En,
enamel. (A2) At 4.3 sec; (B2) at 6.1 sec; (C2) at 7.8 sec; (D2) at 10.6
sec; (E2) at 13.7 sec. Bars 5 25 µm.

217

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proximal side towards the distal side similar to thepattern seen by LMARG at 6.1 sec. The intensity inthe distal portion was approximately 13% comparedwith that of the proximal portion. In the enamel, thelabeled area extended to 15 µm below the enamel surface. However, intense radioactivity was only 10 mmdeep in the surface layer (Figures 3C2 and 4; Table 1).
At 10.6 and 13.7 sec, the intensity of radioactivity
in the stellate reticulum, stratum intermedium, andameloblast layer remained almost constant (Figure 4),
and the distribution patterns were almost the same as
those seen in autoradiographs at 7.8 sec. The strongest
radioactivity was located in the stellate reticulum, thenext strongest radioactivity was in the stratum intermedium, and the weakest radioactivity was in theameloblast layer. The radioactivity in the ameloblast
layer gradually decreased from the proximal side towards the distal side. The radioactivity in the most
distal portion was approximately 30% compared withthe proximal side. In the enamel, the intensity of radioactivity increased with time, but the labeled areaand labeling patterns were almost the same as thoseseen at 7.8 sec (Figure 2; Table 1).
The time-related changes in radioactivity accumulated within the enamel are shown in Figure 11. Uptake of the radiolabeled calcium was not observed until
4.3 sec after the injection. However, the uptake appeared at 6.1 and increased linearly with time thereafter.
Maturation Stage

Smooth-ended Ameloblast Region. CARG at 4.3 sec
after 45Ca injection revealed relatively high radioactivity in the blood vessels in the connective tissue overlying the enamel organ (Figure 5A2). LMARG, in addition to those tissues, showed weak radioactivity in thecapillary vessels of the papillary layer and the entire
thickness of the ameloblast layer (Figures 6A2 and 7).
However, no radioactivity was observed in the enamel.
At 6.1 sec, the radioactivity in the capillary vesselsof the papillary layer and in the ameloblast layer significantly increased (Figures 5B2 and 7; Table 2). In
the enamel, a clearly labeled area was observed (Figures 5B2 and 6B2). The labeled area was more than
50 mm in depth, and the intensity of radioactivity
gradually decreased from the enamel surface towards
the dentin–enamel junction (Figure 7).

Figure 4 Graphic presentation of Table 1. Radioactivity is seen in
the stellate reticulum and stratum intermedium layers at 4.3 sec
and in the enamel at 6.1 sec. The radioactivity in the ameloblast
layer is lower than that in the stratum intermedium layer and decreases from the proximal towards the distal side. In the enamel,
almost all of the radioactivity is located in the surface layer. Abbreviations as in Figure 1.

Kawamoto, Shimizu

Calcium Movement in the Ameloblast Layer

219

Figure 5 The 45Ca contact autoradiographs (right) at the maturation stage of the lower incisor section of 9-day-old rats injected IV with
45CaCl , and the calcein distribution (left) of the section used for the autoradiography. The ameloblast layer was cut away from the enamel2
surface. High radioactivity corresponds to the blackened areas. Radioactivity appears in the blood vessels in the connective tissue overlying
the enamel organ at 4.3 sec, but not in the enamel itself (A2). After 6.1 sec, the radioactivity is seen in the enamel of the SA region(B2,C2,D2,E2). In the RA region, after 10.6 sec, the 45Ca uptake into the enamel is seen (D2,E2), and significant intense radioactivity is localized to the enamel in the apical two thirds of this region at 13.7 sec (E2). PL, papillary layer; Ve, vessel; En, enamel; SA, region of smoothended ameloblasts; RA, region of ruffle-ended ameloblasts. (A2) At 4.3 sec; (B2) at 6.1 sec; (C2) at 7.8 sec; (D2) at 10.6 sec; (E2) at 13.7 sec.
Bars 5 1 mm.

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Figure 6 45Ca light microscopic autoradiographs at the SA region of
the maturation stage. The counted regions are indicated in Figure
1B. Left was focused on the tissues and right was focused on the silver grains. Each autoradiograph is prepared with the section adjacent to that used for contact autoradiography. Small grains are distributed in the papillary and ameloblast layers at 4.3 sec after IV
injection of 45CaCl2 (A2), but not in the enamel. At 6.1, 7.8, 10.6, and13.7 sec, many grains are seen in the enamel. PL, papillary layer; Am,ameloblast layer; En, enamel. (A2) At 4.3 sec; (B2) 6.1 sec; (C2) 7.8 sec;
(D2) 10.6 sec; (E2) 13.7 sec. Bars 5 25 µm.

Kawamoto, Shimizu

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Calcium Movement in the Ameloblast Layer
Table 1 Radioactivity in the cell layers and enamel (secretory stage)aDistance fromenamel surface

Enamel surface
5 µm
10 µm
15 µm
20 µm
25 µm
30 µm
35 µm
40 µm
45 µm
50 µm

Examined
areas

Time after 45Ca injection
4.3 sec

6.1 sec

7.8 sec

10.6 sec

13.7 sec

SR2
SR1
SI1
Am6
Am5
Am4
Am3
Am2
Am1

10.6 6 4.0
10.6 6 3.1
5.2 6 2.1
2.9 6 1.0
2.6 6 0.6
0.9 6 0.0
0.9 6 1.0——

34.3 6 4.1
34.7 6 2.1
20.0 6 1.0
13.3 6 6.6
11.3 6 2.5
5.3 6 0.6
4.3 6 3.2
3.7 6 1.5
2.7 6 1.5

86.7 6 4.7
83.0 6 3.6
60.7 6 3.5
36.7 6 5.1
37.3 6 3.5
15.3 6 4.6
10.0 6 1.0
5.6 6 1.0
4.7 6 0.6

77.6 6 11.0
73.6 6 7.9
49.3 6 4.4
25.0 6 4.0
28.6 6 3.2
20.3 6 3.5
16.3 6 3.6
13.6 6 3.1
8.0 6 3.1

51.0 6 3.8
56.6 6 4.0
36.3 6 9.0
24.3 6 5.3
21.3 6 3.6
12.3 6 3.0
7.6 6 0.6
6.6 6 1.5
7.0 6 1.5

En1
En2
En3
En4
En5
En6
En7
En8
En9
En10

—
—
—
—
—
—
—
—
—
—

2.6 6 0.6
2.0 6 1.0
0.7 6 1.2
0.7 6 1.5
—
—
—
—
—
—

12.3 6 2.2
11.8 6 2.2
3.8 6 2.1
0.5 6 0.6
—
—
—
—
—
—

24.0 6 3.1
16.3 6 4.0
3.3 6 1.7
0.5 6 0.6
—
—
—
—
—
—

49.1 6 6.0
23.6 6 5.1
8.1 6 3.8
0.3 6 1.4
0.6 6 1.5
—
—
—
—
—

a Time-related changes of 45Ca radioactivity in various regions of the stellate reticulum, stratum intermedium, ameloblast, and enamel layers at the secretory
stage of rat lower incisor. Areas for grain counts are diagrammatically indicated in Figure 1A. Each value shows the net grain counts. Values are means 6 SD.

At 7.8 sec, the radioactivity in the papillary andameloblast layers further increased (Figure 7). The radioactivity in the enamel was slightly lower than thatseen at 6.1 sec, but the distribution pattern was almost
the same (Table 2).
At 10.6 and 13.7 sec, the intensity and distribution
patterns of the radioactivity in the ameloblast layer
were almost the same as that seen at 7.8 sec (Figure 7).
The distribution of radioactivity in the enamel was

also the same as that at 6.1 sec. The highest radioactivity was located in the uppermost surface layer andthe intensity gradually decreased toward the dentin–
enamel junction.
Ruffle-ended Ameloblast Region. CARG and LMARG
at 4.3 sec after an iv injection of 45CaCl2 showed thatthe distribution of radioactivity in the connective tissues overlying the enamel organ, the papillary layer,

Table 2 Radioactivity in the cell layers and enamel (SA region of maturation stage)aDistance fromenamel surface

Enamel surface
5 µm
10 µm
15 µm
20 µm
25 µm
30 µm
35 µm
40 µm
45 µm
50 µm

Examined
areas

Time after 45Ca injection
4.3 sec

6.1 sec

7.8 sec

10.6 sec

13.7 sec

PL2
PL1
Am6
Am5
Am2
Am1

23.9 6 1.0
21.9 6 3.5
12.6 6 1.5
16.6 6 3.5
10.9 6 4.4
7.9 6 2.6

21.2 6 3.5
26.6 6 8.3
23.7 6 3.0
26.1 6 7.6
24.1 6 5.9
54.2 6 6.7

51.1 6 9.0
52.4 6 8.3
59.9 6 3.5
55.9 6 0.7
40.9 6 0.7
36.9 6 6.4

112.8 6 10.5
96.5 6 9.6
64.5 6 8.6
56.9 6 6.6
44.8 6 8.0
48.8 6 8.7

46.6 6 7.7
58.6 6 3.7
50.3 6 4.3
57.3 6 8.7
52.3 6 4.5
97.6 6 6.6

En1
En2
En3
En4
En5
En6
En7
En8
En9
En10

—
—
—
—
—
—
—
—
—
—

88.2 6 1.5
68.9 6 8.5
56.2 6 4.9
40.6 6 9.2
29.9 6 4.4
21.5 6 6.4
12.6 6 6.0
9.2 6 1.2
7.9 6 2.6
3.9 6 2.0

49.4 6 2.8
43.9 6 4.9
43.4 6 1.4
39.9 6 2.1
25.9 6 0.7
19.4 6 2.6
16.4 6 1.4
4.9 6 0.7
2.9 6 0.7
1.9 6 0.7

151.1 6 6.8
108.8 6 9.5
48.8 6 7.5
26.1 6 6.6
22.1 6 6.7
14.8 6 2.6
13.1 6 2.1
9.8 6 2.0
7.3 6 3.1
3.1 6 1.2

118.6 6 9.3
85.0 6 9.3
55.6 6 7.5
26.6 6 4.2
13.0 6 3.1
6.3 64.6
3.6 6 1.2
0.6 6 1.2
1.9 6 3.1
0.6 6 3.1

a Time-related changes of 45Ca radioactivity in various regions of the papillary, ameloblast, and enamel layers at the SA region of the rat lower incisor maturation

stage. Areas for grain counts are diagrammatically indicated in Figure 1B. Each value shows the net grain number. Values are means 6 SD.

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Kawamoto, Shimizu
and the ameloblast layer were similar to that seen in
the SA region. Radioactivity was not detected in theenamel (Figures 5A2 and 8).
At 6.1 sec after the injection, LMARG showed thatthe radioactivity in the papillary and ameloblast layers
significantly increased, whereas it decreased from thepapillary layer towards the distal side of the ameloblast layer (Figures 8B2 and 9). The radioactivity inthe enamel was the same as the background, althoughsome grains were dispersed.
At 7.8 sec, the distribution of radioactivity in thecell layer was similar to that seen at 6.1 sec, and theradioactivity in the enamel was almost the same as the
background (Figures 8C2 and 9).
At 10.6 sec, significant changes in the distributionof radioactivity were not observed in the cell layer(Figure 8D2; Table 3). However, in the enamel, afaintly labeled area appeared in an area representing
approximately the apical two thirds of the RA region(arrow R1 in Figure 5D2).
At 13.7 sec, significantly increased radioactivity appeared in the ameloblast layer in the apical two thirdsof the RA region, but the intensity was not the same
(Figures 5 and 9). The radioactivity was stronger thanthat in the papillary layer, and the strongest radioactivity was found in the distal portion (Figure 9; Table3). In the enamel, the area representing approximately
the apical two thirds of the RA region was heavily labeled (Figure 5). The intensity was not the same in thisregion, and the labeling in the enamel just beneath theheavily labeled RA cells was intense. The radioactivity
was distributed to a depth of 50 mm. No radioactivity
was found in the area of approximately the incisal one
third of the RA region, and radioactivity of the ameloblast layer in the area was very weak.
Transitional Area from RA to SA. The distribution patterns of radioactivity in the transitional area from RA
to SA in LMARG at 10.6 sec after a 45Ca injection are
shown in Figure 10A. The contour map representationof the intensity is shown in Figure 10B. In the incisal
one third of the RA region, the radioactivity in theameloblast layer was weak, although relatively strong
radioactivity was observed in the papillary layer. The
radioactivity in the enamel was almost the same asthat in the background. The radioactivity in the ameloblast layer increased close to the transitional area andbecame strongest at the transitional area. The intensity again decreased close to the middle portion of the
SA region. The radioactivity of the ameloblast layer inFigure 7 Graphic presentation of Table 2. Radioactivity appearedin the papillary layer at 4.3 sec and in the enamel at 6.1 sec. The radiolabeled calcium incorporated into the enamel is rapidly diffused
towards the dentin–enamel junction. Abbreviations as in Figure 1.

Calcium Movement in the Ameloblast Layer
Figure 8 45Ca light microscopic autoradiographs at the RA region.
The counted regions are indicated in Figure 1B. Left was focused on
the tissues and right was focused on the silver grains. Each autoradiograph was prepared with the section adjacent to that used in contact autoradiography. Small grains are observed in the papillary andameloblast layers at 4.3 sec after an IV injection of 45CaCl2 (A2), but
not in the enamel. Many grains are seen in the cell layers at 6.1 and7.8 sec (B2,C2) but the grain density in the enamel is almost the sameas the background. At 13.7 sec, many grains are observed in theenamel (E2). PL, papillary layer; Am, ameloblast layer; En, enamel.
(A2) at 4.3 sec; (B2) at 6.1 sec; (C2) at 7.8 sec; (D2) at 10.6 sec; (E2) at
13.7sec. Bars 5 25 mm.

223

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Kawamoto, Shimizu
this region was very weak, whereas strong radioactivity was distributed in the papillary layer. In the enamel,
the strongest radioactivity was found in the transitionalarea, and the intensity decreased closer to the middle
portion of the SA region.

Discussion
When the pathway and the speed of calcium movement in the ameloblast layer are estimated from the
distribution patterns of radiolabeled calcium, the timeof exposure to the label is very important. In a preliminary experiment, radiolabeled calcium injected into
iliac vein was not detected in the lower incisor until
3.5 sec after injection, and the well-known labeling
patterns appeared in the enamel after 15 sec. Therefore, an exposure time of 3.5–15 sec was selected forthis study, and only one animal was examined fromeach time because it was too difficult to expose the animal to the tracer for the same period.
In autoradiography over a very short period using alarge sample, the results are greatly influenced by thetime required to freeze the sample. The 9-day-old rats
began to freeze immediately after being placed in theliquid nitrogen. Naturally, the enamel organ of thelower incisor situated approximately 1.2 mm under
the skin would be frozen very soon after immersion.
All the animals used were frozen under the same conditions. In addition, to measure accurately the exposure time to the radiolabeled calcium, injection of
tracer and measurements were carried out automatically with an apparatus especially designed by us.
Therefore, we believe that the influence of freezingtime on the accuracy of the measurement is veryslight.
Even if such technical difficulties are overcome,there is a risk of false results when radiolabeled calcium in a readily soluble form is used for autoradiography. False results can be caused by translocation of
the tracer during tissue preparation, by the procedureof coating the autoradiographic emulsion on the section, by chemography induced by chemical substancesin tissues, and by miscounting of grains. Translocationof the tracer was minimized by using freeze-sectioning
and dry-emulsion film coating techniques as describedby Kawamoto and Shimizu (1986). Chemography was
also minimized by exposure at 280C. Miscounting ofgrains was solved by using CLSM, by which an in-

Figure 9 Graphic presentation of Table 3. Strong radioactivity isseen in the cell layers at 6.1 and 7.8 sec, but no radioactivity in theenamel. 45Ca uptake into the enamel is seen at 10.6 and 13.7 sec,
and it diffuses rapidly towards the dentin–enamel junction. The examined areas of R1 and R2 at 10.6 sec are indicated by arrows R1
and R2 in Figure 5D2. Abbreviations as in Figure 1.

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Calcium Movement in the Ameloblast Layer
Table 3 Radioactivity in the cell layers and enamel (RA region of maturation stage)aDistance fromenamel surface

Enamel surface
5 µm
10 µm
15 µm
20 µm
25 µm
30 µm
35 µm
40 µm
45 µm
50 µm

Time after 45Ca injection

Examinedareas

4.3 sec

6.1 sec

7.8 sec

10.6 sec (R1)

10.6 sec (R2)

13.7 sec

PL2
PL1
Am6
Am5
Am2
Am1

8.9 6 3.6
6.2 6 0.6
2.9 6 1.0
2.9 6 1.0
2.9 6 2.0
1.2 6 1.2

112.9 6 6.0
89.4 6 7.1
83.6 6 1.2
71.9 6 5.0
51.9 6 6.1
53.6 6 8.0

46.7 6 2.1
47.1 6 7.5
61.7 6 3.2
73.1 6 5.1
63.4 6 2.6
48.4 6 3.6

51.8 6 5.0
50.5 6 11.5
53.1 6 5.5
57.8 6 3.6
56.8 6 1.7
56.8 6 1.7

71.1 6 7.6
58.5 6 4.5
56.5 6 4.9
52.3 6 6.5
47.6 6 3.7
40.1 6 3.4

81.0 6 9.0
90.3 6 5.0
114.6 6 3.2
119.6 6 13.5
121.6 6 3.6
112.6 6 14.2

En1
En2
En3
En4
En5
En6
En7
En8
En9
En10

—
—
—
—
—
—
—
—
—
—

9.2 6 3.0
3.9 6 1.4
1.7 6 1.7
1.2 6 1.7
—
—
—
—
—
—

8.7 6 3.1
4.1 6 1.5
0.7 6 1.5
0.4 6 1.0
0.4 6 1.0
—
—
—
—
—

34.5 6 3.6
27.5 6 1.5
11.8 6 4.0
9.5 6 1.7
4.5 6 2.5
2.8 6 2.1
2.5 6 2.0
2.8 6 1.5
1.5 6 1.0—

8.8 6 3.6
7.8 6 1.1
1.1 6 1.5
—
—
—
—
—
—
—

147.6 6 5.1
107.6 6 3.8
86.0 6 8.5
77.6 6 12.7
70.6 6 7.5
55.6 6 5.1
48.6 6 5.1
41.3 6 4.3
30.3 6 7.2
21.9 6 7.6

a Time-related changes of 45Ca radioactivity in various regions of the papillary, ameloblast, and enamel layers at the RA region of the rat lower incisor maturation

stage. Areas for grain counts are diagrammatically indicated in Figure 1B. The positions corresponding to the results of R1 and R2 are indicated by arrows R1 andR2 in Figure 5E2. Each value shows the net grain number. Values are means 6 SD.

focus image was made with images of different focal
planes on the sample. From a careful performanceof 45Ca autoradiography, four clear results wereachieved: the time for calcium to diffuse across theameloblast layer; the tightness of intercellular junctions of ameloblasts to calcium; the pathway of calcium movement in the ameloblast layer; and the diffusion speed of calcium within the enamel layer.
In the maturation stage, there are two types ofameloblasts: ruffle-ended ameloblasts (RAs), with aruffled border consisting of deep membrane invaginations and smooth-ended ameloblasts (SAs) with flattened distal cell surfaces (Reith and Boyde 1981; Josephsen and Fejerskov 1977; Warshawsky and Smith
1974; Suga 1959). Both cells are arranged on the enamel
in a banded pattern, and their histological characteristics change periodically. The RA cells and the SA cells
can be identified by the distribution of calcein fluorescence (Josephsen 1983). Smith et al. (1987) showedthat these ameloblasts modulate extremely rapidly, on
the order of 1 hr. In this study, although influenced by
the lag between the injection of calcein and radiolabeled calcium, the influence was very little, and bothregions were well identified by the calcein fluorescence.
The time required for calcium to pass through theameloblast layer was estimated from the time at which
the radiolabeled calcium appeared on the stratum intermedium in the secretory stage or on the papillary
layer in the maturation stage, entering into theenamel. Each time is summarized in Figure 12. In thesecretory stage and the SA region, the radiolabeledcalcium injected into the iliac vein was recognized in
the stratum intermedium cell layer and the papillary

layer by autoradiography at 4.3 sec and in the enamel
at 6.1 sec. In the RA region it was recognized in thepapillary layer at 4.3 sec. The grains in the enamel at
6.1 and 7.8 sec appear to represent radiation from thelabel distributed in the ameloblast layer, because it
was distributed only in the surface layer. Therefore,
the relatively intense labeling seen at 10.6 sec indicates
incorporation of radiolabeled calcium into the enamel.
From those results, the time for calcium across theameloblast layer was estimated to be less than 1.8 sec
in the SA region and between 3.5 and 6.3 sec in the
RA region. The time difference is affected mainly by
the distance to which calcium moves and by the calcium transport systems. In the RA region, it takes
much longer than in the SA region. This suggests thatthe transport system of calcium in the RA region isdifferent from that in the secretory stage and the SA
region, because the distance to which calcium moves
from the blood to the enamel in the RA region is considerably shorter than in the secretory stage and the
SA region, as described below.
In the SA region, Josephsen and Fejerskov (1977)
showed that SA cells close the proximal junctions andopen the intercelluar spaces to enamel. Tracer experiments (Kallenbach 1980a,b; Josephsen 1984; Kawamoto and Shimizu 1994) and histochemical studies(Nishikawa and Josephsen 1987) supported the morphological findings. In the autoradiograph of the middle portion of the SA region, the radioactivity in theproximal part of the SA cells was very low, althoughstrong radioactivity was present in the papillary layer.
This suggests that the intercellular junctions of theproximal side of the SA cells also act as a barrier tocalcium movement.

226

Kawamoto, Shimizu
Figure 10 Grain counts (A) of the transitional areafrom the RA to the SA regions in the autoradiograph at 10.6 sec after an IV injection of 45CaCl2,
and a contour map representation (B) of the grain
number in examined areas. Each region represents
a unit area (5 3 25 mm2). Intense radioactivity isseen in the ameloblast layer in the transitional areafrom the RA to the SA region. Intense radioactivity
is distributed in the papillary layer of SA region, but
the radioactivity in the proximal side of the ameloblast layer is weak. Arrows in B show the proposed
calcium flux from the papillary layer to the enamelof the SA region.

Histochemical studies (Takano 1995; Salama et al.
1978) show no Ca-ATPase activity at the distal end of
the SA cells. Kawamoto and Shimizu (1990,1994)
showed that radiocalcium and radiophosphate simultaneously injected iv are incorporated into the enamelof the SA region without cellular regulation, and thatthe ratio of both tracers is not very different from thatin the blood. Accordingly, the calcium that enters theenamel of the SA region initially penetrates the intercellular spaces of the RA cells from the papillary layer.
It subsequently diffuses to the intercellular spaces of
the SA cells and finally enters the enamel of the SA region. The contour map image of radioactivity in thecell layer shown in Figure 10 demonstrates the routeof calcium movement.
In the RA region, well-developed tight intercellularjunctions are formed at the distal side but the intercellular spaces of ameloblasts give access to the intercellular spaces of the papillary layer (Sasaki and Garant1986a,b; Sasaki et al. 1983; Josephsen and Fejerskov
1977). A light microscopic autoradiograph 6.1 sec after 45Ca injection showed clear uptake of radiolabeledcalcium in the enamel of the SA region. However, radioactivity was not found in the enamel of the RA region, whereas strong radioactivity was present in theameloblast layer. These findings indicate that the in-tercellular junctions of the distal side of the RA cells
act as a barrier to calcium movement.
In this region, it has been demonstrated that CaATPase localizes at the distal end of the ameloblast(Takano and Akai 1987; Salama et al. 1978) and thata large amount of calcium is transported into theenamel (Kawamoto and Shimizu 1990; Crenshaw and
Takano 1982; Reith and Boyde 1981; Suga et al.
1970). Takano et al. (1987) showed that the calcium
uptake in enamel is greatly inhibited by perfusion withvanadate, which is an inhibitor of Ca-ATPase. Kawamoto and Shimizu (1990, 1994) showed that the ratioof radiocalcium to radiophosphate incorporated into
the enamel is clearly higher than that in the blood, andthat the high ratio is achieved by active cellular calcium transport. As mentioned above, the time for calcium to diffuse across the ameloblast layer is much
longer than that in the SA region, and this extended
time is the result of movement via cells. The considerably high radioactivity in the RA cell layer supports
the hypothesis that calcium enters the enamel via theRA cells.
Relatively high standard deviations appeared in theameloblast layer and in the enamel at 13.7 sec. Kawamoto and Shimizu (1994) reported a variation in calcium transport function in the RA cells of this region.

Calcium Movement in the Ameloblast Layer

Figure 11 Time-related changes in total radioactivity distributedin all the examined areas (En1–En10 in Figure 1A) in the secretory
stage enamel are shown. After 4.3 sec, the radioactivity increases
almost linearly with time.

Autoradiography showed a variation in the radioactive intensity in those tissues. It is possible that a variation in the calcium transport function of each RA cellis reflected in both the radioactive intensity in thosetissues and the deviation.
Results related to the pathway and the mechanismof calcium transport in the secretory stage are confused compared to those in the maturation stage. Although at first Sasaki et al. (1982) showed complete
zonular tight junctions at the distal end of secretory
ameloblasts by freeze-fracture study, Sasaki (1984)
later revealed the presence of various spaces among thetight junctional strands of the distal junctional complexes. He stated that the interstrand spaces may provide a diffusion pathway for small molecular weight

Figure 12 The time required for calcium to pass through the ameloblast
layer is shown by both arrows. Whiteand black arrows indicate the time
when radiolabeled calcium appearedat the proximal side of ameloblasts
and at the enamel, respectively. At
4.3 sec, radiolabeled calcium is seenat the proximal side in all the developing stages. In the enamel, radiolabeled calcium appeared at 6.1 sec inthe secretory stage and the SA region. In the RA region, it is recognized after 10.6 sec.

227
materials. The extracellular pathway to the enamelhas been well demonstrated by several tracer experiments (Kawamoto and Shimizu 1994; MaKee et al.
1992; Sasaki 1984; Kallenbach 1980a).
It remains to be determined whether the radiolabeled calcium in the most outer layer (En1) of theenamel localizes inside the cell processes (Tome’s process) or outside, because the En1 area includes theprocess in addition to the enamel. The methods employed in this study did not permit us to distinguishbetween the radiocalcium that was within the cells
and that outside them. The time required for calcium
to diffuse across the ameloblast layer is significantly
less than that required in the RA region, although thesecretory ameloblasts are much taller than the RA
cells. In other words, the distance that calcium mustmove in the secretory stage is greater than that in the
RA region. The time required, less than that in RA region, suggests that the calcium in the enamel of theoutermost layer (En1) moves extracellularly to theenamel and localizes outside the processes. The calcium ion concentration in the extracellular fluid adjacent to the proximal cell membrane is 1023 M, and thefree calcium ion concentration in the fluid extracted
from secretory stage enamel is 1025 M (Aoba andMoreno 1987). Therefore, the flux of calcium toenamel would be generated by the gradient of calcium
concentration.
Electron microscopic 45Ca autoradiography (Nagai
and Frank 1975) showed two transfer pathways fromthe blood to the enamel surface, and also demonstrated that calcium passes through intercellular spaces
and reaches the enamel, with the second pathway consisting of an intracellular transfer through the ameloblasts. In autoradiography, it is too difficult to distinguish the radiolabeled calcium that is transported
from inside the cells to the enamel. In our preliminary
autoradiography 3, 5, and 10 min after an ip 45Ca in-

228
jection, the radioactivity of the ameloblast layer adjacent to the enamel in the incisal one third of the RA region, where calcium is not transported, increased withtime. This experiment suggests that autoradiography
for investigating the pathway of calcium movement
must be performed within 3 min after 45Ca injection.
Based on the distribution patterns of Ca-ATPase
activity (Sasaki and Garant 1987; Takano and Crenshaw 1981; Inage and Weinstock 1979; Salama et al.
1978) and the tightness of distal intercellular junctions(Garant et al. 1983; Takano and Ozawa 1980; Warshawsky 1978; Josephsen and Fejerskov 1977; Skobe
and Garant 1974; Kallenbach 1966), Bawden (1989)
described that calcium is transported to the enamelsurface by an ATP-dependent pump, as calcium does
not diffuse by the pericellular route from the extracellular fluid through the ameloblast layer to the enamel
matrix fluid. If calcium is actively transported by secretory ameloblasts, the changes in radioactivity accumulated within the enamel must be expressed as a broken
line, because the speeds of passive and active cellulartransport of calcium are different in the cell layer. In
addition, if the radiolabeled calcium enters the ameloblasts, the radioactivity of the layer must increase.
However, the radioactivity within the enamel increased with the time almost linearly during the period
examined, and the radioactivity of the ameloblast
layer remained low and with little change, decreasingfrom the proximal side towards the distal side. These
results indicate that calcium is not transported to theenamel surface by the secretory ameloblasts. Kawamoto and Shimizu (1994) showed that the amount ofradiolabeled calcium incorporated into the enamelover a unit area is significantly low compared to thatin the RA region, and that the ratio of radiolabeledcalcium to radiolabeled phosphate incorporated by
the enamel was the lowest value among the ratios inthe mineralizing enamel, dentin, and bone. The ratioat the secretory stage enamel was only slightly lower
than that in blood. On the basis of these results, theydescribed that the net flux within the ameloblasts by
the Ca-ATP pump is towards the intercellular spacesof the ameloblasts and the stratum intermedium cells.
It is known that radiolabeled calcium entering thematuration stage enamel rapidly diffuses to the entire
thickness, although its diffusion in the secretory stage
is restricted to the superficial layer of newly depositedmatrix (Takano 1995; Kawamoto and Shimizu 1990;
Kumamoto and Leblond 1956). We confirmed thosefindings and showed the diffusion patterns of calcium
in enamel in more detail. In the maturation stage, calcium diffused more than 50 mm in depth for 1.8 sec,
but only about 15 µm for 9.4 sec in the secretory stage.
Biochemical and histochemical studies (Uchida etal. 1991,1995; Fukae et al. 1993; Robinson et al. 1979)
have shown that much of the organic matrix localizes

Kawamoto, Shimizu
to the surface layer in the secretory stage and that theorganic components suddenly decrease in the maturation stage. Fukae and Tanabe (1987) showed that a
non-amelogenin and non-enamelin protein has a highaffinity for calcium in forming porcine enamel. The
diffusion of calcium to a deeper layer might be restricted by those organic components and/or for unknown physicochemical reasons.
Acknowledgment
This study was supported by research grant no.
08672145 from the Ministry of Education of Japan.

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