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Osteodentin formation in rat incisor as visualized by radioautography after 3H-proline administration.

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THE ANATOMICAL RECORD 216:19-26 (1986)
Osteodentin Formation in Rat Incisor as Visualized
by Radioautography After 3H-Proline Administration
A.C. KARIM AND S.P. PYLYPAS
Department of Anatomy, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3
ABSTRACT
Osteodentin formation was studied in rat incisor pulp after adriamycin administration. Male Sprague Dawley rats (100 5 gm) were injected intravenously with adriamycin (5 m g k g body weight), and after 7 days they were again
injected intravenously with 3H-proline (3 pCi/gm). These animals were killed in
groups of three from 5 minutes to 4 hours after proline injection by perfusion with
3% phosphate-buffered formaldehyde followed by 2.5% phosphate-buffered glutaraldehyde. Control animals injected with only physiological saline, and 7 days later
with 3H-proline (3 pCi/gm), and were killed at the same time intervals. Radioautography on sections showing osteodentin formation revealed that a t 5 minutes after
3H-proline injection the labeling was located over the cells associated with the
osteodentin matrix. At 1hour after injection the labeling was located over the cells
and the matrix, while at 4 hours the labeling was seen only over the matrix. It
therefore appears that at least a proline-containing component of the osteodentin
matrix is synthesized and secreted by the cells associated with it.
Many antineoplastic drugs such as cyclophosphamide
(Koppang, 1978, 1981), vinblastine (Mikkelsen, 1978),
vincristine (Stene, 1978; Stene and Koppang, 19801, colchicine (Noguiera et al., 19811, and adriamycin (Karim
and Eddy, 1984) were shown to cause osteodentin formation in rat incisors. As early as 1935 (Santone, 1935)
it was thought that osteodentin was produced by odontoblasts that lost their processes and became osteoblasts.
Later (Bernick, 1966) it was proposed that osteodentin
was produced by abnormally differentiated pulp mesenchymal cells. Although several investigators (Stene and
Koppang, 1980; Koppang, 1981; Karim and Eddy, 1984)
have shown a close association between differentiated
pulp mesenchymal cells and the osteodentin matrix,
there is no conclusive evidence to show that the osteodentin matrix is synthesized and secreted by the cells
associated with it.
As far as normal dentin formation is concerned, it has
been shown by radioautography using 3H-proline (Weinstock and Leblond, 1974a,b; Josephsen and Warshawsky, 1982) that the odontoblasts are actively
involved in the synthesis and secretion of the dentin
matrix. The initial localization of radioactive proline
over the rough endoplasmic reticulum and its subsequent passage with time to the Golgi apparatus, presecretory and secretory granules, and eventually into the
predentin layer extracellularly substantiate the view
that the dentin precursor is synthesized within the rough
endoplasmic reticulum, packaged into secretory granules via the Golgi apparatus, and eventually secreted
extracellularly. The present study was undertaken to
determine whether a similar relationship exists between the osteodentin matrix and the mesenchymal cells
associated with it.
0 1986 ALAN R. LISS. INC.
MATERIALS AND METHODS
Sprague Dawley rats weighing 100 5 gm were used
in this study. Two groups of nine animals each were
injected intravenously with 3H-proline (3 pCi/gm of body
weight; obtained from Amersham, specific activity 39.7
Ci/mM). One of these groups (experimental) was injected
intravenously with adriamycin (Adria Laboratories
Canada Ltd.) a t a dose of 5 m g k g of body weight 7 days
before injection of proline. All animals were killed in
groups of three at 5 minutes, 1 hour, and 4 hours after
proline injection by perfusion with 3% formaldehyde in
phosphate buffer for 15 minutes followed by a n additional 15-minute perfusion with 2.5% buffered glutaraldehyde (Karim and Warshawsky, 1979).
The incisors were extracted with the alveolar bone of
the jaws and demineralized in a n isotonic EDTA solution which was continuously agitated at 4°C (Warshawsky and Moore, 1967). After 2 weeks of decalcification, the incisors were cut into 1-mm-thick crosssectional segments and washed in several changes of
phosphate buffer. The segments were postfixed in 1%
osmium tetroxide a t 4°C for 4 hours, dehydrated in
graded concentrations of acetone, infiltrated with acetone-Epon mixtures, and embedded in a n Epon mixture
( 5 5 mixture of Epon solutions A and B). The Epon
embedded blocks were polymerized at 60°C for 24 hours.
One-micron-thick sections were collected on cleaned
slides and processed for light microscopic radioautography as follows: After prestaining with 5% iron alum for
15 minutes followed by a n additional 15 minutes with
Regaud's iron hematoxylin, the slides were dipped in
+
Received May 22, 1984; accepted March 19, 1986.
20
A.C. KARIM AND S.P. PYLYPAS
NTBz Kodak emulsion, (Kopriwa and Leblond, 1962),
exposed in the dark for 5 weeks a t 4“C, developed, and
fixed.
Thin sections were prepared for electron microscopic
radioautography according to the method of Kopriwa
(1973) with the following modifications:
Ilford L4 emulsion was melted in a 45°C water bath
for 1 hour, diluted 1:2.75, and transferred to a 32°C
waterbath. The slides, preheated at 45 “C, were dipped
and air dried for half a n hour. After 3 months exposure
the slides were developed for 1 minute in D-19 diluted
1:lO. Poststaining of EM radioautographs involved 1.5
minutes in glacial acetic acid to remove the celloidin,
followed by 2 minutes in 2.5% aqueous uranyl acetate
and 20 minutes in lead citrate. The radioautographs
were viewed under a Hitachi HU-12 electron microscope. The silver grains on the light microscopic radioautographic preparations were quantitatively analysed in
the following manner. A square grid measuring 103 pm
was superimposed over the specimen under oil immersion. The grains over the cells (cellular compartment)
were counted in each grid space. Twenty counts were
made over 20 different sections from each interval.
Grains were also counted over the osteodentin matrix
(extracellular compartment). The counts over all the
specimens were placed into three groups. The first group,
external control, involved animals that received 3H-proline only. The second group, the internal control, involved areas in the pulp not showing osteodentin
formation in animals that received both adriamycin and
3H-proline. The third group of counts, the experimental,
were made over areas showing osteodentin formation.
In the case where osteodentin formation was absent (in
both control groups), the extracellular compartment referred to the extracellular matrix of the pulp.
The grains were analyzed by a mixed factorial designs
program prepared by Dr. E. Chebib of the Biostatistics
Department in the Faculty of Dentistry. This program
compared the groups at various time intervals after 3Hproline injection as follows. The experimental was compared with both the external and internal controls, while
the external control was compared with the internal
control. Thus the graphs (Figs. 7-9) show the mean
number of grains per area of grid over the various compartments at different time intervals.
RESULTS
Qualitative Analysis
Light microscope radioautography
At 5 minutes after 3H-proline injection (Fig. la) the
pulp mesenchymal cells associated with the osteodentin
matrix were heavily labeled. The labeling was seen as
developed black silver grains over the cells. There was
no labelin over the osteodentin matrix. However, at 1
hour after %H-proline injection (Fig. 2a) the labeling was
seen over the cells, as well as over the osteodentin matrix. The labeling over the matrix was confined mainly
to the periphery. At 4 hours after 3H-proline injection
(Fig. 3a) the cells associated with the matrix were completely unlabeled, while the matrix was heavily labeled.
The labeling over the matrix was localized mainly
within the central region. There were very few silver
grains over the cell-matrix interface or over the periphery of the matrix.
Figure 6a and b also show the labeling pattern at 1
hour after 3H-proline injection. Both the cells and the
osteodentin matrix are labeled (Fig. 6a). However, in
sections through a trabecule of the osteodentin matrix
(Fig. 6b), the labeling was actually localized over the
periphery of the trabecule. Thus, the cells and the
“preosteodentin” (asterisks) were labeled, while the
mineralized and older osteodentin (Od) was unlabeled.
At 4 hours after 3H-proline injection (Fig. 6c) the “preosteodentin” shows less labeling than that seen a t 1hour
(Fig. 6b). A very weak labeling was seen over the associated cells (arrowheads).
The external control samples, at various time intervals after 3H-proline injection (Figs. lb-3b1, did not show
any osteodentin formation. However, there was a low
uptake of labeling within the cells of the pulp. The cells
involved in osteodentin formation were much larger
than these cells.
Electron microscope radioautography
At 5 minutes after 3H-proline injection (Fig. 4) many
of the silver grains located within the cells were seen
over the rough endoplasmic reticulum. Very few grains
were also observed over the saccules of the Golgi apparatus. No labelin was seen over the osteodentin matrix.
At 1 hour after 8H-proline injection the silver grains
were seen over both the cells and the matrix. At 4 hours
after 3H-proline injection (Fig. 5 ) all the silver grains
were located over the collagenous osteodentin matrix.
The cells associated with the matrix were unlabeled.
Quantitative Analysis
Light microscopic radioautography
The distribution of grains over the cellular compartment at various time intervals after 3H-proline injection
is shown in Figure 7. The maximum number of grains
within all groups occurred at 1 hour after 3H-proline
injection. However, a t this time there were about 550
grains per unit area over the cells in the experimental
group, while the grains over both the external control
and internal control groups numbered 50 and 100, respectively. At 4 hours after 3H-proline injection the
number of silver grains over the internal control group
had diminished to that over the external control, which
did not change significantly from the level a t 1 hour.
During this time the grains over the experimental group
had diminished to about 175. This was still above the
control level.
Within the extracellular compartments (Fig. 8) the
initial uptake of labeled proline a t 5 minutes was identical in all groups. However, between 5 minutes and 4
hours there was a tremendous incorporation of radioactivity in the experimental group. On the other hand,
both control groups showed a similar uptake of radioactivity a t 1 hour after injection. Between 1 hour and 4
hours the level of radioactivity over the external control
did not change, while that over the internal control
doubled.
The combined total number of grains over the extracellular and cellular compartments is compared among
all groups in Figure 9. Within 1 hour the maximum
number of grains is seen over the experimental group.
Between 1hour and 4 hours the labeling pattern did not
change significantly. Maximum incorporation of radioactivity was also seen at 1hour over the external control
RADIOAUTOGRAPHY OF OSTEODENTIN FORMATION
Figs. 1-3. Light microscope radioautographs of pulp tissue seen after adriamycin and 3H-proline
injections (Figs. la-3a) and after 3H-proline only (Figs. lb-3b). ~ 5 0 0In
. the experimental sample at 5
minutes after 3H-prolineinjections (Fig. la) the labeling, which appears as black grains, is seen only over
the cells (arrowheads) associated with the osteodentin matrix (Od). At 1 hour after 'H-proline injection
(Fig. 2a) both the cells and the osteodentin matrix are labeled. However, by 4 hours after injection of 3Hproline (Fig. 3a) the cells (arrowheads) are unlabeled while the matrix (Od)is heavily labeled. The external
control samples at the same time intervals (Figs. lb-3b) did not show osteodentin formation, but showed
a weak labeling of the cells.
21
22
A.C. KARIM AND S.P. PYLYPAS
Figs. 4 3 . Electron microscope radioautographs of pulp tissue seen after adriamycin and 3H-proline
injections. x 12,600. At 5 minutes after 3H-proline injection (Fig. 4) the silver grains (arrowheads) over
the cells are localized mainly to the rough endoplasmic reticulum (rer). However, by 4 hours after 3Hproline injection (Fig. 5) the silver grains (arrowheads) are located over the collagenous osteodentin matrix
(Od). There are no silver grains over the cells a t this time.
RADIOAUTOGRAPHY OF OSTEODENTIN FORMATION
Fig. 6. Light microscope radioautographs of pulp after adriamycin and 3H-proline injection. x 1,000.
Figure 6a and b show the radioautographic pattern at 1hour after 3H-proline injection. Note in Figure 6a
both the cells (arrowheads) and the matrix (Od) are labeled. However, Figure 6b shows a section through
an osteodentin trabecule. Here one sees that the labeling of the matrix (asterisks) its confined to the
periphery of the trabecule, while the mass of the matrix (Od) within the core is unlabeled. Note that at 4
hours after 3H-proIine injection Pig. 6c) the cells (arrowheads) are unlabeled, while the number of grains
over the periphery of the osteodentin matrix (Od) is less than that seen in Figure 6b.
23
24
A.C. KARIM AND S.P. PYLYPAS
650
CELLULAR COMPARTMENT
1
T
0-0
Experimental
A-A
E x t. Control
0-0
Int. Control
650
N
5
1
550
I-
450
-
0
0
P
E X T R AC E L L U L A R
COMPA R TM E NT
0-0
Experimental
A-A
Ext. Control
0-0
Int. Control
\
Y)
.-C
350
-
250
-
150
-
50
-
:
Y
0
0
Z
C
0
s
I
------
4
L
~
I
.08
2
1
Time
3
4
(hrs)
Fig. 7. The distribution of silver grains over the cellular compartment a t various time intervals after 3H-proline injection is shown
within the experimental, the external control, and the internal control
groups. At 1 hour after injection maximum labeling occurred in all
groups. The experimental group had about five times the number of
grains as the controls. Moreover, the internal control group had twice
the number of silver grains as the external control. At 4 hours after
injection the amount of label over both control groups was the same.
However, although the labeling over the experimental group had decreased tremendously at 4 hours after injection, it was still slightly
higher than that over the controls.
group. This level did not change even at 4 hours after
injection. On the other hand, the labeling pattern over
the internal control was similar to that seen over the
external control at 1hour after injection. However, at 4
hours after injection the amount of radioactivity over
the internal control was almost double that seen over
the external control.
DISCUSSION
Although the osteodentin matrix contains a large
amount of collagen fibers (Takuma et al., 1968; Karim
and Eddy, 1984) there are fewer fibers there than found
in normal dentin (Takuma et al., 1968). Since proline is
a large constituent of collagen, this tritiated amino acid
was used as a labeled precursor to determine the relationship between the collagenous osteodentin matrix and
the cells associated with it. Moreover, this precursor has
been previously used to demonstrate collagen synthesis
and secretion in dentin formation in rat incisors (Weinstock and Leblond, 1974a, 1974b; Josephsen and Warshawsky, 1982).
2
.08
Time
3
4
(hrs)
Fig. 8. The distribution of silver grains over the extracellular compartment at various time intervals after 3H-proline injection is shown
within the experimental, the external control, and the internal control
groups. In both the experimental and internal control groups maximum labeling was seen at 4 hours after injection. However, the slight
increase observed between 5 minutes and 1 hour in the external
control group remained constant between 1and 4 hours.
In the present study the initial uptake of 3H-proline
into the differentiated mesenchymal cells associated
with the osteodentin matrix (Karim, 1984), its passage
through the cells, and its deposition into the matrix
followed a pattern similar to that of collagen synthesis
and secretion observed during dentin formation (Weinstock and Leblond, 1974a,b; Josephsen and Warshawsky, 1982). In the former studies the initial uptake
of label was localized over the rough endoplasmic reticulum 2-5 minutes after injection. As the time after 3Hproline injection increased, the label was found over the
Golgi apparatus, then over the secretion granules, and
finally over the extracellular matrix at 4 hours after
injection. This indicated that the collagen precursor was
synthesized within the rough endoplasmic reticulum,
packaged into granules within the Golgi apparatus, and
eventually secreted extracellularly where it was added
to the preexisting matrix. Although the present study
did not demonstrate all the steps in this biosynthetic
pathway, evidence that the radioactive proline was first
incorporated into the cells and then deposited extracellularly is seen in Figures 7 and 8. It was shown that the
maximum labeling of the cellular compartment occurred at 1 hour after a pulse injection of 3H-proline
RADIOAUTOGRAPHY OF OSTEODENTIN FORMATION
850
-
750
-
N
5
I
650-
0
0
2
<
EXTRACELLULAR & CELLULAR
COMPARTMENTS
I
550-
v)
5
.-
450-
0-0
Experimental
A-A
Ext. Control
0-0
Int. Control
(3
Y
0
0
Z
350-
c
0
2
250-
150
-
50
.08
2
1
Time
3
4
(hrs)
Fig. 9. The distribution of the total number of silver grains over both
the extracellular and cellular compartments combined at various time
intervals after 3H-proline injection is shown within the experimental,
the external control, and the internal control groups. Note that the
experimental groups showed a higher level of activity than the controls
with respect to the mobilization of 3H-proline. However, within the
control groups, the internal control showed a slightly higher level of
activity than the external control.
(Fig. 7). However, between 1 hour and 4 hours after
injection the labeling over the cells diminished rapidly,
while there was a great increase in the labeling over the
extracellular compartment at this time (Fig. 8). Since
the pulse injection made the 3H-proline available only
for a short period of time, the high radioactivity over
the extracellular compartment could only reflect a
transfer of label from the cellular compartment. Therefore it is conceivable that a process similar to that seen
in dentin formation is occurring in osteodentin
formation.
It was observed that the pulp cells involved in osteodentin production were much larger in size than those
not affected by adriamycin (Figs. 1-3, controls). This no
doubt reflects the highly synthetic state of the cells, and
the increased amount of rough endoplasmic reticulum
that was reported to occur in these cells (Karim and
Eddy, 1984; Karim, 1985). Figure 6 (a-c) show the trabecular nature of the osteodentin matrix (Karim and
25
Eddy, 1984) and the pattern of silver grain distribution
at 1and 4 hours after 3H-prolineinjection. Although the
results show that both the cells and the matrix are
labeled at 1 hour after injection (Fig. 6a), a section
through the long axis of a trabecule (Fig. 6b) shows a
pattern similar to that seen in dentin formation. Both
the cells and the “preosteodentin” (asterisks) are labeled
while the presumably mineralized and older osteodentin
(Od) is unlabeled. In Figure 6c the cells are relatively
unlabeled and the number of silver grains over the
“preosteodentin” has diminished. Thus there was an
apparent movement of silver grains toward the center
into the more mineralized area as more unlabeled matrix was added at the cell-matrix interface.
It therefore appears that osteodentin formation occurs
by a mechanism similar to that of dentin formation.
Earlier studies (Weinstock and Leblond, 1974a,b; Josephsen and Warshawsky, 1982)have shown that within
4 hours most of the silver grains were over the predentin
layer, while there was a weak labeling over the odontoblasts. Moreover, labeling of the dentin layers occurred
about 2 days after 3H-proline injection. In the present
study the cells were completely unlabeled within 4
hours, indicating that the label had moved out of the
cells at a more rapid rate than that observed in dentin
formation. This rapid rate of osteodentin formation is
not controlled, since earlier studies (Karim and Eddy,
1984)have shown a complete occlusion of the pulp chamber by its production.
Quantitatively it was shown that the synthetic activity of the apparently normal pulp cells from the adriamycin-treated rats was extremely high. Within the
control groups, although there was no significant difference in the labeling at 4 hours after 3H-prolineinjection
(Fig. 7), the higher labeling of the internal control group
at 1 hour after injection indicated that the adriamycin
had increased the biosynthetic activity of these cells
that were apparently not involved in the formation of
osteodentin.
The passage of 3H-prolinefrom the cellular compartment (Fig. 7) to the extracellular compartment (Fig. 8)
is reflected by the increased labeling of the extracellular
compartment between 1 hour and 4 hours after injection. During this increased labeling of the extracellular
compartment there was a concomitant decrease in the
labeling of the cellular compartment, thus indicating
that a component of the extracellular matrix is synthesized within the cells and secreted extracellularly.
This study has shown that a proline-containing component of the osteodentin matrix is synthesized and
secreted by the mesenchymal cells associated with it.
This precursor is presumably going into the collagenous
osteodentin matrix.
ACKNOWLEDGMENTS
The authors are grateful t o Mr. Roy Simpson for the
illustrations and to Dr. Jean A. Paterson for her assistance and advice with the electron microscope radioautographic technique. This work was supported by the
Medical Research Council of Canada.
LITERATURE CITED
Bernick, S. (1966) Vascular and nerve supply to the molar teeth of
guinea pigs. J. Dent. Res., 45:249-260.
Josephsen, K., and H. Warshawsky (1982) Radioautography of rat
26
A.C. KARIM AND S.P. PYLYPAS
incisor dentin as a continuous record of the incorporation of a
single dose of 3H-labeledproline and tyrosine. Am. J. Anat., 164:4556.
Karim, A.C. (1985) The initiation of osteodentin formation in the rat
incisor after adriamycin administration. Anat. Rec., 213:377-384.
Karim, A.C., and E.L. Eddy (1984) A light and electron microscopic
study of osteodentin formation in the rat incisor after adriamycin
administration. Am. J. Anat., 169~207-219.
Koppang, H.S. (1978)Histomorphologic investigations of dentinogenesis in incisors of offsprings of cyclophosphamide-treatedpregnant
rats. Scand. J. Dent. Res., 86:444-458.
Koppang, H.S. (1981)Effect of cyclophosphamide on dentinogenesis in
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Kopriwa, B.M. (1973) A reliable, standardized method of ultrastructural electron microscope radioautography. Histochemie, 37;l-17.
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Mikkelsen, H.B. (1978) Acute and protracted effects of vinblastine on
odontoblasts and dentinogenesis in rat incisors. Scand. J. Dent.
Res., 86:313-324.
Noguiera, T.D.O., T. Stene, and H.S. Koppang (1981)Colchicine’s effect
on rat incisors odontoblasts and dentinogenesis. Scand. J. Dent.
Res., 89:45-58.
Santone, P. (1935) Studien uber den aufbare die Struktur und die
histogeneses der molaren der sangetiere. 1 Molaren von cavia
caloya. Z tochr. Mikr. Anat. Fersch., 37:49-100.
Stene, T. (1978) Vincristine’s effect on dentinogenesis in rat incisor.
Scand. J. Dent. Res., 87:39-49.
Stene, T., and H.S. Koppang (1980)Autoradiographic investigations of
proliferative responses in the rat incisor pulp after vincristine
administration. S a n d . J. Dent. Res., 88:96-103.
Takuma, S., Y . Kierahashi, and Y. Tsugae (19681 Electron microscopy
of the osteodentin in rat incisors. In: Dentine and Pulp: Their
structure and reactions. N.B.B. Symon, ed. Baltimore, Williams
and Wilkins Co., pp. 169-196.
Warshawsky, H., and G. Moore 1967 A technique for the fixation and
decalcification of rat incisor for electron microscopy.
.. J. Histochem.
Cytochem., 15:542-549.
Weinstock, M., and C.P. Leblond (1974a) Synthesis, migration and
release of precursor collagen by odontobiasts as visudized by radioautography after 3H-proline administration. J. Cell Biol., 60:92127
Weinstock, M., and C.P. Leblond (1974b) Formation of collagen. Fed.
Proc., 33(5):1205-1218.
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