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Electron microscopic study of long-term denervated rat skeletal muscle

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THE ANATOMICAL RECORD 248:339–345 (1997)
Immuno-Scanning Electron Microscope Characterization of Large
Tubules in Human Deciduous Dentin
HIROKO AGEMATSU1*, TAKASHI SAWADA2, HIROKI WATANABE2,
TAKAAKI YANAGISAWA2, AND YOSHINOBU IDE1
1Department of Anatomy, Tokyo Dental College, 1-2-2 Masago Mihamaku Chiba, Japan
2Department of Ultrastructural Science, Tokyo Dental College,
1-2-2 Masago Mihamaku Chiba, Japan
ABSTRACT
Background: This study was undertaken to elucidate the
type and origin of collagen fibrils which construct the large tubules in
deciduous coronal dentin by scanning electron microscope and anti-types
I and III collagen antibody procedures.
Methods: The studies were performed on human deciduous teeth. The
teeth were fixed in 4% paraformaldehyde solution and then fractured
either mesio-distally parallel to the long axis of the tooth or transversely
perpendicular to the long axis of the tooth crown. The specimens were
three-dimensionally observed employing the scanning electron microscope to distinguish the content of large tubules. Polyclonal antibodies of
anti-type I and anti-type III collagen with 20 nm colloidal gold, and
secondary electron imaging and backscatter electron imaging of highresolution field emission scanning electron microscopy were used to
examine the types of collagen fibrils.
Results: The large tubules extended from the vicinity of the incisal edge
of the dentino-enamel junction to the pulp cavity. Inside the large tubules,
fibers in compact bundles run parallel to the longitudinal axis of the
tubules. The fiber bundles consisted of collagen fibrils which were 50-150
nm in diameter with typical cross striation. Immuno-scanning electron
microscopy showed type I collagen-labelling gold particles and type III
collagen-labelling gold particles to be abundant on the fibrils. Types I and
III collagen-labelling gold particles were present on the banded collagen
fibrils regardless of their diameter.
Conclusions: It was found that type III collagen is present together with type
I collagen on the fibrils constructing the large tubules of the human deciduous
dentin. This immunohistochemical study suggested that the fibrils constructing the large tubules were derived from the von Korff fibers, and types I and III
collagens formed copolymers. Anat. Rec. 248:339–345, 1997. r 1997 Wiley-Liss, Inc.
Key words: large tubules; human deciduous dentin; type I collagen; type
III collagen; immuno-scanning electron microscopy
INTRODUCTION
The dentin is penetrated by numerous dentinal tubules that extend through its entire thickness from the
dentino-enamel junction (D.E.J.) to the pulp. The diameter of the dentinal tubules commonly range from 1 to 4
µm. Reports have shown that tubules which were
clearly larger (5-50 µm) in diameter as compared to the
ordinary dentinal tubules extend from the incisal edge
of the D.E.J. to close vicinity of the pulp cavity in the
coronal dentin of cow, red deer and human (Tronstad,
1972; Miller, 1981; Hals, 1983a,b; Hals and Olsen, 1984;
Dyngeland and Fosse, 1986; Dyngeland, 1988; Agematsu
et al., 1990). These tubules with large diameters have been
referred to as giant tubules, large tubules, and microcanals. In this study, the term large tubules will be used.
r 1997 WILEY-LISS, INC.
In a histochemical study, Dyngeland et al. (1984)
reported that in immature unerupted cow incisors, the
large tubules were observed to communicate with the
incisal pulp cavity via its wide openings and vital cells
of the pulp were present in the large tubules. Furthermore, Dyngeland and Kvinnsland (1988) reported that
in bovine dentin, large tubules consisted of a vascularized pulp portion and a large collagen filled portion.
Contract grant sponsor: Ministry of Education, Science, and Culture
(Japan); contract grant number: 08672093.
*Correspondence to: Hiroko Agematsu, D.D.S., Department of
Anatomy, Tokyo Dental College, 1-2-2 Masago Mihamaku Chiba, 261
Japan.
Received 6 August 1996; accepted 10 February 1997.
340
H. AGEMATSU ET AL.
Proceeding fracturing, these fragments were fixed again
in 4% paraformaldehyde solution.
Scanning Electron Microscopy
Mesio-distally and transversely fractured dentin fragments were dehydrated in a series of ethanol, immersed
in t-butyl alcohol, frozen, and dried in an Eiko ID-2, and
gold-palladium coated using a cool sputter coater. The
specimens were observed by a Hitachi S-800 FESEM
operated at 15 kV.
Immunostaining for Scanning Electron Microscopy
Fig. 1. Diagrams of preparation procedure for exposing the large
tubules. a: In the longitudinal plane. b: In the transversal plane.
Recent observations (Agematsu et al., 1990) of the large
tubules in the human deciduous dentin employing the
scanning electron microscope revealed collagen fibers,
but no odontoblast processes or other cells in the large
tubules. It has become accepted in recent years that
collagen is a significant component of the dentinal
tubules. Dai et al. (1991) showed that intratubular
collagen is a significant feature especially at the pulpal
side of human dentin. However, it is uncertain whether
or not the origin and distribution of collagen fibrils in
the large tubules are similar to those of the dentinal
tubules. The nature of the collagen fibrils within the
large tubules have also not yet been defined in human
teeth.
In this study, we investigated the three-dimensional
organization and distribution of collagen fibrils within
the large tubules in human dentin using the highresolution field emission scanning electron microscopy
(FESEM). Furthermore, we attempted to demonstrate
the immunohistochemical characterization of collagen
fibrils by the immunogold technique in order to reveal
the nature of the collagen fibrils within the large
tubules.
MATERIALS AND METHODS
Samples
The experimental material consisted of 35 human
deciduous incisors extracted from children aged 5-7.
The teeth showed slight mobility and the degree of root
resorption was 1⁄2 to 3⁄4 of its original length. All teeth
were clinically caries free and with slight attrition.
Immediately after extraction, the teeth were fixed in
4% paraformaldehyde solution in PBS at 4°C for 24 h.
The mesial and distal surfaces of the teeth were ground
down with a grindstone to expose the pulp cavity.
Specimens were then frozen with liquid nitrogen and
fractured mesio-distally parallel to the long axis of the
tooth with a chisel (Fig. 1a). In several specimens, the
incisal third was fractured transversely perpendicular
to the long axis of the tooth with a chisel (Fig. 1b).
For indirect immunostaining with antibodies using
the colloidal gold procedure, the mesio-distally fractured dentin fragments were used. The labial fragments and lingual fragments were used respectively for
type I collagen and type III collagen immunolabelling.
Test specimens were rinsed in 0.01 M PBS (pH7.2)
and immersed in PBS containing 1% chicken egg
albumin (OVAL, Sigma) for 30 min. This was followed
by incubation with rabbit polyclonal antiserum either
to collagen type I (rabbit anti-bovine type I collagen,
LSL Co. Ltd. Tokyo, 1:200) (Sasano et al., 1996; Kikuchi
et al., 1996) or to collagen type III (rabbit anti-bovine
type III collagen, LSL Co. Ltd. Tokyo, 1:80) (Kikuchi et
al., 1996) for 12 h at 4°C.
Controls were incubated either with PBS only, or
with non-immune serum obtained from rabbits. All
specimens were then rinsed well with PBS, followed by
incubation with goat anti-rabbit IgG conjugated with
20 nm colloidal gold (BioCell Research Lab. UK, 1:50)
for 12 h at 4°C.
After immunostaining, the specimens were fixed in
1% glutaraldehyde buffered solution with PBS, dehydrated in a series of ethanol, and processed by sublimation. The specimens were then platinum coated (2-5
nm) with a cool sputter coater and examined by a
Hitachi S-800 high-resolution FESEM for secondary
electron imaging (SEI). In addition, immunogold particles were identified by the atomic number contrast
which were obtained via backscattered electron imaging (BEI).
RESULTS
Scanning Electron Microscopic Findings
The large tubules which were 10-20 times bigger in
diameter as compared to the dentinal tubules were
either round or ovoid in shape. They were aligned in a
straight line from the mesial to the distal end, at close
to the labio-lingual central portion of the coronal dentin
(Fig. 2). The large tubules which are easily observed as
dark grooves were directed toward the pulp cavity from
the vicinity of the incisal edge of the D.E.J. Running
parallel to the dentinal tubules, in the mesio-distally
fractured dentin in which incisal dentinal exposure was
minimum, they were more or less converged in the pulp
vicinity. The distance between each large tubule was
approximately 100-500 µm (Fig. 3). In its periphery,
there was a wall structure similar to that of the
peritubular dentin of the dentinal tubule. The inner
axis of the large tubules having a diameter of approximately 20 µm was largely composed of fiber bundles
that ran longitudinally and these small spherical bodies were present between these collagen fibers on the
Fig. 2. Low magnification SEM micrograph of transverse fracture.
The large tubules (LT) are arranged linearly in the mesio-distal
direction. Bar scale indicates 100 µm.
Fig. 3. Low magnification SEM micrograph of mesio-distal fracture.
The large tubules (LT) are clearly observed as grooves extending from
the incisal edge towards the pulp cavity. Bar scale indicates 200 µm.
Fig. 4. The large tubule (LT) has a glossy wall (asterisks) similar to
the peritubular dentin. The interior of the large tubule is composed of
bundles of longitudinally oriented fibers and of numerous spherical
shaped bodies (arrows). Bar scale indicates 20 µm.
Fig. 5. Higher magnification of interior of the large tubule. Longitudinally oriented collagen fibrils (Col) with periodical cross striations
and spherical shaped bodies (SB) which are made up of aggregates of
regular parallelepipedical crystals (a) and finely granulated crystals
(b) were also observed. Bar scales indicate 0.5 µm.
342
H. AGEMATSU ET AL.
longitudinally fractured surface (Fig. 4). In addition,
enlargements of the interior of the large tubules in the
longitudinally fractured specimens revealed longitudinally oriented fiber bundles and lateral branches which
have separated from the bundles. Under higher magnification, these fiber bundles appeared to consist of
collagen fibrils that had a cross striation structure with
a periodicity of approximately 60-70 nm and ranged
from 50 to 150 nm in diameter (Fig. 5a,b). The spherical
calcified bodies (0.5-2.5 µm in diameter) were composed
of regular parallel-epipedal crystals (Fig. 5a) and granulated crystals (Fig. 5b) were also observed to be among
the fibers.
Scanning Electron Microscopic Findings on Immunostaining
Immunostaining with type I collagen antibody revealed that gold particles were especially abundant on
the collagen fibrils in the large tubules and appeared
throughout its long axis. Immunogold labelling for type
I collagen was observed on the collagen fibrils regardless of their diameter (Fig. 6). Under higher magnification, gold particles indicating type I collagen were
clearly observed to be on the collagen fibrils which had
approximately 60-70 nm periodicity and were 50-150
nm in diameter. Gold particles could not be found on the
crystals which lie below the collagen fibrils (Fig. 7).
Figure 8a and b show a higher magnification of SEI and
BEI on the large tubule immunostained with collagen
type I at the same location. The 20 nm immunogold
particles attached on to the cross striation of the
collagen fibrils were clearly identified by their shape
and size, as observed by secondary electron imaging
(Fig. 8a). Furthermore, owing to the atomic number
contrast of BEI, the colloidal gold particles could easily
be seen as bright particles (Fig.8b).
Controls using normal rabbit serum resulted in minimum immunostaining and only few randomly distributed gold particles on the collagen fibrils within the
large tubule were visible (Fig.9).
Immunostaining with type III collagen antibody
showed that immunogold labelling overlay the collagen
fibrils within the large tubules (Fig. 10). The 20 nm
colloidal gold particles coated with the platinum could
be identified by SEI (Fig. 11a). This was further confirmed by visualizing the colloidal gold marker with
atomic number contrast obtained through BEI (Fig.
11b).
DISCUSSION
Dyngeland and colleagues (Dyngeland, 1988; Dyngeland et al., 1984; Dyngeland and Fosse, 1986, 1987;
Dyngeland and Kvinnsland, 1988) have made a series
of investigations on the structure of the large tubules
using the bovine dentin. However, reports on the human large tubule are few in number. In this study, our
findings yielded some additional information on the
formation and content of the large tubule in human
dentin.
Our observation which revealed that the large tubules were arranged linearly in the mesio-distal direction and only in the labio-lingual central portion of the
incisal dentin is very interesting. The dentin below the
incisal edge has characteristics slightly different from
those of other dentinal regions (Tronstad, 1972; Mjor
and Fejerskov, 1986). Agematsu et al. (1990) reported
on the continuous vertical alignment of interglobular
dentin under the incisal edge. Their findings are suggestive of dentinogenesis imperfecta of dentin beneath the
incisal edge. Tronstad (1972) observed a slit which
appeared to be located at the junction between the
dentin developing from the buccal and from the lingual
dentinal surfaces, and has assumed that the slit is
probably caused by crowding of odontoblasts during
dentinogenesis. At the initiation of dentin formation,
odontoblasts differentiate at the D.E.J. and shift inward to where surface area is less. Consequently, we
consider that the dentin formed on the labial side and
on the lingual side collide, causing degeneration and
disruption of odontoblasts during dentinogenesis at
directly below the incisal center, and because of this
phenomena, tubules of bigger diameter appear.
As a result of electron microscopic observations on
alterations occurring with increasing age in odontoblasts in the pulp horn directly below the cusp of the
first molar in rats, Shimomura (1979) revealed that, in
this region, cytoplasmic organelles in odontoblasts either degenerate or that the appearance of autophagic
vacuoles result in the destruction or vanishing of some
odontoblasts as dentinogenesis proceeds. From this
report, we made the following hypothesis: during dentin formation, apoptosis of odontoblasts occur in the
incisal dentin. Wright and Gantt (1985) reported that
large tubules appeared more frequently in teeth with
dentinogenesis imperfecta. On the basis of these findings, the authors speculated that the large tubules
appear localized in the labio-lingual junction below the
incisal edge for a reason; partial imperfect dentin
formation occurs as a result of apoptosis induced by
odontoblast concentration in this area.
We observed the content of the large tubule in detail.
As a result, odontoblast processes and other cell component were not found within the large tubule in any
area. The majority of large tubules was seen to be filled
with dense bundles of longitudinally oriented collagen
fibrils with typical cross striations. Thus we attempted
identification of collagen type using immuno-scanning
electron microscopy which is useful in observing the
localization of specific substances in three dimensions
(Takata et al., 1988). We found the presence of type III
collagen together with type I collagen on the collagen
fibrils within the large tubule of human deciduous
dentin.
In general, normal mineralized human dentin is
shown to be composed mainly of type I collagen and to
lack type III collagen (Thesleff et al., 1979; Wang et al.,
1980; Tung et al., 1985). But, type III collagen is present
in human dentin where the organic matrix is newly
formed in dentinogenesis imperfecta (Sauk et al., 1980)
and is reparative dentin (Magloire et al., 1988). Nagata
et al. (1992) described that positive staining for type III
collagen was observed on the cross-banded fibril bundles
running parallel to the dentinal tubules within the
peritubular dentin or in vicinity of the dentinal tubules
in the mice normal root dentin by immuno-electron
microscopy. Recently, it has been reported that in
normal human tooth, type III collagen is occasionally
present in the peritubular dentin (Waltimo et al., 1994).
We were able to detect type III collagen together with
type I collagen only on the collagen fibrils within the
large tubule.
Fig. 6. Immunostaining for type I collagen coupled with 20 nm
colloidal gold. Numerous immunogold particles (arrowheads) were
attached to the banded collagen fibrils within the large tubule. Bar
scale indicates 0.5 µm.
Fig. 7. Higher magnification of collagen fibrils immunostaining for
type I collagen coupled with 20 nm colloidal gold. Immunogold
particles (arrowheads) are clearly observed on the banded collagen
fibrils but are not observed on the crystals (arrows). Bar scale
indicates 0.1 µm.
Fig. 8. Higher magnifications of SEI (a) and BEI (b) of collagen
fibrils immunostaining for type I collagen. Immunogold particles
(arrowheads) detected on the collagen fibrils with SEI (a). Immunogold particles(arrowheads) are further identified by atomic number
contrast with BEI (b). Bar scales indicate 0.2 µm.
Fig. 9. Controls using normal rabbit serum. Immunogold particles can
scarcely be observed on the collagen fibrils. Bar scale indicates 0.2 µm.
Fig. 10. Immunostaining for type III collagen coupled with 20 nm
colloidal gold. Immunogold particles(arrowheads) were attached to the
banded collagen fibrils regardless of the fibril diameter. Bar scale
indicates 0.2 µm.
Fig. 11. SEI (a) and BEI (b) of collagen fibrils immunostaining for
type III collagen. Immunogold particles (arrowheads) are detected on
the collagen fibrils with SEI (a). Immunogold particles (arrowheads)
are further identified by atomic number contrast with BEI (b). Bar
scales indicate 0.2 µm.
IMMUNO-SEM OF LARGE TUBULES IN HUMAN DENTIN
It was reported that the presence of longitudinal
collagen fibrils between odontoblasts, which run from
the mineralized dentin to the odontoblast layer, were
observed in pig molars (Bishop and Yoshida, 1992), and
they regarded these fibrils as corresponding to the von
Korff fiber. It has also been demonstrated that type III
collagen is a major component of the fiber which runs
among the odontoblasts and into the predentin (Ohsaki
and Nagata, 1994). It is presumed that the fibrils which
were observed within the large tubules in this study are
similar to the von Korff fibers in distribution and
collagen type. As a conclusion, we think that type III
collagen positive fibrils within the large tubules were
derived from von Korff fibers.
It has been stated that type III collagen forms
thinner fibrils as compared to type I collagen fibrils,
and that type III collagen is generally considered to
consist of reticular fibers (Romanic et al., 1991). However, Keene et al. (1987) suggested that type III collagen is present on all banded collagen fibrils regardless
of diameter and that type III collagen is codistributed
with type I collagen in most tissues. In this immunoscanning electron microscopic study, we found both
type I and type III collagens on the banded collagen
fibrils within the large tubules regardless of fibril
diameter. We assume that type III collagen is codistributed together with type I collagen and that both of them
probably form copolymers.
ACKNOWLEDGMENTS
This work was supported in part by grants-in-aid for
scientific research (08672093: H.A.) from the Ministry
of Education, Science, and Culture, Japan.
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