Features of formation of the cap enameloid in
the pharyngeal teeth of tilapia, a teleost.
Ichiro SASAGAWA
Department of Anatomy, School of Dentistry at Niigata,
The Nippon Dental University,
1-8 Hamaura-cho, Niigata 951, Japan.
Key words: Enameloid, collagen, teleost, tilapia, tooth development.
Summary−
This study focuses on the morphological changes of the organic matrix
during formation of the cap enameloid in tilapia. At the early and middle
stages of enameloid matrix formation, collagen fibrils in the enameloid
matrix were dispersed and about 15 nm thick. At the late stage, however,
the collagen fibrils became thicker and formed interwoven thick bundles.
Abundant flocculent and/or fine network-like materials, which were probably
glycosaminoglycans or proteoglycans, were located between the collagen fibrils.
The inner dental epithelial (IDE) cells contained large granules enclosing
filamentous substances which were stained with phosphotungstic acid, suggesting
that the IDE cells synthesize collagen fibrils. Morphological evidence implied
that at this stage odontoblasts were also involved in the formation of enameloid
matrix including collagen fibrils. Most of the organic matrix in cap enameloid
was progressively removed by the epithelial cells during the stages of mineralization
and maturation. The process of removal could be divided into two steps.
First, the flocculent and/or fine network-like materials between the collagen
fibrils disappeared, while the collagen fibrils themselves were stable and
their periodic cross-banding remained visible. Second, at the stage of maturation,
the collagen fibrils were completely absorbed and removed from the cap enameloid.
Fine filamentous materials and tubular structures found in the cap enameloid
might be remnants of the organic matrix.
Introduction
The most interesting features during formation of the cap enameloid in
teleost might be the production of the collagen fibers as an enameloid matrix
and their removal from the enameloid at the late stage of formation. Large
numbers of collagen fibers that form thick bundles are present in the cap
enameloid matrix at the stage of formation of the enameloid matrix8 9 12
13 14 15 16 17). At the stage of mineralization of the enameloid, when the
mineralization progresses from the boundary between the enameloid and the
dentine to the surface of the enameloid, fine slender crystals accumulate
along the collagen fibrils9 13 14 15). At the stage of maturation of the
enameloid, when the degree of mineralization of the enameloid markedly increases,
the collagen fibrils are completely degraded and are removed from the enameloid8
14 15 17). In spite of their removal, it seems that the orientation of the
long axes of crystals in mature cap enameloid is the same as that of the
collagen fibrils which have already been removed. Hence, the collagen fibers
likely play an important role not only at the stage of formation of enameloid
matrix but also at the stages of mineralization and maturation of enameloid
in teleosts. However, details of the structure of the collagen fibrils and
other visible organic matrix in cap enameloid are not well known at each
stage of tooth development. The aim of this paper is to present our data
regarding the fine structure of the enameloid matrix, mainly collagen fibrils,
and to summarize several findings concerning the organic matrix during formation
of the enameloid in tilapia.
Materials and Methods
Three adult tilapias, Tilapia nilotica, Cichlidae, Teleostei (total length,
35-37cm), were used in this study.
After decapitation, pharyngeal tooth plates that contained tooth germs
were dissected out and stored in Karnovsky fixative (cacodylate buffer,
pH 7.4) overnight at room temperature. Selected specimens were then demineralized
in a 2.5% solution of EDTA-2Na for about three weeks. The specimens were
fixed again in a 1% solution of osmium tetroxide. After postfixation and
dehydration, these specimens were embedded in Araldite-Epon resin. Semithin
sections were cut with glass knives, stained with toluidine blue and then
examined under a light microscope. Ultrathin sections were cut with glass
or diamond knives, stained with uranyl acetate and lead citrate (U-Pb) or
phosphotungstic acid (PTA), and examined under a transmission electron microscope.
Results and Discussion
Stage of formation of the enameloid matrix
In the early and middle stages of enameloid matrix formation, a defined
layer of enameloid matrix became visible between the inner dental epithelial
(IDE) cells and the odontoblasts, although it was thin. Thin collagen fibrils,
which ranged from 12 to 20 nm in diameter, were found in the enameloid matrix.
The cross-banding pattern of the thin fibrils was often observed and showed
a periodicity of about 67 nm. The spaces between the collagen fibrils were
wide, so that the organic components of cap enameloid seemed to be dispersed
at these stages. These collagen fibrils tended to form loose bundles and
to be arranged along odontoblast processes (Fig.1).
At the late stage of enameloid matrix formation, when the production
of the cap enameloid by the organic matrix was almost complete, the collagen
fibrils were mainly 30-40 nm in diameter. The collagen fibrils were gathered
in thick bundles that were interwoven into a pattern characteristic of cap
enameloid.
It appears that the collagen fibrils in the enameloid matrix are dispersed
and thin, approximately 15 nm in diameter, at the early and middle stages
of enameloid matrix formation, and that the fibrils at the late stage become
almost twice as thick as those at the earlier stages and form thick bundles
of the type that is characteristic of teleost cap enameloid. The increase
in diameter of the collagen fibrils might be due to production of the organic
matrix in cap enameloid. Prostak and Skobe7) reported that the density of
collagen fibrils in cap enameloid was increased in tooth buds that had nearly
achieved their full thickness. It is also possible that the number of collagen
fibrils increases during formation of the enameloid matrix.
Flocculent and/or fine network-like materials were visible in the spaces
between the collagen fibrils at the stage of enameloid matrix formation.
Periodic attachment of electron-dense materials to the cross-banding of
the collagen fibrils was usually observed in the enameloid matrix (Fig.2).
The presence of these fine materials that form a network-like structure
and their periodic attachment to the collagen fibrils suggest that a large
amount of glycosaminoglycans (GAGs) and/or proteoglycans is contained in
the enameloid matrix. Kogaya4) reported that large quantities of chondroitin
sulfates were detected in the developing enameloid of Polypterus, and that
they were situated in close proximity to the enameloid collagen. The enameloid
matrix was intensely stained by PAS-alcian blue, implying that a large quantity
of tissue carbohydrates was present in the enameloid.
Many odontoblast processes were observed in the enameloid matrix from
the surface to the lower region at the early and middle stages of enameloid
matrix formation. Several processes were attached to the lamina densa beneath
the IDE cells. At the late stage, however, the odontoblast processes were
barely seen in the outer two-thirds of the enameloid matrix, while they
remained present in the enameloid matrix near the odontoblasts.
At the middle stage of enameloid matrix formation, matrix vesicles 40-80
nm in diameter with a distinct unit membrane and containing few crystals
were visible. At the late stage of enameloid matrix formation, the matrix
vesicles contained electron-dense, crystal-like structures. Small aggregates
of crystal-like structures that were associated with electron-dense fine
material which was probably derived from matrix vesicles, were scattered
in the apical enameloid matrix. Fine crystals are probably formed at the
matrix vesicles prior to the beginning of defined mineralization along the
collagen fibrils in cap enameloid.
At the late stage, the elongated odontoblasts contained abundant rER
and well-developed Golgi apparatuses in the distal cytoplasm (Fig.3). Long
rER enclosing fine materials were usually arranged almost parallel to the
long axis of each cell. The Golgi apparatus consisted of several lamellae,
many types of vesicles and vacuoles. Elongated granules with fine filamentous
contents that resembled procollagen were usually found in the Golgi area.
This suggests that the odontoblasts synthesize procollagen molecules. In
tilapia, such morphological features of the odontoblasts imply that the
odontoblasts produce considerable amounts of organic matrix including collagen
as part of the enameloid matrix.
Tall columnar IDE cells contained well-developed organelles at the middle
and late stages. Many extensive rER, arranged nearly parallel to the long
axes of the cells, were visible in the cytoplasm. Developed Golgi apparatuses,
which consisted of several lamellae, many vesicles, vacuoles and small electron-dense
granules, were located around the nuclei. Elongated granules with fine filamentous
contents that resembled procollagen were often found in the Golgi area.
Moreover, several large granules contained fibril-like structures, about
150 nm thick with cross-banding at intervals of 32 nm, and fine electron-dense
materials around the fibrils (Fig.4). In the sections stained with PTA,
the contents of the large granules showed a PTA-positive fine filamentous
structure (Fig.5). These two types of granules seen in the IDE cells resembled
the secretory granules that were observed in the odontoblasts of mammals.
This evidence suggests that the IDE cells synthesize a kind of collagen,
and supports the hypothesis proposed by Prostak and his colleagues5 6 7).
It is unclear whether the bulk of collagen fibers in cap enameloid is
of ectodermal origin. It is still uncertain how ectodermal procollagen molecules
and/or collagen fibrils move into the enameloid matrix through the defined
basal lamina beneath the IDE cells. The IDE cells in tilapia probably synthesize
and secrete ectodermal collagen, but the ectodermal collagen might not be
the only major component of the enameloid matrix. It is also probable that
the IDE cells in teleosts secrete other proteins, such as enameline2) and
proteolytic enzymes3). The proportion and distribution of the ectodermal
collagen in the enameloid matrix and the role of ectodermal collagen during
formation of the enameloid are subjects that must be investigated.
Stage of mineralization of the enameloid
In the stage of enameloid mineralization, marked mineralization began
at the boundary between the enameloid and the dentine and advanced toward
the surface side of the enameloid.
In the cap enameloid, crystal deposition that progressed along the collagen
fibrils extended to the apex. An electron-dense substance covered the collagen
fibrils around which fine slender crystals accumulated. In the demineralized
sections, the cross-banding pattern of collagen fibrils was often visible
in the region covered with the electron-dense substance. After the mineralization
reached the apex of the cap enameloid, fine crystals were found at the surface
region of the enameloid, bundles of collagen fibrils were loose and each
collagen fibril tended to be wavy in the non-mineralized region. In this
region, wide spaces were visible among the fibrils and the fine network-like
materials among the collagen fibrils had disappeared. However, the thickness
of collagen fibrils seemed unchanged and their cross-banding pattern remained
(Fig.6). This evidence suggests that GAGs and/or proteoglycans which are
situated between the collagen fibrils are mainly degraded and removed; however,
the structure of collagen fibrils is maintained. The electron-dense substance
begins to cover the collagen fibrils at the stage of mineralization. The
electron-dense substance which was visible around the mineralized collagen
might indicate the presence of non-collagenous proteins that are involved
in mineralization.
In the elongated IDE cells, many long rER and mitochondria were seen
in the cytoplasm. Widely distributed Golgi apparatuses were usually located
at the distal side of the nuclei. Relatively many primary and secondary
lysosomes were visible in the cytoplasm. The distal cell membrane was smooth
and did not exhibit marked infolding. Basal lamina was still present in
the non-mineralized region, but it became progressively indistinct with
the progress of mineralization at the surface layer. It seems that the IDE
cells at the stage of enameloid mineralization are in the transitional state
between secretory cells and absorptive cells.
The outer dental epithelial (ODE) cells seemed to constitute a pseudostratified
epithelial cell layer which was slightly wavy due to indentation by ingrowing
capillaries. The ODE cells contained labyrinthine canalicular spaces that
occupied most of the cytoplasm. A number of organelles were visible in the
narrow spaces between the canaliculi. The morphological features of the
ODE cells support the idea that the ODE cells might be engaged in active
transport of inorganic ions1). It is likely that the ODE cells undergo a
change in phenotype and become capable of active transport prior to the
stage of maturation when the IDE cells have a marked ruffled border and
most of the collagen fibrils disappear from the cap enameloid.
Stage of maturation of the cap enameloid
During the stage of enameloid maturation, the entire enameloid area became
mineralized. The size of each crystal and the number of crystals increased
in the cap enameloid, and the enameloid was packed with large crystals
until the middle stage of maturation.
In demineralized sections, the majority of the organic matrix of the
cap enameloid was dissolved away except for some structures. No collagen
fibrils having the cross-banding pattern were found in the enameloid at
this stage. In the lower region of the enameloid near the dentine, the remnants
of the organic matrix consisted of microfibrils forming a network-like structure
(Fig.7). At the boundary between the enameloid and the dentine, a transition
between collagen fibrils showing the cross-banding pattern and scattered
fine microfibrils was observed. It is certain that the collagen fibrils
in cap enameloid are absorbed and transported from the enameloid at the
stage of maturation8 14 15 17). In the present study, the collagen fibrils
dissociated into scattered microfibrils, suggesting collagenase activity.
However, there are still many uncertain points concerning the mechanism
of unbinding of the collagen fibrils and their removal from the cap enameloid,
as well as the presence of collagenase.
Large numbers of tubular structures, approximately 16 nm in thickness
and 250 nm in observable length, were found at the outer layer of demineralized
enameloid (Fig.8). It is unlikely that the tubular structures are involved
in the mineralization, because their distribution is limited to the outer
layer of the cervical side of enameloid, and they seem to be situated among
large crystals11). It is conversely possible that the tubular structures
are remnants of absorbed collagen fibrils.
Details of the structures of IDE and ODE cells at the enameloid maturation
stage were already reported in the most recent issue of the Proceedings
of ACBTE10). It is certain that the IDE cells having a ruffled border at
their distal end and the ODE cells containing abundant canaliculi are highly
involved in the absorption of organic matrix in the cap enameloid and its
removal from the enameloid. Although the IDE cells having a ruffled border
in teleosts might correspond to the ruffle-ended ameloblasts in mammals,
a clear difference is that the IDE cells in teleosts absorb collagen fibrils.
The collagen fibrils in cap enameloid are probably degraded until they exhibit
no fibrous structure in the spaces in the enameloid cap, since no fibrous
elements are found in the cisternae of the ruffled border of IDE cells.
It is likely that the presence of epithelial cells, that is, IDE and ODE
cells, is essential for the formation of hypermineralized enameloid during
the mineralization and the maturation stages.
Acknowledgement
The author is grateful to the staff at Hokuetsu Mizugiken Inc. for their
kindness
in offering their facilities for the collection of specimens.
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Figure legends
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Fig. 1
Thin collagen fibrils forming loose bundles in the enameloid matrix at the
early and middle stages of enameloid matrix formation. Demineralized by
EDTA-2Na solution, stained with uranyl acetate and lead citrate (U-Pb),
bar=1μm, ×11,040.
Fig. 2
Periodic attachment of electron-dense materials to the cross-banding pattern
of the collagen fibrils in the enameloid matrix. Demineralized, U-Pb, bar=200nm,
×46,000.
Fig. 3
Odontoblasts containing abundant rER, well-developed Golgi apparatuses and
various types of granules at the late stage of enameloid matrix formation.
Demineralized, U-Pb, bar=1μm, ×9,200.
Fig. 4
Large granules including fibril-like structures and fine electron-dense
materials in the distal cytoplasm of IDE cells at the late stage of enameloid
matrix formation. Demineralized, U-Pb, bar=200nm, ×64,400.
Fig. 5
Sections stained with PTA showing the large granules containing PTA-positive
fine filamentous structures. Demineralized, phosphotungstic acid (PTA),
bar=200nm, ×
46,000.
Fig. 6
Fine network-like materials between the collagen fibrils have disappeared
during the stage of enameloid mineralization. Bundles of collagen fibrils
are loose and each collagen fibril tends to be wavy in the non-mineralized
enameloid matrix. Demineralized, U-Pb, bar=500nm, ×27,600.
Fig. 7
Remnants of the organic matrix consisted of microfibrils forming a network-like
structure in the demineralized enameloid near the boundary between the enameloid
and the dentine. Demineralized, U-Pb, bar=500nm, ×27,600.
Fig. 8
Fine tubular structures found at the outer layer of demineralized enameloid.
Demineralized, U-Pb, bar=100nm, ×92,000.