ACBTE Vol. 3:7-18, 1993

Studies on the matrix of enameloid in Paralichtys olivaceus.



Minoru WAKITA, Shigeru TAKAHASHI
and Yosihiro HANAIZUMI


Department of Oral Anatomy II,
Hokkaido University School of Dentistry
Sapporo 060, JAPAN


Introduction
One of the histological characteristics of enamel or enameloid, which is the hard tissue covering tooth crowns in teleosts, is that its organic matrix mostly consists of collagen. Collagen exist in the form of fibrils, which ordinarily aggregate to form thick bundles. These collagen bundles in the enameloid region form a mixed weave pattern displaying a distinctive histological structure (Wakita and Takano, 1989, 1991). In certain fishes, some collagen fibers do not form bundles but arrange parallel to the enameloid surface, and this surface layer continues to the surface of the so-called tooth shaft to form the collar enameloid (Shellis, 1975, 1981).
In the animal body collagen often occupies a given space with a characteristic arrangement and structure, as seen in different kinds of tissue like periodontium, dermis, cornea, tendons, etc.
During formation, collagen is secreted by collagen forming cells which were in the space destined to be filled by this tissue. Subsequently, this collagen becomes integrated in the form of secondary fibrils, maybe by collagen forming cells there, to display the arrangement which is specific to these kinds of tissue.
However, there is little understanding of the morphological and biochemical role of cells on the control mechanisms and processes of the integration of protocollagen into a single fibril in the intercellular space (Trelstad and Silver, 1981; Kivirikko and Myullyl3 1984; Scott, 1990).
The processes with which collagen fibrils gather to become thick fibril bundles and show the characteristic histology of the various kinds of tissue during formation of cementum (Beersten et al. 1974; Beersten, 1975; Yamamoto and Wakita, 1990), tendon (Birk and Trelstad, 1986; Young and Birk, 1986), dermis (Gibson et al., 1965; Meyer and Neurand, 1987; Pierard and Lapiere, 1987), and cornea (Birk and Trelstad, 1984) has been shown.
There is, however, little information on the factors controlling the assembly of matrix fibrils in the enameloid, bone, or dentine, although there are some reports which show that during enameloid formation, thick matrix bundles with fibres running in different directions are piling up alternately from the surface to the deeper regions of enameloid in some teleost fishes (Wakita and Takano, 1989; 1991).
The present study aims to study the role of odontoblasts in the formation and assembly of collagen fibrils in local areas during the enameloid formation stage.


Materials and methods:
The fish, Paralichtys olivaceus , examined in the present studies were supplied from the Fishery Cooperative Association at Yoichi (Yoichi Gyogyou Kumiai). The upper and lower jaws were dissected out and immediately immersed in a 2.5% glutaraldehyde and 4% paraformaldehyde mixture buffered with sodium phosphate at PH7.4 at about 4 ¡C. Jaws were cut into smaller pieces with 3 or 4 teeth each in the same fixative. Specimens were fixed for overnight storage in a refrigerator, then transferred to 0.1M phosphate buffer containing 0.22M saccharose before removing tooth germs. After isolation, the tooth germs were immediately dehydrated in a ascending series of ethanols, substituted by QY-1, and embedded in epoxy resin (Luveak 812). Some tooth germs were decalcified by 2% L-ascorbic acid (Wakita 1983) or 5% EDTA. All semithin sections were stained by Methylene blue-Azur II and the developmental stage of each tooth germ was examined under the light microscope. The tooth germs in the enameloid forming stage were selected out and trimmed, and ultrathin sections were examined by HITACHI H-7000 TEM.


Results
Enameloid in semithin sections from tooth germs just after formation showed the arrangement of matrix fibers clearly when they were stained by Methylene blue-Azur II. In these preparates, the arrangement allowed a distinguishing of two sorts of matrix fibers under the light microscope. One located at the subsurface area. This appeared as a clear and thin layer, parallel to the enameloid surface. They did not show any distinctly bundled shape. The other was located beneath this subsurface layer and occupied the rest of the space of the enameloid of the crown. Fibers in this area appeared mostly as coarse fiber bundles, which were complicatedly interwoven. Particularly the fiber bundles in the area under the crown tip showed extraordinarily thick diameters, and a coarse textile structure. At the area close to the dentine, the matrix fibers were slightly thinner in diameter and arranged more regularly. The matrix fibers in the dentine region became fine, and the textile like structure and regularities in the arrangement disappeared. The border between enameloid and dentine was visibly clear from the morphology of the matrix fibers.(Fig.1).
The electron microscope showed the fibril structure of the subsurface area in detail.(Fig. 2). The subsurface area of enameloid was formed by thin collagen fibrils running roughly parallel to the enameloid surface. These fibrils did not assemble into secondary fibril bundles, but all gathered to form a thin fibril layer beneath the enameloid surface. These fibrils were not arranged completely parallel to each other, but fibrils in many cases crossed at small angles to each other. The fibrils in the deeper portion left this subsurface layer and moved into the inner area with the change in the direction, to where thick bundles were interwoven. Here these fibrils from the surface layer mixed into bundles.
In enameloid areas other than this subsurface area, most collagen fibrils were assembled into thick densely interwoven bundles. The diameter of a single fibril forming the collagen bundles were various. The cross section of a collagen bundle showed that this bundle is a mass of single fibrils with various diameters. The thick bundles were interconnected with few thin fibrils.
Before enameloid formation, ameloblasts layered onto odontoblasts with the basal lamina in between (Fig. 3). The odontoblasts were columnar in shape and showed an undifferentiated stage, except for being relatively rich in rER. Many collagen fibrils were seen between these odontoblasts. These fibrils were located in the intercellular space of odontoblasts as thin bundles containing dozens of fibrils (Fig. 4).
The intercellular collagen continued to the mesenchymal region.
At the area where the enameloid formation started, these collagen bundles beneath the ameloblasts continued as bundles between odontoblasts passing through the odontoblasts layer, to participate in forming the surface area of the enameloid.
It was frequently observed that there were many odontoblast processes lying between these incomplete collagen bundles, but it was rare that the odontoblast processes crossed the enameloid layer up to the ameloblasts or its basal lamina.
The development site of the most complicated region with interwoven collagen bundles were observed by TEM. The present study showed interesting features at the matrix forming site.
Firstly, the newly formed collagen fibrils were not directly on the cell surface of odontoblasts. The newly forming enameloid matrix was never in direct contact with the odontoblast body proper, but indirectly with intervening small cell processes (Fig. 5,). The cell processes always intervened between the forming matrices of enameloid and the odontoblast body proper. These small processes, here named as interposing processes for the present, always appeared in 1 to 3 layers separating the matrix and the cell body. These tiny interposing processes continued till the odontoblasts themselves. When there were two or more interposing processes between matrix and the odontoblast cell, small amounts of collagen fibrils were occasionally seen in the narrow space between these processes. This occasionally appeared, especially when the fibril bundle was adequately thick.
Secondly, there was a relationship between the direction and structure of the intervening processes and the direction of collagen fibrils which were adjacent to the interposing processes. Where collagen fibrils were cross-sectioned, the processes adjacent to these fibrils usually had round or oval contours; where collagen bundles were longitudinally sectioned, processes showed a shape with elongated and narrow saccles. Where collagen fibrils were cut at various angles, the processes appeared with various cross sections.
These morphological relationships between the sectional direction of collagen and the structure of the interposing processes was especially shown at the only site where these processes occupied the location between the collagen bundles and the odontoblastic body. There was not any significant relationship between matrix and processes in other locations. Even if there were rare cases with a few collagen fibrils nearest to the odontoblast, the detailed observations showed the existence of thin processes of odontoblasts (Fig. 6). Where the collagen bundles and the interposing processes piled up alternately, the direction of collagen fibrils in each bundle was different.



Discussion
The development of enameloid matrix in Paralichtys olivaceus was examined by TEM from the surface to deeper parts.
Matrix fibers could be distinguished into two kinds, one was running parallel to the enameloid surface without forming distinct fibril bundles, and the other appeared as thick interweaving collagen bundles. Collagen as the organic matrix of enameloid was formed in the mesenchymal region between ameloblasts and odontoblasts (Wakita, 1974; Ono, 1974; Inage, 1975; Wakita and Takano, 1989; 1991), and since the enameloid matrix was formed in the space between these two cell layers, isolated from other intercellular spaces, the organic matrix of enameloid has clearly different characteristics than other tissue composed of well integrated collagen fibrils like periodontium, tendons, etc. The cells composing the ameloblast layer and the odontoblast layer are closely attached to each other and form dense cell layers. This means that the matrix may be formed in the space between the two cell layers . Recently there have been reports to suggest the intimate participation of the inner dental (enamel) epithelium (IEE or IDE) to the matrix formation in fish enameloid (Postak and Skobe, 1984, 1986a, b; Sasagawa and Ferguson, 1990). However, there are no descriptions which mention the structural ralationship between complicatedly interlaced odontoblasts and matrix fibers.
The mechanisms of aggregation of pre-collagen molecule to the single fibril has not been established biochemically. There are reports showing the involvement of the pericellular matrix in the assembly of collagen molecules (Delvoye et al., 1988 ; Scott, 1990), and it has been suggested that the site of collagen discharge is in the deep recesses of the cell surface (Trelstad and Silver, 1982).
This means that the large molecular weight matrix, which already exists or is formed in the extracellular space, may function as the template or stencil for the arrangement of collagen fibrils.
At the same time, this may help to provide an explanation why collagen assembles into fibrils in the extracellular space and why these fibrils are further assembled into thick bundles. At the early phase of collagen formation it has only been established that the recesses of the cell surface take part in the assembly of the thick bundles of fibrils. It also seems certain that it participates in the wide membranous cell processes which function to assemble fibrils into thick bundles and does not just remain in the recesses of cell surfaces. This participation was reported in relation to the development of tooth cementum (Yamamoto and Wakita, 1990), periodontium (Beersten, et al., 1974; Beersten, 1975), cornea (Birk and Trelstad, 1984, 1986), and tendon (Young and Birk, 1986; Trelstad and Birk, 1984). Young and Birk reported that during tendon formation the fibroblasts enclosed the small sheaf of fibrils with the membranous cell processes to form a compartment, and these compartments fused to form thick fascicles. They further report that fibroblasts play a central role in shaping the matrix architecture by partitioning the extracellular space into functional units. Ploetz et al. (1991) noted that the collagen of the skin changed to thick fibers through a number of compartmentalization steps.
During fibril assembly by compartmentalization, the contribution of cytoskeltons like actin and microtubules has been reported by demonstrating cytoplasm of fibroblast cell processes extending out to and surrounding collagen fibrils. This indicates a close involvement with established fibers (Dahners et al., 1986), or that the overall pattern of microtubule alignment is similar to that of collagen fibers secreted by fibroblasts (Dane and Tacker, 1986). Some experimental in vitro studies have proved an involvement with the assembly of collagen in extracellular space, when the cell contracted (Nakagawa et al., 1989; Welch et al, 1990).
It is clear that the thickness and arrangement of collagen fibers are regulated by collagen forming cells in each kind of tissue. And it is well known that the architecture of collagen is different in different tissue, as is the thickness of a single fibril or bundle.
The matrix fibers in skin are arrayed along the direction resisting externally applied forces (Gibson et al., 1965; Pierard and Lapiere, 1987), and this is also well known from bone tissue (Gerstenfeld et al., 1988; Boyde et al., 1990; Carando et al., 1991), and in tendons (Evanko and Vogel, 1990).
It may then be asked how the situation is in enameloid?
It is very instructive to consider the relationship between the arrangement of collagen fibers and stress lines in enameloid. The geometrical arrangement of matrix fibers in enameloid has been reported to show a close similarity to the matrix arrangement in the dermis (Gibson et al., 1969; Meyer and Neurand, 1987). There are, however, very few descriptions of an analysis of the arrangement of matrix fibers in enameloid or in enamel from a dynamic view point. A description of the functional adaptation of form and structure in shark teeth showed that the positions and orientation of enamel-fibers on fangs and cutting teeth were in perfect accordance with the pattern of trajectories in a homogeneous tooth model under stress (Preuschoft et al., 1974). Although this examination was a dynamic simulation, it may make us expect that the arrangement of matrix fibers of enameloid in other fishes would be in accordance with the resistance against the force working on this tissue.
However, different from dentine but like enamel, the changes in organic matrix in enameloid mostly take place with inorganic substances (usually hydroxyapatite) during its maturation . This means that the matrix fibers do not directly resist forces working to break the functional tooth. It seems unlikely that the arrangement of matrix in enameloid is the result of processes taking place during enameloid formation.
It is well known that fibroblasts may be involved in a reorganization of extracellular fibrils by adhering with fibronectin and a contraction of the cytoskeleton. These experiments are generally done in relation to wound healing (Nakagawa et al., 1989; Welch et al., 1990). It is, however, self-evident that these functions of fibroblasts in vitro cannot apply to the odontoblasts forming the matrix of enameloid. Because odontoblasts do not exist as single free cells but always as components of the single cell layers, and they do not have the freedom to move alone like fibroblasts.
The present study is the first dealing with the correspondence between the direction of matrix fibrils and the structure of odontoblast processes from developmental aspects, at the matrix forming site.
From published electron micrographs it may readily be expected that the three-dimensional structure of these odontoblast processes should be finger like or slender sacs (Domon and Wakita, 1986, 1989). And the distinct positional relationships between the cell processes and extracellular matrix in localization and direction was reported to relate to the structure of dentine (Ichijo et al., 1975; Tanaka, 1989). They described collagen fibrils filling dentinal tubules running parallel to odontoblast processes. This relationship between processes and matrix fibers in dentinal tissue also shows a close resemblance with that in enameloid formation. Both occur in enclosed spaces, the structure and direction of processes and the matrix have an intimate relationship and both are observed in the same situation, odontoblast processes and collagen formed by the cells.
The studies here suggest that the odontoblast processes interposing between the odontoblast cell body and the developing collagen, and showing its peculiar structure and direction, form the enameloid matrix for the most part and decide its arrangement when they are formed. It is also suggested that the cell processes between fibril bundles which run in different directions in the vicinity of the odontoblasts function to gather single fibers into bundles with particular directions, the thin fibrils assembling to become thick bundles, at least in local development. The processes of odontoblasts play the main role to determine the course and position of matrix fibrils just secreted, and also participate in the compartmentalization of those fibrils to form thicker bundles (Young and Birk, 1986; Ploetz et al., 1991).
It also suggests that the piled-up processes often seen near the odontoblast may be the crucial factor determining the histology of the complicatedly interwoven enameloid matrix, with the help of the compartmentalizing function.


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Explanation of figures:

Fig. 1 : Optical micrograph showing enameloid in matrix formation stage of Paralychtis olivaceus, stained with Methylene blue - Azur II. Three areas of matrix are distinguished from their matrix morphology. (a) the area adjacent to the ameloblast layer, which appears as a thin and clear layer. (b) the area beneath the enameloid tip, which is consisted of coarse fibers interwoven irregularly. (c) the area near the dentine, which is occupied by fine textile structures. Ab:ameloblast layer, D:dentine

Fig. 2 :Electron micrograph showing arrangement of matrix fibrils collagen in the subsurface region of Paralychtis olivaceus. Collagen fibrils forming thin area (small asterisk) right under the ameloblast (arrow) composed of thin bundles of fibrils, but do not form thick bun-dles as in the area beneath this layer (large asterisk) . In the latter area many thick bundles of collagen are densely interweaving. Both areas interrelated with thin bundles each other.

Fig. 3 :Electron micrograph in low magnification showing ameloblasts ( Ab ) and odontoblasts ( Ob ). Odontoblast layer as well as ameloblast layer has little intercellular space. Cells in both layers show undifferentiated young stage before enameloid formation. Between those two cell layers there are few collagenous matrix arrows), and sometimes between odontoblasts (small arrow).

Fig. 4 : Electron micrograph showing the formation of superficial layer of enameloid. Matrix fibers run parallel to the enameloid surface to make the subsurface layer. Many collagen bundles (asterisk) passing through the odontoblasts join this layer to make it thicker. The thin processes of odontoblasts interpose between these bundles (arrow head). Ab:ameloblast, Ob:odontoblasts

Fig. 5 : Electron micrograph showing matrix forming site in the middle area of enameloid of Paralychtis olivaceus. There are many small processes to interpose between odontoblasts body (Ob) and enameloid matrix (M). In this scene, the relationship of cutting directions between collagen fibrils and interposing processes are well shown. The alternative piling up of matrices and processes with those relationship appears (large arrow head). Some thin collagen bundles are seen in deeper portion among the interposing process (small arrow head).

Fig. 6. : Electron micrograph showing collagen fibrils passing through the odontoblast layer to take a part of enameloid matrix (arrows). Very thin interposing processes (arrow head) exist between thick matrix bundles and odontoblastic bodies. Ob : odontoblast, M:
enameloid matrix.