Mammals and a few reptiles have evolved their dentition by adapting an organ, known as the periodontal ligament, that links the tooth to the jaw and is the source of power for tooth function. The periodontal ligament plays a part in the attachment and support, nutrition, synthesis and resorption of the teeth, and in proprioception.

The periodontal ligament consists of cells, connective tissue fibres, matrix, vessels, and nerves. The oxytalan fibre is one of the two connective-tissue fibre components in the human periodontal ligament, the other being that collagen fibre. The precise role of oxytalan fibres is unclear. They have been studied with reference to their arrangement and distribution, biomechanical characteristics and so on. In those studies their three-dimensional distribution was conjectural, as only light microscopy or transmission electron microscopy were used. Although Chen et al. (1994) and Shinohara (1996) observed oxytalan fibres by scanning electron microscopy, their findings did not reveal the relation between the fibres and other structures.

Recently, confocal laser scanning microscopy was introduced to reveal the three-dimensional features of fine structures. Our purpose now was to improve the method of confocal laser scanning microscopy to observe the overall three-dimensional distribution of oxytalan fibres in the mouse periodontal ligament and to clarify the relations between oxytalan fibres and blood vessels.

We used 20 male ICR mice weighing between 28 and 35 g. The mice were anaesthetized beyond recovery with diethyl ether, and the mandibles rapidly dissected out and fixed in 10% Lillie’s aqueous neutral calcium acetate formalin for one or more days at room temperature. Specimens were mineralized in 5% formic acid for 1 week and embedded in OCT compound (Tissue-Tek). Serial sections 60-?m thick were cut with cryostat (Leica CM3000, Germany) vertically through the apices of the molars in a mesiodistal plane. Specimens were oxidized with monopersulphate compound (Oxone, Aldrich, Milwaukee, WI, USA) and stained with aldehyde fuchsin according to the method of Löe and Nuki (1964), in which background staining is reduced by treatment for 2 min in 4% hydrochloric acid in 70% ethanol. Adjacent control sections were stained by the same technique without pre-oxidization. The specimens were observed in four regions: the cervical one-third (region A), middle one-third (region B), apical one-third (region C), and furcal areas (region D). These specimens were observed by light microscopy and with a confocal laser scanning microscope (Zeiss LSM310, Germany) with objective lenses of ×40, and ×63 (immersion). The excitation wavelength and barrier filters were varied to obtain fine images of the aldehyde fuchsin-stained fibres. The images obtained by confocal scanning were transferred to an IBM-compatible computer (MS DOS, MS Windows 3.1).

Fig. 1. (a) The four regions (A, B, C, D) where oxytalan fibres were observed. M1, lower first molar; M2, lower second molar; A, cervical one-third; B, middle one-third; C, apical one-third; D, furcal. (b) Confocal laser single-scan image of the cervical one-third (region A) between the mouse lower molar (M1) and second molar (M2). The oxytalan meshwork is clearly seen as opaque white lines. There are several groups of oxytalan fibres. The principal group starts from the subgingival area to form the vertical meshwork along the tooth axis. Aldehyde fuchsin staining with pre-oxidization. Bar=50 ?m. (c) Transmission confocal laser scanning-microscopic image of the same slide as (b). The oxytalan fibres in this figure are the opaque white lines in (b). Aldehyde fuchsin staining with pre-oxidization. Bar=50 ?m. (d) Confocal laser single-scan image of section adjacent to (b) in which pre-oxidization was omitted. No opaque white lines are seen. Aldehyde fuchsin staining. Bar=50 ?m. (e) Transmission confocal laser scanning-microscopic image of the same section as in (d) shows no oxytalan or elastic fibres. Aldehyde fuchsin staining. Bar=50 ?m. Ox, oxytalan fibre; Bo, bone; M1, first molar; M2, second molar; G, gingiva.

Aldehyde fuchsin-stained fibres could be clearly detected when the confocal microscope was operated in the reflection mode at an excitation wavelength of 514 nm and through a 488-nm barrier filter. Oxytalan fibres appeared as opaque white lines in the background, varying in tone depending on their structure. We compared these images with those of transmission confocal laser scanning and light microscopy, and found that the lines coincided with the structures usually called oxytalan fibres [according to the definition of Fullmer (1958)]. The meshwork structure of oxytalan fibres was observed in greater detail with this new technique than with conventional methods. Moreover, the adjacent slides that had not been pre-oxidized with Oxone before staining did not show any fibres that looked like elastic.

Fig. 2. (a) Confocal laser single-scan image in region B shows that the oxytalan meshwork runs in the same direction as in Fig. 1(b). The oxytalan fibres tend to be located around the walls of the vessels (arrows) and interconnect between the vessels. (b) Confocal laser single-scan image in region C shows that the oxytalan fibres are of smaller diameter but more densely located around the walls of blood vessels and envelop the apex. (c) Confocal laser single-scan image in region D shows that the fibres starting from the cementum are located near or around the walls of blood vessels and interconnect between the blood vessels. (d) Three-dimensional depth-coding confocal laser scanning-microscopic image in region D shows fibres finer in diameter embedded only in part of cementum (arrows). The depth is coded from 0 ?m (red) to 10.5 ?m (blue). (e) Three-dimensional depth-coding confocal laser scanning-microscopic image in region B shows that the fibres run around the walls of the vessels. In addition, they branch off where the vessel bifurcates (arrow) and coincide with the blood vessels. The depth is coded from 0 ?m (red) to 19.5 ?m (blue). (f) Light-microscopic image of the same region as in (e). In (e), both oxytalan fibres and the vessel bifurcation can be clearly seen. Ox, oxytalan fibre; Bo, alveolar bone; M1, first molar; M2, second molar; Ap, apex; T, tooth; Ce, cementum; De, dentine; V, vessel. Aldehyde fuchsin staining with pre-oxidization. Bar=50 ?m.
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The meshwork consisted of fine filamentous components ranging from 2.0 ?m dia. to the limit of resolution for the microscope. Larger bundles were consistently brighter than smaller bundles. The fibres could be observed throughout the periodontal ligament, as some were very close to the surface of the specimens and others deeper. The density of oxytalan fibres was not uniform: they were more numerous near the alveolar bone than near the tooth, and were more predominant in the cervical one-third (region A) than in other parts of the tooth. Their number gradually decreased along the root of the molar but then increased again at the apex. The fibre bundles on the tooth side were smaller in diameter than those on the alveolar-bone side. These fibres attached to the tooth only into the part of the cementum below the gingival attachment.

In the cervical one-third, although several fibre groups ran in many directions, the principal fibres mainly started from the subgingival area and cementum to form a meshwork that was aligned occluso-apically. In the middle third, the meshwork from region A and fibres from the cementum in this area formed a meshwork that ran in the same direction as in region A and tended to run along the walls of the blood vessels and interconnected vessels that appeared as black ovals. In the apical area, the fibres formed a meshwork that enveloped the apex but were still located near or around the blood vessels. In the furcal area, the fibres also started from the cementum and formed a meshwork that ran around and interconnected with the blood vessels.