Showa Univ. J. Med. Sci. 6(2), 165'170, December 1994 Original Light and Electron-Microscopic Observation of Attachment between the Periodontal Ligament and Alveolar Bone in Rat Molars Mie KUROIWA,Kenji CHIHARAand Shohei HIGASHI Abstract: The organization of collagen fibers and fibrils on the alveolar bone side in the periodontal ligament of rat molars was observed by scanning electron microscopy after maceration of the cellular elements by sodium hydroxide (NaOH) treatment. Two types of periodontal ligament-to-alveolar bone attach- ments were distinguished. Type A: thick collagen fiber bundles of the periodontal ligament, about 3-8 µm in diameter, embedded in the alveolar bone matrix, which were bound encircling the bone matrix fibrils on the bone surface. Type B: fiber bundles about 0.3-1 pm in diameter, which were combined together on the alveolar bone surface. The Type A fibers were observed mainly on the deposition side of the alveolar bone; the Type B fibers were observed on the resorptive side. These two types of collagen fibril components suggest that they are related to tooth movement. Key words: periodontal ligament, alveolar bone, collagen fiber, NaOH macera- tion, scanning electron microscope (SEM) Introduction Maceration of tissues with NaOH selectively removes connective cellular elements and has been used for observing the structure of various collagen fibril tissues by scanning electron microscopy (SEM)1~. We have previously observed the peridontal ligament in rat molar teeth using this method2~. SEM observations can easily show three-dimensional tissue structure. The periodontal ligament fibers are composed of dense fenestrate networks and consist of bundles of collagen fibrils that branch out and anastomose2). Kraw and Enlow3~ and Kurihara and En1ow4) reported the relation between the different of types of attachment between the periodontal ligament fibers and the alveolar bone sup- porting the tooth. However, the types of attachment have not been distinctly revealed by SEM. The periodontal ligament fiber plays an important role in the functin of tooth eruption and physiological and orthodontic tooth movement5,6). The periodontal ligament fibers may be classified according to their size and orientation. The present study is concerned with the types of attachment between the periodontal liga- ment fibers and the alveolar bone that are revealed by SEM after treatment with NaOH. Materials and Methods Sprague-Dawely rats, 4 and 20 weeks of age were anesthetized with ether and sacrificed. Second Department of Oral Anatomy, Showa University, School of Dentistry, 1-5-8, Hatanodai, Shinaga- ku, Tokyo 142, Japan. 166 Mie KuxoiwA, et al. Figs. 1, 2. Light micrographs of attachment between the periodontal ligament and the alveolar bone of a rat molar, stained with van Gieson stain. Fig. 1, Type A. Fig. 2, Type B. PF, periodontal ligament fibers; B, alveolar bone; Bar=20 ƒÊm. The extracted maxillar and mandibular jaws were fixed by immersion in a fixative containing 2% paraformaldehyde and 2.5% glutaraldehyde in a 0.1 M phosphate buffer (pH 7.4). The jaws were demineralized in 10% ethylenediamine tetraacetic acid (EDTA) at pH 7.4. The jaws were cut or fractured into several pieces mesiodistally and the roots of the teeth were cut crosswise, rinsed in a 0.1 M phosphate buffer at pH 7.4, and again fixed in a buffer of paraformaldehyde and glutaraldehyde solution for 5 days. The specimens were macerated in a 5% aqueous solution of NaOH for 5-10 days at room temperature (20-25•Ž)1,2). The NaOH-treated specimens were rinsed in distilled water for about 1 day until they became transparent. The specimens were treated with 2% tannic acid solution for 2h, washed in distilled water for 1 h, and treated with 1 % OsO, in a 0.1 % phosphate buffer for 2 h. The specimens were dehydrated with ethanol at increasing concentrations and sublimated with t-butyl (ID-2, Eiko Engineering Co., Japan). The sublimated specimens were attached to aluminium stubs, coated with a 50.E thick platinum-palladium layer (5450 Polaron Equip- ment Ltd., England), and observed by an SEM (S-430 and S-700, Hitachi, Japan). Some demineralized specimens were dehydrated with the ethanol series and embedded in paraffin. The serial sections were sliced and stained with van Gieson stain. Results The light micrographs showed the attachment between the periodontal ligament and the alveolar bone of the rat molars stained with van Gieson stain (Figs. 1, 2). Two types of periodontal ligament-to-bone attachments (Types A and B) were observed on the alveolar Periodontal Ligament-to-Bone Attachments 167 Figs. 3, 4, 5. Scanning electron micrographs of attachment of Type A between the periodontal ligament fiber and the alveolar bone of a rat molar. PF, periodontal ligament fibers; BF , bone matrix fibers; arrow, transmigration and combination between the periodontal ligament fibrils and bone matrix fibrils; Bar-0.5 ƒÊm (Fig. 3); 2.5 ƒÊm (Fig. 4); 5 ƒÊm (Fig. 5). bone surface. Type A consisted of a fiber bundle about 3-8 tm in diameter (Fig . 1). The fiber bundle was in a tree-like formation and perpendicularly embedded in the alveolar bone surface. Type B consisted of a fiber bundle about 0 .3-1 .tm in diameter (Fig. 2). The periodontal ligament fiber bundles were disorderly extended and adhered to the alveolar bone in a poorly-defined network. Under SEM the attachment between the periodontal ligament and the alveolar bone was 168 Mie KUROIWA. et al. Figs. 6, 7, 8. Scanning electron micrographs of attachment of Type B between the periodontal ligament fiber and the alveolar bone of a rat molar. PF, periodontal ligament fibres; BF, bone matrix fibers; arrowheads: intertwining of the periodontal ligament fibrils and the bone matrix fibrils; Bar=0.5 ƒÊm (Fig. 6); lƒÊm (Fig. 7); 0.5 µm (Fig. 8). observed three-dimensionally, which showed that the NaOH-treatment had effectively removed the cellular elements (Figs. 3-8). In Type A, the periodontal fiber bundles were in a tree- like formation embedded in the alveolar bone (Figs. 3-5). The matrix collagen fibers of Periodontal Ligament-to-Bone Attachments 169 the alveolar bone surface ran along the bone surface (Fig. 3). The alveolar bone matrix collagen fibers encircled the embedded collagen fiber bundle root of the periodontal ligament on the bone surface. The fibrils of the penetrating fiber bundle combined with the bone matrix fibrils (Figs. 4, 5). The fibrils between the periodontal ligament fiber bundle and the alveolar bone matrix fiber transmigrated themselves (Figs. 3, 4). The bone matrix fibriles encircled the periodontal ligament fiber bundle and also combined with the next fiber bundle (Fig . 5). In Type B, the fiber bundles become frayed, f enestrated and combined with the alveolar bone matrix fiber (Fig. 6). The fibrils of the frayed periodontal ligament fiber bundle inter- wined with the bone matrix fibers (Figs. 7, S). Discussion We discerned two types (Types A and B) of periodontal ligament-to-alveolar bone attach- ments. When the tooth function is altered , the size, thickness and type of attachment of the periodontal ligament fibers embedded within the alveolar bone are also altered7'8. Bernick9~ reported that the stress of functional occlusion caused a thickening of the fibers . The rat molars physiologically drift distally1). The tooth movement was histologically produced by changes throughout the periodontium, while a resorptive and a depository surface was being developed on the alveolar bone. Type A was mainly observed on the depository bone surface . However, Kurihara and Enlow4) described two and/or three types (Types I , II and/or III) of attachments by trans- mission electron microscopy. The characteristic feature of Type I4) ("continuous•h attachment) is the direct, uninterrupted and intact nature of fiber bundle continuity from the bone matrix into the fiberous stroma of the periodontal ligament. Type I resembled the Type A attach- ment observed in the present study. The fibrils of the penetrating periodontal ligament fiber bundle transmigrated and combined with the bone matrix fibrils . This combination would intensify the continuity between the periodontal ligament fibers and the alveolar bone . The fibrils encircled the periodontal ligament fiber bundle root on the alveolar bone surface com- bined with the next fiber bundle and formed the fibril network (Fig . 5). This fibril network would be progressively related to bone reforming. The bone matrix was deposited on the fibril network, which was subsequently calcified. The periodontal ligament fiber bundles then become the component fibers of the alveolar bone . The alveoral bone reforming appeared on the depository bone surface7). The fibrils encircled the periodontal ligament fiber bundles enclosing them as new bone deposits. The periodontal ligament retains its breadth by elongation of the precollagenous linkage fibrils10). Type B was mainly observed on the resorptive bone surface . Type B was characterized by adhesion of the periodontal ligament fibers to the alveolar bone surface on which the fibrous combination had been intertwined with the thin fiber bundle . Type B resembled the •gadhesive•h attachment observed by Kurihara and Enlow4) . This attachment differed from Type A; the periodontal ligament fiber bundle was frayed on the alveolar bone , and the fibrils were intertwined with the fibrils of the bone matrix . The resorption of the alveolar bone occurred from the alveolar bone surface by changing the periodontal ligament fibers . The fibers composing Type B may have remained intact , without being absorbed and/or they may have been reformed. Although the bone on the resorptive side of the alveolar bone underwent progressive resorption, the collagen fiber periodontal connection between the bone and tooth was always maintained. When the resorption process was completed on the 170 Mie KUROIWA, et al. alveolar bone resorptive front, all the fibers were totally severed in the connective region.