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To visualize the cells and fibers of the developing periodontal ligament (PDL) by scanning electron microscope (SEM), we examined a new tissue preparation method including decalcification, sectioning by cryomicrotome, and chemical treatment for removal of cells or collagen fibers. The advantages of this method were as follows: (1) it was possible to expose the restricted area, (2) it caused no damage by heat or various embedding agents such as paraffin or resin, and (3) it was possible to make comparisons the SEM observation with histochemical or immunohistochemical observation using the neighboring sections. We could classify the development of PDL into three stages by alkaline phosphatase (ALPase) activity and observe each stage by this method. Stage I was the zone of dental follicle proper that showed negative ALPase activity. Stage II was the tissue surrounding the disrupted Hertwig's epithelial root sheath (HERS) which evinced intense ALPase activity, and stage III was the further advanced zone of differentiation that displayed moderate ALPase activity. Using this new method for SEM, cells with many processes and thin fibers were seen irregularly at stage II. On the other hand, at stage III, fibers were seen as interconnecting meshworks of thick bundles and cells that showed regularly arranged rows running obliquely to the surface of the root and alveolar bone. At the transition between stages II and III, the thickness and orientation of fibers changed abruptly.

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The study was designed to elucidate the effect of physiological tooth movement on cellular cementum, using the upper molar roots of 10-week-old rats. Paraffin sections stained with haematoxylin and eosin displayed two types of cellular cementum, lightly and darkly staining. The lightly stained was present on the distal half of all molar roots except the mesial root of the first molar. The alveolar bone facing the lightly stained cementum showed resorption lacunae and multinucleated osteoclasts, while the opposite bone surface was lined with osteoblasts. In contact microradiographs of undemineralized ground sections, the X-ray density of the lightly stained cementum was similar to that of the periodontal ligament and pulp, while the X-ray density of the darkly stained cementum was similar to that of alveolar bone. Tetracycline labelling lines were seen at the interface between the two types of cellular cementum as well as on surfaces of bone and cementum located mesially to the root dentine. The results suggest that the mechanical stress of tooth movement differently affects the alveolar bone and cellular cementum; the bone is resorbed whereas the cementum resists resorption and its calcification is inhibited under the compressive force of tooth movement.

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Glycolipids, glycoproteins, glycosaminoglycans and sialoglycoproteins have all been implicated in a number of developmentally significant processes related to complex interactions between cell surfaces and the extracellular matrix. The present study was designed to localize glycoconjugates recognized by peanut agglutinin (PNA) and Maclura pomifera (MPA) lectins during mouse molar root development. Postnatal ICR mice at 10, 15, 21, 28 and 42 days were used. Lower jaws were dissected, fixed in 4% paraformaldehyde, decalcified in 5% EDTA and embedded in paraffin. Serial sections were made and stained with FITC-conjugated PNA or MPA. beta-Lactose was used as an inhibitory sugar for PNA, and alpha-D-melibiose for MPA. PNA specifically stained Hertwig's epithelial root sheath (HERS), whereas MPA stained a number of tissues. The outermost layer of root dentin, forming cellular cementum, alveolar bone and HERS showed positive reactions with MPA. Glycoconjugates localized by the lectins may be functionally related to molecules which contribute to root formation and cemento-genesis.

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These studies were made as a first step in elucidating unknown functions of enamel proteins in odontogenesis. The cytoplasm of ameloblasts and the proteins in dentine matrix before mineralization were stained with this monoclonal antibody. SDS-PAGE and Coomassie blue staining of proteins extracted from enamel showed several protein bands. Immunoblotting revealed that proteins recognized by this antibody were situated between 20-30 kDa. These results indicate that enamel proteins, presumably amelogenins, have an epitope resembling monocyte-macrophage protein. The findings suggest that epithelial-mesenchymal interaction in odontogenesis and preosteoblast-preosteoclast interaction in osteogenesis may be similar.

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Tooth germs of rat incisors were examined by lectin-histochemistry in the portion from the apical end to enamel forming stage. Tissue sections were prepared from paraformaldehyde-fixed and paraffin-embedded tissues with or without EDTA-decalcification. They were stained with fluorescein-isothiocyanate-labeled (F- for short) lectins and observed by a fluorescent microscope. The boundary between inner enamel epithelia and dental papilla cells was stained with F-Con A, F-MPA and F-PNA. The boundary between the epithelia and dentin was stained with F-Con A and F-MPA. Stratum intermedium cells were stained with F-Con A, F-MPA and F-PNA, and were different in the time when they began to be stained with each lectin during their development. Distal cytoplasm of secretory ameloblasts and odontoblasts were stained with F-MPA and F-Con A, respectively. The staining with these lecting became stronger gradually from the apical to the incisal side. These results suggest that F-MPA and F-Con A are useful as a marker indicating the time when enamel and dentin begins to form, respectively. Distal cytoplasm of secretory ameloblasts was also stained with F-Con A and F-PNA. The odontoblastic layer was stained only with F-Con A, but the dental papilla was stained with F-Con A, F-MPA, and F-PNA. Stellate reticulum and outer enamel epithelia were stained with F-Con A and F-MPA. The comparison of the results from decalcified and non-decalcified tissues showed that the EDTA-decalcification scarcely affected these lectin bindings.

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Four about 4-week-old puppies were fixed by perfusion; the secretory ameloblasts of the first molar tooth germs were observed with an electron microscope. The morphology of the puppy secretory ameloblast closely resembles that of man. In addition, the primary cilia and a pair of basal bodies were observed close to Golgi apparatus of the secretory ameloblast. Cytoplasmic microtubules radiated from the basal bodies. It is suggested that a pair of basal bodies may be the microtubule-organizing center of the secretory ameloblast. Further, melanocytes were present in the intermediate cell layer and aggregated melanosomes were present in the cytoplasm of both stratum intermedium cells and secretory ameloblasts.

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Cell death in the epithelial tooth germs of mouse mandibular molars from the 12th to the 14th day of gestation was investigated by electron and light microscopy. Light microscopy revealed granular substances in the epithelial portion of the tooth germ on the 12th day, and an increase in their number to the 14th day when the enamel knot developed. In the areas where granular substances were observed by the light microscope, electron microscopy revealed cells with condensed chromatin. The cytoplasm of these cells increased in electron density, the cisterns of rough endoplasmic reticulum were dilated and the cristae of mitochondria disappeared. These cells were comparable to the physiologically degenerative cells reported in other organs. These degenerative cells are joined to adjacent cells by desmosomes and are believed to be derived from the epithelial cells. Further, degenerative bodies, composed of amorphous structures and enveloped by a limiting membrane, were observed in the epithelial cells. They are presumed to be derived from the degenerated cells engulfed by neighboring cells. From this evidence, phagocytic ability is attributed to the epithelial cells of the tooth germ.

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Observation of the radiographs taken of 37 mandibles from children ten months to seven years of age showed that the unerupted, permanent mandibular central incisors were rotated in 45 of 74 jaw halves (60.8%). The type of rotation in which the mesial aspect is directed lingually was most commonly seen (44.6%). The next most common observation was of jaw halves without any rotation of the central incisor (39.2%). The type of rotation in which the central incisor's distal aspect is turned lingually was the least common observation (16.2%). The rotation seemed symmetric in 25 jaws (67.6%).

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