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The highly polarized secretory ameloblasts in the incisors of rats fixed by perfusion with glutaraldehyde two or three hours after intravenous administration of vinblastine sulfate at a dosage of 5 mg per 100 g body weight were studied in the light microscope and the electron microscope. The following effects were observed: 1. All cytoplasmic microtubules in the ameloblasts had vanished. This was not accompanied by the appearance of paracrystals of microtubular protein or macrotubules. 2. The ameloblasts preserved their external features of polarized cells but lost their ability to maintain normal orderly segregation of the cell constituents; i.e. their normal internal compartmentalization and polarity had vanished. 3. The ameloblasts lost their capability of directional translocation of the secretory granules towards the cell apex. 4. Secretory granules already translocated to the cell apex regurgitated in the cell and a probably delayed discharge of secretory material had started in abnormal site at the surface. 5. The normal arrangement of ribosomes into polyribosomes on the membranes of the rough endoplasmic reticulum was no longer present; the ribosomes were apparently distributed at random. 6. New secretion was inhibited or brought to a standstill but secretory material already present in the Golgi complex appeared to be transported normally. 7. The centriole had started to develop into a cilium in many of the cells. 8. The number of autophagic vacuoles had increased.

The structure of the epithelial-mesenchymal interface of a developing mouse embryonic tooth germ was examined at the time of cell differentiation by using transmission and scanning electron microscopy. During odontoblast differentiation, the basal lamina was continuous beneath the inner enamel epithelium, and the fibrils at the mesenchymal aspect increased in density and length. Numerous cell processes of the preodontoblasts were seen in close contact with the basal lamina. This supports the idea that the basement membrane is involved in the differentiation of mesenchymal cells into odontoblasts, probably by providing special attachment sites for the aligning mesenchymal cells. The rearrangement of intracellular organelles in the cells of enamel epithelium and a change of epithelial morphology were already seen at the time of the initial predentin secretion by odontoblasts. At this time, the basal lamina was still present. The penetration of epithelial microvilli into the epithelial-mesenchymal interface through breaks in the basal lamina related to the onset of predentin mineralization. This suggests that the determination of epithelial cells into ameloblasts already occurs before the disappearance of the basal lamina.

In order to revalue the effects of colchicine on incisor secretory ameloblasts, entire mice were perfused with Krebs solution supplemented with a buffer and amino acids, through the right common carotid artery. The normal ultrastructure of the cells was maintained for 2 h with the perfusate alone. When colchicine (0.3-3.0 micrograms/ml) was added to the perfusate, it induced ultrastructural changes, such as the loss of cytoplasmic microtubules, the loss of secretory granules in Tomes' process, the abnormal accumulation and secretion of secretory granules, disarranged Golgi apparatus and the fragmentation of rough endoplasmic reticulum. Vesicles (150-400 nm in diameter) resembling immature secretory granules also accumulated, the degree of accumulation depending on the duration of colchicine treatment. The accumulation of secretory granules and these vesicles suggests that the intracellular transport system was affected by colchicine but that the production of secretory granules was continuous throughout the experimental period. The present perfusion system has enabled us to treat ameloblasts with an agent that is a useful experimental tool for elucidating cell functions, despite being lethal to animals in vivo.

Trypsin-dissociated dental papillae and enamel organs removed from the first lower molars of day-18 and day-19 mouse embryos were cultivated for 2 to 4 days on Millipore filters in DMEM supplemented with 15 per cent fetal calf serum and chondroitin sulphate and hyaluronic acid, individually or together. Three concentrations of glycosaminoglycans (GAGs) were also added to the media (01, 0.2, 0.4 mg/ml). Control cultures were made in the absence of GAGs, and additional experiments performed in which the presence of GAGs was associated with serum-free medium. Elongated and polarized odontoblasts showing synthetic activity were only observed in the presence of serum containing medium supplemented with 0.1 or 0.2 mg/ml of the GAGs. [3H]-thymidine autoradiography demonstrated that these cells were already post-mitotic at the onset of the culture. Polarized ameloblasts were never observed. These data provide evidence that GAGs are able to maintain the polarized state of cultured odontoblasts.

In order to elucidate the cytochemical properties of the membranous structure between enamel and ameloblasts of the rat incisor at the maturation stage, chromic phosphotungstic acid (Cr-PTA) and periodic acid-silver methenamine (PA-silver) techniques for electron microscopy were employed in combination with a digestion test with hyaluronidase, neuraminidase, collagenase or trypsin. Also, acid phosphatase activity of ameloblasts at the maturation stage was examined with a modified GOMORI's metal salt method. An intensely Cr-PTA reactive band approximately 0.1 micron thick appeared along the surface layer of enamel at the transitional stage, and at the very beginning of the maturation stage another intensely Cr-PTA reactive band which was seen by uran-lead stain to be a delicate electron-dense membranous structure appeared as well between enamel and ameloblasts. A lot of cytoplasmic small vesicles or tubular structures, both intensely reactive to Cr-PTA, were observed near the apical membranes of the overlying ameloblasts indicating that those organelles must have been responsible for the secretion of the latter band. Acid phosphatase activity was clearly demonstrated at Cr-PTA reactive large vesicles in the cytoplasm of those cells. The PA-silver staining technique manifested a band heavily deposited with silver grains along the surface layer of enamel, i.e., where the former band existed, but showed no particular reaction at the latter, the band-like layer between enamel and ameloblasts. Hyaluronidase or neuraminidase treatment remarkably decreased the Cr-PTA reaction of the latter band. Trypsin or collagenase treatment, on the other hand, not only eliminated the Cr-PTA reaction but digested the band itself. These results suggest that the membranous structure between enamel and ameloblasts of a rat incisor is not so-called enamel cuticle but a basal lamina produced by overlying ameloblasts and that the basal lamina contains collagenous components even though it lies on enamel.

Trimetaphosphatase (TMPase) and cytidine-5'-monophosphatase (CMPase) were localized to investigate the lysosomal system, particularly tubular lysosomes, in ruffle-ended ameloblasts associated with maturation of enamel in rat incisor. Demineralized specimens were incubated for TMPase and for CMPase in a modified medium where cerium was used as the capture ion. Ruffle-ended ameloblasts showed distal invaginations and membrane-bound bodies filled with fine granular material, some of which displayed CMPase reaction product. Elongated tubular configurations 80-140 nm wide were distributed throughout the cytoplasm and were reactive with both TMPase and CMPase, thus characterizing these structures as lysosomes. They often contained fine granular material morphologically similar to that present in multivesicular bodies. During late enamel maturation, fewer tubular lysosomes were observed when compared to early maturation. These cytochemical results demonstrate the presence of tubular lysosomes in ruffle-ended ameloblasts, and it is suggested that they are elements of the endosomal system in these cells. These findings are also consistent with a resorptive function for ruffle-ended ameloblasts during enamel maturation.

Ultrastructural changes of secreting rat-incisor ameloblasts after administration of vincristine and vinblastine were observed. Polarity of nuclei and cytoplasmic organellae was remarkably disturbed; consequently, the regular secreting orientation of cellular products was altered greatly, leading to heterotopic deposition of large amounts of enamel matrix.

Ruthenium red staining and tannic acid fixation were used to analyse the fine structure of embryonic mouse dental basement membrane in intact first mandibular molars or in EDTA-isolated dental papillae. Preameloblasts are separated from extracellular matrix proper by a basal lamina that contains regularly arranged proteoglycan granules of about 10 nm in diameter. This distribution pattern is particularly evident in the inner and outer lamina rara of the basal lamina associated with EDTA-isolated dental papillae. The plasmalemma of preameloblasts demonstrates electron dense plaques on the inner leaflet. Ruthenium red positive granules (50 nm in diameter) coat nonstriated and striated fibrils of the matrix. Hyaluronidase treatment digested the ruthenium red positive granules. Tannic acid fixation allowed the demonstration of filaments within the lamina rara interna, connecting the lamina densa with plasmalemma of preameloblasts. These observations are discussed in the context of the terminal differentiation of odontoblasts.

A three dimensional reconstruction of the epithelial tissue at the apical end of the lower rat incisor was made from serial 1 mum thick cross sections. This tissue formed an elongated structure, called the odontogenic organ, which was composed of a bulbous and a "U"-shaped part. Both parts were joined to one another at the posterior aspect of the apical foramen. The bulbous part of the odontogenic organ was situated at the lingual side of the "U"-shaped part and protruded anteriorly over the pulp. It was formed by cells of the outer dental epithelium and stellate reticulum whose organization suggested that the bulbous part was important in the production of cells for renewal of all the epithelia of the incisor. The "U"-shaped part of the odontogenic organ was apparently derived from the bulbous part and delineated the pulp by forming the lateral, mesial and labial sidewalls around the apical foramen. It was composed of all the epithelial cell types recognizable as precursors to (a) cells of the enamel organ which form the enamel, and (b) Hertwig's epithelial root sheath, a part of the odontogenic organ which induces the formation of dentin on the lingual aspect of the incisor.

Five cases of ameloblastic fibro-odontoma were examined by light and electron microscopy. The ultrastructure of the epithelio-mesenchymal interface was compared with the morphology of the normally developing tooth germ. It was noted that the ultrastructure of the tumors was identical with the normally developing odontogenic tissues up to but not including the differentiation of mesenchymal cells into tall columnar odontoblasts. The absence of odontoblasts in the ameloblastic fibro-odontomas results in an absence of tubular dentin in place of which a homogeneous collagen-containing material is synthesized. The failure of mesenchymal cells to acquire the morphology of tall columnar odontoblasts probably is due to the absence of the normally occurring induction of the mesenchyme by the odontogenic epithelium.

First molars from 2-day-old mice were cultured for 7 days in medium supplemented with 0.04, 0.4, 4.0 parts/10(6) selenium (Se) or 10 per cent calf serum. Collagen synthesis, measured after labelling with [3H]-proline for the last 24 h of culture, was most active at 0.4 parts/10(6) Se. Supplementation with 300 parts/10(6) Se for the first 24 h of culture caused ultrastructural changes in newly-deposited enamel and dentine matrices.

Specimens of aldehyde-fixed and glycerol-impregnated tooth germs obtained from 1-2 day old rats were prepared for ultrathin section studies and for freeze-fracture, with the purpose of studying the structural organization of membranes of developing ameloblasts. In this report we describe unusual membrane domains which were found surrounding several ameloblast gap junctions. Developing ameloblasts - when examined in ultrathin sections - exhibit gap junctions which appear straight, curved or invaginated. In freeze-fracture replicas, in addition to their typical appearance, several gap junctions were found to be surrounded by a membrane margin which was undulating and devoid of intramembrane particles (IMP's). We believe that these hitherto unreported particle-free membrane margins are associated with the formation of curved or invaginated gap junctions. It is possible that these membrane margins are particle-free because plasma membrane proteins (presumably IMP's) become transiently detached from the cytoskeleton and move laterally. It is therefore likely that these margins are pure lipid domains which are more flexible, thus providing a transient hinge-like mechanism which facilitates the movement required for the formation of the curved or invaginated ameloblast gap junctions.

We investigated the ultrastructural distribution of calcium in several kinds of hard tissue forming cells (secretory and maturation ameloblasts, odontoblasts osteoblasts, chondrocytes, and osteodentine forming cells) of mammals, amphibians, and fish by use of the potassium pyroantimonate technique. The calcium distribution pattern is compared among these cells, and its biological significance is discussed. Except for mammalian odontoblasts, all types of the hard tissue forming cells exhibited fundamentally the same distribution pattern of calcium; the antimonate reaction product was mainly localized on the inner face of the plasmalemma and inside mitochondria. On the other hand, in mammalian odontoblasts, the reaction product was found within secretory granules and in the intercellular spaces. Thus, the calcium distribution pattern in odontoblasts of lower vertebrates differed from that of mammalian odontoblasts and was similar to that of the osteoblasts or chondrocytes of the vertebrates examined. The differences in calcium distribution pattern among these hard tissue forming cells were not related to their origin, ectodermal or mesodermal (ectomesenchymal). We suggest on the basis of previous studies cited in this paper and of the present data that they are closely associated with the phylogeny and physiological system of Ca-ATPase.

Enamel secretion by ameloblasts was investigated in the incisors of 100 gm normal and colcemid-injected male rats. Morphological studies were done on rats given a single intraperitoneal injection of 0.1 mg (1.25 mM) of colcemid and sacrified 1 to 4 hours after injection. Protein synthesis and secretion were investigated with radioautography in normal and colcemid-treated rats injected with 3H-proline and sacrificed at intervals between 0.5 and 3.5 hours after injection. Colcemid was injected 0.5 hours prior to 3H-proline in each experimental rat. Electron microscopic examination revealed several morphological alterations between 1 and 4 hours after injection of colcemid. These changes included fragmentation of the normally elongated rough endoplasmic reticulum into shorter profiles; a disorganization of the normally tubular configuration of the Golgi apparatus into a number of seples and profiles of smooth endoplasmic reticulum from Tomes' processes; and the accumulation of secretion granules at the mature face of the Golgi stacks, as well as in the infranuclear cytoplasm where thye are normally not found. Radioautography revealed that protein synthesis by the rough endoplasmic reticulum had continued in colcemid-altered ameloblasts. Labeled secretion granules were found at the mature surface of the Golgi stacks and in the infranuclear cytoplasm, however they did not migrate into Tomes' processes. Consequently, labeled enamel matrix did not appear extracellularly at the same time as in normal controls. Quantitative radioautography in the light microscope revealed that the effect of colcemid, although reversed within 4 hours, had temporarily inhibited normal migration, and exocytosis of secretion granules.

The distribution of calcium in relation to secretory ameloblasts of the rat incisor was studied. An experimental model system in which enamel mineralization was temporarily inhibited by injecting sodium fluoride and cobalt chloride was used. Potassium pyroantimonate (PPA) cytochemistry, electron energy loss spectroscopy (EELS), and energy dispersive X-ray spectrometry (EDS) were used to clarify the role of the ameloblast in controlling calcium distribution during normal and experimentally altered enamel mineralization. Secretory ameloblasts chemically-preserved in glutaraldehyde either with or without PPA were analyzed for calcium; those preserved with PPA showed higher concentrations of calcium than did those preserved with glutaraldehyde only. Freeze-dried control and experimental tissues showed an increasing gradient of calcium from stratum intermedium cells to the distal ends of the ameloblasts. Calcium levels were reduced near the distal ends of the cells following fluoride and cobalt injections, while magnesium levels were increased markedly in the same region. This multi-method approach showed correlated calcium localization in specific regions of this cell in relation to changes in function. The study thus provides additional evidence for active involvement of the ameloblasts in enamel mineralization.

The effect of a single injection of sodium fluoride (60 mg/kg) on the development of rat molar enamel beneath fluoride-induced subameloblastic cysts was studied by transmission electron microscopy using undecalcified sections. Three bands of altered enamel were identified and defined as the cyst surface band, the hypoplastic band, and the hypercalcified band. The irregular cyst surface band, not previously described, was found to have two components: electron-dense enamel globules and organic spherules. The electron-dense globules consisted of small, randomly arranged crystals (confirmed by selected area electron diffraction) occurring within a stippled organic matrix. The organic spherules have staining properties similar to stippled material and lack a crystalline component. They may be a form of organic material being extruded from the underlying developing enamel. The critical role of normal matrix production and ameloblast Tomes' process structure on the development of the crystal orientation and rod pattern is discussed.

Differentiation of preodontoblasts to odontoblasts and preameloblasts to ameloblasts during development of the mouse mandibular incisor proceeds in a gradient from the area of the odontogenic organ, where undifferentiated ectomesenchymal and epithelial cells proliferate, toward the incisal tip where mature tooth tissues, dentin and enamel, are present. The freeze-fracture technique has been used in the work presented here to study cell membrane ultrastructure of preodontoblasts and preameloblasts at several stages of differentiation. At early stages of differentiation, cuboidal preameloblasts are joined together distally by numerous gap junctions. Relatively fewer junctions occur elsewhere on the lateral plasma membranes, but gap junctions frequently occur proximally between preameloblasts and stratum intermedium cells. As differentiation proceeds and the cells become columnar, distal and proximal junctions persist. Tight junctions, however, were not observed at any of the stages studied. Intramembrane particle concentration of the lateral preameloblast plasmalemma appears to increase as differentiation proceeds. Odontoblasts are also joined distally by numerous gap junctions which persists through later stages of differentiation. Although odontoblast cell processes were observed to project toward the preameloblast layer, no clear points of cell to cell contact or defined intercellular junctions between the two cell types were observed.

The incorporation of 3H-proline into the secretory ameloblasts of rat molar tooth germs cultured with or without colchicine was studied by light and electron microscope radioautography to determine the function of microtubules in the transport of precursor enamel protein from the rough-surfaced endoplasmic reticulum (rER) to the Golgi cisternae. The grain counts over the transitional vesicles, which accumulated in various cellular regions with colchicine treatment, continued to increase with chase time, unlike in controls. At 30 and 90 min chase, these counts were significantly higher than in controls. Moreover, the total grain count over the organelles (rER, pale granules, and transitional vesicles), which are positioned before the Golgi cisternae in the synthetic pathway, maintained a significantly higher level at 90 min chase in colchicine-treated tooth germs than in controls. The transport of synthesized protein to the Golgi cisternae via transitional vesicles was suppressed in colchicine-treated tooth germs. Some grains appeared with time over pale granular materials that appeared in the intercellular spaces of secretory ameloblasts with colchicine treatment. However, at each chase period, the grain count over pale granular materials was not so high as the count over the enamel in control. The present results indicate that colchicine affects the transport of newly synthesized protein from the rER to the Golgi cisterna via transitional vesicles, probably by interfering with the oriented transport related to microtubular function. It is suggested that the microtubular system may be concerned with the movement of the transitional vesicles.

The distribution of actin filaments in the inner enamel-secretory ameloblast of the rat incisor was examined by labelling with heavy meromyosin. Actin filaments were mainly located in the long axis of the cell beneath the cell membrane. They tended to be more numerous in Tomes' process than in the cell body. Some filaments were associated with microtubules, with other actin filaments, and coated vesicles. Possible relation of the actin filaments to cell motility, stabilization of some, and movement of other cellular components is discussed.

In order to clarify the morphology and function of maturation ameloblasts, kitten tooth germs were examined using an ultrastructural tracer technique. Kitten maturation ameloblasts were divided into two cell types: a ruffle-ended type showing plasma membrane infoldings and invaginations at the distal cell surface and a smooth-ended type showing a non-invaginated, smooth distal cell surface. Both ameloblasts possessed two sets of junctional complexes at their proximal and distal ends. In either type of cell, intravenously injected horseradish peroxidase penetrated from the vascular region into the developing enamel surface through both the proximal and distal junctional complexes, although the tight junction compartment inhibited horseradish peroxidase permeation. Ruffle-ended ameloblasts further incorporated peroxidase from the distal cell surface into the cytoplasm by means of membrane invaginations and coated vesicles. Smooth-ended ameloblasts showed little intracellular peroxidase incorporation. These results show clearly that resorptive and non-resorptive maturation ameloblasts exist in kitten enamel organ and that the two cell types correspond to ruffle-ended and smooth-ended maturation ameloblasts in rat incisors.

Glutaraldehyde-fixed rat incisors were either post-fixed in ferrocyanide-reduced osmium impregnated with the Ur-Pb-Cu technique or incubated in the medium for acid-phosphatase (AcPase) reaction. The Golgi apparatus of the secretory ameloblast was composed of 4-7 cisternae, small vesicles and condensing vacuoles. It formed an elongated, continuous membrane system over from one- to two-thirds of the supranuclear cytoplasm. Condensing vacuoles seemed to be produced from the dilated margins of both Golgi cisternae and GERL. AcPase activity was demonstrated in the inner 2 or 3 Golgi cisternae, in GERL and in some condensing vacuoles and secretory granules within the Tomes process. It thus seemed that primary lysosomes originate from both the Golgi apparatus and GERL. Whereas many lysosomal bodies appeared in the supranuclear cytoplasm, autophagic vacuoles were rare. A well-developed Golgi apparatus and GERL were, therefore, considered to be involved in the digestion of exogeneous materials as well as in the formation of the precursor of enamel matrix. The simple Golgi apparatus consisting of only compactly stacked cisternae and small vesicles, were sometimes observed in the supranuclear cytoplasm and having no clear relationship with rough-surfaced endoplasmic reticula or GERL, may serve as a source of the plasma membranes necessary for continuous renewal of the cisternae of the well-developed Golgi apparatus.

The distribution of approaches of rough endoplasmic reticulum to the cell membrane within the supranuclear region of the secretory ameloblast of the rat incisor was quantitated using a Zeiss MOP-3. In ameloblasts cut in cross section, most of these approaches appear as circular profiles representing cross sections of elongated cisternae, which are aligned parallel to the long axis of the cell. Because of their position, orientation, and distribution of ribosomes, these approaches were consistent with the appearance of subsurface cisternae. Using cross-sectioned ameloblasts, the lengths of apposed plasma membranes either between or within rows of cells were measured from electron micrographs. Along these lengths, matched approaches of rough endoplasmic reticulum from opposite sides of the apposed plasma membranes were counted. Approaches from either side that were unmatched were also counted. Thirteen percent of the approaches were matched between rows of ameloblasts, and 13.5% of the approaches were matched within rows, demonstrating no significant difference between the two sites. Furthermore, mathematical analysis showed that the theoretical probability of two approaches coinciding is 17%. The experimental values are not statistically different from the theoretical probability, and it is concluded that the matching of rough endoplasmic reticulum approaches to the plasma membrane, or subsurface cisternae, occurs at random.

Clinical, radiologic, and histologic studies were performed in eight cases of ameloblastoma in patients younger than 16 years of age. The mean age of the patients was 10.8 years, equal numbers of males and females were affected, and the most common location was the lower molar and ramus areas. Radiologically, a monocystic osteolytic image was observed in all cases. Histologically, two forms of growth were observed: an intramural, infiltrating, plexiform or follicular type, and a polypoid type, prolapsing toward the lumen of a cystic cavity. The ameloblastomas recurred in three cases a year after surgery.

The fine structure of internal enamel epithelium, preameloblasts and secretory ameloblasts in primary tooth germs (bell stage) from four human foetuses was investigated. The characteristics of the differentiation of internal enamel epithelium via preameloblasts to secretory ameloblasts are described. The internal enamel epithelium consists of a row of low differentiated prismatic cells separated from the dental papilla by a distinct even basal lamina. In the preameloblasts the rough endoplasmic reticulum cisterns and mitochondria increase in number, the Golgi complexes become extensive and take up a distal position, and secretory granules are formed. Furthermore, the basal lamina is removed by coated vesicles, and proximally and distally in the cells a complex of zonulae adhaerentes, terminal webs and gap junctions is formed. The secretory ameloblasts make up a layer of highly differentiated cells demonstrating typical merocrine secretion.

Differentiation of the cells of the dental bud which produce the hard tissues of teeth was studied at the ultrastructural level; the activity of these cells (odontoblasts and ameloblasts), the laying down of organic matrices elaborated by them, the mineralization of these matrices, and the maturation of dentine and enamel are described. Injections of tritiated proline enabled us by the autoradiographic method, to confirm and to indicate more precisely the activity of these cells. Dental calcified tissues of Rana exhibit the general properties of these tissues among Vertebrates: high content of apatite, low content of organic materials (collagenous in dentine, non-collagenous in enamel), precise orientation of crystals, and true but non-prismatic enamel. The enamel of Anura as well as enamel of Urodela is a true enamel of epithelial origin.

Human fetal primary tooth germs in the cap stage were fixed with a glutaraldehyde-formaldehyde mixture, and formative processes of tight and gap junctions of the inner enamel epithelium and preameloblasts were examined by means of freeze-fracture replication. Chains of small clusters of particles on the plasma membrane P-face of the inner enamel epithelium and preameloblasts were the initial sign of tight junction formation. After arranging themselves in discontinuous, linear arrays in association with preexisting or forming gap junctions, these particles later began revealing smooth, continuous tight junctional strands on the plasma membrane P-face and corresponding shallow grooves of a similar pattern on the E-face. Although they exhibited evident meshwork structures of various extents at both the proximal and distal ends of cell bodies, they formed no zonulae occludentes. Small assemblies of particles resembling gap junctions were noted at points of cross linkage of tight junctional strands; but large, mature gap junctions no longer continued into the tight junction meshwork structure. Gap junctions first appeared as very small particle clusters on the plasma membrane P-face of the inner enamel epithelium. Later two types of gap junctions were recognized: one consisted of quite densely aggregated particles with occasional particle-free areas, and the other consisted of relatively loosely aggregated particles with particle-free areas and aisles. Gap junction maturation seemed to consist in an increase of particle numbers. Fusion of gap junctions in the forming stage too was recognized. The results of this investigation suggest that, from an early stage in their development, human fetal ameloblasts possess highly differentiated cell-to-cell interrelations.

The epithelial tissue forming the posterior aspect of the apical foramen in the upper incisor of the rat was reconstructed from 1 mum thick serial cross sections. Like the lower incisor, this portion of the odontogenic organ in the upper incisor was composed of a bulbous and a "U"-shaped part. However, the bulbous part was considerably blunter and the "U"-shaped part much larger in circumference in comparison to the lower incisor. Although no differences were found between the upper and lower incisor regarding the contents and the basic organization of cells within each part of the odontogenic organ, specific differences were found within the bulbous part in the upper incisor. There was a more definitive boundary between the outer dental epithelium and stellate reticulum, a more intimate relationship of cell streams to the stellate reticulum, and a noticeable lack of swirling of cells as part of the streams. These features suggest that the activity inside the bulbous part is less intense in the upper incisor than it is in the lower incisor. In addition, the relationships between the bulbous part, the "U"-shaped part and the root sheath part of the odontogenic organ and the enamel organ were described for the upper incisor.

Using horseradish peroxidase (HRP) as a soluble protein tracer, electron microscopic studies were carried out in order to analyze endocytosis in the ruffle-ended ameloblasts of rat incisors. Accumulated HRP was initially incorporated from the ruffled border into the cytoplasm by means of pinocytic vacuoles ( pinosomes ) and pinocytotic coated vesicles. The majority of the HRP was taken up by the large number of pinosomes , which then formed large endocytotic vacuoles by fusing either with each other or with preexisting endocytotic vacuoles. As time passed HRP accumulated, not in the pinosomes and ruffled border but in the endocytotic vacuoles and multivesicular bodies. Frequent connections between HRP-labeled coated vesicles and these cytoplasmic bodies indicate that these vesicles serve as an HRP carrier. These findings strongly suggest that ruffle-ended ameloblasts actively absorb soluble proteins from the enamel matrix during enamel maturation.

The molar tooth germs of newborn mice were isolated and the zone of ameloblast differentiation studied with the electron microscope. Special attention was paid to the changes that occur in the contacts such as desmosomes, tight junctions and close attachments associated with cell interdigitations between the inner enamel epithelium (IEE) and the stratum intermedium (SI) cells from the beginning of differentiation to the onset of enamel secretion. These changes are accompanied by variations in the width and configuration of the intercellular space separating the two cell layers. The terminal web persists from the beginning of cell elongation to the appearance of Tomes's process. Through its attachment to the zonulae adherentes, a terminal web-lateral junction system is created which stabilizes the differentiating ameloblasts. Also the SI cell layer is interpreted as a stabilizer for the IEE layer, particularly during the differentiation phase. This interpretation does not conflict with information that the SI cells transport materials required for enamel secretion.

Calcium was not found in secretory ameloblasts and stratum intermedium cells when treated with OsO4-pyroantimonate or when surfaces prepared by fracturing fresh, rapidly frozen, developing molar tooth germs were subject to electron probe X-ray analysis. Pyroantimonate reaction product, considered to be calcium, was found in mitochondria of enamel organ cells which were first placed in a bath containing calcium and potassium. The plasma membrane was disrupted in cells ehich showed mitochondrial localization of reaction product. The results provide no data which indicates that enamel organ cells have a direct, active role in the movement of calcium into the enamel. Rather, it is suggested that the secretory enamel organ might serve as a selective barrier in regulating the initial mineralization of enamel.

Adult rats received a single dose of HRP intravenously and were killed from 10 min to 6 hr after injection. Following fixation with glutaraldehyde, the enamel organs were treated with a Graham-Karnovsky-type procedure for peroxidase activity, post-osmicated, and embedded in plastic. Sections were studied with light and electron microscopes. Ten minutes after injection, reaction product was found in all extracellular spaces of the enamel organ, at the enamel-ameloblast interface over smooth-ended and intermediate ameloblasts, and in apical surface invaginations and vesicles of the latter cell types. The enamel-ameloblast interface over the ruffle-ended aemloblasts and the extracellular spaces within the ruffled border were free of reaction product and remained so for up to 6 hr. The apical terminal bars of the ruffle-ended ameloblasts functioned as a barrier to HRP. The basal terminal bars of the smooth-ended ameloblasts likewise seemed to prevent the passage of the HRP. Possibly, HRP flows in a lateral direction from groups of ruffle-ended into groups of smooth-ended ameloblasts. Between 10 min and 6 hr, HRP was cleared more rapidly from the extracellular spaces of the papillary layer than from those of the ameloblast layer, and there was little backflow of tracer from the ameloblast into the papillary layer. Eventually, tracer was cleared also from the extracellular spaces of the ameloblast layer, probably mainly through micropinocytosis by the ameloblasts. A working model is proposed regarding the handling of large molecules by the enamel organ in the maturation zone.

Basement membranes generally appear in transmission electron microscopy as thin, cross-sectioned profiles. Face-views are difficult to obtain particularly when their contours are curved or undulated. Such preparations are suited for the study of the distribution of basement membrane constituents through thickness but are limited for examining their spatial distribution. Scanning electron microscopy offers the potential of visualizing surface views. We have applied this approach to examining the three-dimensional distribution of glycoconjugates in a basement membrane. Large, face-views of the basal lamina related to maturation ameloblasts in the region where enamel of the rat incisor is EDTA soluble were exposed. Glycoconjugates were then revealed by incubating the segments in lectin-gold complexes and binding sites were visualized by backscattered electron imaging. This basal lamina was found to contain fucose, mannose and to be extremely rich in N-acetyl-D-galactosamine. The abundance of glycoconjugates containing the latter sugar residue may impart on this basal lamina selective properties which could regulate the movement of material into, and out of, the enamel layer.

Two cases of adenomatoid odontogenic tumor were examined by light and electron microscopy. Morphologically the tumors could be divided into four layers. The ultrastructure of the tumor cells of each layer was revealed to be similar respectively to that of four layers seen in the enamel organ of a normal tooth germ. Duct-like structures and eosinophilic small areas were frequently present in compactly proliferating cell layers. Short columnar cells forming duct-like structures and eosinophilic small areas appeared similar ultrastructurally to ameloblasts during predentin formation. Therefore it seems reasonable to assume that this tumor originates from the enamel organ. The contents in the lumen of each eosinophilic small area varied, and seemed to be secreted into the stroma by circumscribing epithelial tumor cells. A fine filamentous layer was present in both of the duct-like structures and some of the eosinophilic small areas, and the former was different from the latter with respect to the alcian blue (pH 2.5) and toluidine blue (pH 4.4) staining reactions.

Cytodifferentiation of inner enamel epithelium and the adjacent connective tissue from the tip of the cervical loop to the initiation of enamel elaboration in two Macaca species was examined. Ten- to twelve-month-old specimens were fixed by perfusion and the permanent tooth buds were prepared for transmission electron microscopy. At the cervical loop proper, inner enamel epithelium cells have lobed nuclei, a paucity of cytoplasm, and wide extracellular spaces; the basal lamina facing the dental papilla is straight. With increasing distance from the tip of the cervical loop, the following changes occur gradually: (a) preameloblasts elongate from 15 to 45 micrometers, and their organelles, particularly mitochondria and profiles of rough endoplasmic reticulum, become more numerous; (b) extracellular spaces decrease between preameloblasts starting at the basal (infranuclear) end; (c) the basement membrane becomes convoluted and associated with aperiodic fibers; (d) preodontoblast projections penetrate the aperiodic fibers; (e) collagen fibers subjacent to the basement membrane increase in density, with particularly thick fibers paralleling the aperiodic fibers. These modifications occur within three-fourths of the distance from the tip of the cervical loop to the mineralization front. The condensation of preodontoblasts is followed immediately by predentin synthesis. Concomitantly, the basement membrane breaks down and the aperiodic fibers are engulfed by preameloblasts. Preameloblast projections penetrate junctional predentin, contact mineralized dentin, and enamel synthesis ensues. At this stage the ameloblast is 45 micrometers long, the nucleus is central or basal, the Golgi apparatus has migrated apically, but the Tomes' process has not yet formed. The results indicate that odontogenesis in Macaca monkeys more closely resembles human odontogenesis than does that in the murine rodents.

Tetracycline was administered to rats in varying doses in order to study the likelihood of ultrastructural alterations to ameloblasts and to study odontoblasts and dentin as well. The changes caused by tetracycline to the mineralization of dentin and the ultrastructural changes to the odontoblasts and ameloblasts increased with larger doses.

Fluoride given as a high single dose to young rats with developing molars has earlier been shown to cause subameloblastic cysts with disorganized ameloblasts in the cystic wall and an irregular mineralization pattern of the underlying enamel. In the present study immunohistochemistry has been employed to determine if an increased permeability of the enamel organ occurred at the areas of cell disturbances. For this purpose 5-day-old rats were injected with 60 mg sodium fluoride per kg body weight. They were decapitated after 24 h, the maxilla prepared histologically and paraffin sections incubated for the demonstration of IgG according to the avidin-biotin-peroxidase complex method. Staining for IgG was present in the cystic lumina and in areas of disorganized ameloblasts. No reaction was observed in areas of unaffected ameloblasts. It was suggested that the fluoride-induced cell injury increased the permeability of the ameloblastic cell layer. Diffusion of IgG and most likely other substances as well, through the ameloblastic layer may have contributed to cyst formation and to the irregular mineral deposits that have been found in the ameloblastic layer and at the enamel surface.