Effects of zinc deficiency on the distribution of membrane-coating granules in rat buccal epithelium.

Nutritional zinc deficiency causes consistent excessive cell proliferation in the epithelium of the buccal mucosa. The number per cell and the intracellular location of membrane-coating granules in this epithelium were investigated in male rats placed at weanling age for a 4-week period on a diet containing 1.2 ppm of Zn and in their pair fed controls. Membrane-coating granules were identified on electron micrographs following their demonstration in thin sections by the use of an alkaline bismuth technique. Counts of membrane-coating granules in the first 4 rows of spinous cells and the last 4 rows of granular cells showed that in the zinc-deficient group (1) the total number of granules per cell was increased; (2) the proportion of granules displaced to the cell periphery was decreased in favor of a higher proportion persisting in the center and (3) there was a marked reduction in number and proportion of granules positioned near the superficial cell membrane. The greater uniformity in the distribution of the granules in the hyperplastic-hypertrophic epithelium of the zinc deficient group suggests weakening of the surface-oriented polarity characteristic of the control tissue.

Cytochemical localization of calcium and Ca2+,Mg2(+)-adenosine triphosphatase in colchicine-altered rat incisor ameloblasts.

Colchicine is known to affect secretory, transport, and degradative functions of ameloblasts. The effects of colchicine on membrane-associated calcium and Ca2+,Mg2(+)-ATPase in secretory and maturation ameloblasts were investigated cytochemically. The pyroantimonate (PPA) method was used for localizing calcium and a modified Wachstein-Meisel medium was used to localize Ca2+,Mg2(+)-ATPase. Sections representing secretory and early maturation stages were examined by transmission electron microscopy. Morphological changes induced by colchicine included dislocated organelles and other well-established reactions to such anti-microtubule drugs. Calcium pyroantimonate (Ca-PA) deposits in most ameloblast types were markedly reduced, with the greater reduction occurring in those cells more severely altered morphologically. However, the cell membranes of both control and experimental smooth-ended maturation ameloblasts were essentially devoid of Ca-PA. The normal distribution and intensity of Ca2+,Mg2(+)-ATPase was not affected by colchicine. Because the observed reduction of membrane-associated calcium is apparently not mediated by Ca2+,Mg2(+)-ATPase in this case, other aspects of the calcium regulating system of ameloblasts are apparently targeted by colchicine.

Expression of plasma membrane Ca++ pump epitopes parallels the progression of enamel and dentin mineralization in rat incisor.

We investigated the expression of Ca++ pump epitopes during enamel and dentin mineralization in the rat incisor. Secretory and maturation ameloblasts were studied as well as odontoblasts, using a monoclonal antibody (5F10) against human erythrocyte plasma membrane Ca++, Mg(++)-ATPase. A progressive increase in staining intensity in ameloblasts and the odontoblasts was observed beginning with the onset of mineralization. The mainly membrane-related labeling of ameloblasts showed variable intensity depending on the stage of enamel formation, whereas that of the odontoblasts showed even intensity during continued dentinogenesis. Staining of papillary cells was evident only during enamel maturation. Western blot analysis of freeze-dried ameloblasts was also used to determine the molecular weight of the Ca++ pump epitopes as well as the distribution and relative concentration of epitopes at each stage. An immunoreactive band of MW 140 KD and lower molecular weight bands that are more intense in late than in early maturation were demonstrated. Our studies suggest that the expression of plasma membrane Ca++ pump parallels the progression of mineralization in rat incisor enamel and dentin.

Comparison of scanning and transmission electron microscopy of the epithelial pocket wall in juvenile and adult periodontitis.

Using scanning (SEM) and transmission (TEM) electron microscopy, this study compared fine structural features of the pocket walls in both juvenile and adult periodontitis (JP and AP, respectively) in 40 cases. Gingiva was also obtained from a control group consisting of periodontally noninvolved teeth. Clinical parameters were assessed in both JP and AP patients as well as in controls. Clinical findings showed low plaque accumulation, marked periodontal tissue destruction and less gingival inflammation in JP. Bone destruction and attachment loss were more marked in JP than in AP. AP had a higher plaque index and more evident gingival inflammation. SEM observations of JP as compared to AP showed gross distortions in pocket walls, an increased beaded appearance of microridges, and separation between pocket epithelial cells. TEM showed partially desquamated and separated superficial epithelial cells, but only in JP were fine granular precipitates observed in the intercellular spaces. The observations demonstrated structural features indicative of more prominent degenerative changes in JP than in AP. Also, these features were coincidental with a higher plaque index in AP than in JP, where clinical features (including a low plaque index) were not proportional to the epithelial destructive changes present.

Elemental and cytochemical localization of calcium in rat cheek epithelium.

Energy-dispersive X-ray microanalysis, combined with scanning electron microscopy, was applied to 18 microns thick freeze-dried cryosections to determine the level of calcium in successive layers of the epithelium. This indicated low levels of calcium in basal cells, high in spinous cells, moderate in granular cells, lowest in the inner keratin and highest in the outer keratin layers. The potassium pyroantimonate technique was used to study the cytochemical distribution of Ca2+. The reaction product, calcium pyroantimonate (Ca-PA), was generated from cellular and intercellular calcium by perfusion of the anaesthetized rat with a solution of potassium pyroantimonate in glutaraldehyde. Ca-PA was localized in nuclei, mitochondria, lysosomal-type bodies and membrane-coating granules. A denser Ca-PA distribution was found between upper spinous and lower granular cells. The proposed identification of Ca2+ as a major component in Ca-PA was confirmed by EDTA decalcification and X-ray microprobe analysis of the reaction product. Thus X-ray microanalysis in combination with cytochemistry can localize Ca2+ in a growing and differentiating tissue such as stratified squamous epithelium.

Time-related changes induced by zinc-deficient diet in the concentration of rat cheek epithelial membrane-coating granules.

Weanling male Sprague-Dawley rats were fed a diet containing 0.4 parts/10(6) zinc and controls were fed an identical diet supplemented with 40 parts/10(6) zinc. After 9, 18 and 27 days of zinc deficiency, specimens were excised from cheek epithelium and processed for transmission electron microscopy to study the concentration of membrane-coating granules (MCG). Their concentration was increased in the granular-cell layers of the zinc-deficient epithelium and became significantly greater after 18 and 27 days than 9 days of deficiency. MCGs appeared in the parakeratinized layers of zinc-deficient epithelium and their concentration became significantly greater after 27 days in comparison with 9 and 18 days of deficiency. Thus the intracellular retention of MCGs was increased in the granular and parakeratinized layers with the increase in time of zinc deficiency.

Microradiographic and electron microscopic observations of the enamel-covered dentine in rat molars.

The enamel-covered primary and secondary coronal dentine in the molars of 90-day-old and 360-day-old rats was examined using microradiography. Some preparations were subsequently examined by transmission and scanning electron microscopy (SEM). Fractured dentine surfaces and methacrylate casts of the tubular system in the primary dentine were also examined with SEM. No microradiographic evidence of a hypermineralized peritubular matrix, such as that seen in man and other species, was seen in either young or old rats. Transmission and SEM confirmed the microradiographic findings. The tubule obliteration and extensive intra-luminal mineral deposits which have been reported in the enamel-free coronal dentine of the rat molar were not seen in the enamel-covered coronal dentine but some evidence of tubule obliteration was seen in the secondary dentine.

Morphological variations and population density of membrane-coating granules in human gingival sulcular epithelium.

Surgically excised specimens of sulcular wall with minimal inflammatory response as judged by clinical then histological criteria were processed for electron microscopy. The specimens were divided into crestal, middle and cervical areas of the sulcular epithelium. The highest concentration of membrane-coating granules was found in the upper spinous cell layers of sulcular epithelium. The profiles of these granules showed examples of both classical keratinized (lamellated) and non-keratinized (non-lamellated) forms but also other appearances that were not derived from them through differences in the plane of section. The population of granules decreased between the crestal and cervical zones, and the decrease in number was marked for the lamellated granules. This decrease in numbers of membrane-coating granules, together with the wider intercellular spaces, may be the reason why the sulcular epithelium is most permeable in the cervical region.

Smokeless tobacco-induced lamellar body abnormalities.

OBJECTIVES: To compare the morphological changes and quantitative distribution of lamellar bodies (Lb) (membrane coating granules) in the hamster cheek pouch epithelium with smokeless tobacco (ST). MATERIALS AND METHODS: Archives of experimental material from previously published studies [S. Ashrafi, A. Das, R. Worawongvasu, B. Mehdinejad and J. Waterhouse (1992) Scanning Microscopy6: 183] were utilized. Animals in experimental group received most ST (snuff) in their right pouch, 5 days weekly, for 24 months, while no snuff was given to control group. After 24 months, the epithelial tissues were processed for electron microscopic study. Volume densities of Lb were assessed by morphometry. MAIN OUTCOME MEASURES: Densities of Lb in the two groups, experimental vs control. RESULTS: In the control, Lb extruded their contents into the intercellular spaces of upper granular layers and in between the last granular cell layers and keratin layers to form a permeability barrier. Conversely, in the smokeless tobacco-treated epithelium, the majority of the Lb that were formed remained inside and accumulated within the granular cells, without extruding their contents into the intercellular spaces to form a lipid compound permeability barrier. CONCLUSIONS: Commercial alkaline ST may have contributed to the abnormal accumulation of Lb in the granular cell layer and affected the extrusion process of Lb to form an incomplete permeability barrier in the oral epithelium.

Comparative light, scanning, and transmission electron microscopic study of chemically induced premalignant lesions in hamster's cheek pouch.

The early chemically induced epithelial dysplastic changes in the hamster's cheek pouch were studied using light, scanning, and transmission electron microscopy. Epithelial dysplasia was noticed on the light microscopic level by the sixth week of the experiment and became marked by the eighth week. At the scanning electron microscopic level, surface alterations with features previously reported in early epithelial dysplasis in human and oral mucosa were seen by the second week of the experiment and progressed over time. These early changes were also confirmed at the ultrastructural level. The usefulness of scanning and transmission microscopy in detecting early oral epithelial dysplastic changes in this animal model is discussed.

Structure of Retzius lines in partially demineralized human enamel.

Human third molars were partially demineralized in an acid-alcohol solution and embedded in Epon 812. Six-micron sections were cut from regions of the cervical enamel exhibiting prominent Retzius lines. The plastic was removed from the specimens by microincineration and were examined with the scanning electron microscope. The most prominent structural feature seen along the Retzius lines was the cervical translocation of some of the prisms. The scanning electron microscopic images also suggested that prisms were translocating in the transverse plane of the tooth. A series of pores, which appeared to be empty, were observed in association with the translocations occurring along the Retzius lines.

Secretory ameloblasts and calcium distribution during normal and experimentally altered mineralization.

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.

Surface ultrastructural changes in chemically induced premalignant lesions in the hamster cheek pouch.

The histopathological and topographical alterations occurring during the early stages of chemical carcinogenesis in the hamster cheek pouch using 7,12-dimethylbenz (alpha) anthracene (DMBA) were studied by light and scanning electron microscopy. At the scanning electron microscopic level, cellular overlapping and mild disturbance of intercellular bridges were noticed as early as the second week of DMBA application; these changes progressed with time. Clear epithelial dysplastic changes at the light microscopic level were detected by the sixth week of the experiment. The results of this investigation demonstrate the usefulness of scanning electron microscopy as an adjunct tool to study early alterations in cell morphology which occur in the stratified squamous epithelium of the hamster cheek pounch in response to a chemical carcinogen.

Calcium distribution in freeze-dried enamel organ tissue during normal and altered enamel mineralization.

Energy dispersive X-ray microanalysis was applied to freeze-dried blocks of enamel organ tissue to determine levels of calcium in various cellular regions. The tissue blocks were dissected free from adjacent forming enamel following injection of cobalt or fluoride ions, both of which temporarily inhibit enamel mineralization. In all control and experimental specimens there was an increasing gradient of calcium from the stratum intermedium cells to the distal ends of the ameloblasts. Calcium levels were significantly reduced near the distal ends of the ameloblasts following cobalt or fluoride injection as compared with controls. It is suggested that evidence of an intercellular buildup of calcium near the distal ends of the ameloblast supports a controlling function of these cells. The changes in calcium levels are correlated with alterations in mineralization known to occur in the adjacent enamel of the model systems employed.