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.

Mitochondrial volume densities in the smokeless tobacco-treated hamster cheek pouch epithelium.

OBJECTIVES: To compare the morphological changes and quantitative distribution of mitochondria in the hamster cheek pouch (HCP) epithelium treated with smokeless tobacco (ST). MATERIALS AND METHODS: Archives of experimental material from previously published studies (Ashrafi et al., 1992) were utilized. Animals in experimental group received moist 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 microscopy study. Volume densities of mitochondria were assessed by morphometry. MAIN OUTCOME MEASURES: Mitochondrial volume densities in the two groups, experimental vs control. RESULTS: In both control and experimental groups mitochondria were concentrated between the nucleus and basal cell plasma membrane. A decrease in the mean mitochondrial volume density (Vvmit) was observed from the basal layer to the more superficial layers in both groups. The experimental HCP displayed more mitochondria than control, and the granular epithelial cell layer in experimental group showed significantly a higher mean Vvmit than the control group (P = 0.03). It was concluded that greater numbers of mitochondria were retained in ST-treated granular cells of the hyperplastic epithelia than in the normal epithelium.

Zinc deficiency produces time-related ultrastructural changes in rat cheek epithelium.

The subject of this study was whether the ultrastructural changes in cheek epithelium of zinc-deficient rats are time related. Weanling male Sprague Dawley rats were fed a zinc-deficient diet containing 0.4 ppm zinc (ZD) ad libitum and controls were pair-fed zinc adequate diet containing 40 ppm zinc. After 9, 18, and 27 days of zinc deficiency, specimens from cheek epithelium of both groups were processed for transmission electron microscopy. Partial conversion of the orthokeratinized cheek epithelium to parakeratinized was seen as early 9 days. An electron-lucent band surrounding the nucleus was observed in ZD cells. Mitochondria, tonofilaments, keratohyalin granules and ribosomes seemed to be increased with the increase in time of zinc deficiency. There was a thickening of the stratum corneum as well as hyperplasia and widening of the intercellular spaces of the spinous layer cells. Retention of a few membrane coating granules (MCGs) in the parakeratinized layer was seen after 9 days. Parakeratinization was further increased after 18 days of zinc deficiency, and the number of MCG profiles also increased. The epithelium was fully parakeratinized following 27 days of zinc deficiency, and the number of MCG profiles was increased. It was concluded that zinc deficiency affected cell proliferation and differentiation of the epithelium as early as 9 days, and caused a delay in loss of nuclei and MCGs in parakeratinized cells.

Scanning electron microscopy of cyclosporine-induced gingival overgrowth.

Overgrown human gingival specimens were examined histologically and by scanning electron microscopy (SEM) to study structural changes caused by cyclosporine. The biopsy specimens were from organ transplant recipients receiving cyclosporine to suppress the rejection of the transplanted organ. The epithelium of the overgrown gingiva was thickened, acanthotic and parakeratotic. Retepegs were anastomosing and extending into connective tissue. The SEM examination of the outer surface of the attached gingival showed loss of cellular attachments and cells were exfoliating. The normal honeycomb structure formed by interconnecting microvilli surrounding the pits was distorted. Outer gingival cell surface showed numerous round, ovoid and dome-like structures instead of parallel, reticular or fingerprint-like microridges. It was concluded that cyclosporine not only caused hyperplasia but also changed the structure of the outer epithelial cell surface.

In situ hybridization and monoclonal antibody analysis of plasma membrane Ca-pump mRNA and protein in submandibular glands of rabbit, rat and man.

The degree of supersaturation of saliva with calcium (Ca) is related to the mineral phase of enamel in erupted teeth, the incidence of caries, and the formation of calculus. The mechanisms for regulating salivary Ca concentration are therefore of relevance to dentistry. Sections of rabbit, rat and human submandibular gland (SMG) were processed for immuno-histochemistry with a specific anti-plasma membrane Ca-pump antibody, 5F10. Western blots confirm that the molecular weight of the proteins identified by our antibody (135 kDa) is consistent with an appropriate molecular weight for PMCA antigen (135-150 kDa). Tissue sections were also processed for in situ hybridization to study the distribution of the PMCA mRNA isoforms. In mammals, the PMCA1 gene is reported to code for a PMCA protein with a role in maintaining the intracellular Ca levels in both epithelial and non-epithelial cells. Other genes including the PMCA2 and PMCA4 genes may code for PMCA proteins specific to Ca transporting tissues. Our studies demonstrate cytoplasmic labeling of PMCA mRNA with hPMCA-1 and hPMCA-4 specific cDNA probes in humans, and rPMCA-1 and rPMCA-2 specific oligonucleotide probes in rats. Labeling of PMCA protein and all mRNA isoforms was found in the cytoplasm of the interlobular and intralobular ducts (except for intercalated ducts). The demonstrated presence of PMCA in SMGs of rabbit, rat, and man, may suggest a role for PMCA in the regulation of intracellular Ca and in a mechanism for regulating and maintaining the high concentration of Ca in salvia.

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.

Migration of epithelium during phenytoin-dependent gingival overgrowth in mice.

A small cavity was made in the mesiopalatal area of the maxillary first molar adjacent to the gingiva. Mice were maintained on 40 mg/kg phenytoin (or on diluent for control) by daily intraperitoneal injections. After 9 weeks, light microscopic observations revealed that in experimental mice, epithelial cells migrated towards the cavity and covered it. In controls, epithelial cell migration towards the cavity did not occur. For scanning electron microscopic (SEM) studies, specimens were fixed in 4% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for 2 hours, dehydrated, critical point dried and coated with gold. The surface of the outer gingival epithelium of experimental and of control mice showed a honeycomb arrangement of the microridges suggesting their keratinized nature. Epithelial cells lining the cavity showed well marked macroridges along their borders. Parallel microridges were observed on the upper surface of these cells suggesting that they were non-keratinized. It was concluded that the migrating epithelial cells, that covered the cavity during phenytoin-dependent gingival overgrowth, were of the non-keratinized type.

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.

A light, transmission and scanning electron microscope study of snuff-treated hamster cheek pouch epithelium.

The effects of smokeless tobacco (snuff) on hamster cheek mucosa were studied by light microscopy, transmission (TEM) and scanning electron microscopy (SEM). Two grams of commercially available smokeless tobacco were placed into the blind end of the right cheek pouch of each experimental animal, once a day and five days a week for 24 months. The control animals did not receive smokeless tobacco. After 24 months treatment with smokeless tobacco, hamster cheek mucosal epithelium lost its translucency and had become whitish in color. By light microscopy hyperorthokeratosis, prominent granular cell layers with increased keratohyalin granules and hyperplasia were seen. At the ultrastructural level, wider intercellular spaces filled with microvilli, numerous shorter desmosomes, many thin tonofilament bundles, increased number of mitochondria, membrane coating granules and keratohyalin granules were seen in snuff-treated epithelium. The changes in the surface of the epithelium as seen by SEM were the development of an irregular arrangement of the microridges and the disappearance of the normal honeycomb pattern. The microridges were irregular, widened and surrounded the irregular elongated pits. Some smooth areas without microridges and pits were also seen. The long-term histological, TEM and SEM changes induced by smokeless tobacco treatment of the epithelium are well correlated with each other and were similar to those reported in human leukoplakia without dyskeratosis. They imply changes of pathological response resulting from topically applied snuff.

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.

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.

Calcium levels in ruffle-ended and smooth-ended maturation ameloblasts.

Scanning electron microscopy was used to distinguish the topographical characteristics of two maturation ameloblast types in freeze-dried blocks of enamel organ tissue. This distinction was based primarily upon the configuration of the distal ends of the ameloblasts and the presence or absence of wide intercellular spaces. Energy dispersive x-ray spectrometry was applied to compare calcium levels in various regions of tissue identified as constituting either ruffle-ended or smooth ended ameloblasts. Greater levels of calcium were found in the distal ends of the ruffle-ended cells than in their proximal ends. In addition, greater calcium levels were found in the distal ends of the ruffle-ended cells than the distal ends of the smooth-ended cells. The higher calcium levels in ruffle-ended cells correlates with the view that these cells are actively involved in control of movement of calcium to the enamel front.

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.

Effect of fluoride and cobalt on forming enamel: scanning electron microscope and X-ray microanalysis study.

The forming surfaces of enamel of rat incisors were examined by scanning electron microscope one hour after injection of either 5 mg/100 g body weight of sodium fluoride or 12 mg/100 g body weight of cobalt chloride. The cell debris from the surfaces of the separated incisors was either gently wiped off with soft facial tissues or chemically removed by treating with NaOH, NaOCl or trypsin. Best results to remove cell debris were obtained from 0.25% trypsin treatment. SEM studies revealed that the surface of the normal secretory enamel was characteristic in appearance with well-developed smooth prism outlines. In fluoride specimens the prism outlines were feathery in appearance, laced with protruding spine-shaped clusters of mineral crystals. In the case of cobalt treatment, prism outlines were less uniform and in some areas they were incomplete. The calcium concentration of surface enamel was significantly lower in the cobalt-treated specimens than those from control and fluoride-treated animals. The Ca:Mg ratio was also lower in cobalt-treated specimens as compared to control and fluoride-treated ones.

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.

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.

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.

Interface corrosion in amalgam-to-amalgam and amalgam-to-nonprecious metal crown couplings.

This study examined the contact surface area in the coupling of a class II amalgam restoration with another class II amalgam restoration or with a stainless steel or nickel-chrome crown in 1% NaCl solution. The characterization of interfaces was carried out by using SEM and EDX microanalysis. The results indicate that the coupling of an amalgam-stainless steel crown and an amalgam-inconel crown in NaCl solution forms a deposit on the crown surfaces. This deposit contains all the constituents of corrodible phases of amalgam, including Zn. If amalgam restorations in adjoining teeth are contemplated, non-Zn-containing amalgam alloys of the same composition should be considered.