Nanoleakage at the dentin adhesive interface vs microtensile bond strength.

Excessive etching of the dentin may decrease bond strength because the adhesive may fail to completely infiltrate to the base of the over-etched demineralized collagen network. The purpose of the present study was to evaluate the influence of increasing etching times on the microtensile bond strength of Single Bond and the leakage of silver ions within the hybrid layer. After etching occlusal dentin for 15, 30, or 60 seconds with 35% phosphoric acid gel, Single Bond was applied and cured for 10 seconds. Z100 was added and cured for 60 seconds. After 24 hours of water immersion, the teeth were sectioned into slices 0.7 mm thick, and hour-glass-shaped specimens were prepared. Alternate slices were either dried for 30 minutes in air, kept wet, or they were coated with fingernail varnish except for 0.5 mm around the bonded area. Only the varnished samples were then stained with 50% AgNO3. Microtensile bond strength was tested using a Vitrodyne V-1000 universal tester. The samples of the stained group were embedded in self-curing PMMA and polished. All samples were observed with an SEM. Nanoleakage of silver ions was measured by exposure to laser ablation with an inductively connected plasma mass spectrometer and by electron dispersive elemental analysis. Increasing etching times seemed to have a negligible effect on bond strength of Single Bond, producing an average value of ca 38 MPa. However, the silver uptake increased upon prolonged etching times. Short-term results suggest that overetching has no detrimental effect on bond strength values of Single Bond. However, increased silver uptake, depending on the etching time, raises concern about the long-term stability of the bond.

Histopathology of the ocular surface after eye rubbing.

PURPOSE: This study demonstrates the effects of eye rubbing on ocular surface tissue. METHODS: Rabbits (3-4 kg; n = 24) were killed at 0, 4-h, 8-h, and 12-h intervals after a 5-min period of eye rubbing. Ocular surface tissues were studied by light and scanning electron microscopy. Contralateral eyes served as controls. Eye rubbing was accomplished by using digital pressure over the closed eyelid with a force sufficient to appreciate by palpation the orbital rim. Biomicroscopic examination revealed marked vascular injection of the conjunctiva. Ocular surface tissues studied included the lid margins, the upper and lower tarsal conjunctivae, the bulbar conjunctiva, and the cornea. RESULTS: Changes in the ocular surface included dramatic alteration in the upper tarsal conjunctiva when compared with controls. The cornea and bulbar and lower tarsal conjunctiva were not altered when compared with control tissues, except for some increase in exfoliating cells in the cornea. The surface epithelial cells of the upper tarsal conjunctiva had a spheroidal structure and were markedly elevated, the microprojections were altered, and there was evidence of increased cellular exfoliation. These changes were most pronounced at the 0 and 4-h time points, less noticeable at 8 h, and no appreciable changes were observed when compared with control tissues at 12 h. CONCLUSION: This study demonstrates that eye rubbing causes surface alterations in the stratified cuboidal to columnar epithelial surface of the upper tarsal conjunctiva while sparing the stratified squamous epithelial surface of the distal lid margins and cornea.

Computer graphics of SEM images facilitate recognition of chromosome position in isolated human metaphase plates.

There is general agreement that at the time of mitosis chromosomes occupy precise positions and that these positions likely affect subsequent nuclear function in interphase. However, before such ideas can be investigated in human cells, it is necessary to determine first the precise position of each chromosome with regard to its neighbors. It has occurred to us that stereo images, produced by scanning electron microscopy, of isolated metaphase plates could form the basis whereby these positions could be ascertained. In this paper we describe a computer graphic technique that permits us to keep track of individual chromosomes in a metaphase plate and to compare chromosome positions in different metaphase plates. Moreover, the computer graphics provide permanent, easily manipulated, rapid recall of stored chromosome profiles. These advantages are demonstrated by a comparison of the relative position of group A-specific and groups D- and G-specific chromosomes to the full complement of chromosomes in metaphase plates isolated from a nearly triploid human-derived cell (HeLa S3) to a hypo-diploid human fetal lung cell.

Acrylamide arrests mitosis and prevents chromosome migration in the absence of changes in spindle microtubules.

Cultured HT 1080 fibrosarcoma cells were exposed to acrylamide (ACR), an industrial neurotoxicant that disrupts neuronal intracellular transport, to determine if mitosis (another microtubule-based intracellular transport system) was adversely affected. The number of cells arrested in mitosis increased, in a concentration-dependent manner, from 1 to 10 mM acrylamide. A 4-h exposure to 10 mM acrylamide increased the mitotic index by 4.5-fold over control, comparable to the arrest caused by colchicine. In mitotic acrylamide-exposed cells, the chromosomes remained at the metaphase plate; no changes in spindle microtubules (MTs), as seen with tubulin immunofluorescence, were observed. The distance between spindle poles (interaster) was the same in control and experimental cells. The non-neurotoxic analogue methylene bisacrylamide had no effect in the same concentration range. The data suggest potential molecular mechanisms of action for general toxicity and neurotoxicity to be disruption in MT disassembly or MT-kinetochore interactions and/or cellular homeostasis.

Intermediate structures in nuclear morphogenesis following metaphase from HeLaS3 cells can be isolated and temporally grouped.

Previously nuclear reformation following metaphase in HeLaS3 cells was conceptualized in terms of a stepwise process which was continuous throughout anaphase and telophase. This concept was based on a three-dimensional visualization by scanning electron microscopy (SEM) of individual, organically prepared chromatid structures (prenuclei) which could be sequentially arranged. Morphologic analysis revealed unique topographies and morphometric properties which suggested that it should be possible to isolate populations of prenuclei aqueously. Such an isolation using detergents and density centrifugation is presented which yields metaphase plates and two populations of prenuclei with distinctive morphology. Essentially, prenuclei are freed from late mitotic cells in suspension cultures of synchronized HeLaS3 cells by treatment with 0.1% Nonidet-P40 followed by treatment with a mixture of Tween 40-desoxycholate (0.5%). Critical for the isolation is the presence of a divalent cation (5 mM Mg(+)+) and an acid pH (approximately 5.8). After density centrifugation, 2N decondensing structures (late intermediates) are recovered from 42% Percoll, and a mixture of 2N predecondensing (early intermediates) and 4N metaphase plates are recovered from 52% Percoll. The latter intermediates can be further separated into highly enriched populations (greater than 94% pure) by fluorescence-activated sorting. Predecondensing structures are of the same overall morphology as prenuclei isolated previously by organic means, can also be ordered sequentially to demonstrate nuclear morphogenesis, and retain centromere/kinetochore loci. These chromosomal loci based on immunostaining of individual structures appear to be positioned centrally during chromatid reassociation and then appear to be dispersed prior to structural rearrangements leading to formation of a disc-like prenucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatid behavior in late mitosis: a scanning electron microscopy analysis of mammalian cell lines with various chromosome numbers.

Chromatid activity during the process of nuclear reformation following metaphase is a period of mitosis where little precise information is available. Nuclear reformation requires that chromosomes, at metaphase and chromatids during anaphase and telophase align, position and associate in a clearly defined sequence to insure the specific design of each nucleus. Four cell lines with chromosome numbers ranging from seven to almost seventy were chosen to determine whether the process of nuclear assembly is the same throughout. Chromosomal alignment at metaphase is found to be radial in all four cell lines. Chromosome positioning is essentially the same in all four, where the smaller chromosomes are located centrally and longer ones are positioned peripherally in a radial alignment. Chromosomal association is directly related to chromosome number. The more chromosomes in a one dimensional plane occupying a given area, the closer the association. In comparing the HeLaS3 and muntjac chromatids, the former has the closer association at metaphase. Since association is the most important aspect of chromatid behavior in nuclear reformation, chromatid positioning becomes a vital process during anaphase movement. Chromatid positions established during anaphase determines later positioning in the interphase nucleus because of the subsequent interconnection of adjacent chromatids by the formation of a fibrous meshwork. This fibrous meshwork, formed in anaphase and early telophase, functions to stabilize chromatids following their positioning and it also serves as a substrate or matrix for the assembly of nuclear envelope.

A simplified method for culture of oral epithelial cells.

In order to facilitate studies on oral mucosa a simplified method for the culture of oral epithelial cells from adult hamsters was developed. Cheek pouches were excised and epithelial cells isolated by collagenase digestion. These were grown in CM-V medium containing spermine in order to inhibit overgrowth of the epithelial cells by fibroblasts. The epithelial cells were subcultured by routine tissue culture procedures. The cells isolated were examined by light microscopy and scanning and transmission electron microscopy. Morphologically the cells were typical of epithelial cells. Ultrastructural examination showed structures typical of epithelia including filaments, keratohyalin granules and desmosomal junctions. The culture system provides epithelial cells that can be used for a variety of biochemical and morphological studies.

Nuclear reformation following metaphase in HeLa S3 cells: three-dimensional visualization of chromatid rearrangements.

Nuclear reformation from chromatids following metaphase was visualized three-dimensionally for the first time in mammalian cells (HeLa S3) by scanning electron microscopy (SEM). Anaphase and telophase configurations free of mitotic apparatus, cytoskeletal elements and nuclear envelope were prepared using a slightly modified standard cytological procedure which permitted visualization of chromatid position and orientation. Mid-anaphase alignments were observed to be more complex than previously revealed by light and transmission electron microscopy (TEM). One pole consisted of chromatids joined along their lateral length, the other pole consisted of telomeres, apparently of the longest chromatids, aligned in a double concentric layer. As anaphase progressed, re-association of these chromatids appeared to occur progressively along their lateral length toward their telomeres. Morphological evidence is presented suggesting that this lateral re-association may involve interchromatid fibers. After complete joining, structures resembling a hollow half sphere had formed. Based on different preparative procedures for SEM and published TEM analysis, it is this shell-like configuration upon which the nuclear envelope is reestablished in early telophase. As telophase progressed there was loss in depth of the internal chamber resulting in a disc configuration. Following loss of chromatid outline from the surface of this structure, interphase nuclear shape was assumed. Morphometric determinations revealed relative dimensions of chromatid configurations and supported the conclusion that nuclear reformations proceeded by discrete steps. The complexity of such a process, as revealed by SEM analysis, is discussed.

A scanning electron microscopic technique for three-dimensional visualization of the spatial arrangement of metaphase, anaphase and telophase chromatids.

Chromosome and chromatid alignment in mitotic configurations remains a topic of interest because there is little precise information. For example, reconstruction of mitotic configurations from serial sections collected with transmission electron microscopy has proven to be neither practical nor a sensitive method for conceptualizing these arrangements. Similarly light microscopy has been even more unsatisfactory because of its limited resolution and lack of three-dimensional capabilities. These limitations conceivably could be overcome by visualization of mitotic configurations by scanning electron microscopy (SEM). However, SEM has its limitations, of which the most obvious with regard to visualization of mitotic configurations, is that such structures in dividing cells are obscured from the beam by membranes, cellular organelles, and the mitotic apparatus. These "contaminants," we have found, can be removed by the appropriate procedure such that a direct three-dimensional visualization of intact life-like mitotic configurations of chromatids from mammalian cells is possible. We also demonstrate that these configurations, although some artifacts may exist, retain the same basic shape and chromatid arrangements throughout metaphase, anaphase, and telophase when compared to configurations isolated with a non-ionic detergent and neutral buffers.

Chromosome stabilizing structures in mitotic Indian muntjac (Muntiacus muntjak) cells.

A new technique which removes all membranes, cytoskeletal elements, organelles, but preserves intact metaphase, anaphase and telophase configurations is combined with scanning electron microscopy (SEM) as an approach for direct visualization of chromosomal behavior in late mitosis. With this approach we are able to confirm the presence of a centromeric ring which stabilizes the centromeres during the cell cycle and present evidence for a lattice-like sheet of interchromatidic fibers in late mitosis.

Radiation-induced clastogenic plasma factors.

Ionizing irradiation induces chromosomal aberrations in directly exposed cells and is known to have mutagenic and carcinogenic potential for the exposed host. Under controlled conditions, we examined whether such clastogenic effects of irradiation might be due in part to radiation-induced plasma factors. Irradiated cells and sera from CF-Nelson rats were used at 15 min, and 1, 7, 14, and 56-70 days after total body irradiation (250 R, n = 67 or 400 R, n = 39). Control rats (n = 44) served as donors of nonirradiated sera and cells. In addition, sera from six rats were irradiated (250 R or 400 R) in vitro. On the average, 298 metaphases from six rats were studied at each time-point. Cytogenetic abnormalities observed included chromatid- and chromosome-type lesions and hyperdiploidy. The frequency of abnormalities was comparable at both radiation doses. Nonirradiated cells exposed in vitro to irradiated serum (15 min postirradiation) exhibited a 36- to 48-fold increment in hyperdiploidy (p = 0.0001) and a 2.- to 2.2-fold rise in chromatid gaps and breaks (p less than 0.01), but none of the chromosome-type aberrations seen in cells exposed to radiation. The clastogenic activity of irradiated plasma persisted in circulation for the 10-wk duration of the study and was not abrogated by dilution with nonirradiated serum. Serum irradiated in vitro was not clastogenic. This study shows that irradiation of rats results in the prompt appearance of clastogenic activity in their plasma. This activity is not due to radiation-induced depletion of protective factors nor to chemical-physical changes of normal plasma components, but results from circulating factors released by irradiated cells.

Gallocyanin-chromalum for improved scanning electron microscopy of whole nuclei without critical point drying.

Bone marrow nuclei fixed with modified Carnoy's, then stained with gallocyanin chromalum followed by air drying showed no difference in morphology when compared by means of scanning electron microscopy with similar nuclei prepared by critical point drying. Glutaraldehyde at pH 4.0 and 7.1, mercury-containing Zenker's fluid, and chromalum alone, all of which are considered to be nuclear protein cross-linking fixatives, failed to preserve the nuclear morphology as well as gallocyanin-chromalum or critical point prepared bone marro nuclei.

Trisomy 22 in a 20-year-old female.

Trisomy 22 was confirmed in a 20-year-old ambulatory female. Growth and mental retardation plus various dysmorphic features of this syndrome are described and compared with a previous survey. Several interesting unreported findings such as sexual immaturity and gait are discussed in regard to the 22 trisomy syndrome.