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PubMed1979

A method for the ultrastructural examination of cell monolayers cultured in plastic microtitre plates.

A method is presented by which cells growing as monolayers cultured in microtitre plates can be embedded for electron microscopy. The technique has the following advantages: numerous specimens may be prepared with relatively small numbers of cells, cell-cell interactions remain undisturbed and may be enumerated, and morphology can be studied under circumstances identical with those used in cell-mediated cytotoxicity assays.

Animals↗
PubMed1992

Periodic acid incubation can replace hydrochloric acid hydrolysis and trypsin digestion in immunogold--silver staining of bromodeoxyuridine incorporation in plastic sections and allows the PAS reaction.

We have examined the possibility of improving the present methods of detecting bromodeoxyuridine (BrdU) and for combining the PAS reaction with the BrdU detection by means of immunogold-silver staining (IGSS). This was done in testes fixed in Carnoy or Bouin, and in parts of the small intestine which were fixed in Carnoy or periodate-lysine-paraformaldehyde (PLP). All tissues were embedded in a mixture of glycol methacrylate and butanediol-monoacrylate. It was found to be impossible to carry out BrdU detection using HCl hydrolysis and trypsin digestion in combination with a PAS reaction. However, incubation of the plastic sections in periodic acid for a period of 30 minutes appeared to make it possible to eliminate the HCl denaturation step and to carry out a specific PAS reaction. Moreover, after incubation in periodic acid, trypsin digestion was no longer required to make the BrdU label accessible in GMA-embedded sections, nor to re-expose the antigenic sites in plastic sections of tissues fixed with cross-linking fixatives. In this way the loss of cell structures, which is inevitable when trypsin is used, can be avoided. Now a BrdU detection with improved morphology can be combined with the PAS reaction in the same plastic section in order to stain tissue carbohydrates. This is important for tumour diagnosis, where the PAS reaction can be very useful.

Animals↗
PubMed1990

Light microscopic autoradiography for study of early changes in the distribution of water-soluble materials.

An approach using autoradiography for the study of early changes in the distribution of water-soluble materials and the chemography involved was investigated. Radioactive calcium chloride (45Ca) was injected into the iliac vein of a rat. Ten seconds after the injection the rat was frozen in hexane (-90 degrees C). The frozen rat was embedded in 5% sodium carboxymethyl cellulose and blocked in the coolant. A sheet of plastic tape coated with a synthetic rubber glue was fastened to the trimmed block surface, and whole-body sections 2-10 microns thick were cut with a disposable microtome knife. Selected sections were freeze-dried and then covered with a dried autoradiographic emulsion film about 1 microns thick. The autoradiograph clearly showed the distribution of radioactive calcium in the calcification zone of long bones. The samples chosen to assess chemographic artifacts showed positive and negative chemographies on most of the tissues when these were kept at 23 degrees C, and although both chemographic effects were significantly reduced when the samples were kept at -20 degrees C, cells in several tissues still exhibited positive and negative chemographies. The technique can be used for the study of any animal whose size is suitable for whole-body freeze-sectioning.

Animals↗
PubMed1978

A comparison of iron histochemical methods for use on glycol methacrylate embedded tissues.

Four histochemical tests for iron and four procedures for its removal were investigated in regard to their suitability for glycol methacrylate embedded tissues. The HCl-ferrocyanide and chlorate hematoxylin methods were easily modified for plastic sections. The latter does not use iron-containing reagents. Desiderization was complete both after a fifteen minute exposure in 1% Na2S2O4 in 0.1 M acetate-HCl buffer (pH 4.5) and, if an acid method is preferred, after twelve hours in 5% oxalic acid. A six hour treatment in 3.7 N H2SO4 also removed all histochemical iron but was accompanied by a relatively greater loss of tissue basophilia.

Animals↗
PubMed2006

How tough is bone? Application of elastic-plastic fracture mechanics to bone.

Bone, with a hierarchical structure that spans from the nano-scale to the macro-scale and a composite design composed of nano-sized mineral crystals embedded in an organic matrix, has been shown to have several toughening mechanisms that increases its toughness. These mechanisms can stop, slow, or deflect crack propagation and cause bone to have a moderate amount of apparent plastic deformation before fracture. In addition, bone contains a high volumetric percentage of organics and water that makes it behave nonlinearly before fracture. Many researchers used strength or critical stress intensity factor (fracture toughness) to characterize the mechanical property of bone. However, these parameters do not account for the energy spent in plastic deformation before bone fracture. To accurately describe the mechanical characteristics of bone, we applied elastic-plastic fracture mechanics to study bone's fracture toughness. The J integral, a parameter that estimates both the energies consumed in the elastic and plastic deformations, was used to quantify the total energy spent before bone fracture. Twenty cortical bone specimens were cut from the mid-diaphysis of bovine femurs. Ten of them were prepared to undergo transverse fracture and the other 10 were prepared to undergo longitudinal fracture. The specimens were prepared following the apparatus suggested in ASTM E1820 and tested in distilled water at 37 degrees C. The average J integral of the transverse-fractured specimens was found to be 6.6 kPa m, which is 187% greater than that of longitudinal-fractured specimens (2.3 kPa m). The energy spent in the plastic deformation of the longitudinal-fractured and transverse-fractured bovine specimens was found to be 3.6-4.1 times the energy spent in the elastic deformation. This study shows that the toughness of bone estimated using the J integral is much greater than the toughness measured using the critical stress intensity factor. We suggest that the J integral method is a better technique in estimating the toughness of bone.

Animals↗
PubMed2003

[Biomechanical properties (compressive strength and compressive pressure at break) of hyaline cartilage under axial load].

INTENTION OF THE STUDY: Explanations concerning the physical properties of hyaline cartilage are different. It was the intention of this study to determine the material parameters of hyaline cartilage under axial load (elasticity, plasticity, elasticity and module pressure stress to break). METHODS: Specimens from the medial femoral condyle (chondro-cortical ships) from adult female domestic pigs (n=28) were used for the experiments. The specimens were completely embedded in plaster to minimize shearing. Axial load was carried out by an universal mechanical testing machine (Zwick Z2.5/TS1S, Ulm, Germany) to determine elastic and plastic deformation and pressure stress to break. RESULTS: Axial load up to 5 MPa produces an almost elastic deformation, an increasing axial load results in a plastic deformation. In the range of 3 to 5 MPa the principle of Hooke is valid. The elasticity module amounted to 39.2 +/- 11.9 N/mm(2), determined under 3.8 MPa axial load. An axial load of 25.8 +/- 5.2 MPa (sigma max ) causes a break of cartilage. A strong correlation between break resistance and thickness of the chondral slice (r=0.71; p < 0.05) was observed. CONCLUSIONS: The low module of chondral elasticity characterizes this tissue as "soft". Moderate axial load causes an ideal elastic, higher axial load a plastic deformation. The medium pressure to break to amounted 25.8 MPa. The medium pressure to break of 25.8 MPa is comparable with the forces produced by an unrestrained limited downfall from a height of 4.3 m. It must be concluded that isolated chondral fractures are rare consequences of a trauma as long as accompanying ligamentous or osseous damages are not found.

Animals↗
PubMed2001

Role of stromal cells in osteoclast differentiation in bone marrow.

Bone marrow stromal cells have been considered to play an important role in osteoclast differentiation. However, the interaction of these cells in vivo has not been clearly demonstrated. To clarify this, we examined the distribution of alkaline phosphatase (ALPase) and tartrate-resistant acid phosphatase (TRAPase) activities as markers of osteoblastic and osteoclastic cells, respectively. Rat tibiae were fixed and embedded in Technovit 8100 or paraffin. ALPase and TRAPase activities were detected simultaneously on a plastic section by the azo-dye method. ALPase activity was detected on the plasma membranes of osteoblasts and some bone marrow fibroblastic stromal cells. These ALPase-positive cells were connected to each other by cytoplasmic processes, forming a cellular network in bone marrow. The ALPase activity of fibroblastic stromal cells tended to be stronger in those cells close to the bone surface than in the cells in the center of bone marrow. Reticular fibers in bone marrow were found to form a network. The ALPase-positive fibroblastic stromal cells may be reticular cells, because the localization of those cells was in accord with the localization of reticular fibers. The TRAPase-positive mononuclear cells and osteoclasts were mostly observed to be associated with the intensely ALPase-positive fibroblastic stromal cells. Immunoreactivity of osteoclast differentiation factor (ODF) was found in the fibroblastic stromal cells. These findings suggest that the network of ALPase-positive fibroblastic stromal cells in bone marrow serves as a guide for the migration of osteoclast precursor cells toward the bone surface, and may control the differentiation and activity of osteoclasts.

Acid Phosphatase↗
PubMedDate not supplied

In vitro microleakage of dentin adhesives.

This in vitro study measured the microleakage of current dentin bonding agents and glass-ionomer bases. Freshly extracted human molars were prepared to a flat surface, and dentin adhesives and composite resins were applied in a plastic matrix. Samples were stored in water at 37 degrees C, thermocycled, stained with AgNO3, embedded in epoxy resin, and sectioned for evaluation of stain penetration at the composite resin/tooth interface. The reliability index of the dentin adhesives varied significantly between materials. The enamel control had essentially no microleakage, and the aluminum oxalate dentin adhesive on dentin had significantly less microleakage than other dentin adhesives tested. Present dentin adhesives were unable to prevent, but may reduce, microleakage.

Acrylic Resins↗
PubMed1978

Freeze-drying of unfixed monolayer cultures for electron microscopic autoradiography.

A method has been developed for freezing, drying and embedding of unfixed monolayer cultures for electron microscopic autoradiography (EM ARG). Experimental results showed: a) Aclar 33 C was a more suitable substrate than the plastic of petri dishes, b) cultures pressed rapidly against the polished face of a large copper cylinder chilled in liquid nitrogen had better cellular morphology than did cultures dipped in Freon 12 chilled in liquid nitrogen, and c) cultures embedded in Epon alone had finer extracellular ice spaces and lower background grain densities than did cultures embedded in Epon with 1% silicone. This method has been used to evaluate the effect of fixation on the localization of the neurotransmitter, 3H-gamma-aminobutyric acid (3H-GABA), in neurons of dispersed cell cultures. EM ARG results showed that the neuronal cell bodies and vesicle elements were present in similar numbers in both glutaraldehyde fixed and freeze-dried cultures.

Animals↗
PubMed1988

Effect of EGF on human skin fibroblasts is modulated by the extracellular matrix.

The aim of this work was to clarify the reason why a discrepancy exists between the effects of epidermal growth factor (EGF) on fibroblasts in culture repressing collagen biosynthesis and in vivo stimulating wound healing. The effect of EGF on the biosynthetic activity of fibroblasts was measured in various conditions of cultures: on plastic, on plastic coated with various macromolecules of the extracellular matrix, on top of a type I collagen gel, and within a three-dimensional collagen lattice. While the noncollagen protein (NCP) synthesis was not affected by the interactions of the cell with the various coated matrices, collagen synthesis was inhibited. At the surface of a collagen gel, protein synthesis was reduced, while collagen synthesis and degradation were slightly stimulated. When embedded in a lattice, the overall biosynthetic activity of fibroblasts was largely depressed. The addition of EGF to cultures on plastic and on the various coated macromolecules resulted in a further repression of collagen synthesis while cell multiplication was slightly stimulated. On the contrary, the addition of EGF to fibroblasts in a collagen lattice resulted in a stimulation of both NCP and collagen synthesis as observed in vivo. These opposite effects of EGF in a two- or three-dimensional culture system are not related to modification in number or affinity of the EGF receptors at the cell surface. These results further support the similarity in the state of differentiation of fibroblasts in a three-dimensional lattice and in vivo.

Adolescent↗
PubMed1995

Bedside fluoroscopy for the detection of foreign bodies.

OBJECTIVE: To determine the sensitivity and specificity of portable fluoroscopy for foreign-body detection using an ex-vivo experimental model. METHODS: A prospective, randomized masked investigation was performed to characterize foreign-body detection in cubes of beef. Three emergency physicians (EPs) independently used a portable fluoroscope to determine the presence or absence of various foreign bodies in ten cubes of beef (approximately 6 cm on each side). Six different groups of foreign bodies were examined: glass, wood, graphite (pencil lead), metal (needle), plastic, and gravel. An independent observer placed the six objects randomly into each set of ten beef cubes. One hundred observations were made by each physician using sets of ten beef cubes at a time. RESULTS: Fluoroscopy detected 117 of 180 foreign bodies (sensitivity 65%; 95% CI 58-72%), including all cubes of beef embedded with gravel, metal, and glass. Graphite was detected 27 of 30 times (sensitivity 90%; 95% CI 74-98%). None of the plastic or wooden foreign bodies was identified despite magnification techniques and real-time imaging (sensitivity 0%; 95% CI0-12%, each). Of the 120 cubes of meat with no foreign body, two false-positives were recorded (specificity 98%; 95% CI 94-100%). The overall positive predictive value and negative predictive value of fluoroscopy were 98% and 65%, with 95% CIs of 94-100% and 58-72%, respectively. Interobserver agreement between the three EPs was considered excellent (kappa = 0.75). CONCLUSION: Bedside fluoroscopy lacks sufficient sensitivity to rule out many foreign bodies in the ED. Its use should be limited to suspected gravel, glass, pencil lead, and metallic objects, which are known to be radiopaque.

Emergency Medicine↗
PubMed1997

Transmission electron microscopy of chromosomes by longitudinal section preparation: application to fragile X chromosome analysis.

We developed a method for the preparation of ultrathin longitudinal sections of chromosomes enabling TEM studies of whole chromosomes. By using a novel "repeat chill" method of exposing the glass slide and plastic block interface to liquid nitrogen, it was possible to separate consistently hardened epoxy resin-embedded chromosome spreads from glass slides for ultrathin longitudinal sectioning of entire spreads and of individual chromosomes. The method was applied to analyze the fragile X chromosome. The ultrastructure of centromeres, telomeres, fragile sites and other chromosomal areas can now be studied in detail.

Centromere↗
PubMed2006

Langerhans cells in vulvar lichen sclerosus and vulvar squamous cell carcinoma.

INTRODUCTION: Langerhans cells (LCs), specializing in antigen presentation, are a very important part of the skin immune system (SIS). MATERIALS AND METHODS: Skin biopsies from 22 women with vulvar lichen sclerosus (LS): 15 patients with early and 7 with the late stage of the disease, were evaluated. Five women with vulvar squamous cell carcinoma (SCC) were also examined. The control group consisted of 9 women who underwent plastic surgery of the vulvar region. Immunohistochemical staining was performed on formalin-fixed paraffin-embedded tissues samples using antihuman CD1a antibody (NCL-CD1a-235, Novocastra). RESULTS: Increased numbers of LC stainings were present in early LS, whereas decreased numbers of these cells were present in late LS and in SCC compared with the control group. CONCLUSIONS: This study showed that dysregulation of the SIS may lead to suppression of LCs in the vulvar epithelium and may be one of the reasons for a higher tendency for carcinogenesis in the vulvar region.

Aged↗
PubMed2005

Transdifferentiation of mature rat hepatocytes into bile duct-like cells in vitro.

We investigated the mechanism of phenotypic plasticity of hepatocytes in a three-dimensional organoid culture system, in which hepatocytic spheroids were embedded within a collagen gel matrix. Hepatocytes expressed several bile duct markers including cytokeratin (CK) 19 soon after culture and underwent branching morphogenesis within the matrix in the presence of insulin and epidermal growth factor. Cultured hepatocytes did not express Delta-like, a specific marker for oval cells and hepatoblasts. Furthermore, hepatocytes isolated from c-kit mutant rats (Ws/Ws), which are defective in proliferation of oval cells, showed essentially the same phenotypic changes as those isolated from control rats. The bile duct-like differentiation of hepatocytes was associated with increased expression of Jagged1, Jagged2, Notch1, and several Notch target genes. CK19 expression and branching morphogenesis were inhibited by dexamethasone, a mitogen-activated protein kinase kinase 1 (MEK1) inhibitor (PD98059), and a phosphatidyl inositol 3-kinase inhibitor (LY294002). After being cultured for more than 3 weeks within the gels, hepatocytes transformed into ductular structures surrounded by basement membranes. Our results suggest that hepatocytes might have the potential to transdifferentiate into bile duct-like cells without acquiring a stem-like phenotype and that this is mediated through specific protein tyrosine phosphorylation pathways.

Animals↗
PubMed2002

Selective electrical interfaces with the nervous system.

To achieve selective electrical interfacing to the neural system it is necessary to approach neuronal elements on a scale of micrometers. This necessitates microtechnology fabrication and introduces the interdisciplinary field of neurotechnology, lying at the juncture of neuroscience with microtechnology. The neuroelectronic interface occurs where the membrane of a cell soma or axon meets a metal microelectrode surface. The seal between these may be narrow or may be leaky. In the latter case the surrounding volume conductor becomes part of the interface. Electrode design for successful interfacing, either for stimulation or recording, requires good understanding of membrane phenomena, natural and evoked action potential generation, volume conduction, and electrode behavior. Penetrating multimicroelectrodes have been produced as one-, two-, and three-dimensional arrays, mainly in silicon, glass, and metal microtechnology. Cuff electrodes circumvent a nerve; their selectivity aims at fascicles more than at nerve fibers. Other types of electrodes are regenerating sieves and cone-ingrowth electrodes. The latter may play a role in brain-computer interfaces. Planar substrate-embedded electrode arrays with cultured neural cells on top are used to study the activity and plasticity of developing neural networks. They also serve as substrates for future so-called cultured probes.

Action Potentials↗
PubMed1981

A simple method for the preparation of cell cultures for ultrastructural investigation.

A method is described that allows investigation of cultured cells at the light or electron microscopical level without changing the culturing conditions or the in situ situation during dehydration or embedding. Fixed and dehydrated cell culture layers were cut into small pieces and separated from the plastic dish by adding propylene oxide. With this simple method we could obtain orientated sections of selected cell groups.

Animals↗
PubMed1994

Adaptation of the reverse hemolytic plaque assay to electron microscopy: a study of the individual secretory activity in prolactin cell subpopulations.

We used the reverse hemolytic plaque assay (RHPA), a method for detecting secretion by single cells, to demonstrate functional heterogeneity among prolactin (PRL) cells of rat anterior pituitary at the light microscopic (LM) level. We attempted to adapt RHPA for electron microscopy (EM) to define the relationships between fine structure and secretory activity in individual PRL cells. A major modification of the technique, intended to improve preservation of ultrastructure, was allowing the cells to recover after their enzymatic dispersion from the pituitary tissue, through a brief (3-4 hr) culture step before the assay. Adaptation for EM was achieved by the use of plastic slides for construction of specialized Cunningham chambers, permitting application of all the EM procedures (flat embedding, punching of selected areas) usually employed for cultured pituitary monolayers. Moreover, immunocytochemical pre- and post-embedding methods were also applied for cell identification and study of subcellular hormone distribution. Such a modified RHPA enabled us to analyze the ultrastructure of plaque-forming cells surrounded by their companion red blood cell ghosts. The first results with EM RHPA showed that under basal conditions a subpopulation of PRL cells containing small granules (150-200 nm) was actively secreting, whereas PRL cells with large (300-600 nm) and irregular granules appeared to be stimulated by thyrotropin-releasing hormone or KCl. The EM RHPA technique described here might receive more general application and could be utilized for study of many other secretory systems.

Animals↗
PubMed1990

Observations on the contrasting reaction of some electron dense stains applied on epoxy-embedded tissue sections.

Epon sections from glutaraldehyde-fixed rat bone marrow were treated with aqueous solutions of the following electron contrasting agents: uranyl acetate, ruthenium red, potassium permanganate, potassium dichromate, stannous chloride, palladium (II) chloride, sodium molybdate, phosphomolybdic acid, molybdenum heteropolyblue, phosphotungstic acid, iron(II)-phenanthroline, aluminium-hematoxylin, mercurochrome, cuprolinic blue, and sirius light turquoise blue. At the ultrastructural level, a high degree of electron opacity was always observed in mast cell granules and the crystalline inclusion (internum) of eosinophil granules. The chromatin revealed a somewhat lower and variable contrasting reaction, while the matrix (externum) of eosinophil granules appeared with scarce or no contrast. This pattern of electron opacity showed no correlation with the type of agent used; therefore, it can be assumed that binding processes based on the own chemical reactivity of the compounds are rather of secondary importance. The differential epoxy resin embedding of cell structures and the variable access of aqueous reagents through the non-polar plastic could be the predominant factors which account for these contrasting reactions.

Animals↗