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E B Hunziker

Publications and source records attributed to E B Hunziker.

At least 91 records · Page 5Linked to original sources
PubMed1989

Image analysis of electron micrographs relating to mineralization in calcifying cartilage: theoretical considerations.

Biological mineralization kis a cell-mediated process which is believed to be triggered by a "nucleating agent." Various matrix structures, such as matrix vesicles, collagen fibrils and macromolecules, have been claimed to be the source of this substance, since these components have been found by transmission electron microscopy (TEM) of thin sections to be associated with early mineral crystals. Systematic image analysis of the relationships revealed in electron micrographs between specific matrix components and early mineral deposits has shown that unequivocal image interpretation is not possible. This is due principally to the problems posed by overprojection and truncation phenomena, since the structures being analyzed lie within the same dimensional range as thin section thickness. Various examples are illustrated and discussed. The site at which mineral crystals are initially laid down thus cannot be identified with any matrix structure using thin section TEM. Possible technical approaches to resolve this problem of image analysis are discussed.

Animals↗
PubMed1989

Physiological mechanisms adopted by chondrocytes in regulating longitudinal bone growth in rats.

1. Chondrocyte activities within growth plate cartilage are the principal determinants of longitudinal bone growth, and it was the aim of this investigation to assess how these cell activities are modulated under various growth rate conditions. Using proximal tibial growth plates from rats of different ages, growth rate was determined by fluorochrome labelling and incident light fluorescence microscopy. Various cellular parameters contributing to longitudinal bone growth were quantified by light microscopic stereology. The size of the proliferating cell population ('growth fraction') was estimated by autoradiography (using [3H]thymidine labelling). 2. A comparison between data for suckling (21-day-old) and fast-growing (35-day-old) rats revealed that growth acceleration is achieved almost exclusively by cell-shape modelling, namely by an increase in final cell height and a decrease in lateral diameter, whereas final cell volume and surface area are slightly reduced. Cell proliferation rate in the longitudinal direction and net matrix production per cell remain unchanged. The physiological increase in linear growth rate thus appears to be based principally upon a controlled structural modulation of the chondrocyte phenotype. On the other hand, a physiological reduction in growth rate (i.e. growth deceleration) effected during the transition from pre-puberty (35-day-old rats) to maturity (80-day-old rats) is achieved by simultaneous decreases in several chondrocyte parameters, including cell height (i.e. phenotype modulation), cell volume and proliferation rate (in the longitudinal direction). However, chondrocytes continue to produce matrix at a level comparable to that attained during the period characterized by high growth rates (i.e. at 21 and 35 days). Cartilage matrix thus appears to play a subordinate role in regulating longitudinal bone growth rate. The duration of the hypertrophic cell activity (i.e. phenotype modulation) phase remains constant (at approximately 2 days) under the various growth rate conditions. 3. The findings presented in this study indicate that measurement of bulk parameters such as [35S]sulphate incorporation into matrix components, [3H]thymidine uptake by cells and growth plate height are of limited value as estimators of longitudinal bone growth, since changes in the parameters that these measurements quantify bear little relationship to changes in linear growth rate, and may be useful only as indicators of total growth plate activity.

Aging↗
PubMed1988

Quantitation of structural features characterizing weight- and less-weight-bearing regions in articular cartilage: a stereological analysis of medial femoral condyles in young adult rabbits.

The structural organization of articular cartilage from the medial femoral condyle of young adult rabbits has been examined after processing according to an improved fixation procedure. By using recently developed stereological methods, a quantitative analysis of chondrocyte number, surface area, volume, and matrix volume per cell was carried out in the light microscope; at the electron microscopic level, quantities of cytoplasmic components within chondrocytes (including organelles) were estimated. These measurements were made for each of the four zones from the (articular cartilage) surface down to the tidemark, and the results (for each zone) were compared between weight- and less-weight-bearing regions. In general, articular cartilage revealed considerable heterogeneity in structure throughout its depth. The number of cells per unit volume is maximal beneath the surface and decreases toward the tidemark. The size of chondrocytes, and the mean matrix volume surrounding each, increases from the surface toward the deeper zones. Comparison between weight- and less-weight-bearing regions reveals striking differences. The numerical volume density of cells in the superficial zone of regions bearing high physiological load is less than half of that in less-weight-bearing regions, chondrocyte death being principally responsible for this reduction. A comparison between the midzones of weight- and less-weight-bearing areas reveals that the former is characterized by a decrease in cell density and an approximately threefold increase in cell size in relation to the latter. The increase in cell volume is attributable principally to an accumulation of intermediate filaments and glycogen particles, and represents an adaptation to increased functional requirements. Near the tidemark, numerical volume densities of chondrocytes in both weight- and less-weight-bearing locations are similar, but the larger cell size in the former still persists.

Animals↗
PubMed1988

Electron microscopic analysis of mineral deposits in the calcifying epiphyseal growth plate.

Early mineral deposits within calcifying rat epiphyseal growth plates were studied by bright field and selected-area dark field electron microscopy, and X-ray microanalysis. These mineral deposits were prepared in situ by high-pressure freezing, freeze substitution, and low-temperature embedding, and were examined in unstained, stained, and ethyleneglycol tetraacetic acid (EGTA)-treated stained thin sections. On unstained sections mineral rods occur within an amorphous density of calcium and phosphorus (CaP). X-ray microanalysis of stained sections reveals that the location of electron-dense deposits does not always correspond to that of the CaP mineral deposits identified in electron microscopic images. Such an analysis showed a depletion of both Ca and P in stained sections at sites corresponding to high levels of these elements in unstained sections. Staining thus demineralizes early deposition sites of CaP; at the same time lead (Pb) and uranium (U) bind to the organic components of the extracellular matrix formerly associated with Ca and P. This substitution phenomenon alters the overall fine structure of mineral sites by depleting the amorphous density of Ca and P, and by creating isolated rodlike structures that have formerly been interpreted as representing hydroxyapatite (HAP) crystals. Selected-area dark field imaging shows nascent sites of HAP crystals to be associated with the limiting membrane of matrix vesicles, but such crystals were undetectable at these sites with conventional bright field images. Dark field imaging also showed that the typical 30-80 nm crystal rods found in calcified cartilage consist of aggregates of HAP crystals.

Animals↗
PubMed1988

Molecular morphology of fibrin monomers and early oligomers during fibrin polymerization.

The structural features of early fibrin oligomers produced during the initial stages of polymerization were investigated by rotatory shadowing after cryotechnical preparation. The building blocks of polymerization, namely fibrin monomer units (in analogy to fibrinogen itself), were found to exhibit a high degree of flexibility which is independent of fibrinopeptide A and B removal. Early polymers exhibited directed longitudinal growth and were frequently branched. Along the main oligomer axis, fibrin monomer units were randomly orientated. Within early oligomers, a given fibrin monomer unit was found to establish a single contact with each of its two neighbors, suggesting that during the early stages of polymerization, only one polymerization and one binding site are activated per fibrinogen molecule (becoming an AB2 fibrin monomer unit). This morphological feature was corroborated by the finding that early oligomer fractions are deficient in only 50% of releasable fibrinopeptide A. Early associations between AB2 fibrin monomer units were demonstrated to be reversible and to occur in the absence of direct domainal contact; interactions thus presumably occur via fine molecular protrusions on either D or E domains. The arrangement of AB2 fibrin monomer units within early oligomers suggests that, with respect to their structural organization, fibrinogen molecules are radially symmetrical through the E domain (implying an antiparallel organization of polymerization and binding sites). This pattern is inconsistent with a "top-bottom" model, and thus with "half-staggered double-stranded" polymer growth. The methodological problems responsible for the apparent conflict with previous morphological findings are discussed.

Fibrin↗
PubMed1988

Improvement of growth and food utilization by human recombinant growth hormone in uremia.

We compared growth rate, food conversion ratio and morphology of the growth zone in female Sprague-Dawley rats with subtotal nephrectomy or sham operation. Both groups were either given vehicle or 1.4 IU/day recombinant human growth hormone (GH) by s.c. osmotic minipump, or 2.5 IU twice daily intraperitoneally for 14 or 20 days, respectively. Compared to uremic rats infused with vehicle, infusion of GH significantly (P less than 0.01) improved growth; that is, it increased gain of weight (delta 27.0 +/- 7.7 g vs. 11.6 +/- 4.9 g) and length (delta 1.8 +/- 0.3 cm vs. 1.12 +/- 0.44 cm) in ad libitum fed uremic rats. This was accompanied by increased food utilization ratio (0.146 vs. 0.065 g weight gain per g food intake). A similar increment of growth and food utilization ratio was also observed in GH versus solvent infused controls, either pairfed as for the uremic animals or fed ad libitum. Despite administration of GH, growth was not completely restored to normal in uremic animals. Circulating immunoreactive IGF I was not significantly increased by GH infusion in either uremic animals or controls. Histological analyses of the proximal tibia showed increased rate of longitudinal growth, as evaluated by tetracyclin-labeling, and increased volumetric density of primary spongiosa with unchanged width of primary spongiosa trabecules when GH was infused in uremic animals. The data suggest that growth impairment in the uremic rat is partially responsive to GH, and this is not accompanied by an increase of circulating IGF I. Therapeutic trials with recombinant GH in uremic children appear justified.

Animal Nutritional Physiological Phenomena↗
PubMed1987

In situ localization of cartilage extracellular matrix components by immunoelectron microscopy after cryotechnical tissue processing.

Localization and distribution of proteoglycans within rat growth plate cartilage were investigated by immunoelectron microscopy. By use of a mixture of three monoclonal antibodies directed against chondroitin sulfate chains and of post-embedding staining by protein A-gold, the immunosensitivity and resolution achieved by electron microscopy within tissue processed by high-pressure freezing, freeze-substitution, and low-temperature embedding were compared with those in tissue preserved by three alternative procedures (i.e., mild chemical fixation in combination with either low-temperature embedding or conventional embedding, and high-pressure freezing and freeze-substitution followed by conventional embedding). The loss of matrix components incurred during each stage of high-pressure freezing, freeze-substitution, and low temperature embedding was also determined by measuring the loss of [35S]-proteoglycans from tissue labeled in vivo, and the results compared with previously determined estimates for tissue processed using conventional techniques. Immunosensitivity, determined as the number of gold particles per unit area, was highest in tissue processed by high-pressure freezing, freeze substitution, and low-temperature embedding. Comparable results (with a reduction of only 3-7%) were achieved within tissue preserved by mild chemical fixation followed by low-temperature embedding. In both procedures where conventional embedding was adopted, sensitivity was considerably reduced (by 51% for high-pressure freezing and freeze substitution and by 74% for mild chemical fixation). Loss of matrix components was negligible during all stages of high-pressure freezing, freeze-substitution, and low-temperature embedding. Such information, and that derived from morphological inspection of the various matrix compartments in cartilage processed by high-pressure freezing, freeze-substitution, and low-temperature embedding (J Cell Biol 98:277, 1984), together demonstrate that application of this technique results in successful immobilization of proteoglycans in situ within cartilage matrix. Although loss of proteoglycans from mildly fixed cartilage embedded under low-temperature conditions is minor, morphological examination of this tissue reveals considerable shifting of proteoglycans within matrix compartments. Hence, even though immunosensitivity may be high, resolution is poor. The beauty of the high-pressure freezing, freeze-substitution, and low-temperature embedding technique is that it combines high immunosensitivity with precise localization of matrix components at the molecular level.

Animals↗
PubMed1987

Quantitation of chondrocyte performance in growth-plate cartilage during longitudinal bone growth.

The longitudinal growth of bone depends on the activities of individual chondrocytes of the growth plate. Each chondrocyte remains in a fixed location throughout its life, and there accomplishes all of its functions. Although a cell may perform several or all of its activities simultaneously, one of these will usually predominate during a particular phase of its life. The two most prominent stages are those of cellular proliferation and hypertrophy (including the mineralization of matrix) before the resorption of tissue during vascular invasion. By applying recently developed stereological procedures and improved methods for the fixation of cartilage, we compared cellular shape modulation, various ultrastructural parameters (surface areas or volumes of endoplasmic reticulum, Golgi membranes, and mitochondria), the production of matrix, and cellular turnover for proliferating and hypertrophic chondrocytes within the proximal tibial growth plate of the rat. By the late hypertrophic stage, fourfold and tenfold increases in the mean cellular height and volume, respectively, and a threefold increase in the mean volume of the matrix per cell were achieved. The high metabolic activity of hypertrophic cells was reflected by a twofold to fivefold increase in the mean cellular surface area of rough endoplasmic reticulum, the Golgi membranes, and the mean cellular mitochondrial volume. Rates of longitudinal growth were determined by fluorochrome labeling and incident-light fluorescence microscopy. Using these values and the stereological estimators describing cellular height, the rates of cellular turnover were calculated. The rapid progression of the vascular invasion front was found to eliminate, for each column of cells, one chondrocyte every three hours; that is, eight cells a day. The maintenance of a steady-state structure for growth-plate cartilage in rats in a steady state of growth thus necessitates efficient compensation for these losses, which is achieved by a high rate of cellular proliferation and rapid hypertrophy.

Animals↗
PubMed1986

Consistent and efficient delineation of reference spaces for light microscopical stereology using a laser microbeam system.

A serious problem in stereology is to ensure a consistent definition of reference spaces at different levels of magnification whenever the boundaries of such reference spaces are either fuzzy or non-existent, and hence they have to be defined artificially. (It is well known that inconsistent definitions of the reference space leads to unknown amounts of bias in stereological results.) In this paper a new application is found for the laser microbeam system used in microdissection, whereby the required boundaries can be easily and neatly traced (in fact, cut) directly onto uncovered sections for light microscopy. The dangers of bias inherent from inconsistencies of definition are thereby eliminated completely, and alternative, very expensive procedures requiring direct marking of paper prints with a pen are no longer necessary.

Animals↗
PubMed1986

Stereology for anisotropic cells: application to growth cartilage.

A number of either new or recently available stereological methods are described for estimating volume, surface area and number of anisotropic cells. The methods are illustrated with direct reference to the epiphyseal growth plate. Different estimates of a given quantity are obtained by applying alternative methods to the same set of sections, in order to compare the relative merits of the methods. For instance, the surface area of the cells is estimated via the Dimroth-Watson model (which gives a measure of the degree of anisotropy in addition to the surface area estimate) and from vertical sections using cycloid test systems. Cell number is estimated by traditional unfolding methods and by the new disector method. Also, volume-weighted mean cell volume is estimated from vertical sections via point-sampled intercepts using two different kinds of rulers to classify intercept lengths. Finally, nested design statistics is applied to a set of data from twelve animals in order to compare the relative impacts of biological and stereological (sampling) variations on the observed coefficient of error of a group mean estimate. The preferred methods are listed in the final section.

Animals↗
PubMed1986

Differential extraction of proteoglycans from cartilage tissue matrix compartments in isotonic buffer salt solutions and commercial tissue-culture media.

Small tissue blocks of native rat growth plate cartilage were incubated for short periods in one of several generally used isotonic buffer salt solutions or commercial tissue-culture media. The total percentage (approximately 12) of [35S]-labeled proteoglycans (PG) extracted from cartilage matrix under these conditions was not significantly influenced by either the chemical composition of the medium or the presence of a protease inhibitor. Morphological examination of incubated tissue after fixation in the presence of ruthenium hexamine trichloride (RHT) (included to preserve PG in situ) revealed, however, that the PG staining profiles across cartilage matrix varied with the composition of the incubation medium used. The various susceptibilities exhibited by PG within the different matrix compartments to selective extraction was estimated semi-quantitatively. The observed effects may prove useful in extracting these molecules differentially from cartilage matrix compartments.

Animals↗
PubMed1985

Matrix compartments in the growth plate of the proximal tibia of rats.

The cartilaginous matrix in the growth plate of the proximal tibia of rats is subdivided into various compartments according to criteria established by electron microscopic examination. In conventionally fixed specimens, the arrangement of collagen fibrils was analyzed by transmission and scanning electron microscopy. Distribution of proteoglycans and relations between matrix and cells were studied after fixation in media containing cationic dyes. Matrix compartments are best characterized by the density and arrangement of their collagen fibrils. On the other hand, proteoglycans are distributed almost homogenously all over the matrix. Each chondrocyte is surrounded by a thin envelope of proteoglycans, the pericellular matrix. Adjacent to this is a layer dominated by the content and order of its collagen fibrils, the territorial matrix. Its inner part covers the pericellular matrix with a thin net of intersecting fibrils. The outer part unites the cells of each column by a sheath of tightly packed longitudinal fibrils. This distinction is only possible in the longitudinal parts of the territorial matrix, whereas in the transverse septa both layers fuse into a common network. The interterritorial matrix is interposed between the columnar units and thus represents the central part of the longitudinal septa. Mineralization is restricted to the interterritorial matrix and matrix vesicles are coincidentally found in the same compartment. During growth, this structural organization undergoes a permanent and relatively fast remodeling, a process that is discussed in view of possible cell matrix interactions.

Animals↗
PubMed1984

Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. I. Chondrocyte ultrastructure--implications for the theories of mineralization and vascular invasion.

Electron microscopic examination of epiphyseal cartilage tissue processed by high pressure freezing, freeze substitution, and low temperature embedding revealed a substantial improvement in the preservation quality of intracellular organelles by comparison with the results obtained under conventional chemical fixation conditions. Furthermore, all cells throughout the epiphyseal plate, including the terminal chondrocyte adjacent to the region of vascular invasion, were found to be structurally integral. A zone of degenerating cells consistently observed in cartilage tissue processed under conventional chemical fixation conditions was not apparent. Hence, it would appear that cell destruction in this region occurs during chemical processing and is not a feature of cartilage tissue in the native state. Since these cells are situated in a region where tissue calcification is taking place, the implication is that the onset and progression of cartilage calcification are, at least partially, controlled by the chondrocytes themselves. The observation that the terminal cell adjacent to the zone of vascular invasion is viable has important implications in relation to the theory of vascular invasion. This may now require reconceptualization to accommodate the possibility that active cell destruction may be a precondition for vascular invasion.

Animals↗
PubMed1984

Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. II. Intercellular matrix ultrastructure - preservation of proteoglycans in their native state.

The extracellular matrix of epiphyseal cartilage tissue was preserved in a state believed to resemble closely that of native tissue following processing by high pressure freezing, freeze substitution, and low temperature embedding (HPF/FS). Proteoglycans (PG) were preserved in an extended state and were apparent as a reticulum of fine filamentous threads throughout the matrix. Within this network, two morphologically discrete components were discernible and identified with the carbohydrate and protein components of PG molecules. Numerous points of contact were clearly visible between components of the PG network and cross-sectioned collagen fibrils and also between PG components and chondrocytic plasmalemmata. These observations provide direct morphological indication that such relationships may exist in native epiphyseal cartilage tissue.

Animals↗
PubMed1983

Ruthenium hexammine trichloride (RHT)-mediated interaction between plasmalemmal components and pericellular matrix proteoglycans is responsible for the preservation of chondrocytic plasma membranes in situ during cartilage fixation.

Treatment of cartilage tissue with the cationic dye ruthenium hexammine trichloride (RHT) prior to fixation has been shown to prevent the detachment of chondrocytic plasmalemmata from the pericellular matrix and the aqueous extraction of proteoglycans during the subsequent fixation procedures. However, plasmalemmal rupture is prevented only by the simultaneous addition of RHT and the dialdehydic fixative glutaraldehyde. It is proposed that RHT forms an electrostatic cross-linkage between anionic components within the chondrocytic plasmalemma and proteoglycans of the pericellular matrix; experimental support for this hypothesis is presented. The precise nature of the plasmalemmal components with which RHT interacts is unknown. However, since their anionic properties are apparently lost following treatment with chondroitinase ABC, it seems likely that they represent chondroitin sulfate groups of membrane intercalated proteoglycans.

Animals↗