Beta-glucuronidase and hexosaminidase are marker enzymes for different compartments of the endo-lysosomal system in mussel digestive cells.

In environmental toxicology, the most commonly used techniques used to visualise lysosomes in order to determine their responses to pollutants (LSC test: lysosomal structural changes test; LMS test: lysosomal membrane stability test) are based on the histochemical application of lysosomal marker enzymes. In mussel digestive cells, the marker enzymes used are beta-glucuronidase (beta-Gus) and hexosaminidase (Hex). The present work has been aimed at determining the distribution of these lysosomal marker enzymes in the various compartments of the endo-lysosomal system (ELS) of mussel digestive cells and at exploring whether intercellular transfer of lysosomal enzymes occurs between digestive and basophilic cells. Immunogold cytochemistry has allowed us to conclude that beta-Gus is present in every compartment of the digestive cell ELS, whereas Hex is not so widely distributed. Moreover, Hex is intimately linked to the lysosomal membrane, whereas beta-Gus appears to be not necessarily membrane-bound. Therefore, two populations of heterolysosomes with different enzyme load and membrane stability have been distinguished in the digestive cell. In addition, heterolysosomes of different electron density have been commonly observed merging together by contact; we suggest that some might act as storage granules for lysosomal enzymes. On the other hand, beta-Gus seems to be released to the digestive alveolar lumen in secretory lysosomes produced by basophilic cells and endocytosed by digestive cells. Regarding the implications of the present study on the interpretation of lysosomal biomarkers, we conclude that beta-Gus, but not Hex, histochemistry provides an appropriate marker for the LSC test and that, although both lysosomal marker enzymes can be employed in the LMS test, different values would be obtained depending on the marker enzyme employed.

Stimulatory effects of creatine on metabolic activity, differentiation and mineralization of primary osteoblast-like cells in monolayer and micromass cell cultures.

The effects of creatine (Cr) supplementation on primary rat osteoblast-like cells cultured as monolayer and micromass were investigated. Cr was added to the medium at concentrations of either 10 or 20 mM. At various time points, the cell cultures were analyzed morphologically, metabolically and biochemically. The degree of differentiation of primary osteoblast-like cell cultures was higher in micromass cultures compared to monolayer cultures, as judged by alkaline phosphatase (ALP) activity and extent of mineralization. In both culture systems, Cr supplementation showed positive effects, which were dependent on the organizational level of the osteoblast-like cells in such a way that the cells in monolayer culture showed significantly increased metabolic activity, ALP activity and mineralization in the presence of Cr than without the supplement. In micromass cultures, Cr also significantly enhanced ALP activity and mineralization, without affecting metabolic activity. The effect of Cr on ALP activity was more pronounced at higher concentrations of Cr, but 20 mM Cr already showed some adverse effects on cell viability. In conclusion, chemically pure Cr added to low serum cell culture medium has a stimulatory effect on metabolic activity, differentiation and mineralization of osteoblast-like cells indicating that Cr supplementation could also be used as a potential clinical intervention to stimulate cell growth, differentiation and mineralization during bone repair in vivo.

Focal adhesion quantification - a new assay of material biocompatibility? Review.

The development of novel synthetic biomaterials is necessitated by the increasing demand for accelerated healing of tissues following surgical intervention. Strict testing of such materials is necessary before application. Currently, before any material can be marketed, approval by regulatory organisations such as the FDA is required. Presently, in vitro testing is performed as a prerequisite to in vivo evaluation. The in vitro techniques currently employed do not reflect the progress in our understanding of extra and intra-cellular processes, with far more sensitive in vitro evaluations now available. Obtaining quantifiable data is increasingly relevant to evaluating events occurring in vivo. Quantifying cell adhesion to surfaces provides some of this data as an initial assessment method. Major developments in this field are occurring but many investigators still use less than optimal methods for assessing biomaterials. The relevance of using cell adhesion assays to help determine biomaterial biocompatibility is reviewed. Additionally, current in vitro methods of evaluating biomaterials are discussed in the context of novel testing concepts developed by the authors.

The ultrastructure of mouse articular cartilage: collagen orientation and implications for tissue functionality. A polarised light and scanning electron microscope study and review.

Adult mouse articular cartilage (AC) has not been thoroughly described using high resolution imaging techniques, despite the fact that the availability of knockout mice with specific extracellular matrix (ECM) mutations have renewed interest in using the mouse as a model for a variety of different human conditions. With osteoarthritis affecting millions of people worldwide, investigations into the structure and, therefore, the ability of AC to act as a load-bearing tissue, are crucial for developing treatments and prevention techniques to limit the degree of severity in this condition. Cryofixation and formaldehyde fixation as well as chemical digestion of the uncalcified regions of AC were used in combination with bright field light, polarised light and scanning electron microscopy to image the structure of adult mouse AC. Chemical digestion of the tissue revealed unique insights into the structure of mouse AC and the high cellular density of the tissue. Tightly packed sheets of collagen fibrils formed the territorial matrix (TM) of the deep zone. These were observed closely surrounding the chondrons, after applying both chemical and cryofixation techniques. The interterritorial matrix (IM), in contrast, was more isotropically arranged. The results of the study have implications for the interpretation of biomechanical functionality of mouse AC with probable applications to other species.

Variation in cell-substratum adhesion in relation to cell cycle phases.

The quantification of focal adhesion sites offers an assessable method of measuring cell-substrate adhesion. Such measurement can be hindered by intra-sample variation that may be cell cycle derived. A combination of autoradiography and immunolabelling techniques, for scanning electron microscopy (SEM), were utilised simultaneously to identify both S-phase cells and their focal adhesion sites. Electron-energy 'sectioning' of the sample, by varying the accelerating voltage of the electron beam, combined with backscattered electron (BSE) imaging, allowed for S-phase cell identification in one energy 'plane' image and quantitation of immunogold label in another. As a result, it was possible simultaneously to identify S-phase cells and their immunogold-labelled focal adhesions sites on the same cell. The focal adhesion densities were calculated both for identified S-phase cells and the remaining non-S-phase cells present. The results indicated that the cell cycle phase was a significant factor in determining the density of focal adhesions, with non-S-phase cells showing a larger adhesion density than S-phase cells. Focal adhesion morphology was also seen to correspond to cell cycle phase; with 'dot' adhesions being more prevalent on smaller non-S-phase and the mature 'dash' type on larger S-phase cells. This study demonstrated that when quantitation of focal adhesion sites is required, it is necessary to consider the influence of cell cycle phases on any data collected.

Deformation of chondrocytes in articular cartilage under compressive load: a morphological study.

The main function of articular cartilage is to transmit load. The objective of this study was to describe the deformation of chondrocytes under static loading and its relation to collagen matrix deformation. Whole intact rabbit knee joints were loaded statically with either high or low magnitude and long or short duration. Specimens were cryopreserved while under load and prepared for morphological evaluation by field emission scanning electron microscopy. With this method an immediate preservation of the chondrocyte in its loaded state was possible. Static compression of articular cartilage produced a zone-specific deformation of chondrocyte shape, depending on the magnitude and duration of load. Under high-force and long-duration loading, the chondrocytes showed considerable deformation concomitant with the highly deformed collagen fibres. Chondrocyte deformation occurred mostly in the transitional and upper radial zones and less in the lower layers. There was no significant change of the chondrocyte shape in the tangential zone under high- or low-force short-duration loading. These results show that the chondrocytes undergo significant changes in shape ex vivo and that they are sensitive to differences in the magnitude and duration of loads being applied. Chondrocyte deformation is strongly linked to the deformation of the surrounding cartilage collagen matrix.

The rheology of collapsing zeolites amorphized by temperature and pressure.

Low-density zeolites collapse to the rigid amorphous state at temperatures that are well below the melting points of crystals of the same composition but of conventional density. Here we show, by using a range of experimental techniques, how the phenomenon of amorphization is time dependent, and how the dynamics of order-disorder transitions in zeolites under temperature and pressure are equivalent. As a result, thermobaric regions of instability can be charted, which are indicative of polyamorphism. Moreover, the boundaries of these zones depend on the rate at which temperature or pressure is ramped. By directly comparing the rheology of collapse with structural relaxation in equivalent melts, we conclude that zeolites amorphize like very strong liquids and, if compression occurs slowly, this is likely to lead to the synthesis of perfect glasses.

Simultaneously identifying S-phase labelled cells and immunogold-labelling of vinculin in focal adhesions.

A new combination of autoradiography and immunolabelling techniques is presented that allows the simultaneous identification of both S-phase cells and their focal adhesions using scanning electron microscopy. The technique allows both labels to be discerned visually by their unique shapes and location within and on the cell. S-phase cells were radio-labelled with a pulse of tritiated thymidine, selectively incorporated into synthesizing DNA. The cells were then immunogold-labelled for the focal adhesion protein, vinculin, prepared for autoradiography, and embedded in resin. The resin was then polymerized before removing the substrate, to expose the embedded cell undersurface. Electron-energy 'sectioning' of the sample by varying the accelerating voltage of the electron beam allowed separate S-phase cell identification in one electron-energy 'section' and visualization of immunogold label in another 'section', within the same cell. As a result of applying this technique it was possible to positively identify S-phase cells and immunogold-labelled focal adhesions on the same cell simultaneously, which could be used to quantify focal adhesion sites on different substrates.

Novel aspects to the structure of rabbit articular cartilage.

Applying cryo and modified chemical preparation techniques, mainly for scanning electron microscopy, revealed entirely new aspects to the structure of the radial zone of rabbit tibial plateau articular cartilage. The aggrecan component of the extracellular matrix was contained radially in columns, each with a diameter of 1-3 mm, by a tightly packed matrix of collagen fibrils. The collagen fibrils were arranged radially, some straight and others in an opposed spiral arrangement, with regularly repeating patterns. This organization existed in the regions surrounding the columns of chondrocytes, known as chondrons. The load bearing property of the tissue was explained by the directed flow and containment of the interstitial fluid, modulated by the protein-carbohydrate complexes, along these collagen bounded tubular structures. The reason why such a structure has not been described previously may be that it is not retained by aldehyde fixation followed by dehydration, the method commonly used for tissue preparation for electron microscopy.

Fibroblast and osteoblast adhesion and morphology on calcium phosphate surfaces.

Implant loosening in bone fixation is an unresolved complication associated with internal fixation. It is generally accepted that this problem can be overcome by modifying the implant/bone interface for improved osseointegration. This is achieved, in part, by hydroxyapatite (HA) or tricalcium phosphate coatings. Unfortunately, the benefits of these coatings are constrained by not only the generally low strength of their adhesion to the implant surface but also the limited cohesion within their layers. Anodic Plasma-chemical treatment (APC) has been developed to incorporate electrolytes and produce coatings with various microtopographies and strong adhesion to implants. In this in vitro study fibroblast and osteoblast morphologies and adhesion to various substrates were evaluated using qualitative and quantitative methods. The substrates were Thermanox plastic and commercially pure titanium. The latter were surface-treated using several different methods: conventional anodisation, plasma spraying of HA and anodic plasma-chemical (APC) treatment in an electrolyte solution containing either calcium and phosphate (APC-CaP) or phosphoric acid (APC-P). Both osteoblasts and fibroblasts showed extensive cell spreading, total cell area and greatest amount of adhesion, with defined adhesion patterns on the Thermanox plastic, anodised titanium, and the two APC-CaP substrates. With fibroblasts, almost no cell spreading and very low adhesion, was observed in cells cultured on the APC-P and HA surfaces. The extent of cell spreading correlated with the area of focal adhesions as assessed by the amount of vinculin labelling. The Thermanox plastic, anodised titanium, and the two APC-CaP substrates were the most cytocompatible substrates with regard to this in vitro evaluation.

Differentiation of rat osteoblast-like cells in monolayer and micromass cultures.

During intramembranous bone formation, preosteoblasts condense, differentiate into osteoblasts and deposit bone matrix. We compared the differentiation process of rat calvarial osteoblast-like cells inoculated as micromasses, which mimic the in vivo condensation process, with cells inoculated as monolayers. The cells were analysed morphologically at 1,2 and 3 weeks by light microscopy (alkaline phosphatase activity, mineralization), by transmission electron microscopy, and biochemically (collagen typing, alkaline phosphatase activity, protein and DNA content). The cells inoculated as monolayers formed alkaline phosphatase positive and mineralized nodules during the culture period. The cells inoculated as a micromass formed a large mineralized area consisting of smaller nodules. The ultrastructure of the cells in both culture systems showed the typical features of osteoblasts and osteocytes. The main difference between monolayer and micromass cultures was found after 1 week in culture. The cells inoculated as a micromass formed a multilayer of cells. The cytoplasm contained rER, mitochondria, vesicles and ribosomes. There were abundant collagen fibrils in membrane folds and in the extracellular matrix. This was in contrast to the cells in monolayer culture which showed hardly any collagen fibrils in the extracellular matrix. The promotion of the differentiation was also confirmed by biochemical data showing that the DNA content was lower in the micromass than in the monolayer cultures during the culture period. These results show that micromass, as compared to monolayer, culture promotes the differentiation of rat osteoblast-like cells in vitro.

Immunogold labelling of fibroblast focal adhesion sites visualised in fixed material using scanning electron microscopy, and living, using internal reflection microscopy.

A new immunogold labelling method for the visualisation of vinculin, an integral protein in focal adhesions of cells, is reported. Quantification of vinculin is indicative of substrate cytocompatibility (cytocompatibility is one aspect of biocompatibility; it is the cellular response to a biomaterial). For efficient labelling, most of the cell body above the cell-substrate interface was removed with detergent. The antigen blocking procedure, size of label (5 nm) and duration of silver-enhancement (6 min), for visualisation of the labelled sites on the whole cell by scanning electron microscopy (SEM), were determined. Imaging living cells with interference reflection light microscopy, followed by backscattered electron (BSE) imaging of the same fixed and immunolabelled cells confirmed the results. Collecting low voltage BSE images of embedded cells after the substrate had been removed provided 'sectional' views through the cell. This enabled visualisation of vinculin exclusively within the cell-substrate contact zone; the focal adhesions. The method could be of general use in the imaging of protein distribution at biological tissue/substrate interfaces.

Enhancement of immunogold-labelled focal adhesion sites in fibroblasts cultured on metal substrates: problems and solutions.

Visualisation of cell adhesion patterns by scanning electron microscopy requires special preparation and labelling. The membranes and cytoplasm must be removed, without damaging the antigen, to facilitate antibody access to vinculin in the focal adhesions. Low beam energy imaging is used to visualise the cell undersurface (embedded in resin after staining with osmium tetroxide) and immunogold-labelled adhesion sites. The gold probe, must be large enough (>40 nm) for detection, while viewing the whole cell, but large gold markers increase steric hindrance and decrease labelling efficiency. This problem can be overcome by using small gold probes (1-5 nm) followed by enlargement with silver enhancement, but osmium tetroxide stain etches the silver. We demonstrated that metal substrates increased this etching. Reducing the concentration of osmium tetroxide and incubation time reduced the amount of etching. We have demonstrated that gold enhancement was not etched by osmium tetroxide, irrespective of the substrate. Therefore, comparative studies of cell adhesion to different biomaterial substrates can be performed using immunogold-labelling with gold enhancement.

Influence of cell isolation, cell culture density, and cell nutrition on differentiation of rat calvarial osteoblast-like cells in vitro.

The effects of various cell isolation procedures, growth media and the cell culture density on the in vitro differentiation of neonatal rat calvarial osteoblast-like cells were investigated. Cells were isolated by enzymatic treatment, or after explant culture and inoculated as a monolayer or micromass in serum containing BGJb, or Dulbecco's Modified Eagle Medium (DMEM). The cells were kept for up to 3 weeks in culture and were then characterized, both morphologically and biochemically. The isolation technique appeared to have no effect on the differentiation process. The calvaria could be used several times as explant cultures for a reliable source of differentiating osteoblast-like cells. The cultures kept in DMEM had a significantly higher DNA content, but significantly less alkaline phosphatase activity (ALP) per DNA and protein per DNA content than the BGJb cultures. Monolayer cultures had a significantly higher DNA content than the micromass cultures, in both growth media. Furthermore, the micromass culture had a significantly higher ALP per DNA than monolayer cultures at 1 week. The morphology of all cell cultures at 3 weeks reflected the biochemical results. Only the cells grown in BGJb formed abundant ALP positive and mineralized nodules in monolayer cultures. In contrast, cells grown as micromasses formed a dense calcified area, independently of the growth medium used. DMEM promoted the proliferation, whereas BGJb stimulated the differentiation of osteoblast-like cells in monolayer cultures. Micromass cultures were less sensitive to nutritional conditions than monolayer cultures and promoted the differentiation of osteoblast-like cells.

Characterizing fretting particles by analysis of SEM images.

Detailed surface characteristics of the particles produced during fretting may well be significant in determining their biological effects. Apart from a broad size determination very few attempts have been made at devising means of describing profile textures of particles. An approach is presented for describing the nature of particle shape and surface texture, as detected on the particle profile. Careful processing and analysis of the digitised image enabled both the sizes of micro-projections and their relative numbers to be determined. Such processing of images of a large number of particles generated a considerable amount of data. An artificial neural network was used to categorise the data and to compare the nature of fretting particles generated by titanium, titanium-molybdenum and stainless steel. Although showing a tendency towards a spherical form, all three metals produced different results, with titanium showing the greatest diversity of textures and sizes, steel the least.

Freeze-substitution of rabbit tibial articular cartilage reveals that radial zone collagen fibres are tubules.

Investigations of the micromorphology of rabbit tibial articular cartilage using scanning and transmission electron microscopy revealed that the collagenous elements in the tissue form fluid-containing tubular structures. The commonly described radial or deep zone longitudinal fibres were found to be tubular structures with internal diameters of 1-2 microm. The walls of the tubules were composed of tightly packed fibrils of collagen. The tangential zone, close to the tibial plateau, was composed mainly of a spongy arrangement of collagen fibrils, containing bunches of tangentially lying small (< 1 microm) diameter tubules. The application of conventional chemical fixation techniques resulted in the fine detail of this tissue being obscured. When the tissue was frozen, followed by cryo-scanning electron microscopy or freeze-drying, prior to observation in the scanning electron microscope the tubule structures were not obviously present. It was only by applying freeze-substitution techniques, followed by critical point drying or resin embedding, that the structure was revealed clearly. Segregation of water into ice crystals did occur during the freezing process, but the formation of those crystals played no part in creating the tubular morphology observed. A similar structure was still revealed following pre-treatment with glycerol, methanol or Triton X-100, provided that concentration of these additives was not too high. The walls of the tubules in the radial region were composed of straight, longitudinally arranged as well as helically arranged, 30 nm diameter fibrils. The lumen of the tubules appears to be lined by a circumferentially arranged array of approximately 10 nm diameter fibres, spaced at regular intervals of 50-70 nm.

A novel method for viewing heavy metal stained and embedded biological tissue by field emission scanning electron microscopy.

Backscattered electron (BSE) imaging was used to display heavy metal stained biological structures of various embedded specimens. Samples were fixed, stained and embedded in resin blocks as with preparation for the transmission electron microscope (TEM). Blocks were trimmed to center the specimens in a trapezoidal face of up to 5 mm2 and their sides painted with conductive silver paint leaving the face uncovered. Blocks were sputter coated with 6-8 nm of silver, chromium or aluminum, with aluminum providing the best specimen contrast in BSE mode. Samples were examined in a field emission scanning electron microscope operated at a high emission current of 50 microA. Both the fixation protocol and microscope operating parameters were optimized to maximize the number of BSE available from the smallest probe. An accelerating voltage of 10 keV was found optimal for resolution and contrast. The technique allowed the direct visualization of embedded samples at resolutions beyond light microscopy with good contrast, without cutting sections, and avoiding grid bars obscuring areas of interest. The two dimensional images provided averaged information on the internal structures of the specimens in relation to the predicted emission depth of the BSE. The technique could be used for rapid diagnostics in pathological examinations, or for routine preselection of areas of interest within a sample face before final trimming for ultrathin sectioning for higher resolution TEM study.

Microjet impingement followed by scanning electron microscopy as a qualitative technique to compare cellular adhesion to various biomaterials.

Adhesion of cells to biomaterial surfaces is one of the major factors which mediates their biocompatibility. Quantitative or qualitative cell adhesion measurements would be useful for screening new implant materials. Microjet impingement has been evaluated by scanning electron microscopy, to determine to what extent it measures cell adhesion. The shear forces of the impingement, on the materials tested here, are seen to be greater than the cohesive strength of the cells in the impinged area, causing their rupture. The cell bodies are removed during impingement, leaving the sites of adhesion and other cellular material behind. Thus the method is shown not to provide quantification of cell adhesion forces for the metals and culture plastic tested. It is suggested that with highly adherent biomaterials, the distribution and patterns of these adhesion sites could be used for qualitative comparisons for screening of implant surfaces.

Lysosomal origin of the chloragosomes in the chloragogenous tissue of the earthworm Eisenia foetida: cytochemical demonstration of acid phosphatase activity.

The cytochemical localization of the lysosomal marker enzyme acid phosphatase was studied in the chloragogenous tissue of earthworms. The Gomori lead technique and the cerium capture technique were utilized. Both techniques demonstrated the chloragosomal location of this enzyme. Only a small proportion of chloragosomes presented reactivity, which suggests that these organelles are distinctly heterogeneous. The reaction product was localized in the periphery of chloragosomes, suggesting a membrane-bound compartmentalization of acid phosphatase. In addition, degenerating mitochondria and membrane whorls were observed in some chloragosomes, indicating the possibility that these organelles perform autophagosomal functions.

Estimation of gold-labelled macromolecules attached to cell surfaces.

A method for obtaining a semi-quantitative estimation of the amount of colloidal gold label attached to a cell surface is described. The X-ray emission, in a scanning electron microscope, from an even metal coating applied by diode sputter coating is used as an internal standard. The emission from the standard is used to correct for errors which would have arisen due to factors such as variable specimen surface topography. Examples of the semi-quantitative estimation of 10-nm gold-labelled wheat-germ agglutinin to L929 murine fibroblast cells are given.