I. ap Gwynn

Collagen fibre arrangement in the tibial plateau articular cartilage of man and other mammalian species

Experimental animal models are frequently used to study articular cartilage, but the relevance to man remains problematic. In this study animal models were compared by examination of the collagen fibre arrangement in the medial tibial plateau of human, cow, pig, dog, sheep, rabbit and rat specimens. 24 cartilage samples from each species were prepared and maximum cartilage thickness in the central tibial plateau measured. Samples were fixed, dehydrated, freeze‐fractured and imaged by scanning electron microscopy (SEM). At low magnification, 2 different arrangements of collagen fibres were observed: leaf‐like (human, pig, dog) and columnar (cow, sheep, rabbit, rat). The porcine collagen structure was the most similar to that of man. This arrangement was consistent from the radial to the upper zones. Under higher magnification at the surface of the leaves, the collagen was more randomly oriented, whereas the columns consisted of parallel collagen fibrils. The maximum thickness of cartilage did not correlate with the type of collagen arrangement but was correlated with the body weight of the species (r=0.785). When using animal models for investigating human articular cartilage function or pathology, the differences in arrangement of collagen fibres in tibial plateau cartilage between laboratory animals should be considered especially if morphological evaluation is planned.

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 (5nm) and duration of silver‐enhancement (6min), 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.

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 effect of sublethal lead exposure on the ultrastructure and on the distribution of acid phosphatase activity in chloragocytes of earthworms (Annelida, Oligochaeta)

SummaryLaboratory experiments were conducted to study the effects of the exposure to a sublethal concentration (500 p.p.m.) of lead on the ultrastructure and acid phosphatase compartmentalization of the chloragogenous tissue of earthworms, Eisenia foetida. For the cytochemical demonstration of acid phosphatase activity, lead and cerium were used as capturing agents. In both cases there was a change in the compartmentalization of acid phosphatase, the enzyme activity being localized within the chloragosomes in controls, but distributed throughout the cytosol in treated animals. In addition, acid phosphatase activity increased following lead exposure. At the ultrastructural level, disruption of the chloragosomal membranes, an increase in chloragosomal fusion processes and vesiculation of the cytoplasm were evident. Moreover, an enhanced release of chloragosomes to the extracellular space was found in lead-exposed worms.

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

SummaryThe 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.

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 (>40nm) 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 (1–5nm) 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.

On the egg investments and fertilization reaction inPomatoceros triqueter L.

SummaryThe ultrastructure of unfertilized and fertilized egg investments ofPomatoceros triqueter is described. A characteristic feature is the presence of a hitherto unrecorded additional investment layer, inserted between the chorion and the “outer border layer”, which is referred to as the intermediate layer. The fertilization reaction consists of the release of cortical granule material into the perivitelline “space”, and the severing of the microvillous cytosome to “outer border layer” connections, thus allowing an increase in the width of the perivitelline “space”. More ridged corrugations are found on the outer surface of the investments in the fertilized as compared with the unfertilized condition.

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.

Formation and function of cytoplasmic annulate lamellae in the eggs ofPomatoceros triqueter L.

SummaryDuring an ultrastructural study of the eggs of the serpulid wormPomatoceros triqueter L., annulate lamellae were frequently encountered in the cytoplasm. In particular, some observations indicated that they originate by successive outfoldings of the nuclear envelope. Consequently, annulate lamellae must consist of alternating layers of nuclear and cytoplasmic material, each layer being separated by part of the nuclear envelope. It was observed that there was a similarity between nuclear and inter-annulate lamellar material. Moreover tritiated thymidine was shown to be present in the stacks. It is inferred that this system might well function as an efficient means of transporting nuclear material into the cytoplasm.

A new method for investigating the undersurface of cell monolayers by scanning electron microscopy

Glow discharge is commonly used for cleaning the inside of coating units and for cleaning hard surfaces before carbon or metal evaporation procedures. In this study it has been used to remove the embedding medium to reveal, for scanning electron microscope (SEM) study, the undersurfaces of Balb/c 3T3 fibroblastic cells that have been cultured on Thermanox discs and embedded in LR White resin. Ten to twenty‐minute ionization times were found to reveal the largest area of the undersurface without causing damage to the cells. Chemical etching of the resin was also shown to reveal the undersurface of the cells, but caused some damage, preventing successful re‐embedding for transmission electron microscopy, and at higher magnifications revealed less detail. A circular impression within the main outline of the cells was observed in many cells, which is considered to reflect the presence of a nucleus. The undersurfaces of most cells, after applying both methods of etching, displayed a number of very short processes. Subsequent transmission electron microscopy of ultrathin sectioned, re‐embedded, areas of the gold sputter‐coated blocks revealed the depth of ionization that had occurred and confirmed that the specimens observed in SEM were the undersurfaces of cells. This method can be modified to study the attaching surface of any organism to a substratum.