Differential response to compressive loads of zones of canine hyaline articular cartilage: micromechanical, light and electron microscopic studies.

Thin (100 micron) perpendicular slices of canine femoral condylar cartilage were placed horizontally on the stage of a Nachet microscope and viewed by transmitted light in the differential interference contrast mode. Each slice was held on the microscope stage by a loading rig and tested mechanically in compression. Measured loads to a maximum of approximately 2-3 MN/m2 were applied to the end of the slice corresponding to the articular surface. Photographs were taken of the cartilage before and during loading, and the distance by which selected chondrocytes were displaced was used as an index of mechanical strain, i.e., of change in length/original length. Maximum strains were observed in the superficial cartilage zone. Minimum strains were recorded in the mid-zone, at a depth corresponding to approximately 75% of the total cartilage thickness. The relative concentrations of cartilage collagen (COL) and proteoglycan (PG) were assessed by the light and electron microscopic histochemical study of cartilage sections taken from contiguous blocks. Superficial cartilage, which deformed most, had high concentrations of oriented COL fibres, low concentrations of PG. Mid-zone cartilage, which deformed least, had lower concentrations of randomly arrayed COL fibres but relatively high concentrations of PG.

Ultrastructural alterations in glycosaminoglycans of dog femoral condylar cartilage after surgical division of an anterior cruciate ligament: a study with cupromeronic blue in a critical electrolyte concentration technique.

The cationic dye, cupromeronic blue, has been used in a critical electrolyte concentration technique to analyse the ultrastructural changes in cartilage matrix glycosaminoglycans which occur in the dog anterior cruciate ligament division model of osteoarthrosis. Amorphous material appearing at the articular surface of cartilage from the stifle joints of animals subjected to open surgical division of the anterior cruciate ligament has been shown not to comprise glycosaminoglycan. The nature of this material is unknown, but it appears to replace the surface lamina of normal cartilage. It may therefore affect the mechanical properties of the superficial cartilage. The pericellular matrix around single chondrocytes or separating pairs of chondrocytes becomes enriched with sulphated glycosaminoglycan as a response to ligament section. This material is thought to be newly synthesised and secreted and reflects the increased cellular activity resulting from surgically induced canine joint disease.

Observations by light microscopy and transmission electronmicroscopy on intestinal spirochaetosis in baboons (Papio spp.).

Spirochaetes were recognised in the large bowel of 57 of 59 baboons as a basophilic fringe at the microvillous brush border of the epithelial cells. Both caecum and colon were usually affected, but seven animals had spirochaetes in the caecum alone. Examination of three animals by transmission electronmicroscopy revealed only one type of spirochaete; ring forms and cross-walls were present. Inflammatory changes were not seen in association with the infection, and the distribution of spirochaetes in 10 animals with soft or diarrhoeic faeces resembled that in normal animals.

Low temperature and conventional scanning electron microscopic observations of dog femoral condylar cartilage surface after anterior cruciate ligament division.

The left stifle joints of 16 beagle dogs were subjected to surgical division of the anterior cruciate ligament (ACL). The femoral condyles showed surface alterations and damage after 14 days when examined by low temperature (LTSEM) and conventional scanning electron microscopy (SEM). The first change was an increase in the fibrous texture of the articular cartilage surface. Folds and surface roughening were later recognised. After 112 days deformation and disruption gave way to cracking or fracture of the surface layers. Superficial damage was focal in distribution and accompanied by a decrease in the frequency of tertiary elevations. The affected areas of cartilage increased in frequency and size with time and surgery. The observations obtained from LTSEM, in which the tissue remains fully hydrated during examination, may be caused by alterations in water content and a loss of proteoglycan at the surface, together with changes in cell shape and size. Surface cracks may be the en face appearance of histological fibrillation.

Ultrastructural histochemistry of the surface lamina of normal articular cartilage.

Two collagen-poor, ultramicroscopic layers are described at the surface of canine articular cartilage. They are distinguished by staining with an electron-dense cationic dye, Cupromeronic Blue, in a critical electrolyte concentration technique and by digestion with testicular hyaluronidase. The superficial layer, approximately 50 nm thick, stained at low electrolyte concentrations but failed to stain in conditions specific for sulphated glycosaminoglycans. It was hyaluronidase-resistant and may be either glycoprotein or protein in nature. The deeper layer, 100-400 nm thick, stained positively at electrolyte concentrations specific for sulphated glycosaminoglycans but not in conditions specific for keratan sulphate. It was removed by hyaluronidase digestion. This layer probably represents a chondroitin sulphate-rich proteoglycan. These surface layers may be important in the lubrication of the articular surface and in the permeability and compression resistance of the superficial cartilage zone.

Low temperature backscattered electron imaging in the study of human tissues.

A scanning electron microscopic technique for the examination of bulk, fresh, hydrated human tissue is described. Samples of fresh tissue are frozen in liquid nitrogen against a mirror-finished copper block and planed in a cryoultramicrotome before transfer to a low temperature scanning electron microscope. After sublimation of water from the specimen surface, the tissue is examined in secondary electron and backscattered electron modes. Adjacent pieces of tissue, and those retrieved after backscattered electron observation, can be readily prepared for and examined by light and by conventional transmission electron microscopy. The method has been tested with multiple blocks taken from 6 cases of human breast carcinoma. In the backscattered electron mode, the infiltrating columns of neoplastic cells can be distinguished from mammary adipose and fibrous tissue. Within a carcinoma, the collagenous stroma, carcinoma cells, and perivascular and perineural infiltrates can be identified. These features have been contrasted with those obtained by light microscopy, by low temperature scanning, and by transmission electron microscopy. This use of backscattered electron imaging for the investigation of unfixed hydrated tissue offers the possibility that the technique could be of considerable value in the microscopy of very small samples in which, because of a need for subsequent biochemical, histochemical, and immunologic investigation, fixation and dehydration are to be avoided.

Proteoglycan association with collagen d band in hyaline articular cartilage.

Proteoglycans of canine articular cartilage were labelled for transmission electron microscopy using the cationic copper phthalocyanin dye, cupromeronic blue, in a critical electrolyte concentration method. Much of the proteoglycan appeared to be structurally unrelated to collagen but a small proportion was positioned close to fibrils. On demonstrating the characteristic collagen banding pattern with uranyl acetate and phosphotungstic acid, it was evident that proteoglycan interacted with collagen at the d band.

Synovitis after surgical division of the anterior cruciate ligament of the dog.

The synovial reaction to the surgical division of an anterior cruciate ligament in inbred beagle dogs, was studied. The left ligaments of 16 animals were divided by a 'blind' technique: the right stifle joints were also explored under aseptic conditions but the cruciate ligaments were not cut. Operation was followed by the formation of a blood-stained sterile effusion and by acute synovitis, sustained on the left, transient on the right. The effusion gradually diminished, becoming serous, but haemosiderin remained within subsynoviocytic macrophages for at least 84 days and lymphocytic foci and synovial cell hyperplasia persisted for similar times. It is suggested that interpretation of the cartilage changes in this cruciate ligament division model of osteoarthrosis should take account of the extent and duration of the post-operative inflammation.

Demonstration by X-ray microprobe analysis of relationship between chondrocytes and tertiary surface structure of hyaline articular cartilage.

Blocks of hyaline cartilage from the femoral condyles of five young adult beagles dogs were quench-frozen in nitrogen slush at 63 K. The free cartilage surfaces of blocks from three animals were examined in the secondary electron mode; the remaining specimens were cut by cryoultramicrotone (approximately 188 K) tangential to the surface to expose midzone cartilage which was examined in the backscattered electron mode. A random array of gently convex prominences was apparent at the free cartilage surfaces. When X-ray emissions were recorded from tissue immediately below these elevations, the spectra proved to be similar to those derived from midzone chondrocytes. These spectra revealed high count rates for the X-rays characteristic of P and K. By contrast, in areas of free surface remote from these prominences, and in midzone intercellular matrix, larger count rates for the X-rays characteristic of Na, S. Cl and Ca were detected. The evidence supports the hypothesis that the elevations seen on the non-loaded articular cartilage of disarticulated mammalian synovial joints are the surface representations of superficial chondrocytes.

Ultrastructural changes in dog femoral condylar cartilage following anterior cruciate ligament section.

Canine femoral condylar cartilage from stifle joints subjected to surgical division of the anterior cruciate ligament showed conspicuous, widespread alterations in chondrocyte morphology and matrix organisation when compared by transmission electron microscopy with cartilage from sham-operated and non-operated joints. Chondrocyte changes were of a kind associated with increased synthesis of matrix components; zone I cells lost their flattened shape and became rounded, cells in zones I and II developed an extensive endoplasmic reticulum, occurred in pairs more often than usual and were situated in enlarged electron-lucent lacunae. Associated with altered chondrocyte function and increased hydration, the matrix became increasingly disorganised; collagen fibrils were widely separated, disorientated and of small diameter. It is postulated that the disorder is primarily one of inappropriate cell function.

An investigation by transmission electron microscopy of freeze replicas of dog articular cartilage surfaces: the fibre-rich surface structure.

Femoral condylar cartilage blocks from the disarticulated stifle joints of five inbred beagle dogs were rapidly frozen. Single stage platinum and carbon replicas were prepared at 78 K from the unfixed, hydrated frozen material. Transmission electron microscopic investigations of the replicas were then made. Two different forms of fine cartilage surface structure were suggested by the low temperature replicas. The first, amorphous and with few fibres, was thought to represent the normal, superficial lamina obscurans. The second displayed many delicate parallel arrays of collagen fibrils with a periodic structure of 68-71 nm. Between these arrays were gently convex smooth-surfaced elevations, 150-500 nm in diameter, and therefore two orders of magnitude smaller than the tertiary surface undulations previously identified by light microscopy and by scanning electron microscopy. It is suggested that the 150-500 nm elevations seen at the replicated surface may be expanded proteoglycans, restrained laterally and deeply, less restrained superficially because of the loss, during preparation, of the lamina obscurans. Other hemispherical, replicated surface deposits 400-1700 nm in diameter are believed to be lipid.

Dermatan sulphate-rich proteoglycan associates with rat tail-tendon collagen at the d band in the gap region.

Rat tail tendon was stained with a cationic phthalocyanin dye, Cupromeronic Blue, in a 'critical-electrolyte-concentration' method [Scott (1980) Biochem. J. 187, 887-891] specifically to demonstrate proteoglycan by electron microscopy. Hyaluronidase digestion in the presence of proteinase inhibitors corroborated the results. Collagen was stained with uranyl acetate and/or phosphotungstic acid to demonstrate the banding pattern a-e in the D period. Proteoglycan was distributed about the collagen fibrils in an orthogonal array, the transverse elements of which were located almost exclusively at the d band, in the gap zone. The proteoglycan may inhibit (1) fibril radial growth by accretion of collagen molecules or fibril fusion, through interference with cross-linking, and (2) calcification by occupying the holes in the gap region later to be filled with hydroxyapatite.

Proteoglycan-collagen arrangements in developing rat tail tendon. An electron microscopical and biochemical investigation.

1. Developing tail tendons from rats (19-day foetal to 126 days post partum) were examined by electron microscopy after staining for proteoglycan with a cationic copper phthalocyanin dye. Cuprolinic Blue, in a "critical electrolyte concentration" method. Hydroxyproline was measured on papain digests of tendons, from which glycosaminoglycuronans were isolated, characterized and quantified. 2. Mean collagen fibril diameters increased more than 10-fold with age according to a sigmoid curve, the rapid growth phase 2 being during 30-90 days after conception. Fibril periodicities were considerably smaller (50-55 nm) in phases 1 and 2 than in phase 3 (greater than 62 nm). 3. Dermatan sulphate is the main glycosaminoglycuronan in mature tendon. Chondroitin sulphate and hyaluronate preponderate in foetal tissue. 4. Proteoglycan was seen around but not inside collagen fibrils. Proteoglycan and collagen were quantified from electron micrographs. Their ratios behaved similarly to uronic acid/hydroxyproline and hyaluronate/hydroxyproline ratios, which decreased rapidly around birth, and then levelled off to a low plateau coincident with the onset of rapid growth in collagen fibril diameter. 5. Dermatan sulphate/hydroxyproline ratios suggest that the proteoglycan orthogonal array around the fibril is largely dermatan sulphate. In the foetus hyaluronate and chondroitin sulphate exceed that expected to be bound to collagen. 6. An inhibiting action of chondroitin sulphate-rich proteoglycan on fibril diameter growth is suggested. 7. The distributions of hyaluronate, chondroitin sulphate and dermatan sulphate are discussed in the light of secondary structures suggested to be present in hyaluronate and chondroitin sulphate, but not in dermatan sulphate.