Abstract. Matrix vesicles, associated with initial calcification in cartilage, have been isolated from bovine fetal epiphyseal cartilage. Cartilage was digested with collagenase, then partitioned into seven fractions by differential centrifugation. The cellular fractions contained over 80% of the DNA in the digest. The extracellular fraction that contained matrix vesicles, in which apatite crystals were often seen on electron microscopy, also displayed the highest specific activity for alkaline phosphatase, pyrophosphatase, ATPase, and 5'-AMPase (EC 3.1.3

Full-depth plugs of adult human articular cartilage were cut into serial slices from the articular surface and analysed for their glycosaminoglycan content. The amount of chondroitin sulphate was highest in the mid-zone, whereas keratan sulphate increased progressively through the depth. Proteoglycans were isolated from each layer by extraction with 4M-guanidinium chloride followed by centrifugation in 0.4M-guanidinium chloride/CsCl at a starting density of 1.5 g/ml. The efficiency with which proteoglycans were extracted depended on slice thickness, and extraction was complete only when cartilage from each zone was sectioned at 20 microns or less. When thick sections (250 microns) were extracted, hyaluronic acid was retained in the tissue. Most of the proteoglycans, extracted from each layer under optimum conditions, could interact with hyaluronic acid to form aggregates, although the extent of aggregation was less in the deeper layers. Two pools of proteoglycan were identified in all layers by gel chromatography (Kav. 0.33 and 0.58). The smaller of these was rich in keratan sulphate and protein, and gradually increased in proportion through the cartilage depth. Chondroitin sulphate chain size was constant in all regions. The changes in composition and structure observed were consistent with the current model for hyaline-cartilage proteoglycans and were similar to those observed with increasing age in human articular cartilage.

1. Analysis of the purified proteoglycans extracted from normal human articular cartilage with 4M-guanidinium chloride showed that there was an age-related increase in their content of protein and keratan sulphate. 2. The hydrodynamic size of the dissociated proteoglycans also decreased with advancing age, but there was little change in the proportion that could aggregate. 3. Results suggested that some extracts of aged-human cartilage had an increased content of hyaluronic acid compared with specimens from younger patients. 4. Dissociated proteoglycans, from cartilage of all age groups, bind to hyaluronic acid and form aggregates in direct proportion to the hyaluronic acid concentration. 5. Electrophoretic heterogeneity of the dissociated proteoglycans was demonstrated on polyacrylamide/agarose gels. The number of proteoglycan species observed was also dependent on the age of the patient.

Papain has been shown by Thomas (1956) and Potter, McCluskey, Weissmann & Thomas (1960) to have a gross chondrolytic effect on rabbit ear cartilage in vitro and in vivo. It has been shown that the intravenous injection of the blood proenzyme plasminogen into rabbits during experimentally enhanced permeability conditions, which permit the diffusion and activation of the enzyme in carti

A variety of crystals have been identified in both normal and pathological connective tissues. Calcium phosphate 'cuboidal' microcrystal deposition has been found, using transmission electron microscopy (TEM), in femoral articular cartilage, where they are distributed as a band 5-50 µ m beneath the articular surface. These cuboid microcrystals have been identified as magnesium whitlockite. Our objective was to investigate their presence in the human intervertebral disc. In this study, two degenerate and 15 scoliotic discs were examined using light microscopy and TEM techniques to determine the presence of calcium phosphate crystals. Calcium pyrophosphate dihydrate (CPPD) deposition was identified in one degenerate disc specimen. Using TEM and electron probe analysis, cuboid microcrystals were identified in the annulus fibrosus and nucleus pulposus of both degenerate specimens, but not in the discs from young scoliotic patients. Cuboid microcrystal deposition was found predominantly around cells, which were mainly necrotic, with some association with extracellular lipidic/membranous debris. This is the first TEM report of whitlockite in the intervertebral disc. In one specimen coexistence of cuboid and CPPD crystal deposition was found.

Extracellular membranous matrix vesicles, which contain various phosphatases and appear to initiate hydroxyapatite formation in growth cartilage, were isolated and incubated with 45Ca2+ and shown to form mineral in the presence of ATP. There is enhanced calcification in the presence of serum and under alkaline conditions.

The thiol proteinase cathepsin B (EC 3.4.22.1), previously called cathepsin B1, was assayed in human articular cartilage by its hydrolysis of the synthetic substrate alpha-N-benzoyl-DL-arginine 2-naphthylamide. The enzyme was activated by cysteine and EDTA and completely inhibited by iodoacetamide and HgCl2. It was also partially inhibited by whole human serum. Human osteoarthrotic cartilage had increased activity when compared with normal cartilage. Cathepsin B activity of normal cartilage was age-related, being high in juveniles and declining to low values in adult and elderly individuals. Cathepsin D and cathepsin B both exhibited a zonal variation through the cartilage depth; the surface cells appeared to contain more activity than those close to the subchondral bone.

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