A model system of explanted cartilage has been used in vitro to determine whether insulin-like growth factor 1 (IGF 1), which promotes matrix formation is effective in the presence of cytokines such as interleukin 1 (IL1) and tumour necrosis factor (TNF), which induce net matrix depletion. IGF 1 induced a dose-dependent 2.5-fold stimulation of proteoglycan synthesis, with a half-maximal dose of 25 ng/ml. A similar relative increase occurred in response to IGF 1 (10-100 ng/ml) in cartilage cultured also with IL1 or TNF (5-500 pM). There was no detectable qualitative change in the average molecular size or charge of the aggregating proteoglycan synthesized by explants exposed to IGF 1 alone or with IL1 or TNF. The increased production of prostaglandin E2, which is initiated when IL1 or TNF bind to the chondrocytes, was the same in the presence or absence of IGF 1. The time taken for 50% of pre-labelled proteoglycan to be released from the explants (t1/2) increased in the presence of IGF 1 (100 ng/ml) from 21 to 32 days in control cultures and from 8 to 26 days in cartilage cultured with IL1 (50 pM). It is concluded that IGF 1 enhances the synthesis of aggregating proteoglycan in cartilage exposed to cytokines and can directly decrease both the basal and the cytokine-stimulated degradation of proteoglycan in cartilage.

A homogeneous preparation of catabolin from pig leucocytes caused a reversible dose-dependent (0.01-1 nM) decrease in the synthesis of proteoglycan in slices of pig articular cartilage cultured in serum-free medium. The monomers that were synthesized and secreted in the presence of catabolin had the same average hydrodynamic size and ability to aggregate as the controls, and the core protein was substituted with the same number of glycosaminoglycan chains. The chains were the same average length and charge as normal and were sulphated to the same extent as the controls. Newly synthesized extracellular proteoglycan was not preferentially degraded. A 2-3-fold increase in glycosaminoglycan synthesis occurred in control and catabolin-treated cartilage in the presence of beta-D-xyloside (1 mM), more than 80% being secreted into the medium as free chains. Decreased incorporation of sulphate was not reversed in the presence of lysosomal-enzyme inhibitors, and there was no evidence in pulse-chase experiments of increased intracellular degradation of glycosaminoglycan chains before secretion. It is concluded that catabolin-treated cartilage synthesizes a smaller number of normal proteoglycan molecules.

Porcine synovium in organ culture produces a factor that causes chondrocytes to degrade their matrix. A quantitative assay for the factor, for which the cartilage of bovine nasal septum is used, is described. Evidence is presented that the catabolic factor is a protein.

No abstract

Standard resolution MR images with fat-suppressed contrast lead to an objective and reproducible measurement of spatial dimensions of articular cartilage when analyzed semi-automatically using computerized edge-detection methods.

1. Pig synovium in tissue culture secretes a specific collagenase in a latent form. 2. The latent enzyme was concentrated by (NH4)2SO4 precipitation and activated with 4-aminophenylmercuric acetate, and the active enzyme was purified by chromatography on Ultrogel AcA44, DEAE-cellulose, heparin-Sepharose and a zinc-chelate medium to a specific activity of 53 400 units/mg. of protein. 3. The enzyme was shown to be essentially homogeneous by polyacrylamide-gel electrophoresis. 4. The purified collagenase digested collagen to give the characteristic three-quarter and one-quarter pieces.

The degradation of proteoglycan was examined in cultured slices of pig articular cartilage. Pig leucocyte catabolin (10 ng/ml) was used to stimulate the chondrocytes and induce a 4-fold increase in the rate of proteoglycan loss from the matrix for 4 days. Material in the medium of both control and depleted cultures was mostly a degradation product of the aggregating proteoglycan. It was recovered as a very large molecule slightly smaller than the monomers extracted with 4M-guanidinium chloride and lacked a functional hyaluronate binding region. The size and charge were consistent with a very limited cleavage or conformational change of the core protein near the hyaluronate binding region releasing the C-terminal portion of the molecule intact from the aggregate. The 'clipped' monomer diffuses very rapidly through the matrix into the medium. The amount of proteoglycan extracted with 4M-guanidinium chloride decreased during culture from both the controls and depleted cartilage, and the average size of the molecules initially remained the same. However, the proportion of molecules with a smaller average size increased with time and was predominant in explants that had lost more than 70% of their proteoglycan. All of this material was able to form aggregates when mixed with hyaluronate, and glycosaminoglycans were the same size and charge as normal, indicating either that the core protein had been cleaved in many places or that larger molecules were preferentially released. A large proportion of the easily extracted and non-extractable proteoglycan remained in the partially depleted cartilage and the molecules were the same size and charge as those found in the controls. There was no evidence of detectable glycosidase activity and only very limited sulphatase activity. A similar rate of breakdown and final distribution pattern was found for newly synthesized proteoglycan. Increased amounts of latent neutral metalloproteinases and acid proteinase activities were present in the medium of depleted cartilage. These were not thought to be involved in the breakdown of proteoglycan. Increased release of proteoglycan ceased within 24h of removal of the catabolin, indicating that the effect was reversible and persisted only while the stimulus was present.

Pig articular cartilage was maintained in culture for 3 days with and without porcine interleukin 1. The proteoglycans remaining in the cartilage and those released into the medium were analysed by using radioimmunoassays for the hyaluronate-binding region, link protein and keratan sulphate. In interleukin 1-treated cultures after 3 days there was 38% release of total glycosaminoglycans into the medium, 18% release of binding region, 14% release of link protein and 20% release of keratan sulphate epitope, whereas in control cultures the proportions released were much less (16, 9, 10 and 7% respectively). Characterization of the proteoglycans in the media after 1.5 days and 3 days of culture showed that interleukin 1 promoted the release of proteoglycan of large average size and also the release of link protein and of low-Mr binding region which was unattached to proteoglycan. Both the link protein and binding region released were able to bind to exogenously added hyaluronate, whereas the proteoglycan in the medium was not. The proteoglycans extracted from cultured cartilage were similar to those from fresh cartilage: they contained a high proportion of aggregating proteoglycans and some low-Mr binding region. The proportion of this binding region extracted from the interleukin 1-treated cartilage was increased. The presence of interleukin 1 in the cultures therefore appeared to increase the rate of proteolytic degradation of proteoglycan in the matrix and to lead to a more rapid loss of intact binding region, of link protein and of large proteoglycan fragments into the medium.