The degradation of collagen in pig synovium in vitro and the effect of colchicine.

Colchicine induced a rapid destruction of the collagenous matrix of pig synovial explants in culture in the presence of serum. The most efficacious doses were 0.01-0.1 micrograms/ml (2.5 x 10(-8) M - 2.5 x 10(-7) M). The histological progression of the tissue breakdown induced by colchicine was very similar, although faster, to that described for other agents (Fell et al., 1986), with cells having basophilic nuclei accumulating in areas of fibril degradation. The loss of collagen correlated with an increase in collagenase production and at the peak of resorption (6 to 8 days) active collagenase was present in the culture media. Immunocytochemical methods demonstrated active collagenase bound to collagen fibrils after only 4 days in culture, before significant collagen degradation could be observed histologically. Collagen breakdown was completely inhibited by cortisol, and partially inhibited by indomethacin: only the inhibition by indomethacin could be reversed by exogenous prostaglandin E2. Vinblastine at a higher dose was as effective as colchicine but the lumicolchicines, which do not disrupt microtubules, were ineffective. Although the precise mechanism of action of colchicine is unknown, this culture system provides a useful in vitro model for increasing our understanding of the cellular mechanisms of tissue breakdown and for elucidating the roles of active collagenase and related metalloproteinases.

The promotion and inhibition of collagen-breakdown in organ cultures of pig synovium: the requirement for serum components and the involvement of cyclic adenosine 3':5'-monophosphate (cAMP).

In many pathological situations connective tissue cells acquire the ability to degrade the macromolecular components of their extracellular matrix. To study the destruction of collagen we used organ cultures of porcine synovial tissue. In the presence of 15% rabbit serum explants shrink considerably during 10-14 days, owing to early loss of interfibrillar material followed by retraction and local destruction of collagen fibres, partly by phagocytosis. These changes, and the release of latent collagenase into the medium, are largely inhibited by cortisol and partially by indomethacin. Collagen destruction can be greatly accelerated by the addition to the culture medium of one of the following: sodium fluoride, 3-isobutyl-1-methylxanthine, dibutyryl cyclic adenosine 3':5'-monophosphate or forskolin; these agents are known to affect cyclic adenosine monophosphate metabolism and our results suggest strongly that a change in the intracellular levels of cyclic adenosine monophosphate is a key-step in the process leading to the increased catabolism of collagen. With these compounds the destruction of collagen is largely extracellular; the histological changes and the increased levels of collagenase associated with the destruction can be prevented by cortisol and, except in the case of dibutyryl cyclic adenosine monophosphate, at least partially by indomethacin. Without serum only 3-isobutyl-1-methylxanthine sometimes causes drastic breakdown of collagen. This model system should be of great benefit in exploring the mechanisms involved in collagen destruction.

The effect of cortisol on porcine articular tissues in organ culture.

(1) The effects of hydrocortisone succinate (1.0--0.01 micrograms HC/ml of medium) on porcine articular tissues in organ culture have been studied by histological and biochemical methods. (2) 1.0 and 0.1 micrograms HC/ml considerably inhibited the severe breakdown of matrix that occurs in living and dead cartilage explained in contact with synovial tissue. (3) The depletion of matrix in living cartilage cultivated in the same dish as but not in contact with synovial tissue, is much diminished when the medium contains 1.0 or 0.1 micrograms HC/ml. (4) Cartilage grown in the used medium of synovial tissue loses both proteoglycan and hydroxyproline; the addition of HC to the used medium has little (1.0 microgram HC/ml) or 0.1 microgram HC/ml) no inhibitory effect. If, however, the used medium is from synovial tissue that has been cultured in the presence of 1.0 or 0.1 micrograms HC/ml, loss of proteoglycan and collagen from the cartilage is much reduced. (5) In isolated cartilage in normal medium there is outgrowth of cells from the cut surface and some loss of proteoglycan and collagen; outgrowth is completely, and loss of matrix components partially suppressed by 1.0 micrograms HC/ml. (6) Isolated synovial tissue cultured in normal medium shrinks to about one-third of its original size, loses much of its collagen and secretes neutral metallo-proteinases into the medium; in the presence of 1.0 microgram HC/ml these changes are largely prevented. The explants also secrete an inhibitor of metallo-proteinases the production of which is not reduced by HC.

The effect of synovial tissue on the synthesis of proteoglycan by the articular cartilage of young pigs.

Explants of pig articular cartilage were grown in organ culture in the presence of synovial tissue; controls consisted of paired explants that were cultivated in isolation. To find whether the synovial tissue affected synthesis of sulfated proteoglycan by the cartilage, 35SO4 was added to the medium and its incorporation into the cartilage examined by both biochemical assay and autoradiography. The synovial tissue severely inhibited the uptake of 35SO4, but if the synovium was removed after 8 days cultivation and the cartilage was grown in isolation for a further 4 days, incorporation of 35SO4 equalled and sometimes surpassed that of controls which had been grown without synovium continuously for 12 days. Synovium did not prevent the formation of new cartilage on the cut surfaces of the explants, but it reduced the incidence of newly formed cartilage.

The breakdown of collagen by chondrocytes.

The chondrocytes of pure articular cartilage from young pigs display collagenolytic activity when the cartilage is cultivated by an organ culture method in medium containing 5--10 IU retinol/ml. Breakdown of collagen occurs in all regions of the cartilage; in the thicker explants it is greatest and most frequent in those that contain the deepest zone composed of proliferating and hypertrophic chondrocytes; it is also very drastic in thin explants composed mainly of superficial fibro-cartilage.

A cartilage catabolic factor from synovium.

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.

The effect of synovial tissue on the breakdown of articular cartilage in organ culture.

UNLABELLED: Living (pig) cartilage in contact with synovium lost both proteoglycan and collagen and sometimes became reduced to a mass of fibroblast-like chondrocytes without matrix; dead cartilage lost proteoglycan but less collagen. Similar changes appeared in living cartilage grown at a distance from the synovium but in the same dish; dead cartilage was unaffected. CONCLUSION: the synovium has a) a direct, presumably enzymatic action on cartilage matrix and b) an indirect effect mediated through the chondrocytes.

The pig synovium, II. Some properties of isolated intimal cells.

I. The effect of trypsinization. Certain physiological and immunological properties of isolated intimal synoviocytes were studied in various in vitro systems. Suspensions of the synoviocytes were obtained by incubating sheets of joint capsule in a solution of trypsin and then scraping off the intimal cells with a small knife. Synovium was examined immediately after trypsinization, but before scraping, by light and electron microscopy. The intimal cells had withdrawn many of their long processes, acquired lamelliform pseudopodia, and somewhat resembled cells of the A-type (Barland et al. 1962). To test the viability of the trypsinized synovium, fragments of trypsinized joint capsule were explanted in organ culture with the intima in contact with a Millipore substrate (Fell et al. 1976). After two days the intimal synoviocytes had become branched and, as in untreated control explants, now resembled B-cells. II. Phagocytosis and opsonic adherence. In a freshly prepared suspension of synoviocytes scraped from trypsinized synovial tissue many cells were still branched, but during 1 1/4 hours' incubation in serum-containing medium the majority had withdrawn their processes and become rounded. Cells in the branched form had little capacity for phagocytosis, but most of those in the rounded form were actively phagocytic. After suspensions of intimal cells had been incubated with opsonized sheep erythrocytes, most of the rounded, but none of the few remaining branched cells, had formed rosettes. Intimal cells from scraped synovial tissue were maintained in Sykes-Moore chambers for periods of up to 48 hours. Although the young pig synovium contains only a small proportion of macrophage-like (A-type) cells, in the cultures the cell population consisted of cells indistinguishable from macrophages, with a few small colonies of typical fibroblasts. In marked contrast to the fibroblasts, the macrophage-like cells in the Sykes-Moore cultures were highly phagocytic and formed conspicuous opsonic rosettes. Excessive phagocytosis inhibited subsequent rosetting by the macrophage-like cells. III. The effect of antiserum, with and without complement, on synovial cells. In view of earlier work by Fell & Barratt (1973) the effect of rabbit antiserum to pig erythrocytes (AS) with and without serum complement (C') on intimal synoviocytes in a Sykes-Moore chamber was investigated. AS+C' either lysed the macrophage-like cells or caused them to fuse into multinucleate giant cells. In the presence of AS without C', the macrophage-like cells formed large lakes of multinucleated cytoplasm. Fibroblasts in the same cultures were much more resistant to lysis by AS+C' and did not form multinucleate giant cells when exposed to AS either with or without C'.

The pig synovium. I. The intact synovium in vivo and in organ culture.

1. The normal synovium of the metacarpophalangeal joints of young pigs was examined by light and electron microscopy with special reference to the superficial layer (intima). 2. Cells of the macrophage-like or A-type (Barland et al. 1962) constituted only a small proportion of the intimal synoviocytes; the majority were of the intermediate and B-types. 3. Synovial villi were explanted on Millipore filters and maintained as organ cultures. The intimal cells in contact with the Millipore formed long branched processes which penetrated deeply into the substrate; these cells, which had a very well-developed endoplasmic reticulum, resembled those of the B-type. The synoviocytes at the upper (free) surface of the villus withdrew their long processes, acquired lamelliform pseudopodia, and their endoplasmic reticulum regressed; they were similar in appearance to the A-type. 4. In the organ cultures the highly branched cells (B-type) next to the Millipore were less phagocytic than the rounded cells (A-type) at the free surface of the villus.

The capacity of pig articular cartilage in organ culture to regenerate after breakdown induced by complement-sufficient antiserum to pig erythrocytes.

Explants of pig articular cartilage including invading marrow and subchondral bone (together='invasion zone') were cultivated for 10 or 14 days in complement-sufficient rabbit antiserum to pig erythrocytes (AS+C'), and then transferred to heat-inactivated normal rabbit serum (NRS) for a period of recovery. In AS+C' the cartilage matrix lost first proteoglycan and then collagen, but the cells remained viable. The degradation of collagen was accompanied by a fibroblastic transformation of the chodrocytes, first seen in the region immediately above the invasion zone. Immunohistochemical studies showed that after cultivation in AS+C', IgG antibodies entered areas in which the matrix was depleted of proteoglycan and reacted strongly with the majority of chondrocytes; those that had under-gone fibroblastic transformation exhibited little or no reaction. The degree of recovery in NRS depended on the extent to which the matrix had broken down in AS+C'. If degradation of collagen was confined to the region immediately above the invasion zone, and elsewhere only proteoglycan had been lost, new metachromatic material was regenerated in the non-calcified cartilage, and the fibroblast-like chondrocytes resumed their normal appearance and regained their reactivity with the IgG antibodies of AS; new cartilage and chondroid tissue appeared in the cavities of the invasion zone. If degradation of collagen and fibroblastic transformation of chondrocytes had spread throughout the cartilage, breakdown continued in NRS and cartilage disappeared completely above the invasion zone; new cartilage waw sometimes formed in the cavities of the invasione zone.

The effect of complement-sufficient antiserum against pig erythrocytes on pig articular tissues in organ culture.

Pig cartilage devoid of marrow or synovium is virtually unaffected by rabbit complement-sufficient antiserum to pig erythrocytes (AS + C'), probably because normal cartilage matrix excludes immunoglobulins. When synovium or invading marrow is present, AS + C' causes depletion of the proteoglycan, with consequent entry of IgG into the cartilage, followed by the breakdown of collagen; the chondrocytes assume a fibroblastic form and their reaction with the antiserum is greatly reduced. If degradation of the matrix has not advanced too far, new matrix is regenerated when the explant is transferred to control medium. Experiments on the depletion of pure cartilage by exposure to retinol, indicate that articular cartilage produces enzymes capable of degrading protoglycan but not collagen. The work was done in collaboration with colleagues at the Strangeways Laboratory and in the Division of Immunology, Pathology Department, University of Cambridge.

Breakdown of proteoglycan and collagen induced in pig articular cartilage in organ culture.

Explants of articular cartilage from young pigs were maintained in organ culture for 10--16 days, and degradation of matrix was induced by retinol or complement-sufficient antiserum. The percentage breakdown of proteoglycan and collagen (as hydroxyproline release) was measured. The response of the cartilage depended on whether or not the explants were cut so as to include some of the invading marrow ('invasion zone'). In media containing retinol, cartilage lost up to three-quarters of its proteoglycan whether the invasion zone was present or not, but very little of its collagen unless this region was included. In the presence of complement-sufficient anti-serum, however, cartilage without the invasion zone was virtually unaffected, but both proteoglycan and hydroxyproline were released when invasion zone was included; here proteoglycan release began almost immediately, but there was a time-lag of 6--8 days before a substantial amount of hydroxyproline appeared in the medium. Histological examination of sample explants from the experiments supported the biochemical findings. The possible significance of the results in relation to rheumatoid arthritis is discussed.