Pig articular cartilage in organ culture. Effect of enzymatic depletion of the matrix on response of chondrocytes to complement-sufficient antiserum against pig erythrocytes.
Authors: S J Millroy, A R Poole
Journal: Annals of the Rheumatic Diseases (1974)

There is currently much interest in the mechanism fibroblast-like chondrocytes (Fell, Welland, Barratt, responsible for the breakdown of articular cartilage in Poole, and Green, unpublished results).rheumatoid and osteoarthritis and other jointThe following experiments were undertaken to diseases.lft has been suggested (Glynn, 1971) that find whether the influence of soft connective tissue autoallergic factors may be responsible for the on the response of articular cartilage to AS + C' chronicity of the disease, though they are unlikely to could be simulated by first depleting the matrix of be the primary cause.Langer, Gross, and Greaves isolated cartilage, devoid of this tissue, by tryptic (1972) have shown that rat articular cartilage is digestion and then cultivating the cartilage in medium autoantigenic, by injecting syngeneic chondrocytes containing AS + C'.In particular, it was impor- and showing autoreactivity by the leucocyte migration tant to see whether the breakdown of matrix would inhibition test.Moreover, antichondrocyte anti-continue once IgG had reacted with the chondrocytes bodies have also been detected in normal human, rabbit, and pig sera (R.R.A. Coombs, personal Materials and methods communication, 1972).Since normal articular cartilage is impermeable to EXPLANTS IgG (Maroudas, 1970; Lotke and Granda, 1972; Articular cartilage, without the underlying cavities of the Poole, Barratt, and Fell, 1973), except immediately invading bone marrow, was removed from the trotters of below the articular surface, and presumably to IgM young pigs (weight 68-91 kg) and prepared for culture as also in view of its much higher molecular weight described by Fell and Barratt (1973).(c.850,000), such antibodies could not act directly on TRYPSINIZATION the chondrocytes unless the permeability of the carti-Explants were incubated aseptically for 1 hr at 370 in lage matrix were increased, e.g. by enzymatic di-0 25 % trypsin (1 :250 Difco) in Hanks's balanced salt gestion.Recent studies by Fell and Barratt (1973) and solution, and were then washed 3 times in holding medium Poole and others (1973) have shown that in organ (Fell and Barratt, 1973).culture articular cartilage from young pigs is almost unaffected by exposure to complement-sufficient CULTURE MEDIA unafecte by xposre o coplemnt-sfficentThechemicallydefined medium BGJ, described byBiggers, antiserum to pig erythrocytes (AS + C'), though this Gwatkin, and Heyner (1961) supplemented with 5 mg antiserum cross-reacts with the chondrocytes.These sodium acetate and 15 mg ascorbic acid/100 ml (BGJ5) authors have shown, however, that when pig articular was used, as in previous experiments (Fell and Barratt, cartilage in organ culture is associated with soft 1973).This medium was also supplemented with serum to connective tissue either as invading bone marrow or give a final concentration of 20%.A rabbit antiserum to as an adjacent explant ofjoint capsule, in the presence pig erythocytes (AS) was raised and characterized as of AS + C' there is a loss of proteoglycan from the described by Fell and Barratt (1973).Heat-inactivated matrix and penetration of IgG throughout the normal rabbit serum (NRS) and complement (unheated depleted cartilage; AS + C' then reacts with the normal rabbit serum, C) were also prepared as described chondepletedcartiage ASr+pC'rthn rc with hed by these authors.Fragments of cartilage were cultured in chondrocytes, a small proportion of whlch are killed BGJ5 containing either 20% NRS, or 10% C' plus 1, 2*5, and the rest transformed into fibroblast-like cells or 10% AS; where necessary the total serum content which may be actively mitotic.Eventually the matrix was adjusted to 20% with NRS.Explants were otherwise disappears completely, leaving a flat sheet of viable cultured as described by Fell and Barratt (1973).