Explant Cultures Of Articular Cartilage Research Articles

Pharmacological actions of 17β-oestradiol on articular cartilage chondrocytes and chondrosarcoma chondrocytes in the absence of oestrogen receptors

(1) Pharmacological concentrations (greater than 10 −5 M) of 17β-oestradiol inhibited 35S-labelled proteoglycan synthesis in bovine articular cartilage explant cultures. They also inhibited 35S-labelled proteoglycan synthesis and 3H-labelled protein synthesis in cell cultures of chondrocytes from bovine articular cartilage and Swarm rat chondrosarcoma. Maximal inhibition was about 30–50%. Physiological concentrations (10 −9−10 −8 M) of oestradiol had no effect on the synthesis of either protein or proteoglycan. (2) The inhibitory action of high concentrations of oestradiol on these biosynthetic pathways is not common to all steroids since 10 −4 M cortisol had no effect on articular chondrocyte cell cultures. 10 −4 M testosterone had a similar action to oestradiol. (3) Neither physiological nor pharmacological concentrations of 17β-oestradiol had any effect on 35S-labelled proteoglycan turnover in the cartilage explant system. (4) 10 −5 M oestradiol inhibited cell division in cultures of articular chondrocytes which had entered the log growth phase. 10 −7 M oestradiol had no effect on articular chondrocyte growth. (5) In male rats implanted with silastic capsules releasing 17β-oestradiol, increase in body weight was retarded by about 25% over a period of 6 weeks, compared to control rats. Rat chondrosarcoma grew to the same size in oestrogen-treated rats as it did in controls. (6) Oestrogen receptors could not be detected in freshly isolated bovine articular chondrocyctes or in rat chondrosarcoma. (7) In conclusion, neither the mitotic rate of articular chondrocytes nor their proteoglycan metabolism is under the direct physiological control of oestradiol. Growth and biosynthetic activity of the rat chondrosarcoma chondrocytes are independent of either direct control by the hormone or control effected by oestradiol regulation of a second hormone or growth factor.

21] Catabolism and turnover of proteoglycans

Publisher Summary This chapter describes a technique for the measurement of the rate of turnover of proteoglycans in explant cultures of bovine articular cartilage and the subsequent characterization of the proteoglycans lost from and retained within the extracellular matrix of the tissue. When placed in organ culture, the metabolism of bovine articular cartilage is variable, but after approximately 5 days in culture, steady-state rates of proteoglycan synthesis and loss are attained. This chapter discusses explant culture of articular cartilage. The concentration and absolute rate of proteoglycan metabolism within the matrix of cartilage explants is related to the serum concentration of the culture medium. This chapter also presents incubation of cartilage cultures with [ 35 S] sulfate. Using cultures which possess steady-state proteoglycan metabolism it is possible to investigate proteoglycan turnover without the complication of fluctuating rates of proteoglycan metabolism. This chapter also describes measurement of turnover of 35S-labeled proteoglycans. This chapter concludes with analysis of 35 S-labeled proteoglycans retained in the extracellular matrix and appearing in the culture medium.

Stimulation of proteoglycan biosynthesis by serum and insulin-like growth factor-I in cultured bovine articular cartilage

The addition of foetal calf serum to explant cultures of adult bovine articular cartilage is known to stimulate proteoglycan synthesis in a dose-dependent manner. We have now shown the activity in serum responsible for this effect to be heat- and acid-stable, to be associated with a high-Mr complex in normal serum but converted to a low-Mr form under acid conditions. The activity has an apparent Mr approximately 10,000 and isoelectric points similar to those reported for insulin-like growth factors (IGFs). Addition of a monoclonal antibody against insulin-like growth factor-I (IGF-I) prevented foetal calf serum from stimulating proteoglycan synthesis. Physiological concentrations of recombinant IGF-I or pharmacological levels of insulin when added to cartilage cultures mimicked the proteoglycan-stimulatory activity of serum. IGF-I appeared to act by increasing the rate of proteoglycan synthesis and did not change the nature of the proteoglycan synthesized nor the rate of proteoglycan catabolism by the tissue, suggesting that IGF-I may be important in the regulation of proteoglycan metabolism in adult articular cartilage. Furthermore, IGF-I can replace foetal calf serum in the culture medium, thereby allowing the use of a fully-defined medium which will maintain the synthesis and tissue levels of proteoglycan in adult articular cartilage explants for up to 5 days.

Control of proteoglycan biosynthesis. Further studies on the effect of serum on cultured bovine articular cartilage.

Proteoglycan synthesis in explant cultures of adult bovine articular cartilage is stimulated in a dose-dependent manner when the tissue is cultured in the presence of foetal-calf serum. The stimulation of proteoglycan synthesis is paralleled by a similar increase in DNA synthesis; however, when DNA synthesis is inhibited by hydroxyurea the stimulation of proteoglycan synthesis by serum remains essentially the same. The apparent half-life of the pool of proteoglycan core protein precursor was measured in freshly isolated tissue as well as in tissue cultured for 7 days in the presence and in the absence of foetal-calf serum; under all conditions the half-life was the same, suggesting that this value is independent of the net rate of proteoglycan synthesis. In the presence of actinomycin D, an inhibitor of RNA synthesis, there was a difference in the apparent half-life of the available pool of mRNA coding for proteoglycan core protein: 8.5 h for tissue maintained in the presence of serum and 3.8 h for tissue cultured in the absence of serum. It is suggested that proteoglycan synthesis is stimulated by serum factors at the level of DNA-dependent RNA synthesis. Concomitant with an increase in the rate of proteoglycan synthesis induced by the presence of serum in the culture medium, an increase in the concentrations of several glycosyltransferases involved in chondroitin sulphate synthesis was also observed.

Age-related changes in the kinetics of release of proteoglycans from normal rabbit cartilage explants.

The release of proteoglycans from explant cultures of articular cartilage from immature and mature rabbits has been studied with the following results. At both ages the tissue proteoglycan was released in two phases: an initial extensive release (day 0 to 3) and a period of slow release (day 4 to 15). The percentage released in the initial phase was, however, significantly greater for mature (55%) than immature (38%) explants. At both ages the newly synthesized proteoglycans (in vivo labeled) were also released in two kinetic pools. Thus, graphical analysis of release data readily resolved the disappearance curves into two linear components with in vitro half-lives of 1 day and 22 days. Again, the percentage in the short half-life pool was much greater for mature (70%) than immature (40%) explants. At both ages the initial release was largely chondrocyte-mediated since freeze-thawing the tissue before culture markedly reduced proteoglycan release. At both ages the released proteoglycans were smaller than equivalent preparations of extracted proteoglycans and they were much less capable of forming aggregates with hyaluronate. The results show that there are age-dependent changes in rabbit articular cartilage that increase the proportion of proteoglycans, both total and newly synthesized, that are susceptible to rapid chondrocyte-mediated catabolism in explant cultures.

Demonstration of increased proteoglycan turnover in cartilage explants from dogs with experimental osteoarthritis.

The turnover of proteoglycans (assessed by the release into the medium of newly synthesised [35S]-proteoglycan) in explant cultures of articular cartilage from various anatomical sites of the knee joints (stifle) of mature beagles with experimental osteoarthritis has been studied with the following findings: (a) The proportion of newly synthesised proteoglycans released from cartilage explants maintained in vitro was generally increased for cartilage from operated compared with nonoperated control joints. (b) At 3 weeks after surgery there was a significant increase in the release of [35S]-proteoglycans from explants of the lateral and medial tibial plateaux of operated joints compared with sham-operated joints but not from other sites. On the other hand, when this comparison was made at 3 to 6 months after surgery, significant increases in the release of [35S]-proteoglycans were observed from cartilage of all anatomical areas except the patellar groove. (c) The release of [35S]-proteoglycan from cartilage explant cultures was dependent on live chondrocytes, since freeze-thawing the tissue immediately after labelling markedly reduced the release from both normal and osteoarthritic cartilage.