Cultured Bovine Articular Chondrocytes Research Articles

Differences between sub-populations of cultured bovine articular chondrocytes. II. Proteoglycan metabolism.

Sub-populations of bovine articular chondrocytes derived from different depths of the cartilage showed differences in accumulation of proteoglycan-rich extracellular matrix in culture. To extend these morphological studies, the synthesis and catabolism of 35S-labeled proteoglycans have been examined in similar cultures. Chondrocytes from deep zones synthesized significantly more proteoglycans than cells from the superficial zone. While all populations of chondrocytes synthesized predominantly aggregating proteoglycans, a higher proportion of isotope was present in non-aggregating proteoglycans in cultures of superficial chondrocytes, by comparison with those of deep cells. Proteoglycans were degraded more rapidly by superficial cells than by chondrocytes from deeper layers. These results correlate both with previous histochemical studies of similar cultures, and with known depth-related variations in biochemical composition of intact articular cartilage.

Differences between sub-populations of cultured bovine articular chondrocytes. I. Morphology and cartilage matrix production.

Bovine articular chondrocytes cultured in agarose gel comprise a heterogeneous population when judged by morphological and histochemical criteria. The purpose of the present experiments was to compare, under the same conditions of culture, sub-populations of chondrocytes derived from different depths of articular cartilage. Sub-populations of chondrocytes were cultured separately following their isolation from slices of articular cartilage cut from successive depths of the tissue. Chondrocytes derived from superficial and deep zones differed significantly in morphology, rate of proliferation, and activity in secreting a proteoglycan-rich extracellular matrix. The differences are sufficient to account for the heterogeneity observed in cultures of the entire cell population, and the correlate well with known variations with depth in morphology and histochemistry of intact articular cartilage. These results demonstrate that articular chondrocytes continue in culture to express metabolic differences which reflect their original anatomical location; such differences may have important functional significance.

Characterization of a specific insulin-like growth factor-I/somatomedin-C receptor on high density, primary monolayer cultures of bovine articular chondrocytes: regulation of receptor concentration by somatomedin, insulin and growth hormone

In order to obtain a phenotypically stable cell population of chondrocytes, high density primary monolayer cultures of bovine articular chondrocytes were established. Using these cultures, a specific insulin-like growth factor-I/somatomedin-C (IGF-I/SM-C) receptor was demonstrated and characterized. At 15 degrees C steady-state binding was attained by 5 h, and averaged 25% per 2.2 X 10(6) cells. Fifty per cent displacement of 125I-labelled IGF-I/SM-C by unlabelled IGF-I/SM-C occurred at concentrations of only 2.3 ng/ml, whereas IGF-II and porcine insulin were approximately 15- and 1000-fold less potent respectively. Scatchard analysis gave a linear plot, with a calculated association constant of 2.26 X 10(9) l/mol and a receptor number of 15 400 sites per cell. Preincubation of chondrocyte monolayers with either IGF-I SM-C or porcine insulin at 37 degrees C for 20 h resulted in reduction of 125I-labelled IGF-I/SM-C binding in a dose-dependent manner, although higher concentrations were required with insulin. More than 40% down-regulation of the receptor occurred with IGF-I/SM-C at concentrations of 10 nmol/l and nearly 70% reduction at 50 nmol/l. Interestingly, after preincubation with either human (h) or bovine (b) GH, 40% down-regulation of 125I-labelled IGF-I/SM-C binding was observed at concentrations of 10 mumol/l. Local production of IGF-I/SM-C by chondrocytes in response to GH stimulation may have occurred, but, because only 120 pmol IGF-I/SM-C and less than 30 pmol IGF-I/SM-C per litre were recovered from serum-free conditioned media preincubated with bGH and hGH respectively, this was not established.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-inflammatory drugs, prostanoid and proteoglycan production by cultured bovine articular chondrocytes

The effect of various anti-inflammatory drugs on the production of prostaglandins E 2 and F 2α, 6 keto PGF 1α and thromboxane B 2 by bovine articular chondrocytes was measured by radioimmunoassay. While indomethacin and meclofenamic acid caused a dose-dependent inhibition of all prostanoids measured, the effects of hydrocortisone and colchicine varied with respect to different prostanoids. Hydrocortisone (10 −7M – 10 −3M) both in the presence and absence of added arachidonic acid, resulted in an inhibition of prostaglandins E 2 and F 2, and to a lesser extent, 6 keto PGF 1α, but T×B 2 production was only slightly inhibited by the drug in the absenced of arachidonic acid and markedly increased in its presence. Colchicine (10 −7M – 10 −3M) had the opposite effect, causing an inhibition of T×B 2 and stimulating PGE 2 and 6 keto PGF 1α production. These findings suggest that certain anti-inflammatory drugs may, in addition to their action on phospholipase A 2 and cyclo-oxygenase, exert potent effects at the level of the different synthetases. In order to see whether these alterations in relative prostanoid levels affected proteoglycan metabolism, the effect of anti-inflammatory drugs on proteoglycan synthesis by cultured chondrocytes was tested using 35SO 4 labeling methodology. The results showed that the concentrations tested (10 −5M to 10 −7M), indomethacin, dexamethasone, hydrocortisone and colchicine inhibited 35SO 4 incorporation into newly synthesized proteoglycan molecules both in the presence (10 −6M) and absence of exogenous arachidonic acid. In the same concentration range choroquine had no effect. These results do not support the hypothesis of direct prostanoid involvement in the modulation of proteoglycan synthesis in articular cartilage.

Characterization of the metalloproteinase inhibitor produced by bovine articular chondrocyte cultures

Primary cultures of bovine articular chondrocytes release a latent metalloproteinase which is activated by incubation with organomercurials to degrade proteoglycans. All the enzyme present in the culture medium is latent and binds to columns of heparin-Sepharose. The yield of activity from the heparin-Sepharose columns (measured after organomercurial treatment) is approximately 300–1000% depending on the chondrocyte culture batch. Recombination of column fractions shows that the increase in activity is due to the separation of an inhibitor of the metalloproteinase by the chromatographic step. The metalloproteinase inhibitor has a molecular weight of approximately 35 000 (determined by Bio-Gel P-60 chromatography) and binds reversibly to columns of concavalin A-Sepharose. It is relatively heat stable (30 min at 60°C) and resistant to inactivation by trypsin (2 h, 37°C, 10 μg/ml trypsin). The inhibitor is active against rat uterine collagenase and gelatinase but does not affect bacterial metalloproteinases such as thermolysin and Clostridium histolyticum collagenase.

The [formula omitted] production of prostanoids by cultured bovine articular chondrocytes

Bovine articular chondrocytes, cultured as cell suspensions and monolayers, produced prostaglandin (PG) E 2 and PGI 2 (assayed as 6 keto PGF 1α), rather less PGF 2α and irregular quantities of thromboxane (Tx) B 2. Addition of foetal calf serum to the medium greatly stimulated PG production (a sixfold increase in PGE 2 and a twofold increase in 6 keto PGF 1α). Prostanoid production by cell suspension grown in serum-free medium generally plateaued after 24 hours. In the presence of 20% foetal calf serum, prostanoid production in long-term monolayer cultures increased during the first 6 days of culture. Levels of PGE 2α levels remained high. Indomethacin (10- 6M) inhibited chondrocyte PG production both in the presence and absence of added arachidonic acid (10- 4M). Prostanoids produced by chondrocytes may play a role in the modulation of cartilage metabolism in vivo .

Properties of the proteoglycan-degrading enzyme released by primary cultures of bovine articular chondrocytes

Properties of the proteoglycan-degrading enzyme released by primary cultures of bovine articular chondrocytes