Articular Chondrocytes In Monolayer Culture Research Articles

Identification of ecto-nucleoside triphosphate pyrophosphatase in human articular chondrocytes in monolayer culture

In cultured monolayers of human articular chondrocytes we have observed an enzyme activity which catalyzes the extracellular conversion of ATP to AMP and PP i. The enzyme was active at very low concentrations of ATP (μM) and exhibited optimal activity at concentrations of ATP of approx. 100 μM. The enzyme was active in intact cells as judged by measurement of the release of the cytoplasmic marker enzyme lactate dehydrogenase. No increase in production of PP i from ATP was observed on mechanically disrupting the cells and no activity was shed into the medium by intact cells. Activity was stable between days 4 and 8 after subculturing the cells and was not affected by the timing of the final medium change prior to assay. Activity was also observed with other nucleoside triphosphates (GTP, CTP and UTP). We suggest that this activity is attributable to ecto-nucleoside triphosphate pyrophosphatase. This observation may be important in relation to the pathogenesis of the human disease of chondrocalcinosis in which crystals of calcium pyrophosphate dihydrate deposit in articular cartilage.

Experimental studies on the pathogenesis of ochronotic arthropathy

Lapine adult and fetal articular chondrocytes in monolayer culture were employed as an experimental model for studying the effects of homogentisic acid on chondrocyte morphology, proliferation and synthesis of proteoglycans. Concentrations of homogentisic acid in the range from 0.1 to 100 micrograms/ml were investigated and a cytotoxic effect was detected with concentrations of 5 micrograms/ml and above. Thus, with adult articular chondrocytes this effect was seen between 36 and 48 h of subculture with 5 micrograms/ml or more of homogentisic acid present from the beginning of subculture. In fetal articular chondrocyte cultures a concentration of 1 microgram/ml elicited a statistically significant reduction (13%) in cell proliferation without altering sulphated macromolecular synthesis. Experiments with 3H-thymidine autoradiography to measure the pulse labeling index (LI) in fetal chondrocytes in vitro showed that the 5 micrograms/ml concentration of homogentisic acid present for 21 h from the beginning of subculture gave a LI of 2%, compared with 25% for controls. Thus, homogentisic acid can reduce chondrocyte proliferative capacity before morphological alterations can be detected by light microscopy. No significant alterations in the synthesis of proteoglycans were detected at concentrations below the cytotoxic level. The homogentisic acid-induced cytotoxicity and reduction of proliferation in chondrocytes represents a possible pathway by which cartilage is damaged in ochronotic arthropathy.

Effects of Polymethylmethacrylate on Rabbit Articular Chondrocytes in Monolayer Culture

The effects of polymethylmethacrylate (PMMA) on DNA, protein, and sulfated-proteoglycan synthesis by rabbit articular chondrocytes were observed in monolayer cultures. PMMA pellets in ratios of 1:1 and 1:2 (liquid monomer:powder) significantly reduced [3H]thymidine incorporation into DNA during the first 24 h of culture and less so after 48 and 72 h. The reduction in [3H]thymidine incorporation was restricted to the cohort of chondrocytes nearest the PMMA. Consequently, cellular proliferation was unaltered by PMMA. By contrast, PMMA failed to inhibit [3H]leucine or [3H]serine/35SO4 incorporation. Both control and PMMA (1:1)-treated chondrocyte CsCl density gradient medium fraction dA1 eluted as a retarded peak on Sepharose CL-2B under associative conditions. The average partition coefficient (Kav) of PMMA-treated fraction dA1 was 0.41, as compared with 0.27 for control cultures. The Kav of medium fraction dD1 (proteoglycan monomer) was unaltered. Both control and PMMA-treated dA1 fraction elution profiles on Sepharose CL-2B were altered by incubation with Streptomyces hyaluronidase, indicating the presence of proteoglycan aggregate. The PMMA-treated cultures synthesized smaller proteoglycan aggregates. Since PMMA has been a critical factor in the success of total joint arthroplasty, defining interactions of differentiated cells with the cement is imperative for an understanding of the effects of PMMA on the biology of cartilage and bone.

‘Gelatinase-like’ activity from articular chondrocytes in monolayer culture

In addition to releasing collagenase and proteoglycanase activity, rabbit articular chondrocytes in monolayer culture released into the culture medium, latent, neutral enzyme activity which when activated by p-aminophenylmercuric acetate degraded fluorescein-labeled polymeric rat tail tendon Type I collagen and the tropocollagen TC A and TC B fragments of human Type II collagen into smaller peptides at 37°C. Enzyme activity was abolished if p-aminophenylmercuric acetate-activated culture medium was preincubated with 1,10-phenanthroline, a metal chelator. Thus, articular chondrocytes in monolayer culture are capable of producing neutral proteinases which acting together can result in complete degradation of tendon and cartilage collagen to small peptides.

Effect of purified growth factors on rabbit articular chondrocytes in monolayer culture. II. Sulfated proteoglycan synthesis.

The effect of 3 purified peptide growth factors--platelet-derived growth factor (PDGF), epidermal growth factor (EGF), pituitary fibroblast growth factor (FGF)--heat-inactivated fetal bovine serum (FBS), insulin, and 0.2 mM ascorbate on synthesis of sulfated proteoglycan by rabbit articular chondrocytes was studied in monolayer culture. Growth of the cells increased linearly as the concentration of heat-inactivated FBS rose from 0 to 30%. Glycosaminoglycan (GAG) synthesis (35SO4/micrograms DNA) was enhanced as the concentration of heat-inactivated FBS went from 0 to 5%. At higher levels of serum, radiosulfate incorporation declined progressively. Two modes of study of the test factors were used: 1) the dose of the factor was increased while the serum concentration was fixed at a low basal level (1% heat-inactivated FBS); 2) the dose of the test factor was kept constant but the level of heat-inactivated FBS varied from 0 to 10%. There was an inverse relationship between GAG and DNA synthesis when proliferation of cells was increased by EGF and platelet lysate. PDGF (1 U/ml) stimulated radiosulfate incorporation as well as DNA formation in the serum-free medium; the values for GAG synthesis did not increase as the serum concentration increased, but the cell mass did. The action of FGF was intermediate between that of EGF and PDGF: with 50 ng FGF/ml, increasing concentrations of serum caused a large progressive reduction of radiosulfate incorporation as growth was stimulated. In basal medium, however, FGF caused mild enhancement of GAG synthesis. Insulin increased aggregatable proteoglycan production far out of proportion to its growth-promoting activity in the presence of 1% heat-inactivated FBS. The response was effaced when higher concentrations of serum were employed. Ascorbate had a unique anabolic effect, increasing both cell growth and proteoglycan synthesis that is not suppressed by higher concentrations of serum. The content of serum and its several peptide and hormonal components thus have divergent effects on growth and proteoglycan synthesis in cell culture. This phenomenon must be taken into account in studying biochemical processes and pharmacologic reactions of articular chondrocytes in vitro.

Effect of purified growth factors on rabbit articular chondrocytes in monolayer culture. I. Dna synthesis.

The ability of purified growth factors, insulin, ascorbate, and several other compounds to stimulate DNA synthesis by rabbit articular chondrocytes was studied in monolayer culture. Platelet-derived growth factor (1 U/ml), pituitary fibroblast growth factor (1-100 ng/ml), and epidermal growth factor (1-50 ng/ml) were stimulatory in a basal medium supplemented with 1% heat-inactivated fetal bovine serum. Insulin, 1-50 micrograms/ml, has small growth-promoting effects but acted synergistically with platelet-derived, pituitary fibroblast, and epidermal growth factors. Increasing concentrations of serum up to 10% enhanced the growth-promoting action of the purified factors, but not of insulin. There were indications of cooperation between insulin and bovine serum albumin and dexamethasone. Ascorbate (0.2 mM) reduced or had little growth-promoting action in the basal medium. At 5 and 10% serum concentrations, however, ascorbate promoted DNA synthesis as effectively as the purified growth factors. No significant stimulatory effect was shown by transferrin, thrombin, L-glutamine, putrescine, selenous acid, dexamethasone, 7S nerve growth factor, or multiplication-stimulating activity.

The effect of soluble sodium urate on the proliferation and proteoglycan synthesis of lapine articular chondrocytes in monolayer culture.

The effects of soluble monosodium urate (350, 50, and 5 mumol/l) on the proliferation and proteoglycan synthesis of lapine articular chondrocytes were studied in a monolayer culture system. Cellular proliferation in vitro was unaffected by urate treatment. Chondrocytes treated with 50 mumol/l sodium urate showed a 26% reduction in binding of 35SO4 to matrix macromolecules. However, this reduction did not achieve statistical significance. The 350 and 5 mumol/l concentrations did not alter 35SO4 binding. It is proposed that monosodium urate has no marked direct effect on chondrocyte viability, proliferation and proteoglycan synthesis.

Cell-cell interactions in the rheumatoid joint.

Although rheumatoid synovium has been extensively studied in organ culture, particularly with respect to the synthesis of prostaglandins and proteinases, the behaviour of normal human synovium in culture has been much less well characterized. In this study, cultures of fragments of normal synovial tissue produced significantly less prostaglandin E (PGE) than cultures of rheumatoid synovium. The difference, however, did not persist when synovial cells obtained by enzymatic dispersion of normal and rheumatoid tissue were compared in monolayer culture. Production of PGE could be reactivated in both normal and rheumatoid synovial cells by products of mononuclear blood cells and also by factors in culture medium obtained after incubation of fragments of either normal or rheumatoid synovial tissue. These products of mononuclear cells and of synovial tissue also stimulated the production of PGE by human articular chondrocytes in monolayer culture. If these types of cellular interactions observed in vitro also occur in the arthritic joint as a result of the failure of normal control mechanisms, they could play a part in the irreversible destruction of joint structures.

Effect of platelet lysate on growth and sulfated glycosaminoglycan synthesis in articular chondrocyte cultures.

Human platelet lysate (PL) has a strong growth promoting action on rabbit articular chondrocytes in monolayer culture. The responsible factor is heat stable (56 degrees C, 30 minutes) and above 10,000 MW. PL (80 microgram protein/ml) reduces cell protein content and sulfated glycosaminoglycan synthesis. Synthesis of DNA is stimulated within the first 12 hours of culture but the decline in radiosulfate incorporation lags. PL acts to a slight extent on chondrocytes in serum-free media, but its effect is potentiated by "platelet-poor human serum" or fetal bovine serum. PL is one of several agents having such effects on chondrocytes cultured in serum-containing media. Stimulation of growth in this cell type thus reduces nonreplicative biosynthetic activity nonspecifically. In the epiphyseal growth plate and in pathologic alterations of permeability of the matrix of articular cartilage, platelet-derived factors, together with somatomedin or other cofactors in serum, may be the principal mediator of growth of chondrocytes in vivo.

The effect of chondrocyte growth factor on membrane transport by articular chondrocytes in monolayer culture.

Chondrocyte growth factor (CGF), a contaminant of pituitary glycoprotein hormones, stimulates growth of cultured lapine articular chondrocytes while depressing SO4-proteoglycan synthesis. To study its effect on membrane transport, NIH-bTSH and two other preparations with comparable CGF activity were employed. In early log phase (36 hr) cultures CGF (64 microgram/ml) did not alter thymidine (dThd) uptake during the first 5 min. By 15 min however, TCA-precipitable dThd was 4-fold greater than in controls while the TCA-soluble fraction remained the same. CGF increased deoxy-glucose (DG) uptake in 36-hr old cultures. At 66 hr, CGF reduced DG transport. The transport of cycloleucine (CL) and aminoisobutyric acid (AIB) was reduced by CGF in 36 and 66-hr old cultures. There was a dose dependency between CGF concentration, the lowered uptake of DG, CL and AIB, and cell protein content. The effect of CGF on DG transport and dThd incorporation into DNA was not immediate but required prior exposure of the cells to CGF. CGF did not alter DG transport in rabbit or mouse fibrocytes or Chang liver cells. This and the reported finding that pituitary fibroblast growth factor (FGF), increases amino acid transport in other cells suggests that the biological specificity of CGF may not be identical to that of FGF.

The effect of low oxygen concentration on growth, glycolysis, and sulfate incorporation by articular chondrocytes in monolayer culture.

AbstractRadiosulfate incorporation, DNA synthesis, and glycoiysis by rabbit articular chondrocytes and skin fibrocytes in monolayer culture were compared under different concentrations of oxygen (19, 6.8, 1.3, and 0.6%). Cell for cell, chondrocytes under each condition had higher levels of radiosulfate incorporation and glycolysis than did fibrocytes. Growth of the fibrocytes was reduced approximately 20% when the oxygen concentration was reduced to 6.8%, but that of chondrocytes was unaffected. A progressive Pasteur effect was observed with both cell types. Radiosulfate incorporation was reduced at low oxygen concentrations. The findings in this cell culture system are thus at variance with other published evidence that hypoxia is a major histogenetic force favoring chondrogenic expression.

Effect of low oxygen tensions on glucose-metabolizing enzymes in cultured articular chondrocytes.

SummaryThe specific activities of 12 glucose-metabolizing enzymes of rabbit articular chondrocytes in monolayer culture were generally higher than those of cutaneous fibrocytes. A greater LDH:MDH ratio in the former suggests preservation of a chondroid phenotype in vitro. At reduced oxygen concentrations, levels of LDH, MDH, PGM, aldolase, enolase, PFK and PGI increased. IDH and GAPDH decreased under these conditions while HK and G6DPH were unaffected.

Collagen synthesis by articular chondrocytes in monolayer culture

Chondrocytes grown in monolayer culture from the joints of young rabbits synthesized collagen provided that ascorbic acid (50 μg/ml) was present in the nutrient medium. The collagen formed in vitro by whole articular cartilage was composed of α1 chains only, suggesting a chain composition [α1(II)] 3. By contrast the collagen produced by the cultured chondrocytes contained both α1 and α2 chains with a varying chain ratio (α1:α2 usually 2:1; occasionally as high as 4:1). The articular chondrocytes, under these conditions of culture, thus apparently produced a collagen resembling that of skin and cultured cutaneous fibrocytes having a chain composition [α1(I)] 2α2.

A pituitary growth-promoting factor for articular chondrocytes in monolayer culture.

A heat-labile anterior pituitary factor exerts a strong mitogenic action on articular chondrocytes in secondary monolayer culture. At the same time secretion of macromolecular radiosulfate into the medium by the stimulated cells is markedly reduced. This factor was found in NIH bovine and ovine TSH as well as LH but not in more purified (Condliffe-Bates, Pierce) preparations of TSH. The response, measured by the DNA content of the cell pellet, was dose-dependent; the lowest effective concentration was 1 μg NIH TSH/ml culture medium. The effect was fairly selective for chondrocytes, rabbit and human, and was displayed to much lesser degree by skin fibroblasts and 4 other cell types studied. FSH had a smaller effect while GH, prolactin, ACTH and a preparation having high EPS activity were relatively ineffective. Of 19 other hormone preparations examined, only crude HCG gave a comparable mitogenic response. Insulin, 0.1 U/ml, had a consistent but small effect. Estradiol-17β and diethylstilbesterol (10 μg/ml)...

Failure of Rumalon to increase sulfate incorporation by articular chondrocytes in monolayer culture.

Failure of Rumalon to increase sulfate incorporation by articular chondrocytes in monolayer culture.

Sulfate incorporation by articular chondrocytes in monolayer culture.

AbstractArticular chondrocytes in primary monolayers or subcultures synthesize sulfated macromolecules, presumably mucopolysaccharides. Intracellular 35SO4 incorporation by postnatal rabbit chondrocytes reached a steady level by 24 hr of incubation; the nondialyzable extracellular (centrifuged medium + trypsin digest) counts increased over 72 hr, when they were up to 100 times the intracellular value. Cell for cell, radiosulfate counts in each fraction were higher in chondrocytes than fibroblasts. Data from a 3‐year‐old rabbit were comparable to those from recently weaned animals. Secretion of sulfated mucopolysaccharide was also found in cultures of adult human and bovine articular chondrocytes. Although Ham's F12 medium is advantageous in establishing a primary culture of chondrocytes, sulfate incorporation was greater when Dulbecco‐Eagle medium, with or without supplemental L‐proline, was used for subcultures. Ascorbic acid (5 mg/100 ml) added to an already confluent monolayer had no consistent effect.