Effect of osmolarity on glycosaminoglycan production and cell metabolism of articular chondrocyte under three-dimensional culture system

OBJECTIVES:This study examined how physiological levels of extracellular osmolarity influence proteoglycan accumulation in articular chondrocytes in a three-dimensional culture system.METHODS:Cells were obtained from metacarpal phalangeal joints of 18-24 month bovine. They were cultured for 6 days in alginate beads at 4 million cells/ml in DMEM containing 6% FBS under 21% O2. Medium osmolarity was altered by NaCl addition over the range 270-570 mOsm and monitored using a freezing point osmometer. Profiles across intact beads were determined by manual counting using fluorescent probes and transmission electron microscope. Lactate production was measured enzymatically and glycosaminoglycan (GAG) accumulation was measured using a modified dimethylmethylene blue assay. Rate of sulfate GAG synthesis was measured using a standard 35S-sulfate radioactive method.RESULTS:The cell viability was similar for the high and low osmolarity cultures. However, confocal microscopy showed that the cells were the largest under 270 mOsm and became smaller with increasing osmotic pressure. GAG production was largest in the 370mOsm, and the capacity for GAG production and cell metabolism (lactate production) was low under hypo-osmolarity and hyper-osmolarity, and cell deaths were often observed on electron microscopy.CONCLUSIONS:In our model the prevailing osmolarity was a powerful regulator of GAG accumulation by cultured chondrocytes. These results thus indicate GAG synthesis rates are regulated by GAG concentration, with implications both for the aetiology of osteoarthritis and for tissue engineering.     

Lymphokine-mediated suppression of chondrocyte glycosaminoglycan and protein synthesis

Spontaneously released and T cell mitogen augmented lymphokine produced by human mononuclear cells has been shown to induce a concentration dependent reversible suppression of chondrocyte glycosaminoglycan and protein synthesis without significantly enhancing chondrocyte catabolic activity. The modulatory factor(s) is of T cell origin and is trypsin, pronase, and heat sensitive. Prostaglandin inhibitors failed to influence factor formation or activity. Although eluting from Sephadex G-100 over a wide range, peak activity had an approximate molecular weight of 53,000 and appeared distinct from recognized forms of lymphotoxin.     

Alterations in articular chondrocyte growth and proteoglycan synthesis due to prostanoid precursors

Summary The effects of the prostaglandin precursors, dihomo-gamma-linolenic acid and arachidonic acid, on chondrocyte proliferation, proteoglycan synthesis and morphological structure were studied using lapine articular chondrocytes in vitro. Neither substance exerted a cytotoxic effect on chondrocytes. Dihomo-gamma-linolenic acid caused a dose-dependent inhibition of chondrocyte proliferation (8% and 35% reduction at 10 and 100 mol/l respectively) (P<0.01), whereas arachidonic acid failed to cause any significant alteration: 10 mol/l of dihomo-gamma-linolenic acid stimulated proteoglycan synthesis by 14% (P<0.01), whilst 100 mol/l elicited a reduction of 14% (P<0.01); 100 mol/l of arachidonic acid also caused a statistically significant inhibition (31%) (P<0.001) of ³⁵SO₄ incorporation into proteoglycans. The inhibitory effects on proteoglycan synthesis may be mediated by intracellularly synthesized prostaglandins, which are known to exert this effect. This may also help explain the susceptibility of articular cartilage to damage with increasing age, as arachidonic acid is found in increasing concentrations in the superficial layers of articular cartilage.     

Effect of anti-inflammatory drugs on sulphated glycosaminoglycan synthesis in aged human articular cartilage.

The anti-inflammatory drugs, sodium salicylate, indomethacin, hydrocortisone, ibuprofen, and flurbiprofen, were examined for their effects on sulphated glycosaminoglycan synthesis in aged human cartilage in vitro. Cartilage was obtained from femoral heads removed during surgery and drug effects were found to vary significantly from one head to another. Statistical analysis of the results showed that sodium salicylate exhibits concentration-dependent inhibition of glycosaminoglycan synthesis over the concentration range used. Indomethacin, hydrocortisone, and ibuprofen, at concentrations comparable to those attained in man, caused a statistically significant depression of sulphated glycosaminoglycan synthesis in cartilage from some femoral heads but not others, reflecting the variable response of human articular cartilage to anti-inflammatory drugs. Sodium salicylate and indomethacin at higher doses produced significant (Pless than 0-005) inhibition of sulphated glycosaminoglycan synthesis in all femoral heads studied. The results for flurbiprofen were less conclusive; this compound appears not to inhibit glycosaminoglycan synthesis over the concentration range used.     

Some recombinant human cytokines stimulate glycosaminoglycan synthesis in human synovial fibroblast cultures and inhibit it in human articular cartilage cultures

Recombinant human cytokines were compared for their effects on glycosaminoglycan (GAG) synthesis in human synovial fibroblast cultures and human articular cartilage explant cultures. In fibroblast cultures, recombinant human interleukin-1 alpha (rHuIL-1 alpha), rHuIL-1 beta, and recombinant human tumor necrosis factor alpha (rHuTNF alpha) stimulated hyaluronic acid (HA) production and, to a lesser extent, sulfated GAG production, while recombinant human gamma-interferon did not have a significant effect. Half-maximal stimulation of HA by rHuIL-1 beta was 0.14 pM, while stimulation for rHuIL-1 alpha and rHuTNF alpha was 1.6 pM and 32 pM, respectively. Indomethacin (10 micrograms/ml) had no influence on HA stimulation by cytokines, while hydrocortisone (2-10 micrograms/ml) caused a significant reduction. In articular cartilage cultures, the cytokines inhibited production of sulfated GAGs. The activity of rHuIL-1 beta was greater than that of rHuIL-1 alpha (half-maximal inhibition at 0.71 pM and 4.7 pM, respectively) and both were considerably more active than rHuTNF alpha; gamma-interferon again had no significant effect. Neither indomethacin nor hydrocortisone influenced cytokine-induced inhibition by either rHuIL-1 preparation. These studies indicate that cytokines released during an inflammatory process may affect GAG synthesis in human joint tissues and may have opposite effects on GAG synthesis in different types of connective tissues.     

Histologic assessment of lymphokine-mediated suppression of chondrocyte glycosaminoglycan synthesis

An experimental model has been developed to histologically assess the effect of T cell mitogen-induced lymphokines derived from rabbit splenocytes on proteoglycan matrix depleted auricular cartilage explant glycosaminoglycan synthesis. Explant exposure to lymphokine was shown by light and electron microscopy to significantly suppress chondrocyte glycosaminoglycan regenerative capacity. This inhibitory effect was reversible in that synthetic activity could be restored by placement of explants in control media after as long as 12 days of lymphokine exposure.     

Salicylate-induced depletion of endogenous inorganic sulfate. Potential role in the suppression of sulfated glycosaminoglycan synthesis in murine articular cartilage

Sodium salicylate has been shown to suppress glycosaminoglycan (GAG) synthesis by articular hyaline cartilage in vitro. We investigated the in vivo effect of sodium salicylate on murine patellar cartilage, using incorporation of intraperitoneally administered 35S-sulfate as a measure of sulfated GAG synthesis. Our results indicated that a single dose of sodium salicylate (200 mg/kg) inhibited in vivo sulfated GAG synthesis by 56%, compared with controls, and had no effect on sulfated GAG breakdown. A striking finding was that sodium sulfate treatment reduced the serum concentration of inorganic sulfate from 1.1 mM to approximately 0.3 mM, and that this serum reduction was associated with a twofold increase in urinary excretion of sulfate. Using anatomically intact patellar cartilage, in vitro studies clearly showed that, in concentrations reached in vivo (greater than or equal to 1 mM), salicylate suppressed murine chondrocyte GAG synthesis. However, in the presence of serum, the effects of 1 mM salicylate were abolished. We also found that sulfated GAG synthesis was clearly inhibited at low concentrations of sulfate (less than 0.5 mM). Our data indicate that sodium salicylate can suppress articular chondrocyte sulfated GAG synthesis in vivo, and that this effect may particularly be due to a drug-induced reduction of endogenous sulfate.     

Cartilage inorganic pyrophosphate elaboration is independent of sulfated glycosaminoglycan synthesis

Inorganic pyrophosphate (PPi), a product of glycosaminoglycan synthesis, may be cosecreted with matrix proteoglycan to reach the extracellular site where calcium pyrophosphate dihydrate crystals form. To test this hypothesis, sulfated glycosaminoglycan synthesis by articular cartilage in culture was stimulated or inhibited while the effect on extracellular PPi was measured. When stimulated by 0.8 mM xyloside to increase 35SO4 incorporation (mean +/- SEM % of control 183 +/- 16, n = 5), PPi accumulation changed little (from 54 +/- 6 pmoles/mg to 63 +/- 8 pmoles/mg of cartilage wet weight). Inhibition of sulfation with monensin or diethylcarbamazine disproportionately lowered 35SO4 incorporation compared with PPi elaboration. Using 60 mM diethylcarbamazine, PPi production was preserved (105 +/- 8% mean +/- SEM) compared with control cultures, while sulfation was markedly inhibited (7 +/- 1%). This dissociation of sulfate incorporation and PPi secretion indicates that it is not likely that glycosaminoglycan sulfation is the source of the PPi that escapes from chondrocytes to participate in the formation of extracellular crystals.     

Histologic assessment of lymphokine-mediated suppression of chondrocyte glycosaminoglycan synthesis.

An experimental model has been developed to histologically assess the effect of T cell mitogen-induced lymphokines derived from rabbit splenocytes on proteoglycan matrix depleted auricular cartilage explant glycosaminoglycan synthesis. Explant exposure to lymphokine was shown by light and electron microscopy to significantly suppress chondrocyte glycosaminoglycan regenerative capacity. This inhibitory effect was reversible in that synthetic activity could be restored by placement of explants in control media after as long as 12 days of lymphokine exposure.     

The effect of age on glycosaminoglycan synthesis in rabbit articular and costal cartilages

The rates of synthesis of chondroitin 4- and 6-sulfate were compared for articular and costal cartilage from rabbits of three different ages. Cartilages were labeled in vitro with 3H glucosamine and 35SO4. The glycosaminoglycans were isolated and separated and assayed for radioactivity and uronic acid content. The data obtained showed that in immature articular cartilage the rate of synthesis of chondroitin 4-sulfate is considerably greater than for chondroitin 6-sulfate and that this condition is reversed as the animal ages. These results are concordant with the known variation in distribution of the glycosaminoglycans with age and suggest that the chondrocyte alters its synthetic activity with advancing age.     

Effects of diphosphonates on glycosaminoglycan synthesis and proteoglycan aggregation in normal adult articular cartilage

The effects of disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP) and disodium dichloromethylene diphosphonate (Cl₂MDP) on glycosaminoglycan synthesis and macromolecular organization of proteoglycans have been examined in normal adult canine cartilage. One to 500 μM of either compound produced reversible inhibition of glycosaminoglycan synthesis to about 70% of control levels, whereas lower concentrations had no effect. Based on Sephadex G-200 chromatography, the average hydro-dynamic size of glycosaminoglycans in diphosphonate-treated cartilage was similar to that of those in control cartilage. In most cases proteoglycan aggregates from diphosphonate-treated cartilage were smaller in hydro-dynamic size than those from control cartilage, as judged by Sepharose 2B elution profiles. The size of purified proteoglycan subunits, obtained after dissociation of the aggregates with 4 M guanidinium chloride or after incubation of the aggregates with hyaluronic acid β1 ← 3 hydrolase, was not affected by the diphosphonates. Furthermore, proteoglycans from diphosphonate-treated cartilage did not interact in vitro with hyaluronic acid, suggesting that diminished proteoglycan aggregation may have resulted from an abnormality in the hyaluronate-binding region of the proteoglycan core protein.     

Partial reversal by beta-d-xyloside of salicylate-induced inhibition of glycosaminoglycan synthesis in articular cartilage

While net ³⁵S-glycosaminoglycan synthesis in normal canine articular cartilage was suppressed by 10⁻³M sodium salicylate to about 70% of the control value, addition of xyloside (10⁻⁶M–10⁻³M) to the salicy-late-treated cultures led to a concentration-dependent increase in glycosaminoglycan synthesis, which rose to 120–237% of controls. Similar results were obtained when ³H-glucosamine was used to measure glycosaminoglycan synthesis, confirming that salicylate suppresses and xyloside stimulates net glycosaminoglycan synthesis, and not merely sulfation. Salicylate (10⁻³M) did not affect the activity of xylosyl or galactosyl transferase prepared from canine knee cartilage, and net protein synthesis was unaltered by either salicylate or xyloside. The proportion of newly synthesized proteoglycans existing as aggregates when cartilage was cultured with xyloside was similar to that in controls, although the average hydrodynamic size of disaggregated proteoglycans and of sulfated glycosaminoglycans was diminished.     

The influence of hydrocortisone on aggrecan metabolism in human articular chondrocyte cultures: comparison of two different matrices

OBJECTIVE: Numerous reports of negative effects as well as protective effects of glucocorticoids on articular cartilage convinced us to study the influence of hydrocortisone on aggrecan synthesis of isolated phenotypically stable human articular chondrocytes cultured in two different matrices. METHODS: Macroscopically normal human articular cartilage was obtained from femoral condyles within 24 hours postmortem. Chondrocytes were isolated and cultured in gelled agarose or in alginate. After 14 days in culture, hydrocortisone was added for 5 days at concentrations ranging from 0.005 microgram to 1 mg/ml for the agarose cultures and from 0.005 microgram to 1 microgram/ml for the alginate culture system. Aggrecan synthesis was measured by the incorporation of 35Sulphate, and the proportion of neosynthesized aggrecan that bound to hyaluronan to form aggrecan aggregates was analyzed by gel chromatography. RESULTS: At concentrations from 0.005 to 1 microgram/ml, hydrocortisone was found to produce a similar dose-dependent stimulation of aggrecan synthesis in both matrices. The synthesis of aggrecans remained at the same level for concentrations of 1 microgram/ml up to 100 micrograms/ml of hydrocortisone. When supraphysiological concentrations of hydrocortisone were added the aggrecan synthesis rate plateau declined. Simultaneously with the increase in aggrecan synthesis, the proportion of low-molecular weight 35S-proteoglycans decreased in favour of 35S-aggrecan aggregates and monomers in the agarose system. The chondrocytes cultured in alginate showed this increase of aggrecan aggregates and monomeric aggrecans in both the cell-associated and the inter-territorial matrix. CONCLUSION: Hydrocortisone is a stimulator of aggrecan synthesis by normal human articular chondrocytes cultured in vitro. The two culture systems (agarose and alginate) tested in this experiment showed a comparable aggrecan synthesis rate, increasing under the influence of hydrocortisone at concentrations up to 1 microgram/ml. The proportions of 35Sulphate incorporated in aggrecan aggregates and monomeric aggrecan were also higher under the influence of hydrocortisone.     

Effect of loading and prednisolone treatment on the glycosaminoglycan content of articular cartilage in dogs.

The effect of prolonged prednisolone administration on glycosaminoglycan components was studied by means of determinations of galactosamine, glucosamine, uronic acid, sulphate and sialic acid content in normally loaded and spared articular cartilages of the knee-joints in dogs. After 40 days, all glycosaminoglycan components decreased by 58% in the spared cartilage, as compated with the controls. Prolonged administration of 0.5 mg prednisolone/kg body weight (corresponding to a low therapeutic dose), elicited a further decrease in galactosamine, whereas the other components remained practically unchanged. In the normally loaded articular cartilage of prednisolone-treated dogs the glycosaminoglycan components decreased by about 11-31% compared with untreated controls. The quantitative data indicate that in the changes occurring the glycosaminoglycan composition of the articular cartilage due to relief of function and to prednisolone treatment, chondroitin suplphate is mainly involved. The changes due to prednisolone treatment are dependent on the functional demand of the joint.     

A comparison of the effects of two gold-containing therapeutic agents on articular chondrocyte growth in vitro

Chondrocyte structure and function under the influence of two gold-containing therapeutic agents, aurothioglucose and triethylphosphine gold, were studied in a monolayer culture system for cultivating lapine articular chondrocytes. The functional parameters investigated were chondrocyte proliferation and the incorporation of 35SO4 as indicator of glycosaminoglycan synthesis. Aurothioglucose (0.2, 1 and 10 micrograms/ml) failed to affect either parameter and caused no cytotoxic effect detectable with the light microscope. Triethylphosphine gold (1.5 and 3 micrograms/ml) prevented subcultured chondrocytes from forming monolayers and was cytotoxic to chondrocytes present in established monolayers, studied with the light and scanning electron microscope. A 0.3 microgram/ml concentration did not alter chondrocyte proliferation or sulphate incorporation and was not cytotoxic.     

Stimulation of dna synthesis by ascorbate in cultures of articular chondrocytes

The addition of 0.2 mM Na L-ascorbate increased the incorporation of ³H-thymidine by rabbit articular chondrocytes in cell and organ culture. The stimulatory response of explants to ascorbate was potentiated by pretreatment of the cartilage with 0.2% clostridial collagenase (type 1) or trypsin for 15–30 minutes. In explants there was a latent period of 3 to 4 days before increased labeling of the nuclei could be detected. The effect was transient and declined after 8 days of culture. It was more evident in organ cultures of immature (3-month-old) than 2- to 3-year-old rabbits. Age differences were not detected in cell cultures. Explants of adult human articular cartilage were stimulated by ascorbate when the medium was supplemented with 10% fresh human serum but not by fetal bovine serum. The findings indicated that synthesis of DNA by articular chondrocytes in situ is regulated by responsiveness of the cells proper to compounds such as vitamin C, by properties of the extracellular matrix, and by factors in the serum. Ascorbate was cytotoxic at concentrations >0.2 mM in the presence of certain batches of serum and when added to monolayers before the cells attached to the surface of the flask.     

The effect of transforming growth factor-beta 1 on glycosaminoglycan production by human marrow cultures

We have assayed the effect of transforming growth factor-beta 1 (TGF-beta 1), a potent modulator of hematopoiesis, on glycosaminoglycan production in human marrow cultures. Glycosaminoglycans are a component of the extracellular matrix known to affect cell growth and differentiation. TGF-beta 1 and [35S]sulfate were added simultaneously to hematopoietically active human marrow cultures, and radiolabeled glycosaminoglycan production was determined by cetylpyridinium chloride precipitation. TGF-beta 1 at 15 ng/ml for 72 h increased [35S]sulfate incorporation into media glycosaminoglycans to 190% of control levels but did not affect the [35S]sulfate incorporation into cell-associated glycosaminoglycans. Approximately 90% of the glycosaminoglycans in the media fraction and 85% of the glycosaminoglycans in the cell-associated fraction were susceptible to degradation by chondroitin ABCase in both treated and control cultures. Pulse-chase experiments suggested that the increase in glycosaminoglycan [35S]sulfate incorporation was not due to decreased glycosaminoglycan degradation. This concentration of TGF-beta 1 did not alter nonadherent granulocyte-macrophage colony-forming unit (CFU-GM) number per flask but significantly decreased the more primitive adherent CFU-GM number per flask (by 50%-70%). These data suggest that the ability of TGF-beta 1 to modulate hematopoiesis may be due, in part, to its effects on glycosaminoglycan production.     

Insulin-like growth factor-I augments chondrocyte hypertrophy and reverses glucocorticoid-mediated growth retardation in fetal mice metatarsal cultures

The study aims were to improve our understanding of the mechanisms of glucocorticoid-induced growth retardation at the growth plate and determine whether IGF-I could ameliorate the effects. Fetal mouse metatarsals were cultured for up to 10 d with dexamethasone (Dex; 10(-6) m) and/or IGF-I and GH (both at 100 ng/ml). Both continuous and alternate-day Dex treatment inhibited bone growth to a similar degree, whereas IGF-I alone or together with Dex caused an increase in bone growth. GH had no effects. These observations may be explained at the cellular level; cell proliferation within the growing bone was decreased by Dex and increased by IGF-I and these effects were more marked in the cells of the perichondrium than those in the growth plate. However, the most prominent observation was noted in the hypertrophic zone where all treatments containing IGF-I significantly increased (3-fold) the length of this zone, whereas Dex alone had no significant effect. In conclusion, Dex impaired longitudinal growth by inhibiting chondrocyte proliferation, whereas IGF-I stimulated chondrocyte hypertrophy and reversed the growth-inhibitory Dex effects. However, the IGF-I-mediated improvement in growth was at the expense of altering the balance between proliferating and hypertrophic chondrocytes within the metatarsal.     

Effect of nalidixic acid, pipemidic acid and cinoxacin on chondrocyte metabolism in explants of articular cartilage

Explants of immature bovine articular cartilage were exposed to nalidixic acid, pipemidic acid and cinoxacin at one and ten times the human therapeutic plasma level for 7 days. Only nalidixic acid had significant effects on the chondrocyte metabolism. 20 micrograms/ml nalidixic acid caused an increase of 35S-sulfate incorporation into glycosaminoglycans at day 7. Two hundred micrograms/ml nalidixic acid inhibited the incorporation of 3H-thymidine into DNA. The incorporation of 35S-sulfate into glycosaminoglycans was decreased at day 0, while at day 7 the incorporation had returned to the control value. Pipemidic acid and cinoxacin had no significant effects on either the 3H-thymidine or the 35S-sulfate incorporation.