ATP-induced chondrocalcinosis.

OBJECTIVE. To determine whether adult articular cartilage mineralizes in the presence of ATP. METHODS. Intact adult porcine articular cartilage and monolayers of chondrocytes were cultured in physiologic media containing ATP, and mineralization was measured as retention of 45Ca. Cartilage was analyzed by electron microscopy. RESULTS. Articular cartilage sequestered 45Ca when incubated with 100 microM ATP. Use of the ATP analog alpha, beta-methylene ATP did not promote mineralization and addition of pyrophosphatase inhibited mineralization, indicating that hydrolysis of ATP to AMP and inorganic pyrophosphate is necessary for the process to occur. Mineral was concentrated in articular cartilage vesicles in the perichondral area. CONCLUSION. Adult articular cartilage mineralizes in the presence of ATP, in a manner similar to that found with isolated matrix or articular cartilage vesicles. This supports the notion that these structures have a role in chondrocalcinosis.     

Articular Cartilage Vesicles Generate Calcium Pyrophosphate Dihydrate-Like Crystals in Vitro

Objective. To identify the morphology of a mineral-forming fraction of adult porcine hyaline articular cartilage digest and characterize the mineral it forms. Methods. Electron microscopy, Fourier transform infrared (FTIR) spectroscopy, x-ray microanalysis, compensated polarized light microscopy, and biochemical studies including ¹⁴C-labeled UDPG pyrophosphohydrolase radiometric assay. Results. This fraction of articular cartilage digest contained membrane-limited vesicles resembling growth plate cartilage matrix vesicles and formed mineral after only 24 hours in physiologic salt solution containing 1 mM ATP. The mineral contained inorganic pyrophosphate, 95% of which derived from ATP, and phosphate, 93% of which derived from inorganic phosphate in the medium. The FTIR spectrum of this mineral closely resembled the spectrum of standard calcium pyrophosphate dihydrate (CPPD) crystals. Compensated polarized light microscopy showed positively birefringent, rod-shaped crystals morphologically identical to CPPD. Ca:P ratios, defined by energy-dispersive microanalysis, were also consistent with CPPD. Conclusion. The articular cartilage vesicle fraction of porcine hyaline cartilage is capable of generating mineral that strongly resembles CPPD.     

Type II collagen levels correlate with mineralization by articular cartilage vesicles

Objective

Pathologic mineralization is common in osteoarthritic (OA) cartilage and may be mediated by extracellular organelles known as articular cartilage vesicles (ACVs). Paradoxically, ACVs isolated from OA human cartilage mineralize poorly in vitro compared with those isolated from normal porcine cartilage. We recently showed that collagens regulate ACV mineralization. We sought to determine differences between collagens and collagen receptors on human and porcine ACVs as a potential explanation of their different mineralization behaviors.

Methods

ACVs were enzymatically released from old and young human and porcine hyaline articular cartilage. Western blotting was used to determine the presence of types I, II, VI, and X collagen and various collagen receptors on ACVs. Type II collagen was quantified by enzyme‐linked immunosorbent assay. Biomineralization was assessed by measuring the uptake of ⁴⁵Ca by isolated ACVs in agarose gels and by ACVs in situ in freeze‐thawed cartilage.

Results

As previously shown, isolated human ACVs mineralized poorly in response to ATP compared with porcine ACVs, but human and porcine ACVs mineralized similarly in situ in freeze‐thawed cartilage. Type II collagen levels were 100‐fold higher in isolated human ACVs than in porcine ACVs. Type II collagen in human ACVs was of high molecular weight. Transglutaminase‐crosslinked type II collagen showed increased resistance to collagenase, suggesting a possible explanation for residual collagen on human ACVs. Expression of other collagens and collagen receptors was similar on human and porcine ACVs.

Conclusion

Higher levels of type II collagen in human ACV preparations, perhaps mediated by increased transglutaminase crosslinking, may contribute to the decreased mineralization observed in isolated human ACVs in vitro.

     

Promotion of articular cartilage matrix vesicle mineralization by type I collagen

OBJECTIVE: Calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals occur in up to 60% of osteoarthritic joints and predict an increased severity of arthritis. Articular cartilage vesicles (ACVs) generate CPPD crystals in the presence of ATP and BCP crystals with added beta-glycerophosphate. While ACVs are present in normal articular cartilage, they mineralize primarily in cartilage from osteoarthritic joints. The aim of this study was to explore the hypothesis that ACV mineralization is regulated by components of the surrounding extracellular matrix. METHODS: Porcine ACVs were embedded in agarose gels containing type II and/or type I collagen and/or proteoglycans. Mineralization was measured as (45)Ca accumulation stimulated by ATP or beta-glycerophosphate and reflects both nucleation and growth. Synthetic CPPD and BCP crystals were embedded in similar gels to isolate the effect of matrix components on crystal growth. RESULTS: After establishing baseline responsiveness of ACVs to ATP and beta-glycerophosphate in agarose gels, we examined the ability of ATP and beta-glycerophosphate to stimulate mineral formation in gels containing various matrix components. Type II collagen suppressed the ability of ATP to stimulate mineralization, while a combination of type II plus type I collagen increased the effect of ATP and beta-glycerophosphate on mineralization. Type I collagen affected ACV mineralization in a dose-responsive manner. Neither type of collagen significantly affected crystal growth or levels of mineralization-regulating enzymes. Proteoglycans suppressed mineral formation by ACVs in gels containing both type I and type II collagen. CONCLUSION: Cartilage matrix changes that occur with osteoarthritis, such as increased quantities of type I collagen and reduced proteoglycan levels, may promote ACV mineralization.     

Mechanisms of calcium transport in human colonic basolateral membrane vesicles

Human colon has been suggested to play an important role in calcium absorption especially after extensive disease or resection of the small intestine. We have previously demonstrated the presence of a carrier-mediated calcium uptake mechanism in the human colonic luminal membrane vesicles. Current studies were, therefore, undertaken to investigate the mechanism(s) of calcium exit across the basolateral membrane domain of the human colon. Human colonic basolateral membrane vesicles (BLMVs) were isolated and purified from mucosal scrapings of organ donor colons, utilizing a technique developed in our laboratory. ⁴⁵Ca uptake was measured by a rapid filtration technique. ⁴⁵Ca uptake represented transport into the intravesicular space as evidenced by an osmolarity study and by the demonstration of Ca²⁺ efflux from calcium preloaded vesicles by Ca²⁺ ionophore A23187. Calcium uptake was stimulated by Mg²⁺ ATP. The kinetic parameters for ATP-dependent Ca²⁺ uptake revealed saturation kinetics with Michaelis constant (K _m) of 0.22 ± 0.04 M and a maximum rate of uptake (V _max) of 0.38 ± 0.12 nmol/mg protein/min. The K _m of ATP concentration required for half maximal Ca²⁺ uptake was 0.39 ± 0.04 mM. ATP-stimulated calcium uptake into these vesicles was further stimulated in the presence of calmodulin and was inhibited by calmodulin antagonist, trifluoperazine. Uptake of ⁴⁵Ca into BLMVs was markedly inhibited by cis-Na⁺ but was significantly stimulated by trans-Na⁺ (40–50% stimulation). Our results demonstrate the presence of a Mg²⁺/ATP-dependent calmodulin-regulated Ca²⁺ transport system and a Na⁺–Ca²⁺ exchange process in the human colonic basolateral membranes.     

Articular cartilage vesicles generate calcium pyrophosphate dihydrate-like crystals in vitro.

OBJECTIVE. To identify the morphology of a mineral-forming of adult porcine hyaline articular cartilage digest and characterize the mineral it forms. METHODS. Electron microscopy, Fourier transform infrared (FTIR) spectroscopy, x-ray microanalysis, compensated polarized light microscopy, and biochemical studies including 14C-labeled UDPG pyrophosphohydrolase radiometric assay. RESULTS. This fraction of articular cartilage digest contained membrane-limited vesicles resembling growth plate cartilage matrix vesicles and formed mineral after only 24 hours in physiologic salt solution containing 1 mM ATP. The mineral contained inorganic pyrophosphate, 95% of which derived from ATP, and phosphate, 93% of which derived from inorganic phosphate in the medium. The FTIR spectrum of this mineral closely resembled the spectrum of standard calcium pyrophosphate dihydrate (CPPD) crystals. Compensated polarized light microscopy showed positively birefringent, rod-shaped crystals morphologically identical to CPPD. Ca:P ratios, defined by energy-dispersive microanalysis, were also consistent with CPPD. CONCLUSION. The articular cartilage vesicle fraction of porcine hyaline cartilage is capable of generating mineral that strongly resembles CPPD.     

Bradykinin,a new potential mediator of inflammation-induced bone resorption

The effect of bradykinin and desArg⁹-bradykinin on bone was studied in cultures of calvarial bones taken from 6–7-day-old mice. Bradykinin, at and above a 3-nM concentration, caused a dose-dependent stimulation of bone mineral mobilization and matrix degradation. Bradykinin-stimulated resorption was inhibited by calcitonin, an increased concentration of phosphate in the culture medium, hydrocortisone, dexamethasone, indomethacin, meclofenamic acid, naproxen, and 5,8,11,14-eicosatetraenoic acid. The results suggest that bradykinin stimulates osteoclast-mediated bone resorption by a process that is dependent on endogenous prostaglandin production. The stimulatory effect of bradykinin, but not of parathyroid hormone and prostaglandin E₂, was potentiated by the angiotensin-converting enzyme inhibitor, BPP_5a. Treatment with carboxypeptidase B did not affect the capacity of the peptide to stimulated ⁴⁵Ca release. DesArg⁹-bradykinin (1 m̈mole/liter) stimulated ⁴⁵Ca release to the same degree as did bradykinin. Bradykinin (3 m̈M) did not affect the degradation of cartilage proteoglycans, as assessed by the release of ³⁵S-sulfate from prelabeled calf articular cartilage in organ culture. These findings suggest that generation of bradykinin in inflammatory lesions of rheumatoid arthritis and periodontitis may contribute to the bone resorptive process seen in the joints and alveolar bone; however, bradykinin does not directly activate chondrocytes into a catabolic state.     

EFFECT OF SALICYLATE ON PROTEOGLYCAN METABOLISM IN NORMAL CANINE ARTICULAR CARTILAGE IN VITRO

In osteoarthritis, diminished aggregation of articular cartilage proteoglycans affects tissue biomechanics. Since salicylates are commonly employed in treatment of osteoarthritis, we examined the effect of sodium salicylate on proteoglycan metabolism and aggregation in normal canine articular cartilage. At salicylate concentrations of 10⁻³ M, 5 x 10⁻³ M and 10⁻² M, net proteoglycan synthesis in normal canine articular cartilage was 73%, 42%, and 16%, respectively, of control levels. Catabolism of glycosaminoglycans in the presence of 10⁻³ M salicylate (which corresponds to a serum salicylate level of 20–25 mg %) was the same as that in control cartilage, while higher concentrations of the drug increased the rate of degradation. The hydrodynamic size of newly synthesized proteoglycan aggregates and of disaggregated proteoglycans was unaffected by sodium salicylate.     

Transglutaminase activity in aging articular chondrocytes and articular cartilage vesicles

Objective. Transglutaminases (TGases) (E.G. 2.3.2.13) catalyze a posttranslational modification of proteins and are associated with biomineralization in growth plate cartilage. Type II TGase participates in the activation of latent transforming growth factor β (TGFß), a crucial factor for both normal cartilage mineralization and the pathologic mineralization that results in calcium pyrophosphate dihydrate (CPPD) crystal formation in aging articular cartilage. To explore a possible association between TGase levels and CPPD crystal formation in mature articular cartilage, TGase activity in articular chondrocytes from old and young pigs and in the articular cartilage vesicle (ACV) fraction of porcine articular cartilage was examined. In addition, the effects of TGase inhibitors on the production of inorganic pyrophosphate (PPi), a process necessary for CPPD crystallogenesis, were determined. Methods. TGase activity was measured with a radiometric assay in cultured articular chondrocytes from the knee joints of old (3–5 years old) and young (2–6 weeks old) pigs and in the ACVs. PPi levels were measured in chondrocyte-conditioned media in the presence of TGase inhibitors or control compounds. Results. Levels of TGase activity in the cytosolic fraction of old chondrocytes were 7-fold higher than those in identically cultured young chondrocytes. The mean ± SD activity level in the membrane fraction of lysed chondrocytes was 6.0 ± 0.6 units/mg protein in old articular chondrocytes and was undetectable in young chondrocytes. In ACVs, the mean ± SD TGase activity level was 1.23 ± 0.1 units/mg protein. Type II TGase protein was present in chondrocyte cytosol and in ACVs. TGase activity was increased by TGFß to 120% of control values (P < 0.01), and decreased by insulin-like growth factor 1 to 80% of control values (P < 0.01). TGase inhibitors blocked media accumulation of PPi, an essential precursor of CPPD crystal formation, and a sensitive marker of TGFß effect. Conclusion. These data suggest a potential link between TGase activity and processes of pathologic biomineralization that result in CPPD crystal formation in aging articular cartilage.     

Calcification of isolated matrix vesicles and reconstituted vesicles from fetal bovine cartilage.

Ca deposition by isolated matrix vesicles from fetal calf growth plate cartilage and by a deoxycholate extract from matrix vesicles that included their phosphatase was studied under defined in vitro conditions. Electron microscopy showed that after removal of deoxycholate and lyophilization of the vesicle extract, new vesicles were reconstituted, often with multiple membrane layers. Both intact calf vesicles and reconstituted vesicles initiated Ca deposition maximally when supplied with ATP, GTP, CTP, or UTP. Only nucleoside triphosphates supported Ca deposition well; mono- and diphosphoesters, although hydrolyzed, were ineffective as substrates. Nucleoside triphosphates supported Ca deposition even if the final [Ca] X [P] reached in the reaction mixture was below a metastable level (3.5 mM2), suggesting that matrix vesicles or reconstituted vesicles promote calcification by localizing Ca or PO4 or both. ATP or GTP supported Ca deposition readily at concentrations ranging from 0.25 to 1.0 mM but, at 2.5 and 5.0 mM, Ca deposition was inhibited. The ATPase of intact matrix vesicles and reconstituted vesicles was stimulated by addition of Ca2+ and Mg2+. Ca deposition did not require additional Mg2+. These results lend support to the hypothesis that matrix vesicles and their phosphatases play an important role in mineralization.     

Ntp pyrophosphohydrolase in human chondrocalcinotic and osteoarthritic cartilage: some biochemical characteristic

Nucleoside triphosphate pyrophosphohydrolase activity was first detected in articular cartilage in previous studies at our laboratory. In this report, the enzyme is partially characterized with respect to its pH optimum and Km. The enzyme was metal-dependent and was active in the presence of 1 mM Ca⁺⁺. It was inhibited by several substances, including cysteine and dithiothreitol. Its activity was not inhibited by tetramisole at concentrations which inhibited 100% of the pyrophosphatase activity in the same extracts. It functioned most effectively on ATP, but also on UTP, CTP, and GTP. A role for scavenging nucleotides and production of pyro-phosphate in osteoarthritic and chondrocalcinotic cartilage is postulated.     

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.     

Mitochondrial oxidative phosphorylation is a downstream regulator of nitric oxide effects on chondrocyte matrix synthesis and mineralization

OBJECTIVE: Increased chondrocyte nitric oxide (NO) and peroxynitrite production appears to modulate decreased matrix synthesis and increased mineralization in osteoarthritis (OA). Because NO inhibits mitochondrial respiration, this study was undertaken to directly assess the potential role of chondrocyte mitochondrial oxidative phosphorylation (OXPHOS) in matrix synthesis and mineralization. METHODS: We studied cultured human articular chondrocytes and immortalized costal chondrocytes (TC28 cells). We also assessed the effects of antimycin A and oligomycin (inhibitors of mitochondrial complexes III and V, respectively) on chondrocyte mitochondrial respiration, ATP synthesis, and inorganic pyrophosphate (PPi) generation, and the mineralizing potential of released matrix vesicles (MV). RESULTS: Articular chondrocytes and TC28 cells respired at comparable rates. Peroxynitrite and NO donors markedly suppressed respiration and ATP generation in chondrocytes. Because NO exerts multiple effects on chondrocytes, we investigated the primary functions of mitochondrial respiration and OXPHOS. To do so, we identified minimally cytotoxic doses of antimycin and oligomycin, which both induced intracellular ATP depletion (by 50-80%), attenuated collagen and proteoglycan synthesis, and blocked transforming growth factor beta from increasing intracellular ATP and elaboration of PPi, a critical inhibitor of hydroxyapatite deposition. Antimycin and oligomycin also abrogated the ability of the ATP-hydrolyzing enzyme plasma cell membrane glycoprotein 1 (PC-1) to increase chondrocyte PPi generation. Finally, MV from cells treated with antimycin or oligomycin contained less PPi and precipitated >50% more 45Ca. CONCLUSION: Chondrocyte mitochondrial reserve, as NO-sensitive mitochondrial respiration-mediated ATP production, appears to support matrix synthesis and PPi elaboration and to regulate MV composition and mineralizing activity. NO-induced depression of chondrocyte respiration could modulate matrix loss and secondary cartilage mineralization in OA.     

Degradation of human articular cartilage by neutrophils in synovial fluid

Objective. To determine whether surface-adherent immunoglobulins are capable of mediating synovial fluid (SF) neutrophil degradation of proteoglycan and collagen in intact, normal human articular cartilage, and to define the respective roles of neutrophil serine proteases and metalloproteases in degrading these cartilage constituents. Methods. Pellet explants of normal human articular cartilage pretreated with bovine serum albumin (BSA) or IgG were incubated with polymorphonuclear cells suspended in SF (PMN-SF), or with supernatants derived from neutrophils stimulated with surface-associated IgG. Proteoglycan degradation was measured by assaying release of ³⁵S-proteoglycan fragments from cartilage explants prelabeled with ³⁵S-sulfate. Collagen degradation was measured by assaying hydroxyproline content in the PMN-SF preparations or neutrophil supernatants following their incubation with unlabeled explants. Results. Significant release of both ³⁵S fragments and hydroxyproline was noted following incubation of PMN-SF with IgG-treated pellets, compared with pellets treated with BSA. IgG preparations derived from pooled normal serum or rheumatoid arthritis SF were equally efficacious in mediating PMN degradation of cartilage collagens. Explant release of ³⁵S fragments during incubation with PMN supernatant was completely inhibited when serine proteases were inactivated by diisopropyl fluorophosphate (DFP); however, release of ³⁵S fragments was enhanced when metalloprotease activity was present in the supernatant. Release of hydroxyproline during incubation of explants with PMN supernatant was comparable in the presence of DFP or EDTA, but was markedly enhanced when both serine and metalloprotease activity were present in the supernatant. Conclusion. Neutrophils in SF are capable of degrading both proteoglycans and collagens in intact human articular cartilage. Degradation of these cartilage constituents is facilitated by immunoglobulins adherent to the cartilage surface and by the synergistic action of PMN serine and metalloproteases released during activation of neutrophils with surface-associated immunoglobulin.     

The superficial layer of human articular cartilage is more susceptible to interleukin-1–induced damage than the deeper layers

Objective. To compare the responses of chondrocytes from superficial and deep layers of normal human articular cartilage to interleukin-1 (IL-1) and IL-1 receptor antagonist protein (IRAP), and to evaluate the binding sites for IL-1 on these cells. Methods. Cartilage and chondrocytes from superficial and deeper layers of human femoral condyles were cultured with and without IL-1 in the presence and absence of IRAP. The effect of these agents on ³⁵S-proteoglycan synthesis and catabolism and production of stromelysin and tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured by biochemical and immunologic assays. Receptor binding was evaluated using ¹²⁵I-labeled IL-1. Results. IL-1 induced more severe inhibition of proteoglycan synthesis and a lower ratio of secreted TIMP-1:stromelysin in chondrocytes from superficial cartilage than those from deeper cartilage. IRAP blocked responses to IL-1 more effectively in chondrocytes from deep cartilage than those from superficial cartilage. Chondrocytes from the articular surface showed approximately twice the number of high-affinity binding sites for IL-1 as did cells from deep cartilage. Conclusion. Chondrocytes from the surface of articular cartilage show a greater vulnerability to the harmful effects of IL-1 and are less responsive to the potential therapeutic effects of IRAP than cells in the deeper layers of the tissue.     

Investigation of calcium crystals in OA knees

For studies on matrix mineralization in osteoarthritis (OA), a clear analytical approach is necessary to identify and to quantify mineralization in the articular cartilage. The aim of this study is to develop an effective algorithm to quantify and to identify cartilage mineralization in the experimental setting. Four patients with OA of the knee undergoing total knee replacement and four control patients were included. Cartilage calcification was studied by digital contact radiography (DCR), field emission scanning electron microscopy (FE-SEM) X-ray element analysis and Raman spectroscopy (RS). DCR revealed mineralization in all OA cartilage specimens. No mineralization was observed in the control cartilage. Patient I showed rhomboid shaped crystals with a mean Ca:P molar ratio of 1.04 indicated the presence of calcium pyrophosphate dihydrate (CPPD) crystals, while Patients II, III and IV presented carbonate-substituted hydroxyapatite (HA). RS also showed the presence of CPPD crystals in Patient I while Patients II, III and IV revealed spectra confirming the presence of HA crystals. In the corresponding chondrocyte cell culture analyzed with SEM, the presence of CPPD crystals in the culture of Patient I and HA crystals in the culture of Patient II, III and IV was confirmed. No mineralization was found in the cell culture of the controls. The differentiation between BCP and CPPD crystals plays an important role, and the techniques presented here provide an accurate differentiation of these two types of crystals. For quantification of articular cartilage mineralization, DCR is a simple and accurate method.     

Specificity of a porcine 127-kd nucleotide pyrophosphohydrolase for articular tissues

Objective. To determine the tissue specificity of a porcine 127-kd nucleotide pyrophosphohydrolase (NTPPHase) found in vesicles derived from hyaline articular cartilage (ACV). Methods. Homogenates of porcine brain, lung, liver, kidney, urinary bladder, pancreas, spleen, skin, vena cava, marrow, bone (cells), tendon (Achilles), ligament (anterior cruciate), elastic cartilage, meniscus, and hyaline cartilage were analyzed for NTPPHase activity (thymidine monophosphate paranitrophenyl ester substrate) and by Western blot using polyclonal antibodies against 127-kd NTPPHase and against recombinant PC-1, another ecto-NTPPHase. Results. All tissues contained NTPPHase activity; the highest specific activity was found in hyaline articular cartilage, the lowest in brain. ACV-associated 127-kd NTPPHase was expressed in cartilage, ligament, and tendon. PC-1 was also expressed in those tissues and in skin, kidney, bone cells, and (probably) in liver and muscle. Conclusion. The 127-kd NTPPHase appears to be highly specific for articular tissues.     

Antisense oligonucleotides,a novel tool for the control of cytokine effects on human cartilage. focus on interleukins 1 and 6 and proteoglycan synthesis

Objective. To prevent the negative effects of interleukin–1 (IL–1) and IL–1—induced IL–6 on cartilage proteoglycan (PG) synthesis, we used an antisense oligonucleotide (ASO) specific for IL–6 messenger RNA (mRNA) to inhibit IL–6 production. Methods. Explants of human articular cartilage were cultured in the presence or absence of IL–6—ASO, IL–1, and exogenous IL–6. As metabolic parameters, cartilage production of IL–6 was determined in the B9 bioassay and PG as incorporation of ³⁵SO₄. Results. The IL–6—ASO prevented IL–1—induced production of IL–6 in the cartilage explants, as well as IL–1—induced inhibition of PG synthesis. This inhibition was restored, despite the presence of IL–6—ASO, when exogenous IL–6 was added. A control ASO (not specific for IL–6 mRNA) was not effective. Conclusion. The IL–6—ASO used can penetrate the extracellular matrix of articular cartilage, enter the chondrocytes, and inhibit the IL–1—induced production of IL–6. Furthermore, IL–6—ASO can prevent the IL–1—induced inhibition of cartilage PG synthesis. The effect of exogenous IL–6 shows that IL–1 requires IL–6 for inhibition of PG synthesis.     

Steroid hormones strongly support bovine articular cartilage integration in the absence of interleukin‐1β

Objective

Posttraumatic integration of articular cartilage at fracture sites is essential for mechanical stability of cartilage, and ruptured cartilage is a prerequisite for early osteoarthritis. This study was undertaken to investigate effects on articular cartilage integration mediated by steroid hormones, interleukin‐1β (IL‐1β), and combinations thereof.

Methods

Articular cartilage blocks were cultured in partial apposition for 2 weeks with ascorbic acid, testosterone, 17β‐estradiol, and dehydroepiandrosterone (DHEA), with or without IL‐1β. Mechanical integration was measured as adhesive strength, i.e., the maximum force at rupture of integrated cartilage blocks divided by the overlap area. Glycosaminoglycan content was used to study synthesized extracellular matrix.

Results

Culture in medium without supplements did not lead to integration (adhesive strength 0 kPa). With administration of ascorbic acid (100 μg/ml), the median adhesive strength was 49 kPa. In comparison with ascorbic acid alone, all steroid hormones induced a strong, concentration‐dependent stimulation of integration (with maximum values observed with DHEA at 3 × 10⁻⁵M, testosterone at 10⁻⁸M, and 17β‐estradiol at 10⁻¹¹M). For testosterone and 17β‐estradiol, this was also reflected by an increase of glycosaminoglycan content. Adhesive strength was increased with IL‐1β at 10 pg/ml, but not at 1 pg/ml or 100 pg/ml. In the presence of both IL‐1β and sex hormones, integration of articular cartilage was reduced.

Conclusion

This is the first study to demonstrate that steroid hormones such as 17β‐estradiol, DHEA, and testosterone stimulate articular cartilage integration. This effect is abrogated by low concentrations of IL‐1β. In the absence of IL‐1β or after neutralization of IL‐1β, steroid hormones might be favorable adjuvant compounds to optimize cartilage integration.