Morphological and cytoskeletal aspects of cultivated normal and osteoarthritic human articular chondrocytes after cyclical pressure: a pilot study.

OBJECTIVE: This study investigated the effect of hydrostatic cyclical pressure on the cell ultrastructure and cytoskeleton of normal and osteoarthritis (OA) human cultivated chondrocytes in vitro. METHODS: The different effects of pressurization with sinusoidal waves at a minimum pressure of 1 MPa, a maximum pressure of 5 MPa and a frequency of 0.25 Hz for 3 hrs on normal and OA chondrocytes were assessed by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and immunoflurescence microscopy (IF). RESULTS: Structural differences exist between normal and OA chondrocytes at the nuclear, cytoplasmic and cytoskeletal level. Pressurization did not alter the normal chondrocytes, but had a beneficial effect on OA chondrocytes, by increasing the number of cell organelles responsible for synthesis activities. IF examination has shown that the distribution of actin protein in normal chondrocytes is polarized on the apical sides of the cellular cytoplasm. However, in OA chondrocytes the signal of the actin protein is not as well defined. Similarly, the localization of the tubulin protein in normal and OA cells also appears to be different. Hydrostatic pressure did not cause any modification in the cytoskeletal organization of the OA chondrocytes. CONCLUSION: This study confirms the different morphology, structure and cytoskeletal aspect of normal and OA chondrocytes and the important role played by pressure on cell morphology. The recovery of OA chondrocytes observed by an increase of cytoplasmic organelles does not seem to involve the cytoskeleton.

Glucosamine sulfate modulates dysregulated activities of human osteoarthritic chondrocytes in vitro.

OBJECTIVE: The efficacy of glucosamine sulfate (GS) in the symptomatic treatment of patients with osteoarthritis (OA) is suggested to be mediated by still unknown effects on the altered OA cartilage. DESIGN: Using human OA chondrocytes in culture, the effects of GS on protein synthesis, caseinase, collagenase, phospholipase A2 (PLA2) and protein kinase C (PKC) activities as well as production of nitric oxide and cyclic AMP were studied in both cells and culture medium. RESULTS: GS significantly reduced PLA2 activity, and more modestly collagenase activity, in the OA chondrocytes in a dose-dependent manner. By contrast, PLA2 and collagenase activity of the culture medium was not modified. No effects on caseinase activity was seen. GS significantly and dose-dependently increased protein synthesis. GS did not modify nitric oxide and cAMP production but significantly increased PKC production. CONCLUSION: GS modified cultured OA chondrocyte metabolism by acting on PKC, cellular PLA2, protein synthesis and possibly collagenase activation. Extrapolation of the effect to the in-vivo situation remains hypothetical but they might represent some possible mechanisms of action of the drug in human.

Osteoarthritic cartilage fibrillation is associated with a decrease in chondrocyte adhesion to fibronectin.

OBJECTIVE: Cartilage destruction in osteoarthritis (OA) is generally accepted as a failed repair process. Cell adhesion is implicated in tissue repair. Therefore, adhesion of OA chondrocytes to extracellular matrix proteins was investigated. DESIGN: Using chondrocytes from human OA femoral head cartilage, adhesion to fibronectin and type II collagen of cells from distinct areas showing an intact cartilage surface or a fibrillated cartilage surface was studied. Modulation of chondrocyte adhesion by both protein kinase C (PKC) inhibitors and glucosamine sulfate (GS) was also investigated. RESULTS: A significant (P < 0.05) decrease in adhesion to fibronectin of chondrocytes from fibrillated cartilage, relative to those from grossly normal OA cartilage, was demonstrated. Adhesion to type II collagen was not modified by the chondrocyte origins (either from normal or fibrillated OA cartilage). Adhesion to fibronectin of cells from grossly intact cartilage was decreased by the addition of PKC and calmodulin-dependent kinase inhibitors, W7 and sphingosine, to the cell culture. Adhesion to fibronectin of chondrocytes from fibrillated cartilage was significantly (P < 0.05) increased after glucosamine sulfate treatment. CONCLUSION: Fibrillation of cartilage from OA femoral head is associated with a defective adhesion of chondrocytes to fibronectin. The process is suggested to be dependent of PKC and/or calmodulin-dependent kinases and potentially reversible. Conceivably, it could play a role in OA cartilage destruction.

Changes in rat epiphyseal cartilage after treatment with dexamethasone and glycosaminoglycan-peptide complex.

In 3-month-old rats, systemic administration of glucocorticosteroids induced regressive changes in the epiphyseal plate cartilage. Dexamethasone treatment resulted in inhibition of both chondrocyte proliferation and cartilage matrix production. The inhibition of chondrocyte proliferation was determined histologically from the thickness of the epiphyseal plate, the number of cell columns and the ratio of proliferating to hypertrophied chondrocytes. The decrease in cartilage matrix production was measured autoradiographically by incorporation of radioactive 35S-sulphate. Concomitant treatment with glycosaminoglycan-peptide complex (GP-C = RUMALON) overcame the negative effect of dexamethasone. The values in the dexamethasone + GP-C group approached those of the control group and differed significantly from the group given dexamethasone alone.

Amelioration of lapine osteoarthritis by treatment with glycosaminoglycan-peptide association complex (Rumalon).

The chondroprotective potential of glycosaminoglycan-peptide association complex (GP-C) was examined in the medial meniscectomy model of lapine osteoarthritis (OA). Prophylactic treatment with increasing doses of intramuscular GP-C (0.05-0.5 ml/kg) caused a significant reduction in OA lesion area and histologic scores, and the effect on disease activity appeared to be dose related. The DNA and uronic acid contents of OA tissue were unaffected by prophylactic treatment with GP-C. However, levels of hydroxyproline in OA cartilage increased to near control levels with prophylactic treatment. Cartilage levels of active and total metalloproteinases that digest proteoglycans were elevated in rabbits with OA; prophylactic treatment with low-dose GP-C (0.05 ml/kg) produced a significant reduction in active, but not total, enzyme. Cartilage levels of tissue inhibitor of metalloproteinases in animals with OA were comparable with control levels, but rose with increasing doses of GP-C. We also investigated GP-C as a therapeutic treatment in animals that had already developed OA lesions. Carbon black retention and histologic score returned to near-normal after therapeutic treatment with GP-C. Uronic acid and hydroxyproline levels were decreased in OA cartilage. Therapeutic treatment with GP-C had no statistically significant effect on uronic acid levels, but was associated with increased hydroxyproline content in the cartilage. The changes in metalloproteinase and metalloproteinase inhibitor were similar to those found in the studies of prophylactic treatment. The findings in this animal model may help explain some of the beneficial effects of GP-C in human OA.

[Metabolism of cartilaginous tissues of rats under the effects of corticoids. Dose-dependent effect of the glycosaminoglycan-peptide complex]. / Etude du métabolisme des tissus cartilagineux du rat sous l'action des corticoïdes. Effet dose-dépendant du complexe glycosaminoglycanes-peptides.

The incorporation of labelled sulfate in knee articular cartilage and in xiphoid process cartilage was studied by autohistoradiography in the rat. Sulfate incorporation was decreased by the systemic administration of dexamethasone. A dose-dependent correction of this decrease was produced by the simultaneous administration of glycosaminoglycanes-peptides complex.

Ultrastructural analysis of rat articular cartilage following treatment with dexamethasone and glycosaminoglycan-peptide complex.

This ultrastructural study describes a stereological analysis of rat articular cartilage, with and without exposure to dexamethasone and a chondroprotective drug used in the treatment of osteoarthritis. Normal rat cartilage was found to contain 27.6 x 10(4) chondrocytes/mm3 which occupied approximately 10% of the cartilage tissue, and the organelle content of each chondrocyte was calculated to be about 20% of the cytoplasmic volume. After 3 weeks of treatment with dexamethasone the chondrocytic volume was increased by 23% with a proportionate increase in the cellular volume of the whole cartilage, and in addition the organelle content was significantly reduced to about half that of the control chondrocytes. By contrast the administration of GP-C (RUMALON) to dexamethasone-treated animals reduced these steroid effects. No significant change was seen in the total chondrocyte numbers with either of the treatments. Whereas dexamethasone inhibits chondrocyte metabolism and produces concomitant ultrastructural changes, GP-C was found to counteract these effects, a result which supports the contention that GP-C helps to preserve chondrocyte function.

[The dose dependent effect of glycosaminoglycan peptide complex on corticosteroid-induced disordered metabolism in cartilage tissue of rats]. / Der dosisabhängige Effekt vom Glycosaminoglycan-Peptid-Komplex auf den durch Corticosteroide gestörten Stoffwechsel in Knorpelgeweben von Ratten.

Systemic corticosteroid treatment induces morphological and functional changes in the articular cartilage similar to those in human osteoarthritis. In animal experiments the dexamethasone-induced inhibition of chondrocyte metabolism can be reduced in a dose-related manner by concomitant treatment with glycosaminoglycan-peptide complexes (GP-C)***). The metabolic changes in cartilage tissues of the joint and Processus Xiphoideus measured quantitatively by 35S-sulphate incorporation are comparable. The results indicate that GP-C could also have a dose-related effect on human osteoarthritic cartilage.

The mode of action of a glycosaminoglycan-peptide-complex (Rumalon) on articular cartilage of the rat in vivo.

Quantitative ultrastructural morphometry and autoradiography of articular cartilage were used to assess in 3 months old rats the effects of in vivo administration of dexamethasone alone or in combination with a glycosaminglycan-peptide-complex (GAGPC). Dexamethasone treatment (3 mg/kg week for three weeks) induced a decrease of 35S-sulphate incorporation in cartilage and ultrastructural changes of articular chondrocytes, mainly characterized by an increase in cell mortality rate, a decrease in length of endoplasmic reticulum, in the number of Golgi bodies and in mitochondrial pool and size. These autoradiographic and ultrastructural changes were reversed or prevented when GAGPC was administered concomitantly with dexamethasone. These results show that the modifications measured by quantitative ultrastructural morphometry of chondrocytes are consistent with changes in biosynthetic functions and that the GAGPC protects cartilage from the inhibitory effects of corticoids.

[Ultrastructural changes in skeletal muscle in polymyalgia rheumatica]. / Ultrastrukturelle Veränderungen in der Skelettmuskulatur bei Polymyalgia rheumatica.

Skeletal muscle from 21 patients with clinical confirmed polymyalgia rheumatica (PMR) were examined electronmicroscopically. The changes were classified according to 15 ultrastructure criteria. The focal changes have a regressive character and, while they are nonspecific, the systematic assessment of all criteria produced a characteristic pattern in which the ultrastructural picture of skeletal muscles provided a certain profile in cases of PMR. The functionally most important ultrastructural change concerns the mitochondria. Deposition and deformation of crystals result in mitochondrial changes which are structurally definable and are harmful to cell respiration. They are compensated by new formation and accumulation of mitochondria. Comparative analysis of regressive skeletal changes and of processes in the region of the media of the muscular arteries demonstrates analogous morphological changes which point to a common harmful and overlying systemic process in PMR and giant cell arteritis. Changes in the ultrastructure of the kind and degree described cannot be explained by inflammatory or noninflammatory arterial occlusions.

[Rare side effects of gold]. / Seltene Gold-Nebenwirkungen.

Rare side-effects of parenterally administered gold salts in rheumatoid arthritis are reported: metallic gold deposition in the teeth; reversible lymphadenopathies of the neck; IgA loss under gold treatment; gold-induced appearance of ANA and DNA-antibodies and gold deposition in the skin.

The influence of ossein-hydroxyapatite compound ('Ossopan') on the healing of a bone defect.

A study was undertaken in 60 adult rabbits in order to determine the effects of ossein-hydroxyapatite compound on bone healing. Standardized bony defects were produced in the distal femoral epiphyses, after which animals were randomized into four equal groups. An untreated group served as a control. One group received ossein-hydroxyapatite compound, 830 mg per day, a second group received bone mineral (ossein-hydroxyapatite compound reduced to ash, to remove organic constituents), 510 mg per day, and a third group received calcium carbonate, 650 mg per day. A series of fluorescent vital markers was administered to the animals from the 7th to the 32nd day after production of the defect. A third of the animals in each group were sacrificed 35, 56 and 84 days, respectively, after induction of the defect. Histological sections of the region of the bone defect were examined with a fluorescence microscope and resulting photomicrographs were scored with respect to degree of fluorescence, nature and degree of defect filling and structure of the newly formed bone. All three active treatment groups resulted in significantly improved mineralization as compared with the untreated control group. Treatment with ossein-hydroxyapatite compound, but not the other two active treatments, resulted in significant improvements in the pattern and quality of bone healing, particularly when assessed at 56 or 84 days after induction of the bone defect. These results indicate that ossein-hydroxyapatite compound has a beneficial effect on the process of bone healing but that this effect is lost if the organic components of the compound are destroyed or if pure calcium carbonate treatment is substituted. This strongly suggests that organic components of ossein-hydroxyapatite compound have osteogenic effects, enhancing the utilization of the mineral intake, and is consistent with previous experimental findings. It is suggested, therefore, that ossein-hydroxyapatite compound has considerable clinical potential and should be regarded as having specific, sophisticated effects on bone metabolism, rather than as a simple dietary mineral supplement.

A new test method for the standardized evaluation of changes in the ultrastructure of chondrocytes.

By means of a new ultrastructural test system, which is based on standardized morphometry and statistical evaluation, we are able for the first time to quantify changes in the cell metabolism of chondrocytes in the articular cartilage. To compare anti-inflammatory substances of different structure, different absorption characteristics and pharmacokinetics as regards their effect on rat cartilage, we used an equieffective dosage of the different anti-inflammatory drugs. Dexamethasone as steroidal and indomethacin and phenylbutazone as classical non-steroidal anti-inflammatory drugs (NSAIDs) were administered in an ED50 dosage referred to provoked arthritis over 12 weeks, using the same mode of administration. The standardized results of untreated rats weighing 300 and 450 g were used as the controls. Dexamethasone brings about massive degenerative changes in the ultrastructure of the vital chondrocyte. Under indomethacin and phenylbutazone the metabolic activity of the chondrocyte is inhibited to a much lesser extent. The damage to the chondrocyte after treatment with dexamethasone, indomethacin and phenylbutazone cannot be regarded as minimal but in some cases is tolerable as regards the benefit/risk ratio in the treatment of rheumatoid diseases.

[The effect of steroidal and nonsteroidal anti-inflammatory agents on the ultrastructure of chondrocytes in the rat. Electron microscope and morphometric study]. / Einfluss steroidaler und nichtsteroidaler Antiphlogistika auf die Ultrastruktur von Chondrozyten der Ratte. Elektronenoptische und morphometrische Studie.

In order to obtain indications regarding the different influences of antiinflammatory drugs on the articular cartilage, the effects of a steroidal drug (dexamethasone) and of nonsteroidal antiinflammatory substances (indometacin and phenylbutazone) on the ultrastructure of the chondrocyte were examined in comparison with tiaprofenic acid (Surgam). Using an equiefficient dosage of the active principles, referred to the ED50 in the model of adjuvant arthritis in the rat, the synthetic apparatus of the chondrocytes was stimulated by tiaprofenic acid while it was diminished by indometacin and phenylbutazone and particularly by dexamethasone.