Chondrons have recently been extracted from adult articular cartilages and techniques developed to study their structure and composition in isolation. This study introduces methods to immobilize isolated canine chondrons in thin layers of agarose gel for immunohistochemistry and future in vitro studies. An antibody to Type VI collagen which stained the chondron in suspension was used to successfully validate the system and its feasibility for immunoelectron microscopy. Monoclonal and polyclonal antibodies to a variety of epitopes on the proteoglycan molecule were tested on fresh and fixed plugs cored from chondron-agarose gels. Plugs were immunolabeled with peroxidase-diaminobenzidine before or after digestion with testicular hyaluronidase or chondroitinase ABC. Trypsin/chymotrypsin were used to challenge epitopes of the core protein. The results indicate that epitopes to keratan sulfate, chondroitin sulfate, hyaluronate binding region, and core protein are localized in the chondron. Consistent staining was found in the tail and interconnecting segments between chondrons, whereas staining of the pericellular matrix and capsule adjacent to the chondrocyte varied according to the enzyme pre-treatment employed. We conclude that isolated chondrons are rich in proteoglycan monomer, which is particularly concentrated in the tail and interconnecting segments of the chondron where it could function to protect and stabilize the chondrocyte.
Chondrons were isolated from human and canine osteoarthritic cartilage using low-speed homogenization techniques. Changes in chondron morphology were evaluated using differential interference-contrast microscopy, phase-contrast microscopy, and histochemical and ultrastructural methods. Chondrocyte viability was assessed using fluorescein diacetate staining, and chondron metabolism was investigated using autoradiography. The results suggest that initial changes in the collagen and proteoglycan distribution within the chondron are followed by chondrocyte proliferation to form clusters. These techniques offer the potential to study cell matrix interactions in degenerative osteoarthritis.Chondrocytes from hyaline cartilage are surrounded by a specialized microenvironment termed the chondron (1-3). In ultrastructural studies of mature canine and human articular cartilage, chondrons in the middle and deep layers were shown to consist of a single chondrocyte, a pericellular matrix rich in proteoglycans, and a pericellular capsule containing
A chondron rich preparation was isolated from mature canine tibial cartilage using low-speed homogenization techniques. Proteoglycans were extracted from this preparation by exhaustive treatment with 4M guanidine-HCl. A significant proportion of the total proteoglycan, measured as uronic acid, was resistant to extraction and represented 27.9% in intact cartilage chips and 18.6% in the chondron fraction. Histochemical examination of chondrons confirmed that extraction resistant proteoglycans remained within the capsule of the chondron after 4M guanidine-HCl treatment. Electrophoretic analysis of the glycosaminoglycans extracted from intact cartilage chips and the chondron fraction showed approximately equivalent amounts of chondroitin sulphate (79.3%), keratan sulphate (16.3%) and hyaluronic acid (4.3%) present. In contrast, the extraction resistant residue in the chondron fraction was significantly enriched for hyaluronic acid (10.5%, p less than 0.05) but was depleted of chondroitin sulphate (70.9%, p less than 0.05). The major chondroitin sulphate isomer in the resistant fraction was chondroitin 6-sulphate while in the soluble fraction, the quantities of the two isomers were approximately equivalent. Comparison with previously published data suggests a role for minor collagens in the retention of proteoglycans in the cellular microenvironment.
A heterogenous population of intact chondrons extracted from low-speed homogenates of canine tibial cartilage were stained by indirect immunofluorescence methods with a polyclonal antibody to type VI collagen. In each of the four chondron groups examined, anti-(type VI collagen) anti-serum was concentrated in the capsule immediately adjacent to the chondrocyte complex. A constant but weaker fluorescent reaction persists in ‘tail-like’ extensions common to single and double chondrons and in the medial connections between adjacent chondrons in linear columns and aggregated clusters. Frayed collagen bundles typical of chondron preparations did not react with the antibody. Similarly, chondrons reacted with normal rabbit serum, or treated by type VI collagen extraction procedures, showed no staining reaction. The differential localization of type VI collagen in the pericellular capsule is discussed in relation to the maintenance of the chondron's integrity and to the protection of the chondrocyte during dynamic compressive loading.
gene and protein expression of MMP-1, MMP-3 and MMP-13, and also inhibits TNF-a activation in human chondrocytes. However, the effect of Chilean EEP on chondrocytes stimulated with IL-1b is not yet known. The main goal of this study was to evaluate the effect of EEP on oxidative stress, markers of osteoarthritis and autophagy proteins in chondrocytes stimulated with IL-1b. Methods: Chondrocytes of the hyaline cartilage of New Zealand rabbits were cultivated until passage 2 and treated with IL-1b (10 hg/ml), EEP (2.5 mg/ml) and rapamycin (100 hM and 20 hM) for 24 hours. After the treatment, nitric oxide levels and the expression of COL2A1, MMP13, LC3, ATG5 and AKT1 were measured. P-values less than 0.05 were considered significant. Results: The nitrosative stress generated by the inflammatory stimulus with IL-1b decreased significantly with EEP treatment. Also, treatment with EEP showed increased COL2A1 expression and decreased MMP13 expression. Finally, EEP treatment decreased protein expression of LC3I, ATG5 and AKT1 to the values of the control condition. Conclusions: These results suggest that the treatment with EEP in IL-1b-stimulated chondrocytes has beneficial effects by increasing healthy cartilage markers while decreasing those related to OA. EEP treatment is able to reverse the deregulation shown in the autophagy pathway. More studies are needed to reveal the mechanisms involved on how propolis show these effects.
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