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[Ultrastructure of cultured cartilage, articular cartilage, growth plate and meniscus].

OBJECTIVE: To investigate possibility of cartilage cultured in centrifuge tube as graft materials. METHODS: Articular chondrocytes isolated from a 3-week-old rabbit formed cartilage after cultivation for 2 weeks. Articular cartilage of humeral head, growth plate of proximal tibia and meniscus were collected from a 6-week-old rabbit. The ultrastructure of chondrocytes and extracellular matrix in the three kinds of cartilages and cultured cartilage were observed by transmission electronic microscopy. RESULTS: Cartilage cultured in centrifuge tube possessed unique ultrastructure and was similar to articular cartilage and growth plate, but it was markedly different from meniscus. The four kinds of cartilages were characteristic of respectively different chondrocytes and extracellular matrix. Cultured cartilage showed typical apoptosis of chondrocytes and "dark chondrocytes" appeared in growth plate. Condrocyte apoptosis was not seen in articular cartilage and meniscus. CONCLUSION: Cartilage cultured in centrifuge tube has unique ultrastructure and may be used as graft materials for articular cartilage and growth plate.

Animals↗

Del1: a new protein in the superficial layer of articular cartilage.

Articular cartilage contains four distinct zones, extending from the surface to the subchondral bone. Freshly isolated chondrocytes from the superficial zone of articular cartilage retain a collagenase-P-resistant cell-associated matrix. In the studies described here, the protein Del1 was identified as a component of the cell-associated matrix of superficial zone chondrocytes from adult bovine articular cartilage. Very little Del1 was associated with freshly isolated deep zone chondrocytes. Western blot analysis of articular cartilage cell and tissue extracts using polyclonal antibodies specific for Del1 showed Del1 was present in an insoluble cell-associated fraction. Extracts of the superficial zone of articular cartilage were found to be enriched in Del1 compared to the deeper layers of the tissue. Immunohistochemical staining of full-thickness articular cartilage with anti-Del1 antibodies also showed an enrichment of Del1 in the superficial zone. These observations are the first to describe the protein Del1 in a nonendothelial, nonfetal tissue.

Amino Acid Sequence↗

A viscoelastic analysis of the tensile weakening of deep femoral head articular cartilage.

Articular cartilage from below the surface of the femoral head of the hip joint shows a profound age-dependent weakening in its tensile mechanical properties. This ageing is also associated with a reduced viscoelastic response in the older tissue. A constitutive model of the viscoelastic behaviour of deep articular cartilage (as discussed by Egan in 1988) is used to generate a graphical pattern which represents the mechanical behaviour. This constitutive approach suggests that the tensile weakening of the older cartilage is due to an age-related reduction in the recruitment of load-carrying structures as the tissue is deformed. The viscoelastic constitutive model also predicts a reduction in the tensile strength of deep articular cartilage with rate of deformation. This prediction is supported by experimental fracture stress data. A weakening of the tensile integrity of the microstructure of articular cartilage could make the tissue less able to sustain normal compressive physiological loading without damage and thus make the tissue more susceptible to osteoarthritic degeneration. The constitutive approach indicates that the weakening of the older tissue may be related to changes within the microstructure which determine how applied mechanical energy is stored and dissipated.

Age Factors↗

The collagens of articular cartilage.

Articular cartilage contains at least five genetically distinct types of collagen. Types II, IX, and XI are cartilage-specific and are cross-linked together in a copolymeric network that forms the extracellular framework of the tissue. Fibrils of type II collagen provide the basic architecture. Type XI, a quantitatively minor fibril-forming collagen, is probably copolymerized with type II collagen in the matrix. Type IX collagen accounts for approximately 1% of the collagenous protein in adult articular cartilage and its molecules exist in the tissue covalently linked to the surface of type II collagen fibrils. Its suspected functions include regulating fibril diameters and mediating fibril-fibril and fibril-proteoglycan interactions. Stromelysin, a matrix metalloproteinase, was recently shown to degrade type IX collagen. This action may cause the collagen network swelling seen in articular cartilage in early experimental osteoarthritis, (OA). Collagen type X is restricted to the underlying calcified zone of articular cartilage, a zone that exhibits active remodeling in joints with OA. Degradation products of the various cartilage collagens show promise as molecular markers of joint disease.

Amino Acid Sequence↗

Comparison of matrix vesicles derived from normal and osteoarthritic human articular cartilage.

Articular cartilage matrix vesicles (MVs) from normal human adult articular cartilage were examined for protein and enzyme content and biomineralizing capacity for comparison to MVs derived from human osteoarthritic (OA) cartilage. Femoral condylar and tibial plateau cartilage from each of 9 healthy donors ages 17-37 y was enzymatically digested and serially ultracentrifuged to pellet MV's at 3 x 10(6) g-min. MV protein content, nucleoside triphosphate pyrophospho hydrolase (NTPPPH) specific activity (SA) and capacity for 45Ca precipitation were determined. MV precipitated mineral was examined using Fourier transform infrared spectroscopy (FTIR). Normal human cartilage yields 50% less MV protein/g cartilage than OA cartilage (p < .01). Normal human articular MVs possess 30-70x higher NTPPPH SA than cell-free digest. Mean NTPPPH SAs of MVs derived from normal human cartilage are 3x higher than that of OA MVs (p < .05) and normal MV NTPPPH SA appears to decrease with age (p < .01). Normal human MVs support significantly higher calcium precipitation/mg MV protein in both ATP-dependent (p < .01) and -independent (p = .05) systems. The FTIR spectrum of MV mineral generated in the presence of ATP strongly resembles the standard spectrum for calcium pyrophosphate dihydrate (CPPD). The FTIR spectrum of MV mineral generated without ATP resembles that of carbonate-substituted apatite (AP). The fact that isolated MVs from normal cartilage generate pathologically relevant crystal phases in vitro implies that matrix integrity and substrate availability may be crucial factors in the control of pathologic biomineralization.

Adolescent↗

The association of lipid abnormalities with tissue pathology in human osteoarthritic articular cartilage.

Articular cartilage is one of very few body tissues uniquely characterized as having substantial stores of lipid deposits. Lipid droplets are naturally accumulated by chondrocytes and individual fatty acids have been shown to have protective as well as deleterious effects on cartilage degradation in animal models of degenerative joint disease. As a means to better assess the role of lipids in human joint pathology, a comparative analysis of fatty acids was undertaken in small segments of osteoarthritic articular cartilage. The data were assessed in terms of chondrocyte synthetic activity and histological determination of disease severity. The distribution profile of individual fatty acids in normal and osteoarthritic specimens remained constant, with palmitic, oleic, and linoleic acids representing 85% of the total fatty acids. In contrast, levels of total fatty acids were markedly increased in association with increasing degree of lesion severity. Compared with tissue from normal-aged joints, grade 0 to 1 mild lesions had elevated levels of total fatty acids, essential fatty acids, and chondrocyte synthetic activity of 80%, 312%, and 393%, respectively. More severe tissue involvement (grade 6 to 9), was associated with even greater increases of 440%, 1,100%, and 1,150%, respectively. No change was noted in cholesterol content in any tissue. The accumulation of arachidonic acid was greater than the proportional increase in total fatty acid content and was primarily distributed into the neutral lipid fraction, where it constituted almost 62% of the fatty acid level in tissues of moderate lesion severity. There was an association of lipid accumulation in general and arachidonic acid in particular with histological severity.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗

Anatomy, biochemistry, and physiology of articular cartilage.

Articular cartilage serves as a load-bearing elastic material that is responsible for the frictionless movement of the surfaces of articulating joints. Its ability to undergo reversible deformation depends on its structural organization, including the specific arrangement of the matrix macromolecules and the chondrocytes. Interactions between the matrix and chondrocytes are responsible for the biological and mechanical properties of articular cartilage and enable it to respond by effecting a balance between anabolism and catabolism as well as continual internal remodeling. Age-related changes in the function of chondrocytes may contribute to the initiation and progression of osteoarthritis.

Aging↗

Type II collagen-induced arthritis. A morphologic and biochemical study of articular cartilage.

Articular cartilage was obtained from type II collagen-induced arthritic rat joints. Transmission electron microscopy showed a gradual degeneration of chondrocytes, disorganization of the collagenous extracellular matrix, and formation of microscars. Biochemical analyses indicated that type II collagen was the only collagen present and that it was normal in regard to hydroxylation of lysine and glycosylation of hydroxylysine. Analyses of the proteoglycan in the extracellular matrix revealed a 50% loss of chondroitin sulfate and keratan sulfate.

Animals↗

Enzymic heterogeneity of normal canine articular cartilage.

Articular cartilage is generally considered to be an homogeneous tissue. It has now been shown that, although different regions of the medial tibial cartilage of the dog have very similar oxidative enzymic activities, each region is heterogeneous with respect to these activities. The conventional histological delineation of this cartilage has been modified, to take into account a narrow band (designated zone 2a), just below the most superficial spindle-shaped cells, that has higher oxidative enzymic activity than any other. Changes in the activity in this zone might be diluted by the lack of change in other zones if measured by conventional biochemical procedures which could not measure the activities of the different zones separately.

Alcohol Oxidoreductases↗

Nitric oxide and proteoglycan turnover in rabbit articular cartilage.

Articular chondrocytes are known to synthesize large amounts of nitric oxide in response to exposure to interleukin-1, but the role of this radical in proteoglycan turnover remains controversial. In this study, we used two different inhibitors of nitric oxide synthase, NG-methyl-L-arginine and thiocitrulline, to study the effects of nitric oxide on the synthesis and breakdown of proteoglycan in rabbit articular cartilage. Synthesis of nitric oxide by cartilage slices in response to treatment with interleukin-1 and a partially purified mixture of synovial cytokines known as chondrocyte-activating factors peaked during the first 2 days of culture and then fell to low levels, despite daily replenishment with fresh medium and cytokines to the cultures. The production of nitric oxide was completely inhibited by NG-methyl-L-arginine and thiocitrulline. Interleukin-1 and the chondrocyte-activating factors inhibited proteoglycan synthesis and accelerated proteoglycan breakdown in the slices of cartilage. Both nitric oxide synthase inhibitors substantially counteracted the suppression of proteoglycan synthesis but exacerbated proteoglycan catabolism occurring in response to interleukin-1 and the chondrocyte-activating factors. The accelerated catabolism was associated with increased levels of matrix metalloproteinases in the conditioned medium. This dual effect of nitric oxide complicates decision making with regard to the possible clinical applications of nitric oxide agonists or antagonists in diseases of cartilage.

Animals↗

Deposition of amyloid of unknown origin in articular cartilage.

Articular cartilage, obtained from the large toe during hallux valgus operations in 37 patients, was investigated for the presence of amyloid by using the Congo red staining method. Amyloid deposits were demonstrated, particularly in the superficial layer of the cartilage, in 30 cases. This amyloid did not react immunohistochemically with any of the antibodies against the known five major amyloid types (AA, A lambda, A kappa, AF, AB). From these data it is concluded that hyaline cartilage in older individuals is prone to infiltration by an amyloid of a hitherto unidentified class. From the morphological observations there seems to be no correlation between amyloid deposits and the development of osteoarthrosis.

Aging↗

An investigation of biphasic failure criteria for impact-induced fissuring of articular cartilage.

Articular cartilage consists of both solid and fluid phases with fissures observed on the surface occurring in the solid portion. In order to determine which of the solid phase stresses provides the best predictor for the initiation of a fissure, elastic stresses from a series of in vitro impact experiments were used to derive stresses in the solid phase of the cartilage. This stress information was then analyzed using a logistic regression to identify the best predictor of fissuring. The mechanical analysis indicated that low-magnitude tensile solid hoop stress develops in the solid phase within the contact zone in impacts involving the two smaller radius interfaces. The logistic regression, however, indicated that maximum shear stress in the solid (which is equal to the shear stress from the elastic analysis) was the best predictor of the occurrence of a fissure. This study helps support the suggestion that in stress fields dominated by compression, the maximum shear stress from an elastic analysis may be used to predict fissure initiation in cartilage.

Animals↗

Influence of cyclic loading on the nutrition of articular cartilage.

Articular cartilage is avascular. Nutrients are transported to the cells mainly by diffusion from the synovial fluid. Nutrient transport is also sometimes thought to be assisted by movement of fluid in and out of cartilage in response to cyclic loading of the tissue ('pumping'). The influence of pumping on transport of solutes through cartilage was measured by subjecting plugs of human femoral head cartilage immersed in medium containing radioactive solutes to a simulated walking cycle of 2.8 MPa at 1 Hz. The rate of absorption or desorption of tracers from the cycled plugs was compared with that of unloaded control plugs. For small solutes (urea, NaI) fluid transport did not affect the rate of solute transport significantly. Most major nutrients, such as glucose and oxygen, are small solutes and thus nutrition should not be affected by pumping. The rate of desorption of a large solute (serum albumin), however, was increased by 30-100% in plugs subjected to cyclic loading.

Biological Transport↗

Age related changes in the turnover of proteoglycans from explants of bovine articular cartilage.

Articular cartilage in explant culture synthesized 2 proteoglycan types of different size. The proportion of 35S-sulfate incorporated into the small proteoglycan was higher in mature (17%) than in immature cartilage (11%). The chondroitin sulfate chains of both proteoglycans, synthesized by mature cartilage were shorter than those of immature cartilage, with a higher ratio of 6-sulfated over 4-sulfated disaccharides. Radiolabelled macromolecules from tissue of both ages were released in 2 phases: an initial fast release followed by a period of slow release. The half-lives of both proteoglycan populations were shorter in mature cartilage than in immature tissue (-45%). The amount of proteoglycans released during the initial short phase increases with age, from 23-24% in immature cartilage to 28-29% in mature tissue.

Aging↗

Anaerobic and aerobic metabolism in articular cartilage.

Articular cartilage explants were incubated at various oxygen tensions and the results of 35SO4 and 3H-thymidine incorporation indicate that proteoglycan and DNA synthesis is depressed at low oxygen tensions. The studies of oxygen consumption and glucose/lactate metabolism in the cartilage suggest a shift in energy pathways as the oxygen environment is altered. In low O2 tensions chondrocytes follow anaerobic glycolysis while at physiologic oxygen tensions chondrocytes follow a combination of both aerobic and glycolytic metabolism. The data suggest that hypoxia, frequently found in inflammatory arthritides, would have deleterious effects on chondrocyte metabolism and would significantly impair any reparative potential of the injured chondrocytes.

Animals↗

The compressive strength of articular cartilage.

Articular cartilage provides the smooth bearing surfaces in freely moving (synovial) joints. Its mechanical properties are important because structural failure of cartilage is closely associated with joint disorders, including osteoarthritis. Some mechanical properties of cartilage are well characterized, but little is known about its compressive strength. A technique for measuring cartilage compressive strength is evaluated, and an overview of experiments which relate strength to stiffness and tissue hydration is given. Specimens of bovine articular cartilage-on-bone, approximately 15 mm square, were loaded on a hydraulic materials testing machine using flat impermeable indentors. Linear-ramp loading/unloading cycles of 1 s duration, and of increasing severity, were applied until failure was evident on force-displacement graphs. Some specimens were tested following a 30 min period of creep loading. Inkstaining and histology were used to locate the site of initial damage to each specimen. Specimen failure occurred first in the cartilage surface layer at a nominal applied stress of 14-59 MPa (mean 35.7 MPa). Mechanical properties were little affected by specimen or indentor size, provided both remained within defined limits, and compressive strength could be measured to an accuracy of approximately +/- 5 per cent. Compressive stiffness was a significant predictor of strength, but only if it was measured at high levels of stress. Strength increased following creep-induced water loss, and initial mechanical damage could propagate under moderate cyclic loading. This technique for measuring cartilage compressive strength has potential for investigating the causes of cartilage failure in vivo.

Animals↗

Structural consequences of traumatizing articular cartilage.

Articular cartilage-on-bone has been subjected to repeated impact loading in vitro and the associated structural changes occurring in the general matrix examined by optical and transmission electron microscopy (TEM). The study shows that repeated trauma transforms the pseudorandom arrangement of fibrils comprising the general matrix of normal articular cartilage into a structural configuration strongly aligned in the radial direction and displaying a prominent waveform or crimp. This stress-induced structural transformation can be predicted from the application of a recently developed structural model of articular cartilage. Further, this altered structure bears a close resemblance to that commonly observed in articular cartilage exhibiting both non-progressive degeneration and osteoarthritic changes.

Animals↗

The effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the healing of full-thickness defects of articular cartilage.

Articular cartilage has a limited capacity for repair. We investigated the effect of rhBMP-2 (recombinant human bone morphogenetic protein-2) on the healing of full-thickness osteochondral defects in adult New Zealand White rabbits. A single defect, three millimeters wide by three millimeters deep, was created in the trochlear groove of the right femur in eighty-nine rabbits. The defect was either left empty, filled with a plain collagen sponge, or filled with a collagen sponge impregnated with five micrograms of rhBMP-2. The animals were killed at four, eight, or twenty-four weeks, and the repair tissue was examined histologically and evaluated with use of a grading scale. The defects also were examined immunohistochemically for the presence of type-II collagen at four and eight weeks. The rate of bone repair was evaluated with fluorescent labeling of bone at two and four weeks and with use of fluorescence microscopy at eight weeks. Treatment with rhBMP-2 greatly accelerated the formation of new subchondral bone and improved the histological appearance of the overlying articular surface. At twenty-four weeks, the thickness of the repair cartilage was 70 per cent that of the normal adjacent cartilage and a new tidemark usually had formed between the repair cartilage and the underlying subchondral bone. The average total scores on the histological grading scale were significantly better (p < 0.01) for the defects treated with rhBMP-2 than for the untreated defects (those left empty or filled with a plain collagen sponge) at all time-points. Immunostaining with an antibody against type-II collagen showed the diffuse presence of this cartilage-specific collagen throughout the repair cartilage in the treated defects. The untreated defects demonstrated minimum staining with this antibody.

Animals↗