Electromechanical response of articular cartilage in indentation--considerations on the determination of cartilage properties during arthroscopy

A finite element formulation of streaming potentials in articular cartilage was incorporated into a fibril-reinforced model using the commercial software ABAQUS. This model was subsequently used to simulate interactions between an arthroscopic probe and articular cartilage in a knee joint. Fibril reinforcement was found to account for large fluid pressure at considerable strain rates, as has been observed in un-confined compression. Furthermore, specific electromechanical responses were associated with specific changes in tissue properties that occur with cartilage degeneration. For example, the strong strain-rate dependence of the load response was only observed when the collagen network was intact. Therefore, it is possible to use data measured during arthroscopy to evaluate the degree of cartilage degeneration and the source causing changed properties. However, practical problems, such as the difficulty of controlling the speed of the hand-held probe, may greatly reduce the reliability of such evaluations. The fibril-reinforced electromechanical model revealed that high-speed transient responses were associated with the collagen network, and equilibrium response was primarily determined by proteoglycan matrix. The results presented here may be useful in the application of arthroscopic tools for evaluating cartilage degeneration, for the proper interpretation of data, and for the optimization of data collection during arthroscopy.     

13C NMR relaxation studies on cartilage and cartilage components

We have investigated the molecular motions of polysaccharides of bovine nasal and pig articular cartilage by measuring the 13C NMR relaxation times (T1 and T2). Both types of cartilage differ significantly towards their collagen/glycosaminoglycan ratio, leading to different NMR spectra. As chondroitin sulfate is the main constituent of cartilage, aqueous solutions of related poly- and monosaccharides (N-acetylglucosamine and glucuronic acid) were also investigated. Although there are only slight differences in T1 relaxation of the mono- and the polysaccharides, T2 decreases about one order of magnitude, when glucuronic acid or N-acetylglucosamine and chondroitin sulfate are compared. It is concluded that the ring carbons are motion-restricted primarily by the embedment in the rigid pyranose structure and, thus, additional limitations of mobility do not more show a major effect. Significant differences were observed between bovine nasal and pig articular cartilage, resulting in a considerable line-broadening and a lower signal to noise ratio in the spectra of pig articular cartilage. This is most likely caused by the higher collagen content of articular cartilage in comparison to the polysaccharide-rich bovine nasal cartilage.     

Uncalcified cartilage resorption in human fetal cartilage canals

In the human fetus, epiphyses appear as a solid avascular cartilaginous mass until the eleventh week of development. Around the third fetal month of development, vascular canals coming from the perichondrium are recognized in the mineralized epiphyseal cartilage. Whether cartilage canals develop by passive inclusion or active chondrolysis is still a matter of controversy. We studied the relationships between the intracanalar cells and the surrounding matrix on human fetal epiphyses embedded in glycol methacrylate. At the blind end of canals both stellate fibroblast-like cells and vacuolated macrophages are observed. These cellular foci show all characteristics of active chondrolysis (loss of metachromasia, lacunae containing cells intimately associated with matrix, and presence of granular debris). Similar resorptive foci have been observed in the pannus of rheumatoid joints and in the embryonic chick growth plate composed of uncalcified cartilage. A cellular cooperation (fibroblast/macrophage) is necessary for uncalcified cartilage breakdown. In the human fetus, monocytes/macrophages have been recognized in the peripheral blood as early as the twelfth week of gestation. Our observations support the view that chondrolysis due to both fibroblasts (of mesenchymal origin) and macrophages is the basic mechanism for cartilage canal development.     

Effect of insulin on cartilage differentiation in vitro

A consistent chondrogenesis takes place in high-density microcultures derived from bud mesenchymal cells of 4-day-old chicken embryos in a serum-supplemented medium. In serum-free medium DNA level and uronic acid content in the cultures were low, as well as the 35SO4 uptake and release, and only a small mass of cartilage was formed. With the addition of 0.025-10 micrograms/ml insulin to serum-free medium the uronic acid and DNA content in the cultures increased considerably in a dose-dependent way. The intensity of 35SO4 uptake and release exceeded the values measured in serum-containing medium, more cartilage tissue was formed in them also in a dose-dependent manner. With the use of 20-80 micrograms/ml insulin, the increment in DNA content proved to decrease, and with the use of 80 micrograms/ml insulin the uronic acid content and the cartilage mass decreased to a greater extent than in the case of lover doses.     

Swelling of articular cartilage depends on the integrity of adjacent cartilage and bone

Articular cartilage swells when its collagen network is degraded, both in osteoarthritis (OA) and following mechanical trauma. However, most of the experimental evidence actually shows that it is small excised samples of cartilage that swell, implying that the cartilage was not greatly swollen in-situ before it was excised. We hypothesise that degraded cartilage can be prevented from swelling in-situ by restraint from adjacent normal cartilage and subchondral bone. Four adjacent osteochondral specimens, 20 x 20 mm, were obtained from regions of the humeral heads of each of 11 skeletally-mature cows. The central region of each specimen was injured by compressive loading using a 9 mm-diameter flat metal indenter, and cartilage surface damage was confirmed using Indian ink. Damaged cartilage was allowed to swell in physiological saline for 1 h under one of four conditions of restraint: (A) normal in-situ restraint from subchondral bone and surrounding cartilage, (B) restraint from bone only, (C) restraint from cartilage only, (D) no restraint (excised specimen). Cartilage hydration was assessed by freeze-drying to constant weight. Proteoglycan loss from damaged cartilage was quantified by analyzing the GAG content of the surrounding bath using the DMB assay. Hydration of damaged cartilage after swelling depended on restraint (p < 0.001), averaging: (A) 76.8%, (B) 78.2%, (C) 78.0%, (D) 81.3%. GAG loss following cartilage surface damage was insufficient to explain observed differences in hydration. The 6% increase in hydration between (A) and (D) can be attributed to swelling which is prohibited when the cartilage remains in-situ. Swelling of degraded cartilage can be largely prevented if it remains in-situ, supported by adjacent healthy bone and cartilage. Adverse physico-chemical consequences of cartilage degradation and swelling may become apparent only when this support is diminished, either because the affected region is large, or following deterioration of adjacent bone or cartilage.     

Studies on the cathepsins in elastic cartilage

1. The presence of several enzymes in rabbit ear cartilage was examined by a quantitative method that permits the incubation of a fixed weight of cartilage sections (18mum.) with an appropriate exogeneous substrate. 2. As the presence of cathepsins B and D in cartilage has already been established, evidence is now provided to show that cathepsins A and C are also present and are maximally active at pH5. 3. Cathepsin A was recognized by its hydrolysis of benzyloxycarbonyl-glutamyl-tyrosine and cathepsin C by its hydrolysis of glycyl-tyrosine amide; the cartilage also hydrolysed benzyloxycarbonyl-glutamyl-phenylalanine and benzoyl-dl-phenylalanine 2-naphthyl ester at pH5. 4. The acid phosphatase activity and the DNA content of cartilage have also been measured to provide a basis for comparison with the cathepsin activity of cartilage obtained from other sites and species.     

Articular cartilage and changes in Arthritis: Collagen of articular cartilage

The extracellular framework and two-thirds of the dry mass of adult articular cartilage are polymeric collagen. Type II collagen is the principal molecular component in mammals, but collagens III, VI, IX, X, XI, XII and XIV all contribute to the mature matrix. In developing cartilage, the core fibrillar network is a cross-linked copolymer of collagens II, IX and XI. The functions of collagens IX and XI in this heteropolymer are not yet fully defined but, evidently, they are critically important since mutations in COLIX and COLXI genes result in chondrodysplasia phenotypes that feature precocious osteoarthritis. Collagens XII and XIV are thought also to be bound to fibril surfaces but not covalently attached. Collagen VI polymerizes into its own type of filamentous network that has multiple adhesion domains for cells and other matrix components. Collagen X is normally restricted to the thin layer of calcified cartilage that interfaces articular cartilage with bone.     

Articular cartilage and changes in Arthritis: Collagen of articular cartilage

The extracellular framework and two-thirds of the dry mass of adult articular cartilage are polymeric collagen. Type II collagen is the principal molecular component in mammals, but collagens III, VI, IX, X, XI, XII and XIV all contribute to the mature matrix. In developing cartilage, the core fibrillar network is a cross-linked copolymer of collagens II, IX and XI. The functions of collagens IX and XI in this heteropolymer are not yet fully defined but, evidently, they are critically important since mutations in COLIX and COLXI genes result in chondrodysplasia phenotypes that feature precocious osteoarthritis. Collagens XII and XIV are thought also to be bound to fibril surfaces but not covalently attached. Collagen VI polymerizes into its own type of filamentous network that has multiple adhesion domains for cells and other matrix components. Collagen X is normally restricted to the thin layer of calcified cartilage that interfaces articular cartilage with bone.     

Studies on cartilage formation. XXII. Investigations of certain oxidative metabolic processes in regenerating articular cartilage

The distal articular surface of the femur was surgically removed in 57 dogs. Succinate dehydrogenase and cytochrome oxidase activities were assayed on postoperative days 7, 20, 26, 33 and 70 in the regenerating, chondrifying articular surface and in the granulation tissue adhering to the capsule. In the 70-day samples, the cyanide-induced inhibition of oxygen consumption was determined and enzyme histochemical reactions (cytochrome oxidase, monoamine oxidase, xanthine oxidase, peroxidase and "catalase") were performed. The succinate dehydrogenase activity was the highest in the early postoperative stage in both tissues. This was followed by a definite decrease and a subsequent significant increase in activity when chondrification took place. Measurement of cytochrome oxidase activity could not reveal any convincing result, presumably because of the properties of the tissues studied. The oxygen consumption by the chondrifying articular surface at 70 days was inhibited to about 50% by cyanide, and about 90% inhibition was observed in the tissue adhering to the capsule. The cells of the regenerating articular surface possess cytochrome oxidase and a cyanide- (and sodium azide-) resistant oxidase activity. The enzyme activity of the cartilaginous islets exceeded that of their connective tissue environment. The cytochrome oxidase activity increased in the cells during cartilage differentiation. Presumably, some further cyanide-sensitive and cyanide-resistant oxidases are present in chondroblasts and young chondrocytes.     

Characterization of cathepsins in cartilage

The presence of a cathepsin B-like enzyme in rabbit ear cartilage was established by the use of the synthetic substrates benzoyl-l-arginine amide and benzoyl-dl-arginine 2-naphthylamide. This was facilitated by using a technique that permits the incubation of a fixed weight of thin (18mu) cartilage sections with an appropriate exogenous substrate. The enzymic properties of cathepsin B in cartilage have been compared with an endogenous enzyme that liberates chondromucopeptide by degrading the cartilage matrix autocatalytically at pH5. Besides being maximally active at pH4.7, these cartilage enzymes are enhanced in activity by cysteine and inhibited by arginine analogues, iodoacetamide, chloroquine and mercuric chloride. They are not inhibited by EDTA, di-isopropyl phosphorofluoridate and diethyl p-nitrophenyl phosphate. When inhibiting the release of chondromucopeptide from cartilage at pH5, the arginine-containing synthetic substrates are hydrolysed simultaneously. These enzymes also share the same heat-inactivation characteristics at various pH values, being stable at acid pH and unstable at neutral and alkaline pH. The experimental evidence indicates that a cathepsin B-like enzyme may be partly responsible for the autolytic degradation of cartilage matrix at pH5.