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Degenerated human articular cartilage at autopsy represents preclinical osteoarthritic cartilage: comparison with clinically defined osteoarthritic cartilage.

OBJECTIVE: To investigate whether macroscopically fibrillated human articular knee cartilage observed at autopsy can be considered an early, preclinical phase of osteoarthritis (OA). METHODS: Histological and biochemical characteristics of 3 types of articular knee cartilage were compared: macroscopically degenerated knee cartilage obtained at autopsy (6 donors) from donors without clinical history of OA, normal healthy knee cartilage obtained at autopsy (6 donors), and OA cartilage obtained during joint replacement surgery from patients (n = 6) with clinically defined OA of the knee. From the same donors synovial tissue and synovial fluid were obtained and analyzed for features of inflammation. RESULTS: Histological changes of OA were comparable for degenerated and OA cartilage and significantly different from normal cartilage. Content and synthesis of proteoglycans showed intermediate levels for degenerated tissue compared to normal and OA cartilage. Analysis of synovial tissue revealed a low, mild, and moderate degree of inflammation for joints with normal, degenerated, and OA cartilage, respectively. The same sequence was found for metalloproteinase activity in synovial fluid. CONCLUSION: In general, all changes observed in OA joints were, to a lesser extent, observed in the joints with degenerated cartilage and were significantly different from joints with normal cartilage. We conclude that cartilage degeneration observed at autopsy can be considered a preclinical phase of OA, suitable for studying the process of cartilage degeneration in OA.

Aged↗

A monoclonal antibody distinguishes growth cartilage from other types of cartilage: a new probe for osteogenic cartilage.

Monoclonal antibodies (mAbs) were raised by injection of a homogenate of cultured growth cartilage (GC) cells from young rabbit ribs. These mAbs were examined by immunohistochemical staining for their reactivity to paraffin sections of rabbit tissues. The results showed that an mAb reacted preferentially with late hypertrophic and calcified costal GC zones. The mAb also reacted with hypertrophic GC adjacent to bone that existed in sternum and femur, but not to other cartilages, including resting cartilage, articular cartilage, auricular cartilage, nasal cartilage, tracheal cartilage and meniscus cartilage, or with other tissues, including tendon, skin, muscles, lung, liver, heart, thymus, spleen, eye and gut. It reacted with a wider area of the GC zone when the sections were decalcified, although its reactivity with the extended area was much less intensive than that with late hypertrophic and calcified GC zones. On treatment of the sections with bacterial collagenase, neither the reactive area nor its intensity were changed, while when treated with trypsin the reactivity was lost. These results suggest the existence of a certain molecule which distinguishes GC (osteogenic cartilage) from other (non-osteogenic) cartilage. This mAb is a useful probe for distinguishing osteogenic cartilage from non-osteogenic cartilage, and for studying differentiation steps of cartilage cells in endochondral bone formation. The mAb can also be used as a probe for clinical and stored specimens because it reacts with decalcified and paraffin-embedded human specimens.

Animals↗

Cartilage proteoglycan-induced arthritis in BALB/c mice. Antibodies that recognize human and mouse cartilage proteoglycan and can cause depletion of cartilage proteoglycan with little or no synovitis.

Human fetal cartilage proteoglycan (PG) induces the development of an erosive polyarthritis and spondylitis in BALB/c mice. We have examined the properties of 3 monoclonal antibodies (MAb) to human fetal cartilage PG isolated from immunized mice that cross-react with mouse cartilage PG. Compared with sera from arthritic mice, which contain antibodies reactive with keratan sulfate, MAb 202 (IgG1) reacted only with a protein-related epitope that is distributed on both hyaluronic acid-binding and chondroitin sulfate-attachment regions. MAb 813 (IgG1) reacted with the same fragments and recognized an epitope with the immunologic characteristics of keratan sulfate. MAb 945 (IgM) remains to be further characterized. Introduction of hybridomas secreting MAb 202 and MAb 945 into irradiated mice resulted in the loss of PG from articular cartilage and from growth plate cartilage (with MAb 202 only), as revealed by a loss of staining with toluidine blue. There was no synovial hyperplasia with MAb 202, but some hyperplasia and mononuclear cell infiltration was seen with MAb 945. This was accompanied by the binding of immunoglobulins to articular cartilage, as demonstrated by immunofluorescence. The hybridoma secreting MAb 813 produced no cartilage changes or synovitis, and there was no immunoglobulin binding to cartilage. Polymorphonuclear leukocyte infiltration was never observed with these antibodies. These studies indicate that MAb reactive with mouse cartilage PG can cause the depletion of PG from hyaline cartilage by mechanisms that may be both complement dependent and complement independent. Antibodies may serve to release and expose PG antigen to immune cells, as well as causing a loss of the mechanical properties of cartilage that are PG dependent.

Animals↗

Morphology, histochemistry, and differentiation of the cat's epiglottic cartilage: a supporting organ composed of elastic cartilage, fibrous cartilage, myxoid tissue, and fat tissue.

BACKGROUND: In carnivores, the supporting organ of the epiglottis is usually called "epiglottic cartilage" (EC) although it is composed of elastic cartilage and unilocular fat storing cells. We studied the cat's EC in order to decide whether these fat storing cells are true adipocytes or fat storing (dedifferentiated) chondrocytes. METHODS: ECs were studied in cat embryos at gestation days 40 and 60, in newborn, postnatal, and adult cats. We used classical staining methods, immunohistochemistry, and transmission electron microscopy to identify the different kinds of tissues contributing to the EC and to follow their differentiation. RESULTS: The cat's EC was defined by a layer of coarse collagen fibers representing a tunica albuginea. This tunica covered irregularly formed and irregularly sized areas of elastic cartilage, fibrous cartilage, myxoid tissue, and lobules of unilocular fat cells. All these tissue showed regular morphology. Adipocytes were provided with continuous basal laminae and fat lobules were well supplied with capillaries. Alcianophilia of ground substance was observed in all tissue components but was strongest in elastic cartilage. Most islets of elastic cartilage adhered to the tunica albuginea of the EC at one surface and were connected to the opposite surface by coarse strands of connective tissue traversing the organ. Intercalated areas of fibrous cartilage contained fuchsinophilic collagen bundles. Myxoid tissue was characterized by stellate cells in alcianophilic ground substance with intermingled fuchsinophilic bundles. All kinds of supporting tissues combined with each other without clear demarcation. Immunohistochemistry revealed strong reactivity for S-100 of chondrocytes, myxoid cells, and fat cells. Chondrocytes and myxoid cells also stained for glial fibrillary acidic protein, neurofilament protein 200, and neuron specific enolase. During development, condensation of mesenchymal cells indicated the blastema of the EC at gestation day 40. At day 60, delicate collagen fibrils indicated the future tunica albuginea, faint alcianophilia was noted in the ground substance, and multilocular fat cells were scattered throughout the blastema. At birth, alcianophilia was moderate and multilocular fat cells were numerous. Three weeks after birth, single and grouped unilocular fat cells were seen, alcianophilia of ground substance was prominent, and former blastema cells presented as ramified myxoid cells. Eight weeks after birth, the EC primarily consisted of myxoid tissue, but the first islets of cartilage were seen in the center of myxoid areas. Unilocular fat cells already formed lobules. CONCLUSIONS: These results show that in the cat EC a) differentiation of adipocytes precedes differentiation of all the other tissue components, and b) differentiation of myxoid tissue precedes differentiation of cartilage. It is concluded that myxoid tissue may serve as a precursor of fibrous and elastic cartilage.

Adipocytes↗

Osteogenic activity of growth cartilage examined by implanting decalcified or devitalized ribs and costal cartilage zone, and living growth cartilage cells.

The formation of cartilage prior to bone in an endochondral bone formation process suggests that some osteogenic factors exist in the cartilage. This osteogenic activity of cartilage or cartilage cells was examined by implanting ribs and costal cartilage zone into a subcutaneous pocket of abdominal wall, or the growth cartilage (GC) cells into a peritoneal cavity. Rib segments of young (four-week-old) rabbits and rats were decalcified (DCed) in 0.6 N HCl for 24 h at 4 degrees C, or devitalized (DVed) by freezing/thawing and submerged in water for 48 h at 4 degrees C. These specimens were implanted into the subcutaneous pocket of abdominal wall of eight-week-old rabbits and rats. Both the DCed ribs and the DVed ones formed bone at costal GC zone after four to six weeks. Costal cartilage zone of young rabbits and rats was DCed or DVed as stated above, and implanted into the subcutaneous pocket of abdominal wall of eight-week-old rabbits and rats. The DVed costal cartilage zone specimens formed bone after three to four weeks, but the DCed ones did not, even after eight weeks. GC cells were dissociated enzymatically from rat costal GC zone, centrifuged to be made into pellets (5 x 10(5) cells/pellet), and implanted into the peritoneal cavity of eight-week-old syngeneic rats. After four weeks, implanted GC cells formed bone. These results suggest that there are some osteogenic factors in the GC zone produced by GC cells, whose activity is lost by decalcification, and which are different from known BMPs.

Abdominal Muscles↗

The distribution of cartilage oligomeric matrix protein (COMP) in equine carpal articular cartilage and its variation with exercise and cartilage deterioration.

Based on previous studies where tendons receiving the most load have been shown to have the highest levels of cartilage oligomeric matrix protein (COMP), we hypothesized that COMP distribution in articular cartilage may be influenced by mechanical loading. This investigation aimed (a) to describe the pattern of COMP immunoreactivity in middle carpal joint cartilage of two-year-old Thoroughbred horses; (b) to determine topographical variations; (c) to compare high (group 1) and low (group 2) intensity training and (d) to describe COMP immunoreactivity at sites with early osteoarthritis. Group 1 (n =6) underwent a 19 week high-intensity treadmill training programme and group 2 (n =6) were given daily walking until euthanasia. Dorsal and palmar sites on radial and third carpal articular surfaces were prepared. Immunohistochemistry was performed with polyclonal rabbit anti-equine COMP antiserum using a biotin-streptavidin/peroxidase method. Results showed: (a) intracellular immunoreactivity was present in all cartilage zones, but the distribution of COMP staining within the matrix varied between cartilage zones; (b) differences in distribution between sites were not observed, but total COMP levels in exercised horses (n =2) did vary between sites with dorsal sites containing less COMP than palmar sites on the radial, intermediate and third carpal lateral facet; (c) group 1 cartilage showed marked interterritorial distribution in the deep layer compared to group 2 where staining was more generalized throughout the matrix and (d) fibrillated cartilage showed increased local immunoreactivity in the matrix. These findings demonstrate zonal variations in equine COMP distribution which may be influenced by loading.

Animals↗

Articular cartilage cells immortalized by a temperature sensitive mutant of SV40 large T antigen survive and form cartilage tissue in articular cartilage environment.

A chondrogenic cell line, TC6, was established by using cells derived from articular cartilage of transgenic mice harboring a temperature-sensitive mutant simian virus (SV) 40 large T-antigen gene. TC6 cells express genes encoding proteins related to cartilage phenotypes such as type II collagen. To examine the in vivo behavior of the TC6 cells, these cells were implanted into cavity-shaped full-thickness defects made in the articular cartilage of the central part of the patellar grooves of mouse femora. One week after implantation, the morphology of the cells was still fibroblastic but these cells were just about to start to form a cartilage-like matrix. By 6 weeks after implantation, the cells had produced abundant cartilaginous matrix and their morphology became closer to that of authentic chondrocytes. This was in sharp contrast to the fibroblastic morphology of these cells in an in vitro environment even after long-term culture. These observations indicate that a cartilage-matrix environment provides a scaffold for the TC6 cells to form cartilage tissues. Our data show that the genetically engineered chondrocytic cell line, TC6, can form a cartilage-like matrix in vivo.

Animals↗

Inhibition of interleukin-1alpha-induced cartilage oligomeric matrix protein degradation in bovine articular cartilage by matrix metalloproteinase inhibitors: potential role for matrix metalloproteinases in the generation of cartilage oligomeric matrix protein fragments in arthritic synovial fluid.

OBJECTIVE: To determine whether matrix metalloproteinases (MMPs) degrade cartilage oligomeric matrix protein (COMP) to produce fragments similar to those found in synovial fluid (SF) from patients with arthritis. METHODS: COMP fragments were generated in vitro by treating (a) bovine articular cartilage with interleukin-1alpha (IL-1alpha), (b) purified bovine COMP with MMPs, and (c) articular cartilage with MMPs. The fragments generated in each case were analyzed by Western blot, using an antibody to the C-terminal heptadecapeptide of COMP. RESULTS: IL-1alpha stimulation of cartilage resulted in a fragmentation of COMP, which was inhibited by MMP inhibitors CGS 27023A and BB-94. Isolated, recombinant MMPs rapidly degraded purified COMP, as well as COMP residing in cartilage. Several COMP fragments produced in vitro had similar electrophoretic mobility to those in SF of patients with arthritis. CONCLUSION: MMPs may contribute to the COMP fragments found in vivo. Quantitation of MMP-specific fragments may be useful in the evaluation of MMP inhibitors in patients with arthritis.

Animals↗

[Morphological study of the epiphyseal cartilage in cartilage matrix deficiency (CMD) mouse--a consideration on the roles and functions of cartilage-specific proteoglycan].

Epiphyseal cartilages in mouse with cartilage matrix deficiency due to genetic failure to synthesize cartilage-characteristic proteoglycan were examined under light and electron microscope. Chondrogenesis and proliferation of chondrocytes seemed to occur, though extracellular matrix was small in amount and chondrocytes were packed closely without consistent orientation. In diaphysis, hypertrophic change of chondrocytes and perichondral ossification were observed. In the epiphysis, there was neither zone formation nor column formation, but hypertrophic chondrocytes and calcification in matrix were observed in the area adjacent to the bone shaft. Electron microscopy showed dilatation of rough endoplasmic reticulum, swelling of mitochondria and a large amount of lipid deposition in the chondrocyte. In the cartilage matrix, it was characteristic that a large number of thick collagen fibrils was arranged in parallel and few matrix granule was seen. These findings suggested that cartilage-characteristic proteoglycan was not essential for chondrogenesis, proliferation of chondrocytes and ossification, but were important for cytodifferentiation and chondrocyte activity.

Animals↗

Proteoglycan aggregation in injured articular cartilage. A comparison of healing lacerated cartilage with osteoarthritic cartilage.

Canine knee cartilage which had been damaged by laceration with a scalpel down to subchondral bone (Model A) and knee cartilage from dogs in whom osteoarthritis had been induced by transection of the anterior cruciate ligament by arthrotomy (Model B) were compared 7 weeks after the surgical procedures. Cartilage from the operated knees in Model A was hypocellular and showed extensive fibrosis, while samples from Model B showed moderate fibrillation and osteophyte formation. (In both cases water content was increased and uronic acid content decreased in cartilage from the operated knees.) Despite the much greater pathomorphologic changes in Model A, aggregation of 35S proteoglycans synthesized in culture was unchanged by the operation, while in Model B the proportion of proteoglycans existing as aggregates was diminished.

Animals↗

Cartilage and bone formation in repairing Achilles tendons within diffusion chambers: evidence for tendon-cartilage and cartilage-bone conversion in vivo.

Rodent Achilles tendons were subjected to midpoint tenotomy and allowed to recover for various times in situ before the operated tissue was removed, placed into a Millipore diffusion chamber, and inserted intraperitoneally into syngeneic hosts. Diffusion chambers were then removed at weekly intervals, such that the total time after the operation (i.e., time allowed to recover in situ plus time within the diffusion chamber) was up to 8 weeks, and examined histologically. Ectopic cartilage was produced within the diffusion chamber after a total of 4 weeks but only if the first 2 weeks of recovery were in situ. With increasing time, calcified cartilage, osteoid, and bone were also observed. Overall, the evidence suggests that the cartilage forms via a direct conversion from tendon tissue and that the bone may form as a result of differentiative changes of hypertrophic chondrocytes.

Achilles Tendon↗

Collagens--major component of the physiological cartilage matrix, major target of cartilage degeneration, major tool in cartilage repair.

Collagens serve important mechanical functions throughout the body and in particular in the connective tissues. Additionally, collagens exert important functions as cellular microenvironment and partly via binding and release of cellular growth mediators. In articular cartilage, fibrillar collagens are providing most of the biomechanical properties of the extracellular matrix essential for its functioning. The collagenous matrix is one main target of destructive processes in general degenerative joint disease and focal matrix lesions. The development of an adequate collagen framework represents the major aim of therapeutic cartilage repair. In this respect, collagenous matrices or collagen-imitating scaffolds are more and more emerging as highly suitable vehicles for cell and (growth) factor transport into cartilage lesion. Thus, collagens are not only major constituents of connective tissues in terms of integrity and function, they are also major targets of tissue destruction and regeneration and might become major tools to achieve tissue repair.

Absorbable Implants↗

Embryonic chick cartilage collagens. Differences in the low-Mr species present in sternal cartilage and tibiotarsal articular cartilage.

The collagenous polypeptides present in embryonic chick sternal and tibiotarsal cartilages have been solubilised by digestion with pepsin and separated by salt fractionation. Type II collagen, 1 alpha 2 alpha 3 alpha collagen, and two polypeptides (apparent molecular mass 150 and 42 kDa), which were reducible to a number of smaller peptides, were extracted from both tissues. However, also present in the peptic digests of tibiotarsal cartilages was a major non-reducible highly-soluble polypeptide of 45 kDa. This short-chain collagen is apparently identical to the pepsinized product of G collagen (Mr 59 000), a major low-Mr procollagen-like species previously detected in chick chondrocyte cultures.

Amino Acids↗

Comparative effects of azapropazone on cellular events at inflamed sites. Influence on joint pathology in arthritic rats, leucocyte superoxide and eicosanoid production, platelet aggregation, synthesis of cartilage proteoglycans, synovial production and actions of interleukin-1 in cartilage resorption correlated with drug uptake into cartilage in-vitro.

Azapropazone (APZ) has been compared with standard NSAIDs in title systems to establish aspects of its mode of action on cellular events at inflamed sites. APZ (150 mg kg-1 day-1) given for 10-13 days exhibited a reduction in joint pathology in established adjuvant arthritis in rats comparable with that of indomethacin (2 mg kg-1 day-1) and clobuzarit (20 mg kg-1 day-1). APZ was shown to be a potent inhibitor of the production of leucocyte superoxide and synovial interleukin-1 (IL-1)-like activity and stimulated articular cartilage proteoglycan synthesis, but was ineffective as an inhibitor of platelet aggregation or IL-1 induced cartilage degradation in-vitro. These in-vitro effects may have relevance to the mode of action of this weak inhibitor of prostaglandin synthesis.

Animals↗

Increased degradation and altered tissue distribution of cartilage oligomeric matrix protein in human rheumatoid and osteoarthritic cartilage.

We investigated the degradation and tissue distribution of cartilage oligomeric matrix protein in normal, osteoarthritic, and rheumatoid arthritic articular cartilage of the human knee. Cartilage was subjected to sequential extractions with buffers containing neutral salt, with EDTA, and finally with guanidine/HCl and then was analyzed by Western blotting with a polyclonal antiserum to human cartilage oligomeric matrix protein. Western blots of the nine neutral salt extracts from normal cartilage revealed mostly intact pentameric molecules of cartilage oligomeric matrix protein, in contrast to the 13 osteoarthritic and five rheumatoid arthritic cartilage samples that demonstrated marked degradation of cartilage oligomeric matrix protein as noted by a predominance of reduction-sensitive bands at approximately 150 kDa and nonreduction-sensitive bands in the 67-94 kDa range. The EDTA and guanidine/HCl extracts from all groups were similar and showed mostly intact molecules of cartilage oligomeric matrix protein, with smaller amounts of degraded cartilage oligomeric matrix protein identical to those resolved by the Western blots of the neutral salt extracts. Western blots of matched pairs of synovial fluid and cartilage extracts demonstrated cartilage oligomeric matrix protein fragments of the same molecular mass. Competitive enzyme-linked immunosorbent assay revealed significantly less cartilage oligomeric matrix protein in rheumatoid articular cartilage than in either normal or osteoarthritic cartilage. In contrast to normal cartilage, where cartilage oligomeric matrix protein was predominantly localized to the interterritorial matrix throughout all zones of the matrix, with increased staining in the deeper cartilaginous zones, the most intense staining in osteoarthritic cartilage was in the superficial zones of fibrillated cartilage, with little to no immunostaining in the midzones and relatively poor staining in the deeper cartilaginous zones. This distribution was the inverse of that for proteoglycans, as demonstrated by toluidine blue staining, where proteoglycans were depleted primarily from the superficial fibrillated cartilage. In mild to moderately affected rheumatoid cartilage, the tissue distribution of cartilage oligomeric matrix protein was similar to the distribution of proteoglycans, with relatively uniform staining of the interterritorial and territorial matrics. In more severely affected rheumatoid cartilage, the superficial zones demonstrated punctate immunostaining for cartilage oligomeric matrix protein in the interterritorial and territorial matrics, and staining was restricted to the territorial matrix in the deep cartilaginous zones. It is evident from this study that (a) noncollagenous proteins such as cartilage oligomeric matrix protein are greatly affected in arthritis, (b) degradation fragments released from the matrix into the synovial fluid reflect the processes occurring within the matrix, and (c) different zones of the articular cartilage are susceptible to degradation of cartilage oligomeric matrix protein in the different disease processes.

Adult↗

[Experimental study on laryngeal cartilage defects repair with differently preserved allogeneic cartilages transplantation].

OBJECTIVE: To study the effect of laryngeal cartilage defect repair with differently preserved allogeneic cartilage grafts. METHODS: 16 New Zealand white rabbits were used and divided into two groups. A 6 mm x 3 mm x 1 mm whole thickness cartilage defect was made in each side of the thyroid cartilage of each rabbit. In group one, the tissue-cultured cartilage grafts, preserved in RPMI-1640 medium for 30 days, were implanted in the left defects and the 4% formaldehyde preserved cartilage grafts for 30 days were implanted in the right defects. Fresh autogenous and allogeneic grafts were seprately transplanted into the right and left thyroid cartilage defects of the other group. Thyroid cartilages were taken out at 7, 30, 180 and 360 days after implantation. Samples were observed by macroscopy and prepared for hematoxylin and eosin (H&E) staining and immunohistochemical examination. RESULTS: No marked changes in form and volume were found in the fresh allografts and RPMI-1640 cultured cartilage grafts. The same as the autogenous cartilages, the defects of thyroid cartilage were successfully repaired by the fresh allografts and RPMI-1640 cultured cartilages. Whereas 4% formaldehyde preserved cartilage grafts were completely absorbed and replaced by cicatricial tissues in the thyroid cartilage defects. Histological observation showed that severe inflammatory cellular infiltration in the formaldehyde preserved cartilages at 7 and 30 days. The cartilage matrixes were resorpted and the chondrocytes showed degenerative change. Finally they were replaced by fibrous connective tissue at 360 days. In the RPMI-1640 cultured and fresh allogeneic cartilages only a little inflammatory cellular infiltration was observed and the cartilage matrixes were almost normal from 7 to 360 days. CONCLUSION: It is clinically feasible to use allogeneic grafts stored in RPMI-1640 medium or fresh allogeneic cartilage grafts for repairing laryngeal cartilage defects.

Animals↗

Vascularization and cartilage mineralization of the thyroid cartilage of Munich minipigs and domestic pigs.

Thyroid cartilages of Munich minipigs and domestic pigs were investigated by polychrome sequential labeling, radiography, intravascular injections, histologic examination and scanning electron microscopy in order to gain further insight into the process of vascularization and cartilage mineralization. The relationship between vascularization and cartilage mineralization has only been studied in chondroepiphyses of long bones. Vessels branch off the perichondrial vascular network and enter parts of the thyroid cartilage with a large transverse diameter. Cartilage canals, which are perichondral invaginations, contain an arteriole, a venule, a capillary network and connective tissue. The capillaries form a glomerulus-like structure deep in the matrix of the cartilage. Neighbouring cartilage canals do not display any anastomoses. Cartilage mineralization occurs in large areas of the thyroid cartilage. It is only found in the interterritorial extracellular matrix. Mineralization of the cartilage is evident in areas supplied with cartilage canals as well as in non-supplied areas. Mineralized interterritorial matrix is composed of circular structures of different sizes fusing to form plaques. In scanning electron microscopy circular structures appear as globules. It is possible to visualize the dynamic process of cartilage mineralization with polychrome sequential labeling; it proceeds up to 4 microm per week. Distribution of cartilage canals reveals their nutritional role for the cartilage. According to investigations in chondroepiphyses, cartilage mineralization starts adjacent to the glomerular end of cartilage canals. In contrast, no correlation between cartilage vascularization and the beginning of cartilage mineralization of the thyroid cartilage of Munich minipigs and of domestic pigs has been found.

Animals↗