Tenascin-C and the development of articular cartilage

In comparison to the vast literature on articular cartilage structure and function, relatively little is known about how articular cartilage forms during embryogenesis and is endowed with unique phenotypic properties, most notably the ability to persist and function throughout postnatal life. In this minireview, we summarize recent studies from our laboratory suggesting that the extracellular matrix protein tenascin-C is involved in the genesis and function of articular chondrocytes. These and other data have led us to propose that tenascin-C may be part of in vivo mechanisms whereby articular chondrocytes develop at the epiphysis of long bone models, remain functional throughout postnatal life, and avoid the endochondral ossification process undertaken by the bulk of chondrocytes located in the metaphysis and diaphysis of skeletal models.     

In vivo cartilage formation from growth factor modulated articular chondrocytes

Recent procedures for autologous repair of cartilage defects may be difficult in elderly patients because of the loss of stem cells and chondrocytes that occurs with age and the slow in vitro proliferation of chondrocytes from aged cartilage. In this study secondary chondroprogenitor cells were obtained by modulating the phenotype of articular chondrocytes with growth factors and stimulating the proliferation of these cells in culture. Chondrocytes isolated from the articular cartilage of mature New Zealand White rabbits were exposed to a combination of transforming growth factor beta and basic fibroblast growth factor treatment. These cells ceased the production of Collagen II (a marker for the chondrocyte phenotype) and underwent a 136-fold increase in cell number. Next, the cells were placed in high density culture and reexpressed the chondrocyte phenotype in vitro and formed hyaline cartilage in an in vivo assay. Primary chondrocytes obtained from articular cartilage of elderly humans could be manipulated in a similar fashion in vitro. These human secondary chondroprogenitor cells formed only cartilage tissue when assayed in vivo and in tissue bioreactors. This approach may be essential for autologous repair of degenerated articular cartilage in elderly patients with osteoarthritis.     

Repair of large full-thickness articular cartilage defects with allograft articular chondrocytes embedded in a collagen gel

Full-thickness articular cartilage defects are a major clinical problem; however, presently there is no treatment available to regeneratively repair these lesions. The current therapeutic approach is to drill the base of the defect to expose the subchondral bone with its cells and growth factors. This usually results in a repair tissue of fibrocartilage that functions poorly in the loaded joint environment. The use of phenotypically appropriate chondrocytes embedded in a collagen gel delivery vehicle may provide a method that could be used to repair full-thickness articular cartilage defects with functionally satisfactory hyaline cartilage. Allograft articular chondrocytes embedded in a type I collagen gel were transplanted into large (6 x 3 x 3 mm), full-thickness articular cartilage defects in condylar and patellar weight-bearing surfaces to develop clinically applicable methods to repair articular cartilage defects. Chondrocytes were isolated from the articular cartilage of 4-week-old New Zealand rabbits and embedded in type I collagen gels. This composite was transplanted into a full-thickness defect on the medial femoral condyle and patellar groove of adolescent host rabbits. The repair cartilage was assessed histologically by a semiquantitative scoring system and biomechanically with a microindentation technique of specimens 4-48 weeks after chondrocyte transplantation. Defects in both locations were repaired with histologically apparent hyaline cartilage observed from as early as 4 weeks until 48 weeks after transplantation. The repair cartilage in the medial femoral condyle was more irregular than in the patellar groove, but in all other respects was similar. The grafted tissue did not remodel and differentiate into the morphological zones seen in normal articular cartilage. No tidemark or subchondral bony plate formed even 48 weeks after transplantation. Biomechanically, the repaired cartilage demonstrated indentation values similar to normal articular cartilage 12 weeks after transplantation and remained the same 48 weeks after transplantation. By contrast, the control (i.e., empty) defects healed with tissue that exhibited very poor metachromatic staining and exhibited very high indentation values. Incomplete bonding of the repair tissue to the normal cartilage was seen, and the surface was significantly irregular with major discontinuities. These observations provide the basis for considering the use of allograft articular chondrocytes to repair articular cartilage defects in the weight-bearing regions of the knee.     

Expression of anchorin CII (cartilage annexin V) in human young, normal adult, and osteoarthritic cartilage

In its tissue-specific function as a collagen receptor of chondrocytes, cartilage annexin V (anchorin CII) occupies a key position in the organization of the cell-extracellular matrix (ECM) junction for the tissue. The general role of annexin V (Anx V) in other tissues suggests involvement in cellular secretory processes and in regulation of apoptosis. Immunohistochemical analysis of Anx V in growth plate cartilage, confirmed by in situ hybridization, suggests that Anx V is prominently expressed and forms a major constituent of growth plate chondrocytes. Anx V epitopes are also located in the pericellular matrix of hypertrophic cartilage. In adult articular cartilage the expression is downregulated, with the highest levels of immunostaining found in the upper third of the articular cartilage layers and almost no antigen found in the deep layers. Osteoarthritic (OA) cartilage is characterized by a significant upregulation of message and protein throughout the entire depth of the tissue, an accumulation of cytoplasmic annexin V epitopes, and a release of epitopes into the pericellular and interterritorial matrix, in part co-localized with granular structures. Therefore, Anx V expression and tissue distribution may serve as a histological marker for metabolic alterations and for changes in the cellular phenotype associated with OA.     

Fibronectin synthesis in superficial and deep layers of normal articular cartilage

OBJECTIVE: To study the distribution and synthesis of fibronectin (FN) in superficial and deep layers of normal articular cartilage. METHODS: Superficial and deep bovine and human articular cartilage slices were used to extract and quantitate FN by radioimmunoassay. Chondrocytes were also isolated by collagenase digestion for FN extraction and culture. Superficial and deep cartilage explants were cultured with and without stimulation by cytokines. Quantitation of newly synthesized FN was carried out by incubation with 35S-methionine. FN was purified on gelatin-agarose columns and further characterized by polyacrylamide gel electrophoresis. FN messenger RNA (mRNA) was quantitated by Northern blot analysis. RESULTS: Freshly isolated bovine chondrocytes from deep cartilage contained 2.3 +/- 0.2 times more FN than was found in superficial cells (P < 0.025). Deep cartilage explants contained 1.2 times more FN than was found in superficial tissue. Explants obtained from deep cartilage synthesized 2.4 times more FN per cell than did superficial tissues (P < 0.01). FN synthesis as a fraction of total protein synthesis was significantly greater in deep explants (P < 0.01) compared with superficial tissues. Isolated deep chondrocytes in culture synthesized 1.89 +/- 0.33-fold more FN than did superficial cells (P < 0.05). Cytokine-stimulated superficial cartilage explants failed to respond in terms of FN synthesis. FN mRNA quantitation showed no significant differences between superficial and deep populations. CONCLUSION: Since FN plays a major role in cell adhesion to damaged cartilage surfaces, our results suggest that modulation of FN synthesis near the articular surface of cartilage may be one of the factors that impede pannus invasion following an inflammatory insult to the joint.     

Ultrastructural changes in articular cartilage in rheumatoid arthritis

Electron microscope studies of the articular cartilages removed in the course of the operation on 6 patients with rheumatoid arthritis were carried out. The processes of destruction of chondrocytes and the cartilaginous matrix in different regions of the articular cartilage were traced. In the surface areas of the drastically changed cartilage there were observed leucocytes of the synovial fluid, and in deeper areas--disintegration of chondrocytes and extracellular disposition of lysosomes and altered organellas, destroyed cartilaginous cells. In these areas destruction of collagenous fibres was particularly intensive. In areas of the tissue remote from the destuction hypertrophy of chondrocytes due to hyperplasia of various organellas and the Golgi complex in particular were noted. In the Golgi zone granules of glycogen were detected. No mitoses were observed. Apparently, the enzymatic destruction of the cartilaginous matrix in rheumatoid arthritis could proceed at the expense of the activazation of the synovial fluid lysosomes and lysosomes of chondrocytes themselves. A reparative regeneration of the disintegrating matrix was realized mainly because of hypertrophy of the functionally preserved chondrocytes.     

Chondromodulin-I expression in rat articular cartilage

The localization and expression of chondromodulin-I (ChM-I), an angiogenesis inhibitor, in the rat articular cartilage during maturation from 2 to 10 weeks of age were examined by immunohistochemistry, Western blot analysis and ribonuclease protection assay, and the results were compared with those in the epiphyseal cartilage. ChM-I was found to be diffusely immunostained in the inter-territorial space of the cartilage matrix from the intermediate to the deep layers at the immature stage. As the articular cartilage matured, the immunoreactivity was localized around the hypertrophic chondrocytes in the deep layer and the immunoreactivity became weak after maturation. In contrast, the ChM-I immunoreactivity was intense in the epiphyseal cartilage at all ages examined. ChM-I was detected by Western blotting as a broad band or occasionally as a cluster of multiple bands (approximately 25 kDa) in both the articular and the epiphyseal cartilage. The intensity of the bands decreased gradually with age in the articular cartilage, but was unchanged in the epiphyseal cartilage at all ages. Ribonuclease protection assay revealed that ChM-I mRNA also decreased gradually with age in the articular cartilage in parallel with the maturation of the articular cartilage, while no decrease in ChM-I mRNA was found in the epiphyseal cartilage. The expression of ChM-I mRNA in the articular cartilage was less than that in the epiphyseal cartilage at all ages. The decrease in amount of ChM-I in the mature articular cartilage suggests that ChM-I plays a more important role in the maintenance of avascularity in the immature articular cartilage than in the mature one. The avascular condition may be preserved by angiogenic inhibitors or mechanisms other than ChM-I in the mature articular cartilage.     

Cathepsin B in osteoarthritis: cytochemical and histochemical analysis of human femoral head cartilage

OBJECTIVE: To localise the cysteine endopeptidase cathepsin B in chondrocytes and cartilage from normal and osteoarthritic (OA) human femoral heads in order to provide qualitative information on its cellular expression and distribution at possible sites of action. METHODS: OA articular cartilage was obtained at surgery for total hip replacement; control cartilage was obtained at postmortem. Chondrocytes were isolated by sequential enzymatic digestion and cathepsin B analysed by immunocytochemistry and activity staining with a fluorogenic substrate. Lysosomes were visualised by fluorescence microscopy after staining of living cells with acridine orange. Using a histochemical reaction, enzyme activity was measured in cryosections of full thickness cartilage. RESULTS: Chondrocytes from normal cartilage contained very few lysosomes and only a minor cell population was cathepsin B positive. A high proportion of chondrocytes from active OA cartilage contained a large number of lysosomes and an excess of cathepsin B in intracellular organelles; the enzyme was stored in an active form. In this respect, OA chondrocytes closely resembled normal cells that had been phenotypically modulated by serial subcultures. No cathepsin B activity could be detected by histochemistry in either chondrocytes or matrix of normal cartilage. While apparently intact and severely degraded OA cartilage was also cathepsin B negative, tissue at sites of active destruction and, particularly, at repair sites was highly positive. CONCLUSION: The presence and the particular distribution of active cathepsin B in OA cartilage at 'more involved' sites suggest a pathological role for this enzyme in sustaining and perpetuating cartilage degradation. While other stimuli may also be responsible for cathepsin B expression in OA chondrocytes, the similarity with artificially modulated cells indicates fibroblastic metaplasia as a plausible mechanism.     

A unique ectonucleotide pyrophosphohydrolase associated with porcine chondrocyte-derived vesicles

Previous studies have shown increased nucleotide pyrophosphohydrolase (EC 3.6.1.8) (NTPPHase) activity in detergent extracts of degenerated human cartilage containing calcium pyrophosphate dihydrate (CPPD) crystals relative to those from osteoarthritis or normal cartilage. NTPPHase was later shown to be an ectoenzyme and its activity was increased in synovial fluid from patients with CPPD crystal deposits relative to fluids from other types of arthritis. We have purified a soluble 61-kD NTPPHase from conditioned media of organ-cultured porcine articular cartilage to electrophoretic homogeneity. Its NH2-terminal sequence through 26 cycles showed < 30% homology to any previously reported protein sequence. An antibody raised to a synthetic peptide corresponding to this sequence reacted with denatured but not native enzyme. This antibody reacted against a sedimentable vesicle-associated 127-kD protein in conditioned media from cultured articular cartilage or from chondrocytes in primary monolayer culture and against a series of soluble proteins in conditioned media supernatant, including a 61-kD protein representing our original isolate. No reactivity was found in 1% SDS extracts of washed cultured chondrocytes, although these contained greater NTPPHase activity than the conditioned media. Antibody to PC-1, another ectoNTPPHase, reacted with 1% SDS extracts of whole chondrocytes but not against those chromatographic fractions containing the major portion of NTPPHase activity. Release of the vesicle-associated 127-kD enzyme into conditioned medium was stimulated three- to sevenfold by TGF beta 1. The antibody also reacted with a series of soluble proteins and with 127-kD sedimentable protein in human synovial fluid. Kinetic studies supported the existence of a unique vesicle-associated NTPPHase; apparent Km (mM) of chondrocyte membrane NTPPHase was 1.5 and 3.0 at pH 7.3 and 9.88, respectively; apparent Km (mM) of vesicle associated NTPPHase was 0.83 and 1.28 at pH 7.3 and 9.88. The data suggest the existence of a unique ecto-NTPPHase associated with vesicles derived from normal articular cartilage.     

Parathyroid hormone-related proteins is abundant in osteoarthritic cartilage, and the parathyroid hormone-related protein 1-173 isoform is selectively induced by transforming growth factor beta in articular chondrocytes and suppresses generation of extracellular inorganic pyrophosphate

OBJECTIVE: Parathyroid hormone-related protein (PTHrP) is a major, locally expressed regulator of growth cartilage chondrocyte proliferation, differentiation, synthetic function, and mineralization. Because mechanisms that limit cartilage chondrocytes from maturing and mineralizing are diminished in osteoarthritis (OA), we studied PTHrP expression by articular chondrocytes. METHODS: PTHrP was studied in normal knee cartilage samples and cultured articular chondrocytes, and in cartilage specimens from knees with advanced OA, obtained at the time of joint replacement. RESULTS: PTHrP was more abundant in OA than in normal human knee articular cartilage. Both demonstrated PTH/PTHrP receptor expression. PTHrP 1-173, one of three alternatively spliced PTHrP isoforms, was exclusively expressed and induced by transforming growth factor beta in cultured chondrocytes. Chondrocytes mainly used the GC-rich P2 alternative promoter to express PTHrP messenger RNA. Inhibition by PTHrP 1-173, but not by PTHrP 1-146 or PTHrP 1-87, of inorganic pyrophosphate (PPi) elaboration suggested selective functional properties of the 1-173 isoform. Exposure to a neutralizing antibody to PTHrP increased PPi elaboration by articular chondrocytes. CONCLUSION: Increased expression of PTHrP, including the 1-173 isoform, has the potential to contribute to the pathologic differentiated functions of chondrocytes, including mineralization, in OA.     

Expression of cartilage oligomeric matrix protein by human synovium

Human synovium was analyzed for the possible expression of cartilage oligomeric matrix protein (COMP). Immunostaining with polyclonal antiserum to COMP demonstrated positive staining within the synovial cells and immediately subjacent connective tissue, with less intense staining in the deeper connective tissue. Western blot analysis using either polyclonal or monoclonal antibodies to human COMP confirmed the presence of COMP by immunoreactive bands with the same molecular mass (approximately 110 kDa) as purified articular cartilage COMP. PCR using oligonucleotides that span human COMP exons 7-13 revealed identical amplification products from cDNA prepared from either human chondrocytes or synovium. Northern blot analysis using a biotinylated-probe to human COMP, spanning exons 12-13, also reveal an identical hybridization product to either human chondrocyte or synovium total RNA. Human synovium should be considered as a potential tissue source of COMP in any investigation of biological markers of cartilage metabolism.     

Assessment of donor cell and matrix survival in fresh articular cartilage allografts in a goat model

The long-term survival of allografts of articular cartilage has been proposed to be dependent on the survival of the cells that maintain the unique structural and material properties of the allograft. In this study, we assessed cell survival in 24 fresh articular cartilage allografts of the medial plateau in a Spanish-goat model. A DNA-probe technique was used to distinguish clearly between DNA from donor (allograft) and host cells. The intraarticular survival of viable allograft chondrocytes in the transplanted articular cartilage started to diminish as early as 3 weeks after transplantation; however, there was considerable variation in the amount of donor cell DNA detected in the allografts at 6 and 12 months following transplantation. This contrasts with our experience with fresh allografts of ligament, tendon, and meniscus, in which no donor DNA was detected 4 weeks after transplantation. DNA from host cells was present in all articular cartilage allografts, as evidenced by detectable unique host DNA patterns. Histological and histochemical assays showed that none of the transplants demonstrated normal structure and composition at 1 year after transplantation. The grafts in which large quantities of donor DNA were present appeared grossly superior to those with no or reduced remaining demonstrable donor DNA.     

Effect of cultured autologous chondrocytes on repair of chondral defects in a canine model

Articular cartilage has a limited capacity for repair. In recent clinical and animal experiments, investigators have attempted to elicit the repair of defects of articular cartilage by injecting cultured autologous chondrocytes under a periosteal flap (a layer of periosteum). The objective of the present study was to determine the effect of cultured autologous chondrocytes on healing in an adult canine model with use of histomorphometric methods to assess the degree of repair. A total of forty-four four-millimeter-diameter circular defects were created down to the zone of calcified cartilage in the articular cartilage of the trochlear groove of the distal part of the femur in fourteen dogs. The morphology and characteristics of the original defects were defined in an additional six freshly created defects in three other dogs. Some residual noncalcified articular cartilage, occupying approximately 2 per cent of the total cross-sectional area of the defect, was sometimes left in the defect. The procedure sometimes damaged the calcified cartilage, resulting in occasional microfractures or larger fractures, thinning of the zone of calcified cartilage, or, rarely, small localized penetrations into subchondral bone. The forty-four defects were divided into three treatment groups. In one group, cultured autologous chondrocytes were implanted under a periosteal flap. In the second group, the defect was covered with a periosteal flap but no autologous chondrocytes were implanted. In the third group (the control group), the defects were left empty. The defects were analyzed after twelve or eighteen months of healing. Histomorphometric measurements were made of the percentage of the total area of the defect that became filled with repair tissue, the types of tissue that filled the defect, and the integration of the repair tissue with the adjacent cartilage at the sides of the defects and with the calcified cartilage at the base of the defect. In histological sections made through the center of the defects in the three groups, the area of the defect that filled with new repair tissue ranged from a mean total value of 36 to 76 per cent, with 10 to 23 per cent of the total area consisting of hyaline cartilage. Integration of the repair tissue with the adjacent cartilage at the edges of the defect ranged from 16 to 32 per cent in the three groups. Bonding between the repair tissue and the calcified cartilage at the base of the defect ranged from 41 to 89 per cent. With the numbers available, we could detect no significant difference among the three groups with regard to any of the parameters used to assess the quality of the repair. In the two groups in which a periosteal flap was sutured to the articular cartilage surrounding the defect, the articular cartilage showed degenerative changes that appeared to be related to that suturing.     

Immunohistochemical profile of basic fibroblast growth factor and heparan sulphate in adult rat mandibular condylar cartilage

Basic fibroblast growth factor (bFGF) and heparan sulphate (HS) were detected immunohistochemically in mandibular condylar cartilage, and the findings compared with those on epiphyseal articular cartilage. In the condylar cartilage, both bFGF and HS were localized in chondrocytes throughout the various zones including the fibrous, proliferative, mature-cell and hypertrophic zones: bFGF immunostaining was most significant in the proliferative and mature-cell zones, while intense staining for HS was found mainly in the hypertrophic zone. Immunoreaction for bFGF was detected in the nuclei of chondrocytes, whereas HS staining was observed in the cytoplasm. In articular cartilage, only chondrocytes beneath the superficial zone (intermediate zone) demonstrated both bFGF and HS immunoreactivities. Chondrocytes in the deeper calcifying region of the articular cartilage did not immunoreact for either bFGF or HS. These findings suggest that, in contrast to the epiphyseal articular cartilage, a continuous bFGF-mediated remodelling of cells and matrix takes place in mandibular condylar cartilage during the process of endochondral ossification.     

Loss of decorin from the surface zone of articular cartilage in a chick model of osteoarthritis

The objective of this study was to immunolocalize decorin and to assess changes as a result of pyridoxine (PN) deficiency in chick articular cartilage from femoral condyles. After maintenance on a normal diet for the first two weeks after hatching, 15 broiler chickens were deprived of this vitamin for 6 weeks. It was previously shown that the ankle joints of PN-deficient animals are swollen with effusions. They also present an abnormal gait, enlarged bony margins, and fissuring of the articular cartilages. Milder changes (no fissures) were also shown in the knee joints. Data from a previous study were suggestive that sulfated glycosaminoglycans are lost from the knee cartilage surface into synovial fluid. The current study was focused on the small proteoglycan, decorin, which coats the surface of collagen fibrils and may regulate their morphology. To examine decorin in normal and PN-deficient articular cartilage, a monoclonal antibody to an epitope on the protein core of decorin was used for immunohistochemical staining of tissue sections and for Western Blot analysis of cartilage extracts. Reduction of staining with the antibody was demonstrated in the tangential surface zone of PN-deficient cartilage, and Western Blot analysis showed reduced intensity of decorin bands compared to normal controls. These data suggest that a lack of decorin may play a role in the enlargement of collagen bundles in the tangential zone of PN-deficient articular cartilage as observed in a previous electron microscopic study.     

Rabbit articular cartilage defects treated with autologous cultured chondrocytes

Adult New Zealand rabbits were used to transplant autologously harvested and in vitro cultured chondrocytes into patellar chondral lesions that had been made previously and were 3 mm in diameter, extending down to the calcified zone. Healing of the defects was assessed by gross examination, light microscope, and histological-histochemical scoring at 8, 12, and 52 weeks. Chondrocyte transplantation significantly increased the amount of newly formed repair tissue compared to the found in control knees in which the lesion was solely covered by a periosteal flap. In another experiment, carbon fiber pads seeded with chondrocytes were used as scaffolds, and repair significantly increased at both 12 and 52 weeks compared to knees in which scaffolds without chondrocytes were implanted. The histologic quality scores of the repair tissue were significantly better in all knees in which defects were treated with chondrocytes compared to knees treated with periosteum alone and better at 52 weeks compared to knees in which defects were treated with carbon scaffolds seeded with chondrocytes. The repair tissue, however, tended to incomplete the bonding to adjacent cartilage. This study shows that isolated autologous articular chondrocytes that have been expanded for 2 weeks in vitro can stimulate the healing phase of chondral lesions. A gradual maturation of the hyalinelike repair with a more pronounced columnarization was noted as late as 1 year after surgery.     

Experimental studies of mechanically-induced articular cartilage defects following implantation of allogeneic embryonal chondrocytes in a collagen-fibrin gel in chickens

Full thickness defects (diameter 1,7 mm; depth 2,5 mm) were created mechanically in articular cartilage and subchondral bone of the condyles of tibiotarsal joints of 9-month old chickens. This full-thickness defects were repaired with cultured allogenic embryonic chick epiphyseal chondrocytes from the tibiae and femura of 10-days-old chicken embryos. The cells were embedded in a collagen-fibrinogen-matrix. Controls were similarly operated, but received either no treatment or implants the delivery substance only. Healing of the defects was observed macroscopically, histologically, histochemically and histomorphometrically after 3, 12 and 24 weeks. This graft was successfully transplanted in mechanically induced defects in 80%. The resulting hyaline cartilage was structurally reorganized according to the host pattern and under the influence of environmental conditions. The articular zone preserved it's cartilaginous phenotype, whereas the subchondral regions were transformed into bone. 12 weeks after the operation the defects in the experimental group were completely filled. In all instances in this group, there was an initial extreme increase of mitotic rate and cell number. After 24 weeks normal and subnormal values were founded. The defects in the control groups healed with fibrocartilage. Our results showed, that only the defects in the experimental group were completely filled with reparative hyaline cartilage tissue. In the present study the mixture of cultured allogenic embryonic chondrocytes and a collagen-fibrinogen-matrix was used successfully as a transplant for repairing defects in articular cartilage of chickens. Thus allogenic transplantation of cultured embryonal chondrocytes appears to be one of the most promising methods for the restoration of articular cartilage.     

Gene expression of matrix metalloproteinases 1, 3, and 9 by chondrocytes in osteoarthritic human knee articular cartilage is zone and grade specific

OBJECTIVES: Matrix metalloproteinases (MMPs) are thought to be major mediators of cartilage destruction. Osteoarthritis (OA) is characterised by cartilage degradation. This study explores gene expression of three MMPs in articular chondrocytes during the histological development of the cartilage lesion of OA. METHODS: Biopsy specimens of human normal and OA cartilage, classified into four grades on the basis of histology, were probed for MMPs 1, 3, and 9 using 35S-labelled cDNA probes. The signal was measured at four different depths (zones) using an automated image analyser and compared with signal from sections probed with lambda DNA. Rheumatoid synovium was used as a positive control for MMP gene expression. RESULTS: Rheumatoid tissue contained mRNA for all three MMPs. Expression in chondrocytes varied with the depth of the chondrocyte in the cartilage and the histomorphological extent of the OA changes. There was no detectable mRNA signal for these three MMPs in normal cartilage. In general, in OA, MMP-1 gene expression was greatest in the superficial cartilage in established disease. By contrast mRNAs for MMP-3 and 9 were expressed deeper in the cartilage, MMP-9 early in disease and MMP-3 with a biphasic pattern in early and late stage disease, most pronounced in the latter. This was a consequence of differential expression in single cells and chondrocyte clusters in late disease. CONCLUSION: The data indicate that expression of genes for MMPs 1, 3, and 9 is differentially regulated in human articular chondrocytes and, in individual cells, is related to the depth of the chondrocyte below the cartilage surface and the nature and extent of the cartilage lesion.     

The fate of the articular cartilage in intracapsular fracture of the femoral neck (articular cartilage in femoral neck fracture)

The fate of the articular cartilage of the hip joint with intracapsular neck fracture was studied by histological, histochemical and autoradiographic techniques and by using a polarized microscope and a scanning electron microscope. Cartilage specimens from 93 femoral heads and 7 acetabula were obtained from fractured hips 2 days to 4 1/3 years postfracture and from control hips with various disorders. The cartilage degeneration appeared 2 weeks after fracture and advanced steadily with time. The matrix was covered, invaded and ultimately replaced by the fibrous tissue. Chondrocyte viability, though it was lost from the surface, was recognized in the deep matrix even in the oldest fracture examined. It is concluded that the humoral factor directly caused by the injury as well as the biomechanical impairment, i.e. a loss of physical stress, may play an essential role in the pathogenesis of the degeneration. The possibility of regeneration was discussed.     

Temporal pattern of cysteine endopeptidase (cathepsin B) expression in cartilage and synovium from rabbit knees with experimental osteoarthritis: gene expression in chondrocytes in response to interleukin-1 and matrix depletion

OBJECTIVE: To determine the temporal pattern of expression of cathepsin-B in chondrocytes and synovium in experimental osteoarthritis, and to determine possible mechanisms for upregulation and secretion of cathepsin-B from chondrocytes. METHODS: Experimental osteoarthritis was induced with partial medial meniscectomy (PM); sham operated (SH) and normal (N) rabbits were used as controls. Cathepsin-B mRNA expression was assessed with northern blotting with a 32P labelled cDNA probe. Cathepsin-B was measured in conditioned media or cell extracts using a fluorogenic substrate Z-Arg-Arg-AMC. Chondrocyte monolayers were used to determine cathepsin-B expression in response to interleukin-1 beta (IL-1 beta). Cartilage explants were used to test the effect of matrix depletion on cathepsin-B release. RESULTS: Chondrocytes obtained from experimental osteoarthritis knees did not show cathepsin-B mRNA upregulation. However, isolated chondrocytes secreted cathepsin-B into the culture medium. Enzyme release was significantly higher at 8 weeks relative to controls, but not at 12 weeks or 4 weeks. Enzyme released from synovium was significantly higher in PM group compared with SH group at 4 and 8 weeks. IL-1 beta was ineffective in upregulating steady state cathepsin-B mRNA in chondrocytes; however, it upregulated the intracellular enzyme, and this was blocked with cycloheximide. Enzymatic depletion of cartilage matrix after exposure of explants to IL-1 resulted in release of significantly higher amounts of cathepsin-B into the medium by matrix depleted chondrocytes compared with intact explants. CONCLUSIONS: In experimental osteoarthritis, cathepsin-B is upregulated in synovial tissue during the early degenerative phase. Progression of experimental osteoarthritis is accompanied by upregulation of cathepsin-B in cartilage. Cartilage and synovial cathepsin-B levels decline as experimental osteoarthritis advances to more degenerative states. IL-1 upregulates intracellular cathepsin-B by increasing cathepsin-B protein synthesis; it is not an effective stimulus for enzyme secretion. Depletion of cartilage matrix during progression of experimental osteoarthritis may contribute to secretion of cathepsin-B and perpetuation of cartilage destruction.