Osteopontin is expressed by adult human osteoarthritic chondrocytes: protein and mRNA analysis of normal and osteoarthritic cartilage.

Osteopontin, a sulfated phosphoprotein with cell binding and matrix binding properties, is expressed in a variety of tissues. In the embryonic growth plate, osteopontin expression was found in bone-forming cells and in hypertrophic chondrocytes. In this study, the expression of osteopontin was analyzed in normal and osteoarthritic human knee cartilage. Immunohistochemistry, using a monoclonal anti-osteopontin antibody was negative on normal cartilage. These results were confirmed in Western blot experiments, using partially purified extracts of normal knee cartilage. No osteopontin gene expression was observed in chondrocytes of adult healthy cartilage, however, in the subchondral bone plate, expression of osteopontin mRNA was detected in the osteoblasts. In cartilage from patients with osteoarthritis, osteopontin could be detected by immunohistochemistry, Western blot analysis, in situ hybridization, and Northern blot analysis. A qualitative analysis indicated that osteopontin protein deposition and mRNA expression increase with the severity of the osteoarthritic lesions and the disintegration of the cartilaginous matrix. Osteopontin expression in the cartilage was limited to the chondrocytes of the upper deep zone, showing cellular and territorial deposition. The strongest osteopontin detection was found in deep zone chondrocytes and in clusters of proliferating chondrocytes from samples with severe osteoarthritic lesions. These data show the expression of osteopontin in adult human osteoarthritic chondrocytes, suggesting that chondrocyte differentiation and the expression of differentiation markers in osteoarthritic cartilage resembles that of epiphyseal growth plate chondrocytes.     

Development and aging of the articular cartilage of the rabbit knee joint: Distribution of biglycan, decorin, and matrilin-1.

We determined the distributions of the small proteoglycans biglycan and decorin and the glycoprotein matrilin-1 (cartilage matrix protein) during development and aging of articular cartilage in the rabbit knee joint. Before cavitation, the matrices of the interzone and the adjacent epiphyseal cartilage do not contain biglycan or decorin, but some chondrocytes express their mRNAs. Matrilin-1 is found only in the deeper epiphyseal cartilage. After cavitation, biglycan and decorin are detected in the presumptive articular cartilage, but there is no matrilin-1. All are present in the underlying epiphyseal cartilage. In the neonate, the epiphyseal cartilage is ossified and the articular cartilage becomes a discrete layer. Biglycan and decorin accumulate in the articular cartilage, but matrilin-1 remains confined to the residual epiphyseal cartilage. In the adult, the distributions of biglycan and decorin are highly variable. Decorin tends to be confined to the central region; matrilin-1 is absent. The findings indicate that the articular and epiphyseal cartilages are different from the earliest developmental stages. The epiphyseal cartilage can be identified by its possession of matrilin-1. Epiphyseal cartilage is removed during development to leave the articular cartilage. The relationships between the distributions of decorin and matrilin-1 and the fibrillar collagens are discussed. (J Histochem Cytochem 47:1603-1615, 1999)     

Localization of types I, II, and III collagen mRNAs in developing human skeletal tissues by in situ hybridization

Paraffin sections of human skeletal tissues were studied in order to identify cells responsible for production of types I, II, and III collagens by in situ hybridization. Northern hybridization and sequence information were used to select restriction fragments of cDNA clones for the corresponding mRNAs to obtain probes with a minimum of cross-hybridization. The specificity of the probes was proven in hybridizations to sections of developing fingers: osteoblasts and chondrocytes, known to produce only one type of fibrillar collagen each (I and II, respectively) were only recognized by the corresponding cDNA probes. Smooth connective tissues exhibited variable hybridization intensities with types I and III collagen cDNA probes. The technique was used to localize the activity of type II collagen production in the different zones of cartilage during the growth of long bones. Visual inspection and grain counting revealed the highest levels of pro alpha 1(II) collagen mRNAs in chondrocytes of the lower proliferative and upper hypertrophic zones of the growth plate cartilage. This finding was confirmed by Northern blotting of RNAs isolated from epiphyseal (resting) cartilage and from growth zone cartilage. Analysis of the osseochondral junction revealed virtually no overlap between hybridization patterns obtained with probes specific for type I and type II collagen mRNAs. Only a fraction of the chondrocytes in the degenerative zone were recognized by the pro alpha 1(II) collagen cDNA probe, and none by the type I collagen cDNA probe. In the mineralizing zone virtually all cells were recognized by the type I collagen cDNA probe, but only very few scattered cells appeared to contain type II collagen mRNA. These data indicate that in situ hybridization is a valuable tool for identification of connective tissue cells which are actively producing different types of collagens at the various stages of development, differentiation, and growth.     

Immunohistochemical localization of fibroblast growth factor-2 in normal and brachymorphic mouse tibial growth plate and articular cartilage

Epiphyses of the proximal tibiae of 7-week-old normal and homozygous recessive brachymorphic mice (bm/bm) were immunostained using a monoclonal antibody to basic fibroblast growth factor to determine its expression in growth plate cartilage, osteoblasts on the surfaces of the primary spongiosa and articular cartilage. In the normal growth plate, the immunoreactive factor was present in chondrocytes of the proliferating and upper hypertrophic zones but absent from lower hypertrophic chondrocytes. Immunostaining was present only in the territorial extracellular matrix immediately adjacent to the chondrocytes of the proliferating and upper hypertrophic zones. Osteoblasts of the primary spongiosa stained heavily in normal mice. Strong staining was observed in intermediate zone articular chondrocytes. Cells in the superficial layer of articular cartilage were unstained. The extracellular matrix of the articular cartilage was completely free of immunostaining. In contrast, the reduced size of bm/bm growth plates was accompanied by significantly reduced staining intensity in proliferating and upper hypertrophic chondrocytes, and staining was absent from the territorial extracellular matrix of all zones of the bm/bm growth plate. Osteoblasts of the primary spongiosa of bm/bm mice stained less than those of normal mice. Articular cartilage chondrocytes in the intermediate zone stained with less intensity in bm/bm mice, and the cells of the superficial layer were unstained. The extracellular matrix of bm/bm articular cartilage was completely free of staining. Brachymorphic epiphyseal growth plate and articular chondrocytes, and osteoblasts in the primary spongiosa, express reduced amounts of immunoreactive fibroblast growth factor-2. This phenotypical characteristic may be associated with abnormal endochondral ossification and development of bone in brachymorphic mice     

Immunohistochemical localization of fibroblast growth factor-2 in normal and brachymorphic mouse tibial growth plate and articular cartilage

Epiphyses of the proximal tibiae of 7-week-old normal and homozygous recessive brachymorphic mice (bm/bm) were immunostained using a monoclonal antibody to basic fibroblast growth factor to determine its expression in growth plate cartilage, osteoblasts on the surfaces of the primary spongiosa and articular cartilage. In the normal growth plate, the immunoreactive factor was present in chondrocytes of the proliferating and upper hypertrophic zones but absent from lower hypertrophic chondrocytes. Immunostaining was present only in the territorial extracellular matrix immediately adjacent to the chondrocytes of the proliferating and upper hypertrophic zones. Osteoblasts of the primary spongiosa stained heavily in normal mice. Strong staining was observed in intermediate zone articular chondrocytes. Cells in the superficial layer of articular cartilage were unstained. The extracellular matrix of the articular cartilage was completely free of immunostaining. In contrast, the reduced size of bm/bm growth plates was accompanied by significantly reduced staining intensity in proliferating and upper hypertrophic chondrocytes, and staining was absent from the territorial extracellular matrix of all zones of the bm/bm growth plate. Osteoblasts of the primary spongiosa of bm/bm mice stained less than those of normal mice. Articular cartilage chondrocytes in the intermediate zone stained with less intensity in bm/bm mice, and the cells of the superficial layer were unstained. The extracellular matrix of bm/bm articular cartilage was completely free of staining. Brachymorphic epiphyseal growth plate and articular chondrocytes, and osteoblasts in the primary spongiosa, express reduced amounts of immunoreactive fibroblast growth factor-2. This phenotypical characteristic may be associated with abnormal endochondral ossification and development of bone in brachymorphic mice     

Phagocytosis of dying chondrocytes by osteoclasts in the mouse growth plate as demonstrated by annexin-V labelling

Endochondral ossification in the epiphyseal growth plate of long bones is associated with programmed cell death (PCD) of a major portion of the chondrocytes. Here we tested the hypothesis that at the ossification front of the epiphyseal growth plate osteoclasts preferentially phagocytose chondrocytes that are undergoing PCD. We injected biotin-labelled annexin-V (anx-V-biotin, an early marker of PCD) intravenously in young adult mice. After 30 min of labelling, long bones were recovered and the tissue distribution examined of anx-V-biotin-labelled cells in the growth plate using ABC-peroxidase histochemistry. Positive staining for anx-V-biotin was detected in hypertrophic chondrocytes still present in closed lacunae at some distance from the ossification front. At the ossification front, chondrocyte lacunae were opened and close contacts were seen between tartrate-resistant acid phosphatase-positive osteoclasts and hypertrophic cartilage cells. Osteoclasts were significantly more frequently in contact with anx-V-biotin-labelled chondrocytes than with unlabelled chondrocytes. Osteoclasts also contained labelled and unlabelled phagocytic fragments within their cytoplasm. We conclude that in the growth plate osteoclasts preferentially phagocytose hypertrophic chondrocytes that are dying, suggesting these dying cells may signal osteoclasts for their removal.     

Immunohistochemical localization of alkaline phosphatase in the chicken epiphyseal growth cartilage

Summary Alkaline phosphatase of chicken epiphyseal cartilage has been localized by two immunohistochemical methods. Double layer immunofluorescence and peroxidase anti-peroxidase (PAP) methods gave similar results. Alkaline phosphatase in epiphyseal cartilage is extracellular as well as intracellular in the localization. Extracellular reaction was strongest in the lower layers of growth plate and the most intense reaction was noted in the pericellular lacunae of hypertrophic chondrocytes. Also intracellular immunoreaction was noticed through the whole growth plate.     

Changes in leucine-rich repeat proteoglycans during maturation of the bovine growth plate

The primary growth plate of the fetal bovine tibia was studied in order to determine whether changes in the structure, abundance and expression of the leucine-rich repeat proteoglycans were occurring during tissue maturation from reserve cartilage to hypertrophic cartilage. The proteoglycans under study were decorin, biglycan, fibromodulin and lumican. Decorin was readily detectable in both the reserve and proliferating zones of the growth plate, but its abundance decreased markedly in the zones of maturation and hypertrophy where it could not be detected under the same conditions of analysis. In contrast to decorin, fibromodulin and biglycan could be detected throughout the growth plate, though their abundance was decreased in the proliferative and hypertrophic zones. Unlike the other proteoglycans, lumican could not be detected throughout the growth plate. At the message level, the expression of decorin shows a similar trend to that of protein abundance in the extracellular matrix, with its expression dropping markedly in the proliferative and hypertrophic zones. In the case of both biglycan and fibromodulin, message expression continued at a similar level throughout the growth plate. Thus, the leucine-rich repeat proteoglycans are different in the way they behave during growth plate maturation.     

Regulated Production of Mineralization-competent Matrix Vesicles in Hypertrophic Chondrocytes

Matrix vesicles have a critical role in the initiation of mineral deposition in skeletal tissues, but the ways in which they exert this key function remain poorly understood. This issue is made even more intriguing by the fact that matrix vesicles are also present in nonmineralizing tissues. Thus, we tested the novel hypothesis that matrix vesicles produced and released by mineralizing cells are structurally and functionally different from those released by nonmineralizing cells. To test this hypothesis, we made use of cultures of chick embryonic hypertrophic chondrocytes in which mineralization was triggered by treatment with vitamin C and phosphate. Ultrastructural analysis revealed that both control nonmineralizing and vitamin C/phosphatetreated mineralizing chondrocytes produced and released matrix vesicles that exhibited similar round shape, smooth contour, and average size. However, unlike control vesicles, those produced by mineralizing chondrocytes had very strong alkaline phosphatase activity and contained annexin V, a membrane-associated protein known to mediate Ca²⁺ influx into matrix vesicles. Strikingly, these vesicles also formed numerous apatite-like crystals upon incubation with synthetic cartilage lymph, while control vesicles failed to do so. Northern blot and immunohistochemical analyses showed that the production and release of annexin V-rich matrix vesicles by mineralizing chondrocytes were accompanied by a marked increase in annexin V expression and, interestingly, were followed by increased expression of type I collagen. Studies on embryonic cartilages demonstrated a similar sequence of phenotypic changes during the mineralization process in vivo. Thus, chondrocytes located in the hypertrophic zone of chick embryo tibial growth plate were characterized by strong annexin V expression, and those located at the chondro–osseous mineralizing border exhibited expression of both annexin V and type I collagen. These findings reveal that hypertrophic chondrocytes can qualitatively modulate their production of matrix vesicles and only when induced to initiate mineralization, will release mineralization-competent matrix vesicles rich in annexin V and alkaline phosphatase. The occurrence of type I collagen in concert with cartilage matrix calcification suggests that the protein may facilitate crystal growth after rupture of the matrix vesicle membrane; it may also offer a smooth transition from mineralized type II/type X collagen-rich cartilage matrix to type I collagen-rich bone matrix.     

Uncoupling of chondrocyte death and vascular invasion in mouse galectin 3 null mutant bones

Galectin 3 is a beta-galactoside binding protein which localizes to the cytoplasm of proliferative, mature, and hypertrophic chondrocytes in the growth plate cartilage of developing long bones. To elucidate the function of galectin 3 during bone development, we examined the epiphyseal femurs and tibias of fetal mice carrying a null mutation for the galectin 3 gene. Detailed histological and ultrastructural studies identified abnormalities in the cells of the proliferative, mature, and hypertrophic zones and in the extracellular matrix of the hypertrophic zone, as well as a reduction in the total number of hypertrophic chondrocytes. The expression patterns of several chondrocyte and bone cell markers were analyzed and revealed a subtle modification of Ihh expression in the galectin 3 mutant growth plate. A striking difference was observed at the chondrovascular junction where many empty lacunae are present. In addition, large numbers of condensed chondrocytes exhibiting characteristic signs of cell death were found in the late hypertrophic zone, indicating that the rate of chondrocyte death is increased in the mutants. These results suggest a role for galectin 3 as a regulator of chondrocyte survival. In addition, this unique phenotype shows that the elimination of chondrocytes and vascular invasion can be uncoupled and indicates that galectin 3 may play a role in the coordination between chondrocyte death and metaphyseal vascularization.     

Perlecan,the multidomain HS-proteoglycan of basement membranes,is a prominent pericellular component of ovine hypertrophic vertebral growth plate and cartilaginous endplate chondrocytes

The aim of this study was to immunolocalise perlecan in ovine vertebral growth plate (VGP) and cartilaginous endplate (CEP) cartilages using a monoclonal antibody (MAb A76) directed to a core protein epitope in perlecan domain-I, and to compare and contrast its localisation patterns with known cartilage matrix components. Perlecan was a prominent pericellular component of mature hypertrophic chondrocytes in the VGP and CEP in newborn 2- to 5-day-old sheep. Type I, II, VI and X collagen, chondroitin-4 and 6-sulphate, 7-D-4 chondroitin sulphate isomer proteoglycan epitope, keratan sulphate, aggrecan core protein, hyaluronan (HA) and hyaluronan binding proteins (HABPs) each had distinct localisation patterns in the VGP and CEP. Type X collagen was a prominent component of the VGP but was undetectable in the CEP. Aggrecan was strongly localised extracellularly throughout the VGP and CEP but increased cell-associated staining was also evident. In contrast to the aforementioned matrix components, HA, HABPs and perlecan were localised strongly to the pericellular matrices of the hypertrophic VGP and CEP chondrocytes apparently indicating an important role for these components in terminal chondrocyte differentiation.     

Expression of Bcl-2 protein in the epiphyseal plate cartilage and trabecular bone of growing rats

 The protooncogene protein, Bcl-2, protects cells from apoptosis and ensures their survival in vitro by inhibiting the action of the apoptosis-inducer, Bax. Its expression in proliferative and long-lived cells in vivo also indicates that it protects against cell death. The chondrocytes of the epiphyseal plate cartilage undergo a series of maturation steps and deposit mineral in the cartilage matrix before dying. The possibility that Bcl-2 helps protect chondrocytes until mineral deposition is completed was investigated by determining the distribution of Bcl-2 immunoreactivity in the epiphyseal plate cartilage of growing rats and its subcellular localization, using a specific antibody. The involvement of Bax in the triggering of chondrocyte death was checked by immunocytochemistry. Bcl-2 expression in the osteoblasts and the final result of their evolution, the osteocytes, was also examined in trabecular bone. Bcl-2 immunoreactivity was non-uniformly distributed throughout the epiphyseal cartilage. It was maximal in proliferative chondrocytes, decreased in mature chondrocytes, and low in hypertrophic chondrocytes, whereas there was Bax immunoreactivity in all chondrocytes examined. Immunolabeling was intense in osteoblasts but considerably lower in fully differentiated osteocytes. Bcl-2 immunoreactivity was mainly in the cytoplasm of chondrocytes, osteoblasts, and early osteocytes; the nuclei appeared clear. The subcellular distribution of Bcl-2 immunolabeling in chondrocytes, revealed by gold particles in the electron microscope, showed that gold particles were frequently concentrated in the mitochondria in all the cartilage zones and lay mainly within the organelles, not at their periphery. The endoplasmic reticulum contained moderate immunoreactivity and there were few gold particles in the cytoplasm and nuclei. The number of gold particles decreased in all the subcellular compartments from proliferative to hypertrophic chondrocytes. In contrast, Bax immunoreactivity changed little during chondrocyte terminal evolution, and its subcellular distribution mirrored that of Bcl-2. These immunocytochemical data indicate that Bcl-2 helps maintain chondrocytes and osteoblasts until their terminal maturation. Accepted: 19 February 1997     

Parathyroid hormone-related peptide expression in the epiphyseal growth plate of the juvenile chicken: evidence for the origin of the parathyroid hormone-related peptide found in the epiphyseal growth plate

Parathyroid hormone-related peptide (PTHrP) has been shown to be essential for normal endochondral bone formation. Along with Indian hedgehog (Ihh), it forms a paracrine regulatory loop that governs the pace of chondrocyte differentiation. However, the source of PTHrP for this regulatory loop is not clear. While one hypothesis has suggested the periarticular perichondrium as the source of PTHrP for growth plate regulation, other data utilizing immunohistochemistry and in situ hybridization would indicate that growth plate chondrocytes themselves are the source of this peptide. The data described in this report supports the view that postnatal growth plate chondrocytes have the ability to synthesize this important regulatory peptide. Immunohistochemistry of tissue sections showed that PTHrP protein was evident throughout the chick epiphysis. PTHrP was seen in chondrocytes in the periarticular perichondrium, the perichondrium adjacent to the growth plate, the prehypertrophic zone of the growth plate, and the hypertrophic zone of the growth plate. However, cells in the proliferative zone, as well as some chondrocytes in the deeper layers of articular cartilage were predominantly negative for PTHrP. PTHrP was detected by Western blotting as a band of 16,400 Da in extracts from hypertrophic chondrocytes, but not from proliferative cells. RT-PCR detected PTHrP mRNA in both proliferative and hypertrophic growth plate chondrocytes, as well as in articular chondrocytes. PTH/PTHrP receptor mRNA was detected by Northern blotting in growth plate, but not articular chondrocytes. Thus, we conclude that most of the PTHrP present in the epiphyseal growth plate of the juvenile chick originates in the growth plate itself. Furthermore, the presence of large amounts of PTHrP protein in the hypertrophic zone supports the concept that PTHrP has other functions in addition to regulating chondrocyte differentiation.     

Enhanced expression of TGF-beta and c-fos mRNAs in the growth plates of developing human long bones

The expression of mRNAs for type I and type II procollagens, transforming growth factor-beta (TGF-beta) and c-fos was studied in developing human long bones by Northern blotting and in situ hybridization. The cells producing bone and cartilage matrix were identified by hybridizations using cDNA probes for types I and II collagen, respectively. Northern blotting revealed that the highest levels of TGF-beta mRNA were associated with the growth plates. By in situ hybridization, this mRNA was localized predominantly in the osteoblasts and osteoclasts of the developing bone, in periosteal fibroblasts and in individual bone marrow cells. These findings are consistent with the view that TGF-beta may have a role in stimulation of type I collagen production and bone formation. Only a low level of TGF-beta mRNA was detected in cartilage where type II collagen mRNA is abundant. In Northern hybridization, the highest levels of c-fos mRNA were detected in epiphyseal cartilage. In situ hybridization revealed two cell types with high levels of c-fos expression: the chondrocytes bordering the joint space and the osteoclasts of developing bone. These differential expression patterns suggest specific roles for TGF-beta and c-fos in osseochondral development.     

Calcium induces differentiation of primary human salivary acinar cells

We previously reported that connective tissue growth factor/hypertrophic chondrocyte-specific gene product 24 (CTGF/Hcs24) stimulated the proliferation and differentiation of rabbit growth cartilage (RGC) cells in vitro. In this study, we investigated the effects of CTGF/Hcs24 on the proliferation and differentiation of rabbit articular cartilage (RAC) cells in vitro. RAC cells transduced by recombinant adenoviruses generating mRNA for CTGF/Hcs24 synthesized more proteoglycan than the control cells. Also, treatment of RAC cells with recombinant CTGF/Hcs24 (rCTGF/Hcs24) increased DNA and proteoglycan syntheses in a dose-dependent manner. Northern blot analysis revealed that the rCTGF/Hcs24 stimulated the gene expression of type II collagen and aggrecan core protein, which are markers of chondrocyte maturation, in both RGC and RAC cells. However, the gene expression of type X collagen, a marker of hypertrophic chondrocytes, was stimulated by rCTGF/Hcs24 only in RGC cells, but not in RAC cells. Oppositely, gene expression of tenascin-C, a marker of articular chondrocytes, was stimulated by rCTGF/Hcs24 in RAC cells, but not in RGC cells. Moreover, rCTGF/Hcs24 effectively increased both alkaline phosphatase (ALPase) activity and matrix calcification of RGC cells, but not of RAC cells. These results indicate that CTGF/Hcs24 promotes the proliferation and differentiation of articular chondrocytes, but does not promote their hypertrophy or calcification. Taken together, the data show that CTGF/Hcs24 is a direct growth and differentiation factor for articular cartilage, and suggest that it may be useful for the repair of articular cartilage.