Comparative morphological analysis of the articular cartilage, epiphyseal plate, spongy bone, and synovial membrane of the knee joint in children treated and not treated with ciprofloxacin]

Comparative data on he postmortem morphological examination of the knee joint in children treated and not treated with ciprofloxacin while alive are presented. Nine children were ill with mucoviscidosis and 8 children were ill with aplastic anemia. The patients were ill with mucoviscidosis and 8 children were ill with aplastic anemia. The patients were treated with ciprofloxacin in a daily dose of 20 to 40 mg/kg body weight at the average for 148 days. The condition of the articular cartilage, growth area, spongy bone and synovial membrane of the knee joint was examined visually, histologically and morphologically. It was revealed that ciprofloxacin had no chondrotoxic effect. The structure affected in some cases by the ciprofloxacin treatment was the synovial membrane which developed subacute synovitis not clinically manifested (latent) in life. The phenomenon is explained.     

Age-related changes in the tensile properties of human articular cartilage: a comparative study between the femoral head of the hip joint and the talus of the ankle joint

Specimens of articular cartilage from the superficial and mid-depth zones of the human femoral head and the talus of the ankle joint were tested in tension in planes parallel to the articular surface and parallel to the predominant orientation of the superficial collagen fibrils. The tensile fracture stress of cartilage from both the superficial and mid-depth zones of the femoral head decreased considerably with age. The superficial zone decreased from 33 MPa at 7 years to 10 MPa by the age of 90 years, while the mid-depth zone decreased from 32 MPa at 7 years to 2 MPa by the age of 85 years. In contrast the fracture stress of both levels of cartilage from the talus of the ankle did not decrease significantly with increasing age. The tensile stiffness at 10 MPa of both the superficial and mid-depth zones of the femoral head decreased with age. That of the superficial zone decreased from 150 MPa at 7 years to 80 MPa at 90 years, while the mid-depth zone decreased from 60 MPa at 7 years to 10 MPa at 60 years. The stiffness of talar cartilage from the superficial zone decreased by 20%, while that of the mid-depth zone showed a slight increase in stiffness at 10 MPa with increasing age. There was no significant decrease in the tensile stiffness at 1 MPa with age for either the femoral head or talar cartilage. Based on the results of previous studies it is possible to conclude that the decrease in tensile properties seen in the femoral head results from a deterioration in the tensile properties of the network of collagen fibrils. It is suggested that progressive fatigue failure, perhaps with associated changes in the structure of cartilage due to altered chondrocyte metabolism, causes the reduction in tensile properties with age. The results offer a potential explanation for the observation that osteoarthritis commonly occurs in the hip and knee joints at an increasing incidence as age increases, while the condition only rarely occurs in the ankle joint except as a secondary event to trauma.     

Expression of collagen type I,II, X and Ki-67 in osteochondroma compared to human growth plate cartilage

In order to characterize the consequences for the process of endochondral ossification we performed an immunohistochemical study and compared the expression of collagen type I, II and X as markers of cartilage differentiation and Ki-67 as a marker of cell proliferation in solitary (7-26 years, n=9) and multiple (11-42 years, n=6) osteochondromas with their expression in human fetal and postnatal growth plates. In fetal and young postnatal controls, we found a thin superficial layer of articular cartilage that stained positive for collagen type I while collagen II was expressed in the rest of the cartilage and collagen type X was restricted to the hypertrophic zone. Osteochondromas from children showed lobular collagen type II-positive areas surrounded by collagen type I. In adults, the separation of collagen type I- and type II-positive areas was more blurred, or the cartilaginous cap was missing. Collagen type X was detected in a pericellular distribution pattern within hypertrophic zones but also deeper between bone trabecula. The proliferative activity of osteochondromas from children younger than 14 years of age was comparable to postnatal growth plates, whereas in cartilage from individuals older than 14 years of age, we could not detect significant proliferative activity.     

Lead content in the femoral heads of inhabitants of Silesia (Poland).

Lead content was evaluated in spongious and cortical bone as well as in cartilage surface of human femoral heads obtained during hip arthroplasty from 45 inhabitants (11 males and 34 females) of the industrial region of Upper Silesia. The average age of this group was 63.9+/-14.4 years. Lead content was assessed using microwave mineralization with the graphite furnace atomic absorption spectrometry (GFAAS) method. The average lead content in the specimens from spongious bone was 2.56 microg/g, 3.05 microg/g in cortical bone and 3.53 microg/g in cartilage surface. The calculated average Pb/Ca ratio was 1783x10(-8) in spongious bone, 1623x10(-8) in cortical bone, and 2512x10(-8) in the cartilage. Both lead concentration and Pb/Ca ratio increased with the age of the patients. Higher lead content was found in the specimens from male hips and in the group of cigarette smokers.     

Observation on the development of osteochondrosis in young rats

A histopathological study on the development of spontaneous osteochondrosis in the humeral head and medial femoral condyle of rats (6-20 weeks old) was carried out. Findings were classified into three types: normal, transitional and osteochondrotic. In the normal type, the articular cartilage at the caudal region of the humeral head and medial femoral condyle was significantly thinned between 6 and 10 weeks of age (generally the caudal region was thicker than elsewhere at all ages). In the transitional type, the thinning of the cartilage was delayed. In the third type, osteochondrotic lesions were detected in the humeral head from 6 weeks of age and in the medial femoral condyle from 10 weeks of age. The thickness of the cartilage had slightly decreased or had not changed at 20 weeks of age. In the early stages, viable chondrocytes and small destructive foci of cartilage were observed in the basal layer of the thick deep zone. These cells were present in pairs or clusters surrounded by matrix in the large lacunae. Cells and destructive foci were also seen in the surface layer of the deep zone as the rats aged. In the advanced stage, a necrotic area or cleft was formed in the basal layer of the articular cartilage and fibrosis was observed in the subchondral bone.     

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

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

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

Summary 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.     

Localization of a dermatan sulfate proteoglycan (DS-PGII) in cartilage and the presence of an immunologically related species in other tissues

A monoclonal antibody to a core-protein-related epitope of a small dermatan sulfate-rich proteoglycan (DS-PGII) isolated from adult bovine articular cartilage (22) was used to localize this molecule, or molecules containing this epitope, in bovine articular cartilages, in cartilage growth plate, and in other connective tissues. Using an indirect method employing peroxidase-labeled pig anti-mouse immunoglobulin G, DS-PGII was shown to be present mainly in the superficial zone of adult articular condylar cartilage of the metacarpal-phalangeal joint. In fetal articular and epiphyseal cartilages, the molecule was uniformly distributed throughout the matrix. By approximately 10 months of age it was confined mainly to the superficial and middle zones of articular cartilage and the inter-territorial and pericellular matrix of the deep zone. DS-PGII was not detected in the primary growth plate of the fetus except in the proliferative zone, where it was sometimes present in trace amounts. In contrast, it was present throughout the adjacent matrix of developing epiphyseal cartilage. In the trabeculae of the metaphysis, strong staining for DS-PGII was seen in decalcified osteoid and bone immediately adjacent to osteoblasts. Staining was also observed on collagen fibrils in skin, tendon, and ligament and in the adventitia of the aorta and of smaller arterial vessels in the skin. These observations indicate that DS-PGII and/or molecules containing this epitope are widely distributed in collagenous tissues, where the molecule is intimately associated with collagen fibrils; in adult cartilage this association is limited mainly to the narrow parallel arrays of fibrils which are found in the superficial zone at the articular surface. From its intimate association and other studies, this molecule may play an important role in determining the sizes and tensile properties of collagen fibrils; it may also be involved in the calcification of osteoid but not of cartilage.     

Study on the Microstructure of Human Articular Cartilage/Bone Interface

For improving the theory of gradient microstructure of cartilage/bone interface, human distal femurs were studied. Scanning Electron Microscope (SEM), histological sections and MicroCT were used to observe, measure and model the microstructure of cartilage/bone interface. The results showed that the cartilage/bone interface is in a hierarchical structure which is composed of four different tissue layers. The interlocking of hyaline cartilage and calcified cartilage and that of calcified cartilage and subchondral bone are in the manner of “protrusion-pore” with average diameter of 17.0 μm and 34.1 μm respectively. In addition, the cancellous bone under the cartilage is also formed by four layer hierarchical structure, and the adjacent layers are connected by bone trabecula in the shape of H, I and Y, forming a complex interwoven network structure. Finally, the simplified structure model of the cartilage/bone interface was proposed according to the natural articular cartilage/bone interface. The simplified model is a 4-layer gradient biomimetic structure, which corresponds to four different tissues of natural cartilage/bone interface. The results of this work would be beneficial to the design of bionic scaffold for the tissue engineering of articular cartilage/bone.     

Specimen preparation and quantification of collagen birefringence in unstained sections of articular cartilage using image analysis and polarizing light microscopy

To establish an optimal method for analysis of the collagen structures from unstained tissue sections, a computerized image analysis system using a charge coupled device camera coupled to a polarizing light microscope was used. Retardation values of birefringence, which are proportional to the content and fibril orientation of collagen in the extracellular matrix of articular cartilage, were determined from sections prepared in different ways. In the superficial zone of articular cartilage, the highest retardation values were recorded from sections cut parallel to the so-called split lines indicating the anisotropic arrangement of collagen. Complete digestion of glycosaminoglycans reduced the retardation value by approximately 6.0%, suggesting a minor, but not insignificant, contribution of glycosaminoglycans to the birefringence of the matrix. The use of a mounting medium with a refractive index close to that of the collagen (e.g. DPX) increased the specificity of the method, since the optical anisotropy of collagen derives predominantly from the intrinsic (structural) birefringence. In conclusion, analysis of unstained sections after careful removal of paraffin and glycosaminoglycans from the tissues provides a sensitive and rapid quantitative assessment of oriented collagen structures in articular cartilage     

Structure of proteoglycans from different layers of human articular cartilage

Full-depth plugs of adult human articular cartilage were cut into serial slices from the articular surface and analysed for their glycosaminoglycan content. The amount of chondroitin sulphate was highest in the mid-zone, whereas keratan sulphate increased progressively through the depth. Proteoglycans were isolated from each layer by extraction with 4M-guanidinium chloride followed by centrifugation in 0.4M-guanidinium chloride/CsCl at a starting density of 1.5 g/ml. The efficiency with which proteoglycans were extracted depended on slice thickness, and extraction was complete only when cartilage from each zone was sectioned at 20 microns or less. When thick sections (250 microns) were extracted, hyaluronic acid was retained in the tissue. Most of the proteoglycans, extracted from each layer under optimum conditions, could interact with hyaluronic acid to form aggregates, although the extent of aggregation was less in the deeper layers. Two pools of proteoglycan were identified in all layers by gel chromatography (Kav. 0.33 and 0.58). The smaller of these was rich in keratan sulphate and protein, and gradually increased in proportion through the cartilage depth. Chondroitin sulphate chain size was constant in all regions. The changes in composition and structure observed were consistent with the current model for hyaline-cartilage proteoglycans and were similar to those observed with increasing age in human articular cartilage.     

Cartilage interstitial fluid load support in unconfined compression

Under physiological conditions of loading, articular cartilage is subjected to both compressive strains, normal to the articular surface, and tensile strains, tangential to the articular surface. Previous studies have shown that articular cartilage exhibits a much higher modulus in tension than in compression, and theoretical analyses have suggested that this tension–compression nonlinearity enhances the magnitude of interstitial fluid pressurization during loading in unconfined compression, above a theoretical threshold of 33% of the average applied stress. The first hypothesis of this experimental study is that the peak fluid load support in unconfined compression is significantly greater than the 33% theoretical limit predicted for porous permeable tissues modeled with equal moduli in tension and compression. The second hypothesis is that the peak fluid load support is higher at the articular surface side of the tissue samples than near the deep zone, because the disparity between the tensile and compressive moduli is greater at the surface zone. Ten human cartilage samples from six patellofemoral joints, and 10 bovine cartilage specimens from three calf patellofemoral joints were tested in unconfined compression. The peak fluid load support was measured at 79±11% and 69±15% at the articular surface and deep zone of human cartilage, respectively, and at 94±4% and 71±8% at the articular surface and deep zone of bovine calf cartilage, respectively. Statistical analyses confirmed both hypotheses of this study. These experimental results suggest that the tension–compression nonlinearity of cartilage is an essential functional property of the tissue which makes interstitial fluid pressurization the dominant mechanism of load support in articular cartilage.     

Type X collagen expression in osteoarthritic and rheumatoid articular cartilage

Type X collagen is a short chain, non-fibrilforming collagen synthesized primarily by hypertrophic chondrocytes in the growth plate of fetal cartilage. Previously, we have also identified type X collagen in the extracellular matrix of fibrillated, osteoarthritic but not in normal articular cartilage using biochemical and immunohistochemical techniques (von der Mark et al. 1992 a). Here we compare the expression of type X with types I and II collagen in normal and degenerate human articular cartilage by in situ hybridization. Signals for cytoplasmic α1(X) collagen mRNA were not detectable in sections of healthy adult articular cartilage, but few specimens of osteoarthritic articular cartilage showed moderate expression of type X collagen in deep zones, but not in the upper fibrillated zone where type X collagen was detected by immunofluorescence. This apparent discrepancy may be explained by the relatively short phases of type X collagen gene activity in osteoarthritis and the short mRNA half-life compared with the longer half-life of the type X collagen protein. At sites of newly formed osteophytic and repair cartilage, α1(X) mRNA was strongly expressed in hypertrophic cells, marking the areas of endochondral bone formation. As in hypertrophic chondrocytes in the proliferative zone of fetal cartilage, type X collagen expression was also associated with strong type II collagen expression.     

Direct measurement of the Poisson's ratio of human patella cartilage in tension

Articular cartilage has been shown to exhibit large transverse contractions when loaded in tension, suggesting the existence of large values for the Poisson's ratio. Previous studies have suggested that this effect is dependent on amplitude of applied strain, so that a single Poisson's ratio may not be sufficient to describe cartilage behavior. In this study, the Poisson's ratio (v), toe region modulus (Eo), and linear region modulus (E) of human patellar articular cartilage were calculated in simple tension tests from optical analysis of the two-dimensional strain fields at equilibrium. The Poisson's ratio was found to be independent of strain due to the absence of viscoelastic effects during testing. The Poisson's ratio was found to be significantly higher in the surface zone (1.87 +/- 1.11, p<0.01) than in the middle zone (0.62 +/- 0.23), with no significant correlation of v with age of the cartilage. In general, values for Poisson's ratio were greater than 0.5, suggesting cartilage behavior in tension deviates from isotropy. Reported values for the Poisson's ratio of cartilage in compression have been much lower than values measured here in tension, reflecting a mechanical contribution of the collagen fibers to anisotropy in tension but not compression. The toe-region modulus (Eo) was significantly higher in the surface zone (4.51 +/- 2.78 MPa, n=8) compared to the middle zone (2.51 +/- 1.93 MPa, n=10). In addition, the linear-region modulus (E) in the surface zone, but not middle zone (3.42 +/- 2.17 MPa, n=10), was found to correlate with age (R=0.97, p<0.02) with values of surface zone E equal to 23.92 +/- 12.29 MPa (n=5) for subjects under 70 yr of age, and 4.27 +/- 2.89 MPa (n=3) for subjects over 70 yr. Moduli values and trends with depth were consistent with previous studies of human and animal cartilage. From direct measures of two independent material properties, v and E, we calculated a shear modulus, G, which had not been previously reported for cartilage from tensile testing. Calculated values for surface zone G were 3.64 +/- 1.80 MPa for subjects under 70 yr old and 0.96 +/- 0.69 MPa for subjects over 70 yr old, and were significantly higher in the surface zone than in the middle zone (1.10 +/- 0.78 MPa). This study provides an intrinsic measure for the Poisson's ratio of articular cartilage and its dependence on depth which will be important in understanding the nonlinear tension-compression and anisotropic behaviors of articular cartilage.     

The chondrocyte cytoskeleton in mature articular cartilage: structure and distribution of actin, tubulin, and vimentin filaments.

We investigated the structure of the chondrocyte cytoskeleton in intact tissue sections of mature bovine articular cartilage using confocal fluorescence microscopy complemented by protein extraction and immunoblotting analysis. Actin microfilaments were present inside the cell membrane as a predominantly cortical structure. Vimentin and tubulin spanned the cytoplasm from cell to nuclear membrane, the vimentin network appearing finer compared to tubulin. These cytoskeletal structures were present in chondrocytes from all depth zones of the articular cartilage. However, staining intensity varied from zone to zone, usually showing more intense staining for the filament systems at the articular surface compared to the deeper zones. These results obtained on fluorescently labeled sections were also corroborated by protein contents extracted and observed by immunoblotting. The observed cytoskeletal structures are compatible with some of the proposed cellular functions of these systems and support possible microenvironmental regulation of the cytoskeleton, including that due to physical forces from load-bearing, which are known to vary through the depth layers of articular cartilage.     

Collagen I and II mRNA distribution in the rat temporomandibular joint region during growth

The distribution of type I and II collagen synthesis in the temporomandibular joint (TMJ) area of 1- to 28-day-old rats was studied after hybridization with probes to pro alpha1(I) and pro alpha1(II) collagen mRNA, and stain intensity through the various cartilaginous zones of the mandibular condyle and other areas of TMJ was assessed. The pro alpha(I) collagen mRNA was detected in the perichondrium/periosteum, in the fibrous and undifferentiated cell layers of the mandibular condyle, in the articular disc, and in all bone structures and muscles. The pro alpha1(II) collagen mRNA was found in the condylar cartilage and the articular fossa. Intensity in the condyle was highest in the chondroblastic layer and decreased towards the lower hypertrophic layer. In the condylar cartilage of the 21- to 28-day-old rats the chondroblastic cell zone was relatively narrow compared with the younger animals, whereas the reverse seems to be the case in the cartilage of the articular fossa. Changes in the pro alpha1(II) collagen mRNA were observed in the osseochondral junction area of the primary spongiosa, in that at the age of 5 days intense staining was found, whereas no staining was observed by 14 days. In the mineralizing zone, however, the majority of osteoblastic cells gave a positive signal with the pro alpha1(I) collagen probe. In conclusion, type II collagen synthesis of the mandibular condyle is restricted to its upper area. This differs from the long bone epiphyseal plate, where this type of collagen is produced virtually throughout the cartilage. Type II collagen synthesis of the fossal cartilage seems to increase as a function of age.     

The effects of proteolytic enzymes on the mechanical properties of adult human articular cartilage.

The effects of the lysosomal proteinase cathepsin D on the mechanical properties of adult human articular cartilage were examined in detail in 7 joints within the age range 21 to 72 years. The results of a preliminary study on the effects of the lysosomal proteinase cathepsin B1 and clostridial collagenase on the mechanical properties of cartilage are also presented. Cartilage which had been incubated with either cathepsin D or cathepsin B1 showed increased deformation in uniaxial compression perpendicular to the articular surface. The enzyme-treated cartilage also showed decreased tensile stiffness at low values of stress. This effect was more pronounced in specimens from the deeper zone of cartilage than in specimens from the superficial zone. It was also more pronounced in specimens which were aligned perpendicular to the predominant alignment of the collagen fibres in the superficial zone than in specimens which were parallel to the collagen fibres. At higher stresses the tensile stiffness of the treated cartilage was not significantly different from that of the untreated tissue. The tensile fracture stress of the cartilage was also not significantly reduced by the action of cathepsin D. In contrast to the effects observed with the cathepsins, the preliminary results obtained by incubating cartilage for 24 h with clostridial collagenase showed that both the tensile stiffness and the fracture stress were considerably lower than the corresponding values for the untreated tissue. Biochemical analysis of the incubation media, and the specimens, revealed that a large proportion of the proteoglycans was released from the cartilage by each of the three enzymes. The proportion of the total collagen which was released from the cartilage was different for each enzyme: cathepsin D released between 0 and 1.5 per cent, cathepsin B1 released between 2.3 and 4.3 per cent and collagenase released between 5.3 and 27.8 per cent of the collagen after 24 h.     

Lack of chondroitin sulphate epitope in the proliferating zone of the growth plate of chicken tibia

Summary Monoclonal antibodies specific to chondroitin sulphate (CS-56) and keratan sulphate (AH12) were used to localize proteoglycans in the proximal tibial articular cartilage and growth plate of broiler chickens. There was no CS-56 labelling in the proliferative zone of the growth plate. In contrast, intense labelling with this antibody was observed in the transitional and hypertrophic zones of the growth plate and the articular cartilage. This was confirmed by extracting chondroitin sulphate fractions from different zones of the growth plate and articular cartilage, and examining their antigenicities to CS-56 by ELISA inhibition assay. It was suggested that the maturation of chondrocytes in the growth plate is related to the production of chondroitin sulphate with CS-56 epitope, which may be a prerequisite for normal endochondral bone formation in the chicken tibia. The role of chondroitin sulphate recognized by CS-56 in the articular cartilage is unknown.     

Demonstration of fibronectin in human articular cartilage by an indirect immunoperoxidase technique

Summary Fresh frozen tissue sections of human articular cartilage was treated without and with human testicular hyaluronidase (2×10⁶ units/l) for 60 min at 37° C and stained by the indirect immunoperoxidase technique with rabbit antihuman fibronectin. The rabbit antihuman fibronectin was purified by affinity chromatography on human fibronectin-Sepharose. Fibronectin was only found on the acellular surface of the articular cartilage in tissue sections not treated with hyaluronidase. In this surface layer, probably identical to lamina splendens, the arrangement of fibronectin was as a membrane. No collagen was seen in this area by van Gieson staining. No staining for fibronectin was found in the cartilage matrix or in the chondrocytes. Treatment of the cartilage tissue with hyaluronidase resulted in visualization of high amount of fibronectin in the cartilage matrix, with the highest intensity around the chondrocytes. The staining of the acellular surface layer of the articular cartilage was identical with the results obtained without hyaluronidase treatment. These results indicate that articular cartilage is rich in fibronectin probably in complex with hyaluronic acid, and that the chondrocytes produce fibronectin in situ. It also demonstrates the steric hindrance of hyaluronic acid aggregates in diffusion of the antibody and the value of hyaluronidase treatment of tissue before demonstration of fibronectin.     

Ectopic mineralization of articular cartilage in the bullfrog Rana catesbeiana and its possible involvement in bone closure

Mineralization of the articular cartilage is a pathological condition associated with age and certain joint diseases in humans and other mammals. In this work, we describe a physiological process of articular cartilage mineralization in bullfrogs. Articular cartilage of the proximal and distal ends of the femur and of the proximal end of the tibia-fibula was studied in animals of different ages. Mineralization of the articular cartilage was detected in animals at 1 month post-transformation. This mineralization, which appeared before the hypertrophic cartilage showed any calcium deposition, began at a restricted site in the lateral expansion of the cartilage and then progressed to other areas of the epiphyseal cartilage. Mineralized structures were identified by von Kossa's staining and by in vivo incorporation of calcein green. Element analysis showed that calcium crystals consisted of poorly crystalline hydroxyapatite. Mineralized matrix was initially spherical structures that generally coalesced after a certain size to occupy larger areas of the cartilage. Alkaline phosphatase activity was detected at the plasma membrane of nearby chondrocytes and in extracellular matrix. Apoptosis was detected by the TUNEL (TDT-mediated dUTP-biotin nick end-labeling) reaction in some articular chondrocytes from mineralized areas. The area occupied by calcium crystals increased significantly in older animals, especially in areas under compression. Ultrastructural analyses showed clusters of needle-like crystals in the extracellular matrix around the chondrocytes and large blocks of mineralized matrix. In 4-year-old animals, some lamellar bone (containing bone marrow) occurred in the same area as articular cartilage mineralization. These results show that the articular cartilage of R. catesbeiana undergoes precocious and progressive mineralization that is apparently stimulated by compressive forces. We suggest that this mineralization is involved in the closure of bone extremities, since mineralization appears to precede the formation of a rudimentary secondary center of ossification in older animals.