Clusterin expression in adult human normal and osteoarthritic articular cartilage

OBJECTIVE: To characterize the expression pattern of clusterin in adult human normal and osteoarthritic cartilage. METHODS: Clusterin mRNA expression in adult human normal and osteoarthritic cartilage was investigated by analysis of cDNA libraries, TaqMan quantitative RT-PCR, microarray and in situ hybridization. RESULTS: Sequence analysis of ESTs from adult human normal and osteoarthritic cartilage cDNA libraries demonstrated that the abundance of clusterin in these libraries was equivalent to genes which have been more commonly associated with cartilage. To examine tissue distribution, TaqMan Quantitative PCR analysis was performed using RNA from a panel of individual normal tissues. Clusterin was expressed at significant levels in cartilage, brain, liver, and pancreas. The expression of clusterin mRNA was up-regulated in early osteoarthritic vs normal cartilage when analysed by microarray analysis. Using in situ hybridization, chondrocytes of normal cartilage expressed moderate levels of clusterin. Upper mid-zone chondrocytes in cartilage with early stages of osteoarthritic disease expressed high levels of clusterin mRNA. In advanced osteoarthritic cartilage, the overall expression of clusterin was reduced. CONCLUSION: The induction of clusterin has been associated with a variety of disease states where it appears to provide a cytoprotective effect. The increased expression of clusterin mRNA in the early stages of osteoarthritis (OA) may reflect an attempt by the chondrocytes to protect and repair the tissue. In contrast, the decrease in clusterin mRNA in the advanced osteoarthritic cartilage accompanies the final degenerative stages of the disease. An understanding of the expression of clusterin in osteoarthritis may allow consideration of this protein as a marker for cartilage changes in this chronic degenerative condition.     

Ultrastructural localization of type VI collagen in normal adult and osteoarthritic human articular cartilage

OBJECTIVE: Type VI collagen is a major component of the pericellular matrix compartment in articular cartilage and shows severe alterations in osteoarthritic cartilage degeneration. In this study, we analysed the exact localization of type VI collagen in its relationship to the chondrocyte and the (inter)territorial cartilage matrix. Additionally, we were interested in its ultrastructural appearance in normal and osteoarthritic cartilage. DESIGN: Distribution and molecular appearance was investigated by conventional immunostaining, by multilabeling confocal scanning microscopy, conventional transmission, and immunoelectron microscopy. RESULTS: Our analysis confirmed the pericellular concentration of type VI collagen in normal and degenerated cartilage. Type VI collagen formed an interface in between the cell surface and the type II collagen network. The type VI collagen and the type II collagen networks appeared to have a slight physical overlap in both normal and diseased cartilage. Additionally, some epitope staining was observed in the cell-associated interterritorial cartilage matrix, which did not appear to have an immediate relation to the type II collagen fibrillar network as evaluated by immunoelectron microscopy.In osteoarthritic cartilage, significant differences were found compared with normal articular cartilage: the overall dimension of the lacunar volume increased, and a significantly increased type VI collagen epitope staining was observed in the interterritorial cartilage matrix. Also, the banded isoform of type VI collagen was found around many chondrocytes. CONCLUSIONS: Our study confirms the close association of type VI collagen with both, the chondrocyte cell surface and the territorial cartilage matrix. They show severe alterations in type VI collagen distribution and appearance in osteoarthritic cartilage. Our immunohistochemical and ultrastructural data are compatible with two ways of degradation of type VI collagen in osteoarthritic cartilage: (1) the pathologically increased physiological molecular degradation leading to the complete loss of type VI collagen filaments from the pericellular chondrocyte matrix and (2) the transformation of the fine filaments to the band-like form of type VI collagen. Both might implicate a significant loss of function of the pericellular microenvironment in osteoarthritic cartilage.     

Quantitative structural organization of normal adult human articular cartilage

OBJECTIVE: Data pertaining to the quantitative structural features and organization of normal articular cartilage are of great importance in understanding its biomechanical properties and in attempting to establish this tissue's counterpart by engineering in vitro. A comprehensive set of such baseline data is, however, not available for humans. It was the purpose of the present study to furnish the necessary information. DESIGN: The articular cartilage layer covering the medial femoral condyle of deceased persons aged between 23 and 49 years was chosen for the morphometric analysis of cell parameters using confocal microscopy in conjunction with unbiased stereological methods. The height of the hyaline articular cartilage layer, as well as that of the calcified cartilage layer and the subchondral bone plate, were also measured. RESULTS: The mean height of the hyaline articular cartilage layer was found to be 2.4mm, the volume density of chondrocytes therein being 1.65%, the number of cells per mm(3) of tissue 9626 and the mean cell diameter 13 microm. Other estimators (including matrix mass per cell and cell profile density) were also determined. CONCLUSIONS: A comparison of these normal human quantitative data with those published for experimental animals commonly used in orthopaedic research reveals substantial differences, consideration of which in tissue engineering strategies destined for human application are of paramount importance for successful repair.     

Labeling of articular cartilage surface with cationized ferritin: Aged human normal and osteoarthritic cartilage

The labeling of the articular surface with cationized ferritin (CF), an electron–dense marker, visualizes the anionic sites and may disclose abnormal penetration of the large CF molecule into the subsurface layers. Various areas of cartilage selected by unaided eye examination were taken from femoral heads excised in three cases of osteoarthritis and two cases of hip fracture. The fragments were examined by optical microscopy and by electron microscopy after labeling with CF. The labeling with and the penetration of CF were correlated with the morphological features of the surface. The surfaces belonging to the erosion border were disrupted and the CF penetrated approximately 2 μm into the matrix along the collagen fibers and in areas containing a patchy dense material. Prefixation with Karnovsky's fixative prevents CF penetration. The fragments taken at a distance from the erosion border showed at electron microscopical examination either an intact appearance of the surface that was labeled without penetration or a disrupted surface with penetration of the label. The osteophytes and the regeneration buds surface were labeled showing little or no penetration. The fragments from cartilage of hip fractures had either an intact surface regularly labeled or a slightly or moderately disrupted surface with moderate penetration of CF. The penetration of large molecules of CF in damaged cartilage demonstrates important permeability changes that may be significant for the pathogenetic mechanism of osteoarthritis. Similar permeability changes were previously shown in mice femoral heads treated in vitro with collagenase or trypsin and labeled with CF.     

Homeostasis of the extracellular matrix of normal and osteoarthritic human articular cartilage chondrocytes in vitro

OBJECTIVE: In normal articular cartilage cells, the IGFRI/insulin-like growth factor 1 (IGF-1) autocrine pathway was shown to overrule the catabolic effects of the IL-1/IL-1RI pathway by up-regulation of the IL-1RII decoy receptor. The activity of the IGF-1/IGFR1 and IL-1/IL-1R pathways, and of the IL-1RII control mechanism in the synthesis and turnover of the extracellular matrix (ECM) by chondrocytes from normal and osteoarthritic (OA) articular cartilage was compared in order to identify possible therapeutic targets of this disease. METHODS: Phenotypically stable human articular cartilage cells were obtained from normal and OA cartilage of the same knee showing focal OA. The cells were cultured in alginate beads over 1 week to re-establish the intracellular cytokine and growth factors, to reexpress the respective plasma membrane receptors and to reach equilibrium in accumulated cell-associated matrix (CAM) compounds. Following liberation of the cells from the alginate beads, the levels of cell-associated matrix (CAM) aggrecan, type II collagen and fibronectin, of intracellular IGF-1, IL-1alpha and beta and of their respective plasma membrane-bound receptors, IGFR1, IL-1RI and the decoy receptor IL-1RII, were assayed using flow cytometry. RESULTS: Coordinated production and accumulation of CAM aggrecan and type II collagen under the effect of the IGFR1/IGF-1 autocrine pathway-as documented for chondrocytes from healthy controls-was absent when the chondrocytes had been obtained from OA joints. When compared with cells obtained from normal tissues, chondrocytes from fibrillated OA cartilage expressed significantly higher intracellular IGF-1 levels and plasma membrane-bound IGFR1. At the same time, significantly higher intracellular IL-1alpha and beta levels and upregulated plasma membrane-bound IL-1RI were observed. Plasma membrane-bound IL-1RII decoy receptor was downregulated in OA chondrocytes. The levels of CAM aggrecan, type II collagen and fibronectin were significantly reduced in the chondrocytes obtained from pathological tissue. CONCLUSION: Paired analysis of normal and OA chondrocytes from the same knee joint has shown an enhanced capacity of chondrocytes from OA cartilage to produce ECM macromolecules. However, the same cells have increased catabolic signalling pathways. As a consequence of this increased IL-1 activity and the reduced amounts of IL-1RII decoy receptor, less of the produced ECM macromolecules may persist in the CAM of the OA chondrocytes.     

Contents and distributions of the proteoglycans decorin and biglycan in normal and osteoarthritic human articular cartilage

The study was designed to determine the contents and distributions of the proteoglycans decorin and biglycan in adult human femoral condylar cartilage and whether these may change in osteoarthritis. New radioimmunoassays were established using peptides representing the amino-terminal 21 amino acid sequence of each proteoglycan (to which a tyrosine was added for radioiodination) and antibodies in a rabbit antiserum raised to both these molecules. Cartilage was extracted with 4 M guanidine HCl to determine total content, and extracts were analyzed by chromatography to determine molecular sizes. Frozen sections were cut parallel to the articular surface and were extracted to determine distribution within the tissue. Gel chromatography on Sepharose CL-2B under dissociative conditions revealed molecules with a partition coefficient of 0.7–0.75 in both normal and osteoarthritic cartilage. In normal adult cartilage, the mean contents of the core proteins of biglycan and decorin were calculated to be approximately 0.34 and 0.48 mg per gram wet weight, respectively. These represented molar contents similar to that of aggrecan. In osteoarthritic cartilage, there were no overall significant changes in the content and distribution of these molecules. There was, however, considerable individual variation in both distribution and content. Analyses indicated that there was a trend in osteoarthritic cartilage toward a loss of biglycan and decorin from the more superficial layers of intact cartilage, where both these molecules are normally more concentrated. This was accompanied by maintenance of proteoglycan content deeper in the cartilage, regardless of the degree of degeneration.     

Enhanced denaturation of the α1(II) chains of type-II collagen in normal adult human intervertebral discs compared with femoral articular cartilage

The mechanical strength of connective tissues is dependent on the integrity of their fibrillar collagen frameworks. The objective of the present study was to assess type-II collagen damage (denaturation) in the adult human intervertebral disc compared with articular cartilage, in order to determine whether damage to this molecule may vary in different anatomical sites in the same person. A new immunochemical assay was used to measure the amounts of denatured and total type-II collagen in the annulus fibrosus and nucleus pulposus of the L5-S1 disc and in cartilage from the femoral condyles of the same individuals (n = 7). Denaturation of type-II collagen was significantly higher in both the annulus fibrosus and the nucleus pulposus than in articular cartilage. Such increased damage to type-II collagen in the adult disc may have relevance to the more pronounced degenerative changes observed in this tissue compared with articular cartilage.     

骨关节炎软骨细胞凋亡调控基因的研究

目的　比较分析正常人及老年性骨关节炎患者软骨细胞bax和bcl 2的表达及细胞凋亡状况。　方法　取 9例骨关节炎患者的关节软骨做实验标本 ,以 6例无骨关节炎病史的意外死亡者关节软骨作为正常对照 ;采用逆转录 /聚合酶链反应 (RT PCR)方法检测bax和bcl 2mRNA表达 ,免疫组化检测bax和bcl 2蛋白 ;应用TUNEL方法进行凋亡细胞原位检测。　结果　骨关节炎患者和正常对照软骨细胞都能表达bax和bcl 2mRNA ;骨关节炎关节软骨细胞baxmRNA表达量较正常对照显著增高 (P <0 0 1) ,bcl 2mRNA表达量也高于正常对照组 (P <0 0 5 ) ,两组间bax/bcl 2表达量的比值差异无显著性意义 (P >0 0 5 ) ;免疫组化可检测到相应表达水平的蛋白 ;骨关节炎软骨细胞凋亡 (4%～ 14% )多于正常对照 (0～ 2 % )。　结论　软骨细胞凋亡受bax和bcl 2共同调节 ;bax和bcl 2的共同调节结果可能是OA患者软骨细胞凋亡增加 ,但凋亡率不高、病理过程进展缓慢的一个重要的原因     

Expression of type VI collagen in normal and osteoarthritic human cartilage

OBJECTIVE: This study was undertaken in order to study the expression of type VI collagen in normal and osteoarthritic human knee cartilage. METHODS: Seventy-two osteoarthritic cartilage/bone samples were obtained form 29 patients with primary OA undergoing surgery for a total knee replacement. Normal cartilage was collected from five human knees at the time of autopsy. Type VI collagen protein was localized using a polyclonal anti human type VI collagen antibody, the corresponding mRNA was detected with an 310 base antisense probe, specific for the alpha2(VI) collagen chain. RESULTS: In normal cartilage, type VI collagen protein is concentrated pericellularly around the chondrocytes of all cartilage zones. In the middle and deep zones, type VI collagen was also found in the interterritorial matrix. Type VI collagen mRNA expression was detected in chondrocytes of all cartilage zones. In moderately affected osteoarthritic cartilage, type VI collagen expression was increased. An intensive immunohistological interterritorial staining for type VI collagen was observed in the middle and deep cartilage zones. Specific mRNA signals were also increased especially in the middle and deep cartilage zone. In the superficial zone and calcified cartilage of these samples, type VI collagen mRNA expression was restricted to focal areas. In severe osteoarthritic cartilage, an intensive staining for type VI collagen mRNA was found in clusters of proliferating chondrocytes and in the deep cartilage zone. Type VI collagen was localized pericellularly and in the matrix of chondrocyte clusters. Furthermore, chondrocytes from the deep zone showed a territorial distribution of type VI collagen. CONCLUSIONS: These results demonstrate that in normal and osteoarthritic cartilage, type VI collagen is expressed in a zone specific pattern. The observed increase of type VI collagen expression in osteoarthritis suggests a potential role in the disease process.