The distribution of serum albumin in human normal and degenerate articular cartilage.

A method for studying the distribution of a high molecular weight solute (serum albumin) between physiological saline and human articular cartilage is described. Samples of normal and fibrillated articular cartilage from both femoral condyles and femoral heads have been studied. Limited studies have also been performed where the glycosaminoglycan content of normal cartilage has been reduced by chemical or enzymic methods. With naturally occurring cartilage a wide range of partition coefficients (0.3 to less than 0.002) was obtained. The partition coefficients are very dependent upon proteoglycan concentration, with the partition coefficient decreasing with increasing fixed charge density. An attempt is made to interpret the observed partitioning in terms of the steric exclusion by the proteoglycans.     

Type III collagen in normal human articular cartilage

Summary Type III collagen in normal human articular cartilage has been detected biochemically and its location in a diffuse area around the chondrocytes demonstrated by immunofluorescence. It can be found pericellularly throughout the depth of the cartilage and is evident in specimens ranging in age from 17 to 81 years.     

Mesenchymal progenitor cell markers in human articular cartilage: normal distribution and changes in osteoarthritis

Introduction

Similar to matrix metalloproteinases, glycosidases also play a major role in cartilage degradation. Carbohydrate cleavage products, generated by these latter enzymes, are released from degrading cartilage during arthritis. Some of the cleavage products (such as hyaluronate oligosaccharides) have been shown to bind to Toll-like receptors and provide endogenous danger signals, while others (like N-acetyl glucosamine) are reported to have chondroprotective functions. In the current study for the first time we systematically investigated the expression of glycosidases within the joints.

Methods

Expressions of β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, sperm adhesion molecule 1 and klotho genes were measured in synovial fibroblasts and synovial membrane samples of patients with rheumatoid arthritis and osteoarthritis by real-time PCR. β-D-Glucuronidase, β-D-glucosaminidase and β-D-galactosaminidase activities were characterized using chromogenic or fluorogenic substrates. Synovial fibroblast-derived microvesicles were also tested for glycosidase activity.

Results

According to our data, β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, and klotho are expressed in the synovial membrane. Hexosaminidase is the major glycosidase expressed within the joints, and it is primarily produced by synovial fibroblasts. HexA subunit gene, one of the two genes encoding for the alpha or the beta chains of hexosaminidase, was characterized by the strongest gene expression. It was followed by the expression of HexB subunit gene and the β-D-glucuronidase gene, while the expression of hyaluronidase-1 gene and the klotho gene was rather low in both synovial fibroblasts and synovial membrane samples. Tumor growth factor-β1 profoundly downregulated glycosidase expression in both rheumatoid arthritis and osteoarthritis derived synovial fibroblasts. In addition, expression of cartilage-degrading glycosidases was moderately downregulated by proinflammatory cytokines including TNFα, IL-1β and IL-17.

Conclusions

According to our present data, glycosidases expressed by synovial membranes and synovial fibroblasts are under negative regulation by some locally expressed cytokines both in rheumatoid arthritis and osteoarthritis. This does not exclude the possibility that these enzymes may contribute significantly to cartilage degradation in both joint diseases if acting in collaboration with the differentially upregulated proteases to deplete cartilage in glycosaminoglycans.     

Mesenchymal progenitor cell markers in human articular cartilage: normal distribution and changes in osteoarthritis

Introduction  

Recent findings suggest that articular cartilage contains mesenchymal progenitor cells. The aim of this study was to examine the distribution of stem cell markers (Notch-1, Stro-1 and VCAM-1) and of molecules that modulate progenitor differentiation (Notch-1 and Sox9) in normal adult human articular cartilage and in osteoarthritis (OA) cartilage.     

Passive osmotic properties of in situ human articular chondrocytes within non-degenerate and degenerate cartilage

Osteoarthritis is characterized by many factors, including proteoglycan loss, decreased collagen stiffness, and increased cartilage hydration. Chondrocyte swelling also occurs, and correlates with the degree of osteoarthritis, however, the cause is unknown but might be related to alterations to their passive osmotic properties. We have used two-photon confocal laser scanning microscopy to measure the passive osmotic characteristics of in situ chondrocytes within relatively non-degenerate and degenerate human tibial plateau cartilage, and in chondrocytes isolated from relatively non-degenerate cartilage. Explants with bone attached were taken from a total of 42 patients undergoing arthroplasty and graded macroscopically and microscopically into two groups, grade 0 + 1 and grade 2 + 3. There was a significant increase in cartilage hydration between these two groups (P < 0.05), however, there was no change when medium osmolarity was varied over approximately 0-480 mOsm. The passive osmotic behavior of in situ chondrocytes (at 4 degrees C) was identical over a range of culture medium osmolarities ( approximately 0-515 mOsm), however, the maximum swelling of cells within degenerate cartilage and isolated chondrocytes was greater compared to those in non-degenerate cartilage. The swelling in the majority of in situ chondrocytes was accounted for by the reduced interstitial osmolarity occurring with cartilage degeneration. There was, however, a small population of in situ chondrocytes whose volume was in excess (>/=2,500 microm(3)) of that predicted from the decreased interstitial osmotic pressure. These results show that for the majority of cells studied, the differences in passive chondrocyte volume between relatively non-degenerate, degenerate, and isolated cells were entirely accounted for by changes to the extracellular osmolarity (180-515 mOsm).     

Patterns of cartilage stiffness on normal and degenerate human femoral heads

Using the apparatus and technique described in an earlier paper, (Kempson et al. 1971), indentation tests were performed on areas of cartilage of 0·125 in dia., in situ on, and distributed evenly over, the cartilage surface of the human femoral head. Curves of indentation vs. time were plotted for a physiological stress of approximately 400 lbf/in². (28·2 kgf/cm², 2·36 MN/m²). The stiffness of each area of cartilage was calculated from the appropriate indentation value, in the form of a creep modulus at 2 sec, using the equations described in the previous paper (see Appendix). Layered maps and histograms showing the variation of both cartilage stiffness and indentation are presented.     

Glycosyltransferase activities in chondrocytes from osteoarthritic and normal human articular cartilage

Six glycosyltransferases (mannosyl-, glucosyl-, N-acetyl-glucosaminyl-, galactosyl-, sialyl- and fucosyltransferases) are studied and characterized for their optimal conditions and their relations with interfering reactions (glycosyl-nucleotide pyrophosphatases, glycosidases and proteinases) in chondrocytes from osteoarthritic and normal human articular cartilage. Osteoarthritis induces increased activities for five glycosyl-transferases. The observed modifications are not explained by alterations in physico-chemical parameters of the enzymes or by intervention of glycosyl-nucleotide pyrophosphatases, glycosidases or proteolytic enzymes.     

Synthesis of fibronectin in normal and osteoarthritic articular cartilage

The content and the biosynthesis of fibronectin was examined in disease-free articular cartilage and in articular cartilage from osteoarthritic canine joints. Fibronectin content was increased in extracts of cartilage from osteoarthritic joints. Incubation of cartilage in vitro with [³H]phenylalanine and subsequent isolation of [³H]fibronectin from a gelatin affinity column and characterization by SDS-polyacrylamide gel electrophoresis and by immunoprecipitation indicated that disease-free and osteoarthritic cartilage explants synthesized fibronectin. About 50% of the [³H]fibronectin was recovered in the incubation medium. The osteoarthritic cartilage synthesized and accumulated up to 5-fold more [³H]fibronectin than disease-free cartilage.     

Identification and clonal characterisation of a progenitor cell sub-population in normal human articular cartilage

Background

Articular cartilage displays a poor repair capacity. The aim of cell-based therapies for cartilage defects is to repair damaged joint surfaces with a functional replacement tissue. Currently, chondrocytes removed from a healthy region of the cartilage are used but they are unable to retain their phenotype in expanded culture. The resulting repair tissue is fibrocartilaginous rather than hyaline, potentially compromising long-term repair. Mesenchymal stem cells, particularly bone marrow stromal cells (BMSC), are of interest for cartilage repair due to their inherent replicative potential. However, chondrocyte differentiated BMSCs display an endochondral phenotype, that is, can terminally differentiate and form a calcified matrix, leading to failure in long-term defect repair. Here, we investigate the isolation and characterisation of a human cartilage progenitor population that is resident within permanent adult articular cartilage.

Methods and Findings

Human articular cartilage samples were digested and clonal populations isolated using a differential adhesion assay to fibronectin. Clonal cell lines were expanded in growth media to high population doublings and karyotype analysis performed. We present data to show that this cell population demonstrates a restricted differential potential during chondrogenic induction in a 3D pellet culture system. Furthermore, evidence of high telomerase activity and maintenance of telomere length, characteristic of a mesenchymal stem cell population, were observed in this clonal cell population. Lastly, as proof of principle, we carried out a pilot repair study in a goat in vivo model demonstrating the ability of goat cartilage progenitors to form a cartilage-like repair tissue in a chondral defect.

Conclusions

In conclusion, we propose that we have identified and characterised a novel cartilage progenitor population resident in human articular cartilage which will greatly benefit future cell-based cartilage repair therapies due to its ability to maintain chondrogenicity upon extensive expansion unlike full-depth chondrocytes that lose this ability at only seven population doublings.     

Enzymic heterogeneity of normal canine articular cartilage

Articular cartilage is generally considered to be an homogeneous tissue. It has now been shown that, although different regions of the medial tibial cartilage of the dog have very similar oxidative enzymic activities, each region is heterogeneous with respect to these activities. The conventional histological delineation of this cartilage has been modified, to take into account a narrow band (designated zone 2a), just below the most superficial spindle-shaped cells, that has higher oxidative enzymic activity than any other. Changes in the activity in this zone might be diluted by the lack of change in other zones if measured by conventional biochemical procedures which could not measure the activities of the different zones separately.     

Revisiting spatial distribution and biochemical composition of calcium-containing crystals in human osteoarthritic articular cartilage

Introduction

Calcium-containing (CaC) crystals, including basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPP), are associated with destructive forms of osteoarthritis (OA). We assessed their distribution and biochemical and morphologic features in human knee OA cartilage.

Methods

We prospectively included 20 patients who underwent total knee replacement (TKR) for primary OA. CaC crystal characterization and identification involved Fourier-transform infra-red spectrometry and scanning electron microscopy of 8 to 10 cartilage zones of each knee, including medial and lateral femoral condyles and tibial plateaux and the intercondyle zone. Differential expression of genes involved in the mineralization process between cartilage with and without calcification was assessed in samples from 8 different patients by RT-PCR. Immunohistochemistry and histology studies were performed in 6 different patients.

Results

Mean (SEM) age and body mass index of patients at the time of TKR was 74.6 (1.7) years and 28.1 (1.6) kg/m², respectively. Preoperative X-rays showed joint calcifications (chondrocalcinosis) in 4 cases only. The medial femoro-tibial compartment was the most severely affected in all cases, and mean (SEM) Kellgren-Lawrence score was 3.8 (0.1). All 20 OA cartilages showed CaC crystals. The mineral content represented 7.7% (8.1%) of the cartilage weight. All patients showed BCP crystals, which were associated with CPP crystals for 8 joints. CaC crystals were present in all knee joint compartments and in a mean of 4.6 (1.7) of the 8 studied areas. Crystal content was similar between superficial and deep layers and between medial and femoral compartments. BCP samples showed spherical structures, typical of biological apatite, and CPP samples showed rod-shaped or cubic structures. The expression of several genes involved in mineralization, including human homolog of progressive ankylosis, plasma-cell-membrane glycoprotein 1 and tissue-nonspecific alkaline phosphatase, was upregulated in OA chondrocytes isolated from CaC crystal-containing cartilages.

Conclusions

CaC crystal deposition is a widespread phenomenon in human OA articular cartilage involving the entire knee cartilage including macroscopically normal and less weight-bearing zones. Cartilage calcification is associated with altered expression of genes involved in the mineralisation process.     

Differential responses of chondrocytes from normal and osteoarthritic human articular cartilage to mechanical stimulation

Mechanical stimulation is critically important for the maintenance of normal articular cartilage integrity. Molecular events regulating responses of chondrocytes to mechanical forces are beginning to be defined. Chondrocytes from normal human knee joint articular cartilage show increased levels of aggrecan mRNA following 0.33 Hz mechanical stimulation whilst at the same time relative levels of MMP3 mRNA are decreased. This anabolic response, associated with membrane hyperpolarisation, is activated via an integrin-dependent interleukin (IL)-4 autocrine/paracrine loop. Work in our laboratory suggests that this chondroprotective response may be aberrant in osteoarthritis (OA). Chondrocytes from OA cartilage show no changes in aggrecan or MMP3 mRNA following 0.33 Hz mechanical stimulation. alpha5beta1 integrin is the mechanoreceptor in both normal and OA chondrocytes but downstream signalling pathways differ. OA chondrocytes show membrane depolarisation following 0.33 Hz mechanical stimulation consequent to activation of an IL1beta autocrine/paracrine loop. IL4 signalling in OA chondrocytes is preferentially through the type I (IL4alpha/cgamma) receptor rather than via the type II (IL4alpha/IL13R) receptor. Altered mechanotransduction and signalling in OA may contribute to changes in chondrocyte behaviour leading to increased cartilage breakdown and disease progression.     

Regional distribution of water and glycosaminoglycan in immature articular cartilage

The distribution of water and glycosaminoglucan in different functional regions of bovine immature articular cartilage were studied. There was always more water in each articulating than in the corresponding growin zone, but there was less water in both zones in the areas of maximum contact. There was more hyaluronate and much more keratan sulphate in the articulating areas of maximum contact than in the minimum contact areas. In the growing zone the distribution of these two glycosaminoglycans did not vary as significantly but there was slightly more keratan sulphate in the area of maximum contact. Regional variations in chondroitin sulphate were also present although not as striking as those of keratan sulphate. The results suggest that some keratan sulphate may be synthesized as reaction to load.     

A theoretical study of the distribution of insulin-like growth factor in human articular cartilage

We present a mathematical simulation which integrates the mechanisms that are currently believed to govern the concentration of the growth factor, IGF1, in cartilage. Articular cartilage is treated as a two-layer continuum: a thin surface layer, exposed to synovial fluid, with a higher cell density, and a deeper layer with impermeable bony endplate. A system of differential equations accounts for diffusion of IGF1 from synovial fluid into, and throughout, the cartilage; IGF1 synthesis, its reactions with soluble binding protein, with cell receptors, and with immobile binding sites on the extracellular matrix. We have collected all available physiologic data relevant to the solution of these equations and used it to compute numerical solutions that yield time dependent profiles for free and complex IGF1 throughout the depth of normal cartilage. Equations for osteoarthritic cartilage were formulated as well. Numerical results indicate a time-scale of several days for IGF1 profiles to settle down after a disturbance. The number of cell receptors for IGF1 appears to be more important than their rate of internalization. There is a lower bound to the number of cell receptors and of immobile binding sites. Parameters that await experimental determination are identified.     

The inability to prepare high-buoyant-density proteoglycan aggregates from extracts of normal adult human articular cartilage.

High-buoyant-density proteoglycan aggregates could not be prepared from extracts of adult human cartilage by associative CsCl-density-gradient centrifugation with a starting density of 1.68 g/ml, even though proteoglycan subunits, hyaluronic acid and link proteins were all present. In contrast, aggregates could be prepared when extracts of neonatal human cartilage or bovine nasal cartilage were subjected to the same procedure. This phenomenon did not appear to be due to a defect within the hyaluronic acid-binding region of the adult proteoglycan subunit, but rather to an interference in the stability of the interaction between the proteoglycan subunit and hyaluronic acid towards centrifugation. The factor responsible for this instability was shown to reside within the low-density cartilage protein preparation obtained by direct dissociative CsCl-density-gradient centrifugation of the adult cartilage extract.     

Degradome expression profiling in human articular cartilage

Introduction

The molecular mechanisms underlying cartilage destruction in osteoarthritis are poorly understood. Proteolysis is a key feature in the turnover and degradation of cartilage extracellular matrix where the focus of research has been on the metzincin family of metalloproteinases. However, there is strong evidence to indicate important roles for other catalytic classes of proteases, with both extracellular and intracellular activities. The aim of this study was to profile the expression of the majority of protease genes in all catalytic classes in normal human cartilage and that from patients with osteoarthritis (OA) using a quantitative method.

Methods

Human cartilage was obtained from femoral heads at joint replacement for either osteoarthritis or following fracture to the neck of femur (NOF). Total RNA was purified, and expression of genes assayed using Taqman^® low-density array quantitative RT-PCR.

Results

A total of 538 protease genes were profiled, of which 431 were expressed in cartilage. A total of 179 genes were differentially expressed in OA versus NOF cartilage: eight aspartic proteases, 44 cysteine proteases, 76 metalloproteases, 46 serine proteases and five threonine proteases. Wilcoxon ranking as well as the LogitBoost-NR machine learning approach were used to assign significance to each gene, with the most highly ranked genes broadly similar using each method.

Conclusions

This study is the most complete quantitative analysis of protease gene expression in cartilage to date. The data help give direction to future research on the specific function(s) of individual proteases or protease families in cartilage and may help to refine anti-proteolytic strategies in OA.     

Abnormalities of DNA in human osteoarthritic articular cartilage

The normal amount of DNA in human diploid nuclei was determined by the use of the Feulgen reaction measured by microdensitometry. The DNA-content of nuclei in normal human articular cartilage was determined in nuclei of zones 3 and 4 of cartilage of the femoral head removed from osteoporotic fractured necks of femur. Analysis of the results indicated that a degree of synthesis of DNA occurred even in these zones of very elderly persons. Results on these zones in the articular cartilage of osteoarthritic joints indicated that different populations occurred. In some there was DNA-synthesis related to tetraploidy; in others, the DNA was very stable to acid hydrolysis with no sign of biosynthetic activity; in the last group, which contained erosions of the superficial zones, the DNA was unstable to hydrolysis.