[Ankle chondrocytes are more resistant to Interleukin-1 than chondrocytes derived from the knee]. / Sprunggelenkchondrozyten besitzen eine höhere Interleukin-1-Resistenz als Kniegelenkchondrozyten.

BACKGROUND: The incidence of degenerative changes and osteoarthritis is lower in the ankle than in the knee joints. This cannot be explained exclusively with differences in anatomy and biomechanical properties of these two synovial joints. Previous studies have indicated distinct differences in the biochemical composition of the extracellular matrix of articular cartilage from knee and ankle joints. The aim of this study was to identify potential metabolic differences between knee and ankle joint chondrocytes using isolated cells to distinguish the secondary effects of the resident extracellular matrix from the primary matrix-independent effects of cellular differentiation. METHOD: Isolated knee and ankle chondrocytes from the same human donor were cultured in alginate beads and subsequently exposed to a three-day pulse of the catabolic cytokine interleukin-1 (IL-1) as a model of an inflammatory episode. The metabolism of proteoglycans (PG's) was analyzed as expressed changes in 35S-sulfate incorporation into glycosaminoglycans (GAG's). RESULTS: The presence of IL-1 induced an inhibition of PG synthesis in knee and ankle articular chondrocytes. The 50% inhibitory concentration (IC50) of IL-1 was about 5 times lower for knee than for ankle chondrocytes. CONCLUSION: Ankle chondrocytes are more resistant to IL-1 induced inhibition of PG synthesis than chondrocytes from the knee.

Cartilage degeneration in different human joints.

Variations among joints in the initiation and progression of degeneration may be explained, in part, by metabolic, biochemical and biomechanical differences. Compared to the cartilage in the knee joint, ankle cartilage has a higher content of proteoglycans and water, as well as an increased rate of proteoglycan turnover and synthesis, all of which are responsible for its increased stiffness and reduced permeability. Chondrocytes within ankle cartilage have a decreased response to catabolic factors such as interleukin-1 and fibronectin fragments, compared to the chondrocytes of knee cartilage. Moreover, in response to damage, ankle chondrocytes synthesize proteoglycans at a higher rate than that found in knee cartilage chondrocytes, which suggests a greater capacity for repair. In addition to the cartilages of the two joints, the underlying bones also respond differently to degenerative changes. Taken together, these metabolic, biochemical and biomechanical differences may provide protection to the ankle.

Diffraction-enhanced X-ray imaging of articular cartilage.

OBJECTIVE: To introduce a novel X-ray technology, diffraction-enhanced X-ray imaging (DEI), in its early stages of development, for the imaging of articular cartilage. DESIGN: Disarticulated and/or intact human knee and talocrural joints displaying both undegenerated and degenerated articular cartilage were imaged with DEI. A series of three silicon crystals were used to produce a highly collimated monochromatic X-ray beam to achieve scatter-rejection at the microradian level. The third crystal (analyser) was set at different angles resulting in images displaying different characteristics. Once the diffraction enhanced (DE) images were obtained, they were compared to gross and histological examination. RESULTS: Articular cartilage in both disarticulated and intact joints could be visualized through DEI. For each specimen, DE images were reflective of their gross and histological appearance. For each different angle of the analyser crystal, there was a slight difference in appearance in the specimen image, with certain characteristics changing in their contrast intensity as the analyser angle changed. CONCLUSIONS: DEI is capable of imaging articular cartilage in disarticulated, as well as in intact joints. Gross cartilage defects, even at early stages of development, can be visualized due to a combination of high spatial resolution and detection of X-ray refraction, extinction and absorption patterns. Furthermore, DE images displaying contrast heterogeneities indicative of cartilage degeneration correspond to the degeneration detected by gross and histological examination.

The effect of bone remodeling inhibition by zoledronic acid in an animal model of cartilage matrix damage.

OBJECTIVE: The purpose of this work was to test the effect of inhibition of bone remodeling, through the use of the bisphosphonate, zoledronic acid, on cartilage matrix damage in an animal model of cartilage matrix damage. DESIGN: New Zealand white rabbits were divided into four groups for treatment purposes: (1) untreated controls; (2) injected into one knee joint with the cartilage matrix degradation enzyme, chymopapain; (3) injected into one knee joint with chymopapain and also given subcutaneous injections of the bisphosphonate, zoledronic acid, three times per week until sacrifice at either day 28 or 56 post-chymopapain-injection; (4) received only the zoledronic acid injections. At sacrifice, the knee joints were examined grossly and histologically, and biochemically for proteoglycan content. Urine samples were analysed, at intervals, for levels of collagen cross-links which are biochemical markers of cartilage and bone. RESULTS: Animals receiving both intraarticular chymopapain injections and subcutaneous zoledronic acid injections displayed a significantly lower degree of grossly and histologically detectable cartilage degeneration on the tibial articular surfaces (the articular surface displaying the greatest degree of degeneration) than did animals only receiving the chymopapain injections. In addition, urinary levels of collagen cross-links for bone and cartilage were significantly higher in those animals only receiving chymopapain injections. CONCLUSION: The bone resorption observed after chymopapain injection into the rabbit knee joint can be inhibited through the use of the bisphosphonate, zoledronic acid. Furthermore, zoledronic acid does not increase the level of cartilage degeneration and appears to provide some level of chondroprotection in this model.

Molecular basis for differences between human joints.

The molecular program of a cell determines responses including induction or inhibition of genes for function and activity, and this is true of the cells within articular cartilage, a major functional component of the joint. While our studies have previously focussed on differences in the molecular programs of the cells within the superficial and deep zones, we have recently begun to focus on relative differences between joints, such as the knee and ankle. In the human, these joints vary greatly in their susceptibility to joint diseases, such as osteoarthritis (OA). We have predicted that there would be a molecular basis for differences between joints that could lead to differences in susceptibility to OA, if inherent pathways locked into the resident cells induce differences in their response to their environment. We have been able to show that there are differences between the matrix components and water content; these properties correspond to a higher equilibrium modulus and dynamic stiffness but lower hydraulic permeability and serve to make the ankle cartilage stiffer, slowing movement of molecules through the cartilage. In addition to these biochemical differences in the cartilage matrix, we have also identified relative differences in the strength of the response to stimulation of chondrocytes from knee and ankle. The stronger response of the knee chondrocytes includes factors that increase damage to the cartilage matrix, such as a depression of matrix synthesis and increased enzyme activity. This response by the knee chondrocytes results in enzyme damage to the matrix that the cells may not be able to repair, while the weaker response of the ankle chondrocytes may allow the cells to repair their matrix damage.

Molecular basis of osteoarthritis: biomechanical aspects.

The unique biomechanical properties of healthy cartilage ensure that articular cartilage is able to transmit force between the joints while maintaining almost friction-free limb movement. In osteoarthritis, the biomechanical properties are compromised, but we still do not understood whether this precedes the onset of the disease or is a result of it. This review focuses on the physical changes to cartilage with age, disease, and mechanical loading, with specific reference to the increased collagen cross-linking that occurs with age (nonenzymatic glycation), and the response of chondrocytes to physiological and pathological loads. In addition, the biomechanical properties and matrix biosynthesis of cartilage from various joint surfaces of the knee and ankle are compared to elucidate reasons why the ankle is less affected by progressive osteoarthritis than the knee.

Bone density of the human talus does not increase with the cartilage degeneration score.

Osteoarthritis (OA) is a common, disabling condition of synovial joints that can eventually lead to reduced, or lost, mobility. It is characterized by both articular cartilage degeneration and subchondral bone changes. However, a cause-and-effect relationship between the two tissues remains controversial. Increased subchondral bone density has been associated with early degenerative changes in the cartilage of knee, hip, and finger joints-joints in which progressive changes to OA are common. In contrast, the ankle joint is known to exhibit early cartilage changes, but is not prone to the development of OA. In the present study, it was found that cartilage degeneration on the talus is not associated with an increase in bone density, as assessed through peripheral quantitative computed tomography (pQCT).

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.

Detection of superficial zone protein in human and animal body fluids by cross-species monoclonal antibodies specific to superficial zone protein.

In this report we describe the purification of human superficial zone protein (SZP), the generation of cross-species monoclonal antibodies (MAbs) and the detection of this protein in human and animal body fluids. Human SZPs, used as immunizing antigens, were purified either from culture media of human cartilage organ cultures or from human synovial fluids. The immunizing antigens were mixed with RIBI adjuvant in one of three forms: nonmodified SZP, superficial zone protein-keyhole limpet hemocyanin conjugate (SZP-KLH), or a mixture of superficial zone protein and hyaluronic acid (SZP-HA). A panel of MAbs including GW4.23, S6.79, S13.52, S13.233, and S17.109 were generated and characterized. Monoclonal antibody (MAb) S6.79, an IgG2b with K(D) 3.14 x 10(-9) M from SZP-KLH immunization, is of particular interest. It reacts strongly to a large molecular weight form of SZP in both enzyme-linked immunosorbent assay (ELISA) and Western blotting. It stains the most superficial layer of articular cartilage in immunohistochemistry, whereas the middle and deep zones of cartilage are not stained. When MAb S6.79 was applied to Western blots of human body fluids, a strong 345-kDa band was detected in samples of synovial fluid and weaker bands of similar size were detected in samples of plasma and serum. MAb S6.79 also showed cross-species immunoreactivity with SZP in samples of synovial fluids harvested from bovine, dog, guinea pig, and rabbit, as demonstrated by Western blotting and antibody absorption experiments. This cross-species MAb will be a useful tool in human and animal model studies for monitoring SZP levels and tissue distribution. It may help define the roles of SZP in normal articular joints and may be of diagnostic or prognostic value for the measurement of SZP in pathological conditions such as osteoarthritis, rheumatoid arthritis, and camptodactyly-arthropathy-coxa vara-pericarditis.

Comparison of biomechanical and biochemical properties of cartilage from human knee and ankle pairs.

Cartilage was obtained from eight matched knee (tibiofemoral and femoropatellar) and ankle (talocrural) joints of five different donors (both left and right from donors 14, 22, and 38 years of age, and left only from donors 31 and 45 years of age) within 24 hours of death. All cartilage was graded as normal by the macroscopic visual Collins' scale and the histological Mankin scale. Cylindrical disks of cartilage were harvested from 10 sites within the tibiofemoral and femoropatellar joint surfaces and four sites within the talocrural joint, and uniaxial confined compression measurements were performed to quantify a spectrum of physical properties including the equilibrium modulus, hydraulic permeability, dynamic stiffness, streaming potential, electrokinetic coupling coefficient, and electrical conductivity. Matched specimens from the same 14 sites were used for complementary measurements of biochemical composition and molecular interaction, including water content, hypotonic swelling behavior, and sulfated glycosaminoglycan and collagen contents. In comparison of the top 1-mm slices of talar cartilage with the top 1-mm of tibiofemoral cartilage, the talar cartilage appeared denser with a higher sulfated glycosaminoglycan content, lower water content, higher equilibrium modulus and dynamic stiffness, and lower hydraulic permeability. The equilibrium modulus increased with increasing sulfated glycosaminoglycans per wet weight and decreased with increasing water content for all joint surfaces. Multiple linear regression showed that greater than 80% of the variation in the equilibrium modulus could be accounted for by variations in the biochemical parameters (water content, sulfated glycosaminoglycans/wet weight, and hydroxyproline content/wet weight) for each joint surface. Nonhomogeneous depth-dependent changes in the physical properties and biochemical composition of full-thickness distal femoral cartilage were consistent with previous reports. Since the compressive deformation of cartilage during cyclic loading is confined to the more superficial regions, the differences in properties of the upper regions of the talar compared with tibiofemoral or femoropatellar cartilage may be important in the etiology of osteoarthritis.

Molecular characteristics of the inhibition of human neutrophil elastase by nonsteroidal antiinflammatory drugs.

Nonsteroidal antiinflammatory drugs(NSAIDs) are known as clinically effective agents for treatment of inflammatory diseases. Inhibition of cyclooxygenase has been thought to be a major facet of the pharmacological mechanism of NSAIDs. However, it is difficult to ascribe the antiinflammatory effects of NSAIDs solely to the inhibition of prostaglandin synthesis. Human neutrophil elastase (HNElastase; HNE, EC 3.4.21.37) has been known as a causative factor in inflammatory diseases. To investigate the specific relationship between HNElastase inhibition and specificity of molecular structure of several NSAIDs, HNElastase was purified by Ultrogel AcA54 gel filtration, CM-Sephadex ion exchange, and HPLC (with TSK 250 column) chromatography. HNElastase was inhibited by aspirin and salicylate in a competitive manner and by naproxen, ketoprofen, phenylbutazone, and oxyphenbutazone in a partial competative manner, but not by ibuprofen and tolmetin. HNElastase-phenylbutazone-complex showed strong Raman shifts at 200, 440, 1124, 1194, 1384, 1506, and 1768 cm(-1). The Raman bands 1194, 1384, and 1768 cm(-1) may represent evidences of the conformational change at -N=N-phi radical, pyrazol ring, and -C=O radical of the elastase-drug complex, respectively. Phenylbutazone might be bound to HNElastase by ionic and hydrophobic interaction, and masked the active site. Inhibition of HNElastase could be another mechanism of action of NSAIDs besides cyclooxygenase inhibition in the treatment of inflammatory diseases. Different inhibition characteristics of HNE-lastase by NSAIDs such as aspirin, phenylbutazone-like drugs and ineffective drugs could be important points for drawing the criteria for appropriate drugs in clinical application.

Hyaluronan oligosaccharides perturb cartilage matrix homeostasis and induce chondrocytic chondrolysis.

OBJECTIVE: To determine the role of CD44-mediated matrix assembly in maintaining cartilage homeostasis. METHODS: Articular cartilage tissue slices as well as isolated chondrocytes were treated with hyaluronan (HA) hexasaccharides. Tissues and cells were processed for histology, immunohistochemistry, colorimetric assay, reverse transcriptase-polymerase chain reaction, and zymography. RESULTS: HA oligosaccharides induced a dose-dependent state of chondrocytic chondrolysis, including near-total loss of stainable proteoglycan-rich matrix, activation of gelatinolytic activity, and exhibition of the NITEGE epitope. However, HA oligosaccharides also induced an increase of proteoglycan synthesis, including elevation of aggrecan and HA synthase 2 messenger RNA. CONCLUSION: Uncoupling chondrocytes from the matrix results in deleterious changes in matrix structure and modifications in chondrocyte metabolism. The close interaction of chondrocytes with their matrix and their ability to "sense" changes in receptor occupancy are needed for maintenance of cartilage homeostasis.

Osteogenic protein-1 promotes the synthesis and retention of extracellular matrix within bovine articular cartilage and chondrocyte cultures.

OBJECTIVE: We have used recombinant osteogenic protein-1 to investigate our hypothesis that proper repair and maintenance of cartilage requires not only enhanced biosynthesis and replenishment of the extracellular matrix but also the enhancement of components necessary for matrix retention. DESIGN: The effects of osteogenic protein-1 were examined on bovine articular cartilage slices as well as isolated chondrocytes grown in alginate beads. Cartilage slices were examined for accumulation of proteoglycan by incorporation of 35S-sulfate and staining using Safranin O or, a biotinylated probe specific for hyaluronan. Bovine chondrocytes were characterized by use of a particle exclusion assay, in-situ hybridization, quantitative-competitive RT-PCR and a hyaluronan-binding assay. RESULTS: Osteogenic protein-1 treatment substantially enhanced the accumulation of hyaluronan and proteoglycan within cartilage tissue slices. As with the tissue, osteogenic protein-1 enhanced the size of cell-associated matrices assembled and retained by chondrocytes in vitro. This enhanced matrix assembly was paralleled by an increased expression of mRNA for aggrecan, hyaluronan synthase-2 and CD44. Of the two hyaluronan synthase genes expressed by chondrocytes, only hyaluronan synthase-2 was upregulated by osteogenic protein-1. Coupled with the increase in the CD44 mRNA was an increase in functional hyaluronan binding activity present at the chondrocyte cell surface. CONCLUSIONS: These results demonstrate that osteogenic protein-1 stimulates not only the synthesis of the major cartilage extracellular matrix component aggrecan, but also two associated molecules necessary for the retention of aggrecan, namely hyaluronan and CD44.

[Adult human chondrocytes in alginate culture. Preservation of the phenotype for further use in transplantation models]. / Adulte humane Chondrozyten in Alginatkultur. Beibehaltung des Phänotyps für die weitere Anwendung in Transplantationsmodellen.

Successful and cartilage-specific cultivation of chondrocytes requires a stable phenotype during the in vitro culture period. This is based on a differentiated extracellular matrix synthesis. The alginate system as a three-dimensional support is a useful system to culture chondrocytes and to analyze the biochemical processes in this system. Talar cartilage from the talocrural joints of 40 different donors were obtained through the Regional Organ Bank of Illinois within 24 hours of death. In 65% of the tissues the cartilage was classified as being undamaged. In these studies we were interested in the results of short-term culture over 14 days. Cell proliferation, total collagen content and total proteoglycan content were measured in the different matrix compartments and were visualized by histology and immunohistochemistry. Already after 7 days in culture the adult human chondroctes looked intact and formed a stable and cell-associated cartilage-specific extracellular matrix in the presence of 10% calf serum. This could be also demonstrated in the presence of IGF-I. With regards to the collagen content IGF-I at a concentration of 50 ng/ml seemed to induce an equal effect to 10% serum; with regards to the proteoglycan content IGF-I at a concentration of 20 ng/ml was equivalent. These encouraging preliminary results may lead to a new approach in tissue engineering for chondrocyte transplantation in combination with their extracellular matrix.

Osteogenic protein 1 stimulates cells-associated matrix assembly by normal human articular chondrocytes: up-regulation of hyaluronan synthase, CD44, and aggrecan.

OBJECTIVE: To determine the effects of osteogenic protein 1 (OP-1) on hyaluronan (HA), CD44, and aggrecan biosynthesis as well as the contribution of these molecules in promoting matrix assembly by human articular chondrocytes. METHODS: Normal human chondrocytes were cultured with or without OP-1 treatment. Changes in the relative expression of messenger RNA (mRNA) for HA synthases 2 and 3 (HAS-2 and HAS-3), CD44, and aggrecan were determined by competitive quantitative reverse transcriptase-polymerase chain reaction. Accumulation of HA was characterized by indirect staining, CD44 by flow cytometry, and aggrecan biosynthesis by 35SO4 incorporation. RESULTS: OP-1 stimulated the expression of HAS-2, CD44, and aggrecan mRNA in a time-dependent manner, resulting in increased expression of HA, CD44, and aggrecan. Prominent increases in HA-rich cell-associated matrices were also observed. CONCLUSION: OP-1 stimulates not only the synthesis of matrix macromolecules such as aggrecan, but also the synthesis of other molecules required for matrix retention, namely, HA and CD44.

Human articular chondrocytes express osteogenic protein-1.

This study demonstrates for the first time that human articular chondrocytes express osteogenic protein-1 (OP-1). OP-1 was originally purified from bone matrix and was shown to induce cartilage and bone formation. Both OP-1 protein and message were present in human normal and osteoarthritic (OA) cartilages. OP-1 mRNA was upregulated in OA cartilage compared with normal adult tissues. However, the level of mature OP-1 protein in the same OA tissues was downregulated, whereas the pro-OP-1 remained high. Moreover, these two forms of OP-1 were localized in an inverted manner. Mature OP-1 was primarily detected in the superficial layer, whereas the pro-form was mostly in the deep layer of cartilage. The presence of pro- and mature OP-1 in extracts of normal and OA cartilages was confirmed by Western blotting. These findings imply that articular chondrocytes continue to express and synthesize OP-1 throughout adulthood. The observed patterns of the distribution of pro- and mature OP-1 also suggest differences in the processing of this molecule by normal and OA chondrocytes and by the cells in the superficial and deep layers. Distinct distribution of OP-1 and its potential activation in deep zones and regions of cloning in OA cartilages may provide clues to the potential involvement of endogenous OP-1 in repair mechanisms. (J Histochem Cytochem 48:239-250, 2000)

Distribution of cartilage thickness on the head of the human first metatarsal bone.

Articular hyaline cartilage takes on the contours of the subchondral bone on which it lies, but its thickness varies between joints, within a single joint and within a single articular surface. Previous studies have correlated articular cartilage thickness distribution with the degree of stress and weight bearing on joint surfaces, but few studies have considered the thickness of the calcified cartilage in relation to these parameters. Here we report a correlation between the cartilage thickness distribution and weight bearing distribution on the head of the 1st metatarsal bone, a component of one of the major weight bearing joints in the lower extremity during the gait cycle. The greatest total and uncalcified articular cartilage thickness was found on the central and lateral distal aspects of the metatarsal head, a region that receives maximal ground reactive force during the propulsive phase of the normal gait cycle. Although the thickness of the calcified cartilage was correlated with the thickness of the uncalcified cartilage, it varied to a lesser extent across the articular surface than did that of the uncalcified cartilage.

Osteogenic protein-1 (OP-1) blocks cartilage damage caused by fibronectin fragments and promotes repair by enhancing proteoglycan synthesis.

OBJECTIVE AND DESIGN: The abilities of osteogenic protein-1 (OP-1) and TGF-beta1 to affect cartilage damage caused by fibronectin fragments (Fn-fs) that are known to greatly enhance cartilage proteoglycan (PG) degradation were compared. MATERIAL: Articular cartilage was obtained from 18 month old bovines. TREATMENT: To test blocking of damage, cartilage was cultured with or without OP-1 or TGF-beta in the presence of 100 nM Fn-fs. To test restoration of PG, cartilage was first cultured with Fn-fs and the cartilage then treated with factors. METHODS: Cartilage PG content was measured in papain digests using the dimethylmethylene blue assay. PG synthesis was measured by incorporation of 35S labeled sulfate. RESULTS: OP-1 blocked damage and restored PG in damaged cartilage, apparently due to enhanced PG synthesis. However, TGF-beta1 alone decreased PG content. CONCLUSIONS: These results clearly demonstrate differences between OP-1 and TGF-beta1, both members of the TGF-beta superfamily and illustrate the efficacy of OP- in blocking Fn-f mediated damage.

Immunodetection and partial cDNA sequence of the proteoglycan, superficial zone protein, synthesized by cells lining synovial joints.

We have previously described a large proteoglycan named superficial zone protein that was isolated and purified from culture medium of superficial slices of bovine articular cartilage. Monoclonal antibodies were raised against superficial zone protein and used as probes in Western blot analyses for immunohistochemical studies both to determine precisely which cells within the joint synthesize the proteoglycan and to isolate a cDNA fragment from a bovine chondrocyte lambdagt11 library that encodes part of the proteoglycan. The cDNA fragment that was obtained with use of monoclonal antibody 6-A-1 encodes the 3' end of the sequence for superficial zone protein. On Western blots, monoclonal antibody 3-A-4 recognized an epitope on native, but not reduced, superficial zone protein, whereas monoclonal antibody 6-A-1 reacted with both native and denatured antigen. The proteoglycan was immunolocalized with monoclonal antibody 3-A-4 in chondrocytes predominantly within the superficial zone of fetal and adult articular cartilage and in some cells of the synovial lining. However, the proteoglycan was not detected in chondrocytes deep in articular cartilage, in nasal septal cartilage, or in synovial stromal cells. The only matrix staining positively for superficial zone protein was at the articular surface bordering the synovial cavity in adult, but not fetal, joints. Isolated chondrocytes and synovial cells showed intracellular binding of monoclonal antibody 3-A-4, and flow-cytometric analysis with the antibody gave the following percentages of immunopositive cells: 37.4, 52.5, 3.4, and 7.5 from chondrocytes from the full-thickness, superficial, and deep zones and from synovial cells, respectively. Thus, both chondrocytes and synovial cells bordering the joint cavity synthesize superficial zone protein and substantiate its usefulness as a phenotypic marker of particular cellular species lining the articular cavity.