Competing regulation of matrix biosynthesis by mechanical and IGF-1 signalling in elderly human articular cartilage in vitro

This study investigates the separate and combined effects of IGF-1 and mechanical loads on chondrocytes in elderly human femoral head articular cartilage. Full depth biopsies of articular cartilage were subjected to either no load, static or cyclic (2 s on/2 s off) loading in unconfined compression at a stress of 1 MPa for 48 h with or without IGF-1 (300 ng ml(-1)). Chondrocyte biosynthetic activity was measured using 35S-sulphate and 3H-leucine during the last 24 h of loading. IGF-1 alone increased the rates of isotope incorporation, by 80% for 35S-SO4 and 40% for 3H-leucine, whereas loading alone reduced matrix biosynthesis. Applying load (cyclic or static) in the presence of IGF-1 returned the incorporation rates to their unstimulated levels. This study suggests elderly human articular cartilage is responsive to stimulation by IGF-1 but mechanical factors seem to act sufficiently strongly in the opposite direction to cancel this response.     

A role for the interleukin-1 receptor in the pathway linking static mechanical compression to decreased proteoglycan synthesis in surface articular cartilage

Loading of articular cartilage during weight bearing is essential for the maintenance of cartilage function. Although certain cyclic loading protocols stimulate extracellular matrix synthesis, constant or static compression decreases proteoglycan and collagen synthesis in cartilage explants. The goal of this study was to determine whether the compression-induced decrease in proteoglycan synthesis involves an interleukin-1 (IL-1) signaling pathway. Cartilage explants were compressed 50% in the presence of IL-1 receptor antagonist (IL-1ra), and the incorporation of [35S]sulfate into macromolecules was measured. IL-1ra increased sulfate incorporation in compressed cartilage but not in cartilage maintained at the in situ thickness (0% compression). IL-1alpha and IL-1beta mRNAs were detected in cartilage compressed 50% for at least 3h, while nitric oxide synthase II mRNA was only detected in cartilage compressed 50% for 6h. The data support a role for the IL-1 receptor in the pathway linking static compression to reduced proteoglycan synthesis.     

Mechanical and physicochemical regulation of the action of insulin-like growth factor-I on articular cartilage

The development and maintenance of healthy joints is a complex process involving many physical and biological stimuli. This study investigates the interaction between insulin-like growth factor-I (IGF-I) and static mechanical compression in the regulation of articular cartilage metabolism. Bovine cartilage explants were treated with concentrations of IGF-I from 0 to 300 ng/ml in the presence or absence of 0-50% static compression, and the transient and steady-state incorporation of [(3)H]proline and [(35)S]sulfate into matrix components were measured. In parallel studies, cartilage explants were treated with 0-300 ng/ml IGF-I at media pH ranging from 6.4 to 7.2 and the steady-state incorporation of [(3)H]proline and [(35)S]sulfate was measured. The effect of 50% static compression on IGF-I transport was determined by measuring the uptake of (125)I-labeled IGF-I into cartilage explants. Static compression decreased both [(3)H]proline and [(35)S]sulfate incorporation in a dose-dependent manner in the presence or absence of IGF-I. IGF-I increased [(3)H]proline and [(35)S]sulfate incorporation in a dose-dependent manner in the presence or absence of compression, but the anabolic effect of the growth factor was lessened when the tissue was compressed by 50%. The response of cartilage explants to IGF-I was similarly lessened in unstrained tissue cultured in media at pH 6.4, a condition which results in a similar intratissue pH to that when cartilage is compressed by 50%. The characteristic time constant (tau) for IGF-I stimulation of cartilage explants was approximately 24 h, while tau for inhibition of biosynthesis by static compression was approximately 2 h. Samples which were both compressed and treated with IGF-I demonstrated an initial decrease in biosynthetic activity at 2 h, followed by an increase at 24 h. Static compression did not alter tau for (125)I-labeled IGF-I transport into cartilage but decreased the concentration of (125)I-labeled IGF-I in the tissue at equilibrium.     

Altered patterns and synthesis of extracellular matrix macromolecules in early osteoarthritis.

The synthesis and contents of extracellular non-collagenous matrix macromolecules was studied in early and late human osteoarthritic (OA) cartilage obtained at surgery for sarcomas in the lower extremities (normal and early OA) or for total knee replacement (late stage OA). The early OA samples were those that had some fibrillation in the joint by visual examination. One group had fibrillation in the area sampled and the other group had no fibrillation. Cartilage was taken from the same topographical area on the medial femoral condyle in all the samples, labeled with [3H]leucine and [35S]sulfate for 4 h at 37 degrees C and extracted with 4 M guanidine-HCl. Analysis of the extracts showed that the total amount of proteoglycans relative to hydroxyproline content was higher in the early and late OA than in the normal cartilage. These proteoglycans showed a relatively lower [35S]sulfate incorporation into GAG chains and a higher [3H]leucine incorporation. The pattern of newly synthesized proteins was altered similarly in early and late OA. Notably, synthesis of cartilage oligomeric matrix protein (COMP), fibronectin, and cartilage intermediate layer protein (CILP) was increased, also reflected in their abundance as determined by enzyme-linked immunosorbent assay (ELISA). Collagen synthesis appeared significantly increased only in the late stage OA. The observed altered composition and pattern of biosynthesis indicate that the joint undergoes metabolic alterations early in the disease process, even before there is overt fibrillation of the tissue. The early OA samples studied appear to represent two distinct groups of early lesions in different stages of the process of cartilage deterioration as shown by their differences in relative rates of synthesis and abundance of proteins.     

Analysis of the mechanical behavior of chondrocytes in unconfined compression tests for cyclic loading

Experimental evidence indicates that the biosynthetic activity of chondrocytes is associated with the mechanical environment. For example, excessive, repetitive loading has been found to induce cell death, morphological and cellular damage, as seen in degenerative joint disease, while cyclic, physiological-like loading has been found to trigger a partial recovery of morphological and ultrastructural aspects in osteoarthritic human articular chondrocytes. Mechanical stimuli are believed to influence the biosynthetic activity via the deformation of cells. However, the in situ deformation of chondrocytes for cyclic loading conditions has not been investigated experimentally or theoretically. The purpose of the present study was to simulate the mechanical response of chondrocytes to cyclic loading in unconfined compression tests using a finite element model. The material properties of chondrocytes and extracellular matrix were considered to be biphasic. The time-histories of the shape and volume variations of chondrocytes at three locations (i.e., surface, center, and bottom) within the cartilage were predicted for static and cyclic loading conditions at two frequencies (0.02 and 0.1 Hz) and two amplitudes (0.1 and 0.2 MPa). Our results show that cells at different depths within the cartilage deform differently during cyclic loading, and that the depth dependence of cell deformation is influenced by the amplitude of the cyclic loading. Cell deformations under cyclic loading of 0.02 Hz were found to be similar to those at 0.1 Hz. We conclude from the simulation results that, in homogeneous cartilage layers, cell deformations are location-dependent, and further are affected by load magnitude. In physiological conditions, the mechanical environment of cells are even more complex due to the anisotropy, depth-dependent inhomogeneity, and tension-compression non-linearity of the cartilage matrix. Therefore, it is feasible to speculate that biosynthetic responses of chondrocytes to cyclic loading depend on cell location and load magnitude.     

Matrix-matrix interaction of cartilage oligomeric matrix protein and fibronectin.

Recent work indicates that cartilage oligomeric matrix protein (COMP) plays an important role in extracellular matrix assembly and matrix-matrix protein interactions. In order to identify the proteins in extracellular matrix that interact with COMP, we used an ELISA-based solid-phase binding assay, which revealed a specific, high-affinity interaction between COMP and fibronectin. This interaction is concentration-dependent and saturable, and appears to occur under physiologically relevant conditions. Electron microscopy after negative staining and fragment binding analysis using the solid-phase assay revealed a predominant binding site for the COMP C-terminal globular domain to a molecular domain approximately 14 nm from the N-terminal domain of fibronectin, which can be inhibited by the presence of a polyclonal antibody specific for the C-terminal heptadecapeptide of COMP. This interaction is further demonstrated in vivo by colocalization of both COMP and fibronectin in the chondrocyte pericellular matrix by laser confocal microscopy of chondrocytes grown in agarose culture, and by appositional and colocalization of these proteins in the growth plate of primates by immunohistochemistry.     

Distribution of cartilage molecules in the developing mouse joint.

This study describes the precise spatial and temporal patterns of protein distribution for aggrecan, fibromodulin, cartilage oligomeric matrix protein (COMP) and cartilage matrix protein (CMP) in the developing mouse limb with particular attention to those cells destined to form articular chondrocytes in comparison to those cells destined to form a mineralized tissue and become replaced by bone. Mouse glenohumeral joints from fetal mice (12-18 days post coitus (dpc) to the young adult (37 days after birth) were immunostained with antibodies specific for these molecules. Aggrecan staining defined the general chondrocytic phenotype, whether articular or transient. Fibromodulin was associated with prechondrocytic mesenchymal cells in the interzone prior to joint cavitation and with the mesenchymal cells of the perichondrium or the periosteum encapsulating the joint elements of the maturing and young adult limb. Staining was most intense around developing articular chondrocytes and much less abundant or absent in those differentiating cells along the anlage. CMP showed an almost reciprocal staining pattern to fibromodulin and was not detected in the matrix surrounding articular chondrocytes. COMP was not detected in the cells at the articular surface prior to cavitation but by 18 dpc, as coordinated movement of the mouse forelimb intensifies, staining for COMP was most intense around the maturing articular chondrocytes. These results show that the cells that differentiate into articular chondrocytes elaborate an extracellular matrix distinct from those cells that are destined to form bone. Fibromodulin may function in the early genesis of articular cartilage and COMP may be associated with elaboration of a weight-bearing chondrocyte matrix.     

ADAMTS-12 associates with and degrades cartilage oligomeric matrix protein

Loss of articular cartilage because of extracellular matrix breakdown is the hallmark of arthritis. Degradative fragments of cartilage oligomeric matrix protein (COMP), a prominent noncollagenous matrix component in articular cartilage, have been observed in the cartilage, synovial fluid, and serum of arthritis patients. The molecular mechanism of COMP degradation and the enzyme(s) responsible for it, however, remain largely unknown. ADAMTS-12 (a disintegrin and metalloprotease with thrombospondin motifs) was shown to associate with COMP both in vitro and in vivo. ADAMTS-12 selectively binds to only the epidermal growth factor-like repeat domain of COMP of the four functional domains tested. The four C-terminal TSP-1-like repeats of ADAMTS-12 are shown to be necessary and sufficient for its interaction with COMP. Recombinant ADAMTS-12 is capable of digesting COMP in vitro. The COMP-degrading activity of ADAMTS-12 requires the presence of Zn2+ and appropriate pH (7.5-9.5), and the level of ADAMTS-12 in the cartilage and synovium of patients with both osteoarthritis and rheumatoid arthritis is significantly higher than in normal cartilage and synovium. Together, these findings indicate that ADAMTS-12 is a new COMP-interacting and -degrading enzyme and thus may play an important role in the COMP degradation in the initiation and progression of arthritis.     

The influence of cyclic tension amplitude on chondrocyte matrix synthesis: experimental and finite element analyses

While not generally viewed as physiologically significant in articular cartilage, substantial tension can develop in fibrocartilage structures and in articular cartilage injuries. This study examined how different amplitudes of cyclic tension influence chondrocyte matrix synthesis. Bovine articular chondrocytes seeded in fibrin gels were loaded continuously for 48 hours at 1.0 Hz with displacements of 5%, 10%, or 20%. Protein and proteoglycan synthesis were measured by (3)H-proline and (35)S-sulfate incorporation, respectively. A poroelastic finite element model of the fibrin gel was developed to determine the strain distributions, hydrostatic pressures, and fluid velocities within the constructs at the various levels of displacement. Compared to unloaded controls, 10% and 20% displacements inhibited proteoglycan synthesis to the same extent, while 5% displacement had no effect. Tensile loading did not significantly affect protein synthesis. The finite element model predicted a wide range of strains and fluid velocities within the region of the gel analyzed for matrix synthesis, and the ranges overlapped for the different levels of displacement. These results indicate that the cyclic tension amplitude influences chondrocyte proteoglycan synthesis and that there may be a threshold in the response.     

A mechanical apparatus with microprocessor controlled stress profile for cyclic compression of cultured articular cartilage explants

An apparatus was designed for mechanical compression of cultured articular cartilage explants with acylindrical plain-ended loading head (diameter 2-5 mm) driven by a stepping motor. A load cell under the culture dish was applied for feedback regulation utilizing a microprocessor-based control unit. The operating programs allowed either continuous or cyclic loading, the latter with adjustable loading/resting ratio. The improvements in the present design compared with previously described apparatuses for similar purposes include: (1) the accurately controlled compression by a load cell and a rapid feedback circuit; (2) the wide range of selectable stresses (25 kPa-12.5 MPa) with both continuous and cyclic loading modes; (3) the ability to handle cycles as short as 1 s with 15 ms peak loading phase. Using a 4 s cycle and 0.5 MPa load for 1.5 h resulted in a significantly enhanced incorporation of radiosulphate in cultured bovine articular cartilage explants, suggesting a stimulation of proteoglycan synthesis. Light and scanning electron microscopic examinations revealed a slight depression and superficial alterations in cartilage structure at the impact site following high pressures. We expect that this apparatus will help in revealing how articular cartilage tissue and chondrocytes respond to external mechanical stimuli.     

软骨寡聚基质蛋白在关节炎中的作用

研究表明在骨性关节炎和类风湿性关节炎患者血清中软骨寡聚基质蛋白(cartilage oligomeric matrix protein,COMP)可以在出现关节形态学改变以前就在生化代谢方面表现异常,为早期诊断提供了新思路,其可以作为反映早期关节软骨破坏和骨关节疾病急性活动程度的指标。从而能在病变不可逆发展之前及早防治,逆转或延缓病变进展。COMP是关节炎疾病的诊断与判断疾病进展和缓解潜在生物学标志物。本文就软骨寡聚基质蛋白及其在关节炎中的作用作一综述。     

The distribution patterns of COMP and matrilin-3 in septal,alar and triangular cartilages of the human nose

The biomechanical characteristics of septal cartilage depend strongly on the distinct extracellular matrix of cartilage tissue; therefore, it is essential that the components of this matrix are identified and understood. Cartilage oligomeric matrix protein (COMP) and matrilin-3 are localised in articular cartilage. This study was the first to examine all subtypes of mature human nasal cartilages (alar, triangular and septal) with specific attention to the distribution of COMP and matrilin-3. Three whole fresh-frozen noses from human donors were dissected, and exemplary biopsies were examined using histochemical staining (haematoxylin and eosin and Alcian blue) and immunohistochemistry (collagen II, COMP and matrilin-3). The following three zones within the nasal cartilage were identified: superficial, intermediate and central. COMP was detected as highest in the intermediate zones in all three subtypes of nasal cartilage, whereas matrilin-3 was detected with pericellular deposition mainly within septal cartilage predominantly in the superficial zones. The distinct staining patterns of COMP and matrilin-3 underscore the different functional roles of both proteins in nasal cartilage. According to the literature, COMP might be involved with collagen II in the formation of networks, whereas matrilin-3 is reported to prevent ossification or regulate mechanosensitivity. The predominant staining observed in septal cartilage suggests matrilin-3’s modulatory role because of its presence in the osteochondral junctional zone and given that the biomechanical load in septal cartilage is different from that in alar or triangular cartilage. In conclusion, COMP and matrilin-3 were detected in mature human nasal cartilage but displayed different staining patterns that might be explained by the functional roles of the respective matrix protein; however, further research is necessary to identify and define the functional aspects of this morphological difference.     

Static compression is associated with decreased diffusivity of dextrans in cartilage explants

The chondrocytes of adult articular cartilage rely upon transport phenomena within their avascular extracellular matrix for many biological activities. Therefore, changes in matrix structure which influence cytokine transport parameters may be an important mechanism involved in the chondrocyte response to tissue compression. With this hypothesis in mind, partitioning and diffusion of 3-, 10-, and 40-kDa dextrans conjugated to tetramethylrhodamine, and 430-Da tetramethylrhodamine itself, were measured within statically compressed bovine articular cartilage explants using a novel experimental apparatus and desorption fluorescence method. Partitioning and diffusion were examined as functions of solute molecular weight and matrix proteoglycan density, and diffusion was measured versus static compression up to 35% volumetric strain. In general, partition coefficients and diffusivities were found to decrease with increasing solute molecular weight. In addition, for a given solute, diffusivities decreased significantly with increasing static compression. Results therefore suggest a possible role for transport limitations of relatively large molecular weight solutes within the extracellular matrix in mediating the biological response of chondrocytes to cartilage compression.     

Protein modification by deamidation indicates variations in joint extracellular matrix turnover

As extracellular proteins age, they undergo and accumulate nonenzymatic post-translational modifications that cannot be repaired. We hypothesized that these could be used to systemically monitor loss of extracellular matrix due to chronic arthritic diseases such as osteoarthritis (OA). To test this, we predicted sites of deamidation in cartilage oligomeric matrix protein (COMP) and confirmed, by mass spectroscopy, the presence of deamidated (Asp(64)) and native (Asn(64)) COMP epitopes (mean 0.95% deamidated COMP (D-COMP) relative to native COMP) in cartilage. An Asp(64), D-COMP-specific ELISA was developed using a newly created monoclonal antibody 6-1A12. In a joint replacement study, serum D-COMP (p = 0.017), but not total COMP (p = 0.5), declined significantly after replacement demonstrating a joint tissue source for D-COMP. In analyses of 450 participants from the Johnston County Osteoarthritis Project controlled for age, gender, and race, D-COMP was associated with radiographic hip (p < 0.0001) but not knee (p = 0.95) OA severity. In contrast, total COMP was associated with radiographic knee (p < 0.0001) but not hip (p = 0.47) OA severity. D-COMP was higher in soluble proteins extracted from hip cartilage proximal to OA lesions compared with remote from lesions (p = 0.007) or lesional and remote OA knee (p < 0.01) cartilage. Total COMP in cartilage did not vary by joint site or proximity to the lesion. This study demonstrates the presence of D-COMP in articular cartilage and the systemic circulation, and to our knowledge, it is the first biomarker to show specificity for a particular joint site. We believe that enrichment of deamidated epitope in hip OA cartilage indicates a lesser repair response of hip OA compared with knee OA cartilage.     

Altered integration of matrilin-3 into cartilage extracellular matrix in the absence of collagen IX

The matrilins are a family of four noncollagenous oligomeric extracellular matrix proteins with a modular structure. Matrilins can act as adapters which bridge different macromolecular networks. We therefore investigated the effect of collagen IX deficiency on matrilin-3 integration into cartilage tissues. Mice harboring a deleted Col9a1 gene lack synthesis of a functional protein and produce cartilage fibrils completely devoid of collagen IX. Newborn collagen IX knockout mice exhibited significantly decreased matrilin-3 and cartilage oligomeric matrix protein (COMP) signals, particularly in the cartilage primordium of vertebral bodies and ribs. In the absence of collagen IX, a substantial amount of matrilin-3 is released into the medium of cultured chondrocytes instead of being integrated into the cell layer as in wild-type and COMP-deficient cells. Gene expression of matrilin-3 is not affected in the absence of collagen IX, but protein extraction from cartilage is greatly facilitated. Matrilin-3 interacts with collagen IX-containing cartilage fibrils, while fibrils from collagen IX knockout mice lack matrilin-3, and COMP-deficient fibrils exhibit an intermediate integration. In summary, the integration of matrilin-3 into cartilage fibrils occurs both by a direct interaction with collagen IX and indirectly with COMP serving as an adapter. Matrilin-3 can be considered as an interface component, capable of interconnecting macromolecular networks and mediating interactions between cartilage fibrils and the extrafibrillar matrix.     

Effects of damage in the articular surface on the cartilage response to injurious compression in vitro

Macroscopic structural damage to the cartilage articular surface can occur due to slicing in surgery, cracking in mechanical trauma, or fibrillation in early stage osteoarthrosis. These alterations may render cartilage matrix and chondrocytes susceptible to subsequent mechanical injury and contribute to progression of degenerative disease. To examine this hypothesis, single 300 microm deep vertical slices were introduced across a diameter of the articular surface of osteochondral explant disks on day 6 after dissection. Then a single uniaxial unconfined ramp compression at 7 x 10(-5) or 7 x 10(-2) s(-1) strain rate to a peak stress of 3.5 or 14 MPa was applied on day 13 during which mechanical behavior was monitored. Effects of slices alone and together with compression were measured in terms of explant swelling and cell viability on days 10 and 17. Slicing alone induced tissue swelling without significant cell death, while compression alone induced cell death without significant tissue swelling. Under low strain rate loading, no differences in the response to injurious compression were found between sliced and unsliced explants. Under high strain rate loading, slicing rendered cartilage more easily compressible and appeared to slightly reduce compression-induced cell and matrix injury. Findings highlight microphysical factors important to cartilage mechanical injury, and suggest ways that macroscopic structural damage may accelerate or, in certain cases, possibly slow the progression of cartilage degeneration.     

Dynamic unconfined compression of articular cartilage under a cyclic compressive load

To study the effect of dynamic mechanical force on cartilage metabolism, many investigators have applied a cyclic compressive load to cartilage disc explants in vitro. The most frequently used in vitro testing protocol has been the cyclic unconfined compression of articular cartilage in a bath of culture medium. Cyclic compression has been achieved by applying either a prescribed cyclic displacement or a prescribed cyclic force on a loading platen placed on the top surface of a cylindrical cartilage disc. It was found that the separation of the loading platen from the tissue surface was likely when a prescribed cyclic displacement was applied at a high frequency.The purpose of the present study was to simulate mathematically the dynamic behavior of a cylindrical cartilage disc subjected to cyclic unconfined compression under a dynamic force boundary condition protocol, and to provide a parametric analysis of mechanical deformations within the extracellular matrix. The frequency-dependent dynamic characteristics of dilatation, hydrostatic pressure and interstitial fluid velocity were analyzed over a wide range of loading frequencies without the separation of the loading platen. The result predicted that a cyclic compressive force created an oscillating positive-negative hydrostatic pressure together with a forced circulation of interstitial fluid within the tissue matrix. It was also found that the load partitioning mechanism between the solid and fluid phases was a function of loading frequency. At a relatively high loading frequency, a localized dynamic zone was developed near the peripheral free surface of the cartilage disc, where a large dynamic pressure gradient exists, causing vigorous interstitial fluid flow.     

Static and dynamic compression regulate cartilage metabolism of PRoteoGlycan 4 (PRG4)

The boundary lubrication function of articular cartilage is mediated in part by molecules at the articular surface and in synovial fluid, encoded by Prg4. The objective of this study was to determine whether static and dynamic compression regulate PRG4 biosynthesis by cartilage explants. Articular cartilage disks were harvested to include the articular surface from immature bovines. Some disks were subjected to 24 h (day 1) of loading, followed by 72 h (days 2-4) of free-swelling culture to assess chondrocyte responses following unloading. Loading consisted of 6 or 100 kPa of static compression, with or without superimposed dynamic compression (10 or 300 kPa peak amplitude, 0.01 Hz). Other disks were cultured free-swelling as controls. PRG4 secretion into culture medium was inhibited by all compression protocols during day 1. Following unloading, cartilage previously subjected to dynamic compression to 300 kPa exhibited a rebound effect, secreting more PRG4 than did controls, while cartilage previously subjected to 100 kPa static loading secreted less PRG4. Immunohistochemistry revealed that all compression protocols also affected the number of cells expressing PRG4. The paradigm that mechanical stimuli regulate biosynthesis in cartilage appears operative not only for load bearing matrix constituents, but also for PRG4 molecules mediating lubrication.