Expression of PSACH-associated mutant COMP in tendon fibroblasts leads to increased apoptotic cell death irrespective of the secretory characteristics of mutant COMP.

OBJECTIVE: Pseudoachondroplasia (PSACH) is a dominantly inherited chondrodysplasia associated with mutations of cartilage oligomeric matrix protein (COMP), characterized clinically by disproportionate dwarfism and laxity of joints and ligaments. Studies in chondrocytes and cartilage biopsies suggest that the cartilage disease is caused by retention of mutant COMP in the endoplasmic reticulum of chondrocytes and by disruption of the collagen network of the extracellular matrix. The pathogenesis of the tendon disease remains unclear in the absence of a cell culture model, with available tendon biopsies leading to conflicting results with respect to the intracellular retention of mutant COMP. METHODS: We established a cell culture model using adenoviral gene transfer in tendon fibroblast cultures. We compared the effect of expression of three PSACH-associated COMP mutants and the wildtype protein on COMP secretion, matrix composition and cellular viability. RESULTS: Our results show that mutants D475N and D469Delta are retained within the endoplasmic reticulum of tendon cells similar to what is known from chondrocytes, whereas mutant H587R is secreted like wildtype COMP. In spite of this difference, the collagen I matrix formed in culture appears disturbed for all three mutants. All COMP-mutants induce apoptotic cell death irrespective of their differing secretion patterns. CONCLUSION: Pathogenic pathways leading to tendon disease in humans appear to be heterogeneous between different COMP mutants.     

Morphological studies of the epiphyseal growth zone in the brachymorphic (bm/bm) mouse

Light microscopy, including immunohistochemical techniques, and electron microscopy were performed on epiphyseal growth cartilage from brachymorphic (bm/bm) mice and age-matched phenotypically normal siblings aged 5, 16 and 25 days. In the bm/bm mice light microscopy showed a disturbed columnar arrangement and numerous chondrocytes with pronounced regressive changes. The normal development of proliferative cells into hypertrophic cells was halted and thus only a rather small and ill-defined hypertrophic zone was seen. The calcifying zone was irregular and the normal lacunae were replaced by a densely staining matrix. Using immunofluorescence techniques, the presence of considerable amounts of both type II and type V collagen was demonstrated in the bm/bm mice, while the cartilage from controls contained only type II. Ultrastructurally the lacunar matrix contained bundles of fine fibrils without the typical collagen periodicity which might indicate synthesis of a defective procollagen. Our observations together with the previously demonstrated deficiency of 3'-phosphoadenosine 5'-phosphosulphate, illustrate the complexity of the growth cartilage disturbance in the bm/bm mouse. Most of our findings are at variance with those described in the literature and possible pathogenetic mechanisms for the observed alterations in the growth cartilage are discussed.     

Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix

Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro.     

Formation of the sphenomandibular ligament by Meckel's cartilage in the mouse: possible involvement of epidermal growth factor as revealed by studies in vivo and in vitro

In mammals, the midportion of the soft tissue of Meckel's cartilage at the degenerating stage forms a ligament known as the sphenomandibular ligament. To clarify the mechanism of formation of this ligament by Meckel's cartilage in mouse, we examined the effects of epidermal growth factor (EGF) on the chondrocytes in terms of the proliferation and differentiation of cells and calcification of the matrix in vivo and in vitro. The effects of EGF were examined by immunohistochemical staining, with EGF-soaked beads, by electron microscopy, and by general histochemical analysis of proteoglycans and calcification. Analysis of labeling with bromodeoxyuridine (BrdU) and the rate of cell growth revealed that EGF enhanced DNA synthesis and the proliferation of Meckel's chondrocytes. Histological findings in organ culture and in cell culture, with and without the application of EGF-soaked beads, revealed that EGF inhibited the differentiation of cells to chondrocytes and induced phenotypic changes in fibroblastic cells. The inhibition of alkaline phosphatase activity that resulted from exposure to EGF was accompanied by prolonged calcification of the matrix. Whole-mount staining revealed that subcutaneous injection of EGF enhanced the disappearance of Meckel's cartilage. Our results suggest a possible mechanism whereby the midportion of Meckel's cartilage remains uncalcified and is rapidly transformed into the sphenomandibular ligament.     

The immunohistochemical localization of superoxide dismutase activity in the avian epithelial growth plate

Summary Superoxide dismutase (SOD) is a scavenger enzyme which catalyses the dismutation (reduction—oxidation) of the superoxide anion (O₂ ^–), a toxic free radical generated during normal cellular respiration. Light microscopy employing immunohistochemistry was utilized for localizing SOD activity in the chick epiphyseal cartilage. Antibodies to mammalian liver CuZn—SOD were prepared and the avidin—biotin—peroxidase technique (ABC complex) was utilized to localize activity for this enzyme in the growth plate cartilage. The localization of enzyme activity varied in accordance with the characteristic zonation pattern of the growth plate (zone of proliferation, zone of maturation, zone of cell hypertrophy and zone of matrix calcification). In the upper regions of the epiphyseal cartilage (the zones of proliferation and maturation), where the vascularity is poor and the oxygen tension low, SOD activity was localized within the chondrocytes. No extracellular activity was observed. However, in the lower regions of the growth plate (the zones of cell hypertrophy and matrix calcification), where both the vascularity and the oxygen tensions are increased, SOD activity was intense in both the chondrocytes and the surrounding extracellular matrix. Thus, the distribution of SOD enzyme activity in this tissue seems to vary in accordance with the level of oxygen present. The significance of the extracellular SOD activity, seen in the lower aspects of the growth plate cartilage, may indicate the sensitivity of matrix components, especially collagen, to toxic free radicals such as the superoxide anion.     

Response of zonal chondrocytes to extracellular matrix-hydrogels

We investigated the biological response of chondrocytes isolated from different zones of articular cartilage and their cellular behaviors in poly (ethylene glycol)-based (PEG) hydrogels containing exogenous type I collagen, hyaluronic acid (HA), or chondroitin sulfate (CS). The cellular morphology was strongly dependent on the extracellular matrix component of hydrogels. Additionally, the exogenous extracellular microenvironment affected matrix production and cartilage specific gene expression of chondrocytes from different zones. CS-based hydrogels showed the strongest response in terms of gene expression and matrix accumulation for both superficial and deep zone chondrocytes, but HA and type I collagen-based hydrogels demonstrated zonal-dependent cellular responses.     

The biology of cartilage. I. Invertebrate cartilages: Limulus gill cartilage

The endoskeletal structure supporting the gill-books of Limulus polyphemus has been investigated by means of light and electron microscopy, chemical analysis and x-ray diffraction. This tissue is a cartilage which has significant correspondences with both vertebrate cartilage and plant tissues. Morphologically, the Limulus cartilage resembles certain cellular vertebrate cartilages with relatively scant matrix, and also certain plant parenchyme, collenchyme and sclerenchyme tissues. Of particular interest, was the observation that during cytoplasmic division, a phragmasome-like structure appears between the daughter cells of the dividing gill cartilage cells. This phragmasome-like structure appears to be a precursor of new matrix (cell-wall) formation between the young chondrocytes, in much the same fashion as its counterpart in plant tissues. Perichondrial cells and underlying chondrocytes contain lipid droplets, abundant glycogen and ribosomes, as do corresponding vertebrate cartilage cells. In some of the Limulus cells, glycogen and ribosomes appear to be admixed with lipid, forming aggregates in which all three materials are in intimate intraparticulate relationship. During molting, the number of ribosomes seen in chondrocytes increases. The tissue contains both hydroxyproline and hydroxylysine, and gives a weak x-ray diffraction pattern.     

Expression of osteopontin in Meckel’s cartilage cells during phenotypic transdifferentiation in vitro, as detected by in situ hybridization and immunocytochemical analysis

 The localization of osteopontin (OP) was examined in Meckel’s cartilage cells that bipotentially expressed cartilage and bone phenotypes during cellular transformation in vitro. Cultured cells were analyzed by in situ hybridization, immunostaining followed by light and electron microscopy, electron microscopy, and electron probe microanalysis. The combination of ultrastructural analysis and immunoperoxidase staining indicated that OP-synthesizing cells were cells that were autonomously undergoing a change from chondrocytes to bone-forming cells at the top of nodules. Double immunofluorescence staining of 2-week-old cultures revealed that OP was first synthesized by chondrocytic cells at the top of nodules. After further time in culture, the distribution of OP expanded from the central toward the peripheral regions of the nodules. Electron probe microanalysis revealed that the localization of OP was associated with matrices of calcified cartilage and osteoid nodules that contained calcium and phosphorus. Immunoperoxidase electron microscopy revealed that, in addition to the intracellular immunoreactivity in chondrocytes and small round cells that were undergoing transformation, matrix foci of calcospherites and matrix vesicles, in particular, included growing crystals that were immunopositive for OP. An intense signal due to mRNA for OP in 3-week-old cultures was detected in nodule-forming round cells, while fibroblastic cells, spreading in a monolayer over the periphery of nodules, were only weakly labeled. These findings indicate that OP might be expressed sequentially by chondrocytes and by cells that are transdifferentiating further and exhibit an osteocytic phenotype, and moreover, that expression of OP is closely associated with calcifying foci in the extracellular matrix. Accepted: 26 May 1998     

The C5 domain of Col6A3 is cleaved off from the Col6 fibrils immediately after secretion.

In articular cartilage, type VI collagen is concentrated in the pericellular matrix compartment. During protein synthesis and processing at least the alpha3(VI) chain undergoes significant posttranslational modification and cleavage. In this study, we investigated the processing of type VI collagen in articular cartilage. Immunostaining with a specific polyclonal antiserum against the C5 domain of alpha3(VI) showed strong cellular staining seen in nearly all chondrocytes of articular cartilage. Confocal laser-scanning microscopy and immunoelectron microscopy allowed localization of this staining mainly to the cytoplasm and the immediate pericellular matrix. Double-labeling experiments showed a narrow overlap of the C5 domain and the pericellular mature type VI collagen. Our results suggest that at least in human adult articular cartilage the C5 domain of alpha3(VI) collagen is synthesized and initially incorporated into the newly formed type VI collagen fibrils, but immediately after secretion is cut off and is not present in the mature pericellular type VI matrix of articular cartilage.     

Type II achondrogenesis-hypochondrogenesis: morphologic and immunohistopathologic studies.

A 32-wk-gestation female with type II achondrogenesis-hypochondrogenesis has been studied. The clinical features were typical, and radiographs revealed short ribs, hypoplastic ilia, absence of ossification of sacrum, pubis, ischia, tali, calcanei, and many vertebral bodies; the long bones were short with mild metaphyseal flaring. The femoral cylinder index was 6.3. Comparison with previous cases placed the patient toward the mild end of the achondrogenesis-hypochondrogenesis spectrum (Whitley-Gorlin prototype IV). Light microscopy revealed hypercellular cartilage with decreased matrix traversed by numerous fibrous vascular canals. The growth plate was markedly abnormal. Ultrastructural studies revealed prominently dilated rough endoplasmic reticulum containing a fine granular material with occasional fibrils in all chondrocytes. Immunohistologic studies indicated irregular large areas of cartilage matrix staining with monoclonal antibody to human type III collagen. The relative intensity of matrix staining for type II collagen appeared diminished. More striking, however, were intense focal accumulations of type II collagen within small rounded perinuclear structures of most chondrocytes but not other cell types. These results strongly suggest intracellular retention of type II collagen within vacuolar structures, probably within the dilated rough endoplasmic reticulum observed in all chondrocytes by electron microscopy (EM), and imply the presence of an abnormal, poorly secreted type II collagen molecule. Biochemical studies (see companion paper) suggest that this patient had a new dominant lethal disorder caused by a structural abnormality of type II collagen.     

Further evidence for secretion of matrix metalloproteinase-1 by Meckel's chondrocytes during degradation of the extracellular matrix

We examined the possibility that chondrocytes in Meckel's cartilage might secrete matrix metalloproteinase-1 (MMP-1) during degradation of the extracellular matrix. Evidence for the secretion of MMP-1 was obtained by immunohistochemical staining and immunoelectron microscopy, in addition to general histochemical staining for proteoglycans. Not only staining with toluidine blue and alcian blue but also immunostaining for chondroitin sulfate proteoglycan (CSPG) revealed that levels of glycoproteins are rapidly reduced at the late stage of degradation. MMP-1 was detected continuously in cells from chondrocytes at the early stage to hypertrophic chondrocytes at the late stage. Immunoelectron microscopy revealed that the deposition of colloidal golds shifted from an intracellular localization in chondrocytes at the early stage to pericellular spaces at the late stage. The localization of tissue inhibitor of the metalloproteinase-1 (TIMP-1) at the early stage was similar to that of MMP-1, but the level of TIMP-1 decreased significantly in hypertrophic cartilage. These findings suggest that MMP-1 is present continuously in Meckel's chondrocytes but that the active form, which degrades the extracellular matrix, is the MMP-1 that accumulates in the pericellular spaces around hypertrophic chondrocytes.     

In situ localization of lectin-binding glycoconjugates in the matrix of growth-plate cartilage

In the distal hypertrophic zone of growth-plate cartilage, the pericellular matrix surrounding individual chondrocytes and the territorial matrix uniting chondrocytes into columnar groups are invaded by metaphyseal endothelial cells prior to osteogenesis. In the present study, lectin-binding glycoconjugates were analyzed in these two matrix compartments of growth-plate cartilage from Yucatan swine. Nine lectin-fluorescein conjugates were tested by a postembedment method on 1-micron-thick, nondecalcified, Epon-embedded sections. Chondrocytes in all cellular zones were surrounded by a pericellular matrix which showed positive binding for peanut agglutinin (PNA), ricin agglutinin (RCA-I), and soybean agglutinin (SBA). Binding by these lectins was sensitive to digestion with hyaluronidase, chondroitinase, and trypsin. Pericellular glyconconjugtes that bind RCA-I and concanvalin A (CONA) after periodic acid oxidation, and which were sensitive to trypsin but not to chondroitinase or hyaluronidase, were present in the hypertrophic cell zone. Within the territorial matrix, binding of lectins specific for galactose, N-acetylgalactosamine, and fucose showed gradients of intensity which became maximal at the last transverse septum. Lectin-binding histochemistry more precisely differentiated the microheterogeneity of glycoconjugate distribution within these two matrix compartments than has been possible with other histochemical techniques. Lectin-binding affinity is a potentially useful technique by which to isolate cartilage matrix macromolecules unique to specific cellular zones of the growth plate.     

Collagen/annexin V interactions regulate chondrocyte mineralization

Physiological mineralization in growth plate cartilage is highly regulated and restricted to terminally differentiated chondrocytes. Because mineralization occurs in the extracellular matrix, we asked whether major extracellular matrix components (collagens) of growth plate cartilage are directly involved in regulating the mineralization process. Our findings show that types II and X collagen interacted with cell surface-expressed annexin V. These interactions led to a stimulation of annexin V-mediated Ca(2+) influx resulting in an increased intracellular Ca(2+) concentration, [Ca(2+)](i), and ultimately increased alkaline phosphatase activity and mineralization of growth plate chondrocytes. Consequently, stimulation of these interactions (ascorbate to stimulate collagen synthesis, culturing cells on type II collagen-coated dishes, or overexpression of full-length annexin V) resulted in increase of [Ca(2+)](i), alkaline phosphatase activity, and mineralization of growth plate chondrocytes, whereas inhibition of these interactions (3,4-dehydro-l-proline to inhibit collagen secretion, K-201, a specific annexin channel blocker, overexpression of N terminus-deleted mutant annexin V that does not bind to type II collagen and shows reduced Ca(2+) channel activities) decreased [Ca(2+)](i), alkaline phosphatase activity, and mineralization. In conclusion, the interactions between collagen and annexin V regulate mineralization of growth plate cartilage. Because annexin V is up-regulated during pathological mineralization events of articular cartilage, it is possible that these interactions also regulate pathological mineralization.     

Genetically engineered chondrocytes overexpressing elastin improve cell retention and chondrogenesis in a three-dimensional GelMA culture system

Elastic cartilage possesses many elastic fibers and has a high degree of elasticity. However, insufficient elastic fiber production remains unsolved in elastic cartilage tissue engineering. Exogenous elastin is difficult to degrade and violates cell proliferation and migration during cartilage regeneration. Moreover, exogenous elastic fibers are difficult to assemble with endogenous extracellular matrix components. We produced genetically engineered chondrocytes overexpressing elastin to boost endogenous elastic fiber production. After identifying that genetic manipulation hardly impacted the cell viability and chondrogenesis of chondrocytes, we co-cultured genetically engineered chondrocytes with untreated chondrocytes in a three-dimensional gelatin methacryloyl (GelMA) system. In vitro study showed that the co-culture system produced more elastic fibers and increased cell retention, resulting in strengthened mechanics than the control system with untreated chondrocytes. Moreover, in vivo implantation revealed that the co-culture GelMA system greatly resisted host tissue invasion by promoting elastic fiber production and cartilage tissue regeneration compared with the control system. In summary, our study indicated that genetically engineered chondrocytes overexpressing elastin are efficient and safe for promoting elastic fiber production and cartilage regeneration in elastic cartilage tissue engineering.     

Association of an extracellular protein (chondrocalcin) with the calcification of cartilage in endochondral bone formation

We examined bovine fetal epiphyseal and growth plate cartilages by immunofluorescence microscopy and immunoelectron microscopy using monospecific antibodies to a newly discovered cartilage-matrix calcium-binding protein that we now call chondrocalcin. Chondrocalcin was evenly distributed at relatively low concentration in resting fetal epiphyseal cartilage. In growth plate cartilage, it was absent from the extracellular matrix in the zone of proliferating chondrocytes but was present in intracellular vacuoles in proliferating, maturing and upper hypertrophic chondrocytes. The protein then disappeared from the lower hypertrophic chondrocytes and appeared in the adjoining extracellular matrix, where it was selectively concentrated in the longitudinal septa in precisely the same location where amorphous mineral was deposited in large amounts as demonstrated by von Kossa staining and electron microscopy. Mineral then spread out from these "nucleation sites" to occupy much of the surrounding matrix. Matrix vesicles were identified in this calcifying matrix but they bore no observable morphological relationship to these major sites of calcification where chondrocalcin was concentrated. Since chondrocalcin is a calcium-binding protein and has a strong affinity for hydroxyapatite, these observations suggest that chondrocalcin may play a fundamental role in the creation of nucleation sites for the calcification of cartilage matrix in endochondral bone formation.     

COMP mutations, chondrocyte function and cartilage matrix

Cartilage oligomeric matrix protein (COMP) is a large extracellular pentameric glycoprotein found in the territorial matrix surrounding chondrocytes. More than 60 unique COMP mutations have been identified as causing two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED/EDM1). Recent studies demonstrate that calcium-binding and calcium induced protein folding differ between wild type and mutant COMP proteins and abnormal processing of the mutant COMP protein causes the characteristic large lamellar appearing rough endoplasimic reticulum (rER) cisternae phenotype observed in PSACH and EDMI growth plate chondrocytes. To understand the cellular events leading to this intracellular phenotype, PSACH chondrocytes with a G427E, D469del and D511Y mutations were grown in 3-D culture to produce cartilage nodules. Each nodule was assessed for the appearance and accumulation of cartilage-specific proteins within the rER and for matrix protein synthesis. All three COMP mutations were associated with accumulation of COMP in the rER cisternae by 4 weeks in culture, and by 8 weeks the majority of chondrocytes had the characteristic cellular phenotype. Mutations in COMP also affect the secretion of type IX collagen and matrilin-3 (MATN3) but not the secretion of aggrecan and type II collagen. COMP, type IX collagen and MATN3 were dramatically reduced in the PSACH matrices, and the distribution of these proteins in the matrix was diffuse. Ultrastructural analysis shows that the type II collagen present in the PSACH matrix does not form organized fibril bundles and, overall, the matrix is disorganized. The combined absence of COMP, type IX collagen and MATN3 causes dramatic changes in the matrix and suggests that these proteins play important roles in matrix assembly.     

Scanning electrochemical microscopy as a local probe of oxygen permeability in cartilage

The use of scanning electrochemical microscopy, a high-resolution chemical imaging technique, to probe the distribution and mobility of solutes in articular cartilage is described. In this application, a mobile ultramicroelectrode is positioned close ( approximately 1 microm) to the cartilage sample surface, which has been equilibrated in a bathing solution containing the solute of interest. The solute is electrolyzed at a diffusion-limited rate, and the current response measured as the ultramicroelectrode is scanned across the sample surface. The topography of the samples was determined using Ru(CN)(6)(4-), a solute to which the cartilage matrix was impermeable. This revealed a number of pit-like depressions corresponding to the distribution of chondrocytes, which were also observed by atomic force and light microscopy. Subsequent imaging of the same area of the cartilage sample for the diffusion-limited reduction of oxygen indicated enhanced, but heterogeneous, permeability of oxygen across the cartilage surface. In particular, areas of high permeability were observed in the cellular and pericellular regions. This is the first time that inhomogeneities in the permeability of cartilage toward simple solutes, such as oxygen, have been observed on a micrometer scale.     

Solute transport in growth plate cartilage: in vitro and in vivo

Bone elongation originates from cartilaginous discs (growth plates) at both ends of a growing bone. Here chondrocytes proliferate and subsequently enlarge (hypertrophy), laying down a matrix that serves as the scaffolding for subsequent bone matrix deposition. Because cartilage is generally avascular, all nutrients, oxygen, signaling molecules, and waste must be transported relatively long distances through the tissue for it to survive and function. Here we examine the transport properties of growth plate cartilage. Ex vivo, fluorescence photobleaching recovery methods are used in tissue explants. In vivo, multiphoton microscopy is used to image through an intact perichondrium and into the cartilage of anesthetized mice. Systemically introduced fluorescent tracers are monitored directly as they move from the vasculature into the cartilage. We demonstrate the existence of a relatively permissive region at the midplane of the growth plate, where chondrocytes transition from late proliferative to early hypertrophic stages and where paracrine communication is known to occur between chondrocytes and cells in the surrounding perichondrium. Transport in the living mouse is also significantly affected by fluid flow from the two chondro-osseus junctions, presumably resulting from a pressure difference between the bone vasculature and the cartilage.     

Selective intracellular retention of extracellular matrix proteins and chaperones associated with pseudoachondroplasia.

Mutations in the cartilage oligomeric matrix protein (COMP) gene result in pseudoachondroplasia (PSACH), which is a chondrodysplasia characterized by early-onset osteoarthritis and short stature. COMP is a secreted pentameric glycoprotein that belongs to the thrombospondin family of proteins. We have identified a novel missense mutation which substitutes a glycine for an aspartic acid residue in the thrombospondin (TSP) type 3 calcium-binding domain of COMP in a patient diagnosed with PSACH. Immunohistochemistry and immunoelectron microscopy both show abnormal retention of COMP within characteristically enlarged rER inclusions of PSACH chondrocytes, as well as retention of fibromodulin, decorin and types IX, XI and XII collagen. Aggrecan and types II and VI collagen were not retained intracellularly within the same cells. In addition to selective extracellular matrix components, the chaperones HSP47, protein disulfide isomerase (PDI) and calnexin were localized at elevated levels within the rER vesicles of PSACH chondrocytes, suggesting that they may play a role in the cellular retention of mutant COMP molecules. Whether the aberrant rER inclusions in PSACH chondrocytes are a direct consequence of chaperone-mediated retention of mutant COMP or are otherwise due to selective intracellular protein interactions, which may in turn lead to aggregation within the rER, is unclear. However, our data demonstrate that retention of mutant COMP molecules results in the selective retention of ECM molecules and molecular chaperones, indicating the existence of distinct secretory pathways or ER-sorting mechanisms for matrix molecules, a process mediated by their association with various molecular chaperones.