Type X collagen synthesis in human osteoarthritic cartilage. Indication of chondrocyte hypertrophy.

OBJECTIVE: To investigate the appearance of hypertrophic chondrocytes in osteoarthritic (OA) cartilage, using type X collagen as a specific marker. METHODS. The biosynthesis of type X collagen was examined by metabolic labeling of freshly isolated articular chondrocytes with 3H-proline, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the synthesized collagens. Extracellular deposition of types X and II collagen was analyzed immunohistochemically. RESULTS. Immunostaining revealed an irregular distribution of type X collagen, which was localized around chondrocyte clusters in fibrillated OA cartilage, but was absent from the noncalcified region of normal articular cartilage. Freshly isolated OA chondrocytes synthesized predominantly type X collagen, while control chondrocytes synthesized mostly type II collagen. CONCLUSION. Our findings indicate focal premature chondrocyte differentiation to hypertrophic cells in OA cartilage.     

Interactions of synovial fluid immunoglobulins with chondrocytes

Objective. To study the interaction of synovial fluid (SF) immunoglobulins with living chondrocytes, and to evaluate the relative contribution of type II collagen (CII) antibodies. Methods. SF of patients with rheumatoid arthritis (RA), osteoarthritis (OA), and gout were incubated with isolated bovine articular chondrocytes. Ig binding was measured by flow cytometry and by quantitation with ¹²⁵I-labeled anti-IgG and anti-IgM. Complement-dependent cytotoxicity was determined by ⁵¹Cr release. Immunoglobulin binding and cytotoxicity were compared between chondrocytes obtained from the superficial and from the deep cartilage zones. Results. Significantly greater IgG and IgM binding was found with RA SF compared with OA or gout SF. Chondrocytes bound more Ig than did fibroblasts. The relative contribution of anti-CII antibodies to Ig binding was studied following absorption of the SF with bovine CII, and by incubation with bacterial collagenase-treated chondrocytes. There was a small but significant reduction in IgG and IgM binding with SF samples that were positive for anti-CII. RA SF exhibited modest, but significantly greater complement-dependent cytotoxicity than OA SF. Gel chromatography fractionation indicated that IgM antibodies were responsible for the cytotoxic activity. Additional studies showed that SF IgM antibodies bound preferentially to, and killed chondrocytes obtained from, the superficial layers of cartilage. Conclusion. Anti-CII antibodies contained in RA SF represent one of many antibody specificities reacting with chondrocyte membrane antigens. Chondrocyte-reactive SF antibodies may play an important pathogenic role in the processes leading to irreversible cartilage damage in RA. These deleterious effects appear to be exerted particularly on chondrocytes located near the articular surface of cartilage.     

Parathyroid hormone 1–34 inhibits terminal differentiation of human articular chondrocytes and osteoarthritis progression in rats

Objective

Parathyroid hormone 1–34 (PTH[1–34]), a parathyroid hormone analog, shares the same receptor, PTH receptor 1, with parathyroid hormone–related peptide (PTHrP). This study was undertaken to address the hypothesis that PTH(1–34) inhibits terminal differentiation of articular chondrocytes and in turn suppresses the progression of osteoarthritis (OA).

Methods

We studied the effect of PTH(1–34) on human articular chondrocytes with azacytidine (azaC)–induced terminal differentiation in vitro and on papain‐induced OA in the knee joints of rats. In the in vitro study, we measured the levels of messenger RNA for SOX9, aggrecan, type II collagen, type X collagen, alkaline phosphatase (AP), Indian hedgehog (IHH), Bcl‐2, and Bax by real‐time polymerase chain reaction, levels of glycosaminoglycan (GAG) by dimethylmethylene blue assay, and rate of apoptosis by TUNEL staining. In the in vivo study, we evaluated the histologic changes in GAG, type II collagen, type X collagen, and chondrocyte apoptosis in the articular cartilage of rat knees.

Results

AzaC induced terminal differentiation of human chondrocytes, including down‐regulation of aggrecan, type II collagen, and GAG and up‐regulation of type X collagen, alkaline phosphatase, and IHH. Apoptosis was reversed by 3–10 days of treatment with 10 nM PTH(1–34). SOX9 expression was not changed by either azaC or PTH(1–34) treatment. Bcl‐2 and Bax were up‐regulated on day 10 and day 14, respectively, after azaC induction of terminal differentiation, but PTH(1–34) treatment did not reverse this effect. Furthermore, PTH(1–34) treatment reversed papain‐induced OA changes (decreasing GAG and type II collagen, and increasing type X collagen and chondrocyte apoptosis) in the knee joints of rats.

Conclusion

Our findings indicate that PTH(1–34) inhibits the terminal differentiation of human articular chondrocytes in vitro and inhibits progression of OA in rats in vivo, and may be used to treat OA.

     

Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9

OBJECTIVE: Human osteoarthritis (OA) is characterized by a pathologic shift in articular cartilage homeostasis toward the progressive loss of extracellular matrix (ECM). The purpose of this study was to investigate the ability of rAAV-mediated SOX9 overexpression to restore major ECM components in human OA articular cartilage. METHODS: We monitored the synthesis and content of proteoglycans and type II collagen in 3-dimensional cultures of human normal and OA articular chondrocytes and in explant cultures of human normal and OA articular cartilage following direct application of a recombinant adeno-associated virus (rAAV) SOX9 vector in vitro and in situ. We also analyzed the effects of this treatment on cell proliferation in these systems. RESULTS: Following SOX9 gene transfer, expression levels of proteoglycans and type II collagen increased over time in normal and OA articular chondrocytes in vitro. In situ, overexpression of SOX9 in normal and OA articular cartilage stimulated proteoglycan and type II collagen synthesis in a dose-dependent manner. These effects were not associated with changes in chondrocyte proliferation. Notably, expression of the 2 principal matrix components could be restored in OA articular cartilage to levels similar to those in normal cartilage. CONCLUSION: These data support the concept of using direct, rAAV-mediated transfer of chondrogenic genes to articular cartilage for the treatment of OA in humans.     

Chondrocyte AMP‐activated protein kinase activity suppresses matrix degradation responses to proinflammatory cytokines interleukin‐1β and tumor necrosis factor α

Objective

Interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα) stimulate chondrocyte matrix catabolic responses, thereby compromising cartilage homeostasis in osteoarthritis (OA). AMP‐activated protein kinase (AMPK), which regulates energy homeostasis and cellular metabolism, also exerts antiinflammatory effects in multiple tissues. This study was undertaken to test the hypothesis that AMPK activity limits chondrocyte matrix catabolic responses to IL‐1β and TNFα.

Methods

Expression of AMPK subunits was examined, and AMPKα activity was ascertained by the phosphorylation status of AMPKα Thr¹⁷² in human knee articular chondrocytes and cartilage by Western blotting and immunohistochemistry, respectively. Procatabolic responses to IL‐1β and TNFα, such as release of glycosaminoglycan, nitric oxide, and matrix metalloproteinases 3 and 13 were determined by dimethylmethylene blue assay, Griess reaction, and Western blotting, respectively, in cartilage explants and chondrocytes with and without knockdown of AMPKα by small interfering RNA.

Results

Normal human knee articular chondrocytes expressed AMPKα1, α2, β1, β2, and γ1 subunits. AMPK activity was constitutively present in normal articular chondrocytes and cartilage, but decreased in OA articular chondrocytes and cartilage and in normal chondrocytes treated with IL‐1β and TNFα. Knockdown of AMPKα resulted in enhanced catabolic responses to IL‐1β and TNFα in chondrocytes. Moreover, AMPK activators suppressed cartilage/chondrocyte procatabolic responses to IL‐1β and TNFα and the capacity of TNFα and CXCL8 (IL‐8) to induce type X collagen expression.

Conclusion

Our findings indicate that AMPK activity is reduced in OA cartilage and in chondrocytes following treatment with IL‐1β or TNFα. AMPK activators attenuate dephosphorylation of AMPKα and procatabolic responses in chondrocytes induced by these cytokines. These observations suggest that maintenance of AMPK activity supports cartilage homeostasis by protecting cartilage matrix from inflammation‐induced degradation.

     

Chondrokalzinose

Calcium pyrophosphate dihydrate (CPPD) crystals are known to cause acute attacks of pseudogout in joints but crystal deposition has also been reported to be associated with osteoarthritis (OA). Aside from CPPD crystals, basic calcium phosphates (BCPs), consisting of carbonate-substituted hydroxyapatite (HA), tricalcium phosphate and octacalcium phosphate, have been found in synovial fluid, synovium and cartilage of patients with OA. Although CPPD crystals have been found to be associated with OA and are an important factor in joint disease, this has also recently been associated with a genetic defect. However, according to the most recent findings, the association of BCP crystals, such as apatite with OA is much stronger, as their presence significantly correlates with the severity of cartilage degeneration. Identification of BCP crystals in OA joints remains problematic due to a lack of simple and reliable methods of detection. The clinical and pathological relevance of cartilage mineralization in patients with OA is not completely understood. It is well established that mineralization of articular cartilage is often found close to hypertrophic chondrocytes. A significant correlation between the expression of type X collagen, a marker for chondrocyte hypertrophy and cartilage mineralization was observed. In the process of endochondral ossification, the link between hypertrophy and matrix mineralization is particularly well described. Hypertrophic chondrocytes in OA cartilage and at the growth line share certain features, not only hypertrophy but also a capability to mineralize the matrix. Recent data indicate that chondrocyte hypertrophy is a key factor in articular cartilage mineralization strongly linked to OA and does not characterize a specific subset of OA patients, which has important consequences for therapeutic strategies for OA.     

Copper modulation of extracellular matrix synthesis by human articular chondrocytes

OBJECTIVE: The effects of Cu2+ on human articular chondrocytes, arising from both N (normal) and OA (osteoarthritic) cartilage, were investigated "in vitro". METHODS: Chondrocytes, cultured in high density, were incubated with copper chloride (0.01-0.25 microM/mL). Proteoglycan and collagen were assessed by incorporation of [35S]-Sulfate and [3H]-Proline. SDS-PAGE analysis was performed to quantify the ratio of type II to type I collagen. RESULTS: Cu2+ neither increased proteoglycan synthesis by chondrocytes. of origin N or OA, nor influenced their proliferation rate. Collagen synthesis was increased. This effect is time and concentration dependant: in cultures treated for 12 days, collagen synthesis stimulation was +20% and +26% (P < 0.02) in N and OA cultures respectively, the ratio of type II to type I collagen was slightly increased. This effect was more obvious in OA cell lines than in N ones. CONCLUSION: The observations suggest that Cu2+ upregulates collagen anabolism in human articular chondrocytes.     

FAS/FAS ligand expression and induction of apoptosis in chondrocytes

Objective. To examine the expression of Fas/Fas ligand and the role of this ligand/receptor interaction in the regulation of apoptosis in normal human articular chondrocytes and in osteoarthritis (OA) cartilage. Methods. Normal and OA human knee cartilage and cells isolated from these tissues were tested for Fas expression by flow cytometry. Induction of apoptosis by antibody to Fas was analyzed by DAPI staining and electron microscopy. Results. Treatment of freshly isolated normal human articular chondrocytes with an agonistic Fas antibody induced apoptosis in a subpopulation (-20%) of the cells. Apoptosis induced by anti-Fas was not dependent on nitric oxide (NO), and anti-Fas also did not induce NO production. Analysis of isolated cells demonstrated similar levels of Fas expression on normal and OA chondrocytes (28% and 32%, respectively). In normal articular cartilage, Fas-positive cells were located mainly in the superficial and midzones. In contrast, in fibrillated OA cartilage, surface layers were partially absent and Fas-expressing cells were also detected in the deeper layers. Fas ligand messenger RNA was not detectable in resting or activated normal or OA chondrocytes. Analysis by electron microscopy showed the nuclear and cytoplasmic changes typical of apoptosis in cultures treated with antibody to Fas. Conclusion. A subpopulation of chondrocytes expresses Fas and is susceptible to Fas-induced apoptosis. Fas-mediated chondrocyte apoptosis may contribute to cartilage degradation in arthritis.     

Formation of nodular structures resembling mature articular cartilage in long–term primary cultures of human fetal epiphyseal chondrocytes on a hydrogel substrate

Objective. To establish long–term cultures of human fetal epiphyseal chondrocytes under conditions that allow the preservation of a cartilage–specific phenotype. Methods. Chondrocytes isolated from 20—24–week human fetal epiphyseal cartilage were cultured for up to 180 days on plastic dishes previously coated with the hydrogel, poly–(2–hydroxyethyl methacrylate). Morphologic, ultrastructural, and biochemical characteristics of the cultures were examined at various intervals, and the expression of genes encoding types I, II, and IX collagen and aggrecan core protein was determined by Northern hybridizations of total cellular RNA with human–specific complementary DNAs. Results. Human fetal epiphyseal chondrocytes cultured for 180 days under conditions that prevented their attachment to the underlying substratum formed nodular structures with morphologic and structural characteristics resembling mature articular cartilage. The cells in the center of the nodules remained spherical and were surrounded by an abundant cartilaginous extracellular matrix, as evidenced by histochemical and ultrastructural examinations. The cells in the periphery of the nodules acquired a discoid morphology and were surrounded by a sparse extracellular matrix. Biosyn–thetic studies demonstrated the maintenance of a cartilagespecific phenotype throughout the 180 days of culture, with the production of aggrecan and types II, IX, and XI collagens but not type I collagen. Northern hybridizations showed high levels of messenger RNAs (mRNAs) for aggrecan core protein, type II procollagen, and type IX collagen, but type I procollagen mRNA was not detectable even at 180 days of culture. Conclusion. The human chondrocyte culture system described here allows the maintenance of a chondrocyte–specific phenotype for prolonged periods (up to 180 days). The long–term chondrocyte cultures formed nodular structures that resemble mature articular cartilage morphologically, ultrastructurally, biosynthetically, and in the pattern of cartilage–specific gene expression.     

Suppression of cartilage matrix gene expression in upper zone chondrocytes of osteoarthritic cartilage

Objective. To evaluate the anabolic activity of osteoarthritic chondrocytes in situ by investigating the messenger RNA (mRNA) expression of 3 major cartilage components, type II collagen, aggrecan, and link protein. Methods. In situ hybridization experiments and histochemical analysis for proteoglycan content were performed on parallel sections of normal and osteoarthritic (OA) cartilage specimens. Results. Most chondrocytes in the deeper zones of OA cartilage showed an increase in mRNA expression, in particular, of type II collagen and to a lesser extent, aggrecan, compared with normal specimens. However, chondrocytes of the upper zone were largely negative for aggrecan or type II collagen mRNA. The expression of link protein mRNA was low in normal and OA specimens. Conclusion. These observations suggest that suppression of the anabolic activity of chondrocytes in the upper zones contributes to the metabolic imbalance observed in OA cartilage. Stimulation of matrix anabolism in superficial chondrocytes might be a suitable target for therapeutic intervention.     

Increased proteoglycan synthesis in cartilage in experimental canine osteoarthritis does not reflect a permanent change in chondrocyte phenotype

Objective. To determine whether chondrocytes in early experimental osteoarthritic (OA) cartilage continue to show increased synthesis and turnover of proteoglycans (PGs) during explant culture. A comparison was also made between the responsiveness of experimental OA and control cartilage to interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) after 1 day and 3 days in culture. Methods. OA was induced in mature animals by sectioning of the anterior cruciate ligament followed by 3 months of normal exercise. PG synthesis in the articular cartilage was determined by measuring ³⁵S-sulfate incorporation during explant culture over 1–3 days. Inhibition of PG synthesis was also determined with various concentrations of IL-1β and TNFα after 1 and 3 days in culture. PGs extracted from the articular cartilage over 1–3 days in culture were examined by agarose–polyacrylamide gel electrophoresis. Results. Up to 24 hours after excision from the joint, PG synthesis was higher in experimental OA cartilage than in control cartilage. It was also less sensitive to inhibition by TNFα. These differences were no longer detected after 48–72 hours in culture. There were no changes in the relative proportions of aggrecan and decorin/biglycan extracted from and synthesized by control and experimental OA cartilage over the 3 days in culture. Conclusion. Previous results indicated that PG synthesis and turnover in articular cartilage was increased for many months after induction of experimental OA. Our present results show that the enhanced rate of PG synthesis and turnover were evident in freshly explanted tissue, but the differences were lost over 3 days in culture. A decreased responsiveness to TNFα was also lost. The hypermetabolic activity of experimental OA chondrocytes was thus reversible and not a permanent change in chondrocyte phenotype.     

Effects of growth factors and cytokines on proteoglycan and collagen synthesis by chondrocytes in guinea pigs with spontaneous osteoarthritis

We examined the effects of various growth factors and cytokines on proteoglycan (PG) and collagen synthesis by chondrocytes isolated from osteoarthritic and normal articular cartilage of Hartley strain guinea pigs. The guinea pig represents a useful animal model of spontaneous osteoarthritis (OA). Cartilage tissue samples were obtained from the knee joints of under-3-month-old guinea pigs (control group) as well as 5- to 8-month-old guinea pigs with OA changes (OA group). Chondrocytes were isolated enzymatically and maintained in suspension culture. Growth factor addition groups were then prepared from both the OA group and the control group, using the factors TGF-β, bFGF, and IGF-1 (1.25 ng/ml each). Cytokine addition groups were also prepared using IL-1α and IL-1β (10 ng/ml each). An addition group was also prepared for sodium hyaluronate (HA) (500 μg/ml). In each group, ³⁵S was added as a PG metabolic marker, ³H-proline was added as a collagen metabolic marker, and the groups were cultured. Next, ³⁵S and ³H-proline uptake was measured by a liquid scintillation counter. The results revealed that (1) both PG synthesis and collagen synthesis were promoted significantly more in OA chondrocytes than in normal chondrocytes; (2) with the addition of growth factors, PG and collagen synthesis was enhanced in OA chondrocytes; and (3) PG synthesis and collagen synthesis were inhibited in both normal and OA chondrocytes with the addition of IL-1α and -β. This result suggests that the repair function is activated more in OA chondrocytes than in normal chondrocytes, thereby promoting the synthesis of the cartilage matrix by chondrocytes. This synthesizing capability is enhanced and acts to effectively repair degenerative articular cartilage further through the addition of growth factors. Received September 20, 1999 / Accepted December 6, 1999     

Morphology and phenotype expression of types I, II, III, and X collagen and MMP-13 of chondrocytes cultured from articular cartilage of Kashin-Beck Disease

OBJECTIVE: We investigated the characteristics of cell morphology and expression of types I, II, III, and X collagen and matrix metalloproteinase-13 (MMP-13) of chondrocytes from articular cartilage of adult patients with Kashin-Beck Disease (KBD) in vitro to understand the pathogenesis in chondrocytes. METHODS: Samples of articular cartilage were divided into 2 groups: KBD group (8 samples, 8 cases) and the control (8 samples, 8 cases). KBD patients were diagnosed according to "Pathological Criteria to Diagnose KBD in China." Hyaline cartilage was digested with collagenase into cell suspensions and cultured in monolayers. Chondrocyte ultrastructure was observed by electron microscope at 10th day in vitro. Primary articular chondrocytes were seeded on microscope slides and immunostained on 12th day of cultivation for types I, II, III, and X collagens and MMP-13. Positive findings were counted by light microscopy and confirmed by flow cytometric analyses. RESULTS: Considerable amounts of vacuoles and distorted nuclei, as well as thickening and irregular arrangement of collagen fibrils, were seen in the KBD samples by electron microscopy. Types I, III, and X collagen were stained in the KBD, but not in the control cultures. The percentages of positive staining for type II collagen were significantly lower in KBD than those in controls (t col II = -5.54, p < 0.001), and for MMP-13 in the KBD group were significantly higher (t MMP-13 = 3.70, p < 0.01). CONCLUSION:Phenotype expressions of types I, II, III, and X collagen and MMP-13 in chondrocytes cultured in vitro were significantly different between the KBD and control cultures, indicating degenerative and hypertrophic changes in chondrocytes of KBD articular cartilage.     

Increased collagen and aggrecan degradation with age in the joints of Timp3−/− mice

Objective

To investigate the in vivo effect of an imbalance between metalloproteinases and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in mouse articular cartilage.

Methods

Hind joints of Timp3^−/− and wild‐type mice were examined by routine staining and by immunohistochemical analysis using antibodies specific for type X collagen and for the neoepitopes produced on proteolytic cleavage of aggrecan (… VDIPEN and … NVTEGE) and type II collagen. The neoepitope generated on cleavage of type II collagen by collagenases was quantitated in sera by enzyme‐linked immunosorbent assay.

Results

Articular cartilage from Timp3‐knockout animals (ages ≥6 months) showed reduced Safranin O staining and an increase in …VDIPEN content compared with cartilage from heterozygous and wild‐type animals. There was also a slight increase in … NVTEGE content in articular cartilage and menisci of Timp3^−/− animals. Chondrocytes showed strong pericellular staining for type II collagen cleavage neoepitopes, particularly in the superficial layer, in knockout mice. Also, there was more type X collagen expression in the superficial zone of articular cartilage, especially around clusters of proliferating chondrocytes, in the knockout mice. More type II collagen cleavage product was found in the serum of Timp3^−/− mice compared with wild‐type animals. This increase was significant in 15‐month‐old animals.

Conclusion

These results indicate that TIMP‐3 deficiency results in mild cartilage degradation similar to changes seen in patients with osteoarthritis, suggesting that an imbalance between metalloproteinases and TIMP‐3 may play a pathophysiologic role in the development of this disease.

     

Type II collagen levels correlate with mineralization by articular cartilage vesicles

Objective

Pathologic mineralization is common in osteoarthritic (OA) cartilage and may be mediated by extracellular organelles known as articular cartilage vesicles (ACVs). Paradoxically, ACVs isolated from OA human cartilage mineralize poorly in vitro compared with those isolated from normal porcine cartilage. We recently showed that collagens regulate ACV mineralization. We sought to determine differences between collagens and collagen receptors on human and porcine ACVs as a potential explanation of their different mineralization behaviors.

Methods

ACVs were enzymatically released from old and young human and porcine hyaline articular cartilage. Western blotting was used to determine the presence of types I, II, VI, and X collagen and various collagen receptors on ACVs. Type II collagen was quantified by enzyme‐linked immunosorbent assay. Biomineralization was assessed by measuring the uptake of ⁴⁵Ca by isolated ACVs in agarose gels and by ACVs in situ in freeze‐thawed cartilage.

Results

As previously shown, isolated human ACVs mineralized poorly in response to ATP compared with porcine ACVs, but human and porcine ACVs mineralized similarly in situ in freeze‐thawed cartilage. Type II collagen levels were 100‐fold higher in isolated human ACVs than in porcine ACVs. Type II collagen in human ACVs was of high molecular weight. Transglutaminase‐crosslinked type II collagen showed increased resistance to collagenase, suggesting a possible explanation for residual collagen on human ACVs. Expression of other collagens and collagen receptors was similar on human and porcine ACVs.

Conclusion

Higher levels of type II collagen in human ACV preparations, perhaps mediated by increased transglutaminase crosslinking, may contribute to the decreased mineralization observed in isolated human ACVs in vitro.