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.

     

Interleukin‐1α induction of tensile weakening associated with collagen degradation in bovine articular cartilage

Objective

To determine whether interleukin‐1α (IL‐1α) induces tensile weakening of articular cartilage that is concomitant with the loss of glycosaminoglycans (GAGs) or the subsequent degradation of the collagen network.

Methods

Explants of young adult bovine cartilage obtained from the superficial (including the articular surface), middle, and deep layers were cultured with or without IL‐1α for 1 week or 3 weeks. Then, portions of the explants were analyzed for their tensile properties (ramp modulus, strength, and failure strain); other portions of explants and spent culture medium were analyzed for the amount of GAG and the amount of cleaved, denatured, and total collagen.

Results

The effect of IL‐1α treatment on cartilage tensile properties and content was dependent on the duration of culture and the depth of the explant from the articular surface. The tensile strength and failure strain of IL‐1α–treated samples from the superficial and middle layers were lower after 3 weeks of culture, but not after 1 week of culture. However, by 1 week of culture, IL‐1α had already induced release of the majority of tissue GAGs into the medium, without detectable loss or degradation of collagen. In contrast, after 3 weeks of culture, IL‐1α induced significant collagen degradation, as indicated by the amount of total, cleaved, or denatured collagen in the medium or in explants from the superficial and middle layers.

Conclusion

IL‐1α–induced degradation of cartilage results in tensile weakening that occurs subsequent to the depletion of GAG and concomitant with the degradation of the collagen network.

     

Attenuation of osteoarthritis progression by reduction of discoidin domain receptor 2 in mice

Objective

To investigate whether the reduction of discoidin domain receptor 2 (DDR‐2), a cell membrane tyrosine kinase receptor for native type II collagen, attenuates the progression of articular cartilage degeneration in mouse models of osteoarthritis (OA).

Methods

Double‐heterozygous (type XI collagen–deficient [Col11a1^+/−] and Ddr2‐deficient [Ddr2^+/−]) mutant mice were generated. Knee joints of Ddr2^+/− mice were subjected to microsurgical destabilization of the medial meniscus. Conditions of the articular cartilage from the knee joints of the double‐heterozygous mutant and surgically treated mice were examined by histology, evaluated using a modified Mankin scoring system, and characterized by immunohistochemistry.

Results

The rate of progressive degeneration in knee joints was dramatically reduced in the double‐heterozygous mutant mice compared with that in the type XI collagen–deficient mice. The progression in the double‐heterozygous mutant mice was delayed by ∼6 months. Following surgical destabilization of the medial meniscus, the progressive degeneration toward OA was dramatically delayed in the Ddr2^+/− mice compared with that in their wild‐type littermates. The articular cartilage damage present in the knee joints of the mice was directly correlated with the expression profiles of DDR‐2 and matrix metalloproteinase 13.

Conclusion

Reduction of DDR‐2 expression attenuates the articular cartilage degeneration of knee joints induced either by type XI collagen deficiency or by surgical destabilization of the medial meniscus.

     

Osteoarthritis‐like changes and decreased mechanical function of articular cartilage in the joints of mice with the chondrodysplasia gene (cho)

Objective

To investigate whether heterozygosity for a loss‐of‐function mutation in the gene encoding the α1 chain of type XI collagen (Col11a1) in mice (chondrodysplasia, cho) causes osteoarthritis (OA), and to understand the biochemical and biomechanical effects of this mutation on articular cartilage in knee and temporomandibular (TM) joints.

Methods

Articular cartilage from the knee and TM joints of mice heterozygous for cho (cho/+) and their wild‐type littermates (+/+) was examined. The morphologic properties of cartilage were evaluated, and collagen fibrils were examined by transmission electron microscopy. Immunohistochemical staining was performed to examine the protein expression levels of matrix metalloproteinase 3 (MMP‐3) and MMP‐13 in knee joints. In 6‐month‐old animals, fixed‐charge density was determined using a semiquantitative histochemical method, and tensile stiffness was determined using an osmotic loading technique.

Results

The diameter of collagen fibrils in articular cartilage of knee joints from heterozygous cho/+ mice was increased relative to that in control cartilage, and histologic analysis showed OA‐like degenerative changes in knee and TM joints, starting at age 3 months. The changes became more severe with aging. At 3 months, protein expression for MMP‐3 was increased in knee joints from cho/+ mice. At 6 months, protein expression for MMP‐13 was higher in knee joints from cho/+ mice than in joints from their wild‐type littermates, and negative fixed‐charge density was significantly decreased. Moreover, tensile stiffness in articular cartilage of knee joints from cho/+ mice was moderately reduced and was inversely correlated with the increase in articular cartilage degeneration.

Conclusion

Heterozygosity for a loss‐of‐function mutation in Col11a1 results in the development of OA in the knee and TM joints of cho/+ mice. Morphologic and biochemical evidence of OA appears to precede significant mechanical changes, suggesting that the cho mutation leads to OA through a mechanism that does not initially involve mechanical factors.

     

Chondroprotective role of the osmotically sensitive ion channel transient receptor potential vanilloid 4: Age‐ and sex‐dependent progression of osteoarthritis in Trpv4‐deficient mice

Objective

Mechanical loading significantly influences the physiology and pathology of articular cartilage, although the mechanisms of mechanical signal transduction are not fully understood. Transient receptor potential vanilloid 4 (TRPV4) is a Ca⁺⁺‐permeable ion channel that is highly expressed by articular chondrocytes and can be gated by osmotic and mechanical stimuli. The goal of this study was to determine the role of Trpv4 in the structure of the mouse knee joint and to determine whether Trpv4^–/– mice exhibit altered Ca⁺⁺ signaling in response to osmotic challenge.

Methods

Knee joints of Trpv4^–/– mice were examined histologically and by microfocal computed tomography for osteoarthritic changes and bone structure at ages 4, 6, 9, and 12 months. Fluorescence imaging was used to quantify chondrocytic Ca⁺⁺ signaling within intact femoral cartilage in response to osmotic stimuli.

Results

Deletion of Trpv4 resulted in severe osteoarthritic changes, including cartilage fibrillation, eburnation, and loss of proteoglycans, that were dependent on age and male sex. Subchondral bone volume and calcified meniscal volume were greatly increased, again in male mice. Chondrocytes from Trpv4^+/+ mice demonstrated significant Ca⁺⁺ responses to hypo‐osmotic stress but not to hyperosmotic stress. The response to hypo‐osmotic stress or to the TRPV4 agonist 4α‐phorbol 12,13‐didecanoate was eliminated in Trpv4^–/– mice.

Conclusion

Deletion of Trpv4 leads to a lack of osmotically induced Ca⁺⁺ signaling in articular chondrocytes, accompanied by progressive, sex‐dependent increases in bone density and osteoarthritic joint degeneration. These findings suggest a critical role for TRPV4‐mediated Ca⁺⁺ signaling in the maintenance of joint health and normal skeletal structure.

     

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.

     

Pathogenesis of osteoarthritis‐like changes in the joints of mice deficient in type IX collagen

Objective

To examine the pathogenetic mechanisms of osteoarthritis (OA)–like changes in Col9a1^−/− mice, which are deficient in type IX collagen.

Methods

Knee joints and temporomandibular joints (TMJs) from Col9a1^−/− mice and their wild‐type (Col9a1^+/+) littermates were examined by light microscopy. Immunohistochemical staining was performed to examine the expression of matrix metalloproteinase 3 (MMP‐3) and MMP‐13, degraded type II collagen, and the discoidin domain receptor 2 (DDR‐2) in knee joints. Cartilage mechanics were also evaluated for compressive properties by microindentation testing of the tibial plateau and for tensile properties by osmotic loading of the femoral condyle.

Results

Histologic analysis showed age‐dependent OA‐like changes in the knee and TMJs of Col9a1^−/− mice starting at the age of 3 months. At the age of 6 months, enhanced proteoglycan degradation was observed in the articular cartilage of the knee and TMJs of the mutant mice. The expression of MMP‐13 and DDR‐2 protein and the amount of degraded type II collagen were higher in the knee joints of Col9a1^−/− mice than in their wild‐type littermates at the age of 6 months. Changes in cartilage mechanics were observed in the femoral and tibial plateaus of Col9a1^−/− mice at 6 months, including a decrease in the compressive modulus and uniaxial modulus. At 3 and 6 months of age, tibial cartilage in Col9a1^−/− mice was found to be more permeable to fluid flow, with an associated compromise in the fluid pressurization mechanism of load support. All of these changes occurred only at medial sites.

Conclusion

Lack of type IX collagen in Col9a1^−/− mice results in age‐dependent OA‐like changes in the knee joints and TMJs.

     

Chondrocyte‐intrinsic Smad3 represses Runx2‐inducible matrix metalloproteinase 13 expression to maintain articular cartilage and prevent osteoarthritis

Objective

To identify mechanisms by which Smad3 maintains articular cartilage and prevents osteoarthritis.

Methods

A combination of in vivo and in vitro approaches was used to test the hypothesis that Smad3 represses Runx2‐inducible gene expression to prevent articular cartilage degeneration. Col2‐Cre;Smad3^fl/fl mice allowed study of the chondrocyte‐intrinsic role of Smad3 independently of its role in the perichondrium or other tissues. Primary articular cartilage chondrocytes from Smad3^fl/fl mice and ATDC5 chondroprogenitor cells were used to evaluate Smad3 and Runx2 regulation of matrix metalloproteinase 13 (MMP‐13) messenger RNA (mRNA) and protein expression.

Results

Chondrocyte‐specific reduction of Smad3 caused progressive articular cartilage degeneration due to imbalanced cartilage matrix synthesis and degradation. In addition to reduced type II collagen mRNA expression, articular cartilage from Col2‐Cre;Smad3^fl/fl mice was severely deficient in type II collagen and aggrecan protein due to excessive MMP‐13–mediated proteolysis of these key cartilage matrix constituents. Normally, transforming growth factor β (TGFβ) signals through Smad3 to confer a rapid and dynamic repression of Runx2‐inducible MMP‐13 expression. However, we found that in the absence of Smad3, TGFβ signals through p38 and Runx2 to induce MMP‐13 expression.

Conclusion

Our findings elucidate a mechanism by which Smad3 mutations in humans and mice cause cartilage degeneration and osteoarthritis. Specifically, Smad3 maintains the balance between cartilage matrix synthesis and degradation by inducing type II collagen expression and repressing Runx2‐inducible MMP‐13 expression. Selective activation of TGFβ signaling through Smad3, rather than p38, may help to restore the balance between matrix synthesis and proteolysis that is lost in osteoarthritis.

     

Membrane type 1 matrix metalloproteinase is a crucial promoter of synovial invasion in human rheumatoid arthritis

Objective

A hallmark of rheumatoid arthritis (RA) is invasion of the synovial pannus into cartilage, and this process requires degradation of the collagen matrix. The aim of this study was to explore the role of one of the collagen‐degrading matrix metalloproteinases (MMPs), membrane type 1 MMP (MT1‐MMP), in synovial pannus invasiveness.

Methods

The expression and localization of MT1‐MMP in human RA pannus were investigated by Western blot analysis of primary synovial cells and immunohistochemical analysis of RA joint specimens. The functional role of MT1‐MMP was analyzed by 3‐dimensional (3‐D) collagen invasion assays and a cartilage invasion assay in the presence or absence of tissue inhibitor of metalloproteinases 1 (TIMP‐1), TIMP‐2, or GM6001. The effect of adenoviral expression of a dominant‐negative MT1‐MMP construct lacking a catalytic domain was also examined.

Results

MT1‐MMP was highly expressed at the pannus–cartilage junction in RA joints. Freshly isolated rheumatoid synovial tissue and isolated RA synovial fibroblasts invaded into a 3‐D collagen matrix in an MT1‐MMP–dependent manner. Invasion was blocked by TIMP‐2 and GM6001 but not by TIMP‐1. Invasion was also inhibited by the overexpression of a dominant‐negative MT1‐MMP, which inhibits collagenolytic activity and proMMP‐2 activation by MT1‐MMP on the cell surface. Synovial fibroblasts also invaded into cartilage in an MT1‐MMP–dependent manner. This process was further enhanced by removing aggrecan from the cartilage matrix.

Conclusion

MT1‐MMP serves as an essential collagen‐degrading proteinase during pannus invasion in human RA. Specific inhibition of MT1‐MMP–dependent invasion may represent a novel therapeutic strategy for RA.

     

NR2‐reactive antibody decreases cell viability through augmentation of Ca2+ influx in systemic lupus erythematosus

Objective

Anti–N‐methyl‐D ‐aspartate (anti‐NMDA) receptor subunit NR2–reactive antibody may play a crucial role in neuronal manifestations of systemic lupus erythematosus (SLE). However, how NR2‐reactive antibody acts as a critical modulator of the NMDA receptor is unknown. This study was undertaken to investigate the biologic function of NR2‐reactive antibody in patients with SLE.

Methods

The study included 14 patients with SLE, 9 of whom had NR2‐reactive antibody. We analyzed the effects of NR2‐reactive antibody on cell viability and intracellular Ca²⁺ level. We also investigated the efficacy of zinc as a modulator of the intracellular Ca²⁺ level in the presence of NR2‐reactive antibody.

Results

There was a significant inverse correlation between the NR2‐reactive antibody titer and cell viability (R² = 0.67, P < 0.0001; n = 23), and there was a significant association between the NR2‐reactive antibody titer and the intracellular Ca²⁺ level in NR1/NR2a‐transfected HEK 293 cells (R² = 0.69, P < 0.0001). Intracellular Ca²⁺ levels were significantly higher in cells incubated with IgG derived from NR2‐reactive antibody–positive SLE patients than in those incubated with IgG derived from NR2‐reactive antibody–negative SLE patients (P = 0.0002). The addition of zinc decreased the intracellular Ca²⁺ level in a dose‐dependent manner. NR2‐reactive antibody–positive SLE IgG weakened the efficacy of zinc as a negative modulator of the intracellular Ca²⁺ level.

Conclusion

Our findings indicate that NR2‐reactive antibody decreases cell viability by Ca²⁺ influx in SLE through inhibition of the binding capacity of zinc.

     

Early elevation in circulating levels of C‐telopeptides of type II collagen predicts structural damage in articular cartilage in the rodent model of collagen‐induced arthritis

Objective

To investigate changes in the circulating levels of the C‐telopeptide of type II collagen (CTX‐II) with relation to disease onset and structural damage of cartilage in a rodent model of collagen‐induced arthritis (CIA), and to investigate immunolocalization of the CTX‐II epitope in the articular cartilage of affected joints.

Methods

Seven‐week‐old female Lewis rats were immunized with type II collagen and monitored using blood sampling at weekly intervals. At study termination (day 23), the animals were killed, synovial fluid was collected, and the affected joints were scored macroscopically for disease severity and underwent immunohistochemical evaluation.

Results

At the time of disease onset (day 15), which was characterized by redness and swelling of the affected joints (mean ± SD macroscopic severity score 9.1 ± 1.6), there was a 355% increase in serum CTX‐II levels. The early change in serum CTX‐II from day 0 to day 15 showed a significant association with the severity of cartilage damage (r = 0.61, P < 0.01). Immunostaining revealed extensive presence of the CTX‐II epitope in the damaged, uncalcified cartilage tissue.

Conclusion

The elevation in serum CTX‐II concomitant with the onset of disease and proportional to cartilage damage demonstrates that CTX‐II is a sensitive diagnostic tool for monitoring joint disease in the rodent model of CIA. Furthermore, the immunohistochemical findings are consistent with the concept that the major source of serum CTX‐II is the damaged articular cartilage.

     

Class II major histocompatibility complex–associated response to type XI collagen regulates the development of chronic arthritis in rats

Objective

Chronic inflammation of the peripheral joints is a hallmark of rheumatoid arthritis (RA). The autoantibody response in RA has been shown to be directed mainly to ubiquitous antigens, whereas the response to cartilage proteins has been less extensively investigated. This study was undertaken to characterize the immune response in pristane‐induced arthritis (PIA) in the rat to the cartilage‐specific proteins type II collagen (CII) and type XI collagen (CXI) and to genetically fine‐map their underlying major histocompatibility complex (MHC) associations.

Methods

The genetic control of CII and CXI immunity was mapped using intra‐MHC–recombinant inbred strains immunized with the respective collagens. Reactivity with CII and CXI was tested in acute and chronic PIA and in 356 HLA‐typed patients with recently diagnosed RA.

Results

Mapping of arthritis susceptibility within the MHC region revealed a 144–223–kb locus containing <12 genes, including paralogs for HLA–DQ and HLA–DR. Susceptibility to CII and CXI was linked to haplotypes RT1^av1 (DA) and RT1^f (DA.1F), respectively. After injection of pristane, rats of both strains developed weak T cell and IgG responses to CII, but not to CXI. In chronic arthritis, however, collagen reactivity was stronger, specific for CXI, and restricted to rats with RT1^f MHC. Among RA patients, 12% exhibited a specific IgG response to CXI, 6% to CII, and 6% to both collagens.

Conclusion

These findings demonstrate a shift in cartilage recognition in early and chronic arthritis in the rat, suggesting that CXI autoreactivity contributes to the perpetuation of chronic disease. The results provide evidence of the importance of joint antigens in arthritis development.

     

Inhibition of furin‐like enzymes blocks interleukin‐1α/oncostatin M–stimulated cartilage degradation

Objective

To investigate the role of furin‐like enzymes in the proteolytic cascades leading to cartilage breakdown and to examine which collagenase(s) contribute to collagen degradation.

Methods

Bovine nasal cartilage was stimulated to resorb with the addition of interleukin‐1α (IL‐1α)/oncostatin M (OSM) in the presence or absence of a furin inhibitor, Dec‐RVKR‐CH₂Cl, or selective matrix metalloproteinase 1 (MMP‐1) inhibitors. Collagen and proteoglycan levels were determined by assay of hydroxyproline and sulfated glycosaminoglycan, respectively. Collagenase and gelatinase activity were measured using ³H‐acetylated collagen and gelatin zymography, respectively.

Results

The addition of Dec‐RVKR‐CH₂Cl to stimulated cartilage reduced the release of collagen fragments and the levels of active collagenase and MMP‐2, suggesting that furin‐like enzymes are involved in the cascades leading to activation of procollagenases. At MMP inhibitor concentrations that selectively inhibit MMP‐1, no inhibition of collagen release was observed, but increasing the concentration to the 50% inhibition concentration for MMP‐13 resulted in a 50% blockage of collagen release. The addition of Dec‐RVKR‐CH₂Cl to resorbing cartilage also partially blocked proteoglycan release, thus demonstrating a role for furin‐activated enzymes in the pathways leading to proteoglycan degradation.

Conclusion

Furin‐like enzymes are involved in cascades leading to activation of procollagenases and degradation of collagen. MMP‐13, which can be activated by furin‐processed membrane‐type 1 MMP‐1, appears to be a major collagenase involved in collagen degradation induced by IL‐1α/OSM. Furin‐like enzymes also appear to play a role in the pathways leading to proteoglycan degradation. These findings are of importance when considering proteinase inhibition as a target for therapeutic intervention in arthritic diseases.

     

Inhibition of toll‐like receptor 4 breaks the inflammatory loop in autoimmune destructive arthritis

Objective

Degeneration of extracellular matrix of cartilage leads to the production of molecules capable of activating the immune system via Toll‐like receptor 4 (TLR‐4). The objective of this study was to investigate the involvement of TLR‐4 activation in the development and progression of autoimmune destructive arthritis.

Methods

A naturally occurring TLR‐4 antagonist, highly purified lipopolysaccharide (LPS) from Bartonella quintana, was first characterized using mouse macrophages and human dendritic cells (DCs). Mice with collagen‐induced arthritis (CIA) and mice with spontaneous arthritis caused by interleukin‐1 receptor antagonist (IL‐1Ra) gene deficiency were treated with TLR‐4 antagonist. The clinical score for joint inflammation, histologic characteristics of arthritis, and local expression of IL‐1 in joints were evaluated after treatment.

Results

The TLR‐4 antagonist inhibited DC maturation induced by Escherichia coli LPS and cytokine production induced by both exogenous and endogenous TLR‐4 ligands, while having no effect on these parameters by itself. Treatment of CIA using TLR‐4 antagonist substantially suppressed both clinical and histologic characteristics of arthritis without influencing the adaptive anti–type II collagen immunity crucial for this model. Treatment with TLR‐4 antagonist strongly reduced IL‐1β expression in articular chondrocytes and synovial tissue. Furthermore, such treatment inhibited IL‐1–mediated autoimmune arthritis in IL‐1Ra^−/− mice and protected the mice against cartilage and bone pathology.

Conclusion

In the present study, we demonstrate for the first time that inhibition of TLR‐4 suppresses the severity of experimental arthritis and results in lower IL‐1 expression in arthritic joints. Our data suggest that TLR‐4 might be a novel target in the treatment of rheumatoid arthritis.

     

Potent induction of chondrocytic differentiation of human adipose‐derived adult stem cells by bone morphogenetic protein 6

Objective

Recent studies have identified an abundant source of multipotent progenitor cells in subcutaneous human adipose tissue, termed human adipose‐derived adult stem cells (ADAS cells). In response to specific media formulations, including transforming growth factor β1 (TGFβ1), these cells exhibit significant ability to differentiate into a chondrocyte‐like phenotype, expressing cartilage‐specific genes and proteins such as aggrecan and type II collagen. However, the influence of other growth factors on the chondrogenic differentiation of ADAS cells is not fully understood. This study was undertaken to investigate the effects of TGFβ1, TGFβ3, insulin‐like growth factor 1, bone morphogenetic protein 6 (BMP‐6), and dexamethasone, in various combinations, on the chondrogenic potential of ADAS cells in alginate beads.

Methods

The chondrogenic response of alginate‐encapsulated ADAS cells was measured by quantitative polymerase chain reaction, ³H‐proline and ³⁵S‐sulfate incorporation, and immunolabeling for specific extracellular matrix components.

Results

Significant differences in chondrogenesis were observed under the different culture conditions for all outcomes measured. Most notably, BMP‐6 up‐regulated AGC1 and COL2A1 expression by an average of 205‐fold and 38‐fold, respectively, over day‐0 controls, while down‐regulating COL10A1 expression by ∼2‐fold.

Conclusion

These findings suggest that BMP‐6 is a potent inducer of chondrogenesis in ADAS cells, in contrast to mesenchymal stem cells, which exhibit increased expression of type X collagen and a hypertrophic phenotype in response to BMP‐6. Combinations of growth factors containing BMP‐6 may provide a novel means of regulating the differentiation of ADAS cells for applications in the tissue‐engineered repair or regeneration of articular cartilage.

     

Microenvironment regulation of extracellular signal–regulated kinase activity in chondrocytes: Effects of culture configuration,interleukin‐1, and compressive stress

Objective

To compare extracellular signal–regulated kinase (ERK) activity in response to interleukin‐1 (IL‐1) in chondrocytes under various culture configurations designed for the study of cartilage biology and repair, and also in response to dynamic load for chondrocytes in cartilage.

Methods

Isolated bovine articular chondrocytes were maintained in serum‐supplemented medium under 4 culture configurations: high‐density monolayer, attached to a cut surface of cartilage, within tissue‐engineered constructs, or within intact cartilage explants. Samples were subjected to a change of medium with or without IL‐1. Cartilage explants were also subjected to dynamic compression.

Results

In chondrocyte monolayers, both basal and IL‐1–stimulated ERK activities were similarly elevated at 0.5 hours after medium change, diminishing by 74% after 16 hours. In contrast, chondrocytes in other culture configurations exhibited lower basal levels of ERK activity and a moderate activation of ERK in response to IL‐1 that was sustained over the 16‐hour treatment time. The dynamic component of loading of cartilage explants led to a 5‐fold activation of ERK, compared with free‐swelling controls, that was indistinguishable from the effects of IL‐1.

Conclusion

ERK signaling in response to IL‐1 in chondrocyte monolayers exhibited a pattern that was distinct from that in other culture systems, suggesting that the extracellular matrix plays an important regulatory role in modulating the response to extracellular stimuli. Since IL‐1 and dynamic loading have distinct effects on chondrocyte biosynthesis, signaling pathways other than ERK participate in the chondrocyte responses to these stimuli.

     

Tissue‐engineered composites for the repair of large osteochondral defects

Objective

To test the hypothesis that engineered cartilage can provide a mechanically functional template capable of undergoing orderly remodeling during the repair of large osteochondral defects in adult rabbits, as assessed by quantitative structural and functional methods.

Methods

Engineered cartilage generated in vitro from chondrocytes cultured on a biodegradable scaffold was sutured to a subchondral support and the resulting composite press‐fitted into a 7‐mm long, 5‐mm wide, 5‐mm deep osteochondral defect in a rabbit knee joint. Defects left empty (group 1) or treated with cell‐free composites (group 2) served as controls for defects treated with composites of engineered cartilage and the support, without or with adsorbed bone marrow (groups 3 and 4, respectively).

Results

Engineered cartilage withstood physiologic loading and remodeled over 6 months into osteochondral tissue with characteristic architectural features and physiologic Young's moduli. Composites integrated well with host bone in 90% of cases but did not integrate well with host cartilage. Structurally, 6‐month repairs in groups 3 and 4 were superior to those in group 2 with respect to histologic score, cartilage thickness, and thickness uniformity, but were inferior to those in unoperated control tissue. At 6 months, Young's moduli in groups 2, 3, and 4 (0.68, 0.80, and 0.79 MPa, respectively) approached that in unoperated control tissue (0.84 MPa), whereas the corresponding modulus in group 1 (0.37 MPa) was significantly lower.

Conclusion

Composites of tissue‐engineered cartilage and a subchondral support promote the orderly remodeling of large osteochondral defects in adult rabbits.