A single blunt impact on cartilage promotes fibronectin fragmentation and upregulates cartilage degrading stromelysin-1/matrix metalloproteinase-3 in a bovine ex vivo model.

Post-traumatic osteoarthritis (PTOA) is characterized by progressive cartilage degeneration in injured joints. Since fibronectin-fragments (Fn-fs) degrade cartilage mainly through up-regulating matrix metalloproteinases (MMPs) and pro-inflammatory cytokines, we hypothesized that Fn-fs play a key role in PTOA by promoting chondrolysis in and around injured cartilage. To test this hypothesis, we profiled the catabolic events focusing on fibronectin fragmentation and proteinase expression in bovine osteochondral explants following a single blunt impact on cartilage with a drop tower device which created partial-thickness tissue damage. Injured and control explants were cultured for up to 14 days. The presence of Fn-fs, MMPs (-1, -3, -13), ADAMTS-5 in culture media and in cartilage was determined with immunoblotting. The daily proteoglycan (PG) depletion of cartilage matrix was assessed with DMMB assay. The effect of explant-conditioned media on chondrocytes was also examined with immunoblotting. Impacted cartilage released significantly higher amount of native Fn, three chondrolytic Fn-fs and PG than non-impacted controls did. Those increases coincided with up-regulation of MMP-3 both in culture media and in impacted cartilage. These findings support our hypothesis that PTOA may be propelled by Fn-fs which act as catabolic mediators through up-regulating cartilage-damaging proteinases.

Biomechanical modulation of collagen fragment-induced anabolic and catabolic activities in chondrocyte/agarose constructs.

INTRODUCTION: The present study examined the effect of collagen fragments on anabolic and catabolic activities by chondrocyte/agarose constructs subjected to dynamic compression. METHODS: Constructs were cultured under free-swelling conditions or subjected to continuous and intermittent compression regimes, in the presence of the N-terminal (NT) and C-terminal (CT) telopeptides derived from collagen type II and/or 1400 W (inhibits inducible nitric oxide synthase (iNOS)). The anabolic and catabolic activities were compared to the amino-terminal fibronectin fragment (NH2-FN-f) and assessed as follows: nitric oxide (NO) release and sulphated glycosaminoglycan (sGAG) content were quantified using biochemical assays. Tumour necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta) release were measured by ELISA. Gene expression of matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), collagen type II and fibronectin were assessed by real-time quantitative polymerase chain reaction (qPCR). Two-way ANOVA and the post hoc Bonferroni-corrected t-test was used to examine data. RESULTS: The presence of the NT or CT peptides caused a moderate to strong dose-dependent stimulation of NO, TNFalpha and IL-1beta production and inhibition of sGAG content. In some instances, high concentrations of telopeptides were just as potent in stimulating catabolic activities when compared to NH2-FN-f. Depending on the concentration and type of fragment, the increased levels of NO and cytokines were inhibited with 1400 W, resulting in the restoration of sGAG content. Depending on the duration and type of compression regime employed, stimulation with compression or incubation with 1400 W or a combination of both, inhibited telopeptide or NH2-FN-f induced NO release and cytokine production and enhanced sGAG content. All fragments induced MMP-3 and MMP-13 expression in a time-dependent manner. This effect was reversed with compression and/or 1400 W resulting in the restoration of sGAG content and induction of collagen type II and fibronectin expression. CONCLUSIONS: Collagen fragments containing the N- and C-terminal telopeptides have dose-dependent catabolic activities similar to fibronectin fragments and increase the production of NO, cytokines and MMPs. Catabolic activities were downregulated by dynamic compression or by the presence of the iNOS inhibitor, linking reparative activities by both types of stimuli. Future investigations which examine the signalling cascades of chondrocytes in response to matrix fragments with mechanical influences may provide useful information for early osteoarthritis treatments.

Differential effects of fibronectin fragment on proteoglycan metabolism by intervertebral disc cells: a comparison with articular chondrocytes.

STUDY DESIGN: This in vitro study used the alginate bead culture system to probe for differences in the effects of fibronectin fragment on cell proliferation and proteoglycan metabolism by different populations of intervertebral disc cells and articular chondrocytes. OBJECTIVE: To compare the effects of fibronectin fragment on cell proliferation, and proteoglycan synthesis and degradation by cells from the nucleus pulposus, the anulus fibrosus, and articular cartilage. SUMMARY OF BACKGROUND DATA: In articular cartilage, the administration of fibronectin fragment stimulates cartilage degeneration. Fibronectin fragment levels were increased in human intervertebral discs with increased disc degeneration. Fibronectin fragment injected into the central region of the rabbit intervertebral disc induced a progressive degeneration of that disc. METHODS: Bovine tails and metacarpophalangeal joints from 14- to 18-month-old animals were used. Alginate beads containing cells isolated from intervertebral discs and articular cartilage were cultured with (1-100 nmol/L) or without (control) fibronectin fragment in the presence of 10% fetal bovine serum. In these cultures, deoxyribonucleic acid and proteoglycan contents, as well as the rate of proteoglycan synthesis were determined. Proteoglycan degradation was measured in cultures with or without 10 nmol/L fibronectin fragment. RESULTS: In articular chondrocytes, fibronectin fragment strongly suppressed proteoglycan synthesis and stimulated proteoglycan degradation; the total proteoglycan content was diminished in a dose-dependent manner. Compared to articular chondrocytes, nucleus pulposus cells responded to fibronectin fragments in a similar, although less pronounced manner. On the other hand, anulus fibrosus cells treated with fibronectin fragment did not show any significant effects on proteoglycan degradation. A slight but statistically significant up-regulation of proteoglycan synthesis was observed at 10 nmol/L fibronectin fragment in outer anulus fibrosus cells. However, total proteoglycan content was decreased significantly at high concentrations of fibronectin fragment. CONCLUSIONS: Fibronectin fragment has different effects on cell proliferation, proteoglycan synthesis, degradation, and accumulation by articular chondrocytes and intervertebral disc cells. The different effects of fibronectin fragment in those different cell types suggest metabolic differences between these cells, and may further suggest differences in pathways of fibronectin fragment signaling as well as a differential need of these cells to be involved in tissue remodeling in which both anabolic and catabolic pathways might be altered.

High molecular weight hyaluronan promotes repair of IL-1 beta-damaged cartilage explants from both young and old bovines.

OBJECTIVE: The addition of exogenous high molecular weight hyaluronic acid (HA) reverses cartilage damage caused by fibronectin fragments (Fn-fs) added to explant cultures of bovine and human cartilage and by Fn-fs in an experimental in vivo model of rabbit knee joint damage. Our objective was to test whether HA was also effective in an IL-1 damage model and whether this repair was stable and occurred in older bovine cartilage. DESIGN: Bovine cartilage explants from 18-month-old or 6-year-old bovines in 10% serum/Dulbecco's modified Eagle's medium were exposed to Fn-f or to IL-1 and the ability of 1mg/ml HA of 800 kDa to block damage or promote restoration of proteoglycan (PG) after the damage was measured. The damage phase as well as the exposure to HA were varied. RESULTS: Exposure of exogenous HA decreased Fn-f-mediated damage, but did not decrease IL-1 beta-induced cartilage damage. If explants from 18-month-old bovines were damaged by a 7-day exposure to Fn-f or IL-1 beta and then exposed for 7 days to HA, PG was restored. This reparative activity persisted up to 4 weeks after the removal of HA from the culture medium. The restoration of PG did not occur in 0.1% serum-free cultures, was less when the exposure to the Fn-f was doubled and failed when exposure to IL-1 beta was doubled. In explants from 6-year-old bovines damaged with IL-1 beta for 7 days, HA fully restored PG content to normal levels. CONCLUSIONS: The reparative activities of HA occur not only in a Fn-f damage model, but also in an IL-1 damage model and occur with older bovine cartilage.