Proteoglycan degradation after injurious compression of bovine and human articular cartilage in vitro: Interaction with exogenous cytokines

Abstract

Objective. Traumatic joint injury leads to an increased risk of osteoarthritis (OA), but the progression to OA is not well understood. We undertook this study to measure aspects of proteoglycan (PG) degradation after in vitro injurious mechanical compression, including up-regulation of enzymatic degradative expression and cytokine-stimulated degradation.Methods. Articular cartilage tissue explants were obtained from newborn bovine femoropatellar groove and from adult normal human donor knee and ankle tissue. Following injurious compression of the cartilage, matrix metalloproteinase 3 (MMP-3) and MMP-13 messenger RNA (mRNA) expression levels were measured by Northern analysis, and PG loss to the medium after cartilage injury was measured in the presence and absence of added exogenous cytokine (interleukin-1␣ [IL-1␣] or tumor necrosis factor ␣ [TNF␣]).Results. During the first 24 hours after injury in bovine cartilage, MMP-3 mRNA levels increased 10-fold over the levels in control cartilage (n ‫؍‬ 3 experiments), whereas MMP-13 mRNA levels were unchanged. PG loss was significantly increased after injury, but only by 2% of the total PG content and only for the first 3 days following injury. However, compared with injury alone or cytokine treatment alone, treatment of injured tissue with either 1 ng/ml IL-1␣ or 100 ng/ml TNF␣ caused marked increases in PG loss (35% and 54%, respectively, of the total cartilage PG content). These interactions between cytokine treatment and injury were statistically significant. In human knee cartilage, the interaction was also significant for both IL-1␣ and TNF␣, although the magnitude of increase in PG loss was lower than that in bovine cartilage. In contrast, in human ankle cartilage, there was no significant interaction between injury and IL-1␣.Conclusion. The cytokines IL-1␣ and TNF␣ can cause a synergistic loss of PG from mechanically injured bovine and human cartilage. By attempting to incorporate interactions with other joint tissues that may be sources of cytokines, in vitro models of mechanical cartilage injury may explain aspects of the interactions between mechanical forces and degradative pathways which lead to OA progression.

Mechanical injury of cartilage explants causes specific time‐dependent changes in chondrocyte gene expression

“…The effects of mechanical injury on the expression of MMP-3 and MMP-13 ( Figure 3B) are similar to the trends reported by Patwari et al (13) in a study in which Northern analysis was used to determine the expression levels in similar cartilage explants that were subjected to the same injury protocol as depicted in Figure 1C. In that study, injury caused a significant increase in MMP-3 expression (10-fold) above the level in controls but no change in MMP-13 in the first 24 hours.…”

“…A single injurious compression of cartilage has been shown previously to decrease ECM biosynthesis rates, compromise mechanical properties, and reduce chondrocyte viability (12)(13)(14)(15)(16)22). We undertook this study to determine if changes also occur at the level of gene expression, and to determine whether the changes are general or are specific to certain genes.…”

“…After equilibration of the explants, a custom-designed incubator-housed loading apparatus (25) was used ( Figure 1A) to injuriously compress 36 cartilage disks from each joint, while the remaining 12 disks served as free swelling controls. Cartilage samples to be injured were placed individually into a polysulfone chamber ( Figure 1B), which allows radially unconfined compression of the disk by impermeable platens (12,13,15). The measured thickness of the cartilage disk just prior to loading was recorded, and the zero-strain position was identified by the point of first contact between the loading platen and the cartilage surface.…”

“…Color figure can be viewed in the online issue, which is available at http://www.arthritisrheum.org. loss to the medium in similar bovine cartilage explants (12,13,15,22,23).…”

“…Mechanical loads applied in vitro range from single compressions of up to 50% strain (12)(13)(14)(15)(16)(17) to large-amplitude cyclic compression at varying frequencies (ϳ0.05-0.3 Hz) for up to 2 hours (18)(19)(20)(21). Injurious mechanical compression of cartilage in vitro can damage the extracellular matrix (ECM), leading to increased water content (12,16,18,22), decreased stiffness (12,22), increased hydraulic permeability (20), loss of glycosaminoglycan (GAG) to the culture medium (12)(13)(14)(15)17,20,22,23), loss of collagen to the medium (20), and temporary denaturation of collagen in the tissue (16,18,20,21). In addition, injurious mechanical compression can lead to cell death by both apoptosis and necrosis (14,16,19,(21)(22)(23), as well as decreased matrix biosynthesis rates in the remaining viable cells after injury (12).…”