Objective

Tibia vara seen in Japanese patients reportedly influences the tibial component alignment when performing TKA. However, it is unclear whether tibia vara affects the component position and size selection. We therefore determined (1) the amount of medial tibial bow, (2) whether the tibia vara influences the aspect ratio of the tibial resected surface in aligning the tibial component with the tibial shaft axis (TSA), and (3) whether currently available tibial components fit the shapes of resected proximal tibias in terms of aspect ratio.

Destruction of articular cartilage in osteoarthritis (OA) is mediated by proteases and cytokines, which are silenced by epigenetic mechanisms in normal chondrocytes, but aberrantly expressed in OA. This is associated with DNA de-methylation of specific CpGs in the promoter regions (Arthritis Rheum, 2005; 52:3110–24). A widely used in vitro model to study the transcriptional regulation in OA is treating monolayer cultures of normal articular chondrocytes with inflammatory cytokines (IL-1b, TNFa or oncostatin M (OSM)) and investigating gene expression after 8–24 hours. The cytokines up-regulate catabolic, but down-regulate chondrocytic genes. However, whether this up- or down regulation is maintained after cytokine withdrawal is rarely investigated. In OA, the expression of catabolic genes is maintained in absence of cytokines and is transmitted to daughter cells, suggesting that epigenetic changes have resulted in permanent up-regulation. We asked whether it is possible to reproduce the epigenetic changes in vitro. Hence we compared gene expression and DNA methylation status in short-term (24h) versus long-term (2–3 weeks) cultures and, in particular, investigated the effects of cytokine withdrawal on these parameters.

Healthy chondrocytes, harvested from human femoral heads after hemiarthroplasty, were cultured in monolayer and passaged once (P1). For short-term culture, the P1 chondrocytes were divided into control culture or cultures with one-shot of IL-1b/OSM, harvested after 24h and 72h. For long-term culture, the cells were cultured with or without IL-1b/OSM, the latter added twice a week. Half the cells were harvested at confluence (3 weeks) and the other halves were passaged again and cultured without cytokines until confluence (2–3 weeks). RNA and genomic DNA were extracted from the same sample. IL-1b, MMP-3, MMP-13 and COL2A1 expression was quantified by real-time PCR. The percentage of cells with DNA methylation at the CpG site at −299bp of IL-1b promoter (a key CpG site) was quantified by a method we reported previously (Epigenetics, 2007; 2: 86–95).

As expected, expression of IL-1b MMP-3, MMP-13 had increased 100–4500-fold 24h after IL-1b/OSM treatment, but decreased considerably after cytokine withdrawal. COL2A1 expression was virtually abolished by IL-1b/OSM and not regained after 72h. The % DNA methylation did not change during the 72h. Repeated treatment with IL-1b/OSM in long-term culture also increased expression of IL-1b and the MMPs. However, this time expression was maintained or even increased after cytokine withdrawal and passaging. Expression inversely correlated with DNA methylation, which dropped from 59% to 35%. This de-methylation was preserved after passaging and cytokine withdrawal.

Conclusion: The widely used short-term cytokine-treated monolayer cultures of articular chondrocytes do not approximate the in vivo situation, where long-term aberrant expression correlates with DNA de-methylation. However, long-term treatment can mimic the loss of DNA methylation, which results in increased gene expression that is maintained after cytokine withdrawal. This model will facilitate studies on the mechanisms of DNA de-methylation, which might ultimately lead to novel therapeutic approaches for the treatment of OA

Epigenetic DNA de-methylation at specific CpG promoter sites is associated with abnormal synthesis of matrix-degrading enzymes in human osteoarthritis (Arthritis Rheum 52:3110–24), but the mechanisms that trigger or cause loss of DNA methylation are not known. Since inflammatory cytokines are known to induce abnormal gene expression in cultured chondrocytes, we wanted to know whether this induction also involved loss of DNA methylation. If so, the abnormal gene expression would be permanent and transmitted to daughter cells rather than a simple up-regulation. To test this hypothesis, we selected IL-1b as the abnormally expressed gene. Healthy chondrocytes, harvested from human femoral head cartilage following a fracture, were divided into five groups: non-culture; control culture; culture with the de-methylating agent 5-aza-deoxycyti-dine (5-aza-dC); culture with the inflammatory cytokine IL-1b; or with TNF-a/oncostatin M. Total RNA and genomic DNA were extracted at confluency, relative mRNA expression of IL-1b was quantified by Syb-rGreen-based real-time PCR, and a method for quantifying the percent of cells with DNA methylation at a specific CpG site was developed (Epigenetics 2: 86–95).

The methylation status of 16 CpG sites in the promoter of IL-1b was determined by the bisulfite modification method. The two CpG sites important for the epigenetic regulation of IL-1b were at -247bp and -290bp, the latter was selected to quantify DNA methylation. 5-aza-dC halved DNA methylation, which resulted in 4–8 fold increases in IL-1b expression; showing that DNA de-methylation per se increases gene expression. However, far greater effects were seen with the inflammatory cytokines. IL-1b increased its own expression 50–100 fold, whereas TNF-a/OSM increased IL-1b expression 500–1000 fold. DNA methylation varied inversely, IL-1b reducing methylation to ~15% and TNF-a/OSM abolishing DNA methylation almost completely.

This is the first demonstration that inflammatory cytokines have the capacity to cause loss of DNA methylation. We also confirmed previous work that IL-1b induces its own expression in healthy chondrocytes, thus setting up a dangerous positive feed-back mechanism. If true in vivo, both the auto-induction and the heritable expression of IL-1b by a growing number of chondrocytes could explain the unrelenting progression of osteoarthritis.

Osteoarthritis (OA) is characterised by progressive erosion of articular cartilage, which, once started, cannot be halted. The breakdown of cartilage is mediated by proteases, including MMP-3 and -13. These may initially be derived from the synovium but are also produced by OA chondrocytes, particularly in later stages of the disease. In normal articular chondrocytes, the proteases are not expressed and it has previously been shown that this is due, in part, to silencing by epigenetic mechanisms, in particular DNA methylation at so-called CpG sites (Arthritis & Rheumatism 52:3110–24). In OA, chondrocytes increasingly produce the enzymes and stably transmit the abnormal gene expression to daughter cells. This aberrant expression has been shown to be associated with an epigenetic “un-silencing” via demethylation of specific CpG sites within the promoter regions. Why and how this demethylation takes place is not known.

The pro-inflammatory cytokine IL-1beta is of potential importance in OA, where temporary synovitis could provide the cytokine. Moreover, it is well established that IL-1beta upregulates MMPs in chondrocyte monolayer cultures. We investigated whether the IL-1 mediated induction of MMPs was associated with DNA demethylation. Control chondrocytes were isolated from non-OA articular cartilage, obtained with ethical permission from patients with a femoral neck fracture, and expanded in monolayer culture. The cells from each patient were divided into pre-culture control, no-treatment control culture and IL-1 treated culture. When confluent, simultaneous RNA and DNA extraction was carried out. mRNA expression was analysed by RT-PCR and the methylation status of specific CpG sites within the promoters of MMP-3, -13, and IL-1â was determined in the same samples, using methylation-sensitive restriction enzymes and PCR. The pre-culture controls expressed type II collagen and low levels of MMP-3, but not MMP-13 nor IL-1beta. All IL-1 treated samples expressed high levels of MMP-3, -13, and, surprisingly, IL-1beta itself. As predicted, the large increases in MMP-3 and IL-1beta were associated with some loss of methylation at specific CpG sites in the promoter of these mediators with the strongest correlation between IL-1beta expression and promoter demethylation. IL-1beta thus induced its own expression, which was associated with loss of DNA methylation at one specific CpG site in the IL-1 promoter. If these in vitro results have relevance for the in vivo situation, then these findings suggest the following mechanisms for OA progression: An initial inflammatory episode in the synovium could induce IL-1beta in surface chondrocytes. Since this induction is associated with loss of DNA methylation, IL-1beta is now part of the expression repertoire of these chondrocytes and this abnormal expression is stably transmitted to daughter cells. IL-1 then could diffuse deeper into the cartilage to induce its own expression in adjacent chondrocytes, thus providing a continuous supply of IL-1beta even after synovial inflammation had abated. This may explain the unremitting progression of OA.

Osteoarthritis (OA) is characterized by progressive erosion of articular cartilage due to degradation of the cartilage matrix. The major enzymes involved are the matrix metalloproteases and aggrecanases, which are either derived from the synovium or synthesized by chondrocytes as OA progresses. This abnormal enzyme synthesis is part of a phenotypic change from normal to ‘degradative’ chondrocytes. If this change could be prevented, then disease progression might be slowed.

In early OA, degradative chondrocytes are only present in the superficial zone, but with increasing severity of OA, more chondrocytes become degradative cells so that, in high-grade OA, these cells are also located in the deep zone. We hypothesized the existence of a ‘factor X’, which diffuses from the superficial to the deep zone and induces cells to change phenotype and express the pro-teases. We further hypothesize that this factor is released by degradative chondrocytes. To test the hypothesis, we co-cultured explants of human superficial-zone OA cartilage (which contains degradative cells and thus factor X) with explants of deep-zone cartilage from fracture neck of femur patients (#NOF), which contains mostly normal chondrocytes that do not express the proteases. We investigated MMP expression by real time RT-PCR and protein synthesis by immunohistochemistry.

Before culture, MMP-2, -3, -9, or -13 were expressed in the superficial-zone OA cartilage, but not in deep-zone #NOF cartilage, as expected. After 4 weeks with separate culture of superficial zones and deep zones, no MMPs was expressed in deep zone chondrocytes, suggesting that culture per se did not induce expression of these enzymes. Neither did culture abolish expression in the superficial zone, as confirmed by RT-PCR and immunohistochemistry. However, when superficial-zone cartilage was co-cultured with deep-zone cartilage, MMP-3 expression were induced in deep- zone chon-drocytes, suggesting that a diffusible factor X, derived from degradative chondrocytes, had induced normal articular chondrocytes to express MMP-3. These experiments provide evidence for the existence of a factor that, when diffusing through the cartilage matrix, has the potential to induce normal non-enzyme expressing cells to become degradative chondrocytes.