In osteoarthritis (OA), articular chondrocytes undergo a phenotypic change and acquire a gene expression repertoire that is characterized by the aberrant expression of numerous catabolic genes including matrix metalloproteinases 3, 9 and 13, ADAMTS-4 and interleukin-1beta (IL1B = gene, IL-1b=protein). Previous studies (Arthritis Rheum 52;3110-24) have shown that epigenetic DNA demethylation at specific CpG sites in the relevant promoters accounts for the aberrant expression and that inflammatory cytokines (TNF-alpha, oncostatin M, IL-1b) can cause both aberrant expression and loss of DNA methylation, at least in vitro (Arthritis Rheum. 2009, 60,3303-3313). If the mechanisms of DNA de-methylation were understood, they might provide a new molecular target for therapeutic intervention. We hypothesize that nuclear translocation of the transcription factor NF-kB is involved in de-methylation because 1) we and others have demonstrated that cytokine-induced expression of IL1B in healthy chondrocytes requires NF-kB and 2) DNA de-methylation during B cell maturation was crucially dependent on the rel/NF-kB family (Nat Genet. 1996, 13,435-441). The aims of the study were to determine whether the NF-kB inhibitor BAY 11-7082 (BAY) could prevent the cytokine-induced loss of DNA de-methylation and thereby show that NF-kB is required for DNA de-methylation.

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

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.

Tissue loss, as a result of injury or disease, provides reduced quality of life for many and with an increasingly ageing population there is a greater requirement for skeletal repair strategies. An emerging attractive approach, tissue engineering, is based on the use of an appropriate source of progenitor cells, a scaffold conducive to cell attachment and maintenance of cell function and the delivery of appropriate growth factors. As a cell source, mesenchymal stem cells (MSCs) or marrow stromal cells derived from adult human tissues offer tremendous potential for tissue regeneration. However, to date, the plasticity, multipotentiality and characteristics of potential stem cells from fetal skeletal tissue remain poorly defined. We have examined, in preliminary studies, the multipotentiality and phenotypic properties of cell populations derived from human fetal femurs collected at 8–12 weeks post-conception in comparison to adult-derived mesenchymal stem cell populations including those isolated using STRO-1 immunoselection. Fetal cells were culture expanded from explants in basal media then maintained for periods of up to 28 days in monolayer cultures in adipogenic and osteogenic conditions. Cells were also maintained in chondrogenic conditions via the pellet culture method, maintained in established media conditions including TGF-â3, with cultures taken to 7, 14, 21 and 28 days. Adipocyte formation was confirmed by morphology: large amounts of lipid accumulation were observed by Oil Red O staining and aP2 (FABP-3) immunocytochemistry. Osteogenic differentiation was also confirmed by Type I Collagen immunocytochemistry. The growth of fetal cells on biomimetic scaffolds and their osteogenic activity was confirmed by confocal microscopy and Alkaline Phosphatase staining respectively. In chondrogenic conditions, chondrocytes were embedded within lacunae and extensive matrix deposition was observed using Alcian blue/Sirius red staining. The chondrogenic phenotype was confirmed by positive staining via SOX9 immunocytochemistry. Differentiation and proliferation were accelerated in fetal populations compared to adult-derived immunoselected MSCs. Plasticity of fetal cells has been demonstrated by the formation of large numbers of adipocytes within osteogenic populations. In summary we demonstrate the proliferative and multi-potential properties of fetal-derived chondrocytic cells in direct comparison to adult-derived MSCs including STRO-1 immunoselected populations. Given the demographic challenges and ethical issues surrounding current embryonic cell research, fetal cell populations may also provide a unique half-way model to address stem cell differentiation in comparison to adult cells. Elucidation of immunogenecity and selective differentiation will confirm the potential of these fetal cells as a unique alternate cell source for therapeutic approaches in the restoration of damaged or diseased tissue.

In osteoarthritis (OA) there is a loss of matrix components, especially aggrecan, which is a major structural component important for the integrity and function of articular cartilage. The breakdown of aggrecan is mediated by enzymes from the ADAM-TS (a disintegrin and metalloproteinase with thrombospondin motifs) family and recent studies have suggested that, in humans, ADAM-TS4 (aggrecanase-1) plays a major role. Articular chondrocytes do not express ADAM-TS4 in contrast to clonal OA chondrocytes. Since in any somatic cell non-expressed genes are thought to be silenced by DNA methylation in the promoter region, the aims of the project were twofold:

to localize enzyme expression for ADAM-TS4 by immunocytochemistry and

Using immunocytochemistry, we confirmed that there is an increased expression of ADAM-TS4 in OA chondrocytes, which initially occurs in chondrocytes of the superficial zone. As the Mankin score increases, ADAM-TS4 positive chondrocytes were found in duplets, then quadruplets until, at Mankin score > 10, all the cells in a typical OA clone were immunopositive for ADAM-TS4, suggesting that abnormal enzyme expression was inherited by daughter cells. DNA was extracted from femoral head cartilage of 24 patients, who had undergone hip replacement surgery for either symptomatic OA or following a fracture of neck of femur (#NOF). The latter was used as control due to the inverse relationship between OA and osteoporosis. For OA samples, it was important to sample only those regions for which immunocytochemistry had shown the presence of ADAM-TS4 synthesizing cells, i.e. the superficial zones near the weight-bearing region. DNA methylation only occurs at cytosines of the sequence 5′...CG...3′, the so-called CpG sites. To determine methylation status of specific CpG sites, methylation sensitive restriction enzymes were used, which will only cut DNA in the absence of methylation. By designing PCR primers that bracketed these sites, presence or absence of PCR bands could distinguish between methylated and non-methylated CpGs respectively. The ADAM-TS4 promoter contains a total of 13 CpG sites. Using restriction enzyme/primers combinations, it was possible to analyze 7 of these sites for methylation status. In the control group, all 7 CpG sites were methylated, while there was an overall 49% decrease of methylation in the OA group (p=< 0.0001). Some of the CpG sites were more consistently demethylated then others, one site at −753bp upstream from the transcription start site, showed a 86% decrease in methylation in OA compared to the control group (p=0.0005), while at other sites the decrease in methylation ranged from 36–50%. Conclusions. This study confirmed by immunocytochemistry that ADAM-TS4 is produced by OA chondrocytes, contributing to the degradation of their matrix. This abnormal enzyme expression is associated with DNA methylation. If a causal relationship could be proven in the future, then DNA de-methylation might play an important role in the pathogenesis of osteoarthritis and future therapies might be directed at influencing the methylation status.

Joint pain, as a consequence of cartilage degeneration or trauma results in severe pain or disability for millions of individuals worldwide. However, the potential for cartilage to regenerate is limited and there is an absence of clinically viable cartilage formation regimes. Cartilage is composed of only one cell type, is avascular and has a relatively simple composition and structure, thus cartilage tissue engineering has tremendous potential. Therefore, to address this clinical need, we have adopted a tissue engineering approach to the generation of cartilage ex vivo from mesenchymal cell populations encapsulated in polysaccharide templates form alginate and chitosan that favours chondrogenesis, and cultured within perfused or rotating bioreactor systems. To drive the chondrogenic phenotype, alginate beads were encapsulated with isolated human bone marrow cells, human articular chondrocytes or a combination of both in a 2:1 ratio, with the addition of TGF-â3, and placed in either a Synthecon rotating-wall bioreactor, perfused at a flow rate of 1ml/hour, or held in static conditions for 28 days. Alcian Blue and Sirius Red staining indicated ordered, structured and even cell distribution within capsules from the rotating bioreactor system in comparison with perfused and static conditions. Furthermore, alginate beads encapsulated with mixed cell populations that were cultured under static and rotating-wall conditions revealed positive staining for both collagen and proteoglycan, and with areas that closely resembled the formation of osteoid. Cell viability, assessed using the fluorescent dye Cell Tracker Green, indicated a higher proportion of metabolically active cells in capsules from the rotating-wall bioreactor than perfused or static under the conditions examined. Immunohistochemistry indicated the expression of type II collagen, SOX9 and C-MYC in samples from all conditions after 28 days. C-MYC is implicated in cell proliferation and differentiation and type II collagen and SOX9 are cartilage-specific markers. Biochemical analysis revealed significantly increased (p < 0.05) protein in samples encapsulated with mixed cell populations compared with alginate samples that were encapsulated with either bone marrow or chondrocytes. There was also a significant increase in protein in all samples that were cultured in the rotating-wall bioreactor in comparison with perfused or static conditions after 28 days. A significant increase in DNA was observed in the rotating-wall than perfused or static for the bone marrow cultures. Interestingly in chondrocyte cultures perfused conditions were found to result in significantly higher DNA than rotating-wall and static, and static conditions resulted in significantly higher DNA for alginate encapsulated with mixed cell populations. The current studies outline a tissue engineering approach utilising progenitor populations, bioreactors and appropriate stimuli to promote the formation of cartilage within a unique innovative polysaccharide capsule structure, and indicate the potential of rotating-wall systems to promote cartilage formation. Understanding the conditions required for the generation of functional cartilage constructs using such bioreactor systems carries significant clinical potential.

Cohort studies in humans have suggested that the peak bone mass attained at skeletal maturity may be programmed in utero. To investigate which aspects of bone development might be influenced in utero, we utilised a rat model of maternal protein insufficiency, which has previously been used to demonstrate the fetal origin of adult hypertension. In rodents, a growth plate remains present throughout life, even after longitudinal growth ceases. Generally, the height of the growth plate is related to the rate of bone growth. Fast growing bones have maximal height growth plates, and as bone growth slows down the height decreases until it remains stationary.

The aim of this study was to compare the morphology of long bones in aged rats that had been subjected to protein insufficiency in utero with that of controls. Rat dams were fed either an 18% casein control diet or a 9% casein low protein diet from conception until the end of pregnancy. The offspring were fed a normal diet until death (~72 weeks), when bone density was measured by dual energy X-ray absorptiometry (DEXA) and the tibiae and femurs were processed for histology.

The offspring of rats from the low protein group had a significantly lower bone mass, as assessed by DEXA. The major differences in bone structure were found in the growth plates, which were very irregular without the usual zones of resting, proliferating and hypertrophic chondrocytes. A number of unusual cellular events were noted to have taken place subsequent to cessation of growth, including: a) elimination of all chondrocytes in a number of regions, resulting in vast acellular areas; b) formation of chondroid bone and/or transdifferentiation of chondrocytes to bone-forming cells in other regions; c) partial resorption of those latter regions while the acellular regions were not resorbed; d) ‘horizontal’ apposition of bone against a smooth metaphyseal edge of the growth plate.

To compare the growth plates from the low and high protein groups semi-quantitatively, the degrees of the above features were scored. In addition, the heights of the growth plates were were assessed by two independent measurements. In the low protein group, the height of the growth plate were found to be significantly greater (p< 0.001). Additionally, the growth plates from this group of animals were observed to be more irregular with regards to all the features outlined above.

These findings are consistent with the hypothesis that growth trajectory and bone mass are programmed in early life. The increased height of the growth plate in animals undernourished in utero may reflect the cessation of growth at an earlier age. The increased irregularity of the growth plate in this group of animals may infer an earlier onset of age-related changes within the growth cartilage.

Chondrocytes at the lower zone of the growth plate must be eliminated to facilitate longitudinal growth; this is generally assumed to involve apoptosis. We attempted to provide definitive electron-microscopic evidence of apoptosis in chondrocytes of physes and chondroepiphyses in the rabbit. We were, however, unable to find a single chondrocyte with the ultrastructure of ‘classical’ apoptosis in vivo, although such a cell was found in vitro. Instead, condensed chondrocytes had a convoluted nucleus with patchy chromatin condensations while the cytoplasm was dark with excessive amounts of endoplasmic reticulum. These cells were termed ‘dark chondrocytes’. A detailed study of their ultrastructure combined with localisation methods in situ suggested a different mechanism of programmed cell death. In addition, another type of death was identified among the immature chondrocytes of the chondroepiphysis. These cells had the same nucleus as dark chondrocytes, but the lumen of the endoplasmic reticulum had expanded to fill the entire non-nuclear space, and all cytoplasm and organelles had been reduced to dark, worm-like inclusions. Since these cells appeared to be ‘in limbo’, they were termed ‘paralysed’ cells. It is proposed that ‘dark chondrocytes’ and ‘paralysed cells’ are examples of physiological cell death which does not involve apoptosis. It is possible that the confinement of chondrocytes within their lacunae, which would prevent phagocytosis of apoptotic bodies, necessitates different mechanisms of elimination.

Growth plates taken from five- to 20-week-old Japanese white rabbits were immunostained for c-Myc protein. This was localised both in the proliferating zone and upper hypertrophic zone at five weeks, whereas after ten weeks it was found mostly in the lower hypertrophic zone. The proliferating chondrocytes tended to show nuclear staining and the hypertrophic cells cytoplasmic staining, although the terminal hypertrophic chondrocytes sometimes expressed the protein in their nuclei. In the younger rabbits, c-Myc co-localised with proliferating cell nuclear antigen, whereas in the hypertrophic zone of older rabbits, it was present in some chondrocytes the nuclei of which also contained DNA breaks.

Our study suggests that, in the rabbit growth plate, c-Myc is associated with different cellular processes, depending on the age and the developmental stage of the chondrocytes.

Chondrocytes of the growth plate are generally assumed to undergo apoptosis, but the mechanisms which induce this cell death are not known. The Fas receptor is a mediator of the apoptotic signal in some systems. We studied its expression in situ in growth plates of rabbits aged from five to 20 weeks. In addition, we investigated the immunolocalisation in the growth plates of the bone proteins, osteonectin and osteocalcin, and the changes in their expression with age.

The Fas-positive chondrocytes were found mostly in the hypertrophic zone, as were the osteonectin-positive and osteocalcin-positive cells. The percentage of Fas-positive cells increased with age whereas little change was found in the number of osteonectin-positive and osteocalcin-positive chondrocytes.

Many of the Fas-positive chondrocytes were also TUNEL-positive. This strongly suggests that apoptosis in the growth plate is mediated through the Fas system.

Double immunostaining for osteocalcin and Fas showed that not all hypertrophic chondrocytes were of the same cell type. Some chondrocytes stained for osteocalcin only, others for Fas only, while some were positive for both.