ObjectivesOsteophytes are highly prevalent in osteoarthritis (OA) and are associated with pain and functional disability. These pathological outgrowths of cartilage and bone typically form at the junction of articular cartilage, periosteum and synovium. The aim of this study was to identify the cells forming osteophytes in OA.MethodsFluorescent genetic cell-labelling and tracing mouse models were induced with tamoxifen to switch on reporter expression, as appropriate, followed by surgery to induce destabilisation of the medial meniscus. Contributions of fluorescently labelled cells to osteophytes after 2 or 8 weeks, and their molecular identity, were analysed by histology, immunofluorescence staining and RNA in situ hybridisation. Pdgfrα-H2BGFP mice and Pdgfrα-CreER mice crossed with multicolour Confetti reporter mice were used for identification and clonal tracing of mesenchymal progenitors. Mice carrying Col2-CreER, Nes-CreER, LepR-Cre, Grem1-CreER, Gdf5-Cre, Sox9-CreER or Prg4-CreER were crossed with tdTomato reporter mice to lineage-trace chondrocytes and stem/progenitor cell subpopulations.ResultsArticular chondrocytes, or skeletal stem cells identified by Nes, LepR or Grem1 expression, did not give rise to osteophytes. Instead, osteophytes derived from Pdgfrα-expressing stem/progenitor cells in periosteum and synovium that are descendants from the Gdf5-expressing embryonic joint interzone. Further, we show that Sox9-expressing progenitors in periosteum supplied hybrid skeletal cells to the early osteophyte, while Prg4-expressing progenitors from synovial lining contributed to cartilage capping the osteophyte, but not to bone.ConclusionOur findings reveal distinct periosteal and synovial skeletal progenitors that cooperate to form osteophytes in OA. These cell populations could be targeted in disease modification for treatment of OA.

The loss of Sdc4 aggravates the course of experimental colitis, potentially through impaired epithelial cell integrity and regeneration. In view of the development of current treatment approaches involving Sdc4 inhibition for inflammatory disorders like arthritis, particular caution should be taken in case of adverse gastrointestinal side-effects.

Necroptotic cell death is characterized by an activation of RIPK3 and MLKL that leads to plasma membrane permeabilization and the release of immunostimulatory cellular contents. High levels of chondrocyte death occur following intra-articular trauma, which frequently leads to post-traumatic osteoarthritis development. The aim of this study is to assess necroptosis levels in cartilage post-trauma and to examine whether chondrocyte necroptotic mechanisms may be investigated and modified in vitro. Fractured human and murine cartilage, analysed immunohistochemically for necroptosis marker expression, demonstrated significantly higher levels of RIPK3 and phospho-MLKL than uninjured controls. Primary murine chondrocytes stimulated in vitro with the TNFα and AKT-inhibitor alongside the pan-caspase inhibitor Z-VAD-fmk exhibited a significant loss of metabolic activity and viability, accompanied by an increase in MLKL phosphorylation, which was rescued by further treatment of chondrocytes with necrostatin-1. Transmission electron microscopy demonstrated morphological features of necroptosis in chondrocytes following TNFα and Z-VAD-fmk treatment. Release of dsDNA from necroptotic chondrocytes was found to be significantly increased compared to controls. This study demonstrates that cartilage trauma leads to a high prevalence of necroptotic chondrocyte death, which can be induced and inhibited in vitro, indicating that both necroptosis and its consequential release of immunostimulatory cellular contents are potential therapeutic targets in post-traumatic arthritis treatment.

Osteoarthritis (OA) is characterized by cartilage degradation that is induced by inflammation. Sterile inflammation can be caused by damage-associated molecular patterns that are released by chondrocytes and activate pattern recognition receptors. We evaluate the role of toll-like receptor-3-activating RNA in the pathogenesis of OA. Toll-like receptor 3 (TLR3) was detected by semiquantitative reverse transcriptase PCR, western blotting and microscopy. Rhodamine-labelled poly(I:C) was used to image uptake in chondrocytes and full-thickness cartilage. The production of IFNβ in chondrocytes after stimulation with poly(I:C) as well as in the synovial fluid of OA patients was measured using ELISA. Chondrocyte apoptosis was chemically induced using staurosporine. Immunohistochemistry was performed to examine TLR3 expression and apoptosis in human and murine OA cartilage. RNA in synovial fluid was quantified by RiboGreen assay. Destabilisation of the medial meniscus was performed in TLR3−/− and wildtype mice. OA was assessed after eight weeks using OARSI score. TLR3 expression was confirmed by western blot and RT-PCR. Poly(I:C) was internalised by chondrocytes as well as cartilage and caused an increase of IFNβ production in murine (11.46 ± 11.63 (wo) to 108.7 ± 25.53 pg/ml; N = 6) and human chondrocytes (1.88 ± 0.32 (wo) to 737.6 ± 130.5 pg/ml; N = 3; p < 0.001). OA cartilage showed significantly more TLR3-positive (KL0 = 0.22 ± 0.24; KL4 = 6.02 ± 6.75; N ≥ 15) and apoptotic chondrocytes (KL0 = 0.6 ± 1.02; KL4 = 9.78 ± 7.79; N ≥ 12) than healthy cartilage (p < 0.001). Staurosporine-induced chondrocyte apoptosis causes a dose-dependent RNA release (0 ng/ml = 1090 ± 39.1 ng/ml; 1000 ng/ml=2014 ± 160 ng/ml; N = 4; p < 0.001). Human OA synovial fluid contained increased concentrations of RNA (KL0-2 = 3408 ± 1129 ng/ml; KL4 = 4870 ± 1612ng/ml; N ≥ 7; p < 0.05) and IFNβ (KL0-2 = 41.95 ± 92.94 ng/ml; KL3 = 1181 ± 1865ng/ml; N ≥ 8; p < 0.05). TLR3−/− mice showed reduced cartilage degradation eight weeks after OA induction (OARSI WT = 5.5 ± 0.04; TLR3−/− = 3.75 ± 1.04; N ≥ 6) which was accompanied by gradually decreasing levels of TUNEL-positive cells (WT = 34.87 ± 24.10; TLR3−/ = 19.64 ± 7.89) resulting in decreased IFNβ expression (WT = 12.57 ± 5.43; TLR3−/− = 6.09 ± 2.07) in cartilage (p < 0.05). The release of RNA by apoptotic chondrocytes thus activating TLR3 signalling is one possible way of perpetuating inflammatory cartilage changes. The inhibition of TLR3 could be a possible therapeutic target for OA treatment.

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