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Effects of carrageenan induced synovitis on joint damage and pain in a rat model of knee osteoarthritis

Open ArchivePublished:July 19, 2018DOI:https://doi.org/10.1016/j.joca.2018.07.001

      Summary

      Objective

      Knee osteoarthritis (OA) is associated with ongoing pain and joint damage that can be punctuated by acute flares of pain and inflammation. Synovitis in normal knees might resolve without long-term detriment to joint function. We hypothesised that osteoarthritis is associated with impaired resilience to inflammatory flares.

      Design

      We induced synovitis by injecting carrageenan into rat knees with or without meniscal transection (MNX)-induced OA, and measured synovitis, weightbearing asymmetry (pain behaviour), and joint damage up to 35 days after OA induction (23 days after carrageenan-injection).

      Results

      Carrageenan injection induced weightbearing asymmetry for 1 week, transient increase in knee diameter for 2 days, and a sustained increase in synovial macrophages, endothelial cell proliferation and vascular density compared with naive vehicle-injected controls. MNX surgery induced weightbearing asymmetry and histological evidence of OA. Carrageenan-injection in MNX-operated knees was followed for 2 days by increased weightbearing asymmetry compared either to MNX+vehicle or to sham+carrageenan groups. OA structural damage and synovitis at day 35 were greater in MNX+carrageenan compared to MNX+vehicle and sham+carrageenan groups. Carrageenan injection did not induce OA in Sham-operated knees.

      Conclusion

      Intra-articular injection of the pro-inflammatory compound carrageenan in OA and sham-operated control knees induced a short term increase in joint pain. Even though pain flares resolved in both groups and damage was not induced in sham-operated knees, carrageen injection exacerbated long-term joint damage in OA knees. OA knees display less resilience to inflammatory episodes. Preventing inflammatory flares may be particularly important in preventing symptoms and long term joint damage in OA.

      Keywords

      Introduction

      Osteoarthritis (OA) is the most prevalent chronic joint disease affecting half of the world's population over the age of 65 years
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      Inflammation and joint damage are each closely associated with OA pain
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      The association of bone marrow lesions with pain in knee osteoarthritis.
      . Inflammation contributes to pain flares in OA, exacerbates cartilage damage and may lead to sustained sensitisation and thereby exacerbate chronic pain
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      Augmented pain behavioural responses to intra-articular injection of nerve growth factor in two animal models of osteoarthritis.
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      Characterizing pain flares from the perspective of individuals with symptomatic knee osteoarthritis.
      . Clinical trials with nerve growth factor (NGF) blocking agents confirm that peripheral sensitisation is critical to OA pain
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      Tanezumab for the treatment of pain from osteoarthritis of the knee.
      . Angiogenesis, formation of new blood vessels from pre-existing ones is a core component of chronic inflammation
      • Ashraf S.
      • Mapp P.I.
      • Walsh D.A.
      Angiogenesis and the persistence of inflammation in a rat model of proliferative synovitis.
      . In OA, angiogenesis is seen in the synovium and subchondral bone
      • Ashraf S.
      • Walsh D.A.
      Angiogenesis in osteoarthritis.
      • Ashraf S.
      • Mapp P.I.
      • Walsh D.A.
      Contributions of angiogenesis to inflammation, joint damage, and pain in a rat model of osteoarthritis.
      . Increased synovial angiogenesis is also associated with chronic synovitis and can exacerbate OA pain
      • Ashraf S.
      • Mapp P.I.
      • Walsh D.A.
      Angiogenesis and the persistence of inflammation in a rat model of proliferative synovitis.
      • Haywood L.
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      • Pearson C.I.
      • Gill S.E.
      • Ganesan A.
      • Wilson D.
      • et al.
      Inflammation and angiogenesis in osteoarthritis.
      .
      Intra-articular injection of corticosteroids can reduce OA-associated inflammation and pain, but might not be immediately accessible during arthritis flares, and risk adverse events in particular with frequent usage. Oral analgesics such as non-steroidal anti-inflammatory drugs (NSAIDS) and opioids might have limited efficacy and also have potential risk of adverse events. Hence, it is urgent to increase our understanding of OA pain mechanisms in order to improve clinical outcomes.
      Preclinical models of knee OA have the potential to increase our understanding of OA pain. The rat medial meniscal transection (MNX) surgical model of knee OA is associated with pain behaviour and joint pathology similar to those observed in human knee OA
      • Ashraf S.
      • Mapp P.I.
      • Walsh D.A.
      Contributions of angiogenesis to inflammation, joint damage, and pain in a rat model of osteoarthritis.
      • Mapp P.I.
      • Sagar D.R.
      • Ashraf S.
      • Burston J.J.
      • Suri S.
      • Chapman V.
      • et al.
      Differences in structural and pain phenotypes in the sodium monoiodoacetate and meniscal transection models of osteoarthritis.
      . Intra-articular injection of 2% carrageenan into rat knee joints produces synovitis and pain behaviour measured as hindlimb weight bearing asymmetry but the inflammation is not sufficient to induce osteoarthritic changes in the cartilage and bone
      • Ashraf S.
      • Mapp P.I.
      • Walsh D.A.
      Angiogenesis and the persistence of inflammation in a rat model of proliferative synovitis.
      • Fehrenbacher J.C.
      • Vasko M.R.
      • Duarte D.B.
      Models of inflammation: carrageenan- or complete Freund's Adjuvant (CFA)-induced edema and hypersensitivity in the rat.
      . Inflammation therefore might not be sufficient alone to induce OA, even though inflammation might contribute to pain and joint damage in people with OA. The normal joint therefore may display some resilience to acute synovitis. We have previously shown that OA knees are more sensitive to intra-articular injection of NGF compared to non-osteoarthritic knees
      • Ashraf S.
      • Mapp P.I.
      • Burston J.
      • Bennett A.J.
      • Chapman V.
      • Walsh D.A.
      Augmented pain behavioural responses to intra-articular injection of nerve growth factor in two animal models of osteoarthritis.
      . This led us to hypothesise that OA knees might be less resilient to inflammation-induced pain and joint damage, than are non-arthritic knees. Understanding what makes OA-knees more “sensitive” and preventing or reversing this vulnerability might offer novel approaches to controlling OA pain.
      In this study we evaluated the effects of acute induction of synovitis by intra-articular injection of carrageenan in sham-operated and osteoarthritic rat knees in order to explore whether OA knees are more sensitive than non-arthritic knees to the pain and structural effects of synovitis.

      Methods

      In vivo experiments were performed on male Lewis rats (Charles River, Margate, Kent, UK, 250–270 g, age≈8 weeks, n = 8 per treatment group unless otherwise stated) were carried out in accordance with UK Home Office (HO) regulations and following the guidelines of the International Association for the Study of Pain. Severity band of the experiments did not exceed that permissible under the HO licence. Rats were closely monitored throughout the experiments to ensure that knee joint swelling did not exceed 50% of the starting diameter and that they did not exhibit signs of lameness that prevented their free movement around the cage. Rats were housed under standard conditions of 12 h light/dark cycle, with unlimited access to food and water. Rats were anesthetised with isoflurane (2% in O2) prior to surgery and intra-articular injections. All outcome measurements were made by an observer (SA) blinded to treatment.

      MNX model of OA

      OA was induced on day 0 in the left knee by medial meniscal transection (MNX) as previously described
      • Ashraf S.
      • Mapp P.I.
      • Walsh D.A.
      Contributions of angiogenesis to inflammation, joint damage, and pain in a rat model of osteoarthritis.
      . Sham operated rats were used as controls, these rats underwent all aspects of the procedure apart from meniscal transection. We previously demonstrated that sham-operated controls do not display pain behaviour, inflammation or OA structural changes beyond 7 days after surgery
      • Mapp P.I.
      • Avery P.S.
      • McWilliams D.F.
      • Bowyer J.
      • Day C.
      • Moores S.
      • et al.
      Angiogenesis in two animal models of osteoarthritis.
      • Mapp P.I.
      • Walsh D.A.
      • Bowyer J.
      • Maciewicz R.A.
      Effects of a metalloproteinase inhibitor on osteochondral angiogenesis, chondropathy and pain behavior in a rat model of osteoarthritis.
      .

      Intra-articular injection of 2% carrageenan

      Rats were given either a single 50 μl intra-articular injection of 2% carrageenan in sterile 0.9% Normal saline, pH 7.4, or Normal saline alone in the left knee on day 12. Day 12 time-point was used because at this time-point MNX induced changes in knee joint pathology (inflammation, cartilage damage, osteophytes and loss of osteochondral junction [OCJ] integrity) are evident, and significantly higher than sham-operated rats
      • Mapp P.I.
      • Sagar D.R.
      • Ashraf S.
      • Burston J.J.
      • Suri S.
      • Chapman V.
      • et al.
      Differences in structural and pain phenotypes in the sodium monoiodoacetate and meniscal transection models of osteoarthritis.
      . Pathological changes in the sham-operated rats as a result of the surgery have resolved by this time-point and knee-joints resemble non-arthritic naïve controls until day 35. The extent of damage in the MNX-rats seen at day 14 remains similar to that observed at day 35, and no significant difference between joint damage at day 14 and day 35 following meniscal transection surgery is observed
      • Mapp P.I.
      • Sagar D.R.
      • Ashraf S.
      • Burston J.J.
      • Suri S.
      • Chapman V.
      • et al.
      Differences in structural and pain phenotypes in the sodium monoiodoacetate and meniscal transection models of osteoarthritis.
      .

      Treatment groups

      The following treatment groups were analysed in this study:
      • 1.
        Non-arthritic naïve rats were given intra-articular injection of saline in the left knee on day 12 (naive + vehicle).
      • 2.
        Rats undergoing sham surgery (day 0, left knee joint) were injected with saline in the left knee on day 12 (sham + vehicle).
      • 3.
        Rats undergoing sham surgery (day 0, left knee joint) were injected with 2% carrageenan in the left knee on day 12 (sham + 2% carrageenan).
      • 4.
        OA was induced in the left knee by MNX surgery on day 0, followed by saline injection in the left knee on day 12 (MNX + vehicle).
      • 5.
        OA was induced in the left knee by MNX surgery on day 0, followed by 2% carrageenan injection in the left knee on day 12 (MNX + 2% carrageenan).

      Pain behaviour and joint swelling

      Prior to surgery and intra-articular injection, as well as on days 12, 13, 14, 19, 26 and 35 after surgery, pain behaviour was measured as weight bearing asymmetry between the ipsilateral and contralateral hind limbs using an incapacitance meter (Linton Instruments)
      • Ashraf S.
      • Mapp P.I.
      • Walsh D.A.
      Contributions of angiogenesis to inflammation, joint damage, and pain in a rat model of osteoarthritis.
      . Joint swelling was also measured at these time-points using digital electronic calipers (Mitutoyo) as the difference in knee diameter (mm) between the ipsilateral and contra-lateral (uninjected) knees.

      Tissue collection and preparation

      Rats were killed by asphyxiation in CO2 35 days after surgery. Synovia with patellae were collected immediately, embedded in optimal cutting temperature (OCT) compound and snap frozen. Tibiofemoral joints were preserved for 48 h at room temperature (RT) in neutral buffered formalin (containing 4% paraformaldehyde), then decalcified in 10% ethylenediaminetetraacetic acid (EDTA) in 10 mM Tris buffer (pH 6.95) for 4 weeks at RT. Coronal sections of trimmed joint tissues were mounted in paraffin wax.

      Synovial morphology

      Synovial sections (5 μm thick) were cut using a motorized cryostat and visualised using a 20× objective lens. CD31-positive cells and proliferating cell nuclear antigen (PCNA)-immunoreactive CD31-positive cells were used to identify endothelial cells (ECs) and proliferating ECs, respectively, in the synovia as two separate measures of the extent of angiogenesis, as previously described
      • Ashraf S.
      • Mapp P.I.
      • Walsh D.A.
      Contributions of angiogenesis to inflammation, joint damage, and pain in a rat model of osteoarthritis.
      • Waseem N.H.
      • Lane D.P.
      Monoclonal antibody analysis of the proliferating cell nuclear antigen (PCNA). Structural conservation and the detection of a nucleolar form.
      • Male D.
      • Rahman J.
      • Linke A.
      • Zhao W.
      • Hickey W.
      An interferon-inducible molecule on brain endothelium which controls lymphocyte adhesion mediated by integrins.
      . Synovium was delineated according to morphology and synovial area was measured using a KS300 image analysis system (Image Associates, Thame, UK). Within this synovial region, a mask of the endothelial area was created by interactive colour thresholding of CD31-positive blood vessels. This endothelial mask was applied over corresponding images of PCNA-positive and DAPI-positive nuclei. Nuclei falling within endothelium were counted as PCNA-positive endothelial nuclei and total endothelial nuclei respectively. EC proliferating index (EC PCNA Index) was defined as the percentage of endothelial cell nuclei positive for PCNA. Vascular density (EC Fractional Area) and macrophage infiltration (macrophage fractional area) was defined as the percentage of synovial area that was CD31-and ED1-positive respectively
      • Ashraf S.
      • Mapp P.I.
      • Walsh D.A.
      Angiogenesis and the persistence of inflammation in a rat model of proliferative synovitis.
      • Dijkstra C.D.
      • Dopp E.A.
      • Joling P.
      • Kraal G.
      The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2 and ED3.
      . Four fields of view on each synovial section were selected that displayed the highest densities of either CD31-positive blood vessels, or PCNA-positive endothelial nuclei, or ED1-positive macrophages. For vascular density, four consecutive sections per case with four fields of view per section were used. This optimal number was determined in previous experiments to minimise the coefficient of variation and so that the observed mean lies within ± 12.5% of the true mean
      • Ashraf S.
      • Mapp P.I.
      • Walsh D.A.
      Angiogenesis and the persistence of inflammation in a rat model of proliferative synovitis.
      • Walsh D.A.
      • Rodway H.A.
      • Claxson A.
      Vascular turnover during carrageenan synovitis in the rat.
      . Nuclei were counterstained with the fluorescent deoxyribonucleic acid (DNA) ligand 4′,6-diamidino-2-phenylindole (DAPI)
      • Sanna P.P.
      • Jirikowski G.F.
      • Lewandowski G.A.
      • Bloom F.E.
      Applications of DAPI cytochemistry to neurobiology.
      . PCNA and ED1 were developed with diaminobenzidine using the glucose oxidase/nickel-enhanced method. Endothelium markers were developed using Fast Red (Sigma). Staining procedures were as previously described
      • Walsh D.A.
      • Rodway H.A.
      • Claxson A.
      Vascular turnover during carrageenan synovitis in the rat.
      .

      Joint pathology

      The entire medial tibial plateau of the mid-coronal sections was stained with Safranin O and used to assess joint pathology through a 4× objective lens. A single section per knee (taken from the same mid-coronal plane) per joint was analysed.
      Chondropathy was evaluated as previously described
      • Janusz M.J.
      • Bendele A.M.
      • Brown K.K.
      • Taiwo Y.O.
      • Hsieh L.
      • Heitmeyer S.A.
      Induction of osteoarthritis in the rat by surgical tear of the meniscus: inhibition of joint damage by a matrix metalloproteinase inhibitor.
      . Briefly, cartilage damage was scored on a scale of 0 (cartilage of normal appearance) to 5 (severe, full thickness degeneration to the tidemark). Extent of damage was estimated as the proportion (1/3, 2/3 or 3/3) of the section of the medial tibial plateau that was involved. The cartilage damage score was then multiplied by 1, 2 or 3 respectively to give an overall chondropathy score (range 0–15).
      Osteophytes were evaluated by 2 characteristics, maturity and size, as previously described
      • Gerwin N.
      • Bendele A.M.
      • Glasson S.
      • Carlson C.S.
      The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the rat.
      • van der Kraan P.M.
      • van den Berg W.B.
      Osteophytes: relevance and biology.
      . Briefly, osteophyte maturity was scored from 0 to 3, as 0 = no osteophytes, 1 = predominantly cartilage, 2 = mixed cartilage and bone with active vascular invasion and endochondral ossification, and 3 = predominantly bone. Osteophyte size was graded as 0 = marginal zone proliferative changes (<200 μm), 1 = small (200–299 μm), 2 = moderate (300–399 μm), 3 = large (400–499 μm) and 4 = very large (≥500 μm).
      The integrity of the osteochondral junction was measured as the number of channels present in the articular cartilage of a Safranin O–stained section of the medial tibial plateaux, using a 20× objective lens. Osteochondral integrity was measured as the number of channels crossing the osteochondral junction (OCJ) into the cartilage from the underlying subchondral bone
      • Ashraf S.
      • Mapp P.I.
      • Walsh D.A.
      Contributions of angiogenesis to inflammation, joint damage, and pain in a rat model of osteoarthritis.
      .
      All image analyses were performed by two observers (SA and SMS) blinded to experimental details using a Zeiss Axioskop 50 microscope (Carl Zeiss, Welwyn Garden City, UK) and a KS300 image analysis system (Image Associates, Thame, UK).
      Joint damage (osteophytosis, chondropathy and integrity of the osteochondral junction) was evaluated combining two separate experiments (MNX+2% carrageenan: n = 16, MNX: n = 16, Sham+2% carrageenan: n = 8, Sham: n = 8, Naives: n = 16), to increase the number of animals, hence the power of the study.

      Statistical analysis

      Power calculations were performed before the study (based on the primary outcome measure of pain behaviour) to show a difference of 10% (P < 0.05) using a power of 80%. Data were analysed using the Statistical Package for the Social Sciences V.16 (SPSS, Chicago, Illinois, USA) and graphically presented using Prism (GraphPad, San Diego, California, USA). Parametric or non-parametric statistical methods were used to analyse normally distributed and non-normally distributed data sets respectively. Normally distributed data (log transformed EC PCNA Indices, and endothelial and macrophage fractional areas, weightbearing asymmetry and knee diameter) were analysed using one-way analysis of variance (ANOVA). Pairwise comparisons were made using Student's t-test with Bonferroni's correction for multiple comparisons. Non-normally distributed data (joint damage scores and osteochondral vascularity) were analysed using Kruskal–Wallis test followed by Mann–Whitney test for pairwise comparisons with Bonferroni's correction. Data are presented graphically as either mean ± s.e.m. or mean (95% Confidence Interval [CI]) unless otherwise stated and as mean ± s.e.m. in the text. Presented statistics are the adjusted P-values. A two-tailed P < 0.05 was taken to indicate statistical significance.

      Reagents

      Biotinylated rat-adsorbed horse anti-mouse antibody (BA2001) and avidin–biotin complexes (ABC Kits) were from Vector Laboratories (Peterborough, UK). Monoclonal antibodies to macrophages (clone ED1, MCA341GA) and CD31 (clone TLD-3A12, MCA1334GA) were from Bio-Rad AbD Serotec Ltd (Oxford, UK). Antibody to PCNA (clone PC10, M0879) was obtained from Dako. All other chemicals including λ-carrageenan (22049) were obtained from Sigma–Aldrich (Poole, UK).

      Results

      Intra-articular injection of 2% carrageenan induces pain behaviour and synovitis

      Sham operated rats do not display pain behaviour, inflammation, or OA structural changes beyond 7 days after surgery
      • Mapp P.I.
      • Sagar D.R.
      • Ashraf S.
      • Burston J.J.
      • Suri S.
      • Chapman V.
      • et al.
      Differences in structural and pain phenotypes in the sodium monoiodoacetate and meniscal transection models of osteoarthritis.
      • Mapp P.I.
      • Avery P.S.
      • McWilliams D.F.
      • Bowyer J.
      • Day C.
      • Moores S.
      • et al.
      Angiogenesis in two animal models of osteoarthritis.
      • Mapp P.I.
      • Walsh D.A.
      • Bowyer J.
      • Maciewicz R.A.
      Effects of a metalloproteinase inhibitor on osteochondral angiogenesis, chondropathy and pain behavior in a rat model of osteoarthritis.
      . In this study sham-operated and vehicle-injected rats showed pain behaviour (weight-bearing asymmetry) and knee joint swelling (knee diameter) that throughout the study were similar to naïve controls injected with vehicle. Intra-articular injection of carrageenan in non-arthritic knees (sham operated) on day 12, at a time point when sham-operated rats do not show any weight bearing asymmetry (Fig. 1), produced an increase in pain behaviour and joint swelling [Fig. 1(A) and (B)]. Responses returned to those seen in sham-operated and vehicle-injected group within 2 weeks (at day 26). However increases in synovial inflammation and angiogenesis (proliferating ECs and vascular density) were observed at day 35 (Fig. 2, Fig. 3, Fig. 4), at a time point when weight bearing asymmetry and joint swelling had subsided to that of sham-operated and vehicle-injected group (Fig. 1). Intra-articular injection of carrageenan into sham-operated non-arthritic knees did not cause joint damage at day 35 (Fig. 5, Fig. 6). Joint tissue sections resembled those from sham-operated and vehicle-injected group.
      Fig. 1
      Fig. 1Effects of intra-articular injection of 2% carrageen on knee joint pain and swelling following meniscal transection surgery. A, pain behaviour measured as difference in hind-limb weight bearing asymmetry. B, joint swelling measured as difference in knee diameter. Meniscal transection or sham surgeries were performed on day 0. Intra-articular injection of 2% carrageenan or vehicle (saline) was given on day 12 (vertical dotted line). Sham-operated rats showed similar pain behaviour and joint swelling to naïve controls throughout the study. Arthritic (MNX) rats showed increased pain behaviour 12 days after surgery compared with sham-operated or naïve controls and this increase was maintained until day 35. MNX surgery was not associated with significant joint swelling. Sham operated rats injected with carrageenan on day 12 showed increase in pain behaviour until day 19 which was reduced to sham-operated and vehicle-injected control levels by day 35. MNX rats given intra-articular injection of 2% carrageenan on day 12 showed transient increase in pain behaviour, for 2 days until day 14, compared to sham operated and carrageenan injected rats, and when compared to MNX rats. Pain behaviour at day 35 was similar in the MNX groups injected with or without carrageenan. Transient increase in joint swelling was seen following injection of carrageenan in sham operated and MNX operated rats until day 19 (7 days after carrageenan injection). Joint diameters remained higher at day 35 in the MNX operated rats given carrageenan injection, whereas they returned to non-arthritic control (sham+vehicle) levels at day 26 in the sham-operated and carrageenan-injected rats. Graphs show mean ± s.e.m.. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs sham-operated and vehicle injected controls, +P < 0.05, ++P < 0.01, +++P < 0.001, ++++P < 0.0001 vs MNX-operated and vehicle-injected group, #P < 0.05, ##P < 0.01, ###P < 0.001 vs sham-operated and 2% carrageenan-injected groups. n = 8 rats per group.
      Fig. 2
      Fig. 2Synovial macrophage infiltration in rat knee joints 35 days after either meniscal transection or sham surgery with intra-articular injection of 2% carrageenan or vehicle (saline). CD68 positive macrophage fractional area, showing the extent of macrophage infiltration was similar in sham-operated and vehicle injected rats, and naïve controls (A). Macrophage infiltration was increased after meniscal transection surgery (A and C) compared to sham-operated controls (A and B) and further exacerbated after the intra-articular injection of 2% carrageenan into rat knee joints 12 days after meniscal transection surgery (A and D). Sham operated rats injected with 2% carrageenan (E) showed similar levels of macrophage infiltration compared with MNX group given vehicle injection (A and C). Macrophages are delineated by immunoreactivity for ED1. Scale bar = 100 μm *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs sham-operated and vehicle-injected controls. +P < 0.05, ++P < 0.01, +++P < 0.001, ++++P < 0.0001 vs MNX+2% carrageenan group. Data are mean ± 95% CI of n = 8 rats per group.
      Fig. 3
      Fig. 3Synovial angiogenesis in rat knee joints 35 days after either meniscal transection or sham surgery with intra-articular injection of 2% carrageenan or vehicle (saline). EC PCNA Index, a measure of synovial angiogenesis was similar in sham-operated and vehicle injected rats compared with naïve controls (A). Synovial angiogenesis was increased after meniscal transection surgery (A and C) compared to sham-operated and vehicle injected controls (A and B) and further exacerbated after the intra-articular injection of 2% carrageenan into rat knee joints 12 days after meniscal transection surgery (A and D). Sham operated rats injected with 2% carrageenan (E) showed similar levels of synovial angiogenesis compared with MNX and vehicle injected group (A and C). ECs are delineated in red by immunoreactivity for CD31 (blue arrows). Proliferating nuclei are delineated black by immunoreactivity for proliferating cell nuclei antigen (PCNA; green arrows). Black arrows indicate proliferating ECs, which contain PCNA-immunoreactive nuclei. Scale bar = 100 μm *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs sham-operated and vehicle injected controls. +P < 0.05, ++P < 0.01, +++P < 0.001, ++++P < 0.0001 vs MNX+2% carrageenan group. Data are mean ± 95% CI of n = 8 rats per group.
      Fig. 4
      Fig. 4Synovial vascular density in rat knee joints 35 days after either meniscal transection or sham surgery with intra-articular injection of 2% carrageenan or vehicle (saline). Synovial vascular density measured as EC fractional area was similar in sham-operated and vehicle-injected rats when compared to naïve controls (A). Synovial vascular density was not significantly increased after meniscal transection surgery (A and C) compared to sham-operated and vehicle-injected controls (A and B) but was further exacerbated after the intra-articular injection of 2% carrageenan into rat knee joints 12 days after meniscal transection surgery (A and D). Sham operated rats injected with 2% carrageenan (E) showed greater synovial vascular density compared with sham-operated and vehicle-injected controls (A and B). ECs are delineated in red by immunoreactivity for CD31. Scale bar = 100 μm *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs sham-operated and vehicle-injected controls. +P < 0.05, ++P < 0.01, +++P < 0.001, ++++P < 0.0001 vs MNX+2% carrageenan group. Data are mean ± 95% CI of n = 8 rats per group.
      Fig. 5
      Fig. 5Histological changes (chondropathy, osteophytosis and channels at the osteochondral junction) of the medial tibial plateau 35 days after either meniscal transection or sham surgery with intra-articular injection of 2% carrageenan or vehicle (saline) control. Medial tibial plateau from naïve controls (A) and sham operated rats (B) given vehicle injection showing normal smooth cartilage and joint margins with no inflammatory tissue infiltration. The chondrocytes are homogenously distributed throughout the cartilage and there is neither evidence of proteoglycan loss nor channels entering the avascular cartilage. Medial tibial plateau from a sham-operated animal having 2% carrageenan intra-articular injection (12 days after surgery) into the operated knee joint (D), showing inflammatory tissue infiltration (black asterisk), moderate cartilage changes with very few channels crossing into the cartilage (blue asterisk) and some channels that have approached the bone cartilage interface but have not yet crossed into the cartilage (pink asterisk). Medial tibial plateau from MNX rat given vehicle injection on day 12 (C) showing severe cartilage loss (arrows) with a large osteophyte formed at the joint margin (circled) and several channels crossing into or approaching the cartilage (dotted line; separates underlying bone from cartilage). Osteophyte seen in the image is at the intermediate stage of maturity (Grade 2), as it is a mixture of cartilage and bone (C). No inflammatory tissue infiltration is evident. Medial tibial plateau from a rat having intra-articular injection of 2% carrageenan (12 days after surgery) into the operated knee joint (MNX) (E), showing infiltration of inflammatory tissue (black asterisk), and a mature osteophyte (Grade 3), predominantly bone (circled) with enhanced, cartilage loss (arrows). Representative coronal sections of the medial tibial plateaux stained with Safranin-O. Scale bar = 100 μm.
      Fig. 6
      Fig. 6Exacerbation in OA structural changes in medial tibial plateaux following intra-articular injection of 2% carrageenan or vehicle (saline) in the rat MNX model of OA. MNX+vehicle: n = 16, MNX+2% carrageenan: n = 16, sham+vehicle: n = 8, sham+2% carrageenan: n = 8, naive controls given vehicle injection: n = 16. MNX or sham-operated animals were given intra-articular injection into their operated knee joint of 2% carrageenan or vehicle control 12 days after surgery. Intra-articular injection of 2% carrageenan in the rat MNX model of OA was followed by significant increases in osteophyte maturity and cartilage damage score compared to vehicle-injected MNX-operated rats. Structural changes in the joints were more severe in the combined MNX and carrageenan model compared to sham-operated and carrageenan-injected rats. No difference in structural damage was seen between the sham operated and vehicle injected, sham-operated and 2% carrageenan-injected and naive control rat knees. Data are given for individual knees. Graphs show mean ± s.e.m.. **P < 0.01, ***P < 0.001 vs sham-operated and vehicle-injected controls. +P < 0.05, ++P < 0.01, +++P < 0.001 vs MNX-operated, carrageenan-injected (MNX+2% carrageenan) rat knees.

      Effects of OA on carrageenan induced pain behaviour and synovitis

      At day 12, MNX-operated rats showed increased weight bearing asymmetry (60.0 ± 9.0) compared with sham-operated and vehicle-injected rats (7.9 ± 2.1, P < 0.0001) and this increase was sustained until day 35 (mnx+vehicle: 27.2 ± 5.2, sham+vehicle: 1.3 ± 2.2, P < 0.05). Weight bearing asymmetry in the MNX group up to 35 days after surgery was similar to that 24 h following intra-articular injection of 2% carrageenan in sham-operated rats [Fig. 1(A)]. Intra-articular injection of 2% carrageenan into the knees 12 days after MNX surgery, was followed by elevated pain behaviour (93.6 ± 5.3) when compared with vehicle injection after MNX surgery (45.2 ± 5.3, P < 0.0001) as well as when compared with sham-operated and carrageenan-injected rats (55.0 ± 8.0, P < 0.001). This increase was maintained for 2 days after carrageenan-injection, after which weight bearing asymmetry did not significantly differ between treatment groups (MNX+2% carrageenan vs MNX+vehicle vs sham+2%carrageenan) through the end of the study (day 35).
      MNX-operated rats showed no significant joint swelling measured as knee diameter for the duration of the study, whereas carrageenan-injection 12 days after surgery was followed by increases in joint swelling. Early increases in joint swelling up to 7 days after carrageenan injection (19 days after surgery) were similar between MNX- and sham-operated knees. Joint swelling after carrageenan injection into sham-operated knees returned to non-arthritic control (sham+vehicle) levels by 14 days after injection (26 days after surgery), whereas MNX+carrageenan group continued to show a small but significant increase in joint diameter at day 35 compared with non-arthritic (sham+vehicle) controls. Vehicle-injection in sham-operated or naïve controls did not produce significant joint swelling.
      Features of chronic synovitis at day 35, including synovial macrophage infiltration and angiogenesis (proliferating ECs and vascular density) were increased after meniscal transection surgery compared to sham-operated and vehicle-injected knees (Fig. 2, Fig. 3, Fig. 4). ED1-immunoreactive macrophages were localized to the synovial lining and dispersed throughout the synovial sublining regions (Fig. 2). CD31-immunoreactive blood vessels were distributed throughout the depth of synovia, occasionally containing PCNA-immunoreactive nuclei (Fig. 3, Fig. 4). Rats given intra-articular injection of carrageenan into the sham-operated knees showed levels of synovial inflammation and angiogenesis, determined by histology, similar to those seen in the MNX+vehicle group at day 35 (Fig. 2, Fig. 3, Fig. 4). MNX-operated knees injected with carrageenan showed greater synovial inflammation and angiogenesis at day 35 when compared with MNX-operated and vehicle-injected knees, and also the increase was greater compared to the sham-operated, carrageenan injected knees (Fig. 2, Fig. 3, Fig. 4).

      Effects of carrageenan injection on OA structural changes

      The MNX model was associated with the expected OA changes in the medial tibial plateaux at day 35. These OA changes included cartilage damage, proteoglycan loss, reduced chondrocyte density, subchondral bone remodelling including increased numbers of channels breaching the OCJ, and osteophyte formation at joint margins (Fig. 5, Fig. 6). Carrageenan-injection was followed by increased cartilage damage (mean increase in score = 4.19) and increased maturity of the osteophytes (mean increase in score = 0.94) in the MNX model of OA compared to vehicle-injected, MNX-operated knees (Fig. 5, Fig. 6). By contrast sham-operated rats (with or without intra-articular injection of carrageenan on day 12) had normal joint appearance at day 35, similar to vehicle-injected naive rat knees (Fig. 5, Fig. 6).

      Discussion

      We demonstrated a short term pain behavioural and joint swelling response to intra-articular carrageenan injection in rats with knee OA, modelling an inflammatory pain flare. Carrageenan-injection in OA knees also increased cartilage damage and osteophyte maturity, despite carrageenan not causing demonstrable joint damage in non-arthritic knees. Our findings demonstrate that OA knees are more sensitive to carrageenan-induced pain, inflammation and joint damage. Even though this increased pain behaviour associated with transient inflammatory episode eventually subsides, joint damage may last long term.
      Advent of sensitive imaging systems has suggested that, even at its earliest stages, before visible cartilage degeneration has occurred, OA is already an inflammatory disease having a strong association between the presence of synovitis and OA progression
      • Atukorala I.
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      . In a significant subset of patients with OA, chronic low-grade inflammation may be a major driver of ongoing joint damage
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      . In addition, clinical studies demonstrate that chronic synovitis leads to long term joint pain
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      Association of joint inflammation with pain sensitization in knee osteoarthritis: the multicenter osteoarthritis study.
      . Although OA is a chronic disease, people with OA experience different profiles of pain, both in terms of duration and intensity, intermittent or constant. Transient episodes of pain and inflammation known as flares may lead to long term joint damage
      • Ashraf S.
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      Augmented pain behavioural responses to intra-articular injection of nerve growth factor in two animal models of osteoarthritis.
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      • et al.
      Characterizing pain flares from the perspective of individuals with symptomatic knee osteoarthritis.
      .
      Carrageenan injection was followed by increased pain behaviour both in sham- and in MNX-operated rats. However this increase in pain behaviour was short lived, returning to levels in vehicle-injected animals by 1 week, despite persistent increases in histological evidence of inflammation and joint damage in MNX-operated rats. Symptomatic resolution of pain might not be sufficient to indicate resolution of underlying disease.
      Synovitis might also be detected by Magnetic Resonance Imaging
      • Felson D.T.
      • Niu J.
      • Neogi T.
      • Goggins J.
      • Nevitt M.C.
      • Roemer F.
      • et al.
      Synovitis and the risk of knee osteoarthritis: the MOST Study.
      or ultrasound
      • Sarmanova A.
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      • Bhattacharya A.
      • Valdes A.M.
      • Walsh D.A.
      • et al.
      Association between ultrasound-detected synovitis and knee pain: a population-based case-control study with both cross-sectional and follow-up data.
      in asymptomatic human knees, and the potential contribution of subclinical synovitis to ongoing joint damage in human OA deserves further study. More sensitive measures of synovitis might be developed for serial use in rats, for example using radioisotopes and imaging, in order to help elucidate more precisely the time course of carrageenan-induced synovitis in arthritic and non-arthritic knees, and possible effects of subclinical synovitis on joint damage.
      Interestingly, even though synovial inflammation and angiogenesis persisted in the sham-operated group given intra-articular injection of carrageenan, no joint damage was observed. The lower intensity of inflammation, macrophage infiltration, angiogenesis or other qualitative differences in inflammation not identified in the current study, might contribute to this absence of OA joint damage. However, our previous studies have shown that inflammation and angiogenesis at this level significantly contribute to OA structural change, in that inhibition of inflammation or angiogenesis can reduce structural change in the MNX-induced OA model
      • Ashraf S.
      • Mapp P.I.
      • Walsh D.A.
      Contributions of angiogenesis to inflammation, joint damage, and pain in a rat model of osteoarthritis.
      • Mapp P.I.
      • Sagar D.R.
      • Ashraf S.
      • Burston J.J.
      • Suri S.
      • Chapman V.
      • et al.
      Differences in structural and pain phenotypes in the sodium monoiodoacetate and meniscal transection models of osteoarthritis.
      . It seems possible, therefore, that the lack of OA structural change in sham-operated animals injected with carrageenan might be due to a resilience of normal cartilage and bone to synovitis.
      Our data extend findings in a lapine surgical model of OA, in which chronic synovitis induced by weekly intra-articular injection of calcium pyrophosphate dehydrate (CPPD) crystals exacerbated OA lesions
      • Fam A.G.
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      Acceleration of experimental lapine osteoarthritis by calcium pyrophosphate microcrystalline synovitis.
      . Our findings suggest that structural OA exacerbation by synovitis might not be specific to CPPD-induced inflammation, and increased structural damage may occur even with transiently symptomatic inflammatory flares. Together these data demonstrate the susceptibility of OA joints to inflammatory flare episodes, which might not be directly related to the initial cause of OA. Our data indicate that joint damage persists even when the inflammatory pain flare subsides, modelling observations in subsets of human OA where chronic synovitis and transient episodes of inflammation and pain might contribute to long term joint damage
      • Neogi T.
      • Guermazi A.
      • Roemer F.
      • Nevitt M.C.
      • Scholz J.
      • Arendt-Nielsen L.
      • et al.
      Association of joint inflammation with pain sensitization in knee osteoarthritis: the multicenter osteoarthritis study.
      • Ashraf S.
      • Mapp P.I.
      • Burston J.
      • Bennett A.J.
      • Chapman V.
      • Walsh D.A.
      Augmented pain behavioural responses to intra-articular injection of nerve growth factor in two animal models of osteoarthritis.
      • Murphy S.L.
      • Lyden A.K.
      • Kratz A.L.
      • Fritz H.
      • Williams D.A.
      • Clauw D.J.
      • et al.
      Characterizing pain flares from the perspective of individuals with symptomatic knee osteoarthritis.
      • Wyatt L.A.
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      • Mapp P.I.
      • Wilson D.
      • Hill R.
      • Ferguson E.
      • et al.
      Histopathological subgroups in knee osteoarthritis.
      .
      A key finding from this study is that increasing inflammation in the MNX model of OA resulted in exacerbation of both cartilage damage and osteophyte maturity. Osteophytes are innervated by sensory nerves
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      Neurovascular invasion at the osteochondral junction and in osteophytes in osteoarthritis.
      , and can be a source of pain in OA
      • Hunter D.J.
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      The symptoms of osteoarthritis and the genesis of pain.
      . Osteophytes originate from the periosteum covering bone at the cartilage–bone junction
      • van der Kraan P.M.
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      Osteophytes: relevance and biology.
      . Osteophytosis might be driven by inflammation in OA
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      The importance of synovial inflammation in osteoarthritis: current evidence from imaging assessments and clinical trials.
      , selectively removing macrophages from the synovial lining reduced osteophyte formation in murine collagenase-induced OA
      • Blom A.B.
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      • Roth J.
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      Synovial lining macrophages mediate osteophyte formation during experimental osteoarthritis.
      • van Lent P.L.
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      • Vitters E.
      • van Rooijen N.
      • et al.
      Crucial role of synovial lining macrophages in the promotion of transforming growth factor beta-mediated osteophyte formation.
      . Angiogenesis also contributes to osteophyte maturation, and it is possible that inflammation-associated angiogenesis mediates structural progression
      • Ashraf S.
      • Walsh D.A.
      Angiogenesis in osteoarthritis.
      .
      Several non-human OA models show different degrees of inflammation, cartilage damage and bone changes. For example, in the destabilisation of the medial meniscus (DMM) model, synovitis and osteophyte formation are less pronounced compared to the collagenase-induced model
      • Schelbergen R.F.
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      • Noël D.
      • et al.
      Treatment efficacy of adipose-derived stem cells in experimental osteoarthritis is driven by high synovial activation and reflected by S100A8/A9 serum levels.
      . Meniscal transection surgery resulted in more inflammation than that seen following intra-articular injection of monosodium iodoacetate (MIA)
      • Mapp P.I.
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      • Ashraf S.
      • Burston J.J.
      • Suri S.
      • Chapman V.
      • et al.
      Differences in structural and pain phenotypes in the sodium monoiodoacetate and meniscal transection models of osteoarthritis.
      . This heterogeneity between models reflects the heterogeneity of human OA. Subgroups of human knee OA are characterised by different severities of synovitis
      • Wyatt L.A.
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      • Wilson D.
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      Histopathological subgroups in knee osteoarthritis.
      , and Generalised Nodal OA might display greater synovitis than other OA subtypes
      • Kortekaas M.C.
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      • Kloppenburg M.
      In erosive hand osteoarthritis more inflammatory signs on ultrasound are found than in the rest of hand osteoarthritis.
      .
      We used the surgically induced, unilateral MNX model of OA, which resembles many features of human post-traumatic OA (synovitis, cartilage damage, osteophytes and subchondral bone changes). Synovitis and angiogenesis make important contributions to joint damage in this model. Suppression of synovitis by glucocorticoids
      • Mapp P.I.
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      • Ashraf S.
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      • Suri S.
      • Chapman V.
      • et al.
      Differences in structural and pain phenotypes in the sodium monoiodoacetate and meniscal transection models of osteoarthritis.
      , or of angiogenesis using a fumigalin derivative
      • Ashraf S.
      • Mapp P.I.
      • Walsh D.A.
      Contributions of angiogenesis to inflammation, joint damage, and pain in a rat model of osteoarthritis.
      each reduces joint damage in the MNX model. However, synovitis in this model is not as pronounced as, for example, in collagen-induced arthritis. Carrageenan injection in non-arthritic knees (sham-operated) resulted in increased synovial macrophage infiltration, a measure of synovitis, at day 35, compared to sham-operated knees injected with vehicle control. We show here that synovitis at day 35 in the MNX model is of similar intensity to that 23 days after carrageenan injection into sham-operated knees, and that this low level of synovitis in the absence of other OA changes is insufficient in sham-operated rats to produce pain behaviour. We also show that synovitis can be augmented within ethically acceptable limits by carrageenan injection in MNX-operated animals, modelling a painful, inflammatory flare. Differing morphological and biochemical profiles exist between the various animal models of OA
      • Mapp P.I.
      • Sagar D.R.
      • Ashraf S.
      • Burston J.J.
      • Suri S.
      • Chapman V.
      • et al.
      Differences in structural and pain phenotypes in the sodium monoiodoacetate and meniscal transection models of osteoarthritis.
      • O'Brien M.
      • Philpott H.T.
      • McDougall J.J.
      Understanding osteoarthritis pain through animal models.
      or synovitis
      • Billiau A.
      • Matthys P.
      Collagen-induced arthritis and related animal models: how much of their pathogenesis is auto-immune, how much is auto-inflammatory?.
      . Further studies in other models would be needed to determine whether our findings may be generalised to other OA subtypes.
      An important limitation of this study is the single time point used for histological evaluations. The 35 day timepoint (3 weeks after carrageenan injection) was selected for histological and immunohistochemistry analysis to explore our hypothesis that inflammatory pain-flares exacerbated long-term joint damage in OA knees. Long term joint damage is a key clinical outcome for patients. Additional time points might help elucidate how quickly carrageenan-induced synovitis increases cartilage damage and osteophyte formation in the MNX group, whether the observed changes peaked, might increase or might potentially resolve with time beyond day 35. We show sustained changes in cartilage damage and osteophyte maturity following carrageenan-induced inflammation in OA knees, but cannot exclude additional effects of inflammation on structure in joint regions that were not sampled in our study, or that might be detected with more sensitive assessment techniques. We also recognise that alternative statistical approaches such as linear mixed effects models, or more sensitive measures of inflammation or structural change, might have revealed additional differences between groups. However, our findings should focus future attention on interactions between the synovium, articular cartilage and subchondral bone.
      The mechanisms that drive OA pain are likely to include peripheral and central sensitisation, and recent data in rats
      • Ashraf S.
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      • Andrews S.W.
      • Walsh D.A.
      Selective inhibition of tropomyosin-receptor-kinase A (TrkA) reduces pain and joint damage in two rat models of inflammatory arthritis.
      • Nwosu L.N.
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      Blocking the tropomyosin receptor kinase A (TrkA) receptor inhibits pain behaviour in two rat models of osteoarthritis.
      • Xu L.
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      • Millns P.J.
      • Sagar D.R.
      • Mapp P.I.
      • et al.
      The anti-NGF antibody muMab 911 both prevents and reverses pain behaviour and subchondral osteoclast numbers in a rat model of osteoarthritis pain.
      and humans
      • Mayorga A.J.
      • Wang S.
      • Kelly K.M.
      • Thipphawong J.
      Efficacy and safety of fulranumab as monotherapy in patients with moderate to severe, chronic knee pain of primary osteoarthritis: a randomised, placebo- and active-controlled trial.
      • Schnitzer T.J.
      • Ekman E.F.
      • Spierings E.L.
      • Greenberg H.S.
      • Smith M.D.
      • Brown M.T.
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      Efficacy and safety of tanezumab monotherapy or combined with non-steroidal anti-inflammatory drugs in the treatment of knee or hip osteoarthritis pain.
      using NGF-blocking pharmaceutical agents emphasise the key contribution of peripheral sensitisation. Peripheral sensitisation in OA has been associated with synovitis
      • Miller R.E.
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      • Obeidat A.M.
      • Raghu P.
      • Ishihara S.
      • Miller R.J.
      • et al.
      The role of peripheral nociceptive neurons in the pathophysiology of osteoarthritis pain.
      . Further research would be needed to define the underlying mechanism of carrageenan-induced pain in rat OA knees, and how these might overlap with mechanisms of augmented joint damage. Previous research has demonstrated reversal of acute carrageenan induced pain by cyclooxygenase inhibitors
      • Pinheiro R.M.
      • Calixto J.B.
      Effect of the selective COX-2 inhibitors, celecoxib and rofecoxib in rat acute models of inflammation.
      , which can also reduce pain in people with OA. Weightbearing asymmetry occurs in carrageenan-injected, sham-operated rats in the absence of joint damage, suggesting that carrageenan-induced pain from rat knees involves neuronal sensitisation.
      The pathophysiology of pain in OA is complex and similarly the symptomatic presentation in OA diverse and heterogeneous. Understanding why osteoarthritic knees are more prone to damage following painful inflammatory flares will enable us to better understand why different people respond differently to the same interventions and ultimately which subgroup of people might better respond to certain treatments. Our study highlights that knees with post-traumatic OA following meniscal transection are able to recover from carrageenan-induced inflammatory pain flares but are not able to prevent the associated inflammation-induced joint damage.

      Author contributions

      • 1.
        Study conception and design: SA, PIM and DAW. Acquisition of Data: SA, SMS and PIM. Data analysis and interpretation: SA and DAW.
      • 2.
        Drafting of manuscript: SA and DAW. Revision of manuscript: all authors revised the manuscript critically for important intellectual content.
      • 3.
        Final approval of the manuscript: all authors have reviewed the final version of the manuscript and approved the version to be submitted.

      Competing interests

      None.

      Role of funding sources

      The study was funded by Arthritis Research UK (ARUK) , grant reference number 20777

      Acknowledgements

      None.

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