Anne-Marie Zuurmond

The infrapatellar fat pad of patients with osteoarthritis has an inflammatory phenotype

Objectives Obesity is a risk factor for the development of osteoarthritis (OA) in hands and knees. Adipose tissue can secrete different adipokines with powerful immunomodulatory effects. The infrapatellar fat pad (IFP) is an intra-articular organ in the vicinity of the synovium and cartilage. It is hypothesised that IFP-derived soluble factors could contribute to pathological processes in the knee joint. A study was therefore undertaken to compare the release of inflammatory mediators in the IFP and subcutaneous adipose tissue (ScAT) and to characterise the adipocytes and immune cell infiltrate in these tissues. Methods Paired IFP and ScAT samples were obtained from 27 patients with primary OA. The stromal vascular cell fraction (SVF) was isolated and characterised by fluorescence activated cell sorting. Cytokine and adipokine release in fat- and adipocyte-conditioned media was measured by luminex. Results IFP secreted higher levels of inflammatory mediators such as interleukin 6 (IL-6), adipsin, adiponectin and visfatin than ScAT. This could be due to differences in the phenotype of adipocytes and/or in the composition and phenotype of the SVF cells. IFP adipocyte-conditioned media showed a trend towards more IL-6 and adipsin than ScAT. Moreover, the SVF fraction of IFP contained more cells/g tissue, a lower percentage of T cells and a higher percentage of mast cells than ScAT. In addition, T cells had a predominantly pro-inflammatory phenotype while macrophages had a mixed pro- and anti-inflammatory phenotype in the IFP. Conclusion There are profound differences in secreted inflammatory factors and immune cell composition between the IFP and ScAT. These data indicate that IFP-derived soluble mediators could contribute to pathophysiological processes in the OA knee joint.

Metabolic Stress–Induced Inflammation Plays a Major Role in the Development of Osteoarthritis in Mice

OBJECTIVEnObesity is associated with systemic inflammation and is a risk factor for osteoarthritis (OA) development. We undertook this study to test the hypothesis that metabolic stress-induced inflammation, and not mechanical overload, is responsible for the development of high-fat diet-induced OA in mice.nnnMETHODSnHuman C-reactive protein (CRP)-transgenic mice received a high-fat diet without or with 0.005% (weight/weight) rosuvastatin or 0.018% (w/w) rosiglitazone, 2 different drugs with antiinflammatory properties. Mice fed chow were included as controls. After 42 weeks, mice were killed and histologic OA grading of the knees was performed. To monitor the overall inflammation state, systemic human CRP levels were determined.nnnRESULTSnMale mice on a high-fat diet had significantly higher OA grades than mice on chow and showed no correlation between OA severity and body weight. In male mice, high-fat diet-induced OA was significantly inhibited by rosuvastatin or rosiglitazone to OA grades observed in control mice. Both treatments resulted in reduced human CRP levels. Furthermore, a positive correlation was found between the relative individual induction of human CRP evoked by a high-fat diet on day 3 and OA grade at end point.nnnCONCLUSIONnHigh-fat diet-induced OA in mice is due to low-grade inflammation and not to mechanical overload, since no relationship between body weight and OA grade was observed. Moreover, the OA process was inhibited to a great extent by treatment with 2 drugs with antiinflammatory properties. The inflammatory response to a metabolic high-fat challenge may predict individual susceptibility to developing OA later in life. The use of statins or peroxisome proliferator-activated receptor γ agonists (e.g., rosiglitazone) could be a strategy for interfering with the progression of OA.

An explorative study comparing levels of soluble mediators in control and osteoarthritic synovial fluid

OBJECTIVEnSoluble mediators in synovial fluid (SF) are acknowledged as key players in the pathophysiology of osteoarthritis (OA). However, a wide-spectrum screening of such mediators in SF is currently lacking. In this study, the levels of 47 mediators in the SF of control donors and osteoarthritic (OA) patients were compared.nnnMATERIALS & METHODSnSF was collected from control donors (nxa0=xa016) and end-stage knee OA patients (nxa0=xa018) and analysed for 47 cytokines, chemokines and growth factors using several multiplex enzyme-linked immunosorbent assays (ELISAs). A Mann-Whitney U test was used to determine differences between OA and control controls. A principal component analysis (PCA) was performed to cluster the 47 mediators.nnnRESULTSnThe majority of the mediators could be detected in both control and OA SF. Interleukin (IL)-6, interferon inducible protein (IP)-10, macrophage derived chemokine (MDC), platelet derived growth factor (PDGF)-AA and regulated on activation normal T cell expressed and secreted (RANTES) levels were found to be higher in OA compared to control SF (Pxa0<xa00.001). Leptin, IL-13, macrophage inflammatory protein (MIP)-1β, soluble CD40 (sCD40L) levels were higher and eotaxin and granulocyte colony-stimulating factor (G-CSF) levels were lower in OA SF than in control SF, albeit borderline significant (Pxa0<xa00.05). The PCA enabled identification of six clusters of mediators, which explained 76% of the variance.nnnCONCLUSIONSnThe current study provides the first extensive profile of cytokines, chemokines and growth factors present in control and OA SF. Increased levels of mediators such as MDC and IL-6 imply involvement of inflammatory processes and might be associated with the influx of inflammatory cells in OA synovial tissue. Moreover, the performed cluster analysis indicated multiple clusters, which could indicate different pathophysiological pathways in the joint.

Similarities and discrepancies in subchondral bone structure in two differently induced canine models of osteoarthritis

In osteoarthritis (OA), cartilage degradation is accompanied by subchondral bone changes. The pathogenesis and physiology of bone changes in OA are still unclear. The changes in subchondral bone architecture and cartilage damage were compared in differently induced experimental models of OA. Experimental OA was induced bilaterally by anterior cruciate ligament transection (ACLT) or by cartilage trauma (Groove model); bilateral sham surgery served as control. Lysylpyridinoline (LP, bone resorption) and C‐telopeptide of type II collagen (CTX‐II, cartilage breakdown) were measured over time. At 20 weeks after surgery, the subchondral cortical plate and trabecular bone of the tibia were analyzed by micro–computed tomography (µCT) and cartilage degeneration was analyzed histologically and biochemically. In both models, cartilage degeneration and cortical subchondral plate thinning were present. CTX‐II levels were elevated over time in both models. Subchondral trabecular bone changes were observed only in the ACLT model, not in the Groove model. Correspondingly, LP levels were elevated over time in the ACLT model and not in the Groove model. Interestingly, the trabecular bone changes in the ACLT model were extended to the metaphyseal area. The early decrease in plate thickness, present in both models, as was cartilage damage, suggests that plate thinning is a phenomenon that is intrinsic to the process of OA independent of the cause/induction of OA. On the other hand, trabecular changes in subchondral and metaphyseal bone are not part of a common pathway of OA development and may be induced biomechanically in the destabilized and less loaded ACLT joint.

Degree of synovitis on MRI by comprehensive whole knee semi-quantitative scoring method correlates with histologic and macroscopic features of synovial tissue inflammation in knee osteoarthritis

OBJECTIVEnTo evaluate the association between synovitis on contrast enhanced (CE) MRI with microscopic and macroscopic features of synovial tissue inflammation.nnnMETHODnForty-one patients (mean age 60 years, 61% women) with symptomatic radiographic knee OA were studied: twenty underwent arthroscopy (macroscopic features were scored (0-4), synovial biopsies obtained), twenty-one underwent arthroplasty (synovial tissues were collected). After haematoxylin and eosin staining, the lining cell layer, synovial stroma and inflammatory infiltrate of synovial tissues were scored (0-3). T1-weighted CE-MRIs (3 T) were used to semi-quantitatively score synovitis at 11 sites (0-22) according to Guermazi et al. Spearmans rank correlations were calculated.nnnRESULTSnThe mean (SD) MRI synovitis score was 8.0 (3.7) and the total histology grade was 2.5 (1.6). Median (range) scores of macroscopic features were 2 (1-3) for neovascularization, 1 (0-3) for hyperplasia, 2 (0-4) for villi and 2 (0-3) for fibrin deposits. The MRI synovitis score was significantly correlated with total histology grade [r = 0.6], as well as with lining cell layer [r = 0.4], stroma [r = 0.3] and inflammatory infiltrate [r = 0.5] grades. Moreover, MRI synovitis score was also significantly correlated with macroscopic neovascularization [r = 0.6], hyperplasia [r = 0.6] and villi [r = 0.6], but not with fibrin [r = 0.3].nnnCONCLUSIONnSynovitis severity on CE-MRI assessed by a new whole knee scoring system by Guermazi et al. is a valid, non-invasive method to determine synovitis as it is significantly correlated with both macroscopic and microscopic features of synovitis in knee OA patients.

Stimulation of Fibrotic Processes by the Infrapatellar Fat Pad in Cultured Synoviocytes From Patients With Osteoarthritis: A Possible Role for Prostaglandin F2α

OBJECTIVEnStiffening of the joint is a feature of knee osteoarthritis (OA) that can be caused by fibrosis of the synovium. The infrapatellar fat pad (IPFP) present in the knee joint produces immune-modulatory and angiogenic factors. The goal of the present study was to investigate whether the IPFP can influence fibrotic processes in synovial fibroblasts, and to determine the role of transforming growth factor β (TGFβ) and prostaglandin F2α (PGF2α ) in these processes.nnnMETHODSnBatches of fat-conditioned medium (FCM) were made by culturing pieces of IPFP obtained from the knees of 13 patients with OA. Human OA fibroblast-like synoviocytes (FLS) (from passage 3) were cultured in FCM with or without inhibitors of TGFβ/activin receptor-like kinase 5 or PGF2α for 4 days. The FLS were analyzed for production of collagen and expression of the gene for procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2; encoding lysyl hydroxylase 2b, an enzyme involved in collagen crosslinking) as well as the genes encoding α-smooth muscle actin and type I collagen α1 chain. In parallel, proliferation and migration of the synoviocytes were analyzed.nnnRESULTSnCollagen production and PLOD2 gene expression by the FLS were increased 1.8-fold (P < 0.05) and 6.0-fold (P < 0.01), respectively, in the presence of FCM, relative to control cultures without FCM. Moreover, the migration and proliferation of synoviocytes were stimulated by FCM. Collagen production was positively associated with PGF2α levels in the FCM (R = 0.89, P < 0.05), and inhibition of PGF2α levels reduced the extent of FCM-induced collagen production and PLOD2 expression. Inhibition of TGFβ signaling had no effect on the profibrotic changes.nnnCONCLUSIONnThese results indicate that the IPFP can contribute to the development of synovial fibrosis in the knee joint by increasing collagen production, PLOD2 expression, cell proliferation, and cell migration. In addition, whereas the findings showed that TGFβ is not involved, the more recently discovered profibrotic factor PGF2α appears to be partially involved in the regulation of profibrotic changes.

Adipocytes Modulate the Phenotype of Human Macrophages through Secreted Lipids

Previous studies have shown accumulation and an enhanced proinflammatory profile of macrophages in adipose tissue of obese mice, indicating the presence of an interaction between adipocytes and macrophages in this tissue. However, the consequences of this interaction in humans are yet incompletely understood. In this study, we explored the modulating effects of adipocytes on the phenotype of macrophages in humans and studied the possible molecular pathways involved. Adipocyte-conditioned media (ACM) treatment of macrophages for 48 h strongly reduced the LPS-induced IL-12p40 secretion by macrophages, whereas the production of TNF-α and other cytokines remained largely unaffected. This effect was independent of the source of adipocytes. Interestingly, the level of inhibition correlated directly with body mass index (BMI) of the adipocyte donor. Because adipocytes release many different cytokines, adipokines, and lipids, we have separated the protein and lipid fractions of ACM, to obtain insight into the molecular nature of the soluble mediators underlying the observed effect. These experiments revealed that the inhibitory effect resided predominantly in the lipid fraction. Further studies revealed that PGE2 and linoleic and oleic acid were potent inhibitors of IL-12p40 secretion. Interestingly, concentrations of these ACM-derived lipids increased with increase in BMI of the adipocyte donor, suggesting that they could mediate the BMI-dependent effects of ACM. To our knowledge, these results provide first evidence that obesity-related changes in adipose tissue macrophage phenotype could be mediated by adipocyte-derived lipids in humans. Intriguingly, these changes appear to be different from those in murine obesity.

The canine bilateral groove model of osteoarthritis

In studies aimed at local treatment of experimental osteoarthritis (OA) it is optimal to have an internal (untreated) OA control. Such an approach excludes interanimal variation, and allows paired statistical evaluation of treatment efficacy. For this purpose, we developed and characterized a bilateral version of the canine Groove model. We hypothesized that the bilateral version of the canine Groove model would show consistent and clear development of features of OA similar to those found in the unilateral version. In six Beagle dogs, grooves were surgically made in the articular cartilage of the femoral condyles of both knee joints. Six additional dogs underwent bilateral sham surgery. The degree of OA was quantified 20 weeks after surgery and was compared in retrospect to 23 animals that undergone the same procedure in a single knee joint with the contralateral knee serving as a non‐OA control. Bilateral groove surgery resulted in OA. This was based on the observed ineffective repair response in which an increase in proteoglycan synthesis, a diminished retention of these newly formed proteoglycans, and an enhanced loss of resident proteoglycans resulted in a decreased cartilage proteoglycan content. These biochemical effects were corroborated by clear histological features of OA. All these effects were found in femor as well as in the (surgically untouched) tibia. Interestingly, features of OA were slightly more severe in the bilateral model than in the unilateral variant. The bilateral canine Groove model showed consistent and clear development of features of OA, comparable to the unilateral model.

Radiographic progression of knee osteoarthritis is associated with MRI abnormalities in both the patellofemoral and tibiofemoral joint

OBJECTIVEnTo investigate patterns of MRI abnormalities in the patellofemoral (PFJ) and tibiofemoral joint (TFJ) and their association with radiographic progression, using hypothesis free analyses.nnnDESIGNn205 patients from the GARP study with symptomatic OA at multiple sites (mean age 60 years, 80% woman, median BMI 26 kg/m(2)), underwent knee MRI at baseline. Cartilage damage, osteophytes, cysts, bone marrow lesions (BMLs) and effusion/synovitis were scored according to a validated scoring method. Baseline and 6-year TFJ and PFJ radiographs were scored (0-3) for JSN and osteophytes according to OARSI and Burnett atlases, respectively; progression was defined as ≥1 point increase. Baseline patterns of MRI abnormalities derived from principal component analysis (PCA) were associated with progression using adjusted generalized estimating equations (GEE).nnnRESULTSnPCA resulted in extraction of six components, explaining 69% of variance. In 29% and 29% of 133 patients with follow-up the TFJ progressed, whereas in 15% and 9% the PFJ progressed for osteophytes and JSN, respectively. Component 1 (cartilage damage of the PFJ and osteophytes of both joints) was statistically significant associated with TFJ JSN progression and PFJ osteophyte progression. Component 2 (all lateral PFJ abnormalities except osteophytes) was associated with JSN/osteophyte progression in the PFJ alone, whereas component 3 (all medial TFJ abnormalities except osteophytes) was associated with JSN and osteophyte progression in both PFJ and TFJ.nnnCONCLUSIONnBaseline structural damage and bone turnover activity, as reflected by BMLs, seem to be involved in knee OA progression. Moreover, progression in PFJ and TFJ seems to be related.

High Fat Diet Accelerates Cartilage Repair in DBA/1 Mice

Obesity is a well‐known risk factor for osteoarthritis, but it is unknown what it does on cartilage repair. Here we investigated whether a high fat diet (HFD) influences cartilage repair in a mouse model of cartilage repair. We fed DBA/1 mice control or HFD (60% energy from fat). After 2 weeks, a full thickness cartilage defect was made in the trochlear groove. Mice were sacrificed, 1, 8, and 24 weeks after operation. Cartilage repair was evaluated on histology. Serum glucose, insulin and amyloid A were measured 24u2009h before operation and at endpoints. Immunohistochemical staining was performed on synovium and adipose tissue to evaluate macrophage infiltration and phenotype. One week after operation, mice on HFD had defect filling with fibroblast‐like cells and more cartilage repair as indicated by a lower Pineda score. After 8 weeks, mice on a HFD still had a lower Pineda score. After 24 weeks, no mice had complete cartilage repair and we did not detect a significant difference in cartilage repair between diets. Bodyweight was increased by HFD, whereas serum glucose, amyloid A and insulin were not influenced. Macrophage infiltration and phenotype in adipose tissue and synovium were not influenced by HFD. In contrast to common wisdom, HFD accelerated intrinsic cartilage repair in DBA/1 mice on the short term. Resistance to HFD induced inflammatory and metabolic changes could be associated with accelerated cartilage repair.