W. Wei

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

OBJECTIVE Stiffening 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. METHODS Batches 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. RESULTS Collagen 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. CONCLUSION These 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.

Statins and fibrates do not affect development of spontaneous cartilage damage in STR/Ort mice

OBJECTIVE Since statins and fibrates are capable of improving the metabolic profile of patients as well as decreasing inflammation, they are considered as potential drugs for preventing osteoarthritis (OA). The goal of the present study was to investigate the effect of these drugs in the STR/Ort spontaneous OA mouse model. DESIGN Male STR/Ort mice received control diet or control diet containing two different dosages of simvastatin or fenofibrate or a combination of both. Mice were euthanized after 16 weeks of treatment at the age of 24 weeks. Serum analysis for metabolic and inflammatory markers, histologic OA grading and micro computed tomography (μCT) analysis of subchondral bone plate were performed. RESULTS Simvastatin treatment did not have a statistically significant effect on any of the measured parameters. Fenofibrate treated mice gained less body weight (BW) and had lower serum amyloid A (SAA) levels, but higher Interleukin (IL)-1α and MIP1α than other mice. Mice treated with 200 mg/kg BW/day fenofibrate had less subchondral bone plate volume than control, but no statistically significant reduction in cartilage damage. In the combination treatment group, BW and SAA were lower than control. Overall, bodyweight, synovium membrane cell layers and SAA levels correlated to subchondral bone plate changes and subchondral bone plate changes correlated to cartilage damage. CONCLUSIONS Statins and fibrates did not affect development of cartilage damage in the STR/Ort spontaneous OA mouse model. Fenofibrates however, had an effect on BW, serum inflammation markers and subchondral bone plate morphology.

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 24 h 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.

Synovial Fluid Fatty Acid Profiles Differ between Osteoarthritis and Healthy Patients

Objective Free fatty acids (FAs) may influence cartilage metabolism and osteoarthritis (OA) disease progression. It is not clearly studied which FAs are present in the synovial fluid of knee joints and whether there are differences in FA content between nonsymptomatic and OA knee joints. The aim of this study was to investigate the presence of different types of FAs in synovial fluid of both OA- and nonsymptomatic control joints, and to analyze differences between both groups. Design A total of 23 synovial fluid samples were collected from patients with end-stage knee OA undergoing total knee replacement, with approval of the medical ethical committee. As controls, 6 synovial fluid samples were obtained from postmortem donors without any history of joint disease or arthritis. Measurement of free FA concentration was done by mass spectrometry for saturated FAs (SFA), monounsaturated FAs (MUFA), and omega-3 and omega-6 polyunsaturated FAs (n-3 PUFAs and n-6 PUFAs). Results Our measurements demonstrated the presence of SFAs, MUFAs, n-3 and n-6 PUFAs in synovial fluid of both nonsymptomatic and OA knee joints. The n-6/n-3 ratio was significantly lower in the OA group (P = 0.0005). Arachidonic acid (n-6 PUFA) concentrations were also lower in OA synovial fluid (P = 0.01), while tetracosadienoic acid (P = 0.0001) and nervonic acid (P = 0.001) (MUFAs) were higher in synovial fluid of patients with knee OA. Conclusion Synovial fluid contains a broad spectrum of free FAs. The FAs profile differs between OA and control subjects, including a tendency for less n-6 FAs in OA joints.

02.40 Lack of obesity-related features in adipocytes and inflammatory cells in the infrapatellar fat pad (ifp) of oa patients

Background Obesity is associated with the development and progression of osteoarthritis (OA). The infrapatellar fat pad (IFP) could contribute to this association due to its localization in the knee joint and secretion of inflammatory mediators. However, little is known about the effects of obesity on the IFP. Therefore, the aim of this study was to investigate the presence of obesity-related features in adipocytes and infiltrating immune cells in the IFP of OA patients. Materials and methods IFP volume was determined by MRI in 79 knee OA patients. IFP and subcutaneous adipose tissue (SCAT) were obtained from 106 knee OA patients (total n=155: 68% women, mean age 65 years, mean (SD) body mass index (BMI) 29.9 kg/m2 (5.7)) undergoing joint replacement surgery. Crown-like structures (CLS) were determined using immunohistochemistry. Adipocyte size was determined by light microscopy and histology. Stromal vascular fraction (SVF) cells were characterised by flow cytometry. Results IFP volume (mean(SD) 23.6 (5.4) mm3) associated with gender and height, but not with BMI. Likewise, volume and size of IFP adipocytes (mean 271 pl, mean 1933 μm) was not correlated with BMI. Few CLS were observed in IFP and the number did not correlate with BMI. Moreover, high BMI was not associated with higher SVF immune cell numbers in IFP, nor with changes in their phenotype. No molecular differences were observed with BMI, besides an increase in TNFα expression. Extensive characterisation of IFP macrophages revealed that CD206 and CD163, usually associated with an anti-inflammatory phenotype were the most abundantly expressed surface markers on macrophages (81% and 41% respectively), while macrophages produced predominantly IL-6 and TNFα, but little IL-10. Interestingly, surface marker and cytokine expression revealed that CD163+ macrophages had an activated and pro-inflammatory phenotype. Conclusions Obesity-related differences usually observed in SCAT and visceral adipose tissue could not be detected in IFP of OA patients, a fat depot implicated in OA pathogenesis.

SAT0001 Lack of obesity-related features in adipocytes and inflammatory cells in the infrapatellar fat pad (IFP)

Background Obesity is associated with the development and progression of osteoarthritis (OA), both for weight-bearing and non-weight bearing joints. Several lines of research indicate that obesity-related systemic factors, such as adipose tissue-derived factors, could be involved in this association. The infrapatellar fat pad (IFP) is an adipose tissue depot localized in the knee joint. and could mediate obesity-associated effects. However, it is currently unknown whether and how obesity affects IFP. Objectives To investigate the presence of obesity-related features in adipocytes and infiltrating immune cells in the IFP of OA patients. Methods Knee OA patients (N=155: 68% women, mean age 65 years, mean (SD) BMI 29.9 kg/m2 (5.7)) were recruited: IFP volume was determined by MRI in 79 knee OA patients, while IFP and subcutaneous adipose tissue (SCAT) were obtained from 106 patients undergoing arthroplasty. Crown-like structures (CLS) were determined using immunohistochemistry. Adipocyte size was determined by light microscopy and histology. Stromal vascular fraction (SVF) cells were characterized by flow cytometry. Results IFP volume (mean (SD) 23.6 (5.4) mm3) was associated with height, but not with BMI or other obesity-related features such as waist circumference, fat percentage and waist to hip ratio. The volume of IFP adipocytes did not correlate with BMI, in contrast to SCAT adipocytes. Few CLS were observed in IFP and their number did not differ between individuals with high and low BMI. Moreover, high BMI was not associated with higher infiltrating immune cell numbers in IFP, nor with changes in immune cell populations. Likewise, no molecular differences were observed in FCM-secreted factors between high and low BMI, except for an increased TNFa secretion in obesity. Since obesity is usually associated with a shift towards pro-inflammatory macrophages in conventional adipose tissue, we have extensively characterized IFP macrophages. Surprisingly, CD206 and CD163, usually associated with an anti-inflammatory phenotype were the most abundantly expressed surface markers on macrophages (81% and 41% respectively). In contrast, cytokine profiles revealed a pro-inflammatory phenotype of the total macrophage population, with cells producing predominantly IL-6 and TNFα, but little IL-10. Interestingly, the CD163+ macrophages were bigger and had a more activated and pro-inflammatory phenotype than their CD163- counterparts. However, no association with BMI could be observed for different macrophage populations or their cytokines. Conclusions BMI-related features usually observed in SCAT and visceral adipose tissue could not be detected in IFP of OA patients, a fat depot implicated in OA pathogenesis. Disclosure of Interest None declared