Search

Background In this study we investigated the contribution of high-fat diet-induced metabolic overload to osteoarthritis (OA) progression originally caused by mild mechanical trauma to the mouse knee joint. We hypothesized that metabolic stress would induce a proinflammatory environment by altering systemic lipid levels and immune cell populations. Methods Twelve-week-old male C57BL/6J mice (n=20) were given a low-fat diet (LFD, 10%kcal from fat) or high-fat diet (HFD, 45%kcal from fat) for 18 weeks. OA was initiated by transecting the medial meniscotibial ligament of the right knee joint at t=10 weeks. OA severity and changes in M1/M2 polarization of synovial macrophage populations were determined in serial coronal FFPE-mounted sections. Eicosanoid levels and monocyte populations were evaluated before and after ligament transection. Results Diet-induced metabolic stress assessed by body weight, systemic cholesterol levels, and insulin resistance index was significantly higher in HFD mice. This group also showed increased cartilage damage, synovitis, and osteophyte formation compared with LFD controls. High-fat feeding elevated systemic arachidonic acid levels, which mainly resulted in increased levels of its cytochrome P450-catalysed diol metabolites 5,6-dihydroxyeicosatrienoic acid (DHET) and 8,9-DHET. High-fat feeding also triggered an increase in pro-inflammatory intermediate CD43++Ly6Cint monocytes after ligament resection. Ligament resection in addition to high-fat feeding induced increased expression of the activation marker CD11c selectively on non-classical CD43++Ly6Clow monocytes. No significant changes in synovial macrophage polarization were observed. Conclusions Metabolic stress resulted in a proinflammatory environment and aggravated injury-induced OA progression. Our results suggest that a CYP450-focused eicosanoid metabolism and activated circulating monocytes may be drivers of this metabolic stress-induced OA progression, contributing to the mechanistic understanding and potentially serving as future diagnostic and prognostic biomarkers for metabolic OA.

Background High systemic cholesterol levels have been associated with osteoarthritis (OA) development and lowering cholesterol using statins has been suggested as a potential treatment for OA1. Recently, novel therapeutic high-intensive cholesterol-lowering treatment has been shown to regress Western-type diet (WTD)-induced atherosclerosis in dyslipidemic APOE*3Leiden.CETP mice, a translational model with a human-like lipoprotein metabolism2. Objectives Here, we investigated the potency of these therapeutic cholesterol-lowering strategies to attenuate OA development in dyslipidemic APOE*3Leiden.CETP mice. Methods Female mice (n=13-16 per group) were fed a cholesterol-supplemented WTD for 38 weeks. After 13 weeks, mice were matched into a baseline group (sacrificed at t=13w) and 5 groups that received WTD alone or with treatment for 25 weeks: atorvastatin alone (A), in combination with PCSK9 inhibitor alirocumab (AA) or ANGPTL3 inhibitor evinacumab (AE) or triple treatment (AAE). Plasma lipids were monitored to assess metabolic dysfunction. Knee joints were analysed for cartilage degradation, synovial inflammation and osteophyte formation. Aggrecanase activity and S100A8 expression were determined using immunohistochemistry. Results Systemic administration of cholesterol-lowering interventions resulted in a significant reduction of body weight (A: -16%; AA: -8%; AE: -12%; AAE: -18% at end point) without changes in food intake. WTD-feeding manifested itself in dyslipidemia, with high cholesterol levels that were significantly reduced after administration of therapeutic cholesterol-lowering interventions (A: -46%; AA: -74%; AE: -74%; -AAE: 86% at end point). Using detailed OARSI scoring, dyslipidemic mice showed a mild but significant increase in cartilage degeneration at end point (7.8 ± 1.9) compared to baseline controls (3.8 ± 1.8, 2.1-fold increase, p=0.001), and a strong 52-fold increase in NITEGE staining, representing proteolytic activity by aggrecanases3. However, therapeutic cholesterol lowering did not ameliorate further progression of cartilage destruction. This was supported by comparable NITEGE staining in all treatment groups at end point. Dyslipidemic APOE*3Leiden.CETP mice developed minor synovial inflammation which was not affected by cholesterol-lowering interventions. Alarmin S100A8, a marker for macrophage activation, showed minor expression in the synovial lining of all treatment groups, supporting the absence of joint inflammation in this model4. Finally, we determined whether high plasma cholesterol levels promoted osteophyte formation. The total number of osteophytes and osteophyte maturation stage was comparable between all groups despite reduction of systemic cholesterol levels. Conclusion Therapeutic cholesterol-lowering interventions did not slow the progression of cartilage degradation and osteophyte formation in dyslipidemic APOE*3Leiden.CETP mice with minor joint inflammation. These findings suggest that therapeutic cholesterol-lowering interventions in the absence of local inflammation within the knee joint cannot prevent progression of OA. References [1] Gierman, LM, Ann Rheum Dis, 2014; 73, 921-927. [2] Dewey, F.E., New Engl J Med2017; 377, 211-221. [3] van Meurs JB, Arthritis Rheum42, 1999; 42, 1128-1139. [4] van Lent PL, Arthritis & Rheumatism2012; 64, 1466-1476. Disclosure of Interests None declared

Objective: C-reactive protein (CRP) levels can be elevated in osteoarthritis (OA) patients. In addition to indicating systemic inflammation, it is suggested that CRP itself can play a role in OA development. Obesity and metabolic syndrome are important risk factors for OA and also induce elevated CRP levels. Here we evaluated in a human CRP (hCRP)-transgenic mouse model whether CRP itself contributes to the development of ‘metabolic’ OA. Design: Metabolic OA was induced by feeding 12-week-old hCRP-transgenic males (hCRP-tg, n = 30) and wild-type littermates (n = 15) a 45 kcal% high-fat diet (HFD) for 38 weeks. Cartilage degradation, osteophytes and synovitis were graded on Safranin O-stained histological knee joint sections. Inflammatory status was assessed by plasma lipid profiling, flow cytometric analyses of blood immune cell populations and immunohistochemical staining of synovial macrophage subsets. Results: Male hCRP-tg mice showed aggravated OA severity and increased osteophytosis compared with their wild-type littermates. Both classical and non-classical monocytes showed increased expression of CCR2 and CD86 in hCRP-tg males. HFD-induced effects were evident for nearly all lipids measured and indicated a similar low-grade systemic inflammation for both genotypes. Synovitis scores and synovial macrophage subsets were similar in the two groups. Conclusions: Human CRP expression in a background of HFD-induced metabolic dysfunction resulted in the aggravation of OA through increased cartilage degeneration and osteophytosis. Increased recruitment of classical and non-classical monocytes might be a mechanism of action through which CRP is involved in aggravating this process. These findings suggest interventions selectively directed against CRP activity could ameliorate metabolic OA development.

Objective: Human cohort studies have demonstrated a role for systemic metabolic dysfunction in osteoarthritis (OA) pathogenesis in obese patients. To explore the mechanisms underlying this metabolic phenotype of OA, we examined cartilage degradation in the knees of mice from different genetic backgrounds in which a metabolic phenotype was established by various dietary approaches.Design: Wild-type C57BL/6J mice and genetically modified mice (hCRP, LDLr-/-. Leiden and ApoE*3Leiden. CETP mice) based on C57BL/6J background were used to investigate the contribution of inflammation and altered lipoprotein handling on diet-induced cartilage degradation. High-caloric diets of different macronutrient composition (i.e., high-carbohydrate or high-fat) were given in regimens of varying duration to induce a metabolic phenotype with aggravated cartilage degradation relative to controls.Results: Metabolic phenotypes were confirmed in all studies as mice developed obesity, hypercholesteremia, glucose intolerance and/or insulin resistance. Aggravated cartilage degradation was only observed in two out of the twelve experimental setups, specifically in long-term studies in male hCRP and female ApoE*3Leiden. CETP mice. C57BL/6J and LDLr-/-. Leiden mice did not develop HFD-induced OA under the conditions studied. Osteophyte formation and synovitis scores showed variable results between studies, but also between strains and gender.Conclusions: Long-term feeding of high-caloric diets consistently induced a metabolic phenotype in various C57BL/6J (-based) mouse strains. In contrast, the induction of articular cartilage degradation proved variable, which suggests that an additional trigger might be necessary to accelerate diet-induced OA progression. Gender and genetic modifications that result in a humanized pro-inflammatory state (human CRP) or lipoprotein metabolism (human-E3L. CETP) were identified as important contributing factors. (c) 2017 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.

Evidence is growing for the existence of an obesity-related phenotype of osteoarthritis in which low-grade inflammation and a disturbed metabolic profile play a role. The contribution of an obesity-induced metabolic dysbalance to the progression of the features of osteoarthritis upon mechanically induced cartilage damage was studied in a rat in vivo model. Forty Wistar rats were randomly allocated 1:1 to a standard diet or a high-fat diet. After 12 weeks, in 14 out of 20 rats in each group, cartilage was mechanically damaged in the right knee joint. The remaining six animals in each group served as controls. After a subsequent 12 weeks, serum was collected for metabolic state, subchondral bone changes assessed by μCT imaging, osteoarthritis severity determined by histology, and macrophage presence assessed by CD68 staining. The high-fat diet increased statistically all relevant metabolic parameters, resulting in a dysmetabolic state and subsequent synovial inflammation, whereas cartilage degeneration was hardly influenced. The high-fat condition in combination with mechanical cartilage damage resulted in a clear statistically significant progression of the osteoarthritic features, with increased synovitis and multiple large osteophytes. Both the synovium and osteophytes contained numerous CD68 positive cells. It is concluded that a metabolic dysbalance due to a high-fat diet increases joint inflammation without cartilage degeneration. The dysmetabolic state clearly accelerates progression of osteoarthritis upon surgically induced cartilage damage supported by inflammatory responses as demonstrated by histology and increased CD68 expressing cells localized on the synovial membrane and osteophytes. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:881-890, 2018.

Background Obesity is a well-known and important risk factor for osteoarthritis (OA). Moreover, obesity is highly associated with the metabolic syndrome (MetS)1. Growing evidence indicates that both OA and MetS are low-grade inflammatory conditions with elevation in systemic inflammatory markers. Nonetheless, it is unclear whether MetS low-grade inflammation induces OA, or contributes to the disease. Objectives To determine the contribution of metabolic alterations, induced by a High-Fat Diet (HFD), on the onset or progression of OA in a rat model of local cartilage damage. Methods Forty Wistar rats (12 weeks old, male), were randomly divided over two groups: twenty rats were fed a HFD (60% of the kcal contained fat:D12492i, Research Diets Inc.) while the other animals received a standard diet. After 12 weeks, local articular cartilage damage was induced on the femoral condyles, in one knee joint according to the groove model in 14 rats of each diet group. Remaining animals served as a control group in each arm. At week 24, serum was collected, subchondral bone was assessed by μCT scan (Quantum FX, PerkinElmar,USA), OA severity was evaluated by rat OARSI histopathology score and macrophage presence with CD68 immunostaining from histological sections was assessed. Results HFD feeding resulted in metabolic dysregulation as indicated by significantly increased metabolic parameters (weight, fasting insulin and total cholesterol) compared to the standard fed rats. HFD feeding alone resulted in mild cartilage degeneration (2±1.1 vs 0.58 ±0.7; p=0.06) and synovial membrane inflammation (1.0±0.6 vs 0.3±0.5; p=0.075) both subscores of the rat OARSI histopathology score. However, when HFD feeding is combined with the surgical model of applied local cartilage damage, OA severity is statistically significant increased compared to the local cartilage damage group on a standard diet (6.2±2.1 vs 3.4±1.4; p=0.001). Synovial membrane inflammation (1.3±0.9vs 0.5±0.5; p=0.011) and multiple large osteophyte formation, demonstrated by histology (0.9±1 vs 0.2±0.4; p=0.04) and quantified on μCT (328±349 μm3 vs 7±14 μm3; p=0.0001), contributes most to this increased OA severity. Immunohistochemical CD68 expression as observed on both the synovial membrane as well as in the subchondral bone and around the formed osteophytes can explain the increase in selected inflammatory parameters when groove surgery is combined with a HFD (Figure 1). Conclusions This study shows that a HFD induces metabolic alterations and increases the inflammatory state of the joint. This by itself does not result in severe OA. However, when adding a HFD to a mild cartilage damage model of OA, joint degeneration is significantly increased. This progression of joint degeneration appears to be driven mainly by inflammatory responses as demonstrated by an increased CD68 expression in both the subchondral bone and synovium membrane with increased osteophytosis. Hence, our findings indicate that systemic metabolic and subsequent inflammatory factors need an additional trigger to contribute to the progression of the OA. References Zhuo Q, Yang W, Chen J and Wang Y, Metabolic syndrome meets osteoarthritis. Nat Rev Rheumatol, 2012. 8(12): p. 729–37. Disclosure of Interest None declared

s / Osteoarthritis and Cartilage 24 (2016) S63eS534 S377 more physiological setup. Therefore, a culture model consisting of a TEC applied on the top of a bone-component containing OA-OB was developed and its evolution investigated in an in vivo ectopic environment. Methods: Expanded human articular chondrocytes were labelled with GFP and cultured on Chondro-Gide membranes (Geistlich Pharma AG, 6 mm diameter) for 4 days in medium containing TGF-beta to generate TEC. OB were obtained by outgrowth culture from bone chips harvested from tibial plateaus of patients with OA. OA-OB were seeded on cylindrical ceramic scaffolds (Engipore, 6 mm diameter, 4 mm high) and cultured in osteogenic medium for 4 days. TEC were then combined with the bone-cylinders either with (TEC/OB) or without OB (TEC/-) using fibrin glue (Baxter) prior subcutaneous implantation in nudemice for 4 and 8 weeks. Explanted tissues and corresponding in vitro controls were analyzed (immuno-)histologically to assess the maturation capacity of the TEC and its integrative potential under OA-simulating conditions. Results: At the time of implantation the TEC did not contain appreciable amounts of glycosaminoglycans (GAG). After 4 weeks, explants and in vitro control tissues consisted of fibro-cartilaginous tissues faintly stained for GAG, but stably integrated to the bone component irrespective of the presence of OB. GFP-positive chondrocytes were identified in pores of the bone compartment suggesting their participation to enable cartilage-bone integration. After 8 weeks in vivo, TEC/OB grafts contained amoremature cartilage layer intensely stained for GAG and a subchondral layer with dense extracellular matrix expressing nuclear pannexin and C-tail connexin (Cx43) and limited amounts of infiltrated inflammatory murine cells. Contrarily, TEC/were massively infiltrated by inflammatory cells both in the bone and in the cartilage layer that did not express GAG. Conclusions: We demonstrated that TEC can mature and become functionally integrated with a ceramic bone compartment only in case OB even if obtained from OA joints were present. Future investigations will be required to identify the mechanisms by which OB can mediate this evolution. The here developed model, allowing to study the evolution of a human cell-based cartilage graft in an in vivo OA-mimicking environment, can be applied for pre-clinical testing of cell-/tissue based products for OA cartilage repair. 620 METABOLIC DYSREGULATION ACCELERATES PROGRESSION IN EARLY DEGENERATIVE RAT KNEE JOINTS DRIVEN BY LOCAL INFLAMMATORY CHANGES H.M. de Visser, S.C. Mastbergen, K. Coeleveld, M.H. van Rijen, F.P. Lafeber, H. Weinans. Univ. Med. Ctr. Utrecht, Utrecht, Netherlands Purpose: Obesity is a well-known and important risk factor for osteoarthritis (OA). Moreover, obesity is highly associated with the metabolic syndrome (MetS), which is defined as the simultaneous occurrence of the following factors: obesity, insulin resistance, abnormal blood lipid levels and hypertension. Growing evidence is presented that both OA and MetS are low-grade inflammatory conditions with elevation in systemic inflammatory markers. Although, it remains difficult to conclude whether MetS low-grade inflammation induces OA, or contributes. The aim of this study was to determine the contribution of metabolic alterations, induced by a high-fat diet (HFD), on the onset or progression of OA in a rat model of local cartilage damage. Methods: Forty 12 weeks old male Wistar rats (Charles-River, Sulzfeld, Germany), were randomly divided over two groups: twenty rats were fed a HFD, whereof 60% of the kcal contained fat (D12492i, Research Diets Inc., New Brunswick, USA). After 12 weeks groove surgery was performed on the femoral condyles in one knee joint to induce local articular cartilage damage in fourteen rats of each diet group. 24 weeks after the start of the diet serum samples were collected and an ex-vivo (contrast enhanced-)mCT scan (Quantum FX, PerkinElmar, USA) of both knee joints was performed to identify diet and/or surgery related subchondral bone and articular cartilage changes. In addition, histological sections were made OA severity was assessed by the rat OARSI histopathology score by two observers blinded for the study group. To analyze differences between the different study groups, the independent-samples T test is used. The study was approved by the Utrecht University Medical Ethical Committee for animal studies. Results: At 24 weeks after the start of the diet all relevant metabolic parameters, obesity (859± 117 g vs. 751± 61g), hyperinsulinemia (4.2± 1.8 ng/ml vs. 2.8± 1.6 ng/ml), total cholesterol (123± 33 mg/dl vs. 160± 38 mg/dl) were significantly increased in the HFD fed rats compared to the control fed rats (p 0.05) and cartilage attenuation (2046± 257 vs 1834± 266; p>0.05) of the tibia compartment in the HFD fed rats compared to the control fed rats. However, when HFD feeding is combined with the surgical model of applied local cartilage damage, the articular cartilage thickness of the tibia is decreased compared to the local cartilage damage group with the control fed rats (184± 41 mm vs 220± 36 mm; p1⁄40.04). On the other hand, no difference was found in cartilage attenuation (2075± 196 vs 1994± 220; p>0.05) between these groups. Conclusions: Metabolic dysregulation induced by a HFD does not induce joint degeneration or articular cartilage changes on itself. However, when adding a HFD to a mild cartilage damage model of OA, joint degeneration is increased and articular cartilage thickness is decreased 12 weeks post-surgery. Synovial membrane inflammation and osteophyte formation, contribute the highest to this more severe joint degeneration as observed by histology. The present study shows that feeding rats with a HFD, inducing metabolic alterations accelerates the progression of knee joint degeneration in the groove model of local articular cartilage damage, likely due to the increased inflammatory state of the knee joints that leads to both synovitis and osteophytosis.

s / Osteoarthritis and Cartilage 23 (2015) A82eA416 A260 remained unclear whether synovitis is also related to cartilage alterations or other features in patients with end-stage knee OA, where articular cartilage is almost disappeared. In addition, magnetic resonance imaging (MRI) measurements are sensitive to detect of OArelated structural changes. The aim of this study was to investigate the MRI-detected OA-structural changes of the knee joint those were associated with histological synovitis in patients with end-stage knee OA who underwent TKA. Methods: The forty patients (female: 88%, 71.1y on average) who fulfilled the American College of Rheumatology criteria for knee OA and required TKA due to the end-stage knee OA of Kellgren and Lawrence grade 4 were enrolled in this study. All participants were performed with 3.0-Tesla MRI. The OA morphological changes, such as (1) cartilage morphology, (2) bone marrow lesions (BMLs), (3) bone cysts, (4) bone attrition, (5) meniscal pathology, (6) osteophyte, (7) synovitis and (8) ligaments were scored using the whole-organ MRI scoring (WORMS) method. The synovial samples were obtained from five regions of interest of the knee joint at the operation. The sections were stained with hematoxylin and eosin. The histological synovitis scores (HSS) were measured by the methods previously described. Next, the sections were stained with TGF-b, COX-2, IL-1b and IL-6. A semi-quantitative analysis of the immune-histochemically stained sections was conducted. Associations between the HSS and WORMS were examined using Spearman's correlation coefficient. The means of each HSS were compared using the Mann-Whitney U test. Results: Among the eight OA-related morphological changes, the total BML scorewere significantly associated with the total HSS (r1⁄4 0.64, p1⁄4 0.02). BMLs were present in all patients in medial femoro-tibial joint (MFTJ), and were present in lateral femoro-tibial joint (LFTJ) in approximately half (53%) of the patients. The HSS in MFTJ and LFTJ were significantly associated with medial and lateral BML scores [r 1⁄4 0.35, p 1⁄4 0.03 (medial), r 1⁄4 0.49, p < 0.01 (lateral), respectively]. The total HSS in patients with BML in LFTJ were significantly increased compared to those without BML in LFTJ (p < 0.001). The synovial TGF-b expression in the LFTJ in patients with BMLs in the LFTJ was significantly higher than that in patients without BMLs in the LFTJ, while these differences were not observed in COX2, IL-1b or IL-6. Conclusions: The BMLs, but not other structural changes detected by MRI, were associated with the histological synovitis and synovial TGF-b expression in patients with end-stage knee OA. 402 HIGH-FAT DIET FEEDING AFFECTS OSTEOARTHRITIS PROGRESSION IN A SURGICAL MOUSE MODEL WITH ESTABLISHED AND DEVELOPING METABOLIC SYNDROME A.E. Kozijn yz, R. Stoop y, F.P. Lafeber z, H. Weinans zx, A.M. Zuurmond y. y TNO Metabolic Hlth.Res., Leiden, Netherlands; zUniv. Med. Ctr. Utrecht, Utrecht, Netherlands; xDelft Univ. of Technology, Delft, Netherlands Purpose: Metabolic syndrome (MS) is a major risk factor for osteoarthritis (OA) development. The MS-associated low-grade systemic inflammation may underlie this link. We investigated whether a systemic metabolic burden due to high-fat feeding in mice would aggravate OA progression, in both established as well as developing MS. Methods:Metabolic syndromewas induced by providing a high-fat diet (HFD, 45% kcal from fat) to 12-week old male C57Bl/6J mice (n1⁄410) for 10 weeks. Control mice of same sex and age received a synthetic low-fat diet (LFD, 10% kcal from fat) during that time period. At 10 weeks the medial meniscotibial ligament (MMTL) of the right knee joint was transected to destabilize the joint and thereby induce OA. The left knee joint served as an internal control and underwent all the surgical procedures except for the MMTL transection (sham). Half of the control mice were switched to HFD at the time of surgery (LFD-to-HFD), to attain a state of developing metabolic syndrome at end-point. Body weights and changes in body composition were monitored during the entire study period. At endpoint, OA severity was graded on Safranin Ostained histological sections of the knee joints according to OARSI recommendations. Results: Bodyweights of the mice on longand short-term HFD were significantly different at end-point (HFD, 41.1 ± 6.9 g; LFD-to-HFD, 38.8 ± 3.1 g) compared to mice of the control group (LFD, 31.8 ± 2.1 g). Body composition analysis showed that the increase in body weight almost completely resulted from an increase in body fat mass; lean mass remained constant between groups. Severity of OA was greater in the mice fed a HFD, for both the short-term and the long-term regimen. Separate assessment of the longand short-term HFD groups showed a statistically significant difference in cartilage degeneration between the HFD and control groups (Figure 1; one-tailed t-test, p 1⁄4 0.026). This effect was mainly observed at the lateral knee compartments, implying that the observed cartilage degeneration at the lateral knee compartments was predominantly diet-induced (Figure 2). Interestingly, analysis of the lateral knee compartments of the LFD-to-HFD group showed a clear trend towards an increase in OA severity compared to control (one-tailed t-test, p1⁄4 0.064). OA severity did not correlatewith body fat mass at end-point. Conclusions: Our results show that high-fat feeding indeed aggravates OA progression in a surgical mouse model for OA. In this model, the diet-induced OA progression was mainly observed at the lateral knee compartments. No correlation between OA severity and body weight or body fat mass was found, suggesting that the MS-associated inflammation may underlie the diet-induced OA progression. 403 CHONDROGENIC PROGENITOR CELLS PROVIDE A NOVEL INSIGHT TO PHAGOCYTOTIC ACTIVITIES WITHIN CARTILAGE C. Zhou, H. Zheng, J.A. Martin. The Univ. of Iowa, Iowa City, IA, USA Purpose: Chondrogenic progenitor cells (CPCs) have been identified on the cartilage surface post injury, as well as in osteoarthritic cartilage. CPCs serve chondroprotective and regenerative functions, but in initial response to cartilage injury, CPCs overexpress markers associated with dendritic cells. Based on these findings we hypothesized that CPCs carry the potential to clear cell and matrix debris through phagocytosis. To test this we compared phagocytotic capacity of CPCs to chondrocytes. Methods: Scratches on bovine knee cartilage were created to stimulate CPCs migration. After culturing for ten days, CPCs and chondrocytes were isolated and co-cultured with DiO-labeled cell debris for various time periods. Confocal microscopy and flow cytometry analyses were performed to determine phagocytosis events in both cell types. Abstracts / Osteoarthritis and Cartilage 23 (2015) A82eA416 A261s / Osteoarthritis and Cartilage 23 (2015) A82eA416 A261 Results: Confocal microscopy images show CPCs migrating toward acartilage injured (Fig 1A). In CPCs, most DiO-labeled cell debris appeared to be engulfed within the cytoplasm, while in chondrocytes, most of the label appeared to be bound to the cell membrane (Fig 1B). Flow cytometry quantitatively confirmed that DiOþ CPC percentage was significantly higher than chondrocytes at each time point (3hrs, 6hrs, 12hrs and 24hrs) (Fig 2). In addition, DiOþ CPCs increased dramatically (12.58% for 3hrs, 28.63% for 6hrs) and peaked at 12hrs (68.10%), while DiOþ chondrocytes increased slightly (4.18%, 6.66%, 8.08%, and 11.56%, respectively). Conclusions: CPCs showed much higher and sustained uptake of cell debris than chondrocytes. Though not conclusive, these findings support the hypothesis that CPCs play a role in clearing cell debris in damaged cartilage. The physiologic significance of this activity is unclear, but it is conceivable that it neutralizes the pro-inflammatory activity of cell debris, an essential step in wound healing. Figure 1. A) Morphological distinction between CPCs and chondrocytes, CPC (long stretched cells indicated by red arrows) residing on the surface of scratch site. CPCs showed dramatic morphological difference compared to the chondrocytes (rounded cells in most upper and lower portion of the figure) B) Visual comparison between CPCs (upper) and chondrocytes (lower) in terms of phagocytosis under microscopy. Cell debris (stained with green-fluorescent DiO label) was vastly ingested into the cytoplasm of CPC. While for chondrocytes, most cell debris was sticking outside the cell membrane. Flow cytometry quantification assay showed DiOþ cells percentage within CPC population is significantly higher than chondrocytes, along with different rates to phagocytose cell debris for CPCs and chondrocytes (12hr shows highest in CPCs, while gradually increase in chondrocytes). 404 SYNOVITIS AND CHANGES IN THE EXPRESSION OF SYNOVIOCYTES MARKERS CD68 AND CD55, DURING THE PROGRESSION OF OSTEOARTHRITIS IN A RAT MODEL R. Cruz, D. Solis-Garcia, M. Miranda-S anchez, J.B. Kouri. Centro de Investigaci on y de Estudios Avanzados del Inst. Polit ecnico Natl., M exico