The groove model of tibia-femoral osteoarthritis in the rat

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Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534

The walking speed for all groups were 25 m/min. MD: moderate duration walking, HD: high duration walking, EHD extra high duration walking.

cartilage metabolism was poorly characterized. In this study we examined 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f)mediated XT-1 expression mechanism and its signaling pathway, determining the role of 29-kDa FN-f in cartilage matrix synthesis. Methods: Human articular chondrocytes were enzymatically isolated from articular cartilage and cultured in monolayer. In 29-kDa FN-fstimulated chondrocytes, the relative levels of mRNA and protein for XT-1 were analyzed by real-time quantitative reverse transcriptionpolymerase chain reaction and Western blot analysis, respectively. In order to investigate the effects of 29-kDa FN-f on XT-1, human chondrocytes were transfected with small interfering RNAs (siRNAs) targeting TLR-2. Results: The level of aggrecan and XT-1 in human osteoarthritis cartilage was significantly decreased compared to normal cartilage. XT-1 expression in cultured primary articluar chondrocytes showed a periodic oscillation in both mRNA and protein level. 29-kDa FN-f significantly suppressed the mRNA and protein levels of XT-1 at 14 h and 24 h, respectively. Inhibition of mitogen activated protein kinase and nuclear factor-kB signaling pathway restored 29-kDa FN-f-inhibited XT1 expression. Knockdown of toll like receptor-2 (TLR-2) using small interference RNA revealed that the decrease of XT-1 expression by 29kDa FN-f is mediated by TLR-2 signaling pathway. In addition, Sp3, a repressor of XT-1 promoter, was up-regulated by 29-kDa FN-f. Knockdown and overexpression experiments revealed that XT-1 expression was modulated by 29-kDa FN-f-stimulated Sp3 in primary articular chondrocytes. XT- expression was promoted by AP-1 inhibitor and suppressed by AP-1 activator. Both c-jun and c-fos were activated by 29kDa FN-f. Conclusions: These results demonstrated that 29-kDa FN-f plays a detrimental role in the regulation of cartilage extracellular matrix formation including XT-1 expression. 671 THE GROOVE MODEL OF TIBIA-FEMORAL OSTEOARTHRITIS IN THE RAT H.M. de Visser, H. Weinans, K. Coeleveld, M.H. van Rijen, F.P. Lafeber, S.C. Mastbergen. Univ. Med. Ctr. Utrecht, Utrecht, Netherlands

Figure 1. A) Total Modified Mankin Scores: No changes between groups were found (score ranging from zero to 84).B) Partial Modified Mankin Scores: No changes between groups were found (score ranging from zero to 14). C) COX-2 qPCR analysis for synovium: Data were normalized to control group values (dashed grey line). MD: moderate duration walking, HD: high duration walking, EHD extra high duration walking MTP: medial tibial plateau, MFC: medial femoral condyle, LTP: lateral tibial plateau, LFC: lateral femoral condyle, Pat: patella, Grv: femoral grove. * indicated significant difference when compared to control group. 670 EXPRESSION OF XYLOSYLTRANSFERASE-1 IS MODULATED BY FIBRONECTIN FRAGMENT IN HUMAN ARTICULAR CHONDROCYTES M. Lee, M. Choi, H. Hwang, H. Kim. Hallym Univ. Sacred Heart Hosp., Anyang, Republic of Korea Purpose: Xylosyltransferase-1 (XT-1), encoded by xylt1 gene, is an essential anabolic enzyme to catalyze the initial and rate-determining step in glycosaminoglycan chain synthesis. The effect of fibronectin fragments (FN-fs), generated by proteolytic cleavage of FN and known as damage-associated molecular pattern (DAMP) molecules, on

Purpose: Multiple experimental surgical models in small animals like the rat are currently used to study osteoarthritis (OA) and its potential treatment. However, these models have an important limitation as they often induce a permanent trigger during the course of OA, namely instability, counteracting the potential beneficial effects of therapy. Therefore, animal models of intrinsic cartilage damage, without a permanent trigger, have been described to induce joint degeneration and possibly being more sensitive to therapy. An example of such a model is the canine groove model of joint degeneration with features as observed in early OA. In this canine model, damage to the articular cartilage of the weight-bearing areas of the femoral condyles in the knee is the trigger for development of joint changes consistent with early OA. However, longitudinally in-vivo monitoring the disease progression is difficult in canine experiments. Besides, canine experiments can be limited performed due to restricted facilities and costs involved. The groove model can also be applied in the rat, where intrinsic cartilage damage is induced on the femoral trochlea. Here we report a modified groove model of the rat, where intrinsic cartilage damage on the weight bearing surface of the femoral condyles of a rat knee joint was induced, similar to the canine model. The subsequent degenerative joint changes were evaluated. Methods: In Ten male Wistar rats (Charles-River, Sulzfeld, Germany), 16 weeks of age, surgery was performed in one knee joint. During surgery the articular cartilage, of both the trochlear groove and the weight bearing areas of the femoral condyles, is damaged (grooved) without damaging the underlying subchondral bone. The contralateral knee joint served as an internal control. For a period of 12 weeks the subchondral bone parameters (Subchondral plate, trabecular bone and volume fraction) as well as the articular cartilage of the patella are longitudinally monitored by (contrast enhanced-)micro-CT (mCT) (Quatum FX, PerkinElmar, USA). Subsequently, OA severity is assessed, 6 and 12 weeks post-surgery, by the rat OARSI histopathology score by two observers blinded for study group. Directly induced damage by groove surgery was neglected for OA severity assessment. To analyze

Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534

differences between the surgical induced and contralateral control knee joints the paired-samples T test was used. The study was approved by the Utrecht University Medical Ethical Committee for animal studies. Results: Inducing local cartilage damage on the femoral condyles and trochlea by groove surgery in rat knee joints resulted in mild, but statistically significant higher, joint degeneration at 6 weeks (5.2 ± 0.8 vs 0.6 ± 0.5; p<0.0001) and 12 weeks (4.8 ± 0.7 vs 1 ± 0.9; p <0.001) post-surgery compared to the contralateral control knee joint. Although the tibia plateau was not surgically damaged, increased articular cartilage degeneration compared to the tibia plateau of the control knee joints was observed (4.8 ± 1 vs 1 ± 0.9; p<0.01; see Fig. 1). No difference was found in synovial membrane inflammation between surgically damaged and control knee joints (0.7 ± 0.5 vs 0.5 ± 0.7; p>0.05). With contrast enhanced mCT scans clear loss of cartilage thickness could be observed of the patella cartilage 12 weeks post-surgery (121 ± 48 vs 174 ± 57 mm; p<0.05). In parallel, the amount of grey value was enhanced over time, indicating increased uptake of contrast agent in the articular cartilage, and thus more cartilage degeneration (2255 ± 76 vs 1998 ± 80; p<0.05). In addition, mCT imaging detected significant subchondral bone changes on the tibia compartment of the knee joints already 6 weeks after surgery compared to the control knee joints regarding mean subchondral plate thickness (264 ± 19 vs 250 ± 13 mm; p<0.05), mean trabecular bone thickness (199 ± 14 vs 184 ± 9 mm; p<0.001) and volume fraction (0.55 ± 0.04 vs 0.51 ± 0.04; p<0.01). Conclusions: Here we present results of the modified groove model of local cartilage damage in the rat, which leads to early degenerative joint changes with slow onset, in both the directly damaged (femur) as the opposite (untouched tibia) joint compartment. Besides, this model did not lead to local inflammation and permanent destabilization.

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and then studied the consequences of in vitro extracellular PPi exposure on chondrocyte phenotype biomarkers and signaling pathways. Gene expression studies were performed on OA human cartilage samples. Phenotypic biomarkers were studied by quantitative RT-PCR in human OA chondrocytes stimulated with 0.1mM of PPi. Protein activities were measured by ELISA. MAPK signaling was investigated by immunoblotting and the use of selective inhibitors Results: Proteomics and PCR arrays showed that human OA chondrocytes stimulated with 0.1mM of PPi, displayed a decreased expression of collagen components of the matrix and sustained expression of MMPs and integrins. We confirmed that MMPs were increased by ELISA. Proteomics data also showed that PPi increased markers of hypertrophy such as VEGF and MMP13, whose activity was also increased as demonstrated by ELISA experiments At the same time, proteomics and ELISA showed that the expression of IGFBP3, a protein recently involved in OA, was up-regulated by PPi. Interestingly, these results are accentuated in medium supplemented with 100ug/ml of insulin. We demonstrated further that PPi stimulation mostly activates P38 pathway and to a lesser extent ERK pathway to regulate the expression of its target genes. Conclusions: Altogether, in contrast to previous data obtained in rats, the results obtained in this study demonstrate that PPi seems to promote matrix-remodeling and differentiation of OA chondrocytes towards an hypertrophic phenotype, with a potential role of the insulin pathway via IGFBP3. 673 CHARACTERIZING THE ROLE OF PPARDELTA IN THE PROGRESSION OF BEHAVIOURAL AND HISTOLOGIC SECONDARY OSTEOARTHRITIS A. Ratneswaran y, M.M. Sun y, M.A. Pest y, H. Dupuis y, C. Hamilton z, V. Pitelka y, F. Beier y. y Western Univ., London, ON, Canada; z Univ. of British Columbia, Vancouver, BC, Canada

Figure 1. Representative histological overview of safranin-O staining in a control knee joint and experimental knee joint 6 and 12 weeks postsurgery. 672 EFFECT OF PPI STIMULATION ON OSTEOARTHRITIC ARTICULAR HUMAN CHONDROCYTES M. Guibert, J.-B. Vincourt, H. Kempf, A. Bianchi. UMR 7365, Vandoeuvreles-Nancy, France Purpose: Osteoarthritis (OA) is the most common form of chronic joint disease, characterized by cartilage degeneration that results from complex changes in the chondrocyte phenotype. The presence of phosphate-containing microcrystals in the injured cartilage areas suggests the contribution of the phosphocalcic metabolism in the phenotype switch of some chondrocytes during the disease. The balance between the concentrations of inorganic pyrophosphate (PPi) and inorganic phosphate (Pi) present in the joint environment has emerged as an important regulator of the chondrocyte phenotype. If numerous studies have shown that elevated concentrations of extracellular Pi (ePi) or ePPi have, respectively, activating or repressive mineralizing effects on articular cartilage, we recently demonstrated that ePPi also prevents the in vitro dedifferentiation of articular chondrocyte in rats, an effect mostly triggered by Ank-induced release of PPi. This suggests that PPi may be an attractive candidate to prevent the phenotype switch of the articular chondrocyte observed in OA. To confirm and validate our observation in vitro, we analyze the role of PPi in human chondrocyte phenotype alterations during osteoarthritis. Methods: We first determined PPi targets by proteomics, PCR arrays and ELISA analyzes in cartilage samples obtained from OA individuals,

Purpose: Osteoarthritis (OA) is a debilitating chronic joint disorder affecting the entire joint. Current findings from our laboratory indicate that increased activation of a nuclear receptor, PPARdelta, in cultured murine joints results in breakdown of glycosaminoglycans in the cartilage and an OA-like phenotype. In contrast, cartilage-specific inactivation of PPARdelta results in significantly decreased incidence and progression of OA in a model of surgically induced OA in mice. This has prompted us to speculate whether inhibition of PPARdelta could be a viable treatment strategy and to investigate what genes are likely involved in this process. We hypothesize that inhibition of PPARdelta will slow the progression of OA in animal models and that this is mediated through changes in lipid metabolism. Methods: 300-350g male Sprague-Dawley rats were subjected to an anterior cruciate ligament transection (ACLT) and partial medial meniscectomy (PMMx) surgery and were systemically treated with two different inhibitors of PPARdelta (GSK0660, 3787) or vehicle control DMSO for six days per week, starting one day post-surgery, for four weeks. Weekly testing for spontaneous exploratory behavior (Open Field Testing) and load-bearing asymmetry (Incapacitance Testing) was conducted to measure pain associated behaviours. End point blood glucose and liver weight were measured. Rats were compared through measures of OA progression including Safranin-O staining with OARSI scoring, immunohistochemistry for cartilage matrix breakdown products, picrosirius red staining for collagen structure and organization, and Micro-CT analysis for changes in subchondral bone. Concurrently, microarrays were conducted on immature murine articular chondrocytes treated with the PPARdelta agonist GW501516 to identify targets of this nuclear receptor. Lipid assays were also performed on these cells to determine key metabolic pathways changing after nuclear receptor treatment. Results: Rats treated with either PPARdelta inhibitor are not different in physiological profiles (blood glucose, liver weight, body weight) from vehicle treated or SHAM operated animals. Vehicle treated rats demonstrate changes in behaviour such as decreased vertical activity and increased rest time. In contrast, animals treated with PPARdelta