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Summary Objective To determine the mechanisms of meniscal degeneration and whether this varied zonally and from articular cartilage. Design Normal ovine menisci were dissected into inner and outer zones and along with cartilage cultured ±IL-1α and TNFα. Glycosaminoglycan (GAG) and collagen release, and gene expression were quantified. Aggrecan proteolysis was analysed by Western blotting with neoepitope-specific antibodies. Matrix metalloproteinase (MMP)2, MMP9 and MMP13 activity was evaluated by gelatin zymography or fluorogenic assay. Results Inner meniscus was more cartilaginous containing more GAG and expressing more ACAN and COL2A1 than outer zones. Higher expression of VCAN and ADAMTS4 in medial outer and both zones of the lateral meniscus reflected their embryologic origin from cells outside the cartilage anlagen. All meniscal regions released a greater % GAG in response to cytokines; only outer zones had cytokine-stimulated collagenolysis. Cytokine-induced aggrecanolysis was primarily due to increased ADAMTS cleavage in cartilage and inner menisci but MMPs in the outer menisci. Outer menisci always released more active MMP2 than other tissues and more active MMP13 in basal and TNF-stimulated cultures. Expression of ACAN , COL1A1 and COL2A1 was decreased by both cytokines in all tissues, while VCAN was increased by IL-1α in cartilage and inner menisci. Metalloproteinase expression was differentially regulated by IL-1α and TNFα: ADAMTS4 , MMP1 , MMP3 were upregulated more by IL-1α in inner zones whereas ADAMTS5 , MMP13 and MMP9 were more upregulated by TNFα in outer zones. Conclusions Meniscal degeneration mechanisms are zonally-dependent, and may contribute to the enzymatic burden in the joint.

ing, quantification of behavioural change using automated videoanalysis, and electrodermal activity (EDA) used as an indirect quantification of pain. Evaluations were performed at baseline, D28, and D56. General linear model for repeated measures was performed for the analysis of rating scores, PVF, locomotor activity and EDA. Logistic regression for repeated measurements was applied to ordinal and video-analysis data. Results: VAS scores increased from D0 to D28 and remained stable from D28 to D56 for both observers. The overall scores of STAPS and SVAPS increased over time but no difference between groups was observed. The component “Willingness to hold up contra-lateral limb” in SVAPS was lower (p=0.0495), and PVF higher (p=0.05), in Group 2 compared to Group 1 at D56. The criteria “Lameness when walking”, “Lameness when trotting”, and “Reaction to palpation” increased over time in both groups (p

miR-im6 transduced hBMSCs when compared with control. We also confirmed that the down-regulation of hedgehog signals by miR-im6 can suppress expression of wnt signal molecules (Wnt3a, Wnt7a, Wnt6, Wnt9b and b-catenin)(Fig 2b). On the contrary, indian hedgehog was transiently up-regulated by overexpression of SPB-X in hBMSCs (Fig 2c-f). Transcription factor SPB-X and Wnt6 are target genes of hsa-miRim6: As shown in Fig 3c, luciferase activitieswere significantly decreased in Hela cells was co-transfected with luciferase reporter vectors harboring SPB-X or WNT6 3’UTR and hsa-miR-im6 mimic. This result demonstrates that SPB-X and WNT6 are target genes of hsa-miR-im6. SPB-X knockout induce down-regulation of indian hedgehog: We investigated SPB K/O effects using mouse MSCs isolated from SPB K/O