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Purpose/Aim of the study. Collagen type XV (ColXV) was identified, in our previews studies, as a novel component of bone extracellular matrix. The present study aims to investigate ColXV localization during mineralization of osteodifferentiated human mesenchymal stem cells (hMSCs).hMSCs cultured in osteogenic medium have been analyzed at day 14 and 28 for mineral matrix deposition by alizarin red S staining, ultrastructural analysis and ColXV localization by immunogold electron microscopy.Our data show an intimate association between ColXV and fibrillar components in areas localized far from mineralized nodules.We have demonstrated the efficacy of ultrastructural analysis, combined with immunocytochemistry, to establish a temporal and spatial localization of ColXV. This data, added to previous evidences, contribute to validate the negative effects of calcium deposits on ColXV expression.

International audience; ObjectiveTo define whether good manufacturing practice (GMP)-clinical grade adipose stem cell (ASC)-derived conditioned medium (CM) is as effective as GMP-ASC in modulating inflammatory and catabolic factors released by both osteoarthritis (OA) chondrocytes or synoviocytes.MethodsOA chondrocytes and synoviocytes were treated with ASC-CM or co-cultured with ASC. Inflammatory factors (IL6, CXCL1/GROα,CXCL8/IL8, CCL2/MCP-1, CCL3/MIP-1α and CCL5/RANTES) and proteinases, such as metalloproteinase (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS4, ADAMTS5) and their tissue metalloproteinase inhibitors (TIMP1, TIMP3) were evaluated by qRT-PCR or immunoassays. The involvement of prostaglandin E2 (PGE2) was also analyzed.ResultsMost ASC-CM ratios tested did not decrease IL6, CCL2/MCP-1, CCL3/MIP1-α, CCL5/RANTES on basal inflamed chondrocytes or synoviocytes in contrast to what we found using ASC in co-culture. CXCL8/IL8 and CXCL1/GROα were not decreased by ASC-CM on synoviocytes but were only partially reduced on chondrocytes. Moreover, ASC-CM was less efficient both on basal inflamed OA chondrocytes and synoviocytes in reducing proteinases, such as MMP13, ADAMTS4, ADAMTS5 and increasing TIMP1 and TIMP3 compared to ASC in co-culture. The different ratios of ASC-CM contain lower amounts of PGE2 which were not sufficient to reduce inflammatory factors.ConclusionsThese data show that ASC-CM has a limited ability to decrease inflammatory and proteinases factors produced by OA chondrocytes or synoviocytes. ASC-CM is not sufficient to recapitulate the beneficial effect demonstrated using ASC in co-culture with inflamed OA chondrocytes and synoviocytes and shows that their use in clinical trials is fundamental to counteract OA progression.

Increased age is the most prominent risk factor for the initiation and progression of osteoarthritis (OA). The effects of human growth hormone (hGH) combined or not with hyaluronan amide derivative (HAD) were evaluated on human OA chondrocytes, to define their biological action and potentiality in OA treatment.Cell viability, metabolic activity, gene expression and factors released were tested at different time points on chondrocytes treated with different concentrations of hGH (0.01-10 μg/ml) alone or in combination with HAD (1 mg/ml).We found that OA chondrocytes express GH receptor and that the different doses of hGH tested did not affect cell viability, metabolic activity or the expression of collagen type 2, 1, or 10 nor did it induce the release of IGF-1 or FGF-2. Conversely, hGH treatment increased the expression of hyaluronan receptor CD44. HAD combined with hGH reduced metabolic activity, IL6 release and gene expression, but not the suppressor of cytokine signaling 2 (SOCS2), which was significantly induced and translocated into the nucleus. The parameters analyzed, independently of the treatments used proportionally decreased with increasing age of the patients.hGH only induced CD44 receptor on OA chondrocytes but did not affect other parameters, such as chondrocytic gene markers or IGF-1 or FGF-2 release. HAD reduced all the effects induced by hGH partially through a significant induction of SOCS2. These data show that GH or HAD treatment does not influence the response of the OA chondrocytes, thus the modulation of cellular response is age-independent.

Summary Objective To contribute to clarify molecular mechanisms supporting senescence and de-differentiation of chondrocytes in chondrocyte pathologies such as osteoarthritis (OA). Specifically, we investigated the relationship between the nuclear lamina protein Lamin B1 and the negative regulator of chondrogenesis Slug transcription factor in osteoarthritic chondrocytes. Methods Lamin B1 and Slug proteins were analyzed in cartilage explants from normal subjects and OA patients by immunohistochemical technique. Their expression was confirmed on isolated chondrocytes both at passage 0 and passage 2 (de-differentiated chondrocytes) by immunofluorescence and western blot. Subsequently, we explored the " in vivo " binding of Slug on LMNB1 promoter by chromatin immunoprecipitation assay (ChIP). Results In this study we demonstrated that nuclear lamina protein Lamin B1 and anti-chondrogenic Slug transcription factor are upregulated in cartilage and OA chondrocytes. Furthermore, we found that Slug is " in vivo " recruited by LMNB1 gene promoter mostly when chondrocytes undergo de-differentiation or OA degeneration. Conclusions We described for the first time a potential regulatory role of Slug on the LMNB1 gene expression in OA chondrocytes. These findings may have important implications for the study of premature senescence, and degeneration of cartilage, and may contribute to develop effective therapeutic strategies against signals supporting cartilage damage in different subsets of patients.

Summary Objective To define if adipose mesenchymal stromal cell (ASC) treatment mediated switching of the pro-inflammatory profile of M1-like macrophages as a means to develop a tailored in vitro efficacy/potency test. Design We firstly performed immunohistochemical analysis of CD68, CD80 (M1-like) and CD206 (M2-like) macrophages in osteoarthritic (OA) synovial tissue. ASC were co-cultured in contact and in transwell with activated (GM-CSF + IFNγ)-M1 macrophages. We analyzed IL1β, TNFα, IL6, MIP1α/CCL3, S100A8, S100A9, IL10, CD163 and CD206 by qRT-PCR or immunoassays. Prostaglandin E2 (PGE2) blocking experiments were performed using PGE2 receptor antagonist. Results In moderate grade OA synovium we did not always find a higher percentage of CD80 with respect to CD206. M1-like-activated macrophage factors IL1β, TNFα, IL6, MIP1α/CCL3, S100A8 and S100A9 were down-modulated both in contact and in transwell by ASC. However, in both systems ASC induced the typical M2-like macrophage markers IL10, CD163 and CD206. Activated-M1-like macrophages pre-treated with PGE2 receptor antagonist failed to decrease secretion of TNFα, IL6 and to increase that of IL10, CD163 and CD206 when co-cultured with ASC confirming a PGE2 specific role. Conclusions We demonstrated that ASC are responsible for the switching of activated-M1-like inflammatory macrophages to a M2-like phenotype, mainly through PGE2. This evidenced that activated-M1-like macrophages may represent a relevant cell model to test the efficacy/potency of ASC and suggests a specific role of ASC as important determinants in therapeutic dampening of synovial inflammation in OA.

Individual Determinants 1. Patellar osteophytes 2. Femoral trochlear groove periarticular osteophytes 3. Lateral and/or medial femoral condylar periarticular osteophytes 4. Femoral subchondral sclerosis 5. Distal femoral condylar remodeling 6. Subchondral cystic lucencies 7. Sesamoid periarticular osteophytes 8. Femoral intercondylar notch osteophytes 9. Proximal tibial periarticular osteophytes 10. Proximal tibial subchondral sclerosis 11. Proximal tibial subchondral cystic lesions 12. Central tibial plateau osteophytes 13. Joint effusion/capsular thickening 14. Intraarticular mineralized osseous fragments 15. Meniscal mineralization 16. Intercondylar avulsion fracture fragments

s / Osteoarthritis and Cartilage 23 (2015) A82eA416 A383 presence of macrophages, especially in the lining layer, is associated with progression of cartilage damage in OA, whereas synovial expression of MMPs seems related to progression regarding osteophyte formation. 636 ADIPOSE STROMAL CELLS EFFECTS ON OSTEOARTHRITIC SYNOVIOCYTES ARE DEPENDENT BY MACROPHAGES C. Manferdini y, E. Gabusi y, F. Paolella y, A. Piacentini y, S. Fleury-Cappellesso z, C. Jorgensen x, D. Noel x, A. Barbero k, G. Lisignoli y. y Istituto Ortopedico Rizzoli, Bologna, Italy; z EFS-Pyr en e esM editerran e e, Toulouse, France, France; x Inserm U844, Universit e Montpellier 1, Montpellier, France; kDept. of Surgery and Biomedicine, Univ. Hosp., Basel, Switzerland Purpose: Synovium is mainly composed of synovial fibroblast (SF) and macrophage (SM) and a low percentage of other cell types. In osteoarthritis (OA) synovial inflammation is a key factor associated to pain and symptoms and to a unique chemokine signature. Aim of the study was to analyze the role of SF and SM in counteracting the effects due to adipose stem cell co-culture. Methods: Good manufacturing practice (GMP)-clinical grade ASC were isolated from subcutaneous adipose tissue. Synoviocytes were isolated from synovia of OA patients undergoing total joint replacement. Synoviocytes both at passage 1 (p1) and 5 (p5) were characterized for the following markers: CD3, CD14, CD16, CD45, CD68, CD90, vimentin, VCAM-1 by flow cytometry or immunocytochemistry. Secreted inflammatory factors (IL6, CXCL8/IL8, CXCL1/GROa, CCL2/MCP-1, CCL3/ MIP1a, CCL5/RANTES, IL1b, TNFa) were analysed by multiplex beadbased sandwich immunoassay or ELISA tests on synoviocytes p1 and p5 untreated or co-cultured with ASC in transwell. Macrophages type 1 (M1) and 2 (M2) were also isolated and co-cultured with ASC. Results: Synoviocytes at p1 (mainly composed of SM and SF) were approximately 1% positive to CD3, 8% to CD45,10% to CD14,12% to CD16, 8% to CD68, 90% to CD90 while synoviocytes at p.5 (only SF) were negative to all the markers analyzed except for CD90. Moreover, synoviocytes at p. 1 produced a significant higher amount of IL6, IL8/CXCL8, CCL2/MCP-1, CCL3/MIP1a and CCL5/RANTES than at p.5, except CCL3/ MIP1a that was not detected. We confirmed on serial sections of synovial tissue that CCL3/MIP1a was a marker of SM since the same cells were also positive to macrophages markers ( CD68, CD16). Interestingly, we confirmed that ASC reduced the release of inflammatory factors only when cultured with p.1 synoviocytes. By contrast, ASC cocultured with p5 synoviocytes induced the release of all factors, except CCL3/MIP1a that was still not released and expressed. Finally, we demonstrated that ASC effects were strictly dependent by macrophages type 1 and 2 that decreased the release of IL1b, TNFa and CCL3/ MIP1a.These effects were more evident when macrophages type 1 and 2 were co-cultured in contact. Conclusions: These data demonstrate that the GMP-ASC effects on OA synovial inflammation is strictly dependent by macrophages. ASC counteracting effects are mainly mediated by both soluble factors and cells cross talk. 637 UPREGULATION OF GENE EXPRESSION OF MATRIX METALLOPROTEINASES AND PROSTAGLANDIN E-2 PRODUCTION IN BIOMECHANICAL OR BIOCHEMICAL STIMULATION TO THREEDIMENSIONAL TISSUE OF HUMAN SYNOVIAL CELLS K. Nakata, K. Shimomura, T. Mae, Y. Yonetani, Y. Take, M. Kondo, H. Yokoi, H. Yoshikawa;. Osaka Univ., Suita, Japan Purpose: The purpose of this study was to elucidate the alteration of gene expression levels of matrix mettalloproteinases (MMPs), ADAMTS4 and interleukins (ILs) and protein production of PGE-2, IL-6, and IL-8 in cyclic biomechanical or biochemical stimulation of IL-1b to threedimensional cultured tissues of human synovium-derived cells and further investigate the effects of NSAIDs, dexamethasone, and inhibition of MyD88 using IL-1Ra on the biomechanical or biochemical stimulation to three-dimensional cultured tissues. Methods: Human synovium-derived cells were cultured in monolayer, collected and then seeded to a mechanically reinforced collagen scaffold to construct a three-dimensional cultured tissue. The threedimensional cultured tissues were stimulated either biomechanically or biochemically using a cyclic loading bioreactor at 0.5Hz, 40kPa for 1 hour or 10 ng/ml of IL-1b. The mRNA expressions of MMP-1, MMP-3, ADAMTS-4, ADMTS-5, IL-6, and IL-8 in the 3-D tissue were quantitatively determined by real time RT-PCR at 6 hours after mechanical loading or IL-1b stimulation. The media were also assayed for protein production of PGE2, IL-6, and IL-8 by HTRF®. mRNA expression and protein production were also examined in the presence or absence of various concentrations of NSAIDs of celecoxib (CEL), indomethacin (IND) and etodolac (ETD), dexamethasone (DEX) from at a concentration of 1.0 x 10-2 to that of 1.0 x 10 mM or 1mM of IL-1Ra. Results: Both cyclic biomechanical stimulation and biochemical stimulation of IL-1b statistically significantly upregulated gene expressions of MMP-1, -3, ADAMTS-4 and protein production of PGE2, IL-6 and -8 in three-dimensional tissues of human synovium-derived cells. NSAIDs and DEX statistically significantly inhibited the upregulation of protein expression of PGE2 by cyclic biomechanical loading or biochemical stimulation of IL-1Ra. CEL had an inhibitory effect of upregulated gene expression of ADAMTS-4 by biomechanical stimulation even at low concentration of 1.0 x 10-2 mM, whereas IND did in relatively high dose of 1.0 x 10-1 mM or more. ETD had no effects on ADAMTS-4 gene