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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.

Adult stem cells have more restricted differentiation potential than embryonic stem cells (ESCs), but upon appropriate stimulation can differentiate into cells of different germ layers. Epigenetic factors, including DNA modifications, take a significant part in regulation of pluripotency and differentiation of ESCs. Less is known about the epigenetic regulation of these processes in adult stem cells. Gene expression profile and location of DNA modifications in adipose-derived stem cells (ADSCs) and their osteogenically differentiated lineages were analyzed using Agilent microarrays. Methylation-specific PCR and restriction-based quantitative PCR were applied for 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) detection in selected loci. The level of DNA modifications in the POU5F1 locus was quantified with deep sequencing. Expression levels of selected genes were assayed by real-time PCR. ADSCs differentiation into osteogenic lineages involved marked changes in both 5mC and 5hmC profiles, but 5hmC changes were more abundant. 5mC losses and 5hmC gains were the main events observed during ADSCs differentiation, and were accompanied by increased expression of TET1 (P = 0.009). In ADSCs, POU5F1 was better expressed than NANOG or SOX2 (P ≤ 0.001). Both 5mC and 5hmC marks were present in the POU5F1 locus, but only hydroxymethylation of specific cytosine showed significant effect on the gene expression. In summary, the data of our study suggest significant involvement of changes in 5hmC profile during the differentiation of human adult stem cells.

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.

Background Osteoarthritis (OA) is the most common joint disease and the major cause of pain and disability in the aging population [1]. Adipose stromal cells (ASC) are promising candidate for cell therapy in OA, because they have immunomodulatory, trophic and differentiation capacities [2,3]. Synovial inflammation is accepted as important OA feature for the symptoms and disease progression [4]. Synovial tissue is mainly composed of synovial fibroblasts (SF), macrophages (SM) and a low percentage of other cell types [5]. Objectives Aim of the study was to analyze the effects of adipose stromal cells in co-culture with SF and SM. Methods GMP clinical grade ASC were isolated from subcutaneous adipose tissue. Synoviocytes were isolated from synovial tissue of OA patients undergoing total joint replacement. Synovial cells at passage 1 and 5 were analyzed for: 1. different phenotypical markers by flow cytometric analysis, 2. inflammatory factors by multiplex immunoassay, 3.anabolic and degradative factors by qRT-PCR. Both p.1 (mix of SF and SM) and p.5 (only SF) synovial cells, as different cell models, were co-cultured with adipose stem cells (ASC) to define their effects. Furthermore macrophages type 1 (M1) were isolated and co-cultured with ASC. Results Synoviocytes at passage 1 were positive to typical markers of SM (CD14,CD16,CD68, CD80,CD163) and SF (CD55,CD73,CD90,CD105,CD106), whereas at passage 5 were positive only to SF markers and showed a higher percentage of CD55 and CD106. At p.1 synovial cells released a significantly higher amount of all inflammatory (IL6,CXCL8,CCL2,CCL3,CCL5) and anabolic (IL10) factors than those at p.5. Moreover, p.1 synovial cells expressed also higher amount of some degradative factors (MMP13, S100A8, S100A9) than p.5 synovial cells. Co-culture experiments showed that the amount of SM in p.1 synovial cells specifically orchestrate the up or down-modulation of some inflammatory (IL6,CXCL8,CCL2,CCL3,CCL5) and degradative factors (ADAMTS5,MMP13, S100A8,S100A9) analyzed. Interestingly, p.5 synovial cells induced all factors analyzed, except CCL5. Finally, we demonstrated that ASC effects were strictly dependent by M1, that decreased the release of typical macrophages cytokines (IL1β, IL6, TNFα and CCL3/MIP1α) and that ASC effects are responsible for the switching by M1 like inflammatory macrophages to M2 like phenotype mainly due to PGE2 involvement. Conclusions These data demonstrate that the GMP-ASC effects on OA synovial inflammation are strictly dependent by macrophages, that orchestrate the switching activated-M1 inflammatory macrophages to a M2-like phenotype, mainly through PGE2 References Loeser RF (2012) Arthritis Rheum 64:1697–707. C. Manferdini (2013) Arthritis Rheum 65:1271–81. M. Maumus (2013) Stem Cell Research 11, 834–844. Y. Henrotin (2014) Semin Arthritis Rheum 43(5):579–87. MD. Smith (2011) The Open Rheumatology Journal 5:100–6. Acknowledgements This research was supported by Horizon 2020 Programme (project ADIPOA2, grant agreement no: 643809). The materials presented and views expressed here are the responsibility of the authors only. The EU Commission takes no responsibility for any use made of the information set out. Disclosure of Interest None declared

Objective To examine the effect of different sources of Good Manufacturing Practice clinical grade adipose-derived mesenchymal stem cells (AD-MSCs) on inflammatory factors in osteoarthritic (OA) chondrocytes and synoviocytes. Methods AD-MSCs from infrapatellar Hoffa fat, subcutaneous (SC) hip fat, and SC abdominal fat were cocultured in Transwells with chondrocytes or synoviocytes. Inflammatory factors (interleukin-1β [IL-1β], tumor necrosis factor α, IL-6, CXCL1/growth-related oncogene α, CXCL8/IL-8, CCL2/monocyte chemotactic protein 1, CCL3/macrophage inflammatory protein 1α, and CCL5/RANTES) were evaluated by quantitative reverse transcription–polymerase chain reaction or multiplex bead–based immunoassay. The role of different immunomodulators was analyzed. Results All the inflammatory factors analyzed were down-modulated at the messenger RNA or protein level independently by all 3 AD-MSC sources or by allogeneic AD-MSCs used in coculture with chondrocytes or synoviocytes. Inflammatory factor down-modulation was observed only when AD-MSCs were cocultured with chondrocytes or synoviocytes that produced high levels of inflammatory factors, but no effect was observed in cells that produced low levels of those factors, thus highlighting a dependence of the AD-MSC effect on existing inflammation. The immunomodulators IL-10, IL-1 receptor antagonist, fibroblast growth factor 2, indoleamine 2,3-dioxygenase 1, and galectin 1 were not involved in AD-MSC effects, whereas the cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2) pathway exerted a role in the mechanism of antiinflammatory AD-MSC action. Conclusion The antiinflammatory effects of AD-MSCs are probably not dependent on AD-MSC adipose tissue sources and donors but rather on the inflammatory status of OA chondrocytes and synoviocytes. AD-MSCs seem to be able to sense and respond to the local environment. Even though a combination of different molecules may be involved in AD-MSC effects, the COX-2/PGE2 pathway may play a role, suggesting that AD-MSCs may be useful for therapies in osteoarticular diseases.

Background The aim of the study was to characterize synovial cells from OA synovium with low-grade and moderate-grade synovitis and to define the role of synovial macrophages in cell culture. Methods Synovial tissue explants were analyzed for the expression of typical markers of synovial fibroblasts (SF), synovial macrophages (SM) and endothelial cells. Synovial cells at passage 1 (p.1) and 5 (p.5) were analyzed for different phenotypical markers by flow cytometric analysis, inflammatory factors by multiplex immunoassay, anabolic and degradative factors by qRT-PCR. P.1 and p.5 synovial cells as different cell models were co-cultured with adipose stem cells (ASC) to define SM effects. Results Synovial tissue showed a higher percentage of CD68 marker in moderate compared with low-grade synovitis. Isolated synovial cells at p.1 were positive to typical markers of SM (CD14, CD16, CD68, CD80 and CD163) and SF (CD55, CD73, CD90, CD105, CD106), whereas p.5 synovial cells were positive only to SF markers and showed a higher percentage of CD55 and CD106. At p.1 synovial cells released a significantly higher amount of all inflammatory (IL6, CXCL8, CCL2, CCL3, CCL5) and some anabolic (IL10) factors than those of p.5. Moreover, p.1 synovial cells also expressed a higher amount of some degradative factors (MMP13, S100A8, S100A9) than p.5 synovial cells. Co-culture experiments showed that the amount of SM in p.1 synovial cells differently induced or down-modulated some of the inflammatory (IL6, CXCL8, CCL2, CCL3, CCL5) and degradative factors (ADAMTS5, MMP13, S100A8, S100A9). Conclusions We found that p.1 (mix of SM and SF) and p.5 (only SF) synovial cells represent two cell models that effectively reproduce the low- or moderate-grade synovitis environment. The presence of SM in culture specifically induces the modulation of the different factors analyzed, confirming that SM are key effector cells. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-0983-4) contains supplementary material, which is available to authorized users.

Background aims The clinical use of human mesenchymal stromal cells (MSC) requires ex vivo expansion in media containing supplements such as fetal bovine serum or, alternatively, human platelet lysate (PL). Methods Platelet concentrates were frozen, quarantine stored, thawed and sterile filtered to obtain PL. PL content and its effect on fibroblast-colony-forming unit (CFU-F) formation, MSC proliferation and large-scale expansion were studied. Results PL contained high levels of basic fibroblast growth factor (bFGF), soluble CD40L (sCD40L), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB/BB (PDGF-AB/BB), chemokine (C-C) ligand 5 (CCL5; RANTES) transforming growth factor-β1 (TGF-β1) and chemokine (C-X-C) ligand 1/2/3 (GRO), with low batch-to-batch variability, and most were stable for up to 14 days. Inhibition of PDGF-BB and bFGF decreased MSC proliferation by about 20% and 50%, respectively. The strongest inhibition (about 75%) was observed with a combination of anti-bFGF + anti-PDGF-BB and anti-bFGF + anti-TGF-β1 + anti-PDGF-BB. Interestingly, various combinations of recombinant PDGF-BB, bFGF and TGF-β1 were not sufficient to promote cell proliferation. PL from whole blood-derived pooled platelet concentrates and apheresis platelet concentrates did not differ significantly in their growth-promoting activity on MSC. Conclusions PL enhances MSC proliferation and can be regarded as a safe tool for MSC expansion for clinical purposes. \in particular, PDGF-BB and bFGF are essential components for the growth-promoting effect of PL, but are not sufficient for MSC proliferation.

The physiological functions of human TCRVgamma9Vdelta2(+) gammadelta lymphocytes reactive to non-peptide phosphoantigens contribute to cancer immunosurveillance and immunotherapy. However, their regulation by mesenchymal stem cells (MSC), multipotent and immunomodulatory progenitor cells able to infiltrate tumors, has not been investigated so far. By analyzing freshly isolated TCRVgamma9Vdelta2(+) lymphocytes and primary cell lines stimulated with synthetic phosphoantigen or B-cell lymphoma cell lines in the presence of MSC, we demonstrated that MSC were potent suppressors of gammadelta-cell proliferation, cytokine production and cytolytic responses in vitro. This inhibition was mediated by the COX-2-dependent production of prostaglandin E2 (PGE(2)) and by MSC through EP2 and EP4 inhibitory receptors expressed by Vgamma9Vdelta2 T lymphocytes. COX-2 expression and PGE(2) production by MSC were not constitutive, but were induced by IFN-gamma and TNF-alpha secreted by activated Vgamma9Vdelta2 T cells. This regulatory cross-talk between MSC and Vgamma9Vdelta2 T lymphocytes involving PGE(2) could be of importance for the antitumor and antimicrobial activities of gammadelta T cells.

SUMMARY: Since the pioneering work of Alexander Friedenstein on multipotent mesenchymal stromal cells (MSCs), a tremendous amount of work has been done to isolate, characterize and culture such cells. Assay of colony forming unit-fibroblasts (CFU-Fs), the hallmark of MSCs, is used to estimate their frequency in tissue. MSCs are adherent cells, so they are easy to isolate, and they show contact inhibition. Thus, several parameters must be taken into account for culture: cell density, number of passages, culture medium, and growth factors used. The purity of the initial material is not a limiting parameter. Similar but not identical cell populations are found in almost all mammal or human tissues. MSCs seem to be very abundant in adipose tissue but at low frequency in blood from umbilical cord or in adult tissue. The culture conditions are very similar, whatever the source of cells. Because of their favorable properties, MSCs are very promising tools for regenerative medicine.

International audience; Recent literature suggested that cells of the microenvironment of tumors could be abnormal as well. To address this hypothesis in multiple myeloma (MM), we studied bone marrow mesenchymal stem cells (BMMSCs), the only long-lived cells of the bone marrow microenvironment, by gene expression profiling and phenotypic and functional studies in three groups of individuals: patients with MM, patients with monoclonal gamopathy of undefined significance (MGUS) and healthy age-matched subjects. Gene expression profile independently classified the BMMSCs of these individuals in a normal and in an MM group. MGUS BMMSCs were interspersed between these two groups. Among the 145 distinct genes differentially expressed in MM and normal BMMSCs, 46% may account for a tumor-microenvironment cross-talk. Known soluble factors implicated in MM pathophysiologic features (i.e. IL (interleukin)-6, DKK1) were revealed and new ones were found which are involved in angiogenesis, osteogenic differentiation or tumor growth. In particular, GDF15 was found to induce dose-dependent growth of MOLP-6, a stromal cell-dependent myeloma cell line. Functionally, MM BMMSCs induced an overgrowth of MOLP-6, and their capacity to differentiate into an osteoblastic lineage was impaired. Thus, MM BMMSCs are abnormal and could create a very efficient niche to support the survival and proliferation of the myeloma cells.

La production d’un medicament de therapie innovante (MTI) necessite l’etablissement d’un systeme de controle qualite (CQ) performant repondant aux principes des bonnes pratiques de fabrication (BPF). Chaque matiere premiere et consommable (MPC) entrant dans la fabrication d’un MTI doit repondre a des specifications et etre soumis a un controle puis a une liberation de lot par le responsable CQ, conduisant ainsi a la certification du medicament par le pharmacien responsable. Suite a une analyse de risque sur les MPC, une base de donnees a ete etablie pour les differents types de MPC impliques dans le processus de fabrication incluant les differents CQ associes selon les BPF et la pharmacopee europeenne. L‘elaboration d’un outil informatique nous permet de generer une fiche de specifications et d’acceptabilite (FSA) recapitulant les controles a effectuer sur chaque lot de MPC recu. Les FSA pre-remplies sont completees par les techniciens en fonction des resultats obtenus lors des controles, puis validees par le responsable du CQ pour etre ensuite classees. De facon parallele, les MPC receptionnees sont placees physiquement et informatiquement en statut quarantaine, puis basculees en statut libere apres validation par le responsable CQ. Les differents types de controles des MPC ont ete hierarchises et la mise en place de ce circuit a permis sur 169 lots de MPC de deceler 11 non-conformites, toutes observees sur l’integrite du conditionnement ou du produit, conduisant a des actions correctives avec les fournisseurs. Ce nouveau circuit d’aide a permis d’ameliorer considerablement le controle a reception des MPC tel que defini dans les BPF ( Figure 1 ).

Adipose tissue-derived stromal cells (ASCs) are promising tools for the new therapeutic field of regenerative medicine. Many research teams are intent on producing these cells for therapeutic purposes. The cell production must follow strict rules for safety and for constant quality of the cell product to ensure a reliable effect in patients. These rules are grouped under the generic term Good Manufacturing Practices. In this chapter, we describe the general concepts of ASC production for therapeutic use, explaining new terms such as traceability and qualification. We also introduce general requirements for the installation, equipment, material, and staff for the cell production. Then, we outline a general strategy for building a cell culture process. Finally, as an example, we describe the use of CellStack™ chambers and specific tube sets that allow for producing cells beginning with the stromal vascular fraction under near-closed conditions.

Adipose tissue-derived stromal cells (ASCs) are promising tools for the new therapeutic field of regenerative medicine. Many research teams are intent on producing these cells for therapeutic purposes. The cell production must follow strict rules for safety and for constant quality of the cell product to ensure a reliable effect in patients. These rules are grouped under the generic term Good Manufacturing Practices. In this chapter, we describe the general concepts of ASC production for therapeutic use, explaining new terms such as traceability and qualification. We also introduce general requirements for the installation, equipment, material, and staff for the cell production. Then, we outline a general strategy for building a cell culture process. Finally, as an example, we describe the use of CellStack™ chambers and specific tube sets that allow for producing cells beginning with the stromal vascular fraction under near-closed conditions.

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

Because of their multi/pluripotency and immunosuppressive properties, mesenchymal stem/stromal cells (MSCs) are important tools for treating immune disorders and for tissue repair. The increasing use of MSCs, their definition as advanced-therapy medicinal products in European regulations, and the US Food and Drug Administration requirements for their production and use imply the use of production processes that should be in accordance with Good Manufacturing Practices (GMPs). Complying with GMPs requires precisely defining the production process (es) as well as the multiple criteria required for a quality final product. Such variables include the environment, staff training and qualification, and controls. Developing processes based on well-defined or completely defined media and operating in closed systems or bioreactors is important and will increase safety and reproducibility. One of the most challenging issues remains implementation of relevant and reproducible controls for safety and efficacy. A linking of researchers, research and development teams, producers, and clinicians is mandatory to achieve GMP-compliant processes with relevant controls for producing well-defined, safe, and efficient MSCs.

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