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Scaffold-based bone tissue engineering strategies fail to meet the clinical need to fabricate patient-specific and defect shape-specific, anatomically relevant load-bearing bone constructs. 3D bioprinting strategies are gaining major interest as a potential alternative, but design of a specific bioink is still a major challenge that can modulate key signaling pathways to induce osteogenic differentiation of progenitor cells, as well as offer appropriate microenvironment to augment mineralization. In the present study, we developed silk fibroin protein and gelatin-based conjugated bioink, which showed localized presence and sustained release of calcium. Presence of 2.6 mM Ca2+ ions within the bioink could further induce enhanced osteogenesis of Bone marrow derived progenitor cells (hMSCs) compared to the bioink without calcium, or same concentration of calcium added to the media, as evidenced by upregulated gene expression of osteogenic markers. This study generated unprecedented mechanistic insights on the role of fibroin-gelatin-CaCl2 bioink in modulating expression of several proteins which are known to play crucial role in bone regeneration as well as key signaling pathways such as β-catenin, BMP signaling pathway, Parathyroid hormone-dependent signaling pathway, Forkhead box O (FOXO) pathway, and Hippo pathways in hMSC-laden bioprinted constructs.

Knee osteochondritis dissecans (OCD) is a focal disease of the joint characterized by modifications of bone and cartilage tissues. Biomimetic osteochondral scaffolds are used to restore these tissues. The aim of this prognostic prospective cohort study was to evaluate serum biomarkers of cartilage (fragments or propeptide of type II collagen: CTXII, C2C, and CPII) and bone (tartrate-resistant acid phosphatase (TRAP) 5b and osteocalcin (OC)) turnover during follow-up of patients treated with an osteochondral scaffold, to identify which were related to healing outcome and clinical score. We found that cartilage (CPII) and bone (OC) synthetic biomarkers were significantly increased during the first-year follow-up, while the respective degradative markers (CTXII, C2C, and TRAP5b) were not modulated. Only CTXII/CPII and C2C/CPII cartilage ratios were significantly modulated, evidencing a higher remodeling of cartilage compared to bone tissue. Cartilage and bone single biomarkers or ratios at one-year follow-up showed values close to or similar to those of healthy subjects. International Knee Documentation Committee (IKDC) score significantly increased from T0 to T2, while the Tegner score did not. Taking into consideration an IKDC score > 70 as clinical success, we found that all OCD cases with both CPII (> 300 pg/ml) and C2C/CPII (

Background/Aims: Mesenchymal stromal cells (MSCs) hold considerable promise in bone tissue engineering, but their poor survival and potency when in vivo implanted limits their therapeutic potential. For this reason, the study on culture conditions and cellular signals that can influence the potential therapeutic outcomes of MSCs have received considerable attention in recent years. Cell maintenance under hypoxic conditions, in particular for a short period, is beneficial for MSCs, as low O2 tension is similar to that present in the physiologic niche, however the precise mechanism through which hypoxia preconditioning affects these cells remains unclear. Methods: In order to explore what happens during the first 48 h of hypoxia preconditioning in human MSCs (hMSCs) from bone marrow, the cells were exposed to 1.5% O2 tension in the X3 Hypoxia Hood and Culture Combo – Xvivo System device. The expression modulation of critical genes which could be good markers of increased osteopotency has been investigated by Western blot, immunufluorescence and ELISA. Luciferase reporter assay and Chromatin immunoprecipitation was used to investigate the regulation of the expression of Collagen type XV (ColXV) gene. Results: We identified ColXV as a new low O2 tension sensitive gene, and provided a novel mechanistic evidence that directly HIF-1α (hypoxia-inducible factor-1 alpha) mediates ColXV expression in response to hypoxia, since it was found specifically in vivo recruited at ColXV promoter, in hypoxia-preconditioned hMSCs. This finding, together the evidence that also Runx2, VEGF and FGF-2 expression increased in hypoxia preconditioned hMSCs, is consistent with the possibility that increased ColXV expression in response to hypoxia is mediated by an early network that supports the osteogenic potential of the cells. Conclusion: These results add useful information to understand the role of a still little investigated collagen such as ColXV, and identify ColXV as a marker of successful hypoxia preconditioning. As a whole, our data give further evidence that hypoxia preconditioned hMSCs have greater osteopotency than normal hMSCs, and that the effects of hypoxic regulation of hMSCs activities should be considered before they are clinically applied.

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.

Hyaluronic acid (HA) is an ideal material for tissue regeneration. The aim of this study was to investigate whether a hyaluronan amide derivative (HAD) can enhance the mineralization of human mesenchymal stem cells (hMSCs). Osteogenically induced hMSCs cultured with or without HAD at different concentrations (0.5 mg/ml or 1 mg/ml) were analyzed for mineral matrix deposition, metabolic activity, cellular proliferation, and the expression of 14 osteogenic genes. Unmodified HA (HYAL) was used as control. We demonstrated that only cells treated daily until day 28 with 0.5 mg/ml HAD, but not with 1 mg/ml of HAD and HYAL, showed a significant induction of mineralization at day 14 compared to the osteogenic control group. HAD at both concentrations tested, significantly decreased the expression of the proliferating marker MKI67 at day 2. By contrast, increased metabolic activity was induced only by HYAL from day 14. HAD at both concentrations significantly down modulated SNAI2, DLX5, RUNX2, COL1A1, and IBSP genes, while significantly up regulated COL15A1. The induction of mineralization of 0.5 mg/ml of HAD at day 14 was significantly dependent on a specific modulation of RUNX2 and COL1A1. Our data demonstrate that only 0.5 mg/ml of HAD, but not HYAL, modulated hMSCs osteogenic differentiation, suggesting that the physicochemical features and concentration of HA products could differently affect osteogenic maturation.

Cell-based therapies using adipose-derived mesenchymal stromal cells (ADMSCs) have shown promising results for the treatment of osteoarthritis (OA). In fact, ADMSCs are now indicated as one of the most powerful cell sources through their immunomodulatory and anti-inflammatory activities. Recently, an innovative one-step closed device was developed to obtain microfragmented adipose tissue (MF) to avoid the need for good manufacturing practices for ADMSCs expansion while maintaining their regenerative potential. The aim of this study was to assess the mechanisms of action of MF and ADMSCs from MF (MF-ADMSCs) on an inflammatory cell model of OA synoviocytes. We found that MF produced low levels of inflammatory factors such as interleukin 6 (IL-6), CC-chemokine ligand 5/receptor-activated normal T-cell expressed and secreted (CCL5/RANTES), CC-chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1), and CC-chemokine ligand 3/macrophage inflammatory protein-1α (CCL3/MIP-1α), and a higher level only of CXC-chemokine ligand 8/interleukin 8 compared with MF-ADMSCs. Matrix metalloproteinase 9 (MMP-9) degradative factor but released a lower level of its inhibitor tissue inhibitor of the metalloproteinase (TIMP-1). MF in coculture with synoviocytes significantly induced both the metabolic activity and the release of IL-6. In contrast, MF, not MF-ADMSCs, partially decreased CCL5/RANTES. Moreover, MF reduced the release of both macrophage-specific chemokines (CCL2/MCP-1 and CCL3/MIP-1α) and degradative marker MMP-9. Interestingly, MF increased TIMP-1 (the MMP-9 inhibitor) and down-modulated toll-like receptor (TLR4) receptor and key molecules of NFκB pathways. These data evidenced different effects of MF versus MF-ADMSCs on inflamed synoviocytes. MF reduced typical macrophages markers and its potentiality by switching off macrophages activity was strictly dependent on TLR4 and NFκB signaling.

The healing potential of knee osteochondritis dissecans (OCD) focal lesions is not well defined. We performed a cross-sectional study correlating local and systemic biological characteristics with the patients’ characteristics. We evaluated both local tissue markers (CD34, CD146, CD166, and tartrate-resistant acid phosphatase (TRAP)) and systemic serum biomarkers (fragments or propeptide of type II collagen: C2C, CTX-II, CPII, and TRAP5b) on histologically scored osteochondral fragments or serum from OCD patients. These biological features were associated with the patients’ characteristics (IKDC subjective score, age, and body mass index (BMI)). Histological cartilage tissue score correlated with patients’ IKDC and C2C and CPII biomarkers. CPII correlated also with histological bone tissue score. The percentage of CD146 positive cells in cartilage and CD34 positive cells in bone highly correlated with the patient’s age and BMI, respectively. The percentage of TRAP in bone was directly correlated with both IKDC and age. Multivariate statistical analysis evidenced that only four parameters significantly predicted IKDC. In conclusion, a complete picture of OCD knee characteristics, defined by local and systemic markers of cartilage and bone remodeling, together with the patients’ characteristics, might help to better understand the healing potential of each patient and to target and improve current OCD treatments.

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.

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

none 8 no The surgical treatment of knee articular focal lesions may offer heterogeneous clinical results. Osteochondritis dissecans (OCD) lesions showed to heal better than degenerative lesions (DL) but the underlying biological reasons are unknown. We evaluated the basal histological and immunohistochemical characteristics of these lesions analyzing a series of osteochondral fragments from young patients with similar age but presenting different etiology. Osteochondral tissue samples were stained with Safranin O and graded using a histological score. Markers of mesenchymal progenitor cells (CD146), osteoclasts (tartrate-resistant acid phosphatase, TRAP), and vessels (CD34) were evaluated. Histological score showed a higher degeneration of both cartilage and bone compartments in OCD compared to DL fragments. Only CD146-positive cells were found at the same percentage in cartilage compartment of both DL and OCD patients. By contrast, in the bone compartment a significantly higher percentage of CD146, TRAP, and CD34 markers was found in OCD compared to DL patients. These data showed distinct histological characteristics of osteochondral focal lesions located in the same anatomical region but having a different etiology. The higher percentages of these markers in OCD than in DL, mainly associated with a high bone turnover, could help to explain the higher clinical healing potential of OCD patients. Gabusi, Elena; Manferdini, Cristina; Paolella, Francesca; Gambari, Laura; Kon, Elizaveta; Filardo, Giuseppe; Mariani, Erminia; Lisignoli, Gina Gabusi, Elena; Manferdini, Cristina; Paolella, Francesca; Gambari, Laura; Kon, Elizaveta; Filardo, Giuseppe; Mariani, Erminia; Lisignoli, Gina

Mesenchymal stromal cells (MSCs) are key players in the repair or regeneration of the damaged bone tissue. However, heterogeneity exists between MSCs derived from different donors in their bone formation ability both in vitro and in vivo. The identification of markers defining MSCs with different functional phenotypes is fundamental to maximize their clinical potential. In our previous in vivo study, impaired expression in MSCs of cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), the two key enzymes in the catabolic pathway of homocysteine, was associated to decreased bone formation and to the onset of osteoporosis in mice. Here, we investigated whether osteogenic differentiation of human MSCs (hMSCs) modulates the expression of CBS and CSE. The expression of CBS and CSE was also assessed during chondrogenesis to confirm the specificity of their expression during osteogenesis. hMSCs displayed a heterogeneous mineralizing capacity between donors (70% of the samples mineralized, while 30% did not mineralize). Inducible expression of CBS and CSE was found to be associated with a mineralizing phenotype in hMSCs. In particular, up-regulation of CSE was restricted to hMSCs undergoing mineralization. During chondrogenesis, CBS was significantly up-regulated while CSE expression was not affected. Ex-vivo findings confirmed that mature h-osteoblasts (hOBs) show consistently higher expression of CBS and CSE than hMSCs. Our data provide the first evidence that the expression of CBS and CSE in hMSCs closely correlates with the transition of hMSCs toward the osteoblastic phenotype and that CSE may constitute a novel marker of osteogenic differentiation.

We have previously demonstrated that collagen type XV (ColXV) is a novel bone extracellular matrix (ECM) protein. It is well known that the complex mixture of multiple components present in ECM can help both to maintain stemness or to promote differentiation of stromal cells following change in qualitative characteristics or concentrations. We investigated the possible correlation between ColXV expression and mineral matrix deposition by human mesenchymal stromal cells (hMSCs) with different osteogenic potential and by osteoblasts (hOBs) that are able to grow in culture medium with or without calcium. Analysing the osteogenic process, we have shown that ColXV basal levels are lower in cells less prone to osteo‐induction such as hMSCs from Wharton Jelly (hWJMSCs), compared to hMSCs that are prone to osteo‐induction such as those from the bone marrow (hBMMSCs). In the group of samples identified as ‘mineralized MSCs’, during successful osteogenic induction, ColXV protein continued to be detected at substantial levels until early stage of differentiation, but it significantly decreased and then disappeared at the end of culture when the matrix formed was completely calcified. The possibility to grow hOBs in culture medium without calcium corroborated the results obtained with hMSCs demonstrating that calcium deposits organized in a calcified matrix, and not calcium ‘per se’, negatively affected ColXV expression. As a whole, our data suggest that ColXV may participate in ECM organization in the early‐phases of the osteogenic process and that this is a prerequisite to promote the subsequent deposition of mineral matrix.

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.

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

The objective of this study was to define the effects of osteoarthritic (OA) milieu on good manufactured practice‐adipose‐derived mesenchymal stromal cells (GMP‐ASC) that are commonly utilized in cell therapies. Two different OA milieu: OA synovial fluid (SF) and OA‐conditioned medium (CM) from synoviocytes were used to treat GMP‐ASC both in normoxia or hypoxia. GMP‐ASC were tested for cell migration, proliferation, cytokine receptors expression (CXCR1, CXCR2, CXCR3, CXCR4, CXCR7, CCR1, CCR2, CCR3, CCR5, IL6R), and cytokines (CXCL8/IL8, CXCL10/IP10, CXCL12/SDF‐1, CCL2/MCP1, CCL3/MIP1α, CCL4/MIP1β, CCL5/RANTES, IL6) release. Healthy SF was used as controls. We demonstrated that GMP‐ASC show an increase in proliferation, migration, and modulation of CXCR1, CXCR3, CCR1, and CCR5 receptors in hypoxic condition. Moreover, GMP‐ASC migration increased 15‐fold when treated either with OA‐SF or OA‐CM compared with healthy SF both in normoxia and hypoxia. GMP‐ASC treated in both OA milieu showed an increase in CXCR3, CCR3, and IL6R and a decrease in CCR1 and CCR2 receptors. In OA‐SF, we detected higher amount of CXCL10/IP10 than in OA‐CM, while CCL2/MCP1 and CCL4/MIP1β were higher in OA‐CM compared with OA‐SF. CXCL10/IP10 was the only chemokine of the OA milieu, which was down‐modulated after treatment with GMP‐ASC. In conclusion, we demonstrated specific effects of OA milieu on both GMP‐ASC proliferation, migration, and cytokine receptor expression that were strictly dependent on the inflammatory and hypoxic environment. The use of characterized OA milieu is crucial to define the therapeutic effect of GMP‐ASC and indicates that CXCL10/IP10–CXCR3 axis is partially involved in the GMP‐ASC effect on synovial macrophages. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 38:336‐347, 2020, The objective of this study was to define in vitro the effects of osteoarthritic (OA) milieu and hypoxia on good manufactured practice‐adipose‐derived mesenchymal stromal cells (GMP‐ASC) that are commonly utilized in cell therapies. Hypoxia increased GMP‐ASC proliferation. OA milieu‐treated GMP‐ASC showed an increase in cellular migration, as well as in CXCR3, CCR3, and IL6R and a decrease in CCR1 and CCR2 receptors in hypoxia. CXCL10/IP10 was the only chemokine of the OA milieu down‐modulated after treatment with GMP‐ASC.

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