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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., (© 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society.)

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., (© 2018 Wiley Periodicals, Inc.)

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. [ABSTRACT FROM AUTHOR]

Objective: To 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.Methods: OA 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.Results: Most 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.Conclusions: These 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]