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Macrophages play an important role in the development and progression of osteoarthritis (OA). The aim of this study was to identify macrophage phenotypes in synovium and monocyte subsets in peripheral blood in C57BL/6 mice by destabilizing the medial meniscus (DMM), and the association of macrophage subsets with OA features. DMM, sham, and non-operated knees were histologically assessed between 1 and 56 days for macrophage polarization states by immunohistochemistry (IHC), cartilage damage, synovial thickening, and osteophytes (n=9 per timepoint). Naive knees (n=6) were used as controls. Monocyte and polarized synovial macrophage subsets were evaluated by flow cytometry. CD64 and CD206 levels on IHC were higher at early timepoints in DMM and sham knees compared to naive knees. iNOS labeling intensity was higher in DMM and sham knees than in naive knees from d3 onwards. CD163 expression was unaltered at all timepoints. Even though macrophage polarization profiles were similar in DMM and sham knees, only in DMM knees the presence of iNOS and CD206 associated with synovial thickness, and CD163 staining inversely correlated with osteophyte presence. At day 14, monocyte subset distribution was different in peripheral blood of DMM compared to sham mice. In conclusion, monocyte subsets in blood and synovial macrophage phenotypes vary after joint surgery. High levels of iNOS+ , CD163+ , and CD206+ cells are found in both destabilized and sham-operated knees, and co-existence with joint instability may be a requirement to initiate and exacerbate OA progression. This article is protected by copyright. All rights reserved.

Meniscal damage is, despite its major role in knee osteoarthritis (OA), often neglected in OA animal models. We evaluated structural meniscal degeneration during the course of OA in the murine collagenase-induced OA (CIOA) model. To investigate this, OA was induced in the knee joints of 33 male C57BL/6 mice by an intra-articular injection of 10U collagenase. The mice were sacrificed after 1, 3, 7, 14, 28, and 56 days, and the knees were harvested and processed for histological analysis. As control, six knees were obtained from 16-week-old mice in which no OA was induced. Meniscal damage, meniscal extrusion, and articular cartilage damage were evaluated on thionin-stained sections. Associations between parameters of interest were evaluated with Spearman rho correlation tests. When compared to non-OA knees, meniscal extrusion was visible from day 1 onwards and meniscal degeneration had a tendency to increase over time. The meniscus damage appeared around the same time as articular cartilage damage (day 14–28) and was statistically significantly more pronounced anterior than posterior, and no differences were seen between medial and lateral menisci. Meniscus and articular cartilage damage were moderately associated in the CIOA knees (ρ = 0.57; 95%CI [0.23–0.78]). Our findings suggest that the CIOA model is a valuable model to study the role of meniscal damage during OA progression and can support the development of future preventative treatment strategies.

Objective: The aims of this study were to modulate inflammation in synovial explants with the compounds: dexamethasone, rapamycin, bone morphogenetic protein 7 (BMP-7) and pravastatin, and to investigate the modulatory capacity of the compounds on specific macrophage phenotypes. Design: Synovial explants from osteoarthritis (OA) patients were treated with 10(-6) M dexamethasone, 100 ng/mL rapamycin, 500 ng/mL BMP-7 or 50 mu M pravastatin. Half of the explants were pre-stimulated with IFN gamma + TNF alpha to simulate acute inflammation. Inflammatory state of the synovium was assessed with gene expression analysis. Primary human monocytes were isolated and stimulated towards macrophage phenotypes M(IFN gamma + TNF alpha), M(IL-4) and M(IL-10) with the respective cytokines, followed by treatment with the compounds. Results: Dexamethasone had an anti-inflammatory effect on IFN gamma + TNF alpha stimulated and osteoarthritic synovium, likely due to suppression of pro-inflammatory M(IFN gamma + TNF alpha) macrophages while enhancing anti-inflammatory M(IL4) and M(IL10) macrophages. Rapamycin and BMP-7 further enhanced inflammation in stimulated synovium, but rapamycin did not have a clear effect on non-stimulated synovium. Rapamycin suppressed M(IL-4) and M(IL-10) macrophages without affecting M(IFN gamma + TNF alpha). BMP-7 suppressed M(IFN gamma + TNF alpha) and enhanced M(IL-10) in the macrophage cultures. Pravastatin did not affect synovium, but enhanced M(IL-10). Conclusions: These data indicate that macrophage phenotype modulation can be used to guide joint inflammation and thereby contribute to the development of new therapies to delay the progression of OA. The varying effects of the compounds on synovium of different degrees of inflammation, indicate that the modulatory capacity of the compounds depends on OA stage and underlines the importance of identifying this stadium for adequate treatment. (C) 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

BACKGROUND CONTEXT Synthetic bone grafts are a commonly used alternative to autograft in grafting procedures, such as spinal fusions. In pre-clinical spinal fusion models, it has been recently shown that calcium phosphate grafts with a submicron surface topography enhance bone healing potential when compared to conventional calcium phosphate grafts. Immune cells such as macrophages, are pivotal to the (osteo)immunological response upon implantation of a graft as they can polarize into a proinflammatory M1 phenotype or anti-inflammatory M2 phenotype. Therefore, it is hypothesized that calcium phosphates with submicron topography upregulate M2 macrophages, resulting in enhanced bone healing and tissue integration. PURPOSE The purpose of this study was to evaluate M1/M2 polarization of primary human macrophages after in vitro culture on two commercially available calcium phosphate bone grafts, one with a submicron surface topography and the other with a conventional surface. STUDY DESIGN/SETTING Primary monocytes were isolated from buffy coats and cultured on granules of a biphasic calcium phosphate with submicron topography (BCP PATIENT SAMPLE Not applicable. OUTCOME MEASURES To determine macrophage phenotype, enzyme-linked immunosorbent assays (ELISAs) for chemokine ligand 5 (CCL5) as a marker for M1 macrophages and chemokine ligand 18 (CCL18) as a marker for M2 macrophages, were performed on the cell culture supernatants. Protein concentrations were normalized to DNA content. Macrophage morphology on the surface of the bone graft materials was observed by scanning electron microscopy (SEM). METHODS For both materials, 0.4 cc/well of granules were transferred to well plates and preincubated in RPMI culture medium with 10% fetal bovine serum for at least 2 hours. CD14+ monocytes were isolated from buffy coats (n=2) and subsequent CD14 magnetic-activated cell sorting. CD14+ monocytes were seeded at 4.5×105 cells/well on the calcium phosphate granules and supplemented with 10 ng/ml macrophage colony-stimulating factor. After 24h and after 72h of culture, the culture medium was collected for ELISAs. The macrophages were lysed for DNA quantification. For visualization by SEM, macrophages on granules were fixed in glutaraldehyde. RESULTS After 24h and 72h of culture, CCL18 concentrations were 3-3.5 times higher when macrophages were cultured on BCP CONCLUSIONS Higher levels of M2 macrophage marker CCL18 after culture on calcium phosphate with submicron topography, indicate M2 polarization. The initial presence of CCL5 and its subsequent abatement at 72h in both materials suggests early M1 macrophage activity that is reduced thereafter. The stretched-out, elongated macrophage shape on the material with submicron topography may suggest a higher affinity to the submicron surface topography than the conventional material and is consistent with M2 morphology described in literature. These results indicate that upregulation of M2 macrophages at the surface of calcium phosphate with submicron topography, may play a role in its enhanced performance in bone healing compared to that of conventional calcium phosphate bone grafts. FDA DEVICE/DRUG STATUS MagnetOs Granules (Not approved for this indication), Vitoss Granules (Not approved for this indication).

It is well-accepted that articular (ART) cartilage composition and tissue architecture are intimately related to mechanical properties. On the other hand, very little information about other cartilage tissues is available, such as elastin-rich auricular (AUR) cartilage. While thorough investigation of ART cartilage has enhanced osteoarthritis research, ear cartilage reconstruction and tissue engineering (TE) could benefit in a similar way from in-depth analysis of AUR cartilage properties. This study aims to explore the constituent-function relationships of AUR cartilage, and how elastin influences mechanical behavior. Stress-relaxation indentation and tensile tests were performed on bovine ART and AUR cartilage. Elastase incubation was performed to simultaneously deplete elastin and sulfated glycosaminoglycans (sGAG), while hyaluronidase incubation was used to deplete sGAG-only, in order to systematically investigate matrix components in material behavior. ART and AUR cartilages showed different viscoelastic behaviors, with AUR cartilage exhibiting a more elastic behavior. Higher equilibrium properties and limited viscous dissipation of strain energy were observed in AUR cartilage, while ART cartilage exhibited a rapid viscous response and high resistance to instantaneous loading. In conclusion, loss of sGAG had no effect on auricular mechanics in contrast to articular cartilage where GAG loss clearly correlated with mechanical properties. Auricular cartilage without elastin lost all compressive mechanical integrity, whereas in articular cartilage this was provided by collagen. This work shows for the first time the involvement of elastin in the mechanical behavior of ear cartilage. In future, this data can be used in AUR cartilage TE efforts to support reproduction of tissue-specific mechanical properties. (C) 2015 Elsevier Ltd. All rights reserved.

Summary Objective Macrophages play a crucial role in the progression of osteoarthritis (OA). Their phenotype may range from pro-inflammatory to anti-inflammatory. The aim of this study was to evaluate the direct effects of macrophage subtypes on cartilage by culturing macrophage conditioned medium (MCM) on human articular cartilage. Design Human OA cartilage explants were cultured with MCM of pro-inflammatory M(IFNγ+TNFα), or anti-inflammatory M(IL-4) or M(IL-10) human monocyte-derived macrophages. To assess effects of anti-inflammatory macrophages, the cartilage was cultured with a combination of MCM phenotypes as well as pre-stimulated with IFNγ+TNFα cartilage before culture with MCM. The reactions of the explants were assessed by gene expression, nitric oxide (NO) production and release of glycosaminoglycans (GAGs). Results M(IFNγ+TNFα) MCM affected OA cartilage by upregulation of IL1B (Interleukin 1β), IL6 , MMP13 (Matrix Metalloproteinase-13) and ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin Motifs-5), while inhibiting ACAN (aggrecan) and COL2A1 (collagen type II). M(IL-10) upregulated IL1B and Suppressor of cytokine signaling 1 ( SOCS1 ). NO production and GAG release by the cartilage was increased when cultured with M(IFNγ+TNFα) MCM. M(IL-4) and M(IL-10) did not inhibit the effects of M(IFNγ+TNFα) MCM of neither phenotype affected IFNγ+TNFα pre-stimulated cartilage, in which an inflammatory gene response was deliberately induced. Conclusion M(IFNγ+TNFα) macrophages have a prominent direct effect on OA cartilage, while M(IL-4) and M(IL-10) do not inhibit the effects of M(IFNγ+TNFα), or IFNγ+TNFα induced inflammation of the cartilage. Therapies aiming at inhibiting cartilage degeneration may take this into account by directing suppression of pro-inflammatory macrophages or stimulation of anti-inflammatory macrophages.

s / Osteoarthritis and Cartilage 23 (2015) A82eA416 A266 were allocated into four groups (n1⁄49 per group): a) control (explants in DMEM), b) MPA treatment (control for drug), c) inflammation (treatment with interleukin-1b (IL-1b)), and d) inflammation followed by inhibition with MPA (IL-1bþMPA). The MPA (10-7uM) was added following 24 hours of equilibration time in the MPA group. The proinflammatory cytokine IL-1b (10ng/ml) was added to the culture media to initiate inflammation in the IL-1b group. For the IL-1bþMPA group, IL-1b was added following the equilibration time and MPA (10-7uM) was added 24 hours later. All explants were harvested at 48 hours and snap frozen in liquid nitrogen and later used for mRNA analysis using real time qPCR. Matrix metalloproteinase (MMP)-1, MMP-3, and MMP13 were analyzed in the explants. In addition, one set of fat pad and synovial explants from each limb was tested for cellular viability using SYTO13 green fluorescent nucleic acid stain (Invitrogen). ANOVA with Holm-Sidak's post-hoc analysis was used to determine differences in mRNA expression between groups, using Prism 6 Graph Pad software. Results: Prior to being exposed to the experimental conditions, the cells in explants from the synovium and fat pad were stained to ensure they were viable (Figure 1). The mRNA expression levels for MMP-3 and MMP-13 were suppressed in fat pad tissue when treated with MPA following induction of inflammation (p