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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.

Background: Macrophages are crucial in the development and progression of various diseases. To monitor their role, various proteins expressed by macrophages may be used as imaging target. In this preclinical study we investigate the value of the somatostatin receptor subtype 2 (SSTR2) as a novel imaging marker for proinflammatory macrophages, using an experimental osteoarthritis (OA) mouse model. Methods: SSTR2 gene expression levels in pro-inflammatory macrophages and human synovium was determined by qPCR. Tracer binding was determined in macrophages and human osteoarthritic synovium after in vitro stimulation with IFNγ and TNFα. Presence of pro-inflammatory macrophages in OA mice was determined by anti-CD64+ staining. Accumulation of the tracer in OA knees was determined by μSPECT. Results: Human macrophages and synovial tissue stimulated with IFNγ+TNFα had significantly upregulated SSTR2 gene expression and showed increased uptake of SSTR2-targeting tracer. Shortly after OA induction an increase in the presence of pro-inflammatory macrophages was seen as assessed by immunohitochemsitry. Similar findings were obtained with SPECT, with peak uptake of the SSTR2-targeting tracer immediately after surgery followed by a gradual decrease during the course of the next 8 weeks. Conclusions: Pro-inflammatory macrophages have elevated SSTR2 expression which makes it possible to image an inflammatory process in the knee with a radiolabeled somatostatin analog for SPECT.

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).

Macrophages play a key role in the foreign body response. In this study it was investigated whether obesity affects the acute response of macrophages to biomaterials in vitro and whether this response is associated with biomarkers in blood. CD14(+) monocytes were isolated from blood from obese and age and gender matched lean persons. Monocyte subsets were determined based on CD14 and CD16 on their surface. C-reactive protein (CRP) was measured in peripheral blood. The response of monocyte-derived macrophages to polypropylene (PP), polylactic acid (PLA), polyethylene terephthalate (PET) mono filament, and PET-multifilament (mPET) in culture was based on cytokine production. More IL-6 (for PET), less CCL18 (all materials) and IL-1ra (for PLA) was produced by macrophages from obese patients than lean subjects. Body mass index, serum CRP and to a lesser extend percentages of monocyte subtypes correlated with IL-6, TNF alpha, CCL18, and IL-1ra production. Taken together, monocyte-derived macrophages of obese patients respond more pro-inflammatory and less anti-inflammatory to biomaterials than macrophages from lean subjects, depending on the material. These results are a step towards personalized medicine for the development of a model or even a blood test to decide which biomaterial might be suitable for each patient. (C) 2016 Elsevier Ltd. All rights reserved.