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BACKGROUND Many patients with glioma suffer from poorly understood executive functioning deficits before and/or after tumor resection. In the past decade, it has become increasingly clear that there is a complex interplay between glioma and both local and global functional activity and connectivity. Moreover, multilayer network integration of the frontoparietal network across multiple modalities has recently been found to explain individual differences in executive functioning in healthy subjects. OBJECTIVE To test whether multilayer frontoparietal network integration relates to individual differences in executive functioning before and after glioma resection. METHODS Patients with glioma (n = 37) underwent neuropsychological tests assessing word fluency, inhibition, and set shifting as components of executive functioning, and resting-state magnetoencephalography at T1 (at diagnosis, before resection) and T2 (1 year after resection). We constructed binary multilayer networks comprising six layers, with each layer representing frequency-specific functional connectivity between source-localized time series of 78 cortical regions. Average frontoparietal network multilayer eigenvector centrality, a measure for network integration, was calculated at both time points. Regression analyses were used to investigate associations with executive functioning. RESULTS At T1, lower multilayer integration (p = .017) and having epilepsy (p = .006) associated with poorer set shifting. Decreasing multilayer integration (p = .022) between T1 and T2 and not undergoing chemotherapy at T2 (p = .004) related to deteriorating set shifting. No significant associations were found for word fluency or inhibition. CONCLUSION As hypothesized, our results establish multilayer integration of the frontoparietal network as a cross-sectional and longitudinal correlate of executive functioning in glioma patients, further building upon the idea that glioma impacts the whole brain network and general cognitive functioning. However, multilayer integration did not significantly predict postoperative changes in executive functioning, limiting the direct clinical relevance of this measure.

Multiple sclerosis continues to be a challenging and disabling condition but there is now greater understanding of the underlying genetic and environmental factors that drive the condition, including low vitamin D levels, cigarette smoking, and obesity. Early and accurate diagnosis is crucial and is supported by diagnostic criteria, incorporating imaging and spinal fluid abnormalities for those presenting with a clinically isolated syndrome. Importantly, there is an extensive therapeutic armamentarium, both oral and by infusion, for those with the relapsing remitting form of the disease. Careful consideration is required when choosing the correct treatment, balancing the side-effect profile with efficacy and escalating as clinically appropriate. This move towards more personalised medicine is supported by a clinical guideline published in 2018. Finally, a comprehensive management programme is strongly recommended for all patients with multiple sclerosis, enhancing health-related quality of life through advocating wellness, addressing aggravating factors, and managing comorbidities. The greatest remaining challenge for multiple sclerosis is the development of treatments incorporating neuroprotection and remyelination to treat and ultimately prevent the disabling, progressive forms of the condition.

The promotion of spinal fusion using bone autografts is largely mediated by the osteoinductive potential of progenitors/mesenchymal stem cells (MSC) that reside in the marrow spaces of cancellous bone. Iliac crest is the common autograft donor site, but its use presents an increased risk for donor site pain, morbidity and infection. Degenerative bone samples harvested during facetectomy might provide an alternative viable source of osteoinductive autografts. In this study, we conducted an intra-individual comparison of the osteogenic potential of isolated low passage MSC from both sources. Iliac crest and degenerative facet joints were harvested from eight consecutive patients undergoing transforaminal lumbar interspinal fusion due to lumbar spinal stenosis. MSC were isolated by collagenase digestion, selected by plastic adherence and minimally expanded for downstream assays. Clonogenic and osteogenic potential was evaluated by colony formation assays in control and osteogenic culture medium. Osteogenic properties, including alkaline phosphatase (ALP) induction, matrix mineralization and type I collagen mRNA and protein expression were characterized using quantitative histochemical staining and reverse transcription PCR. Spontaneous adipogenesis was analysed by adipocyte enumeration and gene expression analysis of adipogenic markers. Average colony-forming efficiency in osteogenic medium was equal between iliac crest (38 ± 12%) and facet joint (36 ± 11%). Osteogenic potential at the clonal level was 55 ± 26 and 68 ± 17% for iliac crest and facet joint MSC, respectively. Clonogenic and osteogenic potential were significantly negatively associated with donor age. Osteogenic differentiation led to significant induction of ALP activity in iliac crest (sixfold) and facet joint (eightfold) MSC. Matrix mineralization quantified by Alizarin red staining was increased by osteogenic differentiation, yet similar between both MSC sources. Protein expression of type I collagen was enhanced during osteogenesis and significantly greater in iliac crest MSC. Correspondingly, COL1A2 mRNA expression was higher in osteogenically differentiated MSC from iliac crest. Adipocyte numbers showed significant differences between iliac crest (63 ± 60) and facet joint (18 ± 15) MSC under osteogenic conditions. Negative (GREM1) and positive (FABP4) adipogenic markers were not differentially expressed between sources. MSC from iliac crest and degenerative facet joints largely display similar clonogenic and osteogenic properties in vitro. Differences at the molecular level are not likely to impair the osteoinductive capacity of facet joint MSC. Bone autografts from facetectomy would be viable alternatives as bone autografts for intervertebral spinal fusion in lumbar spinal stenosis.

Mitochondrial mutations and dysfunction have been demonstrated in several age-related disorders including osteoarthritis, yet its relative contribution to pathogenesis remains unknown. Here we evaluated whether premature aging caused by accumulation of mitochondrial DNA mutations in PolgD275A mice predisposes to the development of knee osteoarthritis. Compared with wild type animals, homozygous PolgD275A mice displayed a specific bone phenotype characterized by osteopenia of epiphyseal trabecular bone and subchondral cortical plate. Trabecular thickness was significantly associated with osteocyte apoptosis rates and osteoclasts numbers were increased in subchondral bone tissues. While chondrocyte apoptosis rates in articular and growth plate cartilage were similar between groups, homozygous mitochondrial DNA mutator mice displayed elevated numbers of hypertrophic chondrocytes in articular calcified cartilage. Low grade cartilage degeneration, predominantly loss of proteoglycans, was present in all genotypes and the development of osteoarthritis features was not found accelerated in premature aging. Somatically acquired mitochondrial DNA mutations predispose to elevated subchondral bone turnover and hypertrophy in calcified cartilage, yet additional mechanical or metabolic stimuli would seem required for induction and accelerated progression of aging-associated osteoarthritis.

Background Increased nerve growth factor (NGF) levels are associated with chronic pain conditions, including low back pain and osteoarthritis (OA). NGF signalling through its receptor TrkA regulates pro-inflammatory neurotransmitters such as substance P (SP). Inhibition of NGF has shown therapeutic efficacy in knee OA [1] and chronic back pain [2], but trials have revealed rare cases of rapidly progressive OA of peripheral joints. Objectives To describe the tissue distribution of NGF, TrkA, SP and macrophages in facet joint osteoarthritis (FJOA) of the lumbar spine and their association with FJOA grade. Methods FJOA specimens were obtained by facetectomy from patients undergoing intervertebral fusion (n=10, average age 69 years, 5 males). FJOA severity and presence of synovial hypertrophy was graded using preoperative magnetic resonance imaging (MRI). Relative abundance of NGF, CD68 (macrophages), TrkA and SP in capsular synovium (SY), cartilage (CL), subchondral bone (SB) and subchondral bone marrow (BM) was evaluated semi-quantitatively on a scale ranging from 0-3 using immunohistochemistry. Association between imaging parameters and tissue expression was determined using Pearson correlation analysis. Results Synovial hypertrophy as determined by MRI was present in six cases (60%) and median Weishaupt grade of FJOA was 2 (1.5-3) corresponding with moderate to severe OA. NGF was abundantly expressed in SY (3 [0.5-3]) and to a lesser extent in BM tissues (2 [1-3]), whereas TrkA expression was detected in BM exclusively. NGF abundance in SY and BM showed a strong correlation (r=0.94), but did not associate with synovial hypertrophy or FJOA severity. CD68+ macrophages were highly abundant in BM (3 [1.5-3]) and sparse in SY tissues (0.5 [0-1]). The relative abundance of macrophages and NGF was strongly correlated in SY tissue only (r = 0.78). SP as a downstream mediator of NGF signalling was also abundantly expressed in SY, CL and BM tissues. Tissue distributions of CD68, NGF and SP are summarized in the figure. Conclusion NGF expression and signalling is evident in lumbar spine FJOA specimens, but not strongly associated with synovial hypertrophy or disease severity. These results are in agreement with recent studies of human knee OA, which have shown osteochondral NGF expression as a hallmark of symptomatic OA independently of chondropathy or synovitis [3]. References [1] Lane NE, Schnitzer TJ, Birbara CA, Mokhtarani M, Shelton DL, Smith MD, Brown MT.(2010) Tanezumab for the treatment of pain from osteoarthritis of the knee. N Engl J Med. 363:1521. [2] Katz N, Borenstein DG, Birbara CA, Bramson C, Nemeth MA, Smith MD, Brown MT. (2011) Efficacy and safety of tanezumab in the treatment of chronic low back pain. Pain. 152(10):2248 [3] Aso K, Shahtaheri SM, Hill R, Wilson D, McWilliams DF, Walsh DA. (2019) Associations of symptomatic knee OA with histopathologic features in subchondral bone. Arthritis Rheumatol. ePub Disclosure of Interests Matthias Seidel Grant/research support from: Pfizer, Actelion, Consultant for: Pfizer, Lilly, Nathalie Busso: None declared, Veronique Chobaz: None declared, Cordula Netzer: None declared, Thomas Huegle Grant/research support from: AbbVie, Lilly, Novartis and Pfizer, Speakers bureau: AbbVie, Lilly, Novartis and Pfizer, Jeroen Geurts: None declared

Background:Disease heterogeneity, both clinically and molecularly, has been a major hurdle in the development of efficacious disease-modifying osteoarthritis drugs (DMOADs). Biomechanical, inflammatory, osteoporotic and metabolic OA have been proposed as clinically relevant subtypes for stratification of knee OA patients, yet this remains to be included in clinical trial design. Disease heterogeneity does not only occur within, but also between joint types. However, robust data on joint-specific pathomechanisms of OA are still lacking.Objectives:In this study, we performed ex vivo biomarker profiling of human osteochondral tissue of knee and spine OA to identify joint-specific pathomechanisms and DMOAD treatment responses.Methods:Facet joint and tibial plateaus were obtained from patients undergoing lumbar spinal fusion (n=11, mean age 72.8) and total joint arthroplasty (n=8, mean age 73.0) respectively. Osteochondral specimens were cut in equal-sized samples (100-300 mg wet weight) and randomly assigned to treatment groups: control (DMSO), inflammation (1 μg/mL LPS) or inflammation + DMOAD (TGF-betatype I receptor inhibitor,10 μM SB-505124). Explant culture was conducted for one week and biomarkers of bone metabolism (Pro-Col-Ia, SOST, OPG, SPP1), inflammation (MCP-1, IL-6, MMP3, OSM, TIMP1, VEGFA) and cartilage metabolism (ACAN, COMP) were determined by ELISA. Normalized biomarker secretion was analysed using clusteranalyses and ANOVA. Cartilage proteoglycans were assessed by whole mount Alcian blue staining. Expression of Oncostatin-M (OSM) and its receptors OSMR and LIFR in joint tissues was assessed by RT-PCR and immunohistochemistry.Results:Clusteranalyses revealed that LPS stimulation increased IL-6 and MCP-1 secretion by both facet joint (FJ) and knee joint (KJ) tissues. Interestingly, Oncostatin-M (OSM) and its downstream mediators MMP3 and TIMP1 were increased in the majority of FJ, but not KJ specimens. Statistical analyses corroborated increased OSM, MMP3 and TIMP1 levels in a spine-specific fashion (Figure).Whole mount Alcian blue staining revealed heterogeneous effects of LPS treatment on cartilage proteoglycans, which was negatively correlated with OSM (r=-0.54) and TIMP1 levels (r=-0.45) – yet poorly associated with ACAN (r=0.19). Inhibition of TGF-beta type I receptor signalling in osteochondral tissues led to a drastic reduction of Pro-Collagen-Ia and IL-6 secretion in both spine and knee OA specimens. Interestingly, DMOAD treatment significantly reduced OSM, TIMP1 and MMP3 levels in FJ specimens only. Vice versa, KJ tissues revealed a specific upregulation of monocyte chemoattractant protein-1 (MCP-1) and osteopontin (SPP1) upon inhibition of TGF-beta signalling. OSM was exclusively expressed in subchondral bone marrow macrophages. Isolated chondrocytes and osteoblasts expressed both LIFR and OSMR, yet intact cartilage only showed OSMR expression, while OSMR and LIFR was expressed in marrow tissueConclusion:Oncostatin-M expression and signalling was uncovered as specific pathomechanism of spine OA. DMOAD treatment effects suggested interplay of OSM and TGF-beta signalling pathways in facet joint osteoarthritis. Known to be predominantly expressed by macrophages and immune cells, OSM may be an important osteoimmunological mediator of tissue damage and remodelling in spine, but not knee OA. This study also highlights the value of ex vivo human tissue models for OA phenotyping and preclinical evaluation of DMOADs.Disclosure of Interests:None declared

Facet joint osteoarthritis may be a cause of low back pain in degenerative spine diseases including lumbar spinal stenosis. Subchondral bone is regarded as a potential therapeutic target for osteoarthritis treatment. The goal of this study was to characterize subchondral bone histopathology in osteoarthritic facet joints from lumbar spinal stenosis patients. Fifteen patients with degenerative spinal stenosis scheduled for transforaminal lumbar interbody fusion surgery were recruited for this study. Osteoarthritis severity was graded on T1- and T2-weighted MRI images using Weishaupt scoring system. Dissected osteoarthritic facet joints were subjected to histological and immunohistochemistry analyses to study relative abundance of osteoblast, osteoclasts, and macrophages using van Gieson's, tartrate-resistant acid phosphatase and CD68-antibody staining, respectively. Presence of nerve fibers was evaluated by PGP9.5-antibody staining. Differential bone histopathology, independent from radiological osteoarthritis grade, was observed in facet joints. Extensive de novo bone formation was found in subchondral bone tissues of eight of fifteen specimens. Regions of bone formation showed high abundance of blood vessels and CD68-positive macrophages, but were devoid of multinucleated osteoclasts. Additional pathological changes in subchondral marrow spaces, including inflammatory infiltration and enhanced osteoclast activity, were characterized by macrophage-rich tissues. PGP9.5-positive nerve fibers were detected near arterioles, but not in regions displaying bone pathology. Individual histopathological parameters did not associate with clinical features or radiological osteoarthritis severity. Subchondral bone histopathology of facet joint osteoarthritis in lumbar spinal stenosis is characterized by marrow infiltration by macrophage-rich tissues and enhanced de novo bone formation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1475-1480, 2016.

Background Mitochondrial dysfunction has been demonstrated in ageing and osteoarthritic tissues. However it remains unclear whether dysfunctional mitochondria are directly implied in the pathogenesis of osteoarthritis. Objectives We investigated knee joints of prematurely ageing mitochondrial DNA mutator mice ( Polg D275A ) to evaluate a causal relationship between mitochondrial dysfunction and different features of osteoarthritis. Methods Bone structural parameters and chondropathy were evaluated in knee joints of mice displaying increased mtDNA mutations rates and accelerated ageing, due to expression of a proofreading-deficient mtDNA polymerase, using micro-computed tomography and histopathological analysis. Results Homozygous mutants displayed osteopenia of the epiphyseal trabecular bone and subchondral cortical plate in comparison to wild type controls and heterozygous mutants. Osteopenia was associated with a strong increase of osteoclast numbers (0.88±0.30/mm bone perimeter) compared to heterozygous (0.25±0.03/mm) and wild type mice (0.12±0.04/mm). New bone formation was not observed. Wild type mice displayed only low grade cartilage degeneration (OARSI grade ≤1) due to loss of cartilage proteoglycans. Increased tibio-femoral chondropathy was not apparent in hetero- and homozygous mitochondrial DNA mutator mice. Conclusions Mitochondrial dysfunction and premature ageing in mice with somatically acquired mtDNA mutations predisposes to enhanced subchondral bone resorption as potential early step of osteoarthritis, but not to cartilage damage or new bone formation. This phenotype potentially corresponds to an osteoporotic osteoarthritis phenotype in humans. Disclosure of Interest None declared

Background Facet joint osteoarthritis (FOA) is a frequent cause of chronic low back pain and spinal stenosis. Neuron-derived molecules regulate pain sensation and inflammation. Nerve growth factor (NGF) is one of the most important mediators for this mechanism that is termed neurogenic inflammation.1 Among many other effects, it regulates substance P (SP) expression as pivotal downstream peripheral pro-inflammatory molecule. Consequently, NGF inhibitors (NGFi) as a novel class of pain medication have shown significant efficacy in OA and to some extent also low back pain. However, it is unknown which tissue compartments in facet joints are involved in NGF signalling. Objectives To determine expression patterns of NGF, its high affinity transmembrane tyrosine kinase A (TrkA) receptor and SP in cartilage, subchondral bone marrow and capsular tissues of facet joints (FJ). Methods Dissected human FOA specimens of six donors were examined. OA severity was graded on HE-stained tissue sections. NGF, TrkA and SP expression was evaluated by immunohistochemistry with monoclonal antibodies. Similarly, new bone formation was assessed by staining for osteocalcin. Results FJ had low (n=2), high (n=2) and intermediate (n=2) inflammatory bone marrow infiltrates. NGF was strongly expressed in capsular tissue (figure, 40x), focally in degenerated cartilage and differentially in FJ with active bone marrow infiltrates. TrkA was primarily found in all bone marrows and capsular tissues but not in cartilage. TrkA expression colocalized with osteocalcin in subchondral bone, indicating an association with new bone formation. SP strongly positive cells were found in the bone marrow, cartilage and synovial tissue of all FJ. Conclusions Here we define for the first time tissue compartments of the NGF axis in human FOA. The findings indicate that neurogenic inflammation is prevalent in different compartments of FOA. Based on these data NGFi might be efficient by targeting inflamed joint capsule or subchondral bone marrow as a source of pain. NGFi may thus have an impact on bone remodelling. Further studies are needed to more precisely examine this mechanism. References [1] Seidel MF, Herguijuela M, Forkert R, Otten U. Nerve growth factor in rheumatic diseases. Semin Arthritis Rheum. 2010;40(2):109–26. [2] Netzer C, Urech K, Hugle T, Benz RM, Geurts J, Scharen S. Characterization of subchondral bone histopathology of facet joint osteoarthritis in lumbar spinal stenosis. J Orthop Res. 2016;34(8):1475–80. Disclosure of Interest None declared

Facet joint osteoarthritis is a prominent feature of degenerative spine disorders, highly prevalent in ageing populations, and considered a major cause for chronic lower back pain. Since there is no targeted pharmacological therapy, clinical management of disease includes analgesic or surgical treatment. The specific cellular, molecular, and structural changes underpinning facet joint osteoarthritis remain largely elusive. The aim of this study was to determine osteoarthritis-related structural alterations in cortical and trabecular subchondral bone compartments. To this end, we conducted comparative micro computed tomography analysis in healthy (n = 15) and osteoarthritic (n = 22) lumbar facet joints. In osteoarthritic joints, subchondral cortical plate thickness and porosity were significantly reduced. The trabecular compartment displayed a 42 percent increase in bone volume fraction due to an increase in trabecular number, but not trabecular thickness. Bone structural alterations were associated with radiological osteoarthritis severity, mildly age-dependent but not gender-dependent. There was a lack of association between structural parameters of cortical and trabecular compartments in healthy and osteoarthritic specimens. The specific structural alterations suggest elevated subchondral bone resorption and turnover as a potential treatment target in facet joint osteoarthritis.

Subchondral bone tissue plays a key role in the initiation and progression of human and experimental osteoarthritis and has received considerable interest as a treatment target. Elevated bone turnover and remodeling leads to subchondral bone sclerosis that is characterized by an increase in bone material that is less mineralized. The aim of this study was to investigate whether perturbations in subchondral bone-resident progenitor cells might play a role in aberrant bone formation in osteoarthritis. Colony formation assays indicated similar clonogenicity of progenitor cells from non-sclerotic and sclerotic subchondral trabecular bone tissues of osteoarthritic knee and hip joints compared with controls from iliac crest bone. However, the osteogenic potential at the clonal level was approximately two-fold higher in osteoarthritis than controls. An osteogenic differentiation assay indicated an efficient induction of alkaline phosphatase activity but blunted in vitro matrix mineralization irrespective of the presence of sclerosis. Micro-computed tomography and histology demonstrated the formation of de novo calcified tissues by osteoblast-like cells in an ectopic implantation model. The expression of bone sialoprotein, a marker for osteoblast maturation and mineralization, was significantly less in sclerotic progenitor cells. Perturbation of resident progenitor cell function is associated with subchondral bone sclerosis and may be a treatment target for osteoarthritis.

Summary Purpose The cellular component of subchondral bone is thought to be responsible for aberrant bone remodeling in osteoarthritis (OA). Direct phenotypical analysis of the cellular compartment is critical to better understand the OA disease process. This study provides proof-of-principle for flow cytometry-based phenotyping of isolated subchondral trabecular bone (STB) marrow cells without prior use of cell culture techniques. Methods Tibial plateaus were obtained from OA patients undergoing total knee arthroplasty. Subchondral bone chips were digested with collagenase IA and single cell suspensions were directly phenotyped using flow cytometry. Cells were analyzed for the expression of alkaline phosphatase (ALP) as osteoblast/osteoprogenitor marker and monocyte/macrophage markers (CD14, CD68, HLA-DR, CD115). Results MTT staining revealed abundant viable cells in the bone marrow compartment of STB prior to digestion, which were efficiently released by collagenase. Within the CD45-negative cell fraction, approximately 20% of the cells were positive for the early osteoblast/osteoprogenitor marker ALP. Within the CD45+ hematopoietic cell fraction, the majority of cells were of monocytic origin (>80%) displaying strong surface expression of CD14. Discreet macrophage populations (CD14+/HLA-DR+/CD68+) and putative osteoclast progenitors (CD45+/HLA-DR−/CD115+) were unequivocally identified. Osteoblast, macrophage and osteoclast progenitor presence in the subchondral bone unit (SBU) was confirmed by (immuno)histochemical staining for osteocalcin, CD68 and tartrate-resistant acid phosphatase, respectively. Conclusions Flow cytometric analysis is a valuable methodology to study the cellular compartment of STB marrow. This method provides a proof of principle that the whole resident cell population can be directly phenotypically characterized without the prior use of cell culture techniques.

Subchondral osteosclerosis, characterized by an increase of hypomineralized bone material, is a pathological hallmark of osteoarthritis. The cellular components in the subchondral marrow compartment that participate in this aberrant bone remodeling process remain to be elucidated. This study assessed the presence of marrow inflammatory cells and their relative abundance between nonsclerotic and sclerotic tissues in knee osteoarthritis. Bone samples from osteoarthritic knee tibial plateaus were stratified for histological analyses using computed tomography osteoabsorptiometry. Immunohistological analysis revealed the presence of CD20 (B-lymphocyte) and CD68 (macrophage), but not CD3 (T-lymphocyte) immunoreactive mononuclear cells in subchondral marrow tissues and their relative abundance was significantly increased in sclerotic compared with nonsclerotic bone samples. Multinucleated osteoclasts that stained positive for CD68 and tartrate-resistant acid phosphatase, predominantly associated with CD34-positive blood vessels and their abundance was strongly increased in sclerotic samples. Bone-specific alkaline phosphatase activity in outgrowth osteoblasts was induced by conditioned medium from nonsclerotic, but not sclerotic, bone pieces. These results suggest that an interaction between bone-resident cells and marrow inflammatory cells might play a role in aberrant bone remodeling leading to subchondral osteosclerosis. Elevated osteoclast activity in sclerotic bone suggests that bone formation and resorption activities are increased, yet uncoupled, in human knee osteoarthritis.

Background: We analyzed the histopathologic findings in end-stage osteoarthritic ankle joint tissue that display increased uptake of bone-seeking radiotracer in single-photon emission computed tomography–computed tomography (SPECT-CT) imaging. Methods: Six consecutive patients with end-stage osteoarthritis undergoing total ankle replacement received preoperative SPECT-CT imaging using 99mTechnetium dicarboxypropane diphosphonate (99mTc-DPD). Using imaging data for stratification, osteochondral tissue sections were prepared from SPECT-positive (+) and -negative (–) areas of tibial and talar resection specimens. Histomorphometric analyses of osteoblast numbers, collagen deposition, and cartilage degeneration were performed on hematoxylin and eosin, van Gieson’s and Safranin- O stained tissue sections. Osteoclast activity was visualized using tartrate-resistant acid phosphatase (TRAP) staining. Results: Increased 99mTc-DPD uptake was observed exclusively subjacent to the subchondral bone plate of tibial and talar joint compartments. SPECT(–) tissues displayed typical fatty marrow morphology containing mainly collagen-positive blood vessels and few marrow and bone-lining cells. SPECT(+) tissues were characterized by increased numbers of active bone-lining osteoblasts depositing collagen fibers. Collagen area fraction of subchondral bone marrow was significantly increased in SPECT(+) (0.52 ± 0.21) compared with SPECT(–) (0.29 ± 0.13) tissues ( P = .30). Multinucleated TRAP(+) osteoclasts were absent from bone formation sites, but associated with vascular structures invading articular cartilage through the subchondral bone plate. Increased 99mTc-DPD uptake was specifically and strongly correlated with increased osteoblast numbers ( P = .011), and with collagen area fraction ( P = .030) but not with Mankin score ( P = .202), or with osteoclast number ( P = .576). Conclusion: Subchondral bone tissues in SPECT(+) areas of end-stage ankle osteoarthritis were histologically characterized by increased osteoblast-mediated bone formation in the absence of functional osteoclasts, and increased cellularity and collagen deposition in marrow tissues. Clinical Significance: Our findings suggest a pathologic bone-remodeling process in end-stage ankle OA areas with increased 99mTc-DPD uptake. Level of Evidence: Level IV, case series.

The accumulation of senescent cells is implicated in the pathology of several age-related diseases. While the clearance of senescent cells has been suggested as a therapeutic target for patients with osteoarthritis (OA), cellular senescence of bone-resident osteoblasts (OB) remains poorly explored. Since oxidative stress is a well-known inducer of cellular senescence, we here investigated the effect of antioxidant supplementation on the isolation efficiency, expansion, differentiation potential, and transcriptomic profile of OB from osteoarthritic subchondral bone. Bone chips were harvested from sclerotic and non-sclerotic regions of the subchondral bone of human OA joints. The application of 0.1 mM ascorbic acid-2-phosphate (AA) significantly increased the number of outgrowing cells and their proliferation capacity. This enhanced proliferative capacity showed a negative correlation with the amount of senescent cells and was accompanied by decreased expression of reactive oxygen species (ROS) in cultured OB. Expanded cells continued to express differentiated OB markers independently of AA supplementation and demonstrated no changes in their capacity to osteogenically differentiate. Transcriptomic analyses revealed that apoptotic, cell cycle-proliferation, and catabolic pathways were the main pathways affected in the presence of AA during OB expansion. Supplementation with AA can thus help to expand subchondral bone OB in vitro while maintaining their special cellular characteristics. The clearance of such senescent OB could be envisioned as a potential therapeutic target for the treatment of OA.

The demand and growth for published research in the field of multiple sclerosis (MS) has increased exponentially over the last few years. The primary aim of the Multiple Sclerosis Journal (MSJ) has always been to reflect the excellent work of the ever-expanding and diverse clinical and research field of MS, and this role will now be shared by MSJ’s companion journal MSJ Experimental, Translational, and Clinical, an open access journal.

ObjectiveHuman bone marrow mesenchymal stromal cells (hBM-MSC) are being applied in tissue regeneration and treatment of autoimmune diseases (AD). Their cellular and immunophenotype depend on isolation and culture conditions which may influence their therapeutic application and reflect their in vivo biological functions. We have further characterised the phenotype induced by fibroblast growth factor 2 (FGF2) on healthy donor hBM-MSC focusing on the osteoimmunological markers osteoprotegerin (OPG), receptor activator of nuclear factor kB (RANK), RANK ligand (RANKL) and HLA-DR and their regulation of expression by the inflammatory cytokines IL1β and IFNγ.MethodsRANK, RANKL, OPG and HLA-DR expression in hBM-MSC expanded under specific culture conditions, were measured by RT-PCR and flow cytometry. MAPKs induction by FGF2, IL1β and IFNγ in hBM-MSC was analysed by immunoblotting and RT-PCR.ResultsIn hBM-MSC, OPG expression is constitutive and FGF2 independent. RANKL expression depends on FGF2 and ERK1/2 activation. IL1β and IFNγ activate ERK1/2 but fail to induce RANKL. Only IL1β induces P38MAPK. The previously described HLA-DR induced by FGF2 through ERK1/2 on hBM-MSC, is suppressed by IL1β through inhibition of CIITA transcription. HLA-DR induced by IFNγ is not affected by IL1β in hBM-MSC, but is suppressed in articular chondrocytes and lung fibroblasts.ConclusionsRANKL expression and IL1β regulated MHC-class II, both induced via activation of the ERK1/2 signalling pathway, are specific for progenitor hBM-MSC expanded in the presence of FGF2. HLA-DR regulated by IL1β and ERK1/2 is observed on hBM-MSC during early expansion without FGF2 suggesting previous in vivo acquisition. Stromal progenitor cells with this phenotype could have an osteoimmunological role during bone regeneration.

Subchondral bone and the synovium play an important role in the initiation and progression of OA. MRI often permits an early detection of synovial hypertrophy and bone marrow lesions, both of which can precede cartilage damage. Newer imaging modalities including CT osteoabsorptiometry and hybrid SPECT-CT have underlined the importance of bone in OA pathogenesis. The subchondral bone in OA undergoes an uncoupled remodelling process, which is notably characterized by macrophage infiltration and osteoclast formation. Concomitant increased osteoblast activity leads to spatial remineralization and osteosclerosis in end-stage disease. A plethora of metabolic and mechanical factors can lead to synovitis in OA. Synovial tissue is highly vascularized and thus exposed to systemic influences such as hypercholesterolaemia or low grade inflammation. This review aims to describe the current understanding of synovitis and subchondral bone pathology and their connection in OA.

We tested the hypothesis that changes in serum cartilage oligomeric matrix protein (COMP), tumour necrosis factor α (TNF-α), interleukin-6 (IL-6) and high-sensitivity C-reactive protein (hsCRP) concentration after regular endurance training and running a marathon race depend on body mass index (BMI) and/or on marathon performance. Blood samples were collected from 45 runners of varying BMI and running experience before and after a 10-week marathon training programme and before, immediately and 24 h after a marathon race. Serum biomarker concentrations, BMI and marathon finishing time were measured. The mean (95% confidence interval (CI)) changes from before to immediately after the marathon were COMP: 4.09 U/L (3.39-4.79 U/L); TNF-α: -1.17 mg/L (-2.58 to 0.25 mg/L); IL-6: 12.0 pg/mL (11.4-12.5 pg/mL); and hsCRP: -0.08 pg/mL (-0.14 to -0.3 pg/mL). The mean (95% CI) changes from immediately after to 24 h after the marathon were COMP: 0.35 U/L (-0.88 to 1.57 U/L); TNF-α: -0.43 mg/L (-0.99 to 0.13 mg/L); IL-6: -9.9 pg/mL (-10.5 to -9.4 pg/mL); and hsCRP: 1.52 pg/mL (1.25-1.79 pg/mL). BMI did not affect changes in biomarker concentrations. Differences in marathon finishing time explained 32% of variability in changes in serum hsCRP and 28% of variability in changes in serum COMP during the 24 h recovery after the marathon race (P

Item does not contain fulltext OBJECTIVE: To evaluate the applicability of a lentiviral (LV) serum amyloid A3 (Saa3)-promoter luciferase (Luc) reporter for assessing inflammation in experimental arthritis, synovial fibroblasts (SF) from osteoarthritis (OA) and rheumatoid arthritis (RA) patients. METHODS: In mice, synovium was transduced in vivo by cholesterol optimised LV, and two flares of acute joint inflammation were induced by injection of streptococcal cell wall (SCW) material into the knee-joint cavity. The time course of synovial inflammation was assessed using ex vivo luciferase assays, and histology. Uptake of (99m)technetium (Tc) was used to assess oedema. SF (n=12) of RA and OA patients were stratified by hierarchical clustering of whole genome expression profiles. Relative Saa3-promoter responses were determined in cytokine- or toll-like receptor (TLR)-stimulated SF subgroups. RESULTS: In vivo, the Saa3-promoter reporter activity was strongly upregulated at 1 and 2 days after the first and second SCW challenge. The Saa3-promoter activities during acute inflammation correlated with Tc uptake measurements but were more sensitive and able to respond to the ongoing synovitis in the chronic phase of SCW arthritis. Molecular stratification defined two inflammatory SF subtypes, unrelated to disease classification. Relative Saa3-promoter responses to interleukin 1beta, tumour necrosis factor alpha and TLR4 agonist were significantly increased in OA/RA SF with a high compared to a low inflammatory profile subtype. Serum stimulation of the Saa3-promoter reporter cell-line could distinguish between healthy and RA patients. CONCLUSION: The Saa3-promoter reporter demonstrates a robust and feasible tool for assessing the course and severity of experimental arthritis and for distinguishing molecularly distinct inflammatory SF subtypes from a heterogeneous patient population.

Osteoarthritis of the knee and spine is highly prevalent in modern society, yet a disease-modifying pharmacological treatment remains an unmet clinical need. A major challenge for drug development includes selection of appropriate preclinical models that accurately reflect clinical phenotypes of human disease. The aim of this study was to establish an ex vivo explant model of human knee and spine osteoarthritis that enables assessment of osteochondral tissue responses to inflammation and drug treatment. Equal-sized osteochondral fragments from knee and facet joints (both n = 6) were subjected to explant culture for 7 days in the presence of a toll-like receptor 4 (TLR4) agonist and an inhibitor of transforming growth factor-beta (TGF-β) receptor type I signaling. Markers of inflammation, interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), but not bone metabolism (pro-collagen-I) were significantly increased by treatment with TLR4 agonist. Targeting of TGF-β signaling resulted in a strong reduction of pro-collagen-I and significantly decreased IL-6 levels. MCP-1 secretion was increased, revealing a regulatory feedback mechanism between TGF-β and MCP-1 in joint tissues. These findings demonstrate proof-of-concept and feasibility of explant culture of human osteochondral specimens as a preclinical disease model, which might aid in definition and validation of disease-modifying drug targets.

Contains fulltext : 89359.pdf (Publisher’s version ) (Closed access) OBJECTIVE: The levels of both Fcgamma receptor (FcgammaR) and the alarmins S100A8 and S100A9 are correlated with the development and progression of cartilage destruction during antigen-induced arthritis (AIA). This study was undertaken to study the active involvement of S100A8, S100A9, and S100A8/S100A9 in FcgammaR regulation in murine macrophages and synovium during AIA. METHODS: Recombinant murine S100A8 (rS100A8) was injected into normal mouse knee joints, and the synovium was isolated for analysis of FcgammaR messenger RNA (mRNA) expression by reverse transcription-polymerase chain reaction (RT-PCR). Macrophages, including bone marrow macrophages derived from Toll-like receptor 4-deficient (TLR-4(-/-)) mice, and polymorphonuclear cells (PMNs) were stimulated with S100 proteins, and levels of FcgammaR mRNA and protein were measured using RT-PCR and fluorescence-activated cell sorting analyses. AIA was induced in the knee joints of S100A9-deficient (S100A9(-/-)) mice, compared with wild-type (WT) controls, and the extent of cartilage destruction was determined using immunohistochemical analysis. RESULTS: Intraarticular injection of rS100A8 into the knee joints of normal mice caused a strong up-regulation of mRNA levels of activating FcgammaRI (64-fold increase) and FcgammaRIV (256-fold increase) in the synovium. Stimulation of macrophages with rS100A8 led to significant up-regulation of mRNA and protein levels of FcgammaRI and FcgammaRIV, but not FcgammaRIII, while the effects of S100A9 or S100A8/S100A9 complexes were less potent. Stimulation of PMNs (32Dcl3 cell line) with S100 proteins had no effect on FcgammaR expression. Up-regulation of FcgammaRI and FcgammaRIV was abrogated in rS100A8-stimulated macrophages from TLR-4(-/-) mice, indicating that the induction of FcgammaR expression by S100A8 is mediated by TLR-4. FcgammaR expression in the inflamed synovium of S100A9(-/-) mice was significantly lower on day 14 after arthritis induction when compared with WT controls, and these findings correlated with reduced severity of matrix metalloproteinase-mediated cartilage destruction. CONCLUSION: S100A8 is a strong promoter of activating FcgammaRI and FcgammaRIV in macrophages through the activation of TLR-4, and acts as a regulator of FcgammaR expression in inflamed synovium in chronic experimental arthritis. 01 november 2010

Contains fulltext : 80087.pdf (Publisher’s version ) (Closed access) OBJECTIVE: Wnt signaling pathway proteins are involved in embryonic development of cartilage and bone, and, interestingly, developmental processes appear to be recapitulated in osteoarthritic (OA) cartilage. The present study was undertaken to characterize the expression pattern of Wnt and Fz genes during experimental OA and to determine the function of selected genes in experimental and human OA. METHODS: Longitudinal expression analysis was performed in 2 models of OA. Levels of messenger RNA for genes from the Wnt/beta-catenin pathway were determined in synovium and cartilage, and the results were validated using immunohistochemistry. Effects of selected genes were assessed in vitro using recombinant protein, and in vivo by adenoviral overexpression. RESULTS: Wnt-induced signaling protein 1 (WISP-1) expression was strongly increased in the synovium and cartilage of mice with experimental OA. Wnt-16 and Wnt-2B were also markedly up-regulated during the course of disease. Interestingly, increased WISP-1 expression was also found in human OA cartilage and synovium. Stimulation of macrophages and chondrocytes with recombinant WISP-1 resulted in interleukin-1-independent induction of several matrix metalloproteinases (MMPs) and aggrecanase. Adenoviral overexpression of WISP-1 in murine knee joints induced MMP and aggrecanase expression and resulted in cartilage damage. CONCLUSION: This study included a comprehensive characterization of Wnt and Frizzled gene expression in experimental and human OA articular joint tissue. The data demonstrate, for the first time, that WISP-1 expression is a feature of experimental and human OA and that WISP-1 regulates chondrocyte and macrophage MMP and aggrecanase expression and is capable of inducing articular cartilage damage in models of OA.