Intervertebral disc (IVD) degeneration is a pathological process often associated with chronic back pain and considered a leading cause of disability worldwide. 1. During degeneration, progressive structural and biochemical changes occur, leading to blood vessel and nerve ingrowth and promoting discogenic pain. 2. In the last decades, several cytokines have been applied to IVD cells in vitro to investigate the degenerative cascade. Particularly, IL-10 and IL-4 have been predicted as important anabolic factors in the IVD according to a regulatory network model based in silico approach. 3. Thus, we aim to investigate the potential presence and anabolic effect of IL-10 and IL-4 in human NP cells (in vitro) and explants (ex vivo) under hypoxia (5% O2) after a catabolic induction. Primary human NP cells were expanded, encapsulated in 1.2% alginate beads (4 × 106 cells/ml) and cultured for two weeks in 3D for phenotype recovery while human NP explants were cultured for five days. Afterwards, both alginate and explant cultures were i) cultured for two days and subsequently treated with 10 ng/ml IL-10 or IL-4 (single treatments) or ii) stimulated with 0.1 ng/ml IL-1β for two days and subsequently treated with 10 ng/ml IL-10 or IL-4 (combined treatments). The presence of IL-4 receptor, IL-4 and IL-10 was confirmed in human intact NP tissue (Fig 1). Additionally, IL-4 single and combined treatments induced a significant increase of proinflammatory protein secretion in vitro (Fig. 2A-C) and ex vivo (Fig. 2D and E). In contrast, no significant differences were observed in the secretome between IL-10 single and combined treatments compared to control group. Overall, IL-4 containing treatments promote human NP cell and explant catabolism in contrast to previously reported IL-4 anti-inflammatory performance. 4. Thus, a possible pleiotropic effect of IL-4 could occur depending on the IVD culture and environmental condition. Acknowledgements: This project was supported by the Marie Skłodowska Curie International Training Network “disc4all” under the grant agreement #955735. For any figures and tables, please contact the authors directly

Intervertebral disc (IVD) degeneration (IDD) involves imbalance between the anabolic and the catabolic processes that regulate the extracellular matrix of its tissues. These processes are complex, and improved integration of knowledge is needed. Accordingly, we present a nucleus pulposus cell (NPC) regulatory network model (RNM) that integrates critical biochemical interactions in IVD regulation and can replicate experimental results. The RNM was built from a curated corpus of 130 specialized journal articles. Proteins were represented as nodes that interact through activation and inhibition edges. Semi-quantitative steady states (SS) of node activations were calculated. Then, a full factorial sensitivity analysis (SA) identified which out of the RNM 15 cytokines, and 4 growth factors affected most the structural proteins and degrading enzymes. The RNM was further evaluated against metabolic events measured in non-healthy human NP explant cultures, after 2 days of 1ng/ml IL-1B catabolic induction. The RNM represented successfully an anabolic basal SS, as expected in normal IVD. IL-1B was able to increase catabolic markers and angiogenic factors and decrease matrix proteins. Such activity was confirmed by the explant culture measurements. The SA identified TGF-β and IL1RA as the two most powerful rescue mediators. Accordingly, TGFβ signaling-based IDD treatments have been proposed and IL-1RA gene therapy diminished the expression of proteases. It resulted challenging to simulate rescue strategies by IL-10, but interestingly, IL-1B could not induce IL-10 expression in the explant cultures. Our RNM was confronted to independent in vitro measurements and stands for a unique model, to integrate soluble protein signaling and explore IDD. Acknowledgements: European Commission (Disc4All-ITN-ETN-955735)

The development of a representative human, in vitro OA model could deepen understanding of disease mechanisms. Our research aimed to reprogram healthy and OA-derived synoviocytes to induced pluripotent stem cells (iPSCs), thereby generating a novel OA in vitro model. Comparison between the two models shall enable research into underlying processes with potential for clinical translation. A meta-analysis of OA synovial biomarkers was conducted, identifying up to thirteen relevant pathophysiology-related factors, including, amongst others, IL-13, IL-10, IL-6, PIICP, and HA, with PIICP demonstrating the largest effect (SMD 6.11 [3.50, 8.72], p <0.00001). With these findings in mind, human fibroblast-like synoviocytes (HFLS) from healthy and OA patients were transduced using Sendai viral reprogramming. Two clones for each of the resulting iPSC lines were expanded and preliminarily analysed in triplicate by ICC and RT-qPCR for pluripotency characteristics. Healthy HFLS-derived and OA-HFLS-derived iPSC (UoS-B and UoS-C lines, respectively) were generated, indicating successful reprogramming. Morphological observations demonstrated typical iPSC appearance, and ICC confirmed presence of pluripotency markers Tra-1-60, Oct3/4 and Nanog. Expression of Oct3/4, Nanog and Sox2 were confirmed by RT-qPCR with OA-iPSC lines expressing higher levels of all markers compared to non-OA iPSC. In particular, expression of Oct3/4 and Sox2 was 3.5 fold and 4.6 fold higher (p <0.001) in OA-iPSCs (UoS-C) vs. non-OA iPSCs (UoS-B), respectively. Sendai virus clearance was confirmed by passage 4. The successfully obtained OA and non-OA iPSCs can be differentiated towards mesenchymal lineages, including chondrocyte and bone progenitor cells, enabling phenotypic comparison and biomarker analysis as identified in meta-analysis. Cell bank dissemination of these cell lines could deepen further in vitro OA research, with potential impact for clinical translation via the identification of novel cellular and molecular targets

Common tendon injuries impair healing, leading to debilitation and an increased re-rupture risk. The impact of oxygen-sensing pathways on repair mechanisms, vital in regulating inflammation and fibrosis, remains unclear despite their relevance in tendon pathologies. Recent studies show that pulsed electromagnetic field (PEMF) reduce inflammation in human tendon cells (hTDCs) and in hypoxia-induced inflammation. We investigated the hypoxia's impact (1% and 2% oxygen tension) using magnetic cell sheet constructs (IL-1β-magCSs) primed with IL-1β. IL-1β-magCSs were exposed to low OT (1h, 4h,6h) in a hypoxic chamber. To confirm the role of PEMF (5Hz, 4mT, 50% duty cycle) on hypoxia modulation, IL-1β-magCSs, previously exposed to OT, were 1h-stimulated with PEMF. Our results show a significant increase in HIF- 1a and HIF-2a expression on IL-1β-magCSs after exposure to 2%-OT at all time points, compared to 1%- OT and normoxia. TNFa, IL-6, and IL-8 expression increased after 6 hours of 1%-OT exposure. PEMF stimulation of hypoxic IL-1β-magCSs led to decreased pro-inflammatory genes and increased anti-inflammatory (IL-4,IL-10) expression compared to unstimulated magCSs. IFN-g, TNF-α, and IL-6 release increased after 6 hours, regardless of %-OT, while IL-10 levels tended to rise after PEMF stimulation at 2%-OT. Also, NFkB expression was increased on IL-1β-magCSs exposed to 4 h and 6 h of 2%-OT, suggesting a link between NFkB and the production of pro-inflammatory factors. Moreover, PEMF stimulation showed a significantly decreased NFkB level in IL-1β-magCSs. Overall, low OT enhances expression of hypoxia-associated genes and inflammatory markers in IL-1β-magCSs with the involvement of NFkB. PEMF modulates the response of magCSs, previously conditioned to hypoxia and to inflammatory triggers, favouring expression of anti-inflammatory genes and proteins, supporting PEMF impact in pro-regenerative tendon strategies. Acknowledgements: ERC CoG MagTendon(No.772817), FCT under the Scientific Employment Stimulus-2020.01157.CEECIND. Thanks to Hospital da Prelada for providing tendon tissue samples (Portugal), and TERM. RES Hub (Norte-01-0145-FEDER-022190)

Establishing disease biomarkers has been a long-sought after goal to improve Osteoarthritis (OA) diagnosis, prognosis, clinical and pharmaceutical interventions. Given the role of the synovium in contributing to OA, a meta-analysis was performed to determine significant synovial biomarkers in human OA tissue, compared to non-OA patients. Outcomes will direct future research on marker panels for OA disease modelling in vitro/in vivo, aiding clinical research into OA disease targets. A PRISMA compliant search of databases was performed to identify potential biomarker studies analysing human, OA, synovial samples compared to non-OA/healthy participants. The Risk of Bias In Non-Randomised Studies of Interventions (ROBINS-I) tool assessed methodological quality, with outcome analysed by Grading of Recommendations Assessment, Development and Evaluation (GRADE). Meta-analyses were conducted for individual biomarkers using fixed or random effect models, as appropriate. Where three or more studies included a specific biomarker, Forest Plot comparisons were generated. 3230 studies were screened, resulting in 34 studies encompassing 25 potential biomarkers (1581 OA patients and 695 controls). Significant outcomes were identified for thirteen comparisons. Eleven favoured OA (IL-6, IL-10, IL-13, IP-10, IL-8, CCL4, CCL5, PIICP, TIMP1, Leptin and VEGF), two favoured non-OA controls (BMP-2 and HA). Notably, PIICP showed the largest effect (SMD 6.11 [3.50, 8.72], p <0.00001, I. 2. 99%), and TIMP1 resulted critically important (0.95 [0.65, 1.25], p <0.00001, I. 2. 82%). Leptin and CCL4 showed lower effects (SMD 0.81 [0.33, 1.28], p =0.0009; 0.59 [0.32, 0.86], p <0.0001, respectively). Thirteen significant synovial biomarkers showed links with OA bioprocesses including collagen turnover, inflammatory mediators and ECM components. Limitations arose due to bias risk from incomplete or missing data, publication bias of inconclusive results, and confounding factors from patient criteria. These findings suggest markers of potential clinical viability for OA diagnosis and prognosis that could be correlated with specific disease stages

Abstract. OBJECTIVES. Osteoarthritis therapies are limited to symptom management and joint replacement. AMPA/kainate glutamate receptor (GluR) antagonists (NBQX/DNQX, 2.5–20mM) alleviate symptoms and disease in rodent models of osteoarthritis. We hypothesised that poly(lactic-co-glycolic) acid (PLGA) nanoparticles and thermoresponsive hydrogels sustain GluR antagonist release to improve their efficacy in an humanised 3D bone model of inflammation. METHODS. Drug release in PBS (37 °C) was measured by HPLC of samples taken from 2.5mM NBQX/DNQX loaded PLGA nanoparticles (double emulsion) and thermosetting hydrogels (homogenised Pluronic-F127 (22%/25% w/v) and Carbopol 934 (0.5% w/v) with 2.5mM NBQX/DNQX in dH2O)(n=3). Y201 MSCs were cultured in 3D in rat tail collagen type I gels and exposed to IL-6/sIL-6r (5/40ng/ml), free NBQX (200μM) or NBQX loaded PLGA nanoparticles for 24 and 72hrs. Bone turnover, inflammatory and glutamate signalling markers were quantified by immunoassay and RTqPCR. Data analysed using t-test/ANOVA with Tukeys and principal component analysis (PCA)(SPSS). RESULTS. Nanoparticles released 45% encapsulated DNQX over 3hrs followed by sustained release over 5 weeks. Thermoresponsive hydrogels released entire DNQX load over 27 hours (22 and 25% hydrogels). PCA revealed IL-6/sIL-6r treatment affected bone turnover, inflammation and glutamate signalling markers in vitro. Free NBQX treatment increased anti-inflammatory cytokine at 24hrs (IL-4, IL-10, IL-13) levels vs IL-6 treatment groups (p<0.05) and corrected IL-6 induced reduction in ALP expression (24hrs, p<0.05). Nanoparticle NBQX delivery induced increased IL-6 expression vs controls (72hrs, p<0.05) and increased glutamate and IL-6 protein release at (72hrs, p<0.05 and p<0.001 respectively). CONCLUSIONS. Nanoparticles rapid release of DNQX (6.6μM /3 hours), mimics free drug effective in vivo but was followed by sustained release over 5 weeks. hydrogels released 2.5mM DNQX over a short time (27hrs). Free NBQX intervention mitigated bone turnover, inflammation and glutamate signalling changes following IL-6 exposure to bone cells in vitro. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Diffuse noxious inhibitory control (DNIC) works through the “pain-inhibits-pain” principle in which an additional painful (conditioned) stimulus can suppress the initial experienced pain through the descending and inhibiting pathways. Painful stimulation produced less pain inhibition in patients with knee osteoarthritis patients (KOA) than in controls, suggesting an impaired DNIC function and a loss of endogenous pain modulation. Electroacupuncture (EA) is widely used to treat acute pain associated with KOA, but the available evidence of its benefit on chronification of acute pain is scarce. This is a single-arm clinical study aims to evaluate the effect of EA on the chronification of pain associated with KOA and provide a profile of various cytokines underlying the pathogenesis of KOA. Participants are recruited through hospital-based recruitment and advertisements, diagnosis was based upon the criteria formulated by the American College of Rheumatology. Each participant was administered with EA (2 mA < current < 5 mA) at the ipsilateral EX-LE5, ST35, ST34 and SP10 for two weeks (once a day, 30 minutes per session, in 5 sessions per week). Visual Analog Scale (VAS), DNIC function, the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Numerical Rating Scale (NRS), Emotional Scale (ES) and Present Pain Intensity (PPI) are evaluated before treatment and after 5 to 10 sessions of treatment. Cytokines including GRO, TNF-α, VEGF, IP-10, IL-1β, IL-17, IL-8, MCP-1 and IL-10 levels in plasma were measured using a Human Cytokine/Chemokine Magnetic Bead Panel on MAGPIX instrument before and after two weeks of treatment. A total of 39 patients with KOA were enrolled in our study (age: 63.46±9.89 years; height: 1.63±0.07 m; BMI: 22.83±2.89), all of them completed the trial. After 5 sessions of EA treatment, a significant decrease of VAS, WOMAC scores, NRS, ES and PPI was detected, but no significant difference in DNIC was observed. After two weeks' treatment, all clinical parameters (VAS, DNIC, WOMAC, NRS, ES, PPI) reduced significantly when compared with baseline; GRO, IL-17A, IL-1b, IL-8, MCP-1, TNF-a, VEGF levels in plasma reduced significantly while IL-10 and IP-10 concentrations were elevated. This study appeared to provide evidence that EA was effective in improving chronic pain associated with KOA through repairing the impaired DNIC function and down-regulation of OA detrimental cytokines. A randomized controlled prospective study with large sample size is required to clarify the effect of EA in reversing the chronification of pain in KOA

Tendon injuries are associated with the formation of inferior, disorganized scar tissue at the tendon bone insertion site and high failure rates. Two major processes are discussed being key players: the inflammatory reaction upon tear and the remodeling process of the tendon. In a previous study we demonstrated that the profile of MMPs and TIMPs, being key factors of tendon modeling and remodeling, is altered in tenocytes of rotator cuff tears from donors with higher age (>65 years) and degenerative status (high degree of muscle fatty infiltration)[1]. But do these cells also show different expression of inflammatory cytokines or react different upon cytokine stimulation? The aim of our project was to analyze the expression of inflammatory cytokines in human tenocyte-like cells (hTLCs) on mRNA-level and the responsiveness to cytokine stimulation regarding differences between varying donor characteristics such as age, sex and the degenerative status of the tendon. TLCs were isolated from SSP tendon biopsies from 16 male and 14 female donors undergoing arthroscopic or open shoulder surgery. Cells from each donor (passage 1 or 2) were seeded in a 6-well plate and RNA was isolated after 7 days of culture. Quantitative Real-Time PCR was performed to analyze the expression of IL-6, IL-1β, TNF-α, IL-10, IL-33, TGF-β1 and COX-2. Furthermore, hTLCs of 12 male donors were stimulated for 3 days with a combination of TNF-α and IFN-γ (10ng/ml). The effect of the cytokines was analyzed by flow cytometry regarding surface marker expression: ICAM (CD54), VCAM (CD106), and Major Histocompatibility Complex (MHC)-class I and MHC-class II. Statistics: Mann-Whitney-U-Test, Spearman´s-Rho-correlation, p≤0.05. Gene expression analysis revealed high levels of IL-6, TGF-β1 and COX-2 in hTLCs but low expression of TNF-α and IL-10. No differences in the expression of the inflammatory cytokines were found between low and high fatty infiltration or with respect to age. The stimulation of the hTLCs with TNF-α and IFN-γ increased the number of ICAM and VCAM positive cells up to 100% and 97±5%, respectively. MHC-class II was not expressed on unstimulated cells but 77±17% MHC-class II positive cells were present after stimulation. All unstimulated cells were positive for MHC-class I, but the MFI (Mean Fluorescent Intensity) increased after stimulation. No significant difference in the expression of surface markers was detected when comparing tenocytes of donors with low and high muscle fatty infiltration. In contrast to the significant changes in expression levels of MMPs and TIMPs in tenocytes of donors with different age and degenerative status[1], we could not detect any significant changes in the expression of inflammatory cytokines or in the responsiveness of these tenocytes upon cytokine stimulation. All tenocytes showed the potential to respond to inflammatory processes. This indicates that the response of the tenocytes to inflammatory stimuli seems to be independent of donor characteristics, whereas the tendon remodeling might depend on age and degenerative status of the donor

Summary. An in vivo model of implant infection was developed to assess immune response. Titanium and PEEK implants were tested in the presence of an osteotomy and Staphylococcus aureus contamination. Immune response differed yet the outcome of contamination did not. Introduction. The presence of an implant increases infection risk by reducing the number of bacteria required to cause an infection. The nature or magnitude of this risk may be influenced by the implant material. A model of implant associated osteomyelitis was developed based upon the MouseFix. TM. model and the development of infection and immune responses associated with either titanium or PEEK implants was investigated. Methods. MouseFix. TM. titanium plates with or without Staphylococcus aureus contamination were used with a femoral osteotomy in C57bl/6 and BALB/c mice (ethical permission: 2012/15). C57bl/6 mice receiving titanium implants were sacrificed at seven time-points over 35 days after surgery (n=6 per group). In addition, PEEK implants in C57bl/6 mice and both titanium and PEEK implants in BALB/c mice were assessed at 1, 3 and 7 days. Bacteria from the implant, bone and soft-tissue were quantified. Cytokine levels in the bone, soft tissue and spleen were assessed. Lymph node cells were characterised for cytokine production by flow cytometry. Results. Bacterial contamination led to the development of chronic osteomyelitis. The bacterial counts showed an increase at day 1 followed by an initial decrease at early time-points in all conditions. However, after day 21 the bacterial load increased again. The materials caused differences in bacterial counts at day 3. In non-infected bone, IL-4, IL-6, IL-17A and KC production was elevated. In the local lymph node there was an increase of CD3+IL4+ cells. Infected bone presented a decrease in IL-4 levels, an early increase of IL-6 and IL-10 and a late increase in IL-17 and KC; additionally, CD3+IFN-gamma+ cells were increased at early time-points and after day 21. CD3+IL-17+ cells were increased between days 7 and 21. IL-4 and IL-10 producing lymphocytes were also elevated after day 14. When comparing the materials, PEEK stimulated an increase in IL-4 and a decrease in IL-17 type response compared to titanium. Discussion. Our results suggest a Th2-type response in uninfected and a Th17-type response in infected animals. The use of different materials altered the immune response but not the outcome of contamination

Introduction. Although osteonecrosis of the femoral head has been observed in young adult patients with autoimmune diseases such as SLE and MCTD that are treated by corticosteroids, the pathogenesis of the osteonecrosis remains unclear. We established a rat model with osteonecrosis of the femoral head by injecting lipopolysaccharide (LPS) and corticosteroid, and assessed consequences of the histopathological alteration of the femoral head, the systemic immune response, and the lipid synthesis. Methods. Male Wistar rats were given 2 mg/kg LPS intravenously on days 0 and 1 and intramuscularly 20 mg/kg methylprednisolone on days 2, 3, and 4. The animals were sacrificed 1, 2, 3, or 4 weeks after the last injection of the methylprednisolone. Histopathological and biochemical analyses were performed every week. The bone samples were then processed for routine hematoxylin and eosin staining to assess the general architecture and injury of the tissue. The triglyceride and the total cholesterol concentrations in the PRP were measured. The levels of various cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ, TNF-α) in blood samples were measured. Results. The body weight of the rats over time decreased for 2 weeks but had recovered by week 4. The plasma triglyceride concentrations had decreased significantly by weeks 2 and 3. The total plasma cholesterol concentrations had increased significantly by week 1 but then decreased significantly by week 4. The plasma concentrations of IL-1?α, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ and TNF-α had increased significantly by week 1. These cytokines can all be induced by toll-like receptor 4 (TLR4) signaling. We defined osteonecrosis as the diffuse presence of empty lacunae or pyknotic nuclei of osteocytes in the bone trabeculae, accompanied by surrounding bone marrow cell necrosis. Osteonecrosis of the femoral head was observed only in the epiphysis of the femoral head in sacrificed specimen every week. Histological analysis revealed osteocytic death surrounded by necrotic bone marrow with or without repaired tissue. Conclusion. We established a new rat model of corticosteroid-induced femoral head osteonecrosis. The necrosis that is generated in this model is similar to that seen in patients treated with corticosteroid. In particular, the necrotic lesion was exclusively observed in the proximal epiphysis. LPS is known to activate the immune system via the TLR4 signaling pathway. It has been recognized that the unique immunogenic effects of LPS promote autoimmune disease . LPS and methylprednisolone induced osteonecrosis of the femoral head in rats and this was associated with a disruption of the innate immune system and lipid synthesis. These findings suggest that the TLR4 signaling pathway plays an important role in the pathogenesis for osteonecrosis of the femoral head

Human synovium harbours macrophages and T-cells that secrete inflammatory cytokines, stimulating chondrocytes to release proteinases like aggrecanases and matrix metalloproteinases (MMPs) during the development of Osteoarthritis (OA). Inflammation of the synovium is a key feature of OA, linked to several clinical symptoms and the disease progression. As a prelude to testing in an OA mouse model, we have used the tetracycline system (Tet) to modify mouse mesenchymal stem cells (mMSCs) to over-express viral interleukin 10 (vIL10), an anti-inflammatory cytokine, to modulate the osteoarthritic environment and prevent disease development. MSCs isolated from the marrow of C57BL/6J mice expressed CD90.2, SCA-1, CD105, CD140a, and were negative for CD34, CD45 and CD11b by flow cytometry. Adenoviral transduction of MSCs carrying CMVIL10 and TetON as test, and untransduced, AdNull and TetOFF as negative controls was successful and tightly controlled vIL10 production was demonstrated by CMVIL10 and TetON MSCs using a vIL10 ELISA kit. Co-incubation of vIL10MSC CM with lipopolysaccharide activated bone-marrow derived murine macrophages (BMDMs) resulted in reduction of TNF-α, IL-6 levels and elevated production of IL-10 by ELISA and high iNOS release by Griess assay. Co-culture of active macrophages with TetON MSCs, resulted in polarisation of macrophage cell population from M1 to M2 phase, with decrease in pro-inflammatory MHC-II (M1 marker) and increase in regulatory CD206 (M2 marker) expression over time. The PCR profiler array on MSC CM treated BMDMs, also showed changes in gene expression of critical pro-inflammatory cytokines and receptors involved in the TLR4 pathway. The biscistronic TetON transduced MSCs proved to be most immuno-suppressive and therefore feasible as efficient anti-inflammatory therapy that can utilised in vivo

Objectives. To evaluate the in vitro effects of hyaluronic acid (HA) on adipose-derived stem cells (ASC) in order to consider the possibility of their combined used in the treatment of knee arthrosis. Material and methods. The ASC cells were grown both in the presence and absence of AH, and several studies were carried out: proliferation (WST8) and cell viability studies (Alamar Blue and Trypan Blue), possible chondrogenic differentiation (collagen type 2 expression) by RT-PCR, AH receptor expression (CD44) by flow cytometry and RT-QPCR, and expression of inflammatory and anti-inflammatory factors (IL-6, TGFß, IL-10) by RT-QPCR. Results. The number of ASC significantly increased after 7 days with HA (158±39%, p < .05). Additionally, the cell viability of the ASC treated with HA after 1, 3, 5 and 7 days was similar to that of the control cells, being considered non-toxic. There were no changes observed in the expression of CD44 and chondrogenic differentiation. TGFß expression was not modified after AH treatment, but there was a 4-fold decrease in IL-6 expression and IL-10 expression increased up to 2-fold compared to control cells. Conclusions. Hyaluronic acid favours ASC proliferation without causing cellular toxicity, and inducing an anti-inflammatory profile in these cells. Hyaluronic acid appears to be a suitable vehicle for the intra-articular administration of mesenchymal stem cells

Background. Post-traumatic immunosuppression (PTI) after surgery increases vulnerability to nosocomial infections, sepsis, and death. Knee arthroplasty offers a sterile clinical model to characterise PTI and explore its underlying mechanisms. Methods. This prospective non-randomised cohort study of primary total knee arthroplasty was approved by the Local Ethics Committee. Exclusion criteria included revision-arthroplasty, pre-existing infections, blood-transfusions, malignancy, and auto-immune disease. 48 recruited patients fell into two groups, the first received unwashed anti-coagulated autologous salvaged blood transfusions after surgery (ASBT cohort, n=25). The second received no salvaged blood transfusions (NSBT cohort, n=18). Venous blood was sampled pre-operatively and within 3–7 days post-operatively. Salvaged blood was sampled at one and six hours post-operatively. Biomarkers of immune status included: interleukins (IL) or cytokines (x15), chemokines (x3), Damage-Associated-Molecular-Patterns (DAMPS) (x5), anti-microbial proteins (x3), CD24, and Sialic-acid-binding-Immunoglobulin-type-Lectin-10 (Siglec-10). Results were expressed as fold-change over pre-operative values. Only significant changes are described. Results. Certain biomarkers associated with sterile trauma were common to all 43 patients, including supra-normal: IL-6, IL-1-Receptor-Antagonist, IL-8, Heat-Shock-Protein-70 (HSP70), Calprotectin, CD24 and Siglec-10. But, whereas in NSBT patients post-operative pro-inflammatory biomarkers were sub-normal consistent with PTI, they were supra-normal in ASBT patients implying its reversal. These PTI-biomarkers included: IL-1β, IL-2, IL-17A, Interferon-gamma (IFN-γ), Tumour-Necrosis-Factor-alpha (TNF-α), and Annexin-A2. Reversal of PTI by salvaged blood was further endorsed in ASBT by sub-normal levels of the anti-inflammatory biomarkers: IL-4, IL-5, IL-10, and IL-13. Salvaged blood analyses revealed sustained supra-normal levels of DAMPs, CD24 and Siglec-10; and increasingly elevated levels of cytokines and chemokines during the six hour collection period. Interestingly, plasma CD24, Siglec-10, HSP70 and Calprotectin levels were significantly correlated, implying physical association within the circulation. Conclusions. Several anti-inflammatory processes triggered by traumatised tissue induce systemic PTI, thereby increasing vulnerability to infections. Reversal of PTI by re-infusion of anti-coagulated salvaged blood suggests a novel source of immuno-stimulants

Introduction. Tendon healing begins with inflammation and results in an incomplete repair with fibrosis, culminating in tendon pathology along with tissue degeneration. Inflammatory mediators regulate the expression of growth factors, and members of the TGFβ superfamily including BMPs have been suggested to play a key role in the development of fibrosis. In established tendon diseases where inflammation and reparative processes persists, the cellular phenotype of tendon cells has been implied to undergo a transformation from that of normal tissue. This study investigates the inflammation-driven mechanisms of tendon pathology using an in vitro tendon cell model. We hypothesized that cells from diseased tendons will exhibit dysregulation of TGFβ superfamily members in response to inflammatory mediators when compared to cells derived from healthy tendons. Materials and Methods. Diseased human tendon cells were isolated from patients with large to massive rotator cuff tears (n=4). Cells isolated from healthy human hamstring tendons served as control tissue (n=5). Cells were treated with human recombinant IL-1β (5ng/ml), oncostatin M (10ng/ml), IL-6 (10ng/ml), IL-10 (10ng/ml) in serum-free medium, or serum-free medium alone (control) for 24 hours. Cell viability was monitored by Alamar Blue assay, and expression of TGFB1, TGFBR1, TGFBR2, CTGF, BMP2 and BMP7 were quantified by quantitative reverse transcription polymerase chain reaction (RT-QPCR). Results. Cytokine stimulation did not significantly influence cell viability in either group. In diseased cells, IL-1β induced a 4.9-fold increase in BMP2 compared to control cells (p=0.032). There were no significant changes in the expression of other TGFβ superfamily genes after stimulation with other cytokines. CTGF was significantly increased in diseased compared to healthy cells following IL-1β stimulation (p=0.0295). No other genes showed differential regulation by inflammatory cytokines between diseased and healthy cells. Discussion. This work suggests that BMP-2, a growth factor related to cell differentiation, is dysregulated with IL-1β stimulation and plays a key role in the development of tendon diseases. Differences in IL-1β-induced CTGF expression suggests increased responsiveness of diseased cells to this cytokine. BMP-2 could be an important growth factor in the development of tendon diseases and further investigation of its role in chronic inflamed tissue is warranted

The mechanism of adverse tissue reaction to implant derived cobalt and chromium is unknown. It is possible that only one of these metals, cobalt, plays critical role in the failure of MOM implant. Cobalt ions are known to stabilize hypoxia inducible factor (HIF) 1α, which is involved in inflammatory pathway involving upregulation of BNIP3, GLUT1, HO-1 and COX-2 genes. This study used human monocytic cell line U937 to test the cytotoxic and inflammatory response to cobalt and chromium in form of ions and nanoparticles (NP) at clinically relevant doses. MTT assay was used to assess cytotoxic potential of metals for up to 24 hours. Gene expression was studied using qPCR and protein expression using Western Blot technique. Inflammatory cytokine release was studied using ELISA assay. Cytotoxicity study showed similar toxicity cobalt NP throughout the range of concentration 5–100μg/ml. Stabilization of HIF1α protein was observed after stimulation with cobalt ions and NP. This resulted in upregulation of GLUT1, BNIP3, HO-1 and COX-2 genes. Stimulation caused increased release in TNFα and inhibition of IL-10. No significant release of IL-1β was observed. Stimulation with chromium ions or NP did not cause any changes in cell viability, stabilization of HIF or cytokine release profile. Chromium NP caused upregulation of COX-2 after 6 hours of exposure. These results indicate significant role of cobalt in the inflammatory process and its potential as the cause of failure of MOM implants

Objectives

Metabolic syndrome and low-grade systemic inflammation are associated with knee osteoarthritis (OA), but the relationships between these factors and OA in other synovial joints are unclear. The aim of this study was to determine if a high-fat/high-sucrose (HFS) diet results in OA-like joint damage in the shoulders, knees, and hips of rats after induction of obesity, and to identify potential joint-specific risks for OA-like changes.

Objectives

Rotator cuff tears are among the most frequent upper extremity injuries. Current treatment strategies do not address the poor quality of the muscle and tendon following chronic rotator cuff tears. Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor that activates many genes that are important in skeletal muscle regeneration. HIF-1α is inhibited under normal physiological conditions by the HIF prolyl 4-hydroxylases (PHDs). In this study, we used a pharmacological PHD inhibitor, GSK1120360A, to enhance the activity of HIF-1α following the repair of a chronic cuff tear, and measured muscle fibre contractility, fibrosis, gene expression, and enthesis mechanics.

Objectives

The cytotoxicity induced by cobalt ions (Co²⁺) and cobalt nanoparticles (Co-NPs) which released following the insertion of a total hip prosthesis, has been reported. However, little is known about the underlying mechanisms. In this study, we investigate the toxic effect of Co²⁺ and Co-NPs on liver cells, and explain further the potential mechanisms.

Objectives

Rotator cuff tears are among the most common and debilitating upper extremity injuries. Chronic cuff tears result in atrophy and an infiltration of fat into the muscle, a condition commonly referred to as ‘fatty degeneration’. While stem cell therapies hold promise for the treatment of cuff tears, a suitable immunodeficient animal model that could be used to study human or other xenograft-based therapies for the treatment of rotator cuff injuries had not previously been identified.

We studied 51 patients with osteo-articular tuberculosis who were divided into two groups. Group I comprised 31 newly-diagnosed patients who were given first-line antituberculous treatment consisting of isoniazid, rifampicin, ethambutol and pyrazinamide. Group II (non-responders) consisted of 20 patients with a history of clinical non-responsiveness to supervised uninterrupted antituberculous treatment for a minimum of three months or a recurrence of a previous lesion which on clinical observation had healed. No patient in either group was HIV-positive. Group II were treated with an immunomodulation regime of intradermal BCG, oral levamisole and intramuscular diphtheria and tetanus vaccines as an adjunct for eight weeks in addition to antituberculous treatment. We gave antituberculous treatment for a total of 12 to 18 months in both groups and they were followed up for a mean of 30.2 months (24 to 49). A series of 20 healthy blood donors served as a control group.

Twenty-nine (93.6%) of the 31 patients in group I and 14 of the 20 (70%) in group II had a clinicoradiological healing response to treatment by five months.

The CD4 cell count in both groups was depressed at the time of enrolment, with a greater degree of depression in the group-II patients (686 cells/mm³ (sd 261) and 545 cells/mm³ (sd 137), respectively; p < 0.05). After treatment for three months both groups showed significant elevation of the CD4 cell count, reaching a level comparable with the control group. However, the mean CD4 cell count of group II (945 cells/mm³ (sd 343)) still remained lower than that of group I (1071 cells/mm³ (sd 290)), but the difference was not significant. Our study has shown encouraging results after immunomodulation and antituberculous treatment in non-responsive patients. The pattern of change in the CD4 cell count in response to treatment may be a reliable clinical indicator.