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

Aims. To investigate the correlations among cytokines and regulatory T cells (T-regs) in ankylosing spondylitis (AS) patients, and their changes after anti-tumour necrosis factor-α (TNF-α) treatment. Methods. We included 72 AS patients with detailed medical records, disease activity score (Bath Ankylosing Spondylitis Disease Activity Index), functional index (Bath Ankylosing Spondylitis Functional Index), and laboratory data (interleukin (IL)-2, IL-4, IL-10, TNF-α, interferon (IFN)-γ, transforming growth factor (TGF)-β, ESR, and CRP). Their peripheral blood mononuclear cells (PBMCs) were marked with anti-CD4, anti-CD25, and anti-FoxP3 antibodies, and triple positive T cells were gated by flow cytometry as T-regs. Their correlations were calculated and the changes after anti-TNF-α therapy were compared. Results. The frequency of T-regs in PBMCs was positively correlated to ESR and CRP in AS (r = 0.35 and 0.43; p = 0.032 and 0.027, respectively), and there was also a significant correlation between serum level of TNF-α and CRP (p = 0.041). The frequency of T-regs in PBMCs positively correlated to serum levels of TNF-α, IL-10, and TGF-β, while IL-2, IL-4, and IFN-γ showed opposite results. After anti-TNF-α treatment, there were significantly lower serum levels of TNF-α, IL-10, TGF-β, and frequency of T-regs in PBMCs among these AS patients (p = 0.026, 0.032, 0.029, and 0.037, respectively). Conclusion. In AS patients, proinflammatory cytokine may give positive feedback to induce more T-reg production and anti-inflammatory cytokine secretion to suppress this inflammatory status, and they can be reversed by anti-TNF-α therapy. However, the detailed interactions among T-regs and complex cytokine networks in autoinflammatory diseases still need more studies and further functional assay. Cite this article: Bone Joint Res 2023;12(2):133–137

Objectives. Emerging evidence indicates that tendon disease is an active process with inflammation that is critical to disease onset and progression. However, the key cytokines responsible for driving and sustaining inflammation have not been identified. Methods. We performed a systematic review of the literature using MEDLINE (U.S. National Library of Medicine, Bethesda, Maryland) in March 2017. Studies reporting the expression of interleukins (ILs), tumour necrosis factor alpha (TNF-α) and interferon gamma in diseased human tendon tissues, and animal models of tendon injury or exercise in comparison with healthy control tissues were included. Results. IL-1β, IL-6, IL-10, and TNF-α are the cytokines that have been most frequently investigated. In clinical samples of tendinopathy and tendon tears, the expression of TNF-α tended not to change but IL-6 increased in tears. Healthy human tendons showed increased IL-6 expression after exercise; however, IL-10 remained unchanged. Animal tendon injury models showed that IL-1β, IL-6, and TNF-α tend to increase from the early phase of tendon healing. In animal exercise studies, IL-1β expression showed a tendency to increase at the early stage after exercise, but IL-10 expression remained unchanged with exercise. Conclusions. This review highlights the roles of IL-1β, IL-6, IL-10, and TNF-α in the development of tendon disease, during tendon healing, and in response to exercise. However, there is evidence accumulating that suggests that other cytokines are also contributing to tendon inflammatory processes. Further work with hypothesis-free methods is warranted in order to identify the key cytokines, with subsequent mechanistic and interaction studies to elucidate their roles in tendon disease development. Cite this article: W. Morita, S. G. Dakin, S. J. B. Snelling, A. J. Carr. Cytokines in tendon disease: A Systematic Review. Bone Joint Res 2017;6:656–664. DOI: 10.1302/2046-3758.612.BJR-2017-0112.R1

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)

Aims. Osteoporosis is characterized by decreased trabecular bone volume, and microarchitectural deterioration in the medullary cavity. Interleukin-19 (IL-19), a member of the IL-10 family, is an anti-inflammatory cytokine produced primarily by macrophages. The aim of our study was to investigate the effect of IL-19 on osteoporosis. Methods. Blood and femoral bone marrow suspension IL-19 levels were first measured in the lipopolysaccharide (LPS)-induced bone loss model. Small interfering RNA (siRNA) was applied to knock down IL-19 for further validation. Thereafter, osteoclast production was stimulated with IL-19 in combination with mouse macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). The effect of IL-19 was subsequently evaluated using tartrate-resistant acid phosphatase (TRAP) staining and quantitative real-time polymerase chain reaction (RT-qPCR). The effect of IL-19 on osteoprotegerin (OPG) was then assessed using in vitro recombinant IL-19 treatment of primary osteoblasts and MLO-Y4 osteoblast cell line. Finally, transient transfection experiments and chromatin immunoprecipitation (ChIP) experiments were used to examine the exact mechanism of action. Results. In the LPS-induced bone loss mouse model, the levels of IL-19 in peripheral blood serum and femoral bone marrow suspension were significantly increased. The in vivo results indicated that global IL-19 deletion had no significant effect on RANKL content in the serum and bone marrow, but could increase the content of OPG in serum and femoral bone marrow, suggesting that IL-19 inhibits OPG expression in bone marrow mesenchymal stem cells (BMSCs) and thus increases bone resorption. Conclusion. IL-19 promotes bone resorption by suppressing OPG expression in BMSCs in a LPS-induced bone loss mouse model, which highlights the potential benefits and side effects of IL-19 for future clinical applications. Cite this article: Bone Joint Res 2023;12(11):691–701

It has been previously shown that Low-Magnitude High-Frequency Vibration (LMHFV) is able to enhance ovariectomy-induced osteoporotic fracture healing in rats. Fracture healing begins with the inflammatory stage, and all subsequent stages are regulated by the infiltration of immune cells such as macrophages and the release of inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10). Therefore, the aim of this study was to investigate the effect of LMFHV treatment on the inflammatory response in osteoporotic fracture healing. In this study, ovariectomy-induced osteoporotic and sham-operated closed-femoral fracture SD-rats were randomized into three groups: sham control (SHAM), ovariectomized control (OVX-C) or ovariectomized vibration (OVX-V) (n=36, n=6 per group per time point). LMHFV (35Hz, 0.3g) was given 20 min/day and 5 days/week to OVX-V group. SHAM operation and ovariectomy were performed at 6-month and closed femoral fracture was performed at 9-month. Callus morphometry was determined by callus width from weekly radiography. Local expressions of inducible nitric oxide synthase (iNOS) (macrophage M1 marker), CD206 (macrophage M2 marker), TNF-α, IL-6 and IL-10 were detected by immunohistochemistry and quantified by colour threshold in ImageJ, assessed at weeks 1 and 2 post-fracture. Significant difference between groups was considered at p≤0.05 by one-way ANOVA. Callus formation was higher in OVX-V than that of OVX-C as shown by callus width at weeks 1 and 2 (p=0.054 and 0.028, respectively). Immunohistochemistry results showed that CD206 positive signal and the M2/M1 ratio which indicates the progression of macrophage polarization were significantly higher in OVX-V rats (p=0.053 and 0.049, respectively) when compared to OVX-C at week 1. Area fraction of TNF-α positive signal was significantly higher in SHAM and OVX-V rats at week 1 (p=0.01 and 0.033, respectively). IL-6 signal was also significantly higher in SHAM and OVX-V groups at week 1 (p=0.004 and 0.029, respectively). IL-10 expression was significantly lower in SHAM and OVX-V groups at week 1 (p=0.013 and 0.05, respectively). Here we have shown that LMHFV treatment promoted the shift from pro-inflammatory stage towards anti-inflammatory stage earlier. It has been reported that the polarization of pro-inflammatory macrophages M1 to anti-inflammatory macrophages M2 was indicative of the endochondral ossification process in the long bone fracture model. Besides, we found that LMHFV treatment enhanced pro-inflammatory markers of TNF-α and IL-6 and suppressed anti-inflammatory marker of IL-10 at week 1, showing that inflammatory response was enhanced at week 1 post-fracture. These inflammatory cytokines involved in fracture healing were shown to coordinate different fracture healing processes such as mesenchymal stem cell recruitment and angiogenesis. Our previous study has demonstrated that ovariectomized rats exhibit lower levels of inflammatory response after fracture creation. Therefore, we report that LMHFV treatment can modulate macrophage polarization from M1 to M2 at an earlier time-point and partly restore the impaired inflammatory response in OVX bones at the early stage of fracture healing that may lead to accelerated healing of osteoporotic fracture as shown by promoted callus formation

The purpose of this study was to investigate the effects of extra corporeal shock waves (ESW) therapy on the metabolism of healthy and osteoarthritic human chondrocytes, and particularly on the expression of IL-10, TNF-α and β1 integrin. Human adult articular cartilage was obtained from 9 patients (6 male and 3 females), with primary knee osteoarthritis (OA), undergoing total joint replacement and from 3 young healthy donors (HD) (2 males, 1 female) with joint traumatic fracture. After isolation, chondrocytes underwent ESW treatment (Electromagnetic Generator System, Minilith SL1, Storz Medical) at different parameters of impulses, energy levels and energy fluxes. After that, chondrocytes were cultured in 24-well plate in DMEM supplemented with 10% FCS for 48 hours and then β1 integrin surface expression and intracellular IL-10 and TNF-α levels were evaluated by flow-cytometry. At baseline, osteoarthritic chondrocytes expressed significantly lower levels of β1 integrin and higher levels and IL-10 and TNF-α levels. It has been recently reported that ESW may be useful to treat OA in dogs, and veterinarians have begun to use ESW also to treat OA in horses. Following ESW application, while β1 integrin expression remain unchanged, a significant decrease of IL-10 and TNF-α intracellular levels was observed both in osteoarthritic and healthy chondrocytes. IL-10 levels decreased at any impulses and energy levels, while a significant reduction of TNF-α was mainly found at middle energies. Our study confirmed that osteoarthritic chondrocytes express low β1 integrin and high TNF-α and IL-10 levels. Nonetheless, ESW treatment application down-regulate the intracellular levels of TNF-α and IL-10 by chondrocytes, suggesting that ESW might restore TNF-α and IL-10 production by osteoarthritic chondrocytes at normal levels thus potentially interfering with the pathologic mechanisms causing cartilage damage in OA and representing the theoretical rationale for using ESW as therapy of OA

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

Aim. In the current study we aim to characterize the use of cationic host defense peptides (HDPs) as alternative antibacterial agents to include into novel antibacterial coatings for orthopedic implants. Staphyloccous aureus represent one the most challenging cause of infections to treat by traditional antibacterial therapies. Thanks to their lack of microbial resistance described so far, HDPs represent an attractive therapeutic alternative to antibiotics. Furthermore, HDPs have been showed to control infections via a dual function: direct antimicrobial activity and regulation of immune response. However, HDPs functions characterization and comparison is controversial, as changing test conditions or cell type used might yield different effects from the same peptide. Therefore, before moving towards the development of HDP-based coatings, we need to characterize and compare the immunomodulatory and antibacterial functions under the same conditions in vitro of 3 well-known cathelicidins: human LL-37, chicken CATH-2, and bovine-derived IDR-1018. Method. S. aureus, strain SH1000, was incubated with different concentrations of each HDP and bacterial growth was monitored overnight. Primary human monocytes were isolated from buffy coats using Ficoll-Paque density and CD14 microbeads, and differentiated for 7 days to macrophages. After 24h incubation in presence of LPS and HDPs, macrophages cytokines production was measured by ELISA. Macrophages cultured for 24h in presence of HDPs were infected with serum-opsonized S. aureus. 30 min and 24h after infection, bacterial phagocytosis and intracellular killing by macrophages were measured by flow cytometry and colony forming units (CFU) count respectively. Results. All HDPs efficiently inhibit macrophages LPS-mediated activation, as observed by a reduced production of TNF-α and IL-10. Despite a comparable anti-inflammatory action, only CATH-2 shows direct antibacterial properties at concentrations 10-times lower than those needed to stimulate immune cells. Although stimulation with HDPs fails to improve macrophages ability to kill intracellular S. aureus, IDR-1018 decreases the proportion of cells phagocytosing bacteria. Conclusions. In addition to a strong anti-inflammatory effect provided by all HDPs tested, CATH-2 has direct antibacterial effects while IDR-1018 reduces the proportion of macrophages infected by S. aureus. Use of these HDPs in combination with each other or with other conventional antibacterial agents could lead the way to the design of novel antibacterial coatings for orthopedic implants

Polyethylene wear-debris induced inflammatory osteolysis is known as the main cause of aseptic loosening and long term revision total hip arthroplasty. Although recent reports suggest that antioxidant impregnated ultra-high molecular weight polyethylene (UHMWPE) wear-debris have reduce the osteolytic potential in vivo when compared to virgin UHMWPE, little is known about if and/or how PE rate of oxidation affects osteolysis in vivo. We hypothesized that oxidized UHMWPE particles would cause more inflammatory osteolysis in a murine calvarial bone model when compared to virgin UHMWPE. Male C57BL/6 eight weeks old received equal amount of particulate debris overlaying the calvarium of (n=12/group): sham treatment (no particles), 2mg (6,75×107 particles/mg) of endotoxin-free UHMWPE particles (PE) or of endotoxin-free highly oxidized-UHMWPE (OX) particles. In vivo osteolysis was assessed using high resolution micro-CT and inflammation with L-012 probe dependent luminescence. At day 10, calvarial bone was examined using high resolution micro-CT, histomorphometric, immunohistochemistry analyses and qRT-PCR to assess OPG, RANK, RANK-L, IL-10, IL-4, IL-1b and TRAP genes expression using the protocol defined by individual TaqManTM Gene Expression Assays Protocol (Applied Biosystems). In vivo inflammation was significantly higher in the OX (1.60E+06 ± 8.28E+05 photons/s/cm2) versus PE (8.48E+05 ± 3.67E+05) group (p=0.01). Although there was a statistically significant difference between sham (−0.27% ± 2.55%) and implanted (PE: −9.7% ± 1.97%, and OX: − 8.38% ± 1.98%) groups with regards to bone resorption (p=0.02), this difference was not significant between OX and PE (p = 0.14). There was no significant difference between groups regarding PCR analyses for OPG, RANK, RANK-L, IL-10, IL-4, IL-1b and TRAP (p = 0.6, 0.7, 0.1, 0.6, 0.3, 0.4, 0.7 respectively). Bone volume density was significantly decreased in PE (13.3%±1.2%) and OX (12.2%±1.2%) groups when compared to sham (15%±0.9%) (p < 0 .05). Histomorphometric analyses showed a significantly decreased Bone Thickness/Tissue Thickness ratio in the implanted group (0.41±0.01 mm and 0.43±0.01 mm) compared to sham group (0.69± 0.01) (p < 0 .001). However, there were no significant difference between OX and PE (p = 0.2). Our findings suggest that oxidized UHMWPE particles display increased inflammatory potential. Results were not significant regarding in vivo or ex vivo osteolysis. As antioxidant-diffused UHMWPE induce less inflammation activity in vivo, the mechanism by which they cause reduced osteolysis requires further investigation

Background. Mesenchymal stem cells (MSCs) are undergoing evaluation as a potential new therapy for immune and inflammatory-mediated conditions such as IVD degeneration (IDD). Both adipose (ASCs) and bone-marrow (BMSCs) derived MSCs have been widely used in this regard. The optimal tissue source and expansion conditions required to exploit the regenerative capacity of these cells are not yet fully elucidated. In addition the phenotypic response of transplanted cells to the disease environment is not well understood. In this study, ASCs and BMSCs were exposed to a combination of hypoxic conditioning and selected inflammatory mediators, conditions that mimic the microenvironment of the degenerate IVD, in an effort to understand their therapeutic potency for in vivo administration. Methods and Results. Donor-matched ASCs and MSCs were pre-conditioned with either IL-1β (10ng/ml) or TNFα (10ng/ml) for 48 hours under hypoxic conditions (5% O. 2. ). Conditioned media was collected and 45 different immunomodulatory proteins were analysed using human magnetic Luminex® assay. Secreted levels of several key cytokines and chemokines, both pro- and anti-inflammatory, were significantly upregulated in ASCs and BMSCs following the conditioning regime. Under all conditions tested, ASCs expressed significantly higher levels of IL-4, IL-6, IL-10, IL-12, TGF-α, and GCSF compared to BMSCs. Pre-conditioning with TNFα resulted in significantly higher levels of IL-10 while preconditioning with IL-1β resulted in higher levels of IL-6, IL-12 and GCSF. Conclusion. These data suggest that pre-conditioned ASCs may have enhanced therapeutic potential in modulating IVD repair through the increased release of trophic factors that play a role in immunomodulation. Conflicts of interest: None. Sources of funding: Financial support for this research was provided by EU Horizon 2020 RESPINE grant (Project ID# 732163)

We evaluated the contribution of specific gene polymorphisms of IL-1a/IL-1R/IL-1RA/IL-4Ra/IL-1b/IL-12/γIFN/TGF-b/TNF-a/IL-2/IL-4/IL-6/IL-10 cytokines in patients with AVN. DNA was extracted from 112 patients and 238 healthy Greek individuals. DNA analysis was performed by the PCR-SSP method and the use of the Protrans kit. Statistical analysis was performed by χ2 test. In the patients, the TC frequency of the IL-1a (nt-889) was 52% while in normal was 40%. The C/G allele frequency of TGF-b codon 25 in patients was 9% C and 91% G vs 13% C and 87% G in normal. At position −238 of TNFa, 11% of the patients had the GA genotype in contrast to 1% of the controls. The GG/GG haplotype of TNFa gene promoter (nt. −308 and −238) was more frequent in both groups, while the GG/GA haplotype detected in 9% and 1% of the patients and controls, respectively. At the −1082 position of the IL-10 gene, the GG genotype was detected in 15% of the controls and 7% of the patients. Also, the GCC/GCC haplotype in IL-10 (positions -1082/-819/-592) was higher in the controls (15%) than the patients (7%). The genotypes TC (nt-889) of IL-1a, GC (codon 25) of TGF-b, GC (nt-1082) of IL-10 and GA (nt −238) of TNFa, are more prevalent in the patients than the healthy individuals (p< 0.05). Based on our results, the presence of one of the above mentioned polymorphisms or the simultaneous carriage of more than one may contribute to the risk for osteonecrosis

Aim. The aim of this prospective comparative study was to evaluate the serum levels of different cytokines in patients underwent total knee replacement (TKR) and received allogeneic blood transfusion, post-operative auto-transfusion or not transfused. Material and Methods. This was a prospective non-randomized comparative study in 248 patients underwent TKR. Patient's demographic and clinical data including age, gender, body mass index (BMI), preoperative Hb value, complications were documented. The serum levels of IL-1b, IL-6, IL-8, IL-10, and TNF were measure pre-operatively, the 1st, 2nd, 3rd and 5th post-operative day. Patients were categorized in three groups; in Group 0 patients received no blood transfusion, in Group 1 patients received post-operative auto-transfusion and in Group 2 allogeneic blood transfusion was applied. Statistical analysis of the results was performed using repeated measures ANOVA. Results. Significant changes were observed in cytokines levels in Groups 1 and 2. In Group 1 (auto-transfusion) the levels of all cytokines significantly increased the 1st postoperative day, remaining above the pre-operative levels even the 5th post-operative day. In Group 2 (allogenic transfusion), although the levels of IL-6, IL-8 and IL-10 were also significantly increased the 1st postoperative day, they gradually returned to the per-operative levels by the 5th post-operative day. In Group 0 (no transfusion) the only significant increase was observed in IL-6 between pre-operative and 1st and 3rd day values. Furthermore, the area under the curve (AUC) of IL-1b, IL-6, IL-8 and IL-10 levels in Group 1 and AUC of IL-6, IL-8 and IL-10 levels in Group 2, were significantly higher compared to Group 0. There was no significant difference in post-operative patient's complications. Conclusion. According to the results of this study significant elevation of cytokine values were observed during the first five post-operative days in patients received blood transfusion after TKR. These changes were more pronounced in the auto-transfusion group

Aim. To evaluate a panel of peripheral blood and synovial fluid biomarkers for the identification of periprosthetic joint infection PJI. Method. Peripheral blood and synovial fluid measurements of CD64, IL-1a, IL-1b, IL-6, IL-8, IL-10, IL-17, Alpha Defensin and CRP were made on samples collected from patients with suspected PJI using a combination of flow cytometry (CD64), ELISA (Alpha Defensin) and MSD Electrochemiluminescence (IL-1a, IL-1b, IL-6, IL-8, IL-10, IL-17). Receiver operating characteristic (ROC) curves which combine sensitivity and specificity were created for each marker using GraphPad PRISM statistical software. The diagnosis of infection was based on MSIS major criteria. Results. A total of 35 infections were identified (12 acute, 23 chronic). The best performing peripheral blood biomarker in both acute and chronic PJI was CRP with an area under the curve (AUC) of 0.88 (sensitivity 83%, specificity 94%) in acute infection and 0.82 in chronic infection (sensitivity 80%, specificity 85%). In synovial fluid the best performing acute infection marker was CRP with an AUC of 0.94 (sensitivity 87.5%, specificity 95%) and in chronic cases was Alpha defensin with an AUC of 0.98 (sensitivity 100%, specificity 85%). Conclusions. CRP measured in peripheral blood shows excellent diagnostic characteristics in both acute and chronic cases. This is also replicated in synovial fluid from acute PJIs but not in chronic infection where Alpha defensin showed the best performance

The most frequent pathogenic organism in arthroplasty infections is Staphylococcus. The immune response impairment is a frequent finding in elderly people. Objective: to investigate the response of some cytokines and the effect of age in an experimental model of osteomyelitis. Materials and methods. 40 adult male Wistar rats received a stainless steel needle, intramedullarily in the left tibia. Young rats (3 months old) and Old rats (22 months old) were alloted in: Group A: Sterile implant. Group B: Sterile implant + slime producing S. aureus. 9 weeks after surgery, rats were sacrified. Determinations: Cytokines (IL-1b, IL-2, L-4, IL-6, IL-10 and IL-12)(ELISA) in blood (previous to surgery and to sacrifice) and in tibia extract (after sacrifice); the number of bacteria in tibia and implant. The Wilcoxon, Mann-Whitney U test were used (p≤ 0.05 significant). Results. Infection was detected in all the operated tibias in old rats receiving S.aureus, and in 7/10 of young rats. IL-2 levels increased in blood in the S.aureus group after surgery in old and young rats. Pre and postoperative IL-2 levels in blood were higher in old rats in both groups than in the corresponding groups of young rats. There was a decrease with age in blood of IL-4 (previous and after surgery), and a decrease of IL-1. S.aureus groups increased IL-1 levels in the operated tibia independently of age; increased IL-2 and IL-10 levels in young rats in the operated tibia; increased IL-4, IL-6 and IL-12 in old rats in blood, decreased IL-4 and increased IL-2 and IL-10 in blood in young rats. Conclusions. Significant differences in tibia infection were found with age. Old rats presented differences with young rats in cytokine response in an experimental model of osteomyelitis, showing an immune response impairment associated with old age

Introduction : The reason why heterotopic ossification develops after total hip arthroplasty is still not known, but it is assumed that inflammatory reaction is the major driving force. In literature little is known about the cytokine levels at the site of surgery, most measurements are done in serum. This study was conducted to investigate if the levels of different pro- en anti-inflammatory cytokines are measurable in drainage fluid and, when measurable, whether we can find a difference in cytokine concentration between one and six hours postoperatively. Materials and methods: Samples from the drainage system in 30 consecutive patients undergoing primairy total hip replacement were collected at one and six hours after closure of the wound. GM-CSF, G-CSF, IFN-γ, TNF, MCP-1, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13 and MIP-1beta levels were measured in the drainage fluids. Results: Measurable levels of all cytokines studied were found, except for IL-17. A significant elevation of almost all cytokines was observed between the sample after one hour and six hours postoperatively. The elevation was significant for all cytokines except IL-10 and MIP-1b. We found a strong correlation between the different pro-inflammatory cytokines. Levels are much higher than previously shown levels in serum. When computing the IL-6 to IL-10 ratio, this ratio increased from 304 (SD 256) to 12357 (SD 6788) (p< 0,000), which shows an increased predominance of the pro-inflammatory interleukines when comparing the measurements after one and six hours respectively. Conclusion: Detectable levels of numerous cytokines can be measured in drainage fluid post-operatively. The levels of most cytokines in drainage fluid are higher in samples taken six hours after surgery as compared to samples taken after one hour. Further studies are needed to detect the relation between these cytokine concentrations and the heterotopic bone formation

This study investigated the relationship between histological, clinical and radiological features of aseptically loose total joint replacements (TJRs) and synovial fluid levels of interleukin (IL)-1b, IL-6, IL-8 and IL-10. Tissue and synovial fluid samples were retrieved from patients undergoing primary (hip; n=15: knee; n=13), or revision of aseptically loose TJRs (hip; n=14: knee; n=9). The presence of inflammatory cells, blood vessels and wear debris in the tissue were assessed on a relative scale. Revision TJRs were assessed for sepsis, migration of the implant, gross loosening and the degree of radiolucency. Cytokine levels in the synovial fluid samples were determined by ELISA. All cytokines were increased in synovial fluid from revision TJRs compared to primary replacements, as were the degree of macrophage and giant cell infiltration (p< 0.01). There was a significant positive correlation between the presence of macrophages and giant cells with the levels of IL-1b, IL-8 and IL-10 (p< 0.05) but not IL-6. The amount of wear debris was related to the presence of macrophages and giant cells (p< 0.01) but not to any of the cytokines. There were no relationships between any of the clinical parameters and the presence of wear debris or the levels of any cytokine with the exception of IL-6 and gross loosening (p< 0.01). Similarly there were no differences between hips and knees for any of the parameters except IL-6, for which higher levels were found in hips (p< 0.05). The results suggest that macrophages and giant cells are responsible for the majority of IL-1b, IL-8 and IL-10 production but another cell type is contributing to IL-6 production. Furthermore, IL-6 does not fit the pattern of the other cytokines as it is upregulated in hip joints compared to knees and correlates with the presence of gross loosening. This may suggest a unique role for IL-6 that requires further investigation

T cells have been implicated in the pathogenesis of osteolysis. The goal of this study was to compare the ratios of CD4+ T cell populations in total hip arthroplasty (THA) patients with and without osteolysis. We found no significant differences in the frequency of peripheral blood CD4+CD25+ regulatory and effector T cells, serum IL-10 and TGF-β concentrations, and immuno-suppressive ability of regulatory T cells from patients with osteoarthritis prior to THA, and THA patients with and without radiographic evidence of osteolysis. CD4+ T cells are critical in regulating immune-mediated conditions. This study compared the frequency of CD4+ T cell subpopulations in the peripheral blood of patients with and without osteolysis following total hip arthroplasty (THA). Numbers of CD4+CD25hi regulatory T cells, CD4+CD25moderate effector T cells, and CD4+CD25+ T cells in the peripheral blood of thirty patients with osteoarthritis prior to primary THA, thirty patients with asymptomatic THAs and no radiographic evidence of osteolysis, nineteen patients with asymptomatic THAs with radiographic evidence of early osteolysis (not requiring revision surgery) and nine patients scheduled for revision THA for osteolysis were determined by flow cytometry. Serum IL-10 and TGF-β levels were measured using ELISA kits. Results were compared by t-test and rank sum test. CD4+ CD25hi regulatory T cells and CD4+ CD25neg T cells were isolated from blood using a MACS cell isolation kit, co-cultured for three days, and T cell proliferation determined by [3H]-thy-midine uptake. The frequency of CD4+CD25hi regulatory T cells, CD4+CD25moderate effector T cells, and CD4+CD25+ T cells were similar in each study group. Regulatory T cells from patients with and without osteolysis had a normal functional ability to inhibit CD4+ T cell proliferation. Serum levels of the regulatory T cell-derived cytokines IL-10 and TGF-β were also comparable between groups. Our data suggests that CD4+ T cell immune responses are normal in THA regardless of the level of osteolysis, in contrast to previous studies that have implicated T cell hypersensitivity in the pathogenesis of osteolysis surrounding THA