Aims

Synovial fluid white blood cell (WBC) count and percentage of polymorphonuclear cells (%PMN) are elevated at periprosthetic joint infection (PJI). Leucocytes produce different interleukins (IL), including IL-6, so we hypothesized that synovial fluid IL-6 could be a more accurate predictor of PJI than synovial fluid WBC count and %PMN. The main aim of our study was to compare the predictive performance of all three diagnostic tests in the detection of PJI.

Confirming clinical evidence, we recently demonstrated in a rodent model that a severe trauma which induces an acute systemic inflammation considerably impairs fracture healing. Interleukin-6 (IL-6) is a key cytokine in posttraumatic inflammation as its serum level correlates with injury severity and mortality. IL-6 signals are transmitted by the transmembrane glycoprotein 130 (gp130) via two distinct mechanisms: firstly, through classic signalling via the membrane-anchored IL-6 receptor and secondly, through trans-signalling using a soluble IL-6 receptor. Whereas IL-6 trans-signalling is considered a danger signal driving inflammation, classic signalling may mediate anti-inflammatory, pro-regenerative processes. The role of the two distinct pathways in bone healing has not yet been elucidated. Here, we studied the function of IL-6 in the pathophysiology of compromised bone healing induced by severe trauma. Male C57BL/6J mice received an osteotomy of the right femur stabilized with an external fixator. Systemic inflammation was induced by additional blunt chest trauma (TxT) applied immediately after the osteotomy. Mice were injected with either fusion protein sgp130Fc, which selectively inhibits IL-6 trans-signalling, or a neutralizing anti-IL-6 antibody (IL-6 Ab), blocking both signalling pathways. Control mice received vehicle solution. Animals were euthanised 21 days after surgery. Fracture healing was analysed by biomechanical testing, μCT, and histomorphometry (n= 6–9; p=0.05; ANOVA/Fisher LSD post hoc). Thoracic trauma significantly impaired fracture healing [bending stiffness (EI) −57%, p<0.00]. Treatment with sgp130Fc significantly attenuated bone regeneration as demonstrated by an increased EI (+110%, p<0.00) and a trend of augmented apparent Young”s modulus (+69%, p=0.13) compared to TxT control. Histomorphometric analysis could not detect differences in the amount of bone, confirming µCT results, but revealed a significantly decreased cartilage area after treatment with sgp130Fc (−76%, p=0.01). Inhibition of both signalling pathways with IL-6 Ab, however, did not have any effects. In conclusion, severe trauma significantly impaired fracture healing, confirming previous studies. Treatment with sgp130Fc ameliorated the negative effects providing evidence that IL-6 trans-signalling triggers the excessive immune response after trauma impairing bone regeneration. Injection of IL-6 Ab did not improve fracture healing thereby implying that classic signalling may rather have beneficial effects

Background: Infection diagnosis in THA remains difficult in some cases. Intraoperative analysis of frozen sections is related to the high sensitivity, specificity, positive predictive value, negative predictive value and accuracy. However, it is a technically demanding procedure and is not a universally accepted method. In the present study, we compared interleukin-6 (IL6) serum level with the erythrocyte sedimentation rate (ESR), the level of C-reactive protein (CRP) and the analysis of frozen sections of intraoperative specimens (FS). Materials: Sixty-nine patients with a THA needing a reoperation due to a suspected infection or another aseptic failure were studied. Patients with chronic inflam-matory diseases, antibiotic treatment prior to surgery, Paget’s diseases and immunodeficiency syndromes were excluded from the study. The mean age at the time of the operation was 68 years old (range: 39 to 91). ESR, CRP and the serum level of IL6 were measured in blood samples before surgery. The cut-off levels were: ESR: ≥ 32 mm/hr, CRP: ≥ 3.2 mg/dl and interleukin-6 ≥ 12 pg/ml. Intraoperatively, samples of tissues were taken to be analyzed immediately on FS, to be routinely processed at the moment and to be referred for bacteriological cultures and histological study. Results: Eleven (16%) of the 69 hips were infected. ESR showed a sensitivity of 0.72 (0.41 to 1.00), a specificity of 0.86 (0.76 to 0.95), a positive predictive value of 0.50 (0.22 to 0.77), and a negative predictive value of 0.94 (0.84 to 1.00).CRP showed a sensitivity of 0.72 (0.41 to 1.00), a specificity of 0.91 (0.83 to 0.99), a positive predictive value of 0.61 (0.31 to 0.91), and a negative predictive value of 0.94 (0.87 to 1.00). IL6 showed a sensitivity of 0.36 (0.30 to 0.69), a specificity of 0.94 (0.88 to 1.00), a positive predictive value of 0.57 (0.13 to 1.00), and a negative predictive value of 0.88 (0.80 to 0.97). The evaluation of the FS showed a sensitivity of 0.81 (0.54 to 1.00), a specificity of 0.98 (0.94 to 1.00), a positive predictive value of 0.90 (0.66 to 1.00), and a negative predictive value of 0.96 (0.91 to 1.00).The combination of CRP and IL6 identified all patients with deep infection of the implant and showed a sensitivity of 0.57 (0.13 to 1.00), a specificity of 1.00 (0.99 to 1.00), a positive predictive value of 1.00 (0.87 to 1.00), and a negative predictive value of 0.94 (0.87 to 1.00). Conclusion: In this study, we obtained similar results combining CRP and IL6 as with the analysis of the frozen sections, which has been in the past our first option to determine whether a THA is infected or not. IL6 and CRP may be used as a valuable routine diagnostic tool in revision THA

Purpose. Many studies have found associations between laboratory biomarkers and periprosthetic joint infection (PJI), but it remains unclear whether these biomarkers are clinically useful in ruling out PJI. This meta-analysis compared the performance of interleukin-6 (IL-6) versus procalcitonin (PCT) for the diagnosis of PJI. Materials and Methods. In this meta-analysis, we reviewed studies that evaluated IL-6 or/and PCT as a diagnostic biomarker for PJI and provided sufficient data to permit sensitivity and specificity analyses for each test. The major databases MEDLINE, EMBASE, the Cochrane Library, Web of Science, and SCOPUS were searched for appropriate studies from the earliest available date of indexing through February 28, 2017. No restrictions were placed on language of publication. Results. We identified 18 studies encompassing a total of 1,260 subjects; 16 studies reported on IL-6 [Fig. 1] and 6 studies reported on PCT [Fig. 2]. The area under the curve (AUC) was 0.93 (95% CI, 0.91 to 0.95) for IL-6 and 0.83 (95% CI, 0.79 to 0.86) for PCT. The pooled sensitivity was 0.83 (95% CI, 0.74 to 0.89) for IL-6 and 0.58 (95% CI, 0.31 to 0.81) for PCT. The pooled specificity was 0.91 (95% CI, 0.84 to 0.95) for IL-6 and 0.95 (95% CI, 0.63 to 1.00) for PCT. Both the IL-6 and PCT tests had a high positive likelihood ratio (LR); 9.3 (95% CI, 5.3 to 16.2) and 12.4 (95% CI, 1.7 to 89.8), respectively, making them excellent rule-in tests for the diagnosis of PJI. The pooled negative LR for IL-6 was 0.19 (95% CI, 0.12 to 0.29), making it suitable as a rule-out test, whereas the pooled negative LR for PCT was 0.44 (95% CI, 0.25 to 0.78), making it unsuitable as a rule-out diagnostic tool. Conclusion. Based on the results of the current meta-analysis, IL-6 has higher diagnostic value than PCT for the diagnosis of PJI. Moreover, the specificity of the IL-6 test is higher than its sensitivity. Conversely, PCT is not recommended for use as a rule-out diagnostic tool. For any figures or tables, please contact the authors directly

Abstract. Cranial cruciate ligament (CrCL) disease/rupture is a highly prevalent orthopaedic disease in dogs and common cause of pain, lameness, and secondary joint osteoarthritis (OA). Previous experiments investigating the role of glutamate receptors (GluR) in arthritic degeneration and pain revealed that OA biomarkers assessing early bone turnover and inflammation, including osteoprotegerin (OPG) and the receptor activator of nuclear factor kappa-B ligand (RANKL) are more likely to be influenced by glutamate signalling. Moreover, interleukin-6 (IL-6) has a complex and potentially bi directional (beneficial and detrimental) effect, and it is a critical mediator of arthritic pain, OA progression and joint destruction. Objectives. 1) to recruit dogs undergoing CrCL disease/rupture surgery and obtain discarded synovial fluid (SF) and serum/plasma (ethics approval, RCVS:2017/14/Alves); 2) to quantify the biomarkers listed above in the SF and serum/plasma by enzyme linked immunosorbent assay (ELISA); 3) to assess radiographic OA at the time of surgery and correlate it with the biomarkers and clinical findings. Methods. Abnova, Abcam and AMSBIO ELISA kits were tested using a validation protocol relating the standard curve to a dilution series of SF and serum/plasma (1× to 1/50×), with and without SF hyaluronidase treatment to evaluate linearity, specificity and optimal dilutions. Validated ELISA kits were used to measure [IL-6], glutamate [glu], [RANKL] and [OPG] in SF and serum/plasma. For each dog, CrCL disease pre-operative lameness scores were graded as: (1) mild, (2) moderate (easily visible), (3) marked (encumbered), (4) non-weightbearing lameness. Blinded OA scoring was performed on radiographs [15–60, normal-severe OA]. Results. canine population (n=14) was of various breeds, aged between 2–10 years and weighing 17.1–45.5Kg; 42.86% male; 57.14% female; 83.33% males and 62.5% females were neutered. Lameness scores varied from 1 and 4 (average 2.07±1.12) and radiographic OA scores from 18 and 36 (average 27.86±5.11). Individual correlations in concentrations with respect to age, weight, lameness score (1–4) and OA scores (15–60) were tested. SF [glu] and lameness score were inversely correlated with higher levels of lameness corresponding to lower SF [glu] (P=0.0141). SF [RANKL] inversely correlated with weight (P=0.0045) and lameness score (P=0.0135), and serum [RANKL] inversely correlated with weight (P=0.0437). There was also a negative correlation between SF and serum [OPG] and weight (P=0.0165 and P=0.0208, respectively). No other significant correlations were detected. Overall, [glu] and [IL-6] are increased in SF compared to serum/plasma, by 12.84 and 1.28, respectively, whereas all the remaining biomarkers are higher (2–3 times) in the serum/plasma compared to SF. Principal component analysis (PCA) and Pearson correlation coefficient matrix [IL-6/glu/RANKL/OPG] (n=7) showed SF [IL-6] correlates with SF [glu] (rs=0.64) and strong positive correlations between SF/serum [RANKL] and SF/serum [OPG] (rs 0.68–0.96). Conclusions. Dogs with CrCL disease show an association between the bone remodelling markers RANKL and OPG, and the inflammatory cytokine IL-6, and to a lesser extent SF [glu]. Therapeutics targeting bone remodelling, IL-6 or GluR/[glu] may be of interest for the management of OA in dogs. 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

Introduction: Activated peri-prosthetic macrophages release pro-inflammatory cytokines, including interleukin-6 (IL-6), that stimulate osteoclast activation and aseptic loosening. Natural sequence variations (polymorphisms) within the IL-6 gene promoter region are associated with diseases characterised by increased osteoclast activity, including osteoporosis, and affect IL-6 production in-vitro. We tested whether polymorphisms in the IL-6 gene promoter influence the risk of aseptic loosening after total hip arthroplasty (THA). Methods: 614 Caucasians, 292 men and 322 women, mean age 75.8 years who had undergone primary cemented THA for idiopathic osteoarthritis a mean of 13.4 years previously were recruited. Peripheral blood was taken and DNA extracted using standard techniques. Subjects were genotyped for the IL-6 -174, -572, and -597 promoter single nucleotide polymorphisms using the Taqman 5′ nuclease method. Results: The allele frequencies and carriage rates for both alleles at promoter positions −174, −572, and −597 were similar between controls and aseptic loosening subjects (Table, χ. 2. P> 0.05 all comparisons). Discussion: Although Il-6 has been implicated in the pathogenesis of aseptic loosening and the −174, −572, and −597 polymorphisms are associated with bone loosing pathologies, they do not appear to play a major role in aseptic loosening after THA

Aims. Tocilizumab, an interleukin-6 (IL-6) receptor (IL-6R) targeting antibody, enhances the anti-tumour effect of conventional chemotherapy in preclinical models of cancer. We investigated the anti-tumour effect of tocilizumab in osteosarcoma (OS) cell lines. Methods. We used the 143B, HOS, and Saos-2 human OS cell lines. We first analyzed the IL-6 gene expression and IL-6Rα protein expression in OS cells using reverse transcription real time quantitative-polymerase chain reaction (RT-qPCR) analysis and western blotting, respectively. We also assessed the effect of tocilizumab on OS cells using proliferation and invasion assay. Results. The OS cell lines 143B, HOS, and Saos-2 expressed IL-6R. Recombinant human IL-6 treatment increased proliferation of 143B and HOS cells. Tocilizumab treatment decreased proliferation and invasion of 143B, HOS, and Saos-2. Conclusion. In conclusion, we confirmed the production of IL-6 and the expression of IL-6R in OS cells and demonstrated that tocilizumab inhibits proliferation and invasion in OS cells. Cite this article: Bone Joint Res 2020;9(11):821–826

Aims. This study aimed to explore whether serum combined with synovial interleukin-6 (IL-6) measurement can improve the accuracy of prosthetic joint infection (PJI) diagnosis, and to establish the cut-off values of IL-6 in serum and synovial fluid in detecting chronic PJI. Methods. Patients scheduled to have a revision surgery for indications of chronic infection of knee and hip arthroplasties or aseptic loosening of an implant were prospectively screened before being enrolled into this study. The Musculoskeletal Infection Society (MSIS) definition of PJI was used for the classification of cases as aseptic or infected. Serum CRP, ESR, IL-6, and percentage of polymorphonuclear neutrophils (PMN%) and IL-6 in synovial fluid were analyzed. Statistical tests were performed to compare these biomarkers in the two groups, and receiver operating characteristic (ROC) curves and area under the curve (AUC) were analyzed for each biomarker. Results. A total of 93 patients were enrolled. There was no difference in demographic data between both groups. Synovial fluid IL-6, with a threshold of 1,855.36 pg/ml, demonstrated a mean sensitivity of 94.59% (95% confidence interval (CI) 81.8% to 99.3%) and a mean specificity of 92.86% (95% CI 82.7 to 98.0) for detecting chronic PJI. Then 6.7 pg/ml was determined to be the optimal threshold value of serum IL-6 for the diagnosis of chronic PJI, with a mean sensitivity of 97.30% (95% CI 85.8% to 99.9%) and a mean specificity of 76.79% (95% CI 63.6% to 87.0%). The combination of synovial IL-6 and serum IL-6 led to improved accuracy of 96.77% in diagnosing chronic PJI. Conclusion. The present study identified that a combination of IL-6 in serum and synovial IL-6 has the potential for further improvement of the diagnosis of PJI. Cite this article: Bone Joint Res 2020;9(9):587–592

Aims: Hormone replacement therapy (HRT) reverses the menopausal decline in bone mineral density (BMD).We investigate if part of this response is through modulation of Interleukin-6 (IL-6) activity, which is known to be reduced by HRT. Methods: We have examined the association of the -174 G/C functional promoter polymorphism of the IL-6 gene with the BMD response to HRT (Prempak C: 0.625mg oestrogen per day and 0.15mg norgestrel). 65 women were genotyped for the IL-6 polymorphism, and differences in genotype related to changes in BMD over a one year follow up period. Results: Baseline BMD (0.75 g/cm. 2. ) was independent of IL-6 genotype. The rise in BMD with HRT (5% ± 3%, p < 0.00005 by paired t-test) was genotype-dependent, with BMD rising least amongst those of GG genotype (6% ± 3% for ≥1 C allele vs 4% ± 2% GG, p=0.03). In the HRT group, BMD rose most amongst those with the putatively ‘lowest IL-6’ genotype combination- namely ≥ 1 ACE I allele and ≥ 1 IL-6 C allele (n=14) (7% ± 3%), when compared with other genotype combinations (4% ± 2%) (n=16) (p=0.003). Conclusion: These are the first data to demonstrate an influence for IL-6 genotype in influencing response to oestrogen therapy, rather than its physiological withdrawal

Senescent chondrocyte and subchondral osteoclast overburden aggravate inflammatory cytokine and pro-catabolic proteinase overproduction, accelerating extracellular matrix degradation and pain during osteoarthritis (OA). Fibronectin type III domain containing 5 (FNDC5) is found to promote tissue homeostasis and alleviate inflammation. This study aimed to characterize what role Fndc5 may play in chondrocyte aging and OA development.

Serum and macroscopically healthy and osteoarthritic cartilage were biopsied from patients with knee OA who received total knee replacement. Murine chondrocytes were transfected with Fndc5 RNAi or cDNA. Mice overexpressing Fndc5 (Fndc5Tg) were operated to have destabilized medial meniscus mediated (DMM) joint injury as an experimental OA model. Cellular senescence was characterized using RT-PCR analysis of p16INK4A, p21CIP1, and p53 expression together with ß-galactosidase activity staining. Articular cartilage damage and synovitis were graded using OARSI scores. Osteophyte formation and mechanical allodynia were quantified using microCT imaging and von Frey filament, respectively. Osteoclast formation was examined using tartrate-resistant acid phosphatase staining.

Senescent chondrocyte and subchondral osteoclast overburden together with decreased serum FNDC5 levels were present in human osteoarthritic cartilage. Fndc5 knockdown upregulated senescence program together with increased IL-6, MMP9 and Adamts5 expression, whereas Alcian blue-stained glycosaminoglycan production were inhibited. Forced Fndc5 expression repressed senescence, apoptosis and IL-6 expression, reversing proliferation and extracellular matrix production in inflamed chondrocytes. Fndc5Tg mice showed few OA signs, including articular cartilage erosion, synovitis, osteophyte formation, subchondral plate sclerosis and mechanical allodynia together with decreased IL-6 production and few senescent chondrocytes and subchondral osteoclast formation during DMM-induced joint injury. Mechanistically, Fndc5 reversed histone H3K27me3-mediated IL-6 transcription repression to reduce reactive oxygen species production.

Fndc5 loss correlated with OA development. It was indispensable in chondrocyte growth and anabolism. This study sheds light onto the anti-ageing and anti-inflammatory actions of Fndc5 to chondrocytes; and highlights the chondroprotective function of Fndc5 to compromise OA.

Although IL-6 mRNA expression in rat is restricted to the first day post-fracture, the inflammatory phase, the protein has been observed later in the healing process, indicating additional roles. The importance of IL-6 was demonstrated by delayed healing in knockout mice through diminished osteoclast numbers, formation thereof being stimulated by IL-6. The aim of our study was to investigate with which cells this cytokine is associated and when during fracture healing.

A closed fracture of the lower right limb was created in rats. The tibia was obtained from six animals at each of 1, 3, 7, 14 and 28 days post-fracture, decalcified and prepared for standard immunohistochemistry with an IL-6-specific polyclonal antibody. The number and types of cells positively stained for IL-6 along the whole length of the periosteal callus on one surface and in the fracture was evaluated.

Mostly inflammatory cells were initially stained, becoming virtually absent by day 7 when this phase has normally ended. Within the immediate vicinity of the fracture where endochondrial ossification occurred, staining of chondrocytes was significant (69%) by day 7 when this cell was laying down cartilaginous tissue that was also calcified. Distally to the fracture where direct bone formation occurred through intra-membranous ossification by osteoblasts, staining of these cells was observed, peaking at day 14 (56%). As this bone started to take on the appearance of cortex and surviving embedded osteoblasts differentiated to osteocytes, the latter cells were stained, suggesting a role in remodelling. At the fracture as bone replaced the cartilaginous tissue and union occurred, staining of chondrocytes decreased, whereas local osteoblasts were positive.

IL-6 appears to play a role throughout fracture healing, in endochondrial and intra-membranous ossification. The level of staining of each cell type reflected the degree of their activity with respect to production of related tissue.

Aims: To examine the relationship between the Interleukin 6 (IL-6) −174 G> C promoter polymorphism and exercise-induced femoral cortical bone resorption. Methods: The skeletal response to exercise was assessed in 130 male Caucasian army recruits. Five cross-sectional magnetic resonance images of the right femur were obtained before and after a 10 week period of basic physical training, and changes in cross-sectional cortical area calculated. Recruits were genotyped for the −174 G> C IL-6 promoter polymorphism. Results: Genotype frequencies (GG 36%, GC 47%, CC 22 17%) were in Hardy-Weinberg Equilibrium. The mean percentage change in proximal femoral cross sectional cortical area was strongly IL-6 genotype-dependent, with GG homozygotes losing 6.8 ± 3.82% in cortical area, GC gaining +5.5 ± 4.88%, and CC gaining +17.3 ± 9.46% (p=0.007 for linear trend). These changes persisted throughout the right femur and were significant in the femur as a whole (p=0.03). Conclusion: This study demonstrates a linear relationship between a functional polymorphism in the IL-6 gene and femoral cortical remodelling during strenuous physical exercise. Previous studies have suggested an important role for IL-6 in the regulation of bone mass in postmenopausal women, and in the invasion of bone by metastatic tumour deposits. These data extend these observations to the regulation of bone mass in healthy males, supporting a fundamental role for IL-6 in the regulation of bone mass and bone remodelling in humans.

Aims

The study aimed to determine whether the microRNA miR21-5p (MiR21) mediates temporomandibular joint osteoarthritis (TMJ-OA) by targeting growth differentiation factor 5 (Gdf5).

A balanced inflammatory response is important for successful fracture healing. The response of osteoporotic fracture healing is deranged and an altered inflammatory response can be one underlying cause. The objectives of this review were to compare the inflammatory responses between normal and osteoporotic fractures and to examine the potential effects on different healing outcomes. A systematic literature search was conducted with relevant keywords in PubMed, Embase, and Web of Science independently. Original preclinical studies and clinical studies involving the investigation of inflammatory response in fracture healing in ovariectomized (OVX) animals or osteoporotic/elderly patients with available full text and written in English were included. In total, 14 articles were selected. Various inflammatory factors were reported; of those tumour necrosis factor-α (TNF-α) and interleukin (IL)-6 are two commonly studied markers. Preclinical studies showed that OVX animals generally demonstrated higher systemic inflammatory response and poorer healing outcomes compared to normal controls (SHAM). However, it is inconclusive if the local inflammatory response is higher or lower in OVX animals. As for clinical studies, they mainly examine the temporal changes of the inflammatory stage or perform comparison between osteoporotic/fragility fracture patients and normal subjects without fracture. Our review of these studies emphasizes the lack of understanding that inflammation plays in the altered fracture healing response of osteoporotic/elderly patients. Taken together, it is clear that additional studies, preclinical and clinical, are required to dissect the regulatory role of inflammatory response in osteoporotic fracture healing.

Cite this article: Bone Joint Res 2020;9(7):368–385.

Aims. Rheumatoid arthritis (RA), which mainly results from fibroblast-like synoviocyte (FLS) dysfunction, is related to oxidative stress. Advanced oxidation protein products (AOPPs), which are proinflammatory mediators and a novel biomarker of oxidative stress, have been observed to accumulate significantly in the serum of RA patients. Here, we present the first investigation of the effects of AOPPs on RA-FLSs and the signalling pathway involved in AOPP-induced inflammatory responses and invasive behaviour. Methods. We used different concentrations of AOPPs (50 to 200 µg/ml) to treat RA-FLSs. Cell migration and invasion and the expression levels of tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), matrix metalloproteinase-3 (MMP-3), and MMP-13 were investigated. Western blot and immunofluorescence were used to analyze nuclear factor-κB (NF-κB) activation. Results. AOPPs promoted RA-FLS migration and invasion in vitro and significantly induced the messenger RNA (mRNA) and protein expression of TNF-α, IL-6, MMP-3, and MMP-13 in dose- and time-dependent manners. Moreover, AOPPs markedly activated the phosphorylation of nuclear factor-κB (NF-κB) p65 protein, which triggered inhibitory kappa B-alpha (IκBα) degradation, NF-κB p65 protein phosphorylation, and NF-κB p65 translocation into the nucleus. Furthermore, treatment with a neutralizing antibody specific to receptor for advanced glycation end products (RAGE) significantly suppressed aggressive behaviour and inflammation, decreased TNF-α, IL-6, MMP-3, and MMP-13 expression, and blocked AOPP-induced NF-κB pathway activation. Conclusion. The results indicate that AOPPs can enhance aggressive behaviour and the inflammatory response in RA-FLSs via the RAGE–NF-κB pathway. These results present AOPPs as a new class of potentially important mediators of progressive disease in RA patients. Cite this article: Bone Joint Res 2021;10(4):259–268

The interleukin-6/gp130-associated Janus Kinases/STAT3 axis is known to play an important role in mediating inflammatory signals, resulting in production of matrix metalloproteinase-3 (MMP-3). The hip joints with rapidly destructive coxopathy (RDC) demonstrate rapid chondrolysis, probably by increased production of MMP-3 observed in the early stage of RDC. In the recent study, no apparent activation of STAT3 has been shown in the synovial tissues obtained from the osteoarthritic joint at operation. However, no data are currently available on STAT3 activation in the synovial tissues in the early stage of RDC. This study aimed to elucidate STAT3 activation in the synovial tissues in the early stage of RDC. Synovial tissues within 7 months from the disease onset were obtained from four RDC patients with femoral head destruction and high serum levels of MMP-3. RDC synovial tissues showed the synovial lining hyperplasia with an increase of CD68-positive macrophages and CD3-positive T lymphocytes. STAT3 phosphorylation was found in the synovial tissues by immunohistochemistry using anti-phospho-STAT3 antibody. The majority of phospho-STAT3-positive cells were the synovial lining cells and exhibited negative expression of macrophage or T cell marker. Treatment with tofacitinib, a Janus Kinase inhibitor, resulted in a decrease in phospho-STAT3-positive cells, especially with high intensity, indicating effective suppression of STAT3 activation in RDC synovial tissues. Inhibitory effect of tofacitinib could act through the Janus Kinase/STAT3 axis in the synovial tissues in the early stage of RDC. Therefore, STAT3 may be a potential therapeutic target for prevention of joint structural damage in RDC. Acknowledgements: This study was supported by Katakami Foundation for Clinical Research

Total hip replacement (THR) is indicated for patients with osteoarthritis where conservative treatment has failed. Metal alloys used in THR implants such as cobalt-chromium (CoCr) have been known to cause pro-inflammatory reactions in patients, therefore leading to the need for costly revision surgery. This study therefore aimed to investigate the role of TLR4 in the activation of a human osteoblast model in response to CoCr particles in vitro. Human osteoblasts (MG-63 cell line) were seeded at a density of 100,000 cells and treated with 0.5, 5, 50mm3 CoCr particles per cell for 24-hours. Trypan blue and the XTT Cell Proliferation Kit II were then used in conjunction with the cells to assess CoCr-induced cytotoxicity. Cells were pre-treated with a commercially available TLR4-specific small molecule inhibitor (CLI-095) for 6 hours. Untreated cells were used as a negative control and lipopolysaccharide (LPS) was used as a positive control. Following treatment the cell supernatant was collected and used for enzyme-linked immunosorbant assay (ELISA) to measure the secretion of interleukin-8 (IL-8), CXCL10, and interleukin-6 (IL-6). Trypan blue and XTT analysis showed that there was no significant changes to cell viability or proliferation at any dose used of CoCr after 24 hours. There was a significant increase in protein secretion of IL-8 (p<0.001), CXCL10 (p<0.001), and IL-6 (p<0.001) in the cells which received the highest dosage of CoCr. This pro-inflammatory secretory response was ameliorated by TLR4 blockade (p<0.001). CoCr particles are not cytotoxic to osteoblasts but they do induce pro-inflammatory changes as characterised by increased secretion of chemokines IL-8, CXCL10, and IL-6. These responses occur via a TLR4-mediated pathway and upon inhibition they can be effectively ameliorated. This is particularly important as TLR4 could be a potential target for pharmacological intervention used in patients experiencing immunological reactions to metal implant debris

Mesenchymal stem cells (MSC) have potent immunomodulatory and regenerative effects via soluble factors. One approach to improve stem cell-based therapies is encapsulation of MSC in hydrogels based on natural proteins such as collagen and fibrin, which play critical roles in bone healing. In this work, we comparatively studied the influence of collagen and fibrin hydrogels of varying stiffness on the paracrine interactions established by MSC with macrophages and osteoblasts. Type I collagen and fibrin hydrogels in a similar stiffness range loaded with MSC from donants were prepared by modifying the protein concentration. Viability and morphology of MSC in hydrogels as well as cell migration rate from the matrices were determined. Paracrine actions of MSC in hydrogels were evaluated in co-cultures with human macrophages from healthy blood donors or with osteoblasts from bone explants of patients with osteonecrosis of the femoral head. Lower matrix stiffness resulted in higher MSC viability and migration. Cell migration rate from collagen hydrogels was higher than from fibrin matrices. The secretion of the immunomodulatory factors interleukin-6 (IL-6) and prostaglandin E. 2. (PGE. 2. ) by MSC in both collagen and fibrin hydrogels increased with increasing matrix stiffness. Tumor necrosis factor-α (TNF-α) secretion by macrophages cultured on collagen hydrogels was lower than on fibrin matrices. Interestingly, higher collagen matrix stiffness resulted in lower secreted TNF-α while the trend was opposite on fibrin hydrogels. In all cases, TNF-α levels were lower when macrophages were cultured on hydrogels containing MSC than on empty gels, an effect partially mediated by PGE. 2. Finally, mineralization capacity of osteoblasts co-cultured with MSC in hydrogels increased with increasing matrix stiffness, although this effect was more notably for collagen hydrogels. Paracrine interactions established by MSC in hydrogels with macrophages and osteoblasts are regulated by matrix composition and stiffness

Anterior cruciate ligament (ACL) injuries have been increasing, especially amongst adolescents. These injuries can increase the risk for early-onset knee osteoarthritis (OA). The consequences of late-stage knee OA include structural joint change, functional limitations and persistent pain. Interleukin-6 (IL-6) is a pro-inflammatory biomarker reflecting knee joint healing, and increasing evidence suggests that IL-6 may play a critical role in the development of pathological pain. The purpose of this study was to determine the relationship between subjective knee joint pain and function, and synovial fluid concentrations of the pro-inflammatory cytokine IL-6, in adolescents undergoing anterior cruciate ligament reconstruction surgery. Seven youth (12-17 yrs.) undergoing anterior cruciate ligament (ACL) reconstruction surgery participated in this study. They completed the Pedi International Knee Documentation Committee (Pedi-IKDC) questionnaire on knee joint pain and function. At the time of their ACL reconstruction surgery, synovial fluid samples were collected through aspiration to dryness with a syringe without saline flushing. IL-6 levels in synovial fluid (sf) were measured using enzyme linked immunosorbent assay. Spearman's rho correlation coefficient was used to determine the correlation between IL-6 levels and scores from the Pedi-IKDC questionnaire. There was a statistically significant correlation between sfIL-6 levels and the Pedi-IKDC Symptoms score (-.929, p=0.003). The correlations between sfIL-6 and Pedi-IKDC activity score (.546, p = .234) and between sfIL-6 and total Pedi-IKDC score (-.536, p = .215) were not statistically significant. This is the first study to evaluate IL-6 as a biomarker of knee joint healing in an adolescent population, reported a very strong correlation (-.929, p=0.003) between IL-6 in knee joint synovial fluid and a subjective questionnaire on knee joint pain. These findings provide preliminary scientific evidence regarding the relationship between knee joint pain, as determined by a validated questionnaire and the inflammatory and healing status of the patient's knee. This study provides a basis and justification for future longitudinal research on biomarkers of knee joint healing in patients throughout their recovery and rehabilitation process. Incorporating physiological and psychosocial variables to current return-to-activity (RTA) criteria has the potential to improve decision making for adolescents following ACL reconstruction to reduce premature RTA thereby reducing the risk of re-injury and risk of early-onset knee OA in adolescents

Aim. Diagnosis of periprosthetic shoulder infections (PSI) is difficult as they are mostly caused by low-virulent bacteria and patients do not show typical infection signs, such as elevated blood markers, wound leakage, or red and swollen skin. Ultrasound-guided biopsies for culture may therefore be an alternative for mini-open biopsies as less costly and invasive method. The aim of this study was to determine the diagnostic value and reliability of ultrasound-guided biopsies for cultures alone and in combination polymerase chain reaction (PCR), and/or synovial markers for preoperative diagnosis of PSI in patients undergoing revision shoulder surgery. Method. A prospective explorative diagnostic cohort study was performed including patients undergoing revision shoulder replacement surgery. A shoulder puncture was taken preoperatively before incision to collect synovial fluid for interleukin-6 (IL-6), calprotectin, WBC, polymorphonuclear cells determination. Prior to revision surgery, six ultrasound-guided synovial tissue biopsies were collected for culture and two additional for PCR analysis. Six routine care tissue biopsies were taken during revision surgery and served as reference standard. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV; primary outcome measure), and accuracy were calculated for ultrasound-guided biopsies, and synovial markers, and combinations of these. Results. Fifty-five patients were included. In 24 patients, routine tissue cultures were positive for infection. Cultures from ultrasound-guided biopsies diagnosed an infection in 7 of these patients, yielding a sensitivity, specificity, PPV, NPV, and accuracy of 29.2%, 93.5%, 77.8%, 63.0%, and 65.6%, respectively. Ultrasound-guided biopsies in combination with synovial WBC increased the NPV to 76.7% and accuracy to 73.8%. When synovial WBC and calprotectin were combined with ultrasound-guided biopsies, it resulted in a better diagnostic value: sensitivity 69.2%, specificity 80.0%, PPV 69.2%, NPV 80.0%, and accuracy 75.8%. Ultrasound-guided biopsies in combination with calprotectin and ESR yielded a sensitivity of 50.0%, specificity of 93.8%, PPV of 80.0%, NPV of 78.9%, and accuracy of 79.2%. Synovial fluid was obtained in 42 patients. Sensitivities of WBC, PMN, IL-6, and calprotectin were between 25.0% and 35.7%, specificities between 89.5% and 95.0%, PPVs between 60.0% and 83.3%, NPVs between 65.4% and 69.4%, and accuracies between 64.5% and 70.6%. Conclusions. In this prospective study we showed that ultrasound-guided biopsies for cultures alone and in combination with PCR and/or synovial markers are not reliable enough to use in clinical practice for the preoperative diagnosis of low grade PSI