Adolescent idiopathic scoliosis (AIS) is a poorly understood progressive curvature of the spine. The 3-dimmensionnal spinal deformation brings abnormal biomechanical stresses on the load-bearing organs. We have recently reported for the first time the presence of facet joint cartilage degeneration comparable to age-related osteoarthritis in scoliotic adolescents. To better understand the degenerative mechanisms and explore new therapeutic possibilities, we focused on Toll-like receptors (TLRs) which are germline-encoded pattern recognition receptors that recognize pathogens and endogenous proteins such as fragmented extracellular matrix components (alarmins) present in intervertebral discs (IVD) and articular cartilage. Once activated, they regulate the production pro-inflammatory cytokines, proteases and neurotrophins which can lead to matrix catabolism, inflammation and potentially pain. These mechanisms have however not been studied in the context of AIS or facet joints. Facet joints of AIS patients undergoing corrective surgery and of cadaveric donors (non-scoliotic) were collected from consenting patients or organ donors with ethical approval. Cartilage biopsies and chondrocytes were isolated using 3mm biopsy punches and collagenase type 2 digestion respectively. qPCR was used to assess gene expression of the degenerative factors (MMP3, MMP13, IL-1ß, IL-6, IL-8) The biopsies were cut into two equal halves, one was treated for 4 days with a TLR2 agonist (Pam2CSK4, Invivogen) in serum-free chondrocyte media while the other one was cultured in media alone. MMP3, MMP13, IL-6 and IL-8 ELISAs and DMMB assays were performed on the biopsy cultured media. The ex vivo cartilage was then fixed, cryosectionned and also stained with SafraninO-Fast Green dyes. Baseline gene expression levels of TLR1,−2,−4,−6 were all upregulated in scoliotic chondodryctes compared to non-scoliotic. Pearson correlation analysis revealed that all TLR1,−2,−4,−6 gene expression correlated strongly and significantly with degenerative markers (MMP3, MMP13, IL-6, IL-8) in scoliotic chondrocytes but not in non-scoliotic. (Figure 1) When monolayer facet joint chondrocytes were activated with Pam2CSk4, there was a significant upregulation in previously described degenerative markers, TLR2 and NGF, a potent neurotrophin. These findings were strengthened by protein secretion analysis of select markers such as MMP-3, −13, IL-6 and IL-8 which were all upregulated after TLR2 activation. The scoliotic biopsies which were treated with Pam2CSK4 had a significant loss of proteoglycan content as shown by histology, was reflected in the proteoglycan content found in the media by DMMB. TLR gene expression levels were upregulated and correlated with proteases and pro-inflammatory cytokines in degenerating scoliotic cartilage, suggesting they promote cartilage degradation, especially considering the lack of correlations in non-scoliotic healthy cartilage. Furthermore, when TLRs are activated by Pam2CSK4 it triggers the release of the same proteases and pro-inflammatory cytokines in our ex vivo experiment. All this exacerbates the loss of proteoglycan in the cartilage ex vivo model after four days of insult with a TLR2 specific agonist. These results suggest that TLRs are an important pathway partaking in the cartilage degeneration of scoliotic facet joints and potentially all cartilage beyond our scope. Future studies aim at blocking TLRs to alleviate proteolysis and inflammation. For any figures or tables, please contact the authors directly

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

Background. Osteoarthritis (OA) has been described as a non-inflammatory arthritis and yet the choice of drug treatment is NSAIDs. Aim. To test the hypothesis that cytokines and chemokines are associated with inflammation in OA. Methods. Synovium biopsy and synovial fluid of 17 patients undergoing total knee arthroplasty (TKR) were sampled at the onset of their surgery. Histology of synovium and immunoassay of synovial fluid were conducted. A 3 point scale, 3 being the most cellular, was used to assess the cellularity of synovium histology slides, a parameter known to correlate with several markers of OA. Synovial fluid was analysed using a multi-anylate fluorescent immunoassay. In brief, cytokines and chemokines associated with inflammation were quantified, namely IL-12, TNF, IL-10, IL-6, IL-1, IL-8. Results. The 3 point scale used to describe the cellularity of the synovium placed the majority in groups 2 and 3. Low levels (<120 pg/ml) of IL-12, IL-10, IL-1 and TNF were measured in all 3 cellularity groups. Markedly elevated values of IL-6 and IL-8 were measured in the synovial fluid of knees with the most cellular synovium (maximum values were 8325 pg/ml and 1540 pg/ml respectively). Conclusion. Elevated levels of IL-6 are associated with bone resorption, being clinically linked with aseptic loosening. IL-8 is capable of promoting angiogenesis and can act as a chemokine which attracts T cells. T cells were identified in the synovium of OA patients indicating an inflammatory component to the heterogeneous disease of OA

To identify the differences in inflammatory profiles between hip OA, knee OA and non-OA control cohorts and investigate the association between cytokine expression and clinical outcome measurements, specifically pain. A total of 250 individuals were recruited in three cohorts (100 knee OA, 50 hip OA, 100 control). Serum was collected and inflammatory profiles analysed using the Multiplex Human Cytokine Panel (Millipore) on the Luminex 100 platform (Luminex Corp., Austin, TX). The pain, physical function and activity limitations of hip OA cohort were scored using the WOMAC, SF-36, HHS and UCLA scores. All cytokine levels were compared between cohorts individually using Mann–Whitney–Wilcoxon (MWW) test with Bonferroni multiple comparison correction. Within hip OA cohorts, the effect of hip alignment (impingement and dysplasia) and radiographic grade (Kellgren and Lawrence grade, K/L grade) on cytokine levels were accessed by MWW test. Spearman's rank correlation test used to assess the association between cytokines and pain levels. The three cohorts showed distinct inflammatory profiles. Specifically, EGF, FGF-2, MCP-3, MIP-1a, IL-8 were significant different between knee and hip OA; FGF-2, GRO, IL-8, MCP-1, VEGF were significant different between hip OA and control; Eotaxin, GRO, MCP-1, MIP-1b, VEGF were significant different between knee OA and control (p-value < 0.0012). For hip OA cohorts, cytokines do not differ between K/L grade three and K/L grade four or between patients that displayed either impingement or dysplasia. Three cytokines were significant associated with pain: IL-6 (p-value = 0.045), MDC (p-value = 0.032) and IP-10 (p-value = 0.038). We have demonstrated that differences in serum inflammatory profiles exist between hip and knee OA patients. These differences suggest that OA may include different inflammatory subtypes according to affected joints. We also identified that the cytokine IL-6, MDC and IP-10 are associated with pain level in hip OA patients. These cytokines might help explain the inconsistent of presentation of pain with radiographical severity of OA joints. Future studies are needed to validate our findings and then to understand the following questions: (1) how differently affected joints are reflected in systematic biomarkers; (2) how these cytokines are biologically involved in the OA pain pathway

Intervertebral discs (IVDs) degeneration is one of the major causes of back pain. Upon degeneration, the IVDs tissue become inflamed, and this inflammatory microenvironment may cause discogenic pain. Cellular senescence is a state of stable cell cycle arrest in response to a variety of cellular stresses including oxidative stress and adverse load. The accumulation of senescent IVDs cells in the tissue suggest a crucial role in the initiation and development of painful IVD degeneration. Senescent cells secrete an array of cytokines, chemokines, growth factors, and proteases known as the senescence-associated secretory phenotype (SASP). The SASP promote matrix catabolism and inflammation in IVDs thereby accelerating the process of degeneration. In this study, we quantified the level of senescence in degenerate and non-degenerate IVDs and we evaluated the potential of two natural compounds to remove senescent cells and promote overall matrix production of the remaining cells. Human IVDs were obtained from organ donors. Pellet or monolayer cultures were prepared from freshly isolated cells and cultured in the presence or absence of two natural compounds: Curcumin and its metabolite vanillin. Monolayer cultures were analyzed after four days and pellets after 21 days for the effect of senolysis. A cytotoxicity study was performed using Alamar blue assay. Following treatment, RNA was extracted, and gene expression of senescence and inflammatory markers was evaluated by real-time q-PCR using the comparative ΔΔCt method. Also, protein expression of p16, Ki-67 and Caspase-3 were evaluated in fixed pellets or monolayer cultures and total number of cells was counted on consecutive sections using DAPI and Hematoxylin. Proteoglycan content was evaluated using SafraninO staining or DMMB assay to measure sulfated glycosaminoglycan (sGAG) and antibodies were used to stain for collagen type II expression. We observed 40% higher level of senescent cells in degenerate compare to the non-degenerate discs form unrelated individuals and a 10% increase when we compare degenerate compare to the non-degenerate discs of the same individual. Using the optimal effective and safe doses, curcumin and vanillin cleared 15% of the senescent cells in monolayer and up to 80% in pellet cultures. Also, they increased the number of proliferating and apoptotic cells in both monolayer and pellets cultures. The increase in apoptotic cell number and caspase-3/7 activity was specific to degenerate cells. Following treatment, mRNA expression levels of SASP factors were decreased by four to 32-fold compared to the untreated groups. Senescent cell clearance decreased, protein expression of MMP-3 and −13 by 15 and 50% and proinflammatory cytokines levels of IL-1, IL-6 and IL-8 by 42, 63 and 58 %. Overall matrix content was increased following treatment as validated by an increase in proteoglycan content in pellet cultures and surrounding culture media. This work identifies novel senolytic drugs for the treatment of IVD degeneration. Senolytic drugs could provide therapeutic interventions that ultimately, decrease pain and provide a better quality of life of patients living with IVDs degeneration and low back pain

Post operative prosthetic joint infection (PJI) is the most common cause of failure of total joint arthroplasty, requiring revision surgery, but a gold standard for the diagnosis and the treatment of PIJ is still lacking [1]. SuPAR, the soluble urokinase plasminogen activation receptor, has been recently described as a powerful diagnostic and prognostic tool, able not only to detect sepsis but also to discriminate different grade of sepsis severity [2,3]. This study aimed to examine the diagnostic value of SuPAR in post operative PJI, in order to explore the possible application of this new biomarker in the early diagnosis of PJI. The level of SuPAR have been measured in PJI patients and controls (patients undergoing prosthesis revision without infection), and correlated with pro and anti inflammatory markers (CRP C-reactive protein, IL-6, IL-1 TNFα, IL-10, IL-12, IL-8, IL1ra and the chemokine CCL2). Statistical analysis of Receiver Operating Characteristic (ROC) curves and Area Under the Curve (AUC) was performed. As described in Figure 1, serum SuPAR displayed a strongly significative increase in PJI patients compared to not infected controls, and a significative positive correlation with C-reactive protein, IL-6, IL-1 and TNFα and the chemokine CCL2. SuPAR displayed a very good AUC, significantly higher than CRP and IL-6 AUC. This study clearly show that the measure of Serum level of SuPAR provide a extremely important benefit because it is a precise indicator of bacterial infection, and the addition of SuPAR serum level measurement to classical inflammatory markers can strongly improve the diagnosis of prosthesis joint infection. The authors acknowledge ViroGates, Denmark for providing suPARNOSTIC Standard Kit. The authors would also acknowledge the Italian Ministero dell’ Istruzione, Università e Ricerca (MIUR) and Italian Ministero della Salute for providing funds for this research project

Osteoarthritis (OA) is the most common form of arthritis worldwide. It is a major cause of disability in the adult population with its prevalence expected to increase dramatically over the next 20 years. Although current therapies can alleviate symptoms and improve function in early course of the disease, OA inevitably progresses to end-stage disease requiring total joint arthroplasty. Mesenchymal stromal cells (MSCs) have emerged as a candidate cell type with great potential for intra-articular (IA) repair therapy. However, there is still a considerable lack of knowledge concerning their behaviour, biology and therapeutic effects. To start addressing this, we explored the secretory profile of bone marrow derived MSCs in early and end-stage knee OA synovial fluid (SF). Subjects were recruited and categorised into early [Kellgren-Lawrence (KL) grade I and II, n=12] and end-stage (KL grade III and IV, n=11) knee OA groups. The SF proteome of early and end-stage OA was tested before and three days after the addition of bone marrow MSCs (16.5×10^3, single donor) using multiplex ELISA (64 cytokines) and mass spectrometry (302 proteins detected). Non parametric Wilcoxon-signed rank test for paired samples was used to compare the levels of proteins before and after addition of MSCs in early and end-stage knee OA SF. Significant differences were determined after multiple comparisons correction (FDR) with a p<0.05. Gender distribution and BMI were not statistically different between the two cohorts (p>0.05). However, patients in early knee OA cohort were significantly younger (44.7 years, SD=7.1) than patients in the end-stage cohort (58.6 years, SD=4.4; p<0.05). In both early and end-stage knee OA, MSCs increased the levels of VEGF-A (by 320.24 pg/mL), IL-6 (by 826.78 pg/mL) and IL-8 (by 128.85 pg/mL), factors involved in angiogenesis; CXCL1/2/3 (by 103.35 pg/mL), CCL2 (by 1187.27 pg/mL), CCL3 (by 15.82 pg/mL) and CCL7 (by 10.43 pg/mL), growth factors and chemokines. However, CXCL5 (by 48.61 pg/mL) levels increased only in early knee OA, whereas PDGF-AA (by 15.36 pg/mL) and CXCL12 (by 497.19 pg/mL) levels increased only in end-stage knee OA. This study demonstrates that bone marrow derived MSCs secrete angiogenic and chemotactic factors both in early and end-stage knee OA. More importantly, MSCs show a differential reaction between early and end-stage OA. Functional assays are required to further understand on how the therapeutic effect of MSCs is modulated when exposed to OA SF

Peri-prosthetic osteolysis and subsequent aseptic loosening is the most common reason for revising total hip replacements. Wear particles originating from the prosthetic components interact with multiple cell types in the peri-prosthetic region resulting in an inflammatory process that ultimately leads to peri-prosthetic bone loss. These cells include macrophages, osteoclasts, osteoblasts and fibroblasts. The majority of research in peri-prosthetic osteolysis has concentrated on the role played by osteoclasts and macrophages. The purpose of this review is to assess the role of the osteoblast in peri-prosthetic osteolysis.

In peri-prosthetic osteolysis, wear particles may affect osteoblasts and contribute to the osteolytic process by two mechanisms. First, particles and metallic ions have been shown to inhibit the osteoblast in terms of its ability to secrete mineralised bone matrix, by reducing calcium deposition, alkaline phosphatase activity and its ability to proliferate. Secondly, particles and metallic ions have been shown to stimulate osteoblasts to produce pro inflammatory mediators in vitro. In vivo, these mediators have the potential to attract pro-inflammatory cells to the peri-prosthetic area and stimulate osteoclasts to absorb bone. Further research is needed to fully define the role of the osteoblast in peri-prosthetic osteolysis and to explore its potential role as a therapeutic target in this condition.

Cite this article: Bone Joint J 2013;95-B:1021–5.

We examined the usefulness of neutrophil CD64 expression in detecting local musculoskeletal infection and the impact of antibiotics on its expression. Of 141 patients suspected of musculoskeletal infection, 46 were confirmed by microbiological culture to be infected and 95 had infection excluded. The median CD64 count of patients with localised infection was 2230 molecules per cell (interquartile range (IQR) 918 to 4592) and that of the patients without infection was 937 molecules per cell (IQR 648 to 1309) (p < 0.001). The level of CD64 correlated with the CRP level in patients with infection, but not in those without infection (r = 0.59, p < 0.01). Receiver operator characteristic curve analysis revealed that CD64 was a good predictor of local infection. When the patients were subdivided into two groups based on the administration of antibiotics at the time of CD64 sampling, the sensitivity for detecting infection was better in those who had not received antibiotics.

These results suggest that measurement of CD64 expression is a useful marker for local musculoskeletal infection.