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

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

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

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