Introduction. The complex process of inflammation and osteolysis due to wear particles still is not understood in detail. So far, Ultra-high-molecular-weight-polyethylene (UHMWPE) is the bearing material of choice in knee arthroplasty and revision knee arthroplasty, but there is a growing demand for alternative bearing materials with improved wear properties. Lately, increasing interest developed in the use of natural and carbon-fiber-reinforced-poly-ether-ether-ketones (CFR-PEEK). While there is a lack of data concerning the effects of CFR-PEEK particles on human tissue, the effects of such wear debris in vitro and in animal studies is controversially discussed. The aim of this study was to analyze human tissue containing CFR-PEEK as well as UHMWPE wear debris. The authors hypothesized no difference between the used biomaterials because of similar size parameters of the wear particles in a prior knee simulator study of this implant. Methods and Materials. Synovial tissue samples of 10 patients while knee revision surgery of a rotating hinge knee implant design (Enduro®, Aesculap, Germany) were achieved. The tibial inserts of this design were made from UHMWPE (GUR 1020), whereas the bushings and flanges are made of CFR-PEEK containing 30% polyacrylonitrile (PAN) based carbon fibers (PEEK-Optima LT1, Invibio Ltd. Thornton-Cleveleys, UK). In a prior in vitro test most of the released CFR-PEEK particles were in a size range between 0.1 and 2μm. The implant survival until revision surgery was 22 (2.5–48 min.-max.) months. As a control synovial tissue out of a patient also got knee revision surgery without any PEEK components. The tissue was fixed with 4% paraformaldehyde, embedded in paraffin, sliced into 2 µm thick sections. stained with hematoxylin and eosin in a standard process. A modified panoptical staining (preincubation in propylenglycol; >3h; 35°C) was also done which stained the UHMWPE particles turquoise. The study was approved by the ethics committee of the local university. Results. Overall, histologically a “wear-type” reaction was seen in the testing and the control group similar as described for other materials in the common literature. In all samples of the testing group the UHMWPE particles were scattered in the tissue similar to the control. Larger UHWMPE particles were incorporated in giant cells. In contrast to these findings, CFR-PEEK particles were not scattered in tissue but located only as conglomerates. In addition, these conglomerates have been found exclusively near to or in vessels. Furthermore, CFR-PEEK particles were collected in macrophages, no CFR-PEEK particles were seen in giant cells. In conclusion, the hypothesis has to be rejected. Interestingely, different behaviour of UHMWPE and PEEK particles has been found in human synovial tissue. This aspect needs further investigation concerning the cytokine expression and also the surface texture of particles. Acknowledgement. This study was supported by Aesculap, Germany

Aim. The current antibiotic treatment of periprosthetic joint infection (PJI) is optimized by measuring concentrations in plasma. However, it remains unclear whether effective concentrations of the antibiotics are reached at the site of PJI. Nonetheless, adequate target site concentrations are important to achieve effective eradication of the micro-organism. In order to determine the efficacy of cefuroxime and flucloxacillin in synovial fluid, synovial tissue and bone tissue in relation to the minimal inhibitory concentration (MIC) of the pathogen causing the PJI, we perform a pharmacokinetic/pharmacodynamic (PK/PD) study. Therefore, we aimed to develop validated analytical methods for analysis of cefuroxime and flucloxacillin in synovial fluid, synovial tissue and bone tissue. Method. Blank samples of synovial fluid, synovial tissue and bone tissue were obtained by orthopedic surgeons during surgery. For validation the samples of each matrix were spiked with both cefuroxime and flucloxacillin. Synovial tissue and bone tissue was pulverized with a mikro-dismembrator. Samples were kept frozen at −20°C until analysis. After a sample preparation quantification of cefuroxime and flucloxacillin in each matrix was performed on the ultra-performance convergence chromatography-tandem mass spectrometry (UPC2-MS/MS). Stable-isotope-labeled meropenem-d6 served as internal standard. The linearity, limits of quantification, accuracy and precision and carry-over were determined for all methods separately. The methods were validated according to the European Medicine Agency (EMA) and Food and Drug Administration (FDA) guidelines on bioanalytical method validation. Results. These methods were successfully validated for cefuroxime and flucloxacillin quantification in all matrices according to the EMA and FDA guidelines. The limits of quantification were adequate to cover potential cefuroxime and flucloxacillin concentration in synovial fluid, synovial tissue and bone tissue as described in literature, with a range of 1–100mg/L for synovial fluid and 1–20 µg/g for synovial tissue and bone tissue (r >0.995). Accuracy and within-run precision were validated according to acceptance values (RSD <15%). Carry over was less than 20%. Matrix effects and recovery were investigated for synovial fluid. The results were within the range of 80–120%. Conclusions. The results of the validation fall within the limits of quantification according to the EMA and FDA guidelines. Therefore, these methods can be applied during a PK/PD study to discover the exposure of antibiotics in synovial fluid, synovial tissue and bone tissue at the site of infection in patients with a PJI

Aim. Prosthetic joint infections (PJI) are a common reason for revisions in patients that underwent total arthroplasty of the hip (THA) or knee (TKA). Extensive antibiotic treatment follows while a clear understanding of target site concentrations is lacking. The aim is to investigate the target site concentrations, like bone and synovial tissue concentrations, which consequently may lead to an optimisation of the dosing regiments of cefuroxime of PJI patients suffering from pain and immobility. Dosing optimisation may lead to a reduced risk of (re-)infection and adverse effects like renal-insufficiency and therefore lower health-care costs. Method. Patients (n=26) with PJI of hip or knee undergoing a one- or two-stage revision treated with cefuroxime were included as part of the ASTERICS study. During implant removal two samples were collected 15-30 and 60-120 minutes after IV infusion of plasma, bone tissue and synovial tissue and one synovial fluid sample. Samples were analysed using a UltraPerformance Convergence Chromotography – quadruple mass spectrometry system (UPC. 2. -MS/MS). Bone tissue and synovial tissue were pulverized before analysis acquiring for bone tissue a homogenate of cortical and cancellous bone. Using nonlinear mixed effect modelling (NONMEM) a base model was developed to analyse the bone to plasma ratio of cefuroxime in osteomyelitis patients. Results. Mean bone concentrations (mg/L) of cefuroxime at 30-60 min after IV administration in the knee and hip are 21.29 (SD:11.86) and 19.06 (SD: 11.79) respectively and 8.23 (SD:4.90) and 9.67 (SD:9.75) respectively at 90-120 min after IV administration. The penetration of cefuroxime described by the bone:plasma ratio into knee and hip affected by osteomyelitis is 0.3 and 0.4 respectively within 1 hour and 0.1 for both joints within 2 hours. The results mentioned here were collected during knee operations without blood void conditions. Concentration data was used to develop a base pharmacokinetic model using NONMEM and was best described by a two-compartment model. Conclusions. Cefuroxime penetrates osteomyelitis affected bone tissue within the hour proving the usefulness of cefuroxime as prophylaxis of orthopaedic surgery and as treatment option for PJI. However, PK modelling and further simulations need to prove whether repeated cefuroxime dosing in this population is required to reach minimal inhibitory concentrations in target tissue

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

Osteoarthritis (OA) is the fastest growing global health problem, with a total joint replacement being the only effective treatment for patients with end stage OA. Many groups are examining the use of bone marrow or adipose derived mesenchymal stem cells (MSCs) to repair cartilage, or modulate inflammation to promote healing, however, little efficacy in promoting cartilage repair, or reducing patient symptoms over temporary treatments such as micro-fracture has been observed. There is a growing body of literature demonstrating that MSCs derived from the synovial lining of the joint are superior in terms of chondrogenic differentiation and while improvements in clinical outcome measures have been observed with synovial MSCs, results from clinical studies are still highly variable. Based on our results, we believe this variability in clinical studies with MSCs results in part from the isolation, expansion and re-injection of distinct MSCs subtypes in normal vs. OA tissues, each with differing regenerating potential. However, it remains unknown if this heterogeneity is natural (e.g. multiple MSC subtypes present) or if MSCs are influenced by factors in vivo (disease state/stage). Therefore, in this study, we undertook an ‘omics’ screening approach on MSCs from normal and OA knee synovial tissue. Specifically, we characterized their global proteome and genomic expression patterns to determine if multiple MSC from normal and OA joints are distinct at the protein/gene expression level and/if so, what proteins/genes are differentially expressed between MSCs derived from normal and OA synovial tissue. Synovium tissue was collected from OA patients undergoing joint replacement and normal cadaveric knees. The in vitro adipogenic, chondrogenic and osteogenic differentiation potential of the MSCs was analyzed via qPCR and histology. Fully characterized MSC populations where then analyzed through an unbiased shotgun proteomics, and microarray analysis. Synovial MSCs isolated from both OA and normal knees demonstrated similar multipotent differentiation capacity. Likewise, both OA and normal MSCs display the typical MSCs cell surface marker profile in vitro (CD90+, CD44+, CD73+, CD105+). Using shotgun proteomics, 7720 unique peptides corresponding to 2183 proteins were identified and quantified between normal and OA MSCs. Of these 2183 proteins, 994 were equally expressed in normal and OA, MSCs, 324 were upregulated in OA MSCs (with 50 proteins exclusively expressed in OA MSCs), 630 proteins were upregulated in normal MSCs (with 16 proteins exclusively expressed in normal MSCs). Microarray analysis of normal and OA MSCs demonstrated a similar result in where, 967 genes were differentially expressed between normal and OA MSCs, with 423 genes upregulated in OA, and 544 genes upregulated in normal MSCs. In this project, we have demonstrated that although normal and OA synovial derived MSCs demonstrate similar multipotent differentiation potential and cell surface markers expression, these cells demonstrated significant differences at the molecular level (protein and gene expression). Further research is required to determine if these differences influence functional differences in vitro and/or in vivo and what drives this dramatic change in the regulatory pathways within normal vs. OA synovial MSCs

The diagnosis of infection following shoulder arthroplasty is notoriously difficult. The prevalence of prosthetic shoulder infection after arthroplasty ranges from 3.9 – 15.4% and the most common infective organism is Cutibacterium acnes. Current preoperative diagnostic tests fail to provide a reliable means of diagnosis including WBC, ESR, CRP and joint aspiration. Fluoroscopic-guided percutaneous synovial biopsy (PSB) has previously been reported in the context of a pilot study and demonstrated promising results. The purpose of this study was to determine the diagnostic accuracy of percutaneous synovial biopsy compared with open culture results (gold standard). This was a multicenter prospective cohort study involving four sites and 98 patients who underwent revision shoulder arthroplasty. The cohort was 60% female with a mean age was 65 years (range 36-83 years). Enrollment occurred between June 2014 and November 2021. Pre-operative fluoroscopy-guided synovial biopsies were carried out by musculoskeletal radiologists prior to revision surgery. A minimum of five synovial capsular tissue biopsies were obtained from five separate regions in the shoulder. Revision shoulder arthroplasty was performed by fellowship-trained shoulder surgeons. Intraoperative tissue samples were taken from five regions of the joint capsule during revision surgery. Of 98 patients who underwent revision surgery, 71 patients underwent both the synovial biopsy and open biopsy at time of revision surgery. Nineteen percent had positive infection based on PSB, and 22% had confirmed culture positive infections based on intra-operative tissue sampling. The diagnostic accuracy of PSB compared with open biopsy results were as follows: sensitivity 0.37 (95%CI 0.13-0.61), specificity 0.81 (95%CI 0.7-0.91), positive predictive value 0.37 (95%CI 0.13 – 0.61), negative predictive value 0.81 (95%CI 0.70-0.91), positive likelihood ratio 1.98 and negative likelihood ratio 0.77. A patient with a positive pre-operative PSB undergoing revision surgery had an 37% probability of having true positive infection. A patient with a negative pre-operative PSB has an 81% chance of being infection-free. PSB appears to be of value mainly in ruling out the presence of peri-prosthetic infection. However, poor likelihood ratios suggest that other ancillary tests are required in the pre-operative workup of the potentially infected patient

Aberrant infrapatellar fat metabolism is a notable feature provoking inflammation and fibrosis in the progression of osteoarthritis (OA). Irisin, a secretory subunit of fibronectin type III domain containing 5 (FNDC5) regulate adipose morphogenesis, energy expenditure, skeletal muscle, and bone metabolism. This study aims to characterize the biological roles of Irisin signaling in an infrapatellar fat formation and OA development. Injured articular specimens were harvested from 19 patients with end-stage knee OA and 11 patients with the femoral neck fracture. Knee joints in mice that overexpressed Irisin were subjected to intra-articular injection of collagenase to provoke OA. Expressions of Irisin, adipokines, and MMPs probed with RT-quantitative PCR. Infrapatellar adiposity, articular cartilage damage, and synovial integrity verified with histomorphometry and immunohistochemistry. Infrapatellar adipose and synovial tissues instead of articular cartilage exhibited Irisin immunostaining. Human OA specimens showed 40% decline in Irisin expression than the non-OA group. In vitro, the gain of Irisin function enabled synovial fibroblasts but not chondrocytes to display minor responses to the IL-1β provocation of MMP3 and MMP9 expression. Of note, Irisin signaling reduced adipogenic gene expression and adipocyte formation of mesenchymal progenitor cells. In collagenase-mediated OA knee pathogenesis, forced FNDC5 expression in articular compromised the collagenase-induced infrapatellar adipose hypertrophy, synovial hypercellularity, and membrane hyperplasia. These adipose-regulatory actions warded off the affected knees from cartilage destruction and gait aberrance. Likewise, intra-articular injection of Irisin recombinant protein mitigated the development of infrapatellar adiposity and synovitis slowing down the progression of cartilage erosion and walking profile irregularity. Affected joints and adipocytes responded to the Irisin recombinant protein treatment by reducing the expressions of cartilage-deleterious adipokines IL-6, leptin, and adiponectin through regulating PPAR&gamma, function. Irisin dysfunction is relevant to the existence of end-stage knee OA. Irisin signaling protects from excessive adipogenesis of mesenchymal precursor cells and diminished inflammation and cartilage catabolism actions aggravated by adipocytes and synovial cells. This study sheds emerging new light on the Irisin signaling stabilization of infrapatellar adipose homeostasis and the perspective of the therapeutic potential of Irisin recombinant protein for deescalating knee OA development

There is currently no cure for osteoarthritis (OA), although there are ways to manage it, but most require quite invasive surgeries. There is a resident mesenchymal progenitor cell (MPC) population within the synovial membrane of the joint that have the ability to differentiate into bone, fat, and cartilage. We hypothesise that in vivo and in vitro cell surface marker expression comparisons of the MPCs can determine which population has the highest chondrogenic capacity and is best suited for future clinical trials. Method optimisation protocol: Synovial biopsies (2 or 5mm) were obtained from patients undergoing surgery. The biopsies were digested in either collagenase type I, IA, IV or II at a concentration of 0.5 or 1.0 mg/mL. Digestion was conducted at 37°C for 30, 60, 90 or 120min. To assay for the number of MPCs obtained, the cell suspension was stained with CD90 (a synovial MPC marker) and magnetically purified. The purified cells were then assayed by flow cytometry (Co-stained with a live/dead cell marker, BV510) or bright-field microscopy. Study protocol: Synovial tissues were digested in type IV collagenase for two hours to obtain a single cell suspension. The cells were subsequently stained with mesenchymal stem cell markers, including CD 90, CD 271, CD 44, CD73, and CD105, a macrophage marker, CD68. The macrophages were excluded and the remaining cells were index sorted into 96-well plates. The cells were expanded, and underwent 21-day chondrogenic, adipogenic, and osteogenic differentiation. Differentiation was assayed using RT-qPCR and histological methods. Additionally, the cells were re-analysed for marker expression after culturing. Optimisation: Synovial biopsies of 5mm produced a greater number of live CD90+ cells than 2mm biopsies. It was observed that type IV collagenase at 1mg/ML treatment for 120 min (hip) and 90 min (knee) obtained the greatest number of CD90+ MPCs from the synovium. Results: A single cell was isolated from an OA hip biopsy and was positive for the markers CD90, CD44, CD73, and negative for the markers CD68, CD271, CD105. Following differentiation, PCR analysis suggested that the cell line was able to differentiate into chondrocytes and adipocytes, but not osteoblasts. Histology data agreed with the PCR data with the adipocytes and chondrocytes having positive staining, whereas the osteoblasts were negative. FACS analysis following proliferation showed that the expression in vivo versus in vitro was the same except CD105 that became positive after proliferation in vitro. MPCs express cell surface markers that provide information as to populations have the best cartilage regeneration abilities. By determining the properties of the MPCs in OA hips that allow for better chondrogenic differentiation abilities in vitro, selecting the optimal cells for regenerating cartilage can be done more efficiently for novel cell therapies for OA

We present seven patients with recurrent haemarthroses after total knee arthroplasty, caused by an inherent platelet function defect. These patients developed painful knee swelling, persistent bleeding and/or wound breakdown, a platelet factor 3 availability defect being identified in all cases. Surgical exploration, with joint debridement, lavage and synovectomy, was performed in four patients who did not improve with conservative therapy. Histopathological examination of synovium revealed a focal synovial reaction with histiocytic infiltration, and occasional foreign-body giant cells. One patient required an early revision because of aseptic loosening of their tibial component. The condition was treated by single-donor platelet transfusions with good results. The diagnosis, management, and relevance of this disorder are discussed.