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

Total joint replacement (TJR) is by far the most effective therapy for end-stage OA patients. Most of patients achieve joint pain reduction and function improvement following to TJR, however up to 22% of them either do not improve or deteriorate after surgery. The aim of this study was to identify genetic variants to be associated with poor outcome of TJR in primary OA patients by a genome-wide association approach (GWAS). Study participants were primary OA patients from the Newfoundland Osteoarthritis Study (NFOAS) that comprised total knee or hip replacement and recruited before 2016 in St. John's, NL. DNA samples were extracted from patients' blood. Study participants completed their pre-operation and 3.99±1.38 years post-surgery outcome assessment using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). DNA samples were genotyped using the genome-wide Illumina HumanOmni2.58 genotyping microarray containing 2.4 million SNPs. Pre-association quality control filtering was conducted for the raw genotyping data using PLINK 1.7 program, and genotype imputation was performed using the IMPUTE2 algorithm with multiple population reference data from 1000 Genome Project. The imputed data with ∼3.1 million variants was used to test the association with non-responders to TJR using the additive genetic model. Eighty three primary OA patients (44 responders and 39 non-responders) were included in the analysis. Association analysis detected three chromosomal regions on chr5, 7, and 8 to be significantly associated with non-responding to pain. The top SNPs at these loci are intergenic variants that include SNP (rs17118094, p=4.4×10-5) on chr5. This SNP is adjacent to SGCD gene that plays an important role in muscular strength and maintenance. Another associated SNP (rs71572810, p=4.7×10-5) is nearby IMMP2L gene on chr7. This gene is reported to be associated with behavioral abnormalities. Finally, SNP (rs6992938, p=5.8×10-5) on chr8 is located downstream of TRPA1 gene that is known to have a central role in the pain response to endogenous inflammatory mediators. Three loci were also found to be significantly associated with non-responding to function. The lead variant in the locus on chr1 is an intergenic SNP (rs9729377, p=1.7×10-5) falling between CTBS and MCOLN2 genes. CTBS gene is associated with TNF-α, a cytokine that stimulate the inflammation acute phase reaction, and MCOLN2 gene plays a role in the chemokine secretion and macrophage migration in the innate immune response. Other top SNPs in loci on chr2 and 10 harbor CCDC93, INSIG2, and KLF6 genes that are associated with heel bone mineral density, hypercholesterolemia, obesity and BMI. To our knowledge, this project is the first study that investigated the association between genetic factors and TJR non-responders. Our results demonstrated that genes related to muscle strength, behavioral trait, pain response, and inflammation play a significant role in poor outcome of TJR, warranting further investigation

Aims. Bacterial infection activates neutrophils to release neutrophil extracellular traps (NETs) in bacterial biofilms of periprosthetic joint infections (PJIs). The aim of this study was to evaluate the increase in NET activation and release (NETosis) and haemostasis markers in the plasma of patients with PJI, to evaluate whether such plasma induces the activation of neutrophils, to ascertain whether increased NETosis is also mediated by reduced DNaseI activity, to explore novel therapeutic interventions for NETosis in PJI in vitro, and to evaluate the potential diagnostic use of these markers. Methods. We prospectively recruited 107 patients in the preoperative period of prosthetic surgery, 71 with a suspicion of PJI and 36 who underwent arthroplasty for non-septic indications as controls, and obtained citrated plasma. PJI was confirmed in 50 patients. We measured NET markers, inflammation markers, DNaseI activity, haemostatic markers, and the thrombin generation test (TGT). We analyzed the ability of plasma from confirmed PJI and controls to induce NETosis and to degrade in vitro-generated NETs, and explored the therapeutic restoration of the impairment to degrade NETs of PJI plasma with recombinant human DNaseI. Finally, we assessed the contribution of these markers to the diagnosis of PJI. Results. Patients with confirmed PJI had significantly increased levels of NET markers (cfDNA (p < 0.001), calprotectin (p < 0.001), and neutrophil elastase (p = 0.022)) and inflammation markers (IL-6; p < 0.001) in plasma. Moreover, the plasma of patients with PJI induced significantly more neutrophil activation than the plasma of the controls (p < 0.001) independently of tumour necrosis factor alpha. Patients with PJI also had a reduced DNaseI activity in plasma (p < 0.001), leading to a significantly impaired degradation of NETs (p < 0.001). This could be therapeutically restored with recombinant human DNaseI to the level in the controls. We developed a model to improve the diagnosis of PJI with cfDNA, calprotectin, and the start tail of TGT as predictors, though cfDNA alone achieved a good prediction and is simpler to measure. Conclusion. We confirmed that patients with PJI have an increased level of NETosis in plasma. Their plasma both induced NET release and had an impaired ability to degrade NETs mediated by a reduced DNaseI activity. This can be therapeutically restored in vitro with the approved Dornase alfa, Pulmozyme, which may allow novel methods of treatment. A combination of NETs and haemostatic biomarkers could improve the diagnosis of PJI, especially those patients in whom this diagnosis is uncertain. Cite this article: Bone Joint J 2024;106-B(9):1021–1030

Objective. The optimal dosage and timing of tranexamic acid in total hip arthroplasty (THA) still is undetermined. Previous studies showed the hyper-fibrinolysis would last for 18 hours after surgery. The study aimed to examine the efficacy and safety of multiple bolus of intravenous TXA on hidden blood loss and inflammation response following primary THA. Methods. 150 patients were randomly divided into three groups to receive single bolus of 20 mg/kg IV-TXA before skin incision (Group A), or another bolus of 1 g IV-TXA 3 hours later (Group B), or another two boluses of 1g IV-TXA 3 hours and 6 hours later (Group C). All patients received a standard perioperative enhanced recovery protocol. The primary outcomes was hidden blood loss. Other outcome measurements such as hemoglobin level, total blood loss, transfusion rate, inflammation markers (CRP, IL-6), VAS pain score, length of hospital stay (LOH) and venous thromboembolism (VTE) were also compared. Results. The hidden blood loss in group C was 402.13 ± 225.97 ml, which was less than that in group A (679.28±277.16 ml, p< 0.001) and group B (560.62±295.22 ml, p= 0.010). However, such difference was not detected between group A and B (p= 0.072). The mean value of total blood loss in group A, B and C were 1090.78±251.41, 979.42±247.89, 768.71±180.19 ml, respectively, with a significant intergroup difference (p <0.001). The Hb drop on postoperative day (POD) 3 in group A, B and C was 30.82±6.31, 27.16±6.83, 21.98±3.72 g/L, and the difference between groups was significant (p <0.001). Only one patients received red blood cell transfusion. The mean level of CRP in group C was lower than that in group A and B on POD 2 (p= 0.000, p= 0.034), POD 3 (p= 0.000, p= 0.014). The serum level of IL-6 in group C was lower than group A on POD 1, POD 2 and POD 3 (p=0.017, p=0.023, p= 0.005; respectively). The patients in group C had slightly less postoperative pain. The LOH in group C was shorter than those in group A (p= 0.023). No episodes of VTE or other adverse events occurred in any patient. Conclusion. Multiple boluses of IV-TXA can effectively reduce hidden blood loss following primary THA. What is the most important is that, by adding another boluses of IV-TXA, patients can gain a smaller decline of Hb, less postoperative inflammation response, less pain and shorter length of hospital stay

Purpose. There is controversial whether synovectomy must be done in primary total knee arthroplasty (TKA). The objectivity of the study was to compare the effect of synovectomy on inflammation and clinical outcomes after surgical treatment of knee osteoarthritis. Methods. A total of 240 patients who underwent primary unilateral TKR were randomly divided into a group without (Group A) and with synovectomy (Group B). All operations were performed by the same surgeon and follow-up was for 4 year. Clinical outcomes (including American Knee Society score (AKS), SF-36, and HSS scores) serum inflammatory markers (including interleukin-6 (IL-6), CRP and ESR) and the difference in temperature of the affected knee skin, swelling, ROM, patients VAS satisfaction score and VAS pain score were sequentially evaluated until 4 years after surgery. Result. There were no statistically different clinical parameters between the two groups preoperatively. At the 4 years follow-up, both groups had a similarly significantly improved AKS clinical and functional score. Similar changes in serial inflammatory markers were identified in both groups. In addition, no difference was seen regarding drainage-fluid inflammatory markers at any follow-up time. There was no difference in respect to patients satisfaction score from surgery to 1 year, but Group B showed greater patients satisfaction score from 2 year to four year, with less number of patients suffering from anterior pain. There was no difference with regard to other parameters at any follow-up time. Conclusions. Synovectomy in primary TKA does not seem to have any clinical advantage and shorten the duration of the inflammatory response, but it might increase patient satisfaction score and reduce anterior knee pain

Background. The anterior approach for total hip arthroplasty has recently been hypothesized to result in less muscle damage. While clinical outcome studies are essential, they are subject to patient and surgeon bias. We prospectively analyzed biochemical markers of muscle damage and inflammation in patients receiving anterior and posterior minimally-invasive total hip arthroplasty to provide objective evidence of the surgical insult. Methods. 29 patients receiving an anterior and 28 patients receiving a posterior total hip arthroplasty were analyzed. Peri-operative and radiographic data were collected to ensure similar cohorts. Creatine kinase, C-reactive protein, Interleukin-6, Interleukin-1beta, and Tumor necrosis factor-alpha were collected pre-operatively, post-operatively, and on post-operative days 1 and 2. Comparisons between the groups were made using the Student's t-test and Fisher's Exact test. Independent predictors of elevation in markers of inflammation and muscle damage were determined using multivariate logistic regression analysis. Results. Markers of inflammation were slightly decreased in direct anterior group (mean differences in C-reactive protein 27.5 [95% confidence interval −24.7–79.6] mg/dL, Interleukin-6 13.5 [95% confidence interval −11.5–38.4] pg/ml, Interleukin-1beta 42.6 [95% confidence interval −10.4–95.6], and Tumor necrosis factor-alpha 148.6 [95% confidence interval −69.3–366.6] pg/ml). The rise in creatine kinase was 5.5 times higher in the post anesthesia care unit (mean difference 150.3 [95% confidence interval 70.4–230.2] units/L, p < 0.05) and nearly twice as high cumulatively in the miniposterior approach group (305.0 [95% confidence interval −46.7–656.8] units/L, p < 0.05). Conclusion. Anterior total hip arthroplasty caused significantly less muscle damage compared to traditional posterior surgery as indicated by creatine kinase levels. The clinical importance of this rise needs to be delineated by further clinical studies. The overall physiologic burden as measured by markers of inflammation, however, appears to be similar. Objective measurement of muscle damage and inflammation provides an unbiased way of determining the immediate effects of surgical intervention in total hip arthroplasty patients

The cellular mechanisms of tendinopathy remain unclear, particularly with respect to the role of inflammation in early disease. We have previously identified increased levels of inflammatory cytokines in an early human model of tendinopathy and sought to extend these studies to the cellular analysis of tissue. Purpose. To characterise inflammatory cell subtypes in early human tendinopathy we explored the phenotype and quantification of inflammatory cells in torn and control tendon samples. Design. Controlled laboratory study. Methods. Torn supraspinatus tendon and matched intact subscapularis tendon samples were collected from twenty patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from ten patients undergoing arthroscopic stabilisation surgery. Tendon biopsies were evaluated immunohistochemically by quantifying the presence of macrophages (CD68 and CD206), T cells (CD3), mast cells (Mast cell tryptase) and vascular endothelium (CD34). Results. Subscapularis tendon biopsies obtained from patients with torn supraspinatus tendon exhibited significantly greater macrophage, mast cell and T cell expression compared to either torn supraspinatus samples or control subscapularis derived tissue (p< 0.01). Inflammatory cell infiltrate correlated inversely (r=0.5, p< 0.01) with rotator cuff tear size, with larger tears correlating with a marked reduction in all cell lineages. There was a modest but significant correlation between mast cells and CD 34 expression (r= 0.4, p< 0.01) in pre-rupture subscapularis tendon. Conclusion. We provide evidence for an inflammatory cell infiltrate in early mild/moderate human supraspinatus tendinopathy. In particular, we demonstrate significant infiltration of mast cells and macrophages suggesting a role for innate immune pathways in the events that mediate early tendinopathy. Further mechanistic studies to evaluate the net contribution and hence therapeutic utlity of these cellular lineages and their downstream processes may reveal novel therapeutic approaches to the management of early tendinopathy

Aim. Osteomyelitis (OM) is a debilitating infection of the bone that originates from hematogenous spreading of microbes or contamination after surgery/fracture. OM is mainly caused by the opportunistic bacterium Staphylococcus aureus (SA), which can evade the host immune response, acquire antibiotic resistance and chronically colonize the musculoskeletal tissue . 1,2. , yet the underlying molecular and cellular processes are largely unclear. This study aimed to characterize the pathogenetic mechanisms of SA-OM with a focus on the long pentraxin 3 (PTX3), a soluble pattern recognition molecule and bone tissue component that is emerging as a new player in osteoimmunology . 3. and a diagnostic marker of periprosthetic joint infections, a common form of OM. 4. . Method. A murine model of OM based on intra-bone injection of SA was developed that closely mimicked surgery/trauma-related OM in humans and allowed addressing the role of PTX3 in gene-modified (Ptx3-/-) animals. Local and systemic infection and inflammation were assessed via microbiology, flow cytometry, histochemistry and microCT techniques. Results. SA-injected mice developed chronic infection with measurable levels of viable bone-resident bacteria up until 30 days from microbial challenge. The infection was confined to the treated limbs only and accompanied by extensive tissue remodelling. The bacterial load was higher in WT than Ptx3. -/-. animals at 6 and 14 days from SA injection. Accordingly, WT mice had enhanced systemic inflammation with expanded innate immune compartment in the spleen and increased serum levels of inflammatory cytokines and chemokines. PTX3 levels were higher in SA- than vehicle (PBS)-injected WT animals both in the serum and bone tissue. Furthermore, administration of a PTX3-targeting antibody reduced the bacterial burden in the bones of SA-injected WT mice. Conclusions. In a mouse model of SA-OM, genetic deficiency of PTX3 protected from infection and inflammation, pointing to this pentraxin as a crucial player in OM pathogenesis and a novel therapeutic target in bone infections. The study was approved by the Italian Ministry of Health (approval n. 520/2019-PR issued on 19/07/2019) and supported by Fondazione Beppe and Nuccy Angiolini

Osteoarthritis (OA) is a disease of the synovial joint with synovial inflammation, capsular contracture, articular cartilage degradation, subchondral sclerosis and osteophyte formation contributing to pain and disability. Transcriptomic datasets have identified genetic loci in hip and knee OA demonstrating joint specificity. A limited number of studies have directly investigated transcriptional changes in shoulder OA. Further, gene expression patterns of periarticular tissues in OA have not been thoroughly investigated. This prospective case control series details transcriptomic expression of shoulder OA by analysing periarticular tissues in patients undergoing shoulder replacement for OA as correlated with a validated patient reported outcome measure of shoulder function, an increasing (clinically worsening) QuickDASH score. We then compared transcriptomic expression profiles in capsular tissue biopsies from the OA group (N=6) as compared to patients undergoing shoulder stabilisation for recurrent instability (the control group, N=26). Results indicated that top ranked genes associated with increasing QuickDASH score across all tissues involved inflammation and response to stress, namely interleukins, chemokines, complement components, nuclear response factors and immediate early response genes. Some of these genes were upregulated, and some downregulated, suggestive of a state of flux between inflammatory and anti-inflammatory signalling pathways. We have also described gene expression pathways in shoulder OA not previously identified in hip and knee OA, as well as novel genes involved in shoulder OA

The poor prognosis of patients with soft-tissue sarcoma as not changed in the past several decades, highlighting the necessity for new therapeutic approaches. T-cell based immunotherapies are a promising alternative to traditional cancer treatments due to their ability to target only malignant cells, leaving benign cells unharmed. The development of successful immunotherapy requires the identification and characterization of targetable immunogenic tumor antigens. Cancer-testis antigens (CTA) are a group of highly immunogenic tumor-associated proteins that have emerged as potential targets for CD8+ T-cell recognition. In addition to identifying a targetable antigen, it is crucial to understand the tumor immune microenvironment. The level of immune infiltration and mechanisms of immune suppression within the tumor play important roles in the outcome of immunotherapy. The goal of this study is to identify targetable immunogenic antigens for T-cell based immunotherapy and to characterize the tumor immune microenvironment in human dedifferentiated liposarcoma (DDLS) by Nanostring and IHC. To assess the complexity of the human DDLS tumor immune microenvironment and to identify target antigens we used the nCounter NanoString platform to generate a gene expression profile for hundreds of genes from RNA obtained from 29 DDLS and 10 control fat FFPE samples. To classify inflammatory status of DDLS tumors, we performed hierarchical clustering based on expression levels of selected tumor inflammatory signature genes (CCL5, CD27, CD274, CD276, CD8A, CMKLR1, CXCL9, CXCR6, HLA-DQA1, HLA-E, IDO1, LAG3, PDCDILG2, PSMB10, STAT1, TIGIT). To confirm protein expression and distribution of identified antigens, we performed immunohistochemistry on human tissue micro-arrays encompassing DDLPS tumor tissues and matched normal control tissue from 63 patients. IHC for the cancer testis antigens PBK, SPA17, MAGE-A3, NY-ESO-1 and SSX2 was performed, and the staining results were scored by two authors based on maximal staining intensity on a scale of zero to three (absent=0, weak=1, moderate=2, or strong=3) and the percentage of tumor cells that stained. Hierarchical clustering of DDLS tumors based on expression of tumor inflammation signature genes revealed two distinct groups, consisting of 15 inflamed tumor and 14 non-inflamed tumors, demonstrating tumor heterogeneity within the DDLS sarcoma subtype. All antigens were found to be expressed in DDLS at an mRNA level. SPA17 was expressed at the highest levels in DDLS, however, this antigen was expressed at high levels in normal fat. Notably, antigens PBK and TTK had the largest fold change increase in expression in DDLS compared to normal fat controls. Immunohistochemical analysis of selected antigens revealed that PBK was found to be expressed in 96% (52/54) of DDLS samples at high levels. Other antigens were absent or expressed at low levels in DDLS; MAGEA3 in 15.87% (10/63) NY-ESO-1 in 6.35% (4/62) and SSX2 in 12.7% (8/63) and SPA17 in 5.5% (3/54). This data shows considerable inter-tumoral heterogeneity of inflammation, which should be taken into consideration when designing an immunotherapy for DDLS. To date, these results show promising expression of PBK antigen in DDLS, which may be used as a target in the future development of an immunotherapy for sarcoma

Aim. The diagnosis of prosthetic joint infection (PJI) is challenging and relies on a combination of parameters. However, the currently recommended diagnostic algorithms have not been validated for patients with recent surgery, dislocation or other events associated with a local inflammatory response. As a result, these algorithms are not safely applicable offhand in such conditions. Calprotectin is a leukocyte protein that has been shown to be a reliable biomarker of PJI. The purpose of this study was to evaluate the use of calprotectin to rule out PJI within 3 months after surgery or dislocation. Method. We included patients who underwent arthroplasty revision surgery at our institution within 3 months after any event causing inflammation. Calprotectin was measured using a lateral-flow assay. European Bone and Joint Infection Society (EBJIS) criteria were used as gold standard. The diagnostic accuracy of calprotectin was calculated. Results. Twenty-two patients (14 females, 8 males) with a mean age of 65.1 ± 12.3 years with 13 total hip (THA) and 9 total knee arthroplasties (TKA) were included. There were 4 instances of possible early-onset acute infection, 4 dislocations, 2 patella tendon ruptures, 1 local tissue reaction to the sutures, 4 cases of early loosening, 2 component breakages and 1 avulsion of a polyethylene patella button. Using the EBJIS criteria, PJI was confirmed postoperatively in 12 cases. With a cut-off at 50mg/L, the calprotectin lateral flow test was positive in 10 cases. This results in a sensitivity of the calprotectin test of 0.75, a specificity of 0.9, positive and negative predictive values of 0.9 and 0.75, respectively, and a positive and negative likelihood ratio of 7.5 and 0.28, respectively. Conclusions. Aggravating the difficulties of ruling out PJI prior to revision surgery, local inflammation can be caused by some conditions in which the widely accepted PJI definition criteria cannot be applied. Nevertheless, an accurate diagnosis of PJI is just as crucial in these situations as it is in planned revision surgery. This study suggests that calprotectin is a promising diagnostic parameter for ruling out PJI in such cases. The calprotectin lateral-flow assay is readily applicable at the beginning of the procedure, yielding results that can assist in the decision whether to perform septic revision or aseptic partial or component exchange within 15 minutes, and with an overall accuracy of 81.8%

The incidence of PJI in knee replacements is 2.8% and slightly lower with hip replacement surgery. PJI make up 15% (or even more) of knee revisions. To combat PJI, antibiotic laden bone cement has been used for many decades, but antibiotic stewardship dictates more prudent management of antimicrobials. Projected increase in infection rate, due to increased surgery and latent infection to be almost 5-fold up to 2035. Biofilm is a complex structure of bacteria and polysaccharide matrix and, is recognised as a major component in PJI and other orthopaedic infections. Biofilm is responsible for high incidence of resistance to antimicrobials and ineffective host immune response. Method. Stabilized hypochlorous acid has been reported to have a rapid kill rate on all pathogens, including MDR pathogens associated with chronic and acute wound infections. It destroys biofilm on contact, is not cytotoxic, reduces inflammation and stimulates wound healing. 0,038% of Hypochlorous acid was used as prophylaxis against infection and to treat PJI. We report on our experience with hypochlorous acid as a wound irrigation as prophylaxis against infection (more than 600 cases) and for PJI. We also report on a University study where a head to head analysis was done on the anti-biofilm efficacy between hypochlorous acid 0,038% (Trifectiv Surgical Wound Irrigation) and Product X (an industry-standard product for the prevention and treatment of biofilm infection. Hypochlorous acid offers a valuable addition to the armamentarium of wound antiseptics, with added anti-inflammatory value. An in vitro study demonstrated superior efficacy against biofilm when compared to Product X

Aim. Treatment recommendations for periprosthetic joint infections (PJI) include surgical debridement, antibiotic therapy or staged revision. In surgical related foot and ankle infections (SR-FAI), implant removal will lead to instability. Debridement is difficult because the implant is outside the joint. Recommendations regarding PJI treatment can therefore not be extrapolated to the treatment of SR-FAI. Method. We searched PubMed for the etiology and treatment of SR-FAI, taken into account the time of occurrence, causative microorganisms and surgical treatment options. We integrated this knowledge into a treatment algorithm for SR-FAI. Results. Within the first 6 weeks after surgery, it is difficult to distinguish acute osteomyelitis from surgical site infection in which infection is limited to the soft tissue. The predominantly causative microorganism is Staphylococcus aureus. No debridement can be performed, because of the diffuse soft tissue inflammation and the absence of a joint space. If early SR- FAI is suspected without signs of systemic symptoms, fistula or abscess, empirical antibiotic treatment covering Staphylococcus aureus is recommended. If there is suspicion of ongoing SR-FAI after 2 weeks of empirical treatment, samples for culture after an antibiotic free window should be obtained to identify the causative microorganisms. If SR-FAI is confirmed, but there is no consolidation yet, targeted antibiotic treatment is given for 12 weeks without initial implant removal. In all other cases, debridement and samples for culture should be obtained after an antibiotic free window. Staged revision surgery will be performed if there is still a nonunion. Conclusions. Treatment algorithm regarding PJI cannot be extrapolated to the treatment of SR-FAI. Until now, no treatment guideline for SR-FAI is available. We have introduced a treatment algorithm for the treatment of SR-FAI. The guideline will be validated during the next 2 years

Specific and rapid detection methods for spinal tuberculosis, with sufficient sensitivity in HIV-1 co-infected individuals, are needed, to ensure early initiation of appropriate treatment to prevent physical disability and neurological fallout. In addition, understanding the systemic and local pathophysiology of spinal tuberculosis, and its interaction with HIV-1 infection, is crucial to guide future therapeutic interventions. We prospectively enrolled adult patients presenting with signs and symptoms of suspected spinal tuberculosis, at Groote Schuur Hospital, between November 2020 and December 2021. TB diagnostic testing was performed on open and CT-guided spinal biopsies using Xpert MTB/RIF Ultra compared to gold standards TB culture and histology. A highly sensitive droplet digital PCR assay for detecting and quantifying Mycobacterium tuberculosis complex (MTBC) and HIV-1 DNA was tested. Plasma inflammatory proteins were measured to assess systemic inflammation. Xpert Ultra had a high sensitivity of 94.7% and specificity of 100% for STB against TB culture and histology in both open and CT-guided biopsy samples. The ddPCR assay confirmed TB detection in 94% of patients with positive Xpert Ultra results. Four patients with negative TB diagnostic results had MTBC DNA detected by ddPCR. HIV-1 DNA was detected in the spinal tissues from all HIV-1-infected patients. MTBC DNA levels were significantly higher in HIV-1-co-infected spinal tissue samples (p< 0.01). We identified four biomarkers significantly associated with higher bacterial burden at the disease site (p< 0.01). Xpert Ultra and MTBC ddPCR improve the detection of STB. DdPCR can be utilized as an additional, highly sensitive tool for detecting and quantifying Mtb, in pathological samples that may be paucibacillary. These findings provide novel diagnostic and pathophysiologic insight into STB, in the context of HIV-1 infection, and provide rationale to include these tests in hospital and research settings for patients from communities burdened by TB and HIV-1

Prosthetic joint infection (PJI) is a complex disease that causes significant damage to the peri-implant tissue. Developing an animal model that is clinically relevant in depicting this disease process is an important step towards developing novel successful therapies. In this study, we have performed a thorough histologic analysis of peri-implant tissue harvested post Staphylococcus aureus (S. aureus) infection of a cemented 3D-printed titanium hip implant in rats. Sprague-Dawley rats underwent left hip cemented 3D-printed titanium hemiarthroplasty via posterior approach under general anesthesia. Four surgeries were performed for the control group and another four for the infected group. The hip joint was inoculated with 5×10. 9. CFU/mL of S. aureus Xen36 prior to capsule closure. The animals were scarified 3 weeks after infection. The femur was harvested and underwent micro-CT and histologic analysis. Hematoxylin and eosin (H&E), as well as Masson's trichrome (MT) stains were performed. Immunohistochemistry (IHC) using rabbit antibody for S. aureus was also used to localize bacterial presence within femur and acetabulum tissue . The histologic analysis revealed strong resemblance to tissue changes in the clinical setting of chronic PJI. IHC demonstrated the extent of bacterial spread within the peri-implant tissue away from the site of infection. The H&E and MT stains showed 5 main features in infected bone: 1) increased PMNs, 2) fibrovascular inflammation, 3) bone necrosis, and 4) increased osteoclasts 5) fibrosis of muscular tissue and cartilage. Micro CT data showed significantly more osteolysis present around the infected prosthesis compared to control (surgery with no infection). This is the first clinically relevant PJI animal model with detailed histologic analysis that strongly resembles the clinical tissue pathology of chronic PJI. This model can provide a better understanding of how various PJI therapies can halt or reverse peri-implant tissue damage caused by infection

Cryocompression therapy is a non-invasive and non-pharmacological modality used in managing acute post-operative inflammation and pain. A prospective, randomised controlled trial (RCT) was undertaken to evaluate the effectiveness of a post-operative cryocompression protocol using the Game Ready™ (GR) device versus usual care on recovery following total knee arthroplasty (TKA). A single centre RCT was conducted with 70 TKAs (68 patients) randomised to a 2-week intervention period consisting of treatment with GR cryocompression (n=33, 33.3% males) or a usual care protocol of ice with static compression using tubigrip (n=35, 54.3% males). Knee range of movement (ROM) (flexion and extension), a visual analogue pain score (VAS) and limb circumference were documented at day 1, 2 and 14, as well as 6 and 12 weeks post-surgery. ROM was also recorded at day 90, while medication use and length of hospital stay were documented. Patient reported outcome measures (PROMs) including the KOOS and patient satisfaction questionnaire were employed. The GR group demonstrated 2.3° more (p=0.05) knee extension ROM overall, as well as 2. 8° more at day 1 (p=0.048), 3.8° at day 14 (p=0.007) and 5.4° at 3 months (p=0.017). There were no group differences (p>0.05) observed in pain (VAS), flexion ROM, limb circumference, opioid use or other PROMs. Across the full cohort, higher pain levels resulted in increased opioid intake (p=0.002), older patients used significantly less opioids (p<0.001) and males reported significantly less pain (VAS) than females (p=0.048). Using GR following TKA is a safe, non-invasive tool that can be used to aid in the post-operative recovery period. Patients using the GR cryocompression device gained significantly more extension ROM compared to the conventional ice with compression group, despite no other group differences

Aim. The use of medical devices has grown significantly over the last decades, and has become a major part of modern medicine and our daily life. Infection of implanted medical devices (biomaterials), like titanium orthopaedic implants, can have disastrous consequences, including removal of the device. For still not well understood reasons, the presence of a foreign body strongly increases susceptibility to infection. These so-called biomaterial-associated infections (BAI) are mainly caused by Staphylococcus aureus and Staphylococcus epidermidis. Formation of biofilms on the biomaterial surface is generally considered the main reason for these persistent infections, although bacteria may also enter the surrounding tissue and become internalized within host cells. To prevent biofilm formation using a non-antibiotic based strategy, we aimed to develop a novel permanently fixed antimicrobial coating for titanium devices based on stable immobilized quaternary ammonium compounds (QACs). Method. Medical grade titanium implants (10×4×1 mm) were dip-coated in a solution of 10% (w/v) hyperbranched polymer, subsequently in a solution of 30% (w/v) polyethyleneimine and 10 mM sodium iodide, using a dip-coater, followed by a washing step for 10 min in ethanol. The QAC-coating was characterized using water contact angle measurements, scanning electron microscopy, FTIR, AFM and XPS. The antimicrobial activity of the coating was evaluated against S. aureus strain JAR060131 and S. epidermidis strain ATCC 12228 using the JIS Z 2801:2000 surface microbicidal assay. Lastly, we assessed the in vivo antimicrobial activity in a mouse subcutaneous implant infection model with S. aureus administered locally on the QAC-coated implants prior to implantation to mimic contamination during surgery. Results. Detailed material characterization of the titanium samples showed the presence of a homogenous and stable coating layer at the titanium surface. Moreover, the coating successfully killed S. aureus and S. epidermidis in vitro. The QAC-coating strongly reduced S. aureus colonization of the implant surface as well as of the surrounding tissue, with no apparent macroscopic signs of toxicity or inflammation in the peri-implant tissue at 1 and 4 days after implantation. Conclusions. An antimicrobial coating with stable quaternary ammonium compounds on titanium has been developed which holds promise to prevent BAI. Non-antibiotic-based antimicrobial coatings have great significance in guiding the design of novel antimicrobial coatings in the present, post-antibiotic era

Aim. To describe the histopathology of the first and last debrided bone tissue in chronic osteomyelitis and answer the following research question; is the last debrided bone tissue viable and without signs of inflammation?. Method. In total, 15 patients with chronic osteomyelitis were allocated to surgical treatment using a one stage protocol including extensive debridement. Suspected infected bone tissue eradicated early in the debridement procedure was collected as a clearly infected sample (S1). Likewise, the last eradicated bone tissue was collected as a suspected non-infected sample (S2), representing the status of the bone void. In all cases, the surgeon debrided the bone until visual confirmation of healthy bleeding bone. The samples were processed for histology, i.e. decalcification and paraffin embedding, followed by cutting and staining with Haematoxylin and Eosin. Immunohistochemistry with MAC-387 antibodies towards the calprotectin of neutrophil granulocytes (NGs) was also performed and used for estimation of a neutrophil granulocyte (NG) score (0, 1, 2 or 3), by the method described for fracture related infections (1). Results. For the S1 samples the median NG score was 3 which is considered confirmatory for infection. However, following debridement the median NG score was significantly (p = 0.032) reduced to 2. Often NGs were seen as single cells, but in seven S1 samples and in one S2 sample massive NG accumulations were observed. The S1 samples showed a mix of granulation tissue, fibrosis, viable bone, and bone necrosis. The S2 samples contained viable bone tissue and occasionally (10/15) small fragments of necrotic bone or bone debris were seen. Furthermore, a large number of erythrocytes were observed in most S2 samples. Conclusions. The present study shows that the inflammatory response still existents after debridement, although the response fades from the center of infection. Therefore, sampling of debrided bone tissue for histology must be performed initially during surgery, to avoid underestimation of the inflammatory response, i.e. the NG score. The last debrided bone tissue cannot by definition be considered completely viable and caution should be made to remove blood (rinse) before intraoperative evaluation of the viability of debrided cancellous bone. Remnant necrotic bone fragments or debris could represent low-vascular hiding places for leftover bacteria. Application of local antibiotics might have a central role in clearing of these small non-viable bone pieces at the bone void interface

Aim. To develop a new system for antibacterial coating of joint prosthesis and osteosynthesis material. The new coating system was designed to release gentamicin immediately after insertion to eradicate surgical contamination. Method. Steel implants (2×15mm) were coated with a solid nanocomposite xerogel made from silica and the dendritic polymer, hyperbranched polyethyleneimine. The xerogel was anchored inside a porous surface made by pre-coating with titanium microspheres. Finally, gentamicin was encapsulated in the xerogel, i.e. no chemical binding. A total of 50 µg gentamicin was captured into each implant. The efficacy of the new coating was evaluated in a porcine model of implant associated osteomyelitis. In total, 30 female pigs were randomized into 3 study groups (n=10). Group A; plain implants + saline, Group B; plain implants + 10. 4. CFU of Staphylococcus aureus, and Group C; coated implants + 10. 4. CFU of S. aureus. Implant + inoculum was placed into a pre-drilled implant cavity of the right tibia and the pig was euthanized 5 days afterwards. Postmortem microbiology and pathology were performed. Two additional pigs were used in a pharmacokinetic study where microdialysis (MD) catheters were placed alongside coated implants. Extracellular fluid was sampled regularly for 24 hours from the MD catheters and analyzed for gentamicin content. Results. Within Groups A and C, all implants were found sterile by sonication and bacteria could not be identified within the surrounding bone tissue. In contrast, all Group B animals had S. aureus positive implant and tissue microbiology. Macroscopic and microscopic pathological examinations confirmed that Group A and C animals were complete identic, i.e. no pus around implants and only minor peri-implant inflammation related to insertion of implants per se. All Group B animals had pus around their implants and a massive peri-implant inflammatory response dominated by neutrophil granulocytes. Maximum gentamicin release (35 µg /mL) was measured in the first obtained MD sample, i.e. after 30 min, and the concentration stayed above the MIC level for the used S. aureus strain for 8 hours. Conclusions. The new xerogel coating prevented development of osteomyelitis. Prevention was due to a fast gentamicin release immediately following insertion and antimicrobial active concentrations were detectable several hours after implantation. This means that the critical time point of most relevant surgical procedures potentially could be protected by the novel coating. The new coating will be investigated on larger scale implants and full-size prosthesis in the future

Aim. To make an inoculum for induction of Implant-Associated Osteomyelitis (IAO) in pigs based on bacterial aggregates resembling those found on the human skin, i.e. aggregates of 5–15 µm with low metabolic activity. The aggregates were evaluated and compared to a standard planktonic bacterial inoculum. Method. The porcine Staphylococcus aureus strain S54F9 was cultured in Tryptone Soya Broth for seven days. Subsequently, the culture was filtered through cell strainers with pore sizes of 15 µm and 5 µm, respectively. The fraction of 5–15 µm aggregates in the top of the 5 µm filter was collected as the aggregate-inoculum. The separation of aggregates into different size fractions was evaluated by light microscopy. The metabolism of the aggregate-inoculum and a standard overnight planktonic inoculum was evaluated with isothermal microcalorimetry. In total, six female minipigs were allocated into three groups (n=2), receiving different inoculums. Group A: overnight planktonic inoculum; 10. 4. CFU S. aureus (S54F9), Group B: seven days old 5–15 µm aggregate-inoculum; 10. 4. CFU S. aureus (S54F9), Group C: saline. All inoculums were placed in a pre-drilled implant cavity in the right tibia of the pig and a sterile stainless-steel implant was inserted. The pigs were euthanized seven days after surgery. Postmortem macroscopic pathology, microbiology, computed tomography and histopathology were performed. Results. The separation of aggregates into different size fractions was done successfully by the filtering method. Isothermal microcalorimetry showed, a delayed Time-to-peak metabolic activity of the aggregate-inoculum compared to the planktonic inoculum. S. aureus was isolated from subcutis, bone and implants from all animals in groups A and B. Both group A animals showed osteomyelitis at gross inspection with suppuration and sequestration, while groups B and C animals had no macroscopic lesions. From CT scans, both group A animals also showed positive signs of osteomyelitis, i.e., osteolysis, while only one animal in group B did, and none in group C. Histopathological examination of the bones showed more extensive inflammation in group A animals compared to those in group B, which showed more osteoid formation. Conclusions. Formation and separation of low metabolism bacterial aggregates into different size fractions was possible. The aggregates can be used as inoculum in the porcine IAO model, with microbiological re-isolation from both implants and tissue. Furthermore, the aggregates caused a less aggressive IAO, than the planktonic counterparts. Using aggregated bacteria as inoculum appears to be more relevant to the clinical situation of infecting bacteria