Undifferentiated pleomorphic sarcoma (UPS) is one of the most common and aggressive adult soft tissue sarcomas (STS). Once metastatic, UPS is rapidly fatal. Most STS, including UPS, are resistant to conventional immunotherapies as these tumours have low numbers of spontaneous tumour infiltrating lymphocytes (TILs) and are densely populated with immune suppressive macrophages. Intra-tumoural activation of the STimulator of INterferon Genes (STING) pathway is a novel immunotherapeutic strategy to recruit anti-tumour TILs into the tumour microenvironment. In a murine model of UPS, we have demonstrated that intra-tumoural injection of a murine-specific STING agonist, DMXAA, results in profound immune mediated tumour clearance. Recently, molecules capable of activating both human and mouse STING pathways have been developed. In pursuit of clinically relevant therapeutic opportunities, the purpose of this study is to evaluate the anti-tumour potential of two agonists of the human and murine STING receptors: ADU-S100 and MSA-2 as monotherapies and in combination with the immune checkpoint inhibitor, anti-PD1 in a murine model of UPS. Immune competent mice were engrafted with murine UPS cells in the hindlimb muscle. Once palpable, mice in the monotherapy group were treated with a single intra-tumoural dose of 1) ADU-S100 or 2) MSA-2 or 3) DMXAA. In additional experimental groups, mice were treated with the different STING agonists and monoclonal anti-PD1. Tumour volume measurements and tumour bioluminescence were measured over time. To quantify dynamic changes in immune populations and in the tumour immune microenvironment, STING treated UPS tumours were evaluated using flow cytometry and mRNA quantification at various timepoints after therapy. DMXAA monotherapy produced complete tumour eradication in 50% of mice, whereas both ADU-S100 or MSA-2 monotherapy only extended survival but did not result in complete tumour clearance. Flow cytometry and transcriptional profiling of tumours at multiple timepoints post-treatment showed similar inflammatory changes and increased TILs numbers across all STING agonists. The addition of anti-PD1 treatment to STING therapy significantly extended survival times with both ADU-S100 and MSA-2, and resulted in 14% complete tumour clearance with ADU-S100. No complete survivors were observed with MSA-2-anti-PD1 combinations therapy. STING activation is a promising immunotherapeutic strategy for UPS. Recently developed human STING agonists are not as effective as DMXAA despite similar immunologic responses to treatment. STING and anti-PD-1 treatment were therapeutically synergistic for both human STING agonists. These results justify further research around STING activation as a therapeutic modality for STS. DMXAA may possess additional off-target therapeutic properties beyond STING activation which warrants further investigation. Elucidating these differences may be critical to further optimize STING therapy for human STS

The meniscus is at the cornerstone of knee joint function, imparting stability and ensuring shock absorption, load transmission, and stress distribution within the knee joint. However, it is very vulnerable to injury and age-related degeneration. Meniscal tears are reported as the most common pathology of the knee with a mean annual incidence of 66 per 100,000. Knee osteoarthritis progresses more rapidly in the absence of a functional meniscus. Historically, tears extending to the avascular inner portion of the meniscus (white-white zone, “WW”), such as radial tears were considered as untreatable and were often resected, due to the lack of vascularity in the WW zone. Perfusion-based anatomical studies performed on cadaveric menisci in the 1980s shaped the current dogma that human meniscus has poor regenerative capacity, partly due to limited blood supply that only reaches 10 to 25% of the meniscus, commonly referred to as red-red zone (“RR”). Previous studies, including those utilizing animal models have shown mobilization of Mesenchymal Stem Cells (MSCs) upon injury into the WW zone, and successful MSC recruitment when administered externally to the injury site. We and others have recently reported positive outcomes of repaired tears in the inner zone of patients. We hypothesized that the “avascular” white-white zone of the meniscus possesses regenerative capacity due to a resident stem/progenitor cell population. Further, we sought to redefine the presence of microvessels in all meniscal zones using advanced stereology and imaging modalities. Fifteen menisci from fresh human cadaveric knees (mean age: 21.53±6.53 years) without evidence of previous injury were obtained from two tissue banks (JRF, Centennial, CO) and Biosource Medical (Lakeland, FL) and utilized for this study. The use of cadaveric specimens for research purposes was approved by the institutional review board. Tibial plateaus were dissected to harvest medial and lateral menisci along their entire length. The RR, red-white (RW) and WW zones were dissected and separated into three thirds from the inner aspect to the marginal border of the meniscus and their wet weights recorded (Fig.1A). Meniscus tissue cellular content in each zone was obtained from dissociation of meniscus tissue using 0.02% w/v pronase (Millipore) for 1h at 37oC, followed by 18h 0.02% w/v collagenase II (Worthington) at 37oC with shaking. Isolated cells were characterized immediately after harvest using flow cytometry with antibodies against MSCs surface markers (CD105, CD90, CD44 and CD29) as well as respective isotype controls. Further, meniscal cells were cultured and split twice when confluence was reached, characterized at P2 and compared to bone marrow-derived MSCs (BM-MSCs) using the same markers. Self-renewal of cells was assessed using colony forming unit (CFU) assay. Differentiation assays were performed to assess whether colony-forming cells retained multilineage potential. For morphological examination of bigger vessels, samples were fixed in 10% formalin for 1 week, paraffin embedded, sectioned (4 μm thick) and stained with H&E and Masson's trichrome. Presence of microvessels was assessed by CD31 immunofluorescence staining. Further, menisci were cleared using the uDisco protocol labeled with the TO-PRO®-3 stain, a fluorescent dye that stains cell nuclei and imaged using light-sheet microscopy. All continuous data are presented as mean ±standard deviation. Non-repeated measures analysis of variance (ANOVA) and Tukey-Kramer HSD post hoc analysis were performed on sample means for continuous variables. Statistical significance was set at p < 0 .05. Menisci were successfully cleared using a modified uDISCO procedure, imaged and analyzed for total cell density. As expected, bigger vessels were observed in RR but not in WW. However, immunofluorescent staining for CD31 showed a subset of CD31+endothelial cells present in the WW zone, indicating the presence of small vessels, most likely capillaries. In order to assess whether enzymatic digestion had a differential result depending on meniscus zone due to cellular content, we analyzed yields per meniscus per zone. The wet weight of different zones (WW:RW:RR) was at a ratio of ∼1:3:5 respectively, however, the ratio of cells isolated from each zone was at ∼1:4:20, indicating that RR has a denser population of mononuclear cells. However, the difference between all zones in cell yields was not significant. The clonogenic potential of isolated cells was shown to be non-significantly different between the three zones. Differentiation of isolated cells to osteogenic lineage using osteogenic media in vitroshowed no difference between the three zones. Flow cytometry analysis of cells from the three meniscal zones displayed presence of two distinct subpopulations of cells immediately after isolation. One subpopulation was positive to MSC surface markers and the other negative. Additionally, flow cytometry of cultured meniscal cells at P2 displayed that the entire cell population was CD44+CD105+CD29+CD90+, suggesting that culturing meniscal cells results in selection of stem/progenitor cells (plastic adherence). Surface marker expression analysis showed differential expression patterns between markers depending on zone. Similar fraction of cells was detected to express both MSC markers CD90 and CD105 (7–10%) and similar fraction of cells expressed both MSC markers CD29 and CD44 (1–2%) in all three zones, indicating similar density of resident stem/progenitor cells in each zone. Importantly, WW showed significantly higher expression for all four MSC markers compared to the RR zone, indicating higher relative density of stem/progenitor resident cells in the WW zone. Our results determine that CD31-expressing microvessels were present in all zones, including the WW zone, which was previously considered completely avascular. Additionally, stem/progenitor cells were shown to be present in all three zones of the menisci, including the WW zone, showcasing its regenerative potential. For any figures or tables, please contact the authors directly

Introduction. It has been shown in vitro that human monocytes can phagocytose submicron polyethylene wear particles generated from total hip arthroplasties (THA) with highly cross-linked polyethylene inlays. The aim of our study was to detect the presence and possible phagocytosis of such particles in peripheral blood monocytes of patients with respective THA. Patients and methods. All patients were operated using the same implant, the cementless SL Plus stem; Bicon cup and a cross-linked polyethylene insert Rexpol (Smith and Nephew). Besides clinical and radiographic check-up, blood samples were collected at follow-up and analyzed by flow cytometry. Polyethylene can be identified by its auto fluorescence when stimulated by a laser with the wavelength of fluorescein isothiocyanate (FITC). Presence of wear particles in monocytes was identified by determination of their size and granularity. Some samples were scrutinized by confocal laser scanning microscopy to correlate the intracellular position of the particles. Blood samples of patients without total joint replacement served as controls. Results. 18 samples of patients with THA were compared to 18 controls. Flow cytometry didn't show any difference of size, granularity and auto fluorescence of the investigated cells between the two groups. Furthermore confocal laser scanning microscopy was unable to establish the intracellular position of the auto fluorescence. There were 11 female and 7 male patients with a mean age of 70,4 years at the time of surgery and an average body mass index of 32 (23 – 41). Average follow-up time was 6,5 years (6 – 8 years). 2 patients had been revised, one for a periprosthetic fracture postoperatively, the other for cup loosening at 5 years. Radiographically there were no signs of loosening. Conclusion. Flow cytometry and confocal laser scanning microscopy were unable to detect submicron polyethylene wear particles in human monocytes in vivo following THA. This could be due to a lack of sensitivity or/and specificity although the in vitro study showing phagocytosis of submicron particles in vitro applied the same methods. The analysis could be too early if the number of wear particles hasn't possibly reached a critical mass at 6.5 years. Potentially the conclusion of the in vitro study is inapplicable and human monocytes are unable to phagocytose polyethylene wear particles. In any case further research in this field seems necessary

Aim. Periprosthetic joint infections (PJI) are severe complications after total joint arthroplasty (TJA). Up to now, a gold standard in the diagnostics of PJI is missing. Small extracellular vesicles (sEVs) are secreted by all types of cells and play a key role in immune response in presence of infection (1). In this prospective study, the diagnostic accuracy of sEVs in the synovial fluid to detect PJI of knee, hip and shoulder joints was investigated. We hypothesized increased surface markers of sEVs in PJI compared to aseptic complications (e.g. implant loosening, stress shielding related pain). Method. Synovial fluid from 48 patients with painful arthroplasty was examined. The distinction between aseptic and infectious cases was made on the basis of the 2018 Definition of Periprosthetic Hip and Knee Infection (2). 35 (72,9%) probands assigned to aseptic and 13 patients (27,1%) to PJI group. Immuno-fluorescence flow cytometry served to document the concentrations of CD9, CD63, CD66b, CD82 and HLA-DR on sEVs. Results. The concentration of CD9 surface marker on sEVs in synovial fluid was significantly lower (p=0.002) in PJI group than in aseptic group. In contrast, the levels of CD82 on sEVs in synovial fluid was significantly higher (p<0.0001) in the PJI group than in aseptic group. The concentrations of CD63, CD66b and HLA-DR on sEVs in synovial fluid did not differ significantly between the two cohorts (CD63: p=0.372; CD66b: p=0.634; HLA-DR: p=0.558). Conclusions. Overall, the significance of sEVs in the diagnostics of PJI is not well enough understood and the subject of current research and scientific discussion. Our data suggest, that CD82 and CD9 on sEVs in synovial fluid are promising biomarkers to differentiate between PJI and aseptic complications

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

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

Injured skeletal muscle repairs spontaneously via regeneration, however, this process is often incomplete because of fibrotic tissue formation. In our study we wanted to show improved efficiency of regeneration process induced by antifibrotic agent decorin in a combination with Platelet Rich Plasma (PRP)-derived growth factors. A novel human myoblast cell (hMC) culture, defined as CD56 (NCAM)+ developed in our laboratory, was used for evaluation of potential bioactivity of PRP and decorin. To determine the their effect on the viability of hMC we performed a MTT assay. To perform the cell proliferation assay, hMCs were separately seeded on plates at a concentration of 30 viable cells per well. Cell growth medium prepared with different concentrations of PRP exudates (5%, 10%, and 20%) and decorin (10 ng/mL, 25 ng/mL, and 50 ng/mL) were added and incubated for 7 days. After incubation we stained the cells with crystal-violet and measured the absorbance. To study the expression of Transforming Growth Factor Beta (TGF-β) and myostatin (MSTN), two main fibrotic factors in the process of muscle regeneration we performed several ELISA assays in groups treated with all therapeutic agents (PRP, decorin and their combination). Further, we have studied the ability of these agents to influence the differential cascade of dormant myoblasts towards fully differentiated myotubes by monitoring step wise activation of single nuclear factors like MyoD and Myogenin via multicolor flow cytometry. We stained the cells simultaneously with antibodies against CD56, MyoD and myogenin. We acquired cell images of 5,000 events per sample at 40 x magnification using 488 nm and 658 nm lasers and fluorescence was collected using three spectral detection channels. We analysed the cells populations according to expression of single or multiple markers and their ratios. Finally, we examined the treated cell populations using a multicolour laser microscope after staining for desmin (a key marker of myogenic differentiation of hMC), α-tubulin, and nuclei. Optical images were acquired at the center of chamber slides where the cell density is at its highest using a Leica TCS SP5 II confocal microscope and analysed using Photoshop CS6, where a “Color Range” tool was used in combination with a histogram palette to count the pixels that correspond to desmin-positive areas in an image. The mitochondrial activity of cells, as determined by the MTT assay, was significantly increased (p < 0 .001) after exposure to tested concentrations of PRP exudate. Similarly, viability was elevated in all tested concentrations of decorin. PRP exudate enhanced the viability of cells to more than 400% when compared to the control (p < 0 .001). The viability of cells treated with PRP exudates was also significantly higher when compared to decorin (p < 0 .001). Decorin did not show a significant effect on cell proliferation compared to the control, however, cultivation with PRP exudate leads to a 5-fold increase in cell proliferation (p < 0 .001). Decorin was shown to down-regulate the expression of TGF-β when compared to the control by more than 15% (p < 0 .001) but significantly less than PRP exudate p < 0 .005). PRP significantly down-regulated TGF-β expression by more than 30% (p < 0 .001). Similarly, the MSTN expression levels were significantly down-regulated by decorin and PRP. MSTN levels of cells treated with decorin were decreased by 28.4% (p < 0 .001) and 23.1% by PRP (p < 0 .001) when compared to the control group. Using flow cytometry we detected a 39.1% increase in count of myogenin positive cells in the PRP-treated group compared to the control. Moreover, there was a 3.09% increase in cells positive only for myogenin, whereas no such cells were found in the control cell population. The population of cells positive only for myogenin is considered as fully differentiated and capable of fusion into myotubes as well as future mucle fibers and is thus of great importance for muscle regeneration. At the same time 20.6% fewer cells remained quiescent (positive only for CD56). Cells positive for both MyoD and myogenin represent the population that shifted significantly towards mature myocites during myogenesis but are not yet fully committed. Finally, a statistically significant up-regulation of desmin expression (p < 0 .01 for the PRP treated group, p < 0 .005 for the decorin and PRP + decorin treated groups) was present in all therapeutic groups when compared to the control. While no significant difference was found between the PRP and decorin-treated groups, their combination led to a more than 3-fold increase (p < 0 .005) of desmin expression when compared to single bioactives. PRP can be a highly potential therapeutic agent for skeletal muscle regeneration and repair, especially if in combination with a TGF-β antagonis decorin. Achieving better healing could likely result in faster return to play and lower reinjury rate

Osteoporosis accounts for a leading cause of degenerative skeletal disease in the elderly. Osteoblast dysfunction is a prominent feature of age-induced bone loss. While microRNAs regulate osteogenic cell behavior and bone mineral acquisition, however, their function to osteoblast senescence during age-mediated osteoporosis remains elusive. This study aims to utilize osteoblast-specific microRNA-29a (miR-29a) transgenic mice to characterize its role in bone cell aging and bone mass. Young (3 months old) and aged (9 months old) transgenic mice overexpressing miR-29a (miR-29aTg) driven by osteocalcin promoter and wild-type (WT) mice were bred for study. Bone mineral density, trabecular morphometry, and biomechanical properties were quantified using μCT imaging, material testing system and histomorphometry. Aged osteoblasts and senescence markers were probed using immunofluorescence, flow cytometry for apoptotic maker annexin V, and RT-PCR. Significantly decreased bone mineral density, sparse trabecular morphometry (trabecular volume, thickness, and number), and poor biomechanical properties (maximum force and breaking force) along with low miR-29a expression occurred in aged WT mice. Aging significantly upregulated the expression of senescence markers p16INK4a, p21Waf/Cip1, and p53 in osteoporotic bone in WT mice. Of note, the severity of bone mass and biomechanical strength loss, as well as bone cell senescence, was remarkably compromised in aged miR-29aTg mice. In vitro, knocking down miR-29a accelerated senescent (β-galactosidase activity and senescence markers) and apoptotic reactions (capsas3 activation and TUNEL staining), but reduced mineralized matrix accumulation in osteoblasts. Forced miR-29a expression attenuated inflammatory cytokine-induced aging process and retained osteogenic differentiation capacity. Mechanistically, miR-29a dragged osteoblast senescence through targeting 3′-untranslated region of anti-aging regulator FoxO3 to upregulate that of expression as evident from luciferase activity assessment. Low miR-29a signaling speeds up aging-induced osteoblast dysfunction and osteoporosis development. Gain of miR-29a function interrupts osteoblast senescence and shields bone tissue from age-induced osteoporosis. The robust analysis sheds light to the protective actions of miR-29a to skeletal metabolism and conveys a perspective of miR-29a signaling enhancement beneficial for aged skeletons

Aim. “Implant associated Staphylococcus aureus or S. epidermidis infections are often difficult to treat due to the formation of biofilms on prosthetic material. Biofilms are bacterial communities adhered to a surface with a self-made extracellular polymeric substance that surrounds resident bacteria. In contrast to planktonic bacteria, bacteria in a biofilm are in an adherent, dormant state and are insensitive to most antibiotics. In addition, bacteria in a biofilm are protected from phagocytic cells of the immune system. Therefore, complete surgical removal and replacement of the prosthetic implant is often necessary to treat this type of infections. Neutrophils play a crucial role in clearing bacterial pathogens. They recognize planktonic bacteria via immunoglobulin (Ig) and complement opsonisation. In this project, we aim to evaluate the role of IgG and complement in the recognition and clearance of staphylococcal biofilms by human neutrophils. Furthermore, we evaluate if monoclonal antibodies (mAbs) targeting biofilm structures can enhance recognition and clearance of staphylococcal biofilms by the human immune system.”. Method. “We produced a set of 20 recombinant mAbs specific for staphylococcal antigens. Using flow cytometry and ELISA-based methods we determined the binding of these mAbs to planktonic staphylococci and in vitro staphylococcal biofilms. Following incubation with IgG/IgM depleted human serum we determined whether mAbs can react with the human complement system after binding to biofilm. Confocal microscopy was used to visualize the location of antibody binding in the biofilm 3D structure.”. Results. “We show that mAbs directed against several staphylococcal surface targets such as wall teichoic acid (a glycopolymer on the S. aureus/S. epidermidis cell wall) and polymeric-N-acetyl-glucosamine (major constituent of the S. epidermidis biofilm extracellular matrix) bind biofilms in a dose-dependent manner. This interaction was specific since no binding was observed for control antibodies (recognizing the hapten DNP). Furthermore we show that these antibodies can penetrate the complete 3D structure of an in vitro biofilm. Products of complement activation via the classical pathway were detected upon incubation with human serum and the biofilm binding mAbs.”. Conclusions. “Having established that our mAbs can bind biofilms and induce complement opsonisation via C3b deposition, we will now study if we can engineer these antibodies to enhance complement deposition. A combination of enhanced complement and antibody opsonisation may improve recognition and clearance of biofilms by phagocytic immune cells. These mAbs could be used to boost the immune system to clear implant associated infections, without the need to replace the implant via invasive surgical procedures.”

Soft tissue sarcomas (STS) have not demonstrated favourable clinical responses to emerging immunotherapies such as checkpoint inhibitors. Studies in carcinomas and melanoma have demonstrated that tumours lacking T-cell infiltrates are associated with poor responses to immunotherapies. It is postulated that STS lack tumour asscoiated lymphocytes which renders these tumours insensitive to checkpoint inhibitors. Our objective was to develop a novel syngeneic mouse model of STS and characterize the immune phenotype of these tumours. Additionally, we sought to evaluate the therapeutic responses of these sarcomas to checkpoint inhibitors and a Type I interferon agonist. K-ras mutagenesis and p53 deletion was induced using a Lenti-Cre-recombinase injection into the hindlimb of 3 week old C57BL/6 mice. Tumours were harvested and characterized using standard histopathology techniques and whole trascriptome sequencing (RNAseq). Full body necrospy and histopathology was performed to identify metastases. Flow cytometry and immunohistochemistry was used to evaluate tumour immune phenotypes. Tumours were implanted into syngeneic C57BL/6 mice and the therapeutic responses to anti-CTLA4, anti-PD1 and DMXAA (Type I interferon agonist) were performed. Tumour responses were evaluated using bioluminescent imaging and caliper measurements. Soft tissue sarcomas developed in mice within 2–3 months of Lenti-Cre injection with 90% penetrance. Histologic analyses of tumours was consistent with a high-grade myogenic sarcoma characterized by smooth muscle actin, Desmin and Myogenin D positive immunostaining. Using crossplatform normalization protocols, geneexpression signatures of the mouse tumours most closely correlated with human undifferentiated pleomorphic sarcoma (UPS). Collectively, gene expression signatures of this murine sarcoma correlated with all muscle-derived human sarcomas (ERMS, ARMS, Synovial sarcoma, UPS). No lung or other visceral metastases were observed in all mice who developed spontaneous tumours. Immune phenotyping demonstrated a paucity of tumour-infiltrating lymphocytes (TILs, (TAMs). 50% of identified TILs in these murine sarcomas expressed PD-1, yet tumours were not responsive to anti-PD1 therapy or anti-CTLA4 therapy. A single intra tumoural (i.t.) injection of the Type I interferon agonist, DMXAA resulted in 80–90% tumour necrosis 72 hrs post-injection, decreased tumour viability up to 2 weeks post-injection and a marked infiltration of CD8+ T-cells and anitgen presenting dendritic cells and macrophages. Additional longitudinal experiments demonstrate a sustained and progressive anti-tumour effect in 83% (5/6) mice up to 6weeks following a single i.t. injection of DMXAA. All control treated mice (6/6) reached humane endpoint within 14 days. At 3 months post-DMXAA treatment, 4/6 mice were free of disease. We re-injected UPS tumours into these mice and tumours did not grow, suggesting abscopal effects after DMXAA treatment of primary tumours. We have characterized a new orthotopic and syngeneic mouse model of a myogenic soft tissue sarcoma. Like most human STS sub-types, these tumours have an immune inert tumour microenvironment and are not sensitive to checkpoint inhibitors. This model, syngeneic to C56BL/6 mice will enable future opportunities to investigate how various branches of the immune system can be targetted or manipulated to unearth new immunotherapeutic strategies for sarcoma. Using this model we have demonstrated that a single, intra-tumoural injection of a Type I interferon agonist can result in anti-tumour effects, recruit cytotoxic lymphocytes and antigen presenting cells with into the the tumour microenvironment. Abscopal tumour rejection after DMXAA treatement suggest adaptive T-cell responses against UPS are active in this model. Future work is needed to determine if upregulation of Type I inferferon pathways can be used as a therapeutic strategy for sarcoma or as a sensitization strategy for checkpoint inhibitors

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

Bone marrow concentrates are being used to augment soft tissue healing. However, only 0.01% of these cells meet the criteria of a mesenchymal stem cell (MSC), which likely accounts for the variability in reported results. Previous studies using an established rat rotator cuff repair model have demonstrated that bone marrow-derived MSCs had no effect on healing. In this study we evaluated the effect of purified human MSCs on rotator cuff healing in an athymic rat model. Hypothesis: Purified human MSCs added to the repair site will improve biomechanical strength and fibrocartilage formation of the healing tendon. Fifty-two athymic rats underwent unilateral detachment and repair of the supraspinatus tendon with either fibrin glue (control) or fibrin glue with 106 hMSCs (experimental) applied at the repair site. Flow cytometry verified the stem cell phenotype of the cells as CD73+, CD90+, CD105+, CD14-, CD34- and CD45-. Rats were sacrificed at 2 and 4 weeks, with 10 used for biomechanical testing and 3 for histologic analysis from each group. Biomechanical testing revealed a significant increase in failure load (11.5±2.4N vs. 8.5±2.4N, p=0.002) and stiffness (7.1±1.2 N/mm vs. 5.7±2.1 N/mm, p0.17). These data demonstrate the potential for stem cells to augment tendon healing. This is the first study to use purified stem cells, rather than simple bone marrow concentrate. In the future, cell sorting techniques and culture expansion could be used to select and expand the small population of true stem cells in bone marrow. Furthermore, healing could potentially be improved with repeat cell injection at an additional post-operative time point

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

Purpose. Chemokines produced by synoviocytes of the subacromial bursa are up-regulated in subacromial inflammation (bursitis) and rotator cuff disease. SDF-1a is an important chemotactic factor in the subacromial bursa that stimulates recruitment of inflammatory cells; however, its mechanism of induction and regulation in the subacromial bursa is unknown. We hypothesised that SDF-1a production in bursal synoviocytes may be induced by local cytokines such as interleukin IL-1β and IL-6. Methods. Subacromial bursa specimens were obtained following an institutional review board-approved protocol from patients undergoing shoulder surgery. Bursal specimens were stained with anti-human antibodies to IL-1, IL-6 and SDF-1a by immunohistochemistry and compared to normal and rheumatoid controls. Bursal cells were also isolated from specimens and cultured. Cultured cells were labelled with fluorescent probes and analysed by flow cytometry to determine cell lineage. Early-passaged cells were then treated with cytokines IL-1β and IL-6 and SDF-1a production and expression were measured by ELISA and RT-PCR. Results. SDF-1a, IL-1β and IL-6 were expressed at high levels in bursitis specimens from human subacromial bursa compared to normal controls. In bursal synoviocytes, there was a dose-dependent increase in SDF-1a production in the supernatants of cells treated with IL-1β. SDF-1a mRNA expression was also increased in bursal cells treated with IL-1β, with stimulation occurring at 6 hours and increasing to five-fold stimulation by 48 hours. IL-6 caused a minimal but not statistically significant increase in SDF-1a expression. Conclusion. SDF-1a, IL-1β, and IL-6 are expressed in the inflamed human subacromial bursal tissues in patients with subacromial bursitis. In cultured bursal synoviocytes, SDF-1a production is stimulated by IL-1β. These cytokines may stimulate or potentiate the inflammatory response that occurs in subacromial bursitis and rotator cuff disease, and may provide a potential new target mechanism for inhibition of this common clinical problem

Objective. To investigate the effects of trauma and fracture surgery on leukocyte maturation and function. Background. Unbalanced inflammation triggered by trauma has been linked to multiorgan dysfunction (MOD) and death. In animal and cellular models, changes in neutrophil function and failure of monocyte infiltration and resolution have been implicated as possible causes. The investigators combine assays on neutrophil function with surface antigen expression on circulating neutrophils and monocytes. These are correlated with severity of traumatic injury, type of surgery and clinical outcome to help explain the aetiology of distant organ injury, and pose a case for damage control surgery. Results. A total of 20 patients requiring internal fixation of femoral shaft fractures, acetabular fractures and pelvic fractures were recruited. Those undergoing surgery following an interval period were used as control, with blood and plasma samples pre-operatively, and 2 and 5 days post-operatively, whilst patients with acute trauma also had an admission sample. Using flow cytometry, the neutrophils were gated on CD15+ CD14- with high side scatter whilst the monocytes were gated on CD14+ CD15- with low side scatter. Two days following surgery the neutrophils showed reduced CXCR2 expression and increased CXCR1, CD11b and IL-6R expression whilst the monocytes showed reduced CCR2 and HLA-DR receptor expression. The change in receptor expression was enhanced in the trauma patients in comparison to the control patients, and correlated with cellular function, using respiratory burst, elastase release and transmigration assays. Conclusions. This first human trial evaluating the immunologic/anti-inflammatory effects of trauma and trauma surgery on the specific antigen expression helps explain one mechanism for organ damage in the post-trauma patient

Background. Replacing bone lost as a consequence of trauma or disease is a major challenge in the treatment of musculoskeletal disorders. Tissue engineering strategies seek to harness the potential of stem cells to regenerate lost or damaged tissue. Bone marrow aspirate (BMA) provides a promising autologous source of skeletal stem cells (SSCs) however, previous studies have demonstrated that the concentration of SSCs required for robust tissue regeneration is below levels present in iliac crest BMA, emphasising the need for cell enrichment strategies prior to clinical application. Aims. To develop a novel strategy to enrich skeletal stem cells (SSCs) from human BMA, clinically applicable for intra-operative orthopaedic use. Methods. Iliac crest BMA was purchased from commercial suppliers and femoral canal BMA was obtained with informed consent from older patients undergoing total hip replacement. 5 to 40ml of BMA was processed to obtain 2–8 fold volume reductions. SSC function was assessed by assays for fibroblastic colony-forming units (CFU-F). Cell viability and seeding efficiency of processed and unprocessed aspirates applied to allograft was assessed. Results. Iliac crest BMA from 15 patients was enriched for SSCs in a processing time of only 15 minutes. Femoral BMA from 15 patients in the elderly cohort was concentrated up to 5-fold with a corresponding enrichment of viable, functional SSCs as confirmed by flow cytometry, CFU-F assays and histological analysis. The SSC enrichment of bone marrow aspirate significantly enhanced cell seeding efficiency onto allograft confirming the utility of this approach for application to bone regeneration. Conclusion. The ability to rapidly enrich BMA demonstrates the potential of this strategy for intra-operative application to enhance bone healing. The development of this device offers immediate potential for clinical application to reduce morbidity in many scenarios associated with local bone stock loss. Further analysis in vivo is ongoing prior to clinical tests

Aim. Ultrahigh molecular weight polyethylene (UHMWPE) has been used for many years as a bearing surface in total joint replacement (TJR). However, late-state failure in TJR is predominantly caused by osteolysis mediated by wear particles. We tested our hypothesis that UHMWPE nanoparticles are important determinants in activating dendritic cells (DCs). Methods. UHMWPE wear particles generated from a knee simulator were profiled using an atomic force microscopy and fractionated into six fractions: 0.05-0.2, 0.2-0.8, 0.8-1, 1-5, 5-10, and 10-20 micrometer. Effects of each fraction, a mixture of nano-sized fractions, and a mixture of all fractions on the activation of mice spleen DCs were determined using flow cytometry with specific antibodies of anti-CD11c-APC, anti-CD80-PE, anti-CD11b-PerCp, anti-CD86-Biotin and streptavidin-FITC. Supernatant from DCs treated with wear particles were assayed for IL-1beta, IL-6, IL-12/23, TNF-alpha and IFN-gamma. Activation of human osteoclasts (OCs) by wear particles were determined using TRAP stain. Results. DCs treated with a mixture of nanoparticles showed a significant increase in CD80 expression. A similar trend was not observed when DCs were treated with solvent or media, suggesting that the increased expression of CD80 was UHMWPE nanoparticle specific. Macrophages treated with nanoparticles did not show a significant increase in the expression of CD80, suggesting that DCs may be more sensitive to activation than macrophages. These results were further supported by the increased production of cytokines, IL-1beta and IL-6. Furthermore, the mixture of nanoparticles and the mixture of all fractions directly stimulated maturation of OCs. Conclusion. This study identifies a novel mechanism where UHMWPE nanoparticles activate DCs. The high proportion of nanoparticles from prosthetic joints would suggest this mechanism is a likely pathway for cytokine production and OCs maturation, all of which involve osteolysis. The nanoparticles as mediators of periprosthetic inflammation should be considered in developing biomaterials for bearing surfaces

Introduction. Modern processing techniques in bone banking are thought to decrease the presence of allogenic material in bone. This project was performed to observe any changes in peripheral blood lymphocyte subsets in response to allografted bone used in revision hip replacement. Methods. 87 patients were entered into this prospective study and grouped according to whether impaction allograft was used or not. Samples were collected pre-operatively and at set time intervals up to one year post-operatively. Using flow cytometry, analysis of venous blood allowed counts of the following cells: Helper T-lymphocytes, cytotoxic T-lymphocytes, memory T-lymphocytes, naïve T-lymphocytes, Natural Killer cells and B-lymphocytes. Results. All patients had a successful outcome at one year. 50 patients with radiologically defined host-graft union were compared with 37 patients who did not receive an allograft. Pre-operatively, a significant difference (p=0.03) was found between the groups of patients with respect to Natural Killer cells but other subsets showed no significant difference. Post-operatively, the significant difference between Natural Killer cells resolved. T-helper lymphocytes, cytotoxic lymphocytes, memory T-lymphocytes and naïve T-lymphocytes in both groups showed decreases in values immediately post-surgery, recovering to normal values within 6 weeks post-surgery. The allograft group showed significant increases from baseline in cytotoxic T-lymphocytes at 6 months (p<0.01) and memory T-lymphocytes one year post-operatively (p=0.04). B-lymphocyte numbers did not alter significantly from baseline. Discussion. Cytotoxic T-lymphocytes recognise HLA-class I molecules which are present on all nucleated cells and have been implicated in having a role in osteoclast regulation. Memory T-lymphocytes are produced after a naïve T-lymphocyte is exposed to an antigen. The observed increases of these subsets were not observed in the non-grafting group suggesting the allografted bone had elicited an immunological response. At 12 months all grafts appeared radiologically stable and the immunological response may have been beneficial to the outcome

Introduction. Problematic bone defects are encountered regularly in orthopaedic practice particularly in fracture non-union, revision hip and knee arthroplasty, following bone tumour excision and in spinal fusion surgery. At present the optimal source of graft to ‘fill’ these defects is autologous bone but this has significant drawbacks including harvest site morbidity and limited quantities. Bone marrow has been proposed as the main source of osteogenic stem cells for the tissue-engineered cell therapy approach to bone defect management. Such cells constitute a minute proportion of the total marrow cell population and their isolation and expansion is a time consuming and expensive strategy. In this study we investigated human bone marrow stem cells as a potential treatment of bone defect by looking at variability in patient osteogenic cell populations as a function of patient differences. We produced a model to predict which patients would be more suited to cell based therapies and propose possible methods for improving the quality of grafts. Methods. Bone marrow was harvested from 30 patients undergoing elective total hip replacement surgery in Musgrave Park Hospital, Belfast (12 males, 18 females, age range 52-82 years). The osteogenic stem cell fraction was cultured and subsequently analysed using colony forming efficiency assays, flow cytometry, fluorescence activated cell sorting and proteomics. Results. The number and proliferative capacity of osteogenic stem cells varied markedly between patients. Statistical analysis revealed significantly better osteogenic capacity in:. male patients. samples in which the growth hormone Fibroblastic Growth Factor-2 was added to culture medium. patients who used the cholesterol lowering agent simvastatin. Patient use of inhaled steroids and NSAIDs were found to have detrimental effects. A statistical model to predict marrow profiles based on these variables was produced. Conclusions. Stem cell based tissue engineering represents the future of the treatment of bone defect. This study provides evidence that inter-patient variability in marrow cell colony forming and proliferation ability can in some way be explained by patient associated factors. Using this knowledge, we can identify which patients would be best suited to this method of treatment and propose techniques for enhancement of their graft profiles

Purpose. Adenosine triphosphate (ATP) has been implicated as an autocrine/paracrine signal in the mechanotransduction pathway of chondrocytes. In this study, human chondrocytes in a 3D agarose scaffold were cultured with exogenous ATP in varying doses to determine its effect on extracellular matrix synthesis by the cells. Further experiments determined basal ATP release, ATP degradation and expression of P2Y1 and P2Y2 purinoreceptors by the cultured cell constructs. Method. Human chondrocytes were obtained by enzymatic digestion of cartilage samples obtained at the time of total joint arthroplasty. The chondrocytes were cultured in a 3D agarose scaffold using standard tissue culture techniques. Various concentrations of exogenous ATP were added to the cultures, along with the radioisotopes to assess matrix synthesis. The cultures were harvested after a 24 hr incubation and radioisotope incorporation was determined by scintillation counting to determine proteoglycan ([35S]-sulfate) and collagen ([3H]-proline) synthesis, respectively. DNA content was determined by the Hoescht 33258 binding assay, and the proteoglycan and collagen synthesis were normalized to DNA content. Basal ATP release and degradation of exogenous ATP were determined by luciferase assay and luminometry. Expression of P2Y1 and P2Y2 purinoreceptors were determined by flow cytometry. Results. Cartilage was obtained and cultured from 22 patients. We identified responders (16/22) and non-responders (6/22) to ATP stimulation. Patients demographics, co-morbidities and medications were reviewed and no correlating characteristics were identified. The average increase in [3H]-proline incorporation was 242% the control (range 115%–388%, p<0.02) and the average increase in [35S]-sulfate incorporation was 238% (range 124%–711%, p<0.02). The expression of P2Y1 and P2Y2 receptors varied widely between individuals, with a range of 11–76% expression and of 3–67% expression for P2Y1 and P2Y2 receptors, respectively. Almost all cells expressing P2Y2 receptors also expressed P2Y1 receptors, and 4/8 patients also had significant cell populations expressing P2Y1 but not P2Y2 receptors (range of 4–17% of cells). Of the 8 patients studied, only 1 patient had measurable ATP within the culture media. ATP degradation within the culture media was measured, with the measured ATP half-life and elimination rate constants were determined. The ATP elimination rate constant values showed good correlation to P2Y1 receptor expression (R=0.99). Conclusion. P2Y1 and P2Y2 receptors were expressed on a significant proportion of chondrocytes from patients with osteoarthritis and there was a significant correlation of the expression of these receptors to the ATP elimination rate constants. The addition of exogenous ATP increased both the proteoglycan and collagen synthesis of the developing cartilage constructs in a subset of patients and appears to be a promising technique to improve extracellular matrix production in these constructs