Chondrosarcoma responds poorly to adjuvant therapy and therefore, new targeted therapy is required. Animal models have been utilised to test therapeutic candidates, however clinically relevant, orthotopic models are lacking. The aim of this study was to develop such a model. In vitro: two human chondrosarcoma cell lines, JJ012 and FS090, were compared with respect to proliferation, colony formation, invasion, MMP-2 and MMP-9 secretion, osteoclastogenesis, endothelial tube stimulation, and expression of the angiogenic factor VEGF, and the anti-angiogenic factor RECK on western blotting. In vivo: 20,000 cells (JJ012 or FS090) were injected either into the intramedullary canal of the
Title. Longitudinal Intravital Imaging to Quantify the “Race for the Surface” Between Host Immune Cell and Bacteria for Orthopaedic Implants with S. aureus Colonization in a Murine Model. Aim. To assess S. aureus vs. host cell colonization of contaminated implants vis intravital multiphoton laser scanning microscopy (IV-MLSM) in a murine model. Method. All animal experiments were approved by IACUC. A flat stainless steel or titanium L-shaped pin was contaminated with 10. 5. CFU of a red fluorescent protein (RFP) expressing strain of USA300LAC, and surgically implanted through the femur of global GFP-transgenic mice. IV-MLSM was performed at 2, 4, and 6 hours post-op. Parallel cross-sectional CFU studies were performed to quantify the bacteria load on the implant at 2,4,6,12,18 and 24 hours. Results. 1) We developed a high-fidelity reproducible IV-MLSM system to quantify S. aureus and host cell colonization of a bone implant in the
Aim. Implant infections caused by Staphylococcus aureus are difficult to treat due to biofilm formation, which complicates surgical and antibiotic treatment. Herewith we introduce an alternative approach using monoclonal antibodies (mAbs) targeting S. aureus and provide the biodistribution and specificity in a
Aim. Bacteriophages are remerging as alternative and adjunctive therapy for fracture-related infection (FRI). However, current administration protocols involve prolonged retention of a percutaneous draining tube with potential risk of developing superinfection. In this study, we applied a cocktail of in vitro evolved biofilm-targeting phages for Methicillin-resistant Staphylococcus aureus (MRSA) in a hydrogel platform co-delivering vancomycin. In vitro synergy and antibiofilm activity was assessed and a subsequent in vivo study was performed in a
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
Aim. Prosthetic joint infections pose a major clinical challenge. Developing novel material surface technologies for orthopedic implants that prevent bacterial adhesion and biofilm formation is essential. Antimicrobial coatings applicable to articulating implant surfaces are limited, due to the articulation mechanics inducing wear, coating degradation, and toxic particle release. Noble metals are known for their antimicrobial activity and high mechanical strength and could be a viable coating alternative for orthopaedic implants [1]. In this study, the potential of thin platinum-based metal alloy coatings was developed, characterized, and tested on cytotoxicity and antibacterial properties. Method. Three platinum-based metal alloy coatings were sputter-coated on medical-grade polished titanium discs. The coatings were characterized using optical topography and scanning electron microscopy with energy dispersive spectroscopy (SEM/EDS). Ion release was measured using inductively coupled plasma optical emission spectrometry (ICP-OES). Cytotoxicity was tested according to ISO10993-5 using
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
Osteoarthritis is a global problem and the treatment of early disease is a clear area of unmet clinical need. Treatment strategies include cell therapies utilising chondrocytes e.g. autologous chondrocyte implantation and mesenchymal stem/stromal cells (MSCs) e.g. microfracture. The result of repair is often considered suboptimal as the goal of treatment is a more accurate regeneration of the tissue, hyaline cartilage, which requires a more detailed understanding of relevant biological signalling pathways. In this study, we describe a modulator of regulatory pathways common to both chondrocytes and MSCs. The chondrocytes thought to be cartilage progenitors are reported to reside in the superficial zone of articular cartilage and are considered to have the same developmental origin as MSCs present in the synovium. They are relevant to cartilage homeostasis and, like MSCs, are increasingly identified as candidates for joint repair and regenerative cell therapy. Both chondrocytes and MSCs can be regulated by the Wnt and TGFβ pathways. Dishevelled Binding Antagonist of Beta-Catenin (Dact) family of proteins is an important modulator of Wnt and TGFβ pathways. These pathways are key to MSC and chondrocyte function but, to our knowledge, the role of DACT protein has not been studied in these cells. DACT1 and DACT2 were localised by immunohistochemistry in the developing joints of
Aim. In vivo biofilm models play major role to study biofilm development, morphology, and regulatory molecules involve in biofilm. Due to ethical restrictions, the use mammalian models are replaced with other alternative models in basic research. Recently, we have developed insect infection model G. mellonella larvae to study implant associated biofilm infections. This model organism is easy to handle, cheap and ethical restriction free and could be used for the high through put screening of antimicrobial compounds to treat biofilm. To promote the use of this model in basic research we aimed to validate this based on the typical biofilm features such as less susceptible to the antibiotics, complexity of the biofilm structure and gene expression profile of biofilms. Method. G. mellonella larvae are maintained at 30oC on artificial diet in an incubator. Titanium and Stainless steel K-wires were cut into small pieces with size of 4mm. After sterilization with 100% alcohol, these K-wires were pre-incubated in S. aureus bacterial suspension (5×10. 6. CFU/ml) for 30 min, washed in PBS and implanted inside the larva after with help of scalpel. The larvae were incubated at 37. o. C for two day for the survival analysis. To analyze the less susceptibility of the biofilms towards antibiotics, the larvae were treated with gentamicin and compared survival with planktonic infection in G. mellonella. To reveal the complex structure of biofilm, the implants were removed and processed for the MALDI analysis. Whole genome-based transcriptome of biofilm was performed to explore the changes in transcriptional landscapes. Results. The results are very promising to validate the use of G. mellonella as in vivo model to study the biofilm formation on implanted materials. The gentamicin treatment could rescue the larvae from the planktonic infection, but not from the biofilm infection on the implants. Further, the MALDI analysis could reveal the complex structure and components of S. aureus biofilm formed on the implant inside the larvae. Finally, the transcriptomic analysis revealed the gene expression changes that can be compared to normal biofilm expression profile. Conclusions. Further, comparison of these results with other in vivo models such as rat and
The T-lymphocyte secreted pro-inflammatory cytokine, interleukin-17F (IL-17F), was found to be a key mediator in the cellular response of the immune system in the early phase of fracture repair but its intracellular signaling processes are currently not known in osteoblasts. The objective of this study was to identify the signaling proteins and crucial gene targets involved in osteoblast activation via IL-17F. It was hypothesised that IL-17F stimulated osteoblast maturation through a novel GSK3beta / beta-catenin independent pathway.
Construction of a functional skeleton is accomplished
through co-ordination of the developmental processes of chondrogenesis,
osteogenesis, and synovial joint formation. Infants whose movement in
utero is reduced or restricted and who subsequently suffer
from joint dysplasia (including joint contractures) and thin hypo-mineralised
bones, demonstrate that embryonic movement is crucial for appropriate
skeletogenesis. This has been confirmed in
Introduction. Aseptic loosening is a major cause of revision of total joint arthroplasty (TJA). Although crosslinked Ultra-high molecular weight polyethylene (UHMWPE) have improved wear resistance, residual radicals remaining in the material have a possibility to increase bio-reactivity of particles [2]. In this study, we attempt to evaluate the effects of irradiation and residual radicals on bio-reactivity of the material with a new method called the inverse culture method [1]. Material and methods. UHMWPE particles (10µm diameter in average, Mitsui chemicals Co., LTD) along with irradiated particles (RAD, 300kGy electron irradiation) and particles annealed after the irradiation (RAD+ANN, 100°C 72 hours) are co-incubated with
Tungsten has been increasing in demand for use in manufacturing and recently, medical devices, as it imparts flexibility, strength, and conductance of metal alloys. Given the surge in tungsten use, our population may be subjected to elevated exposures. For instance, embolism coils made of tungsten have been shown to degrade in some patients. In a cohort of breast cancer patients who received tungsten-based shielding for intraoperative radiotherapy, urinary tungsten levels remained over tenfold higher 20 months post-surgery. In vivo models have demonstrated that tungsten exposure increases tumor metastasis and enhances the adipogenesis of bone marrow-derived mesenchymal stem cells while inhibiting osteogenesis. We recently determined that when mice are exposed to tungsten [15 ppm] in their drinking water, it bioaccumulates in the intervertebral disc tissue and vertebrae. This study was performed to determine the toxicity of tungsten on intervertebral disc. Bovine nucleus pulposus (bNP) and annulus fibrosus (bAF) cells were isolated from bovine caudal tails. Cells were expanded in flasks then prepared for 3D culturing in alginate beads at a density of 1×10. ∧. 6 cells/mL. Beads were cultured in medium supplemented with increasing tungsten concentrations in the form of sodium tungstate [0, 0.5, 5, 15 ug/mL] for 12 days. A modified GAG assay was performed on the beads to determine proteoglycan content and Western blotting for type II collagen (Col II) synthesis. Cell viability was determined by counting live and dead cells in the beads following incubation with the Live/Dead Viability Assay kit (Thermo Fisher Scientific). Cell numbers in beads at the end of the incubation period was determined using Quant-iT dsDNA Assay Kit (Thermo Fisher Scientific). Tungsten dose-dependently decreased the synthesis of proteoglycan in IVD cells, however, the effect was significant at the highest dose of 15 ug/mL. (n=3). Furthermore, although tungsten decreased the synthesis of Col II in IVD cells, it significantly increased the synthesis of Col I. Upregulation of catabolic enzymes ADAMTS4 and −5 were also observed in IVD cells treated with tungsten (n=3). Upon histological examination of spines from mice treated with tungsten [15 ug/mL] in their drinking water for 30 days, disc heights were diminished and Col I upregulation was observed (n=4). Cell viability was not markedly affected by tungsten in both bNP and bAF cells, but proliferation of bNP cells decreased at higher concentration. Surprisingly, histological examination of IVDs and gene expression analysis demonstrated upregulation of NGF expression in both NP and AF cells. In addition, endplate capillaries showed increases in CGRP and PGP9.5 expression as determined on histological sections of
Tendinopathy is one of the most common orthopaedic pathological conditions characterized by tendon degenerative changes. Excessive mechanical loading is considered as a major causative factor in the development of tendinopathy, but the mechanisms of pathogenesis remain unclear. High mobility group box-1 (HMGB1), a potent inflammatory mediator when released into the matrix, has been identified in the early stage tendinopathy patients. Since the release and contribution of HMGB1 in tendinopathy development due to mechanical overloading is unknown, we investigated the role of HMGB1 in tendinopathy using a
Equilibrative nucleoside transporter 1 (ENT1) transfers nucleosides, such as adenosine, across plasma membranes. We reported previously that mice lacking ENT1 (ENT1-KO) exhibit progressive ectopic calcification of spinal tissues, including the annulus fibrosus (AF) of intervertebral discs (J Bone Miner Res 28:1135–49, 2013, Bone 90:37–49, 2016). Our purpose was twofold: (1) to compare ectopic calcifications in ENT1-KO mice with those in human DISH, and (2) to investigate the molecular pathways underlying pathological calcification in ENT1-KO mice. Studies were performed with age-matched wild-type (WT) and ENT1-KO mice, as well as human cadaveric vertebral columns meeting radiographic criteria for DISH.
Objectives. Salubrinal is a synthetic agent that elevates phosphorylation
of eukaryotic translation initiation factor 2 alpha (eIF2α) and
alleviates stress to the endoplasmic reticulum. Previously, we reported
that in chondrocytes, Salubrinal attenuates expression and activity
of matrix metalloproteinase 13 (MMP13) through downregulating nuclear
factor kappa B (NFκB) signalling. We herein examine whether Salubrinal
prevents the degradation of articular cartilage in a
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
Purpose. Vitamin D is a key regulator of bone homeostasis. The enzyme CYP24A1 is responsible for transforming vitamin D into 24,25(OH)2vitD. The putative biological activity of 24,25(OH)2vitD remains unclear. Previous studies showed an increase in the circulating levels of this metabolite following a fracture in chicks. Our laboratory has engineered a mouse model deficient for the Cyp24a1 gene for studying the role of 24,25(OH)2vitD. We set out to study the role of 24,25(OH)2vitD in endochondral and intramembranous bone formation in fracture repair in this mouse model based on the results of the chick fracture repair study. Method. Wild-type and mutant Cyp24a1 gene deficient mice were subjected to two different surgical procedures to simulate bone development and fracture repair. To mimic endochondral ossification, we devised a modified technique to perform intramedullary nailing of a
Purpose. Limb regeneration as it occurs in amphibians has two basic requirements: a source of multipotent cells capable of generating various tissues, and reorganization of those cells to form the one and only pattern of tissue appropriate to restore the missing parts. In the current biomedical world, there is much work dedicated to tissue engineering and to the differentiation of stem cells into various mature cell types. Neither of these approaches however, will by themselves succeed in regenerating a complex structure such as a limb. In our lab, we decided to focus on the pattern organization side of the equation by testing the potential of mammalian limb bud tissue to change its positional identity, and to manipulate that potential. Method. We used
INTRODUCTION. Loosening is concerned to be the major cause of revision in the artificial prosthesis. Wear debris of UHMWPE dispersed into the implant-bone interface are phagocytosed by macrophages releasing inflammatory cytokines such as TNF-α which leads to osteolysis and loosening eventually. It is known that the size and structure [1] as well as attached substances on particle surface such as endotoxin could affect the amount of cytokines released [2]. An in vivo study using rat femurs showed that the presence of polyethylene particles around implants could result in accumulation of lipopolysaccharide (LPS) from exogenous sources that may affect bone remodeling around implants [3]. It is also reported that LPS is transported throughout the body with lipoproteins or LPS binding proteins [4] and Circulating LPS may originate from local sites of infection or via bloodborne bacteria [5]. In this study, we evaluated the effects of LPS that attached to UHMWPE particle surface by measuring TNF-α released from macrophages. MATERIALS AND METHODS. We cultured
