Protective effects of sweroside on human MG-63 cells and rat osteoblasts

Herbal Fructus Corni is a folk medicine with a long history of safe use for treating osteoporosis in postmenopausal women or elderly men in Asia. Sweroside is a bioactive herbal ingredient isolated from Fructus Corni, which has been widely used for the treatment of osteoporosis in traditional Chinese medicine (TCM). Unfortunately, the working mechanisms of this compound are difficult to determine and thus remain unclear. The aim of the study was performed to determine the potential molecular mechanism of the anti-osteoporotic effect of sweroside on the human MG-63 cells and rat osteoblasts. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used to observe the effect of sweroside on cell proliferation. The activity of alkaline phosphatase (ALP) and the amount of osteocalcin were also assayed the cell differentiation. Sweroside significantly increased the proliferation of human MG-63 cells and rat osteoblasts (P<0.01). It increased the activity of ALP, and osteocalcin was also elevated in response to sweroside (P<0.05). Of note, flowcytometer assay showed that sweroside can attenuate and inhibit apoptosis. Sweroside has a direct osteogenic effect on the proliferation and differentiation of cultured human MG-63 cells and rat osteoblasts in vitro. These data will help in understanding the molecular mechanisms of therapeutic efficacy of sweroside, and highlight insights into drug discovery. In the current study, sweroside has been suggested to be a promising osteoporosis therapeutic natural product.

Evaluation of the matabolic response of osteosarcoma cells to conventional and new anticancer drugs by NMR metabolomics

The main scope of this work was to evaluate the metabolic effects of anticancer agents (three conventional and one new) in osteosarcoma (OS) cells and osteoblasts, by measuring alterations in the metabolic profile of cells by nuclear magnetic resonance (NMR) spectroscopy metabolomics. Chapter 1 gives a theoretical framework of this work, beginning with the main metabolic characteristics that globally describe cancer as well as the families and mechanisms of action of drugs used in chemotherapy. The drugs used nowadays to treat OS are also presented, together with the Palladium(II) complex with spermine, Pd2Spm, potentially active against cancer. Then, the global strategy for cell metabolomics is explained and the state of the art of metabolomic studies that analyze the effect of anticancer agents in cells is presented. In Chapter 2, the fundamentals of the analytical techniques used in this work, namely for biological assays, NMR spectroscopy and multivariate and statistical analysis of the results are described. A detailed description of the experimental procedures adopted throughout this work is given in Chapter 3. The biological and analytical reproducibility of the metabolic profile of MG-63 cells by high resolution magic angle spinning (HRMAS) NMR is evaluated in Chapter 4. The metabolic impact of several factors (cellular integrity, spinning rate, temperature, time and acquisition parameters) on the 1H HRMAS NMR spectral profile and quality is analysed, enabling the definition of the best acquisition parameters for further experiments. The metabolic consequences of increasing number of passages in MG-63 cells as well as the duration of storage are also investigated. Chapter 5 describes the metabolic impact of drugs conventionally used in OS chemotherapy, through NMR metabolomics studies of lysed cells and aqueous extracts analysis. The results show that MG-63 cells treated with cisplatin (cDDP) undergo a strong up-regulation of lipid contents, alterations in phospholipid constituents (choline compounds) and biomarkers of DNA degradation, all associated with cell death by apoptosis. Cells exposed to doxorubicin (DOX) or methotrexate (MTX) showed much slighter metabolic changes, without any relevant alteration in lipid contents. However, metabolic changes associated with altered Krebs cycle, oxidative stress and nucleotides metabolism were detected and were tentatively interpreted at the light of the known mechanisms of action of these drugs. The metabolic impact of the exposure of MG-63 cells and osteoblasts to cDDP and the Pd2Spm complex is described in Chapter 6. Results show that, despite the ability of the two agents to bind DNA, the metabolic consequences that arise from exposure to them are distinct, namely in what concerns to variation in lipid contents (absent for Pd2Spm). Apoptosis detection assays showed that, differently from what was seen for MG-63 cells treated with cDDP, the decreased number of living cells upon exposure to Pd2Spm was not due to cell death by apoptosis or necrosis. Moreover, the latter agent induces more marked alterations in osteoblasts than in cancer cells, while the opposite seemed to occur upon cDDP exposure. Nevertheless, the results from MG-63 cells exposure to combination regimens with cDDP- or Pd2Spm-based cocktails, described in Chapter 7, revealed that, in combination, the two agents induce similar metabolic responses, arising from synergy mechanisms between the tested drugs. Finally, the main conclusions of this thesis are summarized in Chapter 8, and future perspectives in the light of this work are presented.

The importance of hormone receptor analysis in osteosarcoma cells growth submitted to treatment with estrogen in association with thyroid hormone

Bone tumor incidence in women peaks at age 50-60, coinciding with the menopause. That estrogen (E2) and triiodothyronine (T3) interact in bone metabolism has been well established. However, few data on the action of these hormones are available. Our purpose was to determine the role of E2 and T3 in the expression of bone activity markers, namely alkaline phosphatase (AP) and receptor activator of nuclear factor kappa B ligand (RANKL). Two osteosarcoma cell lines: MG-63 (which has both estrogen (ER) and thyroid hormone (TR) receptors) and SaOs-29 (ER receptors only) were treated with infraphysiological E2 associated with T3 at infraphysiological, physiological, and supraphysiological concentrations. Real-time RT-PCR was used for expression analysis. Our results show that, in MG-63 cells, infraphysiological E2 associated with supraphysiological T3 increases AP expression and decreases RANKL expression, while infraphysiological E2 associated with either physiological or supraphysiological T3 decreases both AP and RANKL expression. On the other hand, in SaOs-2 cells, the same hormone combinations had no significant effect on the markers` expression. Thus, the analysis of hormone receptors was shown to be crucial for the assessment of tumor potential growth in the face of hormonal changes. Special care should be provided to patients with T3 and E2 hormone receptors that may increase tumor growth. Copyright (C) 2007 John Wiley & Sons, Ltd.

Roles of additives in the trihexyl(tetradecyl) phosphonium chloride ionic liquid electrolyte for primary mg-air cells

As reported previously, water saturated trihexyl(tetradecyl)phosphonium chloride ([P6,6,6,14][Cl]) ionic liquid (IL) is a promising electrolyte for magnesium-air batteries. The added water plays an important role in enabling high rate and high efficiency Mg dissolution while stabilizing the Mg interphase. In this work, the role of the water was investigated by replacement with other additives such as toluene and tetrahydrofuran to specifically target the assumed roles of water, namely: (i) enhancement of transport properties; (ii) complexation and stabilization of the Mg anode; (iii) provision of active protons for the cathodic reaction. Discharge tests show that ethylene glycol supports comparable performance to that provided by water. Examination of the viscosity and conductivity of different [P6,6,6,14][Cl]/additive mixtures indicates that a simple consideration of solution characteristics cannot explain the observed trends. Rather, other factors, such as the presence of active protons and/or oxygen-donor groups, are also key features for the development of IL electrolytes for practical magnesium-air cells. Finally, the presence of ethylene glycol in the electrolyte results in a complex gel on the Mg interface, similar to that found in the presence of water. This may also play a role in enabling stable discharge of the Mg anode. © 2014 The Electrochemical Society.

The importance of hormone receptor analysis in osteosarcoma cells growth submitted to treatment with estrogen in association with thyroid hormone

Bone tumor incidence in women peaks at age 50-60, coinciding with the menopause. That estrogen (E2) and triiodothyronine (T3) interact in bone metabolism has been well established. However, few data on the action of these hormones are available. Our purpose was to determine the role of E2 and T3 in the expression of bone activity markers, namely alkaline phosphatase (AP) and receptor activator of nuclear factor κB ligand (RANKL). Two osteosarcoma cell lines: MG-63 (which has both estrogen (ER) and thyroid hormone (TR) receptors) and SaOs-29 (ER receptors only) were treated with infraphysiological E2 associated with T3 at infraphysiological, physiological, and supraphysiological concentrations. Real-time RT-PCR was used for expression analysis. Our results show that, in MG-63 cells, infraphysiological E2 associated with supraphysiological T3 increases AP expression and decreases RANKL expression, while infraphysiological E2 associated with either physiological or supraphysiological T3 decreases both AP and RANKL expression. On the other hand, in SaOs-2 cells, the same hormone combinations had no significant effect on the markers' expression. Thus, the analysis of hormone receptors was shown to be crucial for the assessment of tumor potential growth in the face of hormonal changes. Special care should be provided to patients with T3 and E2 hormone receptors that may increase tumor growth. Copyright © 2007 John Wiley & Sons, Ltd.

Characterization of tissue transglutaminase in human osteoblast-like cells

Tissue transglutaminase (tTG) is a calcium-dependent and guanosine 5'-triphosphate (GTP) binding enzyme, which catalyzes the post-translational modification of proteins by forming intermolecular ε(ϒ-glutamyl)lysine cross-links. In this study, human osteoblasts (HOBs) isolated from femoral head trabecular bone and two osteosarcoma cell lines (HOS and MG-63) were studied for their expression and localization of tTG. Quantitative evaluation of transglutaminase (TG) activity determined using the [1,414C]-putrescine incorporation assay showed that the enzyme was active in all cell types. However, there was a significantly higher activity in the cell homogenates of MG-63 cells as compared with HOB and HOS cells (p <0.001). There was no significant difference between the activity of the enzyme in HOB and HOS cells. All three cell types also have a small amount of active TG on their surface as determined by the incorporation of biotinylated cadaverine into fibronectin. Cell surface-related tTG was further shown by preincubation of cells with tTG antibody, which led to inhibition of cell attachment. Western blot analysis clearly indicated that the active TG was tTG and immunocytochemistry showed it be situated in the cytosol of the cells. In situ extracellular enzyme activity also was shown by the cell-mediated incorporation of fluorescein cadaverine into extracellular matrix (ECM) proteins. These results clearly showed that MG-63 cells have high extracellular activity, which colocalized with the ECM protein fibronectin and could be inhibited by the competitive primary amine substrate putrescine. The contribution of tTG to cell surface/matrix interactions and to the stabilization of the ECM of osteoblast cells therefore could by an important factor in the cascade of events leading to bone differentiation and mineralization.

Development of docetaxel and alendronate-loaded chitosan-conjugated polylactide-co-glycolide nanoparticles: In vitro characterization in osteosarcoma cells

Purpose: To develop docetaxel (DTX)- and alendronate (ALN)-loaded, chitosan (CS)-conjugated polylactide- co-glycolide (PLGA) nanoparticles (NPs) to increase therapeutic efficacy in osteosarcoma cells. Methods: Drug-loaded PLGA NPs were prepared by nanoprecipitation and chemically conjugated by the carboxylic group of PLGA to the amine-bearing CS polymer. The nanocarrier was characterized by dynamic light scattering, transmission electron microscopy, scanning electron microscopy, and differential scanning calorimetry as well as by in vitro drug release and cell culture studies. Results: NP size was within the tumour targeting range (~200 nm) with an effective positive charge (20 mV), thus increasing cellular uptake efficiency. Morphological analysis revealed clear spherical particles with uniform dispersion. The NPs exhibited identical sustained release kinetics for both DTX and ALN. CS-conjugated PLGA with dual-drug-loaded (DTX and AL) NPs showed typical time-dependent cellular uptake and also displayed superior cytotoxicity in MG-63 cells compared with blank NPs, which were safe and biocompatible. Conclusion: Combined loading of DTX and ALN in NPs increased the therapeutic efficacy of the formulation for osteosarcoma treatment, thus indicating the potential benefit of a combinatorial drug regimen using nanocarriers for effective treatment of osteosarcoma.

Fatigue and biocompatibility properties of a poly(methyl methacrylate) bone cement with multi-walled carbon nanotubes

Composites of multi-walled carbon nanotubes (MWCNT) of varied functionality (unfunctionalised and carboxyl and amine functionalised) with polymethyl methacrylate (PMMA) were prepared for use as a bone cement. The MWCNT loadings ranged from 0.1 to 1.0 wt.%. The fatigue properties of these MWCNT–PMMA bone cements were characterised at MWCNT loading levels of 0.1 and 0.25 wt.% with the type and wt.% loading of MWCNT used having a strong influence on the number of cycles to failure. The morphology and degree of dispersion of the MWCNT in the PMMA matrix at different length scales were examined using field emission scanning electron microscopy. Improvements in the fatigue properties were attributed to the MWCNT arresting/retarding crack propagation through the cement through a bridging effect and hindering crack propagation. MWCNT agglomerates were evident within the cement microstructure and the degree of agglomeration was dependent on the level of loading and functionality of the MWCNT. The biocompatibility of the MWCNT–PMMA cements at MWCNT loading levels upto 1.0 wt.% was determined by means of established biological cell culture assays using MG-63 cells. Cell attachment after 4 h was determined using the crystal violet staining assay. Cell viability was determined over 7 days in vitro using the standard colorimetric MTT assay. Confocal scanning laser microscopy and SEM analysis was also used to assess cell morphology on the various substrates.

The role of transcription factor Pitx1 and its regulation by hypoxia in Adolescent Idiopathic Scoliosis

La scoliose idiopathique de l’adolescent (SIA) est définie comme une courbure de la colonne vertébrale supérieure à 10 degrés, qui est de cause inconnue et qui affecte de façon prépondérante les adolescents. Des études précédentes sur des modèles murins ont démontré une inactivation partielle du gène Pitx1. Cette inactivation partielle provoque une déformation spinale sévère lors du développement des souris Pitx1+/-, ce qui est grandement similaire au phénotype de la SIA. En se basant sur ces observations, nous postulons que la perte de fonction de Pitx1 pourrait avoir un rôle dans la SIA et pourrait être régulée par des mécanismes moléculaires spécifiques. En effet, des études faites sur l’expression de Pitx1 révèlent une perte de son expression dans les ostéoblastes dérivés de patients SIA au niveau de l’ARNm. Nous émettons l’hypothèse que la perte de Pitx1 dans la SIA pourrait être déclenchée par des facteurs hypoxiques puisqu’il est connu que Pitx1 est réprimé par l’hypoxie et que HIF-2 alpha est surexprimés dans les ostéoblastes des patients SIA même dans des conditions normoxiques. De plus, nous avons découvert une mutation dans le domaine ODD des HIF-1 alpha chez certains patients SIA (3,1%). Une fonction connue de ce domaine est de stabiliser et d’augmenter l’activité transcriptionnelle de HIF-1 alpha dans des conditions normoxiques. Nous avons confirmé, par la technique EMSA, l’existence d’un élément de réponse fonctionnel à l’hypoxie au niveau du promoteur de Pitx1. Cependant, des co-transfections avec des vecteurs d’expression pour HIF-1 alpha et HIF-2 alpha, en présence de leur sous-unité beta ARNT, ont conduit à une activation du promoteur de Pitx1 dans la lignée cellulaire MG-63 ainsi que dans les ostéoblastes des sujets contrôles. Il est intéressant de constater qu’aucune activité du promoteur de Pitx1 dans les ostéoblastes SIA n’a été observée, même après la co-expression de HIF-2 alpha et ARNT, confirmant le fait que l’expression de Pitx1 est abrogée dans la SIA. Dans l’ensemble, nos résultats démontrent un rôle important de Pitx1 dans la SIA et une possible régulation par des facteurs hypoxiques.

IL-4 and IL-13 inhibit IL-1β and TNF-α induced kinin B 1 and B2 receptors through a STAT6-dependent mechanism

Background and Purpose Bone resorption induced by interleukin-1β (IL-1β) and tumour necrosis factor (TNF-α) is synergistically potentiated by kinins, partially due to enhanced kinin receptor expression. Inflammation-induced bone resorption can be impaired by IL-4 and IL-13. The aim was to investigate if expression of B1 and B2 kinin receptors can be affected by IL-4 and IL-13. Experimental Approach We examined effects in a human osteoblastic cell line (MG-63), primary human gingival fibroblasts and mouse bones by IL-4 and IL-13 on mRNA and protein expression of the B1 and B2 kinin receptors. We also examined the role of STAT6 by RNA interference and using Stat6-/- mice. Key Results IL-4 and IL-13 decreased the mRNA expression of B1 and B2 kinin receptors induced by either IL-1β or TNF-α in MG-63 cells, intact mouse calvarial bones or primary human gingival fibroblasts. The burst of intracellular calcium induced by either bradykinin (B2 agonist) or des-Arg10-Lys-bradykinin (B1 agonist) in gingival fibroblasts pretreated with IL-1β was impaired by IL-4. Similarly, the increased binding of B1 and B2 ligands induced by IL-1β was decreased by IL-4. In calvarial bones from Stat6-deficient mice, and in fibroblasts in which STAT6 was knocked down by siRNA, the effect of IL-4 was decreased. Conclusions and Implications These data show, for the first time, that IL-4 and IL-13 decrease kinin receptors in a STAT6-dependent mechanism, which can be one important mechanism by which these cytokines exert their anti-inflammatory effects and impair bone resorption. © 2013 The Authors. British Journal of Pharmacology © 2013 The British Pharmacological Society.

Patogenesi molecolare delle osteomieliti associate all'impianto

L’osteomielite associata all’impianto è un processo infettivo a carico del tessuto osseo spesso accompagnato dalla distruzione dell’osso stesso. La patogenesi delle osteomieliti associate all’impianto si basa su due concetti fondamentali: l’internalizzazione del patogeno all’interno degli osteoblasti e la capacità dei batteri di formare il biofilm. Entrambi i meccanismi consentono infatti di prevenire l’eliminazione del batterio da parte delle difese immunitarie dell’ospite e di ostacolare l’azione della maggior parte degli antibiotici (che non penetrano e non agiscono pertanto su microrganismi intracellulari), così sostenendo ed alimentando l’infezione. Il saggio di invasione messo a punto su micropiastra ha consentito di investigare in modo approfondito e dettagliato il ruolo ed il peso dell’internalizzazione nella patogenesi delle infezioni ortopediche peri-protesiche causate da S. aureus, S. epidermidis, S. lugdunensis ed E. faecalis. Lo studio ha evidenziato che l’invasione delle cellule MG-63 non rappresenta un meccanismo patogenetico delle infezioni ortopediche associate all’impianto causate da S. epidermidis, S. lugdunensis ed E. faecalis; al contrario, in S. aureus la spiccata capacità invasiva rappresenta un’abile strategia patogenetica che consente al patogeno di sfuggire alla terapia sistemica e alla risposta immunitaria dell’ospite. È stato studiato inoltre il ruolo dell’immunità innata nella difesa contro il biofilm batterico. In seguito all’incubazione del biofilm opsonizzato di S. epidermidis con i PMN è stato possibile osservare la formazione delle NETs. Le NETs rappresentano ottime armi nella difesa contro il biofilm batterico, infatti le trappole sono in grado di limitare la diffusione batterica e quindi di confinare l’infezione. La comprensione del ruolo dell’internalizzazione nella patogenesi delle osteomieliti associate all’impianto e lo studio della risposta immunitaria innata a questo tipo di infezioni, spesso caratterizzate dalla presenza di biofilm, sono presupposti per identificare e affinare le migliori strategie terapeutiche necessarie ad eradicare l'infezione.

Cell-Adhesive Responses to Tenascin-C Splice Variants Involve Formation of Fascin Microspikes

Tenascin-C is an adhesion-modulating matrix glycoprotein that has multiple effects on cell behavior. Tenascin-C transcripts are expressed in motile cells and at sites of tissue modeling during development, and alternative splicing generates variants that encode different numbers of fibronectin type III repeats. We have examined the in vivo expression and cell adhesive properties of two full-length recombinant tenascin-C proteins: TN-190, which contains the eight constant fibronectin type III repeats, and TN-ADC, which contains the additional AD2, AD1, and C repeats. In situ hybridization with probes specific for the AD2, AD1, and C repeats shows that these splice variants are expressed at sites of active tissue modeling and fibronectin expression in the developing avian feather bud and sternum. Transcripts incorporating the AD2, AD1, and C repeats are present in embryonic day 10 wing bud but not in embryonic day 10 lung. By using a panel of nine cell lines in attachment assays, we have found that C2C12, G8, and S27 myoblastic cells undergo concentration-dependent adhesion to both variants, organize actin microspikes that contain the actin-bundling protein fascin, and do not assemble focal contacts. On a molar basis, TN-ADC is more active than TN-190 in promoting cell attachment and irregular cell spreading. The addition of either TN-190 or TN-ADC in solution to C2C12, COS-7, or MG-63 cells adherent on fibronectin decreases cell attachment and results in decreased organization of actin microfilament bundles, with formation of cortical membrane ruffles and retention of residual points of substratum contact that contain filamentous actin and fascin. These data establish a biochemical similarity in the processes of cell adhesion to tenascin-C and thrombospondin-1, also an “antiadhesive” matrix component, and also demonstrate that both the adhesive and adhesion-modulating properties of tenascin-C involve similar biochemical events in the cortical cytoskeleton. In addition to these generic properties, TN-ADC is less active in adhesion modulation than TN-190. The coordinated expression of different tenascin-C transcripts during development may, therefore, provide appropriate microenvironments for regulated changes in cell shape, adhesion, and movement.

Inhibition of nuclear factor-kappa B by dehydroxymethylepoxyquinomicin induces schedule-dependent chemosensitivity to anticancer drugs and enhances chemoinduced apoptosis in osteosarcoma cells

Osteosarcoma (OS) is the most common primary malignant bone tumor, usually developing in children and adolescents, and is highly invasive and metastatic, potentially developing chemoresistance. Thus, novel effective treatment regimens are urgently needed. This study was the first to investigate the anticancer effects of dehydroxymethylepoxyquinomicin (DHMEQ), a highly specific nuclear factor-kappa B (NF-kappa B) inhibitor, on the OS cell lines HOS and MG-63. We demonstrate that NF-kappa B blockade by DHMEQ inhibits proliferation, decreases the mitotic index, and triggers apoptosis of OS cells. We examined the effects of combination treatment with DHMEQ and cisplatin, doxorubicin, or methotrexate, drugs commonly used in OS treatment. Using the median effect method of Chou and Talalay, we evaluated the combination indices for simultaneous and sequential treatment schedules. In all cases, combination with a chemotherapeutic drug produced a synergistic effect, even at low single-agent cytotoxic levels. When cells were treated with DHMEQ and cisplatin, a more synergistic effect was obtained using simultaneous treatment. For the doxorubicin and methotrexate combination, a more synergistic effect was achieved with sequential treatment using DHMEQ before chemotherapy. These synergistic effects were accompanied by enhancement of chemoinduced apoptosis. Interestingly, the highest apoptotic effect was reached with sequential exposure in both cell lines, independent of the chemotherapeutic agent used. Likewise, DHMEQ decreased cell invasion and migration, crucial steps for tumor progression. Our data suggest that combining DHMEQ with chemotherapeutic drugs might be useful for planning new therapeutic strategies for OS treatment, mainly in resistant and metastatic cases. Anti-Cancer Drugs 23:638-650 (C) 2012 Wolters Kluwer Health broken vertical bar Lippincott Williams & Wilkins.

In vitro expansion of U87-MG human glioblastoma cells under hypoxic conditions affects glucose metabolism and subsequent in vivo growth

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Novel coatings for hard tissue implants

A novel deposition process named CoBlastTM, based on grit blasting technology, has been used to deposit hydroxyapatite (HA) onto titanium (Ti) metal using a dopant/abrasive regime. The various powders (HA powder, apatitic abrasives) and the treated substrates were characterised for chemical composition, coating coverage, crystallinity and topography including surface roughness. The surface roughness of the HA surfaces could be altered using apatitic abrasives of different particle sizes. Compared to the standard plasma spraying process, the CoBlast surface produced excellent coating adhesion, lower dissolution, higher levels of mechanical and chemical stability in stimulated body fluid (SBF). Enhanced viability of osteoblastic cells was also observed on the CoBlast HA surfaces compared to the microblast and untreated Ti as well as the plasma HA coating. CoBlast offers an alternative to the traditional methods of coating HA implants with added versatility. Apatites substituted with antimicrobial metals can also be deposited to add functionality to HA coatings without cytotoxicty. The potential use of these coatings as an infection preventing strategy for application on hard tissue implants was assessed in vitro and also in vivo. Surface physicochemical properties and morphology were determined in addition to surface cytocompatibility assessments using a MG-63 osteoblast cell line. The antibacterial potential of the immobilised metal ion on the surface and the eluted ion to a lesser extent, contributed to the anticolonising behaviour of the surfaces against a standard bacteria strain (S. aureus) as well as a number of clinically relevant strains (MRSA, MSSA and S. epidermis). The results revealed that the surfaces coated with silver substituted apatites (AgA) outperformed the other apatites examined (apatites loaded with Zn, Sr and both Ag and Sr ions). Assessment of bacterial adherence on coated K-wires following subcutaneous implantation in a nude mouse infection model (S. aureus) for two days demonstrated that the 12% wt surface outperformed the 5% wt AgA coating. Lower inflammatory responses were activated with the insertion of the Ag loaded K-wires with a localised infection at the implantation site noted over the two day study period. These results indicated that the AgA coating on the surface of orthopaedic implants demonstrate good biocompatibility whilst inhibiting bacterial adhesion and colonising of the implant surface.