Mesenchymal stromal cells (MSCs) are regenerative and immuno-privileged cells that are used for both tissue regeneration and treatment of severe inflammation-related disease. For quality control of manufactured MSC batches in regard to mature fat cell contamination, a quantitative method for measuring adipogenesis is needed.
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The introduction of metal-on-metal total disc replacements motivated studies to evaluate the effects of cobalt-chromium (CoCr) nanoparticles on cells of the dura mater. Porcine fibroblasts and epithelial cells isolated from the dura mater were cultured with clinically-relevant CoCr nanoparticles and the ions, generated by the particles over 24 h, at doses up to 121 μm(3)per cell. Cell viability and production of proinflammatory cytokines was assessed over 4 days. The capacity of the particles to induce oxidative stress in the cells was evaluated at 24 h. The CoCr particles and their ions significantly reduced the viability of the dural epithelial cells in a dose-dependent manner but not the fibroblasts. Both cell types secreted IL-8 in response to particle exposure at doses of 60.5 μm(3) (epithelial cells) and 121 μm(3) (fibroblasts, epithelial cells) per cell. No significant release of IL-6 was observed in both cell typesat any dose. Reactive oxygen species were induced in both cell types at 50 μm(3) per cell after 24 h exposure. The data suggested novel differences in the resistance of the dural epithelial cells and fibroblasts to CoCr nanoparticle/ion toxicity and demonstrated the inflammatory potential of theparticles. The data contributes to a greater understanding of the potential biological consequences of the use of metal-on-metal total disc prostheses.
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Vitamin E (VE) has been added to ultrahigh-molecular-weight polyethylene (UHMWPE) acetabular cups and tibial trays primarily to reduce oxidative damage to the polymer. The aim of this study was to investigate the relative wear rates of UHMWPE-containing VE compared with virgin UHMWPE. The ability of VE to reduce the amount of inflammatory cytokines produced from stimulated peripheral blood mononuclear cells (PBMNCs) was also investigated. Stimulation was achieved by exposure of PBMNCs to either lipoplysaccharide (LPS) or VE-containing UHMWPE (VE-UHMWPE). In the present study, results showed that the wear rates of UHMWPE with or without VE were not significantly different. Particles generated by UHMWPE with and without VE were not significantly different in size distribution. The production of osteolytic mediators, tumor necrosis factor-alpha, interleukin 1β (IL-β), IL-6, and IL-8 were significantly reduced in (PBMNCs) stimulated with either LPS + VE compared with LPS or VE-UHMWPE particles compared to virgin UHMWPE particles. This trend was also observed when VE was added as a liquid to UHMWPE wear particle-stimulated PBMNCs. The exact mechanism ofhow VE affects the release of inflammatory mediators from particle-stimulated macrophages is not yet understood. It is likely to involve the anti-inflammatory and/or antioxidant effects of VE.
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Patients with cobalt chrome (CoCr) metal-on-metal (MOM) implants may be exposed to a wide size range of metallic nanoparticles as a result of wear. In this study we have characterised the biological responses of human fibroblasts to two types of synthetically derived CoCr particles [(a) from a tribometer (30 nm) and (b) thermal plasma technology (20, 35, and 80 nm)] in vitro, testing their dependence on nanoparticle size or the generation of oxygen free radicals, or both. Metal ions were released from the surface of nanoparticles, particularly from larger (80 nm) particles generated by thermal plasma technology. Exposure of fibroblasts to these nanoparticles triggered rapid (2 h) generation of reactive oxygen species (ROS) that could be eliminated by inhibition of NADPH oxidase, suggesting that it was mediated by phagocytosis of the particles. The exposure also caused a more prolonged, MitoQsensitive production of ROS (24 h), suggesting involvement of mitochondria. Consequently, we recorded elevated levels of aneuploidy, chromosome clumping, fragmentation of mitochondria and damage to the cytoskeleton particularly to the microtubule network. Exposure to the nanoparticles resulted inmisshapen nuclei, disruption of mature lamin B1 and increased nucleoplasmic bridges, which could be prevented by MitoQ. In addition, increased numbers of micronuclei were observed and these were only partly prevented by MitoQ, and the incidence of micronuclei and ion release from the nanoparticles were positively correlated with nanoparticle size, although the cytogenetic changes, modifications in nuclear shape and the amount of ROS were not. These results suggest that cells exhibit diverse mitochondrial ROS-dependent and independent responses to CoCr particles, and that nanoparticle size andthe amount of metal ion released are influential. © 2013 Elsevier Ltd. All rights reserved.
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Application of a high-level decontamination or sterilisation procedure and cell removal technique to tendon allograft can reduce the concerns of disease transmission, immune reaction, and may improve remodelling of the graft after implantation. The decellularised matrix can also be used as a matrix for tendon tissue engineering. One such sterilisation factor, Peracetic acid (PAA) has the advantage of not producing harmful reaction residues. The aim of this study was to evaluate the effects of PAA treatment and a cell removal procedure on the production of tendon matrix. Human patellar tendons, thawed from frozen were treated respectively as: Group 1, control with no treatment; Group 2, sterilised with PAA (0.1 % (w/v) PAA for 3 h) Group 3, decellularised (incubation successively in hypotonic buffer, 0.1 % (w/v) sodium dodecyl sulphate, and a nuclease solution); Group 4, decellularised and PAA sterilised. Histological analysis showed that no cells were visible after the decellularisation treatment. Theintegrity of tendon structure was maintained after decellularisation and PAA sterilisation, however, the collagen waveform was slightly loosened. No contact cytotoxicity was found in any of the groups. Determination of de-natured collagen showed no significant increase when compared with the control. This suggested that the decellularisation and sterilisation processing procedures did not compromise the major properties of the tendon. The sterilised, decellularised tendon could be suitable for clinical use. © 2013 Springer Science+Business Media Dordrecht.
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The introduction of metal-on-metal total disc replacements motivated studies to evaluate the effects of cobalt-chromium (CoCr) nanoparticles on cells of the dura mater. Porcine fibroblasts and epithelial cells isolated from the dura mater were cultured with clinically-relevant CoCr nanoparticles and the ions, generated by the particles over 24 h, at doses up to 121μmper cell. Cell viability and production of proinflammatory cytokines was assessed over 4 days. The capacity of the particles to induce oxidative stress in the cells was evaluated at 24 h. The CoCr particles and their ions significantly reduced the viability of the dural epithelial cells in a dose-dependent manner but not the fibroblasts. Both cell types secreted IL-8 in response to particle exposure at doses of 60.5 μm (epithelial cells) and 121 μm (fibroblasts, epithelial cells) per cell. No significant release of IL-6 was observed in both cell types at any dose. Reactive oxygen specieswere induced in both cell types at 50 μm per cell after 24 h exposure. The data suggested novel differences in the resistance of the dural epithelial cells and fibroblasts to CoCr nanoparticle/ion toxicity and demonstrated the inflammatory potential of the particles. The data contributes to a greater understanding of the potential biological consequences of the use of metal-on-metal total disc prostheses. © 2013 Elsevier Ltd.
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Vitamin E (VE) has been added to ultrahigh-molecular-weight polyethylene (UHMWPE) acetabular cups and tibial trays primarily to reduce oxidative damage to the polymer. The aim of this study was to investigate the relative wear rates of UHMWPE-containing VE compared with virgin UHMWPE. The ability of VE to reduce the amount of inflammatory cytokines produced from stimulated peripheral blood mononuclear cells (PBMNCs) was also investigated. Stimulation was achieved by exposure of PBMNCs to either lipoplysaccharide (LPS) or VE-containing UHMWPE (VE-UHMWPE). In the present study, results showed that the wear rates of UHMWPE with or without VE were not significantly different. Particles generated by UHMWPE with and without VE were not significantly different in size distribution. The production of osteolytic mediators, tumor necrosis factor-alpha, interleukin 1β (IL-β), IL-6, and IL-8 were significantly reduced in (PBMNCs) stimulated with either LPS + VE compared with LPS or VE-UHMWPE particles compared to virgin UHMWPE particles. This trend was also observed when VE was added as a liquid to UHMWPE wear particle-stimulated PBMNCs. The exact mechanism ofhow VE affects the release of inflammatory mediators from particle-stimulated macrophages is not yet understood. It is likely to involve the anti-inflammatory and/or antioxidant effects of VE. © 2013 Wiley Periodicals, Inc.
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Patients with cobalt chrome (CoCr) metal-on-metal (MOM) implants may be exposed to a wide size range of metallic nanoparticles as a result of wear. In this study we have characterised the biological responses of human fibroblasts to two types of synthetically derived CoCr particles [(a) from a tribometer (30 nm) and (b) thermal plasma technology (20, 35, and 80 nm)] in vitro, testing their dependence on nanoparticle size or the generation of oxygen free radicals, or both. Metal ions were released from the surface of nanoparticles, particularly from larger (80 nm) particles generated by thermal plasma technology. Exposure of fibroblasts to these nanoparticles triggered rapid (2 h) generation of reactive oxygen species (ROS) that could be eliminated by inhibition of NADPH oxidase, suggesting that it was mediated by phagocytosis of the particles. The exposure also caused a more prolonged, MitoQ sensitive production of ROS (24 h), suggesting involvement of mitochondria. Consequently, we recorded elevated levels of aneuploidy, chromosome clumping, fragmentation of mitochondria and damage to the cytoskeleton particularly to the microtubule network. Exposure to the nanoparticles resulted in misshapen nuclei, disruption of mature lamin B1 and increased nucleoplasmic bridges, which could be prevented by MitoQ. In addition, increased numbers of micronuclei were observed and these were only partly prevented by MitoQ, and the incidence of micronuclei and ion release from the nanoparticles were positively correlated with nanoparticle size, although the cytogenetic changes, modifications in nuclear shape and the amount of ROS were not. These results suggest that cells exhibit diverse mitochondrial ROS-dependent and independent responses to CoCr particles, and that nanoparticle size and the amount of metal ion released are influential.© 2013 Elsevier Ltd.
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Surgeons have used cryopreserved vascular allografts successfully for many years to treat arterial occlusive disease and to repair arterial aneurysms. Vascular allografts demonstrate high patency rates but contain viable cells, which may evoke a rejection response following implantation. Removing the cells could prevent such a response and negate the need for cryopreservation and ultra-low temperature storage. The objectives of the study were to characterize human common femoral arteries and develop a decellularization protocol with a view to the generation of biocompatible and biomechanically functional vascular grafts for use in vascular bypass and arteriovenous access. The arteries were decellularized by subjecting the tissue to a single freeze-thaw cycle followed by sequential incubation in hypotonic tris buffer and low concentration sodium dodecyl sulphate. Each artery was disinfected using 0.1% (v/v) peracetic acid. Histological analysis demonstrated a lack of cells following decellularization and confirmed the integrity of the tissue histioarchitecture and retention of major structural proteins. There was a>95% reduction in DNA levels. The acellular tissues and extracts were not cytotoxic to either mouse 3T3 or baby hamster kidney cells. Biomechanical properties were determined by burst pressure, compliance, and tensile tests, which confirmed the retention of biomechanical properties following decellularization. In conclusion the study has developed a suitable protocol for the removal of cells from human common femoral arteries without adversely affecting the biochemical or biomechanical properties. These properties indicate the potential use for acellular human common femoral arteries for vascular bypass or arteriovenous access.
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An in vitro study of the wear rates of the CHARITÉ lumbar total disc replacement (TDR).
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Human mesenchymal stem cells (hMSC) have numerous potential advantages over terminally differentiated cells and embryonic stem cells for use in tissue engineering applications. The aims of this study were to develop methods to test the hypothesis that hMSC could be differentiated using cyclic compressive strain alone. hMSC were successfully isolated, purified using D7-FIB antibody, cloned, and characterized. The cells were subsequently analyzed using fluorescence-activated cell sorting using a panel of antibodies and differentiation into multiple cell lineages. D7FIB-positive cells were then seeded into collagen-alginate scaffolds and subjected to 10% or 15% cyclic compressive strain for 4 out of 24 hours for up to 21 days in a bespoke servo-assisted displacement-controlled device. Cells were analyzed using adenosine triphosphate assay to determine cell number, live-dead cell assay, and quantitative real-time polymerase chain reaction at 7 and 21 days. Cloned D7-FIB-positive hMSCs showed evidence of differentiation to an osteogenic lineage under 10% cyclic compressive strain alone (core binding factor alpha 1 (CBFA-1) was significantly upregulated at 7 and 21 days by a factor of 18.3 and 32.2, respectively) and to an osteo-chondrogenic lineage under 15% cyclic compressive strain alone (increased expression of CBFA-1, Sox9, and aggrecan). A combination of a composite viscoelastic scaffold and controlled cyclic compressive strain may be useful for study of the differentiation of MSC.
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Nuclear magnetic resonance (NMR) spin-lattice relaxation rates were measured in bovine and porcine articular cartilage as a function of water content.
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Thrombosis of synthetic small-diameter bypass grafts remains a major problem. The aim of this article is to review the antithrombotic strategies that have been used in an attempt to reduce graft thrombogenicity.
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Several studies have reported biological vascular grafts to be more resistant to microbial infection than synthetic counterparts in vivo. Indeed, small intestinal submucosa (SIS) materials have previously been reported to be antimicrobial. The aim of this study was to assess the antimicrobial activity and the ability to resist biofilm formation of a novel acellular vascular graft and compare it to commercially available alternatives using a range of organisms: MRSA, MSSA, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa and Candida albicans. This was achieved using a modified disk diffusion assay and extraction of the materials into solution followed by minimum inhibitory concentration assays. To assess resistance to biofilm formation a novel biofilm assay was developed which compared the total colony forming units (CFU) recovered from each material and identification of the percentage of CFU which were loosely attached, residing within the biofilm orattached to the biomaterial. The results indicated a lack of antimicrobial activity for all materials tested, including SIS. The biological materials were more resistant to the formation of a biofilm compared to Dacron.
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An important requirement in thick, high-porosity scaffolds is to maximise cellular penetration into the interior and avoid necrosis during culture in vitro. Hitherto, reproducible control of the pore structure in nonwoven scaffolds has proved challenging. A new, channelled scaffold manufacturing process is reported based on water jet entanglement of fibres (hydroentangling) around filamentous template to form a coherent scaffold that is subsequently removed. Longitudinally-oriented channels were introduced within the scaffold in controlled proximity using 220µm diameter cylindrical templates. In this case study, channelled scaffolds composed of poly(l-lactic acid) were manufactured and evaluated in vitro. Environmental scanning electron microscope and µCT (X-ray microtomography) confirmed channel openings in the scaffold cross-section before and aftercell culture with human dermal fibroblasts up to 14 weeks. Histology at week 11 indicated that the channels promoted cell penetration and distribution within the scaffold interior. At week 14, cellular matrix deposition was evident in the internal channel walls and the entrances remained unoccludedby cellular matrix suggesting that diffusion conduits for mass transfer of nutrient to the scaffold interior could be maintained.
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Several studies have reported biological vascular grafts to be more resistant to microbial infection than synthetic counterparts in vivo. Indeed, small intestinal submucosa (SIS) materials have previously been reported to be antimicrobial. The aim of this study was to assess the antimicrobial activity and the ability to resist biofilm formation of a novel acellular vascular graft and compare it to commercially available alternatives using a range of organisms: MRSA, MSSA, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa and Candida albicans. This was achieved using a modified disk diffusion assay and extraction of the materials into solution followed by minimum inhibitory concentration assays. To assess resistance to biofilm formation a novel biofilm assay was developed which compared the total colony forming units (CFU) recovered from each material and identification of the percentage of CFU which were loosely attached, residing within the biofilm or attached to the biomaterial. The results indicated a lack of antimicrobial activity for all materials tested, including SIS. The biological materials were more resistant to the formation of a biofilm compared to Dacron.© 2012 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.
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Investigations into tissue-preserving orthopaedic treatments should consider the tribology of articular cartilage; where simulations using animal joints are a predominant choice. However, very few studies have investigated the differences between human and animal cartilage. The aim of the present study was to characterise the differences in geometry and mechanical properties of human, porcine, bovine and ovine articular cartilage. Creep indentation was performed on osteochondral plugs taken from the superior region of femoral heads of all these species. Cartilage thickness was measured via the resistive force change of a needle descending through cartilage and bone. A biphasic finite element model was used to derive equilibrium elastic modulus and permeability. Results showed that human cartilage was significantly thicker than all other species tested. A positive correlation was found between femoral head diameter and cartilage thickness when comparing between species of quadrupeds. Human cartilage had the largest equilibrium elastic modulus, which was significant when comparing against porcine and bovine. However, porcine cartilage had significantly lower permeability. Significant differences in geometry and mechanical properties of articular cartilage were found between all species tested. It is necessary to consider these variations when choosing animal tissue to represent human.© IMechE 2011.
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The purpose of this investigation was to develop a decellularised human dermis suitable for allografting. Samples of human skin were obtained from deceased donors and taken through a series of steps to remove all cellular material. The steps were: chemical removal of the epidermis, disinfection, lysing of cells in hypotonic buffer, a detergent treatment and a nuclease buffer to remove residual nuclear material. Histological preparations of the decellularised dermis produced were then investigated. In addition residual DNA content, structural strength, collagen denaturation, cytotoxicity and in vivo tissue reactivity following implantation in a murine model were examined. For all donors tested there was no change in morphology as viewed by light microscopy. Mean DNA removal was evaluated at 92.1 %. There were no significant changes in structural strength or evidence of collagen degradation. The tissue did not appear to be cytotoxic or elicit an immune response when implanted in the mouse model. A decellularised tissue has been developed that would appear to be suitable for a range of surgical procedures. © 2012 Springer Science+Business Media B.V.
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A new Stratified Approach For Enhanced Reliability (SAFER) pre-clinical simulation testing of joint prostheses has been described in a preceding paper in this volume. The application of SAFER in vitro simulation and testing to metal-on-metal bearings is described in this review paper. The review aims to provide further understanding of the reasons for, and causes of, increased wear in metal-on-metal hips in a proportion of patients. Variation in positioning (mal-positioning) of the head and cup in hip prostheses results in the head contacting the rim of the cup and producing increased wear. Variation in both translational and rotational positioning has been investigated. Variation in translational positioning of the centres of the head and cup, which is not detected on radiographs, is a frequent occurrence clinically and can result in a substantial increase in wear rate. The variation in translational positioning acts synergistically with variation in rotational positioning to produce substantial increases in wear. These recent findings are consistent with the wear mechanisms and formation of stripe wear reported for ceramic-on-ceramic bearings over a decade ago, and provide insight into the reasons for the variation and increases in the wear rate found clinically in metal-on-metal hips in specific patients, which may cause premature failure.© 2012 Elsevier Ltd.
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A new Stratified Approach For Enhanced Reliability (SAFER) pre-clinical simulation testing of joint prostheses is presented in this article. The aim of this approach is preclinical systematic testing of wear performance in the much wider envelope of conditions found clinically rather than relying only on the standard testing conditions that are currently used. The approach includes variations in surgical delivery, variations in kinematics, variations in the patient population and degradation of the biomaterial properties. Clinical experience of existing prostheses has been used to validate the new in vitro methods.© 2012 Elsevier Ltd.
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Rational molecular design of self- assembling peptide-based materials that spontaneously form self-supporting hydrogels shows potential in many healthcare applications. Binary peptides based on complementary charged sequences are developed, and the use of biophysical analysis and cell-based studies highlights that the charged interactions can influence the properties of peptide materials and ultimately affect biomaterial applications.
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Current international standards require pre-clinical testing of joint replacements under a standard walking cycle, with a standard patient, and with correctly positioned prostheses. These conditions do not replicate the wide variation in wear rates found with a wider range of conditions clinically. In this paper the effect of rotational and translational mal-positioning of the components of ceramic-on-ceramic and metal-on-metal hip prostheses on wear is reported. An elevated level of wear was found with translational mal-positioning and microseparation in ceramic-on-ceramic bearings, while a substantially elevated level of wear was found with both translational and rotational mal-positioning in metal-on-metal bearings.© 2012 .
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In this study we present a novel approach for predicting tissue growth within the pores of fibrous tissue engineering scaffolds. Thin nonwoven polyethylene terephthalate scaffolds were prepared to characterize tissue growth within scaffold pores, by mouse NR6 fibroblast cells. On the basis of measurements of tissue lengths at fiber crossovers and along fiber segments, mathematical models were determined during the proliferative phase of cell growth. Tissue growth at fiber crossovers decreased with increasing interfiber angle, with exponential relationships determined on day 6 and 10 of culture. Analysis of tissue growth along fiber segments determined two growth profiles, one with enhanced growth as a result of increased tissue lengths near the fiber crossover, achieved in the latter stage of culture. Derived mathematical models were used in the development of a software program to visualize predicted tissue growth within a pore. This study identifies key pore parameters that contribute toward tissue growth, and suggests models for predicting this growth, based on fibroblast cells. Such models may be used in aiding scaffold design, for optimum pore infiltration during the tissue engineering process.
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This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.
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The aim of this study was to develop a technique to decellularize a porcine cartilage bone construct with view to using this as a biological scaffold for cartilage substitution. The decellularization protocol applied freeze/thaw cycles; this was followed by cyclic incubation in hypotonic tris buffer and 0.1% (w/v) sodium dodecyl sulfate in hypotonic buffer plus protease inhibitors. Nucleases (RNase and DNase) were used to digest nucleic acids followed by disinfection using 0.1% (v/v) peracetic acid. Histological analysis confirmed the absence of visible cells within the decellularized tissue. DNA analysis revealed the near-complete removal of genomic DNA from the decellularized tissues. The decellularization process had minimal effect on the collagen content of the cartilage. However, there was a significant reduction in the glycosaminoglycan content in the decellularized tissues. There was no evidence of the expression of the major xenogeneic epitope, galactose-α-1,3-galactose. Biomechanical indentation testing of decellularized tissues showed a significant change in comparison to the fresh cartilage. This was presumed to be caused by the reduction in the glycosaminoglycan content. Biocompatibility of the acellular scaffold was determined using contact cytotoxicity assays and a galactosyltransferase knockout mouse model. Decellularized porcine cartilage tissue was found to exhibit favorable compatibility in both in vitro and in vivo tests. In conclusion, this study has generated data on the production of an acellular cartilage bone matrix scaffold for use in osteochondral defect repair. To our knowledge, this is the first study that has successfully removed whole cells and α-gal from xenogeneic cartilage and bone tissue.
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The use of nanoparticles in medicine is ever increasing, and it is important to understand their targeted and non-targeted effects. We have previously shown that nanoparticles can cause DNA damage to cells cultured below a cellular barrier without crossing this barrier. Here, we show that this indirect DNA damage depends on the thickness of the cellular barrier, and it is mediated by signalling through gap junction proteins following the generation of mitochondrial free radicals. Indirect damage was seen across both trophoblast and corneal barriers. Signalling, including cytokine release, occurred only across bilayer and multilayer barriers, but not across monolayer barriers. Indirect toxicity was also observed in mice and using ex vivo explants of the human placenta. If the importance of barrier thickness in signalling is a general feature for all types of barriers, our results may offer a principle with which to limit the adverse effects of nanoparticle exposure and offer new therapeutic approaches.
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Jean-Pierre Vidalain• TarikAït Si Selmi David Beverland • SteveYoung • Tim Board Jens Boldt • Scott Brumby (Editors) The CORAIL® Hip System ...
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This study investigated the factors influencing the oxygen metabolism of aortic valve interstitial cells (VICs). Porcine VICs in cell suspension at different passages, and adhered to coverslips at different confluencies, as well as fresh porcine valve leaflets, were incubated in an oxygen respiration chamber at 37 degrees C in Dulbecco's modified Eagle's medium. The consumption rates at different oxygen concentrations were evaluated based on the Michaelis-Menten equation, and the corresponding maximum consumption rate (V(max)) and the Michaelis-Menten equation constant K(m) were determined. In all cases, the oxygen consumption rate was relatively constant until the concentration dropped to 5% (v/v). The metabolic activity of VICs in terms of oxygen consumption was dependent upon their in vitro passage number and proliferation status. These findings will provide valuable input to the selection of VICs with respect to their age and proliferation status for tissue engineering applications, as well as important input parameters for developing computational models of oxygen transport and optimization of the bioreactor conditions for heart valve tissue engineering.
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Background: Clinical studies have indicated that the angular position of the acetabular cup may influence wear in metal-on-metal total hip bearings. A high cup angle in comparison to the anatomical position may lead to the head being constrained by the superior lateral surface and rim of the cup, thus potentially changing the location of the contact zone between the head and the cup. The aim of this study was to test the hypothesis that both a steep cup angle and a lateralized position of the head can increase head contact on the superior rim of the cup, with the consequence of increased wear.Methods: Hip-joint simulator studies of metal-on-metal bearings were undertaken with cup angles of 45 degrees and 55 degrees. The femoral head was either aligned to the center of the cup or placed in a position of microlateralization. Wear was measured gravimetrically over 5 million cycles.Results: A steep cup angle of 55 degrees showed significantly higher long-term steady-state wear than a standard cup angle of 450 (p<0.01). The difference was fivefold. Microlateralization of the head resulted in a fivefold increase in steady-state wear compared with a centralized head. The combination of a steep cup angle and a microlateralized head increased the steady-state wear rate by tenfold compared with a standard cup angle with a centralized head.Conclusions: These studies support the hypothesis that both an increased cup angle and a lateral head position increase wear in metal-on-metal hip prostheses.Clinical Relevance: These experimental studies support the recent clinical observations of increased wear with increased cup angle. Furthermore, the experimental studies indicate that the position and alignment of the femoral head may also be an important determinant of wear in vivo.
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objective of this study was to develop this novel tribological simulation and to use it to investigate the effects of choice of biomaterial and contact stress. It has been shown, that even under similar stress levels, materials may also make a difference and this will be investigated in future studies. The development of this tribological simulation of the natural knee is the first step towards the development of a whole knee model, which will be used to investigate the tribology of the whole knee in its healthy state, with hemiarthroplasty and with cartilage defect repairs, using different biomaterials such as hydrogels.
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Wear debris from metal on polyethylene joint replacements causes asceptic loosening as a result of an inflammatory reaction of macrophages to micron-sized particles. Metal on metal implants, which generate nanoparticles, have been reintroduced into surgical practise in order to avoid this problem. There is a current concern about possible long-term effects of exposure to metal particles. In this study, the cytotoxic and genotoxic effects of nanoparticles and micron-sized particles of cobalt chrome alloy have been compared using human fibroblasts in tissue culture. Nanoparticles, which caused more free radicals in an acellular environment, induced more DNA damage than micron-sized particles using the alkaline comet assay. They induced more aneuploidy and more cytotoxicity at equivalent volumetric dose. Nanoparticles appeared to disintegrate within the cells faster than microparticles with the creation of electron dense deposits in the cell, which were enriched in cobalt. The mechanism of cell damage appears to be different after exposure to nanoparticles and microparticles. The concept of nanotoxicology is, therefore, an important consideration in the design of future surgical devices. (C) 2007 Elsevier Ltd. All rights reserved
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The aim of this study was to produce a natural, acellular matrix from porcine bladder tissue for use as a scaffold in developing a tissueengineered bladder replacement. Full-thickness, intact porcine bladders were decellularised by distention and immersion in hypotonic buffer containing 0.1% (w/v) SDS and nuclease enzymes. Histological analysis of the resultant matrices showed they were completely acellular; that the major structural proteins had been retained and that there were some residual poorly soluble intracellular proteins. The amount of DNA per mg dry weight of fresh porcine bladder was 2.8 (70.1) mg/mg compared to 0.1 (70.1) mg/mg in decellularised bladder and biochemical analysis showed proportional differences in the hydroxyproline and glycosaminoglycan content of the tissue before and after decellularisation. Uniaxial tensile testing indicated that decellularisation did not significantly compromise the ultimate tensile strength of the tissue. There was, however, an increase in the collagen and elastin phase slopes indicating decreased extensibility. Cytotoxicity assays using porcine smooth muscle cell cultures excluded the presence of soluble toxins in the biomaterial. In summary, a full-thickness natural acellular matrix retaining the major structural components and strength of the urinary bladder has been successfully developed. The matrix is biocompatible with bladder-derived cells and has potential for use in urological surgery and tissue-engineering applications.
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Although bovine pericardium has been used extensively in cardiothoracic surgery, its degeneration and calcification are important limiting factors in the continued use of this material. The study aims were to decellularize bovine pericardium and to compare the biomechanical properties of fresh and decellularized bovine pericardia to those treated with different concentrations of glutaraldehyde (GA).
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Synovial joints are known for their remarkable load bearing capacity while maintaining very low friction and wear over their lifetime. Although other joint components play a role in the lubrication mechanisms, articular cartilage with its unique biphasic characteristics is believed to be largely responsible for the observed low friction properties in these joints. In the current study, a pin-on-plate machine was used in a cartilage loaded against cartilage configuration to study the effect of load variation on cartilage friction properties. Both static and dynamic loading conditions were tested under three different contact stress levels - 0.2, 0.3, and 0.4 MPa with phosphate buffered saline as the lubricant. Indentation experiments were performed and modelled through finite-element (FE) method to determine the biphasic material properties of cartilage. A second FE model adopting the derived biphasic properties of cartilage was then used to simulate the static friction tests and determine the interstitial fluid load support in the cartilage tissue under different loads. An increase in contact stress was found to reduce the friction levels significantly (p<0.05) between the articulating cartilage surfaces in both dynamic and static models, while the percentage of fluid load support within the cartilage was almost identical. It was shown that while interstitial fluid pressurization of cartilage plays the major role, another extraneous factor or mechanism that influences the friction between cartilage surfaces is needed to completely explain the results observed.
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Crosslinked ultra-high molecular weight polyethylene (UHMWPE) has been developed and introduced into clinical practice in order to reduce wear in the hip. Zero wear of highly crosslinked UHMWPE in vitro has been reported by some groups using lubricants with high concentrations of serum proteins in hip simulators. In contrast, some clinical studies have reported finite wear rates. The aim of this study was to compare the wear rates, wear surfaces, and wear debris produced by UHMWPE with different levels of crosslinking in a hip joint simulator, with lower, more physiologically relevant concentrations of protein in the lubricant. The UHMVVPEs were tested in the Leeds ProSim hip joint simulator against cobalt-chromium (CoCr) femoral heads. The wear particles were isolated and imaged using a field emission gun scanning electron microscope (FEGSEM) at high resolution. The highly crosslinked UHMWPEs had significantly lower wear volumes than the non-crosslinked UHMWPEs. No significant difference was found in the percentage number and percentage volume of the particles in different size ranges from any of the materials. They had similar values of specific biological activity The functional biological activity (FBA), which takes into account the wear volume and specific biological activity, showed that the highly crosslinked UHMWPEs had lower FBAs due to their lower wear volume.
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Tissue engineering offers a promising solution to the replacement of anterior cruciate ligament. A decellularized porcine patella tendon scaffold was produced by immersing whole tissues sequentially in hypotonic buffer, 0.1% (w/v) sodium dodecyl sulfate (SDS) in hypotonic buffer, and nuclease solution prior to sterilization with 0.1% (w/v) peracetic acid. Initial studies revealed that primary human tenocytes would attach to, but failed to penetrate into, the decellularized scaffold. A novel use of ultrasonication was therefore developed to allow extrinsic cells to migrate into the acellular scaffold. Various intensities of ultrasonication were tested in order to produce a microscopically more open porous matrix without damaging the overall architecture of the scaffold. Ultrasonication treatment with the intensity of 360 W and a pulse time of 1 s for a total of 1 min was found to be the optimal treatment. This process did not have a significant effect upon the biochemical constituents (collagen, glycosaminoglycans), nor did it denature the collagen. Moreover, the acellular sonicated scaffold retained the essential biomechanical characteristics of the native tissue. Primary human tenocytes penetrated into the center of whole acellular sonicated scaffolds over a 3-week period in static culture. The viability of the cells in the center of the scaffold (depth of circa 2.5 mm) was, however, compromised. To circumvent the problem of nutrient limitation, acellular sonicated scaffolds were split into fascicular scaffolds (500 mum thick). Cells seeded onto the fascicular scaffolds penetrated throughout the scaffold and remained viable after 3 weeks of culture. This study has shown that an acellular biocompatible tendon scaffold can be produced using 0.1% (w/v) SDS and that ultrasonication can provide a novel method to enhance the recellularization of decellularized natural tissues.
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The performance of novel ceramic-on-metal bearing couples has been compared with metal-on-metal and ceramic-on-ceramic bearing couples in laboratory and short-term clinical studies. Laboratory studies compared ceramic-on-metal with metal-on-metal and ceramic-on-ceramic bearings with diameters of 28 and 36 mm under standard conditions and under adverse conditions with head loading on the rim of the cup. Clinical studies compared metal ion levels in ceramic-on-metal with metal-on-metal, ceramic-on-ceramic, and ceramic-on-polyethylene bearings in a randomized prospective study. In the laboratory studies, friction, wear, and ion levels were lower in ceramic-on-metal bearings compared with metal-on-metal, with results similar to ceramic-on-ceramic couples. Under adverse conditions and rim loading, all bearings showed increased wear with lower wear and absence of stripe wear in ceramic-on-metal compared with metal-on-metal bearings. Short-term studies in 31 patients at 6 months revealed lower metal ion levels (cobalt and chromium) in those with ceramic-on-metal compared with metal-on-metal bearings.
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Background Propionibacterium acnes has been strongly implicated in inflammatory acne. However, its role in the disease is unclear. It has been hypothesized that an immune response to P. acnes and/or P. acnes heat shock proteins (HSPs) may play a role in the pathogenesis of inflammatory acne. Objectives To compare the cell-mediated immune response to P. acnes and HSPs in acne patients, nonacne controls and individuals with resolved acne. Methods The proliferative response of peripheral blood mononuclear cells (PBMC) from acne patients, resolved acne donors and healthy controls to P. acnes, P. acnes HSP60 and HSP70, and mycobacterial HSPs was assessed by lymphocyte transformation assay (LTA). The proliferative response of purified CD4+ T cells was further analysed by limiting dilution analysis (LDA). Contingency tables (G-test) were used to analyse the proportion of individuals in each group showing a positive proliferative response for LTA or data fitting single-hit kinetics for LDA. Results Analysis of stimulation of PBMC with P. acnes, P. acnes HSP60 and HSP70 in the LTA showed the proportion of positive responders to be independent of subject group. However, the proportion of acne patients with a positive response to mycobacterial HSPs was significantly higher than those for the other subject groups. Analysis of LDA data showed the proportion of resolved donors with responses to P. acnes fitting the single-hit kinetics model to be significantly lower than those of the other groups. There were no significant differences in responses to other antigens. Conclusions The significantly lower proportion of resolved donors demonstrating a single-hit kinetics response to P. acnes by LDA may represent negative regulation of the CD4+ T-cell response to P. acnes in these subjects.
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The number of peri-prosthetic femoral fractures in todays aging population is increasing. Fractures of the femur may occur intra-operatively or post-operatively, either at the tip of the femoral stem or in areas where cortical thinning has occurred. This is further enhanced if the patient is osteoporotic. These fractures can be difficult to treat due to the complexity of the mechanical status and further complicated biologically by the presence of ultra high molecular weight polyethylene (UHMWPE) wear debris that can migrate from the articulating surface of the prosthesis to the fracture site. In this study, the effect of PE wear debris on the healing of osteoporotic fractures was investigated using a rodent ovariectomised (OVX) model. One hundred female Sprague Dawley rats were subjected to either bilateral OVX or Sham surgery at 10 weeks of age. Three months later, a closed fracture was created in the right femur using a 3-point bending device and an intramedullary k-wire for fixation. Animals were divided into 4 groups (n=3-8). A 0.2ml suspension of Ceridust (PE wear debris), hyaluronic acid&saline was injected directly into the fracture site at the time of surgery into half the animals. Control animals received comparable injections excluding the Ceridust. Animals were sacrificed at 1, 3 and 6 weeks. The OVX animals had a greater body weight compared to the Sham animals (p<0.05). DEXA analysis revealed that the presence of PE wear debris had no effect on the BMD within the fracture callus at either time-point. Mechanical analysis revealed an increase in bone strength with time. The presence of PE had no statistical effect upon the ultimate peak load or stiffness, however there was a trend towards increased peak load in the PE groups at 3 and 6 weeks following 3-months oestrogen deficiency. Histological analysis showed that the control OVX fractures had more cartilage development than the Sham group at 3 weeks and delayed remodeling at 6 weeks. The PE treated OVX group showed more fibrous tissue at the fracture gap and inside the diaphysis tunnel showing further delayed healing compared to the Sham group with PE. In this study after 3-months of oestrogen deficiency, no differences in mechanical or BMD was found in the fractured limbs between the PE -treated and non-PE treated fractures. However, histologically, PE wear debris induced fibrous tissue at the fracture site which further delayed the healing process. More care should be taken with aged patients receiving revision surgery, as these patients become harder to manage when fractured due to the influence of both osteoporosis and PE wear debris.
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The performance of novel ceramic-on-metal bearing couples has been compared with metal-on-metal and ceramic-on-ceramic bearing couples in laboratory and short-term clinical studies. Laboratory studies compared ceramic-on-metal with metal-on-metal and ceramic-on-ceramic bearings with diameters of 28 and 36 mm under standard conditions and under adverse conditions with head loading on the rim of the cup. Clinical studies compared metal ion levels in ceramic-on-metal with metal-on-metal, ceramic-on-ceramic, and ceramic-on-polyethylene bearings in a randomized prospective study. In the laboratory studies, friction, wear, and ion levels were lower in ceramic-on-metal bearings compared with metal-on-metal, with results similar to ceramic-on-ceramic couples. Under adverse conditions and rim loading, all bearings showed increased wear with lower wear and absence of stripe wear in ceramic-on-metal compared with metal-on-metal bearings. Short-term studies in 31 patients at 6 months revealed lower metal ion levels (cobalt and chromium) in those with ceramic-on-metal compared with metal-on-metal bearings. Level of Evidence: Level II, therapeutic study. See the Guidelines for Authors for a complete description of levels of evidence.© 2007 Lippincott Williams&Wilkins, Inc.
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There is currently considerable interest in the wear debris and osteolytic potential of different types of bearings used in total joint replacements. The aim of this study was first to characterize the wear and wear particles generated from two different grades of UHMWPE acetabular cups with different levels of crosslinking in a hip joint simulator. Secondly, the results for the polyethylenes were compared to an alumina ceramic-on-ceramic hip prosthesis. The wear rates of the two noncrosslinked material types were very similar at 49 8 mm3 per million cycles for the GUR 1020 and 45.6 1.4 mm3 per million cycles for the GUR 1050. Moderate crosslinking 4 MRad significantly P 0.05 reduced the wear rate of the GUR 1020 material by 30 % to 35 9 mm3 per million cycles. High levels of crosslinking of GUR 1050 10 MRad produced a highly significant P 0.01 80 % reduction in wear volume. Although reduced wear volumes were observed with moderate levels of crosslinking for the 4 MRad GUR 1020 material, little benefit was conveyed by crosslinking, in terms of predicted overall biocompatibility and estimated osteolytic potential. Introducing high levels of crosslinking 10 MRad into the GUR 1050 material reduced wear and osteolytic potential by up to five-fold compared to the other GUR 1050 materials. However, compared to the noncrosslinked and moderately crosslinked GUR 1020 materials, the highly crosslinked GUR 1050 UHMWPE had only a two-fold lower osteolytic potential. The alumina ceramic-on-ceramic hip prostheses produced extremely low wear rates under both standard and microseparation simulation conditions, and consequently the osteolytic potential of the alumina bearings was estimated to be 20-fold lower than the highly crosslinked polyethylene.
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Tribology of Hip Joints from Natural Hip Joints, Cartilage Substitution, Artificial Replacements to Cartilage Tissue Engineering
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A clinical need exists for an immunologically compatible surgical patch with a wide range of uses including soft tissue replacement, body wall repair, cardiovascular applications, and as a wound dressing. This study aimed to produce an acellular matrix from human amniotic membrane for future assessment as a surgical patch and a delivery system for epithelial cells. A novel detergent-based protocol was modified to remove all cellular components from amnion to render it non-immunogenic. Amnion was harvested within 24 h after elective caesarean section (n = 12). One sample group remained fresh, whereas the other was treated with 0.03% (w/v) sodium dodecyl sulphate, with hypotonic buffer and protease inhibitors, nuclease treatment, and terminal sterilization, using peracetic acid (0.1% v/v). Fresh and treated amnion was analyzed histologically for the presence of cells, deoxyribonucleic acid (DNA), collagen, glycosaminoglycans (GAGs), and elastin. Quantitative analysis was performed to determine levels of GAGs, elastin, hydroxyproline, denatured collagen, and DNA. The biomechanical properties of the membrane were determined using uniaxial tensile testing to failure. Histological analysis of treated human amnion showed complete removal of cellular components from the tissue; the histoarchitecture remained intact. All major structural components of the matrix were retained, including collagen type IV and I, laminin, and fibronectin. Differences were observed between fresh and decellularized amnion in matrix hydroxyproline (34.7 microg/mg vs 49.7 microg/mg), GAG (42.5 microg/mg vs 85.4 microg/mg), denatured collagen (2.2 microg/mg vs 1.7 microg/mg), and elastin (359.2 microg/mg vs 490.8 microg/mg) content. DNA content was diminished after treatment. Acellular matrices were biocompatible, cells grew in contact, and there was no decrease in cell viability after incubation with soluble tissue extracts. In addition, no significant reduction in ultimate tensile strength, extensibility, or elasticity was found after decellularization. Removal of the cellular components should eliminate immunological rejection. The resulting matrix was biocompatible in vitro and exhibited no adverse effects on cell morphology or viability.
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The wear and wear debris from rotating-platform mobile-bearing knees and fixed-bearing knees were compared in knee joint-simulator studies. The wear rate of the fixed-bearing knees was found to increase as the kinematics were increased because of an increase in internal-external rotation and an increase in anterorposterior (AP) translation. The wear rate of the rotating-platform mobile-bearing knees was found to be significantly lower than that of the fixed-bearing knees. The rotating-platform mobile-bearing knee was able to decouple the complex kinematics to pure rotation at the inferior tibial articulating surface and linear flexion-extension and AP sliding at the superior femoral articulating interface, substantially reducing cross-shear and wear. No difference was found in the wear debris between the rotating-platform and fixed-bearing knees. This resulted in a substantially reduced functional biological activity or osteolytic potential for the rotating-platform mobile-bearing knees due to the lower wear rates.
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A brief introduction to artificial hip joints is followed by a detailed review of both the biotribology of the bearing surfaces and the biological responses to the wear debris for evaluating their performances. Various types of artificial hip joints with different bearing surfaces are considered, including ultrahigh molecular weight polyethylene against metal or ceramic; metal-on-metal and ceramic-on-ceramic. Future developments of artificial hip joints are discussed.
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There is currently considerable interest in the wear debris and osteolytic potential of different types of bearings used in total joint replacements. The biological activity of the wear debris is dependent on the size and volume of the particles produced. Wear volume also plays an important role in the functional biological activity of a joint replacement. In vitro studies have shown that crosslinking of ultra high molecular weight polyethylene (UHMWPE) acetabular cups and tibial trays produces a reduction in wear volume, and crosslinking has now been introduced clinically for both types of prostheses. Previous studies have identified both micron and submicron-sized polyethylene wear particles. The aim of this study was to characterize the wear and wear particles generated from moderately crosslinked GUR 1,020 GVF UHMWPE acetabular cups and tibial trays in hip and knee joint wear simulators down to 10 nanometers in size. The wear rates of the two prosthesis types were very similar at 25.6 +/- 5.3 mm(3) per million cycles for the hip prostheses and 22.75 +/- 5.95 mm(3) per million cycles for the knee prostheses. Nanometer-sized wear particles were isolated and characterized from both hip and knee simulator lubricants for the first time. Significantly higher numbers (p<0.05) of particles in the nanometer (<0.1 microm) size range were produced by the hip prostheses compared to the knee prostheses. The knee prostheses produced larger particles, with the mode of particle size in the 0.1-1.0 microm size range, compared to<0.1 microm size range for the hip prostheses. In addition, the knee prostheses produced a greater volumetric concentration of wear particles in the 1.0-10 microm size range, and consequently lower specific biological activity and functional biological activity indices. These results indicated that the knee prostheses had a lower osteolytic potential compared to the hip prostheses.
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There is currently considerable interest in the wear debris and osteolytic potential of different types of bearings used in total joint replacements. The aim of this study was first to characterize the wear and wear particles generated from two different grades of UHMWPE acetabular cups with different levels of crosslinking in a hip joint simulator. Secondly, the results for the polyethylenes were compared to an alumina ceramic-on-ceramic hip prosthesis. The wear rates of the two noncrosslinked material types were very similar at 49 +/- 8 mm(3) per million cycles for the GUR 1020 and 45.6 +/- 1.4 MM3 per million cycles for the GUR 1050. Moderate crosslinking (4 MRad) significantly (P<0.05) reduced the wear rate of the GUR 1020 material by 30 % to 35 +/- 9 MM3 per million cycles. High levels of crosslinking of GUR 1050 (10 MRad) produced a highly significant (P<0.01) 80 % reduction in wear volume. Although reduced wear volumes were observed with moderate levels of crosslinking for the 4 MRad GUR 1020 material, little benefit was conveyed by crosslinking, in terms of predicted overall biocompatibility and estimated osteolytic potential. Introducing high levels of crosslinking (10 MRad) into the GUR 1050 material reduced wear and osteolytic potential by up to five-fold compared to the other GUR 1050 materials. However, compared to the noncrosslinked and moderately crosslinked GUR 1020 materials, the highly crosslinked GUR 1050 UHMWPE had only a two-fold lower osteolytic potential. The alumina ceramic-on-ceramic hip prostheses produced extremely low wear rates under both standard and microseparation simulation conditions, and consequently the osteolytic potential of the alumina bearings was estimated to be>20-fold lower than the highly crosslinked polyethylene.
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This work is concerned with improving nonwoven scaffold design for the tissue engineering of the anterior cruciate ligament. When designing a scaffold two important design criteria to consider are scaffold's internal structure and biocompatibility, both of which are addressed in this paper.The role of a scaffold is to provide a framework for cells to attach, proliferate and secrete extra cellular matrix. The scaffold also acts as a template, directing the growth of cells and newly formed tissue. It is the scaffold's internal structure, together with polymer surface chemistry and morphology, which directly influence the cellular activities that lead to tissue formation.With regard to scaffold's internal structure, structural parameters are discussed in relation to specific scaffold function; for example the effect of scaffold pore-size on cell proliferation, migration and nutrient supply. Another structural factor discussed is the role of fibre orientation as a means of guiding and organising new tissue growth.With the aim of creating a scaffold of optimum design, for the tissue engineering of the anterior cruciate ligament, nonwoven scaffolds of differing structure have been constructed. In order to understand the relationship between manufacturing method and scaffold structure characterisation techniques have been employed to analyse the structural parameters of these scaffolds. Obtained scaffold structural properties are discussed in relation to manufacturing method.Regarding the second scaffold criteria biocompatibility tests have been conducted by the authors on a range of generic fibre types. The results of these tests are provided in the form of cell attachment, with reference to fibre morphology.
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Objectives: The mechanisms by which antibiotics induce a post-anti biotic effect in susceptible bacteria are poorly understood. To explore the mechanisms more fully we examined the recovery of macromolecular synthesis in Escherichia coli during gentamicin- and rifampicin-induced post-antibiotic effects. Methods: E. coli ATCC 25922 was exposed to rifampicin and to gentamicin at 5x MIC for 60 min to induce post-antibiotic effects. The antibiotics were then removed from the culture medium by washing the cells. The rates of DNA, RNA and protein synthesis during the post-antibiotic effect and recovery periods were subsequently determined by measuring the incorporation of radiolabelled uridine, thymidine and leucine into trichloroacetic acid precipitable material. Results: Recovery of E. coli ATCC 25922 from the rifampicin-induced post-antibiotic effect coincided with the recovery of RNA and protein synthesis. Recovery from the gentamicin-induced post-antibiotic effect coincided with the recovery of protein synthesis. Conclusions: These data support the hypothesis that antibiotic molecules retained in the cell mediate the post-antibiotic effect by suppressing the biochemical activity of their molecular targets.
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There is renewed interest in metal-on-metal (MOM) total hip replacements (THRs), however, variable wear rates have been observed clinically. It is hypothesised that changes in soft tissue tensioning during surgery may alter loading of THRs during the swing phase of gait leading to changes in fluid film lubrication, friction and wear. This study aimed to assess the effect of swing phase load on the lubrication, friction and wear of MOM hip replacements. Theoretical lubrication modelling was carried out using elastohydrodynamic theory. All the governing equations were solved numerically for the lubricant film thickness between the articulating surfaces under the transient dynamic conditions with low and high swing phase loads. Friction testing was completed using a single axis pendulum simulator, simplified loading cycles were applied with low and high swing phase loads. MOM hip replacements were tested in a hip simulator, modified to provide different swing phase loading regimes; a low (100 N) and a high load (as per ISO 14242-1; 280 N). Results demonstrated that the performance of MOM bearings is highly dependent on swing phase load. Hence, changes in the tension of the tissues at surgery and variations in muscle forces may increase swing phase load, reduce lubrication, increase friction and accelerate wear. This may explain some of the variations that have been observed with clinical wear rates.
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The lubricating abilities of different formulations of high molecular weight hyaluronic acid (HA), dipalmitoyl phosphatidylcholine (DPCC) and mixtures of both HA and DPCC were assessed in an in vitro model. Levels of start-up friction were determined using an osteoarthritis (OA) damaged human cartilage model set within a specially designed friction rig. To examine the long term benefits of HA, the extent of penetration of HA into cartilage tissue was investigated using fluorescently labelled HA and confocal microscopy. It was found that in this model, all formulations of HA and the majority of DPCC lubricants reduced friction (HA 5 and 10 mgml 1, DPPC 200 mgml 1 reductions of 51.9%, 46.7% and 46.5% respectively), compared to a Ringers solution control. Lubrication was found not to be concentration dependant for HA formulations, but concentration was key for DPCC lubrication (100mg ml 1 reduced friction by only 15.9%). By combining HA and DPCC (HA/DPPC; 5mgml 1/100mg ml 1 and 10mg ml 1/200mg ml 1), a further improvement was noted (69.5% and 61.9%, respectively) as the mean levels of friction were reduced by up to a further 17% than the most effective individual formulation (HA 5mgml 1). Penetration of HA into bovine cartilage by up to 300 mm from the surface was observed over a 48 h period. It was observed that HA specifically targeted the chondrocytes as it was primarily found within the lacunae surrounding the cells.
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The self-assembly of peptides is explored as an alternative route towards the development of new injectable joint lubricants for osteoarthritis (OA). The versatility of the peptide chemistry allows the incorporation of behavior reminiscent of hyaluronic acid (HA), while the triggered in situ self-assembly provides easy delivery of the samples by injection due to the low viscosity of the peptide solutions (that are initially monomeric). Using design criteria based on the chemical properties of HA, a range of de novo peptides were prepared with systematic alterations of charge and hydrophilicity that self-assembled into nematic fluids and gels in physiological solution conditions. The frictional characteristics of the peptides were evaluated using cartilage on cartilage sliding contacts along with their rheological characteristics. Peptide P(11)-9, whose molecular, mesoscopic, and rheological properties most closely resembled HA was found to be the most effective lubricant amongst the peptides. In healthy static and dynamic friction testing (corresponding to healthy joints) P(11)-9 at 20-40 mg/mL performed similar to HA at 10 mg/mL. In friction tests with damaged cartilage (corresponding to early stage OA) P(11)-9 was a less efficient lubricant than HA, but still the best among all the peptides tested. The results indicate that de novo self-assembling peptides could be developed as an alternate therapeutic lubricant for early stage OA.
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Basic principles of engineering tribology are briefly reviewed, in terms of surface metrology, contact mechanics, friction, lubrication and wear. In each of these topics, applications to artificial hip joints are discussed in detail. Various artificial hip joints with different bearing material combinations are considered, including ultra-high molecular weight polyethylene against metal or ceramic, metal-on-metal and ceramic-on-ceramic.
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The tribological performance and biological activity of the wear debris produced has been compared for highly cross-linked polyethylene, ceramic-on-ceramic, metal-on-metal, and modified metal bearings in a series of in vitro studies from a single laboratory. The functional lifetime demand of young and active patients is 10-fold greater than the estimated functional lifetime of traditional polyethylene. There is considerable interest in using larger diameter heads in these high demand patients. Highly cross-linked polyethylene show a four-fold reduction in functional biological activity. Ceramic-on-ceramic bearings have the lowest wear rates and least reactive wear debris. The functional biological activity is 20-fold lower than with highly cross-linked polyethylene. Hence, ceramic-on-ceramic bearings address the tribological lifetime demand of highly active patients. Metal-on-metal bearings have substantially lower wear rates than highly cross-linked polyethylene and wear decreases with head diameter. Bedding in wear is also lower with reduced radial clearance. Differential hardness ceramic-on-metal bearings and the application of ceramic-like coatings reduce metal wear and ion levels.
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In recent years there has been renewed interest in metal-on-metal. (MOM) bearings for both total hip replacement and surface replacement hip arthroplasty. Short-term clinical results have been encouraging; with low wear rates and few prostheses requiring revision. This review concentrates on the factors that affect the wear of all metal devices such as specification of the alloy, processing techniques, head diameter, clearance between the components, lubrication regime, loading and surface finish. The concerns associated with MOM bearings are also discussed. These include wear particle release and dissemination, and elevated metal ion levels, which may lead to cytotoxicity, hypersensitivity and genotoxicity. (C) 2005 Elsevier Ltd. All rights reserved.
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Background: Tissue engineered heart valves have the potential to overcome the limitations of present heart valve replacements. This study investigated the biocompatibility and recellularization potential of an acellular porcine valve matrix. Methods: Samples of acellular porcine matrix were tested for biocompatibility with cultured porcine dermal fibroblasts and smooth muscle cells using contact and extract cytotoxicity assays. These same cell types were then used to statically seed SDS-decellularized porcine valve leaflets for 24 hours to 4 weeks. The degree of cell attachment was observed using SEM and cell migration assessed by histology. Results: No contact inhibition of cell growth or adverse morphological changes were seen following cell contact with the acellular valve matrix, and limited soluble extract toxicity was found. Both porcine cell types readily attached to the decellularized leaflet matrices. Only the porcine SMC proliferated and migrated throughout the leaflet matrix over the 4 weeks of culture. Conclusion: No contact cytotoxicity was seen and soluble extracts had limited effect. Porcine fibroblasts and SMC readily attached to the SDS-decellularized valve leaflet matrix but only the SMC migrated throughout the matrix. This indicated that the acellular porcine valve matrix was biocompatible in vitro and SMC may be the preferred cell type for leaflet recellularization.
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Despite the early promise of tissue engineering, researchers have faced challenges in regenerating tissues that serve a predominantly mechanical function. Current approaches investigate the use of bioactive or bioresorbable matrices, which rely on the appropriate cellular response in vivo, with the intention of developing biological and physical functionality after implantation. A limitation of this approach is the variability of the patient response in terms of resorption, recellularisation and regeneration, which can result in development of inappropriate implant properties. A step further is the utilisation of passive in vitro recellularisation prior to implantation. The success of this approach is also limited since cell differentiation and tissue remodelling do not progress physiologically. Functional tissue engineering is a more promising approach that employs appropriate in vitro-propagated cells to cellularise scaffolds, coupled with appropriate physical conditioning, with a view to establishing tissue functionality prior to implantation. Studies have produced considerable evidence suggesting that physical stimuli may affect gene expression and significantly increase the biosynthetic activity in a range of different cells. The fact that physical stimuli can modulate cell function has motivated the development of functional simulations systems to recellularise tissues in vitro by exposing them to physical stimuli. The development of such technologies will not only provide tissue engineering solutions, but will also provide important in vitro model systems for the enhancement of understanding into mechanotransduction. This review focuses on how mechanotransduction dictates cell function, as well as on the bioreactor systems that have been developed to investigate this phenomenon.
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Background and aims of the study: Tissue-engineered heart valves offer the potential to deliver a heart valve replacement that will develop with the young patient. The present authors' approach is to use decellularized aortic heart valves reseeded in vitro or in vivo with the patient's own cells. It has been reported that treatment of porcine aortic valve leaflets with 0.1% (w/v) sodium dodecyl sulfate (SDS) in hypotonic buffer produced complete leaflet acellularity without affecting tissue strength. The present study aim was to investigate the effect of an additional treatment incorporating 1.25% (w/v) trypsin and 0.1% (w/v) SDS on the biomechanics and hydrodynamics of the aortic root. This treatment has been shown to produce decellularization of both the aorta and valve leaflets.Methods: Fresh porcine aortic roots were treated to reduce the thickness of their aortic wall, and incubated in hypotonic buffer for 24 h. The leaflets were masked with agarose gel, and the aorta was treated with 1.25% (w/v) trypsin for 4 h at 37 degrees C. The trypsin and agarose were removed and the roots incubated with 0.1% (w/v) SDS in hypotonic buffer for 24 h. Fresh and treated circumferential and axial aortic specimens were subjected to uniaxial tensile testing, while intact porcine aortic roots were subjected to dilation and pulsatile flow testing.Results: Decellularized aortic wall specimens demonstrated significantly decreased elastin phase slope and increased transition strain compared to the fresh control. However, the treatment did not impair tissue strength. Decellularized intact roots presented complete leaflet competence under systemic pressures, increased dilation and effective orifice areas, reduced pressure gradients, physiological leaflet kinematics and reduced leaflet deformation.Conclusion: The excellent leaflet kinematics and hydrodynamic performance of the decellularized roots, coupled with the excellent biomechanical characteristics of their aortic wall, form a promising platform for the creation of an acellular valve scaffold with adequate mechanical strength and functionality to accommodate dynamic cell repopulation in vitro or in vivo. This approach can be used for both allogeneic and xenogeneic tissue matrices.
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The mechanism of the postantibiotic effect (PAE) was examined in Escherichia coli. Drugs exhibited longer-lasting PAEs in an acrAB mutant, suggesting that intracellular drug concentrations influence the duration of the PAE. With specific assays for tetracycline and erythromycin, a direct link between intracellular persistence of antibiotics and maintenance of the PAE was established.
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Background and aims of the study: Tissue-engineered heart valves offer the potential to deliver a heart valve replacement that will develop with the young patient. The present authors' approach is to use decellularized aortic heart valves reseeded in vitro or in vivo with the patient's own cells. It has been reported that treatment of porcine aortic valve leaflets with 0.1% (w/v) sodium dodecyl sulfate (SDS) in hypotonic buffer produced complete leaflet acellularity without affecting tissue strength. The present study aim was to investigate the effect of an additional treatment incorporating 1.25% (w/v) trypsin and 0.1% (w/v) SDS on the biomechanics and hydrodynamics of the aortic root. This treatment has been shown to produce decellularization of both the aorta and valve leaflets. Methods: Fresh porcine aortic roots were treated to reduce the thickness of their aortic wall, and incubated in hypotonic buffer for 24 h. The leaflets were masked with agarose gel, and the aorta was treated with 1.25% (w/v) trypsin for 4 h at 37 degrees C. The trypsin and agarose were removed and the roots incubated with 0.1% (w/v) SDS in hypotonic buffer for 24 h. Fresh and treated circumferential and axial aortic specimens were subjected to uniaxial tensile testing, while intact porcine aortic roots were subjected to dilation and pulsatile flow testing. Results: Decellularized aortic wall specimens demonstrated significantly decreased elastin phase slope and increased transition strain compared to the fresh control. However, the treatment did not impair tissue strength. Decellularized intact roots presented complete leaflet competence under systemic pressures, increased dilation and effective orifice areas, reduced pressure gradients, physiological leaflet kinematics and reduced leaflet deformation. Conclusion: The excellent leaflet kinematics and hydrodynamic performance of the decellularized roots, coupled with the excellent biomechanical characteristics of their aortic wall, form a promising platform for the creation of an acellular valve scaffold with adequate mechanical strength and functionality to accommodate dynamic cell repopulation in vitro or in vivo. This approach can be used for both allogeneic and xenogeneic tissue matrices.
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Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. The study aim was to investigate the biocompatibility and recellularization potential of an acellular porcine valve matrix.
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The osteolysis associated with conventional polyethylene on metal total joint replacements is associated with the formation of an inflamed periprosthetic membrane rich in macrophages, cytokines and implant-derived wear particles. There is a wealth of evidence to indicate that the presence and activation of macrophages in the periprosthetic tissues around joint replacements is stimulated by UHMWPE particles. Particles within the size range 0.1-1.0 mum have been shown to be the most reactive. Animal studies have provided increasing evidence that, of the milieu of cytokines produced by particle-stimulated macrophages, TNF-alpha is a key cytokine involved in osteolysis. Recent advances in the understanding of the mechanisms of osteoclastogenesis and osteoclast activation at the cellular and molecular level have indicated that bone marrow-derived macrophages may play a dual role in osteolysis associated with total joint replacement. Firstly, as the major cell in host defence responding to UHMWPE particles via the production of cytokines and secondly as precursors for the osteoclasts responsible for the ensuing bone resorption. (C) 2004 Elsevier Ltd. All rights reserved.
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The major risk factor for skin cancer is exposure to UV radiation from sunlight, but other environmental exposures may also play a role in combination with UV. We have studied the effects of combined exposure of primary human skin cells in vitro to UVA, UVB or UVC with benzo[a] pyrene (BaP), an environmental carcinogen. Normal human keratinocytes were exposed to 5 microM BaP for 24 h followed by either 1 kJ/m(2) UVA, 100 J/m(2) UVB or 10 J/m(2) UVC. Only BaP + UVA caused increased cell death. BaP or UVA alone did not induce significant DNA damage as measured by comet assay but combined exposure induced 35.1 +/- 6.0% tail DNA, compared with 9.7 +/- 1.3% tail DNA in control cells. After including the Fapy-DNA glycosylase enzyme incubation step to detect oxidized purines, % tail DNA increased another 11.2 +/- 2.9%. Combined exposure of BaP and UVB did not increase damage in the comet assay without Fapy-DNA glycosylase, but in the presence of this enzyme % tail DNA increased by 9.3 +/- 2.2%. BaP + UVB also abrogated the UVB-induced cell cycle G2 arrest. BaP + UVC had no effect on the keratinocytes compared with each treatment alone. These results show a wavelength-dependent difference in the effects of combined exposure on normal human keratinocytes. Both UVA and UVB damage can be enhanced by BaP pre-exposure, although the effects seen with UVA were greater. These findings are important to understanding the role of UVA and UVB in skin carcinogenesis and may have implications for recommended sun exposure limits, especially in polluted areas.
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In recent years there has been renewed interest in metal-on-metal (MOM) bearings for both total hip replacement and surface replacement hip arthroplasty. Short-term clinical results have been encouraging; with low wear rates and few prostheses requiring revision. This review concentrates on the factors that affect the wear of all metal devices such as specification of the alloy, processing techniques, head diameter, clearance between the components, lubrication regime, loading and surface finish. The concerns associated with MOM bearings are also discussed. These include wear particle release and dissemination, and elevated metal ion levels, which may lead to cytotoxicity, hypersensitivity and genotoxicity.© 2005 Elsevier Ltd. All rights reserved.
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A contact mechanics model was developed to investigate the effect of macroscopic and microscopic surface features of the bearing surface of both conventional and cross-linked polyethylene acetabular cups tested in hip simulators. Wear of the bearing surface was found to increase the conformity and to decrease the contact pressure for both materials. The surface roughness on the cup surface was found to increase the contact pressure perturbation, particularly for the conventional polyethylene. Copyright© 2005 by ASME.
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The wear of hip replacements can be dependent on serum protein levels and the swing-phase load during the gait cycle. We hypothesise that these effects may be associated with changes in the lubrication and friction of the joint. The aim of this study was to assess the effect of the lubrication regime and lubricant on the friction of metal-on-metal (MOM) and ceramic-on-ceramic (COC) THRs. Increasing the swing-phase load led to decreased fluid film thickness and an increase in friction, in both MOM and COC bearings. Increasing the protein concentration of the lubricant, decreased the friction of MOM THRs. Friction testing of COC bearings demonstrated an increase in friction as the protein concentration increased. The change in lubricating film thickness explains differences in friction and wear as the swing-phase load is changed for a given lubricant. However, when we change the lubricant composition, protein boundary lubrication effects dominate, this influences friction and wear differently in MOM and COC THRs. Copyright© 2005 by ASME.
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The roles of two adjacent genes in the Staphylococcus aureus chromosome with functions in starvation survival and the response to stressful conditions have been characterized. One of these, hprT, encoding a hypoxanthine-guanine phosphoribosyltransferase homologue, was initially identified in a transposon mutagenesis screen. Mutation of hprT affects starvation survival in amino-acid-limiting conditions and the ability of S. aureus to grow in high-salt concentrations. Downstream of hprT is ftsH, which encodes a membrane-bound, ATP- and Zn2+-dependent 'AAA'-type protease. Mutation of ftsH in S. aureus leads to pleiotropic defects including slower growth, sensitivity to salt, acid, methyl viologen and potassium tellurite stresses, and reduced survival in amino-acid- or phosphate-limiting conditions. Both hprT-lacZ and ftsH-lacZ gene fusions are expressed maximally in the post-exponential phase of growth. Although secretion of exoproteins is not affected, an ftsH mutant is attenuated in a murine skin lesion model of pathogenicity.
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Metal-on-metal hip arthroplasties have demonstrated low wear rates. However, the ion release and toxicity of the metal wear particles remains a concern. Modifying the surface of metal bearings with thick chromium nitride (CrN) coatings has the potential to further reduce wear and ion release, and improve the biocompatibility of wear particles produced. The aim of this study was to investigate the application of surface engineering technology to modify metal-on-metal (MOM) bearings to reduce wear and improve the functional biocompatibility of metal-on-metal prostheses. CrN-on-CrN bearings had lower wear rates in comparison to metal-on-metal bearings, particularly under adverse loading conditions. CrN-on-CrN bearings produced similar nanometer-sized particles to metal-on-metal bearings, however, CrN wear particles were less cytotoxic when cocultured with macrophage and fibroblast cells. Key words: Surface engineering, hip arthroplasty, wear, metal-on-metal, wear particles. (C) 2004 Elsevier Inc. All rights reserved.
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Cross-linked polyethylene currently is being introduced in knee prostheses. The wear rates, wear debris, and biologic reactivity of non cross-linked, moderately cross-linked, and highly cross-linked polyethylene have been compared in multidirectional wear tests and knee simulators. Multidirectional pin-on-plate wear studies of noncross-linked, moderately cross-linked (5 Mrad), and highly cross-linked (10 Mrad) polyethylene showed a 75% reduction in wear with the highly cross-linked material under kinematics found in the hip, but only a 33% reduction under wear in kinematics representative of the knee. In knee simulator studies, with the fixed-bearing press-fit, condylar Sigma cruciate-retaining knee under high kinematic input conditions, the wear of 5 Mrad moderately cross-linked polyethylene was 13 +/- 4 mm(3) per 1 million cycles, which was lower (p<0.05) than the wear of clinically used, gamma vacuum foil GUR 1020 polyethylene (23 +/- 6 mm(3)/1 million cycles). For the low-contact stress mobile-bearing knee, the wear of moderately cross-linked polyethylene was 2 1 mm3 per 1 million cycles, which was lower (p<0.05) than GVF GUR 1020 polyethylene (5 2 mm3/1 million cycles). The wear debris isolated from the fixed-bearing knees showed the moderately crosslinked material had a larger percentage volume of particles smaller than 1 pm in size, compared with GVF GUR 1020 polyethylene. Direct cell culture studies of wear debris generated in sterile wear simulators using multidirectional motion showed a increase (p<0.05) in tumor necrosis factor-alpha levels and reactivity for GUR 1050 cross-linked poly- ethylene debris compared with an equivalent volume of noncross-linked GUR 1050 polyethylene. The use of crosslinked polyethylene in the knee reduces the volumetric wear rate. However, the clinical significance of reduced fracture toughness, elevated wear in abrasive conditions, and the elevated tumor necrosis factor-alpha release from smaller more reactive particles warrant further investigation.
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The response of murine macrophages to clinically relevant polyethylene wear particles generated from different polyethylenes at various time points and volumetric doses in vitro was evaluated. Clinically relevant ultra high molecular weight polyethylene (UHMWPE) wear debris was generated in vitro in a lubricant of RPMI 1640 supplemented with 25% (v/v) foetal calf serum using a multi-directional pin-on-plate wear rig under sterile conditions. Wear debris was cultured with C3H murine peritoneal macrophages at various particle volume (microm(3)): cell number ratios. The secretion of TNF-alpha was determined by ELISA. Initially the effect of molecular weight of UHMWPE was considered. Higher molecular weight GUR415HP was shown to have a lower wear rate than the lower molecular weight GUR1120, however a greater volume of the wear debris produced by the high molecular weight GUR415HP was in the 0.1-1.0 microm size range. Wear debris from GUR415HP produced significant levels of TNF-alpha at a concentration of 1 microm(3)/cell while at least 10 microm(3)/cell of GUR1120 wear debris per cell was needed to produce significant levels of TNF-alpha. Secondly the effects of crosslinking GUR1050 was examined when worn against a scratched counterface. The wear rate of the material was shown to decrease as the level of crosslinking increased. However the materials crosslinked with 5 and 10 Mrad of gamma irradiation produced higher percentages of 0.1-1.0 microm size wear particles than the non-crosslinked material. While the crosslinked material was able to stimulate cells to produce significantly elevated TNF-alpha levels at a particle concentration of just 0.1 microm(3)/cell only concentrations of 10 microm(3)/cell and above of the non-crosslinked wear debris were stimulatory. When the counterface was changed from scratched to smooth the wear rate for all three GUR1050 materials was further reduced. For the first time nanometre size wear particles were observed from polyethylene which reduced the percentage mass of debris in the 0.1-1.0 microm size range. For all three materials on the smooth counterface only concentrations of 50 microm(3)/cell and above were stimulatory. This study has demonstrated that molecular weight, crosslinking and counterface roughness are important factors in determining the biological activity of polyethylene.
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Over the last 10 years there has been increasing concern over wear of polyethylene and osteolytic potential in the knee. During this period there have been considerable developments in both the design, materials and sterilisation techniques used for polyethylene in the knee. This paper reviews wear performance of polyethylene sterilised by different techniques, under a range of kinematics conditions in a single knee joint simulator system. Reducations in polyethylene wear were found with both stabilised polyethylene and cross-linked polyethylene, and with rotating platform mobile bearing designs.
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PheP, a putative amino acid permease in Staphylococcus aureus, contributes to starvation survival under glucose-limiting conditions and virulence. A pheP mutation led to poor growth after microaerobic or anaerobic incubation on pig serum agar, which was recovered by phenylalanine addition. Genetic complementation of pheP restored growth and starvation survival.
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Ultra-high molecular weight polyethylene wear particles have been implicated as the major cause of osteolysis, implant loosening and late aseptic failure in total hip arthroplasties in vivo. This study initially screened 22 carbon-carbon composite materials as alternatives for UHMWPE in joint bearings. New bearing materials should satisfy certain criteria--they should have good wear properties that at least match UHMWPE, and produce wear particles with low levels of cytotoxic and osteolytic activity. Initial screening was based on wear resistance determined in short-term tribological pin-on-plate tests. Three materials (HMU-PP(s), HMU-RC-P(s), and SMS-RC-P(s)) which had superior wear resistance were selected for long-term testing. All materials had very low wear factors and SMS-RC-P(s), which had a wear factor of 0.08 +/- 0.56 x 10(-7) mm3/Nm, was selected for the subsequent biological testing and particle size analysis. SMS-RC-P(s) showed good biocompatibility in bulk material form and also the wear particles had low cytotoxicity for L929 fibroblasts in culture compared to metal wear particles. Wear debris size analysis by transmission electron microscopy showed that the particles were very small, with the vast majority being under 100 nm in size, similar to metal wear particles. The potential osteolytic effect of SMS-RC-P(s) wear particles was investigated by culturing particles with human peripheral blood mononuclear cells and measuring TNFalpha production. SMS-RC-P(s) did not significantly stimulate TNFalpha production at a particle volume to cell number ratio of 80:1, indicating that the debris had a low osteolytic potential. The results of this study suggest that carbon-carbon composites, particularly those composed of PAN-based fibers may be important biomaterials in the development of next generation bearing surfaces for use in total joint replacements that have very low wear rates and reduced osteolytic and cytotoxic potential.
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The post-antibiotic effect (PAE) is an important parameter of antibiotic action that is widely used as a predictor of pharmacodynamic activity. Traditionally, PAE has been determined by a labour-intensive method involving determination of viable cell numbers. New methods using spectrophotometric procedures could offer significant advantages for PAE determinations, particularly in terms of speed. A number of such methods have been described in the literature, but extensive comparison with the classical procedure for determining PAEs has not been carried out. We have now compared PAE values obtained using a rapid microplate method with those achieved by the classical viable count procedure.
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The hyaluronate lyase (HL) gene of Staphylococcus aureus 8325-4 (hysA) was inactivated in vitro with the insertion of the erythromycin determinant, ermC, from plasmid pE194. The hysA::ermC mutation was introduced into S. aureus via a temperature-sensitive shuttle vector, where it underwent homologous recombination with the wild-type (w.t.) allele. The insertion of ermC in the chromosomal hysA locus was confirmed by Southern blot hybridization and the loss of HL activity was demonstrated macroscopically by a plate assay. The importance of HL for pathogenicity was assessed by comparing the virulence of the HL- mutant strain to that of the w.t. in an established mouse abscess model of S. aureus infection. A significantly higher cell recovery was obtained from lesions infected with the w.t. strain compared to the lesions infected with the HL- strain (P =0.01). Although the lesion areas from both groups were not significantly different (P=0.9) they were of different morphology. A colorimetric assay was used to measure HL activity from culture supernatants of the S. aureus 8325-4 strains w.t., WA250 (agr) and PC1839 (sar) grown in a chemically defined medium. HL activity reached a maximum in the w.t. strain during mid-exponential phase (t=5 h) and while it showed a 16-fold decrease in the agr mutant it increased 35-fold in the sar mutant background. These results strongly suggest that HL is a virulence factor which is important in the early stages of subcutaneous infections.
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Tissue engineered heart valves are developed either from artificial polymer scaffolds or natural tissue sources. A detergent-based cell extraction method for generating an acellular valve matrix was developed. Samples of decellularized valve leaflets or aorta were incubated intact or as soluble extracts with confluent monolayers of porcine valve interstitial cells (VIC), smooth muscle cells (SMC), or fibroblasts (Fb). Decellularized heart valve tissues showed good biochemical characteristics, kinematics and hydrodynamic performance, good biocompatibility and reseeding potential.
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The factors that cause failure in artificial hip joints were discussed. It was shown that micron and submicron size polyethylene wear particles stimulated magcrophages and caused adverse inflammatory reactions with the release of osteolytic cytokines. It was observed that highly crosslinked polyethylene had a were rate of 9 cubic millimeters per million cycles. It was also shown that cobalt chrome metal on metal bearings had low wear rates of between 0.1 and 1.0 cubic millimeters per million cycles.
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The wear rates, debris and biological activity of non-crosslinked and crosslinked ultra high molecular weight polyethylene (UHMWPE) hip joint prostheses were studied. The non-crosslinked, slightly crosslinked and highly crosslinked samples of UHMWPE were gamma irradiated in nitrogen and were measurements were taken using a coordinate measuring machine. SEM was used for the analysis of the wear debris from the materials. It was found that the wear rate of the highly crosslinked UHMWPE was lower than for the other materials. While the wear rate of the highly crosslinked materials was low, it only generated a two fold reduction in functional biological activity and osteolytic potential.
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The effects of swing phase load on wear and friction of metal-on-metal (MOM) hip replacements, with regards to the release of metal ions and wear particles, were investigated. Cobalt chrome of about 28mm MOM hip replacements were tested in a physiological hip simulator. Friction testing was conducted using a pendulum friction simulator. Tests were carried out with a peak load of 2kN and swing phase loads of 100N and 280N. The results show that over-tensioning of the tissues may also accelerate wear, which consequently increases the number of wear particles and metal ions released.
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Wear, debris, and functional biocompatibility of third generation Biolox Forte and fourth generation Biolox Delta ceramic bearing couples under standard and micro separation simulator testing were discussed. Microseparation sub-luxation testing was found to be necessary to reproduce clinically relevant stripe wear on both materials. Under standard simulator conditions, the wear of both materials was very low with the wear of Biolox Delta being lower than the wear of Biolox Forte. The low wear, even under the most severe loading conditions, combined with good biocompatibility of the wear debris, indicate that third and fourth generation ceramic on ceramic bearing couples are a good choice for active, high demand patients.
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The use of self-assembling peptide nanomaterials as injectable lubricants for the treatment of osteoarthritic joints was investigated. The purpose of the study was to relate the mesoscopic structure of the materials to their performance as boundary lubricants in in vitro models of the knee, to enable the targeted design of the peptide molecular structure. Samples for the study were prepared by the dissolution of a known mass of lyophilized peptide in HPLC water, then mixed and sonicated for 10 minutes. It was suggested that the ability to design self-assembling peptides to produce different morphologies and physical properties, allows them to be tailored for appropriate roles in different applications and states of disease.
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The effect of femoral condylar lift off on the wear of artificial knee joints was investigated. Twelve PFC Signal cruciate-retaining bearing knees were tested using knee simulators. The mean rate of the sample knees was 8.8± 4.8 mm ÷million cycles under the standard kinematic conditions. The mean wear rate was found to increased to 14.7 ± 3.7 mm ÷million cycles when femoral condylar lift was simulated. It was observed that lateral lift off resulted in a higher wear rate on the medical compartment for the PFC Sigmal cruciate-retaining fixed bearing knee.
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The nanometer sized wear debris of ultra high molecular weight polyethylene (UHMWPE) 1020 GVF generated in hip and knee prostheses was studied using a high resolution field emission gun scanning electron microscopy (FEGSEM). The samples were foil packed and irradiated with 4MRad gamma irradiation in a vacuum. A coordinate measuring machine (CMM) was used to measure the wear of the samples. All tests were carried out in 25% bovine serum diluted with 0.1% sodium azide and were run to 5 million cycles. It was observed that the UHMWPE debris from the knee prostheses was generally larger than the debris from the hip prostheses.
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The use of surface engineering technology to modify MOM bearings to reduce the wear rate, number of wear particles and ion release and to improve the functional biocompatibility of wear particles generated was discussed. It was observed that the wear of CrN-on-CrN bearings was substantially less than the MOM articulations under all swing phase load conditions. The reductions in wear volume with CrN was found to be associated with its increased hardness. Analysis showed that the reduced wear and reduction in number of wear particles reduced the area available for metal ion release.
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An acellular xenogenic tendon with mechanical and biochemical properties of fresh tissue for use in tissue engineering of the anterior cruciate ligament (ACL) was developed. Histological analysis showed that the tendons were decellularized by treatment with 0.1% (w/v) SDS with no effect on the gross matrix structure or major biochemical components of the tendon. Assesssment of the mechanical properties of the de-cellularized tendon showed that there is significant decrease in the ultimate tensile strength of the tendon treated with 0.1% (w/v) SDS due to a decrease in the collagen phase slope. Cell culture on the surface of the tendons matrix after seven days showed that tissue matrix was not toxic.
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There is currently much interest in the characterization of wear debris from different types of hip-joint prostheses. There have been numerous studies on the wear of UHMWPE in hip-joint simulators, but relatively few studies on the wear of alternative materials such as metal-on-metal and ceramic-on-ceramic. The aim of this study was to compare the wear volumes and the functional biological activity (FBA) of the wear debris generated from zirconia ceramic-on-UHMWPE, cobalt-chrome metal-on-metal, and alumina cerarmic-on-ceramic hip joints under standard and micro-separation simulation conditions in the same hip-joint simulator.All prostheses showed a higher 'bedding in' wear rate, which was followed by a lower steady-state wear rate. Under standard simulation conditions, the zirconia ceramic-on-UHMWPE prostheses showed the highest wear rates (31 +/-4.0 mm(3)/million cycles), followed by the metal-on-metal (1.23 +/-0.5 mm(3)/million cycles), with the alumina ceramic-on-ceramic prostheses showing significantly (p<0.01) lower wear rates at 0.05 +/-0.02 mm(3)/million cycles. The alumina ceramic-on-ceramic wear rate was 10-50-fold lower than some clinical retrieval studies, and the wear stripe observed on retrieved components was not reproduced under standard simulation conditions. The mean ( 95 per cent confidence limits) size of the UHMWPE wear debris was 300 +/-200 nm (range 0.1-50 mum), 30 +/-2.25 nm (range 9-66 nm) for the metal particles, and 9 +/-0.5 nm (range 2-27 nm) for the alumina-ceramic wear particles. The UHMWPE and cobalt-chrome wear particles were similar to those described in vivo; however, alumina-ceramic particles up to 3 mum have been identified in periprosthetic tissues from around ceramic-on-ceramic prostheses.When micro-separation of the alumina-ceramic components was introduced into the simulation, clinically relevant wear rates, wear patterns, and wear particles were generated. The wear rate increased to 1.84 +/-0.3 mm(3)/million cycles and the stripe wear feature was reproduced on the alumina femoral heads. In addition to the nanometre-sized wear particles generated under standard simulation conditions, larger micrometre-sized alumina wear particles were also observed.The FBA of the wear particles was determined by integrating measurements from in vitro wear simulations, particle characterization, and in vitro cell culture studies. These studies revealed that the UHMWPE particles produced in this study demonstrated a three-fold higher specific biological activity and a 50-fold higher FBA than the alumina-ceramic particles generated under micro-separation simulation conditions.This study revealed significant differences in the wear volumes, size distributions, and FBAof the wear particles generated from the three different prostheses under clinically relevant conditions in in vitro wear simulations.
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Proponents of the biological theory of aseptic loosening have in recent years tended to concentrate on the production and distribution of particulate ultra-high-molecular-weight polyethylene (UHMWPE) debris around the potential joint space. However, mechanical loading of cemented implants with the differing elastic moduli of metal stems, polymethylmethacrylate (PMMA) cement and bone can result in relative micromotion, implying the potential for production of metal and PMMA particles from the stem-cement interface by fretting wear. In order to investigate the production and biological reactivity of debris from this interface, PMMA and metal particulate debris was produced by sliding wear of PMMA pins containing barium sulphate and zirconium dioxide against a Vaquasheened stainless steel counterface. This debris was characterised by SEM, energy-dispersive analysis by X-ray (EDAX) and image analysis, then added to cell cultures of a human monocytic cell line, U937, and stimulation of proosteolytic cytokines measured by ELISA. Large quantities of PMMA cement debris were generated by the sliding wear of PMMA pins against Vaquasheened stainless steel plates in the method developed for this study. Both cements stimulated the release of pro-osteolytic TNFalpha from the U937 monocytic cell line, in a dose-dependent fashion. There was a trend towards greater TNFalpha release with Palacos cement than CMW cement at the same dose. Palacos particles also caused significant release of IL-6, another pro-osteolytic cytokine, while CMW did not. The particulate cement debris produced did not stimulate the release of GM-CSF or IL1beta from the U937 cells. These results may explain the cytokine pathway responsible for bone resorption caused by particulate PMMA debris. Radio-opaque additives are of value in surgical practice and clinical studies to quantify the relevance of these in vitro findings are required before the use of cement containing radio-opacifier is constrained.
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Extremely low wear rates have been reported for metal-on-metal total hip replacements, but concerns remain about the effects of metal ion release, dissolution rates and toxicity. Surface-engineered coatings have the potential to improve wear resistance and reduce the biological activity of the wear debris produced. The aim of this study was to examine the wear and wear debris generation from surface-engineered coatings: titanium nitride (TiN), chromium nitride (CrN) and chromium carbon nitride (CrCN) applied to a cobalt-chrome alloy (CoCr) substrate. The coatings were articulated against themselves in a simple geometry model. The wear particles generated were characterized and the cytotoxic effect on U937 macrophages and L929 fibroblasts assessed. The CrN and CrCN coatings showed a decrease in wear compared to the CoCr bearings and produced small (less than 40 nm in length) wear particles. The wear particles released from the surface engineered bearings also showed a decreased cytotoxic effect on cells compared to the CoCr alloy debris. The reduced wear volumes coupled with the reduced cytotoxicity per unit volume of wear indicate the potential for the clinical application of this technology.
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Concern over polyethylene wear particle induced aseptic loosening of metal-on-polyethylene hip prostheses has led to renewed interest in alternative materials such as metal-on-metal and alumina ceramic-on-alumina ceramic for total hip replacement. This study compared the effects of clinically relevant cobalt-chromium and alumina ceramic wear particles on the viability of U937 histiocytes and L929 fibroblasts in vitro. Clinically relevant cobalt-chromium wear particles were generated using a flat pin-on-plate tribometer. The mean size of the clinically relevant metal particles was 29.5±6.3 nm (range 5-200 nm). Clinically relevant alumina ceramic particles were generated in the Leeds MkII anatomical hip simulator from a Mittelmieier prosthesis using micro-separation motion. This produced particles with a bimodal size distribution. The majority (98%) of the clinically relevant alumina ceramic wear debris was 5-20 nm in size. The cytotoxicity of the clinically relevant wear particles was compared to commercially available cobalt-chromium (9.87 μm±5.67) and alumina ceramic (0.503±0.19 μm) particles. The effects of the particles on the cells over a 5 day period at differentparticle volume (μm) to cell number ratios were tested and viability determined using ATP-Lite™. Clinically relevant cobalt-chromium particles 50 and 5 μm per cell reduced the viability of U937 cells by 97% and 42% and reduced the viability of L929 cells by 95% and 73%, respectively. At 50 μmper cell, the clinically relevant ceramic particles reduced U937 cell viability by 18%. None of the other concentrations of the clinically relevant particles were toxic. The commercial cobalt-chromium and alumina particles did not affect the viability of either the U937 histiocytes or the L929 fibroblasts. Thus at equivalent particle volumes the clinically relevant cobalt-chromium particles were more toxic then the alumina ceramic particles. This study has emphasised the fact that the nature, size and volume of particles are important in assessing biological effects of wear debris on cells invitro. © 2002 Elsevier Science Ltd. All rights reserved.
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Wear of polyethylene and the resulting wear debris-induced osteolysis remains a major cause of long-term failure in artificial hip joints. There is interest in understanding engineering and clinical conditions that influence wear rates. Fluoroscopic studies have shown separation of the head and the cup during the swing phase of walking due to joint laxity. In ceramic-on-ceramic hips, joint laxity and microseparation, which leads to contact of the head on the superior rim of the cup, has led to localized damage and increased wear in vivo and in vitro. The aim of this study was to investigate the influence of joint laxity and microseparation on the wear of ceramic on polyethylene artificial hip joints in an in vitro simulator. Microseparation during the swing phase of the walking cycle produced contact of the ceramic head on the rim of the polyethylene acetabular cup that deformed the softer polyethylene cup. No damage to the alumina ceramic femoral head was found. Under standard simulator conditions the volume change of the moderately crosslinked polyethylene cups was 25.6 +/- 5.3 mm3/million cycles and this reduced to 5.6 +/- 4.2 mm3/million cycles under microseparation conditions. Testing under microseparation conditions caused the rim of the polyethylene cup to deform locally, possibly due to creep, and the volume change of the polyethylene cup when the head relocated was substantially reduced, possibly due to improved lubrication. Joint laxity may be caused by poor soft tissue tension or migration and subsidence of components. In ceramic-on-polyethylene acetabular cups wear was decreased with a small degree of joint laxity, while in contrast in hard-on-hard alumina bearings, microseparation accelerated wear. These findings may have significant implications for the choice of fixation systems to be used for different types of bearing couples.
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Polyethylene wear debris induced osteolysis is a major cause of failure in artificial hip joints. Sub micrometre size particles are taken up by macrophages which are stimulated to release osteolytic cytokines such as TNFα. This leads to bone resorption, loosening and failure. In vitro cell culture studies have shown particles in the size range 0.1 to 1 micrometre to be at least six times more reactive than larger particles. Studies of historically used gamma irradiated in air polyethylene show increased wear ratewith damaged femoral heads and with aged and oxidised polyethylene. The aged and oxidised polyethylene also produced a greater percentage of smaller particles leading to increased osteolytic potential. Combined tribological and biological simulation models have been developed for pre-clinical assessment of osteolytic potential of artificial hip joints.
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There is now considerable clinical concern about the effect of polyethylene wear debris induced osteolysis in long term failure of hip replacements. This paper compares the wear of stabilised and crosslinked polyethylene to alternative hard on hard bearings. The volumetric wear rates of stabilised and moderately crosslinked polyethylene 50 to 35 mm/million cycles were less than previously reported for historical gamma irradiated in air polyethylene, but still of a level that in the long term could cause osteolysis. The moderately crosslinked polyethylene produced less wear than non-crosslinked polyethylene, but particles were smaller and more reactive resulting in little change in the osteolytic potential. Alumina ceramic on ceramic produced substantially less wear and osteolytic potential. Metal on metal also produced less wear than polyethylene but the particles adversely influence cell viability.
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The wear, wear debris and functional biological activity of non-crosslinked and moderately crosslinked ultrahigh molecular weight polyethylene (UHMWPE) acetabular cups have been com pared when articulating against smooth and intentionally scratched femoral heads. Volumetric wear rates were determined in a hip joint simulator and the debris was isolated from the lubricant and characterized by the percentage number and volumetric concentration as a function of particle size. The volumetric concentration was integrated with the biological activity function determined from in vitro cell culture studies to predict an index of specific biological activity (SBA). The product of specific biological activity and volumetric wear rate was used to determine the index of functional biological activity (FBA). On smooth femoral heads the crosslinked UHMWPE had a 30 per cent lower wear rate, but it had a greater percentage volume of smaller, more biologically active particles, which resulted in a similar index of FBA compared with the non-crosslinked material. On the scratched femoral heads the volumetric wear rate was three times higher for the moderately crosslinked UHMWPE and two times higher for the non-crosslinked UHMWPE compared with the smooth femoral heads. This resulted in a higher wear rate for the moderately crosslinked material on the scratched femoral heads. All the differences in wear rate were statistically significant. There were only small differences in particle volume concentration distributions, and this resulted in similar indices of FBA which were approximately twice the values of those found on the smooth femoral heads. Both materials showed lower wear and FBA than for previously studied aged and oxidized UHMWPE gamma irradiated in air. However, this study did not reveal any advantage in terms of predicted FBA for moderately crosslinked UHMWPE compared with non-crosslinked UHMWPE.
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The aims of this study were to investigate the tissues from uncemented Mittelmeier alumina ceramic-on-ceramic total hip replacements using histological methods and to isolate and characterise the ceramic wear debris using laser capture microdissection and electron microscopy. Tissues from around 10 non-cemented Mittelmeier alumina ceramic on ceramic THRs were obtained from patients undergoing revision surgery. Tissues were also obtained from six patients who were undergoing revisions for aseptic loosening of Charnley, metal on polyethylene prostheses. Tissue sections were analysed using light microscopy to determine histological reactions and also the location and content of alumina ceramic wear debris. Tissue samples were extracted from sections using laser capture microdissection and the characteristics of the particules subsequently analyses by TEM and SEM. The tissues from around the ceramic on ceramic prostheses all demonstrated the presence of particles, which could be seen as agglomerates inside cells or in distinct channels in the tissues. The tissues from the ceramic on ceramic retrievals had a mixed pathology with areas that had no obvious pathology, areas that were relatively rich in macrophages and over half of the tissues had in the region of 60% necrosis/necrobiosis. In comparison, the Charnley tissues showed a granulomatous cellular reaction involving a dense macrophage infiltrate and the presence of giant cells and<30% necrosis/necrobiosis. The tissues from the ceramic prostheses also showed the presence of neutrophils and lymphocytes, which were not evident in the tissues from the Charnley retrievals. There were significantly more macrophasges (p<0.05), and giant cells (p<0.01) in the Charnley tissues and significantly more neutrophils (p<0.01) in the ceramic on ceramic tissues. TEM of the laser captured tissue revealed the presence of very small alumina wear debris in the size range 5-90nm, mean size _+ SD of 24 _+ 19 nm whereas SEM (lower resolution) revealed particles in the 0.05-3.2um size range. This is the first description of nanometre sized ceramic wear particles in retrieval tissues. The bi-modal size range of alumina ceramic wear debris overlapped with the size ranges commonly observed with metal particles (10-30nm) and particles of ultra-high molecular weight polyethylene (0.1-1000um). It is possible that the two size ranges of contributed to the mixed tissue pathology observed. It is speculated that the two types of ceramic wear debris are generated by two different wear mechanisms in vivo; under normal articulating conditions, relief polishing wear and very small wear debris is produced, while under conditions of microseparation of the head and cup and rim contact, intergranular and intragranular fracture and larger wear particles are generated.
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Although the wear of existing metal-on-metal (MOM) hip prostheses (1 mm3/10(6) cycles) is much lower than the more widely used polyethylene-on-metal bearings, there are concerns about the toxicity of metal wear particles and elevated metal ion levels, both locally and systemically, in the human body. The aim of this study was to investigate the possibility of reducing the volume of wear, the concentration of metal debris and the level of metal ion release through using surface-engineered femoral heads. Three thick (8-12 microm) coatings (TiN, CrN and CrCN) and one thin (2 microm) coating (diamond-like carbon, DLC), were evaluated on the femoral heads when articulating against high carbon content cobalt-chromium alloy acetabular inserts (HC CoCrMo) and compared with a clinically used MOM cobalt-chromium alloy bearing couple using a physiological anatomical hip joint simulator (Leeds Mark II). This study showed that CrN, CrCN and DLC coatings produced substantially lower wear volumes for both the coated femoral heads and the HC CoCrMo inserts. The TiN coating itself had little wear, but it caused relatively high wear of the HC CoCrMo inserts compared with the other coatings. The majority of the wear debris for all half-coated couples comprised small, 30 nm or less, CoCrMo metal particles. The Co, Cr and Mo ion concentrations released from the bearing couples of CrN-, CrCN- and DLC-coated heads articulating against HC CoCrMo inserts were at least 7 times lower than those released from the clinical MOM prostheses. These surface-engineered femoral heads articulating on HC CoCrMo acetabular inserts produced significantly lower wear volumes and rates, and hence lower volumetric concentrations of wear particles, compared with the clinical MOM prosthesis. The substantially lower ion concentration released by these surface-engineered components provides important evidence to support the clinical application of this technology.
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The aims of this study were to investigate the tissues from uncemented Mittelmeier alumina ceramic-on-ceramic total hip replacements using histological methods and to isolate and characterise the ceramic wear debris using laser capture microdissection and electron microscopy. Tissues from around 10 non-cemented Mittelmeier alumina ceramic on ceramic THRs were obtained from patients undergoing revision surgery. Tissues were also obtained from six patients who were undergoing revisions for aseptic loosening of Charnley, metal-on-polyethylene prostheses. Tissue sections were analysed using light microscopy to determine histological reactions and also the location and content of alumina ceramic wear debris. Tissue samples were extracted from sections using laser capture microdissection and the characteristics of the particles subsequently analysed by TEM and SEM. The tissues from around the ceramic-on-ceramic prostheses all demonstrated the presence of particles, which could be seen as agglomerates inside cells or in distinct channels in the tissues. The tissues from the ceramic-on-ceramic retrievals had a mixed pathology with areas that had no obvious pathology, areas that were relatively rich in macrophages and over half of the tissues had in the region of 60% necrosis/necrobiosis. In comparison, the Charnley tissues showed a granulomatous cellular reaction involving a dense macrophage infiltrate and the presence of giant cells and<30% necrosis/necrobiosis. The tissues from the ceramic prostheses also showed the presence of neutrophils and lymphocytes, which were not evident in the tissues from the Charnley retrievals. There were significantly more macrophages (p<0.05), and giant cells (p<0.01) in the Charnley tissues and significantly more neutrophils (p<0.01) in the ceramic-on-ceramic tissues. TEM of the laser captured tissue revealed the presence of very small alumina wear debris in the size range 5-90 nm, mean size + SD of 24 +/- 19nm whereas SEM (lower resolution) revealed particles in the 0.05-3.2 microm size range. This is the first description of nanometre sized ceramic wear particles in retrieval tissues. The bi-modal size range of alumina ceramic wear debris overlapped with the size ranges commonly observed with metal particles (10-30 nm) and particles of ultra-high molecular weight polyethylene (0.1-1,000 microm). It is possible that the two size ranges of contributed to the mixed tissue pathology observed. It is speculated that the two types of ceramic wear debris are generated by two different wear mechanisms in vivo, under normal articulating conditions, relief polishing wear and very small wear debris is produced. while under conditions of microseparation of the head and cup and rim contact, intergranular and intragranular fracture and larger wear particles are generated.
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To investigate whether there are differences between members of the Burkholderia cepacia complex in their ability to invade human respiratory epithelial cells, 11 strains belonging to genomovars I-V were studied in an antibiotic protection assay using the A549 cell line. Strains belonging to genomovars II and III were more invasive than those of genomovars I, IV and V. There was also intra-genomovar variation in invasiveness. No correlation between invasiveness and other putative virulence factors of importance in B. cepacia infection in individuals with cystic fibrosis, cable pilus and B. cepacia epidemic strain marker was identified.
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We compared and quantified the modes of failure and patterns of wear of 11 Mittelmeier and 11 Ceraver-Ostal retrieved alumina-alumina hip prostheses with reference to the corresponding clinical and radiological histories. Macroscopic wear was assessed using a three-dimensional co-ordinate measuring machine. Talysurf contacting profilometry was used to measure surface roughness on a microscopic scale and SEM to determine mechanisms of wear at the submicron level. The components were classified into one of three categories of wear: low (no visible/measurable wear), stripe (elliptical wear stripe on the heads and larger worn areas on the cups) and severe (macroscopic wear, large volumes of material lost). Overall, the volumetric wear of the alumina-alumina prostheses was substantially less than the widely used metal and ceramic-on-polyethylene combinations. By identifying and eliminating the factors which accelerate wear, it is expected that the lifetime of these devices can be further increased.
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The long term wear and wear debris generated in HIPed alumina on alumina bearings for hip prostheses with microseparation in vitro is compared to standard simulator conditions and ex vivo specimens. Microseparation studies were completed to five million cycles at two severity levels in attempts to rigorously evaluate the long-term tribological performance of the bearings. During the first million cycles (bedding-in) of the microseparation tests characteristic stripe wear was observed on all of the femoral heads with a matching area on the rim of the acetabular inserts. Under mild microseparation conditions an average wear rate of 0.55 mm3/million cycles was observed during the initial million cycles which reduced to a steady state level of 0.1 mm3/million cycles. Under more severe conditions an average wear rate of 4.0 mm3/million cycles was observed during bedding-in which reduced to a steady state level of 1.3 mm3/million cycles. These compare to a bedding-in wear rate of 0.11 mm3/million cycles and steady-state wear rate of 0.05 mm3/million cycles for the same material under normal simulation with no microseparation. Furthermore, under microseparation the wear mechanisms and wear debris were similar to those observed in previous alumina retrieval studies with debris ranging from 10 nm to 1 microm in size.
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The comparative performance of artificial hip joints has been extensively investigated in vitro through measurements of wear volumes. In vivo a major cause of long-term failure is wear-debris-induced osteolysis. These adverse biological reactions are not simply dependent on wear volume, but are also controlled by the size and volumetric concentration of the debris. A novel model is presented which predicts functional biological activity; this is determined by integrating the product of the biological activity function and the volumetric concentration function with the wear volume over the whole particle size range. This model combines conventional wear volume measurements with particle analysis and the output from in vitro cell culture studies to provide a new indicator of osteolytic potential. The application of the model is demonstrated through comparison of the functional biological activity of wear debris from polyethylene acetabular cups articulating under three different conditions in a hip joint simulator.
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A six station ProSim (Manchester, UK) knee simulator was used to assess the wear of six PFC (DePuy) fixed bearing total knee replacements under two different kinematic conditions defined as low and high kinematic inputs. The high kinematics displacement and rotation inputs were based on the kinematics of the natural knee with ISO standards used for the axial load and flexion. Low kinematics were defined as approximately half the magnitude. The six specimens were run for three million cycles under low kinematics and three million cycles under high kinematics. The mean wear rate found during the low kinematics phase was 7.7 +/- 2mm(3) per million cycles. This then increased significantly to an average wear rate of 41 +/- 14mm(3) during the high kinematics input phase. The wear areas were characterized by a predominant damage mode of burnishing with some abrasive wear occurring during the high kinematics phase. This study supports the findings that introduction of cross-shearing of the polyethylene by introducing both rotational and anterior/posterior displacement increases the wear rate. This has implications for younger patients with higher levels of activity that need knee replacements. (C) 2001 Kluwer Academic Publishers.
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The effects of cyclic mechanical strain and challenge with physiologically relevant doses of submicrometre-size polyethylene (PE) particles on the osteolytic potential of primary human mononuclear phagocytes were investigated. Cells were seeded into a three-dimensional tissue matrix and co-cultured with particles (mean size 0.21 microm) at particle volume to cell number ratios of 7.5, 15, 30 and 100 microm3/cell. Matrices were then either cultured statically or subjected to 20 per cent cyclic compressional strain in the 'ComCell' for 16 h prior to the assessment of cell viability and quantification of the pro-inflammatory cytokine tumour necrosis factor alpha (TNFalpha). The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazdium bromide) assay was shown to be too insensitive to detect changes in cell viability. However, when quantified by the adenosine triphosphate (ATP) assay, cell viability was demonstrated to be reduced following exposure to cyclic strain. Macrophages cultured in the static three-dimensional tissue equivalent model produced very high levels of TNFalpha in response to submicrometre PE particles at a ratio of 100 microm3/cell. Cyclic strain in the absence of particles gave only a small increase in TNFa production. However, the combined effects of strain and particle stimulation at a ratio of 30 microm3/cell resulted in the secretion of significantly more TNFalpha than was produced by macrophages subjected to strain alone, or the cells-only control. This synergy between cyclic strain and PE particle stimulation was only evident when the volume of particles was reduced below the volume that maximally stimulated cells. These results suggest that while cyclic strain may not be the primary factor responsible for macrophage activation and periprosthetic osteolysis, at low particle load, it may contribute significantly to the osteolytic potential of macrophages in vitro or in vivo.
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Malassezia is the aetiological agent of pityriasis versicolor. The mycelial phase of the organism predominates in lesions of pityriasis versicolor.
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The wear debris generated from ultra high molecular weight polyethylene (UHMWPE) have been recognised as one of the major causes of failure in total hip replacements (THR). It is essential to reduce the wear debris generated from UHMWPE acetabular cups in order to minimise this problem. Debris in the submicron size range is believed to have greater osteolytic potential. It is now known that crosslinked UHMWPE acetabular cups have reduced volumetric wear rates but little is known about the influence of crosslinking on the size and morphology of the wear debris. In this study, the wear of grade GUR 1020 crosslinked (vacuum gamma irradiated), GUR 1120 crosslinked (acetylene enhanced irradiated) and non cross linked (ethylene oxide sterilised) GUR 1020 UHMWPE was compared in multidirectional pin-on-plate wear tests under three different counterface conditions (smooth, isotropically rough and scratched counterfaces). Multidirectional motion was chosen because this motion was closer to the relative motion in the natural hip. From this study, better wear resistance of crosslinked UHMWPE compared with non-crosslinked UHMWPE was demonstrated for the smooth counterface conditions. However, in the rough and scratched counterface conditions, the vacuum gamma irradiated crosslinked material produced significantly higher wear rates than the non-crosslinked material. The analysis of the wear debris showed that the majority of the volume of the acetylene enhanced crosslinked UHMWPE wear debris was in the most biologically active size range (0.1 to 0.5 microm). In contrast, the non-crosslinked material and the vacuum gamma irradiated crosslinked material had a greater proportion of the volume of the debris in the larger size ranges which are less biologically active. This has important implications for its osteolytic potential.
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The wear of "Biolox Forte" alumina ceramic bearing couples has been investigated at two different acetabular cup angles in a physiological hip joint simulator. All cups were set in the anatomical position of 45 degrees inclination in the M/L plane for the first two million cycles and then four of the six cups were re-aligned to 60 degrees for a further three million cycles. A "running-in" wear of 0.14 mm3 per million cycles was observed for the first million cycles, after which a steady state wear rate of 0.05 mm3 per million cycles was observed. Increasing the acetabular cup angle to 60 degrees did not significantly affect the wear rate.
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This study isolated and characterized UHMWPE particles from 3 explant groups: early Charnley hip failures (ECE;<10 years), late Charnley hip failures (LCE;>10 years) and early knee failures (EKE;<10 years). Debris isolated from the 3 groups had percentage particle number and percentage volumetric concentration distributions that were not significantly different. The greatest number of particles were found in the 0.1-0.5 microm size range and 19-20.6% of the volumetric concentration was below 1 microm in size in all groups. However, there were significant differences in the total volumetric concentration of debris isolated per g of tissue. LCE had significantly higher volumes of debris than ECE and EKE, there was no significant difference in the volume of debris from the EKE and ECE. The mean aspect ratio and mean irregularity ratio of the LCE group were also significantly higher than the ECE and EKE, suggesting that different wear mechanisms were occurring in the late Charnley group compared to the early Charnley and knee groups. These results also suggest that early knees, with normal surface wear, may have similar wear mechanisms to early Charnley hips and indicate that similar volumes of biologically active micrometer and sub-micrometer UHMWPE particles were produced. This may have important implications in the longer-term outcome of total knee arthroplasties, because it indicates a similar potential for osteolysis induced by wear debris.
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Wear and the biological response to wear debris of artificial joints remain major concerns in total hip arthroplasty (THA). The long-term effects of UHMWPE wear debris are well documented and these have led to interest in alternate bearing materials for THA. Alumina ceramic-ceramic hip joints have been successfully used for more than 30 years with low wear and little incidence of osteolysis. The most common wear pattern observed on retrieved components is an elliptical wear 'stripe' on the heads and a corresponding worn area on the cup with an approximated wear rate of 1-5 mm3 pa. More severe wear has also occasionally occurred, usually in association with an abnormal clinical history. Modern alumina-alumina THAs use an improved HIPed (hot isostatically pressed) alumina ceramic-bearing material which may be more resistant to severe wear. Previous in vitro simulator studies have not replicated in vivo wear rates or mechanisms. The aim of this study was to compare previous generation non-HIPed alumina and modern HIPed alumina in a hip joint simulator under 'normal' and 'harsh' testing conditions. HIPed alumina was found to have a lower wear rate than non-HIPed alumina, although the difference was not statistically significant at the 95% confidence level. Testing in Gelofusine and water lubricants did not elevate the wear rates of either material. Elevated swing phase load testing also had no significant effect on the wear rates of either material. Testing in the absence of any lubricant produced verysevere wear of the non-HIPed material in one specimen only.
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We have examined 26 retrieved, failed titanium-alloy femoral stems. The clinical details, radiological appearances and the histology of the surrounding soft tissues in each patient were also investigated. The stems were predominantly of the flanged design and had a characteristic pattern of wear. A review of the radiographs showed a series of changes, progressive with time. The first was lateral debonding with subsidence of the stem. This was followed by calcar resorption and fragmentation or fracture of the cement. Finally, osteolysis was seen, starting with a radiolucency at the cement-bone interface and progressing to endosteal cavitation. Three histological appearances were noted: granulomatous, necrobiotic and necrotic. We suggest that an unknown factor, possibly related to the design of the stem, caused it to move early. After this, micromovement at the cement-stem interface led to the generation of particulate debris and fracture of the cement. A soft-tissue reaction to the debris resulted in osteolysis and failure of fixation of the prostheses.
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The aim of this study was to investigate whether Burkholderia cepacia is capable of survival and growth within the free-living amoeba Acanthamoeba polyphaga using a differential immunofluorescence assay of bacterial-amoebal cocultures and viable counts of bacteria determined after amoebal lysis. The numbers of intra-amoebal bacteria and the numbers of infected amoebae increased over time; although, when heat-killed bacteria were used, no intracellular bacteria were observed, These findings should be taken into account in future studies of environmental reservoirs of Burkholderia cepacia.
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The second most common major heart operation in the western world is valve replacement. Any one of the four heart valves may become either so stenotic or regurgitant that it needs to be replaced in order to restore normal heart function. Although replacement surgery of dysfunctional heart valves has a very high success rate, it can provide the surgeon with a difficult decision regarding the choice of a suitable prosthesis for the individual patient. Over the years many different types of artificial heart valves have been devised. Surgeons typically deal with a heart valve replacement by installing a mechanical prosthesis or by using a bioprosthetic valve, hand-crafted from animal tissue. Least commonly, valves can be taken from human organ donors. Mechanical valve substitutes have a long fatigue life but the central flow occluders often induce blood cell trauma. Tissue substitutes have an unimpeded central orifice when open, cause minimal cell damage but have a relatively short fatigue life, especially in children where calcification may be a major problem. More recently alternative materials, such as polyurethane, have been used in artificial heart valve design while the new concept of tissue-engineering has enhanced the prospects towards an ideal cardiac valve replacement. Today's artificial valves are designed with a better understanding of the cardiovascular system with the aid of computers. Advances in computer software have allowed simulations of fluid flows through valve substitutes, both in cardiac flow simulators and the heart itself.
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Polyethylene wear debris generated at the bearing surfaces of total artificial hip joints is thought to play an important role in the periprosthetic osteolysis and ultimately the aseptic loosening of these prostheses. The macrophage is believed to be central to this process by releasing various cytokines and other mediators of osteolysis upon phagocytosis of the polyethylene wear debris. This study evaluated the in vitro bone resorption response of C3H murine peritoneal macrophages to clinically relevant GUR 1120 polyethylene particles. Macrophages were co-cultured in vitro with GUR 1120 particles with a mean size of 0.24, 0.45, 1.71, and 7.62, and GUR 1120 polyethylene resin with a mean size of 88μm at various particle volume (μm): macrophage ratios (0.1:1; 1:1; 10:1; and 100:1). The conditioned supernatants were incubated with calcium radio-labeled mouse calvariae, and bone resorption was measured as calcium release. The results showed that the 0.24 μm particles stimulated the macrophages to generate bone resorbing activity at a ratio of 10(μm) per macrophage. The 0.45 and 1.71μm particles were active at a ratio of 100(μm) per macrophage, and the 7.62 and 88 μm particles were inactive at all the doses tested. The co-culture supernatants were also assayed for TNF-α, IL-1β, IL-6, and PGE. The results followed the same trend for particle size and volume dose to that observed for the bone resorbing activity. This study has demonstrated, for the first time, the importance of size and dose of clinically relevant polyethylene particles on the osteolytic response of macrophagesin vitro.
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Submicrometer- and micrometer-sized ultra-high molecular weight polyethylene (UHMWPE) wear particles have been associated with osteolysis and failure of total artificial joints. Previous studies have isolated predominantly submicrometer-sized particles at the expense of larger particles (>10 microm). This study aimed to isolate and characterize quantitatively all sizes of UHMWPE wear particles generated in 18 Charnley hip prostheses. In addition, to analyze the wear debris with respect to the total volumetric wear of the cup and damage to the femoral head. Particle size distributions ranged from 0.1 to ->1000 microm. A significant proportion (3-82%) of the mass of the wear debris isolated was>10 microm. The mode of the frequency distribution of the particles was in the range 0.1-0.5 microm for all patients. However, analysis of the mass of wear debris as a function of its size allowed differentiation of the wear debris from different patients. Femoral head damage was associated with high volumetric wear and increased numbers of biologically active submicrometer-sized particles.
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We have compared the capacity of clinically relevant wear debris from seven different cement types to activate macrophages to produce TNF-alpha, IL-1beta, IL-6 and bone resorbing activity in vitro. The bone cements were: CMW 1 original (PMMA only); CMW 1RO (1 microm BaSO4; 9.2%); CMW copolymer bone cement 1 (10 microm BaSO4; 10%); CMW copolymer bone cement 2 (1 microm BaSO4; 10%); Palacos R (10 microm ZrO2; 15.6%); CMW Calcium phosphate cement 20% (10 microm tri-calcium phosphate; 20%) and CMW calcium phosphate cement 30% (10 microm tri-calcium phosphate; 30%). Cement debris was produced aseptically using a simple configuration wear test. The majority of particles were in the size range 0.1-0.5 microm for each cement type. The cement particles were co-cultured with the U937 macrophage cell line at ratios of 10 and 100 microm3 particle volumes to macrophage cell numbers for 24 h. At the 10:1 ratio the particles had no effect on the cells. At the 100:1 ratio, the major cytokine produced was TNF-alpha and there were no statistical differences between the different types of cement debris. The bone resorption activity of the co-culture supernatants was significantly greater than the control (U937 cells without particles) for particles of CMW 1RO, CMW copolymer bone cement 1, CMW copolymer bone cement 2 and Palacos R (P<0.05, ANOVA). However there were no statistical differences between the levels of bone resoprtion evoked by these four cement types. The CMW1 original and CMW calcium phosphate containing cements failed to induce the macrophages to elaborate bone resorption activity at the 100:1 ratio. These data suggest that the addition of radio-opaque additives to bone cement may increase the capacity of the debris to induce osteolysis.
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The response of primary human peripheral blood mononuclear phagocytes to challenge with clinically relevant ultra-high molecular weight polyethylene (UHMWPE) wear debris of known particle size and dose was evaluated. Particles with a mean size of 0.24, 0. 45, 1.7, 7.6, and 88 microm were cocultured with cells for 24 h before assessment of cell viability and production of the osteolytic cytokines interleukin (IL)-1 beta, IL-6, tumor necrosis factor-alpha, and granulocyte macrophage colony-stimulating factor, and prostaglandin E(2). All particle fractions were evaluated at particle volume (microm(3)) to cell number ratios of 10:1 and 100:1, which had been previously identified as being the most stimulatory and clinically relevant. None of the test fractions had an effect on cell viability. Whereas the heterogeneity of human individuals was clearly evident in the responses of the donors evaluated in this study (the response of donor 3 was between 5 and 20 times greater than the other donors), the most biologically active particles were found to be submicrometer in size. Stimulation with phagocytosable particles (0.24, 0.45, and 1.7 microm) resulted in enhanced levels of cytokine secretion. Macrophages stimulated with particles outside this size range produced considerably less cytokines at the volumes tested. These results confirm earlier findings and suggest that the size and volume of UHMWPE particles are critical factors in macrophage activation. Furthermore, they suggest that the heterogeneity of human individuals may be another important factor in determining implant life.
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The response of primary human peripheral blood mononuclear phagocytes to challenge with polyethylene particles of known size and dose was evaluated. Particles with mean sizes of 0.21, 0.49, 4.3, 7.2, and 88 microm were co-cultured with cells for 24 h prior to the assessment of cell viability and production of the osteolytic mediators IL-1beta, IL-6, TNFalpha, GM-CSF and PGE2. All particle fractions were evaluated at particle volume (microm3) to cell number ratios of 10:1 and 100:1 which were previously identified as being the most biologically active and clinically relevant. The heterogeneity of human individuals was clearly evident both in the profile and the magnitude of the response of the donors evaluated in this study (the response of donor 5 being 2- to 15-fold lower than that of the other donors). Only the sub-micrometre particles stimulated significantly enhanced cytokine secretion at the ratios tested: mean particle sizes of 0.49 and 0.21 microm being the most biologically active. Macrophages stimulated with particles outside this size range produced considerably lower levels of mediator. These results compared favourably with the results of earlier studies, which demonstrated that particles within the phagocytosable size range (0.1-10 microm) were the most biologically active. These results, therefore, confirm earlier findings and suggest that the size and volume of polyethylene particles are critical factors in macrophage activation. Furthermore, they suggest that the heterogeneity of human individuals may be another important factor in determining implant life and could provide the basis for a valuable diagnostic tool to identify those patients most at risk of implant loosening.
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Polyethylene wear debris generated at the bearing surfaces of total artificial hip joints is thought to play an important role in the periprosthetic osteolysis and ultimately the aseptic loosening of these prostheses. The macrophage is believed to be central to this process by releasing various cytokines and other mediators of osteolysis upon phagocytosis of the polyethylene wear debris. This study evaluated the in vitro bone resorption response of C3H murine peritoneal macrophages to clinically relevant GUR 1120 polyethylene particles. Macrophages were co-cultured in vitro with GUR 1120 particles with a mean size of 0.24, 0.45, 1.71, and 7.62, and GUR 1120 polyethylene resin with a mean size of 88 microm at various particle volume (microm)(3): macrophage ratios (0.1:1; 1:1; 10:1; and 100:1). The conditioned supernatants were incubated with (45)calcium radio-labeled mouse calvariae, and bone resorption was measured as (45)calcium release. The results showed that the 0.24 microm particles stimulated the macrophages to generate bone resorbing activity at a ratio of 10(microm)(3) per macrophage. The 0.45 and 1.71 microm particles were active at a ratio of 100( microm)(3) per macrophage, and the 7.62 and 88 microm particles were inactive at all the doses tested. The co-culture supernatants were also assayed for TNF-alpha, IL-1beta, IL-6, and PGE(2). The results followed the same trend for particle size and volume dose to that observed for the bone resorbing activity. This study has demonstrated, for the first time, the importance of size and dose of clinically relevant polyethylene particles on the osteolytic response of macrophages in vitro.
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The aim of this research was to investigate the tribological condition of acetabular tissue before and after bipolar hip surgery. Articular cartilage was taken from the femoral head of patients undergoing primary joint replacement as a control. Tissue was also taken from the acetabular cups of patients undergoing revision hip surgery after primary bipolar surgery and compared with the control cartilage. The biomechanical characteristics of the two tissue types were tested using friction and compression tests. The friction tests were carried out on a sliding friction rig under nominal contact stresses of 0.5 and 4 MPa. The compression tests were carried out under a 0.8 MPa contact stress. The majority of the bipolar patients produced friction coefficients that were significantly higher than those produced by the control group, and the compression tests highlighted that the tissue from the bipolar patients produced a much greater rate of increase in displacement compared with the control cartilage. Histology showed major differences between the control cartilage and the bipolar tissue. The control cartilage showed a healthy collagen structure with a good distribution of proteoglycan whereas the majority of the bipolar tissue had lost tissue architecture and had a sparse fibrous structure. The high friction coefficients with the bipolar tissue imply that the frictional torque at the outer head of the bipolar prosthesis would be large compared with the inner bearing frictional torque. It was therefore predicted that the motion of the bipolar prosthesis should occur at the inner bearing.
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Since the implication of polyethylene wear debris as a major cause of osteolysis in total joint replacements, there has been much interest in polyethylene wear studies and in cell culture studies using ultra-high molecular weight polyethylene (UHMWPE) wear debris. Studies have shown that particles in the 0.1-10 microns size range are particularly important in causing adverse cellular reactions resulting in osteolysis. The morphology, the mass and size distributions, and the number of wear particles produced at the joint surfaces are influenced by the tribological conditions at the joint. Laboratory wear tests are used to investigate the wear properties of prosthetic joint materials and different research groups have used different lubricants in these tests. This paper shows that the volumetric wear and morphology of UHMWPE particles generated in vitro are influenced by the type of lubricant used. This study compared, quantitatively, UHMWPE wear debris generated in deionized water to debris that was generated in a system lubricated by bovine serum which was diluted to 25 per cent. The wear factors of UHMWPE in water and serum lubricants were significantly different (p<0.05). UHMWPE wore 14 times more in water than in serum. Quantitative analysis of the wear particles showed that the debris that was generated in serum was morphologically different from debris that was produced in a water-lubricated system. Furthermore, the particles produced in serum showed a closer similarity to those found in retrieved acetabular tissues.
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The wear debris generated from UHMWPE (ultra high molecular weight polyethylene) has been recognised as one of the major causes of failure in THR (total hip replacement). GUR 1120 (compression moulded) and GUR 4150HP (ram extruded) which are currently the most frequently used materials in THR were studied in pin-on-plate wear test. The wear particles generated from this test were observed by scanning electron micrograph and analysed by image analysis. The results from this study showed that GUR 4150HP had superior wear resistance than GUR 1120 under relatively high wear factor conditions. These results also highlighted the importance of multidirectional motion and its effect on the wear rates of UHMWPE. The multidirectional motion tended to show a higher wear factor than previous studies using unidirectional motion conducted under otherwise similar conditions. The wear debris analysis conducted with the wear particles collected from unidirectional (relatively rough) pin-on-plate wear tests (GUR 1120 and GUR 4150HP) showed that the greatest number of particles had a size range of 0.1-0.5 micron followed by 0.5-1.0 micron, 1.0-5.0 microns and 5.0-10.0 microns, in both GUR 1120 and GUR 4150HP. However, comparing the masses of the wear particles, the bigger size range of greater than 10 microns, had the highest percent mass followed by 1.0-5.0 microns, 0.5-1.0 micron, 0.1-0.5 micron and 5.0-10.0 microns.
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The biological reactions to polyethylene wear debris have been shown to result in osteolysis and loosening of total hip arthroplasties. This has led to renewed interest in the use of metal on metal bearings in hip prostheses. This study employed uniaxial and biaxial multistation pin on plate reciprocators to assess how the carbon content of the cobalt chrome alloy and the types of motion affected the wear performance of the bearing surfaces and the morphology of the wear debris generated. The low carbon specimens demonstrated higher wear factors than both the mixed carbon pairings and the high carbon pairings. The biaxial motion decreased the wear rates of all specimens. Plate wear was significantly reduced by the biaxial motion, compared to pin wear. The metal wear particles isolated were an order of magnitude smaller than polyethylene particles, at 60-90 nm, and consequently, 100-fold more particles were produced per unit volume of wear compared to polyethylene. The low carbon specimens produced significantly larger particles than the other material combinations, although it is thought unlikely that the difference would be biologically significant in vivo. The volumetric wear rates were affected by the carbon content of the cobalt chrome alloy, the material combination used and type of motion applied. However, particle morphology was not affected by the carbon content of the alloy or the type of motion applied.
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This study analyses explanted prostheses from a single surgeon's 16-yr series of Mittelmeier cementless total hip replacements. The patient group was young (patients aged 15-60, average 40) and active. Revision was related to various factors. All components were measured using a Kemco 3D coordinate measurement machine. Wear surfaces and linear wear penetrations of 11 retrieved Mittelmeier Autophor ceramic hip components (average implantation time 8.6 yr, range 1-13 yr) were analysed and the different types of wear identified. Surface analysis was then performed using Talysurf contacting profilometry and scanning electron microscopy. Three types of wear were identified: low wear (1 case), stripe wear (6 cases) and severe wear (4 cases). Stripe wear was characterised as a stripe of worn area on the head up to 150 microns deep whilst the rest of the head showed very low wear. The cups in the stripe wear cases were worn over about 40-50% of the surface. Severe wear cases had very large areas of heavy wear and visible volume loss on both heads and cups. The four cases of severe wear were associated with abnormal clinical histories.
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Ultra-high-molecular-weight polyethylene (UHMWPE) components for total joint replacement generate wear particles which cause adverse biological tissue reactions leading to osteolysis and loosening. Sterilisation of UHMWPE components by gamma irradiation in air causes chain scissions which initiate a long-term oxidative process that degrades the chemical and mechanical properties of the polyethylene. Using a tri-pin-on-disc tribometer we studied the effect of ageing for ten years after gamma irradiation in air on the volumetric wear, particle size distribution and the number of particles produced by UHMWPE when sliding against a stainless-steel counterface. The aged and irradiated material produced six times more volumetric wear and 34 times more wear particles per unit load per unit sliding distance than non-sterilised UHMWPE. Our findings indicate that oxidative degradation of polyethylene after gamma irradiation in air with ageing produces more wear.
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The aim of this research was to investigate the role of the hydrophilic properties of the proteoglycan molecules within the cartilage matrix, on the tribological properties of joint tissues in the mixed and boundary lubricating regime. Bovine articular cartilage, bovine meniscus and bovine cartilage that had been degraded to remove the chondroitin sulphate from its proteoglycans were studied in order to investigate differences in their friction and compression responses. The tissues were tested on a sliding friction rig under nominal contact stresses of 0.5 and 4 MPa. The compression tests were carried out under a 0.8 MPa contact stress. The compression tests showed the cartilage and meniscus deforming at the same rates, but the degraded cartilage deformed more quickly to reach its equilibrium position in a shorter period of time. The friction tests carried out at a constant load revealed the friction of the meniscus rising more rapidly with loading time than the cartilage. The degraded cartilage followed an almost identical curve as the untreated cartilage. Although the reduced proteoglycan content of the degraded cartilage substantially altered the biphasic compression response, it did not have an effect on the frictional properties of the tissue.
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We examined stainless-steel, cobalt-chrome, titanium and alumina and zirconia ceramic femoral heads retrieved at revision surgery. All the heads had articulated against ultra-high-molecular-weight-polyethylene (UHMWPE) acetabular cups. We studied the simulation of third-body damage and the wear of UHMWPE against the various materials used for the heads. The surfaces of the retrieved heads were analysed using a two-dimensional contacting profilometer. Third-body damage was characterised by the mean height of the scratches above the mean line (Rpm). The alumina ceramic and zirconia ceramic retrieved heads were found to have significantly less damage. In laboratory studies the ceramics were also more resistant to simulated third-body damage than the metal alloys. We studied the wear of UHMWPE against the damaged counterfaces in simple configuration tests. The damaged ceramics produced less polyethylene wear than the damaged metal counterfaces. The wear factor of UHMWPE against the damaged materials was dependent on the amount of damage to the counterface (Rp). Our study has shown the benefit of using the harder and more damage-resistant ceramic materials for femoral heads.
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The aim of this study was to investigate the effects of the proteins and the lipids on the frictional properties of articular cartilage in the mixed and boundary lubricating regimes. Bovine articular cartilage that had been treated to remove the lipids, and cartilage that had been treated to remove the proteins, from the surface layer were studied in order to investigate differences in their lubricating abilities. Tests were carried out on a sliding friction rig under contact pressures of 0.5 and 4 MPa. The results of the friction tests carried out under the 4 MPa contact pressure showed a slight increase in friction coefficient for both the lipid and the protein deficient cartilage when compared with the control cartilage. These differences were more apparent at the shorter loading times but none of the differences were found to be statistically significant. The tests at the lower contact pressure of 0.5 MPa showed that removing the lipids by washing the surface with detergent increased the friction coefficient at the shorter loading times but had no effect at the longer loading times. Digesting the cartilage with trypsin to remove the proteins from the surface layer had no effect on the friction coefficient at the shorter loading times but reduced the friction coefficient at the longer loading times. Both these results were confirmed with specimens that had both the lipids and the proteins removed from the surface layer.
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Damage to polished femoral heads in vivo can cause increased wear of acetabular cups. Oxidation and ageing after sterilisation by gamma irradiation in air, can also change the mechanical properties and wear resistance of ultra high molecular weight polyethylene (UHMWPE). This study investigated the combined effect of these changes in material properties on the wear of UHMWPE for different counterface roughnesses, representative of new femoral heads and those damaged in vivo. Wear rates were studied on a tri-pin-on-disc tribometer in a protein-containing solution. A comparison was made of the wear, using three different counterface roughnesses, of specimens that were manufactured from polyethylene acetabular cups of different shelf ages (3-120 months) after gamma irradiation in air but never implanted. These were compared to the wear of control specimens that were manufactured from cups that had not been sterilised. The wear surfaces were tested 1 mm below the initial articulating surface of the cup, the position of high degradation. The wear rate of UHMWPE which had been sterilised by gamma irradiation in air was shown to increase significantly with ageing time on the shelf for all counterface conditions. The wear rate of all materials increased markedly as the counterface roughness increased, but to different extents depending on the age of the material. The combined effect of ageing and increase in counterface roughness had a dramatic effect (as high as 2000 fold increase) on the wear rate. Both ageing of the polymer and damage to the femoral head have been cited as causing increased wear in vivo. The results of this study demonstrate that these variables can act synergistically to markedly effect UHMWPE wear rate.
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The effect of burn patient serum on fibroblast and keratinocyte cell morphology in culture was investigated using the scanning electron microscope. Serum was taken from five patients with burn injuries ranging from 8 to 65 per cent TBSA (10-65 per cent full-thickness). One patient had superficial burns. Pooled serum from 23 volunteers was used as the control serum. The cells were seeded onto collagen-coated glass coverslips and incubated for 5 days with culture medium containing 10 per cent (v/v) control serum or patient serum taken during the early postburn period. Scanning electron micrographs demonstrated a reduction in fibroblast cell density with serum from patients with lull-thickness burns. Furthermore, the spindle shape of the fibroblast cell was greatly exaggerated compared with control cultures. The integrity of the keratinocyte sheet was destroyed when keratinocyte cells were incubated with serum from patients with full-thickness burns. Globular-like structures or membrane protrusions were present in concentrated areas on keratinocyte cells which were not present in control cultures. This study demonstrated the vulnerability of cutaneous cells to systemic factors present in the early postburn serum. The extent of the effect appears to be related to the presence of full-thickness injury. This effect may further explain the frequent aberrant wound healing response to burn injury.
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The purpose of this investigation was to assess the potential of commercially available extracellular matrix proteins, as enhancers of human keratinocyte attachment and proliferation, with a view to their incorporation into a Skin equivalent. The following substrates were studied: type I and type IV collagen, fibronectin, gelatin and laminin. Human keratinocytes were cultured in low-calcium, serum-free medium. The number of cells attached to each substrate, observed under phase-contrast inverted microscopy in randomly selected fields of view, were counted 2 h afterseeding. Measurements of growth rate and colony-forming efficiency were made at 24-h intervals. None of the substrates tested were found to have an effect significant enough to warrant further investigation or inclusion into skin equivalent.
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The objective of this study was to investigate the effect of counterface roughness and lubricant on the morphology of ultra-high molecular weight polyethylene (UHMWPE) wear debris generated in laboratory wear tests, and to compare this with debris isolated from explanted tissue. Laboratory tests used UHMWPE pins sliding against stainless steel counterfaces. Both water and serum lubricants were used in conjunction with rough and smooth counterfaces. The lubricants and tissue from revision hip surgery were processed to digest the proteins and permit filtration. This involved denaturing the proteins with potassium hydroxide (KOH), sedimentation of any remaining proteins, and further digestion of these proteins with chromic acid. All fractions were then passed through a 0.2 micron membrane, and the debris examined using scanning electron microscopy. The laboratory studies showed that the major variable influencing debris morphology was counterface roughness. The rougher counterfaces produced larger numbers of smaller particles, with a size range extending below 1 micron. For smooth counterfaces there were fewer of these small particles, and evidence of larger platelets, greater than 10 microns in diameter. Analysis of the debris from explanted tissues showed a wide variation in the particle size distribution, ranging from below 1 micron up to several millimetres in size. Of major clinical significance in relation to osteolysis and loosening is roughening of the femoral components, which may lead to greater numbers of the sub-micron-sized particles.
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Degenerative changes in a proportion of implanted homologous cardiac valves are considered to be due to the immunologic tissue reactions initiated by the donor valves. Sterilization and storage protocols can be used to modulate the immunogenicity of donor valves prior to implantation. Therefore, it is essential to assess the effect of treatment protocols on the immunogenicity and viability of donor valve tissue. The optimal conditions for two novel in vitro tests to assess the immunogenicity and viability of human cardiac valve tissue, the valve cusp cell/responder lymphocyte reaction to assess immunogenicity and the tetrazolium based colorimetric assay to assess viability of valve tissue, are described. The in vitro tests that have been developed in this study will be helpful in assessing the effect of various treatment protocols on the immunogenicity and viability of homologous human cardiac valves.
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The size of morphology of ultrahigh molecular weight polyethylene (UHMWPE) debris generated at the articulating surfaces of artificial joints are characterized by scanning electron microscopy. Results show that a great number of the particles are in the micron size range, although samples obtained from some patients have sizes in the millimeter range. These findings can be used to improve the tribology of future prosthesis designs.
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Leg ulcers present a common and recurring problem in older people creating discomfort and distress for the patient and a great cost to the health care services. Cultured keratinocyte grafts have been used by many investigators to stimulate healing of chronic venous ulcers. It has been proposed that they may do this by producing cytokines which modulate the healing process. However, the types and levels of cytokines in the leg ulcer fluid before and during healing are not known. Wound fluid was collected from venous leg ulcers in 18 patients beneath occlusive Tegaderm dressing for 4 to 6 h. The leg ulcers were divided on clinical criteria into 'healing' and 'non-healing'. PDGF-AB, GM-CSF, IL-1 alpha, IL-1 beta, IL-6 and bFGF were measured by ELISA and the levels of IL-1 alpha, IL-1 beta and IL-6 were also measured using biological assays. The effect of leg ulcer wound fluid on fibroblast and keratinocyte proliferation was measured indirectly by 3H-thymidine incorporation and MTT assay. Total protein, albumin levels, fibronectin degrading activity and collagenase activity, both active and latent were measured. No statistically significant differences in the levels of cytokines or collagenase were identified between healing and non-healing leg ulcers in the sample of leg ulcers studied. However, this study does give valuable information concerning the levels of cytokines and collagenase in chronic leg ulcer wound fluid.
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The immunogenicity of allogeneic cardiac valves (ACV) has not been previously demonstrated in vitro, though valve failure due to tissue degeneration has been attributed to adverse immunological reactions. A novel in vitro assay has been developed in a Brown Norway (BN; RT1n)-Lewis (RT1; donor-recipient) rat model system that demonstrates the immunogenicity of ACVs. A single cell suspension of viable cardiac valve conduit (CVC) cells was obtained by collagenase treatment of BN rat aortic valve conduits. Brown Norway rat CVC cells (5 x 10(4)) and Lewis responder lymphocytes (10(5)) were co-cultured in 96 well plates in RPMI 1640 plus 2.5% (v/v) non heat-inactivated Lewis rat serum and supplements with appropriate controls. Stimulation of responder lymphocytes by CVC cells was measured by 3H-thymidine incorporation into DNA. The counts obtained between 96-192 h of co-culture in the CVC cell/responder lymphocyte reaction were significantly higher (P<0.05) than those of responder cell controls as assessed by analysis of variance. These results indicate the presence of potent immunostimulatory cells in viable ACVs and the possibility of using a sensitive and reproducible in vitro assay to evaluate ACV immunogenicity.
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The effects of various concentrations of granulocyte macrophage colony stimulating factor (GM-CSF), gamma interferon (gamma IFN) and interleukins 1 alpha (IL-1 alpha), 1 beta (IL-1 beta) and 3 (IL-3) on the anaemic mouse spleen cell bioassay for erythropoietin (EPO) were investigated. Addition of IL-3 and GM-CSF at various concentrations had no effect on EPO stimulated 3H thymidine incorporation. However the addition of IL-1 alpha, IL1-beta and gamma IFN (3.3 x 10(-8) gl-1) caused a significant (P<0.01) inhibition of EPO stimulated thymidine incorporation. This suggests that the EPO bioassay may be influenced by variable levels of some inflammatory cytokines in serum. Previous studies have shown that the bioassay is influenced by serum transferrin levels and thus serum immunoassays remain the technique of choice for specific estimates of EPO. Since EPO bioassays are not specific, they should be reserved for situations in which an estimate of the total erythropoietic activity of serum is required.
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This study examined the humoral immune responses to Malassezia furfur serovars A, B and C of 10 patients with pityriasis versicolor, 10 patients with seborrheic dermatitis and 20 age- and sex-matched controls. A transferable solid-phase ELISA was used to determine titres of total Igs, IgM, IgA and IgG specific to M. furfur serovars A, B and C. The results demonstrated that patients with seborrheic dermatitis had a significantly higher titre of total Igs to serovar A than patients with pityriasis versicolor; and that patients with seborrheic dermatitis had a significantly higher titre of IgA to serovar C than patients with pityriasis versicolor. The titres of total Igs for controls and patients with seborrheic dermatitis were significantly lower to serovar B than to serovar C. A modified TSP ELISA was used to determine the titres of the IgG subclasses. Titres of IgG1,3,4 to serovar B were significantly higher in seborrheic dermatitis patients than pityriasis versicolor patients and titres of IgG3 to serovar A were significantly higher in seborrheic dermatitis patients than pityriasis versicolor patients. However, despite the differences between the patient groups, none of these results was significantly different to those of controls. Thus, this study did not demonstrate any differences in humoral immunity of patients suffering from Malassezia-associated dermatoses when compared to normal controls. These results may suggest that the humoral immune response to M. furfur is not related to the pathogenesis of Malassezia-associated dermatoses, but simply to the carriage of M. furfur on the skin.
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It has been postulated that patients with Malassezia furfur-associated dermatoses have a deficient cell-mediated immune response to M. furfur. This study examined the cell-mediated immune responses to M. furfur serovars A, B and C of 10 patients with pityriasis versicolor and 10 age- and sex-matched controls; and 10 patients with seborrheic dermatitis and 10 age- and sex-matched controls. The responses to each serovar of M. furfur were assessed using the lymphocyte transformation assay and the leukocyte migration inhibition assay. The lymphocyte transformation responses of the patients with pityriasis versicolor to M. furfur serovars A, B and C (0/10, 6/10 and 5/10 respectively) were not significantly different from those of controls (0/10, 2/10 and 1/10). However, for patients with seborrheic dermatitis, significantly more patients' lymphocytes responded to serovars B and C (6/10 and 6/10 respectively) than those of controls (1/10 and 1/10). No patient or control responded to serovar A. In the leukocyte migration inhibition assay, the leukocytes from a greater proportion of patients with pityriasis versicolor (5/7) responded to serovar B than controls (2/10); and the leukocytes from a greater proportion of patients with seborrheic dermatitis (4/10) responded to serovar C than controls (0/9). Thus, this data did not indicate the presence of any cell-mediated immune deficiency to M. furfur in patients with pityriasis versicolor or seborrheic dermatitis, as measured by the lymphocyte transformation assay or the leukocyte migration inhibition assay. The greater responsiveness of T lymphocytes from patients may indicate that T lymphocytes might be involved in the pathogenesis of these diseases.
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Propionibacterium acnes is frequently referred to as a strictly anaerobic skin commensal of low pathogenicity, a description which is misleading. The bacterium is like Dr Jekyll and Mr Hyde, demonstrating both beneficial and harmful effects which are host- and possibly strain-dependent. Although the pilosebaceous follicles of the face and upper trunk are the major habitat of P. acnes, significant populations also exist in the healthy mouth and colon of a minority of individuals. P. acnes can behave as an opportunist pathogen in compromized hosts but the incidence of such infections is low or underestimated. There is considerable evidence that persistence of this phagocytosis-resistant bacterium in a variety of organs and tissues can lead to a chronic inflammatory response with granuloma formation. P. acnes has the capacity to produce enzymes which could contribute to tissue destruction in dental caries and periodontal disease. Whether some strains are more virulent than others or are associated with specific disease syndromes will be ascertained only when an adequate biotyping scheme has been developed. P. acnes should not be dismissed as a culture contaminant without good reason, especially in situations where other bacteria are not recovered. To date, attempts to harness the potent immunomodulating effects of the organism in cancer therapy have been disappointing.
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The aetiological role of Malassezia furfur in various dermatoses is controversial. The role of the three serovars of M. furfur in Malassezia-associated diseases has not been investigated. This study measured population densities of M. furfur serovars A, B and C, propionibacteria and Micrococcaceae on the chest, back, forehead, left and right cheeks of 10 patients with pityriasis versicolor, and 10 age- and sex-matched controls; and 10 patients with seborrhoeic dermatitis, and 10 age- and sex-matched controls. The population densities of M. furfur, propionibacteria and Micrococcaceae did not vary at a given site between patients and the corresponding control subjects. Malassezia furfur serovar A was found to be the predominant isolate on the chest and back of all four groups, but there was no difference in the distribution of serovars on the forehead and cheeks. No serovar was specifically associated with lesional skin in either disease. Thus, this data indicated that there was no difference in either the total population density of M. furfur or the distribution of serovars on lesional skin compared with control skin in either pityriasis versicolor or seborrhoeic dermatitis.
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The use of allografts to effect wound closure on full thickness skin injuries is limited by unpredictable graft rejection times. If the period of graft take could be extended, the use of allografts would reduce the morbidity and mortality associated with these wounds. This study evaluated the effects of variation in the cryopreservation protocol on the viability and immunogenicity of skin using a murine model system. Immunogenicity was assessed by the stimulatory activity of C3H (H-2K) skin-derived epidermal cells (EC) in primary one-way EC/lymphocyte reactions with BALB/c (H-2d) and CBA (H-2K) responder lymphocytes. Viability was determined by measuring tetrazolium reductase activity. The following cryopreservation protocols were assessed: freezing at 1, 30, 64, and>100 degrees C/min in 10 and 15% (v/v) Me2SO and freezing at 30 degrees C/min in 5 to 20% (v/v) Me2SO or glycerol. A cryopreservation protocol of 30 degrees C/min in 15% (v/v) Me2SO proved optimal for murine skin allograft storage and immunomodulation. The viability of skin treated by this protocol was maintained (78% of fresh skin viability, no significant difference analysis of variance). The stimulatory capacity of treated EC for H-2K and H-2d lymphocytes was 5 +/- 4 and 5 +/- 9% (+/- 95% confidence limits) of fresh EC (100%) activity. Langerhans cell numbers in epidermal sheets and EC suspensions did not correlate with the stimulatory capacity of fresh and treated EC for allogeneic lymphocytes. A functional impairment of Langerhans cell immunostimulatory capacity was implied.(ABSTRACT TRUNCATED AT 250 WORDS)
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To understand the basis for the anti-inflammatory activity of tetracyclines in acne, we compared the cytokine profiles [interleukin 1 (IL-1) alpha and beta, tumor necrosis factor (TNF) alpha, and IL-6] and bacterial flora of 66 open comedones removed from eleven patients before and after at least 8 weeks treatment with either tetracycline or minocycline. Pre-treatment, the only cytokine regularly recovered from comedones was bioactive IL-1 alpha-like material. The mean concentration of IL-1 alpha-like bioactivity/mg comedonal material rose from 272.0 +/- 88.6 pg to 844.3 +/- 196.7 pg following treatment (p<0.05, Wilcoxon matched pairs). All six minocycline-treated patients showed an increase in bioactive IL-1 alpha-like material compared with three of five tetracycline-treated patients. The incidence (p<0.001, chi 2) and concentration (p<0.05, Wilcoxon) of immunochemical IL-beta were also raised post-treatment, although significantly more patients assigned to minocycline therapy had detectable levels of this cytokine before therapy was initiated. However, the mean concentration of IL-1 beta/mg comedonal material post-treatment was similar in both groups (72.5 +/- 23.3 pg for tetracycline-treated compared with 78.6 +/- 41.9 pg for minocycline-treated patients). The other cytokines were either absent (IL-6) or present in<10% of comedones (TNF alpha) before and after therapy. Following treatment, only three of 11 patients showed a decrease of>or = 1 log10 in propionibacterial numbers/mg comedonal material, whereas six patients showed an increase of>0.5 log10 in numbers of staphylococci. In eight patients, the increase or decrease in staphylococcal numbers correlated with the change in concentration of IL-1 alpha-like bioactivity. This is the first study to show an effect of antibiotic therapy on cytokine levels in vivo. Increased levels of IL-1 in comedones destined to become inflamed may enhance resolution and promote repair of the damaged follicular epithelium. Hence, these results provide further evidence of the augmentation of immune responses by tetracyclines and support the hypothesis that epidermal IL-1 plays a physiologic role in wound healing.
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A transferable solid-phase (TSP) ELISA was developed for the determination of antibody titres specific to Malassezia furfur serovars A, B and C in human sera. A survey of levels of class-specific antibodies (IgM, IgG and IgA) to M. furfur serovars A, B and C in relation to age (2-64 years; 60 individuals) demonstrated that individuals had immunity to M. furfur by the age of 2-3 years. There was no difference in either IgM or IgG levels into adulthood. The only age-related differences were lower IgM titres to the three serovars in the 60-64 year age-group compared with younger individuals. There was, however, a difference between titres of antibody specific to the three serovars. The mean reciprocal log2 IgM titre to serovar A (6.9) was significantly higher (P<0.05) than that to serovar B (mean reciprocal log2 titre of 5.8), but not to serovar C (6.1). In contrast, the mean reciprocal log2 IgG titre to serovar A (6.5) was significantly lower (P<0.05) than those to serovars B and C (mean reciprocal log2 titre of 8.9 in both cases).
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The factors that initiate the inflammatory response in acne are not known. The presence of pro-inflammatory cytokines in acne comedones was therefore investigated. One hundred eight open comedones were collected from 18 untreated acne patients (10 male, 8 female). Each comedone was homogenized and centrifuged, and the supernatant was analyzed for bioactive and immunochemically detectable IL-1 alpha, IL-1 beta, and TNF alpha. Viable counts of propionibacteria, staphylococci, and Malassezia spp. were determined in the comedone pellet. Bioactive IL-1 alpha-like material was demonstrated in 76% of open comedones (range of 23-4765 pg IL-1 alpha-like bioactivity/mg of comedone material). In 58% of comedones, levels exceeded 100 pg/mg. There was no correlation between IL-1 alpha-like bioactivity and IL-1 alpha determined immunochemically. Bioactive IL-1 beta was not detected in any comedones. Twenty-four percent contained low levels of immunochemical IL-1 beta (range 12-103 pg IL-1 beta/mg comedone material). Bioactive TNF alpha was detected in three comedones with a further five comedones containing immunochemical TNF alpha (range of 61-820 pg TNF alpha/mg comedone material). The majority of open comedones (97%) contained microorganisms. There was, however, no significant correlation (Spearman's rank) between levels of any cytokine, in particular IL-1 alpha-like bioactivity, and numbers of microorganisms. Thus, bioactive IL-1 alpha-like material in the majority of open comedones may be concerned in the initiation of inflammation in acne following spongiosis or rupture of the pilosebaceous follicle wall.
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The effects of minocycline and tetracycline on the mitotic response of human peripheral blood lymphocytes was investigated in vitro. The effects of the antibiotics on the mitotic response of purified lymphocytes stimulated with Interleukin-1 beta varied according to the individual from whom the lymphocytes were obtained. At concentrations above those reported to be present in serum during conventional therapy (2-8 mg/l), there was a tendency for both minocycline and tetracycline to suppress the mitotic response. Minocycline was superior to tetracycline in this respect. However, at physiological concentrations the antibiotics either had no significant effect, suppressed the mitotic response (minocycline at 2 mg/l with one of six donors), or enhanced the mitotic response (tetracycline at 2 and 8 mg/l with four of six donors). The stimulatory effect of tetracycline was not demonstrated when lymphocytes were cultured in whole blood for up to seven days with the antibiotic alone. Similar effects of the antibiotics were observed when mononuclear cell fractions isolated from six donors were stimulated with an optimal concentration of phytohaemagglutinin (PHA). Stimulation of lymphocytes in whole blood cultures with PHA in the presence of minocycline and tetracycline revealed that, under these culture conditions, the antibiotics could suppress the mitotic response of lymphocytes at physiological doses with cells from a majority of donors.
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The capacity of minocycline and tetracycline for modulation of IL-1 secretion by LPS-stimulated human monocytes was investigated in vitro. Both minocycline and tetracycline suppressed the murine thymocyte co-mitogenic bioassay of IL-1 at 2 and 4 mg/l respectively. IL-1 beta secretion by LPS-stimulated human monocytes cultured for 24h at 1 x 10(6)/ml with 0, 5, 10 and 50 mg/l minocycline or tetracycline was therefore determined by ELISA. Monocytes from five different individuals served as replicates. LPS-stimulated monocytes secreted significantly more IL-1 beta in the presence of minocycline (P less than 0.01, 2-way analysis of variance). There was no difference in the enhanced levels of IL-1 beta secreted with 5 mg/l minocycline compared with 50 mg/l minocycline. Loss of viability could only be associated with enhanced IL-1 beta release by monocytes with 50 mg/l minocycline. Although tetracycline enhanced IL-1 beta secretion in four of the five replicate experiments, this did not prove significant owing to the large error variance between individual monocyte cultures. Thus, therapeutic levels of tetracyclines, especially minocycline, modulate mononuclear cell activities in vitro.
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The effects of tetracycline, minocycline and erythromycin on complement activation in vitro were studied. At concentrations of 100 mg/l or less, these antibiotics did not inhibit the capacity of Propionibacterium acnes to cleave C3 in normal human serum or in serum chelated of Ca2+ allowing complement activation by the alternative pathway alone. The antibiotics had no effect (at 100 mg/l) on total haemolytic activity of complement in normal human serum. This study did not provide evidence to support the hypothesis that the efficacy of these antibiotics in the therapy of inflammatory acne vulgaris can be explained by inhibition of complement activation.
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Bioassays and immunoassays for cytokines are now widely available for use in clinical laboratories which may have little or no expertise in cytokine biology. Whilst this facilitates the accumulation of data concerning cytokine levels in body fluids in disease, it is based on the assumption that such assays can be used for this purpose. In many cases, the presence of complex interfering factors in plasma and other body fluids require that assays should be subjected to detailed assay validation before confidence can be placed on the results. It is the purpose of this report to outline the potential problems with cytokine assays and the criteria that should be applied before making measurements in biological fluids.
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Malassezia furfur strains were isolated from the clinically normal skin of 10 volunteers by swabbing four different sites (forehead, ear, back and chest). The strains could be divided into three basic groups on the basis of cultural characteristics. Both unabsorbed and absorbed specific rabbit antisera were prepared against nine of the strains, and both species and group specific antigens could be demonstrated. Serologically, three group specific surface antigens could be identified which corresponded to the three groups identifiable on cultural characteristics. The relevance of these findings to previous in vitro results is discussed.
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Complement C3 deposition around lesions is an early event in the inflammation of acne vulgaris. The aims of this study were to determine the relationship between the capacity of individual comedones to cleave complement C3 and their microbial flora. The contents of 48 open comedones were expressed from the upper back of acne vulgaris patients and each comedo was homogenized individually and assayed for microbial content and capacity to induce cleavage of complement C3 in an in vitro assay system. An association between Propionibacterium population size and extent of C3 cleavage was found, but Staphylococcus and Pityrosporum population sizes did not appear to have an appreciable influence. A strong association between the weight of expressed material and C3 cleavage was apparent, irrespective of microbial population size. This observation suggests that comedones contain non-microbial material having the capacity to induce complement cleavage and hence initiate inflammation.
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The purpose of this communication is to present information which will allow the reader to judge for himself the importance of the microflora in acne and also to express the authors' working hypothesis of the role of the microflora in acne. It is generally considered that the microflora of the sebaceous follicle, whether normal or acne affected, is limited to four major genera of micro-organisms, Propionibacterium, Staphylococcus, Micrococcus and Pityrosporum. Other bacteria, bacteriophage and mites, have been found, but there is no evidence, as yet, of an association with acne and, in consequence, these are ignored in this communication.
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