Modelling of microstructural evolution in multi-layered overlay coatings.

Functionally graded, multi-layered coatings are designed to provide corrosion protection over a range of operating conditions typically found in industrial gas turbines. A model incorporating diffusion, equilibrium thermodynamics and oxidation has been developed to simulate the microstructural evolution within a multi-layered coating system. The phase and concentration profiles predicted by the model have been compared with an experimental multi-layered system containing an Al-rich outer layer, a Cr-enriched middle layer and an MCrAlY-type inner layer deposited on a superalloy substrate. The concentration distribution and many microstructural features observed experimentally can be predicted by the model. The model is expected to be useful for assessing the microstructural evolution of multilayer coated systems which can be potentially used on industrial gas turbine aerofoils.

The use of combined three-dimensional electron backscatter diffraction and energy dispersive X-ray analysis to assess the characteristics of the gamma/gamma-prime microstructure in alloy 720Li.

Multiple three-dimensional reconstructions of a γ/γ' phase structure in Alloy 720Li have been carried out by employing a serial milling technique with simultaneous electron backscatter diffraction (EBSD) and energy dispersive x-ray (EDX) analysis data collection. Combining EBSD data with EDX is critical in obtaining maps to distinguish between the chemically differing, but crystallographically similar γ and γ' phases present in the alloy studied. EDX is shown to allow the differentiation of γ and γ' phases, with EBSD providing increased grain shape accuracy. The combination of data sources also allowed identification of coherent γ/γ' phase interfaces that would not be identified using solely EBSD or EDX. The study identifies a region of grain banding within the alloy, which provides the basis for a three-dimensional comparison and discussion of γ' phase size between coarse and fine grain regions, whilst also identifying coherent γ' phase interfaces, possible only using both EDX and EBSD systems simultaneously. The majority of the γ' phase lies in the range of 1-10 µm in non-banded regions, with a detectable particle size limit of 500 nm being established. The validity of the reconstruction has been demonstrated using an electron interaction volumes model, and an assessment of the validity of EBSD and EDX data sources is discussed showing γ' phase connectivity in all dimensions.

Combined EBSD/EDS tomography in a dual-beam FIB/FEG-SEM.

An automated method for collecting combined three-dimensional (3D) electron backscatter diffraction (EBSD)/energy dispersive spectroscopy (EDS) data sets on a dual-beam focused ion beam (FIB)/field emission gun scanning electron microscope (FEG-SEM) microscope is described. The method uses simple scripting files on the dual beam to move between the EBSD collection and the FIB slicing positions, which are linked to a commercial EBSD data collection programme. The EDS data are collected simultaneously with the EBSD patterns analogous to combined two-dimensional (2D) EBSD/EDS. The technique has been successfully applied to study both the interdiffusion zone between a coating and a substrate and a complex multi-phase coating on a nickel-based superalloy sample. This analysis is shown to enable the complex grain shapes, location of precipitates and phase interconnectivity within these samples to be determined without the ambiguities associated with 2D stereographic analysis.

Expansion and activation of NK cell populations in a gammaherpesvirus infection.

NK cells are an important component of the innate immune response to many virus infections. In particular, they play a major role in control of alpha and beta herpesvirus infections in humans and mice and there is evidence for a protective role in Epstein-Barr virus infection. MHV-68 has been widely used to study gammaherpesvirus pathogenesis and provides a tractable means of investigating the role of NK cells in gammaherpesvirus infections. We have shown that, following MHV-68 infection of mice, the NK cell population is expanded and activated and capable of cytotoxic killing in vitro. However, depletion of NK cells prior to MHV-68 infection did not affect viral loads in vivo. To investigate the possibility that MHV-68 was downregulating NK cell activity in vivo and evading the NK cell response, we infected NK cell-depleted mice with the related virus, MHV-76, which lacks a 9.5 kb region of the genome known to be involved in modulating the host immune response. Infection of NK cell-depleted mice with MHV-76 did not result in increased viral loads indicating that genes within this region do not encode products which modulate NK cell activity.

Microstructural characterization of autogenous laser welds on 316L stainless steel using EBSD and EDS.

This research is concerned with autogenous welding of 316L stainless steel and the microstructure generated by such a process. Autogenous welding does not require a filler material and in this case relies on an initial shallow melt phase to maintain a conduction limited weld. Essentially, a high power laser beam traverses the substrate, with the beam shaped by conventional optics, which produces a Gaussian irradiance distribution; or with a diffractive optical element, used to produce a uniform irradiance distribution. Initial results have shown that due to the nature of the heating cycle, complex microstructures are developed. These fine, complicated microstructures cannot be satisfactorily resolved and quantified using standard optical microscopy techniques. Electron backscatter diffraction (EBSD) and energy dispersive spectroscopy (EDS) have been carried out on a number of different microstructures prepared using a range of welding parameters. It is demonstrated that the simultaneous determination of the chemistry and crystallography is a very useful tool for rapid identification of the different phases formed on solidification as a consequence of varying welding procedures.

Improvements in GORE-TEX vascular graft performance by Carmeda BioActive surface heparin immobilization.

OBJECTIVES: a performance improvement in small-diameter bypass grafts remains a clinical objective. The purpose of the present investigation was to evaluate the potential of enhancing the thromboresistance of ePTFE grafts using a bioactive heparinized graft luminal surface in a canine model. MATERIAL AND METHODS: this study investigated the utility of heparin immobilization onto expanded polytetrafluoroethylene using Carmeda BioActive Surface technology (CBAS-ePTFE) as a means of improving vascular graft thromboresistance. Graft luminal surfaces were covered uniformly with the stably bound, end-point immobilized heparin. RESULTS: acute canine (5 greyhounds) interposition experiments comparing CBAS-ePTFE grafts to control ePTFE grafts showed that CBAS-ePTFE grafts remained patent and had significantly greater thrombus-free luminal surface (p<0.05). In a chronic canine (16 greyhounds) interposition experiment, significantly improved patency (p<0.05) was observed with CBAS-ePTFE grafts compared to controls. Long-term in vivo heparin bioactivity was demonstrated on CBAS-ePTFE grafts explanted between 1 and 12 weeks. On all CBAS-ePTFE grafts, heparin activity levels ranged from 15-25pmol/cm(2) and did not differ significantly (p>0.05). DISCUSSION: these results support the conclusion that a stable, CBAS-ePTFE surface provides improved thromboresistance and improved patency in canine interposition models. Maintenance of heparin catalytic activity on the graft surface in vivo likely contributes to this outcome and holds promise for the utility of this graft surface for clinical applications.

Guided tissue fabrication from periosteum using preformed biodegradable polymer scaffolds.

A successful tissue engineering method for bone replacement would imitate natural bone graft by providing the essential elements for new bone formation using synthetic scaffolds, osteogenic cell populations, and bone induction factors. This is a study of the suitability of various formulations of poly(DL-lactic-co-glycolic acid) (PLGA) foams to provide a tissue conducting scaffold in an ovine model for bone flap fabrication. Three formulations were used of different copolymer ratio and molecular weight. Porous wafers of PLGA were stacked into rectangular chambers (volume 4 cm3) enclosed on five sides. Some chambers also contained autologous morcellized bone graft (MBG). The chambers were inserted with the open face adjacent to the cambium layer of the periosteum in rib beds of seven sheep and harvested after 8 weeks in vivo. Gross and histologic examination of the resulting tissue specimens demonstrated molded units of vascularized tissue generally conforming to the shape of the chambers and firmly attached to the periosteum. Polymer degradation appeared to occur by varying degrees based on polymer formulation. New bone formation was observed only in areas containing MBG. There was no evidence of significant inflammatory reaction or local tissue damage at 8 weeks. We conclude that a PLGA foam scaffold is (1) an efficient conductor of new tissue growth but not osteoinductive, (2) contributes to the shape of molded tissue, and (3) biocompatible when used in this model. Further studies are warranted to develop practical methods to deliver bone induction factors to the system to promote osseous tissue generation throughout the synthetic scaffold.

Hydroxyapatite fiber reinforced poly(alpha-hydroxy ester) foams for bone regeneration.

A process has been developed to manufacture biodegradable composite foams of poly(DL-lactic-co-glycolic acid) (PLGA) and hydroxyapatite short fibers for use in bone regeneration. The processing technique allows the manufacture of three-dimensional foam scaffolds and involves the formation of a composite material consisting of a porogen material (either gelatin microspheres or salt particles) and hydroxyapatite short fibers embedded in a PLGA matrix. After the porogen is leached out, an open-cell composite foam remains which has a pore size and morphology defined by the porogen. By changing the weight fraction of the leachable component it was possible to produce composite foams with controlled porosities ranging from 0.47 +/- 0.02 to 0.85 +/- 0.01 (n = 3). Up to a polymer:fiber ratio of 7:6, short hydroxyapatite fibers served to reinforce low-porosity PLGA foams manufactured using gelatin microspheres as a porogen. Foams with a compressive yield strength up to 2.82 +/- 0.63 MPa (n = 3) and a porosity of 0.47 +/- 0.02 (n = 3) were manufactured using a polymer:fiber weight ratio of 7:6. In contrast, high-porosity composite foams (up to 0.81 +/- 0.02, n = 3) suitable for cell seeding were not reinforced by the introduction of increasing quantities of hydroxyapatite short fibers. We were therefore able to manufacture high-porosity foams which may be seeded with cells but which have minimal compressive yield strength, or low porosity foams with enhanced osteoconductivity and compressive yield strength.

Retinal pigment epithelium cells cultured on synthetic biodegradable polymers.

Alterations in the normal structure or functions of retinal pigment epithelium (RPE) can result in a number of ocular diseases. Implantation of RPE cells cultured on thin, biodegradable polymer films may provide a means of transplanting an organized sheet of RPE cells with distinct apical/basal characteristics for the restoration of normal RPE function. We have investigated the interactions of human RPE cells with different biodegradable polymer films to assess their suitability as substrates for RPE culture. Four biodegradable polymers were used: low molecular weight (MW) 50:50 poly(DL-lactic-co-glycolic acid) (PLGA); high MW 50:50 PLGA; 75:25 PLGA; and poly(L-lactic acid) (PLLA). Polymer film substrates were manufactured using a solvent casting technique. Human fetal RPE cells (10-16 weeks gestational) were plated on the polymer substrates and the cultures assessed with respect to cell attachment and proliferation. Histological and immunohistochemical studies were performed on the cells after 8 days in culture. RPE cells attached to all the polymers studied after 8 h in culture. After 8 h, 80.2 +/- 9.5% and 82.3 +/- 7.9% of the plated cells were attached to substrates of high MW 50:50 PLGA and 75:25 PLGA, respectively. The cells proliferated on all substrates, and there was about a threefold increase in cell number over the 8-day culture period on all the polymers studied. Immunohistochemistry after 8 days in culture demonstrated RPE cells labeled with a distinct reaction product for cytokeratin in the cell cytoplasm. All the polymers studied were suitable for RPE culture; however, high MW 50:50 PLGA and 75:25 PLGA proved to be the best in terms of manufacturing properties, cell attachment, and proliferation. These polymers can provide a suitable substrate for RPE cell culture and hold promise for the subretinal implantation of organized sheets of RPE cells.

Manufacture and characterization of poly(alpha-hydroxy ester) thin films as temporary substrates for retinal pigment epithelium cells.

For many disorders of the retinal pigment epithelium (RPE) for which there are no effective treatments, transplantation of RPE cells may provide a viable means of restoring function. Using a solvent casting technique, we have manufactured thin films of poly(L-lactic acid) and poly(DL-lactic-co-glycolic acid) 75:25 and 50:50. Non-porous, flexible films with controlled thickness as thin as 12 +/- 3 microns and reproducible surface morphologies and flexural properties were produced. Fetal human RPE cells were found to attach to these substrates when cultured in vitro. The films made using this technique may provide a means of transplanting allogeneic RPE cells as a therapy for a number of ocular diseases related to RPE dysfunction.

Fabrication of biodegradable polymer scaffolds to engineer trabecular bone.

We present a novel method for manufacturing three-dimensional, biodegradable poly(DL-lactic-co-glycolic acid) (PLGA) foam scaffolds for use in bone regeneration. The technique involves the formation of a composite material consisting of gelatin microspheres surrounded by a PLGA matrix. The gelatin microspheres are leached out leaving an open-cell foam with a pore size and morphology defined by the gelatin microspheres. The foam porosity can be controlled by altering the volume fraction of gelatin used to make the composite material. PLGA 50:50 was used as a model degradable polymer to establish the effect of porosity, pore size, and degradation on foam mechanical properties. The yield strengths and moduli in compression of PLGA 50:50 foams were found to decrease with increasing porosity according to power law relationships. These mechanical properties were however, largely unaffected by pore size. Foams with yield strengths up to 3.2 MPa were manufactured. From in vitro degradation studies we established that for PLGA 50:50 foams the mechanical properties declined in parallel with the decrease in molecular weight. Below a weight average molecular weight of 10,000 the foam had very little mechanical strength (0.02 MPa). These results indicate that PLGA 50:50 foams are not suitable for replacement of trabecular bone. However, the dependence of mechanical properties on porosity, pore size, and degree of degradation which we have determined will aid us in designing a biodegradable scaffold suitable for bone regeneration.

Mini-review: Islet transplantation to create a bioartificial pancreas.

Donor scarcity precludes the use of pancreatic transplantation to treat type I diabetes. Xenogeneic islet transplantation offers the possibility of overcoming this problem; however, it entails the use of immunoisolation devices to prevent immune rejection of the transplanted islets. These devices consist of a semipermeable membrane, which surrounds the islets and isolates them from the host's immune system, while allowing the passage of insulin and essential nutrients, including glucose. Problems associated with proposed device designs include diffusion limitations, biocompatibility, device retrieval in the event of failure, and mechanical integrity. Microencapsulation appears to be the most promising system of immunoisolation, however, the design of a device suitable for human clinical use remains a challenge. (c) 1994 John Wiley & Sons, Inc.

Relative efficiency of Manitoba traps and adhesive panels for the capture of the common cleg, Haematopota pluvialis (Meigen) (Diptera: Tabanidae).

A comparative study of four traps was made in Argyll, Scotland, over a period of two months for the collection of Haematopota pluvialis. An adhesive trap was shown to be three times more efficient than a standard Manitoba trap, or versions of a Manitoba trap in which the target black ball was replaced by an adhesive black ball or a pair of adhesive panels. The reduced efficiency of the Manitoba trap is attributed to the plastic canopy which appears white in colour, particularly when wet.

Tolerance levels of select stream macroinvertebrates to the Simulium larvicides, Chlorphoxim and decamethrin.

Tolerance levels of six non-target stream macroinvertebrates organophosphorus Simulium lavicide, chlorphoxim, and the synthetic pyrethroid decamethrin, were determined using rapid through-flow laboratory test vessels. A 1-hour exposure followed by 24-hour mortality recorded was adopted as a standard basis for comparison with the target. Simulium larvae. Additional comparisons between the reactions of Simulium larvae and non-targets to chlorphoxim were carried out on the basis of 15-minute exposures in a miniature stimulated stream or experimental channel. At the lowest concentration sufficient to produce 95% mortality in late instar Simulium larvae, decamethrin was highly lethal to all non-targets tested, with the exception of Ephemerella. Baetis and Gammarus proved to have a very high susceptibility to decamethrin, the former still recording 50% mortalities after exposure to concentrations as low as 0.005 parts per billion/1-hour. In the case of chlorphoxim, all the non-targets were found to be significantly more tolerant than Simulium larvae under identical conditions. The margin was narrowest in the case of Baetis (LC95 0.005 ppm as compared with 0.003 ppm for Simulium but wide enough in the case of the predaceous non-targets - Hydropsyche, Rhyacophila and Agrion - to suggest high survival at Simulium larvicide dosages. The results were compared with these previously obtained with other Simulium larvicides such as Abate, chlorpyrifos and permethrin under identical laboratory conditions. The applicability of these techniques to Simulium damnosum and its associated non-targets in West Africa is discussed, as also is the need for a comparable laboratory phase of evaluation in that programme.

Relative toxicity of decamethrin, chlorphoxim and temephos (abate) to simulium larvae.

Tolerance levels Simulium larvae (S. ornatum and S. equinum) to chlorphoxim and decamethrin, relative to those with Abate, were determined in rapid through-flow laboratory test vessels; a 1-hour exposure followed by 24-hour mortality observations being the standard of comparison of the three chemicals. In addition, a further comparison between chlorphoxim and Abate was made on the basis of a 15-minute exposure in a miniature simulated stream, with the same mortality criteria. On the basis of LC95/1-hour data the results showed that decamethrin was approximately 4 times as lethal as chlorphoxim, and approximately 20 times more than Abate. On the basis of a 15-minute exposure, chlorphoxim was found to be even more toxic than Abate--approximately 10 times more--than with the 1-hour exposure--viz 5 times more. Concentration/mortality curves indicate that with both the organophosphorus larvicides there is a critical zone where mortality increases sharply with increase in concentration of toxinant. With decamethrin, there is a more uniform rate of increase in mortality with increasing concentration, these differences being illustrated by comparison at both LC50 and LC95 levels. These findings are discussed firstly, in relation to other laboratory test methods in use, and secondly with reference to the scarcity of comparable laboratory data for Simulium damnosum in the Onchosceriasis Control Programme (OCP) in the Volta River basin area of West Africa.

Relative susceptibility of stream macroinvertebrates to temephos and chlorpyrifos, determined in laboratory continuous-flow systems.

Laboratory techniques are described for evaluating the lethal and behavioral impact of pesticides on a range of stream macroinvertebrates under continuous through-flow and simulated stream conditions. The same basic test unit has been used, with slight modifications, to study the reactions of both Simulium larvae and non-target stream invertebrates. On the basis of a standard 1-hr exposure period to different concentrations followed by a 24-hr holding period in a continuous flow of clean water, different test organisms showed wide and consistent differences in tolerance to each of the two insecticides tested. The widest difference between two organisms occurred in the case of the Amphipod, Gammarus pulex (LC90-95, greater than 1 ppm) which was found to be about 5000 x more tolerant to temephos than are nymphs of the mayfly, Baetis rhodani. (LC 90-95, 0.001-0.002 ppm) The widest difference in the reactions of any one species is shown on the part of Gammarus which is about 100 times more susceptible to chlorpyrifos (LC 90-95, 0.05-0.1 ppm) than to temephos. The susceptibility levels of other indicator species such as Agrion, Hydropsyche, Brachycentrus, Ephemera, etc. are discussed in relation to susceptibility levels of Simulium larvae under the same test conditions, and also in relation to current field dosages of the two insecticides in practical and experimental Simulium control.