Limit case analysis of the "stable indenter velocity" method for obtaining creep stress exponents from constant load indentation creep tests.

This study concerns a commonly-used procedure for evaluating the steady state creep stress exponent, n , from indentation data. The procedure involves monitoring the indenter displacement history under constant load and making the assumption that, once its velocity has stabilised, the system is in a quasi-steady state, with stage II creep dominating the behaviour. The stress and strain fields under the indenter are represented by "equivalent stress" and "equivalent strain rate" values. The estimate of n is then obtained as the gradient of a plot of the logarithm of the equivalent strain rate against the logarithm of the equivalent stress. Concerns have, however, been expressed about the reliability of this procedure, and indeed it has already been shown to be fundamentally flawed. In the present paper, it is demonstrated, using a very simple analysis, that, for a genuinely stable velocity, the procedure always leads to the same, constant value for n (either 1.0 or 0.5, depending on whether the tip shape is spherical or self-similar). This occurs irrespective of the value of the measured velocity, or indeed of any creep characteristic of the material. It is now clear that previously-measured values of n , obtained using this procedure, have varied in a more or less random fashion, depending on the functional form chosen to represent the displacement-time history and the experimental variables (tip shape and size, penetration depth, etc.), with little or no sensitivity to the true value of n .

Cell structure, stiffness and permeability of freeze-dried collagen scaffolds in dry and hydrated states.

Scaffolds for tissue engineering applications should be highly permeable to support mass transfer requirements while providing a 3-D template for the encapsulated biological cells. High porosity and cell interconnectivity result in highly compliant scaffolds. Overstraining occurs easily with such compliant materials and can produce misleading results. In this paper, the cell structure of freeze-dried collagen scaffolds, in both dry and hydrated states, was characterised using X-ray tomography and 2-photon confocal microscopy respectively. Measurements have been made of the scaffold's Young's modulus using conventional mechanical testing and a customised see-saw testing configuration. Specific permeability was measured under constant pressure gradient and compared with predictions. The collagen scaffolds investigated here have a coarse cell size (∼100-150 µm) and extensive connectivity between adjacent cells (∼10-30 µm) in both dry and hydrated states. The Young's modulus is very low, of the order of 10 kPa when dry and 1 kPa when hydrated. There is only a single previous study concerning the specific permeability of (hydrated) collagen scaffolds, despite its importance in nutrient diffusion, waste removal and cell migration. The experimentally measured value reported here (5 × 10(-)(10)m(2)) is in good agreement with predictions based on Computational Fluid Dynamics simulation and broadly consistent with the Carman-Kozeny empirical estimate. It is however about three orders of magnitude higher than the single previously-reported value and this discrepancy is attributed at least partly to the high pressure gradient imposed in the previous study. STATEMENT OF SIGNIFICANCE: The high porosity and interconnectivity of tissue engineering scaffolds result in highly compliant structures (ie large deflections under low applied loads). Characterisation is essential if these scaffolds are to be systematically optimised. Scaffold overstraining during characterisation can lead to misleading results. In this study, the stiffness (in dry and hydrated states) and specific permeability of freeze-dried collagen scaffolds have been measured using techniques customised for low stiffness structures. The scaffold cell structure is investigated using X-ray computed tomography, which has been applied previously to visualise such materials, without extracting any structural parameters or simulating fluid flow. These are carried out in this work. 2-photon confocal microscopy is used for the first time to study the structure in hydrated state.

An accelerated buoyancy adhesion assay combined with 3-D morphometric analysis for assessing osteoblast adhesion on microgrooved substrata.

An accelerated negative buoyancy method has been developed to assess cell adhesion strength. This method has been used in conjunction with 3-D morphometric analysis to understand the effects of surface topology on cell response. Aligned micro-grooved surface topographies (with a range of groove depths) were produced on stainless steel 316L substrates by laser ablation. An investigation was carried out on the effect of the micro-grooved surface topography on cell adhesion strength, cell and nucleus volumes, cell phenotypic expression and attachment patterns. Increased hydrophobicity and anisotropic wettability was observed on surfaces with deeper grooves. A reduction was noted in cell volume, projected areas and adhesion sites for deeper grooves, linked to lower cell proliferation and differentiation rates and also to reduced adhesion strength. The results suggest that the centrifugation assay combined with three-dimensional cell morphometric analysis has considerable potential for obtaining improved understanding of the cell/substrate interface.

Cell adhesion to plasma electrolytic oxidation (PEO) titania coatings, assessed using a centrifuging technique.

The adhesion of bovine chondrocytes and human osteoblasts to three titania-based coatings, formed by plasma electrolytic oxidation (PEO), was compared to that on uncoated Ti-6Al-4V substrates, and some comparisons were also made with plasma sprayed hydroxyapatite (HA) coatings. This was done using a centrifuge, with accelerations of up to 160,000 g, so as to induce buoyancy forces that created normal or shear stresses at the interface. It is shown that, on all surfaces, it was easier to remove cells under normal loading than under shear loading. Cell adhesion to the PEO coatings was stronger than that on Ti-6Al-4V and similar to that on HA. Cell proliferation rates were relatively high on one of the PEO coatings, which was virtually free of aluminium, but low on the other two, which contained significant levels of aluminium. It is concluded that the Al-free PEO coating offers promise for application to prosthetic implants.

Porous materials for thermal management under extreme conditions.

A brief analysis is presented of how heat transfer takes place in porous materials of various types. The emphasis is on materials able to withstand extremes of temperature, gas pressure, irradiation, etc. i.e. metals and ceramics, rather than polymers. A primary aim is commonly to maximize either the thermal resistance (i.e. provide insulation) or the rate of thermal equilibration between the material and a fluid passing through it (i.e. to facilitate heat exchange). The main structural characteristics concern porosity (void content), anisotropy, pore connectivity and scale. The effect of scale is complex, since the permeability decreases as the structure is refined, but the interfacial area for fluid-solid heat exchange is, thereby, raised. The durability of the pore structure may also be an issue, with a possible disadvantage of finer scale structures being poor microstructural stability under service conditions. Finally, good mechanical properties may be required, since the development of thermal gradients, high fluid fluxes, etc. can generate substantial levels of stress. There are, thus, some complex interplays between service conditions, pore architecture/scale, fluid permeation characteristics, convective heat flow, thermal conduction and radiative heat transfer. Such interplays are illustrated with reference to three examples: (i) a thermal barrier coating in a gas turbine engine; (ii) a Space Shuttle tile; and (iii) a Stirling engine heat exchanger. Highly porous, permeable materials are often made by bonding fibres together into a network structure and much of the analysis presented here is oriented towards such materials.

Purification and lipid dependence of the recombinant hyaluronan synthases from Streptococcus pyogenes and Streptococcus equisimilis.

The two hyaluronan synthases (HASs) from Streptococcus pyogenes (spHAS) and Streptococcus equisimilis (seHAS) were expressed in Escherichia coli as recombinant proteins containing His6 tails. Both enzymes were expressed as major membrane proteins, accounting for approximately 5-8% of the total membrane protein. Using nickel chelate affinity chromatography, the HASs were purified to homogeneity from n-dodecyl beta-D-maltoside extracts. High levels of HAS activity could be achieved only if the purified enzymes were supplemented with either bovine or E. coli cardiolipin (CL), although bovine CL gave consistently greater activity. Mass spectroscopic analysis revealed that the fatty acid compositions of these two CL preparations did not overlap. The two HAS enzymes showed similar but distinct activation profiles with the 10 other lipids tested. For example, phosphatidic acid and phosphatidylethanolamine stimulated seHAS, but not spHAS. Phosphatidylserine stimulated both enzymes. spHAS appears to be more CL-specific than seHAS, although both purified enzymes still contain endogenous CL that can not easily be removed. Both seHAS and spHAS were inhibited by phosphatidylcholine, sphingomyelin, and sulfatides and were not substantially stimulated by cerebrosides, phosphatidylglycerol, or phosphatidylinositol. With both HASs, CL increased the Km for UDP-GlcUA, but decreased the Km for UDP-GlcNAc and gave an overall stimulation of Vmax. A kinetic characterization of the two membrane-bound and purified HASs is presented in the accompanying paper (Tlapak-Simmons, V. L., Baggenstoss, B. A., Kumari, K., Heldermon, C., and Weigel, P. H. (1999) J. Biol. Chem. 274, 4246-4253). Both purified HASs became inactive after storage for approximately 5 days at 4 degreesC. Both purified enzymes also lost activity over 4-5 days when stored at -80 degreesC in the presence of CL, but reached a level of activity that then slowly decreased over a period of months. Although the purified enzymes stored in the absence of CL at -80 degreesC were much less active, the enzymes retained this same low level of activity for at least 5 weeks. When both spHAS and seHAS were stored without CL at -80 degreesC, even after 2 months, they could be stimulated by the addition of bovine CL to approximately 60% of the initial activity of the freshly purified enzyme.

Plasma sprayed hydroxyapatite coatings on titanium substrates. Part 1: Mechanical properties and residual stress levels.

Hydroxyapatite (HA) coatings have been sprayed on to substrates of Ti-6Al-4V, using a range of input power levels and plasma gas mixtures. Coatings have also been produced on substrates of mild steel and tungsten, in order to explore certain aspects of the mechanical behaviour of HA without the complication of yielding or creep in the substrate. Studies have been made of the phase constitution, porosity, degree of crystallinity, OH ion content, microstructure and surface roughness of the HA coatings. The Young's moduli in tension and in compression were evaluated by the cantilever beam bend test using a tungsten/HA composite beam. The flexural Young's modulus was determined using a free-standing deposit under the same test. Adhesion was characterised using the single-edge notch-bend test; this is considered superior to the tensile bond strength test in common use. Measured interfacial fracture energies were of the order 1-10 J m(-2). Stress levels were investigated using specimen curvature measurements in conjunction with a numerical process model. The quenching stress for HA was measured to be about 10-25 MPa and the residual stress level in HA coatings at room temperature are predicted to lie in the approximate range of 20-40 MPa (tensile). These residual stresses could be reduced in magnitude by maintaining the substrate at a low temperature (possibly below room temperature) during spraying and it may be worthwhile to explore this. Ideally, the HA coating should have low porosity, high cohesive strength, good adhesion to the substrate, a high degree of crystallinity and high chemical purity and phase stability. In practice, such combinations are rather difficult to achieve by just varying the spraying parameters.

Plasma sprayed hydroxyapatite coatings on titanium substrates. Part 2: optimisation of coating properties.

Heat treatment and the introduction of a Ti bond coat have been applied to hydroxyapatite (HA) coatings sprayed using different plasma powers and gas mixtures. Attempts were made in this way to achieve optimal coating properties for orthopaedic implants. In particular, the effects on the degree of crystallinity, the adhesion, the OH ion content and the purity were evaluated. Heat treatment at 700 C for 1 h in air proved to be effective in increasing the crystallinity, regaining the OH- ion and removing other non-HA compounds, although it caused a significant decrease in the degree of adhesion (interfacial fracture toughness) for those specimens sprayed at high powers. This heat treatment was found to induce significant transformation of amorphous HA to the crystalline form, while not detrimentally changing the properties of the underlying Ti-6Al-4V substrates. Precoating with a 100 microm Ti layer increased the adhesion of the HA coatings on Ti-6Al-4V substrates, primarily by providing a rougher surface and promoting better mechanical interlocking. Changes in coating properties during immersion in biological fluids were also studied and were found to depend critically on the chemical composition of the fluids. Small precipitates formed on the coating surfaces when immersed in Ringers solution. These might account for the apparent drop in the degree of crystallinity when measured using X-ray diffraction. A significant drop in the interfacial adhesion was found for those coatings sprayed at high powers. This could be offset by prior precoating with a titanium bond coat and suitable heat treatment. In summary, the following processing sequence is suggested in order to achieve optimum coating properties: precoating the substrate with a layer of Ti (approximately 100 microm), spraying HA at a sufficiently high-power level (depending on particle size and gas mixture) and heat treatment at 700 degrees C for 1 h in air.