Daryl R. Williams

The use of murine embryonic stem cells, alginate encapsulation, and rotary microgravity bioreactor in bone tissue engineering

The application of embryonic stem cells (ESCs) in bone tissue engineering and regenerative medicine requires the development of suitable bioprocesses that facilitate the integrated, reproducible, automatable production of clinically-relevant, scaleable, and integrated bioprocesses that generate sufficient cell numbers resulting in the formation of three-dimensional (3D) mineralised, bone tissue-like constructs. Previously, we have reported the enhanced differentiation of undifferentiated mESCs toward the osteogenic lineage in the absence of embryoid body formation. Herein, we present an efficient and integrated 3D bioprocess based on the encapsulation of undifferentiated mESCs within alginate hydrogels and culture in a rotary cell culture microgravity bioreactor. Specifically, for the first 3 days, encapsulated mESCs were cultured in 50% (v/v) HepG2 conditioned medium to generate a cell population with enhanced mesodermal differentiation capability followed by osteogenic differentiation using osteogenic media containing ascorbic acid, beta-glycerophosphate and dexamethasone. 3D mineralised constructs were generated that displayed the morphological, phenotypical, and molecular attributes of the osteogenic lineage, as well mechanical strength and mineralised calcium/phosphate deposition. Consequently, this bioprocess provides an efficient, automatable, scalable and functional culture system for application to bone tissue engineering in the context of macroscopic bone formation.

The Use of Inverse Phase Gas Chromatography to Measure the Surface Energy of Crystalline, Amorphous, and Recently Milled Lactose

AbstractPurpose. To assess differences in surface energy due to processing induced disorder and to understand whether the disorder dominated the surfaces of particles. Methods. Inverse gas chromatography was used to compare the surface energies of crystalline, amorphous, and ball milled lactose. Results. The milling process made ca 1% of the lactose amorphous, however the dispersive contribution to surface energy was 31.2, 37.1, and 41.6 mJ m−2 for crystalline, spray dried and milled lactose, respectively. A physical mixture of crystalline (99%) and amorphous (1%) material had a dispersive surface energy of 31.5 mJ m−2. Conclusion. Milling had made the surface energy similar to that of the amorphous material in a manner that was very different to a physical mixture of the same amorphous content. The milled material will have similar interfacial interactions to the 100% amorphous material.

Inverse Gas Chromatographic Method for Measuring the Dispersive Surface Energy Distribution for Particulates

Inverse gas chromatography (IGC) is a widely used method for determining the dispersive component of the surface energy (gamma s (d)) of particulate and fibrous solids. Such measurements are normally conducted at very low solute concentrations (infinite dilution), and they result in a single numerical value of gamma s (d) for homogeneous materials which exhibit Henrys Law adsorption behavior. However, many real solid surfaces are heterogeneous and this may be demonstrated by the nonlinear isotherms obtained at low solute surface coverages resulting in reported gamma s (d) values which are not unique. This paper presents a new method for determining of gamma s (d) distributions as a function of the solute surface coverage using adsorption isosteres for an homologous series of hydrocarbon adsorbates. gamma s (d) distributions reported here were successfully determined using two different solid materials (glass beads and alumina particles) up to typical surface coverages of approximately 10% and clearly show significant variations in gamma s (d) with solute surface coverage. The effects of sample aging and pretreatment also exhibited clear differences in the gamma s (d) distributions obtained. gamma s (d) was determined using both the Dorris-Gray and Schultz methods, with the Dorris-Gray method exhibiting a much lower experimental error. It was established that the errors associated with this statistical measurement of surface energy were strongly dependent on the quality of the experimental data sets obtained. R (2) for the linearity of fit of the retention data to the Dorris-Gray gamma s (d) analysis was found to be a valid criterion for predicting the robustness of gamma s (d) distributions obtained. Detailed discussions of other critical experimental and analysis factors relevant to this methodology, as well as the reproducibility of gamma s (d) profiles are also presented. This paper establishes that IGC can be used for determining the gamma s (d) distributions of particulate solids and is demonstrated that this method is very useful way for studying the surface energy heterogeneity of complex particulate solids.

The use of inverse phase gas chromatography to study the change of surface energy of amorphous lactose as a function of relative humidity and the processes of collapse and crystallisation

The purpose of this study was to assess the effect of relative humidity (RH) on the surface energy of amorphous lactose. Two samples of amorphous lactose were investigated; a spray dried 100% amorphous material and a ball milled sample of crystalline lactose. The milled sample had less than 1% amorphous content by mass, but on investigation at 0% RH, yielded surface energies comparable to those obtained from the 100% amorphous material, indicating that the surface was amorphous. The effect of increasing humidity was to reduce the dispersive surface energy of the two samples from 36.0 +/- 0.14 and 41.6 +/- 1.4 mJ m(-2) at 0% RH for the spray dried and milled samples respectively, to a value comparable to that obtained for the crystalline alpha-lactose monohydrate of 31.3 +/- 0.41 mJ m(-2). The change in surface energy due to water sorption was only reversible up to 20% RH; after exposure to higher RH values subsequent drying did not result in a return to the original surface energy of the amorphous form. This shows that the surface is reorganising as the glass transition temperature (Tg) is reduced, even though the sample has not collapsed or crystallised. It was possible to follow the collapse behaviour in the column with ease, using a number of different methods.

Fourier Transform Infrared Imaging of Human Hair with a High Spatial Resolution Without the Use of a Synchrotron

The cross-section of a human hair has been imaged for the first time using the micro attenuated total reflection (ATR) Fourier transformed infrared (FT-IR) method in combination with a focal plane array (FPA) detector. A rigorous approach was applied to determine the spatial resolution, namely, measuring the distance over which the band absorbance changes from 95 to 5% of the maximum absorbance when passing through a sharp interface. The measured value for IR transmission was ∼16 μm, while the value obtained using ATR imaging was ∼5 μm. The enhanced spatial resolution achieved by this method allows the medulla of the hair (∼8 μm in diameter) to be imaged clearly without the need for a synchrotron source. The spatial resolution of transmission and ATR imaging is compared, and advantages of ATR imaging are discussed.

Effect of Milling on Particle Shape and Surface Energy Heterogeneity of Needle-Shaped Crystals

PurposeMilling and micronization of particles are routinely employed in the pharmaceutical industry to obtain small particles with desired particle size characteristics. The aim of this study is to demonstrate that particle shape is an important factor affecting the fracture mechanism in milling.MethodsNeedle-shaped crystals of the β polymorph of D-mannitol were prepared from recrystallization in water. A portion of the recrystallized materials was ball-milled. Unmilled and milled sieved fractions of recrystallized D-mannitol were analyzed by dynamic image analysis (DIA) and inverse gas chromatography (IGC) at finite concentration to explain the breakage/fracture behavior.ResultsIn the process of ball-milling, D-mannitol preferentially fractured along their shortest axis, exposing (011) plane with increased hydrophilicity and increased bounding rectangular aspect ratio. This is in contrary to attachment energy modeling which predicts a fracture mechanism across the (010) plane with increased hydrophobicity, and small change in particle shape.ConclusionsCrystal size, and more importantly, crystal shape and facet-specific mechanical properties, can dictate the fracture/cleavage behavior of organic crystalline materials. Thorough understanding of the crystal slip systems, combining attachment energy prediction with particle shape and surface characterization using DIA and IGC, are important in understanding fracture behavior of organic crystalline solids in milling and micronization.

Determination of surface heterogeneity of d-mannitol by sessile drop contact angle and finite concentration inverse gas chromatography

The sensitivity of two techniques in tracking changes in surface energetics was investigated for a crystalline excipient, D-mannitol. Macroscopic crystals of D-mannitol were grown from saturated water solution by slow cooling, and sessile drop contact angle was employed to measure the anisotropic surface energy. The facet-specific surface energy was consistent with localised hydroxyl group concentrations determined by X-ray photoelectron spectroscopy (XPS), and was also in excellent agreement with the surface energy distribution of the powder form of mannitol measured via a new methodology using inverse gas chromatography (IGC) at finite concentrations. The gamma(SV)(d) was found to vary between 39.5 mJ/m(2) and 44.1 mJ/m(2) for contact angle and between 40 mJ/m(2) and 49 mJ/m(2) for IGC measurements. We report here, a high level of surface heterogeneity on the native mannitol crystal surfaces. When the surfaces of both D-mannitol samples (powder and large single crystals) were modified by dichlorodimethylsilane to induce surface hydrophobicity, both IGC and contact angle revealed a homogeneous surface due to functionalisation of mannitol crystal surface with methyl groups resulting in gamma(SV)(d) of approximately 34 mJ/m(2). It was shown that both IGC and contact angle techniques are able to detect surface chemical variations and detailed surface energetic distribution.

Anisotropic surface chemistry of crystalline pharmaceutical solids.

The purpose of this study was to establish the link between the wetting behavior of crystalline pharmaceutical solids and the localized surface chemistry. A range of conventional wetting techniques were evaluated and compared with a novel experimental approach: sessile drop contact angle measurements on the individual facets of macroscopic (>1 cm) single crystals. Conventional measurement techniques for determining surface energetics such as capillary rise and sessile drops on powder compacts were found not to provide reliable results. When the macroscopic crystal approach was used, major differences for advancing contact angles, θa, of 0 waterwere observed—as low as 16° on facet (001) and as high as 68° on facet (010) of form I paracetamol. θa trends were in excellent agreement with X-ray photoelectron spectroscopy surface composition and known crystallographic structures, suggesting a direct relationship to the local surface chemistry. Inverse gas chromatography (IGC) was further used to probe the surface properties of milled and unmilled samples, as a function of particle size. IGC experiments confirmed that milling exposes the weakest attachment energy facet, with increasing dominance as particle size is reduced. The weakest attachment energy facet was also found to exhibit the highest θa for water and to be the 0 most hydrophobic facet. This anisotropic wetting behavior was established for a range of crystalline systems: paracetamol polymorphs, aspirin, and ibuprofen racemates. θa was 0 found to be very sensitive to the local surface chemistry. It is proposed that the hydrophilicity/hydrophobicity of facets reflects the presence of functional groups at surfaces to form hydrogen bonds with external molecules.

Computing the Surface Energy Distributions of Heterogeneous Crystalline Powders

Inverse Gas Chromatography (IGC) provides a powerful tool for assessing the surface energy of powders by using different chemicals as probes. Finite Dilution Inverse Gas Chromatography (FD-IGC) allows surface energy measurements in a range of probe surface coverages. For heterogeneous materials the relationship between measured surface energy and surface coverage provides information about the surface energy distribution of the material. However, existing methods of analysing FD-IGC data to determine surface energy distributions are based on inappropriate assumptions. Alternative analysis techniques do not determine the materials surface energy distribution but instead provide information about variations in probe-substrate interactions rather than true material properties of the substrate. A more robust method for computing the surface energy distribution of heterogeneous powders is needed. In the pharmaceutical industry, due to the heterogeneous nature of crystalline powders, the surface energy and its distribution plays an important role in the performance of dry powder inhalers, powder mixing, and the cohesion of compressed tablets. Here a new approach for interpreting IGC data, based on more realistic thermodynamic assumptions, is proposed. Using this approach the surface energy distribution of the substrate is determined by fitting the results of simulated experiments to experimental IGC data. The simulation-fitting approach is demonstrated for mannitol and lactose powders. The addition of fines to coarse lactose powder is shown to have a clear effect on the powders surface energy distribution whilst the surface energy distributions of different particle size fractions of mannitol do not show significant differences in their surface energy distributions.

Characterization of porous materials by finite concentration inverse gas chromatography

Abstract Inverse gas chromatography (IGC) is a well established tool for the measurement of physico-chemical properties of solid materials at infinite dilution as well as for the determination of isotherms and surface heterogeneity at higher concentration. A novel combination of IGC and thermal desorption methods allows an advanced characterization of porous materials with organic vapors. Different porous materials are investigated by a combined IGC/flash thermodesorption. The method is applied to separate the micropore contribution to adsorption from the mesopores and the outer surface area using the difference in the adsorption mechanism.